Multi-gene fluorescence in situ hybridization to detect cell cycle gene copy number aberrations in young breast cancer patients

Abstract Context.—The Topoisomerase IIα (TOP2A) protein is the target of the anthracycline class of chemotherapeutic agents. TOP2A is frequently coamplified with c-erb-B2 and consequently might be a prognostic and/or predictive factor for breast cancer patients when anthracycline-based chemotherapy is a consideration. A total of 20% to 35% of breast carcinomas show amplification of the erb-B2 gene, some of which also have coamplification of the TOP2A gene. Investigation of the prognostic or predictive significance of these gene amplifications requires a reliable and sensitive method for the measurement of gene copy number in clinical tumor samples. Objective.—To assess 2 different assay methods that might allow accurate, reproducible, quantitative, and high-throughput estimation of gene copy number in fresh, frozen, or paraffin-embedded breast cancer specimens. Design.—We developed an assay and analyzed the gene copy numbers of the erb-B2 and TOP2A genes in 8 breast cancer cell lines, 6 fresh frozen samples, and 38 paraffin-embedded breast tumor specimens by a novel real-time polymerase chain reaction (PCR) assay using hybridization probes. The results were compared with standard fluorescence in situ hybridization. Results.—We discovered a 100% concordance between assessment of gene copy number of erb-B2 and TOP2A between quantitative PCR and fluorescence in situ hybridization (FISH). Quantitative PCR also had the additional feature of uncovering an erb-B2 gene polymorphism. Finally, we observed that TOP2A amplification only occurred in conjunction with erb-B2 amplification in our paraffin-embedded cases of invasive breast carcinoma and that this event was present in 5 (42%) of 12 erb-B2 amplified cases. Conclusions.—We conclude that the potentially automatic, real-time PCR analysis using hybridization probes is an efficient method to perform copy number analysis, with results that appear identical to the FISH technique and with the benefit of identifying HER-2 polymorphisms.

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In situ analysis of FGFR2 mRNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients

Gastric Cancer ◽

10.1007/s10120-017-0758-x ◽

2017 ◽

Vol 21 (3) ◽

pp. 401-412 ◽

Cited By ~ 4

Author(s):

Yasutoshi Kuboki ◽

Christoph A. Schatz ◽

Karl Koechert ◽

Sabine Schubert ◽

Janine Feng ◽

...

Keyword(s):

Gastric Cancer ◽

In Situ Hybridization ◽

Cancer Patients ◽

Copy Number ◽

Gene Copy Number ◽

In Situ Analysis ◽

Gene Copy ◽

Fgfr2 Gene ◽

Gastric Cancer Patients

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Use of Chromogenic In Situ Hybridization to Identify MYCN Gene Copy Number in Neuroblastoma Using Routine Tissue Sections

The American Journal of Surgical Pathology ◽

10.1097/01.pas.0000202163.82525.5c ◽

2006 ◽

Vol 30 (5) ◽

pp. 635-642 ◽

Cited By ~ 20

Author(s):

Paul S. Thorner ◽

Michael Ho ◽

Susan Chilton-MacNeill ◽

Maria Zielenska

Keyword(s):

In Situ Hybridization ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Tissue Sections ◽

Mycn Gene ◽

Chromogenic In Situ Hybridization

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Gene copy number of epidermal growth factor receptor (EGFR) by fluorescent in situ hybridization (FISH) in head and neck squamous cell carcinomas (HNSCC) is closely correlated with EGFR protein levels assessed by automated quantitative protein analysis (AQUA)

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.6023 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 6023-6023

Author(s):

P. Weinberger ◽

A. Psyrri ◽

P. Kountourakis ◽

T. Rampias ◽

C. Sasaki ◽

...

Keyword(s):

In Situ Hybridization ◽

Protein Expression ◽

Protein Content ◽

Gene Amplification ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Egfr Gene ◽

Protein Levels

6023 Background: EGFR overexpression correlates with recurrence and with treatment resistance in HNSCC. The mechanisms of EGFR protein overexpression are poorly understood. Nonetheless, previous investigators have not demonstrated a correlation between EGFR gene copy number and protein content, using conventional immunohistochemistry (IHC). The aim of this study was to evaluate the relationship of EGFR gene copy number and protein expression utilizing fluorescence in situ hybridization (FISH) and AQUA, a novel, immunohistochemical method of automated quantitative in situ proteomic analysis which permits subcellular localization. Methods: A tissue microarray composed of 137 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis/Abbot) and EGFR protein expression (DAKO antibody) using AQUA analysis of EGFR staining scored on a scale of 0–255 and by conventional IHC. Agreement was assessed using kappa. Results: Sixteen (15%) of one-hundred six tumors with FISH results demonstrated EGFR high polysomy and/or gene amplification (FISH+). AQUA demonstrated a range of 3.6–102.2; protein levels assessed by AQUA in the FISH amplified cases were significantly higher (p =0.008) than in the FISH non- amplified ones. Using the EGFR 75th percentile as a cut-off, AQUA and FISH showed significant agreement (percentage of overall agreement 82%, kappa=0.458, p=0.003). To the contrary there was no concordance between FISH and conventional IHC results in this series. Conclusions: The discrepancy between EGFR gene amplification rate and protein expression by IHC reported previously may be due to the limitations and nonquantitative nature of conventional IHC. EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. No significant financial relationships to disclose.

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