Predictive, Preventive and Personalized Medicine & Molecular Diagnostics

Biography

Biography: Alain R. THIERRY

Abstract

Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a non-invasive tumor molecular test for cancer patients from a simple blood test. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure and specific determination of cfDNA fragmentation and size, we revisited Q-PCR based method and recently developed the Allele Specific-Q-PCR based-method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. The Intplex test can be adapted to all mutations, genes or cancers and enables rapid, highly sensitive, cost effective and repetitive analysis. It offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a non-invasive attractive tool potentially allowing diagnosis, prognosis, theranostics, therapeutic monitoring and follow-up of cancer patients expanding the scope of personalized cancer medicine.

We carried out two clinical studies in metastatic colorectal cancer patients (i) the clinical real-time evaluation of circulating tumor DNA (ctDNA) analysis for detecting point mutations in comparison with tumor tissue analysis before initiation of anti-EGFR therapy and (ii), the study of the emergence of associated resistance mutation in refractory patients under treatment.

(i) We realized a real-time blinded prospective multicentric clinical study on 140 patients comparing KRAS (exon 2,3 and 4), NRAS (exon é and 3) and BRAF V600E mutations determination by plasma and tissue analysis carried out in the conditions of standard management care. On 121 patients where both analysis were made, 43% were found mutant by tumor tissue analysis while 57% were found KRAS mutant by ctDNA analysis. 7.2% of patients were found BRAF mutant by tumor tissue while 14.4% were determined mutant by ctDNA analysis. 13% of plasma samples carried multiple KRAS point mutations and 4% of plasma samples exhibited at least one KRAS point mutation combined to the presence of BRAFV600E point mutation. Median data turnaround time was 16 [3-273] days for tumor tissue while it was 2 [0.5-10] days for ctDNA analysis. Extensive analysis of discordant samples revealed that use of biopsy, delay between tumor tissue specimen collection and blood draw, and absence of primary tumor at time of blood draw are the main clinical and technical factors potentially affecting concordance. In addition, most of the samples scored mutant by plasma while being tested WT by tumor tissue analysis seem clinically relevant in respect to the anti-EGFR resistance (RAS status) and to the overall survival (BRAF status). By way of observed higher level of mutation frequencies, plasma analysis appears more accurate than tissue analysis consistent with the intra-tumor or inter-tumor heterogeneity and the clonal evolution dynamics. Lastly, we present the first distribution profile of the RAS and BRAF hotspot mutations in non-stratified mCRC patients. Altogether, this is the first initial report stringently demonstrating the clinical utility of using ctDNA analysis for testing the actionable hotspot mutations in a large cohort of mCRC patients.

 (ii) In the other blinded clinical study, we retrospectively analyzed RAS (KRAS and NRAS) and BRAF mutations in serial plasma samples from 42 refractory mCRC patients to Folfox + cetuximab or dasatinib. 98% of the plasma were found mutant before or during treatment. 50% of KRAS mutant samples were missed by tumor tissue analysis before treatment. 4.8% of patients were found BRAF mutant by tumor tissue analysis while 7% were found mutant by ctDNA analysis. Longitudinal plasma analysis shows that 80% of initially WT patients acquire at least one RAS or BRAF mutation during treatment and that 38% of initially mutant patients acquire at least one newly KRAS or BRAF point mutation during treatment. CtDNA analysis allows tracking acquired resistance by studying the real-time clonal evolution of the tumor Patients may harbor mutations at very low frequency on ctDNA down to 0.01% before initiation or during treatment revealing the need of a high sensitive technique to detect mutant subclones. Altogether, our results indicate that plasma analysis could advantageously replace tumor tissue analysis and enables longitudinal examination of tumor molecular profiling.