Detection of KRAS G12/G13 Mutations in Cell Free-DNA by Droplet Digital PCR, Offers Prognostic Information for Patients with Advanced Non-Small Cell Lung Cancer

Abstract

KRAS mutations are found in approximately one third of non-small cell lung cancer (NSCLC) patients. In this study, we aim to investigate whether KRAS G12/G13 mutant allele fraction (MAF) in cell-free DNA (cfDNA) can provide meaningful prognostic information in NSCLC. Multiplex droplet-digital PCR was used to quantitatively assess KRAS G12/G13 MAF in cfDNA from 114 pre-treated advanced disease NSCLC patients. In 14 patients, changes in KRAS G12/G13 MAF were longitudinally monitored during treatment. Plasma KRAS G12/G13 status was associated with poor patients’ outcome in terms of progression-free survival (PFS) (p < 0.001) and overall survival (OS) (p < 0.001). In multivariate analysis, the detection of plasma KRAS mutations was an independent predictor of adverse PFS (HR = 3.12; p < 0.001) and OS (HR = 2.53; p = 0.002). KRAS G12/G13 MAF at first treatment evaluation (T1) was higher (p = 0.013) among patients experiencing progressive disease compared to those with disease control, and increased KRAS MAF at T1 was associated (p = 0.005) with shorter PFS. On the contrary, no association was observed between tissue KRAS mutation status and patients’ prognosis. Our results show that ddPCR-based detection of KRAS G12/G13 mutations in plasma could serve as an independent biomarker of unfavorable prognosis in NSCLC patients. Changes in KRAS MAF can provide valuable information for monitoring patient outcome during treatment.

Circulating Tumor DNA Detection by Digital-Droplet PCR in Pancreatic Ductal Adenocarcinoma: A Systematic Review

“…Similarly, Michaelidou et al used multiplex ddPCR to quantify KRAS G12/G13 MAF in ctDNA from 114 pre-treated advance NSCLC patients. Again, plasma KRAS G12/G13 status was associated with poor patient outcome in terms of PFS and OS ( p < 0.001, respectively) [ 71 ]. Finally, Guibert and collaborators detected KRAS mutations in up to 81% of the patients with a sensitivity of 78% [ 72 ].…”

Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae

“…Recently, we exploited an improved, high‐tech and highly sensitive version of PCR, i.e., droplet digital PCR (ddPCR), to develop diagnostics for the accurate detection and monitoring of emerging low‐frequency insecticide resistance mutations in malaria vector populations 7 . ddPCR is broadly used in human 8 and food diagnostics 9–11 to detect very small amounts of target DNA within a large background of non‐target DNA (e.g., non‐abundant mutations in circulating cell free DNA of cancer patients, 8 or genetically modified (GM) plant specimens) 11 …”

Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR

“…KRAS is an oncogenic driver gene that appears to be mutated in 25–30% of NSCLC patients [ 51 ]. Mutations in codons 12 and 13 of this gene are the most frequent alterations and stand as the principal cause of the development and progression of several cancer types [ 52 ]. Despite all clinical advances in personalized therapies and their proven impact on patient’s clinical outcome, there is only one effective drug (sotorasib) [ 53 ] approved by the Food and Drug Administration (FDA) for KRAS G12C NSCLC patients [ 54 , 55 ].…”

“…The design of ddPCR multiplex assays to detect the most frequent G12/G13 KRAS mutations has allowed for rapid and accurate genotyping of plasma cfDNA with a LOD of at least 0.2% [ 51 , 52 , 59 , 60 ]. As a result of these assays, an association between KRAS mutated concentration and disease stage has been observed.…”

“…Patients with advanced lung cancer stages present greater amounts of detectable KRAS mutations in plasma cfDNA samples, being 8% in stage I, 30% in stage II, 71% in stage III, and 73% in stage IV [ 51 ]. Furthermore, it has been observed that patients with stable disease presented lower KRAS mutations levels than patients who had progressed [ 52 ]. Also, several studies have associated plasma ctDNA KRAS mutations with shorter PFS and OS [ 51 , 52 , 59 , 60 ], and chemotherapy treatment efficacy has been longitudinally monitored, showing a connection between ctDNA KRAS concentration changes and therapy response [ 51 , 61 , 62 ].…”