Individualised multiplexed circulating tumour DNA assays for monitoring of tumour presence in patients after colorectal cancer surgery

Sarah B Ng,Iain Beehuat Tan,Steve Rozen,Polly Sy Poon,Anna Gan,William F Burkholder,Su Pin Choo,Mark Wong,Si-Lin Koo,Alexander Chung,Matthew Ng,Melissa Teo,Danliang Ho,Wei Qiang Leow,Cherylin Fu,Clarinda Chua,Kiat Hon Lim,Patrick Tan

doi:10.1038/srep40737

Abstract

Circulating tumour DNA (ctDNA) has the potential to be a specific biomarker for the monitoring of tumours in patients with colorectal cancer (CRC). Here, our aim was to develop a personalised surveillance strategy to monitor the clinical course of CRC after surgery. We developed patient-specific ctDNA assays based on multiplexed detection of somatic mutations identified from patient primary tumours, and applied them to detect ctDNA in 44 CRC patients, analysing a total of 260 plasma samples. We found that ctDNA detection correlated with clinical events – it is detectable in pre-operative but not post-operative plasma, and also in patients with recurrent CRC. We also detected ctDNA in 11 out of 15 cases at or before clinical or radiological recurrence of CRC, indicating the potential of our assay for early detection of metastasis. We further present data from a patient with multiple primary cancers to demonstrate the specificity of our assays to distinguish between CRC recurrence and a second primary cancer. Our approach can complement current methods for surveillance of CRC by adding an individualised biological component, allowing us not only to point to the presence of residual or recurrent disease, but also attribute it to the original cancer.

Highlights

IntroductionCauses (e.g. infection) or from a different cancer[2]. a more specific and sensitive biomarker is needed to detect the recurrence of Colorectal cancer (CRC)
Causes or from a different cancer[2]
To demonstrate as a proof-of-concept that patient primary-tumour-specific (PPS)-Circulating tumour DNA (ctDNA) can distinguish this situation from a metastatic case, we identified a patient who had undergone synchronous surgery of the primary Colorectal cancer (CRC) and liver metastasis, subsequently developed a liver mass and elevation of carcinoembryonic antigen (CEA) that was initially thought to be due to recurrent CRC, but later revealed to be a cholangiocarcinoma on histopathology upon surgical resection

Summary

Introduction

Causes (e.g. infection) or from a different cancer[2]. a more specific and sensitive biomarker is needed to detect the recurrence of CRC. Most of the plasma samples in these previous studies were assayed only for a single mutation; in cases where a few mutations were assayed, each was a separate assay requiring independent sample aliquots In both instances, this could limit the sensitivity of detection, in the case of ctDNA where input amounts and mutation frequency are low. We and others showed that there is a large degree of genomic concordance amongst somatic mutations (about 80%) in each patient’s primary tumour and corresponding metastatic lesion[10,11] This suggested that we could use somatic mutations identified from a patient’s surgical primary CRC specimen to provide an individualised “nucleic acid thumbprint” for detection in plasma at metastasis, specific to the patient, and to the particular cancer. To detect low frequency variants with a sequencing readout, it is necessary to correct for sequencing noise as well as errors introduced during library amplification

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