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

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.

Genetics and Epigenetics of Gastroenteropancreatic Neuroendocrine Neoplasms

“…A recent case report suggested that it would be more feasible to sequence a tissue sample from the tumor and then design a personalized ctDNA blood-based or even urine-based assay (273). The latter could be applied to monitor the disease, as recently demonstrated by Boons et al (274), who used this approach to correctly identify three metastatic cases in a set of 10 patients with PanNETs. Another promising approach has been proposed for PanNETs, exploiting whole-exome sequencing on ctDNA (275).…”

PDX1 DNA Methylation Distinguishes Two Subtypes of Pancreatic Neuroendocrine Neoplasms with a Different Prognosis

“…We have therefore performed the CNA calling only on the EPIC study cohort, for which good normal control samples and more data points were available, given the large number of probes on the EPIC DNA methylation arrays. To validate CNA calling on the methylation data, we compared the CNA profile based on DNA methylation analysis with the CNA profile based on whole-exome sequencing (WES) analysis for two tumors where both types of data were available [27]. Results were very comparable between CNAs on WES and on the methylation data ( Figure S1).…”

“…Whole-exome sequencing of 22 patients (UZA-01-UZA-10 and Lawrence-001-Lawrence-012) and deep targeted sequencing of 3 patients (Lawrence-013-Lawrence-015) has been performed in previous studies [21,27]. The identified variants in MEN1, DAXX and ATRX were validated using Sanger sequencing, if the allelic ratio was high enough, and are summarized in Table 2.…”

Resistance to targeted treatment of gastroenteropancreatic neuroendocrine tumors

“…Furthermore, up to 43% of the patients have a mutually exclusive mutation in ATRX or DAXX (Jiao et al 2011). In addition, a recent publication demonstrated the presence of DAXX mutations in circulating tumor DNA of pNET patients (Boons et al 2018). Losses of chromosomal regions including MEN1 are observed in 70% of sporadic pNETs (Scarpa et al 2017).…”