Abstract 4211: Detection and quantification of minoritary subclones of KRAS in metastatic colorectal cancers by digital microfluidics: therapeutic implications.
Authors: Pekin Deniz , Normand Corinne , Kotsopoulos Steve , Xinyu Li , Benhaim Leonor , Bouche O. , Lecomte T. , Le Corre D. , Hor T. , El Harrak Z. , Nizard P. , LINK D. , HUTCHISON B. , LAURENT-PUIG P. , Taly Valerie 
Published: August 2014

Abstract
KRAS mutations are associated with an absence of response to EGFR-targeting treatments in colorectal cancer. These therapies are restricted to patients with a KRAS wild type tumor but only 50% of these patients present an objective response. It has been recently demonstrated that secondary resistance was associated with appearance of a KRAS mutation. The presence of a low fraction of KRAS-mutated cells within tumors, not detectable by commonly used procedures, could explain these false negative and these secondary resistances.
To understand the importance of mutated subclones in the apparition of resistance to therapy targeting EGFR, a retrospective study including 95 patients with a metastatic colorectal cancer have been performed. All these patients, refractory to irinotecan were treated either with a combination of anti-EGFR and irinotecan or by anti-EGFR in monotherapy. These patients consisted of 54 men and 41 women with an average age of 62 years ± 11. Thirty-six patients were classified as responders according to RECIST criteria and 59 as non responders. Within this historitical serie, 27 patients were previously characterized as bearing a KRAS mutation by bulk Taqman analysis. DNA extracted from the tumors was analyzed by multiplex droplet-based microfluidics. This method, by segregating individual DNA molecules within million of aqueous microdroplets acting as independent microreactors, allows to perform precise quantitative experiments, for the 7 most frequent mutations of KRAS.
All tumors reported as bearing a mutated KRAS were detected as positives in droplets with proportions of mutated DNA from 10 to 64% (mean of 34,7% ± 17,9%). Two of them (KRAS G12D) were bearing additional subclones (KRAS G12V; KRAS G12S) detected only in droplets. With the 68 negative tumors, 18 were found positive in droplets with KRAS proportions from 0,013% to 86% (mean of 8.6% ± 20). Only 4 patients had a proportion of mutated DNA higher than 10% (86%, 24.5%, 12.7% and 10%), all presented low quantities of amplifiable DNA. We observed an inverse correlation between the proportion of mutated DNA and the frequency of anti-EGFR responses (P&lt; 0.03). The percentage of mutated DNA was 11.6% and 28,4% for responders and non-responders respectively. Survival free progression of patients was significantly different depending on the level of mutated DNA within the tumor. The survival free progression of patients with tumor presenting less than 5% of mutated KRAS was identical to the one of non mutated patients with both technics. HRs were of 0.54±0.2 NS, 3.1±0.9, (P&lt;0.001) 4.9±1.7 (P&lt;0.001) for patients with tumor containing less than 5%, between 5 and 25% or more than 25% of mutated DNA respectively. This work highlights the importance of a quantitative approach for detection of mutation of the KRAS oncogene.
Citation Format: Deniz Pekin, Corinne Normand, Steve Kotsopoulos, Li Xinyu, Leonor Benhaim, O. Bouche, T. Lecomte, D. Le Corre, T. Hor, Z. El Harrak, P. Nizard, D. LINK, B. HUTCHISON, P. LAURENT-PUIG, Valerie Taly. Detection and quantification of minoritary subclones of KRAS in metastatic colorectal cancers by digital microfluidics: therapeutic implications. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2013-4211