Abstract 4246: Promoter hypermethylation and gene expression of FANCF in non-small cell lung cancer (NSCLC).

The Fanconi Anemia (FA) pathway is essential for human cells to maintain integrity following DNA damage. This pathway is involved in repairing double stranded DNA breaks. Cancers with defective FA pathway are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. The FA pathway contains 15 genes, and some of the members have been implicated in susceptibility to a number of cancers by genetic or epigenetic alterations. The methylation of CpG islands in the FANCF gene is thought to plays a role in the occurrence of ovarian cancer. We have recently reported the detection of up to 20% of NSCLC to be FA functionally inactive (lack of FANCD2 foci formation in the nucleus of proliferating cells by triple stain immunofluorescence, FATSI negative). Since epigenetic inactivation can be one of the mechanisms for FA functional deficiency in these tumors, we evaluated a series of NSCLC samples for FANCF methylation. Human lung tumor tissue samples were obtained from The Tissue Procurement Shared Resources of the Ohio State University Comprehensive Cancer Center after IRB approval. FA pathway status was evaluated by FATSI. Genomic DNA and total RNA samples were isolated from frozen lung tumor and matching non-tumor tissues. The methylation status of the FANCF gene promoter was evaluated using methylation-specific PCR (MS-PCR). FANCF gene expression was evaluated by NanoString assays. We screened total of 40 NSCL tumors by the FATSI assay, and the ratio of FANCD2 foci negative tumor was 20% (8/40). Squamous cell carcinoma, adenocarcinoma and large cell carcinoma histologies were all represented in the samples. FANCF promoter methylation was present in 2 of 8 FATSI negative tumors (1 adenocarcinoma and 1 large cell carcinoma) based on MS-PCR analysis, and absent in 8 tested FATSI positive tumors. NanoString analysis was performed in 5 FATSI positive and 5 FATSI negative tumors which the latter including the two tumors with FANCF methylation identified with the MS-PCR. FANCF mRNA level was 1.8-fold lower in tumors with promoter methylation as comparing to matched non-tumor tissues. One of the two tumors containing FANCF methylation was also analyzed with RNAseq, and the results showed a 1.6 fold reduction in FANCF mRNA in the tumor as comparing to matched non-tumor tissue. However the FANCF mRNA level was similar between tumor and non-tumor (tumor/non-tumor = 1.04) in the samples without methylation. The above observation suggests that epigenetic alterations can be the base for FA functional deficiency in NSCLC patients. These findings may have clinical implications, since these tumors may be more sensitive to cross-link based therapy. However, an important caveat is that these changes may not be stable and could revert during treatment. Evaluation of pre and post treatment samples in FATSI negative patients undergoing therapy would be necessary to support this hypothesis. Citation Format: Wenrui Duan, Tyler Rees, Kevin Vu, Brittany Barnwell, Li Gao, Arjun Kalvala, xin wu, Gregory A. Otterson, Miguel A. Villalona-Calero. Promoter hypermethylation and gene expression of FANCF in non-small cell lung cancer (NSCLC). [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 4246. doi:10.1158/1538-7445.AM2013-4246