Mohsen Soosanabadi - Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Reza Mirfakhraie - Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Lilit Atanesyan - MRC-Holland BV, Amsterdam, the Netherlands
Akbar Biglarian - Department of Biostatistics and Computer Sciences, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Fatemeh Aghakhani Moghadam - Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Maryam Rahimi - Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Farkhondeh Behjati - Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Elaheh Keyhani - Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran & Clinical Research Development Center of Rofeideh Rehabilitation Hospital, Tehran, Iran.

Background: The aim of this study was to assess the usability of multiplex ligation-dependent probe amplification (MLPA) for copy number determination of HER gene family members (ERBB1-4) in invasive breast carcinoma and to explore the association of ERBB1-4 gene copy numbers with clinicopathological characteristics of breast cancer (BC) patients. Methods: Clinical and immunohistochemical characteristics were assessed in 104 BC patients and the molecular subtype was determined for each tumor sample. Furthermore, HER-2/neu status was assessed by immunohistochemistry (IHC) and equivocal results were confirmed by Fluorescent in situ hybridization (FISH). The copy numbers of ERBB1-4 genes were determined by MLPA. Results: Twenty-five percent of all patients showed ERBB2 gene-amplification by MLPA, whereas 14.4% of cases showed ERBB-2/neu overproduction at the protein level (IHC). Moreover, only 2.9% and 1.9% of patients showed amplification in ERBB1 and ERBB4, respectively. No copy number changes were observed in ERBB3. Our results indicated a significant association between ERBB2 copy number gain and histological grade (p value= 0.01), stage (p value= 0.02), and tumor subtypes (p value= <0.001). In addition, we found MLPA more accurate in assessing HER2 status with 15.4% and 9.6% gene amplification detection in early stages (1, 2A and 2B) and advanced tumor stages (3A, 3B, and 4), respectively, compared to IHC (early stages= 13.5% and advanced stages= 4.7%). Conclusions: According to our findings, MLPA is a fast, precise and low-cost technique to detect ERBB2 amplification, especially in advanced tumor stages. However, due to infrequent amplification found in ERBB1 and ERBB4 as well as the lack of amplification in ERBB3, their importance in the prognostic evaluation of BC patients remains controversial.  

Breast Cancer, Copy number variation, ERBB1-4, IHC, MLPA.