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PDF file - 121K, Table 1. Supplemental Table 1. Comparison of the Patients Analyzable for VEGFA Amplification to All Patients Enrolled on E2100 Table 2. Estimated Progression Free Survival treatment hazard ratios (arm A vs. arm B) and 95% confidence intervals for various subsets. Comparison between the cases analyzable for VEGFA amplification and all cases in E2100

Purpose: Clinically validated biomarkers for anti-angiogenesis agents are not available. We have previously reported associations between candidate VEGFA single-nucleotide polymorphisms (SNP) and overall survival (OS) in E2100. The associations between tumor VEGFA amplification and outcome are evaluated here.Experimental Design: E2100 was a phase III trial comparing paclitaxel with or without bevacizumab for patients with metastatic breast cancer. FISH to assess gene amplification status for VEGFA was conducted on paraffin-embedded tumors from 363 patients in E2100. Evaluation for association between amplification status and outcomes was conducted.Results: Estrogen receptor (ER)+ or progesterone receptor (PR)+ tumors were less likely to have VEGFA amplification than ER/PR− tumors (P = 0.020). VEGFA amplification was associated with worse OS (20.2 vs. 25.3 months; P = 0.013) in univariate analysis with a trend for worse OS in multivariate analysis (P = 0.08). There was a significant interaction between VEGFA amplification, hormone receptor status, and study arm. Patients with VEGFA amplification and triple-negative breast cancers (TNBC) or HER2 amplification had inferior OS (P = 0.047); amplification did not affect OS for those who were ER+ or PR+ and HER2−. Those who received bevacizumab with VEGFA amplification had inferior progression-free survival (PFS; P = 0.010) and OS (P = 0.042); no association was seen in the control arm. Test for interaction between study arm and VEGFA amplification with OS was not significant.Conclusion:VEGFA amplification in univariate analysis was associated with poor outcomes; this was particularly prominent in HER2+ or TNBCs. Additional studies are necessary to confirm the trend for poor OS seen on multivariate analysis for patients treated with bevacizumab. Clin Cancer Res; 19(5); 1281–9. ©2012 AACR.

PURPOSE: The DBCG 77C trial compared one year of tamoxifen in postmenopausal, steroid-receptor unknown, high-risk breast cancer patients to no adjuvant systemic therapy. After a potential follow-up of 30 years we report overall efficacy for the study and results according to subtypes subsequently assessed by immunohistochemistry and FISH. METHODS: Between 1977 and 1982, 1716 postmenopausal patients with tumors larger than 5 cm or positive axillary nodes were randomly assigned to no systemic therapy or tamoxifen 30 mg daily for one year. The main study is reported as an ITT analysis with the predefined DFS and BCM as endpoint. For multivariate analysis the Cox proportional hazards regression model was applied to assess the adjusted hazard ratio of treatment regimen, and to explore interactions. Formalin-fixed, paraffin-embedded primary breast tumor tissue blocks were available from 1548 (90%) of the 1716 participants enrolled and 1428 were assessable for ER, PR, HER2 and Ki67. The hormone receptor positive (ER and/or PR) cancers were defined as luminal A if Ki67 low and HER2-negative; as luminal B if Ki67 high or HER2-positive; and otherwise as HER2 positive or triple negative. RESULTS: In the intent-to-treat (ITT) population one year of tamoxifen improved the disease-free-survival (DFS) (Hazard ratio (HR) = 0.87; 95% CI 0.77-0.98), the recurrence-free-survival (RFS) (HR = 0.79; 0.69-0.90) and reduced the breast-cancer-specific-mortality (BCM) (HR = 0.83; 0.73-0.93). Recurrence-free survivals were improved significantly by tamoxifen in luminal A (HR = 0.66; 0.53-0.84) and luminal B/HER2- (HR = 0.54; 0.39-0.74) but not in the other subsets, and with similar results for BCM with 30 years follow-up. CONCLUSION: One year of treatment with tamoxifen significantly improves RFS and BCM in postmenopausal patients with ER positive breast cancers. The benefit from tamoxifen was not significantly different in luminal A and B subtypes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-13-03.

Purpose: Clinically validated biomarkers for anti-angiogenesis agents are not available. We have previously reported associations between candidate VEGFA single-nucleotide polymorphisms (SNP) and overall survival (OS) in E2100. The associations between tumor VEGFA amplification and outcome are evaluated here. Experimental Design: E2100 was a phase III trial comparing paclitaxel with or without bevacizumab for patients with metastatic breast cancer. FISH to assess gene amplification status for VEGFA was conducted on paraffin-embedded tumors from 363 patients in E2100. Evaluation for association between amplification status and outcomes was conducted. Results: Estrogen receptor (ER)+ or progesterone receptor (PR)+ tumors were less likely to have VEGFA amplification than ER/PR− tumors (P = 0.020). VEGFA amplification was associated with worse OS (20.2 vs. 25.3 months; P = 0.013) in univariate analysis with a trend for worse OS in multivariate analysis (P = 0.08). There was a significant interaction between VEGFA amplification, hormone receptor status, and study arm. Patients with VEGFA amplification and triple-negative breast cancers (TNBC) or HER2 amplification had inferior OS (P = 0.047); amplification did not affect OS for those who were ER+ or PR+ and HER2−. Those who received bevacizumab with VEGFA amplification had inferior progression-free survival (PFS; P = 0.010) and OS (P = 0.042); no association was seen in the control arm. Test for interaction between study arm and VEGFA amplification with OS was not significant. Conclusion: VEGFA amplification in univariate analysis was associated with poor outcomes; this was particularly prominent in HER2+ or TNBCs. Additional studies are necessary to confirm the trend for poor OS seen on multivariate analysis for patients treated with bevacizumab. Clin Cancer Res; 19(5); 1281–9. ©2012 AACR.

Introduction: HER2 assessment for selection of patients that may benefit from HER2 targeting treatment can be performed by either immunohistochemistry (IHC), fluorescence or chromogen in situ hybridization (FISH or CISH). FISH is a robust and reliable technique for direct visualization and quantitative determination of gene amplifications, deletions and translocations in human cancer cells, but FISH protocols are time-consuming and involve toxic reagents. By introducing a new non-toxic ethylene carbonate based hybridization buffer that perform with very short hybridization times, the total FISH assay time on breast cancer tissue sections can be reduced from the traditional 16–20 hours to 3½–4½ hours. Material and methods: The new Dako HER2 IQFISH pharmDx™ was compared with Dako HER2 FISH pharmDx™ in a comparative study on 120 breast cancer specimens, and reproducibility of the HER2 IQFISH pharmDx™ assay was investigated in a study comprising 3 different sites and a total of 6 different observers. Samples for the comparative study was evaluated by Dako HercepTest™ to include all IHC scoring groups (0, 1+, 2+, 3+). Slides were stained according to manufacturer's instructions using microwave oven for heat pretreatment and RTU pepsin for 3–5 minutes at 37 °C. Hybridization was performed for 2 hours when using HER2 IQFISH pharmDx™ and for 17–20 hours when using HER2 FISH pharmDx™ Kit. All slides were blinded before evaluation. HER2 status was classified as “Non-amplified” when the HER2/CEN17 ratio < 2.0 and “Amplified” when the HER2/CEN17 ratio ≥ 2.0. Results: The new non-toxic hybridization buffer introduces a major safety improvement since formamide is no longer needed. Significantly shorter hybridization times are required to generate the same signal intensity (1–2 hour hybridization versus overnight). HER2 IQFISH pharmDx™ was compared with the traditional HER2 FISH pharmDx™ in a comparative study on 120 breast tissue specimens of human breast carcinoma. The preliminary data on HER2 status for 78 patients obtained by the two assays gave an overall agreement of 98.7% with lower and upper 95% confidence limits at 94.2% and 99.9%. The Kappa value was 0.96 (95% CL: 0.89–1.00). The p-value for McNemars test was 1.00 indicating absence of bias between the two assays. Disagreement between the two assays was observed for one specimen - a heterogeneous tissue with a small amplified area. Data from the reproducibility study that included site-to-site variation, day-to-day variation and inter-observer variation showed that the assay has a high degree of reproducibility. Conclusion: The validation studies of the new HER2 IQFISH pharmDx™ showed a very high concordance to the traditional HER2 FISH pharmDx™ and also that the assay is robust and reproducible. Reduction of the overall assay time from a two-day to a half-day procedure for HER2 FISH, offers more flexible laboratory routines and same day reporting for all working days of the week, which could be used for fast and simultaneous FISH and IHC answers and improved patient care. Taken together, the study demonstrates the potential of a new revolutionary platform that enables optimization and acceleration of FISH analysis to the benefit of cancer patients and laboratory personnel. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-02.

Purpose A Topoisomerase 1 (Top1) poison is frequently included in the treatment regimens for metastatic colorectal cancer (mCRC). However, no predictive biomarkers for Top1 poisons are available. We here report a study on the TOP1 gene copy number in CRC patients and its association with patient prognosis and tumor cell proliferation. Experimental design The study included TOP1 and CEN-20 fluorescence in situ hybridization ( FISH) analyses on formalin fixed paraffin embedded (FFPE) tissue sections from 154 stage III CRC chemonaive patients. The frequencies of aberration in the TOP1 gene copy number, the CEN-20 copy number and the TOP1 /CEN-20 ratio were analyzed and associated with overall survival (OS), time to recurrence (TTR) and in a subgroup analysis of rectal cancer patients only with time to local recurrence (LR in RC). Moreover, the TOP1 and CEN-20 copy numbers were correlated with the tumor Ki67 proliferation index. Results 35.7% of the tumors had an increased TOP1 copy number above 4 n gene copies per cell and 28.6% and 9.7% had a TOP1 /CEN-20 ratio ≥1.5 or ≥2.0, respectively. The TOP1 copy number and the TOP1 /CEN-20 ratios were separately added into multivariate analyses as continuous variables, in which also age, gender, primary tumor location and Ki67 status were added as covariates. In contrast to the TOP1 /CEN-20 ratio, the TOP1 copy number was significantly associated with OS (HR: 0.62; 95% CI: 0.42–0.90; p = 0.01). Neither the TOP1 copy number nor the ratio was significantly associated with TTR and only the TOP1 /CEN-20 ratio was significantly associated with LR in RC (HR: 0.25; 95% CI: 0.08–0.83; p = 0.02). No significant correlation was found between the TOP1 copy number and proliferation, while a weak and inverse correlation between the CEN-20 copy number and proliferation was observed. Conclusions This study showed that increased TOP1 gene copy numbers are frequent findings in cancer cells in stage III CRC tumors but unrelated to the proliferative status of the tumors. The association with prognosis is important to consider when planning and analyzing future studies investigating TOP1 as a potential predictive biomarker for Top1 poisons.

The availability of systemic chemotherapy regimens for the treatment of patients with metastatic colorectal cancer (mCRC) is based on the results from large prospective, randomized studies. The main chemotherapeutic drugs used in treatment of mCRC are the fluoropyrimidines (5-fluorouracil (5-FU); capecitabine) in combination with either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI). The objective response rate to either combination is approximately 50%, where no significant differences with regard to progression free survival or overall survival have been observed. Interestingly, a number of preclinical and clinical studies have indicated lack of full cross resistance between oxaliplatin based and irinotecan based treatment. Therefore, it is possible that certain mCRC patient subpopulations would benefit more from one drug combination rather than the other. To address this clinical problem there has been much focus on development and validation of predictive biomarkers for these three drugs. Here, we present a thorough review on the current status of predictive biomarkers for 5-FU, oxaliplatin and irinotecan treatment of mCRC patients. The overall conclusions were as follows: Several promising biomarker candidates were identified, notably thymidylate synthase for 5-FU, topoisomerase I for irinotecan and ERCC1 for oxaliplatin. However, these candidates warrant further analysis, where assay performance and clinical trial design should be in focus.

A positive relationship between topoisomerase-1 (TOP1) protein and sensitivity toward the TOP1 inhibitor irinotecan has been reported in patients with metastatic colorectal cancer (mCRC). In this study, we analyzed TOP1 gene copy number variation in tumor tissue from CRC patients and CRC cell lines with different sensitivities to the TOP1 inhibitor SN-38 and oxaliplatin.A TOP1 gene probe with a chromosome 20 centromere (CEN-20) reference probe was applied on normal mucosa and on tumor tissue from 50 stage III CRC patients. Additionally, associations between TOP1/CEN-20 ratio and in vitro sensitivity to SN-38 (irinotecan) and oxaliplatin were tested on 10 CRC cell lines. Results. In the malignant epithelium, 84% of the samples demonstrated an increased TOP1 gene copy number and 64% had an increased TOP1/CEN-20 ratio compared with the non-affected mucosa. Sixteen (32%) of the tumors had a ratio of ≥ 1.5 and 9 (18%) of these had a ratio of ≥ 2.0. A positive association was observed between the TOP1 gene copy number and the TOP1/CEN-20 ratio and in vitro sensitivity toward SN-38, but not toward oxaliplatin.A large fraction of the clinical samples demonstrated increased TOP1 gene copy number and increased TOP1/CEN-20 ratio. The cell line study suggested an association between TOP1 gene copy number or TOP1/CEN-20 ratio and sensitivity to irinotecan but not oxaliplatin.

Background: The Estrogen receptor alfa (ER) is an established biomarker that has been studied in details on the protein and RNA level. Recently, the existence of amplifications and deletions of the ESR1 gene have been documented (1-3), although the frequency of the aberrations has been extensively debated. Here we hypothesize that a positive correlation exists between ESR1 gene copy number and ER protein content measured by both a biochemical ligand assay, dextran coated charcoal (DCC), and immunohistochemistry (IHC). Material and Methods: From 289 primary high-risk breast cancer patients, randomized in the DBCG 77C trial between August 1977 and November 1982, ER data from DCC analyses was available. An ER positive tumor was defined as ≥10 fmol ER/mg protein (4). Archival tumor tissue was available from 257 patients. ESR1 copy number was analyzed with Dako Histology FISH Accessory Kit (K5599, DAKO, Glostrup, Denmark) using a probe covering the ESR1 gene at 6q25 and a centromere 6 reference probe (3). IHC analysis for ER was applied on archival paraffin embedded tissue using the antibody ER1D5 (DAKO), 1:200 with a positive cut off value of 10% ER positive tumor cells. Results: ESR1 FISH analysis was performed successfully in 215 (84%) patients. Amplification (ratio ESR1/CEN-6≥2) was observed in 47 of 215 patients (22%) and ESR1 deletion (ratio ESR1/CEN-6 A significant difference p=0.005 was found for the ESR1 deleted tumors compared to the ESR1 amplified tumors. Fig. 1 Box plut showing the ER content according to ESR1/CEN-6 status Discussion: Amplification of the ESR1 gene is associated with higher ER protein content by ER-DCC and more intense immunoreactivity by IHC while ESR1 deletions are associated with decreased content and immunoreactivity compared to tumors with normal ESR1 gene copy numbers. Major variations in ER content and immunoreactivity are however observed within tumors with a normal ESR1 copy number, and other mechanisms than gene aberrations seem to contribute. References: 1. Holst et al. (2007) Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer Nat Genet 39: 655 2. Tomita et al. (2009) Estrogen receptor alpha gene ESR1 amplification may predict endocrine therapy responsiveness in breast cancer patients. Cancer Sci 100:1012 3. Nielsen et al. (2010) Amplification of ESR1 may predict resistance to adjuvant tamoxifen in postmenopausal patients with hormone receptor positive breast cancer. Breast cancer Res Treat, June 17 Epub 4. Thorpe et al. (1993) Short recurrence-free survival associated with high oestrogen receptor levels in the natural history of postmenopausal, primary breast cancer. Eur J Cancer 29A:971. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-01.

Kirkegaard T, Witton C J, Edwards J, Nielsen K V, Jensen L B, Campbell F M, Cooke T G & Bartlett J M S (2010) Histopathology56, 203–211 Molecular alterations in AKT1, AKT2 and AKT3 detected in breast and prostatic cancer by FISH Aims: The AKT family is implicated in cancer progression. There are three mammalian AKT isoforms located on chromosomes 14, 19 and 1, respectively. The aim of the study was to investigate genetic alterations of AKT in breast and prostatic cancers using fluorescence in situ hybridization (FISH). Methods and results: In oestrogen receptor(ER)-positive breast carcinomas, AKT1 was deleted in five (4.8%) and amplified in one (1%) carcinoma. Deletions of AKT2 were seen in 19 (21.1%) cases. No AKT2 amplifications were identified. Ten (9.9%) AKT3 amplifications but no deletions were seen. In prostatic cancer, AKT1 was amplified in one carcinoma (2.6%). No genetic changes were observed for AKT2 and AKT3. High frequencies of aneusomy for all chromosomes were observed in breast and prostatic carcinomas. Conclusions: In breast cancer AKT3 amplifications and AKT1 and AKT2 deletions were seen, which, to our knowledge, have not been shown by FISH before. Although these two cohorts cannot be directly compared, only one AKT1 amplification was identified in prostatic carcinomas. This indicates differences in the genetic changes underlying development of breast and prostatic cancers. To evaluate further the role of genetic changes of AKT in breast cancer progression, a cohort of both ER+ and ER− patients should be evaluated.

A standard procedure for qualitative and quantitative analysis of chromosome slides is presented. The method involves classification of observed metaphases according to chromosome morphology combined with length measurement of human chromosome no. 1. Based on this procedure, the in vitro band inducing effect of daunomycin has been examined and the advantages of the suggested procedure is discussed in relation to other in vitro induction studies.

When human lymphoid cells are exposed to colcemid overnight at 4°C and subsequently incubated at 37.5°–38°C, variation in incubation time offers a simple method to produce chromosome slides with a high frequency of either prophase, prometaphase, or metaphase chromosomes.

Inactivating PTEN mutations are commonly found in prostate cancer, resulting in an increased activation of Akt. In this study, we investigate the role of PTEN deletion and protein expression in the development of hormone-refractory prostate cancer using matched hormone-sensitive and hormone-refractory tumours. Fluorescent in situ hybridisation and immunohistochemistry was carried out to investigate PTEN gene deletion and PTEN protein expression in the transition from hormone-sensitive to hormone-refractory prostate cancer utilising 68 matched hormone sensitive and hormone-refractory tumour pairs (one before and one after hormone relapse). Heterogeneous PTEN gene deletion was observed in 23% of hormone sensitive tumours. This increased significantly to 52% in hormone-refractory tumours (P=0.044). PTEN protein expression was observed in the membrane, cytoplasm and the nucleus. In hormone sensitive tumours, low levels of cytoplasmic PTEN was independently associated with shorter time to relapse compared to high levels of PTEN (P=0.028, hazard ratio 0.51 (95%CI 0.27–0.93). Loss of PTEN expression in the nucleus of hormone sensitive tumours was independently associated with disease-specific survival (P=0.031, hazard ratio 0.52, 95%CI 0.29–0.95). The results from this study demonstrate a role for both cytoplasmic and nuclear PTEN in progression of prostate cancer to the hormone-refractory state.

Aims: CCND1 and EMSY, on 11q13, are frequently amplified in breast cancer. CCND1 is implicated in cell cycle progression and EMSY is a BRCA2-associated repressor protein. The aim was to investigate gene copy numbers of CCND1 and EMSY and to determine if CCND1 amplification is associated with reduced survival of tamoxifen-treated breast cancer patients. Methods and results: Fluorescence in situ hybridization (FISH) was performed on 111 consecutive and 354 oestrogen receptor (ER)+ tamoxifen-treated breast cancers. In the consecutive set, CCND1 and EMSY were amplified in 14.8% and 7.2%, respectively, and deleted in 8.7% and 13.5%, respectively. In the ER+ set, CCND1 and EMSY were amplified in 20.6% and 9.6%, respectively, and deleted in 1.7% and 4.2%, respectively. CCND1 and EMSY gene amplifications were associated with decreased overall survival (OS) (P = 0.03 and P = 0.04, respectively) of patients in the ER+ set. Conclusion: As hypothesized, CCND1 amplifications are associated with poor OS in ER+ patients. EMSY amplification is also associated with poor OS. However, as >70% of EMSY amplifications were CCND1 amplified, EMSY may not have any additional effect on survival of ER+ breast cancer.

Udgivelsesdato: 2008 BACKGROUND: Previous analyses of TOP2A and HER2 in the Danish Breast Cancer Coopererative Group (DBCG) trial 89D suggested that TOP2A amplifications and possible also deletions are predictive markers for the effect of adjuvant epirubicin in patients with primary breast cancer. We present an updated and extended statistical analysis, requested for IVD-labeling of TOP2A testing. MATERIAL AND METHODS: In the DBCG trial 89D 980 Danish patients were randomly assigned to nine cycles of intravenous CMF (cyclophosphamide, methotrexate, and fluorouracil) or CEF (cyclophosphamide, epirubicin, and fluorouracil). Archival tumor tissue was collected retrospectively from 806 of these patients in a prospectively designed, biological sub-study, and was successfully analyzed for TOP2A aberrations and HER2 status in 773 samples (96%). Recurrence-free survival (RFS) was the primary endpoint. RESULTS: TOP2A aberrations (amplifications and deletions) were significantly associated with shorter RFS (p

Depending on the tumour type, a larger or smaller number of cancer patients receive chemotherapy with systemic toxicity as the only effect. In that situation, an alternative, not necessarily medical, treatment would have been a better choice – and toxicity (and financial resources) could have been spared by withholding ineffective drugs. One of the reasons for this apparent paradigm is that the tumour cells of each cancer pa- tient may show different sensitivity/resistance towards different chemotherapeutic drugs, i.e. breast cancer or colorectal cancer is not only breast or colorectal cancer. With our increasing biological insight and understanding, it has become apparent that each patient's tumour tissue is unique and as a consequence, each patient's tumour cell sensitivity/resistance to- wards chemotherapeutic drugs may be different. As of today there is no method in routine clinical use to predict the sensitivity/resistance to chemotherapy in its broad sense in the individual patient. This chapter will describe several different DNA, RNA, protein and cell based assay methodologies and marker molecules that have been brought forward as potential predictive assays/markers to be used to select the most effective drugs for the individual cancer patient.

Purpose: Amplified in breast cancer 1 (AIB1) is a member of the p160/steroid receptor coactivators family and is involved in estrogen-dependent gene transcription by reducing the antagonistic activity of tamoxifen-bound estrogen receptor-α (ER-α). The present study was carried out to test the hypothesis that AIB1 protein expression and/or gene amplification mediates tamoxifen resistance in breast cancer. Experimental Design: Immunohistochemistry using AIB1 antibody and fluorescence in situ hybridization using probes specific for AIB1 and chromosome 20 was done on 402 ER-α–positive tamoxifen-treated breast cancers. Results: AIB1 overexpression was not associated with relapse during treatment with tamoxifen. In contrast, high AIB1 expression in patients with human epidermal growth factor receptor (HER) 2– and HER3-overexpressing tumors or tumors expressing one or more of HER1, HER2, or HER3 (HER1-3 positive) was associated with an increased risk of relapse on tamoxifen [hazard ratio, 2.20; 95% confidence interval, 1.07-3.52 (P = 0.0416); hazard ratio, 2.42; 95% confidence interval, 1.32-4.43 (P = 0.0030), respectively]. AIB1 gene amplification was observed in 18 of 362 (5%) patients. High AIB1 gene copy number had no effect on overall or disease-free survival. Conclusions: Data presented here support a role for AIB1 expression on relapse during tamoxifen treatment in hormone-responsive HER-expressing clinical breast cancers and support clinical evidence, suggesting a cross-talk between ER-α and growth factor receptor pathways through changes in expression of specific coactivator proteins, such as AIB1. This study highlights the potential that tumor profiling, using multiple markers of treatment response, may improve patient selection for endocrine treatment, such as tamoxifen or aromatase inhibitors.

Purpose The aim of the study was to evaluate the predictive value of HER2 and topoisomerase IIα gene (TOP2A) for the efficacy of epirubicin in the adjuvant setting of breast cancer patients. Patients and Methods In the Danish Breast Cancer Cooperative Group trial 89D, 980 pre- and postmenopausal primary patients were randomly allocated to either CMF (cyclophosphamide, methotrexate, and fluorouracil; n = 500) or CEF (cyclophosphamide, epirubicin, and fluorouracil; n = 480) times 9, between January 1990 and November 1999. Tumor tissue was retrospectively identified from 805 patients and was analyzed for HER2-positivity and for TOP2A-amplifications and deletions. Results HER2-positivity was found in 33% of the 805 investigated tumors and was not a predictive marker for epirubicin sensitivity. TOP2A changes were identified in 23% of the 773 investigated tumors: 12% had TOP2A amplifications and 11% had TOP2A deletions. We found that patients with TOP2A amplification had an increased recurrence-free (RFS; hazard ratio [HR], 0.43; 95% CI, 0.24 to 0.78) and overall survival (OS; HR, 0.57; 95% CI, 0.29 to 1.13), respectively if treated with CEF compared with CMF, and that patients with TOP2A deletions had an almost identical hazard ratio (RFS: HR, 0.63; 95% CI, 0.36 to 1.11; OS: HR, 0.56; 95% CI, 0.30 to 1.04). This is in contrast to patients with a normal TOP2A genotype for whom similar outcome was observed in both treatment arms (RFS: HR, 0.90; 95% CI, 0.70 to 1.17; OS: HR, 0.88; 95% CI, 0.66 to 1.17). Conclusion TOP2A amplification—and possibly deletion—seems to be predictive markers for the effect of adjuvant epirubicin containing therapy in primary breast cancer, but a final conclusion has to await a confirmative study or a meta-analysis.

Background: Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene. Methods: Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allele-specific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and immobilized PNA was carried out at 60 degrees C followed by flow cytometric detection. Results: We present a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection used for the correct and fast regenotyping of a Danish basal cell carcinoma cohort. Conclusions: This new assay presents a simple, rapid, and robust method for SNP genotyping for laboratories equipped with a standard flow cytometer. Moreover, this system offers potential for multiplexing and will be operational for middle-scale genotyping. (Less)

New strategies for improving treatment of patients with breast carcinoma have focused on the HER2 oncoprotein with regard to response to traditional therapy regimes and the effect of a new drug specifically directed against the protein. Furthermore, the status of the topoisomerase IIalpha (TOP2A) gene has been suggested as a predictive marker of anthracycline treatment. In this study of 120 tumours, immunohistochemically detected HER2 overexpression with HercepTest has been compared to the HER2 gene amplification investigated with a new HER2 probe for fluorescence in situ hybridization (FISH). In addition, the HercepTest was evaluated as a screening tool for choosing cases for FISH investigation of TOP2A gene aberrations. The HercepTest score 3+ identified HER2 gene amplification in 27 of 30 amplified tumours (sensitivity of 0.90) with a false-negative rate of 0.10 and a false-positive rate of 0.06. TOP2A gene amplification or deletion was found in 20 cases. Sixteen (80%) of these carcinomas were in the HercepTest 3+ group, but four tumours had alterations in the TOP2A gene with normal HER2 status. Traditionally, in the FISH technique the result has been based on counting 60 cells. However, we found that a much less time-consuming method of counting 60 signals gave equally good results.

Platinum chemotherapy remains part of standard therapies in the management of a variety of cancers. Severe side effects and a high degree of resistance to platinum drugs have led numerous researchers to search for predictive biomarkers, which could aid in identifying patients that are the most likely to respond to therapy. The ERCC1-ERCC4 endonuclease plays a critical role in the repair of platinum-DNA damage and has widely been studied in relation to sensitivity to platinum chemotherapy. The standard method to evaluate ERCC1 protein expression is through the use of immunohistochemistry with monoclonal antibody 8F1, an antibody that was recently found to bind an unrelated protein. The present study determines the specificity of a novel antibody, monoclonal antibody 4F9 and presents a method to evaluate ERCC1 expression in colorectal tumor specimens. Using relevant cell lines as controls, the specificity of antibody 4F9 was tested by immunoblotting, immunohistochemistry and immunofluorescence. Scoring guidelines to aid in the evaluation of ERCC1 tumor expression were developed and evaluated in archival formalin-fixed paraffin embedded colorectal cancer specimens. Antibody 4F9 was found to be specific by all methods applied and it was possible to evaluate the ERCC1 expression in the majority (85%) of colorectal cancer tumor specimens.

The immortalized, nontumorigenic human breast epithelial cell line HMT-3522 has been used as a model for premalignant and, eventually, malignant development. During cultivation, the karyotype evolution was followed. At an early stage, a very long constant phase showed a near-diploid karyotype, with only five marker chromosomes. DNA from this phase was used for comparative genomic hybridization (CGH) analysis, confirming a previously known MYC amplification, and the integration sites were subsequently determined by single-locus fluorescence in situ hybridization (FISH). Furthermore, gains of 5q22-qter and 20q11-qter and deletion of most of chromosome 6 (6p23-qter) were detected by CGH. Because of uncertainty about some of the indicated changes, including a deletion of Ip35-pter, the CGH findings were investigated more closely by chromosome painting, leading to a revision of the karyotype: 45,XX,del(I)(p35),-6,dup(8)(pter-->qter::qter-->q24),der(12) t(6;12)(p23; p13),der(14)t(5;14)(q22;q32.3),der(17)t(8;17;20)(17pter-->17q25 ::8qter--> 8q23::8q24-->8qter::8q24-->8qter:: 8q23-->8q24.1::20q11-->20qter). Some karyotypic changes were confirmed by CGH; others had to be revised; and, in the Ip35 region, classical cytogenetics seems superior to CGH. However, CGH revealed a karyotypically unsuspected dup(20q) that might be of special relevance to breast tumor initiation or progression. Our study confirms that CGH is supplementary to current technologies, e.g., karyotyping and Southern analysis, but cannot replace them. In addition, our cell line turned out to be an excellent model for comparison among the different methods. The results imply that future cytogenetic analyses of complex karyotypes should be based on a combination of karyotyping, CGH, and FISH.

BackgroundTopoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH).MethodsNine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN-20) or chromosome 2 (CEN-2). Tissue sections from 154 chemonaive stage III CRC patients, previously studied with TOP1/CEN-20, were analyzed with TOP1/CEN-2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and time to local recurrence (LR; rectal cancer only) were determined.ResultsTOP1 aberrations were observed in four cell line metaphases. In all cell lines CEN-2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN-2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN-2≥1.5). One hundred and three patients (68.2%) had TOP1 gain, of which 15 patients (14.6%) harbored an amplification (TOP1/CEN-20≥2.0). TOP1 gene gain did not have any association with clinical endpoints, whereas TOP1 amplification showed a non-significant trend towards longer TTR (multivariate HR: 0.50, p = 0.08). Once amplified cases were segregated from other cases of gene gain, non-amplified gene increases (TOP1/CEN-2≥1.5 and TOP1/CEN-20ConclusionsTOP1 gene copy number increase occurs frequently in stage III CRC in a mechanism that often includes CEN-20. Using CEN-2 as a measurement for tumor ploidy levels, we were able to discriminate between different mechanisms of gene gain, which appeared to differ in prognostic impact. TOP1 FISH guidelines have been updated.

The Estrogen Receptor (ER) is an established predictive marker for the selection of adjuvant endocrine treatment in early breast cancer. During the 1990s Immunohistochemistry (IHC) replaced cytosol based assays for determination of ER status. This study examined the association between ER protein level determined by two different methods and ESR1 gene copy number. From 289 primary high-risk breast cancer patients, randomized in the Danish Breast Cancer Cooperative Group (DBCG) 77C trial, results from cytosolic ER levels were available from ligand binding assays. Archival tumor tissue was retrieved from 257 patients. ESR1/CEN-6 ratio was analyzed successfully by Fluorescence In Situ Hybridization (FISH) in 220 (86%) patients. ESR1 amplification (ESR1/CEN-6 ≥ 2.00) was observed in 23% of the patients and ESR1 deletion (ESR1/CEN-6

The clinical benefit of anthracyclines has been connected to HER2 status, TOP2A status and centromere 17 copy numbers (CEN-17). Data from a clinical trial randomizing patients to anthracyclines was used to assess whether the number of CEN-17 in breast cancers may predict incremental responsiveness to anthracyclines besides what is obtained when used relatively to TOP2A and HER2. As cut sections of paraffin-embedded tissue are prone to truncation of nuclei, strict definition of ploidy levels is lacking. We therefore used normal breast tissue to assist define ploidy levels in cut sections. Fluorescence in situ hybridization (FISH) with centromere 17 (CEN-17) and TOP2A was performed on 120 normal breast specimens. The diploid CEN-17 copy number was reduced from the expected two signals in whole nuclei to an average of 1.68 signals per nucleus in cut sections of normal breast. Ploidy levels determined in normal breast were applied to data on 767 patients with known HER2 and TOP2A status randomized to anthracyclines in the DBCG 89D trial. CEN-17 ploidy levels were in cut sections from the 767 breast cancer patients established as: Haploid: ≤1.25 (10%), diploid: 1.26-2.09 (60%), triploid: 2.10-2.93 (21%), tetraploid: 2.94-3.77 (5%) or higher ploidy: ≥3.78 (4%). Amplification of HER2 and deletion of TOP2A were frequently observed in tumors with a high ploidy level. In univariate analyses increasing ploidy was associated with decreased disease-free survival (DFS) (P=0.0001) and overall survival (OS) (P

Context.—New guidelines for HER2 testing have been introduced. Objectives.—To evaluate the difference in HER2 assessment after introduction of new cutoff levels for both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) and to compare interobserver agreement and time to score between image analysis and conventional microscopy. Design.—Samples from 150 patients with breast cancer were scored by 7 pathologists using conventional microscopy, with a cutoff of both 10% and 30% IHC-stained cells, and using automated microscopy with image analysis. The IHC results were compared individually and to HER2 status as determined by FISH, using both the approved cutoff of 2.0 and the recently introduced cutoff of 2.2. Results.—High concordance was found in IHC scoring among the 7 pathologists. The 30% cutoff led to slightly fewer positive IHC observations. Introduction of a FISH equivocal zone affected 4% of the FISH scores. If cutoff for FISH is kept at 2.0, no difference in patient selection is found between the 10% and the 30% IHC cutoff. Among the 150 breast cancer samples, the new 30% IHC and 2.2 FISH cutoff levels resulted in one case without a firm diagnosis because both IHC and FISH were equivocal. Automated microscopy and image analysis-assisted IHC led to significantly better interobserver agreement among the 7 pathologists, with an increase in mean scoring time of only about 30 seconds per slide. Conclusions.—The change in cutoff levels led to a higher concordance between IHC and FISH, but fewer samples were classified as HER2 positive.

PurposeTo evaluate whether the combination of HER2 with TIMP-1 (HT) or TOP2A with TIMP-1 (2T) more accurately identifies patients who benefit from cyclophosphamide, epirubicin, and fluorouracil (CEF) compared with cyclophosphamide, methotrexate, and fluorouracil (CMF) than these markers do when analyzed individually.Patients and MethodsThe Danish Breast Cancer Cooperative Group (DBCG) 89D trial randomly assigned 980 high-risk Danish breast cancer patients to CMF or CEF. Archival tumor tissue was analyzed TIMP-1, and HER2-negative and TIMP-1 immunoreactive tumors were classified as HT nonresponsive and otherwise HT responsive. Similarly, the 2T panel was constructed by combining TOP2A and TIMP-1; tumors with normal TOP2A status and TIMP-1 immunoreactivity were classified as 2T-nonresponsive and otherwise 2T-responsive.ResultsIn total, 623 tumors were available for analysis, of which 154 lacked TIMP-1 immunoreactivity, 188 were HER2 positive, and 139 had a TOP2A aberration. HT status was a statistically significant predictor of benefit from CEF compared with CMF (Pinteraction= .036 for invasive disease–free survival [IDFS] and .047 for overall survival [OS]). The 269 (43%) patients with a 2T-responsive profile had a significant reduction in IDFS events (adjusted hazard ratio, 0.48; 95% CI, 0.34 to 0.69; P < .001) and OS events (adjusted hazard ratio, 0.54; 95% CI, 0.38 to 0.77; P < .001). 2T status was a highly significant predictor of benefit from CEF compared with CMF (Pinteraction< .0001 for IDFS and .004 for OS).ConclusionThe 2T profile is a more accurate predictor of incremental benefit from anthracycline-containing chemotherapy than HER2, TIMP-1, or TOP2A individually, and compared with these, 2T classifies a larger proportion of patients as sensitive to anthracyclines.

Udgivelsesdato: 2009-Nov-18 Copy number changes in TOP2A have frequently been linked to ERBB2 (HER2) amplified breast cancers. To study this relationship, copy number changes of ERBB2 and TOP2A were investigated by fluorescence in situ hybridization (FISH) in two cell lines; one characterized by having amplification of both genes and the other by having amplification of ERBB2 and deletion of TOP2A. The characteristics are compared to findings on paired ERBB2 and TOP2A data from 649 patients with invasive breast cancer from a previously published biomarker study. The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. Most gene signals are translocated to abnormal marker chromosomes. ERBB2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher ERBB2 ratio. This observation was confirmed by patient FISH data: among 276 (43% of all patients) abnormal tumors, 67% had different ERBB2 and TOP2A status. ERBB2 amplification with normal TOP2A status was found in 36% of the abnormal tumors (15% of all patients). Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while TOP2A deletion and ERBB2 amplification was observed in 16% of the abnormal cases (8% of all patients). A small number of tumors had TOP2A amplification (4%) or deletion (6%) without simultaneous changes of the ERBB2 gene. ERBB2 deletion was also observed (5%) but only in tumors with simultaneous TOP2A deletion. The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for ERBB2 in the amplified tumors (P

Telomere shortening and chromosomal instability are believed to play an important role in the development of myeloid neoplasia. So far, published data are only available on the average telomere length in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but not on the telomere length of individual chromosomes. We used a new technique, telomere/centromere-fluorescence in situ hybridization (T/C-FISH), which combines fluorescence R-banding and FISH using a probe against the telomere repeats to measure the telomere length of each chromosome arm in 78 patients with MDS. In line with the previous results, patients with MDS showed significantly shorter telomeres than those of healthy controls. Telomere lengths did not differ significantly between distinct morphological subtypes of MDS. However, there was a significant difference in telomere length between patients with an isolated monosomy 7 and patients with a normal karyotype (P < 0.05). Notably, patients with an isolated monosomy 7 showed significantly longer telomeres than patients with a normal karyotype in many chromosome arms, among them 7p and 7q. Neo-telomeres were found in two patients with a complex karyotype, in one case at the fusion site of a dic(14;20). Normal and aberrant metaphases of the same patient did not differ in telomere length, thus indicating to telomere shortening as a basic mechanism affecting all hematopoietic cells in patients with MDS. In some MDS subtypes, like MDS with isolated monosomy 7, telomeres may be stabilized and even increase in length because of the activation of telomerase or alternative mechanisms.

TO THE EDITOR: We read with great interest the joint recommendations from the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) concerning human epidermal growth factor receptor 2 (HER-2) testing in breast cancer. The new recommendations are highly appreciated, and the validation, standardization, and accreditation initiatives are very important for increasing accuracy in HER-2 testing. The joint ASCO/CAP guideline is based on a comprehensive review of published data on HER-2 testing, except for the recommendation for changing the cutoff of stained cells from 10% to 30%. Most published data are based on results obtained using a US Food and Drug Administration (FDA) –approved test that defines a positive 3 staining as being uniform, intensive membrane staining of more than 10% of invasive tumor cells. The new guideline recommends a different cutoff of 30% for 3 . It is not clear whether there are data that support this new cutoff, and we recommend the publication of data from clinical trials to do so. As a point of correction, we wish to bring to your attention that Dako (Glostrup, Denmark) has a US FDA–approved assay, the HER2 FISH (fluorescence in situ hybridization) pharmDx Kit (Dako Denmark A/S). This kit is indicated as an aid in the assessment of patients for whom Herceptin treatment is being considered. The cutoff stated in the package inserts of the approved tests will still be the legally valid cutoff, and it will be impossible to change it without presenting the US FDA with valid data that correlate to clinical outcome. We are concerned that the divergence between the recommendations in the package inserts of the approved HER-2 tests and the ASCO/CAP guideline concerning cutoff will generate confusion rather than clarification.

The selection of therapy for a particular breast cancer patient is traditionally based on average results from randomized clinical trials. Rational pharmacotherapy is in essence about selecting the right drug(s) for the rightpatient,andinordertoguidethisselectionprocess pharmacodiagnostic tests are indispensable. A number of tests have been developed or are under development for targeted therapies, such as antiestrogens, human epidermal growth factor receptor 2 inhibitors, and topoisomerase inhibitors. Based on a biopsy from the tumor, the tests are able to identify patients with a high probability to benefit from these therapies. The detection of the predictive biomarkers is based on different technologies, such as immunohistochemistry, fluorescence in situ hybridization, and chromogenic in situ hybridization. Pharmacodiagnostic tests will play an important role in the further development of targeted therapies and may be seen as a prerequisite for the introduction of individualized medicine in oncology. The Oncologist 2007;12:397–405

Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene.Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allele-specific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and immobilized PNA was carried out at 60 degrees C followed by flow cytometric detection.We present a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection used for the correct and fast regenotyping of a Danish basal cell carcinoma cohort.This new assay presents a simple, rapid, and robust method for SNP genotyping for laboratories equipped with a standard flow cytometer. Moreover, this system offers potential for multiplexing and will be operational for middle-scale genotyping.

The typing of a single nucleotide polymorphism with DNA probes is sometimes problematic because of the limited discriminating power of long DNA probes. As an alternative to existing assays, we have developed a real-time PCR assay for the genotyping of single nucleotide polymorphisms using short peptide nucleic acid (PNA) molecular beacons. A single nucleotide polymorphism in exon 6 of the XPD gene was chosen as the model system. The genotyping experiments were performed in the ABI 7700 using beacons labeled with either fluorescein or JOE, and in the Lightcycler using a fluorescein labeled beacon. QSY-7 was used as the quencher in all the beacons. The result of the genotyping was the same on both instruments and was in agreement with a previously performed RFLP genotyping of 79 samples. The length of PNA molecular beacons is significantly shorter than that of TaqMan or Lightcycler probes, making probe design and genotype discrimination easier.

HMT-3522 is a spontaneously immortalized cell line derived from a fibrocystic breast lesion. After continuous accumulation of genetic changes, the cell line was transformed from a nontumorigenic to a malignant phenotype. One of the earliest genetic aberrations is a missense mutation of codon 179 (His179Asn) in the tumor suppressor gene TP53 leading to outgrowth of a cell type expressing only the mutant form of TP53. In this report, we extend earlier investigations to reveal the genetic background for the evolution from homozygous wild type to hemizygous mutated cells. The status of the TP53 alleles was followed at different stages by fluorescence in situ hybridization (FISH) and allele-specific PCR (ASPCR) on total DNA, as well as flow-sorted chromosomes—taking advantage of a size difference between the two homologues of chromosome 17 that harbor TP53 on 17p. This further allowed us to determine on which of the two chromosomes the mutated allele was located. The results presented here show that the cells have undergone an evolution from homozygous wild type for TP53 to heterozygous (His179Asn mutation in one allele), and finally to a hemizygous mutated state (deletion of the remaining wild-type allele). The finding of a transient period in which heterozygous cells dominate the population before the eventual outgrowth of hemizygous cells strongly indicates that the His179Asn mutation results in a tp53 protein with a dominant negative effect that does not totally abrogate the function of wild type TP53 in vitro.

Background: Platinum-based chemotherapy has long been used in the treatment of a variety of cancers andfunctions by inducing DNA damage. ERCC1 and ERCC4 are involved in the removal of this damage and havepreviously been implicated in resistance to platinum compounds. The aim of the current investigation is todetermine the presence, frequency and prognostic impact of ERCC1 or ERCC4 gene copy number alterations incolorectal cancer (CRC).Methods: Fluorescent in situ hybridization probes directed at ERCC1 and ERCC4 with relevant reference probeswere constructed. Probes were tested in a CRC cell line panel and in tumor sections from 152 stage III CRCchemonaive patients. Relationships between biomarker status and clinical endpoints (overall survival, time torecurrence, and local recurrence in rectal cancer) were analyzed by survival statistics.Results: ERCC1-19q13 copy number alterations were observed in a single cell line metaphase (HT29). In patientmaterial, ERCC1-19q13 copy number gains (ERCC1-19q13/CEN-2 ≥ 1.5) were detected in 27.0% of specimens,whereas ERCC1-19q13 deletions (ERCC1-19q13/CEN-2 < 0.8) were only detected in 1.3%. ERCC1-19q13 gain wassignificantly associated with longer survival (multivariate analysis, HR: 0.45, 95% CI: 0.20-1.00, p = 0.049) in patientswith colon tumors, but not rectal tumors. No ERCC4 aberrations were detected and scoring was discontinued after 50 patients.Conclusions:ERCC1-19q13 copy number gains occur frequently in stage III CRC and influences survival in patientswith colon tumors. Future studies will investigate the effect of ERCC1-19q13 aberrations in a platinum-treatedpatient population with the aim of developing a predictive biomarker profile for oxaliplatin sensitivity in CRC.

475 Background: TOP1 inhibitor treatment is frequently being used in combination therapy of metastatic CRC. This study aims to reveal whether TOP1 gene copy number associates with patient prognosis, since such a relationship may have significant implications for future studies aiming at validating the predictive value of TOP1 gene copy number. Methods: The study included TOP1 and CEN-20 FISH analyses (DAKO A/S Denmark) on FFPE tissue sections from 154 stage III CRC patients who did not receive adjuvant chemotherapy. TOP1 gene copy number, CEN-20 copy number and the TOP1/CEN-20 ratios were analyzed and correlated to overall survival (OS), to time to recurrence (TTR) of patients with CRC and to local recurrence (LR) in patients with rectal cancer (RC). Results: TOP1 copy number counts and the TOP1/CEN-20 ratios, age, gender and primary tumor location were separately added into a multivariate analysis as continuous variables. For OS and LR, TOP1 copy number was significant and the ratio was borderline significant with higher copy number associated with longer OS or longer time to LR. When the patients were dichotomized using the TOP1 median copy number, we found that patients with high TOP1 copy number in their tumor cells had a significant longer OS (HR: 0.68; 95% CI: 0.47-0.98; p = 0.04) compared to patients with low TOP1 copy number. Using the median TOP1/CEN-20 ratio to dichotomize, no significant differences were observed between patients with levels above or below the median ratio number for OS (HR: 0.76; 95% CI: 0.53-1.10; p = 0.14). TOP1 copy number divided the RC patients into two groups with a trend towards a significant difference in time to LR (HR: 0.56; 95% CI: 0.27-1.16; p = 0.11) with higher copy number. If the median ratio was used, a significant association with longer time to LR (HR: 0.43; 95% CI: 0.20-0.92; p = 0.03) was found. No significant associations between TOP1 copy number or ratio and TTR were observed. Conclusions: Increased TOP1 copy number is associated with longer OS in CRC patients and fewer LR in RC patients. Thus, future studies analyzing the association between TOP1 copy number and response to therapy in CRC patients should be planned in such a way that a prognostic and a predictive value of TOP1 can be separated.

The "spontaneously" immortalized cell line HMT-3522, derived from a fibrocystic breast lesion, is used as a model for premalignant breast epithelium. During 205 passages the cytogenetic evolution was followed. The results were compared with our earlier results on oncogene expression and growth factor requirements. During in vitro growth, gain and loss of markers, loss of normal chromosomes, and duplication of the chromosome complement could be demonstrated. The variability increased during in vitro growth, This variability, probably created randomly, leads to cells with different growth capacities, from which sidelines may be selected and become stemlines. The karyotypic evolution (including polyploidization) demonstrated here may be a result of genetic instability and heterogeneity. Although tumorigenicity was not achieved, either due to lack of cancer-specific gene alterations or to lack of proper selection pressure, the results suggest an ongoing process towards malignancy.

10629 Background: Expression of key enzymes of the thymidylate synthase pathway may affect the efficacy of 5-FU and the pro-drug capecitabine (C). The gene copy number of thymidylate synthase (TS), thymidine phosphorylase (TP), and dihydrofolate reductase (DHFR) (high vs low defined by the median for each) was assessed and correlated with time-to progression (TTP) and progression-free survival (PFS). Methods: Adult female patients with pathologically confirmed breast cancer and locally advanced or metastatic disease were treated with C 1000 mg/m2 BID days 1-14 of a 21-day cycle. Human epidermal growth factor receptor 2 (HER2) and estrogen receptor (ER) expression was assessed immunohistochemically. Custom made TS, TP and DHFR FISH probes (Dako, Glostrup, Denmark) along with centromeric reference probes were used to evaluate gene copy number and gene to reference ratios in at least 60 morphologically intact non-overlapping nuclei. Markers dichotomized as high/low by the median were correlated with TTP and ...

Abstract 30 Lenalidomide has been shown to induce transfusion independence and cytogenetic response in a high proportion of patients with MDS and isolated 5q deletion (del5q). However, some of these patients progress into acute myeloid leukemia, particularly those who do not show an erythroid or cytogenetic response. Yet the mechanisms inducing the leukemic transformation in patients with MDS and del(5q) are mainly unclear. Since dysfunctional telomeres play an important role in the development of genetic instability and shorter telomeres are associated with advanced MDS and AML, we reasoned whether telomere shortening may contribute to leukemic progression in patients with MDS and del5q. This study included 14 patients with transfusion-dependent anemia and low- or intermediate-1-risk MDS enrolled in the studies MDS-003 (n=42) or MDS-004 (n=260) who were treated with lenalidomide according to the study protocols. Written informed consent was provided according to the Declaration of Helsinki. Seven patients progressed into RAEB-1 or AML while on study and 7 patients did not. Time span between initial diagnosis and study entry was similar (median 51.3 and 44.4 months respectively in the patients with and without leukemic progression). Combined fluorescence R-banding and T/C-FISH analysis to determine the telomere length of each individual chromosome was performed as described earlier (K Lange et al, Genes Chromosomes Cancer, 2010). Telomere length at study entry was 6.1 kb (range 4.8 to 7.9 kb) in patients with MDS and del5q who later underwent leukemic transformation. Patients without later disease progression had a median telomere length of 9.3 kb (range 8.4 to 10.2 kb). Thus, patients who progressed after a median of 29 months (range 5 to 51 months) had significantly shorter telomeres than patients who had a cytogenetic response or stable disease (p Disclosures: Göhring: Celgene Corp.: Consultancy. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

e21017 Background: The anti-VEGF antibody, bevacizumab, has been FDA approved for the treatment of breast cancer. While germline variability (i.e. single nucleotide polymorphisms) may serve as a predictive marker for anti-VEGF therapy, to date tumor-specific variability has not. More specifically, amplification or deletion of the VEGFA gene has not been studied or evaluated as a predictive or prognostic biomarker for breast cancer. Methods: A VEGFA/centromere enumerization-6 (CEN-6) probe was created and validated using DNA clones and restriction enzyme fragment measurements. The final product contained a RP11-710-L16 probe covering 183 KB including the VEGFA gene and flanking regions. The CEN-6 probe was developed with fluorescein isothiocyanate labeled peptide nucleic acid oligonucleotides. VEGFA and CEN-6 probes were tested on metaphase spreads to exclude cross-hybridization to other chromosomes. A tissue microarray containing 93 breast cancers was analyzed for the presence of VEGFA gene amplification ...

Copy number changes in TOP2A are frequently observed in HER2 amplified breast cancers, and amplification of a whole amplicon containing both genes has been suggested as the underling mechanism. Here, we describe copy number changes of HER2 and TOP2A in two cell lines; one characterized by having amplification of both genes and the other by having amplification of HER2 and deletion of TOP2A. The characteristics are compared to findings in patients with invasive breast cancer.Material and methods: Fluorescence in situ hybridization (FISH) with HER2, TOP2A and centromere 17 (CEN-17) probes was performed on metaphases, interphases and cut sections from breast cancer cell lines. Paired HER2/CEN-17 and TOP2A/CEN-17 data was available from 649 patients from a previously published biomarker study (Knoop et al. J Clin Oncol 2005;23:7483-90).Results: The physical localization of FISH signals in metaphase spreads from cell lines showed that simultaneous amplification is not a simple co-amplification of a whole amplicon containing both genes. HER2 and TOP2A aberrations seem to be due to different mechanisms of amplifications. Most gene signals are translocated to abnormal marker chromosomes. HER2 genes but not TOP2A genes are present in tandem amplicons, leading to a higher HER2 ratio. This observation was confirmed by patient FISH data: 373 tumors (57%) had normal status for both genes. Among the 276 abnormal tumors, 67% had different HER2 and TOP2A status. Simultaneous amplification of both genes was found in 28% of the abnormal tumors (12% of all patients) while deletion of both genes was observed in 5% of the cases (2% of all patients). The average gene/reference ratio was significantly different: 5.0 for TOP2A but 7.2 for HER2 in the amplified tumors (P Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2119.

Purpose: Polysomy of chromosome 17 is frequently described in breast cancer, but firm definition of polysomy is lacking. We have used normal breast tissue to define the ploidy levels in the truncated nuclei of cut sections based on standard cytogenetic definition of ploidy in whole nuclei. Data from patients with invasive breast cancer enrolled in a randomized clinical trial was used to investigate the effect of polysomy of centromere 17 on patient survival. (Ejlertsen B et al., Eur J Cancer 2007:43:877-84).Material and methods: Fluorescence in situ hybridization (FISH) with TOP2A/CEN-17 was performed on 120 normal breast specimens. Centromere 17 (CEN-17) measurements from matching HER2/CEN-17 and TOP2A/CEN-17 FISH data was available from 649 patients from a previously published biomarker study (Knoop A et. al., J Clin Oncol 2005;23:7483-90).Results: Two thirds of the tumors had an average number of CEN-17 signals below two and 10% had more than three average CEN-17 signals. Monosomy (3.78 CEN-17/nucleus). In the aneuploid tumors and especially in the highly polyploidy tumors, amplification is the most frequent event in the HER2 assay whereas deletions increased with the ploidy level in TOP2A assay. Increasing ploidy was associated with decreased overall survival (P=0.0001), but multivariate analysis showed that polyploidy was not an independent prognostic factor neither predictive for treatment outcome.Conclusion: Polysomy 17 is a rare event in tumor samples from patients, but correlate to HER2 amplification and TOP2A deletion. However, inclusion of a reference probe is necessary if deletions are to be revealed and to avoid false positive amplified cases with a high proportion of dividing cells or a doubling of the DNA content. Very high polyploidy is associated with poor survival but is not an independent prognostic factor when other prognostic factor are included in a multivariate analysis. In this large clinical study, polysomy 17 was neither an independent prognostic nor predictive marker. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4035.

597 Background: HER-2, TOP2A, and TIMP-1 have shown predictive properties regarding benefit from anthracyclines in patients with breast cancer. In the present study, TIMP-1 IHC was integrated with TOP2A FISH and HER-2 status in two separate profiles. Methods: The DBCG 89-D trial randomized 980 high-risk Danish breast cancer patients to nine series of CMF or CEF. CEF was superior to CMF in terms of DFS and OS (Ejlertsen et al, EJC 2007). HER-2 status and TOP2A copy number changes were determined as described previously (Knoop et al, J Clin Oncol. 2005). TMA s were constructed and analyzed centrally for TIMP-1 expression by IHC (± tumor cell immunoreactivity). TIMP-1 was combined with HER-2 into a joint HT marker (HT-non-responsive (HT-NR): HER-2 normal and TIMP-1 positive or HT-responsive (HT-R): HER-2 positive and/or TIMP-1 negative) and with TOP2A into a joint 2T marker (2T-NR: TOP2A normal and TIMP-1 positive or 2T-R: TOP2A abnormal and/or TIMP-1 negative). Relationships between IDFS, OS and the HT/2Tprofiles were analyzed using multivariate regression analysis. Results: Among patients with a HT-R profile CEF was superior to CMF in terms of both invasive disease-free survival (IDFS) (HR 0.62; 95% CI 0.45–0.86; p = 0.004) and overall survival (OS) (HR 0.63; 95% CI, 0.45–0.87; p = 0.005). In patients with a HT-NR profile, no significant differences between CEF and CMF were demonstrated for IDFS or OS. A significant HT profile (HT-R or HT-NR) versus treatment (CEF or CMF) interaction was detected by the Wald-test for both IDFS (p = 0.036) and OS (p = 0.047). An even more pronounced separation was observed regarding the 2T profile, and in patients with a 2T-R profile treatment with CEF was superior to CMF in terms of IDFS (HR 0.48; 95% CI 0.34–0.69; p < 0.001) and OS (HR 0.54; 95% CI, 0.38–0.77; p < 0.001). No significant difference was observed in patients with a 2T-NR profile. A highly significant 2T profile versus treatment interaction was detected for IDFS (p < 0.001) and OS (p = 0.004). Conclusions: Profiles created by joining TIMP-1 with either HER-2 status or TOP2A gene status seems advantageous compared to the use of a single marker. [Table: see text]

Abstract #36 Background: Anthracyclins are potent poisons for topoisomerase II, an indispensable enzyme for DNA replication, repair and transcription. Apoptosis is often the consequence of anthracycline induced DNA breaks but cancer cells may escape through activation of the cell survival pathway by Tissue Inhibitor of Metalloproteinases-1 (TIMP-1). The aim of the present study was to determine the predictive value of the combination of the two biomarkers (TOP2A and TIMP-1) for benefits observed with CEF compared to CMF in DBCG trial 89D. Material and Methods: The DBCG 89-D trial randomized 980 high-risk Danish breast cancer patients to nine series of CMF or CEF, without endocrine therapy. Overall CEF was superior to CMF in terms of DFS and OS (Ejlertsen et al, EJC 2007). TOP2A copy number changes were determined with the TOP2A FISH pharmDx™ Kit (Dako A/S, Glostrup) as described previously (Knoop et al, JCO 2005). TMAs were constructed and analyzed centrally for TIMP-1 expression using the anti-TIMP-1 monoclonal antibody VT7 (Sørensen et al, J. Hist. Cytochem. 2006). A combined marker (2T) for anthracycline sensitivity was constructed as follows: 2T non-responsive (TOP2A normal and TIMP-1 positive) and 2T responsive (TOP2A abnormal and/or TIMP-1 negative). Relationships between IDFS, OS and the 2T profile were analyzed using multivariate regression analysis. Results: The assessable 623 patients differed significantly (P Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 36.

Telomere length analysis has aroused considerable interest in biology and oncology. However, most published data are pan-genomic Southern-blot-based estimates. We developed T/C-FISH (telomere/centromere-FISH), allowing precise measurement of individual telomeres at every single chromosome arm. Metaphase preparations are co-hybridized with peptide nucleic acid probes for telomeric sequences and the chromosome 2 centromere serving as internal reference. Metaphase images are captured and karyotyped using dedicated software. A software module determines the absolute integrated fluorescence intensities of the p- and q-telomeres of each chromosome and the reference signal. Normalized data are derived by calculating the ratio of absolute telomere and reference signal intensities, and descriptive statistics are calculated. T/C-FISH detects even small differences in telomere length. Using T/C-FISH we have discovered an epigenetic process occurring in the human male postzygote or early embryo: in umbilical cord blood lymphocytes, telomeres on male Xqs are around 1100 bp shorter than female Xqs.

Cytogenetic analysis was carried out on six passages (pass. 16, 19, 21, 25, 34, and 47) of the nontumorigenic epithelial cell line, HMT-3522, established from a fibrocystic lesion of the human breast. Minor chromosome abnormalities were present in the first passage (pass. 16) available for study, and limited cytogenetic progression was observed during the in vitro growth. A modal chromosome number of 45 chromosomes was found in all passages. Each passage contained 4-5 marker chromosomes. Three markers were consistently present in all passages studied. During in vitro growth two markers were gained and two markers were lost from the stemline karyotype. The two latest passages studied had identical karyotypes: 45,XX, del(1)(q44----p32:),t(5;14)(14p13----14q32::5q22----++ +5q35),t(6;8;12;17)(8p23---- 8q24::6p21.1----6p23;12q24----12p13::6p23- --- 6p25;17p13----17q25::6q11----6q27). The present study demonstrates chromosome abnormalities and karyotypic evolution in a nontumorigenic (in nude mice) and noninvasive (in vitro tested) cell line established from nonmalignant epithelial breast tissue. The results are discussed in relation to gene amplification, double minutes and oncogene localization.