Massively parallel sequencing of cell-free DNA in plasma for detecting gynaecological tumour-associated copy number alteration

Makoto Nakabayashi,Akihiko Sekizawa,Chiaki Iizuka,Akihiro Kawashima,Shingo Miyamoto,Rika Yasuhara,Yosuke Hayakawa

doi:10.1038/s41598-018-29381-y

Abstract

The discovery of circulating tumour DNA molecules created a paradigm shift in tumour biomarkers as predictors of recurrence. Non-invasive prenatal testing (NIPT) to detect circulating cell-free foetal DNA in maternal plasma is increasingly recognised as a valuable substitute to perceive foetal copy number variation (CNV). This study aimed to determine whether the copy number detection in plasma samples using NIPT platform could be used as a prognostic biomarker in patients with gynaecological cancer. We conducted a prospective study using samples containing preoperative plasma from 100 women with gynaecological cancers. Samples were randomly rearranged and blindly sequenced using a low-coverage whole-genome sequencing plasma DNA, NIPT platform. The NIPT pipeline identified copy number alterations (CNAs) were counted in plasma as a gain or loss if they exceeded 10 Mb from the expected diploid coverage. Progression-free survival (PFS) and overall survival (OS) were analysed according to the presence of CNA in plasma using Kaplan–Meier analyses. The NIPT pipeline detected 19/100 cases of all gynaecological cancers, including 6/36 ovarian cancers, 3/11 cervical cancers, and 10/53 endometrial cancers. Patients with CNA in plasma had a significantly poorer prognosis in all stages concerning PFS and OS. Therefore, low-coverage sequencing NIPT platform could serve as a predictive marker of patient outcome.

Highlights

The discovery of circulating tumour DNA molecules created a paradigm shift in tumour biomarkers as predictors of recurrence
One such example involves a patient who was diagnosed with metastatic small cell carcinoma of the vagina that was suggested to account for aneuploidies of chromosome 18 and 13 identified using NIPT2
We found that samples from patients with advanced stages had a higher rate of copy number alterations (CNAs) detection than those with early stages (37.0% for stages III–IV patients versus 12.3% for stages I–II; p = 0.009)

Summary

Introduction

The discovery of circulating tumour DNA molecules created a paradigm shift in tumour biomarkers as predictors of recurrence. Cell-free DNA is released via necrosis, autophagy, apoptosis and other physiological events induced by micro-environmental stress and treatment pressure[10] This phenomenon suggests that ctDNA may be more likely to originate from genomic regions with an increased euchromatic DNA structure resulting in observed differential fragment length distribution in coverage relative to somatic cell-free DNA. Genome-wide detection of CNA can be characterised in ctDNA, acting as tumour biomarkers with excellent sensitivity and specificity[19,20] These methods require deep sequencing that significantly increases the cost and difficulty to use in clinical practice. Several studies using MPS have reported that personalised analysis of rearranged ends was developed to detect unselected genetic events that span across the whole genome in cancer patients[24,25] These findings demonstrate the performance of cancer genome scanning through MPS of plasma DNA

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Conclusion