P3.06 Comparison of High Resolution Melting and Restriction Fragment Length Polymorphism to Genotype Snp in Formalin Fixed Paraffin-Embedded Breast Cancer Tissue

Abstract

ABSTRACT SNPs of NQO1, p53, and MDM2 have been correlated with pathological response or overall survival of breast cancer patients treated with anthracycline based chemotherapy. Therefore, the ability to detect these SNPs may be useful for treatment decision. In this study, we genotyped FFPE breast cancer samples to detect those SNP. Objective The aim of this study was to know the accuracy of HRM (High Resolution Melting) method to genotype SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G's in comparison to RFLP (Restriction Fragment Length Polymorphism) from archived formalin fixed paraffin embedded (FFPE) breast cancer blocks. Materials and Methods FFPE blocks were collected from anatomic pathology laboratory. DNA were isolated, and PCR amplified followed by either RFLP or HRM methods. Restriction enzymes HinfI, BstUI, and MspA1I were used in RFLP to genotype NQO1, p53, and MDM2 SNPs, respectively. The primer design which been used was the same between HRM and RFLP. The results of HRM were analyzed using melting, normalization, and differentiation plot curves. Sequencing technique has been used to resolve discrepancy between RFLP and HRM results. Results We screened SNPs on breast cancer FFPE samples using HRM and RFLP. The rates of genotyping agreement between both methods are 69, 23% (9/13 samples), 80% (8/10 samples), and 100% (7/7 samples) for NQO1, p53, and MDM2 SNPs, respectively. Conclusion HRM is a faster method than RFLP to genotype important breast cancer SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G. Nevertheless, RFLP is more reliable method than HRM when the used primers sets are not optimized for HRM.