Radiol Oncol 2020; 54(2): 135-143. doi: 10.2478/raon-2020-0025 135 review Current and innovative approaches in the treatment of non-muscle invasive bladder cancer: the role of transurethral resection of bladder tumor and organoids Milena Taskovska1,2, Mateja Erdani Kreft3, Tomaz Smrkolj1,2 1 Department of Urology, University Medical Centre Ljubljana, Ljubljana, Slovenia 2 Department of Surgery, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Institute for Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2020; 54(2): 135-143. Received 19 January 2020 Accepted 30 January 2020 Correspondence to: for organoids – Prof. Mateja Erdani-Kreft, Ph.D., Institute for Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia. E-mail: mateja.erdani@mf.uni-lj.si Correspondence to: for monopolar/bipolar TURB – Assoc. Prof. Tomaž Smrkolj, M.D., Ph.D., Department of Urology, University Medical Centre Ljubljana, Zaloška 7, 1000 Ljubljana, Slovenia or Department of Surgery, Faculty of Medicine, University of Ljubljana, Zaloška 7, SI-1000 Ljubljana, Slovenia. Email: tomaz.smrkolj@kclj.si Disclosure: No potential conflicts of interest were disclosed. Background. Bladder cancer is the 7th most common cancer in men. About 75% of all bladder cancer are non- muscle invasive (NMIBC). The golden standard for definite diagnosis and first-line treatment of NMIBC is transurethral resection of bladder tumour (TURB). Historically, the monopolar current was used first, today bipolar current is pre- ferred by most urologists. Following TURB, depending on the tumour grade, additional intravesical chemo- or/and immunotherapy is indicated, in order to prevent recurrence and need for surgical resection. Development of new technologies, molecular and cell biology, enabled scientists to develop organoids – systems of human cells that are cultivated in the laboratory and have characteristics of the tissue from which they were harvested. In the field of urologic cancers, the organoids are used mainly for studying the course of different diseases, however, in the field of bladder cancer the data are scarce. Conclusions. Different currents - monopolar and bipolar, have different effect on urothelium, that is important for oncological results and pathohistological interpretation. Specimens of bladder cancer can be used for preparation of organoids that are further used for studying carcinogenesis. Bladder organoids are step towards personalised medi- cine, especially for testing effectiveness of chemo-/immunotherapeutics. Key words: bladder cancer; transurethral resection of bladder tumour; monopolar/bipolar current; organoids, mito- mycin C; BCG Introduction Bladder cancer is 7th most common cancer in men. In the European Union (EU) age-standardised in- cidence rate is 19.1 for men and 4.0 in women.1 In Europe, bladder cancer incidence is 27.1 and mortality 8.9.2 The highest incidence rate in the EU is reported in Belgium and lowest in Finland. On the global level, incidence and mortality rates vary due to different methodologies and diagnos- tic practices.1,2 Approximately three-quarters of all bladder cancer are non-muscle invasive (NMIBC) – the disease is confined to the mucosa (stage Ta, CIS) and submucosa (stage T1) (Table 1, Figure 1), in patients younger than 40 years this propor- tion is even higher.1,3-5 According to the data from Cancer registry of the Republic of Slovenia, blad- der cancer is the 8th most common cancer in men and the 13th most common when both genders are considered. Age standardised incidence rate is Radiol Oncol 2020; 54(2): 135-143. Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer136 11.74 in men and 2.97 in women. Age-standardised mortality rate is 5.46 in men and 1.52 in women.6 Although many European countries experience incidence rise, the projected growth rate of blad- der cancer incidence rates by 2030 in Slovenia is extremely high, i.e. 92% for men and 256% for women. Standard treatment of NMIBC is transurethral resection of bladder tumour (TURB). Depending on the histopathological tumour characteristics, additional treatment with intravesical chemothera- peutics or immunotherapeutics is indicated. The aim of intravesical therapy is to decrease the rate of recurrence and need for surgical intervention.1 Organoids are 3D models which consist of cells derived from specific tissue or organ and are grown in the laboratory with the aim to study different cell biological mechanisms, homeosta- sis, development of disease and effect of different medications. Organoids have characteristics of the cells from which are derived, although differences could appear because of the effect of microenviron- ment.7 There are some new data about organoids used to study oncogenesis and different treatments in urological cancers (prostate, kidney, bladder).8 Aim of this review is to present different mo- dalities of TURB for treatment of NMIBC, and the role of bladder/urothelium organoids for studying oncogenesis, therapeutic modalities and personal- ised medicine in NMIBC. Transu rethral resection of bladder tumour (TURB) TURB is a golden standard in diagnosis, treatment, and staging of NMIBC as well as in diagnosis of muscle invasive bladder cancer (MIBC).1,9 The first published report involving the application of elec- tric current for endoscopic resection of papillary bladder tumours through cystoscope originates in 1910s. The initial case was performed using water as medium. Since then, TURB has become standard in evaluation and treatment of patients with bladder cancer.9 Guidelines of three urologic associations (European Association of Urology, EAU; American Urology Association, AUA; and Canadian Urology Association, CUA) emphasize the importance of TURB for diagnosis, staging and treatment of NMIBC (1, 9, 10, 11).1,9,10,11 TABLE 1. 2017 TNM classification of urinary bladder cancer 5 T - primary tumour TX Primary tumour cannot be assessed T0 No evidence of primary tumour Ta Non-invasive papillary carcinoma Tis Carcinoma in situ: ‘flat tumour’ T1 Tumour invades subepithelial connective tissue T2 Tumour invades muscle T2a Tumour invades superficial muscle (inner half) T2b Tumour invades deep muscle (outer half) T3 Tumour invades perivesical tissue T3a Microscopically T3b Macroscopically (extravesical mass) T4 Tumour invades any of the following: prostate stroma, seminal vesicles, uterus, vagina, pelvic wall, abdominal wall T4a Tumour invades prostate stroma, seminal vesicles, uterus or vagina T4b Tumour invades pelvic wall or abdominal wall N - regional lymph nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in a single lymph node in the true pelvis (hypogastric, obturator, external iliac, or presacral) N2 Metastasis in multiple regional lymph nodes in the true pelvis (hypogastric, obturator, external iliac, or presacral) N3 Metastasis in common iliac lymph node(s) M - distant metastasis M0 No distant metastasis M1a Non-regional lymph nodes M1b Other distant metastases Radiol Oncol 2020; 54(2): 135-143. Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer 137 TURB is a relatively safe procedure with few complications, in some countries is performed in an outpatient setting. The most common compli- cations are haematuria and urinary tract infection. One of the most important aims of TURB is to re- sect the whole tumour (depending on the size and invasion in muscle layer) and resect muscle layer in order to obtain the correct pathological stage.1,10,11 Since its introduction in the 1910s, technology has developed and so has equipment and technique of TURB.9 In 1900 Joseph Rivere discovered that a spark arcing from an electrode coagulates skin and he used it for treatment of skin lesions. In the fol- lowing decades, this technique was used to treat skin lesions, lesions in the oral cavity, bladder, co- agulation of vascular tumours and haemorrhoids. In 1920s Clark was one of the first who observed the tissues exposed to current under a microscope and found that they shrink from dehydration. Bovie constructed the first diathermy unit that was used for cutting, coagulation, and dissection and was first used on October 1st 1926 in Boston. Since then, this instrument is used in everyday surgical practice (12).12 The clinical effect of electrocautery is a con- sequence of heat. When oscillating current is ap- plied to the tissue, the rapid movement of elec- trons through the cytoplasm causes an increase of intracellular temperature. The effect on the tissue depends upon the amount of thermal energy deliv- ered and the time rate of delivery. Temperature be- low 45°C causes reversible thermal damages, when increased it causes denaturation and loss of protein structure, above 90°C liquid evaporates, resulting in vaporization if heated rapidly or desiccation if heated slowly. Temperatures over 200°C cause car- bonization.12 Electric energy could be monopolar or bipolar. Monopolar energy delivery requires the current to pass from the generator to the active electrode through the patient and out of the body through a dispersive electrode pad which is connected to the generator in order to complete the circuit. On the other hand, bipolar delivery does not require a dispersive return electrode because both active and return electrodes are integrated into energy delivery forceps with target tissue between them (Figure 2).12 Monopolar electrocautery for TURB In urology, TURB was introduced in the 1910s by Beer using monopolar current for fulguration (Figure 2).13,14 Monopolar electrocautery requires high energy and voltage to allow current trans- mission from the loop to the tissue. In monopolar electrocautery glycine is used as a medium that is associated with limitations, such as short resection time due to the risk of development of TURB syn- drome. The heat generated while cutting the tissue causes damage to the surrounding cells. Because of the thermal artefacts, pathologic assessment of the specimen is sometimes difficult.13 There are stud- ies which compared monopolar and bipolar elec- FIGURE 1. Classification of bladder cancer. MIBC = Muscle invasive bladder cancer; NMIBC = Non muscle invasive bladder cancer FIGURE 2. Monopolar vs. bipolar transurethral resection of bladder tumor. Radiol Oncol 2020; 54(2): 135-143. Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer138 trocautery for TURB and concluded that there is very little or no difference in thickness of thermal artefacts.14 One study has shown mean depth of thermal artefacts of 0.237 mm when using bipolar electrocautery and 0.26 mm when using monopo- lar electrocautery.9 Monopolar electrocautery is not inferior to bipolar electrocautery regarding in- tra and postoperative bleeding, and perforation of bladder wall (Table 2).14 Bipolar e lectrocautery for TURB Bipolar electrocautery has been introduced about 30 years ago and a few years after its introduction has gained popularity over monopolar electro- cautery.12 Bipolar electrocautery was first used for transurethral resection of the prostate (TURP) for benign prostate enlargement and obstruction. It was quickly adopted for TURB because of advan- tages: (1) isotonic medium could be used (such as saline), (2) electric circuit is completed using only resection loop and sheath of the device itself, so the patient is not included in the circuit, (3) risk for TUR syndrome is low, (4) time for resection is ex- tended, which is of essential importance in case of large bladder tumours, (5) incidence of obturator jerk is lower, (6) there are fewer bladder perfora- tions. It is also related to fewer postoperative com- plications such as clot retention and contracture of the bladder neck (Table 2).9 One of the main differences between monopolar electrocautery and bipolar electrocautery systems is the coagulation mechanism. In bipolar electro- cautery system voltage is low and energy dissi- pates as heat in the tissue leading to the formation of coagulum and haemostasis.13 In monopolar electrocautery, electrical injury directed into the tissues and electrical resistance creates temperature as high as 400°C which causes tissue desiccation and collateral tissue damage. Radiofrequency energy of bipolar electrocautery systems converts the conductive medium into a plasma field with highly ionised particles that disrupt the organic bonds between tissues and al- lows thermal effect to occur at lower temperatures (between 40 and 70°C). Pathologists sometimes classify thermal damage into three categories: (1) cautery artefact less than 1/3 of the specimen, (2) cautery artefact of 1/3 to 2/3 of the specimen and (3) over 2/3 of the specimen.13 Although different elec- trocautery systems are used for more than 30 years we have no data on the effect of different electro- cautery systems on cellular level. Adjuvant tr eatment of NMIBC Intravesica l chemotherapy TURB is used as definitive therapy in TaT1 tumours. The adjuvant treatment of NMIBC is indicated when dealing with high grade NMIBC. Immediate single intravesical instillation of chemotherapy is used to destroy circulating tumour cells after TURB and has an ablative effect on residual tumour cells at the resection site and on small overlooked tu- mours. Immediate single intravesical instillation should be performed within the first 24 hours after TURB to maximise its effect. Meta-analyses were performed which have shown that after immediate single intravesical instillation the recurrence rate is lower. Recent reviews and meta-analysis have shown that immediate single intravesical instilla- tion also reduces 5-year recurrence rate.1,15 The most commonly used chemotherapeutics for immediate single intravesical instillation are mitomycin C (MMC), epirubicin and pirarubicin.1 In Slovenia, we use MMC for immediate single in- travesical instillation. In literature, there is data that further repeat instillations of chemotherapy also have an impact on the recurrence rate. The length and frequency of chemotherapy instillation are still controversial. According to data available, the length of this treatment should not exceed one year.1,16 There is evidence that intravesical chemotherapy combined with microwave-induced hyperthermia in high-risk patients has enhanced efficacy – it has improved recurrence-free survival at 24 months. There are also undergoing trials regarding use of different methods of hyperthermic intravesical chemotherapy and electromotive drug administra- tion but data about their efficacy is still lacking.1,17 In Slovenia, we are not using combinations with microwave, hyperthermia or electromotive drug TABLE 2. Comparison of monopolar and bipolar current for TURB1.9-14 Variable Monopolar Bipolar Dispersive electrode pad yes no Energy high low Voltage high low Working medium glycine saline Temperature at thermal effect (°C) 400 40–70 Time of resection limited extended (not strictly limited) TUR syndrome common rare Obturator jerk common rare Quality of haemostasis and coagulum poor good Radiol Oncol 2020; 54(2): 135-143. Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer 139 administration, since the use of these combinations is not confirmed by randomised controlled trials. Intravesica l bacillus Calmette-Guerin (BCG) immunotherapy There is evidence in the literature that BCG after TURB is superior to TURB alone or in combina- tion with chemotherapy for preventing recurrence of NMIBC. BCG maintenance therapy reduces the recurrence rate for 32% in comparison to MMC, but increases risk for 28% in patients who did not receive maintenance therapy with BCG.1,18 The main disadvantage of BCG intravesical immuno- therapy is in its side effects. According to data in the literature, serious side effects are encountered in less than 5% of treated patients. Side effects are a consequence of systemic absorption of BCG.1,19 Caution is needed in immunocompromised pa- tients, although some studies have not shown that immunocompromised patients are more prone to experience side effects.1 For BCG instillation is used 6-week schedule introduced by Morales.1,20 Many studies were conducted but none has shown advantages or disadvantages of this schedule com- pared to others.1,21 Meta-analysis has shown that at least one year of maintenance BCG is required to obtain superiority of BCG over MMC for preven- tion of recurrence or progression. Regarding BCG dose, studies have shown that one-third dose is re- quired to be effective for intermediate-risk tumors, full dose is needed for high-risk tumors.1,22 According to the EAU guidelines on NMIBC, BCG intravesical immunotherapy is recommended to patients with intermediate and high-risk tu- mors, three year maintenance therapy is more ef- fective in patients with high-risk tumors to prevent recurrence.1 There are cases when BCG intravesical chemo- therapy fails: (1) muscle invasive bladder can- cer (MIBC) detected during follow-up, (2) BCG- refractory tumor - (a) if high-grade NMIBC is de- tected at three months, (b) CIS is present at three and six months and (c) high grade tumor is de- tected after BCG therapy. Therefore, radical cystec- tomy is indicated.1 Personalize d treatment - precision medicine in the treatment of bladder cancer The efficacy of cancer management is challenging and depends upon genomic, molecular and immu- nologic characteristics of cancer. New discoveries in these fields are making cancer treatment more targeted and efficient. Concerning bladder cancer, mutations in genes of DNA repair pathway (e.g. ERCC2, FANCC, ATM, RB1 and etc.) can predict response to neoadjuvant platinum-based systemic chemotherapies. Therapies that influence the im- mune system such as immune checkpoint inhibi- tors (PDA, PDL1, CTLA4) are approved for use in treatment of bladder cancer and represent renais- sance in medical oncological treatment of this dis- ease.23 Cancer treatment based on the response of blad- der cancer organoid could contribute to more per- sonalized approach in the treatment of this disease. Role of org anoids in studying bladder cancer Organoids I n order to understand the role of cancer-specific genetic alterations in tumorigenesis, maintenance of tumor and sensibility age-standardized response to different therapeutics, development of in vitro and in vivo model systems that accurately reflect genetic diversity and lineage specificity of can- cer was required.7,8 For this purpose, cell lines are used. Their disadvantages is that they are mainly long term 2D cultures, and there is a lack of clini- cal data regarding the organ of origin. To overcome these disadvantages in vivo models are used.8 The advantage of in vivo models is that they are able to recapitulate histological and therapeutic response but there are species-specific differences and inac- curate recapitulation of in vivo human tumour biol- ogy that are main disadvantage.7,8 In combination with organoids derived from normal cells, tumour cell organoids can be used to study transformation from normal to malignant, when exposed to differ- ent carcinogens.8 Sato et al. in 2009 discovered that single leucine- rich repeat containing G-protein coupled recep- tor 5-positive intestinal stem cell is able to gener- ate a continuously expanding, self-organizing, physiological epithelial structure that was similar to normal gut tissue. It was named organoid cul- ture.24 Liu et al. demonstrated that combination of ROCK inhibitor and feeder fibroblast culture conditions enables the infinite growth of multiple primary human epithelial cell types. Based on this, organoids from normal and tumour cells might be able to proliferate indefinitely in vitro, with no need to transduct exogenous viral or cellular genes Radiol Oncol 2020; 54(2): 135-143. Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer140 (Figure 3).8,25,26 Organoids are nowadays used for research of different cancers.7,8 In the field of urol- ogy there are studies/reports on kidney, prostate and bladder organoids.8,27,28 3D organoids could be derived from cell lines, primary tissues, induced pluripotent stem cells, embryonic stem cells and embryonic whole organs such as organ explants that consist of many tissue types. Organoid is composed of multiple cell types and contains multicellular organ structures, which mimic the tissue of origin and functions in a similar manner. Organoids can be generated in different manners.7,8 Cell lines In 3D culture conditions, some immortalized cell lines are able to form polarized 3D structures.8 Smith et al. cultured human bladder cells that were used to investigate how terminally differentiated human urothelial cells interact with uropathogenic E.coli. They cultured 5637 cells under microgravity conditions within a rotating wall vessel bioreactor. Under these conditions, cells remain in suspension and form organoids that reflect characteristics of in vivo tissue-specific determination. Human bladder cells in this model had developed into a model that expresses specific markers and structures, charac- teristic of differentiated human urothelium.8,29 Lee et al. have shown that patient-derived blad- der tumour organoids have most characteristics of parental tumour, although in a culture they may change their marker phenotype. Organoids could be used for studying the biology of the tumour and the effect of different therapies on tumour of indi- vidual patient.7 In the future, these models could be used for personalized treatment of the patient according to tumour characteristics.7 FIGURE 3. Shematic presentation of bladder organoid preparation. Sample of bladder wall (healty and /tumorous) is taken with transurethral resection of bladder tumour. The cells are cultivated under special conditions in laboratory to form organoids. Organoids are used for studying the characteristics of normal and tumorous bladder wall, pathogenesis, response to different treatment approaches. This is step toward personalized treatment of bladder cancer. Radiol Oncol 2020; 54(2): 135-143. Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer 141 Primary adu lt stem cells Primary adult stem cells could also be used for or- ganoids.8 Matulay et al. collected bladder tumour specimens during cystoscopy, enzymatically di- gested them into single cells and cell clusters and then cultured them in organoid-prompting, em- bedded-cell culture conditions. They used these bladder organoids to analyse genetic mutations in bladder cancer.8,30 Pluripotent stem cells Pluripotent stem cells have the ability to form all cellular components of an organ (epithelial, stro- mal and endothelial cells). Organoid could be generated from embryonic or induced pluripotent stem cells (iPSC). Using organoid 3D system, sci- entists are able to induce pluripotent stem cells to develop into organoids of the desired organ. Until 2014 there were two protocols to induce human embryonic stem cells or human iPSC into urothe- lium.8 Osborn et al. have developed in vitro culture system that was matrix-free and cell-contact-free, and was able to induce human embryonic stem cells or human iPSC to differentiate into defini- tive endoderm and then urothelium using directed differentiation in urothelium specific medium.8,31 Kang et al. have developed a protocol that used a chemically defined culture system to induce hu- man iPSC to differentiate into endoderm and blad- der urothelial cells.8,32 Application of organoids in diagnosis and management of bladder cancer Organoids could be used to predict the response to treatment and guide the medicine regimen. For colorectal cancer, 3D organoid based drug sensitiv- ity screen was developed. It identifies molecular signatures associated with altered drug responses. A similar strategy could be used with bladder can- cer organoids.33,34 Resistance to chemotherapy is promoted by cancer stem cells. Models with origin from cancer stem cells may be helpful for identify- ing effective prognostic biomarkers and for indi- vidual treatment.8 In case of prostate cancer, there are already organoids being used to test different drugs, even further, there are models developed to screen for prostate cancer instead of cored biopsy, so-called liquid biopsy.8,30,36 Matulay et al. have established urothelial cancer organoids from patient-derived tissue samples. Using DNA sequencing analysis, they have shown that organoid lines have similar mutational pro- files to those of tumour sample and can provide a platform for personalized drug-response assays in urothelial cancers.8,30 There are few studies aim of which was to identify bladder cancer stem cells, but the results are inconclusive.8 In one study, researchers have described Sonic hedgehog (Shh) expressing and cytokeratin-5 (Ck5) expressing basal urothelial cells from ShhCreER/WT; R26mTmG/WT mice in organoid culture. Those were multipotent stem cells capable of self-renewal and regeneration in- to all cell types within urothelium in response to chemical injury or bacterial infection. Individual Shh-expressing cells formed cyst-like organoids after 5–7 weeks of 3D culture. CK5 was expressed in the outer layer. They also formed a luminal space in which CK5 and Shh were not expressed. Individual cells of these organoids were capable of self-renewal and differentiation, however, the origin and formation of bladder cancer stem cells remain unknown.8,37 Mullenders et al. collected samples of tumours of MIBC and NMIBC patients who underwent rad- ical cystectomy and TURB, and normal macroscop- ically looking urothelium and established a sample of 50 human bladder organoids. Besides histologi- cal and functional investigations, they also used organoids for testing the efficacy of different intra- vesical chemotherapeutics. They applied different concentrations of anticancer drugs for time period of 5 days and found different responses in different organoids.38 Neal et al. purposed an in vitro model of differ- ent cancers for studying immunotherapy. One of them was also bladder cancer and responsiveness to immunotherapy with checkpoint inhibitors.34 Limitations of organoids Organoids have a potentially important role in urological research, clinical decision-making, and treatment of urological cancers. Limitations of or- ganoids are that the spatial orientation of tissues is random, also in many cases, the cellular com- ponents that are present in in vivo systems such as stromal, vascular endothelial and immune cells are missing.8 Conclusions Bladder cancer presents public health problem be- cause incidence and mortality are constant despite Radiol Oncol 2020; 54(2): 135-143. Taskovska M et al. / Current and innovative treatment of non-muscle invasive bladder cancer142 the development of technology, molecular and cell biology, pharmacology and improvement of sur- gical technique. TURB is the golden standard for diagnosis, staging, and treatment of NMIBC. Both monopolar and bipolar current are equally effec- tive, bipolar current having fewer complications. Development of molecular and cell biology leads to the construction of organoids which are a step towards personalized medicine. We expect that they will enable us to treat our patients based on the data acquired from organoids, regarding on- cogenesis, responsiveness to different therapeutic modalities and possibilities for reconstruction. References 1. European Association of Urology. Guidelines. Non-invasive muscle bladder cancer. [cited 2019 Dec 15]. Available at: https://uroweb.org/guideline/ non-muscle-invasive-bladder-cancer/ 2. European Commission. ECIS - European Cancer Information System. Incidence and mortality of bladder cancer in Europe. [cited 2018 Sep 15]. Available at: https://ecis.jrc.ec.europa.eu/explorer.php?$1-All$2- All$4-1,2$3-38$6-0,14$5-2008,2008$7-7,8$0-0$CEstByCountry$X0_8- 3$CEstRelative$X1_8-3$X1_9-AE28 3. 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