Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA

Abstract

Assessment of KRAS status is mandatory in patients with metastatic colorectal cancer (mCRC) before applying targeted therapy. We describe here a blinded prospective study to compare KRAS and BRAF mutation status data obtained from the analysis of tumor tissue by routine gold-standard methods and of plasma DNA using a quantitative PCR-based method specifically designed to analyze circulating cell-free DNA (cfDNA). The mutation status was determined by both methods from 106 patient samples. cfDNA analysis showed 100% specificity and sensitivity for the BRAF V600E mutation. For the seven tested KRAS point mutations, the method exhibited 98% specificity and 92% sensitivity with a concordance value of 96%. Mutation load, expressed as the proportion of mutant alleles in cfDNA, was highly variable (0.5-64.1%, median 10.5%) among mutated samples. CfDNA was detected in 100% of patients with mCRC. This study shows that liquid biopsy through cfDNA analysis could advantageously replace tumor-section analysis and expand the scope of personalized medicine for patients with cancer.

Detection of KRAS Mutations in Plasma DNA Using a fully Automated Rapid Detection System in Colorectal Cancer Patients

“…These data indicate that the anti-cancer effect of chemotherapy containing anti-EGFR antibody is correlated with the mutant load. The detection rate using MBP-QP was relatively low compared to other detection systems previously reported [21,23,24]. It seems to be related to the fact that patients in our study included non-metastatic cancer patients, and sample collection was conducted during chemotherapy in 2/3 of those patients.…”

“…KRAS mutations were frequently detected in systemic metastatic cancers, and these were newly detected after acquired resistance to anti-EGFR antibody. Recently, mutation analysis using peripheral blood-socalled liquid biopsy-has been applied to colorectal cancer [9,10,[23][24][25]. In most published reports, quantitative PCR and BEAMing were used for detection with plasma DNA.…”

“…Mutation status of KRAS with plasma DNA was analyzed after the start of chemotherapy using the MBP-QP and PCR-rSSO methods. White and black arrows in BKRAS mutation in plasma DNA^indicate the peaks for KRAS wild type and mutant type, respectively plasma DNA in from 76% to 98% of cases using these detection methods [21,23,24]. According to results using digital PCR, tumor derived DNA was detected in more than 75% of colorectal cancer patients, and the detection rate was equivalent to advanced pancreas, ovarian, breast, and gastroesophageal cancers [24].…”

PCR digitale en micro-compartiments

“…Il peut s'agir de cellules du microenvironnement tumoral comme de cellules saines de l'organisme [18]. Par exemple, l'ADN libre tumoral circulant peut représenter moins de 0,01 % de l'ADN libre circulant total pour des cancers à un stade précoce [14], et plus de 50 % pour des cancers métastatiques [13,19,20]. Ceci rend indispensable l'utilisation d'outils de détection plus sensibles et plus spécifiques que les techniques conventionnelles (PCR quantitative et séquençage).…”

“…Elle permet à la fois de réali-ser plus efficacement les tests réalisés aujourd'hui par des méthodes telles que la qPCR, mais également de réaliser des expériences au-delà des capacités des procédures conventionnelles. Il est cependant à noter que de nouvelles procédures, plus sensibles que les procédures conventionnelles, basées sur la PCR quantitative spécifique d'allèles, ont été récemment développées pour l'analyse d'ADN libre tumoral circulant [23,24] et validées dans le cadre d'études cliniques [19]. Ainsi, la dPCR est particulièremente pertinente pour la détection et la quantification de marqueurs minoritaires, incluant les sousclones rares au sein de la tumeur ou de l'ADN libre circulant.…”