Abstract 1934: Novel mutation and hypomethylation define distinct biological subgroups of altered KRAS colon tumors.
Authors: Rahi Hamed , Soltani Sohaila , Daremipouran Mohammad , Brim Hassan , Lee Eward L. , Woods Alfreda , Frederick Wayne , Laiyemo Adeyinka O. , Devaney Joe , Leavitt Ron , Sun Xueguang , Ashktorab Hassan 
Published: August 2014

Background: Several driver mutations have been discovered in colorectal cancer (CRC) progressions including KRAS and BRAF that has a practical significant therapeutic and prognostic value. Sequencing technology has advanced and now provides a tool for the discovery of novel driver mutations. In addition, sequencing technologies now provide a tool for whole genome methylation analysis. Here, we performed whole exome sequencing (WES) and whole genome methylation analysis to elucidate the involvement of novel candidate genes the KRAS pathway that may effect colorectal carcinogenesis.

Patients and Methods: WES was carried out on genomic DNA extracted from 8 normal-tumor pairs of frozen biopsies from African Americans patients with CRC. WES and Reduced Representation Bisulfite Sequencing (RRBS) were performed. Pyrosequencing and sanger sequencing used for validation of methylation and single nucleotide variants (SNV) respectively. For WES, base call quality recalibration, realignment around indels, SNV calling and variant call recalibration were carried out using Genome Analysis Tool Kit. Variants were then annotated using Annovar.

Results: WES uncovered somatic mutations alteration in many genes that are known be mutated in CRC including APC, BRAF, KRAS, Notch1, PIK3C2A, and NDRG4. We discovered a number of Novel SNVs in EID3, RGS3, HNRNPF, and GNAS in tumor samples. One GNAS missense mutation was discovered among KRAS mutated tumors. In addition, the tumor with the GNAS mutation was significantly hypomethylated (P&lt;0.05) in the CpG sites of GNAS promoter as compared with paired normal tissue. The tumor was located in the cecum and identified to be invasive adenocarcinoma with stage IV1b (T4b, N2a, M1). Ingenuity pathway analysis (IPA) showed that GNAS were involved in CRC metastasis signaling via APC, BRAF, GSK3A, KRAS, MLH1, MLH2, MLH3, Notch family, and PIK3C family.

Conclusion: This work provides insight into identification of novel somatic mutations in GNAS coupled with promoter hypomethylation at the same locus using WES and RRBS. GNAS SNV resulted in gain of function of G-protein signaling that may play a pivotal role in CRC Identification the biological significance of GNAS in CRC may introduce a new target for colorectal cancer diagnosis and treatment.

Citation Format: Hamed Rahi, Sohaila Soltani, Mohammad Daremipouran, Hassan Brim, Eward L. Lee, Alfreda Woods, Wayne Frederick, Adeyinka O. Laiyemo, Joe Devaney, Ron Leavitt, Xueguang Sun, Hassan Ashktorab. Novel mutation and hypomethylation define distinct biological subgroups of altered KRAS colon tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2013-1934