Detection of Gene Amplification by Multiplex Ligation-Dependent Probe Amplification in Comparison with In Situ Hybridization and Immunohistochemistry.

Sanaz Tabarestani,Hamid Rezvani,Sayyed Mohammad Hossein Ghaderian

doi:10.7314/apjcp.2015.16.17.7997

Abstract

Gene amplification is an important mechanism in the development and progression of cancer. Currently, gene amplification status is generally determined by in situ hybridization (ISH). Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based method that allows copy number detection of up to 50 nucleic acid sequences in one reaction. The aim of the present study was to compare results for HER2, CCND1, MYC and ESR1 gene amplification detected by MLPA with fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as clinically approved methods. Tissue samples of 170 invasive breast cancers were collected. All were ER positive. Tissue samples had previously been tested for HER2 using immunohistochemistry. Amplification of the selected genes were assessed using MLPA, FISH and CISH and results were compared. HER2 MLPA and ISH results were also compared with HER2 immunohistochemistry (IHC) which detects protein overexpression. Amplification of HER2, CCND1, MYC and ESR1 by MLPA were found in 9%, 19%, 20% and 2% of samples, respectively. Amplification of HER2, CCND1, MYC and ESR1 by FISH was noted in 7%, 16%, 16% and 1% of samples, respectively. A high level of concordance was found between MLPA/ FISH (HER2: 88%, CCND1: 88%, MYC: 86%, ESR1: 92%) and MLPA/ CISH (HER2: 84%). Of all IHC 3+ cases, 91% were amplified by MLPA. In IHC 2+ group, 31% were MLPA amplified. In IHC 1+ group, 2% were MLPA amplified. None of the IHC 0 cases were amplified by MLPA. Our results indicate that there is a good correlation between MLPA, IHC and ISH results. Therefore, MLPA can serve as an alternative to ISH for detection of gene amplification.

Highlights

Gene amplification is an important mechanism in the development and progression of cancer
The aim of the present study was to compare the results of Human epidermal growth factor receptor 2 (HER2), CCND1, MYC and ESR1 gene amplification detected by Multiplex ligation-dependent probe amplification (MLPA) with fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and IHC as clinically approved gene amplification methods
Gene amplifications detected by FISH and CISH were highly comparable

Summary

Introduction

Gene amplification is an important mechanism in the development and progression of cancer. CCND1 gene is located on chromosome 11q13 and encodes for cyclin D1 protein which is central to the regulation of G1-S phase transition (Santarius et al., 2010; Musgrove et al, 2011). CCND1 gene is amplified in 15% to 20% and overexpressed in 50% to 70% of breast cancers (Musgrove et al, 2011). Amplification of MYC gene has been shown to be indicative of poor prognosis in breast cancer (Deming et al, 2000, Schlotter et al, 2003). ESR1 gene is located on chromosome 6q25 and encode for estrogen receptor α (ERα). Some investigators have suggested that ESR1 amplification occurs frequently in breast cancer (13% to 20%) and has predictive value for

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Conclusion