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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 < 0.80) was observed in 32%. Further, we identified ESR1 gain (ratio ESR1/CEN-6 from 1.30 to 1.99) in 19% of the patients. A positive correlation of ESR1 FISH with both ER-cytosol and ER IHC was found (p < 0.0001). Amplification and gain of the ESR1 gene are associated with higher ER protein content measured by ligand binding assay and a more intense nuclear staining by IHC compared to tumors with normal ESR1 gene status. Major variations in ER measured by ligand binding assay and IHC are observed within all ESR1 copy number subgroups and other mechanisms than gene copy number seem to contribute to the ER protein content in the tumors., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Aims To check the usefulness of a standardized protocol of PTEN FISH in 31 endometrial carcinomas ( ECs) in comparison with SNP array ( SNPA), multiplex ligation-dependent probe amplification ( MLPA), and immunohistochemistry. Methods and results Fluorescence in-situ hybridization analysis showed two PTEN copies in 17 cases, three copies in nine cases, hemizygous deletion in two cases, and diverse cell populations with different PTEN copy number in three cases. A good correlation was seen between FISH and SNPA, particularly in cases with three copies. FISH identified two cases with entire deletion of chromosome 10, but did not identify a focal deletion of PTEN. Five cases with PTEN deletion and duplication of the second allele by SNPA were interpreted as normal by FISH. Concordance between FISH and MLPA was seen in 15 cases with two copies, and in two cases with PTEN deletion. Six cases were interpreted as amplified by MLPA, but showed polyploidy by FISH. FISH was superior to SNPA and MLPA in assessing the tumours with diverse cell populations with different PTEN copies. Conclusions The results show good concordance between FISH, SNPA and MLPA. SNPA was superior in tumours with deletion of one copy and duplication of the second allele. FISH was superior in assessing tumour heterogeneity. [ABSTRACT FROM AUTHOR]

This abstract was listed as "Withdrawn" in the SABCS Pocket Program and the SABCS Abstract Book and has been reinstated. 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 31/2 -4 1/2hours. 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 ower 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 he 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 ncluded 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 eproducible. 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 mproved patient care. Taken together, the study demonstrates the potential of a new evolutionary platform that enables optimization and acceleration of FISH analysis to the benefit of cancer patients and laboratory personnel. [ABSTRACT FROM AUTHOR]