RADIOLOGY AND ONCOLOGY Radiology and Oncologtj is a journal devoted to publication of original contributions in diagnostic a11d interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, c/inical and experimental oncology, radiobiology, radiophysics and radiation protection. Editor-in-Chief GregorSerša Ljubljana, Slovenia Executive Editor Viljem Kovac Ljubljana, S/ovenia Editor-in-Chief Emeritus Tomaž Benulic Ljubljana, S/ovenia Editorial board Marija Auersperg Bela Fornet Maja Osmak Ljubljana, Slovenia Budapest, Hungary Zagreb, Croatia Nada Bešenski Tullio Giraldi Branko Palcic Zagreb, Croatia Trieste, Italy Vancouve1; Canada Karl H. Bohuslavizki Andrija Hebrang /urica Papa Hamburg, Gennany Zagreb, Croatia Zagreb, Croatia Haris Boka Ltiszl6 Horvath Dušan Pavcnik Zagreb, Croatia Pecs, Hungary Portland, USA Nataša V. Budihna Berta Jereb Stojan Plesnicar Ljubljana, S/ovenia Ljubljana, S/ovenia Ljubljana, S/ovenia Ma1jan Budihna Vladimir Jevtic Ervin B. Podgoršak Ljubljana, Slovenia Ljubljana, S/ovenia Montreal, Canada Malte Clausen H. Dieter Kogelnik Jan C. Roos Hamburg, Germany Salzburg, Austria Amsterdam, Netherlands Christoph Clemm Jurij Lindtner Slavko Šimunic Miinchen, Germany Ljubljana, S/ovenia Zagreb, Croatia Mario Corsi Ivan Lovasic Lojze Šmid Udine, ltaly Rijeka, Croatia Ljubljana,Slovenia Christian Dittrich Marijan Lovrencic Borut Štabuc Vienna, Austria Zagreb, Croatia Ljubljana, S/ovenia Ivan Drinkovic LukaMilas Andrea Veronesi Zagreb, Croatia Houston, USA Gorizia, Italy Gillian Duchesne Metka Milcinski Živa Zupancic Melbourne, Australia Ljublja11a, S/ovenia Ljubljana, Slovenia Publishers Slovenian Medical Association -Slovenian Association of Radiologij, Nuclear Medicine Society, Slovenian Society for Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medical Association -Croatian Society of Radiologij Affiliated with Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups of S.I.R.M. (Italian Society of Medica/ Radiology) Correspondence address Radiologij and Oncologij Institute of Oncology Vrazov trg 4 SI-1000 Ljubljana Slovenia Tei: +386 61 132 00 68 Tel/Fax: +386 61 133 74 10 Reader far English Olga Shrestha Design Monika Fink-Serša Key words Eva Klemencic Secretaries Milica Harisch Betka Savski Printed by Imprint d.o.o., Ljubljana, Slovenia Published quarterly in 700 copies Bank account number 50101 678 48454 Foreign currency account number 50100-620-133-27620-5130/6 NLB -Ljubljanska banka d.d. -Ljubljana Subscription fee far institutions 100 $, individuals 50 $ Single issue far institutions 30 $, individuals 20 $ The publication of this journal is subsidized by the Ministry of Science and Technology of the Republic of Slovenia. According to the opinion of the Government of the Republic of Slovenia, Public Relation and Media Office, the journal Radiology and Oncologij is a publication of informative va/ue, and as such subject to taxation by 5% sales tax. Indexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EMBASE / Excerpta Medica This journal is printed on acid-free paper Radiology and Oncologtj is now available on the internet at: http:/www.onko-i.si/radiolog/rno.htm CONTENTS ULTRASOUND AND NUCLEAR MEDICINE Obstruction of the duodenum due to a billiary calculus (Bouveret's syndrome) Vcev Al, Barbic J, Vccv An, Kovacic D, Vegar M 161 A rare case of symmetric bifemoral fractures in battered child syndrome and overview over the literature Klutmann S, Kroger S, Bolzuslavizki KH, Brenner W, Kmjf C, Oppennann HC, Tibow 1, Hcnzc E 165 EXPERIMENTAL ONCOLOGY Polymerase chain reaction procedures in the diagnosis of lymphoproliferative disorders Griesser H 171 In vivo electroporation of the urinary bladder in mice Veranic P, Jezernik K, Cemažar M, Serša G 187 MDP desmuramyl analogne LK-404 protects bone marrow and spleen cells from cyclophosphamide induced apoptosis Kostanjšek R, Kuralt P, Malovrh T, Škoberne M, Štalc A, Kotnik V 193 CLINICAL ONCOLOGY The cause of testicular cancer Kovac V Lymphotropic staining of the sentinel lymph nodes in breast cancer ­with what, when, how? Bayclzev G, Dclijsky T, Penkova R, Stojanov R 213 221 Carcinoma of the thyroid: Postoperative radiotherapy Mayer R, Stuecklschweiger GF, Preidler KW, Pakisch B, Langsteger W, Oechs A, Prettenhofer U, Hackl A Pectoralis major flaps for reconstruction of the head and neck defects Yildirim E, Turanli M, Sancaktar S, Berberoglu U REPORT Report on the Workshop on Strategy of lnvestment in Radiological Equipment for the Central and Eastern European countries held in Baden bei Wien 27-28 September 1997 European Association oj Radiology (EAR) 225 SLOVENIAN ABSTRACTS 229 NOTICES 234 Radio/ 011col l998; 32(2): 161-3. Obstruction of the duodenum due to a billiary calculus (Bouveret' s syndrome) Aleksandar Vcev1, Jerko Barbic--I, Andrijana Vcev1, Damir Kovacic2 , Miroslav Vegar2 1 Department oj Interna/ Medicine, 2 D epartment oj Surgery University Ho spital Osijek, Osijek, Croatia Duodenal obstruction by a gallstone is a very uncommon clinica/ condition. The gallstone obstruction oj duodenum is usually discovered by gastrointestinal endoscopy ar x-ray examination oj the upper gastroin­testinal tract. Here, we report the case oj a 76-year-old woman with a gastric outlet obstruction due to gall­stone. The obstruction was initially diagnosed by the ultrasound oj the upper abdomen and was later con­jirmed by gastroscopy. The attempt oj endoscopic extraction oj gallstone jrom the duodenum was unsuc­cessjul and was consequently removed by surgica/ procedure. Key words: choletiasis-complications; duodenal obstruction Introduction The intestinal obstruction secondary to a gall­stone is an unusual complication of cholelithiasis and accounts for 1 % of ali cases of intestinal obstruction, however the inci­dence of this condition rises up to 25% in those over age 70.1 The stone usually lodges at the terminal ileum, the narrowest portion of normal gut, but stones can be impacted in pylorus, duodenum, jejunum or colon. The duodenal obstruction by a gallstone is a very rare condition.2, 3 In recent years, diagnosis has been facilitated by endoscopy and severa! 8 cases have been reported.4-Here, we report a case of duodenal obstruction in which the Correspondence to: Aleksandar Vcev, M.O., Depart­rnent of Interna! medicine, University Hospital Osi­jek, J. Huttlera 4, 31 000 Osijek, Croatia. Phone: +385 31 101 101; Fax: +385 31 101 311. diagnosis of gallstone obstruction was made endoscopically and relieved by surgical pro­cedure. Case report A 76 year-old woman was admitted to hospi­tal because of nausea and vomiting. The nau­sea and vomiting were lasting for the last three weeks. Since than, she !ost appetite and 4 kg of body weight. During last seven days vomiting was persistent. The patient stopped eating and she just drank milk and yoghurt. The patient had been well until about six months earlier when she had pain in the upper abdomen. At that tirne, the pain was treated symptomatically with H2-receptor antagonist, antacids and diet. The peptic ulcer disease as the cause of this pain was 162 Vcev A el al. not confirmed by endoscopic examination at that time. However, with this treatment she felt better until the admission to our hospital. She had no previous history of cholelithiasis attacks. At the admission, physical examination disclosed dehydration, tenderness and epi­gastric distension. Laboratory tests were per­formed. The laboratory analysis revealed nor­mal !iver function and prerenal azotemia with hypokalemia, hypochloremia and alka­losis. Ultrasound examination of the upper abdomen was performed. It showed a high density mass of 5 cm in diameter at the pro­jection of bulbus duodeni. The endoscopic examination revealed one large, round, hard, black-green mass projecting through pylorus (Figure 1). Moreover, by endoscopic examina- Figure l. Gallstone projecting through pylorus at the endoscopy. tion we observed an ulcer of the anterior wall of the duodenal bulb with bulb deformation, dilatation of stomach and reflux esophagitis "Savari Miller gr III". This mass was inter­preted as a gallstone obstructing duodenum and the x-ray examination of the upper abdomen was considered unnecessary. We were not able to remove the gallstone by endoscopic mechanical lithotripsy. After correction of dehydration, metabolic alkalo­sis and hypokalemia, a successful removal was performed by surgical procedure. The surgical procedure was performed by entero­tomy proximal to the stane, and removing offending calculi with closure of the intes­tine. The patient's condition allowed us to perform concomitant cholecystectomy with fistula closure. The opening of cholecystoen­teric fistula was on the anterior wall of the duodenal bulb. The patient's post-operative condition was good. Discussion Bouveret described duodenal obstruction due to biliary stane in 1896. Bouveret's syndrome is characterised by signs of ileus like abdomi­nal pain, vomiting and dehydration. Patients usually had a prior history of symptomatic biliary tract disease. However, the patient has not mentioned abdominal pain, biliary colic as well as fever. Fifty-eight cases of this rare clinical entity were described by Simonian in 19682 and since then others4 -8 have reported severa! new cases. It is important to note that most reported new cases were diagnosed by endoscopic examination of stomach and duo­denum. Bouveret' s syndrome may occur in 1 % to 3% of patients with cholecystoenteric fistula.9 The occurrence of cholecystoenteric fistulas has been reported in 0.09% to 3.2% of patients with biliary disease.9 Gallstone ileus is a complication in 0.3% to 0.5% of all cases of cholelithisais.3 It is important to have in mind that ileus could be caused by a gall­stone. Moreover, the incidence of gallstone ileus rises significantly in patients over 65 years of age.1•3 Here, we showed that the ultrasound examination of the upper abdo­men could give the first indication of a gall­stone. This is to our knowledge the first report that this easy and fast diagnostic Duodenal ohslruction due to a bil/iary calculus approach can be used. The usual initial diag­nostic step is the x-ray examination of the abdomen, which must comprise, in differen­tial diagnosis, other causes of intestinal obstruction like duodenal neoplasm, duode­nal polyp and finally foreign body.2 However, here the fina! diagnosis of Bouveret' s syn­drome was established by the endoscopic examination of the stomach, as it was 8 described by others.4-It is possible that the stone may be endoscopically removed or, at least, manipulated in stomach so that after relieving the obstruction, the patients will go for definitive surgical treatment with fistula closing and cholecystectomy, especially in patients in good health condition where is likely that gallstone ileus occur again.5, 6 Sur­gical procedure can be considered as only definitive treatment for Bouveret's syndrome. References 1. Rosato FE. Gallstone ileus and fistula. In: Sabiston DC Jr, editord. Davis-Clzrisloplzer textbook of surgery. Philadelphia: WB Saunders; 1982. p. 1274-8. 2. Simonian SJ. Gallstone obstruction of the duode­nal bulb. Lancel 1968; 1: 893-95. 3. Day EA, Marks C. Gallstone ileus review of the lit­erature and presentation of thirty-four cases. Am] Surg 1975; 129: 522. 4. Ayub A, Michalko CH. Gallstone obstruction of the pylorus. Gastrointest Endosc 1982; 28: 25-6. 5. Bedogni G, Contini S, Meinero M, Pedrazzoli C, Piccinini GC. Pylorodudenal obstruction due to a biliary stone (Bouveret's syndrome) managed by endoscopic extraction. Gaslroinlest Endosc 1985; 31: 36-8. 6. Moriai T, Haegawa T, Fuzita M, Kimura A, Tani T, Makino l. Successful removal of massive intragsatric gallstone by endoscopic electrohy­draulic lithotripsy and mechanical lithotripsy. Am J Gastroenlerol 1991; 85: 627-9. 7. Bottari M, Pallio S, Scribano E, Certo A. Pyloro­duodenal obstruction by a gallstone Bouveret's syndrome. Gastroinlesl Endosc 1988; 34: 440-2. 8. Gallstone duodenal obstruction or Bouveret's syn­drome. Apropos of two cases [French]. J Clzir 1991; 121: 34-8. 9. Torgerson SA, Greening GK, Juniper K, Farrell RL. Gallstone obstruction of duodenal cap (Bouverct's syndrome) diagnosed by cndoscopy. Am J Gas­lroenterol 1979; 72: 165. 10. Deckoff SL. Gallstone ileus. Ann Surg 1955; 142: 52. Radio/ Oncol 1998; 32(2): 165-9. A rare case of symmetric bifemoral fractures in battered child syndrome and overview over the literature Susanne Klutmann1, Sabine Kroger1, Karl Heinz Bohuslavizki4 , Winfried Brenner1 , Claudia Korff1 , Hans-Conrad Oppermann2 , Indra Tibow3 , Eberhard Henze1 Clinics of 1Nuclear Medicine, 2Diagnostic Radiology and 3Pediatrics, Christian-Albrechts-University, Kiel, 4Department oj Nuclear Medicine, University Hospital Eppendorf, Hamburg, Gennany We report on a 1-year-old girl who was suspected far battered child syndrome. A conventional X-ray showed a symmetric bifemoral fracture close to the distal growth plates and a left-sided parietal fracture. Howeve1; bone scintigraphy revealed only a slight symmeh·ic widening of the corresponding growth zones in the axial direction when compared with the proximal tibial growth areas while the parietal fracl:ure was missed. A diagnostic difficulty is sh'essed, and a criteria far bone scanning in battered child syndrome are reviewed. Key words: battered child syndrome, femoral fractures; radionuclide imaging Case history and clinical findings A one-year-old girl with a history of fever and diarrhea for three days was admitted to the children's hospital. The girl's left leg was in a cast due to a distal femoral frac­ture. She suffered from pain in her right leg so that she refused to get in a standing posi­tion for a week. This drew attention to a suspected fracture of the right femur. More­over, she had severa] hematomas of differ­ent age ali over the body and a blood-crust­ed exanthema at the occiput. A bilateral Correspondence to: Dr. Karl H. Bohuslavizki, Depart­ment of Nuclear Medicine, University Hospital Eppendorf, Martinistr. 52, D-20250 Hamburg, Ger­many. Phone: +49 40 47 17 40 47; Fax: +49 40 47 17 67 75; E-mail: bohu@medsph2.uke.uni-hamburg.de periocular hematoma was noticed by her pediatrician three weeks ago which was explained by her mother to be caused by falling twice against a table. She was notice­ably frightened about foreign people. Her general, nutritional and care conditions were poor. The examinations were undertaken in order to prave a suspected battered child syndrome. Laboratory results C-reactive protein was markedly increased at 2.9 mg/dl (normal: < O.S mg/dl). Ali further standard laboratory findings, i.e. blood celi count of erythrocytes, leucocytes and plate­lets were in the normal range. 166 Klutnwnn Se/ ni. X-ray An X-ray of both femurs showed a bilateral transverse fracture at both Figure 3. Conventional whole-body bone scintigraphy in anterior and posterior views showing increased tracer uptake of both distal growth plates of the left and right femur and bilateral slightly widened distal femoral growth plates as compared to proximal tibial growth plates. Furthermore, a diffusely higher uptake in the dia­physis of the right femur is seen in this patient. More­over, focal tracer uptake is evident at the costo-vertebral junction of the left 10th rib. Marginal enlarged asymmet­ric tracer uptake in the left side of the skull. Discussion A child abuse and a battered child syndrome show an increasing incidence with a high estimated number of unreported cases in the last few years.1, 2 Characteristic clinical find­ings of a battered child syndrome are hema­tomas, retina! hemorrhage, skin burns and fractures of different ages situated predomi­nantly in the skull, ribs and metaphyses of the long bones.3 -14 For forensic consequences a suspected battered child syndrome has to 19 be proved carefully.15-Thus, severa] diag­nostic methods have been established as screening procedures in order to prove bone involvement, e.g. bone scintigraphy and X­ray studies.20, 21 However, there is still a high 168 Klutnrnnn Sel al. was turned slighly to the left. A high number of false-negative bone seans in the detection of skull fractures is well-known. 23, 25 Focal tracer accumulation at the costovertebral junction of the 10th left rib was shown by a conventional X-ray to be related to a slight dislocation of the rib. With respect to our findings we would like to emphasize some points that should be kept in mind in differential diagnosis of a battered child syndrome: • Bone scintigraphy in these patients is both demanding and not frequently performed. Thus, a special attention should be paid to it. • An exact symmetric positioning should be ensured in order to allow an accurate side­to-side comparison. • Images with excellent count statistics should be acquired in order to enable the detection of the minor increased focal tracer uptake. Thus, sometimes the sedation of the patients may be necessary for the scintigraphic imag­ing to avoid misleading artefacts. • In addition to a whole-body scintigraphy spot images should be obtained, e.g. lower extremities, vertebral spine, skull from lat­erni views or from a parietal top view. • Even under most accurate conditions there is a decent number of false-negative results in either bone scintigraphy or conventional X-ray. • Therefore, the fina! evaluation of bone lesions should include ali lesions found either by the bone scintigraphy or by the conventional X-ray. References l. DM, Albert DM. Recognizing child abuse. Arch Ophtalmol 1992; 110: 766-8. 2. Merten DF, Radkowski MA, Leonidas JC. The abused child: a radiological reappraisal. Radiologij 1983; 146: 37 7-81. 3. Richter E. Riintgenmorphologische skelettveran­derungen bei kindesrrtiBhandlung. Aktuel Radio/ 1992; 2: 358-62. 4. Triiger J, Stegen P. Kindesmilshandlung. Wichtige befunde der bildgebenden diagnostik. Radiologe 1995; 35: 401-5. 5. Granry JC, Chapotte C, Monrigal JP, Benetreau D, Penneau M. Syndrome de l'enfant secoue. Ann Fr Aneslh Reanim 1994; 13: 133-4. 6. Rogers LF, Poznanski AK. Imaging of epiphyseal injuries. Radiologij 1994; 191: 297 -308. 7. Rodenwaldt J, Wigel W, Grabbe E. Shaken baby trauma: a case report on a problematic differential diagnosis in an infant. Rofo Fortschr Geb Rontgenstr Neuen Bildgeb Ve,fahr 1997; 167: 204-6. 8. Buys YM, Levin AV, Enzenauer RW, Elder JE, Letourneau MA, Humphreys RP, Mian M, Marin JD. Retina! findings after head trauma in infants and young children. Ophtlwlmology 1992; 99: 17 18-23. 9. Waterhouse W, Enzenhauer RW, Parmley VC. Inflammatory orbita! tumor as an ocular sign of a battered child (letter). Am J Ophlhalmo/ 1992; 114: 510-2. 10. Marcus DM, Albert DM. Recognizing child abuse (editoral). Arch Ophtha/111011992; 110: 166-7 8. 11. Poepel B, Seiberth V, Knorz MC, Kachel W. Augenbefunde beim shaken-baby-syndrom. Eine falldemonstration. Ophtha/1110/oge 1994; 91: 380-2. 12. Budenz DL, Farber MG, Mirchandani HG, Park H, Rorke LB. Ocular and optic nerve hemorrhages in abused infants with intracranial injuries. Opthal­mologij 1994; 101: 559-65. 13. Giese MJ. Ocular findings in abused children and infants born to drug abusing mothers. Oplom Vis Sci 1994; 71: 184-91. 14. Loder RT, Bookout C. Fracture patterns in bat­tered children. J Orthop Trauma 1991; 5: 428-33. 15. Weissgold DJ, Budenz DL, Hood I, Rorke LB. Rup­tured vascular malforrnation masquerading as bat­tered/shaken baby syndrome: A nearly tragic mis­take. Surv Opthahnol 1995; 38: 509-12. 16. Berkowitz CD. Pediatric abuse. New patterns of injury. Eni.erg Med Ciin Norih Am 1995; 13: 321-41. 17. Faure C, Kalifa G, Sellier N. Les reponses de l'irn­agerie medicale chez l'enfant battu Syndrorne de Silverrnan-Ambroise Tardieu. J Radio/ 1994; 75: 619-27. 18. Brown GR, Runyan DK. Diagnosing child rnal­treatment. N C Med J 1994; 55: 404-8. Battered clzild syndrome 19. Shaw DG, Hall CM, Carty H. Osteogenesis imper­fecta: the distinction from child abuse and the recognition of a variant form (letter). Am J Med Genet 1995; 56: 116-8. 20. Tan TX, Gelfand. Battered child syndrome. Uncommon pelvic fractures detected by bone scintigraphy. Ciin Nucl Med 1997; 22: 321-2. 21. Van Winckel M, Robberecht E, Afschrift M, Smets A, Wood BP. Radiological case of the month. Bat­tered child syndrorne. Are/z Pediatr Adolesc Med 1997; 151: 621-2. 22. Halle JO. Diagnostic imaging of child abuse. Pedi­atrics 1991; 87: 262-4. 23. Kleinhans E, Kentrup H, Alzen G, Skopnik H, Buli U. Falsch negatives skelettszintigramm bei einer biparietalen schadelfraktur beim "battered child"-syndrorn. N11k/ear111edizin 1993; 32: 206-7. 24. Strouse PJ, Owings CL. Fractures of the first rib in child abuse. Radiology 1995; 197: 763-5. 25. Sty JR, Starshak RJ. The role of bone scintigrapy in the evaluation of the suspected abused child. Radiology 1983; 146: 369-75. 26. Alzen G, Wildberger JE, Gunther RW. Bildgebung beirn traumatisierten kind. Radiologe 1995; 35: 373-7. 27. Tri.iger J, Stegen P. Das mifshandelte kind. Radi­ologe 1978; 18: 233-238. 28. Gilday DL. Specific Problerns and Musculoskele­tal Imaging in Children. In: Sandler MP, Patton JA, Coleman RE, Gottschalk A, Wackers FJT, Hof­fer PB editors. Diagnostic nuclear medicine. 3th ed. Volume two. Baltimore: Williams & Wiliarns; p. 1404-5. 29. Treves ST, Conolly LP, Kirkpatrick JA, Packard AB, Roach P, Jaramillo D. Bone. In: Pedriatic nuclear medicine. Treves ST editor. Berlin: Springer; 1995. p. 233-301. 30. Hahn K, Fischer S, Gordon l. Atlas oj bone scintig­raphy in tlze developing paediatric skele/011. Berlin: Springer; 1993. Radio! 011col l998; 32(2): 171-85. review Polymerase chain reaction procedures in the diagnosis of lymphoproliferative disorders Henrik Griesser Division of Applied Cytology, Department of Pathology, University of Wiirzburg, Germany Immunohistochemistry and 1110/ecular genetic techniques greatly contribute to our understanding of lym­phoproliferative disease. The majority of lymphomatous lesions can be diagnosed by morphology alone but additional diagnostic tests have to be employed when cel/ lineage and c/onality are not obvious. Morpholog­ic distinction of Hodgkin's from 11011-Hodgkin's lymplwma, ar of inflammatory lesions from malignant lym­phoma, can be challenging. Non-random chromosoma/ translocations may help to recognize lymphoma sub­groups with distinct biological characteristics. This review focuses on the polymerase chain reaction (PCR) techniques that have become an important diagnostic tool applicable to limited cellular material and paraf­fin-embedded tissues. The effectiveness of PCR in rearrangement analyses of T celi receptor g and immunoglobulin heavy chain genes is well documented. Some oj the known translocations, such as t(14;18) ar t(2;5) can be routinely assessed by genomic PCR ar by reverse-transcription PCR. Those involving the bc/-1, bc/-6, and c-myc genes require more elaborate and sophisticated PCR procedures. Molecular genetic analyses by PCR, besides their immediate diagnostic value, bear the potential to identify new criteria far lymphoma diagnosis in conjunction with cytomorphology and immunophenotyping. Key words: lymphoproliferative disorders-diagnosis; polymerase chain reaction; lymphoma-diagnosis Introduction Clonality of B cell proliferations, B or T lin­eage assignment, and maturation stage of Correspondence to: Henrik Griesser, M.D., Depart­ment of Pathology, University Medica! Center, Divi­sion of Applied Cytology, Josef-Schneider-Str. 2, D­97080 Wiirzburg, Germany. Tei: +49 (O) 931 -201 5427; Fax: +49 (O) 931 -201 3440; E-mail: path023@lrz­box.uni-wuerzburg.de The paper was presented at: Tutorial on the use of new techniques in diagnosis of malignant lym­phomas. February 2-4, 1998, Ljubljana, Slovenia. lymphoid tumor cell populations are mainly established by immunophenotyping. This method largely fails to determine clonality in T cell tumors and to assign lineage to either very immature cells, which do not yet express these markers, or abnormally activat­ed cells with loss of surface antigen expres­sion. Lymphoma tissues may also be difficult to classify based on morphology and immuno­histochemistry alone when the malignant done is obscured by reactive lymphoid cells. The discovery of immunoglobulin (IG) gene 172 Griesser H rearrangements and the application of molec­ular probes for these genes has opened a new avenue to the diagnosis and biological under­standing of lymphoproliferative disease. The discovery that rearrangement processes in T celi receptor (TCR) genes parallel the recom­bination events in immunoglobulin genes, led to the development of molecular tools applicable to the search for clonality in T­lymphoproliferative lesions.1 Cloning of mol­ecular breakpoints for tumor-specific chro­mosomal translocations permits the identifi­cation of a malignant done using molecular probes for analysis of DNA simply extracted from lymphoid tumor tissue.2 Data from mol­ecular investigations with such probes now provide information about the origin of tumor lymphocytes as well as clues to molec­ular mechanisms involved in blastic transfor­mation and tumor progression. Gene rearrangement analyses by South­ern blot procedures have become a valuable adjunct in the diagnosis of lymphoprolifera­tive disorders.3 This approach has serious limitations. The DNA has to be cut with sev­era! restriction enzymes. Hybridization with severa! probes is often required to obtain reliable and reproducible results. Large quantities (usually more than 20 µg) of high molecular weight DNA have to be extracted from fresh / fresh-frozen tissue or celi sus­pensions for these studies. The whole proce­dure takes several days, often weeks. Appli­cation of the polymerase chain reaction (PCR) techniques overcomes these limita­tions.4 Specific DNA fragments can be rapid­ly amplified with oligonucleotide primers binding to both ends of a gene sequence of interest. Genomic DNA amplification tech­niques are not restricted to the analysis of fresh-frozen tissue samples with their limit­ed morphological quality. DNA extracted from the more readily available formalin­fixed, paraffin-embedded tissue samples, even tiny areas of interest from a stained sec­tion or single cells with approximately 10 pg of DNA provide enough template for amplifi­cation. This review focuses on well characterized PCR protocols for routine analysis of B and T celi neoplasms. Diagnostically important chromosomal aberrations will be considered and the advantages and limitations of molec­ular genetics in lymphoma diagnosis are dis­cussed. IG and TCR gene rearrangement Mechanisnzs and methods The only known rearranging genes are lym­phocyte receptor genes for antigen, coding for either IG or TCR chains. Rearrangement involves random assembly of different vari­able (V), sometimes diversity (D), and joining CT) gene segments, which are discontinuously spread out within a chromosomal location in germ line configuration.5 The recombinase activity is at least in part initiated by prod­ucts of two recombinase activating genes, RAGl and RAG2. Expression of these genes strictly correlates with V(D)J recombinase activity. Their transcripts occur in pre-B and pre-T cells, and are re-expressed during B celi selection in germinal centers where they may be involved in V gene replacement of 6 rearranged IG genes.A set of conserved nucleotides, heptamers and nonamers flanks the germline V, D, and J segments. They function as recombination signal sequences for V-D, D-J, or V-J joining, and are recog­nized by a recombinase enzyme system. These signal sequences are separated by a nonconserved spacer. The spacer situated 3' of the V or D gene segment is 21-23 bp long (about two turns of the DNA helix). It is 11­12 bp long (about one turn of the double helix) when located 5' of the D or J gene seg­ment. Flanking sequences with a one-turn spacer signal can only rearrange to a two­turn signal. This probably ensures joining of l'CR i11 ly111p'10111a ding11osis appropriate gene segments. Thus, one V and one J can recombine, but more than one D segment can join. TCR vp and JP heptamers, however, are virtually indistinguishable from those found next to immunoglobulin genes suggesting that the recognition devices for IG and TCR gene rearrangements are very simi­lar. This explains the observation that T cells have occasionally D-J rearrangements of the IG heavy chain genes. Most commonly, the coding joint stays in the chromosome and a circular DNA molecule containing the signal joint and intervening sequences is excised. Intervening DNA stretches are retained if the two segments are joined in an opposite tran­scriptional orientation (conversion). At the coding ends, the joining is commonly impre­cise. This happens through differential trim­ming of recombining gene termini by exonu­cleases and through duplication of one or two nucleotides at the recombination cleav­age sites (P-nucleotides). Introduction of up to 15 nucleotides between V-D, D-D, D-J or V-J junctions in every possible random sequence generates non-template (N-) diver­sity. The enzyme terminal desoxynucleotidyl transferase (TdT) probably mediates addition of these nucleotides. N diversity contributes most significantly to the variability of the immune receptors, but it may also result in the generation of stop codons at the coding junctions.7 In B cells, affinity maturation of the IG receptor to antigen in germinal centers is managed by extensive base substitutions in the rearranged V segments. Most of these mutations accumulate in the three comple­ment-determining regions (CDRs). This process of somatic hypermutation apparently does not occur in TCR genes. Rearrange­ments involve different TCR and IG chains at different stages of lymphocyte ontogeny as defined by immunophenotyping. The first step in B cell development involves the D to J joining of the IG heavy chain genes (incom­plete rearrangement) which precedes the V to DJ joining (complete rearrangement) forming a functional V region gene on one allele of the pre-B cell with cytoplasmic IGµ expres­sion. Later during B cell ontogeny, the IG light chain (IGL) genes rearrange. lmmature B cells, expressing either µk or µ1c on the sur­face, result from the successful completion of heavy and light chain gene rearrangements. Similarly, a stepwise rearrangement of the immune receptor genes is found in cells com­rnitted to T lineage. Ninety to 95% of mature T cells carry the TCRap receptor on their sur­face, the remainder having TCRyo chain het­erodimers. Studies of precursor T-cell leu­kemia suggest that the TCRo genes rearrange before the TCRy genes. It is known that an incomplete DJ joining of the TCRP chains is the next event and that a complete rearrange­ment of this gene locus follows. Finally, the TCRa chains rearrange. T cells expressing the TCRyo receptors may not have rearranged their TCRa chain genes on both alleles, since the TCRd locus is nested between the Va and the Ja segments on chromosome 14q11, and rearrangement of these segments would result in a deletion of the TCRo locus. As a result of the maturation of lympho­cyte progenitor cells, individual T cells with uniquely rearranged TCR genes and individ­ual B cells with uniquely rearranged IG genes arise. Antigenic stimulation generates a poly­clonal or oligoclonal lymphoproliferation under control of the immune system. Clonal populations will emerge if immune surveil­lance fails to control the lymphoproliferation. Clonality indicates autonomous growth of tumor cells which is an important diagnostic criterion for malignant lymphomas. TCR and IG gene rearrangement studies are therefore extremely valuable in the diagnosis of lym­phoproliferative disease. PCR primers for IG and TCR genes PCR amplification requires less than 1 µg of template DNA. Degradation of the DNA as 174 Griesser H present in formalin-fixed, paraffin-embedded material, stained sections or archival cytology smears does not seriously affect the reaction as long as the cell morphology is sufficiently preserved. Therefore, even small areas of interest in a stained tissue section can be 8 scraped off and used as DNA source.Rearranged IGH genes of single lymphoid cells from sections of fresh-frozen or fixed tissue specimens have been successfully amplified with V gene family-specific and consensus J regi on primers. 9 Prolonged over a week long with formalin or with BS general­ly results in a less reliable amplification when compared to shorter formalin fixation times. Certain fixatives, such as Bouin's solution, or decalcification of tissue with ethylenedi­aminetetraacetic acid (EDTA) results in extensive DNA degradation so that, at best, only very short sequences can be amplified. Visualization of PCR amplification products on a high percentage agarose or a polyacry­lamide gel normally identifies a clonal popu­lation, and rarely is Southern blat analysis or sequencing of the gene products necessary for routine applications. Because of its speed and simplicity, the PCR analysis is given pri­ority for the diagnostic assistance in a diffi­cult case. The rate of false-negative results is, however, higher than in Southern blat stud­ies, so that the latter technique is still valu­able in the case of unsatisfactory PCR results. The CDR3 region of the VH gene seg­ments, formed by the D gene segment and the V-D as well as the D-J junctions with the variable nucleotide deletions/insertions, can be amplified by PCR with primers that bind to consensus gene regions in IGH V frame­work regions (FRl, 2 or 3) and FR4 in the J regions.10 Only complete VDJ rearrangements with the V and J sequences in the right orien­tation are amplified. Clonal incomplete DJ rearrangements, readily detectable by South­ern blat using appropriate J and C region gene probes, cannot be amplified by PCR since the V regions are separated from the J segments by intervening sequences which are too long to be amplifiable. The use of other primers residing in the FR2 region is helpful in those cases where the FR3 primers fail to bind. V gene family-specific primers derived from the FRl region are used in separate PCR reactions or combined in one so-called multiplex PCR, but they require a template DNA of reason­ably good quality to allow for the longer PCR products (about 350 bp as compared to 120 bp when CDR3 region primers are used). Similar to Southern Blat analyses, a back­ground smear of amplified DNA is detected when polyclonal B cells are analyzed, since the length of the amplified fragment varies between individual B cells due to the different D gene sequences and the diversity of the flanking N regions. PCR products of one or two predominant sizes separated from the polyclon­al background smear by gel electrophoresis are evidence of a clonal B cell population. This implies that the sensitivity of the (quantitative) PCR approach to recognize clonal immune receptor rearrangements at least matches the sensitivity of Southern Blat analyses. TCR rearrangements are detected by an approach based on the same principles as the IGH gene analysis.11 Mostly TCRy gene rearrangements have been studied in T cell lymphomas. Even though it is impossible to find consensus primers for all the different V and J regions, the limited repertoire of these genes allows the use of primer mixes in a multiplex PCR which identifies most rearrangements of this gene. Given the lack of D region genes in the TCRy locus, the size variability of the amplified V gene segments is based on the imprecise V-J joining only and therefore considerably lower than in IGH V segments. Another strategy is to use consen­sus primers and differentially cut the amplifi­cation products with restriction enzymes or determine the differences of the individual PCR products by gradient gel electrophoresis or single-strand conformational polymor­phism (SSCP) analyses. PCR i11 ly111pho111a diag11osis Primer sequences have also been estab­lished for the amplification of TCR. genes. Severa! primers have been developed which recognize a set of different V, D and J region genes. Even though a substantial number of cases with clonal TCR. gene rearrangements are missed, this methods seems to be useful as an additional test for clonally expanded T cells expressing the a. heterodimer. TCRo PCR has widely been used for the detection of clone-specific rearrangements in acute lymphoblastic leukemia. Primers established from the unique sequences of the VDJ or VDDJ junctions of leukemia blasts are clone­specific. 12 With such a clone-specific, quanti­tative PCR approach a sensitivity leve! of one in 106 cells can be reached which is well suit­ed for minimal residual disease (MRD) detec­tion. Amplification of TCRo gene sequences has not been performed in a large series of lymphoma cases since the TCRo genes are frequently deleted in mature T celi neo­plasms. Consensus Va and Ja region primers for genomic amplification of TCRa gene recom­binations are not used. Reverse transcribed cellular RNA can be amplified using C region oligonucleotides and degenerate 5' end primers for the cDNAs in a so-called RT­PCR.13 Cloning and sequencing of the ampli­fication products or separation of the frag­ments on a gradient gel or by SSCP analysis allows the detection of a predominant, clonal rearrangement. The PCR procedures for TCRa gene rearrangement detection are hence more sophisticated than TCRy/o PCR, require celi suspensions or fresh/fresh-frozen tissue and may not be widely applicable for routine diagnostic purposes. PCR detection of IGH and TCR rearrange­ments in lymphoproliferative disease The sensitivity and specificity of the PCR approach for the detection of T and B celi clonality has been tested extensively in the past. In severa! large series encompassing severa! hundred B celi non-Hodgkin's lym­phoma (NHL) specimens 70-80% of cases studied with IGH PCR revealed clonal ampli­fication products. Serial dilution experiments of clonal with polyclonal DNA suggest a clon­al detection limit of 1 % or lower. False posi­tive results seem to be exceedingly rare when cross-contamination is avoided and appropri­ate controls are run with the clinical samples. Even though the amount of target DNA need­ed for successful amplification is low, it is advisable to use DNA concentrations corre­sponding to more than a hundred B cells. This helps to avoid preferential primer bind­ing which may lead to a pseudoclonal ampli­fication result with the IGH primers.14 Immunohistochemical analysis of the speci­men which should always be done prior to molecular analysis, provides an estimate of the proportions of T and B lymphocyte popu­lations in the lymphoma tissue. Additional use of primers directed against the FR2 region of the IGH variable gene results in a higher detection rate of clonality (greater than 85%) in B-cell lymphoma cases than the employment of FR3-region primers only (about 75%). This is explained by a lack of primer binding due to deletions and exten­sive hypermutations occuring predominantly in the FR3 and less so in the FR2 region sequences. Alternatively, the consensus V region primers may not anneal to some rare or unknown VH genes participating in the IGH rearrangement. With FR3 primers, low­grade B-NHL with the exception of centrob­lastic/ centrocytic lymphomas and those of mucosa-associated lymphoid tissue (MALT) nearly always have detectable clonal PCR products. The detection rate in high-grade B celi lymphomas ranges from 75 to more than 80%. The percentage of positive cases tends to be lower among centroblastic lymphomas (60 -70%) but not in large celi anaplastic lym­phomas of B-type (B-LCAL) [Griesser, unpub­ 176 Griesser H lished]. Using both FR3/FR4 and FR2/FR4 primer sets we also detected clonal IGH rearrangements in 2 of 12 cases of nodular sclerosing and 4 of 8 mixed cellularity Hodgkin's disease (HD) in a recent study.15 Among 7 samples of nodular lymphocyte predominant HD, only one case with a high­grade B celi lymphoma component showed clonal IGH rearrangement. Detection of B­cell clonality thus helps to distinguish B-NHL from lymphocyte predominant HD and favors a B-NHL over nodular sclerosing HD, but it is certainly not a reliable criterion in the differential diagnosis of mixed cellularity HD and T-cell rich B-cell lymphoma (TCRBL). The finding of more than three clonal PCR products indicates that more than one B-cell done is present. Such biclonal or oligoclonal rearrangements are detectable in rare cases of follicular center cell-derived lymphomas and acute lymphoblastic leukemias (ALL).16 The majority of B-NHL, however, has only one predominant clonal band in routine PCR examinations detectable with simple size selection procedures on agarose or non-dena­turing polyacrylamide gels. Amplification of short PCR products with FR3 and FR2 primers is as effective with DNA extracted from well-preserved formalin-fixed tissues as with cellular DNA from fresh/fresh frozen samples. Even though clonality is detected in nearly ali B-cell lymphoma cases by Southern Blot but missed by PCR in 10-20% of cases, we have seen rare cases that were PCR-posi­tive and Southern Blot negative.16 Primers established from the unique sequences of the VDJ junctions of leukemic lymphoma cells are clone-specific. With such a allele-specific primers PCR detection of MRD reaches a sensitivity leve! of one in 106 cells.17 TCRy PCR findings have been reported for a few hundred cases of T-NHL. Clonality is found in more than 85% of the lymphoma samples with no false-positive results. A combined investigation of T celi neoplasms with primers for both, TCRy and TCR., may lead to even superior results. Our studies on 48 LCAL cases has shown that TCRy-PCR helps to identify T lineage in more than half of the tumor celi populations lacking surface expression of T celi markers in routinely­fixed samples.15, 18 Investigations of ALL sam­ples were mostly performed for MRD detec­tion. A positive result in bone marrow­derived DNA seems to have clinical relevance in predicting relapse. Detection of circulating tumor cells, however, appears clinically insignificant for lymphoma patients in other­wise complete remission.19 Biclonal or oligoclonal TCRg rearrange­ments are sometimes detected by PCR in angioimmunoblastic lymphadenopathy (AIL)­like T-cell lymphomas and rarely in cuta­neous T-cell lymphomas. A considerable pro­portion of CD3-, CD56+ natura! killer celi-like large granular lymphocyte (NK-LGL) leu­kemias/lymphomas Jack TCR rearrange­ments by PCR as well as Southern blot stud­ies. At least some cases from female patients can be studied for clonality with alternative PCR methods, such as analyses for methyla­tion patterns of the human androgen recep­tor genes.20 Unexpected results in rearrangement analyses by PCR Failure to detect clonal rearrangements can have technical reasons. A clonal celi popula­tion may not be well represented in the speci­men used for DNA extraction; the DNA may be severely degraded and not even suitable for PCR amplification; DNA preparations from routinely processed tissue may contain PCR inhibitors. In the latter two instances PCR amplification of ubiquitous genes will fail in control experiments. Some AIL-type T­NHL and stage I and II mycosis fungoides may not contain molecularly detectable T celi clones. The absence of a clonal gene rearr­angement in the presence of a histologic pic­ PCR in ly111p/10111a diagnosis ture typical for malignant lymphoma is diag­nostically irrelevant. Examinations can be repeated in subsequent biopsies or speci­mens taken from a different site if molecular genetical support of the diagnosis is crucial. Complete IGH rearrangements are a fea­ture of B-cells, complete TCR rearrangements highly characteristic of T-cells. Cross-lineage rearrangements detected in Southern blot experiments mostly represent incomplete erroneous immune receptor gene rearrange­ments or, more rarely, translocation events involving the IGH or TCR gene locus. The detection of clonal IGH rearrangements by PCR has an advantage over the Southern blot procedure since only complete VDJ rearr­angements will be amplified enzymatically, but not incomplete rearrangements or rearrangements resulting from chromosomal translocations involving the IGH gene locus. Incomplete cross-lineage IGH rearrangements in neoplastic T cells, which are detectable in Southern Blot analyses, will not be amplified by PCR which makes lineage assignment to the B cell series straightforward. However, complete TCR cross-lineage rearrangements may be detectable by PCR in cases of com­mon ALL and pre-B lymphoblastic lym­phoma. In addition to the IG genes, severa! TCR loci frequently undergo rearrangement and these illegitimate rearrangements may be complete. This renders genotypic lineage determination in such cases by Southern blot procedures or PCR unreliable. In AIL-type T cell lymphomas IGH rearr­angements are not infrequently detected by PCR and are likely to be due to the existence of a true B cell done coexisting with the neo­plastic T cell population. Recently we found some cases among TCRBL samples with clon­al IGH as well as TCRy rearrangement in PCR studies. Morphology and immunohisto­chemical results suggested that these cases were indeed composite lymphomas with a high-grade B-cell and a low-grade T-cell com­ponent. 21,22 The occurrence of clonal TCR rearrange­ments is diagnostically challenging in lym­phoproliferative T cell disorders which are clinically considered non-malignant. Activat­ed T cell clones especially in lymphoprolifera­tions of the skin may expand to a point where they become detectable by rearrange­ment studies but remain localized and con­trolled by the immune system. Clonal cuta­neous lymphoproliferations such as lym­phomatoid papulosis (LYP) lesions, however, may coincide with or transform into a malig­nant T cell lymphoma. A clonal TCR rearr­angement is of limited value for the differen­tial diagnosis between LYP and a cutaneous LCAL. T lymphocyte clones may also expand due to persistent antigen exposure. These lymphoproliferations are mainly oligoclonal but it was shown that single reactive cytotox­ic T celi clones may be detectable in healthy, elderly individuals. 23 Molecular genetical studies require a dedi­cated laboratory with sufficient volume of samples and significant test experience. Fail­ure to detect clonality, or the finding of unex­pected or illegitimate TCR rearrangements is rare under these circumstances. Diagnostic problems arise particularly when molecular results are not correlated with histological and immunophenotypical findings. Problems with unexpected results are mostly avoided when molecular studies in a routine laborato­ry setting are restricted to diagnostically chal­lenging cases and the search for minimal dis­ease. Common chromosomal translocations in malignant lymphomas Different from stepwise rearrangement processes during B celi ontogeny, chromoso­mal translocations involving bcl-2, bcl-1, bcl­6 and c-myc can be viewed as an accident during B cell development and activation. They most frequently involve the IGH gene 178 Griesser H locus on chromosome 14q32 and sometimes the IGL gene loci. In the majority of cases involving bcl-1 and bcl-2, translocations can be pinpointed to an early stage of B cell dif­ferentiation before the heavy chain genes have completed their rearrangement. Translo­cations of the bcl-6 gene can involve a num­ber of chromosomes besides those harboring immune receptor genes. These chromosomal abnormalities not only serve as clonal mark­ers but also aid in the classification of lym­phoma subtypes.24 Bcl-2/JH recombination is highly characteristic of germinal center cell derived lymphomas and bcl-1/JH recombina­tion is mainly detected in mantle zone cell­derived lymphomas. The t(11;14) is not observed together with the t(14;18) transloca­tion and bcl-2 rearrangements are unde­tectable in mantle cell lymphomas. The find­ing of one of these translocations is therefore useful in the differential diagnosis of follicu­lar lymphomas and mantle cell lymphomas with a nodular growth pattern. Bcl-1 rearr­angement analyses also aid in the identifica­tion of blastic variants of mantle cell lym­phoma. 25 Among the high-grade NHL, c-myc rearrangements are a constant feature in Burkitt's lymphoma and bcl-6 translocation is frequently detected in centroblastic lym­phomas. The only common and consistent translocation in T-lineage lymphomas results in the NPM/alk fusion which is almost exclu­sively found in T-LCAL. Molecular probes are being generated through isolation and sequence analysis of regions flanking the chromosomal break­points. These probes are useful for the detec­tion of chromosomal translocations in South­ern blot analyses if the breakpoints are clus­tered and not spread out over a large chromo­somal region. After identification and cloning of specific chromosomal breaks their sequences can be analyzed and tumor-specif­ic PCR primers flanking the breakpoint be designed. This PCR approach is highly suit­able for molecular follow-up studies in a par­ticular patient since amplification products are only generated from cellular DNA carry­ing this translocation. Fusion gene tran­scripts from reciprocal chromosomal translo­cations are detectable by PCR amplification of cDNA (RT-PCR). Mechanisms and methods The bcl-2 oncogene is located on chromo­some 18q21. The breakpoints on chromo­some 18 in the t(14;18) translocation, which is very characteristic of germinal center cell derived lymphomas irrespective of the growth pattern and blast cell content, are clustered around two regions.26, 27 In about 70% of cases the breakpoint occurs in the major breakpoint region (MBR) which is located in the 3' untranslated region of bcl-2 exon III. In most of the remaining cases it occurs in the minor cluster region (mer) locat­ed more than 20 kb 3' of the mbr. The break­point on chromosome 14q32 is found within the JH gene cluster. Translocation of the bcl-2 gene is found rarely in chronic B-lymphocytic leukemia involving predominantly IGL gene loci rather than the IGH gene locus; the breaks on chromosome 18 tend to be outside and usually S' of the mbr/mcr. The bcl-2a and bcl-2. proteins are involved in cell death regulation and have been shown to block apoptosis. Bcl-2 overexpression as a result of the translocation t(14;18) may lead to inade­quate survival of B cells and render them sus­ceptible to additional genetic aberrations. The bcl-1 locus resides on chromosome 11q13 and is involved in the translocation t(11;14). The chromosome 11 sequences join within the J region cluster of the IGH gene locus on chromosome 14q32. Again, one pre­dominant breakpoint region is described (major translocation cluster, mtc) on chromo­some 11.28 At least two more sites have been identified where breakpoints occur within chromosome 11q13. Aside from occasional PCR in ly111p/10111a diagnosis cases of B-CLL and multiple myelomas this translocation is characteristic of mantle zone derived B-cell lymphomas where it is found in about 50% of the mantle celi lymphoma cases with mtc probes. With the additional use of probes for the minor breakpoint clus­ter regions the percentage may be even high­er than 70%. In contrast to the bcl-2 onco­gene, the bcl-1 locus does not harbor an oncogene but likely is linked to a regulatory gene sequence of PRAD1, which is telomeric to the breakpoint region. The translocation leads to constitutive expression of the gene product cyclin Dl which promotes passage through the Gl phase of the celi cycle. Translocations involving the c-myc locus on chromosome 8q24 are somewhat more complex.29 They are a constant finding in sporadic and endemic Burkitt' lymphoma cases and most frequently involve the IGH genes in a t(8;14). Rarely the IGK genes on chromosome 2p12 or IGA genes on chromo­some 22q11 can also be translocated to the myc locus. The breakpoint on chromosome 8 in the t(8;14) generally lies 5' or within the c­myc gene, whereas in variant translocations t(2;8) or t(8;22) involving IGL chain loci the break occurs 3' of the c-myc gene at distances up to 300 kb. Translocations t(8;14) are char­acteristic of Burkitt's lymphoma. Two differ­ent breakpoints occur depending on the type of Burkitt's lymphoma. In most endemic cases (eBL), the break occurs more than 20 kb upstream of the myc locus and involves either the JH or the switch m region on chro­mosome 14. In most sporadic types (sBL) exon I of the c-myc gene or 5' flanking sequences are involved, and more often the switch m or switch g regions than the J region take part on chromosome 14. Translo­cation into the JH or JL region suggest that the translocation occurs at a pre-B celi stage whereas translocation into the IG switch sequences potentially take place throughout the B celi differentiation process. Expression of c-myc is high in proliferating cells and rapidely induced in quiescent cells on mito­genic stimuli. In addition to mediating celi proliferation, c-myc is also implicated in blocking the cellular programs of differentia­tion. Highly proliferating cells with a differ­entiation block are prone to apoptosis. Translocation of c-myc gene generally results in constitutive expression of this otherwise tightly regulated oncogene. Besides translo­cations, mutations in certain cluster regions of the c-myc gene have a similar effect and are detected in more than 50% of Burkitt's lymphomas.30 Chromosomal alterations affecting the bcl­6 gene at band 3q27 are a frequent recurrent abnormality in high-grade B-cell lympho­mas. 31 Severa! partner chromosomes are involved including the sites of the IGH genes at 14q32, the IGk genes at 2p11 and the IGA genes at 22q11. Bcl-6 seems to function as a transcription factor that binds specific DNA sequences and represses transcription from linked promoters.32 High Bcl-6 expression is restricted to mature B-cells inside the germi­nal centers and has been shown to be a key regulator of germinal center formation and B­cell immune response. Chromosomal translo­cations usually disrupt the gene in and around the first exon leaving the coding domain intact. On the partner chromosome the bcl-6 coding domain is juxtaposed to a heterologous promoter resulting in a new chimeric transcript which encodes a normal bcl-6 protein. The promoter substitution pre­vents bcl-6 downregulation and blocks post­germinal center maturation of B-cells. Bcl-6 rearrangements are highly specific for high­grade B-NHL of centroblastic type where they are detected in 35% of cases. Additional­ly they are found in a small fraction of follicu­lar lymphomas. Additionally, in about 70% of the high-grade B-NHL and 50% of follicular lymphomas the bcl-6 gene is affected by mul­tiple, often biallelic mutations clustering within the 5' non-coding sequences.33 It has been suggested that those large celi lym­ 180 Griesser H phomas only with bcl-6 rearrangement may represent de-novo high-grade B-NHL where­as those with bcl-6 and/or bcl-2 alterations may be secondary high-grade lymphomas with a less favorable prognosis.34, 35 Chromosomal translocations may result in a novel fusion transcript that can be detected by RT-PCR. This is the case in LCALs with the translocation t(2;5) (p23; q35).36 The nucleolar phosphoprotein nucleophosmin (NPM) gene on chromosome 5q35 is translocated to the anaplastic lymphoma kinase (alk) on chromo­some 2p23 in about 60% of adult and over 80% of childhood LCAL of T-or 0-type.37 The result is a chimeric protein with the amino terminus of the gene coding for NPM and the carboxy terminus coding for alk. Replacement of the 5' alk sequences switches the transcrip­tion regulation of the catalytic sequences of alk to the NPM promoter. Regulatory sequences of the NPM gene, which is tran­scriptionally most active before the cell entry into the S phase, may activate the alk gene. This kinase gene is physiologically not tran­scribed in lymphocytes. The fusion protein of the two genes could potentially phosphory­late intracellular substrates which are normal­ly under control of lymphoid lineage-specific kinases only. This deregulation eventually triggers the malignant transformation of T-lin­eage cells. PCR detection of chromosomal transloca tions The molecular detection of these abnormali­ties is most reliably done by conventional Southern blot technique that identifies abnormal restriction fragment gel mobilities. The use for routine analysis, however, is lim­ited since the variability of the breakpoints requires the application of severa! probes and DNA restriction with severa! enzymes. This also explains why rearrangements are less frequently detected by PCR, which is per­formed with a limited set of primers (since not all the sequence data for the exact break­point locations are available) and can only amplify short DNA stretches in routine appli­cations. On the other hand, once a transloca­tion is detected by the PCR approach in indi­vidual patients, it is far more sensitive as a clonal marker than the Southern Blot proce­dure. Translocations t(14;18) involving the IgH gene locus and the bcl-2 gene region are detected by PCR with consensus primers for the JH region of the mbr or the mer region.3840 Sensitivity and specificity are enhanced if sets of outer and inner primers are sequen­tially applied in the so-called nested PCR reaction. Alternatively, Southern blotting of PCR products with subsequent probing can be done using a radioactive-labeled interna! oligonucleotide. Chromosomal rearrangements of the bcl-1 gene locus have been amplified by PCR using mtc breakpoint oligonucleotides.41A2 This approach is not reliable enough for routine diagnostic purposes since various minor breakpoint sites are also involved in mantle celi lymphomas. Designing, testing and using many different primer sets for these addition­al breakpoint locations is impracticable in a routine laboratory setting. A limited number of cell lines and some Burkitt's lymphoma samples have been ana­lyzed by PCR using primers for the switch region of the µ heavy chain gene, and sequences flanking and inside exon I of c­myc. With this approach it is possible to detect some of the translocations t(8;14) in sBL cases.43 With new generations of Taq polymerases it has become possible to ampli­fy sequences of more than 10 kb length. Using primers for IG constant region genes and primers flanking sequences 5' to break­point cluster regions it is possible to amplify a considerable proportion of those c-myc and bcl-6 translocations by long-distance (LD-) PCR that were formerly only detectable by PCR i11 lyrnp/10111a diag11osis Southern blot.44 However, template DNA has to be extracted from fresh/fresh-frozen tissue samples or cell suspensions and routinely­processed specimens cannot be analyzed. The presence of chimeric mRNA from the NPM/alk fusion is detected by RT-PCR.45 Even though RNA extracted from routinely­processed specimens is strongly degraded reverse transcribed cDNA fragments of 300 bp are readily amplifiable. This leads to the detection of fusion transcripts in 40-60% of T­LCAL cases by RT-PCR. Advantages and drawbacks of translocation analyses by PCR Most of the experience with the detection of translocations by PCR in malignant lym­phoma has been accumulated for the t(14;18) abnormality. About 80% of the Southern blot­positive follicular lymphomas are PCR posi­tive suggesting that PCR analysis for bcl-2/IGH translocation is a good molecular marker for the t(14;18) abnormality in follicu­lar lymphomas. The PCR technique is more sensitive in the detection of qualitative rather than quantitative abnormalities such as clon­al immune receptor gene rearrangement on a background of polyclonal cells. It is therefore well suited for analysis of minimal disease in lymphomas carrying the t(14;18). Reports about positive PCR results in follicular hyper­plasia should be considered when looking for MRD.46A7 The (overly) high sensitivity may be achieved by using high amounts of tem­plate DNA (> lµg) in the reaction and very efficient primers and cycling conditions. Sim­ilarly, the high degree of sensitivity may also be responsible for some reports about sur­prisingly frequent detection of t(14;18) PCR products in Hodgkin's lymphomas. False­positive PCR results have to be avoided in MRD detection. DNA from the original tumor should be run in parallel when looking for minimal infiltration. Identity of the ampli­ fication products is confirmed by identical size on polyacrylamide gels and/or by sequencing. A quantitative PCR approach would also help to establish the relative num­ber of cells in the sample which contain the rearrangement, but this approach usually goes beyond routine analysis. It is even better not only to detect chromo­somal abnormalities with high sensitivity but also to identify the cells carrying the genetic abnormality. This is possible using another fast molecular diagnostic approach: the detection of translocations by non-radioac­tive, usually fluorescence-based, in-situ hybridization of chromosomes (FISH). The probes detect chromosomal DNA either from metaphase spreads or non-mitotic cells.48 The latter so-called interphase cytogenetic analysis can be done even on previously stained cells. Simultaneous immunopheno­typing of the cells helps to focus the interpre­tation of the results on the tumor celi popula­tion. 49 Genomic DNA probes flanking the breakpoint on both sides and derived from different chromosomes are labelled with two different fluorochromes.50 The two signals would be located on different chromosomes in a metaphase spread or found far apart in interphase cells if the translocation is absent. Joining of genetic material from the two chro­mosomal localizations, in contrast, leads to a doublet fluorescence signal after hybridiza­tion. Results from FISH analysis are usually available within two days, which favourably compares to the Southern blot procedures. When more translocation-specific probes become available, molecular cytogenetics promises to be a fast and simple technique for the detection of nonrandom chromosomal abnormalities. Though less sensitive than PCR detection, FISH is potentially of higher specificity in tracing single tumor cells carry­ing a specific anomaly. The hybridization effi­ciency of FISH probes in sections from archival material depends on the fixative and the age of the paraffin blocks. A negative 182 Griesser H result in these instances is diagnostically not helpful. Conclusions Molecular genetic analysis has become rou­tine for laboratories involved in lymphoma diagnosis. If precautions against sample cont­amination are taken, the PCR technique gen­erates reliable results within one or two days when applied to routinely processed biopsy samples and cytology specimens containing only a hundred cells. Standard PCR proce­dures include assessment of lymphocyte clonality using primers for TCRy and IGH genes as well as the search for a t(14;18) in B celi neoplasms of putative follicular center celi origin. If the results are unsatisfactory, additional primer combinations may be used and/or fresh/fresh-frozen tissue has to be used for DNA studies with Southern Blot techniques. Since no primer sequences suit­able for PCR amplification of TCRa and IGL rearrangements are available, these tests are performed by Southern blotting. This is also the method of choice for an in-depth analysis of chromosomal translocations involving the c-myc, bcl-1, bcl-6 or bcl-2 loci, for example in the setting of prospective clinical studies. RT­PCR is successfully applied for the detection of t(2;5) translocations in LCALs because of the fusion transcripts generated by this abnormality. Limitations of the molecular techniques are well recognized and can be handled in an experienced laboratory. The diagnostic process should begin with the cytomorphological evaluation of the speci­men. Even if microscopic examination does not lead to a conclusive diagnosis, it provides a hypothesis for immunohistochemical and, ultimately, molecular genetic testing. Molecu­lar test results have to be viewed in the con­text of morphology and immunohistochem­istry. The detection of a clonal lymphocyte population in clinical samples is always abnormal but it should never be considered a proof of malignancy. Similarly, genomic instability may result from abnormal lympho­cyte activation or destabilizing influences upon chromosomal DNA leading to translo­cations. Some of these translocations seem to be necessary but are not sufficient for lym­phomagenesis. Thus, the detection of translo­cations in rare cells by a highly sensitive PCR procedure should not be taken as evidence of malignant lymphoma out of the cytomorpho­logical context. On the other hand, a PCR result can be negative for clonality for many reasons besides the absence of a clonal lym­phocyte population, such as lack of primer binding, suboptimal PCR conditions or prob­lems related to the extraction of suitable tem­plate DNA or RNA. Keeping the limitations in mind, immune receptor gene rearrangement studies are well suited to define lineage and clonality of a lymphoproliferation. Analyses of chromoso­mal translocations, in addition, provide infor­mation about the lymphoma subtype, its bio­logic behavior and mechanisms of lym­phomagenesis. Furthermore, the high sensi­tivity and specificity of the PCR-based molec­ular tests are most helpful for monitoring lymphoma therapy, identification of MRD and early diagnosis of relapse. In an optimal laboratory setting, cytomorphologic, immunophenotypic and molecular results are interpreted jointly by individuals with expertise in ali three methods in order to achieve the best diagnosis possible for the patient. PCR in lymphoma diagnosis References l. Griesser H, Feller AC, Lennert K, Minden MD, Mak TW. Rearrangement of the beta chain of the T-cell receptor and immunoglobulin genes in lym­phoproliferative disorders. J Ciin Invest 1986; 78: 1179-84. 2. Kagan J. Molecular biology of chromosomal aber­rations in leukemia/lymphoma. Hematol Pathol 1993; 7: 159-201. 3. Griesser H, Tkachuk D, Reis MD, Mak TW. Gene rearrangements and translocations in lymphopro­liferative diseases. Blood 1989; 73: 1402-15. 4. Saiki RK, Gelfand OH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer-directed enzy­matic amplification of DNA with thermostable DNA polymerase. Science 1988; 239: 487-91. 5. Griesser H, Mak TW. Developmental and func­tional biology of B and T lymphocytes. In: Canel­los GP, Lister TA, Sklar JL, eds. The Lymphomas. Philadelphia: W.B.Saunders Company; 1998, p. 19-42. 6. Schatz DG, Oettinger MA, Schlissel MS. V(D)J recombination: molecular biology and regulation. Annu Rev Immwwl 1992; 10: 359-83. 7. Alt FW, Oltz EM, Young F, Gorman J, Taccioli G, Chen J. VDJ recombination. Immunol Today 1992; 13: 306-14. 8. Zhuang Z, Bertheau P, Emmert-Buck MR, Liotta LA, Gnarra J, Linehan WM, Lubensky IA. A microdissection technique for archival DNA analysis of specific celi populations in lesions <1 mm in size. Am J Patlwl 1995; 146: 620-5. 9. Kiippers R, Rajewski K, Zhao M, Simons G, Lau­mann R, et al. Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sec­tions show clonal immunoglobulin rearrange­ments and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci USA 1994; 91: 10962-66. 10. Diaz-Cano S. PCR-based alternative for diagnosis of immunoglobulin heavy chain gene rearrange­ment. Diagn Mol Pathol 1996; 5: 3-9. 11. Griesser H. Gene rearrangements and chromoso­mal translocations in T celi lymphoma -Diagnos­tic applications and their limits. Virchows Arch 1995; 426: 323-38. 12. Hansen-Hagge TE, Yokota S, Bartram CR. Detec­tion of minimal residual disease in acute lym­phoblastic leukemia by in vitro amplification of rearranged T-cell receptor l\ chain sequences. Blood 1989; 74: 1762-7. 13. Broeren CPM, Verjans GMGM, van Eden W, Kusters JG, Lenstra JA, Logtenberg T. Conserved nucleotide sequences at the 5' end of T celi recep­tor variable genes facilitate polymerase chain reac­tion amplification. Eur J Immunol 1991; 21: 569-75. 14. Griesser H. Applied molecular genetics in the diagnosis of malignant non-Hodgkin's lymphoma. Diagn Mol Pathol 1993; 2: 177-91. 15. Griesser H. Presented at the Tutorial on the use of new techniques in diagnosis of malignant lym­phomas. Ljubljana, Slovenija: February 2-4,1998. 16. Heinemeier C. Rearrangementuntersuchungen der Immunglobulinschwerketten-Gene mit der Polymerase-Ketten-Reaktion -Grenzen und Moglichkeiten als diagnostisches Hilfsmittel. Doc­toral Thesis. Kiel: University of Kiel, 1996. 17. Billadeau D, Blackstadt M, Greipp P, Kyle RA, Oken MM, et al. Analysis of B-lymphoid malig­nancies using allele-specific polymerase chain reaction: a technique for sequential quantitation of residual disease. Blood 1991; 78: 3021-29. 18. Griesser H, Henry M, Boie C, Banerjee D. Large celi anaplastic lymphoma of the gastrointestinal tract -An immuno-and genotypic study on archival material. Hematol Pathol 1994; 8: 121-34. 19. Gribben JG, Neuberg D, Barber M, Moore J, Pesek KW, et al. Detection of residual lymphoma cells by polymerase chain reaction in peripheral blood is significantly less predictive for relapse than detection in bone marrow. Blood 1994; 83: 3600-7. 20. Mashal RD, Lester SC, Sklar J. Clonal analysis by study of X chromosome inactivation in formalin­fixed paraffin-embedded tissue. Cancer Res 1993; 53: 4676-9. 21. Bierwolf S. Zytomorphologische, immunhisto­chemische und molekulargenetische Unter­suchungen an T-Zell-reichen B-Zell-Lymphomen und Composite Lymphomen. Doctoral Thesis. Kiel: University of Kiel, 1997. 22. Lennert K. Borderlands of pathological entities. In: Magrath IT, editor. The 11011-Hodgkin's lym­phomas. London: Arnold; 1997, p. 133-67. 23. Posnett DN, Sinha R, Kabak S, Russo C. Clonal populations of T cells in normal elderly humans: the T celi equivalent to "benign monoclonal gam­mopathy". J Exp Med 1994; 179: 609-18. 184 Griesser H 24. Kluin PhM, Vaandrager JW, van Krieken JHJM, Schuuring E. Chromosomal markers in lymphoma diagnosis. Curr Diagn Pathol 1996; 3: 187-99. 25. Grosso LE, Kelley PD. Bcl-1 translocations are fre­quent in the paraimmunoblastic variant of small lymphocytic lymphoma. Modern Pathol 1998; 11: 6-10. 26. Kneba M, Eick S, Herbst H, Willigerath S, Pott C, Bolz I, et al. Frequency and structure of t(14;18) major breakpoint regions in non-Hodgkin's lym­phomas typed according to the Kiel classification: analysis by direct DNA sequencing. Cancer Res 1991; 51: 3243-50. 27. Ngan B-Y, Nourse J, Cleary ML. Detection of chro­mosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reac­tion and direct genomic sequencing of the enzy­matically amplified DNA in follicular lymphomas. Blood 1989; 73: 1759-62. 28. Williams ME, Meeker TC, Swerdlow SH. Rearr­angement of the chrornosome 11 bcl-1 locus in centrocytic lymphoma: Analysis with multiple breakpoint probes. Blood 1991; 78: 493-8. 29. McKeithan TW. Molecular biology of non-Hodg­kin's lymphomas. Semin Oncol 1990; 17: 30-42. 30. Shiramizu B, Barriga F, Neequaye J, Jafri A, Dalla­Favera R, et al. Patterns of chromosornal break­point locations in Burkitt's lymphoma: relevance to geography and Epstein-Barr virus association. Blood 1991; 77: 1516-26. 31. Dalla-Favera R, Ye BH, Cattoretti G, Lo-Coco F, Chang CC, Zhang J, et al. BCL-6 in diffuse large­cell lymphomas. Important Adv Oncol 1996: 139­48. 32. Chen W, Iida S, Louie DC, Dalla-Favera R, Cha­ganti RSK. Heterologous promoters fused to BCL6 by chromosomal translocations affecting band 3q27 cause its deregulated expression during B­cell differentiation. Blood 1998; 91: 603-7. 33. Migliazza A, Martinotti S, Chen W, Fusco C, Ye BH, Knowles DM, et al. Frequent somatic hyper­mutation in the 5' noncoding region of the BCL-6 gene in B-cell lymphorna. Proc Natl Acad Sci USA 1995; 92: 12520-4. 34. Offit K, Lo Coco F, Douie DC, Parsa NZ, Leung D, Portlock C, et al. Rearrangement of the BCL-6 gene as a prognostic marker in diffuse large-cell lyrnphoma. New Engl J Med 1994; 331: 74-80. 35. Muramatsu M, Akasaka T, Kadowaki N, Ohno H, Yamabe H, Edamura S, et al. Rearrangement of the BCL6 gene in B-cell lymphoid neoplasms: compari­ son with lymphomas associated with BCL2 rearrangement. Br J Haematol 1996; 93: 911-20. 36. Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro ON, Saltrnan DL, Look AT. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 1994; 263: 1281-4. 37. Lamant L, Meggetto F, Al-Saati T, Brugieres L, de Pailleretes BB, Dastugue N, et al. High incidence of the t(2;5)(p23;q35) translocation in anaplastic large celi lyrnphoma and its Jack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction and p80 immunostaining. Blood 1996; 87: 284-91. 38. Ngan B-Y, Nourse J, Cleary ML. Detection of chro­mosomal translocation t(14;18) within the minor cluster region of bcl-2 by polyrnerase chain rection and direct genomic sequencing of the enzymati­cally arnplified DNA in follicular lymphomas. Blood 1989; 73: 1759-62. 39. Ladanyi M, Wang S. Detection of rearrangernents of the bcl2 major breakpoint region in follicular lyrnphomas. Diagn Mol Pathol 1992; 1: 31-5. 40. Poteat HT, Sklar J. A simplified polymerase chain reaction assay for detection of chromosomal translocations in hematologic rnalignancies. Diagn Mol Pathol 1997; 6: 3-9. 41. Rimokh R, Berger F, Delsol G, Digonnet I, Rouault JP, Tigaud JO. Detection of the chromosornal translocation t(11;14) by polyrnerase chain reac­tion in mantle celi lymphoma. Blood 1994; 83: 1871-5. 42. Lim L-C, Segal GH, Wittwer CT. Detection of bcl-1 gene rearrangement and B-cell clonlity in mantle-cell lymphoma using formalin-fixed, paraffin-ernbedded tissues. Am J Ciin Pathol 1995; 104: 689-95. 43. Shiramizu B, Magrath I. Localization of break­points by polymerase chain reactions in Burkitt's lymphoma with 8;14 translocations. Blood 1990; 75: 1848-52. 44. Akasaka T, Muramatsu M, Ohno H, Miura I, Tata­sumi E, Fukuhara S, et al. Application of long-dis­tance polymerase chain reaction to detection of junctional sequences created by chrornosomal translocation in mature B-cell neoplasrns. Blood 1996; 88: 985-94. 45. Downing JR, Shurtleff SA, Zielenska M, Curcio­Brint AM, Behm FG, Head DR, et al. Molecular detection of the (2;5) translocation in non­Hodgkin's lyrnphorna by reverse transcriptase­polyrnerase chain reaction. Blood 1995; 85: 3416-22. PCR in ly111phoma diagnosis 46. Limpens J, de Jong D, van Krieken JHJM, Price CGA, Young BO, van Ommen GJB, et al. Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia. 011cogene 1991; 6: 2271-6. 47. Aster JC, Kobayashi Y, Shiota M, Mori S, Sklar J. Detection of the t(14;18) at similar frequencies in hyperplastic lymphoid tissues from American and Japanese patients. A111 J Pathol 1992; 141: 291-9. 48. Taniwaki M, Ueda Y, Nishida K, Takashima T, Kashima K, Matsuda F, et al. Rapid detection of lymphoma-specific translocations in interphase nuclei of non-Hodgkin's lymphoma by fluores­cence in situ hybridization. Leukemia 1997; 11 Suppl 3: 291 -3. 49. Schlegelberger B, Weber-Matthiesen K, Himmler A, Bartels H, Sonnen R, Kuse R, et al. Cytogenetic findings and results of combined immunopheno­typing and karyotyping in Hodgkin's disease. Leukemia 1993; 8: 72-80. 50. Mathew P, Sanger WG, Weisenburger DO, Valen­tine M, Valentine V, Pickering D, et al. Detection of the t(2;5)(p23;q35) and NPM-ALK fusion in non­Hodgkin's lymphoma by two-color fluorescence in situ hybridization. Blood 1997; 89: 1678-85. Radio/ 011col 1998; 32(2): 187-91. In vivo electroporation of the urinary bladder in mice Peter Veranic1 , Kristijan Jezernik1 , Maja Cemažar2, and Gregor Serša2 1 Institute of Celi Biology, Medica/ Faculty, University of Ljubljana, 2Department of Tumor Biology, Institute of Oncology, Ljubljana, 5/ovenia Celi membrane is the major obstac/e to incorporation of different substances into cells. Especially in the urothclium of the 111a111111alian urinary bladde1; plasma membrane of supe1ficia/ cel/s acts as a strong and thick barrie1; preventing penetration oj exogenous 1110/ecules into the cytoso/. Electroporation is one of the methods which enable access oj different cytochemica/ labels to tlze cytoso/; so fm; howeve1; it has not been used on the urothclia/ cel/s in vivo. Thereforc, thc aim oj this study was to detennine whether in vivo e/ec­troporation of thc urinary bladder is a suitable mcthod for introduction of /abels into the cytosol of urothe­lial cel/s. Labe/s of various molecular masses were introduced: trypan blue, TRITC-phalloidin and FITC­labelled antibody (IgG). The results demonstrated that electroporation in vivo was a suitable method far introduction oj labels into the cytosol of urothelial cclls and could be used as a technique for detection oj intracellular 1110/eculcs and studying biochemical reactions. Key words: bladder; e/ectroporation, trypan blue, fluorescent dyes Introduction Electroporation is a method used for intro­duction of different molecules into the cytosol in vitro and in vivo.1, 2 Exposure of cells or tissues to short intense electric pulses induces transient electropores in the mem­brane, which, under suitable conditions, does not affect viability of cells. Recently, electro­poration has been used for drug delivery as well as gene delivery in vivo.2•3 Based on pre­clinical studies, successful employment of electroporation for delivery of chemothera- Correspondence to: Dr. Peter Veranic, Institute of Celi Biology, Medica] Faculty, Lipiceva 2, Ljubljana, Slove­nia. Tei: +386 61 320 985; Fax: +386 61 317 959; E-mail: veranic(l',>ibmi.mf.uni-lj.si peutic drugs such as bleomycin and cisplatin has been confirmed also in clinical trials on cancer patients.4, 5 Electroporation is widely used for in situ biochemical studies in cells. It has proved its efficiency for introducing mol­ecules of different molecular weight either for labelling intracellular components or for studying biochemical pathways.6, 7 Urothelial celi membrane is asymmetric, with specific protein uroplakins on the outer surface of the membrane. This asymmetric unit membrane protects the underlying epi­thelium from the toxic urine and is believed to play a role in strengthening the urothelial apical surface in order to prevent tissue rup­ture during bladder distension.8 Functioning as a strengthening and a barrier, the plasma 188 Vernnic Pet al. membrane of uroepithelial cells is highly impermeable. In order to penetrate this strong barrier, severa! immunocytochemical as well as electron microscopical methods were employed.9, 10 Due to its broad application electroporation could also be used for intro­duction of molecules into the urothelial cells. Further, this method has potential applica­tion in the treatment of urothelial tumors since introduction of chemotherapeutic drugs into the tumor cells by electric pulses (elec­trochemotherapy) was demonstrated to be effective. However, those tumors were not grown in situ, but transplanted subcutaneou­sly into the back of the mice.11 The aim of this work was to determine whether in vivo electroporation of the urinary bladder was a suitable method to introduce molecules of various molecular masses into the cytosol of urothelial cells. If electropora­tion proves suitable in the present study, it could also be used for cytochemical studies, i.e. the introduction of labels into the cells to detect molecular constituents as well as chemotherapeutic drugs. Material and methods Mice In the experiments, an inbred strain of NIH mice was used, purchased from Krka d.d. (Novo Mesto, Slovenia). Mice were main­tained at a constant room temperature (24 °C) and natura! day/night light cycle, in a con­ventional animal colony. Before the experi­ments, the mice were subjected to an adapta­tion period of at least 10 days. In vivo electroporation Adult female mice were anesthetised with an intraperitoneal injection of a mixture of Ketanest 150µg, Rumpun 10µg and atropine 0.lµg per lg of body weight (Parke-Davis GMBH). Abdomen of the mice was opened and the urinary bladder exposed. After rins­ing the bladder with saline, 0.2 ml of the label was injected intravesically. As labels, molecules of various molecular masses were used: 10 mg/ml trypan blue (MW 961) (Sigma), 7.8 µglml TRITC-phalloidin (MW 1305) (Sigma) and 10µglml FITC-anti rabbit IgG (155 kDa) (Sigma). Application of electric pulses was per­formed as described previously.1 2 Briefly, electric pulses were delivered by two fiat parallel stainless-steel electrodes, 8 mm apart from each other (two stainless-steel strips, length 35 mm, width 7 mm with rounded edges) to the exposed bladder. Eight square wave electric pulses of 720 V or 1040 V amplitude, pulse width 100 µs and repetition frequency 1 Hz, were generated by an electropulsator Jouan GHT 1287 Oouan, France). Electron microscopy Ten minutes after the application of electric pulses, the bladders were fixed with 4% paraform aldehyde and 2% glutaraldehyde in cacodylate buffer, postfixed in 1 % osmium tetroxide in 0.1 M cacodylate buffer, preem­bedding stained with uranyl acetate, dehy­drated and embedded in Epon. Ultrathin sec­tions were stained with lead citrate and exam­ined in a Jeol 100 CX electron microscope. Light microscopy Ten minutes after the application of electric pulses, the bladders were fixed in 4% formaldehyde. After rinsing, the tissue was frozen and sectioned with a cryotom. The sections mounted in a Slow fade mounting solution (Molecular Probes) were examined under a conventional or fluorescent micro­scope (Laborlux Leiz). In vivo electroporation of the urinnry bladder in mice Results Electroporation Light microscopy examination of tissue in the bladder exposed to 8 electric pulses of amplitude 1040 V established that large areas of the urothelium were detached from the basal lamina. Undetached urothelial cells were loosely connected to each other (Figure 1). -./·, .... f'J ,":.'\ . ··­ ':.\. ;:)'i.. -..-',t;.. Figure l. The bladder exposed to electric pulses at an amplitude of 1040 V. Large areas of the urothelium were detached from the basal lamina, 32x. It was obvious that on the ultrastructural level the most severe changes took place at the layer of basal cells. Many of these cells were split into half, parallelly to the basal lamina (Figure 2). In undetached superficial urothelial cells, facing the bladder lumen, the membranes of fusiform vesicles were torn at the edges where the normal membrane linked two asymmetric unit membrane plaques. In order to avoid damage to the cells by electroporation, lower amplitudes of electric pulses were tested in the second set of exper­iments. No morphological changes were observed in the bladder urothelium when the bladder was exposed to electric pulses of amplitude 720 V (Figure 3). Therefore, all the following experiments were performed at this amplitude of electric pulses. '\ . .. Figure 3. Trypan blue staining of urothelial nuclei (i). The bladder was exposed to the electric pulses, at an amplitude of 720 V, 128x. Labelling of the urothelial cel/s In the preliminary experiments of this study, it was proved that none of the labels that we used could penetrate the urothelial cells. After the application of electric pulses to the bladder (amplitude 720 V), trypan blue (MW 961) dye entered the urothelial cells and was evenly distributed over the whole urothe­lium. Nuclei of all the three epithelial layers were stained intensively. In the connective tissue only sporadic nuclei were stained (Fig­ure 3). TRITC-phalloidin (MW 1305) entered only the superficial urothelial cells and labelled the actin filaments at the basolateral membranes. The actins in the intermediary and basal layer of cells were not stained (Fig­ure 4). Loading of cells with FITC-IgG (MW 190 Ver11ic Pel ni. Figure 4. Phalloidin labelling of actin observed mainly at the celi borders of superficial cells. The bladder was exposed to electric pulses at an amplitude of 720 V, 128x. 155 kDa) was successful in approximately 2/3 of superficial cells. The fluorescence was distributed equally in heavy loaded cells. No labelling was found in the cells beneath the superficial ones (Figure 5). Figure 5. FITC-IgG entered most superficial cells (1) after the application of electric pulses at an amplitude of 720 V to the bladder, 128x. Discussion In this study labels of various molecular masses, trypan blue, TRITC-phalloidin and FITC-labelled antibody (IgG), were intro­duced into the urothelial cells by electropora­tion. Our results demonstrate that electropo­ration enables penetration of the label through a thickened plasma membranes and could therefore be used as an additional tech­nique for the detection of molecular con­stituents inside the cells and studying bio­chemical reactions. Electroporation in vitro has already proved its usefulness for labelling intracellular con­stituents with specific labels which can not enter intact cells.-i For example, actin net­work and intermediate filaments were visual­ized after the introduction of FITC-phalloidin and FITC-vimentin by electroporation of human gingival fibroblasts.6 Our results demonstrate that electroporation is also a suitable method for introduction of labels into the urothelial cells in vivo. Ali the labels entered the superficial cells at electric pulses amplitude 720 V without visible changes in celi morphology. The effec­tiveness of label penetration depended on their molecular masses. The smallest label trypan blue (MW= 961) entered ali the urothelial cells, but was mainly prevented from staining nuclei of fibroblasts in the lam­ina propria. In such cases, basal lamina may act as a barrier. Phalloidin, a molecule that is much larger (MW 1305), labelled the actin fil­aments only in superficial cells. The label was bound to actin filaments only in basolat­eral region. Such pattern of actin filaments distribution is also known in urothelial cells which have not been electroporated. The results show that phalloidin has not entered intermediate and basal cells. We can not exclude the possibility that actin from super­ficial cells has bound most of the phalloidin and so reduced the concentration of phal­loidin passing through this dense layer of actin towards intermediate cells, below the sensitivity of fluorescence microscope. Load­ing of most superficial cells with FITC-IgG provides the possibility of using electric puls­es as a new method for labelling cell's con­stituents in vivo with labels that have a high molecular weight. In conclusion, this study expands the area of application of electric pulses in vivo from electrogene therapy,3 electrochemothera­ 1H vivo electroporntio11 of tile 11ri11ary /J/adder in 111ice 2A,5,12 pytransdermal drug delivery1 ,3 to in , situ studies of cellular constituents and bio­chemical pathways in the bladder. Acknowledgement This work was supported by the Ministry of Science and Technology of the Republic of Slovenia. References 1. Orlowski S, Mir LM. Celi electroperrneabilization: a new tool for biochemical and pharrnacological studies. Bioclzim Biophys Acta 1993; 1154: 51-63. 2. Mir LM, Orlowski S, Belehradek J]r, Teissie J, Rols MP, Serša G, et al. Biornedical applications of elec­tric pulses with special ernphasis on antitumor electrochernotherapy. Bioelectroch Bioener 1995; 38: 203-7. 3. Heller R, Jaroszeski M, Atkin A, Moradpour D, Gilbert R, Wands J, Nicolau C. In vivo electroin­jection and expression in rat !iver. FEBS Le/11996; 389: 225-8. 4. Mir LM, Glass LF, Serša G, Teissie J, Domenge C, Miklavcic D, et al. Effective treatment of cuta­neous and subcutaneous malignant tumors by electrochemotherapy. Br J Cmzcer 1998; in press 5. Serša G, Štabuc B, Cemažar M, Jancar B, Miklavcic D, Rudolf Z. Electrochemotherapy with cisplatin: Potentiation of local cisplatin antitumor effectiveness by application of electric pulses in cancer patients. Eur J Cmzcer 1998; in press 6. Glogauer M, McCulloch CAG. Introduction of large rnolecules into viable fibroblasts by electro­poration: optimization of loading and identifica­tion of Iabeled cellular compartments. Exp Celi Res 1992; 200: 227-34. 7. Neumann E, Schaefer-Ridder M, Wang Y, Hof­schneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1982; 1: 841-5. 8. Staehelin LA, Chlapowski FJ, Bonneville MA. Lurnenal plasma membrane of the urinary blad­der. J Celi Biol 1972; 53: 73-91. 9. Okada CY, Rechsteiner M. lntroduction of macro­molecules into cultered mammalian cells by osmotic lysis of pinocytic vesicles. Celi 1982; 29: 33-41. 10. Miller MR, Ulrich RG, Shu-Fong Wang T, Korn D. Monoclonal antibodies against human DNA poly­merase-a inhibit DNA replication in permeabi­lized human cells. J Biol Chem 1985; 260: 134-8. 11. Yamaguchi O, lrisawa C, Baba K, Ogihara M, Yokota T, Shiraiwa Y. Potentiation of antitumor effect of bleomycin by local electric pulses in mouse bladder tumor. Toho/cu J Exp Med 1994; 172: 291-3. 12. Serša G, Cemažar M, Miklavcic D. Antitumor effectiveness of electrochemotherapy with cis-dia­minedichloroplatinum (II) in mice. Cmzcer Res 1995; 55: 3450-5. 13. Prausnitz MR, Bose VG, Langer R, Weaver JC. Electroporation of rnamrnalian skin: A rnechanism to enhance transdermal drug delivery. Proc Na// Acad Sci USA 1993; 90: 10504-8. Radio/ Onco/ 1998; 32(2): 193-200. MDP desmuramyl analogne LK-404 protects bone marrow and spleen cells from cyclophosphamide induced apoptosis Rok Kostanjšek1, Petra Kyralt1 , Tadej Malovrh1, Mojca Škoberne1 , Anton Stalc2, Vladimir Kotnik1 1Institute oj Microbiology and Immunology, Medica/ Faculty, Ljubljana, 2Research and Development, Lek d.d., Ljubljana, Slovenia In this article, we present the data on induction oj apoptosis in mouse bone marrow cells and spleen cells ajter treatment with dijjerent concentrations oj cytostatic cyclophosphamide in vivo and in vitro. Increas­ing apoptosis rate oj the cells was observed with the increasing concentration oj cyclophosphamide, and with the prolongation oj incubation time ajter in vivo as well as in vitro administration oj the drug. When apoptosis inducing activity was established, the immunomodulatory and jeasible protective ejject oj desmuramyl analogue oj MDP (LK-404) against cyclophosphamide induced apoptosis in mouse bone mar­row and spleen cells was studied. Cultivating the cells with cyclophosphamide and LK-404 simultaneously revealed the same apoptosis rate as cultivating with cytostatic only. Treatment oj cells with LK-404 prior to treatment with cyclophosphamide decreased apoptosis oj bone marrow and spleen cells which suggests potential protective role oj LK-404 against cyclophosphamide induced apoptosis. Key words: apoptosis, bone marrow, spleen, cytostatic, immunomodulator Introduction Apoptosis is a genetically controlled process 5 of cell death.1-It has significant value as counter-weight to cell division and prolifera­tion, thus keeping the number of cells in tis­sue constant.6-9 Apoptotic cells present unique changes of DNA molecule. Apoptotic process occurs spontaneously, but can also be indu­ced. lmportant inducers of programmed cell Correspondence to: Prof . Vladimir Kotnik, MD, PhD, Institute of Microbiology and Immunology, Medica! Faculty, Zaloška 4, SI-1000 Ljubljana, Slovenia. Te!: +386 61 14-03-042; Fax: 14-23-518; E-mail: vladi­mir.kotnik@mf.uni-lj.si death are deduction of nutrients, regulatory molecules as for instance cytokines, hor­mones and growth factors, switching on of severa! distinctive genes, treatment with radi­ation or with cytostatics.10,11 Abnormalities in the process of apoptosis may have a large influence on beginning and development of diseases like cancer, vira! infections, autoim­mune disease and central nervous system disease.12-18 Apoptotic cell death can be detected by severa! methods.19 In the presented experi­ments, the apoptosis rate was determined using a specific ELISA (Boehringer Mann­heim, Germany) detecting apoptosis specific Kostanjšek R et al. DNA fragments with enzyme labelled anti­bodies. Cyclophosphamide is a cytostatic drug widely used in anti-tumor therapy. It has cytotoxic effect in all phases of the cel! cycle, although it has stronger influence on activat­ed or already dividing cells. Cyclophos­phamide is a DNA-alkylating drug able to cross-link DNA chains, thus causing cel! death. 20 It is generally known that also the cells of the immune system die as a side effect of such tumor treatment. The reduc­tion in number of immune cells results in a decreased immune response which can lead to severe and more frequent infections. Apoptosis in mouse marrow and spleen cells was studied in described experiments using cyclophosphamide as the inductor of 21, 22 cel! death . The aim of the study was to examine the apoptosis of bone marrow and spleen immune cells as an inducible process which can be stopped by appropriate treat­ment. It would be well appreciated, if selec­tive reduction of apoptosis of immune cells and potentiation of toxicity for the tumor cells could be achieved. U sing immunomod­ula ting MDP derivatives (N-acetyl-L-alanyl­D-isoglutamine) with hemopoiesis restoring 25 activity may be effective.23-Therefore the attempt was done to restore the cyclophos­phamide induced apoptosis in bone marrow and spleen cells with LK-404 one of MDP analogues. LK-404 (N-(7-oxododekanoyl)-L­alanine-D-isoglutamine) an desmuramyl ana­logue synthesized at Faculty of Pharmecy, University of Ljubljana, in cooperation with Lek d.d., it is in opposite to original MDP molecule without disadvantageous pyro­genicity, and less toxic. The potential protec­tive effect of LK-404 against cyclophos­phamide induced apoptosis was studied using LK-404 simultaneously and consecu­tively to treatment with cyclophosphamide. Materials and methods Anima/s and the preparation of cel/s Han NmRi mice were used in ali the descri­bed experiments. The animals were provided by Lek Research and Development Animal Care, Ljubljana, Slovenia. After delivery from Animal Care they were kept under standard conditions in our facilities. The mice, we used as a source of bone marrow and spleen cells, were at the beginning of the experiment, sacrificed with an ether over­dose anesthesia. The femurs and spleens were isolated. The epiphyses were cut off the femur and 5 ml RPMI 1640 Medium (Sigma Chemicals; St. Louis, USA) was squirted through the bone. Washed out cells were sucked into the syringe again and then flushed to the wall of Petri dish. Spleen cells were isolated from the spleen using sintered microscope slide glass plates and afterwards suspended in growth medium RPMI 1640. Contaminating erythrocytes were removed from both celi suspensions by adding 2 ml of 0.85% IRIS buffered ammonium chloride (NH4 CI). The remaining mononuclear cells were washed three times with MEM medium (Sigma Chemical Co., St.Louis, USA). Cells were suspended (1:10) in trypan blue (0.1 %) and then counted in haemocytometer. A con­centration of 5x105 cells per ml was prepared for the experiment. Apoptosis induction Lyophilized cyclophosphamide (Endoxan, Asta Medica AG Frankfurt, Germany) was dissolved in sterile bidistilled water and solu­tions giving concetrations of 6.25, 12.5, 25, 50 and 100 mg of cyc!ophosphamide per kg of mouse weight were prepared. In in vivo experiments, 200 µL of cyclo­phosphamide at a dose of 50 mg/kg of mouse body weight or 100 mg/kg of mouse body MDI' des11111rn111yl n11nlog11e LK-404 nnd npo11tosis weight was injected intraperitoneally. Treat­ment was repeated on days one, four and six. On the seventh day of experiment, the mice were sacrificed. The control group of mice was injected with sterile physiological saline instead of with cyclophosphamide at the same days. In in vitro experiments, cyclophos­phamide was used in appropriate equiva­lents at concentrations of 6.25 mg/kg, 12.5 mg/kg, 25 mg/kg and 50 mg/kg of mouse weight. The cell samples were taken 5, 10, 15, 30, 60 and 90 minutes after the addition of cyclophosphamide to the cells used in the experiment. LK-404 The compound LK-404 was prepared in RPMI 1640 at concentrations of 0.525 µM, 5.25 µM and 52.5 µM and then used in the experi­ments described below. In jirst series oj experiments, isolated bone marrow and spleen cells were treated with LK-404 and cyclophosphamide simultaneous­ly. Aliquoted samples were taken for testing the apoptosis 5, 10, 15, 30, 60 and 90 minutes after incubation. All three concentrations of LK-404 were tested. In the second series oj experiments, consecu­tive applications of LK-404 and cyclophos­phamide were studied. The cells were first incubated at ali three concentrations of LK­404 for 90 minutes. Ninety minutes after the beginning of the experiment, a 25 mg/kg of cyclophosphamide was added to each of the samples. Aliquots were taken for testing 5, 10, 15, 30, 60 and 90 minutes after the cyclophosphamide addition. Celi death detection ELISA The apoptosis rate was determined using Celi Death Detection ELISA (Boehringer Mann­heim, Germany). The assay is based on the quantitative sandwich-enzyme-immunoassay principle using monoclonal antibodies direct­ed against DNA and histones, respectively. This permits a specific determination of mono and oligonucleosomes in the cytoplas­mic fraction of celi lysates. In the first step, anti-histone antibody is fixed to the wall and the bottom of the microtiter plate module. During the second step, the nucleosomes contained in the sample bind, via their his­tone components, to the immobilized anti­histone antibody. In the third step, anti-DNA­peroxidase labeled antibody (POD) reacts with the DNA part of the nucleosome. The amount of peroxidase retained in the immuncomplex is determined spectrophoto­metrically with ABTS (2,2' -azino-di-(3-ethyl­benzthiazoline sulfonate)) as substrate. Desig11 oj the experi111ents pe1fom1ed Treatment oj bone marrow and spleen cel/s with cyclophosphamide in vivo: 9 mice were divided into 3 subgroups. Three mice from the first subgroup were injected with 200 µl of cyclophosphamide at a dose of 100 mg/kg, three from the second subgroup with 200 µl of cyclophosphamide at a dose of 50 mg/kg, and the remaining three with 200 µl of physi­ological saline on the first, fourth and sixth day. The mice were sacrificed on the seventh day. Bone marrow cel\s and spleen cells were isolated and divided into two portions. Apop­tosis of the cells from the first aliquot was measured the same day. The second aliquot was first incubated for 24 hours at room tem­perature; the apoptosis was measured on the second day. -freah11e11t oj bone marrow and spleen cells with cyclophosphamide in vitro: Isolated bone mar­row and spleen cells were treated with the following concentrations of cyclo­phosphamide: 25 or 50 mg/kg. The sample treated with RPMI 1640 alone instead of with cyclophosphamide; was regarded as a Kostanjšek R ct a/. control sample. The samples were incubated for 15, 30, 60 or 90 minutes. Aliquots were taken at indicated times. The number of live cells was determined and amount of apopto­sis measured in each of the samples at each tirne point. Simultaneous application of LK-404 and cyclo­phosphamide to bone marrow and spleen cel/s in vitro: Bone marrow and spleen cells were iso­lated. Cyclophosphamide at a concentration of 25 mg/kg and LK-404 at concentrations of 0.525 µM, 5.25 µM or 52.5 µM were added to cell suspensions. The first control sample was incubated only with 25 mg/kg cyclophos­phamide, whereas the second one only with medium RPMI 1640. Aliquots for testing were taken 5, 10, 15, 30, 60 and 90 minutes after the beginning of the experiment and the amount of apoptosis was measured. Consecutive application of LK-404 and cyclo­phosphamide to bone rnarrow and spleen cel/s in vitro: Bone marrow and spleen cells were iso­lated and incubated with LK-404 at concen­trations of 0.525 µM, 5.25 µM or 52.5 µM. After 90 minutes, a 25 mg/kg of cyclophos­phamide was added to each of the samples. Aliquots were taken for testing 5, 10, 15, 30, 60 and 90 minutes after adding the cyclophosphamide. Two samples of cells were prepared with­out LK-404 and, after 90 minutes of incuba­tion, the cells of the first sample were treated with 25 mg/kg of cyclophosphamide. The cells of the second sample were treated nei­ther with LK-404 nor with cyclo­phosphamide. Aliquots for testing were taken after 5, 10, 15, 30, 60 and 90 minutes. Statistica/ analysis Student's t-test was used to determine the difference between samples and p<0.05 was regarded as significant. Results Treatment of bone marrow and spleen cells with cyclophosphamide in vivo The apoptosis of bone marrow cells and spleen cells depends on the concentration of cyclophosphamide and tirne of incubation. The bones from the mice treated with cyclophosphamide at a dose of 50 and 100 mg/kg contained less marrow and the weight of the spleen was reduced as compared to the control nontreated mice. Treatment of bone marrow and spleen cel/s with cyclophosphamide in vitro In previous experiment, we noticed that cyclophosphamide reduced the number of bone marrow and spleen cells during the incubation. To get a real insight in the process of apoptosis, we compared the num­ber of bone marrow and spleen cells with the amount of apoptotic products in each of the sample. The rate of apoptosis in Figure 1 is therefore presented as a quotient between the apoptosis (O.D.) and the number of cells in the sample. Apoptosis of bone marrow and spleen cells treated with cyclophos­phamide depends on the concentration of cyc!ophosphamide and incubation tirne. Influence of simultaneous application of LK-404 and cyclophosphamide on apoptosis in bone mar­row and spleen cel/s in vitro Bone marrow and spleen cells were treated in four different ways. In the control group, the cells were treated with cyc!ophos­phamide at a dose of 25 mg/kg. The first group was treated with 25 mg/kg of cyclophosphamide plus 0.525 µM of LK-404, the second with 25 mg/kg of cyclophos­phamide plus 5.25 µM of LK-404, and the third with 25 mg/kg of cyclophosphamide plus 52.5 µM of LK-404. In all eight groups MDP des111ura111yl mzalogue LK-404 and apoptosis Bone marrow cells A 5 4 1 0 41 35 30 25 20 15 10 5J 1 __ .,__i._ L O•l.--=--1--. ---t 15min 30min 60min 90min INCUBATION TIME Spleen cells B 15 min 30min 60min 90min INCUBATION TIME Figure l. Panel A: Treatment of bone marrow cells with cyclophosphamide i11 vitro. In the control group (cells were not treated with cyclophosphamide), the apoptosis quotient was slowly increasing with the prolongation of incubation tirne. In the group where bone marrow cells were incubated with 25 mg/kg of cyclophosphamide, the apoptosis quotient was steeply increasing during the prolongation of incubation tirne. In the group where cells were incubated with 50 mg/kg cyclophosphamide, the apoptosis quotient was exponentially increasing with the prolongation of incubation tirne. Panel B: Treatment of spleen cells with cyclophosphamide in vitro. The apoptosis quotients of the control group (cells not treat­ed with cyclophosphamide) were slowly increasing with incubation tirne. The apoptosis quotients of the group where cells were treated with 25 mg/kg of cyclophos­phamide shows a tendency towards a steep increment with incubation tirne. The apoptosis quotients of the cells in the group where cells were treated with 50 mg/kg of cyclophosphamide increase rapidly with incubation tirne. Legend: 1111 control, cyclophosphamide 25 mg/kg, D cyclo­phosphamide 50 mg/kg (four groups of bone marrow cells and four groups of spleen cells), apoptosis increased by the same rate with the prolongation of incubation tirne without significant influ­ence of treatment with LK-404. Influence oj consecutive application oj LK-404 and cyclophosphamide on apoptosis in bone mar­row and spleen cel/s in vitro The rate of apoptosis of bone marrow and spleen cells in the first control sample (cells incubated in RPMI 1640 only) was slowly increasing during the incubation tirne due to spontaneous apoptosis of incubated bone marrow and spleen cells. In the second con­trol sample (cells treated with cyclophos­phamide after 90 minutes of incubation, and not pretraeted with LK-404) apoptosis of bone marrow and spleen cells rapidly increased after cyclophosphamide had been added, due to cyclophosphamide induced apoptosis. The difference in apoptosis between both control samples was significant (p=0.015). Apoptosis in other samples (treated with LK404 prior to cyclophosphamide) increased only slowly in comparison to the control sam­ples. The treatment of bone marrow and spleen cells with the highest concentration of LK-404 (52.5 µM) slowed down the apopto­sis. The decreasing effect calculated between the second control sample (cells treated with cyclophosphamide after 90 minutes of incu­bation and not pre-treated with LK-404) and the sample pre-treated with 52.5 µM of LK­404 was significant (p=0.004 for bone mar­row cells and p=0.042 for spleen cells). The results of these experiments are shown in Figure 2. Discussion The importance of apoptosis in the develop­ment and differentiation of immune cells is Kostanjšek R c/ al. Bone marrow cel1s A INCUBATI N TIME O Spleen cells B 1.2 0.8 tj 0.6 o 0.4 0.2 Srnin 10min 1.2 p----. 0.8 o 0 0.6 1 021 C) ••• o! O min -O· --. ­---­-Srnin 10min .. -0­······0' 15 min 30 min 60 min 90 min Figure 2. Panels A and B: Influence of consecutive appli­cations of LK-404 and cyclophosphamide on apoptosis in bone marrow and spleen cells in vitro. Apoptosis of bone marrow (A) and spleen (B) cells incubated with RPMI 1640 only (--O --) was slowly increasing with the prolongation of incubation time. After addition of cyclo­phosphamide in the second control sample (--D --) (cells treated with cyclophosphamide after 90 minutes, and not pre-treated with LK-404) the apoptosis increased steeply. Apoptosis in other samples (of bolh types of cells) treated with 0.525 µM (--ľ--), 5.25 µM (-+-), and 52.5 µM LK-404 (----) prior to cyclophosphamide addi­tion, decreased in comparison to the second control sam­ple (--. --) of bone marrow and spleen cells. The most effective was pre-treatment with 52.5 µM LK-404 (-1111-). not doubtful any more. At present, the focal question concerning the immune system is the involvement of apoptosis in regulation 10 11 21 22 and functioning of the immune cells.5,, ,, However, the immune response is a self-lim­iting process under the influence of antigen burden, stability of the antigenic structure, activity of macrophage phagocytic system, antigenicity, capability of the antigen presen­tation and recognition, function of B and T cells and competence of effector functions of antibodies and cytotoxic lymphoid cells, it may also be limited by induction of apopto­sis. Interference with apoptosis leaving cells alive for longer period of tirne, as determined by the genetically determined cel! life span, would give an interesting tool to manipulate disease which depends also on immune sys­tem activity.15, 18 In malignant disease treated with cytostatics, immune cells are not prane to respond effectively. The consequences are oportunistic infections which may be the cause of death of the patient. Our experimen­tal approach to study the importance of apoptosis in regulation of immune system function is a copy of the treatment of patients with alkylating drugs. Cyclophos­phamide treatment is accompanied by a decreased function of immune system and followed by increased susceptibility to infec­tion. 20 Protection of the immune cells against cytostatics or adjuving depressed lymphocyte function would be of great value. MDP mole­cule, an important component of the peptido­glycan, has been proved to have an immuno­adjuvant activity. Unfortunately, the original MDP molecule is highly pyrogenic and a good inducer of autoimmunity and as such of no use as an immuno-adjuvant. By changing the structure of MDP molecule, analogues may be prepared with a preserved adjuvant activity with no unpleasant side effects.26 Desmu­ramyl analogues of MDP are able to increase the functioning of the immune cells.27 The exact way how they do it is unknown. It would be therefore interesting to know whether such preparations would be able to interfere with the process of apoptosis. For the induction of apoptosis we applied the widely used cyclophosphamide, a cytosta­tic with not yet describet apoptosis activity. MDP des11111ra111yl analoguc LK-404 and apoptosis We were able to induce apoptosis with cyclo­phosphamide as presented in the results sec­tion. Apoptotic function of cyclophospha­mide was time and 1 cm) patients, and quadrantectomy with axillar lymph dissection -in the rest 32 (T.l cm) patients. The intraoperatively found sentinel lymph nodes were biopsied, submitted to his- Table l. Dyes used in patients with breast cancer Dyes Producer Number of cases independently with carrier total Drimaren Bri!liant Blue Fluka -Cat N 44582 1 % solution in PBS 10 Methylen Blau VITIS Neopharma amp. 1 % 5 ml 8 31 39 Patent Blue V BYK Gulden amp. 2,5% 2ml 25 22 47 Tota! 43 92 135 To enhance dye lymphotropism, severa! carriers were applied together with the dye in ratio 1:1 or 1:2 (Table 2). The quantity of dye applied around the tumor independently or by carriers varied from 1.0 to 3.0 ml, and immediately after application, the site was gently massaged for severa! minutes. Based on studies proving the role of Hyaluronidase for increasing the macromole­cule transportation from interstitial space to lymph vessels, 1, 7 39 patients were treated with Hylase (Hylase Dessau, Germed). A total 40 years of age T N N 2 1 3 1 1 3 3 (1)** 3 o 1 o 2 4 4 (1) 4 3 5 1 5 8(2) 14(1) NX* : No lyrnph nodes surgically rernoved (1)**: Patients with distant rnetastases at diagnosis tumour turned out to be macroscopically incomplete, in 23 (33.1%) patients microscop­ically. A histopathological examination of the specimens revealed 39 papillary carcinomas, 17 folliculary carcinomas, 7 medullary carci­nomas, and 6 anaplastic carcinomas. Papil­lary cancers included both, purely papillary and mixed papillary-follicular tumours. After the surgery, the ablative radioiodine treatment was performed in 56 patients (80.6%) using a mean 50 Gy compared to those with a conventional x-ray treatment with lower doses. Other pre­vious reports describe better Iocal control rates after a high-dose radiotherapy resulting 10 in the improved survival rates.9, the better survival rates only in patients with papillary thyroid cancer, 11 , 12 or the improved local con­trol rates which did not translate into a sur­vival benefit.2 In 1990 Benker et aI.18 observed no beneficial effect of adjuvant radiotherapy on the survival in patients with Tl-T4 tumours. The same bbs.infos­quare.it Radiotherapy and Oncology September 25-28, 1998. The "19th Annual ESTRO Meeting" will be held in Prague, Czech Repub-lic. Contact the ESTRO office, Av. E. Mounierlaan, 83/4, B-1200 Brussles, Belgium; or call +32 2 775 9344; or fax +32 2 779 5470. E-mail:germaine.heeren«"est­ro.be Haematology September 28-30, 1998. "3rd The Educational Forum on Leukaemia and Haematological Malignancies" will be held in Berg­amo, Italy. Contact European School of Oncology, Viale Beat­rice d'Este 37, 20122 Milan, ltaly; or call +39 2 5831 7850; or fax +39 2 5832 1266. E-mail: comprevtum@>bbs.infos­quare.it Radiotherapy Octabe1; 1998. The ESTRO teaching course "Evidence Based Radia­tion Oncology: Principles and Methods" will take place in lzmir, Turkey. Contact the ESTRO office, Av. E. Mounierlaan, 83/4, B-1200 Brussles, Belgium; or call +32 2 775 9344; or fax +32 2 779 5470.E-mail:germaine.heeren@>estro.be Radiobiology Octabe1; 1998. The ESTRO teaching course "Basic Clinical Radiobi­ology" will be held in Como, Italy. Contact the ESTRO office, Av. E. Mounierlaan, 83/4, B-1200 Brussles, Belgium; or call +32 2 775 9344; or fax +32 2 779 5470.E-mail:germaine.heeren@estro.be Surgical oncology Octaber 1-3, 1998. The ESO advanced course "Breast Reconstructive and Cancer Surgery II" will take place in Milan, Italy. Contact European School of Oncology, Viale Beatrice d'Este 37, 20122 Milan, ltaly; or call +39 2 5831 7850; or fax +39 2 5832 1266. E-mail: comprevtum(albbs.infos­ quare.it Oncology October 7-9, 1998. The ESO training course "lnnovation in Cancer Management" will be offered in Cairo, Egypt. Contact ESO Balkans and Middle East Office, Egnatia Epirus Foundation, 7 A Tzavella St., 453 33 loannina, Greece; or call +30 651 72315/76992; or fax +30 651 36695. Oncology October 8-9, 1998. The ESO training course "Rare Tumours: Diagnosis and Treatment" will be held in Sofia, Bulgaria. Contact ESO Balkans and Middle East Office, Egnatia Epirus Foundation, 7 A Tzavella St., 453 33 loannina, Greece; or call +30 651 72315/76992; or fax +30 651 36695. Oncology October 15-21, 1998. The ESO training course "Liver Pathology-Oncolo­ gy" will be offered in Ioannina, Greece. Contact ESO Balkans and Middle East Office, Egnatia Epirus Foundation, 7 A Tzavella St., 453 33 loannina, Greece; or call +30 651 72315/76992; or fax +30 651 36695. Haematology and Oncology October 19-21 1998 The "3rd Educational Forum on Leukaemias: State of art and hot issues" will be held in Bergamo, Italy Contact European School of Oncology, Viale Beatrice d'Este 37, 20122 Milan, Italy; or call +39 2 5831 7850; or fax +39 2 5832 1266. E-mail: comprevtum«Dbbs.infos­quare.it Notices 237 Radiation Oncology October 19-22, 1998. The "Annual Meeting of American Society for Ther­apeutic Radiology and Oncology ASTRO" will take place in Phoenix, Arizona, USA, Contact Vicky Carroll, ASTRO office, 1891 Preston White Drive, Reston, VA 22091, USA; or call +1 703 716 7588; or fax +1 703 476 8167. Laser medicine & surgery October 21-25, 1998. The "7th Congress of Asian Pacific Association for Laser Medicine & surgery" and the "lOth international YAG Laser Symposium" will be offered in Ho Chi Minh City, Vietnam. Contact Dr. Ha Viet Hien, Secretariat officer, 109A Pasteur St, Dist 1, Ho Chi Minh City, Vietnam; or call +84 8 829 9322/ 824 1958; or fax +84 8 824 1959. E­mail: biomed@bdvn.vnd.net Colorectal cancer October 22-24, 1998. The ESO advanced course "Digestive Tract: Col­orectal Cancer" will be held in Milan, Italy. Contact European School of Oncology, Viale Beatrice d'Este 37, 20122 Milan, Italy; or call +39 2 5831 7850; or fax +39 2 5832 1266. E-mail: comprevtum@bbs.infos­quare.it Haematology and oncology October 25-28, 1998. The Annual Meeting of German and Austrian Association of Haematology and Oncology will be offered in Frankfurt, Germany. Contact Prof. Dr. Dieter Hoelzer, Universitaetklinik Frankfurt, Medizinische Klinik III, Theodor Stern Kai 7, 60590 Frankfurt, Germany; or call +49 39 6301 5194; or fax +49 69 6301 7324; e-mail: hoelzer@lem.uni-frank­furt.de Paediatric Oncology October 27-31, 1998. The S.1.O.P. teaching course will take place in Moscow, Russia. Call P.A. Voute +31 20 566 5655; or fax +31 20 691 2231. Lung cancer October 31 -November 4, 1998. The 3rd interantional congress on Jung cancer will be offered in Rhodes, Greece. Contact Congress Secretariat of the 3rd interantion­al congress on lung cancer, Amphitrion Congress Organising Bureau, 7, Sygrou Avenue, 117 43 Athens, Greece; or call +30 1 924 9701; or fax +30 1 924 9836 / 924 9671. E-mail: amphitrion@travelling.gr FONDACIJA DR.]. CHOLEWA FONDACIJA "DOCENT DR. J. CHOLEWA" JE NEPROFITNO, NEINSTITUCIONALNO IN NESTRANKARSKO ZDRUŽENJE POSAMEZNIKOV, USTANOV IN ORGANIZACIJ, KI ŽELIJO MATERIALNO SPODBUJATI IN POGLABLJATI RAZISKOVALNO DEJAVNOST V ONKOLOGIJI. MESESNELOVA 9 61000 LJUBLJANA TEL 061 15 91 277 FAKS 06 1 2 1 8 1 1 3 :ŽR: 501 00-620-1 33-05-1 0331 15-214 779 Activity of "Dr. J. Cholewa" foundation for cancer research and education -report for the second quarter of 1998 In the first and the second quarter of 1998 the "Dr. J. Cholewa" foundation for cancer research and education sought to ensure the continuation of its activities in the view of overcoming ever presenting financial constraints as conditioned by the general level of the economic activity in the republic of Slovenia. With this in mind, better coordination with other similar institutions in Slovenia has been of paramount importance. The Foundation continues to support regular publication of "Radiology and Oncology" international scientific journal, and the regular publication of the "ESO Challenge", the newsletter of the European school of Oncology, both being published and edited in Ljubljana, Slovenia. It actively supported the organisa­tion of the first education "Oncological weekend" meeting held in 1998, that was intended to ali in the medica! profession interested in problems connected with pediatric oncology. The meeting was held in the city of Postojna and has since been regarded as very successful by its participants and organizers. The prepara­tion proceedings for the traditional Hepatobiliary School in Ljubljana are now gathering momentum, and the Foundation hopes to provide its modest contribu­tion. For the 1998 the Foundation also plans to continue to provide grants for the various European School of Oncology courses, research and educational grants for the study in Slovenia and abroad, to provide support for educational and sci­entific meetings and symposia, and to support publishing and editorial activity from the various fields of oncology in Slovenia. It can thus be seen that the " Dr. J. Cholewa" Foundation for Cancer Research and Education continues to pursue its stated goals, as defined by its statute and recent meetings of the Board of directors and the Assembly of the Foundation. Tomaž Benulic, MD Borut Štabne, MD, PhD Andrej Plesnicar, MD EUROPEAN CONFERENCE th ON GENERAL THORACIC SURGERY PORTOROŽ, SLOVENJA THE OCTOBER 22-24, 1998. Main topics: Oesophageal reconstruction procedures Diagnosis and treatment of N2 lung cancer Identifying high -risk patients in lung surgery Complications following lung surgery Mediastinal tumours -diagnosis -surgery and other treatment modalities Free papers Call for abstract and registration Contact: Organizing Secretariat of The "6th European Conference on Thoracic Surgery" Department of Thoracic Surgery University Medica! Center Ljubljana Zaloška 7 1525 Ljubljana, Slovenia Phone: +386 61 317 582 Fax: +386 61 13 16 006 E-mail: joze.jerman@mf.uni-lj.si ANNUAL MEETING OF THE EUROPEAN SOCIETY OF th MUSCULOSKELETAL RADIOLOGY BLED, SLOVENIA THE OCTOBER 30-31, 1998. The fifth Annual Meeting of the European Society of Musculoskeletal Radiolo­gy (ESSR) will be held in Bled, Slovenia, October 30-31, 1998. Our young society celebrates its fifth anniversary, having a short but very sec­cessful history. The society was founded on March 20th, 1993 in Bonn, Germany by thirteen representatives from various European countries. This was a modest beginning of the ESSR, which, today, is an established society with 218 members from 29 European countries. Our main task is to promote musculoskeletal radiol­ogy in Europe, with a particular emphasis on education and research. One of the most important activitiesof the ESSR is the organization of our annual meeting which is an opportunity to share research experience and to refresh the knowl­edge of diferent areas in musculoskeletal radiology. We have a great honour and pleasure to organize the meeting in Slovenia for the first tirne. Two refresher courses and fourteen parallel scientific sessions are scheduled. Introductory lectures will be given by distinguished musculoskeletal radiologists. Young colleagues are encouraged to present the results of their research work. On behalf of the Organizing Committee I cordialy invite you to attend the Fifth Annual Meeting of the ESSR. V, Jevtic, President of the ESSR INFORMATION: Professor V. Jevtic, M.D. Clinical Radiology Institute University medica! Centre Zaloška 7 SI-1525 Ljubljana, Slovenia Zdravljenje pomenopavzalnih bolnic z napredovalo ali ponovljeno obliko raka dojk je bolj uspešno z novim selektivnim zaviralcem aromataz rimidex anastrozol -odmerjanje je enostavno, bolnica vzame eno tableto Arimidexa enkrat dnevno -koncentracije estradiola se hitro zmanjšajo do meje merljivosti -Arimidex se dobro prenaša, prekinitve zdravljenja so redke -povecanje telesne teže je znatno manjše kot po sedanji terapiji -nadomestno zdravljenje s steroidi ni potrebno ZENECA Podrobnejše informacije so na razpolago pri: Zeneca lnternational Ud Podružnica v Sloveniji Einspielerjeva 6, Ljubljana tel.: 061 132 20 74 fax: 061 132 12 08 5RLU5 LJUBLJANA, d.d., MAŠERA-SPASICEVA ul. 10, tel. 061 189 91 00, faks: 061 168 10 2 .AKO CD79a, B Cell Marker Monoclonal Mouse Anti-Human B Cell The two antibodies were clustered at the Fifth lnternational Workshop on Leucoczte Differentiation Antigens, Boston, 1993. ¦ Highly specific tor B cells at all stages of maturation ¦ Well documented antibodies ¦ lmportant new addition to the range of DAKO B and T Cell Markers ¦ Unique products, only from DAKO ¦ Available as two products from the clones JCB117 and HM57 ¦ Applicable on DAKO TechMate ™ immunostainers ¦ Work well on formalin-fixed, paraffin-embedded material DAKO -Your Reference in lmmunohistochemistry Antibodies available: Manaelanal Mause Anti-Human B Celi, CD79a, elane JCB 117 Manaelanal Mause Anti-Human B Celi, CD79a, elane HM57 [s@§@ullinJ§[Q) .i.. LABORMED d.o.o. Tei;..(). .)12 .;vs!er:1!fo/ii\76.f 11 s ZASTOPA HIGIEA d.o.o., Trzin Blatnica 12, 1236 Trzin tel.: (061) 1621 101 fax: (061) 1621 043 Nycomed Imaging is proud oj its role in providing jor the early, accurate diagnosis oj disease, thus improving patients' quality oj life and prospects jor effective treatment. The company is committed to the continuous development oj innovative imaging products to enhance diagnostic procedures. ..NYCOMED Nycomed lmaging AS, P.O.Box 4220 Torshov, N-0401 Oslo, Norway DISTRIBUCIJA SALUS d.d. Mašera Spasica ul. 1 O 1000 Ljubljana tel.: (061) 1681 144 fax: (061)1681 420 HOFFMANN-LA ROCHE Diagnostic Systems Clinical Chemistry Hematology Immunology Polymerase Chain Reaction (PCR) Official distributor: ADRIA MED d.o.o., Luize Pesjakove 8a 1000 Ljubljana, Slovenia tel.: --386 61 1379200, tel./fax: --386 61 1376450 ,_ \:.,. \.--:. ,_ l.... . \.--:. c., l.., G \: . -III ,,, ­ ,_ -III '< ',, 'C v ,-.: .... l.., .......... SIEMENS Rešitve po meri Mammomat 3000 modular Mammomat 3000 modular • univerzalni sistem za vse vrste mamografije • optimizacija doze in kompresije z OPDOSE in OPCOMP sistema • modularna zgradba zagotavlja posodabljanje sistema • servis v Sloveniji z zagotovljenimi rezervnimi deli in garancijo • izobraževanje za uporabnike SIEMENS d.o.o. Dunajska 22 1511 Ljubljana Telefon 061/1746 100 Telefaks 061/1746 135 I:SANOLABOR :J ­ .<:IQQ Pri nas dobite vse za rentgen! • KODAK • SIEMENS • GENERAL ELECTRIC • • PHILIPS • BENNETT • HITACHI • POLAROID • • XENOLITE • MA VIG • CA WO • • rentgenski filmi in kemikalije • kontrastna sredstva • rentgenska zašcitna sredstva • rentgenski aparati, aparati za ultrazvocno diagnostiko, stroji za avtomatsko razvijanje, negatoskopi in druga oprema za rentgen !: 5/lNOU\.BOR, Leskoškova 4, l l 03 Ljubljana , Tei,: 061 185 42 11 Fax: 061 140 13 04 Povrne mik življenja tramadol je registriran tudi pri ameriški zvezni upravi za hrano in zdravila (FDA) Kontraindikacije: Otroci do l leta starosti, akutna zastrupitev z alkoholom, uspavali, analgetiki in drugimi zdravili, ki vplivajo na CŽS, zdravljenje z inhibitorji MAO. Interakcije: Pri socasni uporabi zdravil, ki delujejo na osrednje živcevje, je možno sinergisticno delovanje v obliki sedacije pa tudi mocnejšega ana!geticnega delovanja. Opozorila: Pri predoziranju lahko pride do depresije dihanja, Previdnost Je potrebna pri bolnikih, ki so preobcutljivi za opiate, in pri starejših osebah. Pri okvari jeter in ledvic je potrebno odmerek zmanjšati. Bolniki med zdravljenjem ne smejo upravljati strojev in motornih vozil. Med nosecnostjo in dojenjem predpišemo tramadol le pri nujni indikaciji. Bolnike s krci centralnega izvora skrbno nadzorujemo. Doziranje: Odrasli in otroci, starejši od 14 lcl: 50 do 1 00 mg 3-do 4-krat na dan. Otrokom od / lcla do 14 let dajemo v odmerku I do 2 mg na kilogram telesne mase 3-do 4-krat na dan. Stranski ucinki: Znojenje, vrtoglavica, slabost, bruhanje, suha usta in utrujenost. Redko lahko pride do palpitacij, ortostaticne hipolenzije ali kardiovaskularnega kolapsa. Izjemoma se lahko pojavijo konvu!zije. Oprema in nacin izdajanja: 5 ampul po 1 ml (50 mg/ml), 5 ampul po 2 m! ( ! 00 mg/2 m!), uporaba je dovoljena samo v javnih zdravstvenih zavodih ter pravnim in fizicnim osebam, ki opravljajo zdravstveno dejavnost. 20 kapsul po 50 mg, 1 O m! raztopine (100 mg/ml), 5 sveck po 100 mg, na zdravniški recept. 11/97. Podro/Jn(jšc informacije so na voljo pri proizvajalcu. . .. l