Abstract 508: Prognostic value of circulating tumor DNA in advanced colorectal cancer patients: quantification of hypermethylated or mutant sequences using picoliter-droplet digital PCR
Authors: Garlan Fanny , Laurent-Puig Pierre , Siauve Nathalie , Didelot Audrey , Perkins Geraldine , Blons Hélène , Taieb Julien , Taly Valerie , Zaanan Aziz 
Published: July 2016

Abstract
Background: Circulating tumor DNA (ctDNA) has been suggested as a new marker in different cancer types including colorectal cancer (CRC). Its prognostic role needs to be validated in prospective clinical studies using precise and robust procedures. In this context, picoliter-droplet digital PCR has arisen as a highly sensitive and quantitative approach offering a broad dynamic range of detection.
Patients and methods: All consecutive advanced CRC patients receiving first-line chemotherapy were included in this monocentric prospective study. Plasma samples were collected before the 1st cycle of chemotherapy, then at 14 and 28 days after the beginning of the chemotherapy. Both, carcinoembryonic antigen (CEA) dosages and computed tomography (CT) were performed at baseline and every 8 weeks. For each patient, tumor DNA from biopsies was tested for the presence of KRAS, BRAF and TP53 mutations either by conventional qPCR or Next-Generation Sequencing. When no mutation was identified in the tumor, ctDNA was screened for hypermethylated sequences of WIF1 and NPY genes. CtDNA sequences (mutated or hypermethylated) were quantified (concentration, ng/mL) using picoliter-droplet digital PCR coupled to Taqman probes. Associations of ctDNA concentration with progression-free survival (PFS) and overall survival (OS) were analysed using a Cox proportional hazards model. Multivariate models were adjusted on age, gender, Kohne's score, CEA and type of genetic alteration.
Results: Up to now 43 patients were included (mean age: 66±1.8years [62.6-69.6], gender ratio: M/F 1.15). At baseline ctDNA was detectable in 38/43 (88%) patients based on KRAS, BRAF or TP53 mutation (n = 27), or hypermethylation of the WIF1 or NPY genes (n = 16). Values ranged from 0 to 208 ng/mL, mean and median ctDNA concentration were 12.7 ng/mL and 2.05 ng/mL, respectively. After adjustment on prognostic covariates, the concentration of baseline ctDNA was significantly positively associated with a worse PFS (HR: 1.01 CI95% [1-1.02]; padj = 0.03) and OS (HR: 1.02 CI95% [1.01-1.035] padj = 0.004). The median PFS were 8.6 and 2.3 months in patients with less or more than 5 ng/mL of baseline ctDNA, respectively (HR: 6.7, CI95% 2-22; padj = 0.001). When ctDNA concentration was considered at 14 or 28 days, patients with a ctDNA ≥ 0.2 ng /mL have a significantly worse PFS (HR: 4.12 CI95% [1.41-12.0]; padj = 0.009) than the others independently of the baseline concentration of ctDNA.
Conclusion: This study suggests that ctDNA is a strong prognostic factor highlighting the clinical relevance of quantifying circulating tumor DNA by picoliter-droplet digital PCR in first-line advanced colorectal cancer treatment.
Citation Format: Fanny Garlan, Pierre Laurent-Puig, Nathalie Siauve, Audrey Didelot, Geraldine Perkins, Hélène Blons, Julien Taieb, Valerie Taly, Aziz Zaanan. Prognostic value of circulating tumor DNA in advanced colorectal cancer patients: quantification of hypermethylated or mutant sequences using picoliter-droplet digital PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 508.