Oral Presentations: Molecular Carcinogenesis I

Molecular Carcinogenesis I . 303

Oral Presentations: Molecular Carcinogenesis I

105

107

QUANTITATIVE EVALUATION OF HER21neu PROTEIN EXPRESSION IN BREAST CANCER BY IMMUNOCYTO· CHEMISTRY (ICC) WITH PHOTOSHOP-BASED IMAGE ANALYSIS HA Lehr, TW Jacobs, M Barnes, SJ Schnitt, AM Gown. University of Mainz, Medical Center, Germany, Beth Israel Deaconess Medical Center (BIOMC), Harvard Medical School, Boston MA, PhenoPath Laboratories (PPL) and IRIS, Seattle, WA. Backeround: Detection of Her2lneu overcxpre ssion in breast cancer cells has value for determining both prognosis and response to therapy. We quantified HER2/neu protein expression by ICC with image analysis and compared the results with semi-quantitative ICC scoring and with HER2Ineu amplification as assessed by fluorescence in situ hybridization (FISH). ~: Her2/neu detection was performed in a blinded fashion on 40 invasive breast cancers by ICC using a polyclonal antibody (DAKO; PPL) with and without heat-induced antigen retrieval (HIER). The immunostaining intensity of tumor cells and non-neoplastic breast tissue was quantitatively evaluated by Photoshop-bascd image analysis techniques (J Histochem Cytochem 45:1559, 1997). Likewise, ICC results were semiquantitatively scored in tumor cells and non-neoplastic breast tissue on a scale of 0 to 4. FISH was done using the Oncor Inform" kit (BIDMC) and results were quantified as number of fluoresc ent signals per tumor cell nucleus (0-20). ~: We found a high degree of correlation between ICC results by image analysis and by semiquantitative scoring (r 2=0.76). FISH results correlated with ICC results when the immunostaining intensity of only tumor cells was assessed (r2=O.43 for ICC scoring and r2=0.38 for ICC image analysis), and significantly better when the difference in staining intensity between tumor cells and non-neoplastic breast tissue was assessed (r 2=O.52 for both ICC scoring and image analysis ). The correlation with FISH results was further improved when ICC was combined with HIER (r2 0.60 for ICC scoring and r2=O .58 for ICC image analysis). P values of all given rZ-values were <0.00 1, as assessed by Spearman 's regression analysis. Conclusion: Although ICC and FISH assess different aspects of the Her2/neu gene (overexpres sion vs. amplification), we found a good correlation between the two diagnostic techniques. The most accurate assessment of HER2Ineu overexpression is obtained when ICC is combined with HIER and assessed by image analysis or semiquantitative scoring as the difference between tumor cells and non-neoplastic breast tissue.

PROGNOSTIC SIGNIFICANCE OF GENOMIC ALTERATIONS IN pTalpTl BLADDER CANCER 1. Richter (a.G.), J. Zhaou (a.G.), U. Wagner (a.G.), Th.C.Gasser* (a.G.), H.Moch, MJ. Mihatsch, G.Sauter Institute of Pathology and Urologic Clinics*, University of Basel, Switzerland

=

106 Specific genetic alterations reflect morphologically different types of ductal carcinoma in situ (DCIS) and invasive breast cancer. Horst BUrger (a.G.), Friedrich Otterbaeh (a.G.), Ronald Simon (a.G.), Raihanatou Diallo (a.G.), Christopher Poremba (a.G.), Barbara DockhornDwomiczak (a.G.), Werner Boeker, Institute of Pathology, University of Muenster, Germany Aims: DCIS is presumed to be a precursor lesion of invasive breast carcinoma. Yet a genetic correlation between DCIS and morphologically different subtypes of invasive breast cancer could not be established so far. Material and Methods: We investigated 50 cases of DCIS after microdissection and 80 cases of invasive breast cancers of different subtypes by means of comparative genomic hybridization (CGH). 10 cases of ductal invasive carcinoma G 3 were also investigated by fluorescencein-situ-hybridization (FISH) with probes for centromere II and Ilq13 . Results : The loss of chromosomal material of 16q associated with well and intermediately-differentiated DCIS is statistically significant. Grade 1 and 2 invasive breast cancer also revealed a much higher proportion of 16qlosses. Poorly-differentiated DCIS and invasive tumours with higher tumour grades in contrast, revealed a larger amount of amplifications (I1ql 3, 17qI2). Cases of ductal invasive carcinoma G3 with 16q-losses were mostly aneuploid and showed a much higher average number of aberrations per cases in contrast to ductal invasive carcinoma G 3 without l6q-losses. Tubular and tubulo-Iobular invasive breast cancer showed a similar pattern of quantitative and qualitative genetic alterations as welldifferentiated DCIS. Conclusions: There is evidence of multiple genetic pathways in the tumorigenesis of DCIS and invasive breast cancer. 16q-losses seem to be a hallmark in the pathogenesis of well and intermediately-differentiated DCIS and breast tumours of lower tumour grade. Poorly-differentiated DCIS seem to represent a distinct entity rather than the final stage of a evolutionary progress via well and intermediately-differentiated DCIS and genetic alterations show a high degree of homology with ductal invasive carcinoma Grade3.

Aims: Progression of "superficial" urinary bladder cancer (stage pTalpTl) is driven by a malfunction of specific genes, most of which are yet unknown . Cytogenetic changes are of interest since they may pinpoint the location of novel oncogenes and tumor suppressor genes . It was the aim of this study to evaluate the prognostic significance of cytogenetic changes in superficial bladder cancer. Methods: 95 pTa tumors and 54 pTI carcinomas were examined by comparative genomic hybridization (CGH). CGH allows the identification of all relative DNA copy number gains and losses of a tumor in one examination. Clinical follow up information was available for all patients. Results: pTa tumors had a low number of alterations (pTaGI: meanl.9, pTaG2 : 3.1) with 9q- (44%), 9p- (36%), and -Y (21%) being most prevalent. Neither the total number of alterations nor any of the individual changes were linked to patient prognosis in pTa tumors. pTI carcinomas (mean: 6.7) had markedly more alterations than pTa tumors. The most prevalent changes included Iq+ (37%), 2q- (19 %), 6p+ (15%), 8p(24%), 8q+ (28%), 9p- (3 1%), 9q- (28%), IIp- (28 %), IIq- (26 %), l3q(19 %), 17q21+ (28%), 2Oq+ (33%), and -Y (26 %). 3p+ , 4p-, 5p+ , 5q- , 6q-, IOq-, and 18q- were significantly associated with subsequent tumor progression to muscle invasion (stage pT2-4) . Conclusions: A malfunction of genes located at 3p, 4p, 5p, Sq, 6q, IOq, and 18q may contribute to bladder cancer progression. The dramatic genetic differences between the stages pTa and pTi challenge the concept of combining these stages in one group as "superficial" bladder cancer .

108 COPY NUMBER CHANGES OF CHROMOSOME I I IN SUPERFI· CIAL BLADDER CANCER R. Simon (a.G.), Christian Steidl (a.G.), H. BUrger (a.G.), E. Eltze (a.G.),

L. Hertle (a.G.), W. Boeker and H.-1. Terpe '

Gerhard-Domagk-Institute of Pathology , University of Munster I Institute of Pathology, Klinikum Leverkusen Aim: Alterations of chromosome I I , especially deletions of IIpl5 indicating the presence of one or more suppressor loci in this region and amplifications of I IqI3 have been frequently identified in bladder cancer. Methods: To localize the lip deletion target(s) more precisely and to analyse a possible role of lip and I Iq13 for invasive tumor behaviour, 46 non-invasive (pTa) and invasive (pT!) transitional cell carcinomas of the bladder were analyzed for copy number changes of these loci by means of comparative genomic hybridization (CGH), fluorescence in-situ hybridization (FISH) and microsatellite analysis (loss of heterozygosity, LOH). Results: I) Losses of chromosome lip material were significantly more frequent in pTa (2/20;10%) than in pTi (15/26; 58%) tumors (p
304 . Oral Presentation

109 EVALUATION OF PROGNOSTIC MARKERS IN URINARY BLADDER CANCER USING TUMOR TISSUE ARRAYS. U.Wagner (a .G .), H.Moch, J.Kononen (a .G .), L.Bubendorf (a .G.), T .C.G asser (a .G .), M .J. Mihatsch , O.P.Kallion iemi (a.G.) , G .Sauter. Institut e of Pathology and Clinics of Urol ogy, Unive rsity of Basel , Switzerland. National Hu man Genome Research Institute, Lab oratory for Cancer Genetics, Bethesda, MD .

111

Aims: A rapid ly growing number o f gen es is known to have an altered function in at least a fraction of bladder carcinomas. The evalu atio n of their potential prognostic significanc e would be time consuming and expens ive using traditi on al method s. The tissue microarray approach (" tissue chips") co uld drastically faci ltate such studies (Kononen et aI, Na t. Med , 4: 844 -7) . In this techn ique , 0.6 mm d iameter tum or biop sies are retrie ved from selected regions of arch ival tissue block s, and hundreds of such cylindrical samples are subsequently arra yed in a new paraffin block. Sec tions of arrayed tum or samples permi t a high throughput analysis of multiple target s at the DNA, RNA , or protein level. Th e aim of this study was to evaluate the potential of tissue arrays to identify progn ostic molecular markers in urinary bladder cancer. Methods: A tumor array was co nst ruc ted co ntai ning 127 pTa, 81 pT I , and 128 pT2 -4 bladder carci nomas with follow up information. Both con ventional large sec tions from each individual tumor block and an array section were imm unostained for p53 (0 0-7), a protein known to be link ed to adva nced tum or stage and poor prognosis in bladder canc er. Results: On co nve ntional sections a po sitive p53 imm unostaining was found in 105 of 3 15 tum ors, and p53 positi vity was strongly link ed to poor outco me (p
UROT HELIAL CARCINOMAS OF TH E URET ER SHO W A HIGH FREQUENCY OF MICRO SATELLITE INSTABILITY AND LOSS OF THE MISMATCH REPAI R PROTEINS HMSH2 AND HM LHl A. Hartmann ', L. Zanardo', W. Dietm aier ', H. Blaszyk' , J. Ruschoff", F. Hofstaedter' , R. Knuechel'. 'In st. of Patho logy, Universi ty Regensburg; ' Dept. Lab. Medicine and Pathology. Mayo Clinic, Rochester and ' lnst. of Pat hology, Kassel. Aims : Urothelial carcinomas of the upper urinary tract occur frequently in patients with the HNPCC syndrome. The aim of this study was to determi ne the prevalence of microsatellite insta bility and loss of mismatch repair protein expression in unselected carcinomas of the ureter and renal pelvis. Methods: The study compared in total 68 patients with renal pelvis tumors (n=37), ureteral tumors (n=21) and simultaneous tumors on both sites (n= IO). Normal and tumor tissue was microdissected from deparaffiniz ed slides using manual or laser microdi ssection (PALM). A set of 6 microsatellite markers (BAT25, BAT26, BAT40, APC, D2S 123, 0 17S250) was amplified by PCR and visualized by PA gel electrophoresis and silver staining. Expressi on ofhMSH2 (Ab-2,cloneFE I I.Calbiochem ) and hMLH I (cloneGI68-15,Pharmingen) was determined by immunohistochemi stry. Results: Microsatellite instability (MS I) at two or more mark ers was found in 39% of ureteral carcinomas (12/31), but only in 8% of renal pelvis tumors (3/37 -p=O,002) . All MSI-positive renal pelvis tumors had loss of hML HI expression (n=3). 10 of 12 MSI-pos itive ureteral tumors showed loss of hMSH2 (n=8) or hMLH I (n=2). All MSI-nega tive tumors show ed normal protein expression. Mononuc leotide poly-A repeats were the most informative markers and detected all MSI-positive cases . There was a family history of multiple colorectal andlor ovarian carcinomas in 4 MSIpos itive cases, and in one patient a gennline mutation in hMSH2 was found. Conclusions: Microsat ellite instability and loss of hMSH2 or hML H I is a frequent event in carcinomas of the ureter. This is the highest frequency found in spora dic unselected tumors of any histo logic type. Screenin g of these patients including a detailed family history and applying microsatellite analyses and immunohistoc hemistry for hMSH2 and hMLHI is warranted.

110

112 Detection of 9p deletions in different renal carcinoma subtypes by microsatellite analysis P. Schraml (a. G.)*, R. Bednar (a.G.)*, D. Muller (a.G .)*, Th . Gasser (a.G.)**, J. Richte r (a.G.)*, G. Saute r*, M J . Mihatsch*, H. Moch* *Institute for Path ology, **C li nics for Uro logy , University Basel , Schweiz Aims : Th e clinical beh avior of rena l cell ca rci noma (RCC) cann ot be predicted by histology alone. Recentl y, it has bee n sugges ted that ge nes on c hromoso me 9p are invo lved in the pa thogen esis o f RCC. T umor supp ressor gene ca ndida tes on these ch romosoma l arms include the pl 5 and p l6 genes on 9p21. The ai ms o f thi s stu dy were to de term ine prevalence and progn ostic significance of 9p delet ions in RCC . Methods: DNA was extracted from form alin-fix ed and paraffin embedded mate rial of 88 clear cell and 37 papillary RCC. The tum ors have been examin ed for allelic loss at chro mosome 9p I2- 13, 9p21 and 9p22 regions by four micr osa tellites (09S 156 , 09S171 , D95970, 0951748). The prognostic sig nifi ca nce of 9p deletion s was eva lua ted by the Kapl anMeier statistic (log rank test). Results: C hro moso me 9p deletions were detected in 2 1 of 88 clea r cell (24% ) and in 7 of 37 papillary RCC (19 %) . 9p de letions we re associated with high histo logic grade (p<0.05) and poor patient progn osis (p=O.OI) in papillary RCC. There was no assoc iation bet ween 9p delet ion and tumor stage, histologic grade or patient prognosis in cle ar cell RCC. However , 9p deletions were associated with poor patie nt prognosis in the subgroup of pT3 clea r ce ll RCC (p