Clinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancer

doi:10.1016/j.dld.2015.05.023

Digestive and Liver Disease

Volume 47, Issue 10, October 2015, Pages 884-890

https://doi.org/10.1016/j.dld.2015.05.023 Get rights and content

Abstract

Background

The detection of circulating DNA is considered a promising strategy in cancer patients. Digital PCR has emerged as a sensitive method able to quantify both circulating free and tumour DNA.

Aim

The aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA.

Methods

Digital PCR was used in 34 metastatic colorectal cancer patients to detect and quantify circulating free and tumour DNA based on K-ras mutational status. Clinical outcomes were analyzed according to circulating DNA measurements.

Results

Digital PCR yielded a detection rate of 69% for circulating tumour DNA. The median concentrations of circulating free and tumour DNA were 20 and 6.8 ng/mL, respectively, with significant correlation between both biomarkers (p < 0.001). Median overall survival was 4.8 months in patients with high circulating free DNA (>75% quartile) versus not reached in patients with a low level (<25% quartile) (p = 0.029). Moreover, median overall survival was significantly decreased in patients with detectable circulating tumour DNA compared to those without (respectively 11.8 months versus not reached, p = 0.04).

Conclusions

Chip-based digital PCR is a simple and non-invasive method allowing the efficient detection of circulating DNA. Our results highlight that levels of these circulating markers may have a potential prognostic value.

Introduction

The detection of somatic alterations in tumours is integrated into the daily practice of cancer patient management [1], [2]. In metastatic colorectal cancer (MCRC), somatic K-ras and N-ras mutations have been shown to be markers of resistance to anti-EGFR therapies, and these drugs are now restricted to patients without detectable mutations in these genes [3]. Although cancer genotyping is commonly performed using tumour tissues, this process has inherent limitations due to repetitive tumour samplings for disease and therapy monitoring.

Circulating tumour DNA (ctDNA), resulting from tumour cell apoptosis and/or necrosis, can be detected in serum or plasma and is considered a potential “liquid biopsy” that can be used instead of tumour tissue DNA to identify prognostic and predictive alterations in cancer patients [4], [5], [6], [7], [8], [9]. Using ctDNA, different types of somatic alterations, such as point mutations, rearrangements, amplifications, aneuploidy or methylations, can be detected [10], [11], [12], [13], [14]. Circulating free DNA (cfDNA) comprises both ctDNA originates from tumour cells and nucleic acids released by non-tumour cells surrounding tumour tissue and also peripheral blood cells. The level of circulating free DNA (cfDNA) fragments is also a marker of potential clinical value because it correlates with tumour stage and survival [15], [16], [17]. Metastatic disease is commonly considered to be the best clinical situation in which to perform cfDNA and ctDNA measurements in cancer patients. The sensitivity and specificity of these measurements are dependent on the methods used. Several efficient methods have been reported in recent years such as NGS-based or quantitative PCR-based assays [18], [19]. For instance, the sensitivity and specificity of the NGS-based Safeseq^® technology and of the optimized clamp-PCR Intplex^® test for K-ras detection have been recently estimated to be 87.2% and 99.2% for the NGS-based method and 92% and 98% for the quantitative PCR-based method, respectively [5], [20]. Nevertheless, these promising methods are currently relatively time-consuming or require specific expertise, such as in bioinformatics analysis, and are not ready for implementation in routine practice. Digital PCR (dPCR), which was developed in 1999 and has subsequently been optimized, is an ultrasensitive method that combines compartmentalization and PCR amplification of a single DNA fragment [21], [22], [23]. This compartmentalization is carried out into hundreds or even millions of separate reaction chambers using either chip-based dPCR or droplets-based dPCR. Droplets-based dPCR has been shown to be an efficient method for K-ras mutation detection in plasma of MCRC patients with a sensitivity of 80% [24]. However, the clinical value of ctDNA and cfDNA measurements in MCRC using another type of dPCR platform is not yet clearly established. The aim of this pilot study was to prospectively evaluate a chip-based dPCR platform for the detection of circulating DNA (ctDNA and cfDNA) in MCRC patients.

Section snippets

Patient samples

From April to June 2013, two 4-mL blood samples were collected from 34 consecutive patients undergoing a chemotherapy regimen for MCRC. Samples were systematically collected immediately before a chemotherapy course and the timing during the treatment sequence was not standardized. The mean number of previous chemotherapy lines was 1.3 ± 1.3. All patients, except two, received at least one course of chemotherapy before sample collection with a median of 4 courses (range 0–16). The details of

Patient samples

Thirty-four MCRC patients were included, and their characteristics are presented in Table 1. The mean age was 60.6 years, and 22 patients (65%) had received at least two previous lines of chemotherapy. Among the 34 patients, tumour analysis revealed a somatic K-ras mutation in 16 patients (47%). The five following mutations were detected: p.G12C (c.34G>T) (n = 1; 6.2%), p.G12A (c.35G>C) (n = 3; 18.7%), p.G12D (c.35G>A) (n = 8; 50%), p.G12V (c.35G>T) (n = 2; 12.5%) and p.G13D (c.38G>A) (n = 2; 12.5%).

CtDNA and cfDNA analysis by dPCR

Discussion

This pilot study shows that chip-based dPCR efficiently quantifies cfDNA and ctDNA in the plasma of MCRC patients in approximately five hours, from blood collection to the results (Fig. 1). The simplicity and rapidness of the workflow, as well as the relatively low cost of analysis per sample, suggest that the chip-based dPCR platform may be an appropriate method for the detection of ctDNA and cfDNA. Our results also suggest that cfDNA and ctDNA levels may have prognostic value for MCRC

Conflict of interest

None declared.

Funding

This study was supported by grants from the French National Cancer Institute and Charles Nicolle's Foundation.

Acknowledgements

The authors are grateful to Prof. Pierre-Laurent Puig for providing probe and primer sequences. This study was supported by grants from the French National Cancer Institute and Charles Nicolle's Foundation.

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¹: These authors contributed equally to this work.

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OncologyClinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancer

Abstract

Background

Aim

Methods

Results

Conclusions

Introduction

Section snippets

Patient samples

Patient samples

CtDNA and cfDNA analysis by dPCR

Discussion

Conflict of interest

Funding

Acknowledgements

Modern Pathology

Atherosclerosis

Implementing personalized cancer genomics in clinical trials

Nature Reviews Drug Discovery

Implementing prognostic and predictive biomarkers in CRC clinical trials

Nature Reviews Clinical Oncology

Colorectal cancer in 2013: towards improved drugs, combinations and patient selection

Nature Reviews Clinical Oncology

Prognostic value of circulating mutant DNA in unresectable metastatic colorectal cancer

Annals of Surgery

Detection of circulating tumor DNA in early- and late-stage human malignancies

Science Translational Medicine

The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers

Nature

Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

Nature

Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA

Nature

Analysis of circulating tumor DNA to monitor metastatic breast cancer

New England Journal of Medicine

Detection of tumor PIK3CA status in metastatic breast cancer using peripheral blood

Clinical Cancer Research

Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors

Genes, Chromosomes and Cancer

Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing

Science Translational Medicine

Oncology
Clinical value of chip-based digital-PCR platform for the detection of circulating DNA in metastatic colorectal cancer