Analysis of circulating cell-free DNA identifies KRAS copy number gain and mutation as a novel prognostic marker in Pancreatic cancer

Sumitra Mohan,Cong Zhou,Sakshi Gulati,Mahmood Ayub,Hui Sun Leong,Mairéad G Mcnamara,Juan W Valle,Antoine Hollebecque,Angela Lamarca,Dominic G Rothwell,Tine Descamps,Ged Brady,Nigel K Smith,Caroline Dive,Sudhakar Sahoo,Richard A Hubner,Bedirhan Kilerci

doi:10.1038/s41598-019-47489-7

Abstract

Serial biopsy of pancreatic ductal adenocarcinoma (PDAC), to chart tumour evolution presents a significant challenge. We examined the utility of circulating free DNA (cfDNA) as a minimally invasive approach across a cohort of 55 treatment-naïve patients with PDAC; 31 with metastatic and 24 with locally advanced disease. Somatic mutations in cfDNA were detected using next generation sequencing in 15/24 (62.5%) and 27/31 (87%) of patients with locally advanced and metastatic disease, respectively. Copy number changes were detected in cfDNA of 10 patients of whom 7 exhibited gain of chromosome 12p harbouring KRAS as well as a canonical KRAS codon 12 mutation. In multivariable Cox Regression analysis, we show for the first time that patients with KRAS copy number gain and KRAS mutation have significantly worse outcomes, suggesting that this may be linked to PDAC progression. The simple cfDNA assay we describe will enable determination of the presence of KRAS copy number gain and KRAS mutations in larger studies and clinical trials.

Highlights

We see a relatively short median survival of 7.99 months compared to the 19.77 months reported in a TCGA study[7], this most likely reflects differences in staging with the TCGA cohort comprising operable localised disease whereas our cohort includes patients with locally advanced and metastatic disease, resulting in a shorter median survival, in line with those reported by other groups[8,9]
Analysis of cell free DNA (cfDNA) from each patient revealed the presence of a canonical KRAS somatic mutation, which was determined by Droplet digital PCR (ddPCR) and was found to be 38% (21/55) overall; 48% (15/31) in metastatic disease and 25% (6/24) in locally advanced disease, in keeping with other published studies[6]
Our detection rate of 38% for the presence of a KRAS mutation in patient cfDNA is in line with other reports, there is considerable variation in reported frequencies (27~93%)[10,11] which may reflect the methodologies employed, as well as the variability of KRAS allelic ratios in the tumour[10] and the low ctDNA burden associated with pancreatic cancer[12]

Summary

Introduction

No significant differences were observed in yield of cfDNA detected between the 31 patients with metastatic and 24 with locally advanced PDAC (p-value = 0.19; Fig. 2). From cfDNA NGS, both CNA and somatic mutations were elevated in the patients with metastatic disease compared to the patients with locally advanced disease (p-values of 0.0164 and 0.0049, respectively, Fig. 2B,C).

Results

Conclusion