International CEEPUS Summer School on Complex Diseases 2016 Malignant Diseases August 17th – August 23rd, 2016 Ljubljana, Slovenia Editors-in-chief: Irena Prodan Žitnik Janja Marc Editorial Board: Jerka Dumić Klemen Kodrič Jelena Kotur-Stevuljević Tilen Kranjc Jana Nekvindova Besim Prnjavorac CIP - Kataložni zapis o publikaciji Narodna in univerzitetna knjižnica, Ljubljana 616-006(082) INTERNATIONAL CEEPUS Summer School on Complex Diseases (2016 ; Ljubljana) Malignant diseases / International CEEPUS Summer School on Complex Diseases, August 17th - August 23rd, 2016, Ljubljana, Slovenia ; [urednici Irena Prodan Žitnik, Janja Marc]. - El. knjiga. - Ljubljana : Fakulteta za farmacijo, 2016 ISBN 978-961-6378-72-7 (pdf) 1. Gl. stv. nasl. 2. Prodan Žitnik, Irena 286565376 Table of Contents Table of Contents ....................................................................................................................... 1 Molecular mechanisms in carcinogenesis .................................................................................. 3 Biochemical and inflammatory aspects of tumorigenesis ......................................................... 8 Tumor markers in clinical practice ........................................................................................... 14 Targeted therapies and predictive markers in cancer ............................................................. 16 Therapeutic approaches to different cancer diseases ............................................................. 20 Clinical Pharmacy Services in Oncology ................................................................................... 22 Immunotherapy of cancer ........................................................................................................ 24 Life science research from a postdoc prospective ................................................................... 28 Breast cancer ............................................................................................................................ 30 Luminal A and luminal B type of breast cancer .................................................................... 30 HER2 positive type of breast cancer ..................................................................................... 34 Basal-like type of breast cancer ............................................................................................ 37 Gastrointestinal cancer ............................................................................................................ 41 Gastric cancer (GC) ............................................................................................................... 41 Colon cancer ......................................................................................................................... 43 Liver cancer – hepatocellular carcinoma .............................................................................. 47 Lung cancer .............................................................................................................................. 53 Epidemiology and Molecular Basis of Lung Cancer .............................................................. 53 Diagnosing lung cancer ......................................................................................................... 55 Therapy of lung cancer ......................................................................................................... 57 Melanoma ................................................................................................................................ 61 Epidemiology and diagnostics of malignant melanoma ...................................................... 61 Treatment of malignant melanoma ..................................................................................... 67 Melanomas: resistance to RAF inhibition; paradoxical activation of MAPK pathway; adverse effect of BRAF inhibition ......................................................................................... 70 Bone metastatic disease .......................................................................................................... 74 Bone metastatic disease – Epidemiology and molecular mechanisms ................................ 74 Bone metastatic disease – Diagnosis and treatment ........................................................... 78 Leukemias and lymphomas ...................................................................................................... 82 Leukemias ............................................................................................................................. 82 Lymphomas........................................................................................................................... 89 1 Prostate cancer ........................................................................................................................ 94 What you need to know about prostate cancer and current ways of its diagnostics ......... 94 Molecular basis of prostate cancer .................................................................................... 100 Treatment of prostate cancer ............................................................................................ 104 Summer school lecturers and participants ............................................................................ 107 SUPPLEMENT – Lectures handouts ........................................................................................ 109 2 Molecular mechanisms in carcinogenesis Prepared by Jerka Dumić University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Biochemistry and Molecular Biology, Ante Kovačića 1, HR-10000 Zagreb, Croatia; jdumic@pharma.hr Cancer is a complex genetic disease and it develops as a multi-step process during which accumulation of nonlethal mutations results in malignant transformation of the cells. Due to the fact that each cancer is characterized by distinctive set of mutations and consequently it is phenotypically specific, it should be considered as a unique disease. That genetic/phenotypic specificity of cancers makes lot of troubles for clinicians and seeks for personalized approach in treatment and therapy. This is why, understanding of molecular mechanisms underlying carcinogenesis is of utmost importance for development new drugs and personalized therapeutic approaches. Consequently, personalized medicine at the moment achieves the biggest success in oncology. Carcinogenesis is complex, multi-step process that leads to transformation of normal cells which acquire physiological characteristics that together determine malignant phenotype. Accumulation of mutations may last for years, thus the tumor incidence increases with age and the prevalence of tumors is much higher in population over 60 years of age. Mutations are the consequence of the action of environmental agents ( e.g. chemicals, radiation, or biological agents) or may be inherited in the germ line. In some cases, mutations may be spontaneous and stochastic. Thus, it is difficult to point out the precise cause of cancer, yet it is know that the tumor initiation is a result of genetic change that leads to proliferation and uncontrolled growth. Tumor progression continues and by time particular cells may acquire additional mutations which make them more malignant. Finally, the cell with highest capacity to proliferate and to invade, in addition to other characteristics, become a source of all cells which lately form tumor (clonal selection), i.e. tumors are monoclonal. In addition, tumor cells acquire ability to escape from immune system attack. As it was mentioned above, nonlethal mutations are the primary cause of carcinogenesis. There are three main classes of carcinogenic agents: ( i) chemicals; ( ii) radiation ( iii) microbial agents. While chemicals and radiation are well documented causes of cancer in humans, oncogenic viruses are involved in development of at least some human tumors. Chemical carcinogens: Most chemical carcinogens are mutagenic and considered to be initiators of carcinogenesis. They contain highly reactive groups that interact with DNA (as well as proteins and RNA). Important targets of chemical carcinogens are oncogenes and tumor suppressors (such as Ras and p53) ( e.g. aflatoxin B1 causes characteristic mutations in the p53 gene). Another group of chemicals, promotors ( e.g. , hormones, phenols, and drugs), which by themselves are non- tumorigenic (mutagenic), may induce cell proliferation and thus augment carcinogenicity of some chemical carcinogens. It seems that an initiator may cause the mutational activation of an oncogene while subsequent application of promoters leads to clonal expansion of initiated (mutated) cells. There are two types of chemical carcinogens ( i) direct-acting carcinogens which require no metabolic conversion to become carcinogenic. They are in general weak carcinogens but are important because some of them are cancer chemotherapeutic drugs (e.g., alkylating agents); ( ii) indirect-acting agents - chemicals that require metabolic conversion to an ultimate carcinogen (polycyclic hydrocarbons e.g. benzopyrene, formed in the high-temperature combustion of tobacco in cigarette smoking or during the process of broiling meats; aromatic amines and azo dyes e.g. β-naphthylamine; aflatoxin B1 produced by some strains of Aspergillus, a mold that grows on improperly stored grains and nuts; vinyl chloride, arsenic, nickel, chromium, and insecticides are potential carcinogens in the workplace and about the house; nitrites - used as food preservatives. 3 Radiation: Radiation (UV rays of sunlight, x-rays, nuclear fission, radionuclides) causes chromosome breakage, translocations, and, less frequently, point mutations. Biologically, DNA breaks seem to be the most important form of DNA damage caused by radiation. UV light has the ability to damage DNA by forming pyrimidine dimers, which can be repaired by the nucleotide excision repair pathway. In case of extensive UV light exposure, due to overwhelmed repair systems, it can cause skin cancer. Microbial agents: Several viruses have been linked with human cancer ( i) oncogenic RNA viruses e.g. human T-cell leukaemia virus-1 (HTLV-1) , associated with a form of T-cell leukaemia/lymphoma; ( ii) oncogenic DNA viruses (human papillomavirus (HPV), Epstein-Barr virus (EBV), Kaposi sarcoma herpesvirus (human herpesvirus 8, HHV8), hepatitis B virus (HBV); and (iii) Helicobacter pylori. The precise molecular mechanisms underlying the carcinogenetic effects of different virus type are complex and still under investigation. Once transformed to malignant form, cells comprise several fundamental phenotypic characteristics: Autocrine stimulation of growth and/or self-sufficiency in growth signals: Tumours have the capacity to proliferate without external stimuli, usually as a consequence of oncogene activation or they can produce all needed growth factors, by them self. Insensitivity to growth-inhibitory signals: Tumors may not respond to the molecules that inhibits normal cells growth such as transforming growth factor β (TGF-β) and direct inhibitors of cyclin- dependent kinases (CDKIs). Evasion of apoptosis: Tumours may avoid programmed cell death, by inactivation of tumor suppressor p53 or activation of anti-apoptotic genes. Limitless replicative potential and disturbed cell differentiation: Tumor cells have unrestricted proliferative capacity, avoiding cellular senescence and mitotic catastrophe. In some cases, they lose ability to differentiate, so undifferentiated cells uncontrolledly proliferate. Sustained angiogenesis: Tumor cells, like normal cells, need nutrients and oxygen for their growth, thus they have to stimulate angiogenesis. Ability to invade and metastasize: Spreading into environment is essential for tumor growth, so tumor cells produce high quantity of proteases. Tumor metastases depend on processes that are intrinsic to the cell or are initiated by signals from the tissue environment. Due to a loss of contact inhibition and reduced adhesiveness tumor cells can easily detach from tumor mass and enter into the lymph or blood circulation, thus spreading around the body. Defects in DNA repair: Tumours may fail to repair DNA damage caused by carcinogens or incurred during unregulated cellular proliferation, leading to genomic instability and mutations in proto- oncogenes and tumor suppressor genes. Four classes of normal regulatory genes are the principal targets of genetic damage. – proto-oncogenes – tumor suppressor genes – genes that regulate programmed cell death (apoptosis) – DNA repair genes. While mutant alleles of proto-oncogenes are considered dominant, both alleles of the tumor suppressor genes must be damaged before transformation can occur. However, in some cases loss of 4 a single allele of a tumor suppressor gene reduces level or activity of the protein enough that the brakes on cell proliferation and survival are released. Genes that regulate apoptosis may behave as proto-oncogenes or tumor suppressor genes. Mutations of DNA repair genes do not directly transform cells by affecting proliferation or apoptosis. Instead, DNA-repair genes affect cell proliferation or survival indirectly by influencing the ability of the organism to repair nonlethal damage in other genes, including proto-oncogenes, tumor suppressor genes, and genes that regulate apoptosis. Damage of DNA-repair genes can predispose cells to widespread mutations in the genome and thus to neoplastic transformation. Here, we should also mentioned microRNAs (miRNAs), molecules recognized to be involved in tumor development process, which can act as either oncogenes or tumor suppressors by affecting the translation of other genes. Acquired (environmental) DNA damaging agents (chemicals, radiation, microbes) NORMAL CELL Successful repair DNA damage Failure of DNA repair Inherited mutations in:  genes affecting DNA repair  genes affecting cell growth or Mutations in the apoptosis genome of somatic cells Activation of Inactivation of Alterations in genes growth-promoting tumour suppressor that regulate oncogenes genes apoptosis Unregulated cell proliferation Decreased apoptosis Clonal expansion Additional mutations Angiogenesis Escape immune attacks Tumour progression Malignant neoplasm Invasion and metastasis 5 Genes that promote autonomous cell growth in cancer cells are called oncogenes, and their unmutated cellular counterparts are called proto-oncogenes. Oncogenes are created by mutations in proto-oncogenes and are characterized by the ability to promote cell growth in the absence of normal growth-promoting signals. Their products, oncoproteins, resemble the normal products of proto-oncogenes except that oncoproteins are often devoid of important internal regulatory elements, and their production in the transformed cells does not depend on growth factors or other external signals. In this way cell growth becomes autonomous, freed from checkpoints and dependence upon external signals. Several growth factors, such as PDGF-β chain, fibroblast growth factors, and TGF-α can be mutated in a way that they act as oncoproteins as well as their receptors, e.g. EGF-receptor family members, PDGF receptor, receptor for neurotropic factors. Some growth factor receptors are transmembrane proteins with an external ligand-binding domain and a cytoplasmic tyrosine kinase domain. In the normal forms of these receptors, the kinase is transiently activated by binding of the specific growth factors, followed rapidly by receptor dimerization and tyrosine phosphorylation of several substrates that are a part of the signalling cascade. The oncogenic variants of these receptors are associated with constitutive dimerization and activation without binding to the growth factor. Hence, the mutant receptors deliver continuous mitogenic signals to the cell, even in the absence of growth factor in the environment. There are several examples of oncoproteins that mimic the function of normal cytoplasmic signal- transducing proteins. Most such proteins are strategically located on the inner leaflet of the plasma membrane, where they receive signals from outside the cell ( e.g. , by activation of growth factor receptors) and transmit them to the nucleus. Biochemically, the signal-transducing proteins are heterogeneous. The most well-studied example of a signal-transducing oncoproteins is the RAS family of guanine triphosphate (GTP)-binding proteins (G proteins). It becomes activated by binding of ATP molecules, while the hydrolysis of ATP to ADP turns it into inactivated form. The activated RAS stimulates downstream regulators of proliferation, such as the mitogen-activated protein (MAP) kinase cascade, which floods the nucleus with signals for cell proliferation. Several distinct point mutations of RAS have been identified in cancer cells. The affected residues lie within either the GTP- binding pocket or the enzymatic region essential for GTP hydrolysis, and thus markedly reduce the GTPase activity of the RAS protein. Mutated RAS is trapped in its activated GTP-bound form, and the cell is forced into a continuously proliferating state. Mutations that release latent oncogenic activity also occur in several non-receptor-associated tyrosine kinases, which normally function in signal transduction pathways that regulate cell growth. As with receptor tyrosine kinases, in some instances the mutations take the form of chromosomal translocations or rearrangements that create fusion genes encoding constitutively active tyrosine kinases. An important example of this oncogenic mechanism involves the c-ABL tyrosine kinase. In chronic myeloid leukaemia (CML) and some acute lymphoblastic leukaemias, the ABL gene is translocated from its normal position on chromosome 9 to chromosome 22, where it fuses with the BCR gene. The resultant chimeric gene encodes a constitutively active, oncogenic BCR-ABL tyrosine kinase. Whereas oncogenes drive the proliferation of cells, the products of tumor suppressor genes apply brakes to cell proliferation. Many tumor suppressors, such as Rb and p53, are part of a regulatory network that recognizes genotoxic stress from any source, and responds by shutting down proliferation. There are many other proteins involved in regulation of cell proliferation as transcription factors, cell cycle inhibitors, signal transduction molecules, cell surface receptors, and regulators of cellular responses to DNA damage (e.g. TGF-β receptor, E-cadherin, APC/β-catenin, PTEN, SMAD2 and SMAD4, BRCA1 and BRCA2). Mutated forms of these genes were found to be present in many tumors resulting in reduced level or disturbed activity of these proteins. For instance, p53 protein functions as a critical gatekeeper against the formation of cancer. It acts as a 6 “molecular policeman” that prevents the propagation of genetically damaged cells. p53 is a transcription factor, positioned in the centre of a large network of signals that sense cellular stress, such as DNA damage, shortened telomeres, and hypoxia. It prevents neoplastic transformation by three interlocking mechanisms: activation of temporary cell cycle arrest (quiescence), induction of permanent cell cycle arrest (senescence), or triggering of programmed cell death (apoptosis). In response to DNA damage, p53 is phosphorylated by proteins that sense the damage and are involved in DNA repair. p53 contributes to DNA repair by causing G1 arrest and inducing DNA-repair genes. A cell with damaged DNA that cannot be repaired is directed by p53 to undergo apoptosis. This is why, p53 has been rightfully called a “guardian of the genome.” With loss of p53 function, DNA damage goes unrepaired, mutations accumulate in dividing cells, and the cell continues to malignant transformation. Accumulation of neoplastic cells may result not only from activation of growth-promoting oncogenes or inactivation of growth-suppressing tumor suppressor genes, but also from mutations in the genes that regulate apoptosis. A cell with genomic damage can be induced to die, preventing the accumulation of cells with mutations. Consequently, mutations in genes encoding proteins involved in regulation of apoptosis may also result in impaired apoptosis and allow propagation of mutated cells. Taken together, many molecular events underlie the initiation and promotion of malignant transformation and proliferation of mutated cells. This is why it is of utmost importance to elucidate them in details with aim to provide the platform which will contribute to better diagnostic and therapeutic approaches for malignant diseases in the future. 7 Biochemical and inflammatory aspects of tumorigenesis Jelena Kotur-Stevuljević Faculty of Pharmacy – University of Belgrade, Serbia, Department for Medical Biochemistry Metabolism of the cancer cells Cancer cells certainly have abnormal metabolism, govern by oncogenic mutations in several metabolic enzymes which affects the whole metabolic pathway. There are several existing and emerging therapies aim to target this abnormal metabolism in various ways. Cell metabolism describes the group of intracellular chemical reactions that convert nutrients and endogenous molecules into the energy and biomolecules (proteins, nucleic acids and lipids) thatsustain organisms survival. Adenosine triphosphate (ATP), the principal molecule that drives all energy dependent cellular processes, is mainly generated by two metabolic pathways: glycolysis andoxidative phosphorylation (Fig. 1). In glycolysis, glucose is converted to pyruvate, generating two net ATP molecules. When oxygen levels are low, anaerobic glycolysis continues, turning pyruvate into lactate. If oxygen is plentiful, however, the much more efficient processof oxidative phosphorylation occurs, in which pyruvate is routed to the tricarboxylic acid (TCA), or Krebs, cycle in the mitochondria, generating theoretically 36 ATP molecules per molecule of glucose (Figure 1). Figure 1. Main metabolic pathways in glucose metabolism of normal cells GLUT, glucose transporter, MCT – monocarboxylate transporter If oxygen levels are low pyruvat is converted to lactate even in the cytoplasm. Cancer cells drive pyruvat conversion to lactate even in the presence of oxygen. Metabolism of the nutrient glutamine is also mi+odified in cancer. The transcription factors HIF (yellow) and MYC (green) seem to affect these metabolic pathways at various steps. Cancer cells are wasteful. Compared with normal cells, they use a disproportionate share of the nutrients in their environment. This is partly because cancer cells metabolize glucose by anaerobic glycolysis (that is, they avoid oxidative phosphorylation even when oxygen is abundant — the Warburg effect). Moreover, cancer cells make inefficient use of glutamine, the most common amino acid. In normal cells, glutamine is used to donate amine and amide groups for the synthesis of amino acids, andnitrogen for de novo nucleotide formation. Cancer cells, by contrast, secrete a significant fraction of glutamine-derived nitrogen and carbon as waste rather than incorporating them into macromolecule synthesis. 8 Normal cells can carry out anaerobic glycolysis and inefficient glutamine breakdown when they are induced to proliferate. But if their supply of intracellular nutrients becomes depleted, the cells adapt by reactivating oxidative phosphorylation and ceasing proliferation. This ability of normal cells to switch between proliferation and steady state, according to their nutritional status depends on the integrity of a number of tumor suppressors, including the p53 and LKB1 proteins. Cancer cells, which often lack these tumor-suppressor proteins, exhibit ‘addiction’ to glucose and/or glutamine consumption. Increased activity of the PI3K–AKT signaling pathway — due, for example, to activating mutations in receptor tyrosine kinases— mediates excessive nutrient uptake. AKTactivates the HIF and MYC transcription factors, which enhance the expression of glucose transporters (GLUTs) and glycolytic enzymes(Fig. 1) in order to preserve survival of the cells even in a devastating conditions such as hypoxia. Moreover, HIF diverts pyruvate from the TCA cycle. Some of these actions can be reinforced by the loss of p53. Oncogenic MYCcan also directly induce glutamine uptake in excess of nitrogen or carbon demand. Cells must eliminate the breakdown products of this metabolism by secreting the excess carbon and/or nitrogen as lactate, alanine and ammonia. As anaerobic glycolysis is inefficient, cancer cells maintain ATP levels by burning more glucose. Besides, too high a yield of ATP is probably not a good thing for a cancer cell. Cell growth depends on a continuous glycolytic and mitochondrial flux of metabolites to generate supplies of the macromolecular precursors NADPH, acetyl-CoA, ribose and glucose-derived nonessential amino acids. To maintain this high rate of glycolysis, a ready supply of the phosphate acceptor ADP is needed. If cellular metabolism is too efficient, all the ADP will be phosphorylated to ATP, and further glucose metabolism will be inhibited. Cancer cells therefore divert glucose-derived carbon into aerobic glycolysis and uncouple ATP production from mitochondrial electron transport. This means that they can consume the metabolic cofactor NADH without producing ATP, and promote the diversion of glycolytic intermediates into non-ATP-generating bypass pathways. Cancer cells are engaged extensivelyin net lipid synthesis — another feature of cancer metabolism — to generate phospholipids for membrane production and lipid-derived signalling intermediates such as prostaglandins. It would be inefficent to simultaneously break down their newly synthesized fatty acidsto generate energy.A fundamental problem for solid tumors is that they often outgrow their blood supplies,and face low oxygen levels (hypoxia). Under hypoxic conditions, HIF accumulates and triggersthe transcription of about 200 genes, many of which support cell survival. These includegenes that promote glycolysis and suppress oxidative phosphorylation. HIF accumulation is suppressed by the activity of oxygen-dependent PHD (prolyl hydroxylase domain) enzymes(Fig. 2). The HIF response can be further magnified by mutations that promote HIF translation, such as those leading to AKT activation, or by mutations that compromise HIF degradation, such as those leading to loss of the tumor-suppressor protein pVHL.Furthermore, changes in the levels of reactive oxygen species or TCA-cycle metabolites. Glutamine is essential for cell growth - abnormal increase in biomass is a hallmarkof cancer. It is the metabolite that acts as an intermediate in the transport of reduced nitrogen through the bloodstream. To produce nucleotides and non-essential aminoacids, cancer cells need a robust supply of reduced nitrogen. Furthermore, glutamine plays a crucial part in the uptake of essential amino acids, can maintain the TCA cycle when glucose levels are limiting (Fig. 1), and can support NADPH production, which is necessary for lipid and nucleotide biosynthesis. Oncogenic activation of MYC promotes glutamine use but at the expense of glutamine addiction. Otto Warburg and colleagues were the first to report that cancer cells continue to engage in glycolysis and lactate production even when oxygen levels are abundant. This is known as the Warburg effect. Despite extensive efforts, however, Warburg and others could not find mutations that impaired oxidative metabolism in cancer cells. Consequently, many biologists questioned whether Warburg’s observations truly reflected tumor metabolism or were instead an artefact of inadvertent hypoxia arising during his in vitro experiments. Imaging studies described below support the in vivo relevance of the Warburg effect. The Pasteur effect describes the reciprocal relationship between anaerobic glycolysis and oxidative phosphorylation: when oxygen is present, glucose 9 consumption to produce lactate should be suppressed in favour of oxidative phosphorylation. The Warburg effect is a loss of the Pasteur effect. It is clear that HIF is essential for both the Pasteur and the Warburg effects. In normal cells, oxygen suppresses HIF activity, leading to diminished glycolysis and increased entry of pyruvate into the TCA cycle. In cancer cells, oxygen can no longer effectively decrease HIF activity. Moreover, mutations affecting crucial cancer targets such as MYC and p53 can conspire with HIF activation to maintain a highly glycolytic state. Metabolic pathways are independently regulated at various steps, and mutations in a single enzyme are usually insufficient to affect a nutrient’s uptake or metabolism. Rather, it seems that tumors seize control of their own metabolism by selecting for mutations that affect metabolic pathways rather than individual pathway components. In vitro, many cancer cells die in response to glucose withdrawal. But blocking glycolysis has not been useful in the clinic. For instance, the hexokinase enzyme mediates the first step of glycolysis (Fig. 1), but clinical trials with the hexokinase inhibitors 2-deoxyglucose and lonidamine were terminated, presumably owing to their untoward toxicity or inefficacy. The specificity of other hexokinase inhibitors under investigation, such as 3-bromopyruvate, has also been questioned. Clinical trials on TLN-232 (or CAP-232), an inhibitor of the glycolytic enzyme pyruvate kinase, are continuing. A theoretical concern with glycolysis inhibitors is that glucose is an essential fuel source for red blood cells and, under non-starvation conditions, for the brain. Drugs that indirectly target glycolysis are also being pursued. mTOR inhibitors, for example, indirectly reduce glycolysis by decreasing levels of the HIF-1α subunit and perhaps other HIF-α family members. Several agents that block mTOR or receptor-tyrosine-kinase signalling upstream of mTOR have been approved for cancer treatment. The efficacy of these drugs can be rapidly assessed by their ability to reverse tumor-associated 18FDG uptake. Whether their antiproliferative and pro-cell- death effects are also due to changes in metabolism is unclear, however. An exciting finding is that germline mutations that affect the TCA-cycle enzymes fumarate hydratase and succinate dehydrogenase can produce cancer, by causing HIF stabilization and a state of pseudo- hypoxia (Figs 1, 2). Defective succinate dehydrogenase can also mediate the development of neural- crest tumors independently of HIF. Similarly, mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been reported in brain tumors and acute leukaemia. Instead of their normal product (the TCA-cycle intermediate 2-oxoglutarate), these mutant enzymes produce 2-hydroxyglutarate, which might alter the activity of 2-oxoglutarate-dependent enzymes such as those that modify HIF levels. Several histone demethylase enzymes also seem to work as 2-oxoglutarate-dependent dioxygenases, raising the intriguing possibility that oxygen, reactive oxygen species, and metabolites such as 2-oxoglutarate may have relatively direct roles in influencing DNA-dependent processes including transcription and cell behaviour. Clearly, metabolic pathways are highly interconnected with pathways that govern the hallmarks of cancer, such as unrestrained proliferation and resistance to cell death. The many metabolic enzymes, intermediates and products involved could be fertile ground for improving cancer diagnostics and therapeutics. Regulation of HIF function HIF is a heterodimer consisting of an unstable α-subunit and a β-subunit. In the presence of oxygen, HIF-α is hydroxylated on one (or both) of two prolyl residues by members of the PHD enzyme family. In addition to oxygen, these enzymes require the TCA-cycle intermediate 2-oxoglutarate and reduced iron (Fe2+), and are inhibited by high concentrations of some other TCA-cycle metabolites such as fumarate, succinate and perhaps reactive oxygen species (ROS). A ubiquitin ligase enzyme complex containing the tumour-suppressor protein pVHL recognizes prolyl-hydroxylated HIF-α and earmarks it for degradation by proteasomes by adding a chain of ubiquitin (Ub) proteins. Dashed lines depict components that regulate PHD enzymes. In several congenital disorders, such as Fanconi anaemia, xeroderma pigmentosum, ataxia telangiectasia, Bloom syndrome, Down syndrome and cystic fibrosis the cells show evidence of increased oxidative stress. Affected individuals show an increased incidence of cancer. Chromosomal instability is also a common feature of the first four disorders. Taken together these data suggest that the increased oxidative stress may contribute to 10 development of genomic instability that is a hallmark of cancer cells. The continuos production of oxidants at the site of chronic inflammation may cause cancer. Under a sustained environmental stress, ROS are produced over a long time, and thus significant damage may occur to cell structure and functions and may induce somatic mutations and neoplastic transformation. Indeed, cancer initiation and progression has been linked to oxidative stress by increasing DNA mutations or inducing DNA damage, genome instability, and cell proliferation. The skin, for example, is chronically exposed to both endogenous and environmental prooxidants due to its interface function between the body and the environment, and to protect the skin against this overload of oxidant species, it needs a well-organized system of both chemical and enzymatic antioxidants. The lungs, which are directly exposed to oxygen concentrations higher than in most other tissues, are protected against these oxidants by a variety of antioxidant mechanisms. Furthermore, aging, which is considered as an impairment of body functions over time, caused by the accumulation of molecular damage in DNA, proteins and lipids, is also characterized by an increase in intracellular oxidative stress due to the progressive decrease of the intracellular ROS scavenging. Acting to protect the organism against these harmful pro-oxidants is a complex system of enzymatic antioxidants [e.g., superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase, catalase] and nonenzymatic antioxidants [e.g., glutathione (GSH), vitamins C and D. 11 On the other hand, inflammatory cells also produce soluble mediators, such as metabolites of arachidonic acid, cytokines and chemokines, which act by further recruiting inflammatory cells to the site of damage and producing more reactive species. These key mediators can activate signal transduction cascades as well as induce changes in transcription factors, such as nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), hypoxia-inducible factor-1α (HIF1-α), activator protein-1 (AP-1), nuclear factor of activated T cells (NFAT) and NF-E2 related factor-2 (Nrf2), which mediate immediate cellular stress responses (Figure 2). Induction of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), aberrant expression of inflammatory cytokines [tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and chemokines [IL-8; CXC chemokine receptor 4 (CXCR4)], as well as alterations in the expression of specific microRNAs, have also been reported to play a role in oxidative stress-induced inflammation. This sustained inflammatory/oxidative environment leads to a vicious circle, which can damage healthy neighboring epithelial and stromal cells and over a long period of time may lead to carcinogenesis. 12 Under normal conditions, anti-oxidants outbalance pro-oxidants, but under oxidative conditions, pro- oxidants prevail over anti-oxidants, which can lead to many inflammatory diseases including cancer. To control the balance between production and removal of ROS, a variety of DNA repair enzymes exist, although antioxidants are more specific and efficient in protecting cells from radicals. This antioxidant system includes both endogenous and exogenous and enzymatic and non-enzymatic antioxidants. Glutathione (GSH), is a tripeptide and the major endogenous antioxidant produced by the cells, which helps to protect cells from ROS such as free radicals and peroxides. It is now well established that ROS and electrophilic chemicals can damage DNA, and that GSH can protect against this type of damage. GSH can also directly detoxify carcinogens through phase II metabolism and subsequent export of these chemicals from the cell. On the other hand, elevated GSH levels are observed in various types of cancerous cells and solid tumors, and this tends to make these cells and tissues more resistant to chemotherapy. Cytotoxicity of different anticancer-agents depend almost exclusively on ROS production. Treatment succes depends on tumor cells sensitivity to oxidative stress. Chemo-therapeutics which have this kind of mechanism of action are: doxorubicin, daunorubicin, mitomycin C, etoposid, cisplatin, imatinib, ionizing radiation and photodynamic therapy. 13 Tumor markers in clinical practice Prof. Joško Osredkar,PhD University Medical Centre Ljubljana, Clinical Institute of Clinical Chemistry and Biochemistry, Slovenia In recent years, research on cancer has focused on the discovery of new tumor markers that would be useful in clinical practice, and could contribute to the early detection of cancer (1). Measurement of these markers in the serum of patients is today one of the fastest developing areas of laboratory biomedicine (2). Tumor marker was first described 160 years ago, when Bence Jones noticed the presence of abnormal proteins in the urine of patients with multiple myeloma. Tumor markers are described as products of malignant cells, or substances occurring in other cells under the influence of malignant cells (5). They are useful for the diagnosis of cancer, monitoring the progression of the tumor mass, predicting prognosis and for monitoring the success of therapy 4). The ideal tumor marker comprises a protein, which is synthesized only in a malignant tumor, and is characterized by a certain organ and the type of tumor. It can be demonstrated in all patients with the same type of tumor forms in sufficient quantities early in the development of malignant disease and its serum concentrations reflect the size of the malignant tissue amended (3). Despite much research in this area could be used in clinical practice, only a few of them (2). The main reason is their lack of specificity and increased production in the physiological and non-malignant conditions. The false-positive results may contribute inflammatory processes, benign tumors, liver disease, extensive tumor necrosis, the consequences of diagnostic tests and treatments, pregnancy and renal dysfunction. False negative results can lead to poor blood flow to the tumor, the formation of immune complexes with tumor antigens or rapid elimination of tumor antigen in the serum (3). Today known tumor markers are less suitable for the detection of malignant change, but they are potentially useful for patients with a known diagnosis for predicting the prognosis thereof. They help in the choice of treatment, and are used for the prediction of therapeutic outcome and are of great importance for the early detection of relapse (2, 3). Clinically useful markers of malignant transformation of the α-fetoprotein (AFP) for the control of patients with hepatocellular carcinoma, carcino embryonic antigen (CEA), to monitor patients diagnosed with cancer of the colon and rectum, CA 125 to control therapy in female patients with cancer of the ovary and hCG to control patients with trophoblastic and non-seminoma germ tumors. In breast cancer estrogen receptors are used for prediction of response to hormonal therapy, while HER-2, to enable the identification of patients who are suitable for treatment with trastuzumab (2). Maximum usability expresses prostate specific antigen (5). The first tumor marker for prostate cancer was the enzyme acid phosphatase. Serum acid phosphatase may originate from different organs of the bone, prostate, liver, kidneys, the red blood cells. Diseases of other organs may therefore cause elevated levels of an enzyme in the blood, which leads to poor specificity of the analyte. In 1966 we can find the first known record for gama semino - protein, protein in seminal fluid, which is today known as prostate specific antigen (6). In 1980, we find for the first time documented the presence of elevated levels of PSA in the serum of patients with prostate cancer (4). In 1994, FDA approval was recorded for the use of PSA for early detection of prostate cancer (7). The role and usefulness of individual tumor markers and methods for a given type of cancer is defined by the statistical concepts of sensitivity and specificity (4). The sensitivity of the test is the% 14 of patients with certain tumors, which have a positive test. The specificity of the test represents% healthy, with negative results (3). For optimal cancer treatment we need reliable prognostic and predictive markers. Prognostic markers are factors that predict the final outcome of the disease in the absence of therapy. Predictive markers are associated with the response or resistance to a particular therapy. Traditional prognostic factors for malignancy are the tumor size, stage of disease and the number of metastases in regional lymph nodes (2). Traditional tests for the evaluation of tumor markers are ELISA - type tests for serum indicators and immunohistochemical methods for markers of malignant tissue of transformation (2). 1. Chatterjee K.S, Zetter R.B: Cancer biomarkers: knowing the present and predicting the future. Future Oncology 2005; 1(1): 37-50 2. Duffy J.M: Role of tumor markers in patients with solid cancers: A critical review. European Journal of Internal Medicine 2007;18:175-184 3. Novakovič S: Tumorski označevalci v klinični onkologiji. Onkologija/pregled, Ljubljana 2000 4. Montie E.J, Meyers E.S: Defining the ideal tumor marker for prostate cancer. Urologic Clinics of North America 1997; 24: 247-252 5. Stephen C, Cammann H, A. Meyer H, Lein M, Jung K: PSA and new biomarkes within multivariate models to improve early detection of prostate cancer. Cancer Letters 2007; 249: 18-29 6. Makarov V.D, Carter B: The Discovery of prostate Specific Antigen as a Biomarker for the Early Detection of Adenocarcinoma of the Prostate. The Journal of Urology 2006; 176: 2383- 2385 7. Loeb S, Catalona J.W: Prostate-specific antigen in clinical practice. Cancer Letters 2007; 249: 30-39 15 Targeted therapies and predictive markers in cancer Jana Nekvindová, PhD University Hospital Hradec Králové, Institute for clinical biochemistry and diagnostics, Czech Republic nekvindova.j@seznam.cz Specific differences in cancer cells (or other cells in their vicinity) help them grow and thrive. Targeted therapy is a special type of chemotherapy that takes advantage of (i.e. „targets“) these differences between normal cells and cancer cells. Cancer cells typically have many changes in their DNA/ genes, some of these gene changes might allow the cell to stop working the right way and/or grow and divide very quickly. These changes are what make it a cancer cell, but is a plethora of different types of cancer, and they vary significantly. For example, lung cancer, melanoma and colon cancer cells have different gene changes that help them grow and/or metastasize, even if the same signalling pathway. Another example is breast cancer with various histopathological and genetic subtypes. Basically, we can say that each tumour is unique. Most standard chemo drugs do not consider genetic or subsequent metabolic and signalling changes, they work by killing cells in the body that grow and divide quickly. Cancer cells divide quickly, which is why these drugs often work against them. But chemotherapy drugs also affect other cells in the body that divide quickly, which can sometimes lead to serious side effects. Although targeted drugs are technically considered chemotherapy like other drugs used to treat cancer, they don’t work the same way as standard chemotherapy drugs. These drugs also tend to have side effects different from standard chemotherapy drugs. To choose the most appropriate treatment for an individual patient/ tumour, predictive markers are the best help where they exist. A predictive marker is a particular protein or gene that indicates sensitivity or resistance to a specific therapy. The use of predictive markers is becoming increasingly relevant in cancer therapy as it allows for better identification of patients who will respond positively to the therapy. And frequently, predictive marker (or its product) is the target at the same time. In August 2016, the FDA has already approved dozens of targeted drug cancer therapies, and many more are being studied in clinical trials either alone or in combination with other treatments. In general targeted drugs work to: • block or turn off grow /proliferation signals that tell the cancer cell to grow and divide • stop making new blood vessels to nurture the cancer cells • stimulate the immune system to kill the cancer cells • carry a toxin to kill cancer cells, but not normal cells • change proteins within the cancer cells to induce apoptosis There are there major classes of targeted drugs: • small molecules • antibodies • vaccines Of course, some targeted drugs are more “targeted” than others, affecting only a single change in cancer cells, while others can target several different changes. Others enhance the way the organism fights the cancer cells itself. Besides that, targeted therapy is only rarely used alone, most often it is applied with other cancer treatments such as chemotherapy, surgery and/or radiation therapy. 16 As an example, R-CHOP immunochemotherapy regimen which is used to treat both indolent and aggressive forms of non-Hodgkin lymphoma, consists of rituximab, cyclophosphamide, hydroxydaunorubicin hydrochloride (doxorubicin hydrochloride), vincristine (Oncovin) and prednisone. Targeted cancer agents are broadly classified as either monoclonal antibodies or small molecules. Therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellularly, such as growth factors. In some cases, monoclonal antibodies are conjugated to radio-isotopes or toxins to allow delivery of these cytotoxic agents specifically to the intended cancer cell target. Small molecules can penetrate the cell membrane to interact with targets inside a cell, typically a cell signalling cascade proteins. Small molecules are usually designed to interfere with the enzymatic activity of the target protein. As with any drug, targeted cancer therapies typically have several different names. One or more is used to designate the chemical compound during development; afterwards the drug receives a generic name and then a brand name used by the pharmaceutical company for marketing. E.g. the small molecule STI-571, known as imatinib (generic name) is marketed by Novartis under the brand name Gleevec™. The generic name of a targeted agent provides clues to the type of agent and its cellular target: - small molecules end with the stem "-ib" (indicating protein inhibitory properties) - monoclonal antibodies end with the stem "-mab" (monoclonal antibody) and have an additional substem designating the source of the antibody, e.g "-ximab" for chimeric human-mouse antibodies, "-zumab" for humanized mouse antibodies, and "-mumab" for fully human antibodies. Both monoclonal antibodies and small molecules contain an additional stem in the middle of the name describing their target; e.g. for monoclonal antibodies include "-ci-" for a circulatory system target and "-tu-" for a tumour target, or for small molecules include "-tin-" for tyrosine kinase inhibitors and "-zom-" for proteasome inhibitors. At the beginning of the generic name there is a unique prefix for each agent. Examples: bevacizumab = humanized monoclonal antibody with a circulatory system target (VEGF-A) cetuximab = chimeric monoclonal antibody with a tumour target (EGFR) ipilimumab = fully human antibody with an immune system target (CTLA-4) bortezomib = small molecule proteasome inhibitor imatinib = small molecule tyrosine kinase inhibitor seliciclib = small molecule cyclin-dependent kinase inhibitor There are many important targeted therapies in the clinical use, to present some examples: Rituximab - a chimeric mouse-human anti-CD20 antibody destroys B cells (CD20 is primarily found on their surface) and is therefore used to treat diseases which are characterized by overactive, dysfunctional, or excessive numbers of B cells. This includes many lymphomas, leukaemia, autoimmune disorders and transplant rejections. Monoclonal antibodies cetuximab and panitumumab are epidermal growth factor receptor (EGFR) inhibitors used for treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer and head and neck cancer in cases where downstream signalling protein of the EGFR pathway called K-RAS is not constitutively activated by mutation (otherwise blocking of the receptor above would not be effective). 17 Vemurafenib (V600E mutated BRAF inhibitor) causes programmed cell death in melanoma by interrupting the B-Raf/MEK step on the B-Raf/MEK/ERK pathway − if the B-Raf has the V600E or V600K mutation (about 60% of melanomas do). Melanoma cells without these mutations are not inhibited; vemurafenib paradoxically stimulates normal BRAF and may promote tumour growth in such cases! Ipilimumab is a new agent in melanoma therapy for enhancing the immune response by targeting CTLA-4, a protein receptor that downregulates the immune system. CML – chronic myelogenous leukaemia is best treated with tyrosine-kinase-targeted inhibition. The first of this new class of drugs was imatinib mesylate, approved by FDA in 2001. Since then, other drugs, e.g. dasatinib, nilotinib, radotinib and bosutinib have been introduced in practice. EML4-ALK translocation (predictive marker), which is present in 3-5% of NSCLC (non-small cell lung cancer), leads to formation of a fusion gene coding for an abnormal tyrosine kinase receptor (target) which exaggerated activity can be inhibited by crizotinib or alectinib. There is a list of new drugs being tested. About 25% of breast cancer cases are associated with an amplification of genes coding for a cell surface receptor called HER2/neu (receptor tyrosine-protein kinase erbB-2, also known as CD340 (cluster of differentiation 340) or proto-oncogene Neu). HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Amplification or over-expression (predictive marker) of this oncogene plays an important role in the development and progression of aggressive types of breast cancer. These patients have poor prognosis, however, the tumours can be targeted by trastuzumab (Herceptin), monoclonal humanized antibody against overexpressed (target) surface HER2 receptor. Side effects of targeted cancer therapy drugs Although targeted therapy drugs don’t affect the patients the same way that standard chemotherapy drugs do, they still cause various side effects. As there are many different types of targeted drugs, the side effects from these drugs depend largely on what each drug targets. The side effects have also variable incidence and severity. Because many targeted drugs are still quite new, it’s hard to say how long you can expect side effects to last. We do know that some of the side effects from standard chemo drugs can last a lifetime, such as when the drug causes long-term damage to the heart, lungs, kidneys, or reproductive organs. In many cases we still don’t know if targeted therapy drugs cause these kinds of long-term changes. Common and serious side effects of targeted drugs are: hypertension, bleeding or blood clotting problems, bruising and bleeding (namely with anti-angiogenic drugs). Bleeding, such as from the stomach and intestines, can be severe and even life threatening. Some drugs can also cause blood clots in the lungs and legs, as well as heart attacks and strokes, skin rash, hives, slow wound healing, gastrointestinal perforations, heart damage, autoimmune reactions, swelling, nausea and vomiting, diarrhoea or constipation, mouth sores, shortness of breath or trouble breathing or cough, dizziness, fatigue, headache, hair loss, damage to organs such as the thyroid gland, liver, or kidneys, allergic reactions, increased risks of certain infections and secondary cancers! 18 Recommended literature: My Cancer Genome website by Vanderbilt University (https://www.mycancergenome.org/content/) ClinicalTrials.gov - a service of the U.S. National Institutes of Health, provides a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world (www.clinicaltrials.gov) Meyskens FL Jr, Mukhtar H, Rock CL, Cuzick J, Kensler TW, Yang CS, Ramsey SD, Lippman SM, Alberts DS. Cancer Prevention: Obstacles, Challenges and the Road Ahead. J Natl Cancer Inst. 2015 Nov 7;108(2). pii: djv309. doi: 10.1093/jnci/djv309. Print 2016 Feb. Review. Moriarity A, O'Sullivan J, Kennedy J, Mehigan B, McCormick P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther Adv Med Oncol. 2016 Jul;8(4):276-93. doi: 10.1177/1758834016646734. Epub 2016 May 29. Review. PubMed database (http://www.ncbi.nlm.nih.gov/pubmed) 19 Therapeutic approaches to different cancer diseases Prof Dr Besim Prnjavorac, internist, clinical immunologist University of Sarajevo, Bosnia and Hezegovina E-mail: pbesim@bih.net.ba Mobil +38761166850 Possibilities of treatment of cancer diseases are just in tendency of spreading. Despite of bulky investigations stratification of importance of different approach was not substantilal changed. Surgery is the best choice up to date for treatment of solid tumors. Irradiation as treatment’s possibility was reestablished in recent twenty years. Chemotherapeutics are most common in use with many mechanisms of action, but pharmacological classification was not substantial changed for the long time. Many new chemotherapeutic drugs were synthesized in recent years, but patient’s survival was not so much changed. In treatment of Non Small Lung Cancer (NSLC) survival was pronged for 3-4 mounts in past twenty years. It’s necessary to establish new approach for lung cancer. Up to date discovered therapies are unsuccessful to be well strategy for the future. In calculation of cost-benefit no substantial improvement of general clinical status, or general improvement in the commonest cancers. Widespread application of testing for chromosomal mutation, using PCR method was clarified the most important possible mechanism of tumor cells promotion. Many other dilemmas are resolved for the process of decision making for the of chemotherapy of cancer. Using PCR method it is possible to prognosis efficacy of chemotherapy for different cancers. So, many chemotherapeutic agents, now used for some cancer, proved ineffective. Guidelines for cancer chemotherapy quite accurately suggest the effectiveness of chemotherapy after chromosomal analysis by PCR technique. But guidelines are not literally administered in all countries. Therefore, a lot of money was invested in ineffective chemotherapy, but also a variety of side effects of these drugs are present. The chemotherapeutic drugs can be divided as follows: alkylation agents, antimetabolites, mitotic inhibitors, antibiotics, hormones and hormonal antagonist and enzymes and other antitumor agents. All of these drugs affect cell division or DNA synthesis and function in some way. Alkylating agents, or their reactive intermediates, form covalent bonds with deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein to form an adduct in which a methyl or ethyl group is added. Some of the drugs of this group: (Busulfan (Myleran), Carboplatin (Paraplatin), Chlorambucil, Cisplatin, Cyclophosphamide (Cytoxan), Dacarbazine (DTIC-Dome), Estramustine Phosphate, Ifosphamide, Mechlorethamine (Nitrogen Mustard), Melphalan (Phenylalanine Mustard), Procarbazine, Thiotepa, Uracil Mustard. Antimetobolites alter the synthesis of DNA or RNA. Antimetabolites that are structural analogues of nucleotides are incorporated into cell components as if they were the essential pyrimidine or purine, and as a consequence, disrupt the synthesis of nucleic acids. (Molecular mimicries). Other antimetabolites disrupt essential enzymatic processes of metabolism like the folate antagonist, 5- fluorouracil, which disrupts vital folic acid metabolism. Some of antimetabolites: Cladribine, Cytarabine (Cytosine), metotrexsate, 5-fluorouracil, mercaptopurin. Mitotic inhibitors (Mitotic spindle inhibitors) bind to microtubular proteins and block their ability to polymerize or depolymerize, a process which halts nuclear division, but without impairment of DNA synthesis. DNA topoisomerase II inhibitors block religation of double strand DNA breaks (i.e., sister chromatid separation or cleaved DNA). Etoposide (VP-16), Teniposide (VM-26m Vumon), Vinblastine, Vincristine, Vindesine are the examples of mitotic inhibitors. 20 Antitumor antibiotics interfere between DNA base pairs and disturb the synthesis or function of nucleic acids. Some drugs: Aclarubicin, Bleomycin, Dactinomycin (Actinomycin D), Daunorubicin, Doxorubicin (Adriamycin), Epirubicin, Idarubicin, Mitomycin C, Mitoxantrone, Plicamycin (Mithramycin). Hormones or hormone-blocking agents either exert a corticosteroid effect, such as prednisone, or manipulate the hormone environment in hormone-responsive tumors. The antiandrogenic agent, flutamide, which is used to treat prostate cancer, is believed to block androgen receptor sites. The antiestrogenic agent, tamoxifen, binds to intracellular estrogen receptors, then enters the nucleus where the tamoxifen-estrogen-receptor complex inhibits DNA and protein synthesis. Some of the drugs: Equine Estrogen (Premarin), Cortisone, Chlorotriansene , Dexamethasone , Diethylstilbestrol Ethinyl Estradiol, Fluoxymesterone, Hydroxyprogesterone Caproate, Medroxyprogesterone Acetate (Provera), Megestrol Acetate (Megace), Prednisone, Tamoxifen (Nolvadex), Testosterone. Anti tyrosine kinases ((TK) was introduced after discovery of monoclonal antibody, and thereafter start era of ”anti TK” as targets for cancer therapy. The landscape was changed radically by the success of imatinib mesylate, an inhibitor of the BCR-ABL TK in chronic myeloid leukemia (CML) — a result heralded as a proof-of-principle and a triumph of targeted cancer therapy. TKs are now regarded as excellent targets for cancer chemotherapy, but reality lies somewhere between the extremes of triumph and tribulation. Therapy against tumor stroma and vascularisation. EGFR is activated by binding specific ligands, including epidermal growth factor and transforming growth factor-α, endothelial vascular growth factor (EVGF). Activation of EGFR promotes cell proliferation and survival, as well as angiogenesis, leading to tumor growth and metastasis. Anti-hEGFR-hIgG4 (S228P) features the constant region of the human IgG4 (S228P) isotype and the variable region of cetuximab. Cetuximab is a chimeric human/mouse IgG1 monoclonal antibody that targets epidermal growth factor receptor (EGFR), a cell surface receptor over expressed in many types of cancer. VEGF overexpression in tumors is associated with increased angiogenesis, proliferation and metastasis. Phosphorylated VEGFR-2 (KDR) expression in numerous solid tumors including three lung carcinomas, three breast carcinomas, Non Hodgkin's lymphomas, and melanoma. Therapy against tumor stroma and vascularisation is oriented against primary against interleukins. Induction of higher activity of interleukins is just opposite approach than previous one. Application of bacillus Calmette-Guérin (BCG) is introduced in antitumor therapy thirty years ago, and reestablished in some recent years. The goal of therapy is to stimulate production of Tumor necrosis factor (TNF), and interleukine-2 (IL-2) with idea to slow-down tumor growth. Actually this method is used mostly in treatment of urinary bladder tumors. Different approachs were in use for antitumor therapy. There some substantial success in this treatment, like hormone-sensitive tumors, some type of leukemia, etc. But chemotherapy is to be revised in essential postulate if the good perspective of this therapy could be reestablished. 21 Clinical Pharmacy Services in Oncology Aleš Mrhar, University of Ljubljana, Faculty of Pharmacy, Slovenia Lea Knez, University Clinic Golnik, Slovenia Samo Rožman, Institute of Oncology Ljubljana, Slovenia Clinical pharmacy includes all services performed by pharmacists practising in clinics and hospitals, community pharmacies, community health centres, nursing homes and other settings where medicines are prescribed and used. Its main mission is to move the focus of attention from the drug to the single patient or population receiving drugs. Clinical pharmacists are following the overall goal of clinical pharmacy activities, i.e., to promote the correct and appropriate use of medicinal products and devices. These activities aim to: • maximize the clinical effect of medicines, i.e., using the most effective treatment for each individual patient, • minimize the risk of treatment-induced adverse events, i.e., monitoring the therapy course and the patient's compliance with therapy, and • minimize the expenditures for drug treatments, i.e., trying to provide the best treatment alternative accompanied by the cost-effective use of drugs for the greatest number of patients. In this contribution two places of good clinical pharmacy services in oncology are presented, i.e. University Clinic Golnik and Institute of Oncology Ljubljana. First, clinical pharmacists at the University Clinic Golnik are integrated in the treatment of patients at higher risks for adverse drug reactions (those with decreased renal function, prescribed with strong inhibitors or inducers of drug enzymes and drug transporters, prescribed with strong opioids, prescribed with drugs where TDM is performed and prescribed medicines with a feeding tube), oncology patients, patients with tuberculosis and patients with hereditary angioedema. Moreover, clinical pharmacists successfully implemented on a full scale a medication reconciliation model for all oncological patients admitted to the clinic. As anti-cancer drugs belong to a group of high-risk medications, in guaranteeing the quality, safety and efficacy of therapy with these drugs pharmacists’ role should not be restricted to dispensing only. A study has been designed at the University Clinic Golnik to describe the implementation of a new pharmacy service – chemotherapy prescription screening – and evaluate its benefits by analysing the recorded interventions. The study was conducted immediately after the centralized chemotherapy preparation was introduced and chemotherapy prescription screening implemented. The performed interventions were recorded over a five-month period in 2009. The results showed that chemotherapy prescription screening by pharmacists was integrated successfully into routine clinical practice and pharmacists’ responsibilities and duties were defined. During the study period, 506 cancer prescriptions were reviewed by five pharmacists, who recorded 211 interventions. The high rate of interventions emphasizes the role that pharmacists play in oncology services. The interventions involved cancer drugs (31%), antiemetics (41%) and other support care drugs (12%). The identified problems were related to the following categories: “dose, frequency and regimen” (64%), “drug selection” (21%) and “administrative” (14%). Clinicians accepted most pharmacists’ recommendations (76%), thereby confirming the need for the proposed interventions also from a medical point of view. However, the study provides no information on the clinical significance of the 22 recorded interventions, whether accepted or not. It turned out at the end that the high rate of interventions recorded in this study stresses the importance of integrating pharmacists’ clinical roles into oncology services in order to maintain high quality standards of cancer treatment. Another study has been performed at the University Clinic Golnik to show the importance of drug interactions for patient safety as cancer patients are prescribed numerous medications. The study was designed as a retrospective study and reviewed drug interactions in patients, where anticancer drug therapy was initiated in the year 2012. As part of routine clinical practice, in all patients, drug interactions were reviewed by a pharmacist, who discussed those judges to be clinically important with the patient’s oncologist. As part of the study, drug interactions were reassessed using three different drug interaction databases (Lexi-comp, Stockley’s Drug Interaction, Drugs.com) to record all possible interventions. Only drug interactions between drugs in systemic cancer therapy (including anticancer drugs and support care drugs) and prescription drugs for comorbidities were evaluated. Overall, the study included 223 lung cancer patients. Most patients were older (median 63 years), were taking a median of 4 drugs for treatment of comorbidities, and were prescribed a median of 6 drugs for cancer treatment. Review of drug interaction databases revealed 1416 drug interactions between drugs in systemic cancer therapy and other drugs, only 18 % of detected interventions involved anticancer drugs and only 19 % would affect the outcomes of anticancer treatment; the overwhelming number of possible drug interactions emphasises the importance of identifying clinically relevant drug interactions. The need for the critical appraisal of possible drug interactions is further evidenced by the low number of interactions (52/1416; 4 %), judged by the pharmacist as clinical important. Pharmacists more often identified as important interactions involving anticancer drugs (85 %) and those affecting the outcomes of anticancer therapy (79 %). To prevent the manifestation of drug interactions, pharmacists most often suggested a change in the treatment of comorbid conditions. To conclude, in the treatment with high risk drugs, all efforts should be invested to prevent adverse drug events and avoiding drug interactions falls within this aim. Review of drug interactions at initiation of anticancer drug treatment was successfully implemented into routine clinical practice. The study revealed a large number of possible drug interactions, showing the need for their critical appraisal in order to identify and prevent clinically relevant drug interactions. Second, clinical pharmacists at the Institute of Oncology Ljubljana are involved occasionally in the treatment of patients at surgical ward. They are expected to check oncologic, supportive and chronic drug therapy and propose appropriate solutions. A clinical case of a patient who developed symptomatic urinary tract infection caused by yeasts after surgery due to colorectal carcinoma and relapse of the disease in abdomen is presented. Anidulafungin (a member of echinocandins) was indicated on the basis of microbiological review, however, its pharmacokinetics is not favourable as it is excreted in urine in less than 1%. As a result, symptoms of urinary tract infection persisted and treatment was not efficient. There were two options available: amphotericin B and fluconazole. Clinical pharmacist complained against the use of amphotericin B due to nephrotoxicity (patient had chronic kidney injury), high cost (1300 €/day) and better treatment alternatives. Understanding pharmacokinetics of fluconazole, one can see that the most of the drug is excreted in urine, fluconazole is concentrated in the urine – yielding urine levels > 100 µg/mL, the expected concentrations in urine exceeded the MIC not only for susceptible yeasts (MIC≤ 8 µg/mL), but also for organisms that are susceptible but dose-dependent (MIC 16-32 µg/mL) and sometimes even those that are resistant (MIC ≥ 64 µg/mL). Clinicians accepted pharmacist’s recommendation, after initiation of fluconazole 400 mg/day, symptoms and signs of urinary tract infection improved, treatment was continued for 16 days. 23 Immunotherapy of cancer Assoc. Prof. Matjaž Jeras, Ph.D., M.Pharm., University of Ljubljana, Faculty of Pharmacy, Slovenia e-mail: matjaz.jeras@ffa.uni-lj.si Immune system is involved in pathogenesis, maintenance and progression of cancer and therefore represents an important therapeutic target. Like cancer itself, the immune system is extremely complex. It reacts differently, according to (patho)-physiological conditions within the body. The key factors needed for induction of specific anti-tumor effector T cells are fully functional immune system and specific tumor (TA) and/or tumor-associated antigens (TAA) which may be more or less immunogenic and are often subjected to mutations, due to high genetic instability and deficient mismatched DNA repair in tumor cells. However, tumors, via numerous mechanisms, are able to establish microenvironments that actively protect them against specific anti-tumor immune responses. In general tumors can be divided in those that are highly (melanoma), intermediately (prostate cancer) or weakly immunogenic (thyroid cancer), which corresponds to the degree of their heterogeneic mutational potential (1). Picture1: Somatic mutation frequencies detected in exomes from 3.083 tumor - normal pairs (1). Seen from another perspective, some tumors are considered to be inflammed and therefore more prone to immunological intervention, while others are not. Therefore microenvironment, especially of the latter should be first modulated to provide proinflammatory conditions in order to enhance the existing specific antitumor immune responses and to allow the adoptively transferred anti-tumor immune effector cells to be efficient (2, 3). There are numerous factors and mechanisms that tumor cells are exploiting for prevention of anti- tumor immunity, for example, they (4, 5):  can lose the capability to express certain major histocompatibility (MHC) molecules or even a whole MHC haplotype;  lack costimulatory molecules (CD80, CD86, and others);  produce immunosuppressive (anti-inflammatory) cytokines (VEGF, TGF-, IL-10, IL-4), chemokine CCL22, which attracks and cumulates regulatory T cells (Treg), and indolamine- 2,3-dioxygenase (IDO);  are additionally protected against the immune system by various types of immunosuppresive cells:  besides Treg, also other CD4+ (Tr1, Th3) and CD8+ (Ts) T cells;  tumor-associated macrophages (TAMs or M2) and tolerogenic dendritic cells (DC2);  myeloid-derived suppressor cells (MDSCs) and even disregulated NK cells. 24 In order to change this tumor protective microenvironment and to improve the effects of immunotherapeutic cellular drugs, two main approaches are used, i.e. the non-myeloablative pre- conditioning and the immune checkpoint blockade. With the first one, by applying selected chemotherapeutic drugs (e.g. fludarabine, cyclophosphamide) and γ-irradiation, the numbers of immunosuppressive lymphocytes, MDSCs, TAMs and DC2 are greatly reduced. Consequently more favourable conditions are re-installed that enable maturation of effector DCs (DC1) and thereby effective activation of anti-tumor T cell clones with TA/TAA, as well as higher production of proinflammatory cytokines. Additionally, irradiation induces apoptosis of TCs which are better targets for specific anti-tumor effector CD8+ cytotoxic T cells (CTLs) (5). On the other hand, for the immune checkpoint blocking, specific antibodies that prevent negative, immunosuppressing co- stimulatory receptor-ligand interactions, are used, e.g. anti-CTLA-4, anti-PD1 in case of T cells, and for example anti-KIR, anti-NKG2A in case of NK cells (3, 6, 7). Different types of cellular anti-cancer immunotherapies enable personal, patient-specific treatments, and are based on:  ex vivo selection and expansion of anti-tumor CD8+ T lymphocyte clones (CTLs), isolated from tumor biopsies (TILs – tumor infiltrating lymphocytes) of patients and their subsequent adoptive transfer back to them, together with high dose human recombinant IL-2 (autologous adoptive immunotherapy) (8);  in vitro preparation of TA/TAA-specific effector CD8+ CTLs, using autologous DCs or artificial antigen presenting cells (APCs), and their subsequent adoptive transfer, together with high dose human recombinant IL-2 (autologous adoptive immunotherapy);  in vitro preparation of anti-tumor cellular vaccines based on autologous peripheral blolod monocyte-derived DCs, loaded with TA/TAA;  ex vivo isolation, expansion and activation of patient's autologous NK cells and their subsequent adoptive transfer back to him/her (7). Adoptive immunotherapy with autologous TILs has proved to be effective especialy in non- myeloablatively pre-conditioned metastatic melanoma patients (8, 9). The first ever approved (FDA) autologous cellular immunotherapy drug Sipuleucel-T for treating castrate-resistant prostate cancer, is based on the immunostimulatory capacities of DCs, presentig prostate acid phospatase (PAP) antigens, enhanced by granulocyte and monocyte colonies stimulating factor (GM-CSF) (10). We have optimized two methods for providing TA/TAA to autologous DCs, i.e. their transfection with native or amplified total tumor RNA and their electrofusion with lethaly irradiated TCs (preparation of immunohybridomas) (11, 12). Immunohybridomas are wery efficient in presenting a large array of TA/TAA within the context of patient's MHC molecules and are able to evoke strong anti-tumor CD8+ CTL responses in vitro (Picture 2). Currently we are performing a clinical study using autologous immunohybridomas for treating patients with castrate-resistant prostate cancer. 25 Picture 2: a and b – electrofused immunohybridomas (grey columns) evoke statistically more potent anti-tumor-specific cytotoxic immune responses than mixed DCs and TCs (12). Currently, also other types of anti-cancer vaccinations are being evaluated, for example the Prostvac VF vaccine, using viral vectors packed with genes coding for specific TA (PSA - prostate specific antigen) and several costimulatory molecules, as well as T cells bearing TA/TAA-specific chimeric antigen receptors (CARs) (13, 14, 15). It is expected that in the future cell-based immunotherapies will be used early in the onset of malignant diseases and will be applied together with combinations of different classical (cytotoxic drugs, irradiation) and new (immune checkpoint blockade) anti-cancer therapies. Literature: 1. Lawrence MS, Stojanov P, Polak P, et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 2013; 499:214-218 2. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in tumor microenvironment. Nat Immunol 2013; 14(10):1014-1022 3. Sharma P, Allison JP. The future of immune checkpoint therapy. Science 2015; 348:56-61 4. Joyce J, Fearon DT. T cell exclusion, immune privilege, and the tumor microenvironment. Science 2015; 348:74-80 5. Gattinoni L, Powell DJ Jr, Rosenberg SA, et al. Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol 2006; 6:383-393 6. Kyi C, Postow MA. Checkpoint blocking antibodies in cancer immunotherapy. FEBS Letters 2014; 588:368-376 7. Tarazona R, Sanchez-Correa B, Casas-Avilés, et al. Immunosenescence: limitations in natural killer cell-based immunotherapy. Cancer Immunol Immunother 2016; DOI 10.1007/s00262- 016-1882-x 8. Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 2015; 348:62-68 9. Rosenberg SA, Dudley ME. Adoptive cell therapy for the treatment of patients with metastatic melanoma. Curr Opin Immunol 2009; 21(2):233-240 10. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-Z immunotherapy for castration-resistant prostate cancer. NEJM 2010; 363(5):411-422 11. Bergant M, Meden L, Repnik U, et al. Preparation of native and amplified tumour RNA for dendritic cell transfection and generation of in vitro anti-tumour CTL responses. Immunobiology 2006; 211:179-189 12. Gabrijel M, Bergant M, Kreft M, et al. Fused late endocytic compartments and immunostimulatory capacity of dendritic-tumor cell hybridomas. J Membrane Biol 2009; 229:11-18 26 13. Drake CG. Prostate cancer as a model for tumor immunotherapy. Nat Rev Immunol 2010; 10(8):580-593 14. Kantoff MD, Schuetz TJ, Blumenstein BA, et al. Overall survival analysis of a phase II randomized controlled trial of a Poxviral-based PSA-targeted immunotherapy in metastatic castration-resistant prostate cancer. J Clin Oncol 2010; 28(7):1099-1105 15. Minagawa K, Zhou X, Mineishi S, et al. Seatbelts in CAR therapy: how safe are CARs? Pharmaceuticals 2015; 8:230-249 27 Life science research from a postdoc prospective Janja Zupan, University of Ljubljana, Faculty of Pharmacy, Slovenia Discovering new drugs, treatment possibilities, developing new methods or new models in various disorders that would benefit lives of the people all round the world is a dream every researcher in life science dreams off. Many groundbreaking advances in life science of the 20th and 21st century such as discovery of the penicillin, DNA helix and polymerase chain reaction as the basis for molecular biology and later on recombinant technology, have completely changed the way that the modern world lives and thrives. Life science is a broad field of diverse sciences that share researching live species, either humans (medicine) or animals (biology) with the ultimate aim to improve quality and standards of their lives. Since the discoveries in biology, i.e. basic science or preclinical studies aim to translate to humans, both branches intercross and the term biomedicine is used most of the time. When it comes to funding and money consumption, there is only space science that requires more resources and financial support than the life science. In comparison to a massive space science project such as generating international space station that costed 150 billion USA dollars, 5 billion every year are going into cancer research according to USA National cancer research institute (1). This should not be of any surprise as the highest levels of ethics (bioethics) and standards are required when researching live organisms in order to protect more lives. Given the fact that millions of people in developed countries die of cancer, cancer research is the most funded area in life science with billions of funding every year, most frequently split 50, 30 and 20% into basic, translational and clinical research pipelines (2). United Kingdom (UK) has one of the strongest and most dynamic life science industries in the world with the annual turnover of 56 billion pounds (3). There is a solid and clear support from the government stating that life sciences is a priority sector for the UK economy with 2 billion pounds in public investment in health life science research via the Research Councils and National Institute for Health Research Programme. The final piece of the puzzle is the very supportive charitable sector consisting of over 130 medical research charities funding a third of all publicly funded research (4).The UK public rank top in the G7 (and fourth in the world) for charitable donations (4).The largest single non-government, non-business source of funds in the UK is the Wellcome Trust, which was established from a bequest by the co- founder of Wellcome, one of the first pharmaceutical businesses in the UK later being sold to Glaxo (5). Cancer Research UK is another importer supporter of research with an income of 621 million pounds in 2014, spending 393 million on research activities, funding over 4,000 scientists in the UK. UK Arthritis research, the fourth largest medical charity having budget of around 37 million pounds, presents the main charity funding musculoskeletal disorders research (6). There comes a time in life of every life science researcher when you want to progress from either observational to functional studies or switching from vitro to in vivo experiments that can only be achieved in preclinical research using animal models. It might also be the case, in particularly if you are only part-time researcher, of striving for better research possibilities, such as protected time to do research only. However, most of the time it is just the matter of finding a job after finishing PhD in life science. The most common way to boost up your career as a life scientist is applying for a postdoctoral research position on a specific life science project. Having been given the chance to work as a life science researcher, there is an enormous potential to develop new skills, learn new methods and put your own ideas to life. On the other hand, life science is highly demanding, time- consuming and most of the times unrewarding, hence it requires strong motivation and perseverance. Creating cutting-edge technologies and producing high impact results are outcomes every postdoc endeavors for in order to apply for your own grants and funding that are becoming more and more competitive in the life science. 28 In the current talk my experience working as a UK Arthritis research postdoctoral research fellow at the Institute of medical Sciences University of Aberdeen, Scotland will be presented with the aim to stimulate young researchers eager enough to be challenged in a competitive field of life science. References: 1. https://financesonline.com/10-worlds-most-expensive-science-experiments/ 2. http://www.cancerresearchuk.org/funding-for-researchers/facts-and-figures-about-our- research-funding 3. Elizabeth Klein, The state of the UK helalthcare and life sciences sectors, 2016, http://www.euromedica.com/wp-content/uploads/2016/02/Final_Report_- _State_of_the_UK_Healthcare_Sector_.pdf 4. A Report by the All-Party Parliamentary Group on Global Health. Hasan, N. et al “The UK’s Contribution to Health Globally: Benefiting the country and the world” Published 29th June 2015. 5. Information from www.wellcome.ac.uk. Downloaded 10/10/16 6. http://www.publications.parliament.uk/pa/cm201415/cmselect/cmhealth/401/401we10.ht m 29 Breast cancer Luminal A and luminal B type of breast cancer Tamara Antonić, University of Belgrade, Faculty of Pharmacy, Serbia Alex Peternel, University of Ljubljana, Faculty of Pharmacy, Slovenia Miron Sopić, University of Belgrade, Faculty of Pharmacy, Serbia Introduction Cancer is one of the leading medical conditions in the western world, among which breast cancer has one of the highest percentage of occurrence [1, 2]. There are many subtypes of breast cancer: basal- like, Claudin-low, human epidermal growth factor 2 (HER2) overexpressing, Luminal A and Luminal B [1, 2]. The luminal A breast cancer is the most common subtype, representing 50–60% of all the subtypes [1, 2]. It is characterized by the expression of genes activated by the estrogen receptor (ER) transcription factor that are typically expressed in the luminal epithelium lining the mammary ducts [1, 2]. In addition, what is typical for luminal A is also the expression of progesterone receptor (PGR) and an absence of HER2 expression [3]. Moreover, the GATA binding protein 3 (GATA3) marker expresses its highest level in the luminal A subgroup [3]. GATA-3 belongs to the GATA family of transcription factors. It regulates luminal epithelial cell differentiation in the mammary gland [4]. GATA-3 is one of the three genes mutated in >10% of breast cancers [5]. Tumors with the luminal B molecular profile make up between 10% and 20% of all breast cancers [1, 2]. Compared to the luminal A they have a more aggressive phenotype, higher histological grade and proliferative index [3]. Usually, patients with luminal B type of breast cancer also have worse prognosis then patients with luminal A type [3]. The main biological difference between the subtypes A and B is an increased expression of proliferation genes in the luminal B subtype such as MKI67 and cyclin B1, as well as overexpression of HER2 [6,7]. Molecular basis ER signaling pathway is a complex biological pathway that controls a variety of functions such as cell proliferation, apoptosis, and angiogenesis which serve as a major survival pathway exploited by breast cancer cells [8]. ER modulates the expression of hundreds of genes, some by upregulation of expression and others by downregulation [8]. Upon estrogen binding, ER dimerizes with another receptor monomer and attracts a complex of co-activators and co-repressors to specific sites on DNA, called estrogen receptor elements (EREs). Consequence of the cascade is induction or modulation of gene transcription including coding growth factors (GFs) and receptor tyrosine kinases (RTKs) [9, 10, 11]. ER can also bind to other transcription factors such as activator protein 1 (AP-1) and specificity protein (SP-1) at their specific sites on DNA, thereby functioning as a co-regulator [10, 12]. ER may also work by non-transcriptional mechanisms. A small subset of the cellular pool of ER localized outside the nucleus and/or at the cell membrane associates in response to estrogen with growth factor RTKs and with additional signaling and co-activator molecules. This interaction activates multiple downstream kinase pathways which in turn phosphorylate various transcription factors (TFs) and co-regulators, including components of the ER pathway that enhance gene expression on EREs and other response elements (RE) [9]. The stress kinase pathway via p38 and c- Jun N-terminal kinases (JNK) can also modulate ER function by phosporilation of ER and its co- regulators [13,14]. The microenvironment and its associated integrin signaling may exert a similar activity [15]. The ER signaling pathway is also regulated by membrane receptor tyrosine kinases, which activate signaling pathways that eventually result in phosphorylation of ER as well as its co- activators and co-repressors influencing their specific function [16-19]. Estrogens could cause de novo breast cancer trough either receptor dependent or independent mechanisms [20]. The most widely accepted theory holds that estradiol (E2), acting through estrogen 30 receptor alpha (ERα), stimulates cell proliferation and initiates mutations arising from replicative errors occurring during pre-mitotic DNA synthesis. The promotional effects of E2 then support the growth of cells harboring mutations. Over a period of time, sufficient numbers of mutations accumulate to induce neoplastic transformation. Laboratory and epidemiological data also suggest that non-receptor mediated mechanisms resulting from the genotoxic effects of estrogen metabolites are involved in breast cancer development [21]. In addition, estrogen metabolites act as possible modulators of stem cell functionality and cancer progression [21]. Diagnosis Luminal A subtype has been defined as ER+/PR+/HER2- [22]. Fewer than 15 % of luminal A tumors have p53 gene mutations [23, 24]. Detection of ER and HER2 is immunohistochemical, but the results are checked and compared with fluorescence in situ hybridization (FISH) [25]. Luminal A and B both express ER, but, since luminal B prognosis is worse, a strong effort to find biomarkers that distinguish between these two subtypes has been made [26]. One of the candidate markers for differencing between these two subtypes of breast cancer is Ki67 protein that may be necessary for cellular proliferation [22, 27]. The Luminal A subtype has been defined as ER+/PR+/HER2- and low level of Ki67, while the luminal B subtype has tumors with ER+/PR+/HER2- and high Ki67 or ER+/PR+/HER2+ [22]. One should bear in mind that this definition does not include all luminal B subtype tumors (up to 6% of the luminal B tumors are clinically ER-/HER2-). Also, technique used to determine Ki67 (cut- off point to distinguish luminal A and B set at 13.25%) has not been standardized which adds a variability factor in the assessment of this marker [22]. Therapy Patients with luminal A subtype of cancer have a good prognosis; the relapse rate is 27.8% being significantly lower than that for other subtypes [3]. They have a distinct pattern of recurrence with a higher incidence of bone metastases (18.7%) and with respect to other localizations such as central nervous system, liver and lung which represent less than 10%. The treatment of this subgroup of breast cancer is mainly based on third-generation hormonal aromatase inhibitors (AI) in postmenopausal patients, selective estrogen receptor modulators (SERMs) like tamoxifen and pure selective regulators of ER like fulvestrant [28]. Luminal B also has a chance of relapse, but the pattern differs of that for Luminal A. It is still a tumor with relatively good prognosis in general, but much worse than Luminal A subtype [6, 7]. But on the bright side they respond better to neoadjuvant chemotherapy achieving pathological complete response (pCR) in 17% of the luminal B tumors (7% in luminal A). For these reasons, treatment of this subtype of breast cancer is currently challenging. Many questions about what mechanisms lead to their survival, proliferation and metastatization remain unanswered. Numerous clinical trials are testing inhibitory molecules of the PI3K/AKT/mTOR pathway at different levels, focusing on the treatment of luminal B tumors [26]. References: [1] Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge Ø. Molecular portraits of human breast tumours. Nature. 2000 Aug 17;406(6797):747-52. [2] Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, Van De Rijn M, Jeffrey SS, Thorsen T. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences. 2001 Sep 11;98(19):10869-74. [3] Kennecke H, Yerushalmi R, Woods R, Cheang MC, Voduc D, Speers CH, Nielsen TO, Gelmon K. Metastatic behavior of breast cancer subtypes. Journal of clinical oncology. 2010 Jul 10;28(20):3271- 7. 31 [4] Kouros-Mehr H, Slorach EM, Sternlicht MD, Werb Z. GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell. 2006 Dec 1;127(5):1041-55. [5] Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature. 2012 Oct 4;490(7418):61-70. [6] Loi S, Haibe-Kains B, Desmedt C, Lallemand F, Tutt AM, Gillet C, Ellis P, Harris A, Bergh J, Foekens JA, Klijn JG. Definition of clinically distinct molecular subtypes in estrogen receptor–positive breast carcinomas through genomic grade. Journal of clinical oncology. 2007 Apr 1;25(10):1239-46. [7] Fan C, Oh DS, Wessels L, Weigelt B, Nuyten DS, Nobel AB, Van't Veer LJ, Perou CM. Concordance among gene-expression–based predictors for breast cancer. New England Journal of Medicine. 2006 Aug 10;355(6):560-9. [8] Frasor J, Stossi F, Danes JM, Komm B, Lyttle CR, Katzenellenbogen BS. Selective estrogen receptor modulators discrimination of agonistic versus antagonistic activities by gene expression profiling in breast cancer cells. Cancer research. 2004 Feb 15;64(4):1522-33. [9] Osborne CK, Schiff R. Mechanisms of endocrine resistance in breast cancer. Annual review of medicine. 2011;62:233. [10] Sperduto PW, Kased N, Roberge D, Xu Z, Shanley R, Luo X, Sneed PK, Chao ST, Weil RJ, Suh J, Bhatt A. Effect of tumor subtype on survival and the graded prognostic assessment for patients with breast cancer and brain metastases. International Journal of Radiation Oncology* Biology* Physics. 2012 Apr 1;82(5):2111-7. [11] Klinge CM. Estrogen receptor interaction with estrogen response elements. Nucleic acids research. 2001 Jul 15;29(14):2905-19. [12] Kushner PJ, Agard DA, Greene GL, Scanlan TS, Shiau AK, Uht RM, Webb P. Estrogen receptor pathways to AP-1. The Journal of steroid biochemistry and molecular biology. 2000 Nov 30;74(5):311-7. [13] Wu RC, Qin J, Yi P, Wong J, Tsai SY, Tsai MJ, O'Malley BW. Selective phosphorylations of the SRC- 3/AIB1 coactivator integrate genomic reponses to multiple cellular signaling pathways. Molecular cell. 2004 Sep 24;15(6):937-49. [14] Lee H, Bai W. Regulation of estrogen receptor nuclear export by ligand-induced and p38- mediated receptor phosphorylation. Molecular and cellular biology. 2002 Aug 15;22(16):5835-45. [15] Pontiggia O, Rodriguez V, Fabris V, Raffo D, Bumaschny V, Fiszman G, de Kier Joffé EB, Simian M. Establishment of an in vitro estrogen-dependent mouse mammary tumor model: a new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal–epithelial interactions. Breast cancer research and treatment. 2009 Jul 1;116(2):247-55. [16] Schiff R, Massarweh SA, Shou J, Bharwani L, Mohsin SK, Osborne CK. Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clinical Cancer Research. 2004 Jan 1;10(1):331s-6s.] [17] Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H, Schiff R. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2–positive breast cancer. Journal of the National Cancer Institute. 2004 Jun 16;96(12):926-35. [18] Wu RC, Smith CL, O’Malley BW. Transcriptional regulation by steroid receptor coactivator phosphorylation. Endocrine reviews. 2005 May 1;26(3):393-9. [19] Schiff R, Massarweh S, Shou J, Osborne CK. Breast cancer endocrine resistance how growth factor signaling and estrogen receptor coregulators modulate response. Clinical Cancer Research. 2003 Jan 1;9(1):447s-54s. 32 [20] Preston-Martin S, Pike MC, Ross RK, Jones PA, Henderson BE. Increased cell division as a cause of human cancer. Cancer research. 1990 Dec 1;50(23):7415-21. [21] Yue W, Yager JD, Wang JP, Jupe ER, Santen RJ. Estrogen receptor-dependent and independent mechanisms of breast cancer carcinogenesis. Steroids. 2013 Feb 28;78(2):161-70. [22] Cheang MC, Chia SK, Voduc D, Gao D, Leung S, Snider J, Watson M, Davies S, Bernard PS, Parker JS, Perou CM. Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. Journal of the National Cancer Institute. 2009 May 20;101(10):736-50. [23] Harris JR, Lippman ME, Osborne CK, Morrow M. Diseases of the Breast. Lippincott Williams & Wilkins; 2012 Mar 28. [24] Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature. 2012 Oct 4;490(7418):61-70. [25] Hanley KZ, Birdsong GG, Cohen C, Siddiqui MT. Immunohistochemical detection of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression in breast carcinomas. Cancer Cytopathology. 2009 Aug 25;117(4):279-88. [26] Eroles P, Bosch A, Pérez-Fidalgo JA, Lluch A. Molecular biology in breast cancer: intrinsic subtypes and signaling pathways. Cancer treatment reviews. 2012 Oct 31;38(6):698-707. [27] Bullwinkel J, Baron‐Lühr B, Lüdemann A, Wohlenberg C, Gerdes J, Scholzen T. Ki‐67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells. Journal of cellular physiology. 2006 Mar 1;206(3):624-35. [28] Guarneri V, Conte P. Metastatic breast cancer: therapeutic options according to molecular subtypes and prior adjuvant therapy. The oncologist. 2009 Jul 1;14(7):645-56. 33 HER2 positive type of breast cancer Angela Milanović, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia Amar Gušić, University of Sarajevo, Faculty of Pharmacy, Bosnia and Herzegovina Miron Sopić, University of Belgrade, Faculty of Pharmacy, Serbia Introduction The most common type of cancer in women is breast cancer. Prevalence of this type of cancer is growing highly worldwide from 0.0193% women in region of Eastern Africa to 0.0897% women in the region of Western Europe (1). It is well known that there are a significantly higher percentage of black women that have this malignant disease in comparison to white women (2). It is estimated that HER2 is amplified at about 20-30% cases of breast cancers. Types of breast cancers which are HER2 positive include Ductual Carcinoma in situ (DCIS) and also at Inflammatory Breast Cancer (IBC) and Paget disease of the nipple (3). Survival rates are different worldwide ranging from 80% and above in Sweden, Japan and North America to 60% in middle-developed countries and below 40% in low- developed countries. Low survival rate in these countries is mainly consequence of poor chance for early detection, as well as lack of right diagnostic procedure and treatment conditions (1). Molecular basis The human epidermal growth factor receptor 2 oncogene (HER2) is protein that encodes the type 1 receptor tyrosine kinase (4). Activation of HER2 leads to proliferation, migration and invasion in breast cancer (5). HER2 is receptor-like tyrosine kinase, also a member of the membrane-spanning type I. This family of receptors dimerize after being stimulated by ligand which leads to subsequent autphosphorylation and recruitment of many different downstream signaling cascades. Hyper- activated signaling trough this pathway results with dysregulation of the cell cycle homeostatic system. This can lead to dysregulated growth, oncogenesis, metastasis and potential chemorestistance (3). Mechanism that includes tumorigenic ability of HER2 is related to high basal autophosphorylation activity of this receptor-like tirosin kinase. In process of overexpression at the surface of the cell homodimers may be spontaneously formed, and may increase its availability for heterodmiers formation when a ligand binds to its direct receptor (3). HER2 is significantly enhanced when it is co-expressed with ErbB1 and ErbB3. Overall overexpressed HER2 can promote carcinogenesis primarily in the context of a ligand driven heterodimer and also interacts with MAPK and PI3K pathways. MAPK signaling is being prolonged in situation of HER2 overexpression (3). One of the co-receptors that seem to have a pivotal role in HER2 signaling is ErbB1. Its overexpression correlates inversely with ER status and also correlates with poor prognosis of treatment (3). ErbB3 and ErbB4 have positive correlation with ER status and increased survival prognosis (3). Cyclin D plays a pivotal role in process of breast cancer where in process of HER2 overexpression results with upregulation of cyclin D protein expression and that upregulation involves SP1 and E2F transcription factors (3). Diagnose Right diagnostic procedure of human HER2 status is crucial for obtaining wanted breast cancer outcome. Tumors that have HER2 overexpression are labeled as HER2 positive while those with normal HER2 levels are labeled as HER2 normal or HER2 negative (4). HER2 positive tumors have higher mortality prevalence in early-stage disease, reduced relapse time and increased indices of metastases (4). The most commonly used technique to detect HER2 status is immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). IHC assesses the expression of HER2 protein in cell membranes while FISH method assesses HER2 gene amplification (6). There 34 are many commercially available testing units approved by FDA. They included IHC tests, FISH assays and colorimetric ISH assays. Interpretation of a immunohistochemistry test is based on a 0, 1+, 2+, 3+ scoring system. Results that demonstrate strong complete membrane staining in > 10% of tumor cells are classified as 3+. Specimens which show weak-to-moderate staining in >10% of tumor cells are scored 2+ and are labeled as equivocal and require confirmation of HER2 status by alternative method, usually ISH. Specificity is reported at level of 100%, sensitivity of 70% and accuracy of 85-89 %. (8) HER2 FISH testing was approved based on the high rate of concordance reported between the IHC CTA and FISH assays of 82% to 92% (9). These two methods are considered as gold standard in this area of interest. FISH method is more accurate, reproducible and robust compared to IHC. Generally IHC is widely used as the primary test for HER2 status. IHC is comparatively quick while results can be viewed using a conventional microscope and strained tissue do not degrade over time. IHC also allows parallel viewing of tumor cell morphological features (4). CISH and silver-enhanced in situ hybridization (SISH) are new technologies that are being developed. CISH uses a peroxidase enzyme- labeled probe for chromogenic detection by diaminobenzidine, while SISH uses the same system with a silver-based detection system (4). Therapy Generally, woman diagnosed with breast cancer are being treated with adjuvant systemic therapies to reduce the risk of recurrence. (10) The main focus in the treatment of HER2-positive breast cancer in recent years has been based on developing therapeutic agents to either potentiate the effect of trastuzumab or target cells which have become resistant to trastuzumab. Preclinical evidence suggest that co-inhibition of HER2 as well as other members of the HER family and/or the downstream pathway by giving trastuzumab/lapatinib in combination with other targeted therapies might prevent or prolong time to resistance and treatment failure (11). Trastzumab is a recombinant, humanized monoclonal antibody which effects through binding to the extracellular domain IV of HER2. It is combined usually with lapatinib who is reversible dual HER1/EGFR and HER2 tyrosine kinase inhibitor (TKI). Trastazumab can also be combined with pertzumab. Pertzumab is fully humanized antibody binding to domain II of HER2 (12). Antibodies conjugated to toxins, radionucleotides and prodrugs are showing promising results in animal models (3). In the recent time, the outcome for patients with HER2- positive breast cancer has improved significantly. Clinical guidelines recommend HER2-directed therapies as backbone therapy for these patients. Otherwise, resistance to HER2-directed therapies remains a challenge. Evidence suggests that combinations of HER2-directed agents may show additive or synergistic effect and lead to better outcome (12). However, target populations for specific HER2-directed therapies remain to be defined. Also the ongoing concern regarding side effects especially cardiotoxicity needs to be addressed. There is an urgent need for prospective biomarker-driven trials to identify patients for whom dual targeting is cost-effective (12). References: 1. Acharya UR, Ng EY, Tan JH, Sree SV. Thermography based breast cancer detection using texture features and support vector machine. Journal of medical systems. 2012 Jun 1;36(3):1503-10. 2. Cervival Cancer Facts and Stats. [Online]. April 26, 2016. avaliable on: https://qap.sdsu.edu/screening/cervicalcancer/facts.html. [accessed 20.08.2016]. 3. Harari D, Yarden Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene. 2000 Dec 11;19(53). 4. Perez EA, Cortés J, Gonzalez-Angulo AM, Bartlett JM. HER2 testing: current status and future directions. Cancer treatment reviews. 2014 Mar 31;40(2):276-84. 35 5. Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN. The HER-2 receptor and breast cancer: ten years of targeted anti–HER-2 therapy and personalized medicine. The oncologist. 2009 Apr 1;14(4):320-68. 6. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, McShane LM. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Archives of pathology & laboratory medicine. 2007 Jan;131(1):18-43. 7. Food and Drug Administration. In vitro companion diagnostics devices. 2013. http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/uc m301431.htm. [accessed 20.08.2016]. 8. Press MF, Slamon DJ, Flom KJ, Park J, Zhou JY, Bernstein L. Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. Journal of Clinical Oncology. 2002 Jul 15;20(14):3095-105. 9. Perez EA, Press MF, Dueck AC, Jenkins RB, Kim C, Chen B, Villalobos I, Paik S, Buyse M, Wiktor AE, Meyer R. Immunohistochemistry and fluorescence in situ hybridization assessment of HER2 in clinical trials of adjuvant therapy for breast cancer (NCCTG N9831, BCIRG 006, and BCIRG 005). Breast cancer research and treatment. 2013 Feb 1;138(1):99-108. 10. Dhesy-Thind B, Pritchard KI, Messersmith H, O’Malley F, Elavathil L, Trudeau M. HER2/neu in systemic therapy for women with breast cancer: a systematic review. Breast cancer research and treatment. 2008 May 1;109(2):209-29. 11. Baselga J. Treatment of HER2-overexpressing breast cancer. Annals of Oncology. 2010 Oct 1;21(suppl 7):vii36-40.. 12. Kümler I, Tuxen MK, Nielsen DL. A systematic review of dual targeting in HER2-positive breast cancer. Cancer treatment reviews. 2014 Mar 31;40(2):259-70. 36 Basal-like type of breast cancer Klara Habartova, Charles University, Faculty of Medicine, Hradec Kralove, Czech Republic Mojca Hunski, University of Ljubljana, Faculty of Pharmacy, Slovenia Miron Sopić, University of Belgrade, Faculty of Pharmacy, Serbia Introduction Basal-like breast cancer (BLBC) is one of the five major breast cancer intrinsic subtypes based on gene expression profiling [1, 2]. It was named so because the cancer cells consistently express genes that are expressed in the normal basal/myoepithelial breast cells [3]. It is a particularly aggressive subtype and is associated with larger tumour size and more rapid tumour growth. Patients with these tumours often relapse rapidly [4]. Basal-like breast cancers account for 15-25% of all breast cancer cases. They are more frequent among premenopausal women. Patients with BLBC are in average younger than patients with other subtypes of breast cancer. It is also known that BLBC is more prevalent in African American women than non-African American women [5]. Molecular basis Basal-like breast cancers have a specific genetic pattern. The majority of them is triple-negative, i.e. the tumours lack or show low expression of estrogen receptor (ER), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (HER2) [4]. On the other hand, neoplastic basal-like cells express abnormally large amounts of genes that encode proteins with different functions (signal transduction, cell growth and division, angiogenesis, apoptosis, DNA replication and recombination) [4] such as cytokeratins (5/6, 14 and 17), epidermal growth factor receptor (EGFR), P-cadherin, caveolins 1 and 2, tyrosine kinase receptors, etc [6]. Some of them (cytokeratin5/6, EGFR) are associated with poor prognosis in breast cancer [4]. Furthermore, basal-like tumours are similar to the majority of tumours arising in BRCA1 mutation carriers regarding morphological phenotype, molecular genetic profile and clinical outcome (high proliferation, triple negativity, expression of basal cytokeratins, genome instability, poor prognosis, etc.) [7, 8]. Because of all these similarities and BRCA1 mutations that are very frequent within the basal subtype [1] scientists assume that there is a defect in the BRCA1 DNA-repair pathway in sporadic basal-like breast cancers (triple-negative), but it is not clear whether BRCA1 is a cause for or a consequence of basal phenotype [9]. BRCA1 is a gene that encodes a nuclear phosphoprotein which acts as a tumour suppressor and also plays a very important role in transcription, recombination and maintaining genomic stability by combining with other proteins to mend double- stranded breaks in DNA caused by different factors [10]. First step in repairing double-stranded breaks involves protein kinases ATM and ATR which sense and recognise the damaged DNA and transduce this signal onwards by phosphorylating particular proteins, including BRCA1 [11]. Phosphorylated BRCA1 can activate proteins BRCA2, RAD51, BARD1 and other components and form complex termed BRCA1-BRCA2-Containing Complex (BRCC) which has E3 ubiquitin ligase activity associated with DNA repair [12]. On the other hand, activated BRCA1 can be a part of BRCA1-Associated Genome Surveillance Complex (BASC) which includes tumour suppressors, signal transducers and DNA damage sensors. BASC is associated with DNA replication or DNA-replication repair [11, 12]. Because BRCA1 plays a significant role in sustaining genomic integrity of the cell, mutations, that prevent the production of BRCA1 protein or cause the production of abnormal and inactive protein, can be the reason for developing a cancer especially breast and ovarian cancer [11]. 37 Diagnosis Basal triple negative carcinoma is usually diagnosed by mammography or ultrasound where is visible ill-defined oval, round or lobulated mass lesion with partially indistinct margins. Ultrasound may show pseudocystic component due to necrosis in larger lesions. Biopsy of most basal carcinomas display distinctive morphologic features like circumscriptions with pushing borders, sheets of phleomorphic tumour cells with syncytial-like grow pattern, high nuclear rate and mitotic index [14]. Immunohistochemical testing is negative for estrogen receptors, progesteron receptors and epidermal growth factor receptor 2 [15]. Testing for BRCA1 mutation by genetic screening is also necessary [14]. Therapy Among the subtypes basal triple negative tumours are particularly challenging. In contrast to ER- positive or HER2-positive tumours, no targeted therapy is currently available for these tumours [4]. The triple negativity of basal breast cancer tumours does not render them candidate to hormone therapy and anti-HER2 therapies (e.g. trastuzumab), and until now, surgery in combination with radiation therapy and chemotherapy was the sole available treatment [16]. Recent insights in the pathogenesis of these tumours are being translated into development of new strategies targeting molecular alterations. Some researchers have shown that hormone-receptor-negative breast cancers, actually respond better to chemotherapy than hormone-receptor-positive breast cancers [4]. This concept is supported by most neo-adjuvant anthracycline or taxane-based chemotherapy studies, which reported a higher rate of pathological complete response in the basal subtype than in others [17, 18]. However, despite relatively high rate of pathological complete response, basal tumours are associated with a relatively poor prognosis [19]. Most patients relapse within first and third year and the majority of deaths occur within five years following therapy [20]. This high relapse rate calls for the development of more effective chemotherapy and clinical trials are underway, which undoubtedly, will contribute to enlarge therapeutic possibilities in a near future [21, 22]. The first strategy utilizes the defect in double-strand DNA break repair mechanism, which should confer sensitivity to certain drugs (e.g. platinum compounds, anthracyclines or bleomycine) [23]. The other way to profit from the DNA repair defect is the use of poly (ADP-ribose) polymerase (PARP1) inhibitors. This enzyme is crucial in the base excision repair of single-strand DNA breaks. In its absence, single-strand breaks degenerate to double-strand breaks, which are not able to repair, because BRCA1 is deficient [24]. Several PARP1 inhibitors (e.g. iniparib, veliparib) are, alone or in combination with chemotherapy, in clinical development [25, 26]. Several other potential targets for triple negative tumours are connect with signal pathways and secondary messengers, including everolimus, a mammalian Target of Rapamycine (mTOR) inhibitor. mTOR activation is frequent in triple-negative breast cancers and has been associated with cisplatinum resistance [27]. Dasatinib, which inhibits ABL and SRC family kinases, is another possible drug [28]. Another study reports the phase I/II clinical trial of the drug IMMU-132, which was not long ago granted "breakthrough" status by the FDA, meant to speed the approval process of the most promising new drugs. IMMU-132 is an antibody-drug conjugate, which is composed of an irinotecan and an antibody that binds to the protein Trop2, which is overexpressed in about 80 percent of all triple-negative breast cancers. The approach is similar to the mechanics of the immune system in which an antibody recognizes the surface proteins of a bacterium or virus and then directs a T cell to target invaders. Today no targeted or cytotoxic agent has yet been registered in triple-negative breast cancer, but many drugs are under development, and more trials will be soon activated in the metastatic, neo- adjuvant and adjuvant settings, which may improve patients’ life. 38 References 1. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, Van De Rijn M, Jeffrey SS, Thorsen T. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proceedings of the National Academy of Sciences. 2001 Sep 11;98(19):10869-74. 2. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge Ø. Molecular portraits of human breast tumours. Nature. 2000 Aug 17;406(6797):747-52. 3. Jones C, Mackay A, Grigoriadis A, Cossu A, Reis-Filho JS, Fulford L, Dexter T, Davies S, Bulmer K, Ford E, Parry S. Expression profiling of purified normal human luminal and myoepithelial breast cells identification of novel prognostic markers for breast cancer. Cancer research. 2004 May 1;64(9):3037-45. 4. Bertucci F, Finetti P, Birnbaum D. Basal breast cancer: a complex and deadly molecular subtype. Current molecular medicine. 2012 Jan 1;12(1):96-110. 5. Bertucci F, Finetti P, Cervera N, Charafe‐Jauffret E, Buttarelli M, Jacquemier J, Chaffanet M, Maraninchi D, Viens P, Birnbaum D. How different are luminal A and basal breast cancers?. International Journal of Cancer. 2009 Mar 15;124(6):1338-48. 6. Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez-Boussard T, Livasy C, Cowan D, Dressler L, Akslen LA. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clinical cancer research. 2004 Aug 15;10(16):5367-74. 7. Foulkes WD, Stefansson IM, Chappuis PO, Bégin LR, Goffin JR, Wong N, Trudel M, Akslen LA. Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. Journal of the National Cancer Institute. 2003 Oct 1;95(19):1482-5. 8. Kriege M, Seynaeve C, Meijers-Heijboer H, Collee JM, Menke-Pluymers MB, Bartels CC, Tilanus-Linthorst MM, van den Ouweland A, van Geel B, Brekelmans CT, Klijn JG. Distant disease-free interval, site of first relapse and post-relapse survival in BRCA1-and BRCA2- associated compared to sporadic breast cancer patients. Breast cancer research and treatment. 2008 Sep 1;111(2):303-11. 9. Turner NC, Reis-Filho JS, Russell AM, Springall RJ, Ryder K, Steele D, Savage K, Gillett CE, Schmitt FC, Ashworth A, Tutt AN. BRCA1 dysfunction in sporadic basal-like breast cancer. Oncogene. 2007 Mar 29;26(14):2126-32. 10. NCBI: http://www.ncbi.nlm.nih.gov/gene/672 (available in August 2016) 11. Gudmundsdottir K, Ashworth A. The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability. Oncogene. 2006 Sep 25;25(43):5864-74. 12. Dong Y, Hakimi MA, Chen X, Kumaraswamy E, Cooch NS, Godwin AK, Shiekhattar R. Regulation of BRCC, a holoenzyme complex containing BRCA1 and BRCA2, by a signalosome- like subunit and its role in DNA repair. Molecular cell. 2003 Nov 30;12(5):1087-99. 13. Wang Y, Cortez D, Yazdi P, Neff N, Elledge SJ, Qin J. BASC, a super complex of BRCA1- associated proteins involved in the recognition and repair of aberrant DNA structures. Genes & development. 2000 Apr 15;14(8):927-39. 14. Lester SC, Hicks D. Diagnostic Pathology: Breast. Elsevier Health Sciences. 2016: 326-328. 15. Korsching E, Jeffrey SS, Meinerz W, Decker T, Boecker W, Buerger H. Basal carcinoma of the breast revisited: an old entity with new interpretations. Journal of clinical pathology. 2008 May 1;61(5):553-60. 39 16. Badve S, Dabbs DJ, Schnitt SJ, Baehner FL, Decker T, Eusebi V, Fox SB, Ichihara S, Jacquemier J, Lakhani SR, Palacios J. Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists. Modern pathology. 2011 Feb 1;24(2):157-67. 17. Rouzier R, Perou CM, Symmans WF, Ibrahim N, Cristofanilli M, Anderson K, Hess KR, Stec J, Ayers M, Wagner P, Morandi P. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clinical Cancer Research. 2005 Aug 15;11(16):5678-85. 18. Bertucci F, Finetti P, Rougemont J, Charafe-Jauffret E, Cervera N, Tarpin C, Nguyen C, Xerri L, Houlgatte R, Jacquemier J, Viens P. Gene expression profiling identifies molecular subtypes of inflammatory breast cancer. Cancer research. 2005 Mar 15;65(6):2170-8. 19. Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, Collichio F, Ollila DW, Sartor CI, Graham ML, Perou CM. The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clinical Cancer Research. 2007 Apr 15;13(8):2329-34. 20. Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA. Triple-negative breast cancer: clinical features and patterns of recurrence. Clinical Cancer Research. 2007 Aug 1;13(15):4429-34. 21. Rodler E, Korde L, Gralow J. Current treatment options in triple negative breast cancer. Breast disease. 2010 Jan 1;32(1, 2):99-122. 22. Carey LA. Directed therapy of subtypes of triple-negative breast cancer. The oncologist. 2011 Jan 1;16(Supplement 1):71-8. 23. Quinn JE, Kennedy RD, Mullan PB, Johnston PG, Harkin DP. 211 BRCA1 functions as a differential modulator of chemotherapy induced apoptosis. European Journal of Cancer Supplements. 2004 Sep 30;2(8):65. 24. Turner N, Tutt A, Ashworth A. Targeting the DNA repair defect of BRCA tumours. Current opinion in pharmacology. 2005 Aug 31;5(4):388-93. 25. Tutt A, Robson M, Garber JE, Domchek SM, Audeh MW, Weitzel JN, Friedlander M, Arun B, Loman N, Schmutzler RK, Wardley A. Oral poly (ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial. The Lancet. 2010 Jul 30;376(9737):235-44. 26. Isakoff SJ, Overmoyer B, Tung NM, Gelman RS, Habin K, Qian J, Giranda V, Shepherd S, Garber JE, Ellisen LW, Winer EP. P3-16-05: A Phase II Trial Expansion Cohort of the PARP Inhibitor Veliparib (ABT888) and Temozolomide in BRCA1/2 Associated Metastatic Breast Cancer. Cancer Research. 2011 Dec 15;71(24 Supplement):P3-16. 27. Tanaka C, O’Reilly T, Kovarik JM, Shand N, Hazell K, Judson I, Raymond E, Zumstein-Mecker S, Stephan C, Boulay A, Hattenberger M. Identifying optimal biologic doses of everolimus (RAD001) in patients with cancer based on the modeling of preclinical and clinical pharmacokinetic and pharmacodynamic data. Journal of Clinical Oncology. 2008 Apr 1;26(10):1596-602. 28. Finn RS, Bengala C, Ibrahim N, Roché H, Sparano J, Strauss LC, Fairchild J, Sy O, Goldstein LJ. Dasatinib as a single agent in triple-negative breast cancer: results of an open-label phase 2 study. Clinical Cancer Research. 2011 Nov 1;17(21):6905-13. 40 Gastrointestinal cancer Gastric cancer (GC) Karla Komšić, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia Veronika Novak, University of Ljubljana, Faculty of Pharmacy, Slovenia Jelena Kotur-Stevuljević, University of Belgrade, Faculty of Pharmacy, Serbia Epidemiology The stomach is divided into several anatomic subsites, including the cardia, fundus, body, pylorus, and the antrum. These areas are distinguished by anatomic demarcations, histological differences, or both. Most important is the difference between adenocarcinomas arising from the cardia (cardiac GC) and other parts of the stomach (noncardiac GC), as they have different epidemiologic patterns and causes. Risk factors for cancers arising from cardia and noncardia regions of the stomach may be different. Common risk factors for both are older Figure 1: Stages of gastric cancer (9) age, male sex, tobacco smoking, radiation, and genetics. Intake of aspirin and statins may prevent against both of these cancers. While race is a risk factor for each, the direction differs by site. Factors associated with cardiac GC, but not noncardiac GC, include obesity and gastroesophageal reflux disease which may transform to Barrett's Esophagus. On the other hand, risk factors that are exclusive for noncardiac GC include H. pylori infection (especially in Western countries and Japan), low socioeconomic status, and perhaps diet factors such as low consumption of fruits and vegetables and high intake of salty and smoked food (1,2). Molecular basis Increased mutations in specific genes has impact on the expression of factors connected with the tumor etiology such as: epidermal growth factor (EGF), EGF receptor, K-sam, human epidermal growth factor treceptor (HER-2), interleukin (IL)-8, vascular endothelial growth factor (VEGF), cyclin E, p27, E-cadherin, CD44v6, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) GC. Microsatellite instability status (MSI) is defined as the presence of replication errors in simple repetitive microsatellite sequences due to DNA mismatch repair deficiency. It is classified as high- frequency (MSI-H), low-frequency (MSI-L), or stable (MSS). All those pathological changes and pathways are often found in patients with the H. pylori infection (3,4,5). Diagnostics Nowadays, GC might be identified by various diagnostic biomarkers, for example: p53, adenomatous polyposis coli (APC – tumor suppressor protein), and CD44 have been used as markers for differential diagnosis. Epidermal growth factor receptor (EGFR), c-met, and c-erbB2 have been used to show the stage of malignancy, and MSI has been used as a marker for the screening of genetic instability. A recent report showed that MSI and inactivation of hMLH1 by hypermethylation were more used in early gastric cancers than in gastric adenomas. Techniques of detecting MSI are very efficient because they can diagnose tumors before they become microscopically seen. (2) 41 Therapy Chemotherapy The goal of chemotherapy can be to destroy cancer remaining after surgery, slow the tumor’s growth, or reduce cancer-related symptoms. It also may be combined with radiation therapy. Currently, there is no single standard chemotherapy treatment regimen that is used worldwide. However, most chemotherapy treatments for GC are based on the combination of at least 2 drugs, 5- fluorouracil(5-FU), and cisplatin. Newer drugs similar to 5-FU, such as capecitabine, and similar to cisplatin, such as oxaliplatin, appear to work equally well. In addition, the adjuvant therapy may be applied which consist of leucovorin: in these patients thymidylate synthetase inhibition rate (TSIR) was significantly higher. Targeted therapy This type of treatment blocks the growth and spread of cancer cells while limiting damage to healthy cells. Recent studies show that not all tumors have the same targets. Imatinib, as a tyrosin kinase inhibitor, is widely used due to its high selectivity on proliferation of maligant cells in GIST. However, there are patients that tend to have primar resistance to imatinib, develop secondary resistance or have introlerance on imatinibe therapy. Thus, in these cases, sunitinbe malate is used. Patients with later-stage cancer whose stomach tumor produce too much of the protein Figure 2: Chemical structure of HER2, called HER2-positive cancer, may benefit from imatinib mesylate (10) receiving trastuzumab with chemotherapy. For patients whose tumor has grown while receiving initial chemotherapy, the drug called ramucirumab was approved in 2014 as an additional treatment (6,7,8,9). References 1. Karimi P, Islami F, Anandasabapathy S, Freedman ND, Kamangar F. Gastric cancer: descriptive epidemiology, risk factors, screening, and prevention. Cancer Epidemiology Biomarkers & Prevention. 2014 May 1;23(5):700-13. 2. L. Zheng, L. Wang, J. Ajani, K. Xie: Molecular basis of gastric cancer development and progression. Gastric cancer, June 2004, Volume 7, Issue 2, p: 61–77. 3. Shi J, Qu YP, Hou P. Pathogenetic mechanisms in gastric cancer. World journal of gastroenterology: WJG. 2014 Oct 14;20(38):13804. 4. W. Yasui, N. Oue, P. Aung, S. Matsumura, M. Shutoh, H. Nakayama: Molecular-pathological prognostic factors of gastric cancer: a review. Gastric Cancer, May 2005, Volume 8, Issue 2, p: 86–94. 5. Zheng L, Wang L, Ajani J, Xie K. Molecular basis of gastric cancer development and progression. Gastric cancer. 2004 Jun 1;7(2):61-77. 6. C. E. Kogel, J. M. Schellens: Clinical Pharmacology: Concise Drug Reviews: Imatinib. The Oncologist, 2007, 12: 1390-1394. 7. Blay JY. Pharmacological management of gastrointestinal stromal tumours: an update on the role of sunitinib. Annals of oncology. 2009:mdp291. 8. Cancer.Net: http://www.cancer.net/cancer-types/stomach-cancer/treatment-option, available: August, 2016. 9. http://cdn1.medicalnewstoday.com/content/images/articles/257/257341/stages-of- stomach-cancer.jpg 10. 10 https://upload.wikimedia.org/wikipedia/commons/9/9e/Imatinib_mesylate.png 42 Colon cancer Draškovič Tina, University of Ljubljana, Faculty of Pharmacy, Slovenia Dejanović Miloš, University of Belgrade, Faculty of Pharmacy, Serbia Jelena Kotur-Stevuljević, University of Belgrade, Faculty of Pharmacy, Serbia Introduction Colon canceris a malignant tumor that starts in the colon, more specifically in the inner wall of the large intestine. Symptoms can be very different according to the specific location within the colon where the tumor is located or are even not present in early stages. Most common problems, which are not tending to be specific only for this condition and are therefore sometimes hardly recognizable, are:  Rectal bleeding or blood in the stool  Dark-colored stool  Change in bowel habits  Change in stool consistency  Constipation  Diarrhea  Narrow stools(1)  Weight loss  Feeling tired all the time(2)  Anemia (3). Picture 1 After Duke’s Classification, colon cancer can be divided in 4 stages. In firstone, stage A, cancer is located in the inner lining of the intestine. In stage B cancerextend in the muscle layer.After the invasion of the lymph nodes, we talk about stage C and when it metastasized it is classifiedas stage D (2). Epidemiology Colon cancer isthe fourth leading cause of cancer death, the second most common cancer in women and the third most common in men. It occurs more often in developed countries. We found highest incidence of colon cancer in Australia, New Zealand, Europe and the US and lowest rates in Africa and South-Central Asia. Data shows that more than 1 million people are diagnosed with colon cancer every year (2). Picture 2 43 Molecular basics/mechanism It typically originates in the secretory cells lining the gut (4).The disease begins as a benign adenomatous polyp, which may form on the inner wall of the colon or rectum. Polyp can develop into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer (5).In most cases this process is slow, taking at least 8 to 10 years to develop as clinically developed disease (1). In a younger personit is often associated with hereditary syndromes like Peutz-Jegher's, hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis (3). Risk factors for developing colon cancer arefamily history of cancer of the colon or rectum,certain hereditaryconditions such as familial adenomatous polyposis and hereditary nonpolyposis colon cancer (HNPCC; Lynch Syndrome), history of ulcerative colitis or Crohn disease, personal history of cancers, personal history of polyps (1). Chromosomal instability, DNA-repair enzymes defects and aberrant DNA methylation can lead to the development of colon cancer by facilitating the acquisition of multiple tumor-associated mutations. Cause of the colon cancer can also be a mutational inactivation of tumor-suppressor genes (APC, TP53, TGF-β) or activation of oncogene pathways (RAS and BRAF, Phosphatidylinositol 3-kinase) (5). Diagnostic In the early stages of colon cancer it can be detected because of the bleeding of a polyp, colloquial bowel pain or a bowel obstruction (3).The following tests and procedures can be used as a diagnostic approach: 1) Physical exam and history 2) Digital rectal exam 3) Fecal occult blood test 4) Barium x-rays 5) Sigmoidoscopy 6) Colonoscopy 7) Virtual colonoscopy/colonography or CT colonography 8) Biopsy (1) 9) Stool DNA screening test (2) Therapy Colorectal cancer has a comparatively Picture 3 good prognosis when detected early (3). Most colorectal cancers develop from polyps which can be removed surgically. With thatthe development of the colon cancer could be prevented(1). As a therapy surgery and/or targeted therapy drugscan be used.One possibility is anti-angiogenic therapy which is a new standard of care in advanced colon cancer. This kind of therapy uses monoclonal antibodies that target either vascular epithelial growth factor(VEGF), vascular epithelial growth factor receptor(VEGFR) or inhibit VEGFR tyrosine kinase enzyme. There was a 44 breakthrough in this kind of therapy during some clinical trials with bevacizumab, a monoclonal antibody directed against VEGF (6). Some colon cancers can be stromal tumors (5-10%). Most of gastrointestinal stromal cancers (GIST) have activating mutations in stem cell factor receptor (KIT). Approximately 5% of GIST's have activating mutations in the related gene encoding platelet-derived growth factor receptor alpha (PDGFRα). They are mutually exclusive. Drug imatinibmesylatecan be usedfor the treatment. It selectively blocks KIT and PDGFRα, and it acts as a receptor tyrosine kinase (RTK) inhibitor. Sunitimib malate can also be used after disease progression or when the patient is intolerant to imatinib. It is also an inhibitor of RTK (7). Anti-EGFR (endothelial growth factor) antibodies (cetuximab, panitumumab) can be used for treatment, except if the patient has the V-Ki-ras2 Kirstenratsarcomaviraloncogenhomolog (KRAS) mutation. Recommendations Lifestyle is really important. It is advisable that people exercise, quit smoking, drink less, eat properly and avoid stressful situations. Studies have shown that people who are physically active cope easier with the troubles of cancer therapy (8). People shouldfollow diets that contain a lot of vegetables, high-fiber foods and that have a low fat content which products can act as carcinogens (1). Some medications and supplements, like aspirin, celecoxib, calcium and vitamin D, could be associated with reduced risk of colon cancer (2). It is also important to have a regular check up at the doctor’s office and taking screening test. Picture 4 45 Picture references 1. http://www.webmd.com/colorectal-cancer/ss/slideshow-colorectal-cancer- overviewAccessed August 20, 2016 2. https://en.wikipedia.org/wiki/File:Colon_and_rectum_cancers_world_map- Deaths_per_million_persons-WHO2012.svgAccessed August 20, 2016 3. Blay JY. Pharmacological management of gastrointestinal stromal tumours: an update on the role of sunitinib. Annals of oncology. 2009:mdp291. 4. http://www.beseengetscreened.com/get-involved/be-an-advocateAccessed August 20, 2016 References 1. http://www.medicinenet.com/colon_cancer/article.htm Accessed August 20, 2016 2. https://en.wikipedia.org/wiki/Colorectal_cancerAccessed August 20, 2016 3. https://en.wikipedia.org/wiki/Gastrointestinal_cancerAccessed August 20, 2016 4. http://www.cancer.gov/types/colorectalAccessed August 20, 2016 5. Markowitz SD, Bertagnolli MM. Molecular basis of colorectal cancer. New England Journal of Medicine. 2009 Dec 17;361(25):2449-60. 6. Iwasaki J, Nihira SI. Anti-angiogenic therapy against gastrointestinal tract cancers. Japanese journal of clinical oncology. 2009 Jun 16:hyp062. 7. Blay JY. Pharmacological management of gastrointestinal stromal tumours: an update on the role of sunitinib. Annals of oncology. 2009:mdp291. 8. http://www.cancer.org/ Accessed August 20, 2016 46 Liver cancer – hepatocellular carcinoma Petra Hadžidaova, University Ljubljana, Faculty of pharmacy, Slovenia Eva Peterová, Charles University, Faculty of Medicine, Hradec Kralove, Czech republic Jelena Kotur-Stevuljević, University of Belgrade, Faculty of Pharmacy, Serbia INTRODUCTION Liver carcinoma is charakterized, such as odher carcinoma, by excessive proliferation of the mutated cells. Hepatocarcinogenesis is a complex process associated with accumulation of genetic and epigenetic changes that occur during initiation, promotion, and progression of the disease. Liver cancers can be divide into the primary carcinomas or secundary carcinomas – metastasis. In this abstract we want to focus on primary carcinoma. The most common tumor of liver is hepatocellular carcinoma (HCC). It is the third most common cause of cancer-related death.The pathophysiology of HCC is not understood clearly, but underlying liver dysfunction, especially cirrhosis, is a predisposing condition (Mittal at all 2013, Aravalli at all 2008). EPIDEMIOLOGY The distribution of HCC varies according to geographic location (Fig. 1). The disease burden is highest in areas with endemic Hepatitis B Virus (HBV) infection, such as in sub-Saharan Africa and Eastern Asia, with incidence rates of over 20 per 100,000 individuals. In regions of high incidence the most common cause is HBV transmitted at birth. In this areas is HCC diagnosed about one decade earlier as compared with North America and Europe with incidence rates of over 3 per 100,000 individuals (Mittal at all 2013). Fig. 1 Distribution of HCC in the world (taken from Mittal at all 2013) A variety of risk factors have been associated with HCC. They include exposure to hepatitis viruses, vinyl chloride, tobacco, just food contaminated with aflatoxin B1, heavy alcohol intake, nonalcoholic fatty liver disease, diabetes, obesity, diet, coffee, oral contraceptives and hemochromatosis(Fig.2). This risk factors are dependent on geographical region (Aravalli at all 2008). 47 Fig. 2 Risc factor for development HCC (taken from Aravalli at all 2008) MOLECULAR BASIC Cellular action are often accompanied by increased expression of several factors that influence the survival of cancerous cells by suppressing apoptosis and regulating cell cycle. Some cell signaling pathway activated by risk carcinoma factor are known (Tab. 1). The overwiev of cell signaling pathways is shown on Fig. 3. Tab. 1Cell signaling pathway associated with risc factor (taken from Aravalli at all 2008) Wnt/β-Catenin signaling pathway has a function as a key regulator in tumor development and differentaition. Members of Wnt protein family bind to cell surface receptor and their co-receptor and iniciate signaling pathway. Complicated cascade leads to increases expression of β-Catenin reaching the nucleus. A variety of researchs summarize that the p53 tumor supressor gene has role in carcinogenesis. Tumor supressor gene is inactivated by a single point mutation, that leads to cell cycle arrest and subsequent apoptosis is defective. Mitogen-Activated Protein Kinase Pathway (MAPK) affects most cellular process such as differenciation, proliferation or adhesion. Proteins of HBV, HCV, and hepatitis E virus modulate MAPK signaling by targeting multiple steps along the signaling pathway. Rb protein is critical for the development of several cancer types. Rb protein binding nuclear transcription factors and prevent the cell from replicating damaged DNA. Cells stay in G1 phase. 48 Mutation of Rb leads to reduced control in cell cycle progression subsequently resulting in the development of cancer. Ras signalling proteins are localized at the inner surface of the plasma membrane. They are molecular switches that transduce extracellular signals into the cytoplasm to protein signaling pathway,in order to perform an effect on cell growth, diferentiation and apoptosis. Less common signaling pathways that are involved in carcinogenesis are: JAK/STAT Pathway, Epidermal Growth Factor Receptor and Transforming Growth Factor- Pathways, additional pathwaysof physiological processes such as alcohol metabolism,cellular transport, and ubiquitins, stress Response Signaling. Fig. 3 Overwiev of cell signaling pathway (taken from wikimedia) DIAGNOSTICS When it comes to cancer, time of diagnosis is of utmost importance. The story is no different with HCC, because long-term survival requires detection of small tumors before the disease advances into its more advanced stages. Unfortunately HCC is usually diagnosed when clinical manifestations have already developed at which time survival is measured in months. Since early detection of HCC is so important, surveillance of high-risk individuals is performed by measuring serum alfa-fetoprotein (AFP) and ultrasonography. Diagnosis of HCC on the other hand requires more sophisticated imaging methods, among which CT and MRI are the most frequently used. Liver biopsy is also a viable option, but it is only used if the diagnosis of HCC remains unclear, because of its invasive nature. (4) Surveillance of high-risk individuals. As already mentioned, surveillance of high-risk individuals is preformed through screening test by measuring serum alfa-fetoprotein (AFP), usually complemented 49 by ultrasonography, since it can, combined with AFP values, improve predicted positive value up to 94 %. Successful surveillance heavily depends on properly identifying which individuals have a high- risk of developing HCC. Among those are patients that are older, of male gender, have a family history of HCC, or have cirrhosis. (4) Alpha-fetoprotein (AFP). AFP is a serum glycoprotein, which consists of 591 amino-acids and is produced in high concentrations in fetal yolk sac and fetal liver. The values fall to low levels within 300 days of birth, so elevated numbers are therefore a good indication of pathological changes, which could very well be malignant. AFP is unfortunately not an ideal marker, since other conditions can also result in elevated AFP concentrations and even in patients with HCC the values can range from normal to >100 000 ng/ml (reference values for non-pregnant subjects are < 6ng/mL). Attempts to improve the accuracy of AFP have been made through identifying different isoforms. (4) Fig. 4 The structure of human alpha-fetoprotein (taken from PhosphoSitePlus) Ultrasonography. Ultrasound is widely used for detecting tumors that are smaller than 3 cm. It's commonly used because it is noninvasive, inexpensive and easily available. But just like all other methods and techniques, ultrasound also has its limitations. They are dependent on operator and imaging of obese and cirrhotic patients can be problematic. (4) Diagnosis. The diagnosis of HCC is a multi-step procedure which usually requires different diagnostic tools, of which computer tomography (CT) and magnetic resonance imaging (MRI) are most commonly used. If a liver mass, which is greater than 2 cm in diameter, is discovered on ultrasound and it is known that the patient has pre-existing cirrhosis, there is a greater than 95% chance that the lesion is a HCC. The next step in line is measuring AFP, and if its values are raised the diagnosis is confirmed and further investigation is only required to establish the most appropriate therapy. On the other hand if AFP is normal, further radiological imaging is required. In these cases CT, MRI or lipoidol angiography with follow up CT will give us a reliable answer without the need for biopsy. Liver biopsy is only really required and recommended if doubts about the diagnosis still persists and should only be applied if truly necessary. (5) THERAPY A multidisciplinary setting in which patients are cooperatively managed by a wide number of different specialist is the most effective way of treating HCC. Specialist can choose from surgical or non-surgical therapies, depending on the patient’s condition. (6) Surgical therapy. Among surgical therapies surgical resection and liver transplantation are viable options. The definitive treatment for HCC is surgical resection. It is also the only therapy that offers 50 the prospect of cure or at least long-term survival, however most patients do not have resectable diseases and the recurrence rate is up to 80 % in 5 years. (8) Liver transplantation is actually the most efficient treatment in patients with small tumours, but unfortunately less than 30 % of patients are eligible because of restrictive criteria, graft unavailability and high-cost. (7) Non-surgical therapy. Non-surgical therapies are used if patients are not candidates for resection or liver transplantation. (3) Transcatheter oily chemoembolization (TOCE) is the most commonly offered non-surgical therapy, in which an emulsion of a cytotoxic drug and lipiodol is slowly injected through a catheter that is placed in the appropriate tumour-feeding artery. As a result an embolization takes place. Other therapies such as percutaneous alcohol injection, thermal and laser ablation, radiotherapy and systemic chemotherapy can also be used. There are several chemotherapeutics that can be used such as doxorubicin-based regimens, tamoxifen, antiandrogens (eg, cyproterone, ketoconazole), interferon, interleukin (IL)-2, octreotide, gemcitabine, oxaliplatin, sorafenib and others. (6) Fig. 5: Principle of conventional transarterial chemoembolization (taken from Wang YXJ et al ) CONCLUSSION If we conclude, the pathophysiology of HCC, the most common tumor of liver and third most common cause of cancer-related death, is not completely understood at the present moment. Different risk factors have been associated with HCC, of which by far the most common one is infection with HBV. These risk factors activate or change different pathways, which usually lead to increased expression of several factors that influence the survival of cancerous cells by suppressing apoptosis and regulating cell cycle. Since long-term survival requires detection of small tumors before the disease advances into its more advanced stages, surveillance of high-risk individuals is performed by measuring serum alfa-fetoprotein (AFP) and ultrasonography. Diagnosis of HCC on the other hand is a multi-step process which requires different diagnostic tools through which we can come to a proper conclusion. When it comes to treating HCC choosing a multidisciplinary setting in which patients are cooperatively managed by a wide number of different specialist is the most effective way of treating HCC. Therapies that are available can be of a surgical or non-surgical nature. The definitive treatment for HCC is surgical resection also the only therapy that offers the prospect of cure or at least long-term survival, unfortunately most patients do not have resectable diseases. 51 REFERENCES 1. Mittal S,El-Serag HB. Epidemiology of HCC: Consider the Population. Clin Gastroenterol. 2013 July; 47(0): S2–S6. doi:10.1097/MCG.0b013e3182872f29. 2. Aravalli NR, Steer CJ. Molecular Mechanisms of Hepatocellular Carcinoma. HEPATOLOGY. 2008; Vol. 48, No. 6 3. Teufel A, Staib F. Genetics of hepatocellular carcinoma. World J Gastroenterol. 2007 April 28;13(16): 2271-2282 4. Bialecki ES, Bisceglie AM. Diagnosis of hepatocellular carcinoma. HPB. 2005; 7: 26-34 5. Ryder SD. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut. 2003; 52(Suppl III):iii1–iii8 6. Cicalese L. Hepatocellular carcinoma treatment & management. Upd Sep 28, 2015. Available on website: http://emedicine.medscape.com/article/197319-treatment 7. Belghiti J, Kianmanesh R. Surgical treatment of hepatocellular carcinoma. HPB (Oxford). 2005; 7(1): 42-49 8. Johnson PJ. Non-surgical treatment of hepatocellular carcinoma. HPB (Oxford). 2005, 7 (1): 50-55 REFERENCES FOR PICTURES AND TABLES Tab. 1: Aravalli NR, Steer CJ. Molecular Mechanisms of Hepatocellular Carcinoma. HEPATOLOGY. 2008; Vol. 48, No. 6 Fig. 1: https://upload.wikimedia.org/wikipedia/commons/f/fb/Signal_transduction_pathways.png Fig. 2: https://upload.wikimedia.org/wikipedia/commons/f/fb/Signal_transduction_pathways.png Fig. 3: https://upload.wikimedia.org/wikipedia/commons/f/fb/Signal_transduction_pathways.png Fig. 4: The structure of human alpha-fetoprotein. Available on world wide web: http://www.phosphosite.org/proteinAction?id=14609&showAllSites=true Fig. 5: Wang YXJ et al. Transcatheter embolization therapy in liver cancer: an update of clinical evidences. Chinese journal of cancer research 2015; 27 (2) 52 Lung cancer Epidemiology and Molecular Basis of Lung Cancer Matea Dvorščak, University of Zagreb, Faculty of Pharmacy and Biochemistry,Croatia Eva Kozjek, University Ljubljana, Faculty of pharmacy, Slovenia Neža Levičnik, University Ljubljana, Faculty of pharmacy, Slovenia Besim Prnjavorac, University of Sarajevo, Faculty of Pharmacy, Bosnia and Herzegovina Epidemiology Lung cancer is one of the most common malignant diseases and leading cause of cancer death around the world. Each year, more people die of lung cancer than of colon, breast, and prostate cancers combined (1). Lung cancer can be divided into two major forms: non–small cell lung cancer (NSCLC) that accounts for 85% of all lung cancers and small cell lung cancer (SCLC) that accounts for the remaining 15% of cases. NSCLC can be further divided into 3 subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma (1). At the time of the diagnosis (average age is about 70 years) most of the lung cancer cases are inoperable. The majority of lung cancer patients also do not respond well to chemotherapeutics or radiation therapy and more than 85% of them die during the first 5 years. There has been some improvement in survival during the past years but in the last three decades the mortality rate has not changed significantly (2). Lung cancer is the second most common cancer in both men and women. The probability that a man will develop lung cancer in his lifetime is about 7,1%, while for a woman, the risk is about 5,9%. This include both smokers and non-smokers. For smokers the risk is much higher. It has been estimated that active smoking is responsible for about 90% of all lung cancer cases (3). There is also a significantly increased risk for lung cancer in people with a family history of early- onset lung cancer. Genetic predisposition include polymorphisms of the tumor suppressor genes and the allelic variants of the genes involved in detoxification. Some nonmalignant diseases have been associated with an increased risk for lung cancer, such as COPD and asthma. Enviromental pollution and carcinogenes also increase risk for lung cancer (asbestos, radon, arsenic, chromium, nickel,vinyl chloride etc.) (2). Molecular Basis of Lung Cancer Molecular basis of lung cancer is very complicated and complex. Many different factors (environment, genetics, hormonal factors, viral factors …) have been linked to carcinogenesis. Genetics and environment play a very important role in lung cancer development. An important process in tumor progression is also angiogenesis in which vascular endothelial growth factor (VEGF) is the major mediator (2, 4). Genetics of lung cancer: Genetic changes in lung cancer usually involve both oncogenes and tumor suppressor genes and they may or may not be rate-limiting events. Most often genetic alterations that occur in early phase of lung cancer involve loss of some genetic material on short arms of chromosomes 3 and 9, deletion of 5p and mutations of K-Ras and p53 (5). 53 Major tumor suppressor genes that are involved in lung cancer are TP53, RB1, CDKN2 and several genes located on 3p. Mutations of TP53 are associated with smoking and more aggressive tumors. Tumor suppressor genes are usually inactivated not only by one factor but a combination of genetic changes like point mutations, chromosomal rearrangements and mitotic recombination and non genetic changes (epigenetics). Methylation and acetylation (epigenetics processes) affect on expression of tumor suppression genes and oncogenes (5, 6). Many proto-oncogenes are activated by chromosomal rearrangements (translocations, inversions etc.), amplifications of genetic material (MYC, EGFR, HER2 etc.) and genetic mutations (EGFR, KRAS etc.) (5). Environment Very important environmental factor for development of lung cancer is tobacco. Tobacco carcinogens are in human body firstly metabolised by cytochrome P-450 and then are the oxygenated intermediate metabolites further metabolised by different enzymes (glutathiones, sulfatases etc.) in subsequent transformations (detoxification and secretion). Some of metabolites that were generated then bind with the DNA and form so called DNA adducts. That process is called metabolic activation and it is necessary for carcinogenic effect of tobacco metabolites (polcyclic aromatic hydrocarbons etc.). Formation of DNA adducts can result in miscoding leading to permanent mutations such as K-Ras, p16, p53 or other mutations. Consequently, expression of tumor suppression genes and oncogenes is changed which can result in lung cancer. However our body can protect itself by activating repairing mechanisms or apoptosis of damaged cells. Metabolic activation and detoxification of potential carcinogens both influence the development of lung cancer. Because of that not all smokers develop lung cancer but up to 20% do (7). Angiogenesis and VEGF Tumor needs to develop more blood vessels in order to spread (more than 2 mm3) because of a need for oxygen and nutrients. Major mediator in angiogenesis is VEGF, which is normally expressed by endothelial cells that are generating blood vessels. VEGF needs a receptor to affect. The most important one is VEGFR 2. The binding of VEGF to its receptor causes cascade of many signaling pathways, i.e. dimerization of the receptor, leading to activation of the PLCɣ-PKC-Raf kinase-MEK- MAPK and cell growth. VEGF expression in tumor cells is influenced by several different factors (hypoxia, low pH, growth factors, inflammatory cytokines (e.g., interleukin-6), sex hormones etc.) and genetic changes (activation of oncogenes, inactivation of tumor suppressor genes etc.). Tumor cells do not express VEGFR (so, VEGF from tumor cells cannot initiate signaling pathways) however tumor angiogenesis is still possible because of VEGFR of the other normal host cells (4). References: (1) Dela Cruz C, Tanoue L, Matthay R. Lung Cancer: Epidemiology, Etiology, and Prevention. Clin Chest Med. 2011 December; 32(4). (2) Panov S. Molecular biology of the lung cancer. Radiol Oncol 2005; 39(3): 197-210. (3)Alberg AJ, Samet JM. Epidemiology of Lung Cancer. Chest.2003; 123:21-49. (4) Kerbel RS. Molecular origins of cancer: Tumor angiogenesis. N Engl J Med 2008; 358:2039-49. (5) Varella-Garcia M. Chromosomal and genomic changes in lung cancer. Cell Adh Migr. 2010 Jan- Mar; 4(1):100-106. (6) Massion PP, Carbone DP. The molecular basis of lung cancer: molecular abnormalities and therapeutic implications. Respir Res. 2003; 4(1):12. (7) Furrukh M. Tobacco Smoking and Lung Cancer. Sultan Qaboos Univ Med J. 2013 Aug; 13(3): 345– 358. 54 Diagnosing lung cancer Kity Požek, University Ljubljana, Faculty of pharmacy, Slovenia Matea Dvoršćak, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia Besim Prnjavorac, University of Sarajevo, Faculty of Pharmacy, Bosnia and Herzegovina Lung cancer is often diagnosed in the advanced stage, after metastatic spread and thus has poor prognosis (1). New approaches are desired to achieve an early and accurate diagnosis, which is crucial for improvement of lung cancer survival rates (2). Lung cancer is suspected in patients with risk factors (current/former/passive smokers, 55-80 years old with a smoking history of 30 or more years, individuals exposed to environmental carcinogens, individuals with a family history of lung cancer etc.) and/or symptoms such as a cough, bloody sputum, shortness of breath, wheezing, fatigue, weight loss, headache etc. (2, 3). These patients undergo physical examination and chest X-ray. If the chest radiograph is abnormal patients should proceed with diagnostic procedures. The course of diagnosis is dependent on the type (i.e., small cell or non-small cell lung cancer), the size and location of the primary tumor, and the presence of metastasis and overall clinical status of the patient. The least invasive approach with the minimum steps possible should be used. Nevertheless, bronchoscopy is often essential for a lung cancer diagnosis (3, 4). In the case of an abnormal chest radiograph, the possibility of other medical conditions such as pneumonia, must be excluded. The next step is a Computed Tomography scan (CT scan) of the chest and abdomen which provides a more detailed picture of the tumor. Another option is a combination of a CT scan and a Positron Emission Tomography scan (PET CT scan) which uses radioactive sugar to identify the location of cancer cells which appear brighter on the PET CT scan. If we detect a tumor at its metastatic phase, an additional Magnetic Resonance Imaging (MRI) is required. In case of enlarged nodes, neck or endobronchial (EBUS), an ultrasound should be done. If all of these tests are positive, a lung biopsy is the next step. There are several different possibilities for collecting a tissue sample, depending on the location of the tumor, including a needle biopsy and bronchoscopy. Open surgery is sometimes the only option to collect suspicious tissue mass. If pleural effusion is present, thoracentesis can be used (5). If cancer cells are found in sputum, we can avoid invasive procedures by performing a sputum cytology. If this provides a negative result, a possibility of lung cancer must not be excluded, as the results of this test can often come up as false negative (4). There are some downsides to the current diagnostic methods such as high radiation exposure when performing a chest X-ray and CT scans, even when lower doses of radiation are used (1, 6). Other problems include high cost, overdiagnosis and false positive results (1). The future of early diagnosis and a reduction in mortality might be successful screening tests. These are tests designed to detect lung cancer in patients with risk factors but no obvious symptoms or history of that disease. This approach provides a diagnosis at its earliest stages when lung cancer is most treatable (1). Screening tests such as a chest X-ray, sputum cytology, Computed Tomography (CT), fluorescence endoscopy and low-dose spiral CT (LDCT) had little success improving survival rates over the course of the last fifty years. Today novel screening methods are being developed on the basis of serum biomarkers. Free circulating DNA and RNA, exosomal microRNA, circulating tumor cells and different lung cancer specific antigens are being extensively studied for this purpose (7, 8). Currently, there are no validated biomarkers available for early detection of lung cancer. Potentially useful lung cancer biomarkers are for example progastrin-releasing peptide (ProGRP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cytokeratin 19 (CYFRA-21-1), carbohydrate antigen-125 (CA-125), carbohydrate antigen-19.9 (CA-19.9) and alpha-fetoprotein (6, 7, 55 8). For example CYFRA 21-1 proteins indicate an increased level of cytokeratin 19 fragments which implies the presence of lung cancer. ProGRP is produced by the nueroendocrine tissues of the gastrointestinal and respiratory tracts and is also an available lung cancer biomarker. NSE is a glycolytic enzyme produced in central and peripheral neurons that is also increased in case of lung cancer. These protein biomarkers usually lack their lung-cancer specificity which is why their use in clinical practice is limited (9). However, a better outcome was achieved using a combination of biomarkers. Different combinations have shown to have higher sensitivity for a certain type of lung cancer (10). Developing new lung cancer screening tests and diagnostic methods is challenging and plays an important role in lung-cancer-mortality reduction. With further research, we can expect new screening tests that will allow for the early diagnosis and treatment of lung cancer both of which are vital for improving lung cancer survival rates. References: 1. Midthun DE. Early detection of lung cancer. F1000Res. 2016; 25:5. doi:10.12688/f1000research.7313.1. 2. Polanski J, Jankowska-Polanska B, Rosinczuk J, et al. Quality of life of patients with lung cancer. Onco Targets Ther. 2016; 9: 1023–1028. 3. Latimer KM, Mott TF. Lung Cancer: Diagnosis, Treatment Principles, and Screening. Am Fam Physician. 2015;91(4):250-256. 4. Rivera MP, Mehta AC, Wahidi MM. Establishing the Diagnosis of Lung Cancer: Diagnosis and Management of Lung Cancer. Chest. 2013; 143(5 Suppl):e142S-65S. doi: 10.1378/chest.12-2353. 5. National Collaborating Centre for Cancer (UK). The Diagnosis and Treatment of Lung Cancer (Update). Cardiff (UK): National Collaborating Centre for Cancer (UK); 2011 Apr. (NICE Clinical Guidelines, No. 121.). http://www.ncbi.nlm.nih.gov/books/NBK99021/ (Acessed August 21, 2016). 6. Ruano-Ravina A1, Pérez Ríos M, Fernández-Villar A. Lung cancer screening with low-dose computed tomography after the National Lung Screening Trial. The debate is still open. Arch Bronconeumol. 2013;49(4):158-65. doi: 10.1016/j.arbres.2012.10.003. 7. I H, Cho JY. Lung Cancer Biomarkers. Adv Clin Chem. 2015; 72:107-70. doi:10.1016/bs.acc.2015.07.003. 8. Xiang D, Zhang B, Doll D, et al. Lung cancer screening: from imaging to biomarker. Biomarker Research. 2013; 1:4. doi: 10.1186/2050-7771-1-4. 9. Sung H-J. Cho JY. Biomarkers for the lung cancer diagnosis and their advances in proteomics. BMB reports. 2008; 615-625. 10. Li X, Asmitananda T, Gao L, et al. Biomarkers in the lung cancer diagnosis: a clinical perspective. Neoplasma. 2012; 59(5): 500-7. doi: 10.4149/neo_2012_064. 56 Therapy of lung cancer AdnaAlilović, University of Sarajevo, Faculty of Pharmacy, Bosnia and Herzegovina Tamara Janković, University of Belgrade, Faculty of Pharmacy, Serbia Milica Belić, University of Belgrade, Faculty of Pharmacy, Serbia Besim Prnjavorac, University of Sarajevo, Faculty of Pharmacy, Bosnia and Herzegovina CHEMOTHERAPY OF LUNG CANCER When choosing a therapy, many factors must be considered such as likelihood of treatment achieving a cure and fitness of the patient. Those two are also linked – a patient whose fitness is borderline may not be able to tolerate a more extensive resection needed to achieve the cure. Chemotherapy improves survival in small-cell carcinoma and can even cure a small fraction of patients with limited disease. Surgery is the treatment of choice in 25 to 30 percent of patients with NSCLC who are stage I or II. Another 25 to 30 percent of patients, who are stage IIIa or IIIb are usually treated with radiotherapy or, occasionally, surgery. For the remaining 40 to 50 percent of patients, who are stage IV non-small- cell lung cancer at the time of diagnosis, palliative symptomatic treatment, most often irradiation of the chest, brain or bone is standard care. With these approaches, survival rates are respectively 40 percent, 4-8 percent and < 1 percent in a three year period1. Though moderate improval in survival and increased rates of tumor regression are possible, there is no group of patients with non-small- cell lung cancer in which chemotherapy is unequivocally effective. Drug therapy of small-cell lung cancer Chemotherapy is the main treatment for small-cell lung cancer (SCLC). It’s known that this type of cancer produce the metastases very soon, so when metastases are present, surgery is not right choice. SCLC responds very well to chemotherapy. Single-Agent Chemotherapy Chemotherapeutic agents, including cyclophosphamide, etoposide, doxorubicin, vincristine, carboplatin and methotrexate induce tumor regression in at least 30 percent of patients. Single- agent therapy may still be useful in certain groups of patients, especially elderly and those with cardiopulmonary or renal dysfunction. Patients with impaired ability to ambulate, and patients in whom the likelihood of severe toxicity from combination chemotherapy is high. Combination Chemotherapy Combination chemotherapy is superior to single-agent treatment. Myelosuppression is the common dose-limiting toxicity, therefore combination therapy usually consists of four or fewer agents.The most commonly used combinations are currently etoposide and cisplatin (CAVE); cyclophosphamide, doxorubicin and vincristine (CAV); cyclophosphamide, doxorubicin and etoposide (ACE)1. People who are not able to have CAV or ACE therapy because of their medical conditions, may have topotecan chemotherapy capsules. Drug therapy of non-small-cell lung cancer There are no survival advantages of chemotherapy in patients with non-small-cell lung cancer. Due to that and the toxicity of chemotherapy, only fully ambulatory patients should be offered treatment. Chemotherapy is used to treat NSCLC after surgery for early stage cancer; before, after or alongside radiotherapy and for locally advanced lung cancer. Chemotherapy after surgery can reduce relapse of the disease. Most common used combinations include cisplatin or carboplatin with at least one other drug (vinorelbine, gemcitabine, paclitaxel, docetaxel, doxorubicin, etoposid, pemetrexed). Chemotherapy before or after radiotherapy can help eliminate early stage NSCLC in people who 57 cannot have surgery and prolong life of people with advanced NSCLC. Chemotherapy can help shrink or slow the growth of locally advanced or metastatic NSCLC and also help control the symptoms for some people. Some cancers respond very well to biological therapy, mostly patients with EGFR or VEGFR positivity. BIOLOGICAL THERAPY OF LUNG CANCER Biological or immuno-therapies are derived from, or target substances that occur naturally in the body. Actually most biological therapies were performed by monoclonal antibody (immunotherapy of the cancer). They target and destroy particular types of cancer cells. Possible side effects of biological therapy are: tiredness, diarrhea, skin changes (rashes, discoloration), a sore mouth, weakness, feeling sick, loss of appetite, low blood counts, swelling of parts of the body, due to build- up of fluid. Erlotinib (Tarceva) works by blocking epidermal growth factor receptors (EGFR) in the cancer cells. It is used for locally advanced or metastatic non-small-cell lung cancer that is EGFR positive, if a patient has not had drug treatment before (a first line treatment) or if a patient has already had chemotherapy, because there was a delay in finding out if the cancer is EGFR positive. Gefitinib (Iressa) works by blocking epidermal growth factor receptors. It is used as a first treatment for NSCLC lung cancer that is locally advanced or has spread. A meta-analysis of trials comparing gefitinib with chemotherapy found that there was no difference in how long people lived, but there was a difference in the side effects: less tiredness, sickness and effects on the blood cells, more skin problems (rashes), diarrhoea, and irritation of the lung. Crizotinib (Xalkori) can help to control advanced NSCLC in people whose cancer cells have an overactive version of anaplastic lymphoma kinase (ALK). Afatinib (Giotrif) is a protein tyrosine kinase inhibitor (TKI). It blocks tyrosine kinases and also blocks the epidermal growth factor receptor proteins in cancer cells. It is used for people with advanced not-small-cell lung cancer who had not already had any other type of cancer growth blocker. Nintedanib (Vargatef) is a treatment for adenocarcinoma. It is multi kinase inhibitor which works by blocking particular proteins called protein kinases on cancer cells. It also stops the cancer tissue neovascularisation. . Cetuximab (Erbitux) is a monoclonal antibody. It blocks growth factor receptors on cells. It has been used, with chemotherapy, in trials for advanced non- small- cell lung cancer. Bevacizumab (Avastin) is a monoclonalantibody (MAB) that stops cancer cells making the blood vessels (neovascularisation) they need so that they can grow. RADIATION THERAPY Radiotherapy commonly means external radiotherapy, conventionally delivered as one fraction per day, 5 days a week, for 5 to 7 weeks. Radiotherapy is suitable for treating a wide variety of NSCLC patients. This treatment should be the choice for patients with early stage lung cancer and co- morbiditety who present a high surgical risk or where patient makes an informed choice not to have surgery. Radiotheraopy is indicated for patients with stage I, II or III NSCLC who have good performance status. All patients sould undergo pulmonary function test before having radical radiotherapy of 58 NSCLC. One meta-analysis showed 14% reduction in mortality rate in patients who were treated with radiotherapy compared to chemotherapy. Brachytherapy Lung brachytherapy can be used almost any time when the tumor can be seen in an airway on bronchoscopy. During bronchoscopy, a thin plastic tube is placed down the nose, and down into the airways of the lung, into the diseased bronchus. The bronchoscope is then removed, but the thin tube will stay comfortably in place for about 45 minutes. During this time, a brachytherapy treatment will be given through the segment of the tube which is lying against the cancer. This treats the cancer from the inside-out. Brachytherapy places radioactive sources inside the patient on a temporary or permanent basis to damage cancer cells DNA and destroy their ability to divide and grow. This treats cancer from the inside-out. TARGETED THERAPY With current treatment, the disease is rarely curable, and prognosis is poor, as patients are often diagnosed with metastatic disease. Current systemic therapies have limited effectiveness. Thus, novel strategies for the treatment of lung cancers are necessary. Knowing which genetically and epigenetically changed molecules activate signaling pathways important in carcinogenesis made it possible to develop molecularly targeted therapies which hold promise for improving lung cancer outcomes. Only two of these have been approved until now, for advanced NSCLC: EGFR pathway inhibitors The EGF pathway is frequently disregulated in human cancers, making it an attractive target for anticancer therapy. The EGFR family of receptors consists of transmembrane tyrosine kinase receptors and its ligands are frequently overexpressed in NSCLC tumors, but rarely expressed in SCLCs. Binding of ligand to the EGFR leads to proliferation, inhibition of apoptosis, angiogenesis and invasion, all resulting in tumor growth and spread. Agents targeting EGFR include the TK inhibitors, such as gefitinib and erlotinib, and the monoclonal antibodies. Angiogenesis inhibitors The monoclonal antibodies against VEGF and the VEGFR TKIs are among the agents aimed at targeting the VEGF/VEGFR signaling network. Bevacizumab is a monoclonal antibody that binds to all isoforms of VEGF-A. Its addition to the classic first-line treatment of patients with advanced nonsquamous NSCLC provides a significant survival benefit and has been approved for use in NSCLC. Pulmonary hemorrhage associated with bevacizumab treatment is an occasional but serious life- threatening side effect, highlighting the importance of patient selection and monitoring for this therapy. Patients with squamous cell histology are particularly at risk of bleeding. VEGFR TKIs are small molecules that bind to the ATP pocket of the TK residues of the intracellular domain of VEGFR, thus inhibiting downstream pathways. One of the most promising lung cancer treatments that are still in clinical development are cancer stem cell-specific therapeutic approaches. Cancer stem cell-specific therapeutic approaches Most malignancies may arise from a rare subpopulation of stem-like tumor cells, i.e. ''cancer stem cells''. They may be inherently resistant to the common therapeutic protocols due to their low proliferation rate and drug transporter expression. Thus, the development of therapies speciffically targeting those cells represents a strategy to completely eradicate tumors and potentially lead to cure, even in advanced-stage disease. One of the targets is the telomerase, which is upregulated in cancer stem cells. Telomerase inhibitors can potentially target both cancer stem cells and more mature cancer cells. Human telomerase contains two essential components, a telomerase reverse transcriptase (hTERT) catalytic subunit and a functional telomerase RNA (hTR) component. Activation 59 of telomerase plays a role in the immortalization of cells as an early step in tumorigenesis. Detectable telomerase levels are found in approximately 80% of NSCLCs and 100% of SCLCs. Agents targeting telomerase include telomerase antagonists the RNA template region of hTR (whuch inhibit anchorage-independent growth of lung cancer cells), immunotherapy, gene therapy and hTERT inhibitors. References 1. Alastair J. J. Wood. Drug Therapy: Chemotherapy of Lung cancer. The New England Journal of Medicine. 1992; 327:1434-41. 2. Roy S. Herbst, John V Heymach, Scott M. Lippman. Molecular Origins of Cancer: Lung Cancer. The New England Journal of Medicine. 2008;. 2008;59:1367-80. 3. Vince D. Cataldo, Don L. Gibbons, Roman Perez-Soler, Alfonso Quintas-Cardama. Clinical Therapeutics: Treatment of Non-Small-Cell Lung Cancer with Erlotinib or Gefitinib. The New England Journal of Medicine. 2011; 364:947-55. 4. FortunatoCiardiello, GiampaoloTortora. EGFR Antagonists in Cancer Treatment. The New England Journal of Medicine. 2008; 358:1160-74. 5. Sophie Sun, Joan H. Schiller, Monica Spinola, John D. Minna. New Molecularly Targeted Therapies for Lung Cancer. The Journal of Clinical Investigation. 2007; 117: 2740-48. 60 Melanoma Epidemiology and diagnostics of malignant melanoma Tina Košuta, University Ljubljana, Faculty of pharmacy, Slovenia Luka Planina Medin, University Ljubljana, Faculty of pharmacy, Slovenia Martina Tomažin, University Ljubljana, Faculty of pharmacy, Slovenia Jana Nekvindová, University Hospital Hradec Králové, Institute for clinical biochemistry and diagnostics, Czech republic Epidemiology Melanomas are common cancers arising from the pigment cells of the skin. Local melanomas are curable by surgery, but advanced disease is hard to treat and could be lethal. [1] Approaches to control the disease are mainly focused on prevention and early detection. To apply these approaches, it is crucial that the epidemiology of melanoma is understood. Melanoma mainly affects Caucasian populations (10-20-fold higher than dark-skinned people). The incidence varies greatly depending on geographic location of a certain population. In fact, melanoma incidence variation among populations is one of the greatest in any cancer. [2] This is the most convincing evidence regarding the role of environment (sunlight) as the cause of this cancer. International Agency for Research on Cancer (IARC) shows that the highest incidence rates for melanoma occur in Australia (39:100,000) and New Zealand. Next is USA, followed by Scandinavian countries (15:100,000). Non-Caucasian populations in Africa, Asia and Pacific have rates lower than 3:100,000 per year. People living at low latitudes experience higher rates of melanoma and the difference is clear even within Australia. [3] This latitude effect, however, is not observable in northern hemisphere. Mediterranean countries experience lower rates of melanoma than Scandinavian ones, presumably because of darker skin of Mediterranean people compared to Scandinavians. [4] With rising incidence rates, mortality also rises, but at a slower pace. [5] In all populations, melanoma is rare before the age of 40 and it peaks in the seventh or eighth decade of life, with incidence rising with age. [6, 7, 8, 9, 10] Historically, melanoma was more common in women, but recently the occurrence pattern has changed and rapid increases of melanoma in males changed that. [8, 11] Risk factors Sunlight and especially its UV spectrum is the leading cause of melanoma. Some people, however, believe that sunlight is not the main cause of melanoma. Most melanomas occur on the back and shoulders, which are normally protected by clothing, whereas face and hands rarely produce melanomas, yet are constantly exposed to sunlight. A comprehensive study is difficult to perform, because everyone has been subjected to different amounts of sunlight and other risk factors. There is no reliable and objective method for determining past exposures to the sun. Besides environmental risk factors, there are also genetic risk factors. Number of nevi each person has also plays a role in risk assessment. Persons with >100 nevi have up to sevenfold higher risk compared to <15 nevi. [13] Clinical observation that 10 % of people with melanoma had a family history of the disease hint at a genetic cause. Genetic epidemiologists identified a region on the short arm of chromosome 9, linked to melanoma. The deleted locus was a CDKN2A gene. Germ line mutations in this gene are “melanoma-prone”. Two other genes were since found within the same locus, P14ARF and CDKN2B. Proteins coded by these genes are all potential tumor-suppressors, playing a role in cell-cycle arrest. [14, 15, 16] 61 Previous studies were designed under the assumption that all melanomas are a single homogenous group. But recent findings show that this may not be the case and melanomas are actually heterogeneous and may arise through several different pathways. Diagnosis Melanoma is often discovered by routine health check-ups or by patients itself who come to see a doctor because of suspicious mark on their skin. It is essential to make diagnosis as early as possible to enable detection and removal of the abnormal growth before it can progress to more advanced stage of disease that is more difficult to treat and result in significant drop of survival rate. From almost 100 % if discovered early to 10-20 % if distant metastasis is presented. (1) The most common diagnostic technique is visual inspection with good lighting and magnification. To improve the sensitivity (detection of melanomas) and specificity (percentage of non-melanomas correctly diagnosed as benign) the used technique is skin surface microscopy (dermatoscopy). First symptoms of melanoma are usually changes in the size, colour, shape or sensation of an existing mole or presence of dark area that can look like a new mole. Figure 1: Stages of gastric cancer (9) ..................................................................................................... 41 Figure 2: Chemical structure of imatinib mesylate (10) ........................................................................ 42 Figure 3: A 99m Tc bone scientoscan of a patient with bone metastasis. (5) .......................................... 79 Figure 4: Bisphosphonate and Denosumab target osteolytic bone metastases. They decrease the survival of osteoclasts. Bisphosphonates cause loss of resorptive function in osteoclasts (left), whereas Denosumab specifically antagonizes RANKL, thus blocks osteoclast formation and function (right). Osteoprotegerin (OPG) competes with Denosumab for binding to RANKL and must be considered as negative predictive biomarker for Denosumab efficiency. (12) ............................................................ 80 ABCDE system of diagnosis These features have been used as the so-called ABCDE system of diagnosis. Each letter presents different characteristic described in picture below. Letter A stands for asymmetry which means that the two sides do not match compared to symmetrical benign lesion. B stands for border which is irregular meanwhile the benign mark has even borders. C stands for colour. Malignant lesion is two- or multi-coloured and benign is homogenously coloured. D stands for diameter. Moles that are wider than 6 mm are more likely to be malignant but not necessary. They can be even smaller. E means evolution. Malignant lesion is changing over time in its size, colour, shape or another trait. When the diagnosis is uncertain the excision biopsy is performed for histological examination which is golden standard in diagnosis of melanoma. The biopsy will provide details of the thickness of the tumor and any unfavourable features such as ulceration, regression, level of invasion, mitotic rate, vascular invasion. All this information is important to define the melanoma stage, to decide for proper 62 treatment and to estimate prognosis at five years after diagnosis. (2) To support a diagnosis of melanoma histochemical staining of the S100 protein family has been used. Especially one member so called S100B is used in staging malignant melanoma, in establishing prognosis, in evaluating treatment success and in predicting relapse. Survival rate of melanoma patients with normal S100B levels is significant longer compared to those with elevated level. (3) Depending on the stage of melanoma additional tests are performed. Besides visual examination other various tests are ordered to confirm a diagnosis of melanoma and/or determine if or where the disease has spread. All patients with a primary melanoma greater than 1 mm in depth should be offered lymph biopsy. Other tests include blood exam, liver tests, lactate dehydrogenase test (high levels often indicates metastatic spread to the liver), x-ray and special scanning tests such as ultrasound, CT (computed tomography), MRI (magnetic resonance imaging) and PET (positron emission tomography). (4) Poor prognosis for individuals with metastatic or advanced melanoma and poor treatment results, either due to imprecise determination of melanoma stage at time of diagnosis or inappropriate therapy, or both, are leading towards increasing importance to targeted therapies. In order to sufficiently target specific parts of malignant cell mechanism, adjusted cell signaling pathways have to be well known. [21, 23] The most important molecular pathways in melanoma are: [21, 23]  Tyrosine kinase receptors (TRK),  Ras/Raf (BRAF)/MEK/MAPK/ERK/Mitf pathway  Ras/ /Phosphotidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapcamycin (mTOR) pathway with phosphatase and tensin homolog (PTEN) affecting PI3K and negatively regulating the pathway mentioned above  Melanin synthesis pathway (MC1R pathway)  Pathways of cell cycle regulation such as p53/Rb/p14ARF/p16INKA as a consequence of CDKN2A mutation (very common in inherited melanomas)  Gene expression regulation (histone acetylation, DNA methylation and RNA interference)  Apoptosis pathways and apoptosis effectors High number of somatic mutations and deviations is observed in melanoma.http://cco.amegroups.com/article/view/6721/7550 - B26 Most well- known and frequent among them are BRAF (occurrence in 35-45% cases), NRAS (occurrence in 15-25% cases) and KIT (occurrence in 3-4% of western population cases). Firstly, BRAF mutation is present on the gene coding the serine–threonine protein kinase and is responsible for most melanomas arising from skin without chronic sun damage. This mutation, which most common mutated form is BRAF V600E, is also present in melanomas arising from acral surfaces and melanomas deriving from skin chronically exposed to sunlight. Secondly, NRAS mutations are present in 20% of melanomas with higher prevalence in superficial and spreading melanomas deriving from chronic sun exposure cases melanomas. Nonetheless, NRAS seemed to be involved in melanomas arising from giant congenital nevi and mucosal surfaces. KIT (receptor protein tyrosine kinase) mutation is most commonly present in mucosal and acral melanomas. [24, 25] Apart from targeted therapies, knowledge of these pathways is giving us a wider range in possible assays in field of diagnosis and prognosis. Several techniques are being used nowadays to specify the features of pathology in order to define the characteristic of individual melanomas, to diagnose and set prognosis, to introduce the appropriate therapy and to monitor the therapy. 63 Aside from histologic examination, which is widely available and inexpensive, functional proteomic analysis and gene expression arrays are increasing the specificity and sensitivity of the melanoma’s diagnosis. These techniques comprise of immunohistochemistry, comparative genomic hybridization (CGH), chromogenic (CISH) and fluorescent (FISH) in situ hybridization, multiplex ligation-dependent probe amplification (MLPA), ligase detection reaction and reverse transcriptase in situ polymerase chain reaction (RT in situ PCR). [22, 23] 64 References 1. Ries, L.A.G., Eisner, M.P., Kosary, C.L., Hankey, B.F., Miller, B.A., Clegg, L., et al.: SEER Cancer Statistics Review, 1973–1997. National Cancer Institute, Bethesda (2000) 2. Fraumeni Jr., J.F.: Genes and the Environment in Cancer Causation. National Cancer Institute, Washington (2007) 3. Australian Institute of Health and Welfare (AIHW). ACIM (Australian Cancer Incidence and Mortality) Books. AIHW. Canberra (2010) 4. Armstrong, B.K.: Epidemiology of malignant melanoma: intermittent or total accumulated exposure to the sun. J. Dermatol. Surg. Oncol. 14, 835–849 (1988) 5. Jemal, A., Devesa, S.S., Fears, T.R., Hartge, P.: Cancer surveillance series: changing patterns of cutaneous malignant melanoma mortality rates among whites in the United States. J. Natl. Cancer Inst. 92(10), 811–818 (2000) 6: Coory, M., Baade, P., Aitken, J., Smithers, M., McLeod, G.R., Ring, I.: Trends for in situ and invasive melanoma in Queensland, Australia, 1982–2002. Cancer Causes Control 17(1), 21–27 (2006) 7. Jemal, A., devesa, S.S., Fears, T.R., Hartge, P., Tucker, M.A.: Recent trends in cutaneous melanoma incidence among whites in the United States. J. Natl. Cancer Inst. 93, 678–683 (2001) 8. MacKie, R.M., Bray, C.A., Hole, D.J.: Incidence and survival from malignant melanoma in Scotland. Lancet 360, 587–591 (2002) 9. Lachiewicz, A.M., Berwick, M., Wiggins, C.L., Thomas, N.E.: Epidemiologic support for melanoma heterogeneity using the surveillance, epidemiology, and end results program. J. Invest. Dermatol. 128(5), 1340–1342 (2008) 10. Stang, A., Stabenow, R., Eisinger, B., Jockel, K.H.: Site- and gender-specific time trend analyses of the incidence of skin melanomas in the former German Democratic Republic (GDR) including 19351 cases. Eur. J. Cancer 39(11), 1610–1618 (2003) 11. Bulliard, J.L., Cox, B.: Cutaneous malignant melanoma in New Zealand: trends by anatomical site, 1969–1993. Int. J. Epidemiol. 29(3), 416–423 (2000) 12. Osterlind, A., Hou-Jensen, K., Moller-Jensen, O.: Incidence of cutaneous malignant melanoma in Denmark 1978–1982. Anatomic site distribution, histologic types and comparison with non- melanoma skin cancer. Br. J. Cancer 58, 385–391 (1988) 13. Gandini, S., Sera, F., Cattaruzza, M.S., Pasquini, P., Abeni, D., Boyle, P., et al.: Meta-analysis of risk factors for cutaneous melanoma: I. Common and atypical naevi. Eur. J. Cancer 41(1), 28–44 (2005) 14. Gruis, N.A., van der Velden, P.A., Sandkuijl, L.A., et al.: Homozygotes for CDKN2 (p16) germline mutation in Dutch familial melanoma kindreds. Nat. Genet. 10, 351–353 (1995) 15. Harland, M., Meloni, R., Gruis, N., Pinney, E., Brookes, S., Spurr, N.K., et al.: Germline mutations of the CDKN2 gene in UK melanoma families. Hum. Mol. Genet. 6(12), 2061–2067 (1997) 16. Peters, G.: Tumor suppression for ARFicionados: the relative contributions of p16INK4a and p14ARF in melanoma. J. Natl. Cancer Inst. 100(11), 757–759 (2008) 17. Smith Y. 2016. Melanoma diagnosis. Used: 21.8.2016 at http://www.news- medical.net/health/Melanoma-Diagnosis.aspx 18. J. F. Thompson, R. A. Scolyer, R. F. Kefford, Cutaneous melanoma, Lancet 2005; 365: 687–701 19. Harpio R, Einarsson R. S100 proteins as cancer biomarkers with focus on S100B in malignant melanoma. Clin Biochem. 2004 Jul;37(7):512-8. 65 20. Ocvirk J, Hočevar M. Melanom. Informacije o bolezni in zdravljenju. Narodna in univerzitetna knjižnica 2005; 11-12. 21. J. F. Thompson, R. A. Scolyer, R. F. Kefford, Cutaneous melanoma, Lancet 2005; 365: 687–701 22. J. A. Carlson, J. S. Ross, A. J. Slominski, New techniques in dermatopathology that help to diagnose and prognosticate melanoma, Clinics in Dermatology 2009; 27, 75–102 23. J. A. Carlson, J. S. Ross, A. Slominski, G. Linette, J. Mysliborski, J. Hill, M. Mihm, Molecular diagnostics in melanoma, The Journal of the American Academy of Dermatology 2009; 52 (5), 743- 755 24. S. M. Goldinger, C. Murer, P. Stieger, R. Dummer, Targeted therapy in melanoma – the role of BRAF, RAS and KIT mutations, EJC Supplements, September 2013; 92–96 25. S. Ramanujam, D. Schadendorf, G. V. Long, Systemic therapies for melanoma brain metastases: which drug for whom and when? , Chinese Clinal Oncology 2015; 4(2): 25 Figure 1: HC Marbela: http://www.marbellahighcare.com/en/abcde-melanoma/ (accessed: Aug 2016) 66 Treatment of malignant melanoma Emina Abaz, University of Sarajevo, Faculty of Pharmacy, Bosnia and Herzegovina Nives Bebek, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia Jana Nekvindová, University Hospital Hradec Králové, Institute for clinical biochemistry and diagnostics, Czech republic Treatment Treatment of malignant melanoma depends on stage. In stages II and less, the recommended therapy is surgical excision. In stage III, the best treatment is a complete lymphadenectomy, and in stages IIIB and IIIC also a local therapy applying imiquimod, an immune response modifier, can be considered. [1] Stage IV demands systemic chemotherapy. Pharmacotherapy in malignant melanoma includes targeted drugs, dabrafenib and vemurafenib, for patients with inoperable or metastatic BRAF V600 mutation positive melanoma. According to the fact that malignant melanoma is the most immunogenic cancer, targeted immunotherapy drugs such as ipilimumab are also recommended for previously treated advanced melanoma. Classic cytotoxic chemotherapy with dacarbazine should be considered only if immunotherapy and targeted therapy are not suitable (NICE guidelines – Melanoma: assessment and management). Other drugs that may be considered are also checkpoint therapy monoclonal antibodies such as pembrolizumab or nivolumab, and a MEK inhibitor trametinib. Ipilimumab Ipilimumab (trade name Yervoy) is a new agent in melanoma therapy which enhances the immune response by blocking negative signaling from cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4). CTLA-4 is an immune checkpoint molecule that down-regulates pathways of T-cell activation. Generation of an immune signal requires presentation of tumor antigen by major histocompatibility complex (MHC) class I or II molecules on an antigen presenting cell (APC). However, T cell activation and proliferation requires a second signal, typically generated when CD28, a receptor on the T cell surface simultaneously binds to a costimulatory B7 molecules on the APC. Following activation, T cells upregulate and translocate CTLA-4 receptor molecules to the surface, which bind B7 with a higher avidity than CD28. CTLA-4 successfully outcompetes with CD28 to generate an opposing signal that inhibits T cell proliferation and IL-2 secretion. CTLA-4 is therefore a key negative regulator of endogenous T cell-mediated responses, serving as a natural braking mechanism and allowing for a return to homeostasis following an immune response. [3] Ipilimumab, a fully human monoclonal antibody (IgG1) blocks CTLA-4 expressed by activated T-cells to promote antitumor immunity (Figure 1). Pembrolizumab Pembrolizumab (trade name Keytruda) is a potent, highly selective humanized monoclonal antibody (IgG), designed to directly block the interaction between PD-1 (programmed cell death 1) receptor, expressed on T cells and its ligands, PD-L1 and PD-L2, without antibody-dependent cell mediated cytotoxicity (Figure 1). The PD-1 receptor is a cell surface receptor, belonging to the CD28 family of T cell regulators, within the immunoglobulin superfamily of receptors. It is expressed upon activation in mature hematopoietic cells, such as T and B cells, natural killer cells and monocytes, after prolonged antigen exposure. Through its binding to PD-L1 and PD-L2. PD-1 receptor downregulates T-cell activation and proliferation, along with downregulation of the expression of the anti-apoptotic molecule Bcl-xL, cytokine expression, and the mTOR pathway. PD-1 receptor is highly expressed on tumor-infiltrating lymphocytes (TIL) in response to tumor antigen expression, and its binding to PD-L1 on either tumor cells or APCs, functions as an immune checkpoint to curb persistent immune response. [4] 67 Figure 1. Mechanisms of action - ipilimumab (anti CTLA-4) and pembrolizumab (anti-PD-1) Target therapy Target drugs act in a way that they detect and react with the part of melanoma cells that differ them from normal cells. They can be used when chemotherapy doesn’t work and they usually have less side effects. (NICE guidelines – Melanoma) BRAF inhibitors Vemurafenib, Dabrafenib About half of all melanomas have changes in BRAF gene on the position 600, Valin being exchanged with Glutaminic acid. These changes result in altered BRAF protein which helps these cells grow. Therefore, in order for therapy to be effective, biopsy samples should be tested to see if cancer cells have previously mentioned BRAF mutation. [8] Drug is taken two times daily in amount of 940 mg per day. Adjustment of doses is required in case of liver or kidney damage as well as in case of geriatric and paediatric population. Common side effects include sensitivity to the sun, skin tightening, fatigue, hair loss and nausea. Less common but more dangerous ones include severe allergic reactions and heart rhythm problems. [7] MEK inhibitors Trametinid, Cobimetinib These drugs are reversible inhibitors of activation and activity of Mitogen-activated Extracellular Signal-regulated kinase 1 (MEK1) and MEK2. MEK enzymes furthermore regulate extracellular signal- related kinase (ERK) which promotes cellular proliferation. These drugs are commonly combined with BRAF inhibitors and are taken once per day. Common side effects of these drugs are rash, nausea, swelling and sensitivity to sunlight. Even though, heart damage, excessive bleeding and lung problems also may occur. [9] 68 Figure 2. Mechanism of action of vemurafenib and trametinib References [1] Miller RL, Gerster JF, Owens ML, Slade HB, Tomai MA. Imiquimod applied topically: a novel immune response modifier and a new class of drug. Int J Immunopharmacol; 1999. 21 (1): 1–14 [2] Melanoma. (n.d.). Retrieved August 21, 2016, from https://www.nice.org.uk/guidance/indevelopment/gid-cgwave0674 [3] Wolchok JD, Hodi FS, Weber JS, Allison JP, Urba WJ, Robert C, O'Day SJ, Hoos A, Humphrey R, Berman DM, Lonberg N. Development of ipilimumab: a novel immunotherapeutic approach for the treatment of advanced melanoma. Annals of the New York Academy of Sciences. 2013 Jul 1;1291(1):1-3. [4] Improta G, Leone I, Donia M, Gieri S, Pelosi, G, Fraggetta F. New developments in the management of advanced melanoma – role of pembrolizumab. Onco Targets Ther. 2015 Sep 14; 2535. doi:10.2147/ott.s72823 [5] Melanoma. (n.d.) https://www.nice.org.uk/guidance/ng14/chapter/1- Recommendations#managing-stage-iv-melanoma (Accessed Aug, 2016) [6] Target therapy, http://www.cancer.org/cancer/skincancer-melanoma (Accessed Aug, 2016) [7] Vemurafenib, http://www.cancerresearchuk.org/about-cancer/cancers-in- general/treatment/cancer-drugs/vemurafenib (Accessed Aug, 2016) [8] Cancer.org http://www.cancer.org/cancer/skincancer-melanoma/detailedguide/melanoma-skin- cancer-treating-targeted-therapy (Accessed Aug, 2016) [9] Cancer.org http://www.cancer.org/cancer/skincancer-melanoma (Accessed Aug, 2016) 69 Melanomas: resistance to RAF inhibition; paradoxical activation of MAPK pathway; adverse effect of BRAF inhibition Marija Banićević, University of Belgrade, Faculty of Pharmacy, Serbia Jelena Janać, University of Belgrade, Faculty of Pharmacy, Serbia Jana Nekvindová, University Hospital Hradec Králové, Institute for clinical biochemistry and diagnostics, Czech republic Resistance to RAF inhibition: paradoxical activation of MAPK pathway BRAF mutations, V600E, alternatively V600K/D, are the major driver mutations that lead to malignant melanoma development (Chapman, 2013). Selective RAF inhibitors vemurafenib (formerly designated as PLX4032) and dabrafenib are effective therapy in patients with BRAF V600-driven melanoma. Those two are one of the most compelling examples of targeted therapy knowing that BRAF gene mutations are responsible for tumorigenic behaviour of over 50% of all cutaneous melanomas. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance and patients show signs of disease progression after 6- 7 months after the initialisation of therapy with RAF inhibitors (Poulikos et al., 2014; Gibney et al., 2013). Identification of resistance mechanisms is important for generating alternative ‘druggable’ targets and better treatment for patients with melanoma. (Gibney et al., 2013) In order to do that, understanding of signalling pathways that are implicated in melanoma is substantial. There have been eight pathways identified in malignant melanoma development: MAPK, AKT/PI3K, c-KIT, CDK, GNAQ/GNA11, MITF, NRAS and P53/BCL* (Vidwans et al., 2011). There is a lot of cross- talk between these pathways and their downstream effectors. However, two signaling pathways implicated in melanoma are major: the MAPK pathway and the AKT/PI3K pathway which regulate cell growth, proliferation and cell death (Vidwans et al., 2011). Several mechanisms mediating resistance to BRAF inhibitors have been described and they can be divided into paradoxical MAPK reactivation and MAPK-independent mechanisms. Paradoxical activation is a rapid recovering of MAPK signalling after BRAF inhibition as a result of depressed feedback inhibition. It includes the up-regulation of bypass pathways mediated by cancer Osaka thyroid kinase (COT), development of de novo NRAS or MEK mutations and variant splicing and/or dimerization of mutant BRAFV600 etc. MAPK-independent signaling that has been associated with BRAF resistance comprises receptor tyrosine kinases (TRK) such as platelet derived growth factor receptor β (PDGFβ), insulin-like growth factor 1 receptor (ILGF-1R), and hepatocyte growth factor receptor (HGFR) (Flaherty et al., 2012). Mechanisms of resistance seem to be BRAF-inhibitor-specific. NRAS mutations and splice variants of BRAFV600 mRNA are the two most common mechanisms identified so far. (Chapman et al., 2013) Expression of BRAFV600E splicing variants is the first resistance mechanism identified. It involves a structural change in BRAF. Different splicing variants of mutated BRAF were identified in cell lines resistant to vemurafenib. The 1.7kb cDNA band that was not present at the parent-cells lacks exons 4-8, compared to full length cDNA of BRAFV600E and codes 61kD protein, called p61BRAFV600E. Exons 4- 8 include domains critical for RAF activation-RAS-binding domain (RBD) and cysteine rich domain (CRD). Dimerization of p61BRAFV600E is significantly elevated compared to full- length BRAFV600E which implies that deletion of exons 4-8 promotes dimerization even if the RAS is inactive (Poulikos et al., 2011). These findings have been confirmed in tumours of patients which were treated with vemurafenib as proven by analyzing tumours from 19 melanoma patients with acquired resistance to vemurafenib. 70 BRAFV600E transcripts from resistant tumours lacked exons 4-10, 4-8, 2-8 or 2-10 compared to the vemurafenib- naïve tumours. (Poulikos et al., 2011). Another mechanism of resistance in B-RAFV600E-positive melanomas involves activation of RTK (PDGFRβ)-dependent survival pathway in addition to MAPK or reactivating the MAPK pathway via N- RAS upregulation. These two mechanisms accounted for acquired vemurafenib resistance in 5 of 12 patients in study cohort of Nazarian et al., 2010 and in 4 of 19 patients in the study of Poulikos et al., 2011. MAPK3K8 (COT/TPL2) also drives resistance to RAF inhibition in BRAF V600E cell lines. Cancer Osaka thyroid kinase (COT) expression is sufficient to activate ERK primarily through MEK-dependent mechanisms that do not require RAF signaling. Cell lines expressing elevated COT exhibit de novo resistance to PLX4720 treatment (Johannessen et al., 2010). Adverse effects of BRAF inhibition Besides the resistance, as a complication that interferes with positive outcomes in BRAF-inhibition therapy of patients with malignant melanoma, significant toxicity as well as development of secondary malignancies stands out. Toxicity of dabrafenib and vemurafenib is relatively mild in majority of patients as compared to classical chemotherapy (Gibney et al. 2013). Common adverse effects include skin events, gastrointestinal symptoms, headache, fatigue, pyrexia (Gibney et al. 2013:4). These adverse effects in most cases lead to dose interruptions or modifications, but the patients usually continue the therapy (Gibney et al. 2013). In only 3% of patient adverse effects are the cause of drug discontinuing (Gibney et al. 2013). Cutaneous events are more serious and include hyperkeratosis, keratoacanthomas and squamous cell carcinomas (Gibney et al. 2013). Squamous cell carcinoma (SCC) is the second most common skin cancer among Caucasians (Alam et Ratner, 2001). Risk factors for developing SCC are numerous: the most common is exposure to UV- radiation which causes DNA damage and dysfunction of p53 tumour-suppressor gene (Alam et Ratner, 2001). The SCCs related to the treatment with RAF inhibitors are more differentiated than those developed as a consequence of sun damage (Gibney et al. 2013). In the most cases, SCC develop rapidly after the initiation of the therapy (median 8 weeks) (Gibney et al. 2013). Metastases are not reported and in most cases, the therapy is continued and lesions are managed by excision (Gibney et al. 2013). The key relation between development of SCC and BRAF inhibitor therapy is a paradoxical activation of MAPK pathway which occurs through the BRAF-inhibitor-mediated formation of RAF dimers (Gibney et al. 2013). This theory is supported by the results of many preclinical studies (Gibney et al. 2013). Also, studies results indicated that MAPK signaling could be a SCC initiator, but it is not required for tumour maintenance (Gibney et al. 2013). This is demonstrated by the ability of MEK inhibitor to suppress SCC development following the BRAF inhibitor therapy, but lack of its effectiveness against established SCC (Gibney et al. 2013). In the cases where paradoxical activation of MAPK pathway occurs, an oncogenic event might arise in cells with pre-existing mutations (Gibney et al. 2013). Rarely, adverse effects of BRAF inhibitor therapy include the development of secondary melanomas, colonic adenomas and gastric polyps (Gibney et al. 2013). 71 Considering aforementioned adverse effects, it is a challenge to develop the strategy for prevention of secondary malignancies. Several approaches are proposed:  Concurrent downstream inhibition (MEK inhibitors combined with BRAF inhibitors) (Gibney et al. 2013).  Usage of a new class of pan-RAF inhibitors, so-called paradox breakers. This class of drugs blocks the signaling without paradoxical MAPK pathway activation (Gibney et al. 2013; Zhang et al. 2015).  Usage of synthetic retinoids: these drugs promote cell maturation and differentiation and decrease cell growth and malignant transformation (Gibney et al. 2013).  Usage of cyclooxygenase-2 (COX-2) inhibitors: investigations of SCC carcinogenesis revealed that the COX-2 inhibitors therapy abrogated increased COX-2 expression and prostaglandin production and mitigates SCC development (Gibney et al. 2013). Despite the paradoxical activation of MAPK pathway, which is leading to secondary malignicies, the development of BRAF inhibitors represents a major milestone in the therapeutic managemant of diseminated melanoma. With rationally designed drug combinations, the future of patients with BRAF V600E-driven melanoma continues to look increasingly optimistic (Gibney et al., 2013). MAPK = Mitogen-activated Protein Kinase, AKT/PI3K = Protein Kinase B/Phosphoinositide 3-kinase, c-KIT = CD11, CDK = Cyclin Dependant Kinase, GNAQ/GNA11 = Guanine nucleotide-binding protein G(q) subunit alpha/ Guanin nucleotide- binding protein alfa 11, MITF = Microphthalmia-associated transcription factor, NRAS = Neuroblastoma RAS, BCL = B-cell Lymphoma. 72 References 1. Alam M, Ratner D. (2001). Cutaneous squamous-cell carcinoma. The New England Journal of Medicine 29;344(13):975-83 2. Chapman PB. Mechanisms of resistance to RAF inhibition in melanomas harbouring a BRAF mutation (2013) American Society of Clinical Oncology Educational Book/ASCO. American Society of Clinical Oncology. Meeting 3. Flaherty KT, Jeffery R, Daud A,Gonzalez R, Kefford RF, Sosman J, Hamid O,Schuchter L, Cebon J, Ibrahim N, Kudchadkar R, Burris HA, Falchook G, Algazi A, Lewis K, Long GV, Puzanov I, Lebowitz P, Singh A, Little S. Sun P, Alicia Allred A, Ouellet D, Kim KB, Kiran Patel K, Weber J. (2012) Combined BRAF and MEK Inhibition in Melanoma with BRAF V600 Mutations. The New England Journal of Medicine; 367:1694-1703 4. Gibney GT, Messina JL, Fedorenko IV, Sondak VK, Keiran S.M. Smalley KSM. (2013) Paradoxical oncogenesis-the long term consequences of BRAF inhibition in melanoma. Nature Reviews. Clinical Oncology; 10(7): 390–399. 5. Johannessen CM, Boehm JS; So Young Kim SY, Thomas SR, Leslie Wardwell L, Johnson LA, Emery CM, Stransky N, Cogdill AP. Barretina J Caponigro G, Hieronymus H, Murray RR, Salehi- Ashtiani K,Hill DE, Vidal M, Zhao JJ, Yang X, Alkan O, Kim S,Jennifer L. Harris JL, Wilson CJ, Myer VE, Peter M. Finan PM, Root DE. Roberts TM, Golub T, Flaherty KT, Dummer R, Weber B, William R. Sellers WR, Schlegel R, Jennifer A. Hahn WC, Garraway LA. (2010) COT/MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature; 468(7326): 968–972. 6. Nazarian R, Hubing Shi H, Wang Q, Kong X, Koya RC, Lee H, Zugen Chen2,4, Mi-Kyung Lee1,2,Attar N, Sazegar H, Chodon T, Nelson SF, Grant McArthur G, Sosman JA, Ribas A, Lo RS. (2010) Melanomas acquire resistance to B-RAF (V600E) inhibition by RTK or N-RAS upregulation Nature; 468(7326): 973–977. 7. Poulikos P.I., Yogindra Persaud Y., Janakiraman M., Kong X, Ng C., Moriceau G., Hubing Shi H., Atefi M., Titz B., Gabay M. T, Salton M.S., Dahlman K.B., Tadi M., Jennifer A. Wargo10, Keith T. Flaherty K.T., Kelley M.C.9, Misteli T.4, Paul B. Chapman P.B., Jeffrey A. Sosman J.A.8, Graeber T.G.6, Antoni Ribas A., Lo R.S., Rosen N., Solit D.B. (2011) RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF (V600E). Nature; 480(7377): 387–390. 8. Vidwans SJ, Flaherty KT, Fisher DE, Tenenbaum JM, Travers MD, et al. (2011) A Melanoma Molecular Disease Model, PLoS One. 30;6(3):e18257. 9. Zhang C, Spevak W, Zhang Y, Burton EA, Ma Y, Habets G, Zhang J, Lin J, Ewing T, Matusow B, Tsang G, Marimuthu A, Cho H, Wu G, Wang W, Fong D, Nguyen H, Shi S, Womack P, Nespi M, Shellooe R, Carias H, Powell B, Light E, Sanftner L, Walters J, Tsai J, West BL, Visor G, Rezaei H, Lin PS, Nolop K, Ibrahim PN, Hirth P, Bollag G. (2015) RAF inhibitors that evade paradoxical MAPK pathway activation. Nature; 526(7574):583-6 73 Bone metastatic disease Bone metastatic disease – Epidemiology and molecular mechanisms Ema Muller, University Ljubljana, Faculty of pharmacy, Slovenia Jovana Arandjelovic, University of Belgrade, Faculty of Pharmacy, Serbia Meta Zupančič, University Ljubljana, Faculty of pharmacy, Slovenia Klemen Kodrič, University Ljubljana, Faculty of pharmacy, Slovenia EPIDEMIOLOGY Bone metastatic disease (BMD) is a type of metastatic cancer in which primary cancer cells have disseminated from the original location and start to interact with bone cells. The consequence is development of bone metastases that cause skeletal-related events (SREs) or skeletal complications like bone pain, hypercalcemia, pathologic fractures and spinal cord or nerve root compression. The pain generally appears at the bottom of the skull, in the neck, lower back, legs and pelvis. (1) When patients are diagnosed with bone metastasis, 22 % have a SRE at that time and almost half of them develop it later in progress of the disease. (1, 2) Picture 1: Tumor metastasis in bones (3) Almost all types of cancers can develop bone metastasis. Only in renal cancer bone metastases occur in only one location, whereas in other types of cancer multiple bone metastases are present. (2) Patients with breast or prostate cancer are most prone to bone metastases with the incidence of about 70 %. These two cancers also have quite high prevalence worldwide and since the patients can now live longer because of better disease management, the prevalence of BMD in these cancers is increasing. (2, 4) Lung cancer is the third most prevalent cancer for BMD. It causes bone metastases in approximately 35 % of the cases. Patients with bladder cancer and renal cell carcinoma have 40 % and around 20 – 25 % chance to develop bone metastases, respectively. Although thyroid cancer has a 60 % incidence for BMD, the prevalence is not as high as in former mentioned cancers. The widest range of incidence for bone metastasis has melanoma, with the stretch of 14 – 45 %. Incidence of BMD caused by gastrointestinal cancer is less than 10 %. (1, 4) In the USA with a population of over 300 million people, around 1.4 million people are diagnosed with cancer every year; half of them have a type of cancer that commonly spreads to bones. Consequently, more than 400.000 patients per year develop BMD. Metastases generally appear in people older than 50 years, which corresponds to the class of primary cancer. (2, 5) The prognosis of the disease is mostly dependant on the pathology of the primary cancer and the presence of visceral metastasis. Due to this reason, the life expectancy of cancer patients at the time of bone metastasis diagnosis varies from only months to a few years. (1) The most variable is prostate cancer (patients live from 1 to 4.5 years); breast cancer has a survival rate of around 2 years after diagnosis. Higher mortality rates have 74 melanoma, renal cell carcinoma, bladder and lung cancer, visceral metastases and bone metastases of unknown origin with patients surviving 1 year or less. (2, 4) MOLECULAR MECHANISMS Metastases are a selective process in which cancer cells from primary site undergo the process of invasion, embolization, survival in the circulation, arrest in a distant capillary bed, extravasation, and re-growth in the microenvironment of the secondary organ, so that only a few phenotypically changed cells survive to form metastases in other organs and tissues in the body. Four stages of metastases development in bone have been described: colonization, survival or dormancy, reactivation and growth. (6, 3) Disruptions in signalling pathways in bone cells have the most important role in etiology of the BMD. The balance between two functions of the bone cells – lysis and genesis, is disturbed, causing different patterns in the metastatic bone lesion, ranging from mostly destructing (osteolytic) to mostly bone forming (osteoblastic) activity. (3) Mechanisms are explained with vicious cycle hypothesis, explaining reciprocal interactions between cancer cells, osteoblasts, osteoclasts and the mineralized bone matrix, that contribute to progression. There is no data whether cancer cells colonize haematopoetic stem cells (HSCs) and/or progenitor niches. (6) Premetastatic niche expresses cell surface ligands and receptors that provide a permissive environment for the migrating cancer cells, supported by growth factors, cytokines and chemotactic proteins presented in the bone marrow. (7) Picture 2: Process of bone metastasis development (6) Osteolytic metastasis: Secondary cancer cells can secrete factors for direct and indirect stimulation of osteoclasts activity – differentiation, proliferation and activation from pluripotent osteoclast precursor cells, causing increased osteolysis. Indirect stimulation is based on the OPG/RANK/RANKL pathway, which regulates osteoclast formation and survival. RANKL (receptor activator of nuclear factor kappa B ligand) is a member of tumor necrosis factor ligand superfamily and is expressed on different types of cells, including osteoblasts, and exists in two isoforms – bound and soluble. RANK is a receptor that is expressed on osteoclasts and dendritic cells that after activation with RANKL activates multiple intracellular signalling processes in osteoclasts to stimulate their proliferation and survival. OPG (osteoprotegerin) is a soluble TNF receptor and is a negative regulator of RANK/RANKL pathway – it binds RANKL and decreases osteoclastogenesis and promotes osteoclast apoptosis. In BMD, tumor cells secrete numerous cytokines and growth factors which stimulate OPG/RANK/RANKL pathway, for example parathyroid hormone-related peptide (PTHrP), interleukins (1, 3, 8, 11) and TNF-α. Two possible mechanisms exist for bone degradation: up regulation of RANKL expression or down regulation of OPG secretion and consequential increase of osteoclasts activity and bone degradation. Certain cytokines (IL-6, TNF- α, IL-8, etc.) can stimulate osteoclastogenesis independently of RANKL, but the pathways involved are not clear. (7, 10, 11) In response to factors described, osteolytic bone can secrete factors that can attract cancer cells to the bone surface and facilitate their growth and proliferation. (8) Cancer cells invade surrounding tissues to enlarge the tumor mass by secreting proteolytic enzymes such as aspartic, cysteine and serine proteases and matrix degrading metalloproteinases (MMPs). (9) Breast cancer is the main representative of cancer with osteolytic 75 bone metastases. Breast cancer cells have the ability to secrete PTHrP, a powerful stimulator of bone resorption. Due to TGF stimulation, tumor cells excrete PTHrP. (9) Osetoblastic metastases: Tumor cells secrete various pro-osteoblastic factors – cytokines, transcription factors and growth factors that transform normal bone remodelling into predominantly bone forming state. These factors play a role in differentiation, proliferation and maturation of osteoblasts and can also inhibit the activity of osteoclasts. Osteoblasts also secrete growth factors (TGF- β, BMP, VEGF). These factors interact with cancer cells and potentiate their survival and growth, so that the metastatic growth is amplified in a circle. There are three important pathways for osteoblastic metastasis: Wnt signal transduction cascade, the ET axis and the BMP pathway. Wnt glycoproteins promote embryonic and postnatal bone formation and can, when bound to the receptor complex, initiate a number of intracellular signalling cascades with effects on differentiation, survival and activity of osteoblasts. The ET-1 protein (endothelin) is formed by primary cancer cells and, when bound to its receptor, stimulates proliferation of osteoblasts, promotes mineralization, inhibits osteoclast motility and potentiates pro-osteogenic effects of other growth factors. Bone morphogenetic proteins (BMP) are growth factors in skeletal development and postnatal bone repair which stimulate osteoblasts proliferation, activity and survival in BMD. (7, 12) The prostate cancer produces factors like TGF-β, BMPs, thymosin 15 and ET-1, which stimulate osteoblastic bone formation. Most cancers have a mixed activity on the bone. Prostate cancer metastases are also osteoclastic in the beginning, in order to initiate osteolysis and subsequently communicate to osteoblasts to develop osteosclerosis. (9) CONCLUSION Bone is the most common site for breast and prostate cancer metastases, which probably means that the bones have a suitable environment and provide conditions that favor colonization and growth of disseminated cancer cells. LITERATURE 1. Coleman R. Clinical Features of Metastatic Bone Disease and Risk of Skeletal Morbidity. Clinical Cancer Research. 2006;12(20):6243s-6249s. 2. Randall R. Metastatic bone disease: An Integrated Approach to Patient Care. New York: Springer; 2016. 3. Larry J. Suva, Charity Washam, Richard W. Nicholas & Robert J. Griffin: Bone metastasis: mechanisms and therapeutic opportunities. Nature Reviews Endocrinology 7. 2011; 208–218. 4. Coleman R. Bisphosphonates: Clinical Experience. The Oncologist. 2004;9(suppl_4):14-27. 5. Metastatic Bone Disease: Practice Essentials, Background, Pathophysiology and Etiology [Internet]. Emedicine.medscape.com. 2016 [cited 20 August 2016]. Available from: http://emedicine.medscape.com/article/1253331-overview#a6 6. Croucher PI, McDonald MM, Martin TJ. Bone metastasis: the importance of the neighbourhood, Nat Rev Cancer. 2016;16(6):373-86. 7. M.S. Virk, J.R. Lieberman. Tumor Metastasis to bone. Arthritis Research & Therapy; 2007. 8. R.E. Coleman. Metastatic bone disease: clinical features, pathophysiology and treatment strategies. Cancer Treatment Reviews, 2001; 27; 3; 165-176 9. T. Yoneda. Cellular and Molecular Mechanisms of Breast and Prostate Cancer Metastasis to Bone. European Journal of Cancer, 1998; Vol. 34, No. 2; 240±245. 10. W. C. Dougall, I. Holen and E. Gonzalez Suarez. Targeting RANKL in metastasis. BoneKEy Reports 3, 2014; 519. 11. J. Zhang, J. Dai, Z. Yao,Y. Lu, W. Dougall, E.T. Keller. Soluble receptor activator of nuclear factor kappaB Fc diminishes prostate cancer progression in bone. Cancer Res, 2003; 63:7883- 7890. 76 12. J.B. Nelson, S.H. Nguyen, J.R. Wu-Wong, T.J. Opgenorth, D.B. Dixon, L.W. Chung, N. Inoue. New bone formation in an osteoblastic tumor model is increased by endothelin-1 overexpression and decreased by endothelin A receptor blockade. Urology, 1999; 53:1063- 1069. 13. R. von Moos, I. Haynes. Where Do Bone-Targeted Agents RANK in Breast Cancer Treatment? J. Clin. Med., 2013; 2(3), 89-102, 2013. 77 Bone metastatic disease – Diagnosis and treatment Lisa Gabler, Institute for Cancer Research, Medical University of Vienna, Centre for Molecular Biology, University of Vienna, Austria Jovana Radovanovic, University of Belgrade, Faculty of Pharmacy, Serbia Nejra Džemidžić, University of Sarajevo, Faculty of Pharmacy, Bosnia and Herzegovina Klemen Kodrič, University Ljubljana, Faculty of pharmacy, Slovenia INTRODUCTION Bone metastases are a common consequence of advanced-stage cancer originating from various organs, most commonly from breast and prostate cancer in females and males, respectively (1). Furthermore, occurrence of bone metastases represents a negative prognostic factor regarding survival and quality of life (2, 3). DIAGNOSIS Bone metastases are classified according to their pathogenesis and growth pattern. Characteristically, metastases are either termed osteolytic, hence the cancer cells destroy and reduce the bone structure, or osteoblastic/sclerotic, if the tumor results in gain of bone mass (4, 5). Correct diagnosis of cancer as early as possible is essential for treatment and prognosis. Therefore, aims of diagnosing bone metastases include early detection, preventing pathological fractures and neurologic problems as a result of spinal impairment, and stopping the tumor progress (4, 5). Diagnostic methods like imaging techniques (radiography, computer tomography (CT), magnetic resonance imaging (MRI), nuclear imaging and angiography), biochemical markers of bone turnover in blood and urine, and histological analysis after biopsy are commonly used (5, 6, 7). Radiography (electromagnetic radiation) is commonly used when the symptoms, such as bone pain (ostalgia), have already appeared (4). The method results in 2D images and provides information about the tumor site and localization of tumor-associated fragile bone structure (8, 9). Unfortunately, sometimes bone lesions are not visible by using this method and also lytic bone metastases can sometimes be misdiagnosed as osteoarthritis (5). CT scanning is more accurate and sensitive than radiography for diagnosing lytic metastatic lesions (5, 7). It is used for visualization of bone structure changes due to metastases of certain tumors (4). Results are shown as 3D images that are useful for assessment of tumor size, shape and stability. However, even advanced lesions of the impaired bone may sometimes not be detected by this method (5, 7). MRI is a commonly used method for the detection of metastases in the bone marrow and extra osseous soft tissues (4). In contrast to conventional radiography and CT, MRI does not involve radiation, but rather magnetic field for visualization of bones and other tissues (5). It is the method of choice for the detection of metastatic lesions, and also for evaluating the stage of disease (6, 10). Moreover, it provides information about the tumor mass and eventual bone impairment caused by infection, not by a malignancy. For better detection and illustration of the tumor, MRI as well as CT is commonly combined with conventional radiography (7). 99m Tc skeletal scintigraphy, positron emission tomography (PET) and single photon emission computed tomography (SPECT) are nuclear imaging methods for evaluation of local bone metabolism in early phases of different types of cancer. 99m Tc skeletal scintigraphy enables detection of metabolic deposits formed by enhanced osteoblastic activity (5). Therefore, it is convenient for detecting asymptomatic osteoblastic bone metastases, as well as for tracking the response to treatment (4, 7). This cost-efficient high sensitivity method allows whole-body examination (5). 99mTc skeletal scintigraphy in combination with PET and CT is suitable for diagnosing metabolically active bone and soft tissues tumors and small lesions (4, 5). PET with 18F- fluorodeoxyglucose, combined with CT (18F-FDG-PET-CT), allows the visualization of increased glucose metabolism in cancer cells. SPECT is usually combined with CT in order to increase sensitivity and specificity of the test (4, 5). 78 Figure 3: A 99m Tc bone scientoscan of a patient with bone metastasis. (5) Bone metastases often result in hypercalciemia and increased ALP. There are some blood and urine tests that point to possible bone lesions, such as calcium or ALP in blood and N – telopeptide in urine. However, these tests alone are insufficient to prove the presence of metastasis and require additional methods for clarification (7). Biopsy is a method used only if the non-invasive detection methods do not give a clear image in diagnosing bone metastases (7). THERAPY AND TREATMENT Bone-resident metastatic cancer cells are considered as highly malignant cells and have the ability to interfere with the tightly controlled intercellular signaling network, mainly composed of interactions between osteoblasts (bone-forming cells), osteoclasts (bone-resorbing cells) and other stromal cells. Therefore, bone metastases can either cause osteogenesis or osteolysis (2). Interestingly, much research is conducted based on the so called ‘vicious cycle’ theory, mainly dealing with the pathogenesis of lytic bone metastases. Thus, osteolysis induces liberation of a variety of growth factors including TGF-β, a promoter of tumor cell proliferation. In turn, tumor cells induce osteolysis, again resulting in growth factor release (1). Currently, bisphosphonates represent the gold standard therapy for lytic bone metastases, while radiopharmaceuticals are a better choice for patients with osteoblastic bone metastases (1,3,10). Bisphosphonates are synthetic analogs of pyrophosphate (2) that inhibit osteoclast activity and decrease bone turnover caused by bone metastases (1,3,10) (Fig.2). The agents significantly decrease skeletal-related events (SRE) including bone pain (ostalgia), pathological fractures, spinal cord compression and hypercalcemia. Avoiding SRE is one of the main goals in bone metastases management (1). Zoledronic acid (ZA) is a second-generation bisphosphonate agent with direct antitumor effects in addition to its antiresorptive functions. With respect to postmenopausal lytic breast cancers, ZA is considered to lower the recurrence of bone metastasis as well as to increase patients’ survival (2). Currently, there are several ongoing clinical trials dealing with the combination of bisphosphonates of different potencies and administration routes (2). Recent gene expression studies revealed several other target sites for bone metastases therapy. Drugs targeting these sites include receptor activator of nuclear factor κB ligand (RANKL) inhibitor Denosumab and Cathepsin K inhibitor Odanacatib, as well as chemokine receptor 4 (CXCR4) inhibitors and TGF-β inhibitors targeting the ‘vicious cycle’ (1,2). RANKL is a crucial factor for osteoclasts differentiation and activation. Furthermore, RANKL affects the maturation and activation of various immune cells including dendritic cells (DC) (Fig.2). Denosumab is a fully human monoclonal antibody against RANKL and is approved for the treatment of osteoporosis, rheumatoid arthritis and cancer, especially osteolytic cancer. Denosumab is discussed to interfere with the biological 79 functionality of DC, thus to negatively affect the patients’ immunity (2). The Cathepsin K inhibitor Odanacatib was developed for the treatment of osteoporosis, but seems to be beneficial for reduction of tumor burden in bone metastases too. The drug specifically targets bone resorption while maintaining the number of osteclasts and therefore the bone formation (1,2). With respect to breast and prostate cancer, representing the most common origins of bone-metastases, inhibition of the hormones driving the tumors is a novel therapy strategy. Inhibition of androgen synthesis like estrogen and testosterone for estrogen receptor positive breast and prostate cancer, respectively, are important issues in the management of these diseases. Beneficial response to this therapy is limited to patients with local tumors in the primary site; however, use of hormone inhibitors to treat advanced cancers with bone metastases doesn’t seem to be sufficient (2). Figure 4: Bisphosphonate and Denosumab target osteolytic bone metastases. They decrease the survival of osteoclasts. Bisphosphonates cause loss of resorptive function in osteoclasts (left), whereas Denosumab specifically antagonizes RANKL, thus blocks osteoclast formation and function (right). Osteoprotegerin (OPG) competes with Denosumab for binding to RANKL and must be considered as negative predictive biomarker for Denosumab efficiency. (12) Besides the management of bone metastases, targeting the underlying mechanisms and curing of the disease, prevention and treatment of ostalgia, the most common symptom of this disease, is a main goal in order to positively influence the patients’ quality of life (10). Maximal surgical resection and radiation are included in the general palliate pain treatment. In cases with multiple ostoeblastic bone metastases, radiation therapy can be combined with other treatment options like radiopharmaceuticals, chemical molecules coupled to radioactive isotopes. This therapy mainly targets those sites in the bone with highly active turnover, hence the tumor sites, resulting in reduction of ostalgia and shrinkage of tumor size and mass (11). Bones weakened from malignancies tend to break, resulting in pathological fractures. Artificial cementation of the bones in combination with mechanical bone fixation and radiotherapy is a commonly used technique to prevent or fix pathological fractures. Amongst all analgesic drugs, opioids count as the most effective ones in alleviation of ostalgia. However, the side effects include drug addiction, thus patients must be supervised very precisely (10). CONCLUSION To conclude, all diagnostic techniques have their flaws. Radiograpy and CT are relatively insensitive in the detection of early or small metastatic lesions; CT also covers only a small area of the bone. On the other hand, bone scintiscan findings are sensitive but unspecific. Whole-body MRI and PET 80 scanning are accurate, but expensive. Therefore, combination of different methods is the best option in order to diagnose correctly and efficiently (5). The therapy solely targeting the underlying mechanisms of the tumor is not sufficient in the management of bone-metastases, but treatment of the symptoms is also necessary to achieve better life quality for the patients. (10) REFERENCES 1. Croucher PI, McDonald MM, Martin TJ. Bone metastasis: the importance of the neighbourhood. Nat Rev Cancer 2016 May 25;16(6):373-386. 2. Rose AA, Siegel PM. Emerging therapeutic targets in breast cancer bone metastasis. Future Oncol 2010 Jan;6(1):55-74. 3. Piccioli A, Maccauro G, Spinelli MS, Biagini R, Rossi B. Bone metastases of unknown origin: epidemiology and principles of management. J Orthop Traumatol 2015 Jun;16(2):81-86. 4. Heindel W, Gübitz R, Vieth V, Weckesser M, Schober O, Schäfers M. The diagnostic imaging of bone mestastases. Dtsch Arztebl Int. 2014 Oct; 111(44): 741–747 5. Imaging in Bone Metastases: Overview, Radiography, Computed Tomography. Emedicine.medscape.com. 2016 [cited 20 August 2016]. Available from: http://emedicine.medscape.com/article/387840-overview 6. Lipton A, Uzzo R, Amato RJ, Ellis GK, Hakimian B, Roodman GD et al. The science and practise of bone health in oncology: Managing bone loss and metastasis in patients with solid tumors. J Natl Compr Canc Netw. 2009 Oct; 7(Suppl 7): S1–S30 7. 2016 [cited 20 August 2016]. Available from: http://www.cancer.org/acs/groups/cid/documents/webcontent/003087-pdf.pdf 8. O’Sullivan G, L Carty F and Cronin CG. Imaging of bone metastasis: An update. World J Radiol. 2015;7(8):202 9. Miller T. Bone Tumors and Tumorlike Conditions: Analysis with Conventional Radiography. Radiology. 2008;246(3):662-674. 10. Zhu XC, Zhang JL, Ge CT, Yu YY, Wang P, Yuan TF, et al. Advances in cancer pain from bone metastasis. Drug Des Devel Ther 2015 Aug 18;9:4239-4245. 11. Goyal J, Antonarakis ES. Bone-targeting radiopharmaceuticals for the treatment of prostate cancer with bone metastases. Cancer Lett 2012 Oct 28;323(2):135-146. 12. Baron R, Ferrari S, Russell RG. Denosumab and bisphosphonates: different mechanisms of action and effects. Bone 2011 Apr 1;48(4):677-692. 81 Leukemias and lymphomas Leukemias Biljana Blagojević, University of Belgrade, Faculty of Pharmacy, Serbia Katarina Nemec, University Ljubljana, Faculty of pharmacy, Slovenia Branko Subošić, University of Belgrade, Faculty of Pharmacy, Serbia Jerka Dumić, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia INTRODUCTION Leukemias belong to a broader group of tumors that affect the blood, bone marrow, and lymphoid system, known as tumors of the hematopoietic and lymphoid tissues (1). The type of leukemia depends on the blood cell type that has become cancerous. Any of the blood- forming myeloid or lymphoid cells from bone marrow can turn into a leukemia cell (2). Fig. 1. Hematopoiesis Source: https://www.hmrn.org/about/classification Leukemia is either acute or chronic. Acute leukemia is a fast-growing cancer that usually gets worse quickly. Chronic leukemia is a slower-growing cancer that gets worse slowly over time. The treatment and prognosis for leukemia depend on the type of blood cell affected and whether the leukemia is acute or chronic. Therefore, there are four main types of leukemia — acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML) — as well as a number of less common types (3). 82 EPIDEMIOLOGY Fig. 2: Distribution of main types of leukemia by men and women and increase of incidence by age Source: https://www.hmrn.org/statistics/incidence Leukemia occurs most often in adults older than 55 years, and it is the most common cancer in children younger than 15 years. Incidence for almost all leukemias increases with age. Acute myeloid leukemia (AML) is a relatively rare cancer, the median age at diagnosis is 63 years. AML accounts for about 90% of all acute leukemias in adults, but is rare in children. Acute lymphoblastic leukemia (ALL) hits both children and adults, but its incidence peaks between ages 2 and 5 years. The median age at presentation of chronic myeloid leukemia is 45 to 55 years, although some series report a median age of up to 67 years. Chronic lymphocytic leukemia is primarily a disease of older adults, with a median age of 70 years at the time of diagnosis. The incidence of CLL increases very quickly with increasing age (3). MOLECULAR BASIS The core of the pathogenesis of leukemia, like in the other types of cancers, is genetic disorders. Genetic changes can be introduced by two distinctive mechanisms: 1. The first involves the structural alteration of a normal gene (a proto-oncogene) to generate a novel gene (an oncogene) whose protein product is involved in cellular proliferation, differentiation or survival. 2. The second mechanism involves the loss or inactivation of genes, known as tumor- suppressor genes or anti-oncogenes, whose proteins suppress cancer. 83 Different types of leukemias are differentiated by the alterations in members of specific genes. In general, certain oncogenes are activated in a wide variety of cancers, whereas others are restricted to certain tissues and tumor types. The genes potentially leukemogenic in hematopoietic cells can be grouped into five families: 1. Genes that convey growth-stimulating signals from cell membrane to the nucleus; 2. Genes that activate transcription (the protein products of these genes bind to specific DNA sequences near target genes and enhance the synthesis of messenger RNA); 3. Genes involved in tissue differentiation; 4. Genes involved in programmed cell death; 5. Anti-oncogenes that may normally function to suppress tumor development (4). Myelogenous leukemias The genetic basis for myelogenous leukemias is chromosomal translocation. Approximately 90% of patients with chronic myelogenous leukemia have an acquired genetic abnormality, the Philadelphia chromosome (Ph). Fig. 3: Philadelphia chromosome Source: Abl tyrosine kinase inhibitors for overriding Bcr-Abl/T315I: from the second to third generation. The Ph is a shortened chromosome 22 resulting from a reciprocal translocation between the long arms of chromosome 9 and 22 t(9;22q34;q11). The proto-oncogene c-ABL is transposed from its normal position on chromosome 9 to M-BCR region on chromosome 22. By this translocation the new, fusion gene BCR-ABL is formed. The new gene encodes p210BCR/ABL, an oncoprotein that has increased tyrosine kinase (TK) activity and increased binding to the actin cytoskeleton compared with the p145 Abelson protein, both of which contribute to transformation. The presence of p210BCR/ABL causes growth factor independence and leukemic cell growth in hematopoietic cell lines. Increased p210BCR-ABL protein levels in advanced disease further increases the resistance of the leukemic clone to apoptosis, resulting in tolerance to the genetic errors accumulating in the malignant clone. Subsequent dominance of one or more clones finally leads to culmination in fatal blast crisis (5). 84 Specific M3 subtype of acute myelogenous leukemias is acute promyelocytic leukemia (APML, APL). In APL there is an abnormal accumulation of immature granulocytes called promyelocytes. Over 95% of cases are characterized by a balanced translocation between chromosome 17q21, which encodes the retinoic acid alpha receptor gene ( RARA) and chromosome 15q22. This leads to an abnormal fusion protein called PML-RARA. The fusion protein binds with enhanced affinity to sites on the cell's DNA, blocking transcription and differentiation of granulocytes. It does so by enhancing interaction of nuclear co-repressor (NCOR) molecule and histone deacetylase (HDAC) (6). The retinoic acid alpha receptor gene ( RARA) is mainly expressed in hematopoietic cells and has an important role in regulating gene expression. In the absence of retinoid acid, RARA is bound by nuclear corepressor factor, and this causes transcriptional repression. In the presence of retinoic acid, RARA is activated and terminal differentiation of promyelocytes occurs (7). Lymphocytic leukemias Lymphocytic leukemias have different genetic pathogenesis comparing to the myeloid leukemias. Chromosomal translocations are rare and no unifying mutations have been identified. However, mutation status of V genes is used for the categorization of lymphocytic leukemias, as well as for the prognosis of the disease. Unfavorable prognosis is associated with the expression of non- mutated immunoglobulin heavy variable genes (UM-IgH-VH) and high level of 70 kD zeta-associated protein (ZAP-70). Two different cell profiles of B lymphocytes are possible: with mutated and non- mutated V-genes. In both cases, interactions between antigens and B-cell receptors of adequate affinity induce clonal amplification. The initial inducing lesion provides the marked cell with a growth advantage over other clones stimulated by the same or other antigens. Additional DNA mutations cause the cells to cross the boundary from “normality” to “leukemia”. Continued cycling leads to other genetic changes ( e.g. , deletions at 13q, 11q, and 17p or duplication of chromosome 12) that determine the course of the disease. These changes appear to occur more frequently in patients with non-mutated CLL (8). Chromosomal alterations are detected in >80% of cases and can discriminate patients with different outcomes:  low risk, normal karyotype or 13q deletion;  intermediate risk, 11q deletion or trisomy 12 and  high risk, 17p deletion or complex karyotype. Recent studies demonstrated that microRNAs are involved in an intricate interplay with B-cell receptors (BCR) signaling and microenvironmental stimuli. BCR signaling and immunoglobulin production can be regulated by microRNAs while the expression of certain microRNAs can be altered via BCR stimulation. Researchers have focused on the molecular impact of deregulation of microRNA expression in CLL. miR-15/16 cluster, miR-34b/c, miR-29, miR-181b, miR-17/92, miR-150, and miR- 155 family members, the most deregulated microRNAs in CLL, were found to regulate important genes, helping to clarify molecular steps of disease onset/progression (9). 85 Fig. 4: Interplay between microRNA expression and chromosomal aberration in CLL Source: Balatti V, Pekarky Y, Croce CM. Role of microRNA in chronic lymphocytic leukemia onset and progression. J Hematol Oncol 2015; 8:12 DIAGNOSIS Due to many types of leukemias, diagnosis of this disease is not easy at all. Right diagnosis is the most important for the successful treatment of each type of leukemia. There are many different methods which help in diagnosing leukemia such as blood count (where we expect access of abnormal white blood cells, also sometimes leukemic blast are seen, and there is decreases in platelets and red blood cells), marrow aspiration and biopsy (enables measurement of percent of blast and basophils), cytogenetics or fluorescent in situ hybridization (FISH). FISH is a test that “maps“ the genetic material in human cells, including specific genes or parts of genes which helps us to propose a diagnosis CML (1). The most used techniques are: flow cytometry and microscopy of bone marrow or blood. Light microscopy is used for examinations of either marrow or blood. Flow cytometry, or one of its modification called fluorescence-activated cell sorting (FACS) is sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. The cell suspension enters in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter, difference in their granulation and fluorescence of the labeled antibodies bound to the specific antigenes. An advantage of this method is that various antigens can be marked by fluorescence labeled antibodies (10). 86 Fig.5: Scheme of mechanism of flow cytometry. Source: http://pubs.acs.org/subscribe/archive/mdd/ v07/i11/html/1104feature_willis.html TREATMENT Therapy of leukemias combines more types of treatment: conventional chemotherapy, immunotherapy, allogeneic stem-cell transplantation, radiation etc. Different drugs can be combined in therapy such as antracycline, glucocorticoids, vinca alkaloids, hydroxyurea and tyrosine-kinase inhibitor (imatinib). The most used ones are antimetabolites (cytarabine). Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine) or a pyrimidine, chemicals that become the building-blocks of DNA. They prevent endogenous substances to incorporate into DNA during the S phase (of the cell cycle), stopping normal development and division (11). There is a special therapy approach is for acute promyelocytic leukemia M3. Reduced affinity of the retinoic acid receptor alpha (RARα or RARA) for retinoic acid binding can be overcome with increased intake of retinoic acid, thus the retinoic acid is used as a drug. LITERATURE 1. Vardiman JW, Thiele J, Arber DA et al.The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114 (5): 937–51. 2. National Cancer Institute. What You Need To Know About Leukemia. 23 December 2013. Retrieved 18 June 2014. 3. World Cancer Report 2014. World Health Organization. 2014. pp. Chapter 5.13 4. Cline MJ. The molecular basis of leukemia. N Engl J Med. 1994;330(5):328-36 5. Shet AS, Jahagirdar BN, Verfaillie CM. Chronic myelogenous leukemia: mechanisms underlying disease progression. Leukemia 2002;16(8):1402-11 6. Coombs CC, Tavakkoli M, Tallman MS. "Acute promyelocytic leukemia: where did we start, where are we now, and the future".Blood Cancer Journal. 2015;5(4): e304 87 7. Kotiah SD, Besa EC, Sarkodee-Adoo, C; Talavera, F; Sacher, RA; McKenna, R; Besa, EC, eds. "Acute Promyelocytic Leukemia". Medscape Reference. WebMD. Retrieved 14 January 2014. 8. Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. New England Journal of Medicine. 2005; 352(8):804-815 9. Balatti V, Pekarky Y, Croce CM. Role of microRNA in chronic lymphocytic leukemia onset and progression. J Hematol Oncol. 2015; 8:12 10. Julius MH, Masuda T, Herzenberg LA. Demonstarion that antigen-binding cells are precursors of antibody-producing cells after purification with a fluorescence-activated cell sorter. PNAS. 1972; 69(7); 1934-1938 11. Takimoto CH, Calvo E. "Principles of Oncologic Pharmacotherapy" in Pazdur R, Wagman LD, Camphausen KA, Hoskins WJ (Eds) Cancer Management: A Multidisciplinary Approach. 11 ed. 2008. 88 Lymphomas Tina Levstek, University Ljubljana, Faculty of pharmacy, Slovenia Barbara Pem, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia Tamara Rojnik, University Ljubljana, Faculty of pharmacy, Slovenia Jerka Dumić, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia Introduction Lymphomas are a group of malignant tumors of lymphoid tissue. There are two main types of lymphomas: Hodgkin and non-Hodgkin. Hodgkin lymphoma is a less common type and is characterized by the presence of Reed-Sternberg cells. Every other type is classified as non-Hodgkin lymphoma, which is further divided into more than 60 subtypes.(1) Both Hodgkin and non-Hodgkin lymphomas can occur in children and adults, and prognosis and treatment depend on the stage and type of cancer. Signs and symptoms The primary manifestation of lymphomas is lymphadenopathy, or swollen lymph nodes. Swelling of lymph nodes is one of the differences between lymphomas and leukaemias, and it is also important to note that the lymph nodes may be painless, while pain is usually present in infections. Systemic symptoms (or B symptoms) are common for both Hodgkin and non-Hodgkin lymphoma. Those are fever, night sweats and weight loss. Other possible symptoms include tiredness, loss of appetite, itchiness of skin and cough and shortness of breath.(2) Symptoms of lymphoma are usually non- specific and resemble greatly the symptoms of other illnesses, especially viral infections. The main difference is persistence. Epidemiology International Agency for Research on Cancer has compiled the data about the incidence and mortality of lymphomas. According to their research from 2012, in Europe age standardized rate of incidence per 100,000 people for Hodgkin lymphoma (HL) is 2.3, while for non-Hodgkin (NHL) it's 9.8. European mortality rate for Hodgkin lymphoma is 0.5, and for non-Hodgkin 3.5. Out of the European countries, Croatia has the highest incidence rate of Hodgkin lymphoma (3.3 out of 100,000 people), but Greece has the highest mortality rate (1.1 out of 100,000 people). For non-Hodgkin lymphoma, the incidence rate is the highest in Finland (15.6) and the mortality rate is the highest in Malta (5.3). The incidence rate for HL is roughly the same for men and women (2.5 and 2.1 respectively), while NHL is more common in men (incidence rate of 11.9, whereas for women it's 8.0).(3) Hodgkin lymphoma affects both children and adults, but it is very rare in children under the age of 5. It is most common in two age groups: ages 15 to 40 (particularly young adults in their 20s) and after the age of 55.(4) NHL is far more common in adults than in children. It is the sixth most common cancer in both men in women. The average age of the diagnosis of NHL is 65 years.(5) Diagnosis and staging The diagnosis of lymphomas is usually made by a lymph node biopsy. Most commonly, the biopsy will be executed by removal or excision of a lymph node in the neck, under the arm or in the groin.(6) When performing a biopsy, it is important to remove a large sample of tissue, preferably an entire lymph node, to ensure that the malignant cells are captured and correctly identified. Immunophenotypization of the excised tissue is performed using a panel of antibodies. Flow cytometry is used in the assessment of cell type and lineage. In most lymphoid proliferations, morphological assessment and immunophenotyping are sufficient to establish a diagnosis. In a minority of difficult cases molecular investigation may be required. The techniques used are polymerase chain reaction (PCR), real-time quantitative PCR (RQ-PCR), conventional cytogenetics and fluorescence in situ hybridisation (FISH).(7) 89 Various scans are used as secondary diagnostic tools, such as computed tomography (CT or CAT), magnetic resonance imaging (MRI), positron emission tomography (PET) or integrated PET-CT scan.(8) Because lymphomas often spread to the bone marrow, bone marrow aspiration and biopsy may be important in diagnosis and staging. Blood tests are often performed as additional tests. They may include a complete blood count (CBC), erythrocyte sedimentation rate (ESR or "sed rate") and liver and kidney function tests. Staging helps us to identify the location of the tumor and if the disease has spread from original site to other parts of the body. Stage 1 means that lymphoma is in only one group of lymph nodes or in one body organ. Stage 2 means there is lymphoma in 2 or more groups of lymph nodes or an organ and 1 or more groups of lymph nodes. But all lymphomas must be on the same side of the diaphragm. Stage 3 means that lymphoma is on both sides of the diaphragm. Stage 4 means that many groups of lymph nodes contain lymphoma and it has spread to other body organs ( e.g. liver, kidney, lungs). If patient has B-symptoms (night sweats, fever and loss of weight), the letter B is put after the stage. If the patient does not have these symptoms, the letter A is put after the stage.(9) Staging is usually performed by PET-CT scanning. Hodgkin lymphoma HLs are clonal B cell neoplasms. We recognize two types of HL: classical HL, which is further divided into 4 subtypes based on the appearance of the cells and the composition of background cellular infiltrate, and nodular lymphocyte-predominant HL (NLPHL). It is important to emphasize that the malignant cells are rare in the involved tissue, accounting for only around 1% of the tumor mass. The malignant cells are embedded in an infiltrate of reactive hematopoietic cells, which are considered to be non-malignant. But the clinical features, cell of origin and molecular pathogenesis of NLPHL and cHL are different. Classical HL (cHL) is the most common type of Hodgkin lymphoma. It occurs in about 95 % of cases. cHL is diagnosed when characteristic abnormal lymphocytes, known as Reed-Sternberg cells, are found. Reed-Sternberg cells are giant multinucleated tumor cells with unusual immunophenotype that do not resemble any other normal cell in the body. If cell is mononucleated, they are called Hodgkin cells.(10) The most common type of cHL is nodular sclerosis HL. It affects up to 80 % of people diagnosed with cHL. In addition to Reed-Sternberg cells, there are also bands of connective tissue in the lymph nodes. About 6% of people diagnosed with cHL have lymphocyte-rich cHL. The lymph nodes contain Reed-Sternberg cells and many normal lymphocytes. Mixed cellularity HL usually develops in the abdomen and consists of many different cell types including large numbers of Reed-Sternberg cells. Only about 1% of people with cHL have lymphocyte-depleted Hodgkin lymphoma subtype. The lymph nodes contain almost all Reed-Sternberg cells. The second type of HL is nodular lymphocyte-predominant HL which is present in about 5% of patients with HL. Nodular lymphocyte-predominant Hodgkin lymphoma is more similar at the protein and genetic level to B-cell non-Hodgkin lymphoma because cells have marker CD20 on the surface of the lymphoma cells. CD20 is protein usually found in people diagnosed with B-cell NHL. The malignant cell is the lymphocyte-predominant cell or ‘popcorn’ cell, so-named because of its characteristic multilobated morphology.(11) Different from B-non-Hodgkin lymphomas, in Hodgkin lymphomas single cytogenetic abnormalities have not been found. There are many chromosomal imbalances, including recurrent gains of 2p, 9p, 16p, 17p, 17q and 22q and loss of 13q. Wealso know other minimally gained and lost regions ( e.g. genes involved in NF-κB signaling). Patients who have gains of 16p11.2-13.3, which contain the multidrug resistance gene ABBC1, have poorer disease survival. 90 Approximately one third of HL is associated with EBV, so the virus is believed to play an important role in disease pathogenesis. In EBV-associated tumors, EBV is detected in all the Reed-Sternberg cells. The proof that infection has occurred prior to the transformation is that viral infection is clonal. Reed-Sternberg cells also express EBV proteins (EBNA-1, LMP-1, LMP-2 antigens) and EBV-encoded RNAs. EBNA-1 and LMP-1 are essential for transformation of B cells by EBV. EBV gene products contribute to Reed-Sternberg cell survival, proliferation and reprogramming.(12) A lot of signaling pathways are deregulated in the Reed-Sternberg cell. The most known is the NF-κB pathway which plays essential role in Reed-Sternberg cell survival. Infection with EBV probably leads to NF-κB activation.(13) Non-Hodgkin lymphoma Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignant tumors which includes many subtypes, each with distinctive features. The WHO classification from 2008 has divided NHL into two groups based on morphology and cell lineage: those of B-cell origin and those of T-cell/natural killer (NK)–cell origin. Within those categories, lymphomas are subdivided according to the stages of differentiation.(14) NHLs may result from chromosomal translocations, infections, environmental factors, immunodeficiency states and chronic inflammation. The accumulation of mutations affecting proto- oncogens and tumor suppressor genes results in formation of immortal rapidly proliferating cells. Proto-oncogens are transformed into oncogens by chromosomal translocations, and tumor suppressors are inactivated by chromosomal deletion or mutation. In addition, certain viruses can introduce oncogenic genes into the genome of the cell. Some genetic modifications are associated with specific NHL subtypes, and are reflected in the presence of specific markers used for classification. The most common chromosomal abnormality is the t(14;18)(q32;q21) translocation, in which the bcl- 2 apoptotic inhibitor oncogene at chromosome band 18q21 is joined with heavy chain region of the immunoglobulin (Ig) locus within chromosome band 14q32. This translocation occurs in 85% of follicular lymphomas. The t(11;14)(q13;q32) translocation results in the overexpression of bcl-1 (cyclin D1/PRAD 1), a cell-cycle regulator on chromosome band 11q13, which is associated with mantle cell lymphoma. The 8q24 translocations that lead to c-myc deregulation occur in Burkitt's lymphoma and other types caused by HIV infection. Mucosa-associated lymphoid tissue (MALT) lymphomas are commonly caused by two chromosomal translocations. T[11;18][q21;q21]) translocates the apoptosis inhibitor AP12 gene with the MALT1 gene, resulting in the creation of an aberrant fusion protein. T(1;14)(p22;132) involves the translocation of the bcl-10 gene to the immunoglobulin gene enhancer region. Viruses can cause uncontrolled B- or T-cell stimulation because of their ability to induce chronic antigenic stimulation and cytokine deregulation. Epstein-Barr virus (EBV) is associated with Burkitt's lymphoma, Hodgkin disease, lymphomas in immunocompromised patients and sinonasal lymphoma. Other viruses associated with various subtypes of NHL are Human T-cell leukemia virus type 1 (HTLV- 1), Hepatitis C virus (HCV) and Kaposi sarcoma–associated herpesvirus (KSHV). The only bacteria known to cause lymphomas is Helicobacter pylori, which is associated with gastric mucosa-associated lymphoid tissue (MALT) lymphomas. Other factors in the development of NHL include exposure to chemicals ( e.g., pesticides, herbicides, solvents, organic chemicals, wood preservatives, dust and hair dye), chemotherapy, radiation, immunodeficiency states, Celiac disease and autoimmune disorders.(15) NHL can be divided into two prognostic groups according to growth rate. Slow-growing types of lymphoma are called indolent or low-grade. This group includes follicular lymphoma (the most 91 common kind), mantle cell lymphoma, marginal zone lymphomas (such as MALT lymphoma) and various others. Quickly growing NHL are referred to as aggressive or high grade. They include diffuse large B cell lymphoma (DLBCL), Burkitt's lymphomas, peripheral T cell lymphoma, lymphoblastic lymphoma etc. Over time, low grade lymphomas may change into a high grade type lymphoma.(16) Treatment Treatment of lymphoma in most cases consists of either chemotherapy, radiotherapy or the combination of the two. Oncologist (after discussion with the patient) chooses the therapy based on the stage of the disease, the patientś age, location of lymphoma, general health, other possible diseases and the blood level of lactate dehydrogenase (LDH), which increases with amount of lymphoma. In chemotherapy cytotoxic drugs are used to destroy cancer cells or prevent their proliferation. Normally several chemotherapeutic drugs are used together and the combination usually includes antimetabolites, because of their success in inhibiting proliferation of cells. Radiotherapy is a local treatment in which diseased areas are exposed to ionizing rays. Another way of treatment is immunotherapy with monoclonal antibodies. They bind to the specific antigen on the surface of lymphocytes and cause their death through immune mechanisms. Unlike chemotherapy, this type of treatment is more specific and consequently, we can avoid the side effects. For most patients with HL the treatment is very successful. Patients with early favorable HL (stage 1 or 2 and no adverse risk factors such as B symptoms, extranodal disease or bulky disease) are treated with radiation therapy alone. Radiation may also be used in the early stages of the HL type of nodular lymphocyte predominance, in order to achieve recovery. Patients with early unfavorable HL are prone to relapses and are treated with a combination of radiotherapy and chemotherapy. The most commonly used drug combinations are ABVD: doxorubicin, bleomycin, vinblastine, and dacarbazine and BEACOPP: bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone. Advanced (stage 2 and 3) HL is treated with ABVD and MOPP: mechlorethamine, vincristine, procarbazine and prednisone, but the treatment is more aggressive, higher doses are used and the dosing intervals are shorter. Stem cell transplantation may be used in cases when lymphoma relapses.(17) Treatment of non-Hodgkin lymphoma depends on the histologic type and stage. Asymptomatic patients with indolent NHL are usually not treated but continually monitored, and the treatment begins when they develop symptoms or problems associated with the disease. Radiation therapy is usually the first choice. Early stage (stage 1 and 2) indolent NHL can be treated effectively with radiation therapy alone. When radiation treatment is not preferred, patients can be treated with rituximab (an anti-CD20 monoclonal antibody), alone or in combination with standard chemotherapy. Optimal treatment of advanced stages of indolent NHL is controversial because of low cure rates with the current therapeutic options. Patients in advanced stages show a continuous rate of relapse, but they usually respond well to treatment and achieve long term remissions. The aim of the treatment is to prolong, as much as possible, periods of life which can be lived fully, without medications. Therapy options include rituximab, obinutuzumab, idelalisib, bendamustine, purine nucleoside analogs, alkylating agents, combination chemotherapy or radiolabeled monoclonal antibodies (yttrium-90–labeled ibritumomab tiuxetan). Numerous clinical trials are in progress to settle treatment issues. The approaches currently under trial are intensive therapy with chemotherapy and total-body irradiation (TBI) followed by autologous or allogenic bone marrow transplantation (BMT) or peripheral stem cell transplantation (PSCT), and the use of idiotype vaccines and radiolabeled monoclonal antibodies. Aggressive lymphomas have to be treated immediately. The aim of such treatment is recovery and it depends on the stage of the disease. Standard treatment for aggressive stage 1 and 2 NHL is R-CHOP 92 (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) with or without radiotherapy. R-CHOP is also standard for advanced stages of aggressive NHL, but other combination chemotherapy is also used. Bone marrow transplantation (BMT) is the treatment of choice for patients whose lymphoma has relapsed.(18) References: 1. http://www.lymphoma.org/site/pp.asp?c=bkLTKaOQLmK8E&b=6299689 Accessed 21.8.2016. 2. Lukemia and Lymphoma society (2013) The Lymphoma guide 12-15 3. http://eco.iarc.fr/eucan/Cancer.aspx?Cancer=37 Accessed 21.8.2016. 4. http://www.cancer.net/cancer-types/lymphoma-hodgkin/statistics Accessed 21.8.2016. 5. http://www.cancer.net/cancer-types/lymphoma-non-hodgkin/statistics Accessed 21.8.2016. 6. http://www.cancer.net/cancer-types/lymphoma-hodgkin/diagnosis Accessed 21.8.2016. 7. Australian Cancer Network Diagnosis and Management of Lymphoma Guidelines Working Party (2005) Guidelines for the Diagnosis and Management of Lymphoma. The Cancer Council Australia and Australian Cancer Network, 67-144 8. http://www.cancer.net/cancer-types/lymphoma-non-hodgkin/diagnosis Accessed: 21.8.2016. 9. http://www.cancerresearchuk.org/about-cancer/type/hodgkins-lymphoma/treatment/the- stages-of-hodgkins-lymphoma Accessed: 21.8.2016. 10. Küppers RHansmann M. The Hodgkin and Reed/Sternberg cell. The International Journal of Biochemistry & Cell Biology. 2005;37(3):511-517. 11. http://www.cancer.net/cancer-types/lymphoma-hodgkin/overview Accessed: 21.8.2016. 12. Farrell KJarrett R. The molecular pathogenesis of Hodgkin lymphoma. Histopathology. 2011;58(1):15-25. 13. Janz Mathas S. The pathogenesis of classical Hodgkin's lymphoma: what can we learn from analyses of genomic alterations in Hodgkin and Reed-Sternberg cells?. Haematologica. 2008;93(9):1292-1295. 14. Patel et al. (2015) Non-Hodgkin lymphoma guidelines, Accessed at: http://emedicine.medscape.com/article/2500022-overview#a1, 21.8.2016. 15. Patel et al. (2015) Non-Hodgkin lymphoma guidelines, Accessed at: http://emedicine.medscape.com/article/203399-overview#a4, 21.8.2016. 16. http://www.cancerresearchuk.org/about-cancer/type/non-hodgkins- lymphoma/about/types/the-most-common-types-of-non-hodgkins-lymphoma, Accessed 21.8.2016. 17. PDQ® Adult Treatment Editorial Board. PDQ Adult Hodgkin Lymphoma Treatment. Bethesda, MD: National Cancer Institute. Updated MM/DD/2016. Available at: http://www.cancer.gov/types/lymphoma/hp/adult-nhl-treatment-pdq. Accessed: 21.8.2016. [PMID: 26389492] 18. PDQ® Adult Treatment Editorial Board. PDQ Adult Non-Hodgkin Lymphoma Treatment. Bethesda, MD: National Cancer Institute. Updated MM/DD/2016. Available at: http://www.cancer.gov/types/lymphoma/hp/adult-nhl-treatment-pdq. Accessed: 21.8.2016. [PMID: 26389492] 93 Prostate cancer What you need to know about prostate cancer and current ways of its diagnostics Vintar Staša, University Ljubljana, Faculty of pharmacy, Slovenia Ðunisijević Vuk, University of Belgrade, Faculty of Pharmacy, Serbia Ličen Anja, University Ljubljana, Faculty of pharmacy, Slovenia Tilen Kranjc, University Ljubljana, Faculty of pharmacy, Slovenia INTRODUCTION The most widely diagnosed male cancer in the Western world and the second leading cause of cancer-related deaths is prostate cancer. There are many risk factors involved in its development that are both environmental and genetic. Prostate cancer is highly prevalent and clinically heterogeneous, therefore clinicians must differentiate between indolent and more aggressive forms. Risk classification system characterizes seriousness of the disease and help guide appropriate treatment recommendations. Different diagnostic approaches will be mentioned, from screening methods to various biomarkers and genomic-based tests that are currently being researched. EPIDEMIOLOGY Prostate cancer (PCa) is the fifth most common cancer worldwide, the second most common cancer among men in the United States and most common non-skin cancer diagnosed among American males, affecting one in six men over the course of their lifetime. More than 200 000 patients are diagnosed annually and it is the second leading cause of cancer-related deaths in American men. The incidence spiked in the early 1990s because of the prostate-specific antigen (PSA) screening with which they have successfully diagnosed most of the previously undetected prostate cancer patients in the population. At the same time they have become able to diagnose PCa earlier-that is at local stages and incidence of metastatic diseases has decreased [1]. Mortality rates have been declining because of earlier detection, more aggressive treatment [2] and screening overdiagnosis of preclinical prostate cancers which may never progress clinically. The major risk factors for the development of PCa are race/ethnicity and geographical location, advanced age, inherited susceptibility, hormonal factors and environmental factors such as diet. Incidence is highest in Scandinavia and North America (236.0 per 100000 men) and lowest in Asia (1.9 cases per 100000 annually) [3,4]. The highest mortality rates are found in the Caribbean and the lowest in Asia. Race is a major risk factor with respect to both incidence and mortality, and is for unknown reasons the highest in African-Americans. 90% of diagnoses occur at the age of 55 years and above (around 65 on average) with life expectancy of about 17 years. Older men are more likely to be diagnosed with high-risk PCa. Risk of PCa doubles for a male with one affected first-degree relative and is further increased with more than one affected relative [5-9]. In terms of diet, vitamin E, lycopene (a carotenoid with potent antioxidant properties found in tomato based products), selenium and soy may exert a protective effect. On the other hand, diets rich in fat and red meat, especially well-done meats, may exert a promotional effect [10-13]. Positive associations between high calcium intake and increased incidence and mortality from PCa have also been reported [14-16]. Androgens have an important role in development of PCa. The incidence is significantly decreased by inhibiting conversion of testosterone to dihydrotestosterone. Another important risk factor are higher concentrations of insulin-like growth factor-1 (IGF-1), which normally promote proliferation and apoptotic inhibition of normal prostate cells [17]. IGF-1 levels can be influenced both by diet or 94 genetics, hence the reason for higher or lower rates of PCa in certain populations and countries. BRCA1 and BRCA2 mutations increase the risk of developing PCa. BRCA1 (17q21) double the risk and BRCA2 (13q12) mutation carriers have a five to sevenfold increase in risk, an early onset of disease, a worse prognosis and a higher Gleason score [18-23]. It is very important that clinicians differentiate indolent tumours from those that are more aggressive. This way there is no overtreatment with the first and no undertreatment of the latter tumours. The current classification system was formed by the National Comprehensive Cancer Network (NCCN) which stratifies men into very-low, low-, intermediate-, high-, and very-high-risk groups, each having a different treatment approach. It is based on general tumour stage, Gleason score and PSA level. Gleason score is based upon PCa microscopic appearance and it ranges from 2 to 10, with 2 representing the most well-differentiated tumours and 10 the least-differentiated tumours. For example, men with low risk PCa comprise the majority of patients. PCa is localized to the prostate with a Gleason score ≤6, low-volume disease and serum PSA ≤10 ng/mL. Treatment options for low-risk PCa would be radical prostatectomy, external beam radiotherapy, brachytherapy and less commonly cryotherapy and high intensity focused ultrasound [24-29]. We have to determine on an individual basis whether treatment is at all appropriate. A 50-year-old man has a lifetime risk of 42% of developing PCa, but only a 9.5% risk of developing the disease clinically and being diagnosed and a 2.9% risk of dying from PCa [30]. If the treatment has not begun until the man develops clinical signs of progression, it is called watchful waiting and is sometimes used for older men with shorter life expectancies or with comorbidities, where curative treatment could pose a risk to person’s health. It is appropriate for men who have less than 15 years of life expectancy, as PCa seems to rapidly progress after 15 years. DIAGNOSTICS Current course of diagnostics Most prostate cancers are found during screening with a prostate-specific antigen (PSA) blood test, a digital rectal exam (DRE) or transrectal ultrasound (TRUS). DRE is a part of basic examination in men after 50 years of age. Doctor describes size and consistence and deviation from the normal is indication for biopsy [31,32]. PSA is secreted into the seminal fluid by luminal epithelial cells of the ducts and acini in the prostate. In small quantities it is present in the serum of men with healthy prostates, but is often elevated in the presence of prostate cancer or other prostate disorders such as prostatitis and BPH (benign prostatic hyperplasia). PSA allows finding prostate cancer when the disease is not yet tactile with DRE [33,34]. In USA FDA has approved the PSA test for annual screening of prostate cancer in men of age 50 and more. This kind of testing has resulted in earlier PCa detection, potentially in more curable stage. But there is also a negative side, because usage of PSA screening has led to overdetection, overtreatment and to an increase in the rate of negative biopsis. In Slovenia there is no national programme for screening, diagnostic approaches are used only when clinical signs are presented [38]. TRUS is a diagnostic procedure where doctor inserts a small probe in your rectum to the intestine and examines the prostate. The probe gives off sound waves that enter the prostate and create echoes. The probe picks up the echoes and a computer turns them into a black and white image of the prostate. Ultrasonicly suspicious changes are visible only in half of the patients with prostate cancer [31,32]. 95 If cancer is suspected based on results of screening tests, biopsy is needed to confirm the diagnosis. Biopsy is the removal of small pieces of the prostate for microscopic examination. They are routinely done and usually do not require hospitalization. The most common is core needle biopsy which is usually done by urologist. Most of them take about 12 core samples from different parts of the prostate. Samples are send to the lab where are examined if they contain cancer cells or not [32]. TUMOUR MARKERS It has been mentioned before that PSA – mostly used tumour marker enabled earlier PCa detection at potentionally more curable stage. Nevertheless PSA screening has led to overdetection and overtreatment, because PSA is not cancer-specific or a compensation for the behaviour of prostate cancer [38]. Companies all over the world have developed and are still developing different biomarkers and genetically-based tests for prostate cancer diagnosis, staging, prognostication and monitoring. A lot of molecular tests and algorithms have been evolved to improve pretreatment, to enhance diagnostics accuracy and to distinguish among aggressive and indolent disease to facilitate therapeutic decisions [38]. Nowadays blood and urine-based prostate cancer biomarkers help to decide if biopsy is necessary or not. This kind of tests are inexpensive and analytes can be measured by automated methods as is multi-analyte immunoassay. Such biomarkers are urine prostate cancer antigen 3 (PCA3) score, prostate health index (phi) and the four kalikrein panel (4K score) that can also be combined with other assays (Tabel 1) [38]. Numerus genomics and proteomic test are already commercially available but majority of them have not been approved by FDA. Based on the results of this tests doctors can more easily decide who to rebiopsy and to reduce the number of unnecesary biopsies. Some of this test are: confirm MDx, PCMT, Oncotype DX, Prolaris, ProMark, etc. Assays base on quantitative RT-PCR, multi-gene RT-PCR and other molecular techniques. Objects of interest are different genes, mtDNA or proteins (Table 1). Some of this tests are also useful to distinguish between aggressive and indolent tumours and to help doctors decide who to keep under surveillance and who to treat [38]. Table 1: Novel tests for prostate cancer detection Assay Marker Assay type FDA approved description PCA3 score PSA and PCA3 In vitro RNA TMA Only when mRNA assay repeat biopsyis considered phi PSA, FPSA, p2PSA Multi-analyte No immunoassay 4K score Total PSA, fPSA, Multi-analyte No intact PSA, hK2 immunoassay Confirm MDx 3 genes Quantitative No methylation specific PCR PCMT mtDNA deletions Quantitative PCR No Oncotype DX 17 genes RT-PCR No Prolaris 46 genes RNA expression No ProMark 8 proteins Immunofluorescent No imaging 96 Samples For discovery of biomarkers in prostate cancer different kind of biological sources can be used. 1. Blood Serum and plasma are the fractions that are most frequently used for routine blood testing in hospitals and clinics as they contain many proteins that are synthesized, secreted or lost from the cells and tissues throughout the body. In fact, the majority of serum or plasma proteins are made up of a few high abundant proteins such as albumin, immunoglobulins, alpha-1-antitrypsin and haptoglobins which can mask the presence of potentially significant low abundant proteins. This is the reason why several fractionations, depletions and enrichments are used to enhance detectability of low abundant proteins [33,35,36,37]. 2. Tissue In tissue and cell culture models it can be directly investigated the expression and the role of certain protein. Also it is possible to analyse single or mixed cell populations. The biggest problem with identifying protein biomarkers in tissue and cell cultures is that they cannot provide accurate insight into disease progression in vivo [33,39,40]. 3. Urine Urine is ultrafiltrate of blood and is commonly used for diagnostics and biomarker discovery. Urine has a lot of positive characteristics: it can be obtained non-invasively, it contains proteins and peptides of low molecular weight and it is also very stable body fluid. Like others also urine has some negative characteristics. It is difficult to control variability in sample collection if samples are not taken by a trained personnel, also there are other proteins besides biomarkers and urine composition is dependent on collection time, diet, exercise and stage of disease [33, 41]. LITERATURE [1] Derweesh IH, Kupelian PA, Zippe C, et al. Continuing trends in pathological stage migration in radical prostatectomy specimens. Urol Oncol 2004;22(4):300– 306 [2] Walsh PC. Cancer surveillance series: interpreting trends in prostate cancer–part I: evidence of the effects of screening in recent prostate cancer incidence, mortality, and survival rates. J Urol 2000;163(1):364–365 [3] Howlader N, Noone AM, Krapcho M, et al. (eds). 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[37] Ray, S.; Reddy, P.J.; Jain, R.; Gollapalli, K.; Moiyadi, A.; Srivastava, S. Proteomic technologies for the identification of disease biomarkers in serum: Advances and challenges ahead. Proteomics 2011,11,2139–2161. [38] Falzarano, S.M.; Ferro, M.; Bollito, E.; Klein, E.A.; Carrieri, G.; Magi-Galluzzi, C. Novel biomarkers and genomic tests in prostate cancer: A critical analysis. Minerva Urol. Nefrol. 2015, 67, 211–231. [39] Ahmad, Y.; Lamond, A.I. A perspective on proteomics in cell biology. Trends Cell Biol. 2014, 24, 257–264. [40] Lexander, H.; Hellman, U.; Palmberg, C.; Auer, G.; Hellström, M.; Franzén, B.; Jornvall, H.; Egevad, L. Evaluation of two sample preparation methods for prostate proteome analysis. Proteomics2006, 6, 3918–3925. [41] Rodríguez-Suárez, E.; Siwy, J.; Zürbig, P.; Mischak, H. Urine as a source for clinical proteome analysis: From discovery to clinical application. Biochim. Biophys. Acta 2013, 1844, 884–898. 99 Molecular basis of prostate cancer Helena Kovarikova, Institute for Clinical Biochemistry and Diagnostics, Charles University, Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove, Czech Republic Dominik Kirchhofer, Institute for Cancer Research, Medical University of Vienna, Centre for Molecular Biology, University of Vienna, Austria Tilen Kranjc, University Ljubljana, Faculty of pharmacy, Slovenia Prostate cancer (PCa) is the most frequent cancer type in males in Europe and other continents. Although, its high occurrence in male population, its mortality rate is lower than in other cancers like lung cancer. In the late 1980s a screening method for the prostate specific antigen (PSA) was introduced. As a cause of the better diagnostic screening, the incidence rate of the disease has risen. However, the mortality rates remained the same or decreased [1]. In order to find new therapeutic targets for PCa it is necessary to understand the molecular drivers of cancer progression and tumorigenesis. PCa is caused by many alterations. Not only alterations in molecular signaling pathways but also DNA rearrangements and epigenetic modifications such as microRNA (miRNA) regulation are factors. Alterations in DNA repair mechanisms are found in genes involved in DNA double strand break (DSB) repair such as ATM, as well as in genes involved in homologous recombination such as BRCA1/2 and RAD51B/C. Moreover, Androgen Deprivation Therapy (ADT) is thought to interact with Ku70 which is known to bind DSB and initiate non homologous end joining (NHEJ) [2]. Whereas most activated pathways like the mitogen activated protein kinases (MAPK), Phosphoinositide 3-kinase (PI3K) and the Wnt signaling are commonly altered in many tumor entities, the Androgen Receptor (AR) signaling plays a very prominent role especially in PCa. Therefore, AR is involved in tumorigenesis, recurrence and resistance mechanisms of malignant PCa. Characteristically, alterations in AR signaling are found on gene-, transcription- and protein levels [3]. A multitude of copy number variations are found for the AR coding regions on Xq11-12. Amplifications of AR are described in 2% of primary PCa, but not in benign prostatic hyperplasia (BPH). Interestingly, up to 23.4% of CR-PC harbor extensive copy number amplifications of AR [2]. The amplified AR gene does not only result in elevated gene expression and protein levels, but also has an important impact on protein stability and half life time. Alterations in Posttranscriptional modifications as well as the interaction with chaperones are associated with stabilization of AR molecules [2]. With respect to the AR transcription factor, phosphorylation of the mutated N-terminal domain (NTD) of AR is described as an important stabilizing factor. Especially MAPK and PI3K are thought to play a prominent role in this stabilization. Experimental data suggest that Akt signaling is the primary activator of Ser213 phosphorylation and AR activation. Mutations in PTEN are frequently found in PCa which leads Akt signaling stimulation. Therefore, double mutants are connected to worse outcome of the disease [4][5]. Furthermore, mutations in the Ligand binding domain (LBD) are frequently found in PCa. Yet, it remains unclear how these mutations can contribute to the castration resistant prostate cancer (CR-PCa) formation. Over 14 different splice variants of AR are described. Some splice variants lack the LBD and are therefore hormone independent. These ligand independent AR are thought to drive CR-PCa. Moreover, AR interacts direct with several DNA repair mechanisms like base excision repair, mismatch repair, homologous recombination and NHEJ [2]. In addition, recent data suggest that AR signalling is not only involved in CR-PCa formation but also involved in ADT resistance [4][2]. 100 Despite the signaling pathways, epigenetic factors are found to play a prominent role in PC tumor formation. miRNAs are short non-coding RNA molecules, that are involved in post-translation regulation in various types of diseases including cancer. They are key epigenetic mechanisms involved in tumorigenesis of prostate cancer [6]. Calin et al. [7] was the first one to describe the relationship between miRNAs and cancer. He discovered that genes for miR-15 and miR-16 (located chromosome 13q14) are downregulated or deleted in majority of chronic lymphocytic leukemia (CLL) patients (65%). There are several major signaling pathways targeted by miRNAs in PCa such as apoptosis, cell invasion, migration, transforming growth factor-β, (TGF-β) pathway, extracellular signal-regulated kinases (ERKs), phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR, cell cycle, and epithelial- mesenchymal transition (EMT) [8] (Figure 1). Figure 1: The scheme of key pathways involved in the molecular progression of prostate tumors and miRNAs specifically targeting molecules in those pathways [6]. Extensive research is done on miRNAs expression profile in prostate cancer in recent years. It is focused not only on deregulation of miRNAs in tumor tissue but also in bodily fluids, so the miRNAs could be used as predictive biomarkers and therapy agents (targeting molecules involved in PCa tumorigenesis pathways) (Figure 2) in the future [8]. 101 Figure 2: The scheme of key pathways involved in the molecular progression of prostate tumors and miRNAs specifically targeting molecules in those pathways [6]. Various miRNAs are upregulated in prostate cancer tissue such as miR-21, which is the most well studied oncoMir. It has been reported to be highly overexpressed in prostate cancer tissue [9], blood [10], or urine [11]. Its high overexpression was observed especially in drug resistant PCa [12]. Other miRNAs proved to be deregulated in tumor tissue an also in plasma: let-7c, let-7e, miR-103, miR- 106a, miR-130b, miR-141, miR-16, miR-200b, miR-200c, miR-20a, miR-20b, miR-221, miR-223, miR- 24, miR-26b, miR-30c, miR-346, miR-429, miR-92a and miR-93. Moreover, altered levels of several miRNAs were also detected urine samples of patients with PCa: miR-107 miR-205 miR-214 miR-375 miR-484 miR-574 [6]. A few miRNAs have been associated with AR. Some of them are affected by AR the others regulate AR mRNA or protein levels. 71 miRNAs are likely o by involved in AR regulation in prostate cancer [13]. For example, here is an androgen-response element incorporated into the promoter region of miR-21. As a result, miR-21 and AR regulates each other in a positive feedback loop. miR-21 can effect cell growth, cell migration, apoptosis and androgen insensitivity by influencing various pathways and it is upregulated by androgen [14]. Other miRNAs deregulated by androgen are miR- 141, which is acting as oncomiR [15] or miR-34a/b/c cluster. AR is downregulated by miR-185 which is involved in cell cycle deregulation [16] and Let-7 family which targets c-Myc and thus suppresses AR expression and activity of PCa cells [17]. There many molecular alteration involved in PCa such as androgen receptor signaling pathway, DNA rearrangements and epigenetic modifications such as miRNA regulation. Understanding major molecular regulators involved in prostate cancer development and progression is important in order to find new biomarkers therapeutic approaches for the disease. 102 Literature 1. Jemal, A.; Lortet-tieulent, J.; Ward, E.; Ferlay, J.; Brawley, O.; Bray, F. International Variation in Prostate Cancer Incidence and Mortality Rates. 2012, 61, 1079–1092. 2. Wadosky, K. M.; Koochekpour, S. Molecular mechanisms underlying resistance to androgen deprivation therapy in prostate cancer. 2016. 3. Spratt, D. E.; Zumsteg, Z. S.; Feng, F. Y.; Tomlins, S. A. Translational and clinical implications. 2016. 4. Bayani, J.; Zielenska, M.; Marrano, P.; Kwan Ng, Y.; Taylor, M. D.; Jay, V.; Rutka, J. T.; Squire, J. a Molecular cytogenetic analysis of medulloblastomas and supratentorial primitive neuroectodermal tumors by using conventional banding, comparative genomic hybridization, and spectral karyotyping. J. Neurosurg. 2000, 93, 437–448. 5. Ph, D.; Nelson, W. G.; Ph, D.; Marzo, A. M. De; Ph, M. D. D. Molecular Alterations in Prostate Cancer as Diagnostic, Prognostic and Therapeutic Targets. 2011, 15, 319–331. 6. Kumar, B.; Lupold, S. E. MicroRNA expression and function in prostate cancer: a review of current knowledge and opportunities for discovery. 2016, 559–567. 7. Calin, G. A.; Dumitru, C. D.; Shimizu, M.; Bichi, R.; Zupo, S.; Noch, E.; Aldler, H.; Rattan, S.; Keating, M.; Rai, K.; Rassenti, L.; Kipps, T.; Negrini, M.; Bullrich, F.; Croce, C. M. Frequent deletions and down- regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. 2002, 99, 13–18. 8. Gill, B. S.; Alex, J. M.; Kumar, S. Missing link between microRNA and prostate cancer. Tumor Biol. 2016, 5683–5704. 9. Li, T.; Li, D.; Sha, J.; Sun, P.; Huang, Y. Biochemical and Biophysical Research Communications MicroRNA-21 directly targets MARCKS and promotes apoptosis resistance and invasion in prostate cancer cells. Biochem. Biophys. Res. Commun. 2009, 383, 280–285. 10. Leidinger, P.; Hart, M.; Backes, C.; Rheinheimer, S.; Keck, B.; Wullich, B.; Keller, A.; Meese, E. Differential blood-based diagnosis between benign prostatic hyperplasia and prostate cancer: miRNA as source for biomarkers independent of PSA level, Gleason score, or TNM status. 2016. 11. Stuopelyt, K.; Dani, K.; Jankevi, F.; Jarmalait, S. ScienceDirect Detection of miRNAs in urine of prostate cancer patients. 2016, 2. 12. Fendler, A.; Stephan, C.; Yousef, G. M.; Jung, K. MicroRNAs as Regulators of Signal Transduction in Urological Tumors CONTENT: SUMMARY: 2011, 968. 13. Chunjiao, S. Critical Review Uncovering the Roles of miRNAs and Their Relationship with Androgen Receptor in Prostate Cancer. 379–386. 14. Ramberg, H.; Alshbib, A.; Berge, V.; Svindland, A.; Taskén, K. A. Regulation of PBX3 expression by androgen and Let-7d in prostate cancer. 2011, 1–10. 15. Waltering, K. K.; Porkka, K. P.; Jalava, S. E.; Urbanucci, A.; Kohonen, P. J.; Latonen, L. M.; Kallioniemi, O. P.; Jenster, G.; Visakorpi, T. Androgen Regulation of micro-RNAs in Prostate Cancer. 2013, 614, 604–614. 16. Qu, F.; Cui, X.; Hong, Y. MicroRNA-185 suppresses proliferation, invasion, migration, and tumorigenicity of human prostate cancer cells through targeting androgen receptor. 2013, 121–130. 17. Nadiminty, N.; Tummala, R.; Lou, W.; Zhu, Y.; Zhang, J.; Chen, X.; White, R. W.; Kung, H.; Evans, C. P.; Gao, A. C. MicroRNA let-7c Suppresses Androgen Receptor Expression and Activity via Regulation of Myc Expression in Prostate Cancer Cells. 2012, 287, 1527–1537. 103 Treatment of prostate cancer Vesna Mišić, University of Zagreb, Faculty of Pharmacy and Biochemistry, Croatia Aleksandra Blagojević, University of Belgrade, Faculty of Pharmacy, Serbia Tilen Kranjc, University Ljubljana, Faculty of pharmacy, Slovenia Introduction Prostate cancer is a tumor whose growth is promoted by male hormons and androgens. Despite the fact, that characterizing the molecular landscape of prostate cancer is challenging it is important of understanding the process leading to development and progression of prostate tumors. It is even more important to study genomic basis of castration resistant prostate cancer, which is resistant to androgen deprivation therapy. This facts can help us to find new ways of diagnosis and therapy for prostate cancer. Ways of treating prostate cancer:  Castration - With castration we can block androgens. There are two types of castracion – prostatectomy and chemical castration.  Antiandrogens - There are two types of antiandrogens. Antiandrogens which block androgen syntesis and antiandrogens which are connecting with androgen receptors. Some of antiandrogens: ciproterone acetate, nonsteriodic - flutamide, nilutamide, bicalutamide and enzalutamide.  Hormon therapy – is the first step metastatic prostate cancer treatment. Usually therapy is antiandrogen with castration (completed androgenic block). It gives patients oportunity to live longer but still metastatic prostate cancer is not curable in this type of therapy.. Also if patient become rezistant tothis type of therapy it develops rezistant prostate cancer (CRPC). Some of medications in that moment are abirateron and enzalutamid.  Chemotherapy - It is usually used with CPRC patients who don`t have good treatment results on hormon therapy or patinets who have metastasis. Chemotherapeutics in those cases are docetaxel, cabazitaxel, abirateron acetate, enzalutamide and zoledronat acid. When patient has bone metastazis then we use223Ra and 89Sr.  Cell and imuno therapy - Immunotherapy becomes very important part for the metastatic prostate cancer treatment. We have evidence from phase III studies of sipuleucel-T (antigen- presenting cell vaccine. That study showed increased survival in hormone-refractory prostate cancer patients [1] Patiens with CRPC with some bone metastazis and no sympthoms were in that study. We can also use Sipuleucel-T for the metastatic castration-resistant prostate cancer (mCRPC). Treatment also extends survival. „ In 2010, sipuleucel-T became the first US Food and Drug Administration (FDA)-approved immunotherapy following the demonstration of significant overall survival (OS) improvement in patients with primarily asymptomatic or minimally symptomatic mCRPC [2, 3, 4].“ Mechanisam of Sipuleucel T – imunostimulans inhibitor of prostatic acid phosphatase (PAP). PAP is expressed by prostate cells so it is target therapy. Sipuleucel-T is composed by the patients own cells. CAR (Chimeric Antigen-Receptor) - One of the new approaches in cell therapy is using CAR (Chimeric Antigen-Receptor). It can be used for treatment CRPC. It is the part of target therapy. We have first, secong and third generation of CARs. First-generation has only the CD3-ζ chain and second- generation has CD3-ζ and a domain from a costimulatory molecule. Third-generation has CD3-ζ and two costimulatory molecule domains. Ipilimumab is human anti CTLA-4 monoclonic antibody. It has been produced by DNA recombinate tehnology. Ipilimumab inhibits CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). After 104 inhibiation we have a grow number of reactive T cells. They can mobilize to the tumor and then we have direct imune attack of T cells to the tumor cells. T cells are infiltrating in prostate cancer tumor in most cases. Blocade of CTLA-4 can reduce function of regulatory T-cells and that can cause antitumor imune answer. When we reduce function of regulatory cells we are increasing ratio of intratumor T efector and regulatory T cells and that is the causing death of the tumor cell. There are aproches where we can combine ipilimumab with immune checkpoint blockade antibodies. Ipilimumab has two types of side effects. Life threatnig side effects (diarea or colitis 3. or 4. level, increased hepatic enzymes such as AST or ALT, skin rash 4. level and neuropatics 3. or 4. level) and moderate or minor side effects (diarea for few days, little increased ALT or AST, minor skin rash, minor neuropatic problems and endrochrinologic problems). In the case of major side effect patient should stop using this medication. Ipilimumab has serious major interactions with anticoagulans, antiagregatic, imunosupresive and medications for hypercholesteremia. List of the medications which are in major interactions: • ardeparine, argatrobane, bivalirudine, anisindione, apixabane, rivaroxabane, edoxabane, fondaprinux, enoxaparine, desirudine, dabigatrane, dalteparine, danaparoid, dicumarol, warfarine and lepirudine (gastrointestinal hemorhage). • heparine and tinazaparine (gastrointestinal hemorhage). • leflunomide and teriflunomide (hepatotoxic). • lomitapide and mipomersen (hepatotoxic). {5} {6} BRCA therapy Mutations in the BRCA genes, mainly in BRCA2 increase the risk of developing PC. They also can give us information about prognosis. BRCA genes are in general autosomal dominant cancer-susceptibility genes and they are tumor suppressor genes. BRCA1 have a role in repairing DNA damage, regulation of transcription, and epigenetic effect on genomic stability. BRCA2 role is repairing the DNA damage by regulating the function of other protein such as RAD51. Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme which is important in base repairing of DNA breaks. Inhibition of PARP leads to double strand breaks. Cells are not able to repair those double strand breaks and it leads to a cell death. PARP inhibitor (olaparib) is used to treat BRCA mutation carriers. {9} {10} {11} {12} Olaparib is one of the PARP inhibitors. It has major side effects (chest pain, chills, cough,ear congestion or pain, fever, head congestion, hoarseness or other voice changes, nasal congestion, pale skin,runny nose, sneezing, sore throat, troubled breathing with exertion, unusual bleeding or bruising and unusual tiredness or weakness) and minor side effects (acid in stomach, back pain, belching, blistering, crusting, irritation, itching, or reddening of the skin, blurred vision, burning, numbness, tingling, or painful sensations, cracked, dry, or scaly skin, decreased appetite, diarrhea, difficulty with moving, dry mouth, fear or nervousness, flushed, dry skin, headache, heartburn, increased hunger, increased thirst, increased urination, indigestion, lack or loss of strength, loss of bladder control, muscle pain or stiffness, nausea, pain in the joints, stomach discomfort, upset, or pain, sweating, swelling or inflammation of the mouth, trouble sleeping, unexplained weight loss, unsteadiness or awkwardness, weakness in the arms, hands, legs, or feet). {13} List of the medications which are in major interactions: • CYP 3A4 interactions: dexametasone, amprenavire, netupitant, phenobarbital, clarihromycin, amprenavir, aprepitant, itraconazole, lumacaftor, mibefradil, mitotone, modafinil, primidone, nafcilin, ketoconazole, atazanavir, boceprevir, tenofovir, crizotinib,delaviridine, flucoconazole, imatinib, etavirine, darunavir, indinavir, verapamil, diltiazem • high risk of infections – adalimumab, leflunomide, teriflunomide, golimumab {14} 105 Conclusion In the past few years there was a significant discover of new ways of treatment prostate cancer. Mostly monoclonal and polyclonal antibodies made most of the target therapy which is now the best way of treating prostate cancer. References: {1} Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363(5):411–422. {2} Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363:411–422. doi: 10.1056/NEJMoa1001294. {3} Small EJ, Schellhammer PF, Higano CS, et al. Placebo-controlled phase III trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. J Clin Oncol. 2006;24:3089–3094. doi: 10.1200/JCO.2005.04.5252. {4}Higano CS, Schellhammer PF, Small EJ, et al. Integrated data from two randomized, double-blind, placebo-controlled, phase 3 trials of active cellular immunotherapy with sipuleucel-T in advanced prostate cancer. Cancer. 2009;115:3670–3679. doi: 10.1002/cncr.24429. {5} B. S. Carter, T. H. Beaty, G. D. Steinberg, B. Childs and P. C. Walsh: Mendelian inheritance of familial prostate cancer. Proc Natl Acad Sci U S A, 89(8), 3367-71 (1992) {6} R. A. Eeles: Genetic predisposition to prostate cancer. Prostate Cancer Prostatic Dis, 2(1), 9-15 (1999) {7}. K. Gudmundsdottir and A. Ashworth: The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability. Oncogene, 25(43), 5864-74 (2006) {8}. S. J. Boulton: Cellular functions of the BRCA tumour-suppressor proteins. Biochem Soc Trans, 34(Pt 5), 633-45 (2006) {9}J. J. Park, R. A. Irvine, G. Buchanan, S. S. Koh, J. M. Park, W. D. Tilley, M. R. Stallcup, M. F. Press and G. A. Coetzee: Breast cancer susceptibility gene 1 (BRCAI) is a coactivator of the androgen receptor. Cancer Res, 60(21), 5946-9 (2000) {10} H. Schayek, K. Haugk, S. Sun, L. D. True, S. R. Plymate and H. Werner: Tumor suppressor BRCA1 is expressed in prostate cancer and controls insulin-like growth factor I receptor (IGF-IR) gene transcription in an androgen receptor-dependent manner. Clin Cancer Res, 15(5), 1558-65 (2009) {11}. J. C. Francis, A. McCarthy, M. K. Thomsen, A. Ashworth and A. Swain: Brca2 and Trp53 Deficiency Cooperate in the Progression of Mouse Prostate Tumourigenesis. Plos Genetics, 6(6) (2010) {12} L. Moro, A. A. Arbini, J. L. Yao, P. A. Di Sant'Agnese, E. Marra and M. Greco: Loss of BRCA2 promotes prostate cancer cell invasion through up-regulation of matrix metalloproteinase-9. Cancer Science, 99(3), 553-563 (2008) {13} Dhawan M, Ryan CJ, Ashworth A. Oncologist. 2016 Aug;21(8):940-5. doi: 10.1634/theoncologist.2016-0135. Epub 2016 Jun 17. Review. {14} EMEA. European Medicines Agency "EPARs. European Union Public Assessment Reports. Available from: http://www.ema.europa.eu/ema/index.jsp?curl=pages/includes/medicines/medicines_landingpage.j sp&mid." 106 Summer school lecturers and participants Name e-mail address Country Abaz Emina emina.abaz@gmail.com Bosnia and Herzegovina Alilović Adna adna.alilovic@gmail.com Bosnia and Herzegovina Antonić Tamara antonic.tamara93@gmail.com Bosnia and Herzegovina Aranđelović Jovana jovana.arandjelovicc@gmail.com Serbia Banićević Marija marijabanicevic@hotmail.com Serbia Bebek Nives nives.bebek@gmail.com Croatia Belić Milica mimamima89@yahoo.com Serbia Blagojević Biljana blagojevicbiljana@yahoo.com Serbia Bogavac-Stanojevic, Nataša naca@pharmacy.bg.ac.rs Serbia Bogojević Aleksandra aleksandra.pharmacy@yahoo.com Serbia Dejanović Miloš milosbioh@gmail.com Bosnia and Herzegovina Draškovič Tina tina.draskovic@gmail.com Slovenia Dumić Jerka jdumic@pharma.hr Croatia Dvorščak Matea dvorscak.matea1@gmail.com Croatia Džemidžić Nejra nejra.dze@gmail.com Bosnia and Herzegovina Đunisijević Vuk vucina19931906@gmail.com Serbia Gabler Lisa lisa.gabler@meduniwien.ac.at Austria Gušić Amar amar_gusic1@hotmail.com Bosnia and Herzegovina Habartova Klara habartova.klara@gmail.com Czech Republic Hadžidaova Petra petra.hadzidaova@gmail.com Slovenia Hunski Mojca mojca.hunski@hotmail.com Slovenia Janać Jelena jelena.janac@gmail.com Serbia Janković, Tamara tasha9933@yahoo.com Serbia Jeras Matjaž Matjaz.Jeras@ffa.uni-lj.si Slovenia Kirchhofer Dominik dominik.kirchhofer@meduniwien.ac.at Austria Kodrič Klemen klemen.kodric@ffa.uni-lj.si Slovenia Komšić Karla karlakomsic@gmail.com Croatia Košuta Tina kosuta.tin@gmail.com Slovenia Kotur-Stevuljevic Jelena jkotur@pharmacy.bg.ac.rs Serbia Kovarikova Helena kovarikovahe@lfhk.cuni.cz Czech Repubic Kozjek Eva ekozjek@gmail.com Slovenia Kranjc Tilen tilen.kranjc@ffa.uni-lj.si Slovenia Levicnik Neza neza.levicnik@gmail.com Slovenia Levstek Tina levstekt@gmail.com Slovenia Ličen Anja anja.licen94@gmail.com Slovenia Marc Janja janja.marc@ffa.uni-lj.si Slovenia Milanović Angela angelamilanovic95@gmail.com Croatia Mišić Vesna vesna.misic1988@gmail.com Croatia Mrhar Aleš Ales.Mrhar@ffa.uni-lj.si Slovenia Müller Ema ema.muller210@gmail.com Slovenia Nekvindova Jana nekvindova.j@seznam.cz Czech Republic 107 Name e-mail address Country Nemec Katarina nemec.katarina@gmail.com Slovenia Novak Veronika veronika.novak1@gmail.com Slovenia Osredkar Joško josko.osredkar@kclj.si Slovenia Pem Barbara pembarbara1@gmail.com Croatia Peternel Aleks alex.peternel@gmail.com Slovenia Peterova Eva PETEROVE@lfhk.cuni.cz Czech Republic Planina Medin Luka lukapmedin@gmail.com Slovenia Pozek Kity kity.pozek@gmail.com Slovenia Prnjavorac Besim pbesim@bih.net.ba Bosnia and Herzegovina Prodan Žitnik Irena irena.prodan-zitnik@ffa.uni-lj.si Slovenia Radovanović Jovana jovana_radovanovic@yahoo.com Serbia Rojnik Tamara tamararojnik@hotmail.com Slovenia Sopić Miron miron@pharmacy.bg.ac.rs Serbia Subošić Branko dellabranko1993@gmail.com Serbia Tomažin Martina martina.tomazin@gmail.com Slovenia Vintar Stasa stasa.vintar@gmail.com Slovenia Zupan Janja Janja.Zupan@ffa.uni-lj.si Slovenia Zupančič Meta meta.zup@gmail.com Slovenia 108 SUPPLEMENT – Lectures handouts 109 Cancer Molecular mechanisms in • a complex genetic disease • it develops as a multi‐step process during which accumulation of carcinogenesis nonlethal mutations results in malignant transformation of the cells • each cancer is characterized by distinctive set of mutations and consequently it is phenotypically specific and consequently, it should be considered as a unique disease Jerka Dumić • genetic/phenotypic specificity of each cancer seeks for personalized University of Zagreb Faculty of Pharmacy and Biochemistry approach in treatment and therapy Department of Biochemistry and Molecular Biology Cancer Cancer • the consequence of breakage of the regulatory mechanisms that role • any cell type can be malignantly transformed cell behaviour • tumour – every abnormal proliferation of the cells • cell proliferation, differentiation and survival are precisely regulated • benign tumour vs. malignant tumours (cancers) • in cancer cells regulation is broken and they grow, divide and spread around uncontrolledly and disturb normal functions of healthy cells • benign tumour – remains limited on the location where it developed, no ability to metastasize • disturbance of the level/activity of proteins that play crucial roles in cell signalling, cell cycle regulation and control of programed cell • malignant tumour – it may spread into surrounding tissues or distant death (apoptosis) leads to the uncontrolled proliferation, main parts of the body using lymphatic or blood circulation characteristic of cancer cells Cancer occurence and mortality Cancer classification • any cell type can be malignantly transformed • tumour – every abnormal proliferation of the cells • benign tumour vs. malignant tumours (cancers) • benign tumour – remains limited on the location where it developed, no ability to metastasize • malignant tumour – it may spread into surrounding tissues or distant parts of the body using lymphatic or blood circulation • malignant tumours: CARCINOMAS – malignant diseases of epithelial cells (90%) SARCOMAS – malignant diseases of connective tissues (3%) LEUKAEMIAS & LYPHOMAS – malignant diseases of haematopoietic or immune cells (7%) • usually classified according the tissue (colon cancer) or the cell type (fibrosarcoma) The incidence of cancer increases with age Cancer – a multi‐step process • multi‐step process that includes mutation and selection of the • carcinogenesis is complex, multi‐step cells with pronounced ability to proliferate, survive, spread process that leads to transformation of and metastasize normal cells which acquire • TUMOUR INITIATION – result of the genetic change that leads physiological characteristics that illion) to the proliferation of one cell together determine malignant m phenotype (in • proliferation leads to the excessive growth of monoclonal s population of tumour cells • accumulation of mutations may last for ath years, thus the tumour incidence de • TUMOUR PROGRESSION – accumulation of additional of increases with age and the prevalence mutations in a population of tumour cells er of tumours is much higher in bm • selective advantage of the cells with greater potential for population over 60 years of age uN proliferation ‐ its descendants eventually become the dominant population in the tumour = clonal selection • clonal selection is ever‐present ‐ all tumours grow faster and Age (years) become more malignant Carcinogenic agents • mutations are the consequence of the action of environmental agents (e .g. chemicals, radiation, or biological agents) or may be inherited in the germ line • in some cases, mutations may be spontaneous and stochastic • three main classes of carcinogenic agents: • chemicals • radiation • microbial agents (mainly viruses) • while chemicals and radiation are well documented causes of cancers in humans, oncogenic viruses are involved in development of at least some human tumours Carcinogenic agents Chemical carcinogens • mutations are the consequence of the action of environmental agents (e .g. chemicals, • most chemical carcinogens are mutagenic and considered to be initiators of radiation, or biological agents) or may be inherited in the germ line carcinogenesis • in some cases, mutations may be spontaneous and stochastic • contain highly reactive groups that interact with DNA (as well as proteins and RNA) • three main classes of carcinogenic agents: • important targets of chemical carcinogens are oncogenes and tumour suppressors (such • chemicals as Ras and p53) ( e.g. aflatoxin B1 causes characteristic mutations in the p53 gene) • radiation • promotors ( e.g. , hormones, phenols, and drugs), which by themselves are non‐ tumorigenic (mutagenic), may induce cell proliferation and thus augment • microbial agents (mainly viruses) carcinogenicity of some chemical carcinogens • while chemicals and radiation are well documented causes of cancers in humans, • oncogenic viruses are involved in development of at least some human tumours it seems that an initiator may cause the mutational activation of an oncogene while subsequent application of promoters leads to clonal expansion of initiated (mutated) cells Chemical carcinogens Radiation (continued) • radiation (UV rays of sunlight, x‐rays, nuclear fission, radionuclides) causes • there are two types of chemical carcinogens chromosome breakage, translocations, and, less frequently, point mutations – direct‐acting carcinogens which require no metabolic conversion to become • biologically, DNA breaks seem to be the most important form of DNA damage caused carcinogenic. They are in general weak carcinogens but are important because some by radiation of them are cancer chemotherapeutic drugs ( e.g. , alkylating agents) • UV light has the ability to damage DNA by forming pyrimidine dimers, which can be – indirect‐acting agents ‐ chemicals that require metabolic conversion to an ultimate repaired by the nucleotide excision repair pathway carcinogen • in case of extensive UV light exposure, due to – polycyclic hydrocarbons e.g. benzopyrene, formed in the high‐temperature combustion of tobacco in overwhelmed repair systems, it can cause cigarette smoking or during the process of broiling meats skin cancer – aromatic amines and azo dyes e.g. β‐naphthylamine – aflatoxin B1 produced by some strains of Aspergillus, a mold that grows on improperly stored grains and nuts – vinyl chloride, arsenic, nickel, chromium, and insecticides are potential carcinogens in the workplace and the house – nitrites ‐ used as food preservatives. Microbial agents Characteritics of cancer cells • several viruses have been linked with human cancer • Autocrine stimulation of growth and/or self‐sufficiency in growth signals • the viral agents causing cancer in eukaryotic cells by integrating in host genom • Insensitivity to growth‐inhibitory signals – oncogenic RNA viruses e.g. human T‐cell leukaemia virus‐1 (HTLV‐1), associated with • Evasion of apoptosis a form of T‐cell leukaemia/lymphoma • Limitless replicative potential and disturbed cell differentiation – oncogenic DNA viruses (human papillomavirus (HPV), Epstein‐Barr virus (EBV), Kaposi • Sustained angiogenesis sarcoma herpesvirus (human herpesvirus 8, HHV8), hepatitis B virus (HBV) • Defects in DNA repair • the precise molecular mechanisms underlying the carcinogenetic effects of different • Ability to invade and metastasize virus type are complex and still under nvestigation – Helicobacter pylori ‐ the first bacterium classified as a carcinogen (increased epithelial cell proliferation in a background of chronic inflammation) Autocrine stimulation of growth and/or self‐sufficiency in Insensitivity to growth‐inhibitory signals growth signals • Tumours have the capacity to proliferate without external stimuli, usually as a • Tumours may not respond to the molecules that inhibits normal cells growth such as consequence of oncogene activation or they can produce all needed growth factors, by transforming growth factor β (TGF‐β) and direct inhibitors of cyclin‐dependent them self. kinases (CDKIs). Limitless replicative potential and disturbed cell Evasion of apoptosis differentiation • Tumours may avoid programmed cell death, by inactivation • tumour cells have unrestricted proliferative capacity, avoiding cellular senescence and of tumour suppressor p53 or activation of anti‐apoptotic mitotic catastrophe genes. • in some cases, they lose ability to differentiate, so undifferentiated cells uncontrolledly proliferate • most terminally differentiated cell do not devide • cancer cells remain arrested in the early stage of differentiation (leukemias) Sustained angiogenesis Ability to invade and metastasize • Spreading into environment is essential for tumour growth, so tumour cells produce • Tumour cells, like normal cells, need nutrients and oxygen for their growth, thus they high quantity of proteases. Due to a loss of contact inhibition and reduced have to stimulate angiogenesis adhesiveness tumour cells can easily detach from tumour mass and enter into the lymph or blood circulation, thus spreading around the body. • most cancer cells is not sensitive to inhibition dependent on the cell density (many cancer cells have a reduced need for growth factors) • loss of contact inhibition ‐ contact with neighbour cells inhibits movement and proliferation of normal cells, but not tumour cells • cell interactions and interactions between cells and the extracellular matrix in cancer cells are reduced Defects in DNA repair • Tumours may fail to repair DNA damage caused by carcinogens or incurred during unregulated cellular proliferation, leading to genomic instability and mutations in proto‐ oncogenes and tumour suppressor genes. Cancer – a concequence of demage of good guys • Four classes of normal regulatory genes are the principal targets of genetic damage • proto‐oncogenes • tumour suppressor genes • genes that regulate programmed cell death (apoptosis) • DNA repair genes • while mutant alleles of proto‐oncogenes are considered dominant, both alleles of the tumour suppressor genes must be damaged before transformation can occur • in some cases loss of a single allele of a tumour suppressor gene reduces level or activity of the protein enough that the brakes on cell proliferation and survival are released • genes that regulate apoptosis may behave as proto‐oncogenes or tumour suppressor genes • mutations of DNA repair genes do not directly transform cells by affecting proliferation or apoptosis • instead, DNA‐repair genes affect cell proliferation or survival indirectly by influencing the ability of the organism to repair nonlethal damage in other genes, including proto‐oncogenes, tumour suppressor genes, and genes that regulate apoptosis Proto‐oncogenes and oncogenes How good guys become bad guys? • oncogenes are created by mutations in proto‐oncogenes • growth factors can be mutated in a way that they act as oncoproteins • characterized by the ability to promote cell growth in the absence of normal growth‐ • PDGF‐β chain promoting signals • fibroblast growth factors, • their products, oncoproteins, resemble the normal products of proto‐oncogenes • TGF‐α except that oncoproteins are often devoid of important internal regulatory elements, • growth factor receptors, as well and their production in the transformed cells does not depend on growth factors or • EGF‐receptor family members other external signals • PDGF receptor • in this way cell growth becomes autonomous, freed from • receptor for neurotrophic factors checkpoints and dependence upon external signals ras oncogenes • ras protoonkogene is present in normal cells, whereas ras onkogene is not • the reason of transformation from ras protoonkogene to ras onkogene are mutations occurring during tumour development • the difference between ras oncogene and ras proto-oncogene is a mutation of individual amino acids at key locations • mutation glycine  valine at the position 12, or some other • mutated Ras is constitutively active (binds GTP) since GAP (protein that activates GTPase) has not effect on mutated Ras c‐myc oncogenes abl oncogenes • Many cancer cells have disturbed structure of chromosomes (translocations, • translocation of protooncogene abl from chromosome 9 to 22 is associated with duplications, deletions) chronic myeloid leukaemia (CML) • gene re-arrangements caused by chromosomal translocations often lead to the • fusion of abl with bcr gene  the fusion protein Bcr/Abl in which the N-terminus formation of oncogenes of normal protoonkogenic protein Abl is replaced by N-sequence of the Bcr  • c-myc in human Burkitt lymphoma and murine myeloma dysregulation of the Abl tyrosine kinase activity and cell transformation • chromosomal translocations that affect loci of immunogloubuline genes • translocation of the part of chromosome 8 on one of the loci of immunoglobulines (chromosomes 2, 14 or 22) • at the breakage point (chromosome 8) - protoonkogene c-myc Tumour suppressor genes Rb – tumour suppressor protein • inhibit cell proliferation and tumour formation • retinoblastoma - a rare eye cancer in children; treatment is available • in many tumours, tumour-suppressor genes are missing or have been inactive  • retinoblastoma occur in 50% of the offspring of diseased  for tendency for negative regulation of cell proliferation does not function the formation of tumours is responsible one gene that is inherited in an autosomal dominant way • Rb gene - a negative regulator of tumorigenesis • deletions of chromosome 13q14 • lack of Rb gene is responsible for the occurrence of cancer of the bladder, breast, lung ... • Rb protein is a key target of oncogenic proteins of some DNA viruses (SV40, adenovirus, human papilloma virus, herpes virus ...) • Rb binds to the transcription factor E2F and prevents protein synthesis required for cell division International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 Metabolism in cancer cells Biochemical and inflammatory aspects of tumorigenesis • Cancer cells - abnormal metabolism • Oncogenic mutations in metabolic enzymes Jelena Kotur-Stevuljević Faculty of Pharmacy – University of Belgrade, • Existing and emerging therapies aim to target Serbia this abnormal metabolism in various ways Department for Medical Biochemistry International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 What is normal cell metabolism? • Intracellular chemical reactions • ATP – which two methabolic pathways? • Oxygen level influences metabolic destiny of different substrates International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 How is cancer-cell metabolism Do any normal cells share these different? properties? • Quantity of nutrients • Proliferation phase • Nutritients quantity dependent proliferation • Glucose metabolism • Ability to change proliferation status depends on tumor supressors proteins: p53 and LKB1 • Cancer cells don’t have tumour-suppressor • Glutamin use proteins – “addiction” to glucose and/or glutamine consumption. International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 How do these abnormalities occur? How do cancer cells benefit from their abnormal metabolism? • Suply of building blocks for biomolecules synthesis – • PI3K-AKT signalling pathway NADPH and acetyl-CoA • Tyrosin-kinases receptor mutation • Stealing nutrients from neighboring cells • Excessive nutrients uptake • ROS generation – promotion of cancer cells proliferation by inactivating growth-inhibitory • Role of HIF and MYC phosphatase enzymes • Role of p53 • DNA damage International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 As anaerobic glycolysis is inefficient, how Can cancer cells use fat as a fuel? do cancer cells maintain ATP levels? • Metabolising more glucose • Intensive lipid synthesis • Need for NADPH, acetyl-CoA, ribose and nonessential amino acids. • Phospholipids – why? • Uncoupling ATP production from mitochondrial electron • Prostaglandins – why? transport • FA synthesis • Using NADH without ATP production i.e. “diversion of glycolytic intermediates into non-ATP-generating bypass pathways” International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 What is the effect of changing Why is glutamine crucial for oxygen levels? cancer cells? • Tumor blood supply – hypoxia • Glutamine is essential for cell growth • HIF-200 genes • Transport of reduced nitrogen • PHD enzymes • Synthesis of nucleotides and AA • AKT activation • Essential AA uptake • ROS, TCA metabolities • TCA cycles roles • MYC role International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 How does the Warburg effect come And how does it relate to the in? Pasteur effect? • Reciprocal relationship between anaerobic • Cancer cells continue to perform glycolysis and glycolysis and oxidative phosphorylation lactate production even when oxygen levels are • The Warburg effect is a loss of the Pasteur abundant effect • Warburg effect is proofed by imaging studies • HIF and oxygen • Mutations at MYC and p53 International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 How are cancer-associated metabolic Are metabolic enzymes frequently changes detected? mutated in cancer? • Mass spectrometry • NO • Magnetic resonance imaging • Metabolic pathway vs. single enzyme • Positron emission tomography (PET) -18F- fluorodeoxyglucose International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 Could targeting cancer-associated Do any anticancer agents exploit the glycolysis be used therapeutically? metabolic addictions of cancer cells? • In vitro vs. clinical studies • YES • Inhibition of the HK enzyme (2-deoxyglucose, • Aminopterin – dihydrofolate reductase – Ionidamin) – toxic, ineffective nucleic acid synthesis • PK inhibitors • Activation of this enzyme in anaerobic • Problem: red blood cells, brain glycolisis conditions • HIF inhibitors • Asparaginase – L-asparagine International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 What about other potential What are the latest advances? therapies? • LDH inhibition • Glutaminase inhibitors • FA synthase inhibitor Fumarat-hydratase Ubiquitin ligase complex - pVHL – Succinate-dehydrogenase tumour suppressing protein International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 Disorders Sharing Oxidative Stress and Cancer Proneness OXIDATIVE STRESS, INFLAMMATION •Fanconi anaemia # AND CANCER •Xeroderma pigmentosum •Ataxia telangiectasia •Bloom syndrome •Down syndrome •Cystic fibrosis #Petrovic SZ, Leskovac AR, Kotur-Stevuljevic J, Joksic J et al. Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes BIOL CHEM 2011; 392 (7): 625-632. International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 Chronic Inflammation is Associated Oxidative stress and cancer with Malignancy CANCER INFLAMMATORY CONDITION • Lymphoma HIV, Epstein-Barr and Herpes 8 virus, host vs. graft disease • Colon Ulcerative colitis • Lung Asthma, chronic bronchitis, emphysema • Ovarian Ovarian epithelial inflammation • Bladder Eosinophilic cystitis, schistosomiasis • Pancreatic Pancreatitis • Esophago-gastric junction carcinoma Barret’s esophagus • Gastric Heliobacter pylori infection • Liver Sarcoidosis, hepatitis B virus • Cervical Human papilloma virus • Mesothelioma Asbestos fiber exposure International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 Activators and inhibitors of reactive Transcription factors that are oxygen species modulated by reactive oxygen species International CEEPUS Summer School 2016 International CEEPUS Summer School 2016 Model of a balance between Oxidative stres and anti-cancer pro-oxidants and anti-oxidants therapy • Anticancer agents Doxorubicin Daunorubicin Mitomycin C Etoposid Cisplatin Arsenic trioxide Ionizing radiation Photodynamic therapy Incidence Cancer is a leading cause of disease world wide. An estimated 14.1 million new cancer cases occured in 2012. An estimated 7.4 million males were diagnosed with cancer worldwide in 2012. Lung cancer was TUMOR MARKERS IN the most common, accounting for almost a fifth (17%) of all cases diagnosed. Prostate cancer was CLINICAL PRACTICE the second most common cancer diagnosed in males worldwide (15%). Prof.Joško Osredkar, PhD Prostate cancer is the most common cancer in Europe for males, and the third most common University Medical Centre Ljubljana cancer overall, with around 417,000 new cases Clinical Institute of Clinical Chemistry and Biochemistry diagnosed in 2012 (3% of male cases and 12% of the total). In Europe (2012), the highest World age-standardised incidence rates for prostate cancer are in Norway; the lowest are in Albania. 2 Mortality Cancer is a leading couse of deaths worldwide, with 8,2 million deaths in 2012. More than half of all cancer death each year are due to lung, stomach, liver, colorectal and female breast cancers. In 2012, there were an estimated 8.2 million deaths from cancer in the world: 4.7 million (57%) in males and 3.5 million (43%) in females, giving a male:female ratio of 10:8. World cancer trends Projections to 2030 The World age-standardised (AS) mortality Approximately 44% of cancer cases and 53% If recent trends in major cancers are rate shows that there are 126 cancer deaths of cancer deaths occur in countries at a low seen globally in the future, the for every 100,000 men in the world, and 83 or medium level of the Human Development burden of cancer will increase to for every 100,000 females. Index (HDI) 23.6 million new cases each year by 2030. This represents an increase of 68% compared with 2012 [3] 3 4 The World AS mortality rates in males vary more than two-fold across the different world regions, ranging from 173 per 100,000 in Central and Eastern Europe to 68 per 100,000 in Western Africa (2012). In females, rates vary more than three-fold, ranging Prevalence from 119 per 100,000 in Melanesia to 65 per 100,000 in South-Central Asia (2012). 32.5 million people diagnosed with cancer within the five years previously were alive at the end of 2012. most were women after their breast cancer diagnosis (6.3 million), men after their prostate cancer diagnosis (3.9 million), and men and women after their colorectal cancer diagnosis (3.5 million). 5 6 The Five Most Commonly Diagnosed Cancers in Males Average Percentages and Numbers of New Cases, by Age, UK, 2011-2013 Prostate cancer (PCa) is the most common cancer in elderly males (> 70 years of age) in Europe. It is a major health concern, especially in developed countries with their greater proportion of elderly men in the general population. The incidence is Mortality rates also vary by Human Development Index (HDI) value. In males, highest in Northern and Western Europe (> 200 per 100,000), while rates in Eastern mortality rates are 51% higher in very high HDI countries (132 cases per 100,000) and Southern Europe have showed a continuous increase. There is still a survival compared to low HDI countries (low/medium HDI) (87 cases per 100,000) (2012). difference between men diagnosed in Eastern Europe and those in the rest of Europe. In females, rates vary only slightly between very high HDI countries (85 cases per 100,000) and low HDI countries (87 cases per 100,000) (2012). 7 Prostate Cancer (C61): 1979-2012 European Age-Standardised Incidence Rates per 100,000 Population, Males, Great Britain DIAGNOSTICS IN ONCOLOGY RADIOLOGY European AS incidence rates increasing more than eight-fold between 1979-1981 and 2010-2012. The rapid increase during the early 1990s was most pronounced for the 50-59 and 60-69 age groups, with increases of 76% and 58%, respectively, between 1989-1991 and 1994-1996. Rates have continued to rise for the 50-59, 60-69 and 70-79 age groups, but for men aged 80+, rates have decreased since the early 2000s, dropping by 21% between 2002-2004 and 2010-2012. DIAGNOSTICS IN ONCOLOGY DIAGNOSTCS IN ONCOLOGY RADIOLOGY RADIOLOGY PATHOLOGY PATHOLOGY LABORATORY MEDICINE WE ARE STILL FAR FROM WHAT WE REALITY OF BIOMARKERS IN ONCOLOGY WOULD WISH... Specificity 100% Sensitivity 100% HEALTHY CANCER HEALTHY CANCER REALITY OF BIOMARKERS IN ONCOLOGY REALITY OF BIOMARKERS IN ONCOLOGY Sensitivity Specificity Higher Higher BENIGN ILLNESS BENIGN HEALTHY CANCER HEALTHY CANCER Cut-off MATEMATICAL AND STATISTICAL BASES USEFULNESS OF THE TEST Magic of big samples Extensive shift of results Not so extensive shift of results Median Normal distribution of results WHAT IS THE RESULT SENSITIVITY True positive a Sensitivity = = All cases a + c Cancer Healthy True FalsE positive positive a + b Positive a b c d False True c + d negative negative Negative a + c b + d Sensitivity is a proportion of THOSE, WHO REALY HAVE CANCER (TP + FN) and the results are in TP SPECIFICITY POSITIVE PREDICTIVE VALUE Specificity True negative d = = True positive a All healthy b + d PPV = = All positive a + b Cancer Healthy Cancer Healthy True False True False positive positive a + b Positive positive positive a + b Positive a b a b c d c d False True c + d False True c + d negative negative Negative negative negative Negative a + c b + d a + c b + d Specificity is the proportion of THOSE, WHO ARE Positive predictive value is the proportion of THOSE, HEALTHY (TN + FP) and the results are in TN TESTED POSITIVE (TP + FP) and realy have cancer - TP PROBLEMS NEGATIVE PREDICTIVE VALUE NPV True negative d = = • Sensitivity and specificity are test All negative c + d dependent • Predictive values are dependent of the Cancer Healthy prevalence of the disease True False positive positive a + b Positive a b c d False True c + d Population values negative negative Negative a + c b + d Negative predictive value is a proportion of THOSE, TESTED NEGATIVE (TN + FN) and are healthy - TN Our data HOW TO CHOOSE A CERTAIN TUMOR MARKERS - USE TUMOR MARKER? Tumor markers are used to help detect, diagnose, and Basic research manage some types of cancer. Although an elevated level of a • Diagnosis of illness - tumor marker may suggest the presence of cancer, this alone Base evidence medicine • Prognosis - is not enough to diagnose • Staging - cancer. Therefore, measurements of tumor • Monitoring of therapy - markers are usually combined with other tests, such as • Early detection of recurrence - biopsies, to diagnose cancer. Changes after treatment Clinical praxis • Screening - Tumor marker levels may be measured before treatment to help doctors plan the • Predictive factor - appropriate therapy. In some types of cancer, the level of a tumor marker reflects the stage (extent) of the disease and/or Changes in the treatment the patient’s prognosis (likely outcome or course of disease). TUMOR MARKERS - DETERMINATION TUMOR MARKER - IDEAL • Concentration is dependent of: • Present only in tumor cells; – Number of cells where TM is synthesized, stadium; • Specific for organ and tumor; – Speed of synthesis; • Determined in biological samples all patients with the – Rate of release from the tumor cell; same type of tumor; – Activity of TM; • Present in sufficient concentration; – Type of tumor; • Detected in the early stage; – Blood supply of the tumor; – Degree of necrosis of tumor tissue; • Serum concentrations dependent of tumor growth or – Half time of TM; mass of the tumor; – Role of antibodies. • Serum concentrations as a prognostic factor of the disease. TUMOR MARKERS - WEAKNESSES TUMOR MARKERS - WHEN? • Unsufficient specificity; • Before surgery, before the begining of the treatment (radiation, chemotherapy, hormone or biological therapy); • Synthesis of high concentrations in non cancer • After surgery, during the treatment and after the treatment. illnesses (inflamation, benign tumors); Once in a 3 to 6 months in the first two years, and later • Synthesis in different physiological states once a year or at regular check ups; (pregnancy, menstriation, lactation); • In case of suspected recurrence or progression of the • Synthesis in healthy tissues. disease; • Before the change in the treatment; • 2-3 weeks after elevated values of TM. CHANGE IN THE CONCENTRATION OF TM THROUGH THE TREATMENT MEASURING OF TUMOR MASS Tumor marker (tumor not present) Clinicaly detected tumor Initial high Chemotherapy n Remission, o succesful treatment Tumor growth ati Fall, response to tr treatment cenno Low in remission C Relaps, elevation Tumor Mass Months 1µg 1mg 1g 10-100g 1kg Number of cells Tumor markers may also be measured periodically during cancer therapy. A 10³ 106 109 1010-11 1012 decrease in the level of a tumor marker or a return to the marker’s normal level may indicate that the cancer is responding to treatment, whereas no change or SCREENING an increase may indicate that the cancer is not responding. “DIAGNOSTIC WINDOW“ TUMOR MARKERS - DIVISION ”not detected” ”early” diagnose ”late” diagnose Months to years • Upon chemical structure 1 mg • Place of the synthesis 103 1 mg 10-100 g 106 1010-1011 1 g • Type of tumor disease, etc. 1 kg tumor mass 109 1012 tumor cells • The most common is biochemically, place of Immunological methods origin and functionality. Biophisycal methods TUMOR MARKERS TUMOR MARKERS – Epitelial markers • Cytokeratins • Epitelial membrane antigenes (EMA) – Tumor antigens • Desmoplacyn • Carcinom antigen 15-3 – CA 15-3 • Onkofekal proteins • Mucin carcinom antigen – MCA – Carcinoembrional antigen – CEA • Carcinom antigen 125 – CA 125 – Alpha-fetoprotein - AFP • Carcinomi antigen 19-9 – CA 19-9 – Mezenhimal markers • Prostate specific antigen - PSA • Muscle antigens – Special serum proteins – Desmin • Ferritin – Actin • Tyroglobulin – Myoglobin – Myozin • Beta-2-mikroglobulin • Vascular antigens • Glicoprotein – CD 34 • Immunoglobilins – CD 31 – Mixed markers • Neuronal antigens – S 100 • Tissue polypeptid antigen – TPA – Neuron specific enolase (NSE) • Spermin – Glialni fibrilar acid protein (GFAP) • Spermidin – Synaptophisyn • Putrescin – Receptor for neuron growth factor TUMOR MARKERS – Prognostic markers SCREENING • Adhesion molecules – Kadherins – Integrins – Selektins • Proliferative markers PRIMARY DIAGNOSIS : DIFFERENCIAL – PCNA – Ki67 DIAGNOSIS – AgNOR – Hormones, hormone receptors and/or carcinoplacental antigens • Chorionic gonadotropin –hCG • Estrogen receptor CONTROL OF THERAPY • Progesteron receptor – Biochemical markers • Enzymes and izoenzymes – Prostatic acid phosphatase FOLLOW UP – EARLY DETECTION OF – Alkaline phosphatase – Lactat dehydrogenase METASTASES – Gama GT – Tymidin kinase – Lyzozim CUT OFF (PSA) SCREENING 100% specificity PRIMARY DIAGNOSIS : DIFFERENCIAL HEALTHY (no false positive)…. DIAGNOSIS CANCER CONTROL OF THERAPY FOLLOW UP – EARLY DETECTION OF 0.0 10.0 100 µg/l METASTASES CUT OFF (PSA) CUT OFF (PSA) Only 70% specificity! Only 70% sensitivity (30% false positive) (30% false negative)! HEALTHY HEALTHY PROSTATE CANCER PROSTATE CANCER 0.0 10.0 100 µg/l 100% specificity 0.0 2.0 100 µg/l CUT OFF (PSA) Signs, symptoms and diagnostic procedures At 4.0 µ g/l, 95% specificity HEALTHY and 95% sensitivity PROSTATE CANCER 0.0 100 µg/l 44 Signs, symptoms and diagnostic procedures Prostate Cancer Screening Elevated prostate-specific antigen (PSA) level No PSA level guarantees the absence of prostate cancer. The risk of disease increases as the PSA level increases, from about 8% with a PSA level of 1 ng/mL to about 25% with a PSA level of 4-10 ng/mL. Abnormal digital rectal examination (DRE) findings DRE is examiner-dependent, and serial examinations over time are best Most patients diagnosed with prostate cancer have normal DRE results but abnormal PSA readings Biopsy Biopsy establishes the diagnosis False-negative results often occur, so multiple biopsies may be needed before prostate cancer is detected 45 46 Prostate Cancer Screening Prostate Cancer Screening The Prostate Intervention Versus Observation Trial (PIVOT), which randomized men DRE and PSA evaluation are the two components used in prostate cancer screening. with localized prostate cancer to watchful waiting or radical prostatectomy, found no Transrectal ultrasonography (TRUS) has been associated with a high false-positive significant difference in either all-cause or prostate cancer mortality between the two rate, making it unsuitable as a screening tool, although it has an established role in groups through at least 12 years of follow-up directing prostatic biopsies. Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial was a trial Screening for prostate cancer is a controversial topic. Screening offers the comparing high-intensity PSA screening to less-intensive screening. This trial found no opportunity to find cancers at a more curable stage. reduction in mortality with screening after 10 years Guidelines on prostate cancer screening have been issued by the following organizations: American Cancer Society (ACS) European Randomized Study of Screening for Prostate Cancer (ERSPC) trial found National Comprehensive Cancer Network (NCCN) that, over a median follow-up of 9 years, PSA-screening at an average of every 4 years US Preventive Services Task Force (USPSTF) resulted in a 20% reduction in the rate of death from prostate cancer. However, the American Urological Association (AUA) risk of overdiagnosis was high American College of Physicians (ACP) 47 48 Prostate Cancer Screening Biochemistry: from PSA to PHI Conclusions: After about 10 years, PSA-based screening results in small or no reduction in prostate cancer-specific mortality and is associated with harms related to subsequent evaluation and treatments, some of which may be unnecessary. Data Sources: MEDLINE (2002 to July 2011), the Cochrane Library Database (through the 2nd quarter of 2011) and reference lists. Study Selection: Randomized trials of PSA-based screening; randomized trials and cohort studies of prostatectomy or radiation therapy versus watchful waiting for localized prostate cancer; and large (n>1000), uncontrolled observational studies of perioperative harms. 49 50 Biochemistry: from PSA to PHI Biochemistry: from PSA to PHI PSA exists in serum in multiple forms: complexed to alpha-1-anti-chymotrypsin (PSA-ACT complex), unbound (free PSA), and enveloped by alpha-2- macroglobulin (not detected by immunoassays). Prostate-specific antigen The use of PSA as a serum marker has revolutionised PCa diagnosis. PSA is organ - but not cancer specific, therefore, it may be elevated in benign prostatic hypertrophy (BPH), prostatitis and other nonmalignant conditions. As an independent variable, PSA is a better predictor of cancer than DRE or transrectal ultrasound (TRUS). 51 52 [-2]proPSA levels were significantly higher in the Biochemistry: from PSA to PHI Biochemistry: from PSA to PHI cancer group (n=208) than in the benign group (n=173). Already 4 years PSA level (ng/ml) Risk of Pca (%) Risk of Gleason > 7 Pca (%) before diagnosis [- 2]proPSA differed 0.0 – 0.5 6.6 0.8 significantly between PCa 0.6 – 1.0 10.1 1.0 and benign prostate in all 1.1 – 2.0 17.0 2.0 measured time points, 2.1 – 3.0 23.9 4.6 however, highest prediction 3.1 – 4.0 26.9 6.7 value was 2 and 1 years Table demonstrates the occurrence of Gleason >7 PCa at low PSA levels, precluding an optimal PSA threshold before diagnosis (P<0.001). for detecting non-palpable but clinically significant PCa. When stratified [- 2]proPSA levels according Free:total PSA 50-59 years 60-69 years > or =70 years to GS of RP specimens, [- < or =0.10 49.2% 57.5% 64.5% 2]proPSA was highest in patients with GS8 and 0.11-0.18 26.9% 33.9% 40.8% lowest in those with GS6. 0.19-0.25 18.3% 23.9% 29.7% >0.25 9.1% 12.2% 15.8% Based on free:total PSA ratio: the percent probability of finding prostate cancer on a needle biopsy by age in years 53 54 Biochemistry: from PSA to PHI Biochemistry: from PSA to PHI p2PSA appears to have the highest predictive ability when associated with other variables. Beckman Coulter Inc. Developed the PHI, a mathematical algorithm that is defined as 55 56 Results of our study and comparison with Results of our study and comparison with others others It is the right time, I visit an Number of cases: 115 urologist! •Pca: 36 •BHP: 79 PSA form Area under the curve - ROC PSA 0,64 free PSA 0,51 free PSA/PSA 0,36 pro PSA 0,68 pro PSA/free PSA 0,73 PHI 0,75 57 PSA velocity 0,69 58 Alpha-fetoprotein (AFP) AFP in different cancers • Alpha-fetoprotein (AFP) is a protein produced primarily by the liver in 100 a developing baby (fetus). AFP levels are typically elevated when a [%] baby is born and then decline rapidly. Liver damage and certain 80 cancers can increase AFP concentrations significantly. • AFP is produced whenever liver cells are regenerating. With chronic 60 liver diseases, such as hepatitis and cirrhosis, AFP may be chronically elevated. Very high concentrations of AFP may be 70 40 produced by certain tumors. This characteristic makes the AFP test useful as a tumor marker. Increased amounts of AFP are found in 20 7 many people with a type of liver cancer called hepatocellular 3 3 1 0,5 2,5 3 0 0 2 0 0 0 0 carcinoma and in a liver cancer occurring in infants called a n lč r U č jka ica lo o ec u hepatoblastoma. They are also found in some people with cancers lju o varij o Ž d -T o eh vica stata T P D O erviks K Jetra el kreas M ed ro N of the testicles or ovaries. C atern Ž an L P E M P Cut off AFP: 15 µg/l Carcinoembryonic Antigen (CEA) CEA in different cancers • The carcinoembryonic antigen (CEA) test measures the 100 amount of this protein that may appear in the blood of [%] some people who have certain kinds of cancers, 80 especially cancer of the large intestine (colon and rectal 60 cancer). It may also be present in people with cancer of the pancreas, breast, ovary, or lung. 40 63 • CEA is normally produced during the development of a 56 52 56 20 38 33 32 38 fetus. The production of CEA stops before birth, and it 31 22 24 30 16 22 usually is not present in the blood of healthy adults. 0 a n lč r U č jka ica lo o ec u d -T • The carcinoembryonic antigen (CEA) test is used to: lju o varij o Ž o eh vica stata T P D O erviks K Jetra kreas M C el ed N atern Ž an L roP E M P • Find how widespread cancer is for some types of the disease, especially colon cancer. Cut off CEA: 3 µg/l CA 19-9 CA 19-9 in different cancers • Cancer antigen 19-9 (CA 19-9) is a protein that exists on 100 the surface of certain cancer cells. CA 19-9 does not [%] cause cancer; rather, it is shed by the tumor cells, 80 making it useful as a tumor marker to follow the course 60 of the cancer. • CA 19-9 is elevated in 70% to 95% of people with 40 77 61 64 advanced pancreatic cancer, but it may also be elevated 20 in other cancers, conditions, and diseases such as 27 26 25 30 15 12 14 7 colorectal cancer, lung cancer, gallbladder cancer, bile 4 5 6 0 a n lč r U č jka ica o ec u duct obstruction (e.g., gallstones), pancreatitis, cystic lo lju o varij o Ž d -T o eh vica stata T P D O erviks K Jetra kreas M C el ed N atern Ž an L ro E fibrosis, and liver disease. Small amounts of CA 19-9 are P M P present in the blood of healthy people. Cut off CA 19-9: 37 kU/l CA 15-3 CA 15-3 in different cancers • Cancer antigen 15-3 (CA 15-3) is a protein that is produced by normal breast cells. In many people with 100 [%] cancerous breast tumors, there is an increased production 80 of CA 15-3. CA 15-3 does not cause cancer; rather, it is shed by the tumor cells and enters the bloodstream, 60 making it useful as a tumor marker to follow the course of 40 the cancer. 45 • CA 15-3 is elevated in only about 10% of women with 20 42 33 32 35 33 24 early localized breast cancer but is elevated in about 80% 18 14 11 14 14 17 10 0 of those with metastatic breast cancer. a n lč r U č jka ica lo o ec u lju o varij o Ž d -T o eh vica stata T P D O erviks K Jetra kreas M N • CA 15-3 may also be elevated in healthy people and in C el ed atern Ž an L roP E M P individuals with other cancers (e.g., colon, lung, pancreas, ovary, or prostate malignancies) or certain conditions Cut off CA 15-3: 28 kU/l (e.g., cirrhosis, hepatitis, and benign breast disease). HER2/neu HER2-neu in different cancers • HER2-positive breast cancer is a breast cancer that tests 100 positive for a protein called human epidermal growth [%] factor receptor 2 (HER2), which promotes the growth of 80 cancer cells. 60 • In about 1 of every 5 breast cancers, the cancer cells have a gene mutation that makes an excess of the 40 HER2 protein. HER2-positive breast cancers tend to be 50 20 more aggressive than other types of breast cancer. 11 12 13 11 9 8 8 9 11 7 10 7 10 They're less likely to be sensitive to hormone therapy, 0 a n lč r U č jka ica o ec u though many people with HER2-positive breast cancer lo lju o varij o Ž d -T o eh vica stata T P D O erviks K Jetra kreas M C el ed N atern Ž an L ro E can still benefit from hormone therapy. P M P Cut off Her2-neu: 10 µg/l CA 125 Pogostnost izločanja CA 125 pri različnih rakih • Cancer Antigen 125 (CA-125) is a protein that is present on the surface 100 of most, but not all, ovarian cancer cells. This makes the test useful as [%] a tumor marker in specific circumstances. 80 • Significantly elevated concentrations of CA-125 may be present in the blood of a woman who has ovarian cancer. 60 • Currently, less than 20% of ovarian cancers are found in the early stages before they have spread outside the ovary. The primary reason 40 81 they go undetected is that the symptoms of ovarian cancer are fairly 55 54 20 39 45 non-specific. 28 26 29 14 • CA-125 is not recommended as a screening test for asymptomatic 9 8 12 6 4 0 women because it is non-specific. Small quantities of CA-125 are a n lč r U č jka ica lo o ec u lju o varij o Ž d -T Jetra o eh vica stata T produced by normal tissues throughout the body and by some other P D O erviks K kreas M C el ed N atern Ž an L roP E cancers. Levels in the blood may be moderately elevated with a variety M P of non-cancerous conditions, including menstruation, pregnancy, and pelvic inflammatory disease. Cut off CA 125: 35 kU/l HE 4 HE4 in different cancers • Human epididymis protein 4 (HE4) is a protein that is produced by 100 most, but not all, epithelial ovarian cancer cells. This makes the test [%] useful as a tumor marker in specific circumstances. 80 • Significantly elevated concentrations of HE4 are frequently present in the blood of a woman who has epithelial ovarian cancer. 60 • There are several different subtypes of epithelial ovarian cancer, 40 79 including: serous, endometrioid, mucinous, and clear cell, with 65 serous being the most common. Some studies have shown that HE4 46 52 47 20 37 is elevated in more than 90% of serous and endometrioid epithelial 22 25 28 20 ovarian cancers and about 50% of clear cell tumors, but it is not 14 5 7 0 usually elevated in mucinous cancers. a n lč r U jka ica lo o ec u ljuč o varij o Ž d -T Jetra o eh vica stata T • HE4 is not recommended as a screening test for asymptomatic P D O erviks K kreas M C tern el n ed N a Ž a L roP E women because it is non-specific. Small quantities of HE4 are M P produced by normal tissues throughout the body and mild elevations may be seen with a variety of non-cancerous conditions. Cut off HE4: 70 pM Biomarker release in primary ovary cancer Correlation CA 125 and HE4 CA 125 HE4 HE4 9.6 nM 100 CEA CA 19-9 10000 CA 72-4 CA 125 ravih [%] 80 CA 15-3 v zd CYFRA 21-1 NSE tilo 1000 60 SCCA ProGRP ercen HER2/neu p HE4 l 40 /m 95% U > 5 100 2 ca 1AC 20 frekven CA 125 12.5 kU/l Zdrave, 0 10 A E A P 4 benigna obolenja. E S C R eu E C 19-9 72-4 125 15-3 21-1 N C G H N=505; r=0.073 A A A A A S ro 2/n C C C C R R F P E Y H Mejni tumor C 1 Rak ovarija 1 10 100 1000 10000 N=146; r=0.216 HE4 pM marker CEA AFP CA 19-9 CA 72-4 CA 125 CA 15-3 CYFRA 21-1 NSE SCC ProGRP HER2/neu HE4 Cut off 3 µg/l 15 µg/l 35 kU/l 3 kU/l 35 kU/l 30 kU/l 3 µg/l 20 µg/l 2 µg/l 30 ng/l 14 µg/l 70 pM Correlation CA 125 and HE4 NPV CA 125 in HE4 HE4 1120 nM 10000 CA 125 <12.5 kU/l 23% (n=147/651) TN 1000 CA 125 416 kU/l HE4 <9.6 nM l /m U 9% (n=59/651) TN 5 100 2 1AC Zdrave CA 125 <12.5 kU/l and/or HE4 <9.6 nM in benigna obolenja 10 N=505; r=0.073 29% (186/651) TN => Mejni tumor Rak ovarija benign gynecologic illness 1 N=146; r=0.216 1 10 100 1000 10000 HE4 pM Correlacija CA 125 with HE4 PPV CA 125 in HE4 HE4 > 9.6 nM in < 1120 nM 10000 CA 125 >416 kU/l 1000 10% (n=64/651) TP CA 125 > 12.5 in < 416 kU/l l /m U5 100 2 1 HE4 >1120 nM AC 3% (n=21/651TP Zdrave 10 benigna gin.obolenja N=505; r=0.073 CA 125 >416 kU/l and/or HE4 >1120 nM Mejni tumor 11% (n=69/651) TP 1 rak ovarija 1 10 100 1000 10000 N=146; r=0.216 HE4 pM Regression algoritem Sensitivity HE4, CA 125 and ROMA index (Benigna ginekološka obolenja proti raku ovarija glede na stadij) Specifificity 75% Risk of Ovarian Malignancy Algorithm 100 (ROMA) [%] 80 Premenopauzal women PI = -12.0 + 2.38*LN[HE4] + 0.0626*LN 60 [CA 125] Stage I Stage II Stage III Postmenopauzal women 40 Stage IV PI = -8.09 + 1.04*LN[HE4] + 0.732*LN [CA 125] 20 ROMA = exp(PI) / [1 + exp(PI)]x 100 0 HE4 CA 125 ROMA PI: Predictive Index Sensitivity HE4, CA 125 and ROMA index Sensitivity HE4, CA 125 and ROMA index (Benigna ginekološka obolenja proti raku ovarija glede na stadij) (Benigna ginekološka obolenja proti raku ovarija glede na stadij) Specificity 90% Specificity 95% 100 100 [%] [%] 80 80 60 Stage I 60 Stage I Stage II Stage II Stage III Stage III 40 40 Stage IV Stage IV 20 20 0 0 HE4 CA 125 ROMA HE4 CA 125 ROMA NSE in different cancers NSE in different cancers • NSE: Neuron-specific enolase (NSE) is a substance that 100 has been detected in patients with certain tumors, namely: neuroblastoma, small cell lung cancer, 80 medullary thyroid cancer, carcinoid tumors, endocrine 60 tumors of the pancreas, and melanoma. • Studies of NSE as a tumor marker have concentrated 40 primarily on patients with neuroblastoma and small cell 51 20 38 42 42 42 37 35 33 lung cancer. Measurement of NSE levels in patients with 29 24 13 10 13 6 these two diseases can provide information about the 0 a n lč r U č jka arij iks ica o tra ec as u ica extent of the disease and the patient's prognosis lo lju o v o Ž d eh v -T stata T P D O erv K Je kre M C elo ed N Ž L ro E (outlook), as well as about the patient's response to atern an P M P treatment. Cut off NSE 12,5 µg/l CA 72-4 CA 72-4 in different cancers • Diagnosing gastric carcinoma is often complicated and 100 can be extremely difficult due to presentation of non- specific symptoms that are sometimes associated with 80 non-malignant disease. 60 Although endoscopy, coupled with histologic evaluation of biopsy specimens, is most often used to make definitive 40 diagnosis, the search for additional non-invasive 48 20 42 diagnostic procedures has longed continued: strong new 35 39 36 22 26 23 clinical evidence has recently emphasized the clinical 12 10 10 8 14 12 0 a n r value of the CA 72-4 serum tumor marker assay in lč U č jka ica lo o ec u lju o varij o Ž d -T o eh vica stata T P D O erviks K Jetra kreas M N diagnosis and monitoring of gastric cancer.. C el ed atern Ž an L roP E M P We use it, to be largely complete, with CEA and CA 19-9. Cut off CA 72-4: 3,0 kU/l Cyfra 21-1 in different cancers Cyfra 21-1 in different cancers • The “cytokeratin fragment” is the most stunning marker ever 100 discovered. Very few labs monitor it at present. It is specially interesting for lung cancer, diagnostic and follow-up of 80 epidermoidis forms. 60 Among all forms of lung cancer Cyfra 21-1 delivers an appreciated sensitivity of 65%, and an exceptional specificity 40 58 of 95%. 46 48 44 20 34 33 Regular evaluation of the Cyfra allows easier follow-up 26 11 14 16 17 14 7 6 treatment, and even better when associated with other TMs. 0 a n lč r U jka iks ica lo o ec u d vica -T CA 15-3, Cyfra are now used in breast cancer: its elevation ljuč o varij o Ž o eh stata T P D O erv K Jetra l kreas M d C e e N atern Ž an L roP E in follow-up demonstrates, very early, the apparition of M P metastases. Cyfra can also be used in uterine cancer, oesophagus Cut off CYFRA 21-1: 3,0 µg/l cancer, bladder cancer. Lung cancer ProGRP • Lung cancer is one of the most common cancers in the • Pro-gastrin releasing peptide (ProGRP) is a novel tumor world with 1.35 million new cases diagnosed every year marker with benefits for lung cancer patient • The two main histological types of the disease are small management. It is the tumor marker of choice for SCLC cell lung cancer (SCLC) and non-small cell lung cancer as it supports quick and accurate discrimination between (NSCLC). It is important to distinguish between these SCLC and NSCLC for a faster decision on patient two subtypes as they have different treatments and treatment prognoses • ProGRP can also be used to assess response to therapy • NSCLC (approx. 80% of cases) is curable with surgery in and monitor recurrence of the disease the early stages. SCLC, however, is an aggressively • The 85.7 pg/mL cut-off value is based on a 95 % spreading neoplasm of rapid growth that is usually only specificity from the NSCLC collective. treatable with chemo- and radiotherapy. ProGRP in different cancers Biomarkers in lung cancer 100 CEA CA 19-9 100 CA 72-4 [%] ravih [%] zd 80 CA 125 80 v CA 15-3 tilo CYFRA 21-1 60 NSE 60 ercen SCCA p ProGRP 40 66 95% 40 > ca 20 20 10 7 7 8 9 6 5 9 8 8 6 4 5 frekven 8 0 C C n r U 0 L L jka ica lč lo ec u A E A P C C o varij žo jetra d eh vica -T E S C R S S d o ko kreas stata T C 125 C G N cerviks N 19-9 72-4 15-3 21-1 N atern elož an i m led rop E A A A A A S ro m p n C C C C RF P seč YC marker CEA CA 19-9 CA 72-4 CA 125 CA 15-3 CYFRA 21-1 NSE SCCA ProGRP Cut off ProGRP: 30 ng/l Raz.vr. 2.3 µg/ml 28.4 kU/l 5.9 kU/l 31.5 kU/l 23.1 kU/l 1.3 µg/l 20.0 µg/l 1.5 µg/l 30.0 ng/l Biomarkers in NSCLC Biomarkers in SCLC phase I-IIIA Phase LD (limited growth) 100 CEA 100 CEA CA 19-9 [%] CA 19-9 CA 72-4 ravih [%] ravih CA 72-4 zd 80 CA 125 zd v 80 CA 125 v CA 15-3 tilo CA 15-3 CYFRA 21-1 tilo CYFRA 21-1 60 NSE ercen 60 NSE ercen SCCA p p SCC ProGRP ProGRP 95% 40 95% > > 40 ca ca 20 20 frekven frekven 0 A E A P 0 E S C R A E C P C 19-9 72-4 125 15-3 21-1 N C G E S C R A A A A A S C 19-9 72-4 125 15-3 21-1 N S G C C C C R ro A A A A A ro F P C C C C R P Y F C YC marker CEA CA 19-9 CA 72-4 CA 125 CA 15-3 CYFRA 21-1 NSE SCCA ProGRP marker CEA CA 19-9 CA 72-4 CA 125 CA 15-3 CYFRA 21-1 NSE SCCA ProGRP Raz.vr. 2.3 µg/ml 28.4 kU/l 5.9 kU/l 31.5 kU/l 23.1 kU/l 1.3 µg/l 20.0 µg/l 1.5 µg/l 30.0 ng/l Raz.vr. 2.3 µg/ml 28.4 kU/l 5.9 kU/l 31.5 kU/l 23.1 kU/l 1.3 µg/l 20.0 µg/l 1.5 µg/l 30.0 ng/l CEA < cut of for different cancers The use of only one TM for screening 100 i [%] tivn 80 zi is not recomended because of high o p 60 onič FP and FN results 40 esn 63 R 56 52 56 20 38 33 32 38 Razm.vr.: 22 24 30 31 16 22 CEA 3 µg/l 0 -20 -37 -44 i -48 -44 -62 -67 -68 -62 -40 -78 -76 -70 -69 ativn -84 -78 eg n -60 o ažnL -80 -100 a n lč r U jka ica lo o ec u ljuč o varij o Ž d -T o eh vica stata T P D O erviks K Jetra kreas M C el ed N atern Ž an L roP E M P CA 19-9 < cut off for different cancers CA 15-3 < cut off for different cancers 100 i 100 i [%] [%] tivn tivn 80 zi 80 zi o o p p 60 o o n 60 n ič ič 40 77 esn 40 esn 61 64 R R 20 Razm.vr.: 45 20 42 Razm.vr.: 27 26 35 25 30 7 33 33 5 6 32 10 15 4 12 14 CA19-9 37 kU/l 18 24 14 11 14 14 17 CA15-3 28 kU/l 0 0 -23 -20 -39 -36 i -20 i -55 -58 -73 -74 -65 -75 -70 -67 -68 -67 -40 ativn -76 ativn -85 -86 -40 -82 -86 -86 -86 -83 -96 -88 -93 -95 -94 eg -89 -90 eg n n -60 o -60 o ažn ažn L L -80 -80 -100 -100 a n lč r U a n lč r U jka ica lo o ec u jka ica lo o ec u ljuč o varij o Ž d eh vica -T d -T stata T ljuč o varij o Ž o eh vica stata T P D O erviks K Jetra kreas Jetra M P D O erviks K kreas M C elo ed N el ed N atern Ž an L ro ro P E C atern Ž an L P E M P M P CA 125 < cut off for different cancers HE4 < cut off for different cancers 100 i 100 i [%] [%] tivn tivn 80 zi 80 zi o o p p 60 o o n 60 n ič ič 40 81 esn 40 79 esn R 65 R 55 54 52 20 39 45 46 47 20 9 Razm.vr.: Razm.vr.: 28 37 26 29 8 6 4 25 14 22 12 CA125 35 kU/l 14 20 28 HE4 70pM 0 5 7 0 -19 -21 -20 -35 -45 i i -46 -20 -48 -61 -55 -53 -63 -54 -72 -74 -71 -40 ativn -40 -78 -75 -80 -72 ativn -91 -86 -92 -88 -94 -86 -96 eg -95 -93 eg n n -60 o -60 o ažn ažn L L -80 -80 -100 -100 a n lč r U a n lč r U jka ica lo o ec u jka ica lo o ec u ljuč o varij o Ž d eh vica -T d -T stata T ljuč o varij o Ž o eh vica stata T P D O erviks K Jetra kreas Jetra M P D O erviks K kreas M C elo ed N el ed N atern Ž an L ro ro P E C atern Ž an L P E M P M P Can we diagnose cancer on SCREENING the base of tomor non specific marker? PRIMARY DIAGNOSIS : DIFFERENCIAL DIAGNOSIS Higher concentrations CONTROL OF THERAPY are leading to increased FOLLOW UP – EARLY DETECTION OF specificity for the presence of METASTASES tumor Biomarkers in different cancers - MODERATE Tumor specificity > 99% CEA: > 20 µg/l 500 HE4 CA 19-9: > 1000 kU/l HER2/neu a vr. ProGRP 450 SCCA AFP: > 1000 en NSE µg/l 400 CYFRA 21-1 CA 125: > 1000 U/l CA 15-3 ejitv 350 CA 125 CA 72-4 300 CA 15-3: > 100 kU/l CA 19-9 AFP razm 250 CEA CA 72-4: > 100 kU/l ] > 200 [% CYFRA21-1: > 20 je 150 µg/l an 100 SCC: > 10 šč µg/l rop 50 S NSE: > 50 µg/l 0 C ec C C jke L L ProGRP: > 200 ng/l RC d o varij kreas Jetra C C elo D O erviks S S an Ž C N S100: > 1 P µg/l PSA: > 50 marker CEA AFP CA 19-9 CA 72-4 CA 125 CA 15-3 CYFRA 21-1 NSE SCC ProGRP HER2/neu HE4 µg/l Raz.vr. 3 µg/l 15 µg/l 35 kU/l 3 kU/l 35 kU/l 30 kU/l 3 µg/l 20 µg/l 2 µg/l 30 ng/l 14 µg/l 70 pM Biomarkers in different case - HIGH Biomarkers in different cancers – VERY HIGH 150 HE4 HE4 HER2/neu 50 HER2/neu ProGRP ProGRP SCC SCC .vr. 125 NSE NSE CYFRA 21-1 .vr. 40 CYFRA 21-1 CA 15-3 ff CA 15-3 ff 100 CA 125 o CA 125 o CA 72-4 t t CA 72-4 CA 19-9 30 CA 19-9 AFP 75 AFP CEA 100 x razm CEA 10 x razm 100x cu] > 20 ] > 10x cu 50 ] >[% [% ] > je je an 10 25 an šč šč elease [%ro ro p elease [% R 0 p S 0 S R C C C R ec jke L L C ec C C jke L L C d o varij R d kreas Jetra C C C o varij C C elo D O erviks S S kreas Jetra D O erviks S S an Ž C N elo C N P an Ž P marker CEA AFP CA 19-9 CA 72-4 CA 125 CA 15-3 CYFRA 21-1 NSE SCC ProGRP HER2/neu HE4 marker CEA AFP CA 19-9 CA 72-4 CA 125 CA 15-3 CYFRA 21-1 NSE SCC ProGRP HER2/neu HE4 Raz.vr. 3 µg/l 15 µg/l 35 kU/l 3 kU/l 35 kU/l 30 kU/l 3 µg/l 20 µg/l 2 µg/l 30 ng/l 14 µg/l 70 pM Raz.vr. 3 µg/l 15 µg/l 35 kU/l 3 kU/l 35 kU/l 30 kU/l 3 µg/l 20 µg/l 2 µg/l 30 ng/l 14 µg/l 70 pM CEA, CYFRA 21-1, NSE, ProGRP Benign vs malign lung disease Diagnosis of tumor with biomarkers Razm.vr.: Mediana zdravih 100 > 80 .vr.] CEA Raz.vr.: 1.0 µg/L Is a combination of biomarkers able st/razm 60 on CYFRA 21-1 red Razm.vr.: to incerase diagnostic capacity? [V 0.7 40 µg/L NSE Razm.vr.: 20 6.9 µg/L 0 ProGRP Razm.vr.: Benign LC+M1 Benign LC+M1 Benign LC+M1 Benign LC+M1 10 ng/L (N=318) (N=1426) (N=318) (N=1376) (N=318) (N=1369) (N=171) (N=649) ROC curves Benign vs malign lung diseases From certain point all 100 markers become tumor Občutljivost [%] 80 specific CEA AUC=0,740 60 CYFRA 21-1 AUC=0,830 NSE AUC=0,674 40 ProGRP AUC=0,543 Score MalAL AUC=0,902 20 0 100 80 60 40 20 0 Specifičnost [%] ROC - TPA, C EA, C YFRA TM in stomach cancer in i TPS pri kolorekta t lne ln m raku t 1.0 vosji TPA Sensitivity at 95 % specificity tl 80 uč CEA 0.8 70 ob CYFRA ) 60 0.6 TPS (% st 50 66 66 72 o 0.4 tljiv 40 ču b 30 O 40 0.2 20 27 29 10 0.0 0 0.0 0.2 0.4 0.6 0.8 1.0 CEA CA 19-9 CA 72-4 CEA+ CA 19-9+ CEA+ 1-specifičnost CA 19-9 CA 72-4 CA 72-4 AFP and CEA in l iver c ancer Sensitivity TPA, CYFRA, CA 15-3 and TPS in breast cancer 100 Specifičnost (%) Občutljivost (%) AFP CEA 90 TPA 95 17 90 22 ) 80 (% CYFRA 95 11 70 st CEA 90 21 60 jivotl CA 15.3 95 11 u 50 90 15 bč 40 O TPS 90 10 30 95 19 20 AFP 10 0 PRJ JM PRJ JM PRJ – primary liver cancer JM – liver metastases ROC curve i e n b reast s t c ancer c EARLY DETECTION 100 Early detection of metastases in CA 15-3 breast cancer and relaps 90 MCA % 80 st CEA TO Sensitivity Specificity jivo % % tlu 70 bčO TPA 63 98 60 CA 15-3 46 98 n = 191 CEA 7 99 50 CA15-3+TPA 83 96 100 90 80 70 60 50 CEA+TPA 70 98 Specifičnost % Meaning of individual basical values SCREENING of concrete patient PRIMARY DIAGNOSIS : DIFFERENCIAL BREAST CANCER – BEFORE SURGERY DIAGNOSIS CEA 2,4 µg/l CONTROL OF THERAPY CA 15-3 23,1 kU/l FOLLOW UP – EARLY DETECTION OF METASTASES TM are normal After surgery fall of CA 15-3 in breast cancer After surgery fall of CEA in breast cancer Mediana 25% -75% Per P ze e n c teile nt n ile 25%-75% Perzentile Mediana Pr P ec e e c n e t n ile 35 6 CEA CA 15-3 [ [kU/l] µ 30 g/l]5 25 4 20 3 15 2 10 1 5 0 0 pre op post op pre op post op pre op post op pre op post op pre op post op pre op post op pre op post op pre op post op n=1046 n=871 n=558 n=443 n=457 n=402 n=31 n=26 n=1046 n=740 n=558 n=362 n=457 n=355 n=31 n=23 All N N 0 N 1 N 2 All N N 0 N 1 N 2 Concentration of Biomarkers falls What is (depending on half time) „normal“ after succesful surgery or therapy for a certain patient to individual basic values of the patient can be established solely by following individual basic values This Thus the control measurement of TM individual basic values after first stage of treatment is ABSOLUTELY NECESSARY of a certain patient are real results - for the interpretation during the irrespective of whether the primary treatment value is within or outside the reference range SCREENING Common thinking about tumor PRIMARY DIAGNOSIS : DIFFERENCIAL markers: DIAGNOSIS Tumor markers have been “normal” at the time of diagnosis, and can’t be CONTROL OF THERAPY used to assess the effectiveness of therapy FOLLOW UP – EARLY DETECTION OF METASTASES ….. 100% elevation of CEA, CA 15-3, CA 125 BUT: Kirurška Diagnostic use of biomarkers odstranitev 100% zvišanje: primarnega indikator metastaz tumorja for the early recurrence detection or vr.*] 3 metastases is not dependent of the release of st/raz.on tumor biomarkers from the primary tumor! [vred 2 100% zvišanje 100% elevation from the individual base value 1 means almost 100% tumor specificity individualne bazične vrednosti: Dosežene 4 tedne po kirurškem posegu, radioteraoija in/ali kemoterapija 0 Raz.vr. = individualna bazična vrednost * Breast cancer, 869 patients in 91 patients 91 patients with metastases metases present 66 patients with elevated TM When metastses were detected: In 50% within first 4,3 years 25 patients without elevation of TM in 75% within first 7 years in 100% in the period of 23 years Metastases and elevation of TM Elevation of TM without metastases (n=66) (4% patients) Which marker was elevated? CA 15-3 30 CEA 16 CA 15-3 0 CA 125 10 CEA 13 HER2 1 CA 125 14 CA 15-3 + CEA 1 CEA + CA 125 2 CA 15-3 + CA 125 4 CEA + CA 125 2 All 2 Secundary cancers during the study (n=24) What elevation do we need to have tumor specificity > 99% : Ovariy CA 6 (3+) Colorectal CA 6 (1+) CEA 100% Stomach CA 3 (1+) Limfom 3 CA 15-3 75% Kidney CA 1 Anus CA 1 CA 125 150% Lung CA 1 GIT 1 (1+) S-HER-2/neu 50% Endometrium CA 1 (1+) Clitoris CA 1 RULE: • Serial determinations of TM are comarable • If the identical method and analyzer are used • Beside the result laboratory should report the method (analyzer) • Method should be if possible the same in the laboratory • In the case of the change the new method has to be validated POSSIBILITIES FOR CANCER TREATMENT (choice of optimal approach) • Surgery – any time as the best choice • THERAPEUTIC APPROACHES TO Radiotherapy – Not many invasive – Brachitherapy DIFFERENT CANCER DISEASES • Chemotherapy – Different invasive levels • Immunotherapy – wide spectrum fo therapeutic possiblities Prof Dr Besim Prnjavorac Faculty of Pharmacy, Sarajevo BiH – Decrease of immune response (IMMUNOSUPRESSION) General Hospital Tešanj – Increase of immune response (IMMUNOSTIMULATION) – IMMUNOMODULATION (targeted to different cytokines) • Combination of different approach 1 2 CANCER PROMOTION AND GROWTH –MATHER OF TIME (One milion mistakes of DNA replication daily – tumor promotion is CLASIFICATIONS OF CHEMOTHERAPEUTICS the result of cummulativ mistake – yes or no ...) (immunologic surweilance – recognition of cancer cell...) (Classic historical approach) • The majority of chemotherapeutic drugs can be divided in to: – Alkylating agents, – Antimetabolites, – Mitotic inhibitors, – Antracyclines (adriamycin) – Antibiotics – Vinca – Plant alkaloids (Vincristine) – Taxanes – Hormons, hormonal antagonist and enzymes – Other antitumour agents. • All of these drugs affect cell division or DNA synthesis and function in some way. Carcinogenesis, 16 3 4 (3): 437-441 There are three basic treatment possibilities for PROBLEMS ASSOCIATED WITH CHEMOTHERAPY cancer: surgery, radiotherapy, and chemotherapy. (Resistance to chemotherapy) (imperative to change approach) Some cancers where chemotherapy works very well: • Childhood leukemia Resistance to chemotherapy may develop by several • Retinoblastoma mechanisms: • Osteosarcoma • Testicular cancer(hormone sensitive cancers) • Decrease in the amount of drug uptake by cancer cells • Hodgkin’s Disease E.G. Methotrexate (some kind of adaptation) • Some lymphomas • Increase in the amount of drug removed by cancer cells. • Some early breast cancers (hormone sensitive cancers) E.G. Vinblastine ,doxorubicin, bleomycin ,etapsoid…. • Decrease or alteration in target molecule sensitivity – Cancers that are very difficult to treat with chemotherapeutics this is caused by mutation in the molecule targeted by (need surgery or radiotherapy first): (combination of the drug – drug receptors approach) E.G. Methotrexate,Mercaptopurine,doxorubicin • Colon • Increase in DNA repair ability of the cell via an • Lung increased expression of DNA repairing enzymes. • Pancreatic cancer E.G. Alkylating agent • (late stage of breast cancer –the same tumor cell, but stroma...) different approach of the same cancer. (WHY???). Cell cycle – approach fot target phase of cell cycle POINT OF ACTION OF ANTINEOPLASTIC DRUGS Cell cycle can be divided into:. Gap 0 (G0): There are times when a cell will leave the cycle and quit dividing. This may be a temporary resting period or more permanent. An example of the latter is a cell that has reached an end stage of development and will no longer divide (e.g. neuron). Gap 1 (G1): Cells increase in size in Gap 1, produce enzymes needed for DNA synthesis S Phase: To produce two similar Mitosis or M Phase (formation daughter cells, the complete DNA of mitotic spindle): Cell instructions in the cell must be growth and protein duplicated. DNA replication occurs production stop at this during this S (synthesis) phase. stage in the cell cycle. All of Gap 2 (G2): It is the gap between the cell's energy is focused DNA synthesis and mitosis, the cell on the complex and orderly will continue to grow and produce division into two similar new proteins & RNA. daughter cells. 8 SECOND MESSENGER FORMATION Cytostatic works as antibiotics (Anti Bios) (Forforilation is protein kinase (PK) job... necessair for cell (Better term for use as usual-anti microbics) function) (influence to cell function...) • Chemotherapeutics impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. • Mechanismof action depend of phase of cell cycle 9 10 ENTRY OF GLUCOSE INTO THE CELL (Cancer promotion and growth is mather of cell metabolism) CLASSIC APPROACH FOR TREATMENT OF CANCER 1. Linkage of inzulin with receptor 2. Autofosforilation of receptor. • Chemotherapy – up to date in common use • Oriented to cellmetabolism and growth 3. Activation of moleculs – second • No targeted messengers 4. Induction of protein synthesis (GLUT-4) 5. Activation of fosfatidilinozitol-3- kinaze (PI3K) lead to movement of glucose transporter (GLUT-4) to cell membrane. 6. Entry glucose into the cell . 7. Activation of PI3K in cell matrix and stimulation of glicogene 12 synthesis and storage. ALKYLATING AGENTS: ALKYLATING AGENTS Alkylating agents are so • DNA adducts (Complex of named because of their two compounds) are ability to alkylate many believed to play a major role nucleophilic functional in mutagenesis and groups under conditions blastogenesis, as well as in present in cells. carcinogenesis Alkylating agents, or their • Common locations include reactive intermediates, the N-7 and O-6 of guanine form covalent bonds with which are shown to be deoxyribonucleic acid associated with mutagenesis (DNA), ribonucleic acid and carcinogenesis. (RNA), and protein to form • Brake of DNA synthesis an adduct in which a methyl develop or ethyl group is added. 13 DNA Cross Linkage ALKYLATING AGENTS: • As a result of this “alkylation”, there are a few consequences: 1) Miscoding (In transcription) • Arrests DNA replication 2) Cross linking- this only occurs if the drug is bifunctional • Can result in DNA damage The net result is cancer cell undergo apoptosis (Cell death) and chromosome breaks – Some of the drugs: • Also mutagenic! (Busulfan (Myleran), Carboplatin (Paraplatin), Chlorambucil, Cisplatin, Cyclophosphamide (Cytoxan), Dacarbazine (DTIC-Dome), Estramustine Phosphate, Ifosphamide, Mechlorethamine (Nitrogen Mustard), Melphalan (Phenylalanine Mustard), Procarbazine, Thiotepa, Uracil Mustard 16 ANTIMETABOLITES ANTIMETABOLITES: • Some of antimetabolites: Cladribine, Cytarabine (Cytosine • An antimetabolite is a chemical with a similar structure to Arabinoside), Floxuridine (FUDR, 5-Fluorodeoxyuridine), a metabolite required for normal biochemical reactions, Fludarabine, 5-Fluorouracil (5FU), yet different enough to interfere with the normal Gemcitabine, Hydroxyurea, 6-Mercaptopurine functions of cells, including cell division. (6MP), Methotrexate (Amethopterin), 6- • All antimetabolites are used in cancer treatment, as they Thioguanine, Pentostatin, Pibobroman, Tegafur, interfere with DNA production and therefore cell division and the growth of tumors (mainly in S-phase specific). Trimetrexate, Glucuronate Clinical uses: leukemia, non-Hodgkin's lymphoma • • They are classified into: inflammatory bowel disease such as Crohn's Disease and ulcerative colitis. 1- Folic acid analogues • It is widely used as immunosuppressant in transplantations 2- Purine analogues to control rejection reactions, autoimmune disese,like 3- Pyrimidine analogues Rh.art. • Overlapp of tumor and autoimmune disease developent • Purin and pyrimidine antagonists are phosphorelated inside the body into nucleotid form in order to be cytotoxic 18 CYTOSINE ARABINOSIDE (CYTARABINE; ARA-C) - MAJOR CHEMOTHERAPY DRUG TO TREAT ACUTE LEUKEMIAS NUCLEOSIDE ANALOGUES - BIOCHEMICAL PHARMACOLOGY MIRRORS THE UPTAKE AND METABOLISM OF NORMAL NUCLEOSIDES ARA-CTP COMPETE WITH NORMAL DNA INHIBITION OF THYMIDYLATE SYNTHESIS METABOLISM AND INHIBIT DNA POLYMERASE. INCORPORATION INTO DNA PRODUCES STRAND BRAKS AND INDUCE APPOPTOSIS. • Pyrimidine base 5- Fluorouracil (5FU) inhibits thymidylate synthase • Methotrexate inhibits dihydrofolate reductase, reducing flow of methyl group carried by reduced folate 21 FOLIC ACID ANTIBIOTICS: ANALOGUES (Classic approach – antibiotics-chemotherapeutics) (Antimetabolit) 1-Methotrexate compete with folic acid for DHFR • Antitumor antibiotics interfere between DNA base pairs and (dihydrofolate reductase) disturb the synthesis or function of nucleic acids. and inhibits it . Therefore, it inhibits the synthesis of • Bleomycin is some different bindings to DNA results in DNA, RNA and proteins. single-strand breaks and double-strand scissions, thereby K1 2-Also,DHFR catalyses the disrupting DNA synthesis. conversion of dihydrofolate • Doxorubicin intercalates between base pairs, and also to the active tetrahydrofolat alkylates macromolecules. which is needed for the de • Daunorubicin, doxorubicin, and their derivatives, belong to novo synthesis of the a subclass of antitumor antibiotics called anthracyclines. deoxynucleoside thymidine • Some drugs: phosphate DTMP (required Aclarubicin, Bleomycin, Dactinomycin (Actinomycin D), Daunorubicin, for DNA synthesis) Doxorubicin (Adriamycin), Epirubicin, Idarubicin, Mitomycin C, Mitoxantrone, Plicamycin (Mithramycin) 24 ANTIMETABOLITE BASICS MITOTIC INHIBITORS (Ribonucleotid approach) (Inhibition of formation of mitotic spindle) (Protein oriented approach) • Mitotic inhibitors include among others the vinca alkaloids (vincristine and vinblastine) which are mitotic spindle inhibitors and the epipodophyllotoxins (teniposide and etoposide) which are DNA topoisomerase II inhibitors. • Mitotic spindle inhibitors bind to microtubular proteins and block their ability to polymerize or depolymerize, a process which brake nuclear division. DNA topoisomerase II inhibitors block religation of double strand DNA breaks (i.e., sister chromatid separation or cleaved DNA) • Examples: Etoposide (VP-16, VePesid), Teniposide (VM-26, Vumon), Vinblastine, Vincristine, Vindesine 25 MITOSIS (M phase) Prophase Impair of cytoskeleton formation (microtubules in Metaphase Anaphase Telophase cell division- mitotic spindle) • Plant alkaloids and terpenoids (L01C) These alkaloids are derived from plants and block cell division by preventing microtubule function. Microtubules are vital for cell division, and, without them, cell division cannot occur. The main examples are vinca alkaloids and taxanes. Antimitotic agents bind to microtubuli Supression of microtubuli dynamics Metaphase arrest 27 HORMONES, HORMONAL ANTAGONISTS AND ENZYMES (exept imunosupression of steroids) TAXANES • Hormones or hormone-blocking agents either exert a corticosteroid effect, such as prednisone, or manipulate the • The prototype taxane is the natural product paclitaxel, hormone environment in hormone-responsive tumors. originally known as Taxol and first derived from the bark of • The antiandrogenic agent, flutamide, which is used to treat the Pacific Yew tree. Docetaxel is a semi-synthetic analogue prostate cancer, is believed to block androgen receptor of paclitaxel. Taxanes enhance stability of microtubules, sites. preventing the separation of chromosomes during • The antiestrogenic agent, tamoxifen, binds to intracellular anaphase. (brake cell division) estrogen receptors, then enters the nucleus where the tamoxifen-estrogen-receptor complex inhibits DNA and • Used drug - paclitaxel protein synthesis. • Some of the drugs: • Equine Estrogen (Premarin), Cortisone, Chlorotriansene , Dexamethasone , Diethylstilbestrol Ethinyl Estradiol , Fluoxymesterone , Hydroxyprogesterone Caproate , Medroxyprogesterone Acetate (Provera), Megestrol Acetate (Megace), Prednisone, Tamoxifen (Nolvadex), Testosterone 29 30 TYROSINE KINASES AS TARGETS FOR CANCER THERAPY TYROSINE KINASES AS TARGETS FOR CANCER (Approach to influence on second messenger) (Tumor is the mather of metabolism) THERAPY • • Protein tyrosine kinases (TKs) are enzymes that catalyze Discavery of monoclonal antibody give the new (great) the transfer of phosphate from ATP to tyrosine residues in influence in chemotherapeutic strategy. polypeptides. (including energy transfer) • The landscape was changed radically by the success of • The human genome contains about 90 TK and 43 TK-like imatinib mesylate (gleevec), an inhibitor of the BCR-ABL TK genes, the products of which regulate cellular proliferation, in chronic myeloid leukemia (CML) — a result heralded as a survival, differentiation, function, and motility. proof-of-principle and a triumph of targeted cancer therapy. • • In human genome only 3% of gene are “structural genes”, TKs are now regarded as excellent targets for cancer and 97% are “regulatory” (their function is only in chemotherapy, but reality lies somewhere between the chromosome). extremes of triumph and tribulation. • “Very high level” of metabolic and functional role is just a • (N Engl J Med 2005;353:172-87) challenge for therapeutic targets (Statins in the begenning...) 31 32 TYROSINE KINASES AS TARGETS FOR CANCER IMPAIR OF CYTOSKELETON FORMATION THERAPY (MICROTUBULES IN CELL DIVISION) • Some newer agents do not directly interfere with DNA. • Vinca alkaloids (L01CA) • These include monoclonal antibodies and the new tyrosine kinase inhibitors e.g. imatinib mesylate (Gleevec), which Vinca alkaloids bind to specific sites on tubulin, inhibiting directly targets a molecular abnormality in certain types of the assembly of tubulin into microtubules (M phase of the cancer (chronic myelogenous leukemia, gastrointestinal cell cycle). They are derived from the Madagascar stromal tumors). These are examples of targeted therapies. periwinkle, Catharanthus roseus (formerly known as Vinca rosea). In addition, some drugs that modulate tumor cell behaviour • The vinca alkaloids include: without directly attacking those cells may be used. o Vincristine Hormone treatments fall into this category. (Not so o Vinblastine agressive) o Vinorelbine o 34Vindesine 33 34 THERAPY AGAINST TUMOR STROMA AND VASCULARISATION Anti-hEGFR-hIgG4 (S228P) • Binding of cetuximab to EGFR blocks ligand/receptor • EGFR (epidermal growth factor) is activated by binding binding and induces receptor internalization and specific ligands, including epidermal growth factor and subsequent degradation. Consequently, cetuximab blocks transforming growth factor-α. Activation of EGFR promotes downstream pathways which regulate cell growth and cell proliferation and survival, as well as angiogenesis, angiogenesis. leading to tumor growth and metastasis. • Cetuximab induces cell death through antibody-dependent • Anti-hEGFR-hIgG4 (S228P) features the constant region of cell-mediated cytotoxicity (ADCC) (with activation of the human IgG4 (S228P) isotype and the variable region of complement-very impotynt for all immunity) . cetuximab. Cetuximab is a chimeric human/mouse IgG1 • Cetuximab has been approved by the FDA for the treatment monoclonal antibody that targets epidermal growth factor of metastatic colorectal cancer and metastatic squamous receptor (EGFR), a cell surface receptor overexpressed in cell carcinoma of the head and neck. many types of cancer. Anti-hEGFR-hIgG4 (S228P) was generated by recombinant DNA technology. It has been produced in CHO cells Clin Cancer Res. (Hamster Chinese ovary-cell line) and purified by affinity 2007: 3(5):1552-61 chromatography with protein G. 35 36 Vascular Endothelial Growth Factor (VEGF) and It’s Role in Non-Endothelial Cells: Autocrine Signaling Anti-hEGFR-hIgG4 (S228P) THERAPY by VEGF (Potential target for tumor cells) • Gefitinib(Antibody for EGFR), a quinazolin derivative that • Vascular endothelial growth factor (VEGF) is a potent specifically inhibits the activation of EGFR tyrosine kinase angiogenic factor and was first described as an essential through competitive binding to the ATP-binding domain of growth factor for vascular endothelial cells. the receptor, has received approval of FAD for patients with • VEGF is up-regulated in many tumors and its contribution to advanced NSCLC refractory to chemotherapy tumor angiogenesis is well defined. • VEGF and VEGFR receptors are expressed on numerous non- endothelial cells including tumor cells. Vincenzi B. et al., 2010. Cetuximab: from bench to bedside. • So, it is to considered it as well target for cancer therapy. Curr Cancer Drug Targets. 10(1):80-95. • Anti-VEGF strategies to treat cancers were designed to target the pro-angiogenic function of VEGF and thereby inhibit neovascularization. (Cancer Res. 1995;55: 3964–3968 ) 37 38 Left side: -Gene transcription – after -Cancer promotion and signal transduction – growth is mather of time second messenger -- Changes of activation (PIP3, DAG, metabolism PKC) - Cytoskeleton and membrane receptors pass -Target for cancer cell throgh complete cell (only) membrane, intracelluar - Consideration of TM space and throgh next marker and oncogens neibghur cell. -(“silent speah of cells”) (intercellular comunications- integrin,cytoskeleton, receptors) (No strenght connestion between tm cells) 39 40 Inhibition of normal repair of the cell – to destroy cancer cells. • VEGF AS TARGET... Tumor suppressor gene (TSG) therapies, inhibitors of antiapoptotic proteins (Bcl-2), HDAC (histone-deacetylase), proteasome , pyruvate dehydrogenase kinase isozyme 1; PI3K (second messenger) For tumors of 2-3 mm - to growth - angiogenesis is essential for adequate oxygenation and nutrition. VEGF is the most important growth factor for angiogenesis in normal and tumor cells. - The VEGF family consists of six growth factors (VEGF-A, -B, -C, - D, and -E and placental growth factor [PlGF]) and three receptors J. Natl. Cancer Inst. Negation of negation as 82:4–6. J. Clin. Invest. 117:2740– approach – apoptosis of tm 41 42 2750 (2007 cell is well job Cancer stem cell–specific therapeutic approaches. Hedgehog (HH), Notch, and Wnt signaling - key stem cell self-renewal pathways that The likely cells of origin for the three common histologic are deregulated in lung cancer and thus represent potential subtypes of lung cancer — adenocarcinoma, squamous-cell therapeutic targets (Role of regulatory genes /97%/) carcinoma, and small-cell carcinoma — are depicted. (Note - nearly anytime in point of junction of two cell types) (Neuroendocrin cells near to β2 receptor) Breast Cancer Res. 7:86–95. NEJM 374;19 May 12, 2016 43 44 Intravesical BCG instillation – immune response. Triggers release of INDUCING OF LOCAL INFLMAMATION cytokines and chemokines from these cells. Differentiation of naïve CD4+ T (as possible antitumor therapy) cells into TH1 and/or TH2 cells that direct immune responses toward cellular or humoral immunity, respectively. The ffect of BCG depends on TH1 immune (Approach to proinflammatory and responses. IL-10 inhibits TH1 immune responses, whereas IFN- inhibits TH2 antiinflammatory cytokines –modulation of action) immune responses. Blocking IL-10 or inducing IFN- lead to a TH1-dominated immunity that is essential for BCG-mediated bladder cancer destruction. • Idea – activate inflamatiomn – cytokine sturm • Bacill Calmetet Guerin – in use since 1976. 45 46 BCG for bladder cancer EVALUATION OF BCG AND IFN-α α THWRAPY • This complex and robust immune reaction evoked by BCG is • In vivo monotherapy with IFN-α2b for bladder cancer in evidenced by a massive transient secretion of cytokines in humans has been explored by multiple groups. voided urine, including interleukin (IL)-1, IL-2, IL-5, IL-6, • In 1990, Glashan published data from a randomized IL-8, IL-10, IL-12, IL-15, IL-18, interferon-inducible protein controlled trial evaluating high dose (100 million unit) and (IP)-10, tumor necrosis factor (TNF)-α, granulocyte- low dose (10 million unit) IFN-α2b regimens in patients monocyte colony stimulating factor (GMCSF), and interferon with CIS [29]. Patients were treated weekly for 12 weeks (IFN)-γ. and monthly thereafter for 1 year. • Given that the effect of BCG is immune mediated, decades • The high and low dose groups had complete response rates of research have focused on adjunctive immunotherapies of 43% and 5%, respectively. Of the high dose patients including IFN-, IL-2, IL-10, and IL-12 achieving a complete response, 90% remained disease-free • Combination of BCG anf IFN-α α has the better results than at a notably short 6 months of follow-up any of them alone. 47 48 APPROACH WITH INTERFERONS APPROACH AD IL-2 AS POTENTIAL TARGET • Interferons (IFNs) are glycoproteins initially isolated in the 1950s and valued for their antiviral properties. • Three types have been isolated, IFN- (which is actually a • The cytotoxic antitumor capabilities induced in lymphocytes family of interferons), IFN-β, and IFN-γ. IFN- and IFN-β are by IL-2 make it a potential cancer immunotherapeutic grouped as “Type I” interferons, whereas IFN-γ is a “Type agent. To date, multiple studies have demonstrated II” interferon. regression of metastatic disease following systemic IL-2 • The Type I interferon receptor has 2 components, IFNAR-1 treatment in some cancers and IFNAR-2, which subsequently bind and phosphorylate Jak molecules initiating a cascade resulting in gene A. Rosenberg, M. T. Lotze, and L. M. Muul, “A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer transcription . cells and interleukin-2 or high-dose interleukin-2 alone,” New England • The IFN-α family is well known to stimulate natural killer Journal of Medicine, vol. 316, no. 15, pp. 889–897, 1987 (NK) cells, induce MHC class I response, and increase antibody recognition . • They have antineoplastic properties by direct antiproliferative effects and complex immunomodulatory effects 49 50 MONOCLONAL ANTIBODIES PRODUCTION OF MONOCLONAL ANTIBODY Monoclonal antibodies are proteins produced in the laboratory from a single clone of a B−cell. When used as a treatment for cancer, there are three general strategies with monoclonal antibodies: • Human-mouse kimera 1. Uses the ability of the antibodies to bind to the cancer cells (Lethal irradiation of mouse and than reconstruction having the tumor antigens on their surface. The immune system will see the cancer cells marked with bound immune system by human bone marrow). After antibodies as foreign and destroy them. revitalisation - injection of Ag to wich production of Ab is 2. A second strategy is to use the antibodies to block the looking for.) binding of cytokines or other proteins that are needed by the cancerous cells to maintain their uncontrolled growth. • Recombinnat DNA technology Monoclonal antibodies designed to work like this bind to the cytokine receptors that are on the tumor cell surface. 3. A final strategy involves special antibodies that are linked (conjugated) to a substance that is deadly to the cancer cells. E.G. radioactive isotopes, cytostatics, have been successfully conjugated to antibodies. (monoclonal antibody as the carriers) 51 52 WHICH APPROACH IS THE BEST ONE • Syrgery of solid tumors • Target therapy for leukemia-like tumors (no solid) • Which one is the most promising? THANK A LOT FOR YOUR ATTENTION • Targeted therapy if possible. • Biotechnology for production of cancer cell line, with manipulation of them after successful culture could be promising. • Have you better idea? 53 54 CLINICAL PHARMACY Goals of clinical pharmacy SERVICES IN ONCOLOGY Invited lecture at the The aim of clinical pharmacists is to ensure the correct and safe use of International CEEPUS Summer School 2016 medicines and other medical products through the provision of various clinical pharmacy services. Aleš Mrhar Faculty of Pharmacy, University of Ljubljana Their objective is to: Aškerč eva 7, 1000 Ljubljana, Slovenia • maximise treatment efficacy by choosing the most appropriate medication for ales.mrhar@ffa.uni-lj.si each condition and for each individual patient Asist. Prof. Dr. Lea Knez, mag.pharm.,spec.clin.pharm., University Clinic Golnik • minimise the risk for adverse drug events by providing drug monitoring and lea.knez@klinika-golnik.si Samo Rožman, mag.pharm.,spec.clin.pharm., Institute of Oncology Ljubljana assessing patient‘s medication adherance srozman@onko-i.si • reduce drug expenditure by choosing the most cost-effective among all suitable strategies Clinical outcomes of clinical pharmacists interventions Clinical pharmacist Number of clinical pharmacists is in correlation with clinical outcomes Increase of clinical Clinical pharmacist’s role is to optimise drug treatment pharmacists from 0,34 to across the institutions of health care system, i.e. in: 3,23/100 beds decreases number of deaths - hospitals, clinics (secondary and tertiary level) and in the hospital for 43% and - pharmacies, community health centres, nursing decreases length of homes (primary level) stay in the hospital for 47% Clinical pharmacist is entitled a pharmacist with Master of 1,00 dollar spent on Pharmacy degree (MPharm) and a three-year specialisation clinical pharmacist brings 4,86 dollars saving in clinical pharmacy to the system Bond et al. Pharmacotherapy 2001; 21 (2): 129-141 Clinical and economic outcomes of clinical pharmacists interventions in USA: a systematic review Positive in 15,9% studies, Mixed in 42,1% studies, Outcomes of clinical pharmacists interventions in No effect in 4,8% studies, Uncertain in 37,3% studies Slovenia: a clinical study T Roblek, A Detiček, B Leskovar, S Šuškovic, M Horvat, A Belič, A Mrhar, M Lainščak Clinical-pharmacist intervention reduces clinically relevant drug–drug interactions in patients with heart failure: A randomized, double-blind, controlled trial, Int J Cardiol 2016; 203: 647–652 Pharmacist intervention significantly reduces the number of patients with clinically relevant drug-drug interactions, but not clinical endpoints 6 months from discharge. Study performed at University Clinic Golnik, Golnik, Slovenia Burns et al. Am J Health-Syst Pharm 2010; 67: 1624-1634 Outcomes of clinical pharmacists interventions in Methodological issues Slovenia: a clinical study R Režonja Kukec, I Grabnar, T Vovk, A Mrhar, V Kovač, T Čufer Febrile neutropenia in chemotherapy treated small-cell lung cancer patients, Radiol Oncol 2015; 49: 173-180 Neutropenic events are assumed to be related to increased etoposide plasma concentrations after a standard etoposide and cisplatin dose. While the mean etoposide peak plasma concentration in the first cycle of chemotherapy was 17.6 mg/l, the highest levels of 27.07 and 27.49 mg/l were determined in two patients with febrile neutropenia. Study performed at University Clinic Golnik, Golnik, Slovenia Cases of clinical pharmacy services in Tertiary hospital: oncology in Slovenian University Clinic Golnik settings A. Mrhar, L. Knez The beginning (10 years ago)! What has been done so far in the hospital? CLINICAL PHARMACY Clinical pharmacists are integrated in the treatment of: SERVICES IN GENERAL: Patients at higher risks for adverse drug reactions Oncology patients EXPERIENCES & RESULTS Patients with tuberculosis Patients with hereditary angioedema Advanced medication review (Pharmacotherapeutic review) is obligatory in patients at higher risks for adversed drug reactions: Decreased renal function (<30 mL/min) Prescribed with strong inhibitors or inducers Prescribed with strong opioids Prescribed with drugs where TDM is performed Prescribed drugs with a feeding tube Prescribed medicines with restrictions in their use OVERVIEW CLINICAL PHARMACY I. Implementation of clinical pharmacy services SERVICES IN ONCOLOGY: II. Evaluation of implemented services EXPERIENCES & RESULTS III. Current activities & future plans I. IMPLEMENTATION Self-initiated clinical screening of chemotherapy (CT) prescriptions Review of dose calculation Review of laboratory investigations Review of support therapy ADE expended the service & made it obligatory Prevention of application of CT to neutropenic patient Prevention of ADE (paclitaxel hypersensitivity) occured due to lack of supportive therapy (dexamethasone) Joint commitment towards quality improvement Standardisation of CT protocols (basic) Improvement of CT order form (upgrade) I. IMPLEMENTATION CLINICAL SCREENING OF CT PRESCRIPTIONS BY PHARMACISTS IS OBLIGATORY & IMPLEMENTED INTO ROUTINE CLINICAL PRACTICE. Pharmacists have to check: Physician's signature & completness of all administrative data Calculation of CT doses & need for dose adjustment Prescription of support therapy Drug formulation and administration II. EVALUATION II. EVALUATION: ALL INTERVENTIONS 211 INTERVENTIONS PER 506 CT ORDERS (41,7%) TO EVALUATE CLINICAL INTERVENTIONS MADE BY PHARMACISTS DURING PRESCRIPTION SCREENING DRUGS INVOLVED: Cancer drugs 65/211 (30,8%) Antiemetic drugs 87/211 (41,2%) PERFORMED A CROSS-SECTIONAL OBSERVATION Other support therapy drugs 26/211 (12,3%) STUDY DURING A 5-MONTH PERIOD PROBLEMS INVOLVED: Administrative 32/211 (15,2%) Drug (omission) 45/211 (21,3%) Dose (frequency, regimen) 133/211 (63,0%) IMPLEMENTATION: Implemented 160/211 (75,8%) Knez L, Jošt M, Toni J, Triller N, Čufer T. Implementing new clinical pharmacy services parallel to the Implemented with changes 10/211 (4,7%) introduction of centralized preparation of anti-cancer drugs. Slovenian Journal of Public Health 2011; 50: 12-23 Not implemented 35/211 (16.6%) II. EVALUATION INTERVENTIONS ON CANCER DRUGS II. EVALUATION The results of this study confirm the Problem category importance of integration of pharmacists’ DRUG RELATED: 7/211 (3,3%) clinical roles for the provision of high quality DOSE, FREQEUNCY & DURATION: 56/211 (26,5%) oncology services Doses differ >10% 20/211 (9,5%) Need for dose adjustment 14/211 (6,6%) Actions to tackle identified problems Chosen drug dose not written 10/211 (4,7%) should be taken Other 12/211 (5,7%) ADMINISTRATIVE & ADMINISTRATION: 1/211 (0,5%) Clinical (and economic) significance of the recorded interventions should be Implemented 45/65 (69,2%) defined III. PRESENT & FUTURE MANAGEMENT OF COMPREHENSIVE MEDICATION HISTORY (CHM) Methods: Patients admitted to Universitiy Clinic Golnik were randomly selected and included in the study. For each patient a CMH was obtained by a research pharmacist using various sources of information. Pharmacists participation in obtaining Next, the medication history in the hospital medical record was reviewed. COMPREHENSIVE MEDICATION HISTORY The prescribed drugs were assessed for completeness of information, and possible discrepancies between both medication & histories were recorded and classified. Results: check for DRUG INTERACTIONS with cancer drugs Overall, 108 patients with a median age of 73 years were included in the study. prior to the start of treatment The research pharmacist recorded the use of 651 medicaments, with all relevant details being available for 94.9% of these drugs. Of the 464 medicines listed in the hospital medical record, only 42.0% were considered complete. A comparison of the medication history and the medical record with the CMH revealed at least one discrepancy in 72.4% of the drugs listed. The majority of the identified discrepancies were often present both in the medication order on the Continue the collaboration in IMPROVING THE drug chart (76.2%) and in the discharge letter (69.9%). Most medication discrepancies were due to drug omissions (20.9%) and commissions (6.5%). QUALITY OF CARE and in ONCOLOGY RESEARCH Conclusion: The high rate of discrepancies between the recorded drug history and CMH reported in our study stresses the need for the implementation of medication reconciliation. The participation of pharmacists in the reconciliation process, described in this study, resulted in more complete and accurate drug histories acquired. Režonja R, Knez L, Šuškovič S, Košnik M, Mrhar A. Comprehensive medication history: the need for implementation of medication reconciliation processes. Slovenian Journal of Public Health 2010; 49: 202-210 MANAGEMENT OF DRUG INTERACTION EVALUATION OF OUR MANAGEMENT OF DRUG INTERACTIONS DRUG INTERACTIONS ARE REVIEWED FOR EACH PATIENT THE MANAGEMENT OF DRUG INTERACTIONS WAS REVIEWED STARTING A NEW LINE OF SYSTEMIC CANCER THERAPY IN ALL LUNG CANCER PATIENTS IN WHOM ANTICANCER THERAPY WAS INITIATED IN 2012 (n = 223) • COMPREHENSIVE MEDICATION HISTORY IS OBTAINED • DRUG INTERACTIONS WITH PLANNED CANCER THERAPY • DRUG INTERACTIONS IDENTIFIED BY THE PHARMACISTS WERE (ANTICANCER DRUGS AND SUPPORT CARE DRUGS) ARE CHECKED REVIEWED, DESCRIBED AND ANALYSED • CLINICALLY IMPORTANT DRUG INTERACTIONS ARE IDENTIFIED • A SEPARATE SEARCH FOR DRUG INTERACTIONS WAS PERFORMED FOR THE SAME PATIENTS, USING 3 DIFFERENT COMPENDIA (Stockley‘s Drug • CHANGES TO DRUG THERAPY ARE PROPOSED Interaction, Lexi-Comp, Drugs.com) AND REVIEWING THE RELEVANT SmPCs • THE OBTAINED INFORMATION IS RECORDED IN THE MEDICAL RECORD AND PRESENTED TO THE PATIENT‘S MEDICAL ONCOLOGIST CHALLENGES IN 1st CHALLENGE: THE MANAGEMENT OF DRUG INTERACTIONS OBTAIN PATIENT‘S MEDICATION HISTORY • OBTAINING A COMPREHENSIVE MEDICATION HISTORY IS CHALLENGING… 1st CHALLENGE: OBTAIN PATIENT‘S MEDICATION HISTORY Drug information on hospital admission is very often 2nd CHALLENGE: SEARCH FOR DRUG INTERACTIONS • INCOMPLETE in 72 % • INCORRECT in 71 % 3rd CHALLENGE: IDENTIFY RELEVANT DRUG INTERACTIONS • …BUT OF PARAMOUNT IMPORTANCE FOR FUTURE DRUG TREATMENT WHAT DID OUR AUDIT REVEAL? 1st CHALLENGE: 2nd CHALLENGE: COMPLEX PATIENTS AND TREATMENTS SEARCH FOR DRUG INTERACTIONS PATIENTS OF HIGHER AGE • median 63 years, IQR: 56 - 69 • THE NUMBER OF POSSIBLE DRUG INTERACTIONS IS DIAGNOSED WITH LUNG CANCER OVERWHELMING • 78 % NSCLC • 22 % SCLC TAKE MANY Rx DRUGS • DRUG INTERACTION COMPENDIA ARE „SAME SAME BUT • median 4, IQR: 2-6, range: 0-13 DIFFERENT“ USE OTC, FOOD SUPPLEMENTS, HERBAL REMEDIES • INFORMATION IN SmPC IS VAGUE ARE TREATED WITH COMPLEX ANTICANCER REGIMENS WITH • 8 drugs in 29 % • 6 drugs in 64 % • 1 drug in 7 % AND THESE REGIMENS INCLUDE HIGH RISK DRUGS WHAT DID OUR AUDIT REVEAL? OUR PATIENTS ARE AT HIGH RISK FOR ADVERSE DRUG EVENTS! 2nd CHALLENGE: 2nd CHALLENGE: SEARCH FOR DRUG INTERACTIONS SEARCH FOR DRUG INTERACTIONS THE NUMBER OF POSSIBLE DRUG INTERACTIONS IS OVERWHELMING DRUG INTERACTION COMPENDIA ARE „SAME SAME BUT 1416 DRUG INTERACTIONS IN 223 PATIENTS DIFFERENT“ • excluding drug interactions between chronic therapy drugs! NOT ALL DRUG INTERACTIONS ARE INCLUDED IN ALL COMPENDIA • median: 5, IQR: 2 – 10, range: 0 – 23 int/pt • only 34 % DI included in all 3 compendia 82 % INTERACTIONS WITH SUPPORT CARE DRUGS • whereas 44 % DI included in only 1 compendia • glucocorticoids: 42 %, aprepitant: 27 %, setrons: 13 % MORE IMPORTANT INTERACTIONS Drugs.com 81 % INTERACTIONS WOULD AFFECT OUTCOMES OF CHRONIC RECORDED IN MORE COMPENDIA 263 THERAPY DRUGS • Chi2, p < 0,01 • most often from ATC C (30 %: statins, antihypertensive, diuretics), ATC N (30 %: analgesics, anxiolytics • still, 367 DI of moderate importance were and sedatives), ATC A (14 %: PPI, antidiabetics), ATC M (11 %: NSAR) recorded in only 1 compendia 103 132 484 75 % OF AT LEAST MODERATE CLINICAL IMPORTANCE Lexi- Stockley‘s Comp • in 1 % combined use is contraindicated ALWAYS CHECK 2 COMPENDIA & 100 75 257 LOOK FOR THE RELEVANT DRUG INTERACTIONS! DO NOT RELY ON CLASSIFICATIONS 2nd CHALLENGE: 3rd CHALLENGE: SEARCH FOR DRUG INTERACTIONS IDENTIFY RELEVANT DRUG INTERACTIONS INFORMATION IN SmPC IS VAGUE • HOW TO IDENTIFY RELEVANT DRUG INTERACTIONS FOR MOST INTERACTIONS (77 %) ARE DESCRIBED IN SmPC… THAT VERY PATIENT? • still 325 drug interactions are not included • HOW TO MANAGE THAT VERY DRUG INTERACTION BEST? …BUT IN 90 % ARE DESCRIBED AS GENERAL DRUG INTERACTIONS • e.g. strong CYP 3A4 inhibitors may increase plasma concentration of drug A • HOW TO ENSURE THAT VERY DRUG INTERACTION IS BEING MANAGED AS AGREED? SmPC CAN SERVE ONLY AS A STARTING POINT… …THAT TAKES YOU TO A VERY LONG JOURNEY WHAT DID OUR AUDIT REVEALED? 3rd CHALLENGE: 3rd CHALLENGE: IDENTIFY RELEVANT DRUG INTERACTIONS IDENTIFY RELEVANT DRUG INTERACTIONS HOW TO IDENTIFY RELEVANT DI FOR THAT VERY PATIENT? HOW TO MANAGE THAT VERY DRUG INTERACTION BEST? PHARMACISTS IDENTIFIED ONLY 4 % (52/1416) OF POSSIBLE DRUG • IN 75 % (39/52) OF CASES A CHANGE IN CHRONIC THERAPY WAS INTERACTIONS AS CLINICALLY RELEVANT SUGGESTED • median: 0 DI; 1 DI in 16 % pts, 2 DI in 3 % pts, 3 DI in 1 pt • in 2 cases it was suggested to reconsider the choice of anticancer agent: • Because carbamazepine increases the clearance of vinorelbine, gemcitabine was chosen over PHARMACISTS IDENTIFIED AS RELEVANT MORE OFTEN DRUG vinorelbine. INTERACTIONS… • Because nephrotoxicity of cisplatin is worsen during furosemide therapy, carboplatin was chosen over cisplatin. • …involving anticancer drugs (85 % of identified DI; Chi2, p < 0,01) • pharmacists offered no suggestion in 7 cases • …that affected outcomes of anticancer therapy (79 % of identified DI; Chi2, p < 0,01) • …recorded in all 3 reviewed compendia (50 % of identified DI; Chi2, p < 0,01) …BUT FAILED IN IDENTIFYING 16 MAJOR DRUG INTERACTIONS • concurrent treatment with granisetron and escitalopram increase risk for arrythmia (10 DI) • aprepitant increases fentanyl plasma concentrations (6 DI) 3rd CHALLENGE: CONCLUSIONS IDENTIFY RELEVANT DRUG INTERACTIONS HOW TO ENSURE THAT THE VERY DRUG INTERACTION IS PHARMACISTS DO HAVE TO TAKE AN ACTIVE ROLE IN BEING MANAGED AS AGREED? THE PREVENTION OF DRUG INTERACTIONS IN CANCER • 59% PATIENTS! (23/41) IMPLEMENTED • IN 41 % (17/41) OF CASES THE PROPOSED CHANGE IN THERAPY WAS NOT IMPLEMENTED • OUR PATIENTS ARE AT INCREASED RISK FOR DRUG • the reasons for this were not recorded INTERACTIONS • remember: drugs are not being prescribed only by medical oncologists, but can be prescribed also by GPs and bought as OTC by patients • IDENTIFYING CLINICALLY RELEVANT DRUG INTERACTIONS REQUIRES EXPERTISE (≠ drug interaction compendia!) • ADEQUATE MANAGEMENT OF DRUG INTERACTIONS DEMANDS GOOD COLLABORATION BETWEEN PHARMACISTS, MEDICAL ONCOLOGIST, GENERAL PRACTITIONERS AND PATIENTS Implementation of medication reconciliation model for all oncology patients Physician performs Physician clinical Physician and prescribes examination pharmacist systemic and evaluates evaluate therapy medication Physician intended and Pharmacist Patient history and unintended gives admitted pharmacist discrepancies advice to to the take steps to in drug patients on systemic Pharmacist reduce risk therapy medicines therapy examines of drug use Pharmacist drug interactions Pharmacist Then, drug examines obtains interactions therapy is prescribed medication between ordered to the systemic history systemic pharmacy therapy and chronic therapy Tertiary hospital: Treatment of Candida species urinary tract infection in an Institute of Oncology Ljubljana oncological patient A. Mrhar, S. Rožman (Case report) Patient details Patient details GM - 71 year old female patient, 163 cm, 94 kg Allergy to penicillin History of present illness 6th October 2015 – transfer from the ICU to surgical ward Hemodynamically stable History of present illness Without vasoactive and oxygen support 10/2012: surgery (abdominoperineal excision) due to colorectal carcinoma after adjuvant Spontaneous diuresis chemo-radiotherapy Inflammation parameters in decline 09/2015: relapse of disease in the abdomen, according to PET/CT pathological nodes also Normal stoma output around the aorta Feeding per mouth with the addition of intravenous amino acids After mild acute on chronic kidney failure, serum creatinine (SCr) and blood urea nitrogen (BUN) 10/2015: explorative surgery – radical excision was not performed due to disease return to preoperative values progression SCr = 160 µmol/L [Normal value: 38-80 µmol/L]) 10/2015: after surgery → transfer to the Intensive Care Unit (ICU) BUN = 15 mmol/L [Normal value: 2.8–7.5 mmol/L]) Supportive therapy in the ICU: EGFR ~ 35 mL/min [Estimated glomerular filtration rate, normal value: >90 mL/min] Parenteral and enteral feeding Intravenous fluids Antithrombotic prophylaxis with low-molecular weight heparin 13th of October 2015 – yeasts found in urine (1.000.000 CFU/mL), patient developed Epidural and intravenous analgesia symptomatic urinary tract infection, which prompted treatment Antiulcer prophylaxis Antibiotic prophylaxis Yeast infection Yeast infection Yeast infection – typically caused by Candida species ( Candida albicans Due to yeast isolate, the therapy chosen was anidulafungin (a member of being most common) echinocandins), as it covers all Candida sp. Summary of suggested antifungal drugs against treatable pathogenic fungi (The Sanford Guide to Antimicrobial Therapy, 44th Ed., 2014) Afterwards, the pathogen isolated was Candida glabrata – anidulafungin Fluconazole Itraconazole Voriconazole Posaconazole Echinocandin Amphotericin B was continued, as it is the most active drug against resistant Candida species Candida albicans +++ +++ +++ +++ +++ +++ Candida glabrata ± ± + + +++ ++ Candida tropicalis +++ +++ +++ +++ +++ +++ Candida krusei - + ++ ++ +++ ++ Definition: ± possible activity, + active (3rd line therapy), ++ active (2nd line therapy), +++ active (1st line therapy) Echinocandins: anidulafungin, micafungin, caspofungin History of present illness 6th October 2015 – transfer from the ICU to surgical ward Is an echinocandin a reasonable option? 13th of October 2015 – yeasts found in urine (1.000.000 CFU/mL) 15th of October 2015 – patient was seen by clinical pharmacist 1st Intervention Susceptibility results 15th of October – antifungal susceptibility testing results available Treatment of Candida glabrata urinary tract infection with anidulafungin is not appropriate Candida glabrata, 1.000.000 CFU/mL , if we understand pharmacokinetic properties of the drug: Antifungal MIC Sensitivity Excretion of anidulafungin: less than 1% in urine All echinocandins are excreted primarily in the feces. They do not excrete into urine and do Fluconazole 16 µg/mL Sensitive dose-dependent not achieve measurable concentrations in the urine. Therefore, we cannot treat urinary tract infection with anidulafungin (or other echinocandins). Amphotericin B 0.38 µg/mL Sensitive Symptoms of urinary tract infection persisted, treatment was not efficient 1st intervention – cessation of anidulafungin (the cost of drug 410 € per day!) According to Clinical and Laboratory Standards Institute, the breakpoints for fluconazole in Candida sp. are: Susceptible: MIC ≤ 8 µg/mL Susceptible-dose dependent: MIC: 16 – 32 µg/mL Resistant: MIC ≥ 64 µg/mL Treatment of Candida UTI 2nd Intervention Pharmacist recommends against the use of amphotericin B due to: Nephrotoxicity Pharmacist recommends fluconazole 400 mg/day for Candida glabrata High cost (1300 €/day) UTI. Initially, the recommendation is not accepted, as it is believed that we Better treatment alternatives are not able to achieve appropriate levels in urine. Understanding pharmacokinetics of fluconazole, one can see: Candida glabrata has MIC of 16 µg/mL, while fluconazole plasma levels Most of the drug excreted in urine typically achieved are around 8 µg/mL. Usually, we do not treat Candida sp. Fluconazole is concentrated in the urine – yielding urine levels > 100 µg/mL, which is 10-fold with fluconazole, if the MIC is > 8-16 µg/mL. the simultaneous plasma levels. Therefore, the expected concentrations in urine exceed the MIC not only for susceptible Physician recommends treatment with amphotericin B, even though the yeasts (MIC≤ 8 µg/mL), but also for organisms that are susceptible but dose-dependent (MIC 16-32 µg/mL) and sometimes even those that are resistant (MIC ≥ 64 µg/mL) patients has chronic kidney injury and amphotericin B is known to be nephrotoxic (as there is no other option). Reference: Fisher JF et al. Candida urinary tract infections – Treatment. Clinical Infectious Diseases 2011:52 (Supp 6) Rationale for the dose selection Conclusion In normal renal function, we would recommend 800 mg of fluconazole per day. Due to EGFR ~ 35 mL/min, a 50% reduction in the dose is recommended → 400 mg/day After initiation of fluconalzole 400 mg/day, symptoms and signs of urinary tract infection improved Anticipated success is 86% Fluconazole dose, Candida MIC and anticipated success Treatment was continued for 16 days Dose (mg/day) MIC (µg/mL) Dose/MIC Anticipated success (%) 8 100 95 Lessons learned 800 16 50 86 Pharmacokinetics – pharmacist`s strength. Use it! 32 25 72 Echinocandins not excreted in urine Fluconazole concentrated in urine 8 50 86 Fluconazole the drug of choice for Candida urinary tract infection (also for organisms that are 400 16 25 72 susceptible but dose-dependent, and sometimes even those that are resistant) 32 12.5 68 Reference: Pfaller MA et al. Interpretive breakpoints for fluconazole and Candida revisited: a blueprint for the future of antifungal susceptibility testing. Clinical Microbiology Reviews 2006: 435-447. Take-home message Clinical pharmacy services in oncology is of utmost importance Clinical pharmacy developed a number of services specific for oncology patients Clinical pharmacists contribute significantly in improving clinical, humanistic and economic outcomes of pharmacotherapy in cancer patients IMMUNE RECEPTORS OF INNATE AND ADAPTIVE IMMUNITY IMMUNOTHERAPY OF CANCER Assoc. Prof. Matjaž Jeras, Ph.D., M.Pharm. Receptors expressed on or within the cells of innate immunity are evolutionary old, non- University of Ljubljana, Faculty of Pharmacy polymorphic and encoded en-block. They are called Pattern Recognition Receptors (PRRs), because they recognize conserved Pathogen-Associated Molecular Patterns (PAMPs) and cell E-mail: matjaz.jeras@ffa.uni-lj.si Death Associated Molecular Patterns (DAMPs). Receptors expressed od natural killer (NK) cells are not reactive to normal cells, but attack virus infected, tumor and stress-altered cells. NK CELLS MAJOR NK RECEPTORS AND THEIR LIGANDS Chester C, et al. Frontiers in immunology 2015; 6 (Article 601) MECHANISMS OF NK CELL CYTOTOXICITY ANTIGEN PRESENTING CELLS (APCs) ADCC – Antibody Dependent Cell Cytotoxicity DCs ARE LINKING INNATE AND ADAPTIVE IMMUNITY DCs ARE PROFESSIONAL APCs AND CAN ACT IN A BIPOLAR WAY Immature DCs reside in peripheral tissues and are not able to activate T cells (antigen uptake, processing and binding of peptides to MHC molecules, cytokine production). Only mature DCs can fully activate T cells (antigen presentation, expression of costimulatory molecules, cytokine production). Gi M, Im W, Hong S. Sensors 2009; 9(9), 6730-6751 MHC (HLA) MOLECULES HLA POLYMORPHISM Class I HLA molecules: A, B and C (classical) are expressed on all nucleated somatic cells. They bind and present intracellular antigenic peptides (8 – 10 aa) to TCRs present on 16. 8. 2016 Number of alleles CD8+ cytotoxic T-cells (CTLs). HLA class I alleles 11.100 Class II HLA molecules: DR, DQ and DP are constitutively expressed on APCs. They bind HLA class II alleles 3.920 and present extracellular antigenic peptides (at least 13 aa) to TCRs present on CD4+ T- Total 15.020 cells (T helper cells, Th). ANTIGEN PROCESSING AND PRESENTATION EXTREME POLYMORPISM OF ANTIGEN-SPECIFIC T-CELL RECEPTORS (TCRs), ANTIBODIES AND BCRs FUNCTIONAL TCR AND BCR COMPLEXES ACTIVATION OF T LYMPHOCYTES BY MATURE APCs & IMMUNOLOGICAL SYNAPSE ITAM = Immunoreceptor Tyrosine-based Activation Motifs T-CELL ACTIVATION IS A COMPLEX MULTIPLE-SIGNAL CYTOKINES PRODUCED BY APCs INDUCE FUNCTIONAL PROCESS DIFFERENTIATION OF CD4+ Th CELLS Mahoney KM, et al. Nat Rev 2015; 14:561-584 DIFFERENT EFFECTOR ROLES OF T LYMPHOCYTE CD4+ Th CELLS ARE NEEDED FOR FUNCTIONAL SUBTYPES MATURATION OF CD8+ CYTOTOXIC T CELLS (CTLs) CD8+ CYTOTOXIC T CELLS (CTLs) POSITIVE AND NEGATIVE REGULATION OF T CELL IMMUNE RESPONSES Mature effector CTLs kill their target cells only after they recognize specific antigenic peptides bound to class I HLA molecules on their surfaces, via TCRs. CTLs can use several different killing mechanisms: - Ca++ dependent release of modified lysosomes, i.e. lytic granules, that contain perforin, granzymes (a family of serine proteases – proapoptotic actin) and granulysin (antimicrobial and proapoptotic). - binding of Fas ligands (FasL or CD178), members of the TNF (Tumor Necrosis Factor) molecular family, to Fas (CD95) molecules, expressed on target cells → activation of CTLA-4: Cytotoxic T-Lymphocyte Antigen-4 PD-1: Programmed Death-1 caspases (cisteine proteases), leading to apoptosis. IMUNOSSUPRESSIVE FUNCTION OF PD-1 CONSEQUENCES OF INAPPROPRIATE HOMEOSTASIS OF THE IMMUNE SYSTEM While CTLA-4 negatively regulates T-cell immune responses following their initial activation, thereby PD-1 inhibits effector functions of already activated T cells (for example tumor specific T cells). Unlike CD28 molecules, the CTLA-4 ligands, PD-1 ligands, i.e. PDL-1 and PDL-2 are abundantly expressed in tumors, where they inhibit effector T cells that are in a long- lasting contact with tumor antigens. TUMORS CAN EXPRESS BOTH, CO-INHIBITORY AND IMMUNE EVASION MECHANISMS IN INFLAMED VS. CO-STIMULATIORY LIGANDS NON-INFLAMED CANCERS Inflamed Non-inflamed Mahoney KM, et al. Nat Rev 2015; 14:561-584 Gajewski, Schreiber, Fu. Nat Immunol 2013 IMMUNOLOGICAL PROPERTIES OF SOMATIC MUTATION PREVALENCES IN DIFFERENT TUMOR TISSUES TYPES OF CANCER (IMMUNOGENICITY) Tumors are more or less immunogenic. Some tumor specific (TA) and tumor associated antigens (TAA) are well defined, and many are not. Melanoma is the most immunogenic cancer. Often, the immunogenicity of TA and especially TAA is weak,. Additionally their expression is largely fluctuating and their coding genes are prone to freequent mutations. Tumor cells seldom lose the capability to express certain HLA molecules or even a whole HLA haplotype, they lack costimulatory molecules and produce suppressive (anti- inflammatory) cytokines (VEGF, TGF-β, IL-10, IL-4), chemokine CCL22, which attracks and cumulates regulatory T cells (Tregs) expressing its receptor (CCR4), and indolamine- 2,3,dioxyigenase (IDO). Tumors are additionally protected against the immune system by various types of immunosuppresive cells: Treg, Tr1, Th3, Ts (CD8+), tumor-associated macrophages (TAM or M2) and tolerogenic DCs (DC2). Important immunosuppressors are also myeloid-derived suppressor cells (MDSCs) and even NK cells (IL-10, TGF-β, arginase, iNOS). Schumacher TN, Schreiber RD. Science 2015; 348:69-74 MYELOID-DERIVED SUPPRESSOR CELLS (MDSCs) DC POLARIZATION DEPENDS ON THE NATURE OF THEIR MICROENVIRONMENT MDSCs are not completely differentiated/matured myeloid cells which are morphologically and phenotypically similar to monocytes and neutrophils. They are being attracted into tumors, where they actively protect tumor cells against immune responses, remodulate microenvironment, establish pre-metastatic niches and via direct interactions with tumor cells induce their stemness. MDSCs produce numerous immunosuppressive factors (arginase, iNOS, TGF-β, IL-10, COX2) and induce immunosuppressive natural regulatory T cells (Treg). They are susceptible to gemcitabine, 5-fluorouracil and irradiation. IMUNOSUPPRESSIVE ACTION OF ARGINASE AND IDO CELLULAR IMMUNOTHERAPIES OF CANCER Ex vivo selection and expansion of T lymphocytes isolated from tumor biopsies (TILs – Tumour Infiltrating Lymphocytes) → adoptive cell transfer/ adoptive immunotherapy. In vitro preparation of antigen-specific effector CD8+ CTLs → adoptive cell transfer/adoptive immunotherapy. In vitro preparation of anti-tumor cellular vaccines Indolamine-2,3-dioxygenase (IDO) is an enzyme based on DCs. that degrades typtophan, an essential aminoacid , into kinurenine. In vitro preparation of autologous NK cells. Lymhocytes T are highly susceptible to tryptophan levels → when these are low, they + NONMYELOABLATIVE CONDITIONING stop proliferating. + IMMUNE CHECKPOINT BLOCKADE! NONMYELOABLATIVE CONDITIONING OF PATIENTS THE IMMUNE CHECKPOINT BLOCKADE THERAPY PRIOR TO CELLULAR IMMUNOTHERAPY A: in a non-pre-conditioned patient adoptive transfer of tumor-specific CD8+ CTLs is ineffective → TA/TAA are presented by immature DCs lacking costimulatory elements → only very small amounts of proinflammatory cytokines are available and consumed by B, T and NK cells (cytokine sinks) → suppressive action of Treg, MDSCs and NK cells via IL-10, TGF-β, arginase, iNOS and direct intercellular contacts. B: in a pre-conditioned patient (chemotherapy, tumor irradiation) the numbers of lymphocytes, MDSCs, APCs (TAMs, DC2), therefore the pro-inflammatory conditions enable maturation of DCs into DC1 → more effective activation of T cells with TA/TAA and higher amounts of proinflammatory cytokines → apoptotic TCs are better targets for specific anti-tumor effector CD8+ CTL clones. PREPARATION OF NATURALLY OCCURING THE IMMUNE CHECKPOINT BLOCKADE AUTOLOGOUS TILs FOR ADOPTIVE CELL TRANSFER At least 1cm3 + rhIL-2 e.g. ELISPOT Kyi C, Postow MA. FEBS Letters 2014; 588:368-376 Rosenberg SA, Restifo NP. Science 2015; 348:62-68 LYMPHODEPLETION PRIOR TO T-CELL ADOPTIVE CLINICAL RESPONSES TO ADOPTIVE TRANSFER OF TRANSFER AUTOLOGOUS TILs INTO METASTATIC MELANOMA PATIENTS Rosenberg SA, Restifo NP. Science 2015; 348:62-68 Rosenberg SA, Dudley ME. Current Opinion in Immunology 2009, 21:233–240 SURVIVAL OF TIL-TREATED METASTATIC MELANOMA IN VITRO PREPARATION OF TA/TAA-SPECIFIC CD8+ PATIENTS DEPENDS ON THE INTENSITY OF CTL CLONES FOR ADOPTIVE CELL TRANSFER THEIR PRE-CONDITIONING For in vitro stimulation of autologous lymphocytes we can use peripheral blood monocyte-derived DCs or artificial APCs, which express defined HLA class I molecules presenting specific TA/TAA peptides and costimulatory molecules. Artificial APCs Effector CTLs are expanded following multiple antigen re- stimulations in appropriate cell culture medium containing proinflammatory cytokines (e.g. IL-2). In case of DCs, specific TA/TAA can be provided in different ways: • by incubating immature DCs with tumor cell (TC) lysates or apoptotic TCs; • by incubating them with defined TA/TAA-specific immunogenic peptides (considering HLA alleles); • by transfecting them with total tumor RNA, either native or amplified; • by preparing immunohybridomas → electrofusion of Legend: immature DCs and lethaly irradiated TCs. NMA – non-myeloablative chemotherapy (cyclophosphamide, fludarabine) TBI – total body irradiation Finally antigen-loaded DCs are fully matured, using different activation factors and applied as stimulators of autologous lymphocytes. Rosenberg SA, Dudley ME. Current Opinion in Immunology 2009, 21:233–240 PREPARATION OF T-CELLS RECOGNIZING TUMOR- STRATEGIES FOR TARGETTING PATIENT-SPECIFIC SPECIFIC MUTATIONS TUMOR NEOANTIGEN REPERTOIRE Rosenberg SA, Restifo NP. Science 2015; 348:62 Schumacher TN, Schreiber RD. Science 2015; 348:69-74 INTERACTIONS OF NK AND TUMOR CELLS PREPARATION OF AUTOLOGOUS NK CELLS FOR IMMUNOTHERAPY NK cells are obtained from leukapheretic units after removal of lymphocytes and other cells. Then they are expanded and activated in vitro. This process lasts for 2-3 weeks while the cells are being cultivated in an appropriate medium containing NK-activating cytokines (e.g. IL-2). Tarazona R, et al. Cancer Immunol Immunother 2016; DOI 10.1007/s00262-016-1882-x T CELLS BEARING CHIMERIC ANTIGEN RECEPTORS DEVELOPMENT OF NEW GENERATIONS OF CARs (CARs) Minagawa K, et al. Pharmaceuticals 2015; 8:230-249 PROPERTIES OF CHIMERIC ANTIGEN RECEPTOR PSMA-SPECIFIC CAR- ENGINEERED T CELLS (CAR) ENGINEERED T CELLS ERADICATE DISSEMINATED PC IN NOD/SCID MICE Living drugs. Long-living cells (months/years). Traffic everywhere (including CNS). Make bioactive cytokines/engage other cells. Activity (on target and off target) occurs anywhere and at time of peak expansion. Multiple different CAR designs - new ones to reduce toxicity and extend to different tumors are on the horizon. Zuccolotto G. et al. PLoS One. 2014; 9(10):e109427 IN VITRO PREPARATION OF DCs FROM HUMAN PERSONALIZED CELLULAR IMMUNE THERAPY PERIPHERAL BLOOD MONOCYTES Providing TA/TAA to immature DCs Tolerogenic DCs (TA/TAA pulsing, RNA transfection , immunohybridomas) IL-10; TGF-β; GM-CSF, IL-4 corticosteroids, ... Immature DCs Monocytes Immature DCs Mature DCs LPS; TNF-α; MCM, … GM-CSF & IL-4 Monocytes 5-7 days Antigen uptake DC maturation: TA/TAA: IL-1 , TLR agonists tumor RNA, TCs, TC lysates β, IL-6, TNF-α, PGE2 Fully matured DCs PBMCs Vaccine application Leukapheresis Tumor biopsy SIPULEUCEL IMMUNOTHERAPY (FDA APPROVED) A minimum of 50 x 106 autologous CD54+ cells (T-cells, B-cells, APCs, eosinophiles). Legend: PAP – prostate acid phosphatase; GM-CSF – granulocyte and monocyte colonies stimulating factor Drake et al, NatureReviews/Immunology, 2010 SIPULEUCEL-T: RESULTS OF THE “IMPACT” PHASE 3 ANTI-CEA EFFECTOR CTLs, GENERATED BY DCs TRIAL TRANSFECTED WITH COLON CANCER RNA Target cells: HLA A*02:01 positive T2 cells, pre-incubated with the CEA-specific peptide YLSGANLNL, that fits to HLA A*02:01 antigen-binding groove. 4.1 month survival benefit Reduction in risk of death: 22.5% HR = 0,775 (95% Cl: 0.614, 0.979) P=0.032 Percentages of anti-CEA specific CTL clones, assesed by using pentamers (HLA A*0201/ YLSGANLNL) and flow cytometry. Kantoff MD. et al. N Engl J Med 2010; 363(5):411-422 Immunobiology 2006; 211:179-189 Caco-2 SPECIFIC CTLs INDUCED IN VITRO BY USING IMMUNOHYBRIDOMA YIELDS ASSESSED WITH IMMUNOHYBRIDOMAS MADE OF DCs AND Caco-2 CELLS CONFOCAL MICROSCOPY AND FLOW CYTOMETRY J Membrane Biol 2009; 229:11–18 BBRC 2004; 314:717-723 NON-CELLULAR PROSTVAC VF VACCINE: A PSA-SPECIFIC PROSTVAC EFFICACY IN PATIENTS WITH CRPC - A PHASE 2 IMMUNOTHERAPY CLINICAL STUDY 8.5 month survival benefit Reduction in risk of death: 44% HR: 0.56 (95% CL: 0.37-0.85) P=0.0061 Viral vectors are injected intradermally to infect and destroy EC. Legend: PSA – prostate-specific antigen; LFA3 and ICAM1 – adhesion molecules Drake et al, NatureReviews/Immunology, 2010 Kantoff MD. et al. J Clin Oncol 2010; 28(7):1099-1105 THERAPEUTIC ANTI-TUMOR VACCINES VS. CONVENTIONAL CYTOTOXIC THERAPY Future: Immunotherapies should be used early in the onset of malignant disease. Combinations of different therapies to be applied at the right time and in the right sequence. Outline • Background: life science Life science research from a • UK Arthritis research postdoc: my experience postdoc prospective • Conclusions: tips for applying for a postdoc Janja Zupan CEEPUS Summer school 2016 Ljubljana, 21.08.2016 Life science Biology Biomedicine Medicine Francis Crick and James D. Watson, Alexander Fleming, Penicillin discovery, 1928 Nobel Prize for DNA double helix structure, 1962. Translational medicine Kary Banks Mullis, Nobel Prize for the PCR, 1989 Johnson IS, Human insulin from recombinant DNA technology, Science 1983. Publicly funded research Governmental sector Research councils construction $150 billion $5 billion per year Charitable sector National Institute for > 130 medical research charities Health Research Programme Life science in UK Privately funded research https://financesonline.com/10-worlds-most-expensive-science-experiments/ Elizabeth Klein, The state of the UK helalthcare and life sciences sectors, 2016, http://www.euromedica.com/wp-content/uploads/2016/02/Final_Report_-_State_of_the_UK_Healthcare_Sector_.pdf http://www.cancerresearchuk.org/funding-for-researchers/facts-and-figures-about-our-research-funding Working in life science Searching for a postdoc My experience in finding postdoc UNIVERSITY OF ABERDEEN COLLEGE OF LIFE SCIENCES AND MEDICINE SCHOOL OF MEDICINE AND DENTISTRY DIVISION OF APPLIED MEDICINE RESEARCH FELLOW YMD033RX • Started working as a researcher in 2007 FURTHER PARTICULARS FOR APPLICANTS • 2007-2012 Osteoimmunology and ECTS conferences: active participation • 2011 ECTS PhD course Ljubljana • 2011 ECTS PhD exchange scholarship grant at IGMM, University of Edinburgh • Finished PhD in Clinical biochemistry and laboratory medicine University of Ljubljana 2012 • 2012-2014 applying for several postdoc positions • Largest research institute at University of Aberdeen • 411 researchers and support staff: – 84 principal investigators – 106 post-doctoral scientists and academics – 75 technicians – 140 PhD students • £30 million in research funding raised in the last 12 months • published 415 peer-reviewed papers Regenerative medicine group Arthritis and MSK research groups: • musculoskeletal pharmacology • bone and cartilage cell biology • imaging and joint function • musculoskeletal regenerative medicine and tissue engineering • muscle physiology • cohort studies and clinical trials • nutrition • genetics of bone diseases Mesenchymal stem cells (MSCs) in adult joint Understanding the ontogeny of mesenchymal stem cells in the joint to develop tools to study osteoarthritis • a painful and debilitating disease that causes irreversible damage to cartilage and bones • cartilage ‘cushion’ between the bones of the joint gradually erodes, leading to rubbing of bone on bone • 1/6 people are affected by osteoarthritis in the UK • the most commonly affected joints are knees, hips, spine and hands • no cure or preventive treatment • age, obesity and joint injury are known risk factors • pain killers and joint replacement therapy De Bari et al Arthritis Rheum. 2001 Aug;44(8):1928-42 Kurth et al Arthritis Rheum. 2011 May;63(5):1289-300 Ontogeny of MSCs in the adult knee joint Gdf5-Cre;Tomato mouse model • developmental origin of MSCs in adult knee joint • synovial and BM MSCs De Bari et al. Birth Defects Res C Embryo Today 2010 Dec;90(4):257-71 Hypothesis: MSCs in the adult knee joint synovium descend from the Gdf5- expressing mesenchymal cells of the embryonic joint interzone. Aim: fully characterize the subpopulations of the MSCs that are the Gdf5-Cre;lacZ mouse progeny of Gdf5 expressing mesenchymal cells. Rountree et al, Plos Biol. 2004 Nov;2(11):e355 Koyama et al, Dev Biol. 2008 Apr 1;316(1):62-73.2008 Immunophenotyping of freshly isolated In vitro analysis of whole knee joint sorted cells Gdf5-Cre;Tomato mouse whole knee joint cells Gdf5-Cre;Tomato Gdf5-Cre;Tomato whole knee joint 50mins collagenase digestion Whole knee 50mins collagenase digestion dissection joint dissection hindlimbs hindlimbs Whole knee joint cell isolation: Armaka et al Nat Protocol 2009 Whole knee joint cell isolation: Armaka et al Nat Protocol 2009 FACS unsorted for Tom Tom + Tom - Single live cells Single cells Whole knee joint cells Cell culture In vitro multipotency of Gdf5 progeny cells The fate of the Gdf5 progeny cartilage pellets in vivo sisenegrodnohC TB orit sis v en In eg Col2 o Tom ip DAPI dA TB o sis iv e v n In ego Col2 Tom ste DAPI O Transplantation of Gdf5 progeny cells after JSI Triple transgenic mouse model Gdf5-Cre;Tom; Nes-GFP moT GFP Tips for finding a postdoc • Attend conferences and meetings • Professional networking • CV, cover letter, PPT Acknowledgements Professor Janja Marc Professor Cosimo De Bari Professor Darko Černe Dr Anke Roelofs Professor Barbara Ostanek Susan Clark Dr Simona Mencej-Bedrac Anna Riemen Dr Vid Mlakar Dr Ana Sergijenko Dr Peter Vrtacnik Dr Tiziana Franceschetti Dr Jana Dragojevic Dr Sharon Ansboro Dr Darja Bitenc-Logar Dr Hui Wang Dr Zoran Trost Dr Shahida Shahana Professor Janez Prezelj Denise Tosh Professor Radko Komadina Ausra Lionikiene Asist. Franci Vindisar Agathe Moraine Asist. Rihard Trebse Simon Herman Iain Fraser Cytometry Centre Gregor Haring Microscopy and Histology Core facility Professor Andrej Cör Professor Rob van 't Hof Thank you! Document Outline Malignant diseases - CEEPUS Summer school 2016.pdf Dumić Kotur-Stevuljevic biochemistry of cancer cells Osredkar - Tumor markers in clinical practice - August 2016 Prnjavorac - CEPUS Ljiubljana Prnjavorac Mrhar - CEEPUS Summer School 2016 Mrhar Presentation JERAS - IMMUNOTHERAPY OF CANCER Summer School 2016 Zupan - Life science research from a postdoc prospective