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ADIOLOGY AND NCOLOGY Editorial office Radiologij and Oncology September 2004 Institute oj Oncology Vol. 38 No. 3 Zaloška 2 Pages 165-250 SI-1000 Ljubljana ISSN 1318-2099 Slovenia UDC 616-006 Phone: +386 1 5879 369 CODEN: RONCEM P/wne/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Aims and scope Radiology and Oncology is a joumal devoted to publication oj original contributions in diagnostic and interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. 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Indexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EMBASE / Excerpta Medica Sci Base This journal is printed on acid-jree paper Radiologi; and Oncology is available on the internet at: http://www.onko-i.si/radiolog/rno.html ISSN 1581-3207 Ljubljana, Slovenia ISSN 1318-2099 September 2004 UDC 616-006 Vol. 38 No. 3 CODEN: RONCEM CONTENTS RADIOLOGY, UL TRASONOGRAPHY Computer assisted diagnosis of benign bone tumours Samardziski M, Zafiroski G, Janevska V, Miladinova D, Popeska Ž 165 Spontaneous perirenal and subcapsular haematoma -report of 5 cases Vukelic-Markovic M, Huzjan R, Marušic P, Brkljacic B 171 Sonographically guided fine-needle aspiration biopsies of adrenal masses in lung cancer patients, eleven-year experience Kocijancic I 177 CLINICAL ONCOLOGY Choroid plexus carcinoma: A case report Strojan P, Popovic M, Šurlan K, Jereb B 181 Keratocysts in the jaws Lipovec A, Ihan Hren N 187 Psychological distress and intervention in cancer patients treated with radiotherapy Šoštaric M, Šprah L 193 EXPERIMENTAL ONCOLOGY Use of preneoplastic lesions in colon and liver in experimental oncology Ehrlich VA, Huber W, Grasl-Kraupp B, Nersesyan A, Knasmiiller S Diagnosis and classification of spontaneously developed and radiation-induced murine haematopoietic neoplasms. The murine models for the research on the human haematopoietic neoplasms Szymanska H, Piskorowska ], Krysiak E, Skurzak H, Czarnomska A, Demant P Comparison of Wistar vs. Fischer rat in the incidence of 1,2-dimethylhydrazine induced intestinal tumors Veceric Ž, Cerar A RADIOPHYSICS 217 227 Multileaf collimator in radiotherapy Jeraj M, Robar V 235 SLOVENIAN ABSTRACTS 241 NOTICES 249 Radiol Oncol 2004; 38(3): 165-9. Computer assisted diagnosis of benign bone tumours Milan Samardziski1, George Zafiroski1, Vesna Janevska2, Daniela Miladinova3, Žaneta Popeska4 1University Clinic for Orthopaedic Surgery, Skopje, 2Pathology Institute, Skopje, 3Institute for Pathophysiology and Nuclear Medicine Skopje, 4Faculty of Natural Sciences and Mathematics, Institute for Computer Sciences, Skopje, Macedonia Background. The aim of this study is to determine the correlation between computer-assisted diagnosis (CAD) of benign bone tumours (BBT) and their histological type. Patients and method. Altogether 120 patients were included in two groups. The retrospective group com­prised 68 patients in whom the histological type of BBT was known prior to computer analysis. The prospec­tive group comprised 52 patients in whom the histological type of BBT was unknown prior to computer analysis. Computer program was efficient and easy to use. Results. Average percent of histological type confirmed with CAD in the retrospective and prospective groups was 72.06% and 76.92%, respectively. Histological confirmation of CAD in specific BBT was 91.42% for enchondroma, 96.15% for osteoid-osteoma, and 98.08% for osteochondroma. Significantly low­er percentage of CAD confirmation of fibroma, chondromixoid fibroma, osteoclastoma, desmoplastic fibro-ma and osteobalstoma due to their adverse biological character or complex anatomic localization is under­standable. Conclusions. The results speak in favour of the assumption that computer assisted diagnosis of bone tu­mours program may improve the diagnostic accuracy of the examiner. Key words: bone neoplasms — pathology; diagnosis, computer - assisted Introduction Diagnosis and treatment of benign bone tu­mours (BBT) is a multidisciplinary task. Teams of diverse subspecialists are involved Received 8 April 2004 Accepted 6 May 2004 Correspondence to: Milan Samardziski, MSc, Clinic for Orthopaedic Surgery, Vodnjanska 17, 1000 Skopje, Macedonia; Phone: +389 02 314 7626; Fax: +389 02 3165 137; E-mail: milan_samardziski@yahoo.com in the process. Good quality plain X-rays may be most helpful in 9 of 10 cases. Bone scan, CT and MRI are additionally needed for the diagnosis, staging and decision making on the management of BBT. The diagnosis of his­tological type can be done exclusively by a pa­tohistyologist.1 In the second half of the 20th century, a digital revolution started in the USA. This led to a great advance in technology and data management. A new approach in diagnostics and decision-making process in medicine was inevitable. Warner was the pioneer in com­puter assisted diagnosis (CAD) of congenital heart diseases in 1961.2 Lodwick in 1963 gave his preliminary results with computer assist­ed diagnosis of primary bone tumors.3 Many others followed him soon after: Hall in 1971, Buzdon in 1978, Virtama in 1979, Zafiroski in 1986.4-6 Our task in this study was to deter­mine the correlation between computer-as­sisted diagnosis (CAD) of benign bone tu­mours (BBT) and their histological type. Patients and methods In this study, 120 patients with BBT were in­cluded. The observation period was 7 years. The patients were treated at the Clinic for Orthopaedic Surgery in Skopje. They were di­vided in two groups. The retrospective group comprised 68 patients in whom the histologi­cal type of BBT was known prior to computer analysis. The prospective group comprised 52 patients in whom the histological type of BBT was unknown prior to computer analysis. Of the total of 120 patients, 66 were males and 54 females. The age of patients ranged from 6 to 79 years old (mean 27.4 years). Two thirds (78 patients) were in the second or third decade of their life. The follow-up was from 2 to 5 years (Table 1). Osteochondroma was diagnosed in 34.16% (41) of patients and osteoid-osteoma in 35.0% (42) of patients. Enchondroma was found in 13.33% (16) of patients and 7.5% (9) patients were diagnosed with giant cell tumours. Fibroma, desmoplastic fibroma, chondroblas­toma, chondromixoid fibroma, osteoblas­toma, lipoma and hemangioma were found in 12 patients (10.0%). Enchodromas were 3 times more frequent in female patients while osteohondromas, osteoid-osteomas and giant cell tumours were more often diagnosed in male patients (Table 1). Most of the authors are using Bayes' theo­rem of inverse probability as a basic tool for the mathematical model in the computer pro­gram. Thomas Bayes (1702-1761) was a min­ister who gave the basic mathematical values to the outcome and risk, thereby founding a scientific approach to forecasting.7 (1- Px5 y1) .... Pxj y1 Py1 Px1 y1 Py1 (x1, x5...xj) = —————————————————— (1- Px5yk) .... Pxi yk . Pyk Px1 ykall k Table 1. Patients included in the study and average follow-up Benign bone tumors Age Gender Number of % Follow-up (mean yrs) M F cases (yrs) Osteoma 30 0 2 2 1.66 4.5 Osteoid-osteoma 18.3 30 12 42 35.0 5.3 Osteoblastoma 36.5 1 1 2 1.66 5 Enchondroma 40.7 4 12 16 13.33 3.6 Osteochondroma 21.7 24 17 41 34.16 3.3 Chondrobalastoma 22 1 0 1 0.83 4 Chondromyxoid fibro. 24.5 0 1 1 0.83 3.5 Osteoclastoma (GCT) 33.8 5 4 9 7.50 4.4 Hemangioma 30 0 1 1 0.83 2 Fibroma 18.7 1 2 3 2.50 3.7 Desmoplastic fibroma 14 0 1 1 0.83 3 Lipoma 39 0 1 1 0.83 3 Mean Total Total Total Mean 27.4 66 54 120 100% 3.8 On y axis of probability matrix, all possible diagnoses (y1, y5,...yj) are given, on x axis, all radiological characteristics of the tumours (x1, x5,...xj) are shown. P is probability, and k is the number of possible diagnosis included in the matrix. For an absolutely correct prob­ability, indefinite number of cases are needed (i), and all variables included should be com­pletely independent. An adequate vocabulary, based on the ra­diographic manifestations of BBT, is required for the communication with the computer program.3 The program is capable of predict­ing 34 different histological types of primary bone tumours and tumour like lesions.6 The greatest task with CAD is to achieve a correct histological type of the BBT and to follow two basic principles: (1) the prediction of the di­agnosis must be correct in the highest possi­ble number of cases (ideally in all of them), and (2) if there is a mistake in the prediction, it must not influence further treatment of the lesion in a way that could harm the patient. In the decision-making algorithm, both princi­ples are included.8 We compare our prior experiences of radi­ographic manifestations of BBT with the radi­ographic manifestations of the new cases. The next task in the algorithm is to eliminate as many data (diagnosis) as possible during the decision-making process. In this process, the strongest criteria for eliminating or in­cluding a certain diagnosis are the radiologi­cal grade of tumour growth. Many lesions are seen only in the radiological grades of tumour growth Ia, Ib or Ic (Figure 1).4 During the analysis of the x-ray, the following data were included: age and gender, localisation of the Figure 1. (a) enchondroma in the proximal phalanx of the third finger of the hand, presented with moderate pain until the fracture occurred; (b) CT imaging of osteoid-osteoma in the proximal femur, with typical “nidus”; (c) plain radiograph of the forearm showing osteochondroma of distal radius (almost not seen in frontal plane). BBT, bone destruction, destruction of the cor­tex, periostal proliferation, tumour matrix mineralisation and size of the tumour. Results In this study CAD were compared to the final histological type of BBT. The results showed high statistical significance between the radi­ographic manifestations of BBT and histolog­ical type. The percentage of confirmed CAD in the retrospective study was 72.06% and in the prospective study 76.92%. There was no sta­tistically significant difference between these results (.2 = 0.36; for r =0.34) (Figure 2). The analysis of different radiographic manifestations in correlation with confirmed CAD was made on a joined number of cases from both studies (retrospective and prospec­tive); so, the results gave greater statistic sig­nificance. Highest percentage of CAD was seen in the lesions localised in the cortex of the bone (83.10%) compared to the lesions lo­calized in the bone medulla (61.36%) and oth­er localizations (60.00%). Analysed parame­ters showed high values of .2 test: .2= 7.244455; r = 0.026723 for r < 0.05. The highest percentage of confirmed CAD in correlation with expansion of the cortex under the pressure of growing BBT showed lesions without expansion (78.89%). The highest percentage of unconfirmed CAD showed lesions with the expansion of the cor­tex greater than 10 mm (77.78%). Analysed data revealed high statistic significance (.2= 13.76689; r = 0.001025 for r < 0.05) (Figure 3). Size of the tumours was measured in mil-limetres of their longest diameter. Tumours were divided in the group with the confirmed CAD and the group with unconfirmed CAD. Standard error and standard deviation were higher in the group with unconfirmed CAD and average size of 41.8 mm. The values showed statistical significant difference for .2 test -21.68123; r = 0.010638 for r < 0.005. Discussion Most of the bone tumours originate from the medullar bone, destructing it prior to the growth of the lesion in the cortex. Unfor­tunately, this is not seen until 40-50% of the medullar bone is lost. In contrast to the medullar bone, the cortex shows even slight­est destruction when appropriate x-ray pro­jection is made. Slow growing and benign bone tumours produce a sclerotic reaction of the surrounding bone.9 Analysing these ma­nifestations together with bone tumour ma­trix one can easily determine the radiological grade of tumour growth. Active, aggressive and malignant should be immediately treated and latent (“live me alone”) bone tumours should be regularly inspected and followed.10 Working with this program for computer-as­sisted diagnosis of BBT appears to be easy, understandable and can be used by relatively inexperienced examiner. The use of the pro­gram improves diagnostic accuracy signifi­cantly and results in improved patient man­agement and cost-saving.5 CAD of BBT should be confirmed in the highest possible number of cases (ideally 100%). The average percent of confirmed CAD in retrospective study is 72.06% and in prospec­tive study is 76.92%. This is slightly lower than those in previous studies of Enneking (77.9%) and Bumbasirevic (81.2%).4,9 In our study, for some specific benign bone tumours as en-chondroma, osteochondroma and osteoid-os­teoma, the confirmation is higher than 83.33%. There was no significant influence of the examiner on the results of CAD. The analysis of the results of fibroma, chon­dromixoid fibroma, osteoclastoma, desmo-plastic fibroma and osteoblastoma and lesions localized on scapula and pelvis was inconclu­sive due to their adverse biological character, low number of cases or complexity of the analysis of the specific anatomic localization. Best results of CAD were shown when le­sions were localized in the cortex, in tumours without expansion of the bone and tumours with average size of 27 mm in diameter. The results support the assumption that the com-puter-assisted diagnosis of bone tumours pro­gram may improve the diagnostic accuracy of the examiner. This is due to an analytic, sys­tematic and logic approach to the analysis of the radiographic manifestations of BBT. A slightly lover percentage of confirmed CAD in the retrospective versus prospective study speaks in favour of that conclusion. References 1. Sundaram M. Magnetic resonance imaging for solitary lesions of bone: when, why, how useful? J Orthop Sci 1999; 4: 384-96. 2. Warner HR, Toronto AF, Veasey LG, Stephenson RE. A mathematical approach to medical diagno­sis. Application to congenital heart disease. J Am Med Ass 1961; 177: 177-83. 3. Lodwick GS, Haun CL, Smith WE, Keller RF, Robertson ED. Computer diagnosis of primary bone tumors. A prelim.report 1963; 80: 273-5. 4. Bumbasirevic Z, Buzdon P. Jedna od mogucnosti primene kompjutera u dijagnostici kostnih tumora. Medicinska Istrazivanja 1981; 14 (Suppl 1-2): 75-7. 5. Virtama P, Katevuo K, Makela P, Makinen EO. Computer aided diagnosis of bone tumors. Acta Radiol Diagn (Stockh). 1979; 20: 70-4. 6. Zafiroski G. Maligni koskeni timori. Skopje: Studentski zbor-Skopje; 1986. p. 11-14. 7. Mercer R. The caring doctor. Chapter 1. In: Art and practice of children’s orthopedics; Raven Press, NY, 1992. 8. Ledley R S, Lusted LB. Reasoning foundations of medical diagnosis. Science 1959; 130: 9-21. 9. Enneking WF. A system of staging musculoskele­tal neoplasms. Clin Orthop 1986; 204: 9-24. 10. Simon MA, Springfield D. Surgery for bone and soft-tissue tumors. Philadelphia: Lippinkot-Raven; 1997: 119-207. Radiol Oncol 2004; 38(3): 171-5. Spontaneous perirenal and subcapsular haematoma – report of 5 cases Mirjana Vukelic-Markovic, Renata Huzjan, Petar Marušic, Boris Brkljacic Department of Radiology, University Hospital “Dubrava”, Zagreb, Croatia Background. Spontaneous perirenal and subcapsular haemorrhage is a rare but important clinical condition and is diagnostically very challenging. Sometimes, the aetiology of bleeding remains unclear; when all avail­able diagnostic possibilities are exhausted, therapeutic approach still remains controversial. Case reports. We present a series of 5 patients with perirenal and subcapsular bleeding. In two of among our patients, the initial or control CT scan suggested angiomyolipoma and renal cyst as the cause of the bleeding that was confirmed by pathological analyzes. In other three patients, no pathology other than haematoma itself was visualized on CT scans, nor it was discovered on pathological analyzes in two of the patients. Our CT findings closely correlated with pathological findings – whether positive or negative for the pathological substrate. Interestingly, we found not one case of renal cell carcinoma. Conclusions. In literature, in as many as 50% of cases of perirenal and subcapsular bleeding, a malignant tumour is found. Therefore, by some authors, nephrectomies in all patients are recommended, but others take more expectative approach with long-term close surveillance. We believe, that with new imaging modalities, if using optimal examination technique and follow-up protocols, the patients with bleeding due to benign dis­ease should be recognized and unnecessary nephrectomies avoided. Key words: kidney diseases; haematoma, tomography, X-ray computed Introduction Spontaneous perirenal and subcapsular haemorrhage is a rare but important clinical condition and is often diagnostically very challenging. The appropriate treatment of Received 19 April 2004 Accepted 5 May 2004 Correspondence to: Renata Huzjan, MD., Department of Radiology, University Hospital “Dubrava”, Avenija G. Šuška 6, 10000 Zagreb, Croatia; Phone: +385 1 290 3255; E-mail: renata.huzjan@zg.htnet.hr these patients is based on making a fast and correct diagnosis of subcapsular and perire­nal haemorrhage. Clinical symptoms are of­ten non-specific and misleading and the radi­ological methods, based on ultrasound (US) and CT imaging are crucial in making the cor­rect diagnosis. Diagnosing the haematoma it­self, its extent and location is rather simple with mentioned imaging modalities, but de­termining the source of bleeding and defining the underlying pathological condition that caused the bleeding is more complex task. As sometimes the aetiology of the bleeding still remains unclear though all available di­agnostic possibilities are exhausted, the ther­apeutic approach to these patients is still con­troversial.1-4 In clinical approach, it is neces­sary first to exclude trauma, anticoagulation medication, bleeding diathesis, arteritis, tuberous sclerosis or whether the patient is undergoing a long-term haemodialysis, as all these conditions are known to be associated with perirenal bleeding. The most common underlying kidney conditions include renal cell carcinoma, angiomyolipoma, AV malfor­mation, arterial aneurysm, renal cyst, infarc­tion and abscess. 2,5-7 We report our experience on a series of 5 patients with perirenal and subcapsular bleeding. In our diagnostic algorithm, after the US examination that was used as a first method, the key examination was CT scan­ning. All examinations were performed on conventional CT scanner (Shimadzu Intellect). CT was performed [(after an i.v. contrast medium bolus administration)] from dia­phragm to symphisis with the slice thickness of 10 mm and pitch of 10 mm using native se­quences and sequences.7 When needed, a se­lective angiography was also performed. Case reports Patient No. 1 was male, aged 37 years. He pre­sented with the acute right-sided flank pain. On US examination, a perirenal haematoma was suspected and CT finding confirmed the haematoma with angiomyolipoma as a proba­ble bleeding source (Figure 1a). Angiography (DSA) was performed and revealed patholog­ical vascular pattern characteristic for an-giomyolipoma. Nephrectomy was performed and pathohistological diagnosis confirmed the clinically suspected angiomyolipoma (Figure 1b). Patient No. 2 was female, aged 53 years. The initial CT scan showed subcapsular renal haematoma on the left side without any other pathology (Figure 2a). Two months later, the follow-up CT scan showed substantial regres­sion of the bleeding and renal cyst that was suspected to be a bleeding source (Figure 2b). Three months later, the follow-up CT scan showed complete regression of the haema­toma (Figure 2c). Surgical exploration and pathohistological analyses confirmed the di­agnosis of the renal cyst and the kidney was preserved. Patient No. 3 was male, aged 75 years. The patient presented with an acute lumbar pain. US examination showed a heterogenic mass in the kidney that was suspected to be a bleeding renal tumour. CT scan was per­formed and revealed only a huge left-sided perirenal haematoma (Figure 3). Laboratory findings indicated liver cirrhosis (elevated liv­er enzymes, coagulation disorder). As the pa­tient became haemodynamically unstable ur­gent nephrectomy was performed. Patho-histological analyses revealed no pathological findings apart from the haematoma. We be­lieve that coagulation disorder due to liver disease caused the bleeding in this patient. Patient No. 4 was male, aged 63 years. He presented with a flank pain in the right lum­bar region lasting for 15 days. He had similar symptoms 2 months before and was diag­nosed with renal colic. US and CT examina­tions showed subcapsular haematoma and calculus in the right kidney with no other pathological findings (Figure 4a). Surgical ex­ploration was performed and as the bleeding source could not be identified, the kidney was preserved. Three months later, CT scan and US was normal, as well as CT scan one year later (Figure 4b). We believe that the re­nal colic caused the bleeding in this patient. Patient No. 5 was female, aged 46 years. The patient was diagnosed with rheumatoid arthritis 6 years prior to the current illness. She was admitted due to right-sided lumbar pain lasting for several months. US examina­tion showed hyperechogenic renal mass and renal tumour was suspected. CT scan showed subcapsular haematoma and also retroperi­toneal lymphadenopathy (Figure 5). Occult Figure 4a. The initial CT scan showed subcapsular haematoma without identifying the bleeding source. renal cell tumour was suspected and nephrec­tomy was performed. Pathohistological diag­nosis showed reactive hyperplasia of the lymph nodes and mononuclear infiltration with no malignant disease. Discussion In our patients, the initial or control CT scan suggested the cause of the bleeding in two pa­tients (patients No. 1 and No. 2), angiomyi­olipoma and bleeding renal cyst, respectively. These diagnoses were confirmed by postoper­ative pathohistological analyses. In other three patients no substrate other than haema­toma itself could be visualized on CT scans. In two among them (patients No. 3 and No. 5) nephrectomy was performed and no malig- Figure 5. The initial CT scan showed subcapsular haematoma and retroperitoneal lymphadenopathy. nancies were found on pathological analyses. In the last patient (patient No. 4), neither the initial CT nor the intraoperative examination showed bleeding source and it was decided to preserve the kidney. Even repeated CT scans, done over the period of one year, did not identify any pathological substrate; we there­fore believe that the renal colic caused the bleeding in this patient. In our series of patients, CT findings close­ly correlated with pathohistologic findings – whether positive or negative for the patholog­ical substrate. Interestingly, we did not find any case of renal cell carcinoma among our patients. In our opinion a correct CT exami­nation technique is crucial for making a cor­rect diagnosis. A careful search for small tu­mours after i.v. contrast administration is mandatory. Areas of fat within the kidney and diagnostic for angiomyolipoma should be noticed as angiomyolipomas are common causes of spontaneous haematoma.8 If CT scan is negative for the tumour in order to ex­clude vascular abnormality, a selective an­giography should be performed.9 If the diag­nosis of the cause of haematoma is still un­clear the repeated CT scanning is advised, preferably every 6-8 weeks. It will allow enough time for the haematoma to resorb and, possibly, also for finding a small tumour that might have been present, but hidden by the blood in the initial study. The follow-up is needed until the haematoma completely re­solves or until the diagnosis is made.2,10 Even today, with all our sophisticated tech­nology, the therapeutic approach to sponta­neous perirenal and subcapsular haema­tomas is controversial. The malignant tu­mour, often small in size, is reported in 30% to over 50% of the patients and, according to several authors, radical nephrectomy in the absence of apparent cause of bleeding is rec­ommended in all patients.11-13 On the other hand, as the haemorrhage can be idiopathic or due to benign lesions, other authors2,3,9 propose more expectative approach with long-term close surveillance in order to avoid unnecessary surgery and nephrectomias. We believe that, with new imaging modal­ities, especially spiral and multidetector CT and using optimal examination technique as well as follow-up protocols, we should recog­nize the patients with perirenal bleeding due to benign disease and avoid unnecessary nephrectomias. References 1. Mantel A, Sibert L, Thoumas D, Pfister C, Guerin JG, Grise P. Spontaneous perirenal hematoma: di­agnostic and therapeutic approach. Prog Urol 1996; 6: 409-14. 2. Bosniak MA. Spontaneous subcapsular and perirenal hematomas. Radiology 1989; 172: 601-2. 3. Moudouni SM, Ennia I, Patard JJ, Guille F, Lobel B. Spontaneous subcapsular renal hematoma: diag­nosis and treatment. Two case reports. Ann Urol 2002; 36: 29-32. 4. Štimac G, Dimanovski J, Reljic A, Spajic B, Cus­tovic Z, Klaric-Custovic R, et al. Extensive sponta­neous perirenal hematoma secondary to ruptured angiomyolipoma: case report. Acta Clin Croat 2003; 42: 55-8. 5. Meyers MA. Dynamic radiology of the abdomen: nor­mal and pathologic anatomy. New York: Springer-Verlag; 1994. 6. Brkovic D, Moehring K, Doersam J, Pomer S, Kaeble T, Riedasch G, et al. Aetiology, diagnosis and management of spontaneous perirenal hematomas. Eur Urol 1996; 29: 302-7. 7. Sebastia MC, Perez-Molina MO, Alvarez-Castells A, Quiroga S, Pallisa E. CT evaluation of underly­ing cause in spontaneous subcapsular and perire­nal hemorrhage. Eur Radiol 1997; 7: 686-90. 8. Bulto Monteverde JA, Talens A, Navalon P, Garcia Novales JR, Cubells ML, Mendez M. Renal an-giomyolipoma. Ultrasonography and computer­ized tomography findings. Arch Esp Urol 1999; 52: 1043-50. 9. Beville JS, Morgentaler A, Loughlin KR, Tumeh SS. Spontaneous perinephric and subcapsular re­nal hemorrhage: Evaluation with CT, US and an­giography. Radiology 1989; 172: 733-8. 10. Shih WJ, Pulmano C, Han JK, Lee C. Spontaneous subcapsular and intrarenal hematoma demon­strated by various diagnostic modalities and mon­itored by ultrasonography until complete resolu­tion. J Natl Med Assoc 2000; 92: 200-5. 11. Kendall AR, Seney BA, Coll ME. Spontaneous sub-capsular renal hematoma: diagnosis and manage­ment. J Urol 1988; 139: 246-50. 12. Boumdin H, Ameur A, Lezrek M, Atioui D, Beddouch A, Idrissi Oudghiri A. Spontaneous sub-capsular hematoma of the kidney. Report of 6 cas­es. Ann Urol 2002; 36: 357-60. 13. Touiti D, Zrara I, Ameur A, al Bouzidi A, Beddouch A, Oukheira H, et al. Sponatenous perirenal hematomas: report of 3 cases. Ann Urol 2001; 36: 319-22. Radiol Oncol 2004; 38(3): 177-80. Sonographically guided fine-needle aspiration biopsies of adrenal masses in lung cancer patients, eleven-year experience Igor Kocijancic Department of Radiology, Institute of Oncology, Ljubljana, Slovenia Purpose. The aim of this retrospective study was to define the accuracy and safety of the ultrasonographi­cally (US) guided fine-needle aspiration biopsy (FNAB) of the enlarged adrenals in the patients with lung cancer. Patients and methods. In eleven-year period 64 patients with cytologically proven lung cancer underwent US-guided FNABs of adrenal masses. The accuracy of the method was assessed on the basis of cytology findings and the safety on the number of complications reported after the procedure. Results. US-guided aspiration biopsy turned out to be accurate in 58/64 cases (91%), and very safe with only 4/64 (6%) minor complications. In 52/58 (90%) cases, the cytology sample was found to be malignant. In 6 cases (10%), isolated adrenal masses were adenomas. Conclusions. We recommend US-guided FNAB as a safe and reliable diagnostic method that has many ad­vantages over computer tomography (CT)-guided FNAB, such as safety, patient-friendliness, no X-rays and its reproducibility. Key words: lung neoplasms – secondary – ultrasonography – pathology; adrenal gland neoplasms; biopsy, needle Introduction The adrenal glands are a common site for the metastatic spread of lung cancer.1 The metas­tases in the adrenal glands are often detected at the time of setting the basic diagnosis. As Received 12 August 2004 Accepted 18 August 2004 Correspondence to: Assist. Prof. Igor Kocijancic, MD, PhD; Department of Radiology, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia; Phone: +386 1 5879 505; Fax: +386 1 5879 400; E-mail: ikocijancic @onko-i.si they are usually asymptomatic,2 the only reli­able method to confirm the metastases is di­agnostic imaging. The majority of them are detected by chest and upper abdomen CT or by abdominal ultrasound (US) examination. The improvement of US technology allows visualization of slightly enlarged adrenal glands, with the exception of the scans in obese and meteoristic persons. Any enlarge­ment of the adrenal glands should be ex­plained because benign changes could be de­tected in healthy individuals.3,4 In addition, there are many reports on high percentage of the benign cytology findings of fine-needle as­piration biopsies (FNABs) from the enlarged adrenal glands in lung cancer patients.5,6 In the literature, the results of CT-guided aspiration biopsies7-9 are more often analysed than the results of US-guided ones.8-10 The purposes of these retrospective study were to obtain data on the accuracy and safety using US guided FNABs in the evaluation of en­larged adrenal glands in patients with lung cancer. Patients and methods In the 11-year period, from 1991 to 2001, we performed 64 US-guided biopsies of the en­larged adrenal glands in 64 patients with cy­tologically confirmed lung cancer. Forty-six of them were men and 18 were women, with the average age of 59 years, ranging from 42 to 82 year (SD ±9.6). Lung cancer was cytologically confirmed (by bronchoscopy, transthoracic FNAB, or by sputum examination). The most frequent type was adenocarcinoma (28 patients), fol­lowed by epidermoid (16), microcellular (6) and macrocellular lung cancer (6) and not otherwise specified in 8 cases. In 40 patients the tumour was localized in the right lobe and in 24 patients in the left lobe. Sonographic examination and US-guided FNABs had been performed with Toshiba SSA 240A ultrasound scanner before 1997 and a Toshiba SSA 340A afterwards (Tokyo, Japan). A convex probe with the radius of 25 or 50 mm was chosen for subcostal or tran­shepatic approach, whereas the probe of 15 mm was used for intercostal approach. US-guided aspiration biopsies were per­formed by flexible “Chiba” needles with the length of 16 to 20 cm and 22 G (0.7 mm) thick. A metal probe adapter with a 5 cm long guide helps to maintain the needle aligned with the scan and allowed it to hit the target easier. In all cases, the radiologist carried out the whole process by himself, holding the probe with his left hand while inserting the needle and aspirating with the right one (Figure 1). Prior to biopsy, coagulation tests had been performed in order to check that the patient’s platelets counts and prothrombin time (PT) were within normal values. Patients with platelet count of less than100x109/l and PT ratio of less than 0.60 had been excluded from the biopsy procedure. In the patients in whom a bilateral en­largement of the adrenal glands was observed by US we anticipated the same aetiology of changes in both glands; therefore, FNAB was performed in one gland only. Results We performed 38 biopsies (60%) of the en­larged adrenal glands on the right side and 26 biopsies (40%) on the left side. The mean longest diameter of the enlarged glands was 5.6 ±2.7 cm (range, 2.5-13.0 cm). The material for cytology analysis was as­sessed as appropriate in 58/64 cases (91%), while in 6/64 cases (9%), it was inappropriate. Cytology examination confirmed a malignant process in 52/58 patients (90%) and a benign process in 6/58 (10%) patients. Of 52 malig­nant cases, 40 (77%) were diagnosed as defi­nitely a metastatic process, and the remaining 12 were found to be malignant without fur­ther characterization. The mean longest diameter of benign adrenal masses, which were all unilateral, was 4.2 ± 2.4 cm (range, 2.5-7.0 cm) and that of the malignant ones was 6.3 ± 2.8 cm (range, 3.5-13.0 cm). The mean longest diam­eter of unsuccessfully biopsied lesions (inap­propriate cytology samples) was 5.7 ± 2.1 cm (range, 4.0-8.0 cm). We carried out the majority of aspiration biopsies in the outpatient department; 50 pa­tients (78%) were called in on the day of biop­sy. Four patients complained of pains and of sensitivity to palpation that developed imme­diately after the biopsy, persisted for some hours and faded away spontaneously. In these patients sonographic examination per­formed approximately two hours after the biopsy revealed no abnormalities. Discussion In view of the accuracy and safety, the litera­ture recommends more CT than US-guided FNABs of the enlarged adrenal glands.7,9,11 No comparative studies have been done. The stud­ies report 80-83% primary and 90-93% sec­ondary accuracy of CT-guided FNABs.7,12,13 In our study, the sample was appropriate for the cytology analysis in 58 cases, which means 91% primary accuracy of the method. In 6 of our patients, cytology confirmed a benign enlargement of the adrenal gland (adenoma or hyperplasia). In these patients during follow-up period of, no metastatic spread into the adrenal glands was observed. Due to the small group conclusions referred on these data are not reliable. CT-guided FNAB is considered to be safe;7,11,12 the most frequent complications are pneumothorax13 and bleeding.7 No dis­tinct correlation was made between the nee­dle thickness and localization of the lesion, although Price11 reported that no complica­tions occurred by transhepatic approach CT-guided FNABs of the right adrenals. Most of our patients had the biopsy per­formed as outpatients. After the procedure, they were observed for 2 hours. Only 14 pa­tients (22%) underwent the FNAB as inpa­tients. Four of all patients complained of pe­riodic pains at the puncture site. A key to the success of the procedure is to perform it as quickly as possible. US-guided aspiration biopsies are more flexible and adjustable if performed by a single person holding the probe with his left hand while inserting the needle and aspirating with the right one (Figure 1). It also offers several approaches that could be chosen with regard to the anato­my. This allows better coordination, easier to hit the mass, and shortens the procedure. The needle is thus remained inside the body for only one or two breath holds (less than 1 minute), kept still and thus does not cause any additional impairments that cannot be avoided in CT-guided aspiration biopsies. CT-guided aspiration biopsy is specifically recommended for small adrenal masses.10 In our series, 13 biopsies were successfully per­formed on the glands that were smaller than 4 x 2.5 cm - the size that is considered as the limit of the anatomically normal adrenal glands (Figure 2). In summary, the results of our study con­firm that sonographically guided FNABs of the adrenal glands are just as safe and reliable as the CT-guided ones. Nevertheless, US-guided aspiration biopsy has some advan­tages over the CT-guided one. We believe that CT-guided aspiration biopsy should be per­formed only if US-guided aspiration biopsy is not safe anymore, e.g. low visibility, non-co­operative patient, or really small size of the le­sion. References 1. Salvatierra A, Baamonde C, Llamas JM, Cruz F, Lopez-Pujol J. Extrathoracic staging of bron­chogenic carcinoma. Chest 1990; 97: 1052-8. 2. Bernardino ME. Management of the asymptomatic patient with a unilateral adrenal mass. Radiology 1988; 166: 121-23. 3. Kokko JP, Brown TC, Berman MM. Adrenal ade­noma and hypertension. Lancet 1967; 1: 486-90. 4. Glazer HS, Weyman PJ, Sagel SS, McClennan BL. Nonfunctioning adrenal masses: incidental dis­covery on computed tomography. AJR Am J Roentgenol 1982; 139: 81-5. 5. Oliver TW. Isolated adrenal masses in nonsmall-cell bronchogenic carcinoma. Radiology 1984; 153: 217-8. 6. Gillams A, Roberts CM, Shaw P, Spiro SG, Goldstraw P. The value of CT scanning and percu­taneous fine needle aspiration of adrenal masses in biopsy-proven lung cancer. Clin Radiol 1992; 46: 18-22. 7. Bernardino ME, Walther MM, Phillips VM, Graham SD Jr, Sewell CW, Gedgaudas-McClees K, et al. CT-guided Adrenal Biopsy: Accuracy, safety and indications. AJR Am J Roentgenol 1985; 144: 67-9. 8. Silverman SG. Mueller PR, Pinkney LP, Koenker RM, Seltzer SE. Predictive value of image-guided adrenal biopsy: analysis of results of 101 biopsies. Radiology 1993; 187: 715-8. 9. Porte HL, Ernst OJ, Delebecq T, Metois D, Lemaitre LG, Wurtz AJ. Is computed tomography guided biopsy still necessary for the diagnosis of adrenal masses in patients with resectable non­small-cell lung cancer? EJ Cardio-thoracic Surg 1999; 15: 597-601. 10. Montali G, Solbiati L, Bossi MC, De Pra l, Di Donna A, Ravetto C. Sonographically guided fine-needle aspiration biopsy of adrenal masses. AJR Am J Roentgenol 1984; 143: 1081-4. 11. Price RB, Bernardino ME, Berkman WA, Sones PJ, Torres WE. Biopsy of the right adrenal gland by the transhepatic approach. Radiology 1983; 148: 566. 12. Heaston DK. Narrow gauge needle aspiration of solid adrenal masses. AJR Am J Roentgenol 1982; 138: 1143-8. 13. Pagani JJ. Normal adrenal glands in small cell lung carcinoma: CT guided biopsy. AJR Am J Roentgenol 1983; 140: 949-51. Radiol Oncol 2004; 38(3): 181-5. Choroid plexus carcinoma: A case report Primož Strojan1, Mara Popovic2, Katarina Šurlan3, Berta Jereb1 1Department of Radiotherapy, Institute of Oncology, 2Institute of Pathology, Medical Faculty University of Ljubljana, 3Institute of Radiology, University Clinical Centre, Ljubljana, Slovenia Background. The opinions on the value of adjuvant therapy in choroid plexus carcinomas vary. The aim of present report is to present a case of successful therapy of this rare tumor. Result. A fourteen-year-old girl with third ventricle tumor had non-radical surgery and adjuvant chemother­apy and irradiation. She is alive with no evidence of disease 8.5 years after diagnosis. The role of adjuvant therapy in the context of literature data is discussed. Conclusion. For choroids plexus carcinomas, adjuvant multiagent chemotherapy and craniospinal radio­therapy following surgery should be considered. Key words: choroid plexus neoplasms; chemotherapy, adjuvant; radioteraphy; survival analysis Introduction Choroid plexus tumor (CPT) is a rare neo­plasm, arising from the neuroepithelial lin­ing.1 After its first description in 1832, more than 500 CPT patients have been described in literature.2 Three quarters of the patients are children, with tumors most often found in the lateral ventricles. In adults, the fourth ventri­cle and its recesses are the most common sites of origin.1 The histopathology of CPT ranges from a well - demarcated benign pa-pilloma (WHO grade I) to highly anaplastic, Received 8 June 2004 Accepted 5 July 2004 Correspondence to: Assist. Prof. Primož Strojan, M.D, Ph.D., Department of Radiotherapy, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 110; Fax: +386 1 5879 400; E-mail: pstrojan@onko-i.si infiltrative carcinoma (WHO grade III, choroid plexus carcinoma [CPC]). Surgical re­section alone is curative for benign tumors, but the optimal adjuvant therapy for the ma­lignant ones has not yet been defined and the prognosis is poor.1,3 Our experience in suc­cessfully treating such a patient is therefore of interest. Case report The patient was previously healty 13.8-year old girl who was admitted to the hospital in January 1996, with a six months complaint of headache and double vision when reading. Two months prior to admission she became progressively lethargic and dull resulting in a deterioration of school performance. Neurologic examination revealed bilateral pa-pilledema, ataxia, positive bilateral Babinski Figure 1. T1-weighted transverse postcontrast MRIs of the patient. (A) Before surgery: partially cystic tumor mass of the third ventricle with the extension to thalamus, and dilatation of ventricular system. (B) Two weeks after surgery and before chemotherapy: residual tumor in the third ventricle. (C) After the second cycle of multiagent chemotherapy: marked regression of residual tumor. (D) Five months after radiotherapy: even after gadolinium application, there is no pathologic enhancement suspi­cious for residual tumor mass. Figure 2. Histologic characteristic of resected tumor. (A) Solid growth of carcinoma with focal papillary structures. Mitotic figures are present (arrowheads). H&E, mag­nification 190x. (B) Strong cytokeratin immunoreaction is evident in all tumor cells. Immunohistochemistry, DAKO’s CK 18 mon­oclonal antibody, magnification 190x. signs and right-sided hyperreflexia. Two days before surgery, Parinaud syndrome devel­oped. Computed tomography (CT) and mag­netic resonance (MR) scans of the brain showed a contrast-enhancing lesion in the pineal region with extension to the thalamus and the lamina quadrigemina and dilatation of the supratentorial ventricles (Figure 1A). External ventricular drainage was intro­duced first. No tumor cells were found in the cerebrospinal fluid. Tumor resection was per­formed one week later and a reddish granular tumor tissue overgrowing the posterior part of the third ventricle was found. It infiltrated locally and extended to the lamina quadrige­mina. Histologic examination revealed a highly cellular, focally necrotic tumor compo­sed of polygonal cells with moderately poly­morphic nuclei and scattered mitoses. Tumor cells were organized in sheets or formed pap­illary structures lined with multiple epithelial layers. Due to pineal location, embryonal car­cinoma was also taken into consideration. Strong cytokeratin and NSE labelling of tu­mor cells, in addition to negative AFP and PLAP, confirmed the diagnosis of CPC (Figure 2). Two days after surgery, external ventricular drainage was removed. On post­operative imaging, tumor residue was seen in the postero-inferior part of the third ventricle (Figure 1B). Postoperatively, multiagent chemotherapy was introduced according to the so-called BEP protocol (bleomycin 15 mg/m2 I.V., days 1-3; etoposide 100 mg/m2 I.V., days 1-3; cisplatin 20 mg/m2 I.V., days 4-8). After the second cycle of chemotherapy, the MRI showed a marked re­gression of the residual tumor (Figure 1C), and an additional two cycles of these drugs resulted in further tumor reduction. The treatment con­cluded with craniospinal radiotherapy of 31.5 Gy/21 fx and a local tumor boost of 22 Gy/20 fx b.i.d. The patient was irradiated five days per week, using 5 MV linear accelerator photon beams. The treatment technique consisted of posterior spinal fields with moving junction and a combination of two lateral opposing portals and one posterior portal for the brain. Two-field technique was used for boosting the tumor. Complete disappearance of the tumor was con­firmed by a subsequent MR scan (Figure 1D). To date, 8.5 years after diagnosis, there has been no evidence of local tumor recurrence or metastasis. She has no severe neurologic im­pairment and her Karnofsky index is 100. She finished high school, got married, and gave birth to her first child. So far, the child is nor­mal as was the endocrine testing of his mother. Discussion Experience with this extremely rare malig­nant tumor is scanty. Surgery is, however, unequivocally considered to be the first-line therapy for all histological variants of CPTs. Surgical techniques and possible complica­tions have been widely described in litera­ture.1 In CPCs, the most significant predictive factor for survival is the extent of surgery. This has been confirmed in several single-in­stitution analyses4-6 as well as in literature re­views.2,3 To obtain complete tumor removal, Ellenbogen et al. and others have advocated as many surgical procedures as required.4-6 One of the key debates in CPC therapy re­lates to the value of adjuvant therapy after gross tumor resection. There are both oppo­nents and proponents of the combined treat­ment approach.2-5,7,8 A recent literature re­view by Wolff et al. found adjuvant radiother­apy to be of benefit over surgery alone.2 Similar conclusions with regard to radiother­apy and/or chemotherapy can be drawn from numerous single-institution reports.5,9,10 The need for aggressive adjuvant therapy is widely recognized in the patients with resid­ual CPC following surgery since the expected survival is half of that after gross tumor resec-tion.2,3,8 Even though there are anecdotal re­ports on successful adjuvant radiotherapy7,9 or chemotherapy,10 it is our impression that aggressive combined radio-chemotherapy of­fers the best chance for survival. Examples of beneficial effect of combined therapy, some­times incorporating second-look surgery, can be found in the literature.8,9,11 The radiotherapy target volume is dictated by the propensity of CPC for subarachnoid seeding which, when confirmed, calls for ad-juvant therapy per se, irrespective of the de­gree of completeness of surgical procedure. Subarachnoid seeding was found in 43% of those reported cases that were investigated for dissemination.3 Thus, craniospinal axis ir­radiation to a dose of 30 Gy and a boost to the tumor bed up to 50 Gy is indicated if radio­therapy is used,12 and seems to be more ef­fective than chemotherapy.3 Due to the high risk of severe adverse in­tellectual and endocrinologic sequelae in very young children, early radiotherapy is an op­tion in older age groups only. Two treatment strategies were described in the literature for young patients, both placing emphasis on multiagent chemotherapy, although this also is not entirely free of long-term side-effects.13 To facilitate complete tumor resection as a main prognostic determinant in CPCs, St Clair et al. used preoperative chemotherapy to reduce tumor vascularity and bulk after initial biopsy or limited surgery. Not specifying the postresection therapy, if any, two out of four children from this program were reported to be free of disease for 30 and 39 months from diagnosis.6 The second option was described by Duffner et al. Using chemotherapy for 24 months in children 0-23 months of age and 12 months in those 24-36 months of age and delayed radiotherapy, the autors reported a 3-year survival of 75%, with five of the eight patients surviving a minimum of 48 months following diagnosis.10 The variety of chemo­therapeutic agents and their combinations used by these two groups as well as in other reports indicates that an optimal chemothera­py regimen is yet to be defined. In conclusion, our recommendation would be that for CPCs, after as extensive surgery as possible, adjuvant therapy consisting of mul­tiagent chemotherapy and craniospinal radio­therapy, delayed in very young children, should be considered. The BEP regimen de­tailed above plus radiotherapy gave a good re­sult in the patient described. References 1. Ellenbogen RG, Scott RM. Choroid plexus tumors. In: Kaye AH, Laws ER Jr, editors. Brain tumors: An encyclopedic approach. 2nd ed. London: Churchill Livingstone; 2001. p. 552-62. 2. Wolff JEA, Sajedi M, Brant R, Coppes MJ, Egeler RM. Choroid plexus tumours. Br J Cancer 2002; 87: 1086-91. 3. Geerts Y, Gabreëls F, Lippens R, Merx H, Wesseling P. Choroid plexus carcinoma: A report of two cases and review of the literature. Neuropediatrics 1996; 27: 143-8. 4. Ellenbogen RG, Winston KR, Kupsky WJ. Tumors of the choroid plexus in children. Neurosurgery 1989; 25: 327-35. 5. Pencalet P, Sainte-Rose C, Lellouch-Tubiana A, Kalifa C, Brunelle F, Sgouros S, et al. Papillomas and carcinomas of the choroid plexus in children. J Neurosurg 1998; 88: 521-8. 6. St Clair SK, Humphreys RP, Pillay PK, Hoffman HJ, Blaser SI, Becker LE. Current management of choroid plexus carcinoma in children. Pediatr Neurosurg 1991-1992; 17: 225-33. 7. Packer RJ, Perilongo G, Johnson D, Sutton LN, Vezina G, Zimmerman RA, et al. Choroid plexus carcinoma of childhood. Cancer 1992; 69: 580-5. 8. Fitzpatrick LK, Aronson LJ, Cohen KJ. Is there a requirement for adjuvant therapy for choroid plexus carcinoma that has been completely resect-ed? J Neuro-Oncol 2002; 57: 123-6. 9. Pierga JY, Kalifa C, Terrier-Lacombe MJ, Habrand JL, Lemerle J. Carcinoma of the choroid plexus: A pediatric experience. Med Pediatr Oncol 1993; 21: 480-7. 10. Duffner PK, Kun LE, Burger PC, Horowitz ME, Cohen ME, Sanford RA, et al. Postoperative chemotherapy and delayed radiation in infants and very young children with choroid plexus car­cinomas. Pediatr Neurosurg 1995; 22: 189-96. 11. Aricň M, Raiteri E, Bossi G, Giordana MT, Corbella F, Locatelli D, et al. Choroid plexus carci­noma: report of one case with favourable response to treatment. Med Pediatr Oncol 1994; 22: 274-8. 12. Palazzi M, Di Marco A, Campostrini F, Grandinetti A, Bontempini L. The role of radio­therapy in the management of choroid plexus neo­plasms. Tumori 1989; 75: 463-9. 13. Duffner PK, Krischer JP, Horowitz ME, Cohen ME, Burger PC, Friedman HS, et al. Second malignancies in young children with primary brain tumors fol­lowing treatment with prolonged postoperative chemotherapy and delayed irradiation: A Pediatric Oncology Group study. Ann Neurol 1998; 44: 313-6. Radiol Oncol 2004; 38(3): 187-92. Keratocysts in the jaws Aleksander Lipovec, Nataša Ihan Hren Clinical Department of Maxillofacial and Oral Surgery, University Medical Centre Ljubljana, Slovenia Background. Jaw cysts are a common pathology; among them, the odontogenic keratocysts (OKC) repre­sent a special group because of their aggressive growth and recurrence. Patients and methods. We established retrospectively the epidemiology and clinical characteristics of OKC among all the pathohistologically confirmed jaw cysts that had been surgically cured in the past ten years (from 1994 to 2003) at the Clinical Department of Maxillofacial and Oral Surgery, University Medical Centre in Ljubljana. Results. Among 992 surgically removed jaw cysts, 106 were OKC (10.6% of all). Pathohistological diagno­sis of OKC was confirmed in 90 patients, in 51 men (56.7%) and 39 women (43.4%). Mean age of patients with OKC at the time of treatment was 36 years. The youngest one was 7 years old, the oldest one 83 years. Seventy-four (69.8%) of OKC were removed from the lower jaws, 32 (30.2%) from the upper jaws. As to the location, OKC (49 cases - 46.2%) predominated in the angles and vertical branches of the lower jaw. Recurrence rate after the first removal of OKC was 22.2% (in 20 patients). First recurrence occurs most fre­quently (in 70%) within the first 5 years after primary treatment; the mean time till the first recurrence was 4 years and 7 months. Multiple recurrences of OKC were observed in 9 patients (10% of all patients with OKC). Five of the patients with OKC had the syndrome of basal cell carcinoma (Gorlin–Goltz Sy.). We found out that one third of OKC were clinically unexpected in regard to their untypical locations in the jaws. Conclusions. It is critical for the jaw cysts to be pathohistologically examined. The number of cases of OKC among all jaw cysts in our study is significantly larger (3.5-time) than in the previous epidemiological study in Slovenia; but this may be the consequence of previous underdiagnosis of that pathology entity. Our study is comparative with other similar foreign studies in literature in respect to the patient’s sample and epi­demiological results. Key words: jaw cysts – pathology; odontogenic cysts - epidemiology Received 27 May 2004 Accepted 17 June 2004 Correspondence to: Assist. Prof. Nataša Ihan Hren, MD, PhD, Department of Maxillofacial and Oral Surgery, University Medical Centre, Zaloška 2, Ljubljana, Slovenia; Phone: +386 1 522 43 54. Introduction Cysts in the facial bones are similar to other cystic changes elsewhere in the human skele­ton regarding their morphology and struc­ture. Because of the presence of the develop­mental dental and nondental epithelium and the teeth with surrounding tissues in the jaw­bone, the cysts are much more common in the jaws than anywhere else. They occur as pathological unicentric or multicentric cavity, where the liquid, solid masses or gas accu­mulate, but not as the consequence of pus ac­cumulation.1 The cysts are usually, but not al­ways, surrounded with the epithelium, which differs in morphogenesis and structure.2 The most common characteristic of cysts is the bone defect – the radiolucent area on X-rays, which can cause different clinical signs pre­dominantly because of their sizes or sec­ondary infections. Other causes of bone de­fects are odontogenic and bone tumours, but they are much more rare in a jawbone than in other parts of human skeleton.3 Among them are also very rare bone defects caused by tu­mour metastases, mostly of adenocarcinomas (tumours in the breasts, bronchus, kidneys).4 They are so important because they can be the first clinical sign. Jaw cysts are nonodontogenic and odonto­genic; the latter are more common. Two thirds of odontogenic cysts are of inflamma­tory origin (radicular, residual cysts); follicu­lar cysts (in approximately 15% of all cysts), keratocysts or a few others are rarer.3 Odontogenic keratocysts (OKC) were rec­ognized as a special pathologic entity very late. Among the cysts, they are considered as a peculiar group because of their aggressive growth and great recurrence; they therefore require more radical surgical treatment (the removal with surrounding bone) and pro­longed clinical follow-up. Pathohistologically, they are divided into para- and ortho-kerati-nous types of OKC. Regarding their patho­genesis, they are benign neoplasms.5 Figures 1a and 1b show 2 typical panoramic X-rays of OKC. In 1945, Robinson described primordial cysts as the consequence of impaired dental enamel organ. Later, their appearance was as­sociated with the developmental disturbance of dental lamina. These cystic bone lesions were first named OKC by Philipsen in 1956, which became also their official term in the WHO classification of cysts in 1971.1,2 Among the jaw cysts, the frequency of OKC in different studies ranges from 3.2 to 11.25%.2,6 The factors which influence the growth and development of OKC are still un­known; they can be either in the epithelium of a cyst or in the connective tissue of its cap­sula.6 As OKC are detected in the patients Table 1. The incidence of the pathohistologicaly proved cysts from 1994 to 2003 Cyst / Year 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 . % Radicular 17 14 11 18 33 57 86 82 98 114 530 53.5 Dentigerous 9 5 2 2 9 17 16 5 8 16 89 9.0 Residual 2 2 2 1 6 13 19 13 8 15 81 8.2 Eruption 1 0 0 0 0 0 0 0 1 0 2 0.2 Odontogenic keratocysts 7 1 5 9 7 6 9 16 21 25 106 10.6 Gingival cyst of adults 0 0 0 0 2 0 0 0 1 0 3 0.3 Lateral periodontal 0 1 0 0 1 0 0 0 1 0 3 0.3 Calcifying odontogenic 0 0 0 0 0 0 0 0 0 1 1 0.1 Solitary bone 1 0 1 0 2 1 2 1 1 1 10 1.0 Pseudocyst 0 1 0 0 0 0 1 0 0 0 2 0.2 Nasolabial 0 0 0 0 0 0 0 1 0 0 1 0,1 Nasopalatine 0 0 0 2 2 1 1 0 3 1 10 1.0 Unknown 16 18 27 21 15 19 11 9 11 7 154 15.5 . 53 42 48 53 76 114 145 127 153 180 992 100.0 aged between 7 and 93 years, they can arise in any life period.7 The size of OKC is from 0.1 to 10.0 centimetres in diameter, the mean size at the time of their detection is 4 to 5 cm.8 They grow for 2 to 14 millimetres per year; their growth is slower after the age of 50.9 In literature, a great possibility of recurrence af­ter the removal of the primary OKC in the same place or in their neighbourhood was de­scribed (3 to 62%, mean possibility is 30%).6 In 1960, Gorlin and Goltz first described the syndrome of three mean signs: multiple cells-cells carcinomas of the skin, multiple jaw OKC and rib changes. OKC are a perma­nent and clinically important sign. Later, this syndrome was named nevoid basal-cells car­cinoma syndrome. It is inherited dominantly autosomally and has a high level of genetic penetrance with prevalence of 1 : 60 000.6,10 In the differential diagnosis of OKC before pathohistologic examination, we must also consider tumour ameloblastoma, whose simi­larity arises from the same origin – the dental lamina; the ameloblastoma can also arise from OKC epithelium.7 In addition, the trans­formation in orthokeratinous epithelium of OKC to planocellular carcinoma has also been described.8 The purpose of our study was to establish the frequency and characteristics of OKCs, which had been surgically cured in the past ten years (from 1994 to 2003) in Clinical Department of Maxillofacial and Oral Surgery, University Medical Centre in Ljubljana. Patients and methods We removed and pathohistologically exam­ined 992 cysts in the lower and upper jaws from 930 patients from the beginning of 1994 to the end of 2003. On the basis of pathohis­tologic examinations, we retrospectively sur­veyed all medical documentation of these pa­tients (all diagnostic procedures, treatment and post-operative controls). We determined the basic statistical charac­teristics. Table 1 presents the frequency of different cysts in the observed years. Results In the period from 1994 to 2003, we surgical­ly removed 992 pathohistologically proved cysts at our department. Of them, 105 were OKC, which represented 10.6% of the cysts of oromaxillofacial region (Figures 1a, 1b). In the observed period, pathohistological di­agnoses of OKC were confirmed in 90 patients. Of them, 51 were in men with the mean age of 39 years and 39 in women with the mean age of 33 years. The mean age of all patients with OKC at the time of treatment was 36 years. The youngest one was 7 years old, the oldest one 83 years. Figure 2 represents the distribution of the observed patients by sex and age. Seventy four OKC were removed from the lower jaws (69.8% of all OKC) and 32 from the upper jaws (30.2% of all OKC). Regarding Figure 2. Patients with OKC by the sex and age inter­vals. the location, they predominated in the angles and vertical branches of the lower jaw (in 49 cases, 46.2% of all OKC). The characteristic cases of that predilection location are shown in Figures 3a, 3b and 3c. Recurrence rate after the first removal of OKC was 22.2% (20 of 90 patients). The first recurrence is the most frequent (in 70%) with­in the first 5 years after the primary treat­ment, the mean time until the first recurrence being 4 years and 7 months. The longest time between the first treatment of OKC and diag­nosis of its first recurrence was 15 years. Multiple recurrences of OKC occurred in 9 patients (10% of all patients with OKC). Among the patients with OKC, 5 had the syndrome of basal cell carcinoma (NBCCS, Gorlin–Goltz syndrome). Two of them had multiple basal-cells carcinomas. One patient, 10 years old at the time of diagnosis NBCCS, was hereditarily predisposed, as his mother had the same illness. We established that, in 31 patients, OKC was clinically unexpected before pathohisto-logic examination; the pathohistologic exami­nation then confirmed it was OKC. Discussion All the patients examined here were treated at the Clinical Department of Maxillofacial and Oral Surgery, University Medical Centre Ljubljana. At this institution, orodental pathologies of the patients from Ljubljana re­gion are treated and it is also the Slovenian Figure 3a. The characteristic tomogram radiograph (panoramic x-ray) of OKC on predilection location (the angles and vertical branches of the lower jaws) in the right side. referral clinic that treats the most complicat­ed cases of orodental pathologies of the pa­tients from the whole country. All patients treated at our clinic were included into this study, viz. also the patients with extremely large OKC or those with OKC in the neigh-bourhood of vitally important structures. The consequence of this is that the percentage of OKC among the jaw cysts reported here is greater than that in Slovenia's population. The smallest OKC in our study are from 0.5 to 1 centimetre in diameter and they pre­dominate in the upper jaws. In clinical and ra­diological aspects, this means that these OKC are commonly removed as inflammatory odontogenic pathology without being patho­histologically examined. In these cases, OKC are often overlooked. This also means that these overlooked OKC are not treated and controlled adequately. At our Department, we were already studying the jaw cysts for a ten-year period from 1984 to 1993. Our research was based on pathohistological diagnostics.11 The pre­sent study from 1994 to 2003 is the continu­ation of that one. We observed 3.5-times more OKC in the last decade than in the for­mer one. During the first study, the OKC pathologies were rarely recognised; they were recognised in fewer cases than expect­ed from the literature data. Interestingly, OKC were first pathohistologically recog­nised only in 1982. Among the jaw cysts, the frequency of OKC was 10.6% in the period 1994 -2003. This percentage agrees well with the litera­ture data of 3.2 to 21.8%;2,6,8 in fact, it is ap­proximately in the middle. The ratio of men to women with OKC is 1.28 to 1; the predominance of men with OKC is mentioned also by other authors.7,12,13 The age structure of the patients with OKC in our study is similar to other’s data. In 1992, Shear reported that OKC in elderly popula­tion was often discovered late because of a few and delayed clinical signs.2 In our study, 34.4% of patients came in our institution without any clinical signs; later, the cavities in the jaws were accidentally found on the ra­diographs done by their dentists. The ratio of OKC in the lower against the upper jaw is 2.3 to 1. The higher occurrence of OKC in the lower jaws is reported through­out the literature. The ratios of OKC location in the lower to the upper jaws are from 1.9 : 1 to 4.9 : 1 according to different authors and our findings are within the described range.7,14 The recurrence rates of OKC after the first surgical removal differ from one author to an­other and are within the range of 10.4%15 to 39%.13 During the observed period, the recur­rences of OKC were observed in 20 of our pa­tients (22.2% of all OKC patients). The multi­ple recurrences were observed in 9 patients (in 10% of cases). According to the data from literature, multiple recurrences are ranging from 2.8%7 to 4.6%.14 The mean time until the first recurrence of OKC in our study is 4 years and 7 months, while Brannon reported that this time was 4 years and 10 months.7 The presence of OKC in children or ado­lescents before the age of 19 is, in 75% of cas­es, the first sign of NBCCS.6 Four patients in our study with NBCCS had OKC before the age of 19. According to literature, the first recur­rence is the most frequent within the first 5 years after primary treatment in 75% of cases, whereas according to our data, it occurs in 70% of cases.3,5 Some authors described the recurrence even 20 years after primary treat­ment.2 In our study, the longest time until the first recurrence was 15 years. So, while the regular clinical and radiograph controls of patients with OKC in the first 5 years after the primary surgery are necessary, the pa­tients with NBCCS have to be checked once a year throughout their lifetime. The treatment of OKC is more radical than of the other jaw cysts. Because of the aggres­sive nature of OKC, it is necessary to remove the wall of the cyst and all bone adjacent to the wall colonised by the cysts epithelium is­lands and buds. When the bone is totally de­structed by the cyst, this can spread to the soft tissue, which must be removed entirely. In order to diminish the cases of unrecog­nised OKC, the pathohistologic examinations of all cyst tissue is always necessary, as in our study, there were 37 surprise cases of OKC (34.8% of all OKC) discovered through the di­agnostic procedures, radiographs and clinical diagnosis of other odontogenic cysts. Maxillofacial and oral surgeons in Slovenia have approximately 10% possibility that the jaw cyst is OKC. These cysts must be recog­nised by the pathohistologic examinations to be adequately treated and to assure to the pa­tients to receive adequate clinical follow-up. References 1. Kramer IRH, Pindborg JJ, Shear M. Histological typ­2nd ing of odontogenic tumours. edition. Berlin: Sprienger-Verlag; 1992. p. 35-6. 2. Shear M. Odontogenic keratocyst (primordial cyst). In: Shear M, editor. Cysts of the Oral Regions. 3rd edition. Oxford: Butterworth-Heinemann; 1992. p. 4-45; 246-8. 3. Cawson RA, Odell EW. Essentials of oral patholo­gy and oral medicine. Toronto: Churchill Livingstone; 1998. p. 132. 4. Sapp JP, Eversole LR, Wysocki GP. Contemporary oral and maxillofacial pathology. St Louis: Mosby; 2004. p. 203-4. 5. Shear M. Odontogenic tumors and cysts with both unicystic and multicystic variants. Are the cysts benign tumors? In: Cystic lesions of the maxillofacial area. 6th Salzburg weekend seminar 1998. p. 47-51. 6. Budnick S. Odontogenic cysts. In: Gale N, Luzar B, editors. 19th European Congress of Pathology Pre-meeting. Head and neck pathology proceed­ings. Ljubljana; 2003. p. 122-32. 7. Brannon RB. The odontogenic keratocyst. A clini­copathologic study of 312 cases. Part I. Clinical features. Oral Surg Oral Med Oral Patho 1976; 42: 54-72. 8. Verbin RS, Barnes L. Cysts and cyst-like lesions of the oral cavity, jaws and neck. In: Barnes L, editor. Surgical pathology of the head and neck. 2nd edi­tion. New York: 2001. p. 1439-555. 9. Shear M. The odontogenic keratocyst. In: Cystic le­sions of the maxillofacial area. 6th Salzburg weekend seminar 1998. p. 34-7. 10. Williams TP, Hellstein JW. Odontogenic cysts of the jaws and other selected cysts In: Fonseca RJ, editor. Oral and maxillofacial surgery - 5. Surgical pathology. 1st edition. Sunders Co; 2000. p. 310-6. 11. Ihan-Hren N. Pogostnost cist v celjustih zadnjih 10 let. Zobozdr Vest 1995; 50(1-2): 13-5. 12. Woolgar JA, Rippin BDS, Browne RM. The odon­togenic keratocyst and its occurrence in the nevoid basal cell carcinoma syndrome. Oral Med Oral Pathol 1987; 64: 727-30. 13. Hodgkinson DJ, Woods JE, Dahlin DC, Tolman DE. Keratocysts of the jaw: clinicopathologic study of 79 patiets. Cancer 1978; 41: 803-13. 14. Browne RM. The odontogenic keratocyst clinical aspects. Br Dent J 1970; 128: 225-31. 15. Kakarantza-Angelopoulou E, Nicolatou O. Odonto­genic keratocysts: clinicopathologic study of 87 cases. J Oral Maxillofac Surg 1990; 48: 593-9. Radiol Oncol 2004; 38(3): 193-203. review Psychological distress and intervention in cancer patients treated with radiotherapy Mojca Šoštaric, Lilijana Šprah Institute of Medical Sciences, Slovenian Academy of Science and Arts, Ljubljana, Slovenia Background. Common side effects of treatment with radiation therapy (RT) often cause psychophysical distress in cancer patients. Anxiety, adjustment disorders and depression (which are according to many stud­ies experienced in about half of the oncological population) might originate some serious psychiatric forms of mood disorders and can even culminate in suicide, if not treated appropriately. There are some groups of cancer patients who are especially vulnerable and among them are cancer patients undergoing RT –they should receive special attention from medical staff. The purpose of this review is to present a variety of psy­chosocial interventions and illustrate some methods that are (or could be) used in psycho-oncology practice. Conclusions. A large body of literature suggests that the first intervention step should be effective screen­ing for patients in distress. In regard to these proposals the development of (computerized) screening pro-grammes is the first measure that ought to be taken. Moreover, further systematical research of traditional, non-traditional and complementary intervention strategies in cancer patients in distress would be necessary in order to provide reliable empirical results about the effectiveness of different approaches. Key words: neoplasms –radiotherapy –psychology; mood disorders; adjustment disorders; distress, inter­vention, screening. Introduction The psychosocial oncology research studies indicated that significant proportion of can­cer patients at all stages of the disease have been confronted with psychosocial dis­tress.1,2 The prevalence of some psychiatric Received 16 August 2004 Accepted 23 August 2004 Correspondence to: Mojca Šoštaric, BSc, Institute of Medical Sciences, Slovenian Academy of Science and Arts, Novi trg 2, P.O. Box 306, Sl –1001 Ljubljana, Slovenia; Phone: +386 1 470 64 47; Fax : +386 1 426 14 93; E-mail: smojca@zrc-sazu.si illnesses (major depression, generalized anxi­ety, adjustment disorder) is higher in some groups of cancer patients (patients undergo­ing radiotherapy (RT) or palliative treatment, terminally ill patients and patients experienc­ing uncontrollable pain), which also implies an increased risk for suicidal behaviour.3,4 Some studies estimated that approximate­ly 20% of cancer patients need a psychiatrist for treating major depression or anxiety dur­ing their cancer experience. Additional 15% of cancer patients need the services of a psy­chologist for treating distress and 25% of pa­tients require the services of social workers to deal with financial and practical issues.5,6 Since some reports emphasized that psy­chosocial interventions in cancer patients are not only effective but also economical, more attention should be focused on establishing routine psychosocial screening programs in order to asses psychological functioning, pri­marily anxiety and depression and overall Quality of Life (QoL).7-9 Cancer patients should be targeted around the time of initial diagnosis and treatment. They should be screened for distress and common problems during the treatment trajectory. The purpose is the identification of those cancer patients who experience significant distress at early stages of therapy in order to treat them proac­tively and avoid future psychosocial prob­ lems.10-14 RT was one of the earliest cancer treat­ments and it still plays an important role in the care of cancer patients. It has been used in curative as well as in palliative cancer treat­ment in order to achieve a local control over the tumour with minimum side effects. Despite astonishing progress of modern RT technique (safe doses of radiation which are skin sparing in comparison to old techniques), the treatment causes some common physical side effects, for example: fatigue, nausea, di­arrhoea, gastrointestinal symptoms and skin irritation. Some of them can persist even after the treatment. Studies revealed that cancer patients may also face some psychological problems during the treatment with RT.11,15 A wide range of different aspects of psychologi­cal functioning and well-being can be im­paired in cancer patients prior to, during, and after RT.16,17 Therefore a better insight into the psychosocial functioning of patients un­dergoing RT could facilitate the identification of those who are at higher risk of developing mood disturbances. These patients should re­ceive a full psychosocial support to manage the coping process with cancer and adopt ap­propriate coping strategies.18,19 There are many methods and techniques of psychosocial interventions that are com­monly used in psycho-oncological practices. Most of them are useful in treating cancer pa­tients undergoing RT as well.20,21 A variety of psychosocial approaches (education in group, education on pain management, meditation training, biofeedback, relaxation training, vi­sualization, creative therapies such as music, art or role play, peer supportive group thera­py, family counselling, etc.) have been stud­ied and approved to significantly contribute to patient’s QoL. Nevertheless, many of them are ineffective if medical staff is not ac­quainted with the dynamic of psychological functioning of cancer patients undergoing RT and well trained for recognizing the patient’s crisis. In the following review some aspects of psychosocial functioning and distress in can­cer patients treated with RT will be discussed. In addition some psychosocial interventions will be described. Distress induced by experience with cancer Several studies clearly demonstrate that psy­chosocial distress occurs in one-third to one-half of all cancer patients.3,10,11 There are some groups of cancer patients that are espe­cially vulnerable to psychosocial distress. Particularly patients with history of chronic depression, patients with breast and genitalia cancer, patients using specific coping strate­gy (hopelessness/helplessness), patients without social support, children and elderly patients should be recognized at earlier stages of cancer diagnosis and treatment.3,5,22 Although severity of emotional distress is more closely related to a patient’s pre-exist­ing vulnerability than to the characteristics of the cancer, it is more likely to occur at the fol­lowing stages of patient’s experience with cancer:23-25 Diagnosis Investigation and diagnosis can induce anger, shock, disbelief and emotional distress in cancer patients. Whereas these can be re­solved without interventions in the most cas­es, especially high levels of distress at this time are predictive for onset of later emotion­al problems. During treatment Side-effects of hospital attendance, unpleas­ant surgery, RT and/or chemotherapy are pre­vailing reasons for the distress. Patients might become particularly distressed during apparent treatment failure. End of treatment Some patients can experience "rebound" dis­tress associated with the fear that cancer could spread or occur again. The sense of loss and vulnerability may also appear at this point as outcome of ending the prolonged re­lationship with the cancer service staff. After treatment Many patients who survive cancer may re­order their life priorities. On the other hand others need help to overcome worries, preoc­cupation with loss and illness, a tendency to avoid reminders of cancer, difficulties coping with intimacy and return to work. A form of health anxiety with misinterpretation of physiological sensations and anxious seeking of reassurance may develop as a form of fears of cancer recurrence. Recurrence Patients who believe that they have been cured are at greater risk of severe distress if recurrence occurs. For some of them recur­rence of cancer may be more distressing than receiving the initial diagnosis. Terminal disease Depression is common in the terminal phase, particularly in cancer patients with poorly controlled physical symptoms. Majority of cancer patients experience fear of uncon­trolled pain and the process of dying. They worry what happens after death and are con­cerned about loved ones. Negative or positive psychological states Studies engaged with adjustment to cancer focused primarily on negative psychological states like depression, anxiety and general distress.26-28 Many of them concluded that cancer could be understood as an event and an ongoing process that may physically and emotionally weaken the individual. On the other hand, the number of studies on positive consequences of the cancer experience is growing. These studies suggested that experi­ence with cancer may result also in some pos­itive outcomes, for example: increased self–esteem and more optimistic look, im­proved interpersonal relationships, re-evalua­tion of prior goals, altered priorities in life and new pursuits.29-33 Radiotherapy and psychosocial functioning in cancer patients The results of studies comparing RT to other cancer treatments revealed few differences in psychological functioning among different treatment modalities.11,34 The patients under­going RT experienced a common distress –similar to patients without RT. Some studies indicated a great variability in reported re­sults, but the global trends in psychological responses to RT prevailed. The most common reactions reported by patients before starting a course of RT, were feelings of anxiety rather than depressive symptoms. During the course of treatment, most studies indicated a decline in feelings of anxiety. An increase of depressive symptoms and negative mood was found during and after RT. It was also em­phasized that the field lacks a systematic overview of the empirical data regarding psy­chological functioning prior to, during and af­ ter RT. 11,16,35 Although RT is not painful it induces many side effects, which can continue long after completion of treatment. Nevertheless, the non-invasive nature of RT can make it easier for patients to adapt, which could ex­plain why in spite of anxiety, patients often experience RT much more positive than ini­tially expected.11,36 Some studies aimed to en­lighten the psychosocial functioning in can­cer patients undergoing RT since some un­pleasant side-effects could interfere with their quality of life, namely: fatigue, skin irri­tation, nausea, diarrhoea, genitourinary and gastrointestinal symptoms, long-term cogni­tive disability in patients undergoing RT of brain, eating problems and oral complica­tions in patients undergoing RT of head and neck, etc.37-44 Some recent studies reviewed the impact of RT on neuropsychological functioning in cancer patients.38,45-48 Neuropsychological side-effects of different cancer treatments may include difficulty concentrating, im­paired verbal and visual memory, difficulty organizing information, decreased motor skills, and language problems. It remains un­clear to which extent intellectual and cogni­tive impairment in cancer patients could be linked with RT, since some patients could have already been experiencing some cogni­tive decline due to the effects of stress, fa­tigue or the sometimes toxic by-products of the cancer process itself. It has been emphasized, that the assess­ment of RT side-effects either by the doctor or the patient remains subjective and often leads to different results.49 This implied a need for establishing reliable screening sys­tem with objective parameters in assessment of psychophysical and social distress of ra­diooncological patients.50-54 To provide effec­tive psychosocial support for cancer patients undergoing RT, the screening for psychoso­cial status of patients should be performed during diagnostic and therapeutic proce­dures. A distress inventory composed of in­terviews; checklists and questionnaires should be applied before, during and after the course of RT. First intervention step: screening for cancer patients in distress Depressive and anxiety disorders often re­main unrecognised. In this regard an active screening by simply asking patients about symptoms of anxiety and depression may be helpful. How can an oncologist screen for pa­tients in distress? One research group report­ed that single-item screening was sensitive and specific for depression – an oncologist must simply ask a question: "Are you depressed most of the time?"3 Usually the questionnaire for assessing patients' anxiety and depression includes questions like: "How are you feeling in yourself?"; "Have you felt low or worried?"; "Have you ever been troubled by feeling anxious, ner­vous, or depressed?"; "What are your main con­cerns or worries at the moment?"; "What have you been doing to cope with these?"; "What effects do you feel cancer and its treatment will have on your life?"; "Is there anything that would help you cope with this?"; "Who do you feel is helping you at the moment?" Doctors should also be aware that patients might be distressed because of fac­tors unrelated to cancer.23,55,56 Additional check-up for some risk factors that underlie psychiatric disorders is also re­quired in cancer patients. Risk factors associ­ated with patient (history of psychiatric dis­order, social isolation, dissatisfaction with medical care, poor coping), cancer (limitation of activities, disfiguring, poor prognosis) and treatment (disfiguring, side-effects) should be taken into consideration.8,57 It is common to use diagnostic tools or (computerized) screening programs to pro­vide a better insight into patient’s crisis. Several agencies developed screening guide­lines or books of standards in order to pro­vide distress screening for each patient. Most widely used tools are: Brief Symptom Inven-tory (BSI), Hospital Anxiety and Depression Scale (HADS), General Health Question­naire, QoL Questionnaires and distress ther­mometer.9 A large amount of studies on psy­chometrical characteristics of these screening tools is available on-line. Review of literature showed that more than 45 tools/instruments have been used to measure psychological distress.58 Unfortu­nately none of them is able to identify pa­tients who are highly distressed without clin­ical symptoms of anxiety and depression. Therefore, the development of a reliable screening mechanism seems appropriate and may help in identifying patients who specifi­cally warrant the intervention. Distress-screening tool may also assist health profes­sionals to provide patient-specific-interven­tion processes if the distress level and cause could be identified. Several groups of scientists are dealing with development of screening mechanism for distress induced by RT. A group of German scientists (Klinikum Grosshadern, Munchen) developed questionnaire SIRO (The Stress Index RadioOncology).50 A group in Canada has been developing a computer­ized QoL program for clinical use in palliative RT.5,59 In recent studies two instruments were tested for benefits in screening process: a short and structured interview procedure PRIME-MD (Primary Care Evaluation of Mental Disorders) and BCD (Brief Case-Find for Depression).10,60 Both instruments were found to quickly and reliably identify the prevalence and types of mood disorders (de­pressive disorders: major depression, minor -subsyndromal depression/adjustment disor­der, dysthymia, bipolar disorder; anxiety dis­orders: panic disorder, generalized anxiety disorder). When compared to the PRIME-MD diagnosis of depression the BCD had greater sensitivity. No specific training is needed to administer PRIME-MD and BCD; their appli­cation is quick and therefore convenient for screening by oncologist. Second intervention step: post-screening intervention strategies Interventions usually assume the following common strategies: psycho-education, cogni­tive-behavioural training (group or individ­ual), supportive therapy (group or individ-ual).5 They target different points on the ill­ness trajectory: diagnosis/pre-treatment, im­mediately post-treatment or during extended treatment (such as RT or chemotherapy) and disseminated disease or death. Certain moda­lities of intervention strategies have been proven to be more efficacious at one or more of these time periods. For example, psycho-education may be most effective during the diagnosis/pre-treatment period, when patient searches for information. Group support may be more effective at later stages to cope with more advanced disease.61,62 Cognitive-behav­ioural techniques such as relaxation, stress management and cognitive coping could be most useful during extended treatments.63,64 Relaxation and imagery have been shown to be effective in controlling nausea and vomit­ing associated with chemotherapy treat-ment,65 and furthermore, these techniques can also help patient to decrease the usage of pain medications.66 Many studies have focused on the efficacy of group interventions. It seems that group therapies have repeatedly been shown to be as effective as individual treatment.63,67 Because of the reduced cost of group thera­pies (the greater number of patients who can be treated at the same time) many researchers identify group therapy as the preferred route for treating distress in cancer patients. Several specific group therapy interventions have been standardized and proven effica­cious, for example: supportive expressive therapy for metastatic early stage breast can­cer,68,69 mindfulness-meditation based stress reduction70,71 and standardized group psy-cho-education for patients with different types of cancer diagnoses.72 On the other hand recent surveys confirm the popularity of non-traditional therapies among cancer patients. 23% – 81 % of U.S. and Canadian, 22% – 52% of Australian, 16% – 32% of British and 10% – 61% of mainland European cancer patients used at least one such thera­py.20,73,74 Psychological therapies (e.g., relax­ation, meditation, visual imagery, and hyp­notherapy) are among most popular non-tradi­tional therapies. More than 50% of Australian and up to 29% of U.S. and 10% of European and Canadian cancer patients have reported the use of at least one type of psychological therapy.75,76 Patients have high expectations of these therapies: in one study, up to 25% of par­ticipants expected the psychological therapy to cure their cancer and 75% – 100% expected it to assist their traditional therapies.76 Moreover, alternative/complementary thera­pies are increasingly used to reduce side ef­fects of cancer treatment, without convincing evidence of their effectiveness.77 Some stud­ies are in favor of using complementary ther­apy in order to reduce the stress and anxiety in cancer patient.78,79 A study of patient’s per­ceptions of the benefits of reflexology on their QoL revealed that reflexology interven­tions were perceived to impact positively on psychophysical functioning.80,81 Further indications for effective acupunc­ture treatment of patients with radiation-in­duced xerostomia came from study with pa­tients undergoing RT for head and neck can­ cer.82,83 Some national institutes of health support the use of acupuncture for chemo-therapy-induced nausea and vomiting.84 The nurse practitioners are obligated to be knowl­edgeable about the use of these and other ef­fective complementary treatments in order to provide the best care. Used in conjunction with current antiemetic drugs, acupuncture and acupressure have been shown to be safe and effective for relief of the nausea and vom­iting resulting from chemotherapy. Application of psychological interventions has been found in many studies to improve the QoL in cancer patients. These is hypothesized to be mainly due to reducing their psychologi­cal symptoms and distress, enhancing psycho­logical and functional adjustment and rehabili­tation as well.85 However, the relevance of psy­chosocial interventions on survival from cancer has been widely criticized. In the recent review all identifiable publications about psychologi­cal therapies used by cancer patients have been critically and systematically analysed.20 Despite of extensive body of literature authors could not make strong recommendations about the effectiveness of psychological intervention strategies at improving cancer patient’s out­comes. The results of this review were consid­erably less enthusiastic about the likely bene­fits of psychological therapies for cancer pa­tients compared to the results of other recent reviews. While many studies recommended widespread and routine use of psychological therapies to improve patient’s psychosocial, side effect, survival and immune outcomes, this study emphasized that the most beneficial are group therapy, education, structured and unstructured counselling and cognitive behav­ioural therapy. Furthermore, some long-term cost studies have proven that psychosocial in­tervention programs have also beneficial eco­nomical value.5,86 Cancer patients undergoing RT can engage in all above described intervention programs. Some findings about psychosocial side ef­fects of RT, discussed in previous section have also got practical implications for med­ical staff in oncology practice.87 Namely, it is very important that patient is well informed what to expect during the treatment period. The aim of the RT should be explained and misperceptions eliminated (role models that underwent the RT could be used for educa­tion of patients). This is not important only because of the cooperation between the pa­tient and medical staff, but also because talk­ing often reduces the anxiety that ascends from specific, unknown and uncontrollable situation. In addition, after completed treat-ment with RT patients show high need for in­formation – particularly about the psy­chophysical changes they could experience after completing RT. They are also interested how they could best ask their physicians questions, which agencies they could call when they need help and how they could cope with painful emotions. Therefore pro­viding an information booklet in form of a self-management package seems to be an ef­fective intervention.88 Conclusions Although recent studies comparing different cancer treatments suggested that the psycho­logical impact of RT is not superimposed to other treatments, some measures have to be taken to avoid further psychiatric complica­tions (anxiety, depression, adjustment disor­ders and suicide) in cancer patients undergo­ing RT. 3,10,11,34 The first step is to effectively screen for patients in distress throughout the treatment process (patients should be screened at the initial visit and at appropriate intervals).10-14 A multidisciplinary approach that includes psychological as well as medical assessment and intervention should be car­ried out. Otherwise psychological care might be neglected by the medical focus on cancer treatment. Consequently screening program-mes for those patients who are likely to show psychological dysfunction and helping them to cope with treatment and cancer related problems should be the constituent part of cancer managment.5,36,86 The knowledge of the coping process with cancer and some fundamental strategies in screening for distress is substantial for medi­cal staff working with patients undergoing RT.86 What is expected before starting a course of RT? The majority of patients will experience anxiety (which will probably de­cline during the treatment).11,16,35 They will be distressed mostly by fears of possible side-effects and by the fact of being irradiated. An effective intervention strategy at this stage is psycho-education (providing information about RT and its side effects). The visualiza­tion and the relaxation training are also use­ful for reducing the anxiety. In contrast to the anxiety an increase of depressive symptoms is expected during and after RT. At this point the group-supportive therapy might be ap­propriate.61-69 To conclude: different psycho­logical interventions evoke different effects according to the specific stages of the cancer treatment. In general, five types of therapies were es­tablished due to increasingly active participa­tion by the recipient: providing information, emotional support, behavioral training in coping skills, psychotherapy and spiritual/ex­istential therapy.72 The widespread usage of nontraditional psychological and comple­mentary therapies20,73-84 is a big challenge to the traditional medical and psychological ap­proaches to cancer experience. The field lacks a systematic overview of different approach­es and their contributions to the QoL as well as to the cancer prognosis. Patients are most often willing to partici­pate in the therapeutic process of cancer, yet the psychological aspect of medication might be underestimated by the patient. Therefore oncologist should also notice if a patient needs some further psychological or psychi­atric consultations. Quick and reliable screening tools could be useful for this pur­pose. Although there are some available in­struments for such purposes (BSI, HADS, PRIME-MD, etc.),9,10,58 many findings suggest that the screening process for cancer patients experiencing distress while undergoing RT requires additional empirical analysis. References 1. Sivesind D, Baile WF. The psychologic distress in patients with cancer. Nurs Clin North Am 2001; 36: 809-25. 2. Parker PA, Baile WF, de Moor C, Cohen L. Psychosocial and demographic predictors of qual­ity of life in a large sample of cancer patients. Psychooncology 2003; 12: 183-93. 3. Chochinov HM. Depression in cancer patients. Lancet Oncol 2001; 2: 599-606. 4. Filiberti A, Ripamonti C. Suicide and suicidal thoughts in cancer patients. Tumori 2002; 88: 193­9. 5. Carlson LE, Bultz BD. 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Stiegelis HE, Ranchor AV, Sanderman R. Psychological functioning in cancer patients treat­ed with radiotherapy. Patient Educ Couns 2004, 52: 131-41. 12. Roth AJ, Modi R. Psychiatric issues in older cancer patients. Crit Rev Oncol Hematol 2003, 48: 185-97. 13. Zabora JR, Loscalzo MJ, Weber J. Managing com­plications in cancer: identifying and responding to the patient's perspective. Semin Oncol Nurs 2003; 19: 1-9. 14. Chow E, Tsao MN, Harth T. Does psychosocial in­tervention improve survival in cancer? A meta-analysis. Palliat Med 2004; 18: 25-31. 15. Janda M, Newman B, Obermair A, Woelfl H, Trimmel M, Schroeckmayr H, et al. Impaired qual­ity of life in patients commencing radiotherapy for cancer. Strahlenther Onkol 2004; 180: 78-83. 16. Sehlen S, Hollenhorst H, Schymura B, Herschbach P, Aydemir U, Firsching M, et al. Psychosocial stress in cancer patients during and after radio­therapy. Strahlenther Onkol 2003; 179: 175-80. 17. 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Klein M, Heimans JJ Aaronson NK, van der Ploeg HM, Grit J, Muller M, et al. Effect of radiotherapy and other treatment-related factors on mid-term to long-term cognitive sequelae in low-grade gliomas: a comparative study. Lancet 2002; 360: 1361-8. 39. Caffo O, Amichetti M, Mussari S, Romano M, Maluta S, Tomio L, et al. Physical side effects and quality of life during postoperative radiotherapy for uterine cancer, prospective evaluation by a di­ary card. Gynecol Oncol 2003; 88: 270-6. 40. Larsson M, Hedelin B, Athlin E. Lived experiences of eating problems for patients with head and neck cancer during radiotherapy. J Clin Nurs 2003; 12: 562-70. 41. Sehlen S, Hollenhorst H, Lenk M, Schymura B, Herschbach P, Aydemir U, et al. Only sociodemo­graphic variables predict quality of life after radi­ography in patients with head-and-neck cancer. Int J Radiat Oncol Biol Phys 2002; 52: 779-83. 42. Chandra PS, Chaturvedi SK, Channabasavanna SM, Anantha N, Reddy BK, Sharma S, et al. Psychological well-being among cancer patients receiving radiotherapy--a prospective study. Qual Life Res 1998; 7: 495-500. 43. Jacobsen PB, Thors CL. Fatigue in the radiation therapy patient: current management and investi­gations. Semin Radiat Oncol 2003; 13: 372-80. 44. Montgomery C, Lydon A, Lloyd K. Psychological distress among cancer patients and informed con­sent. J Psychosom Res 1999; 46: 241-5. 45. Anderson-Hanley C, Sherman ML, Riggs R, Agocha VB, Compas BE. Neuropsychological ef­fects of treatments for adults with cancer: a meta-analysis and review of the literature. J Int Neuropsychol Soc 2003; 9: 967-82. 46. Syrjala KL. The neuropsychology of cancer treat­ment. Introduction. Semin Clin Neuropsychiatry 2003; 8: 197-200. 47. Wefel JS, Kayl AE, Meyers CA. Neuropsycho-logical dysfunction associated with cancer and cancer therapies: a conceptual review of an emerg­ing target. Br J Cancer 2004; 90: 1691-6. 48. Costello A, Shallice T, Gullan R, Beaney R. The early effects of radiotherapy on intellectual and cognitive functioning in patients with frontal brain tumours: the use of a new neuropsychologi-cal methodology. J Neurooncol 2004; 67: 351-9. 49. Goldner G, Wachter-Gerstner N, Wachter S, Dieckmann K, Janda M, Poetter R. Acute Side Effects during 3-D-Planned Conformal Radio­therapy of Prostate Cancer. Strahlenther Onkol 2003; 5: 320-7. 50. Sehlen S, Fahmuller H, Herschbach P, Aydemir U, Lenk M, Duhmke E. Psychometric properties of the Stress Index RadioOncology (SIRO)--a new questionnaire measuring quality of life of cancer patients during radiotherapy. Strahlenther Onkol 2003; 179: 261-9. 51. Thomas BC, Mohan VN, Thomas I, Pandey M. Development of a distress inventory for cancer: preliminary results. J Postgrad Med 2002; 48: 16-20. 52. Katz MR, Kopek N, Waldron J, Devins GM, Tomlinson G. Screening for depression in head and neck cancer. Psychooncology 2004; 13: 269-80. 53. Lee-Preston V, Steen IN, Dear A, Kelly CG, Welch AR, Meikle D, et al. Optimizing the assessment of quality of life after laryngeal cancer treatment. J Laryngol Otol 2004; 118: 432-8. 54. Monfardini S, Ferrucci L, Fratino L, del Lungo I, Serraino D, Zagonel V. Validation of a multidi­mensional evaluation scale for use in elderly can­cer patients. Cancer 1996; 77: 395-401. 55. McDaniel JS, Musselman DL, Porter MR, Reed DA, Nemeroff CB. Depression in patients with cancer. Diagnosis, biology, and treatment. Arch Gen Psychiatry 1995; 52: 89–99. 56. Sheard T, Maguire P. The effect of psychological interventions on anxiety and depression in cancer patients: results of two meta–analyses. Br J Cancer 1999; 80: 1770–80. 57. Forester BM, Kornfeld DS, Fleiss J. Psychiatric as­pects ofradiotherapy. Am J Psychiatry 1978; 135: 960-3. 58. Thomas BC, Mohan VN, Thomas I, Pandey M. Development of a distress inventory for cancer: preliminary results. J Postgrad Med 2002; 48: 16-20. 59. Bezjak A, Skeel R, Depetrillo AD, Comis R, Taylor KM. Oncologist’s use of quality of life informa­tion: results of a survey of castem cooperative on­cology group physicians. Qual Life Res 2001; 10: 1­13. 60. Jefford M, Mileshkin L, Richards K, Thomson J, Matthews JP, Zalcberg J, et al. Rapid screening for depression - validation of the Brief Case-Find for Depression (BCD) in medical oncology and pallia­tive care patients. Br J Cancer 2004 [in print]. 61. Blake-Mortimer J, Gore-Felton C, Kimerling R, Turner-Cobb JM, Spiegel D. Improving the quality and quantity of life among patients with cancer: a review of the effectiveness of group psychothera­py. Eur J Cancer 1999, 35: 1581-6. 62. Clark MM, Bostwick JM, Rummans TA. Group and individual treatment strategies for distress in cancer patients. Mayo Clin Proc 2003; 78: 1538-43. 63. Bottomley A. Where are we now? Evaluating two decades of group interventions with adult cancer patients. J Psychiatr Ment Health Nurs 1997; 4: 251­65. 64. Fawzy FI. A short-term psychoeducational inter­vention for patients newly diagnosed with cancer. Support Care Cancer 1995; 3: 235-8. 65. Fawzy FI, Fawzy NW, Arndt LA, Pasnau RO. Critical review of psychosocial interventions in cancer care. Arch Gen Psychiatry 1995; 52: 100-13. 66. Sloman R, Brown P, Aldama E, Chu E. The use of relaxation for the promotion of comfort and pain relief in persons with advanced cancer. Contemp Nurse 1994; 3: 6-12. 67. Fobair P. Cancer support groups and group thera­pies: Part I. Historical and theoretical background and research on effectiveness. Journal of Psycho­social Oncology 1997; 15: 63-81. 68. Classen C, Butler LD, Koopman C, Miller E, DiMiceli S, Giese- Davis J. Supportive-expressive group therapy and distress in patients with metastatic breast cancer: a randomized clinical in­tervention trial. Arch Gen Psychiatry 2001; 58: 494­501. 69. Spiegel D, Morrow GR, Classen C, Raubertas R, Stott PB, Mudaliar N. Group psychotherapy for re­cently diagnosed breast cancer patients: a multi-center feasibility study. Psychooncology 1999; 8: 482-93. 70. Carlson LE, Ursuliak Z, Goodey E, Angen M, Speca M. The effects of a mindfulness meditation based stress reduction program on mood and symptoms of Stress in cancer outpatients: six month follow-up. Support Care Cancer 2001; 9: 112­23. 71. Speca M, Carlson LE, Goodey E, Angen M A. Randomized, waitlist controlled clinical trial: the effect of a mindfulness meditation-based stress re­duction program on mood and symptoms of stress in cancer outpatients. Psychosom Med 2000; 62: 613-62. 72. Cunningham AJ, Edmonds VI, Hampson AW, Hanson H, Hovenac M, Jenkins G. A group psy­choeducational program to help cancer patients cope with and combat their disease. Advances 1991; 7: 41-56. 73. Begbie SD, Kerestes ZL, Bell DR. Patterns of alter­native medicine use by cancer patients. Med J Aust 1996; 165: 545–8. 74. Miller M, Boyer MJ, Butow PN, Gattellari M, Dunn SM, Childs A. The use of unproven methods of treatment by cancer patients. Frequency, expecta­tions and cost. Supp Care Cancer 1998; 6: 337–47. 75. Maher EJ, Young T, Feigel I. Complementary ther­apies used by patients with cancer. BMJ 1994; 309: 671–2. 76. Sollner W, Zingg-Schir M, Rumpold G, Fritsch P. Attitude toward alternative therapy, compliance with standard treatment, and need for emotional support in patients with melanoma. Arch Dermatol 1997; 133: 316–21. 77. Post-White J, Kinney ME, Savik K, Gau JB, Wilcox C, Lerner I. Therapeutic massage and healing touch improve symptoms in cancer. Integr Cancer Ther 2003; 2: 332-44. 78. Keegan L. Therapies to reduce stress and anxiety. Crit Care Nurs Clin North Am 2003; 15: 321-7. 79. Fellowes D, Barnes K, Wilkinson S. Aromatherapy and massage for symptom relief in patients with cancer. Cochrane Database Syst Rev 2004; 2: CD002287. 80. Wright S, Courtney M, Donnely C, Kenny T, Lavin C. Client’s perceptions of the benefits of reflexol­ogy on their quality of life. Complement Therap Nurs Midwifery 2002; 8: 69-76. 81. Luebbert K, Dahme B, Hasenbring M. The effec­tiveness of relaxation training in reducing treat-ment-related symptoms and improving emotional adjustment in acute non-surgical cancer treat­ment: a meta-analytical review. Psychooncology 2001; 10: 490-502. 82. Blom M, Dawidson I, Fernberg JO, Johnson G, Angmar-Mansson B. Acupuncture treatment of patients with radiation-induced xerostomia. Eur J Cancer B Oral Oncol 1996; 32B: 182-90. 83. Blom M, Lundeberg T. Long-term follow-up of pa­tients treated with acupuncture for xerostomia and the influence of additional treatment. Oral Dis 2000; 6: 15-24. 84. Collins KB, Thomas DJ. Acupuncture and acupres­sure for the management of chemotherapy-in­duced nausea and vomiting. J Am Acad Nurse Pract 2004; 16: 76-80. 85. Ross L, Boesen E H, Dalton SO, Johansen C. Mind and cancer: does psychosocial intervention im­prove survival and psychological well-being? Eur J Cancer 2002, 38: 1447-57. 86. Šprah L, Šoštaric M. Psychosocial coping strate­gies in cancer patients. Radiol Oncol 2004; 38: 35­42. 87. Fritzsche K, Liptai C, Henke M. Psychosocial dis­tress and need for psychotherapeutic treatment in cancer patients undergoing radiotherapy. Radiother Oncol 2004; 72: 183-9. 88. Jenkins V, Fallowfield L, Saul J. Information needs of patients with cancer: results from a large study in UK cancer centres. B J Cancer 2000; 84: 48-51. Radiol Oncol 2004; 38(3): 205-16. review Use of preneoplastic lesions in colon and liver in experimental oncology Veronika A. Ehrlich, Wolfgang Huber, Bettina Grasl-Kraupp, Armen Nersesyan, Siegfried Knasmüller Institute of Cancer Research, Medical University of Vienna, Austria The present article gives a brief overview on the use of altered hepatic foci (AHF) and aberrant crypt foci (ACF) in the colon in experimental cancer research. These foci are easily detectable preneoplastic lesions, which have been discovered approximately 30 years ago. AHF and ACF are valuable tools for the detection of cancer - initiating and promoting compounds, and for the detection of chemoprotective agents. They were also successfully used in numerous studies aimed at elucidating the molecular mechanisms of early neopla­sia, such as alterations of the expressions of oncogene and tumor suppressor genes, and changes in the ac­tivities of cancer associated enzymes. Key words: preneoplastic lesions; liver neoplasms; colonic neoplasms Introduction Preneoplastic lesions are used in experimen­tal research since more than thirty years. They consist of morphologically or functio­nally altered populations of cells that are pre­cursors of neoplasms. In contrast to long term experiments in which tumor formation is used as an endpoint, they have the advan­tage that they can be detected after compara- Received 9 July 2004 Accepted 25 July 2004 Correspondence to: Siegfried Knasmüller Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a, 1090 Vienna, Austria. This paper was presented at the “3rd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. tively short time periods (after 2-5 months) and that the number of animals, which are re­quired are relatively small (usually 8-10 ani­mals are used per experimental group). Preneoplastic lesions have been identified in a number of organs, for example in the skin (epidermal dysplasia and hyperplasia, epithe­lial papillomas), lung (alveolar and focal hy­perplasia, nodular lesions), pancreas (atypical acinar foci), kidney (tubules with irregular ep­ithelium), mammary gland (hyperplastic alve­olar nodules) and also in liver and colon (for overview see2). The present article is focused on hepatic altered foci (AHF) and aberrant crypt foci (ACF) in the colon, which have been used extensively in the last years for the detection of carcinogens, for the identifica­tion of chemoprotective agents and also in mechanistic studies. It describes their mor­ A B Figure 2A,B. Different treatment schedules for the detection of initiating and promoting carcinogens for experi­ments in which AHF are used as biological endpoint. phology and molecular characteristics and their use in the identification of initiating, promoting and protective agents and the de­velopment of new techniques. Altered hepatic foci – morphology and phenotypes The use of altered hepatic foci started in the 1970’s. In the early years, a classification sys­tem was developed, which was based on the staining behaviour and included clear, aci­dophilic, intermediate, tigroid, basophilic and also mixed cells of AHF.2 In subsequent years, it was shown that the expression of a variety of enzymes of AHF differs from that of the nor­mal tissue, and based on this observations, his­tochemical methods were developed which en­able the detection of enzymatically altered AHF (for review see 1). An overview on the dif­ferent markers is given in the article of Pitot.3 At present, the most widely used endpoint is the expression of the placental form of glu­tathione-S-transferase (GSTp+), which can be detected by immunohistochemistry. About 3 80% of all foci stained positive for GSTp+. Another frequently used marker is .-glutamyl­transpeptidase. Figure 1 depicts a GSTp+ foci. Methodological aspects AHF can be used to detect tumor initiating (Figure 2A) and promoting properties (Figure 2B) of chemicals. To distinguish between these characteristics, the test animals are treated with the compounds according to dif­ferent schedules.4 Initiators and promoters of AHF Numerous synthetic and natural compounds have been identified, which either initiate or promote the formation of AHF.4 Typical ex­amples for initiators are nitrosamines (which are the most frequently used carcinogens in mechanistic studies), urethane, aflatoxin B1, heterocyclic aromatic amines, and haloe­thans.5 Also polycyclic aromatic hydrocar­bons such as benzo(a)pyrene cause formation of AHF in rats, although the liver is not a tar­get organ for tumor induction of this com­pound. Typical examples for compounds which promote the growth of AHF in the liver are barbiturates (phenobarbital etc.), steroid hor­mones such as dexomethazone and testos­terone, hypolipidemic drugs and polychlori­nated biphenyls (for review see4). Inhibition of foci formation Numerous investigations have been conduc­ted to identify compounds, which prevent the formation of liver foci. These agents were ei­ther protective at the initiation level (i.e. when administered before and/or simultaneously with the carcinogen) or at the promotion le­vel (after carcinogen treatment). Examples for anti-initiators are food additives such as buty-lated hydroxyanisole, which protects against AFB16 and butylated hydroxytoluene, which inhibited the foci formation caused by 2­acetyl-aminofluorene.7 Also glucosinolates, contained in cruciferous vegetables were found protective towards AFB1 and crucife­rous plants themselves inhibited foci forma­tion induced by the heterocyclic aromatic amine (HAA) IQ.8-13 A number of compounds were identified which prevent the development of foci when administered after the carcinogen treatment. For example acetaminophen and aminophe­nol were protective against formation of foci that had been induced by a nitrosamine in the liver4 and flavonone reduced significantly ar­eas of placental GSTp+ foci induced by afla-toxin B1 during the phenobarbital- induced promotion step.14 A very interesting observation was made in experiments with rats in which the restric­tion of dietary calories reduced the number and volume of AHF by 85% in 3 month; food restriction lowered DNA replication but in­creased apoptosis. When treated with a tu­mor promoter (nafenopin) after food restric­tion, only half as many hepatocellular adeno-mas were found as in animals fed ad libitum throughout their lifetime. The authors con­cluded that restricted calorie intake preferen­tially enhances apoptosis of preneoplastic cells.15 Mechanistic aspects It is well documented that AHF increase in number and size with continued exposure to both, genotoxic and non-genotoxic carcino­gens.16-18 Some of the phenotypical abno­rmalities of AHF are stable, however under specific conditions some phenotypical char­acteristics are lost ("phenotypic reversion").19 In rats, it is well documented that AHF devel­op by the clonal expansion of individual cells.19 As a result of sustained growth, AHF develop into nodular lesions.20,21 If these no­dules are neoplasms, as suggested by some studies,22 AHF truly represent preneoplastic lesions. A number of studies have been conducted in which the ratio between cell division and programmed cell death during development of liver cancer was investigated. It was shown, that the cell division rates are in­creased in AHF compared to normal tissue; in adenomas and carcinomas even higher divi­sion rates were observed. Also the death rates (apoptosis) increased gradually from normal to preneoplastic to adenoma and carcinoma tissue.23 Further studies showed, that the prenoplastic tissue is more susceptible to sti­mulation of cell replication and cell death;24,25 and that tumor promoters evidently act as survival factors by inhibiting apoptosis in preneoplastic liver cells, thereby stimulating growth of preneoplastic lesions. Interes­tingly, withdrawal of tumor promoters led to excessive elimination of preneoplastic le­sions, whereas normal tissue was less affec­ted.24 New developments Grasl-Kraupp and coworkers25 developed re­cently an ex vivo cell culture model, with ini­tiated rat hepatocytes. Following treatment of the rats with a nitrosamine (N-nitrosomor­pholine), hepatocytes were isolated after 22 days (maximal occurrence of GSTp+-cells) and cultivated in vitro. Then the cells were either treated with the mitogen cypoterone acetate or with transforming growth factor (TGF-ß) for 1-3 days. In culture, the rate of DNA-repli­cation of GSTp+-cells was compared to that of normal hepatocytes. It was found, that GSTp+-cells show an inherent growth advan­tage and a preferential response towards the effects of TGF-ß and cypoterone acetate as in the in vivo situation. Based on these results, the authors stress that this ex vivo system may provide a useful tool to elucidate biologi­cal and molecular changes during the initia­tion stage of carcinogenesis. Aberrant crypts in the colon – morphology In 198727, Bird discovered that the treatment of rats with a colon carcinogen (dimethylhy­drazine, DMH) leads to formation of morpho­logically aberrant foci, which can be visual­ized with methylene blue stain. ACF consist of altered cells, which exhibit cytoplasmic ba­sophilia, a high nuclear to cytoplasmic ratio, prominent nucleoli, loss of globlet cells, loss of polarity, and in the upper part of the crypt they exhibit increased proliferative activity.28 Figure 3A and 3B depict typical aberrant crypts, which are abnormally large, darkly Table 1. Biochemical and immunohistochemical alterations of ACF.30,33-42 Endpoint Comment Reference Hexosaminidase increased gene closely located to the APC gene Boland et al., 1992 95% of ACF in rats stain positive, Pretlow et al., 1993 not a marker for human ACF Carcinoembryonic antigen intracellular adhesion molecule in human ACF Pretlow et al., 1994 (CEA) altered (93%) but not a marker for dysplasia P-Cadherin cell adhesion molecules P-c expressed in ACF Hardy et al., 2002 E-Cadherin prior to and independent from E-c and ß-catenin ß-Catenin transcriptional activator Hao et al., 2001 in ACF nuclear expression increased (see also chapter: development for new markers) Inducible nitric oxide increased in dysplastic but not in Takahashi et al., 2000 synthase (iNOS) hyperplastic ACF Cyclooxygenase 2 (COX-2) overexpression in ACF Takahashi et al., 2000 Cell proliferation markers several studies show altered patterns in ACF Renehan et al., 2002 Ki-67, proliferating cell Cheng et al., 2003 nuclear antigen (PNCA) P16INK4a Placental form of GST might be associated in humans with K-ras Miyanishi et al., 2001 expression, induced in human ACF and CRC Changes in mucin production alteration of mucin-patterns seen in ACF in Uchida et al., 2001 rats and in humans Bara et al., 2003 Figure 3A,B. A- An aberrant crypt focus with a high level of dysplasia , which is microscopically elevated with a slit-shaped luminal opening. B- A crypt with oval openings. A GSTp+ focus. staining and slightly elevated. Dysplastic crypts with a slit-shaped luminal opening are shown in Figure 3A; Figure 3B depicts non-dysplastic crypts with a larger pericryptical zone.29 ACF show variable features – ranking from mild hyperplasia to dysplasia, and are gener­ally divided into three groups, namely dys-plastic, non-dysplastic (atypic) and mixed type (for details see30). In ACF without dys­plasia, the crypts are enlarged (up to 1,5-fold) and have slightly enhanced nuclei, no mucin depletion and crypt cells staining positive for PNCA and Ki-67 remain in the lower part of the crypts. In ACF with dysplasia, crypts are more elongated, and the nuclei enlarged. PN­CA and Ki-67 stain is extended to the upper Table 2. Epigenetic and genetic alterations in ACF.43-50 part of the crypts. Mixed type ACF show com­binations of the features of pure adenomatous pattern (with dysplasia) and hyperplastic characteristics. In humans, ACF were first described in 1991.31,32 They resemble those seen in ro­dents induced by carcinogens27 and several lines of evidence support the assumption that they are precursors of colorectal tumors (for details see Cheng et al.).30 Biochemical and immunohistochemical alterations of ACF A number of biochemical alterations are typi­cal for ACF. The most important features are listed in Table 1. Alteration Remarks Reference K-ras mutation in ACF in rats, identified in many studies Stopera et al., 1992 also in humans Losi et al., 1996 APC mutation deleted in human ACF – but lower rates as in Smith et al., 1994 adenomas/carcinomas Nascimbeni et al., 1999 hMSH2 mutation mismatch repair gene alteration in ACF Reitmair et al., 1996 in mice colons CpG island methylation in 53% of ACF of humans with sporadic CRC Chan et al., 2002 but only in 11% of FAP patients Microsatellite instability detected in animal models and in humans in ACF Augenlicht et al., 1996 Fragile histidine triad lost in CRC (40%) – only few ACF showed Hao et al., 2000 (FHIT) candidate tumor reduced expression; the loss correlated with suppressor gene the extent of dysplasia Table 3. Compounds, which act as tumor promotors in the colon and cause increased formation of ACF.58-66 Compound Result Reference Thermolysed protein increasing thermolysis of casein increases AOM induced foci numbers and size Zhang et al., 1992 Thermolysed sucrose (5-hydroxymethyl-2-furaldehyde) increases the size of AOM induced foci weakly initiating carcinogens Zhang et al., 1993 Fat (beef tallow) AOM experiments with mice:increases 3-5 times the size of chemically induced foci Corpet et al., 1990 Refined sugars (sucrose, fructose, dextrin) induced foci in rats increased formation of AOM induced foci sucrose and dextrin enhance no. of AOM Stamp et al., 1993 Poulsen et al., 2001 Progastrin (PG) ACF significantly more common in AOM treated mice overexpressing PG Cobb et al., 2004 Haemoglobin, haemin especially haemin but also haemoglobin were potent ACF promoters in AOM treated rats, when fed a low-calcium diet Pierre et al., 2003 Chenodeoxycholic acid (CDCA) AOM induced foci as well as crypt multiplicity significantly increased in rats Ghia et al., 1996 Sutherland et al., 1994 Genetic and epigenetic alterations Different genetic alterations have been identi­fied in ACF in humans and also in chemically induced ACF in rats; a detailed overview is gi­ven in the article of Cheng et al.30 Many genes, which are considered to be involved in colon carcinogenesis, were found to be al­tered in ACF; this supports the assumption that they (ACF or specific subpopulations) represent indeed preneoplastic lesions. Table 2 lists up different alterations which were identified in ACF. Methodological aspects As in AHF-experiments, ACF-studies allow to discriminate between initiating and promo­ting compounds. The treatment schedule is more or less identical as that used for the de­tection of liver foci, but other model chemi­cals are used. Only a few compounds have been detec­ted, which are initiators of colon cancer and aberrant crypts. The most frequently used agents are DMH and its metabolite azoxy-methane (AOM).51 Both compounds lead to DNA methylation and to formation of ACF, which become apparent5-7 weeks after the ad­ministration.52 Also heterocyclic aromatic amines (HAs), which are found in fried meat cause formation of ACF28,53,54 and were used in a number of chemoprevention studies (for review see Dashwood55 and Schwab et al.56). Other agents which cause ACF are N-methyl-N-nitrosurea (MNU) and 3,2-dimethyl-4­aminobiphenyl (DMABP), but these com­pounds were hardly ever used in mechanistic and chemoprevention studies.57 Use of the ACF-model to detect factors which act as tumor promoters in the colon The ACF-model was intensely used in studies aimed at detecting dietary factors which cause tumor promotion in the colon. Table 3 lists up a number of studies. Use of the ACF-model for the detection of chemo-protective compounds Numerous studies have been conducted aimed at identifying compounds which are protective towards colon cancer with the ACF model. Recently, Corpet and Tache57 have published a review on this topic. They found in total 137 articles and results for about 186 complex mixtures and individual compounds are available (the data can be downloaded from: http://www.inra.fr/re­seau-nacre/sci-memb/corpet/indexan.html). The establishment of a ranking order of pro­tective potency showed, that the most potent were pluronic, polyethylene glycol, perilla oil containing ß-carotene and indole-3-carbinol (for details see57). In addition, many other di­etary constituents were found protective, for example vitamins, lactobacilli in fermented foods, different glucosinolates in Brassica vegetables, carotinoids and fibers to name only a few.57 In most of the studies, DMH or AOM were used to cause foci formation and the putative protective compounds were added either be­fore or after administration of the carcinogen. The prevention during the foci “initiation” phase might be either due to inactivation of DNA-reactive molecules, inhibition of meta­bolic activation or induction of DNA-repair processes67 and is compound specific. Since humans are not exposed to DMH and its metabolite AOM, chemoprotective effects seen in such experiments cannot be extrapo­lated to the human situation. On the other hand, it is assumed that the further develop­ment of preneoplastic cells (promotion, pro­gression) is triggered by molecular processes which are independent from the chemical carcinogen used.68 Therefore antipromoting effects seen in the AOM/DMH ACF model might be considered relevant for humans. HAs are formed during cooking of meats.69 They cause cancer in the colon of ro­dents, and in other organs as well70 and evi­dence is accumulating that HAs are involved in the etiology of colon cancer in humans.71 HAs were used in a number of chemopreven­tion studies in which inhibition of ACF for­mation was used as an endpoint55,56, and a number of dietary components such as fibers, chlorophyllins, Brassica vegetables and lacto-bacilli were found protective. In this context it is interesting that epidemiological studies indicate that consumption of these factors is also inversely related with the incidence of colon cancer in humans. One of the problems of the use of HAs in ACF studies is that the foci yield is relatively low, even when the animals are treated with high doses (up to 100 mg/d for several days). The foci frequency could be substantially in­creased by feeding the animals a high fat and fiber free diet, which facilitates the detection of putative protective effects.72 In contrast to AOM or DMH it is not possible to induce ACF with a single HA-dose, therefore it is not possible to distinguish clearly between anti-initiating and anti-promoting effects in these experiments. Corpet and Pierre51 published an article on the correlation between the results of chemo-prevention studies using ACF as an endpoint, and data from experiments with the ApcMin/+ mouse model (these animals have a mutated Apc-gene and therefore highly increased rates of intestinal spontaneous tumors, and are of­ten used as a model for human hereditary colon cancer). Comparison of the efficacy of protective agents in the ApcMin/+ mouse and in the ACF rat model showed a significant correlation (p<0,001). Furthermore, the au­thors also compared the results of rodent studies with clinical intervention studies. For a number of compounds, which were protec­tive in the animal models, also chemopreven­tive properties were seen in humans. New developments Although numerous studies show that ACF detect colon carcinogens and have been used extensively for the identification of dietary factors enhancing or reducing the risk for colorectal cancer, some results suggest that misleading results may be obtained with cer­tain compounds.73 For example it is well docu­mented that cholic acid, a primary bile acid, is a strong tumor promoter in the colon, where­as it significantly decreases the number of ACF.74,75 A similar contradiction was seen with the xenoestrogen genistein.76-78 It was repeatedly postulated by Japanese groups79-82 that ß-catenin acculmulating crypts (BCAC), which are independent from ACF, are more reliable biomarkers for colon cancer development. They show that cholic acid increases the frequency of AOM-induced BCAC in rats. In a critical comment of Pretlow and Bird83 it is stated that BCAC rep­resent in fact specific dysplastic ACF. In a subsequent paper of Hao et al.37, human ACF were analyzed for ß-catenin expression and in approximately 56% of dysplastic ACF, cy­toplastic ß-catenin was increased, whereas in ACF with atypia, ß-catenin in the cytoplasm was only seen in 2% of the total number. As mentioned above, Magnuson and coworkers74 also found that the number of ACF at early time points did not predict tu­mor incidence in rats treated with cholic acid. Therefore the authors suggest that crypt mul­tiplicity should be measured in future stu­dies, due to the fact that it was a consistent predictor of tumor outcome in their study. Another potential short-term endpoint for colon cancer might be mucin-depleted foci (MDF). In AOM-treated rats such foci could be visualised with high-iron diamine Albicon blue.84 Their frequency was lower than that of ACF and they were histological more dys-plastic than mucinous ACF. In a recent arti­cle, it was shown that the number of MDF-fo­ci declined in AOM treated rats, after piroxi-cam (a colon cancer inhibiting drug) adminis­tration, whereas their frequency increased af­ter treatment with cholic acid.84 Conclusions In the last years, highly effective molecular techniques (e.g. microarray based methods) have been developed, which can be employed to elucidate the mechanisms of carcinogene-sis. These approaches can be used to analyze gene expression patterns in vitro in cell cul­ture models, and also in tumors and can be compared with histological endpoints related to neoplasia. The predictive values of results obtained in in vitro models is often restricted, since the indicator cells which are used lack often characteristic features which are impor­tant for the in vivo situation. Typical exam­ples are chemoprevention studies in which metabolically incompetent cell lines may give misleading results, as they do not reflect the activation/detoxification of DNA-reactive carcinogens.85 On the other hand, the use of tumor formation in animal experiments as endpoints is hampered by the high costs and the time requirement and in case of human studies additionally by the limited availability of material. These shortcomings underline the value of preneoplastic foci models, which represent early stages of the neoplastic process. It has been shown that many com­pounds, considered as human carcinogens, can be detected with these models in rodents and also that protective agents which were identified in such foci experiments prevent specific forms of cancer in humans. Furthermore, the foci models are also useful to monitor the time course of biochemical and genetic alterations in neoplasia. 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The murine models for the research on the human haematopoietic neoplasms Hanna Szymanska1, Joanna Piskorowska1, Elnbieta Krysiak1, Henryk Skurzak2, Alina Czarnomska1, Peter Demant3 1Department of Genetics and Laboratory Animal Breeding, The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland; 2Department of Immunology, The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland; 3Department of Cellular and Molecular Biology, Roswell Park Cancer Institute, Buffalo, N.Y., USA The Haematopathology Subcommittee of Mouse Models of Human Cancer Consortium (MMHC) proposed a classification that can be readily compared with the human WHO classification 2001 1 and appropriate­ly delineates the diseases that occur in mice. The mouse lymphoid and nonlymphoid neoplasms develop spontaneously in certain strains and in genetically engineered mice (GEM) or follow induction with ionis­ing radiation or chemical carcinogens or viruses. In the study, the haematopoietic neoplasms that developed in the three investigated mouse strains were identified according to the above classification. They can be use­ful as mouse models of human lymphoid and nonlymphoid haematopoietic neoplasms. Key words: T-cell lymphoma, B-cell lymphoma; models, mice Received 18 March 2004 Accepted 29 July 2004 Correspondence to: Hanna Szymanska, PhD, The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics and Laboratory Animal Breeding, Roentgen Str 5, Warsaw, Poland; E-mail: hanszym@yahoo.com This paper was presented at the “3rd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. Introduction Uniform classification of tumours of murine haematopoietic system has been extensively studied.1,2 The precise diagnosis will make it possible to compare and contrast murine dis­eases with human lesions and enable model-ling human haematopoietic neoplasms in mice. Classifications of murine haematopoietic neoplasms has been changing over the last decades. The first classification formulated by Dunn (1954) was based on the morpholo­gy of neoplastic cells and showed similarities to Rappaport's (1966) nomenclature of hu­man lymphomas.3,4 In 1981, Pattengale and Taylor proposed a histopathological and immunological scheme of murine lymphomas based on the concept stating that human and murine lymphoid cells of neoplasms represent neoplastic con­version of T or B cell lineage.5,6 That classifi­cation showed a very close relation to Lukes and Collins' (1974) and Kiel's classification (1981).7,8 Pattengale and Taylor adopted the term of "lymphoid neoplasm" for all haemato­poietic neoplasms containing transformed cells that have fully or partially differentiated into T-cells or B-cells or natural killer cells and showing monoclonal proliferation. The latest classification of murine lym­phoid neoplasms was recommended by Haematopathology Subcommittee of MMHCC (Mouse Models of Human Cancers Consor­tium) and published by Morse et al. (2002).9 It is worth stressing that the classification can be compared with the latest WHO classifica­tion of human haematopoietic neoplasms.1 The classification is also known as "Bethesda proposals for classification of lymphoid neo­plasms in mice".9 In 2002, the same Subcommittee of MMHCC formulated the latest classification for murine nonlymphoid haematopoietic neo­plasms. It was published by Scott C. Kogan et al. and it is known as "Bethesda proposals for classification of nonlymphoid neoplasms in mice".10 The term "nonlymphoid haematopoi­etic neoplasm" was adopted for haematopo­ietic neoplasms arising from other lineages than lymphoid ones. The Haematopoietic Subcommittee of MMHCC recommends recognition of the following types of murine lymphoid neoplasms: B-cell neoplasms Precursor B-cell neoplasm Precursor B-cell lymphoblastic lym­ phoma/leukaemia Mature B-cell neoplasms Small B-cell lymphoma Splenic marginal zone B-cell lymphoma Follicular B-cell lymphoma Diffuse large B-cell lymphoma - Centroblastic - Immunoblastic - Histiocyte associated Classic Burkitt's lymphoma Burkitt's-like lymphoma Plasma cell neoplasm B natural killer cell lymphoma T-cell - neoplasms Precursor T-cell neoplasms Precursor T-cell lymphoblastic lym­ phoma/leukaemia Mature T-cell neoplasm Small T-cell lymphoma T-natural killer cell lymphoma T-cell neoplasm, character undetermined Large cell anaplastic lymphoma The marked types were not recognized in Pattengale and Taylor classification as sepa­rate categories. E.g., the diffuse large B-cell lymphoma was a subtype of follicular centre cell lymphoma from large cells. It is also worth pointing out that some types of murine lymphomas occur only in mice as special models. E.g. B-natural killer lymphomas can develop only in thymec­tomized (SL/Kh x AKR/Ms) F1 mice.11 The Haematopoietic Subcommittee of MMHCC recommends also four categories of murine nonlymphoid haematopoietic neo­plasms with subtypes: 1. Nonlymphoid leukemias - Myeloid leukemias (granulocytic leukemia) - Erythroid leukemia - Megakaryocytic leukemia - Biphenotypic leukemia 2. Nonlymphoid haematopoietic sarcomas - Granulocytic sarcoma - Histiocytic sarcoma - Mast cell sarcoma 3. Myeloid dysplasias 4. Myeloid proliferations (nonreactive) The diseases represented in that classifica­tion are not very readily compared with hu­man lesions. It is due to the fact that some nonlymphoid haematopoietic neoplasms have not been clearly described in mice. Additional murine models of human nonlym­phoid haematopoietic neoplasms are still an­ticipated. The purpose of this paper is to show that the murine haematopoietic neoplasms devel­oped in our three investigated mouse strains and identified according to the latest classifi­cation can be recognized as murine models of human haematopoietic neoplasms. Material and methods Tumours The murine haematopoietic neoplasms devel­oped: - spontaneously in recombinant congenic strain OcB/Dem – 20 cases. The OcB/Dem mice were bred from the pairs of mice sent to Cancer Centre in Warsaw from the Nether­lands Cancer Institute,12 - spontaneously in AKR/W mice carrying endogenous ecotropic provirus which in­duced potential lymphomas – 46 cases, 13 - in back-cross (CcS17 x CcS2) x CcS/Dem mice exposed to . radiation – 72 cases. Mice were exposed to four whole-body . irradia­tion with the doses of 1.7 Gy at one week in­tervals.14 All mice were sacrificed when they were visibly sick with poor grooming, hunched posture, weight loss and enlargement of the thymus, lymph nodes or/and spleen detected by palpation. Pathological procedures Autopsies were done on each animal. The thymus, mesenteric lymph nodes, spleen, liv­er as well as other organs with visible neo­plastic lesions were fixed in EAFS (ethanol, acetic acid, formol, 0.9% NaCl), and embed­ded in paraffin; 4 µm thick paraffin sections were stained with H&E or prepared for im­munohistochemistry. Immunophenotyping was performed us­ing two techniques of immunohistochemistry ABComplex and MOM® (Mouse on Mouse) – (immunodetection kit designed to localize murine primary antibodies on mouse tissue), and flow cytometry with the appropriate monoclonal antibodies. The specific monoclonal antibodies (MAbs) for flow cytometry were conjugated with FITC (CD90.1, CD90.2, CD3e, CD8, CD5, CD19, CD45R) or PE (CD4, TCRaß, RAM KAPPA – (rat anti mouse .), and for im­munohistochemistry, they were biotin or pure (anti IgM, anti IgD, anti Ig., anti Ig.1.2.3, Gr-1).15 All antibodies were pro­duced by PharMingen Germany. Additionally, two histochemical stainings were performed on the air-dried imprints of tumours, of the spleen, liver or of other or­gans with neoplastic lesions: naphtol ASBI method - acid phosphatase focal staining is considered to be a specific marker for T lym­phoblasts and ASD method - assessment of chloroacetate esterase activity is needed if granulocytic leukemia is to be diagnosed. Blood smears were stained with Giemsa and the number of neoplastic haematopoietic cells was estimated. Results We recognized three types of lymphoid and two types of nonlymphoid haematopoietic neoplasms among 138 classified haemato­poietic neoplasms: LYMPHOID NEOPLASMS: T-cell derived lymphoma: -precursor T-cell lymphoblastic lym­phoma/leukaemia B-cell derived lymphoma: - follicular B-cell lymphoma - diffuse large B-cell lyphoma subtype centroblastic (CB) NONLYMPHOID HAEMATOPOIETIC NEOPLASMS: - granulocytic leukaemia - granulocytic sarcoma Precursor T-cell lymphoblastic lymphoma Human counterpart of mouse precursor T-cell lymphoblastic lymphoma is the lym­phoma of the same nomenclature. The micro­scopic characteristic of examined lymphomas was as follow: - cells were monomorphic, medium size with scant cytoplasm; - nuclei were round with fine immature chromatin; - cells exhibited numerous mitosis; - 1-2 nucleoli were placed in the centre of the nucleus; - the spleen was filled up with sheets of neoplastic lymphoid cells; - in the liver - sheets of neoplastic cells were placed in sinusoids and/or around the vessels. The detailed immunophenotypes of the ex­amined precursor T-cell lymphoblastic lym­phomas are demonstrated in Table 1. The lymphomas exhibited one out of three im­munophenotypes (CD4/CD8)+; CD4+/CD8–; CD4–/CD8+. The double positive phenotype was the most frequent conversely phenotype CD4–/CD8–. The expression of CD4 or/and CD8 and CD90 demonstrated by FACS scan analyses is shown in Figures 1A, B, C. Those lymphomas were positive for acid phos­phatase staining. Representative images of T-cell lymphoblastic lymphoma are shown in Figures 2A, B, C. Follicular B-cell lymphoma Human counterpart is also follicular B-cell lymphoma. Microscopic changes were as follow: - diffuse pattern of lymphoma; - neoplastic cells were small – centrocytic or large – centroblastic; - cytoplasm was scant; - nuclei were cleaved, usually with charac­teristic "heart" shape or noncleaved, round; - 2-3 nucleoli were prominent and adher­ent to the nuclear membrane; - mitoses were visible only among centrob­lastic cells. Follicular B-cell lymphomas showed im­munophenotype of B-cells – sIg+, B220+, CD19+. Clonality was confirmed by the ex- Table 1. Immunophenotypes of tested mouse haematopoietic neoplasms Marker T-cell derived B-cell derived Granulocytic leukemia / lymphomas lymphomas Granulocytic sarcoma CD90/Thy.1. + – – CD3e + –– CD4 + or – – – CD8 + or – – – CD5 +–– TCRaß + –– IgM –+– IgD – +/–– Ig. / RAM KAPPA – + – Ig.1.2.3 – –/+– CD19 – +/–– CD45R B220 – + – Gr-1 – –+ + - positive +/– - more often positive than negative –/+ - more often negative than positive – -negative Figure 1. FACS analysis: Tumor of thymus, T-cell lymphoma (A), (B), (C). Positive reaction with CD 90.1 (Thy 1.1) FITC (left) co-expression (CD4/CD8)+ or expression CD4+ CD8– or CD4– CD8+ (right). Tumor of mesenteric lymph nodes, B-cell lymphoma (D). Negative with CD90 FITC (left) and positive reaction with RAM KAPPA PE (right). Figure 2. Precursor T-cell lymphoblastic lymphoma/leukaemia (A), (B), (C). (A) Tumor of thymus - uniform population of medium sized cells with central prominent nucleoli (red ar­row) and numerous mitosis (green arrow). H&E x400. (B) Tumor of mesenteric lymph nodes - focal positive acid phosphatase reaction on air-dried imprint ASBI method x1000. (C) Tumor of thymus - immunohistochemical staining on air-dried imprints positive for CD90. ABC method x1000. Follicular B-cell lymphoma (D). Tumor of mesenteric lymph nodes. Population of large centroblasts (green arrow). and cleaved "heart" shaped centrocytes (red arrow) H&E x1000. Granulocytic leukaemia (E), (F), (G) (E) Tumor of mesenteric lymph nodes – infiltration of ring forms of granulocytes (red arrow) H&E x1000. (F) Tumor of mesenteric lymph nodes – ASD positive staining for chloroacetate esterase activity on air-dried imprint. ASD method x1000. (G) Spleen – numerous ring shaped forms of granulocytes; positive reaction with Gr-1. ABC method x1000. pression of one or two heavy chains of im­munoglobulin µ, d (IgM or IgD) and one of light chains ., . (Ig. or Ig.). The detailed immunophenotype of follicu­lar B-cell lymphoma is shown in Table 1. Those lymphomas exhibited the expression of RAM KAPPA and no expression of CD90 (Thy 1.) (Figure 1D). Representative image of follicular lymphoma is shown in Figure 2D. Diffuse large B-cell lymphoma The human counterpart is also diffuse large Bcell lymphoma – variant centroblastic. We classified B-cell derived lymphomas with more than 50% of neoplastic lymphoid cells as diffuse large B-cell lymphoma – centrob­lastic. Those lymphomas showed im­munophenotype of mature B-cell lymphoma sIg+, B220+, CD19+, Ig./RAM KAPPA+. Granulocytic leukemia The most severely affected organs were the spleen and mesenteric lymph nodes. The thy­mus was not involved. The immature forms of granulocytes were found in the peripheral blood and in the periportal, sinusoidal liver and around the liver vessels. Infiltration into the kidney and lung was not observed in our material. Leukaemic cells were bean or, more often ring-shaped and were in one stage of maturation. Representative images of granulo­cytic leukemia are shown in Figures 2E, F, G. Granulocytic sarcoma The lesion was primarily a solid tumour de­veloped in a mesenteric lymph node mass without "spillover" to the peripheral blood. Granulocytic leukaemia and granulocytic sar­coma exhibited positive staining for chloroac­etate esterase and showed the expression of Ly-6G Gr-1 in ABC method (Table 1). Assessment of murine tumours of the investigated mouse strains as a model of human haematopoiet­ic neoplasms The vast majority of examined haemato­poietic neoplasms were derived from lym­phoid lineages -127 cases, while nonlym­phoid haematopoietic neoplasms developed only in 11 cases. The occurrence of haemato­poietic neoplasms in examined strains is shown in Table 2. In all examined strains, a T-cell derived lymphoma – a precursor T-cell lymphoblastic lymphoma - was the prevalent type of lym­phoid neoplasms - 106 cases. As expected, in the AKR/W mice that type occurred in significantly higher proportion than in the other strains, due to the fact that the AKR/W mice are the most appropriate mouse models of the human precursor T-cell lymphoblastic lymphoma. Two types of B-cell derived lymphomas, follicular B-cell lymphoma and diffuse large B-cell lymphoma occurred in 21 cases. Those lymphomas developed in the OcB and irradi­ated Bc(CcS17x CcS2)xCcS2/Dem strains more frequently than in the AKR/W mice. Both mouse strains could be recommended as mouse models for studying human counter­part of those B-cell derived lymphomas. Granulocytic leukaemia and granulocytic sarcoma were diagnosed mainly in the Bc(CcS17xCcS2)xCcS2/Dem strain. Those Table 2. The occurrence of neoplasms with given immunophenotype in examined mouse strains Mouse strains No of tumours T-cell derived B-cell derived Nonlymphoid lymphoma lymphoma haematopoietic neoplasms OcB/Dem 20 12 7 1 AKR/W 46 45 1 0 Bc (CcS17 x CcS2) x CcS2/Dem 72 49 13 10 TOTAL 138 106 21 11 animals were prepared especially to test the genetic control of susceptibility to radiation induced B-cell derived lymphomas and gran­ulocytic leukaemia. Therefore, the nonlym­phoid haematopoietic neoplasms developed in that strain could be the potential mouse model of human neoplasms of the same nomenclature. Discussion The mouse precursor T-cell lymphoblastic lymphoma is a very appropriate and well-known counterpart of human lymphoma of the same nomenclature, observed especially in children and young adults.16 However, it should be stressed that the nuclear convolu­tion, often observed in the human lym­phomas, is not seen in the analogous murine lymphomas. Those lymphomas have been extensively studied in the AKR mice. Follicular B-cell lymphomas, which devel­oped spontaneously in the OcB/Dem mice and, as a result of irradiation, in the Bc (CcS17 x CcS2) x CcS2/Dem mice, showed a diffuse pattern. While that pattern is com­mon in mice, the follicular structure is usual­ly recognised in human, and the diffuse vari­ant is seen very rarely. Despite those differ­ences, the cytology of these murine and hu­man lymphomas is similar. The other described type of B-cell derived lymphoma – diffuse large B-cell lymphoma in mice always requires a differential diagnosis with the progression of follicular B-cell lym­phoma or progression of splenic marginal zone lymphoma.17 It is worth stressing that there is an obsta­cle in confirmation of clonality of B-cell de­rived lymphomas in mice, due to the fact that most (approximately 95%) murine light chains are the . type. Therefore, more infor­mative is the restriction to IgM or IgD than the restriction to light chain .. There is also the difficulty in the diagnosis of granulocytic leukemia and granulocytic sarcoma because, in mice, extramedullary haematopoiesis continues in the spleen throughout life. The infiltration in the liver parenchyma by immature forms of granulo­cytes is more informative than the sheets of immature granulocytes in the spleen. Unfortunately, in our material, in some cases, the liver was not involved and there were no immature granulocytes in the peripheral blood. In those cases, the restriction to one stage of maturation of granulocytes in the spleen was the only criterion for diagnosis. Due to the fact that the investigated strains developed haematopoietic neoplasms that can be identified according to the classifica­tion proposed by the Haematopathology Subcommittee of Mouse Models of Human Cancer Consortium, they can be used as murine models of the human diseases. The observations made on these haematopoietic neoplasms can be translated to their human counterparts. References 1. Jaffe ES, Harris NL, Stein H, Vardiman J, eds. Pathology and genetics of tumours of haematopoi­etic and lymphoid tissues: WHO Classification of tumours. Lyon, France 2001: IARC Press. 2. Jaffe ES, Banks PM, Nathwani B, Said J, Swerdlow SH. Recommendations for the reporting of lym­phoid neoplasms: A report from the Association of Directors of Anatomic and Surgical Pathology. Mod Pathol 2004; 17: 131-5. 3. Dunn TB. Normal and pathologic anatomy of the reticular tissue in laboratory mice, with classifica­tion and discussion of neoplasms. J Natl Cancer Inst 1954; 14: 1281-433. 4. Rappaport H. Tumours of the haematopoietic sys­tem. [In:] Atlas of tumour pathology, Sec. 3. fasc.8. Washington 1966. 5. Pattengale PK, Taylor CR, Immunomorphologic classification of murine lymphomas and related leukemias. Proceedings of the Rodent Lymphoma Workshop. 1981: 22-3. 6. Pattengale PK, Taylor CR, Experimental models of lymphoproliferative disease. The mouse as a mod­el for human Non-Hodgkin's lymphomas and re­lated leukemias. Am J Pathol 1983; 113: 237-65. 7. Lukes R, Collins RD, Immunological characteriza­tion of human malignant lymphomas. Cancer 1974; 34: 1488-503. 8. Lennert K. Histopathology of Non-Hodgkin's Lymphomas: Based on the Kiel Classification. New York 1981. 9. Morse HC III, Anver MR, Fredrickson TN, et al. Bethesda proposals for the classification of lym­phoid neoplasms in mice. Blood 2002; 100: 246-58. 10. Kogan SC, Ward JM, Anver MR, Berman JJ, Brayton C, Cardiff RD, et al. 3rd. Bethesda pro­posals for classification of nonlymphoid neo­plasms in mice. Blood 2002; 100: 238-45. 11. Lu L-M, Hiai H. Mixed phenotype lymphomas in thymectomized (SL/Kh x AKR/Ms) F1 mice. Jpn J Cancer Res 1999; 90: 1218-23. 12. Demant P, Hart AA. Recombinant congenic strains - a new tool for analyzing genetic traits de­termined by more than one gene. Immunogenetics 1986; 24: 416-22. 13. Klein O, Staroselsky A, Huszar M, Hiss J, Kay S, Donin N, et al. Biological behaviour and cell prop­erties of new AKR/W lymphoma malignancy vari­ants. Tissue Cell 1998; 30: 95-103. 14. Szymanska H, Sitarz M, Krysiak E, Piskorowska J, Czarnomska A, Skurzak H, et al. Genetics of sus­ceptibility to radiation-induced lymphomas, leukemias and lung tumours studied in recombi­nant congenic strains. Int J Cancer 1999; 83: 674-8. 15. Lai L, Alaverdi N, Maltais L, Morse HC. Mouse cell surface antigens: nomenclature and im­munophenotyping. J Immunol 1998; 160: 3861-8. 16. Panke TW, Langlinais PC, Vriend J, McCue MJ. An animal model for childhood convoluted T-cell lym­phoma. Am J Pathol. 1978; 92: 595-610. 17. Fredrickson TN, Lennert K, Chattopadhyay SK, Morse HC, Hartley JW. Splenic marginal zone lymphomas of mice. Am J Pathol 1999; 154: 805-12. Radiol Oncol 2004; 38(3): 227-34. Comparison of Wistar vs. Fischer rat in the incidence of 1,2-dimethylhydrazine induced intestinal tumors Željka Veceric, Anton Cerar Medical Experimental Center, Institute of Pathology, University of Ljubljana, Slovenia Background. Many investigators have observed differences in the susceptibility to induce intestinal tumors by 1,2-dimethylhydrazine (DMH) between various strains of rodents. The results are difficult to compare because of the different regimes used for induction. The purpose of our study was to evaluate the influence of strain on DMH-induced intestinal tumors between Wistar and Fischer rats. Materials and methods. We used 29 Fischer and 30 Wistar male rats that were injected subcutaneously DMH, weekly, at a dosage of 25 mg/kg-body weight for 20 weeks. After 25 weeks from the beginning of the experiment, the animals were sacrificed and autopsied. The complete length of colorectum and all macro­scopic changes were examined histologically. Results. The induction of intestinal tumors was 97% in Fischer rats and 100% in Wistar rats. In Wistar rats 184 tumors were found: 133 adenomas, 50 tubular adenocarcinomas and 1 signet-cell carcinoma. 77% of carcinomas were found in colorectum and 23% in the small intestine. In Fischer rats, 126 tumors were found: 94 adenomas, 26 tubular adenocarcinomas, 5 signet-cell carcinomas and 1 mucinous carcinoma; 42% of carcinomas were found in the colorectum and 58% in the small intestine. The strain difference in the in­cidence of all induced tumors was statistically significant (P=0.001). The differences in the occurrence of the malignant and benign tumors was also significant (P<0.001; P=0.011). Extra intestinal tumors were not found. Conclusions. Wistar rats showed greater percentage of colorectal tumors, and also the distribution of tu­mors in colorectum resembled more the distribution found in human pathology. That is why we recommend Wistar rat rather than Fischer rat for the research work on the colorectal tumors. Key words: intestinal neoplasms – chemically induced; 1,2 dimethylhydrazine; rats, inbred F344; rats Wistar Received 17 May 2004 Accepted 10 June 2004 Correspondence to: Željka Veceric, Robiceva 2, 1000 Ljubljana, Slovenia. Tel: +386 31 580 325; E-mail: zelj­ka.veceric@email.si Introduction Colorectal carcinoma (CRC) is one of the leading causes of cancer mortality in the USA.1 With respect to its incidence as well as mortality rate, CRC takes the second place in Slovenia.2 This was the reason for much in­terest in the research of this disease and for highlighting the need for animal models that would be comparable to human disease and would help in the study of etiology, patho­genesis and therapy of the human disease. Some studies compared the incidence of experimentally induced intestinal tumors be­tween different species of experimental ro­dents and different strains among the species and demonstrated that susceptibility to car­cinogen and the incidence and distribution of tumors which developed is species-, strain-, and sex-dependant.3-8 Wistar and Fischer rats are among the most commonly used strains of rats in the research of intestinal cancer.9 The published information on the strain-re­lated differences between them is scarce. Besides the results are difficult to compare because different carcinogenic substances, doses, application regimes and application sites are used. So, we decided that this issue is worth of further studies. Materials and methods Animals We used 29 Fischer (344) and 30 Wistar (Hannover) male rats from The Medical Experimental Center, Ljubljana, Slovenia. They were 8-10 weeks old. The experiment was carried out in accordance with the per­mission of The Veterinary Administration Board of The Republic Slovenia. At the onset of the experiment the weight of Wistar rats ranged between 170–340 g and that of Fischer rats between 180–290 g. The experiment was carried out at a room tem­perature of 20–23°C, humidity 40–70%, and at a natural light cycle. The animals were pro­vided pelleted M-K-02 food (Biotechnical Faculty, Ljubljana) and tap water ad libitum. Carcinogenic agent CRC was induced by means of 1,2-dimethyl-hydrazine (DMH) (Fluka Chemie, Switzer­land) prepared according to the standard method10: DMH-HCl was dissolved in 0.001 M EDTA and pH value adjusted to 6.5 using 0.1 M NaOH solution. Fresh solutions were prepared once weekly. Study design The dose of DMH was adjusted accordingly, so that it always amounted to 25 mg/kg of body weight. The solution was injected sub­cutaneously into the skin fold on the hip once weekly throughout a period of 20 weeks. The animals were left to live four weeks after completed DMH injection and thereupon sac­rificed by CO2 inhalation. The body weight was controlled every two weeks. Morphology During autopsy, all internal organs except the central nervous system were examined. Attention was paid also to the possible pres­ence of tumors in the outer auditory canal. The stomach was opened via the major curve while the intestine was approached longitudi­nally on the antimesenterial side. After open­ing, the organs were rinsed with water. The distal part of the ileum, large intestine, anus and neoplasms in the small intestine were spread over a polystyrene board, with intesti­nal mucosa facing upwards, and fixed in 10% buffered formaldehyde. The total length of colorectum and all macroscopically visible le­sions were sampled for the histological exam­ination. The tissue samples were paraffin em­bedded and cut into 4.5 µm thick histological sections. The sections were stained by Kreyberg trichrome method. In the cases when histological picture or tumor stage could not be determined from a single sec­tion, stepwise deeper sections were made. All intestinal lesions were assessed according to histological criteria used in human pathology and the stages of carcinomas defined follow­ing Duke’s staging system: - Stage A: tumor is limited to the intestinal wall; Table 1. Distribution and number of intestinal tumors TUMOR LOCATION No (%) Small intestine Colon ascendens Colon transversum Colon descendens with rectosigmoid FISCHER 21(17) 16(13) 47(37) 42(33) WISTAR 7(4) 14(8) 57(31) 106(57) - Stage B: tumor grows through the lamina muscularis propria; - Stage C: tumor grows through the lamina muscularis propria and disseminates into the lymph nodes; - Stage D: distant metastases.11 Histological criteria for the diagnosis of adenoma were: (1) cytological – increased mi­totic activity, polymorphism and hyperchro­matism of the nuclei, basophilia of the cyto­plasm, decreased mucine excretion and (2) histological – stratification of the nuclei, ir­regular proliferation of the glandular forma­tions. Smaller tumor lesions composed of 2-5 crypts (microadenomas), seen only histologi­cally, were also statistically processed. The criterion for diagnosis of carcinoma was the evidence of tumor growth through the muscularis mucosa. In the case the lesion was suspected of being malignant while there was no clear evidence of tumor growth through the muscularis mucosa, the follow­ing additional histological criteria for carcino­ma were used: a sharp transition of normal epithelium to severely dysplastic epithelium, the presence of significant necrosis on the surface of tumor and desmoplastic stromal reaction. Statistical methods The significance of strain-related difference in the numeric results was tested for the dif­ference between proportions by computer software StatGrafics®Plus. Results Number and distribution of intestinal tumors All animals survived throughout the duration of the experiment. In the intestine of Fischer and Wistar rats 126 and 184 tumors were found, respectively. The tumors were in­duced in 97% of Fischer rats and 100% of Wistar rats, while the tumors of the colorec-tum were induced in 48% of Fischer and in 83% of Wistar rats (Table 1). The strain dif­ference in the incidence of all induced tumors was statistically significant (P=0.001). Extra-intestinal neoplasms were not found. The microadenomas that were evaluated by the systematic histological examination of the whole length of the colorectum represent­ed 70% of tumors in Fischer and 60% of tu­mors in Wistar rats. In Fischer rats, 25% of the induced tumors were carcinomas that were mostly found in the small intestine (58%), followed by the as- Table 2. Histological types and stages of intestinal tumors according to Duke’s system HISTOLOGIC TYPES OF TUMORS Adenomas Tubular Signet-ring cell Mucinous adenocarcinomas carcinomas carcinomas A B C D A B C D A B C D FISCHER 94 19 7 0 0 3 2 0 0 0 1 0 0 WISTAR 133 41 8 0 1 1 0 0 0 0 0 0 0 cending colon (23%), descending colon with rectosigmoid (16%) and the transverse colon (3%). In Wistar rats, 30% of tumors were carci­nomas and were located in the transverse colon (39%), descending colon with rectosig­moid (37%), small intestine (14%) and ascend­ing colon (10%). The most of the small intesti­nal tumors were found in duodenum. Macroscopic appearances and histological exami­nation Macroscopically, tumors grew as plaques or as polypoid lesions on stalk or formed ''nap­kin ring'' masses. In 73% of Wistar rats, mul­tiple colorectal tumors were found. The ma­jority of those tumors was strung closely to- 93% Figure 2. Comparison of colonic tumor stages in Wistar and Fisher rats. gether and gave the appearance of ''chain of tumors'' (Figure 1). The latter consisted of 3-8 tumors and were mostly located in the trans­verse and the descending colon with rectosig­moid. Five Wistar rats presented such chains, while none was found in Fischer rats. In the review of histological samples in Wistar rats 133 adenomas, 50 tubular adeno-carcinomas and 1 signet-ring cell carcinoma were found. In Fischer rats, the histological examination revealed 94 adenomas, 26 tubu­lar adenocarcinomas, 5 signet ring-cell carci­nomas and 1 mucinous carcinoma (Table 2). Most tubular adenocarcinomas were well-differentiated lesions. They grew mostly as polypoid or papillary growths into the lumen. On the contrary, signet-ring cell carcinomas were mostly small, plaque-like lesions with prominent invasion into the deeper levels of the bowel wall. In both strains, signet-ring cell carcinomas were found in the ascending colon with only one tumor being located in the small intestine. The difference in the occurrence of the ma­lignant and benign tumors between the strains was statistically significant (P<0.001; P=0.011). We also found a relation between intesti­nal lymphoid tissue and tumor location. More than a half of the carcinomas were found in the vicinity of the lymphoid follicles. In one Wistar rat, intussusception connected with tumor in the transverse colon was observed. Figure 4. Distribution of intestinal carcinomas in Fischer vs. Wistar rats (each spot represents one tumor). Staging of the intestinal carcinomas In Wistar rats, the majority of tumors (82%) were found in stage A, 16% were in stage B (Figure 3), and 2% in stage D according to Duke’s system (Table 2). Only 3% of tubular adenocarcinomas stage A were found in the small intestine, while 46% were found in the transverse colon, 41% in the descending colon with rectosigmoid and 10% in the as­cending colon. The adenocarcinomas stage B were found mostly (63%) in the small intes­tine, 25% in the descending colon with rec-tosigmoid and 12% in the transverse colon. Only one signet-ring cell carcinoma was found in the ascending colon. In one rat, we found a carcinosis of the liver and peri­toneum (Dukes D). The comparison of stages of only colorectal carcinomas between differ­ent strains of rats is shown in Figure 2. In Fischer rats, 68% of carcinomas were stage A, 32% stage B, while other stages were not found; 56% of adenocarcinomas stage A and 75% stage B carcinomas were found in the small intestine, while others were located in the descending colon with rectosigmoid (22%), transverse (10%) and the ascending colon (10%). The adenocarcinomas in stage B were similarly distributed: small intestine (75%), descending colon with rectosigmoid (12.5%), and ascending colon (12.5%). The signet-ring cell carcinomas presented 12.5% of all carcinomas in Fischer rats and all were found in the ascending colon. The distribu­tion of induced intestinal carcinomas in Fischer and Wistar rats is schematically pre­sented in Figure 4. Discussion DMH injected subcutaneously is one of the most effective CRC inducers in small rodents. This substance has been studied in large-scale experiments12-19, but the incidence of experimentally induced tumors in different strains of animals was not clearly defined. No data can be found in literature comparing DMH-induced tumors in Wistar and Fischer strains, although those are most often used for experimental purposes.9 The intestinal tumors were induced in 97% of Fischer and 100% of Wistar rats. Though there was no significant difference in the share of animals affected with tumors be­tween the two strains , the incidence of in­testinal tumors was significantly higher in Wistar rats. Nevertheless, we have to emphasize that the microadenomas containing 2-5 aberrant crypts were also included in analysis. Micro-adenomas presented almost 70% of all tumors found and their frequency supports the likeli­hood that CRC develop from adenomas.5,6,21-23 Because of the inclusion of microadenomas, the total number of induced tumors in our study somewhat exceeded the number of tu­mors induced by the same dose and number of applications by other authors.6,7,23-25 Fischer rats developed markedly less carci­nomas than Wistar rats; 58% of them were found in the small intestine, others were equally distributed in the ascending and de­scending colon. There were, however, less tu­mors found in the transverse colon than re­ported by other authors.9,23-25 The tumors of the small intestine, which were mainly well differentiated adenocarcinomas, developed most often in the proximal part of the small intestine. Macroscopically, both strains de­veloped polypoid, cauliform lesions and also ring-like lesions with elevated edges that were comparable with human disease. Sessile tumors exhibiting endophytic growth pattern were rare. In our study, the histological types of in­testinal tumors in rats are consistent with those of other authors who report the greatest number of well differentiated adenocarcino-mas and some signet-cell carcinomas, while poorly differentiated adenocarcinomas were rarely found.20-23,26 Our results of tumor stage analysis were comparable with those obtained by other au­thors, according to which a majority of col-orectal tumors (75%) were in stage A.20-23,26 Our comparison of carcinoma stage by strain has shown noteworthy differences. Fischer rats developed twice as much stage B tumors than Wistar rats. The tumors found in Fischer rats were showing more invasiveness and were usually growing deeper in the bowel wall. Likewise some other authors, we also found a case of stage D tumor, with peritoneal carcinosis and distant metastases.5, 20-22 The analysis of the small intestinal tumors revealed differences between the two strains in regard to stages: well or moderately differ­entiated adenocarcinomas stage B predomi­nated in Wistar strain and well differentiated adenocarcinomas stage A in Fischer rats. Most of the macroscopically visible small in­testinal tumors were located in the proximal part (duodenum, proximal jejunum), which is consistent with the carcinoma of the small in­testine in humans. An association of DMH-induced rat col-orectal tumors with colorectal lymphoid folli­cles was observed previously, but not quanti­fied. Our experiment revealed that more than 50% of carcinomas developed in the immedi­ate proximity of the intestinal lymphoid tis­sue. This is supposed to be an immunologic answer to antigenic components present in the tumor and simultaneously because of the more rapid replication of the epithelial cells in the vicinity of lymphatic tissue.27-29 Tumors found in both Wistar and Fischer strain histologically resembled those found in human pathology. Wistar rats have developed a greater incidence of colorectal tumors and distribution of tumors resembled more the dis­tribution as it is seen in human pathology than those in Fischer rats. Therefore we recom­mend Wistar rats rather than Fischer rats for the research work on the colorectal tumors. Acknowledgements The authors wish to thank Mrs. Tadeja Klemenc and Mrs. Majda Prebil for the histo­logical samples preparation, Mrs. Ana Zebic, DVM for her valuable assistance with the ex-periment and Mrs. Martina Perše, DVM for her help with references, comments and sug­gestions. References 1. Boring CC, Sqires TS, Tong T, Montgomery S. Cancer statistics. CA Cancer J Clin 1994; 44: 9. 2. Register raka za Slovenijo. Incidenca raka v Slo­veniji. Ljubljana: Onkološki inštitut v Ljubljani, 1994: 13. 3. Ishiguro Y, Ochinai M, Sugimura T, Nagao M, Nakagama H. Strain differences of rats in the sus­ceptibility to aberrant crypt foci formation by 2­amino-1-metyl-6-phenylimidazo-[4,5-b]pyridine: no implication of Apc and Pla2g2a genetic poly­morphisms in differential susceptibility. Carcino-genesis 1999; 20(6): 1063-8. 4. Evans JT, Hauschka TS, Mittelman A. Differential susceptibility of four mouse stains to induction of multiple large-bowel neoplasms by 1,2-dymethyl-hydrazine. J Natl Cancer I 1974; 52: 999-1000. 5. Melhem MF, Kunz HW, Gill TD. Genetic control of susceptibility to diethylnitrosamine and di­methylbenzanthracene carcinogenesis in rats. Am J Pathol 1991; 139: 45-51. 6. Teague CA, Gavin JB, Heridson PB. The response of three inbred strains of rat to the cancerogen 1,2­dimethylhydrazine. Pathology 1981; 13: 473-85. 7. Evans JT, Shows TB, Sproul EE, Paolini NS, Mittelman A, Hauschka TS. Genetics of colon car-cinogenesis in mice treated with 1,2-dimethylhy­drazine. Cancer Res 1977; 37: 134-40. 8. Breskvar L, Cerar A. A role of gender in the oc­curence of dimethylhydrazine induced colorectal tumors in Wistar rats. Radiol Oncol 1997; 31: 374­9. 9. Martin MS. Experimental intestinal carcinogene-sis. Cancer 1992; 5: 1-10. 10. Shamsuddin AKM. Carcinoma of the large intes­tine: animal models and human disease. Hum Pathol 1986; 17: 451-3. 11. Dukes C. The classification of cancer of the rec­tum. J Pathol Bacteriol 1932; 35: 313-32. 12. Toth B. Synthetic and naturally occuring hy­drazines as possible cancer causative agents. Cancer Res 1975; 35: 3693-7. 13. Fiala ES. Investigations into the metabolism and mode of action of the colon carcinogens 1,2-di­methylhydrazine and azoxymethane. Cancer 1977; 40: 2436-45. 14. Fiala ES, Bobotas G, Kulakis C, Wattenberg LW, Weisburger H. Effects of disulfiram and related compounds on the metabolism in vivo of the colon carcinogen 1,2-dimethylhydrazine. Biochem Phar­macol 1977; 26: 1763-8. 15. Fiala ES, Sthathopoulos C. Metabolism of methy­lazoxymethanol acetate in the F344 rat and strain­2 guinea pig and its inhibition by pyrazole and disulfiram. J Cancer Res Clin Oncol 1984; 108: 129­34. 16. Swenberg JA, Cooper HK, Bucheler J, Kleihues P. 1,2-dimethylhydrazine-induced methylation of DNA bases in various rat organs and the effect of pretreatment with disulfiram. Cancer Res 1979; 39: 465-7. 17. Zedeck MS, Stemberg SS, Pynter RW, McGowan J. Biochemical and pathological effects of methyla­zoxymethanolacetate, a potent carcinogen. Cancer Res 1970; 30: 801-12. 18. Fiala ES, Kulakis C, Christiansen G, Weisburger JH. Inhibition of the metabolism of the colon car­cinogen, azoxymethane by pyrazole. Cancer Res 1978; 38: 4515-21. 19. Fiala ES, Caswell N, Sohn OS, Felder MR, McCoy D, Weisburger JH. Non-alcohol dehydrogenase-mediated metabolism of methylazoxymethanol in the deer mouse Peromyscus maniculatus. Cancer Res 1984; 44: 2885-91. 20. Day DW. The adenoma-carcinoma sequence. Scand J Gastroenterol 1984; 19(Suppl 104): 99­107. 21. Hill MJ, Morson BC, Bussey HJR. Aetiology of ade­noma-carcinoma sequence in large bowel. Lancet 1978; i: 245-7. 22. Sunter JP. Cell proliferation in gastrointestinal car-cinogenesis. Scand J Gastroenterol 1984; 19(Suppl 104): 45-9. 23. Newberne PM, Rogers AE. Adenocarcinoma of the colon. Am J Pathol 1988; 72: 541-4. 24. Hagihara PF, Yoneda K, Sachattelo CR, Hedgecock H, Flesher JV, Ram MD, Griffen WO, Goldenberg DM. Colonic tumorigenesis in rats with 1,2-DMH: Dis Colon Rectum 1980; 23: 137-40. 25. Maskens AP. Histogenesis and growth pattern of 1,2-dimethylhydrazine-induced rat colon adeno-carcinoma. Cancer Res 1978; 36: 1585-92. 26. Lipkin M, Deschner E. Early proliferative changes in intestinal cells. Cancer Res 1976; 36: 2665-8. 27. Rubio CA, Nylander G, Sveander M, Duvander A, Alun ML. Minimal invasive carcinoma of the colon in rats. Am J Pathol 1986; 123: 161-5. 28. Park HS, Goodland RA, Wright NA. The inci­dence of aberrant crypt foci and colonic carcinoma in dimethylhydrazine-treated rats varies in a site-specific manner and depends on tumor histology. Cancer Res 1997; 57(20): 4507-10. 29. Hardman WE, Cameron IL. Colonic crypts located over lymphoid nodules of 1,2-dimethylhydrazine-treated rats are hyperplastic and at high risk of forming adenocarcinomas. Carcinogenesis 1994; 15(10): 2353-61. Radiol Oncol 2004; 38(3): 235-40. Multileaf collimator in radiotherapy Matjaž Jeraj, Vlado Robar Department of Radiotherapy, Institute of Oncology, Ljubljana, Slovenia Background. Basic goal of radiotherapy treatment is the irradiation of a target volume while minimizing the amount of radiation absorbed in healthy tissue. Shaping the beam is an important way of minimizing the absorbed dose in healthy tissue and critical structures. Conventional collimator jaws are used for shap­ing a rectangular treatment field; but, as usually treatment volume is not rectangular, additional shaping is required. On a linear accelerator, lead blocks or individually made Cerrobend™ blocks are attached onto the treatment head under standard collimating system. Another option is the use of multileaf collimator (MLC). Conclusions. Multileaf collimator is becoming the main tool for beam shaping on the linear accelerator. It is a simple and useful system in the preparation and performance of radiotherapy treatment. Multileaf col-limators are reliable, as their manufacturers developed various mechanisms for their precision, control and reliability, together with reduction of leakage and transmission of radiation between and through the leaves. Multileaf collimator is known today as a very useful clinical system for simple field shaping, but its use is getting even more important in dynamic radiotherapy, with the leaves moving during irradiation. This en­ables a precise dose delivery on any part of a treated volume. Intensity modulated radiotherapy (IMRT), the therapy of the future, is based on the dynamic use of MLC. Key words: radiotherapy dosage; radiotherapy; multileaf collimator, field shaping Introduction Basic goal of radiotherapy treatment is the ir­radiation of a target volume while minimizing the amount of radiation absorbed in healthy tissue. Shaping of the beam is an important Received 2 March 2004 Accepted 4 April 2004 Correspondence to: Matjaž Jeraj, BSc. (Radiol.), Teleradiotherapy Unit, Department of Radiotherapy, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia; Phone +386 1 522 3749; Fax: 386 1 4319 108; E-mail: matjaz-jeraj@siol.net way of minimizing the absorbed dose in healthy tissue and critical structures. Conventional collimator jaws are used for shaping a rectangular treatment field; but, as usually the treatment volume is not rectangu­lar, additional shaping is required. On a lin­ear accelerator, lead blocks or individually made Cerrobend™ blocks are attached onto the treatment head under standard collimat­ing system. Another option that will be de­scribed in detail here is the use of multileaf collimator (MLC). The MLC has movable leaves, which can block some fractions of the radiation beam. Typical MLCs have 40 to 120 leaves, arranged in pairs (Figure 1). By moving and controlling a large number of narrow, closely abutting in­dividual leaves, one can generate almost any desired field shape.1 The advantages of MLCs are simple and less time consuming preparation, use without needing to enter the treatment room, and sim­ple change or correction of field shape. The therapy expenses are lower because individual shielding blocks are not needed, thus eliminat­ing the need to handle the Wood’s alloy, which is toxic. With MLC, we shorten the therapy time, and thus also the period during which patient must remain in still position. Other ad­vantages are constant control and continuous adjusting of the field shape during irradiation in advanced conformal radiotherapy.1-5 MLC has also some disadvantages, which include a stepping edge effect, radiation leak­age between leaves, wider penumbra, and problems with generating some complex field shapes.2 MLC Configurations MLC configurations may be categorized as to whether they are total or partial replacements of the upper jaws, the lower jaws, or as ter­tiary collimation configurations. Upper jaw replacement This configuration entails splitting the upper jaw into a set of leaves. In this design (used by Elekta™), the MLC leaves move in the y-direction (parallel to the axis of rotation of the gantry). A “back-up” collimator located be­neath the leaves and above the lower jaws augments the attenuation provided by the in­dividual leaves. The back-up diaphragm is es­sentially a thin upper jaw that can be set to follow the leaves if they are arranged togeth­er to form a straight edge, or else, set to the position of the outermost leaf if the leaves form an irregular shape. The primary advantage of the upper jaw re­placement configuration is that the range of motion of the leaves required to traverse the collimated field width is smaller. This allows a shorter leaf length and therefore a more com­pact treatment head diameter. The disadvan­tage of having the MLC leaves so near the source of radiation is that the leaf width must be somewhat smaller and the tolerances on the dimensions of the leaves and the leaf travel must be tighter than in other configurations.1 Leaves of this collimator have total travel distance 32.5 cm, which means they can ex­tend 12.5 cm across the centre line. Lower jaw replacement The lower jaws can be split into a set of leaves as well. This design is used by Siemens™ and is double-focused. Both leaf ends and leaf sides match the beam divergence. That means that the collimator leaves move along the cir­cumference of a circle centred at the x-ray tar­get of the linear accelerator, such that the end of the collimator is always tangential to the radius of the circle.1 The leaves of Siemens MLC can extend 10 cm across the field centreline, which allows a maximum leaf travel of 30 cm. Third level configurations MLC can be positioned just below the level of the standard upper and lower adjustable jaws (Figure 2). This design is used by Varian™ and was chosen to avoid lengthy downtime in the event of a MLC system malfunction. Using this approach, it is possible to move leaves manually out of the field should a fail­ure occur. The treatment can continue after the replacement Cerrobend™ individual blocks have been manufactured. The major disadvantage of placing the MLC below the standard jaw system is the added bulk and clearance to the mechanical isocentre. Moving the MLC further away from the x-ray target requires increasing the leaves size and a longer travel distance.1 The leaves in the Varian collimator travel on a carriage that serves to extend their movement across the field. However, the dis­tance between the most extended leaf and the most retracted leaf on the same side can only be 14.5 cm. Materials and properties The material of choice for leaf construction is tungsten alloy because it has one of the high­ est densities of any metal. Tungsten alloys are also hard, simple to fashion, reasonably inex­pensive, and have a low coefficient of thermal expansion. Interleaf transmission There are two situations to consider for inter-leaf transmission: (1) between the sides of ad­jacent leaves, and (2) between the ends of the leaves. In order to minimize the leakage between the sides, it is necessary to overlap the leaves usually by specially shaped side profile that steps out and then steps back again.6 To minimize leakage between the ends of closed opposite ends, it is important to know that the transmission decreases with increas­ing the off-axis distance.7 Leaf end shape Multileaf collimators that are double focused (Siemens design) have flat leaf ends that fol­low the beam divergence. The leaf ends of Elektra and Varian MLC design are rounded. There are two concerns over collimation by non-focused leaf ends. First, the penumbra width is larger than the penumbra generated by a focused or divergent edge. Second, the penumbra width might change as a function of the distance of the leaf end from the field midline (Figure 3). The measurements on the Elekta and Varian configurations have shown that these designs result in a little variation in the penumbra width as a function of leaf po­sition and that the penumbra at any position is within 1-3 mm of that obtained with a fo­cused system or with alloy blocks with diver­gent sides.6-10 MLC control features MLCs produced by various manufacturers employ special mechanisms to move the leaves accurately to their prescribed posi­tions. Detection of the leaf position The leaf position must be detected in real-time to achieve a safe and reliable position control. Linear encoders and video optical systems are most commonly used for detection. Linear encoders We can use many different linear encoders, but for detection of leaf positions in MLC sys­tems high precision potentiometers are com­monly used. These potentiometers can detect positions of any individual leaf in the system. For safer work two potentiometers with correlated readings are used in this system. Video-optical system This system of detection uses the same light source for patient positioning and for leaf po­sition recognition. A retro-reflector is mount­ed near the end of each leaf, and the light is reflected from it back to the camera. The ob­tained signal is digitized and processed with an image processor in the MLC controller. The mechanism that drives a leaf Each leaf has a small motor, which drives it precisely in the directions from the main unit. These rotations must than be translated to linear motion which moves the leaf to the de­sired position. Linear screw bars are normal­ly used to translate rotations to linear motion. The speed of the leaf travel varies between 0.2 mm/s to as high as 50 mm/s, depending on the design.1 Clinical applications Leaf placement strategies To realize potential benefits of MLC, it is im­portant that its use is incorporated into the Figure 4. Three leaf coverage strategies in relation to the PTV, (a)“out-of-field” strategy; (b)“in-field” strategy; c)“cross-boundary” strategy. Figure 5. IMRT techniques with the use of MLC. treatment planning process as efficiently as possible. During the treatment planning process, manual placement of each of the 40-120 leaves is not acceptable due to time con­straints. Therefore some automated method must be used in a treatment planning system (TPS). That way in TPS, the position of each leaf is defined so that the field encompasses the planning target volume (PTV). More specifically, the determination of the MLC positions is carried out by means of the fol­lowing steps: Definition of target area Treatment planning system facilitates shap­ing leaves around PTV, as defined by a radia­tion oncologist. An accurate definition of PTV is crucial for the success of the therapy.11 Optimization of MLC conformation To place automatically the leaves of MLC in conformity with the target contour shape, three leaf coverage strategies can be used (Figure 4). Each strategy uses different position of the leaf in relation to the contour of the field that we want to irradiate. The “out of field” strategy (4a) avoids shiel­ding any part of the planning target volume-PTV, which is than irradiated completely. When using the “in field” strategy (4b), PTV is not irradiated completely, but any part out of PTV stays shielded. The most widely used method is cross-boundary technique indicated in panel (c) of Figure 4. One condition for optimizing the leaf positions was criterion that the in-field area was equal to the out-of-field area.4 Optimization of collimator rotation One can optimize matching the leaf shape to target volume by rotating the collimator, and therefore, the direction of leaf travel. An ex­ample is the alignment of the leaf faces with the spinal cord axis, when the cord is close to the target volume. Brahme, in his conclusion of work said, that optimal direction for the leaf motion is the direction along the narrow­er axis. For a simple ellipse, the optimal leaf direction is parallel to the short axis.12 Intensity modulated radiotherapy (IMRT) with multileaf collimator The basic goal of IMRT treatment is precise dose delivery on any part of treated area thus avoiding the surrounding healthy tissue. In IMRT treatment, the leaves of MLC, while moving during the irradiation, ensure the ap­propriate dose that is delivered on the parts of treated area (Figure 5). From the differences between the dose vol­umes delivered during the whole treatment and the dose volumes in which the leaf is shielding some part of the treated area, we can determine what dose has been delivered on this particular part. MLC for intensity modulation should be very precise, motion of leaves must be fast and constant, leaves should be precisely controlled and must have a long reach in the field. Three dimension (3D) treatment planning systems must be used for IMRT. Two strategies of IMRT with multileaf col­limator are used. One is dynamic technique, with continuous movement of leaves during the treatment; the second is step and shot technique with moving the leaves when radi­ation is stopped. Both strategies with this travel determine dose delivered on the parts of treatment volume.2 References 1. Boyer A, Biggs P, Galvin J, Klein E, LoSasso T, Low D, et al. for the Radiation Therapy Committee. Report of Task Group No. 50. Basic applications of multileaf collimators. AAPM Report No. 72. 2001. 2. Jeraj M. Veclistni kolimator v radioterapiji. Diplomska naloga. Ljubljana: Visoka šola za zdravstvo; 2003. 3. Frazier A, Du M, Wong J, Vicini F, Matter R, Joyce M, et al. Effects of treatment setup variation on beam's eye view dosimetry for radiation therapy using the multileaf collimator vs. the cerrobend block. Int J Radiat Oncol Biol Phys 1995; 33: 1247­56. 4. LoSasso, T, Chui CS, Kutcher GJ, Leibel SA, Fuks Z, Ling CC. The use of a multi-leaf collimator for conformal radiotherapy of carcinomas of the prostate and nasopharynx. Int J Radiat Oncol Biol Phys 1993; 25: 161-70. 5. Brahme A. Optimization of radiation therapy and the development of multi-leaf collimation. Int J Radiat Oncol Biol Phys 1993; 25: 373-5. 6. Jordan TF, Williams PC. The design and perfor­mance characteristics of a multileaf collimator. Phys Med Biol 1994; 39: 231-51. 7. Galvin JM, Smith A, Lally B. Characterization of a multi-leaf collimator system. Int J Radiat Oncol Biol Phys 1993; 25: 181-92. 8. Galvin JM, Smith AR, Moeller RD, Goodman RL, Powlis WD, Rubenstein J, et al. Evaluation of mul­tileaf collimator design for a photon beam. Int J Radiat Oncol Biol Phys 1992; 23: 789-801. 9. Boyer AL, Ochran TG, Nyerick CE, Waldron TJ, Huntzinger CJ. Clinical dosimetry for implemen­tation of a multileaf collimator. Med Phys 1992; 19: 1255-61. 10. Huq MS, Yu Y, Chen ZP, Suntharalingam N. Dosimetric characteristics of a commercial multi-leaf collimator. Med Phys 1995; 14: 268-9. 11. International Commission on Radiation Units and Measurements (ICRU). Report 50. Prescribing, recording, and reporting photon beam therapy. (Allisy A, chairman). Bethesda: ICRU; 1993. 12. Brahme A. Optimal setting of multileaf collimators in stationary beam radiation therapy. 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