Clinical Relevance of KRAS-Mutated Subclones Detected with Picodroplet Digital PCR in Advanced Colorectal Cancer Treated with Anti-EGFR Therapy

Abstract

Purpose: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis.Experimental Design: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRASmutated allele fraction.Results: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors.Conclusions: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies.

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies

“…Our data are consistent with other studies reported recently (3,24). LaurentPuig and colleagues suggested that patients with mCRC with KRAS exon2-mutated subclones (lower or equal to 1%) had a benefit from anti-EGFR therapy (24).…”

“…Our data are consistent with other studies reported recently (3,24). LaurentPuig and colleagues suggested that patients with mCRC with KRAS exon2-mutated subclones (lower or equal to 1%) had a benefit from anti-EGFR therapy (24). Similarly, data reported on RAS extension mutations analyzed by BEAMing in the KRAS exon 2 wild-type cohort of patients enrolled in the CRYSTAL trial showed different outcomes depending on the cutoff value of mutated/wild-type.…”

“…Subsequently, we established that 14 were the optimal number of pre-amplification cycles needed to obtain robust results maintaining its quantitative power when using dPCR. It is noteworthy that with the modified approach we have validated the 1% threshold value that was initially reported with no preamplification step (24).…”

“…Recently, LaurentPuig and colleagues (24) has evaluated the role of minor mutated KRAS sub-clones in patients with mCRC treated with anti-EGFR drugs using picoliter multiplex droplet dPCR. A threshold of 1% of KRAS exon 2-mutated clones was determined as optimal to distinguish responder from nonresponder populations.…”

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

“…The LOB and LOD determination are described in the Materials and Methods sections. The dynamic range and sensitivity of the assays were determined as described previously (32)(33)(34). The measured concentration of hypermethylated DNA matched the anticipated concentration from 10% to 0.1% (see online Supplemental Fig.…”