- Email: [email protected]
Predictive array analysis of breast cancer
Symposium: Breast Pathology / Pathology - Research and Practice 200 (2004) 299-302
16 7
Testicular Dysgenesis and Carcinoma in situ Testis
N. E. SKAKKEBYEK, Copenhagen
168 Prostate carcinoma. A continuing diagnostic problem B. HELPAP, Singen
169 Molecular pathology of the prostate carcinoma. Academic playground or guide for diagnosis or therapy planning? L, BUBENDORF, Basel
170
Breast cancer in the male. Thinking about it!
A. REINER, Wien
1 7 1 I G F and IGFBP expression in human seminoma and normal testicular tissue T.P. NEUVIANS, C.G. SAUER, U. BLEYL, R. GROBHOLZ Pathologisches lnstitut, Universit~itsklinikum Mannheim der Universit~it Heidelberg
Aims: Insulin-like growth factors (IGF) are known to stimulate growth and cell proliferation. 1GF 11 has been shown to exert mitogenic effects on several cancers such as ovarian, breast and prostate cancer. Little is known about IGFs and their binding proteins (IGFBP) in germ cell cancer. Methods: Tissue of 9 normal testis were gained from autopsy cases, and classical pure seminoma specimens were obtained from 22 patients. Real-time RT-PCR was used to examine the mRNAexpression of IGF I, IGF 11, IGF-receptor type 1 (IGFR-1), IGFR-2 and 1GFBP 1-6. Further, 1GF I, 1GF II and IGFBP 5 were localised in normal tissue and seminoma by immunohisfochemistry. Results: IGF I, IGFR-1, 1GFR-2 and IGFBP 2 showed no significant changes in m R N A expression. Both in normal testicular tissue and seminomas 1GF II was markedly stronger expressed than IGF I, but was down-regulated by a factor of 16 in seminomas. The IGF I1 scavenger receptor 1GF-R2 was only very weakly expressed in both groups. IGFBP 1,4, 5 and 6 expressions were down-regulated in seminomas. 1GFBP 5 was maximally down-regulated in pT2seminomas by a factor of 4. When comparing pT1- to pT2-seminomas the expression of the mainly inhibiting IGFBP 3 and the potentially proapoptotic IGFBP 5 were down-regulated in pT2-tumours. Conclusions: In conclusion, all investigated members of the 1GFsystem are expressed in human testicular tissue and seminomas. IGF II seems to have a major impact on the function of normal testicular tissue. The marked down-regulation of 1GF II may have an impact on the favourable prognosis of human seminoma. IGFBP 3 and 5 seems to be involved in the progression to an angioinvasive stage.
299
172 Ofiling of differentially expressed proteins in normal and malignant breast tissue by high throughput antibody microassay - a pilot study G. HUDELIST 1'2,M. PACHER-ZAVISIN l , C. SINGER 1, T. HOLPER 3, E. KUBISTA 1, M. SCHREIBER 1, M. BILBAN 4, K. CZERWENKA 3 1Abteilung ftir Spezielle Gyngkologie, UFK Wien 2Abteilung ftir GynS~kologie u. Geburtshilfe, LKH Villach 3 Institut ftir Klinische Pathologie, Universit~it Wien 4 Institut ftir Labormedizin, Universit~it Wien
Aims: Genes work at the protein level and, since the transcriptional activity of a gene does not necessarily reflect cellular protein expression, the identification and quantification of proteins is essential for the understanding of molecular events leading to malignant transformation. Methods: We have employed a high-throughput protein microarray system which contains 378 well-characterized monoclonal antibodies in order to compare the gene expression pattern of malignant and adjacent normal breast tissue in a patient with primary breast cancer. Results: We have identified a number of proteins that show increased expression levels in malignant breast tissues such as casein kinase Ie, p53, annexin XI, CDC25C, eIF-4E and MAP kinase 7. The expression of other proteins, such as the the multifunctional regulator 14-3-3e was found to be decreased in malignant mammary tissue, whereas the majority of proteins remained unchanged when compared to the corresponding normal breast tissue. The protein expression pattern was confirmed by immunohistochemistry for 8 representative proteins known to be involved in carcinogenesis. Conclusions: Taken together, we have described for the first time a tumor cell specificity protein expression pattern by use of a novel commercially available antibody microarray. We hypothesize that the use of protein arrays will not only increase our understanding of the molecular events but could prove useful in evaluating prognosis and in determining optimal antineoplastic therapy.
1 7 3
Predictive array analysis of breast cancer
K. SPECHT, J. SMIDA 1, J. NAHRIG, U. REICH t, U. SCHNITZBAUER 1 , M. MADER 2 , J. BUDCZIES 2 , S. TORNOW 2 , N. HARBECK 3 , M. KIECHLE 3 , H. HOFLER 2 Insfitut ftir Pathologie, Technische Universit~it Mtinchen 1Institut ftir Pathologie, GSF-Forschungszentrum, Neuherberg 2Institut fur Bioinformatik, GSF Forschungszentrum 3Franenklinik und Poliklinik, Klinikum Reehts der lsar der TU Miinchen
Aims: Breast cancer patients with the same stage of disease can have different treatment responses and overall clinical behavior and outcome. We aimed to predict disease outcome by looking at gene expression patterns of primary breast tumors. Methods: Fifty-nine cases of primary breast tumors were analyzed whose clinical course was followed for 6 up to 13 years. Samples were microdissected and aRNA was hybridized to Affymetrix U133A and B oligonucleotide arrays. Data were analyzed using 3 different statistical algorithms to assess groups of genes that predict
300
Symposium: Breast Pathology / Pathology - Research and Practice 200 (2004) 299-302
the clinical phenotype of individual patients. A customized cDNA microarray chip, containing the predictor set, and in addition, 1200 sequence verified, mostly cancer-associated cDNA clones was used to validate Affymetrix gene expression data. Results: A set of 169 genes was identified that is capable of predicting disease recurrence in lymph-node negative breast cancer patients with about 83% accurracy. The set includes genes associated with cell cycle, cell growth and genomic instability. Interestingly, this predictor set shows only minimal overlap with one reported recently by another recurrence study on breast cancer. The prognosis set is now being validated by custom made cDNA microarray analysis of an independent set of breast tumors with known clinical follow-up. Conclusions: Gene expression patterns are a powerful predictor of disease outcome in breast cancer patients and may be more accurate than clinical and histological criteria.
174
A polymorphic sequence in the egfr gene is a genetic modifier of breast cancer risk in young women H. BI[IRGER, S. HERMANN a, K. STRAIF2, N. TIDOW 3, J. CHANG-CLAUDE 1, B. BRANDT 3 Institut ftir Pathologie, Universit~it Mtinster 1Deutsches Krebsforschungszentrnm Heidelberg 2International Agency for Cancer Research, WHO, Lyon 3Institute for Klinische Chemie und Laboratoriumsmedizin, Universit~it MiJnster Aims: Our recent data on a polymorphic CA repeat within intron 1 of the egfr gene (CA-SSR) pointed to a possible inheritance of cancer risk associated with the egfr gene. Material: The relationship between the egfr polymorphism and breast cancer risk was assessed. Cases with a first primary breast cancer by age 50 years and age-matched population controls were interviewed to record known and suspected risk factors. The allelic length of the egfr CA SSR was determined in 616 cases and 1072 population sampled controls. Genotypes were categorized for analysis by allele size. Multivariate logistic regression was used to compare genotype distributions, accounting for other risk factors, and to investigate gene-environment interactions. Results: We did not find an association of the allelic length of the egfr polymorphism,with breast cancer risk. However, we observed a significantly elevated odds ratio of 10.4 (95%CI 1.85-58.70) for the presence of two long alleles (predominantly 19 CA repeats) of the egfr polymorphism among women with a first-degree family history of breast cancer but not among women without family history (p = 0.015 for interaction). The risk increase associated with high red meat consumption and the protective effect of high vegetable intake was also most pronounced among carders of two 19 CA alleles. Conclusions: The length of the egfr CA SSR may be a causal factor for familiar breast cancers and its effect could be modulated by dietary factors.
175
Comparative methylation profiling of lobular and ductal breast cancer U. LEHMANN, P. AHREND, F. LANGER, H. KREIPE Institut ftir Pathologie, Medizinische Hochschnle Hannover, D30625 Hannover, Germany Aims: The relationship between epigenetic inactivation of growth regulatory genes and the histomorphological phenotype of tumour
specimens is still poorly understood. Therefore, a systematic comparative methylation analysis of lobular-invasive and invasive-ductal breast cancer was set up. Methods: Real-time PCR-based quantitative methylation assays were developed for the following genes: p16 zNK4a, cyclinD2, RASSF1A, GSTM, RIZ-1, HIN-1, APC, DAP-kinase. Bisulfit treated genomic DNA isolated from snap-frozen and formalin-fixed paraffin-embedded breast cancer specimens were analyzed. Intraductal tumor cells were isolated by laser-assisted rnicrodissection. Altogether 41 lobular breast cancer specimen and 50 invasive ductal carcinoma of the breast were analyzed. Results: In both groups of carcinomas the frequency of promoter hypermethylation varied between very low (e.g. p16 INK4a,<10%) and very high (RASSF1A, >80%). Also the quantitative methylation levels varied between less than 1% and close to 100%. In addition, characteristic differences between intraductal and invasive tumour cells could be observed. The DAP kinase gene methylation showed a statistically significant association with the lobular subtype, GST~tl hypermethylation only a trend towards the lobular subtype (64% versus 36%). The methylation of the other genes did not show any preference for a histological subtype. Conclusions: In addition to genetic events lobular and ductal breast cancer show characteristic differences in some genes inactivated by hypermethylation of promoter regions. Epigenetic inactivation in breast cancer is a very gene-specific phenomenon displaying characteristic quantitative changes during progression.
176
MMP expression analysis in human breast cancer by real-time quantitative RT-PCR: methodological and Biological aspects A. LEBEAU, H. HESSEL, P. Mf,)LLER, U. SAUER, U. L~)HRS Pathologisches Institut der LMU Mtinchen Aims: Matrix metalloproteinases (MMPs) are assumed to play an important role in the progression of human breast cancer due to their ability to degrade collagen type IV. In previous in situ hybridisation studies we observed different expression patterns of distinct MMPs in intraductal and invasive tumour growth. Therefore our aim was to quantify MMP gene expression in human breast cancer by real-time quantitative RT-PCR. The use of one house-keeping gene is a common practise for norrnalisation of quantitative RTPCR on human tissue. However, in vitro housekeeping gene expression has proven to vary considerably. We compared different housekeeping genes to study their influence on the quantification of MMP expression in human breast cancer. Methods: Relative expression analysis for MMP-2 and -3 was performed by using the LightCycler~ system (Roche Diagnostics) on m~crodissected paraffin sections of 31 human breast carcinomas. Various housekeeping genes including GAPDH and Cyclophilin B were tested for normalisation. Results: The calculated relative expression ratios for MMP-2 and 3 were depending on the used housekeeping genes. Comparison between the different internal controls revealed no consistent pattern, e.g. we observed a great variability between individuals. Consequently, the correlation between MMP-2 and -3 relative expression ratios and clinical or pathomorphological parameters hinged on the choice of housekeeping gene. Conclusion: Our results suggest that the use of internal standards comprising single housekeeping gene is inappropriate for studies involving tissue biopsies. Normalization against a panel of internal reference genes seems to be a suitable alternative.
Supported by grants from the DFG (SFB 469) and the CurtBohnewand-Fond (LMU Miinchen).
16 7
Testicular Dysgenesis and Carcinoma in situ Testis
N. E. SKAKKEBYEK, Copenhagen
168 Prostate carcinoma. A continuing diagnostic problem B. HELPAP, Singen
169 Molecular pathology of the prostate carcinoma. Academic playground or guide for diagnosis or therapy planning? L, BUBENDORF, Basel
170
Breast cancer in the male. Thinking about it!
A. REINER, Wien
1 7 1 I G F and IGFBP expression in human seminoma and normal testicular tissue T.P. NEUVIANS, C.G. SAUER, U. BLEYL, R. GROBHOLZ Pathologisches lnstitut, Universit~itsklinikum Mannheim der Universit~it Heidelberg
Aims: Insulin-like growth factors (IGF) are known to stimulate growth and cell proliferation. 1GF 11 has been shown to exert mitogenic effects on several cancers such as ovarian, breast and prostate cancer. Little is known about IGFs and their binding proteins (IGFBP) in germ cell cancer. Methods: Tissue of 9 normal testis were gained from autopsy cases, and classical pure seminoma specimens were obtained from 22 patients. Real-time RT-PCR was used to examine the mRNAexpression of IGF I, IGF 11, IGF-receptor type 1 (IGFR-1), IGFR-2 and 1GFBP 1-6. Further, 1GF I, 1GF II and IGFBP 5 were localised in normal tissue and seminoma by immunohisfochemistry. Results: IGF I, IGFR-1, 1GFR-2 and IGFBP 2 showed no significant changes in m R N A expression. Both in normal testicular tissue and seminomas 1GF II was markedly stronger expressed than IGF I, but was down-regulated by a factor of 16 in seminomas. The IGF I1 scavenger receptor 1GF-R2 was only very weakly expressed in both groups. IGFBP 1,4, 5 and 6 expressions were down-regulated in seminomas. 1GFBP 5 was maximally down-regulated in pT2seminomas by a factor of 4. When comparing pT1- to pT2-seminomas the expression of the mainly inhibiting IGFBP 3 and the potentially proapoptotic IGFBP 5 were down-regulated in pT2-tumours. Conclusions: In conclusion, all investigated members of the 1GFsystem are expressed in human testicular tissue and seminomas. IGF II seems to have a major impact on the function of normal testicular tissue. The marked down-regulation of 1GF II may have an impact on the favourable prognosis of human seminoma. IGFBP 3 and 5 seems to be involved in the progression to an angioinvasive stage.
299
172 Ofiling of differentially expressed proteins in normal and malignant breast tissue by high throughput antibody microassay - a pilot study G. HUDELIST 1'2,M. PACHER-ZAVISIN l , C. SINGER 1, T. HOLPER 3, E. KUBISTA 1, M. SCHREIBER 1, M. BILBAN 4, K. CZERWENKA 3 1Abteilung ftir Spezielle Gyngkologie, UFK Wien 2Abteilung ftir GynS~kologie u. Geburtshilfe, LKH Villach 3 Institut ftir Klinische Pathologie, Universit~it Wien 4 Institut ftir Labormedizin, Universit~it Wien
Aims: Genes work at the protein level and, since the transcriptional activity of a gene does not necessarily reflect cellular protein expression, the identification and quantification of proteins is essential for the understanding of molecular events leading to malignant transformation. Methods: We have employed a high-throughput protein microarray system which contains 378 well-characterized monoclonal antibodies in order to compare the gene expression pattern of malignant and adjacent normal breast tissue in a patient with primary breast cancer. Results: We have identified a number of proteins that show increased expression levels in malignant breast tissues such as casein kinase Ie, p53, annexin XI, CDC25C, eIF-4E and MAP kinase 7. The expression of other proteins, such as the the multifunctional regulator 14-3-3e was found to be decreased in malignant mammary tissue, whereas the majority of proteins remained unchanged when compared to the corresponding normal breast tissue. The protein expression pattern was confirmed by immunohistochemistry for 8 representative proteins known to be involved in carcinogenesis. Conclusions: Taken together, we have described for the first time a tumor cell specificity protein expression pattern by use of a novel commercially available antibody microarray. We hypothesize that the use of protein arrays will not only increase our understanding of the molecular events but could prove useful in evaluating prognosis and in determining optimal antineoplastic therapy.
1 7 3
Predictive array analysis of breast cancer
K. SPECHT, J. SMIDA 1, J. NAHRIG, U. REICH t, U. SCHNITZBAUER 1 , M. MADER 2 , J. BUDCZIES 2 , S. TORNOW 2 , N. HARBECK 3 , M. KIECHLE 3 , H. HOFLER 2 Insfitut ftir Pathologie, Technische Universit~it Mtinchen 1Institut ftir Pathologie, GSF-Forschungszentrum, Neuherberg 2Institut fur Bioinformatik, GSF Forschungszentrum 3Franenklinik und Poliklinik, Klinikum Reehts der lsar der TU Miinchen
Aims: Breast cancer patients with the same stage of disease can have different treatment responses and overall clinical behavior and outcome. We aimed to predict disease outcome by looking at gene expression patterns of primary breast tumors. Methods: Fifty-nine cases of primary breast tumors were analyzed whose clinical course was followed for 6 up to 13 years. Samples were microdissected and aRNA was hybridized to Affymetrix U133A and B oligonucleotide arrays. Data were analyzed using 3 different statistical algorithms to assess groups of genes that predict
300
Symposium: Breast Pathology / Pathology - Research and Practice 200 (2004) 299-302
the clinical phenotype of individual patients. A customized cDNA microarray chip, containing the predictor set, and in addition, 1200 sequence verified, mostly cancer-associated cDNA clones was used to validate Affymetrix gene expression data. Results: A set of 169 genes was identified that is capable of predicting disease recurrence in lymph-node negative breast cancer patients with about 83% accurracy. The set includes genes associated with cell cycle, cell growth and genomic instability. Interestingly, this predictor set shows only minimal overlap with one reported recently by another recurrence study on breast cancer. The prognosis set is now being validated by custom made cDNA microarray analysis of an independent set of breast tumors with known clinical follow-up. Conclusions: Gene expression patterns are a powerful predictor of disease outcome in breast cancer patients and may be more accurate than clinical and histological criteria.
174
A polymorphic sequence in the egfr gene is a genetic modifier of breast cancer risk in young women H. BI[IRGER, S. HERMANN a, K. STRAIF2, N. TIDOW 3, J. CHANG-CLAUDE 1, B. BRANDT 3 Institut ftir Pathologie, Universit~it Mtinster 1Deutsches Krebsforschungszentrnm Heidelberg 2International Agency for Cancer Research, WHO, Lyon 3Institute for Klinische Chemie und Laboratoriumsmedizin, Universit~it MiJnster Aims: Our recent data on a polymorphic CA repeat within intron 1 of the egfr gene (CA-SSR) pointed to a possible inheritance of cancer risk associated with the egfr gene. Material: The relationship between the egfr polymorphism and breast cancer risk was assessed. Cases with a first primary breast cancer by age 50 years and age-matched population controls were interviewed to record known and suspected risk factors. The allelic length of the egfr CA SSR was determined in 616 cases and 1072 population sampled controls. Genotypes were categorized for analysis by allele size. Multivariate logistic regression was used to compare genotype distributions, accounting for other risk factors, and to investigate gene-environment interactions. Results: We did not find an association of the allelic length of the egfr polymorphism,with breast cancer risk. However, we observed a significantly elevated odds ratio of 10.4 (95%CI 1.85-58.70) for the presence of two long alleles (predominantly 19 CA repeats) of the egfr polymorphism among women with a first-degree family history of breast cancer but not among women without family history (p = 0.015 for interaction). The risk increase associated with high red meat consumption and the protective effect of high vegetable intake was also most pronounced among carders of two 19 CA alleles. Conclusions: The length of the egfr CA SSR may be a causal factor for familiar breast cancers and its effect could be modulated by dietary factors.
175
Comparative methylation profiling of lobular and ductal breast cancer U. LEHMANN, P. AHREND, F. LANGER, H. KREIPE Institut ftir Pathologie, Medizinische Hochschnle Hannover, D30625 Hannover, Germany Aims: The relationship between epigenetic inactivation of growth regulatory genes and the histomorphological phenotype of tumour
specimens is still poorly understood. Therefore, a systematic comparative methylation analysis of lobular-invasive and invasive-ductal breast cancer was set up. Methods: Real-time PCR-based quantitative methylation assays were developed for the following genes: p16 zNK4a, cyclinD2, RASSF1A, GSTM, RIZ-1, HIN-1, APC, DAP-kinase. Bisulfit treated genomic DNA isolated from snap-frozen and formalin-fixed paraffin-embedded breast cancer specimens were analyzed. Intraductal tumor cells were isolated by laser-assisted rnicrodissection. Altogether 41 lobular breast cancer specimen and 50 invasive ductal carcinoma of the breast were analyzed. Results: In both groups of carcinomas the frequency of promoter hypermethylation varied between very low (e.g. p16 INK4a,<10%) and very high (RASSF1A, >80%). Also the quantitative methylation levels varied between less than 1% and close to 100%. In addition, characteristic differences between intraductal and invasive tumour cells could be observed. The DAP kinase gene methylation showed a statistically significant association with the lobular subtype, GST~tl hypermethylation only a trend towards the lobular subtype (64% versus 36%). The methylation of the other genes did not show any preference for a histological subtype. Conclusions: In addition to genetic events lobular and ductal breast cancer show characteristic differences in some genes inactivated by hypermethylation of promoter regions. Epigenetic inactivation in breast cancer is a very gene-specific phenomenon displaying characteristic quantitative changes during progression.
176
MMP expression analysis in human breast cancer by real-time quantitative RT-PCR: methodological and Biological aspects A. LEBEAU, H. HESSEL, P. Mf,)LLER, U. SAUER, U. L~)HRS Pathologisches Institut der LMU Mtinchen Aims: Matrix metalloproteinases (MMPs) are assumed to play an important role in the progression of human breast cancer due to their ability to degrade collagen type IV. In previous in situ hybridisation studies we observed different expression patterns of distinct MMPs in intraductal and invasive tumour growth. Therefore our aim was to quantify MMP gene expression in human breast cancer by real-time quantitative RT-PCR. The use of one house-keeping gene is a common practise for norrnalisation of quantitative RTPCR on human tissue. However, in vitro housekeeping gene expression has proven to vary considerably. We compared different housekeeping genes to study their influence on the quantification of MMP expression in human breast cancer. Methods: Relative expression analysis for MMP-2 and -3 was performed by using the LightCycler~ system (Roche Diagnostics) on m~crodissected paraffin sections of 31 human breast carcinomas. Various housekeeping genes including GAPDH and Cyclophilin B were tested for normalisation. Results: The calculated relative expression ratios for MMP-2 and 3 were depending on the used housekeeping genes. Comparison between the different internal controls revealed no consistent pattern, e.g. we observed a great variability between individuals. Consequently, the correlation between MMP-2 and -3 relative expression ratios and clinical or pathomorphological parameters hinged on the choice of housekeeping gene. Conclusion: Our results suggest that the use of internal standards comprising single housekeeping gene is inappropriate for studies involving tissue biopsies. Normalization against a panel of internal reference genes seems to be a suitable alternative.
Supported by grants from the DFG (SFB 469) and the CurtBohnewand-Fond (LMU Miinchen).