Abstract 2103: High resolution melting analysis: a sensitive screening method for the detection of MDM2 promotor SNP309

Abstract

Abstract Background: Murine double minutes-2 (MDM2) protein plays a key role in physiological processes like growth arrest, senescence and apoptosis. MDM2 negatively regulates key proteins like p53 by inhibiting its transcriptional activity and promoting its proteosomal degradation through ubiquitinylation. In turn, the MDM2 expression is regulated in part by a p53-responsive promotor. This negative feedback control mechanism assures that both p53 and MDM2 are kept at very low levels in proliferating cells. However, in about half of all human tumors, the normal regulation of p53 might be disrupted through direct overexpression of MDM2 caused by e.g. MDM2 gene amplification or a T to G substitution (SNP309) in the promotor region of MDM2. High resolution melting analysis (HRMA) provides a valid approach to efficiently detect DNA mutations. The current study aimed at validating and implementing HRMA for screening of colorectal cancer patients (CRC) to detect MDM2 promotor SNP309. Pyrosequencing was used to confirm and characterize HRMA results. Materials and Methods: First, HRMA sensitivity was established using a cell line and a FFPE (formalin-fixed paraffin embedded) dilution model. Then, the sensitivity of pyrosequencing to detect MDM2 promotor SNP309 was evaluated using the same dilution models. Next, HRMA was validated on 10 cell lines. Since the MDM2 SNP309 status for these cell lines was unavailable, all HRMA results were confirmed and characterized by pyrosequencing. Results: The cell line dilution model revealed a detection limit of 6% while the detection limit in a background of FFPE wild-type DNA was found to be 3%. The detection limit of pyrosequencing to analyze SNP309 in a background of wild-type cell line DNA seems to be between 20% and 15%. In a background of FFPE wild-type DNA, the detection limit seems to be 3%. HRMA revealed abnormal melting patterns in 5/10 cell lines and pyrosequencing confirmed the presence of 2 homozygous (G/G) and 3 heterozygous (G/T) genotypes. Preliminary results on FFPE material from CRC patient samples showed abnormal melting patterns in 6/13 samples. These are currently being confirmed using pyrosequencing. Discussion: HRMA was found to be a fast, efficient, sensitive and reproducible screening method for MDM2 promotor SNP309 detection with a detection limit between 3% and 6%, which is higher than that of conventional sequencing methods. Preliminary results indicate that HRMA can be used as a screening method for MDM2 SNP309 by which DNA from FFPE tissues can be tested. Currently, a cohort of CRC patients is being tested for the presence of SNP309 using HRMA and pyrosequencing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2103. doi:1538-7445.AM2012-2103