Abstract 2820: Clinical significance of low frequency KRAS and BRAF subclones for advanced colon cancer management
Authors: Taly Valerie , Laurent-puig Pierre , Pekin Deniz , Normand Corinne , Kotsopoulos Steve K. , Nizard Philippe , Olson Jeff , Srinivasan Preethi , Corre Delphine L. , Li Xinyu , Zhong Qun , Link Darren R. , Bouché Olivier , Emile Jean-François , Landi Bruno , Boige Valérie , Hutchison Brian J. 
Published: May 2015

Abstract
Alongside conventional chemotherapies, targeted treatments have significantly improved the survival of patients with metastatic colorectal cancer (mCRC) from 6 months with best supportive care treatment, to 24 months with a combination of conventional and targeted chemotherapies. However, patients with KRAS mutated tumors have little or no benefit from these anti-EGFR antibodies based therapies (Panitumumab, Cetuximab) as single agents or combined with chemotherapy. These findings led to an amendment of the Marketing Authorization of these drugs that are now restricted to patients with KRAS wild-type tumor. However, the response of these patients ranged from 40 to 60%. Recent works have demonstrated that secondary resistance to such therapies was associated with emergence of KRAS mutated subclones in KRAS wild-type patients at the diagnosis time. To understand the importance of KRAS mutant subclones at the diagnosis time and to evaluate the consequence of their presence in terms of management of mCRC patients, a highly sensitive and quantitative procedure is required. Droplet-based digital PCR has recently emerged as a highly sensitive and quantitative approach for rare sequence detection.
A retrospective study was set up where tumor samples from 177 mCRC patients were analyzed using a multiplex highly sensitive droplet-based procedures and ultra-deep sequencing using a new droplet-based target enrichment. Results are compared with data obtained by conventional qPCR. Sixty-two patients were classified as responders according to RECIST criteria. All tumors (41) detected as positive using conventional procedures were also positive with our procedure. Among the samples detected as negative with conventional procedures, 23 presented a KRAS or a BRAF mutation only detected by droplet-based procedures and 6 presented additional subclones. For 20 patients, additional biopsy samples were available and presented comparable results. 167 samples were also submitted to deep sequencing and, for alleles detected at fractions superior to 1%, the mutational status of the sample were in agreement for 94% of the samples (157/167) and we observed a correlation with the observed fraction of mutant alleles with the two procedures (R2= 0.7).
We observed an inverse correlation between the proportion of mutated DNA and the frequency of anti-EGFR responses (P&lt; 0.0001). The mean percentage of mutated DNA was 0.45% and 12.7% for responders and non-responders respectively. Progression Free Survival of patients was significantly different depending on the percentage of KRAS mutated allele within the tumor. The Progression Free Survival of patients with tumor presenting less than 5% of mutated KRAS was comparable to the one of non-mutated patients. HRs were of 1.07 (CI95% 0.6-2, NS), 2.9 (CI95% 1.7-4.9, P&lt;0.001), 3.6 (CI95% 2.2-5.8, P&lt;0.001) for patients with tumor containing less than 5%, between 5 and 25% or more than 25% of mutated DNA respectively.
Citation Format: Valerie Taly, Pierre Laurent-puig, Deniz Pekin, Corinne Normand, Steve K. Kotsopoulos, Philippe Nizard, Jeff Olson, Preethi Srinivasan, Delphine Le Corre, Xinyu Li, Qun Zhong, Darren R. Link, Olivier Bouché, Jean-François Emile, Bruno Landi, Valérie Boige, Brian J. Hutchison. Clinical significance of low frequency KRAS and BRAF subclones for advanced colon cancer management. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2820. doi:10.1158/1538-7445.AM2014-2820