EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations

Abstract

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinase has been extensively studied in breast cancer; however, systematic studies of EGFR gene amplification and protein overexpression in breast carcinoma are lacking. We studied EGFR gene amplification by chromogenic in situ hybridization (CISH) and protein expression by immunohistochemistry in 175 breast carcinomas, using tissue microarrays. Tumors with 45 EGFR gene copies per nucleus were interpreted as positive for gene amplification. Protein overexpression was scored according to standardized criteria originally developed for HER-2. EGFR mRNA levels, as measured by Affymetrix U133 Gene Chip microarray hybridization, were available in 63 of these tumors. HER-2 gene amplification by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemistry were also studied. EGFR gene amplification (copy number range: 7-18; median: 12) was detected in 11/175 (6%) tumors, and protein overexpression was found in 13/175 (7%) tumors. Of the 11 tumors, 10 (91%) with gene amplification also showed EGFR protein overexpression (2 þ or 3 þ by immunohistochemistry). The EGFR mRNA level, based on Affymetrix U133 chip hybridization data, was increased relative to other breast cancer samples in three of the five tumors showing gene amplification. Exons 19 and 21 of EGFR, the sites of hotspot mutations in lung adenocarcinomas, were screened in the 11 EGFR-amplified tumors but no mutations were found. Three of these 11 tumors also showed HER-2 overexpression and gene amplification. Approximately 6% of breast carcinomas show EGFR amplification with EGFR protein overexpression and may be candidates for trials of EGFR-targeted antibodies or small inhibitory molecules.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

“…Proteolytic digestion: We 4,27 and others 15,18 demonstrated that the tissue digestion solution from SPoT-Light Tissue Pretreatment Kit (Zymed) provides consistent results. For all commercially available probes tested, we observed that digestion for o4 min leads to total absence of signals in the majority of tissues, whereas digestion for 410 min results in poor morphological features and blurred signals.…”

“…In addition, with the advent of tissue microarrays, ISH experiments can now be performed in a high throughput fashion. [13][14][15][16] This has been elegantly demonstrated by several groups using both fluorescent (FISH) 13,14 and chromogenic ISH (CISH). 15,16 Although FISH provides accurate copy number assessment, this technique is not ideally suited for TMAs, as fluorescent microscopy does not allow an optimal correlation between copy numbers and cytomorphological aspects of the cells and it proves difficult and time consuming to navigate through the tissue microarray section under a fluorescent microscope.…”

Association of epidermal growth factor receptor and mitogen-activated protein kinase with cystic neoplasms of the pancreas

“…In colorectal and breast cancers, amplification of the EGFR gene is invariably associated with overexpression of EGFR. 18,19 However, cancers that overexpress EGFR do not always show amplification of the EGFR gene, 19 indicating that amplification of the EGFR gene causes EGFR overexpression, but EGFR overexpression is not always caused by EGFR gene amplification. Our study indicated that EGFR overexpression in the serous cystic neoplasms is associated with an increase in the copy number of transcripts without amplification of the EGFR gene.…”

“…Aberrant signaling involving the EGFR and its downstream molecules is implicated in the development of cancers of the head and neck, breast, lung, colon, and pancreas. [16][17][18][19][20][21] Recently, EGFRs have come to be regarded as clinically important because of the development of a number of anti-EGFR drugs, including extracellular domain-targeted antibody-based drugs such as cetuximab, panitumumab, and zalutumumab, and intracellular kinase domain-targeted smallmolecule-based drugs such as erlotinib and gefitinib. These drugs enable the medical treatments of some cancers.…”

Fhit Expression Protects Against HER2-Driven Breast tumor Development: Unraveling the Molecular Interconnections

“…EGFR amplification and activating mutations are rare events in breast cancer and differences in EGFR expression appear to be controlled largely at the transcriptional level. 45,48 By contrast, it is well-established that about 20% of breast carcinomas are characterized by HER2 gene amplification, leading to an excess of HER2 protein in cancer cells and a high risk of recurrences (reviewed in ref. 49).…”