Clinical Sequencing Analysis of the Mutation Profiles Associated with Extreme Radioresistance

Abstract

Precision medicine using genetic data of individual tumors is emerging in the field of cancer treatment. However, mutation profiles associated with the response of tumors to radiotherapy have not been fully elucidated. We hypothesized that the patients who exhibited extreme resistance to radiotherapy carry relevant mutation profiles. The patient with uterine cervical cancer that repeatedly locally-recurred after multiple rounds of curative radiotherapy, was selected as analytical subject. Biopsy samples were obtained from treatment-naïve tumor and the tumors that recurred after initial and secondary radiotherapy. Exon sequencing of 409 cancer-related genes was performed using a next-generation sequencer. Prevalence of candidate mutation profiles in clinical malignancy specimens was investigated using cBioportal. Contribution of candidate mutation profiles on radioresistance was examined by (i) meta-analysis of published in vitro radiosensitivity data combined with genomics data of Cancer Cell Line Encyclopedia (CCLE), and (ii) isogenic cell-based experiments using gene knock-in and siRNA techniques. The Differences between two groups were examined by Mann–Whitney test. The sequencing analysis identified activating mutations in PIK3CA (E545K) and KRAS (G12D), and putative biallelic inactivating mutations in SMAD4 (R361C and R261H) as trunk mutation signatures that persisted over the clinical course. Mining of published genomics data revealed that simultaneous mutations of KRAS and SMAD4 have not been reported previously for uterine cervical cancer (n = 0/343); the mutation signature was also rare among various types of malignancies (1.2%, n = 279/22928). Interestingly, this mutation signature was most prevalent in pancreatic cancer (23.7%, n = 195/820 cases) that is known to be clinically radioresistant. These data indicated that simultaneous mutations of KRAS and SMAD4 contributed to extreme radioresistant nature of the tumors analyzed in this study. Mining of CCLE genomics data identified 12 cancer cell lines carrying simultaneous mutations of KRAS and SMAD4. Meta-analysis of 94 publications identified by systemic literature review demonstrated that SF2, surviving fraction after 2 Gy-irradiation as assessed by clonogenic assays, was significantly higher for the KRAS/SMAD4-mutated cell lines compared to that for the background-matched control cell lines (P = 0.010). Experiments using KRASG12D-knock in- and SMAD4-knock down-SW48 cells demonstrated that SF2 for the KRAS/SMAD4-modulated cells was significantly higher than that for the parental cells (P = 0.034). These data indicate that simultaneous mutations in KRAS and SMAD4 are associated with radioresistance. The mutation signature is potentially useful as the biomarker for radioresistance in the practice of precision medicine.