Molecular Profiling and Prognostic Value of KRAS Gene Mutations in Tumor Samples and Liquid Biopsies from Patients with Pancreatic Ductal Adenocarcinoma

Abstract

Introduction: The lack of sensitive, cheap, and fast molecular technologies for detecting KRAS mutations in pancreatic ductal adenocarcinoma (PDAC) patients has hampered its translation into clinical practice. We validated the fast single-step methodology based on amplification-refractory mutation system(ARMS) coupled with high resolution melting analysis(HRMA) to assess KRAS mutations and its prognostic value in PDAC patients. Methods: Prospective multicentric cohort study including PDAC patients with codon-12 KRAS mutational status determined by Sanger Sequencing (SS) on tumor and plasma samples collected at diagnosis. ARMS/HRMA was further applied to assess the two most frequent mutations (G12D/G12V). Primary endpoint was the frequency of KRAS mutations by both techniques and their concordance. Secondary endpoints were the association of KRAS mutations with progression free survival (PFS) and overall survival (OS). Results: Of 55 patients, 29 underwent resection surgery and 26 underwent palliative chemotherapy (ChT) or best supportive care. ARMS/HRMA was performed in 38/55 tumor samples (6 with G12R/G12C mutations by SS and in 11 there was no DNA left after SS). SS detected KRAS mutations in 23/38(60%) and ARMS/HRMA in 33/38(87%) tumor samples (G12D=18, G12V=12, G12V/G12D=3). DNA was successfully extracted in 32/55 plasma samples: SS genotyping was conclusive in 22/32(69%) and only detected a G12V mutation, while ARMS/HRMA was conclusive in all samples and detected mutations in 7 (G12V=3, G12D=3, G12V/G12D=1). Operated patients with KRAS-mutated tumors had lower PFS (5 vs 13 months, p<0.01) and OS (9 vs 14 months, p<0.01). ChT treated patients with KRAS-mutated ctDNA had lower PFS (1 vs 6 months, p=0.02) and lower OS (1 vs 8 months, p<0.01). Conclusions: ARMS/HRMA seems to be highly sensitive and more accurate than SS to assess KRAS mutations in PDAC tumor and plasma samples, with advantages of fastness and low cost. KRAS mutations were associated with lo