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

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

Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors

Background:The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described.Methods:QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 μM everolimus, respectively, 1000-fold and 250-fold initial IC 50. Using total DNA content as a measure of cell number, growth inhibitory dose-response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT-qPCR).Results:Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10-12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed.Conclusions:Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1

Large-scale copy number analysis reveals variations in genes not previously associated with malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor that is often causally associated with asbestos exposure. Comparative genomic hybridization techniques and arrays demonstrated a complex set of copy number variations (CNVs) in the MPM-genome. These techniques however have a limited resolution, throughput and flexibility compared to next-generation sequencing platforms. In this study, the presence of CNVs in the MPM-genome was investigated using an MPM-cohort (N = 85) for which genomic microarray data are available through 'The Cancer Genome Atlas' (TCGA). To validate these results, the genomes of MPMs and matched normal samples (N = 21) were analyzed using low-pass whole genome sequencing on an 'Illumina HiSeq' platform. CNVs were detected using in-house developed analysis pipelines and frequencies of copy number loss and gain were calculated. In both datasets, losses on chromosomes 1, 3, 4, 6, 9, 13 and 22 and gains on chromosomes 1, 5, 7 and 17 were found in at least 25% and 15% of MPMs, respectively. Besides the well-known MPM-associated genes, CDKN2A, NF2 and BAP1, other interesting cancer-associated genes were listed as frequently involved in a copy number loss (e.g. EP300, SETD2 and PBRM1). Moreover, four cancer-associated genes showed a high frequency of copy number gain in

Epigenomic analyses in sub-populations of spermatozoa from infertile men with short anogenital distance

The key developmental steps in the male germ cell lineage include the epigenetic reprogramming of the primordial germ cells, the large-scale replacement of histone by protamine in spermatids and some degree of apoptosis. In past experiments, we prenatally exposed mice to di(2-ethylhexyl)phthalate (DEHP, CAS number 117-81-7). At adulthood, the exposed mice were characterized by reduced anogenital distance (AGD), poor semen quality, and epigenomic abnormalities in their gametes' DNA. However, the phenotype heavily depended on the genetic background of the mouse strain. We identified SNP-dependent binding motifs for sex hormones signaling pathways varying between the susceptible and the resistant mouse strains. These SNP-dependent motifs were located in the genes that were the most affected one by DEHP, at both DNA methylation and RNA transcription levels. We then tried to replicate these observations into human. We selected 6 infertile patients with short AGD and 5 fertile sperm donors. Testicular volume was significantly reduced in infertile men with shorter AGD. Sub-populations of spermatozoa were FACS-sorted using the chromomycin A3 dye (protamine vs. histone) and with the YO-PRO-1 dye (apoptotic vs. non-apoptotic). In the four sorted sperm populations, we analyzed the epigenomes through reduced-representation bisulfite sequencing (RRBS). The results demonstrated both lower CpG methylation and higher DNA fragmentation in infertile patients. Interestingly, numerous de-methylated regions localized within families of Alu repeats containing estrogen response elements (ERE). We are now in the process of replicating the entire study with a new cohort of five infertile patients and five fertile controls. The aim is to understand if there is a relationship between these epigenomic alterations within Alu repeats containing ERE and fertility, and whether exposures to anti-androgenic endocrine disruptors may alter methylation marks within these Alu-ERE repeats

Altered DNA methylation in estrogen-responsive repetitive sequences of spermatozoa of infertile men with shortened anogenital distance

Background: It has been suggested that antenatal exposure to environmental endocrine disruptors is responsible for adverse trends in male reproductive health, including male infertility, impaired semen quality, cryptorchidism and testicular cancer, a condition known as testicular dysgenesis syndrome. Anogenital distance (AGD) is an anthropomorphic measure of antenatal exposure to endocrine disruptors, with higher exposure levels leading to shortened AGD. We hypothesized that exposure to endocrine disruptors could lead to changes in DNA methylation during early embryonic development, which could then persist in the sperm of infertile men with shortened AGD. Results: Using fluorescence activated cell sorting based on staining with either YO-PRO-1 (YOPRO) or chromomycin-3 (CMA3), we isolated four sperm fractions from eleven infertile men with short AGD and ten healthy semen donors. We examined DNA methylation in these sorted spermatozoa using reduced representation bisulfite sequencing. We found that fractions of spermatozoa from infertile men stained with CMA3 or YOPRO were more likely to contain transposable elements harboring an estrogen receptor response element (ERE). Abnormal sperm (as judged by high CMA3 or YOPRO staining) from infertile men shows substantial hypomethylation in estrogenic Alu sequences. Conversely, normal sperm fractions (as judged by low CMA3 or YO-PRO-1 staining) of either healthy donors or infertile patients were more likely to contain hypermethylated Alu sequences with ERE. Conclusions: Shortened AGD, as related to previous exposure to endocrine disruptors, and male infertility are accompanied by increased presence of hormonal response elements in the differentially methylated regulatory sequences of the genome of sperm fractions characterized by chromatin decondensation and apoptosis.</p

pyAmpli : an amplicon-based variant filter pipeline for targeted resequencing data

Abstract Background Haloplex targeted resequencing is a popular method to analyze both germline and somatic variants in gene panels. However, involved wet-lab procedures may introduce false positives that need to be considered in subsequent data-analysis. No variant filtering rationale addressing amplicon enrichment related systematic errors, in the form of an all-in-one package, exists to our knowledge. Results We present pyAmpli, a platform independent parallelized Python package that implements an amplicon-based germline and somatic variant filtering strategy for Haloplex data. pyAmpli can filter variants for systematic errors by user pre-defined criteria. We show that pyAmpli significantly increases specificity, without reducing sensitivity, essential for reporting true positive clinical relevant mutations in gene panel data. Conclusions pyAmpli is an easy-to-use software tool which increases the true positive variant call rate in targeted resequencing data. It specifically reduces errors related to PCR-based enrichment of targeted regions

Additional file 3: of pyAmpli: an amplicon-based variant filter pipeline for targeted resequencing data

pyAmpli CPU runtime. (DOCX 132 kb