Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour dna Mutations.

Galbiati Galbiati,Burgio Burgio,Damin Damin,Ferraro Ferraro,Chiari Chiari,Soriani Soriani,Ronzoni Ronzoni,Ferrari Ferrari,Gianni Gianni

doi:10.3390/cells8080769

Abstract

It has now been established that in biological fluids such as blood, it is possible to detect cancer causing genomic alterations by analysing circulating tumour DNA (ctDNA). Information derived from ctDNA offers a unique opportunity to enrich our understanding of cancer biology, tumour evolution and therapeutic efficacy and resistance. Here, we propose a workflow to identify targeted mutations by a customized microarray-based assay for the simultaneous detection of single point mutations in different oncogenes (KRAS, NRAS and BRAF) followed by droplet digital PCR (ddPCR) to determine the fractional abundance of the mutated allele. Genetic variants were determined in the plasma of 20 metastatic colorectal cancer (mCRC) patients previously genotyped on tissue biopsy at the diagnosis for medication planning (T0) and following the tumour genetic evolution during treatment phase (T1 and T2) with the objective of allowing therapy response prediction and monitoring. Our preliminary results show that this combined approach is suitable for routine clinical practice. The microarray platform enables for a rapid, specific and sensitive detection of the most common mutations suitable for high-throughput analysis without costly instrumentation while, the ddPCR, consents an absolute quantification of the mutated allele in a longitudinal observational study on patients undergoing targeted therapy.

Highlights

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in Europe and a leading cause of death both in Europe and worldwide [1,2]
The outcome for patients with metastatic CRC has been improved by targeting pathways involved in CRC development, such as epidermal growth factor receptor (EGFR) and vascular epidermal growth factor (VEGF) pathways [1,3,4]
DdPCR is not an expensive technique, its major weakness is that a priori knowledge of the mutations to quantify is required. In this prospective pilot study, we proposed a combined pipeline in which a customized microarray-based assay approach enables the identification of the most frequent mutations involved in metastatic CRC (mCRC) followed by a targeted droplet digital PCR (ddPCR) analysis to quantify the fractional abundance of the mutated allele as an useful and implementable workflow in tumour patient management

Summary

Introduction

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in Europe and a leading cause of death both in Europe and worldwide [1,2]. In recent years there has been an increase in the diagnosis of CRC and a contextual decrease in mortality, due to screening programs, early diagnosis and improved therapies, which are increasingly targeted and personalized [3]. About 20–25% of patients with CRC have advanced disease at diagnosis and about 35% of patients treated with curative intent will develop an advanced disease. The outcome for patients with metastatic CRC (mCRC) has been improved by targeting pathways involved in CRC development, such as epidermal growth factor receptor (EGFR) and vascular epidermal growth factor (VEGF) pathways [1,3,4]. Antiangiogenic drugs and EGFR inhibitors (the so-called target therapies) are placed next to the supporting skeleton represented by chemotherapy and their use in the management of patients affected by mCRC. Selecting the most appropriate first-line treatment is the first important issue in the management of mCRC, because this choice will guide subsequent treatments and will define the treatment which will be able to achieve disease control, to obtain clinical benefit and to allow further loco-regional or systemic interventions [4]

Methods

Results

Conclusion