Abstract
BackgroundTesting for human epidermal growth factor receptor-2 (HER-2) in breast cancer is performed by either immunohistochemistry (IHC) or in situ hybridization (ISH). The growth factor receptor-bound protein-7 (GRB7) gene is in close proximity to HER-2 on chromosome 17q11-12 and codes a signal transduction molecule shown to be an independent adverse marker in breast cancer.MethodsHER-2 and GRB7 protein expression from 613 frozen breast tumors was determined by Western analysis. HER-2 protein results were confirmed with IHC. Commercial HER-2 FISH was performed on a subset of tumors with multi-probe FISH used to assess the extent of HER-2 gene amplification. mRNA expression was determined by Multi-plex RT-PCR.ResultsSeven tumors with GRB7 protein over-expression scored HER-2 FISH amplified but had no HER-2 protein over-expression. Four of the 7 tumors showed elevated GRB7 but not HER-2 mRNA over-expression. The breast cancer cell line HCC3153 did not over-express HER-2 protein but showed HER-2 FISH amplification of a limited segment around the HER-2 gene. Ten breast cancer tumors from the TCGA database had gene copy number increases around HER-2 without HER-2 mRNA or protein over-expression.ConclusionsA subset of human breast cancers that test positive with FISH for HER-2 gene amplification do not over-express HER-2 protein. One mechanism for this discordance is the incomplete amplification of the smallest HER-2 region of chromosome 17q11-12, which includes GRB7. HER-2 gene amplification without protein over-expression is clinically significant because patients with such tumors are unlikely to benefit from HER-2 targeted therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-386) contains supplementary material, which is available to authorized users.
Highlights
Amplification of chromosome 17q11-12 occurs in about 20-25% of breast tumors leading to over-expression of the human epidermal growth factor receptor 2 gene (HER-2 or ERBB2) (Slamon et al 1987; Slamon et al 1989; Ross et al 2003)
Previous results of Western analysis (Figure 1a and Additional file 1: Table S1) of primary breast cancer extracts from the Oregon Health and Science University (OHSU) Knight Cancer Institute Breast Cancer Tumor Repository for the presence of growth factor receptor-bound protein-7 (GRB7) and human epidermal growth factor receptor-2 (HER-2) proteins reported that GRB7 over-expression was found in the absence of HER-2 over-expression in 30 of 564 or 5% of tumors (Ramsey et al 2011)
When tumors with this discordant GRB7/HER-2 protein expression pattern were analyzed for HER-2 gene amplification with an FDA approved, commercially available fluorescence in situ hybridization (FISH) assay (PathVysion, Abbott Labs), 6 of 27 samples scored amplified for HER-2 (Table 1)
Summary
Cell culture, and databases Tumor specimens analyzed in this study were derived from the Oregon Health and Science University (OHSU) Knight Cancer Institute Breast Cancer Repository (Ramsey et al 2011; Christianson et al 1998; Saez et al 2006). BAC probe #3079 begins in the middle of GRB7 gene and extends further downstream and away from the HER-2 gene towards the telomere Both ends of the BAC insert have been sequenced and verified as reported (AQ121537 and AQ 121760). HER-2 immunohistochemistry (IHC) HER-2 IHC was performed by the College of American Pathologist (CAP) certified OHSU Clinical Immunohistochemistry Laboratory using an FDA approved kit with modifications for frozen tissues. HER-2 (human epidermal growth factor receptor 2) forward primer 5′ - GGA AAC CTG GAA CTC ACC TA- 3′, reverse primer 5′ - TTG GTG TCT ATC AGT GTG AGA - 3′, product size 388 bp
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