Cell-Free DNA From Metastatic Pancreatic Neuroendocrine Tumor Patients Contains Tumor-Specific Mutations and Copy Number Variations.

Gitta Boons,Guy Van Camp,Matthias Beyens,Katrien Janssens,Marc Peeters,Ken Op De Beeck,Ann Driessen,Karen Zwaenepoel,Timon Vandamme,Geert Roeyen

doi:10.3389/fonc.2018.00467

Abstract

Background: Detection of tumor-specific alterations in cell-free DNA (cfDNA) has proven valuable as a liquid biopsy for several types of cancer. So far, use of cfDNA remains unexplored for pancreatic neuroendocrine tumor (PNET) patients.Methods: From 10 PNET patients, fresh frozen tumor tissue, buffy coat and plasma samples were collected. Whole-exome sequencing of primary tumor and germline DNA was performed to identify tumor-specific variants and copy number variations (CNVs). Subsequently, tumor-specific variants were quantified in plasma cfDNA with droplet digital PCR. In addition, CNV analysis of cfDNA was performed using shallow whole-genome sequencing.Results: Tumor-specific variants were detected in perioperative plasma samples of two PNET patients, at variant allele fractions (VAFs) of respectively 19 and 21%. Both patients had metastatic disease at time of surgery, while the other patients presented with localized disease. In the metastatic patients, CNV profiles of tumor tissue and cfDNA were significantly correlated. A follow-up plasma sample of a metastatic patient demonstrated an increased VAF (57%) and an increased chromosomal instability, in parallel with an increase in tumor burden.Conclusions: We are the first to report the presence of tumor-specific genetic alterations in cfDNA of metastatic PNET patients and their evolution during disease progression. Additionally, CNV analysis in cfDNA shows potential as a liquid biopsy.

Highlights

Pancreatic neuroendocrine tumors (PNETs) are rare tumors with an incidence rate of 0.48 per 100,000 according to the Surveillance, Epidemiology, and End Results (SEER) program [1]
By applying the filters described in the methods section, we were able to select a single variant for every patient, which was validated with Sanger sequencing to confirm tumor-specificity
In tumor DNA, both mutant and WT targets could be detected by droplet digital PCR (ddPCR), with variant allele fraction (VAF) showing a significant correlation with VAFs detected by whole-exome sequencing (WES) (Pearson’s r = 0.8786; p < 0.001)

Summary

Introduction

Pancreatic neuroendocrine tumors (PNETs) are rare tumors with an incidence rate of 0.48 per 100,000 according to the Surveillance, Epidemiology, and End Results (SEER) program [1]. Follow-up and treatment are based on imaging, tumor (re)biopsies and biomarker assessment. Circulating tumor DNA (ctDNA) is the proportion of cell-free DNA (cfDNA) in the blood plasma that is released by a tumor as a result of apoptosis, necrosis and active secretion [5]. The ctDNA can be detected and quantified in cfDNA through tumorspecific genetic alterations. CtDNA has been extensively studied in cancer patients as an alternative for tissue biopsies and for its biomarker potential in different stages of disease, as summarized by Wan et al [6]. Detection of tumor-specific alterations in cell-free DNA (cfDNA) has proven valuable as a liquid biopsy for several types of cancer. Use of cfDNA remains unexplored for pancreatic neuroendocrine tumor (PNET) patients

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