Evaluation of the recently established Dutch nationwide Archipelago of Ovarian Cancer Research biobank.

Fundamental and translational research in ovarian cancer aims to enhance understanding of disease mechanisms and improve treatment and survival outcomes. To support this, we established the Dutch multicenter, interdisciplinary Archipelago of Ovarian Cancer Research (AOCR) infrastructure, which includes a nationwide biobank. In this study, we share our experiences in establishing the infrastructure, offer guidance for similar initiatives, and evaluate the AOCR patient cohort.

A Multiplex Assay for Fast PIK3CA Hotspot Mutation Characterization in a Single Specimen by 3-Color Digital PCR Analysis.

Background: Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have been detected often in solid tumors. Targeted therapy for mutant PIK3CA is now available in the clinic, making molecular diagnostics pivotal. Our aim was to design a multiplex digital PCR (dPCR) assay to evaluate the 4 most common PIK3CA hotspot mutations simultaneously to characterize and quantify these in liquid biopsies.

The Information Technology (IT) Infrastructure of the Multicenter Archipelago of Ovarian Cancer Research Biobank: A Potential Blueprint for Other Biobanks.

Biobanks play a crucial role in fundamental and translational research by storing valuable biomaterials and data for future analyses. However, the design of their information technology (IT) infrastructures is often customized to specific requirements, thereby lacking the ability to be used for biobanks comprising other (types of) diseases. This results in substantial costs, time, and efforts for each new biobank project.

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium.

Background: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands).

Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer.

Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer.

Mutation Analysis of Pancreatic Juice and Plasma for the Detection of Pancreatic Cancer.

Molecular profiling may enable earlier detection of pancreatic cancer (PC) in high-risk individuals undergoing surveillance and allow for personalization of treatment. We hypothesized that the detection rate of DNA mutations is higher in pancreatic juice (PJ) than in plasma due to its closer contact with the pancreatic ductal system, from which pancreatic cancer cells originate, and higher overall cell-free DNA (cfDNA) concentrations. In this study, we included patients with pathology-proven PC or intraductal papillary mucinous neoplasm (IPMN) with high-grade dysplasia (HGD) from two prospective clinical trials (KRASPanc and PACYFIC) for whom both PJ and plasma were available.

Machine learning-based somatic variant calling in cell-free DNA of metastatic breast cancer patients using large NGS panels.

Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants.

Establishment of the Dutch Nationwide, Interdisciplinary Infrastructure and Biobank for Fundamental and Translational Ovarian Cancer Research: Archipelago of Ovarian Cancer Research.

Objectives: Ovarian cancer has the worst overall survival rate of all gynecologic malignancies. For the majority of patients, the 5-year overall survival rate of less than 50% has hardly improved over the last decades. To improve the outcome of patients with all subtypes of ovarian cancer, large-scale fundamental and translational research is needed.

CABA-V7: a prospective biomarker selected trial of cabazitaxel treatment in AR-V7 positive prostate cancer patients.

Background: Metastatic castration-resistant prostate cancer (mCRPC) patients with positive AR-V7 expression in their circulating tumour cells (CTCs) rarely derive benefit from abiraterone and enzalutamide.

Design: We performed a prospective, multicenter, single arm phase II clinical trial (CABA-V7) in mCRPC patients previously treated with docetaxel and androgen deprivation therapy.

Objective: In this trial, we investigated whether cabazitaxel treatment resulted in clinically meaningful PSA response rates in patients with positive CTC-based AR-V7 expression and collected liquid biopsies for genomic profiling.

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling.

Background: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples.

Methods: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories.

The COMPLETE trial: HolistiC early respOnse assessMent for oroPharyngeaL cancEr paTiEnts; Protocol for an observational study.

Introduction: The locoregional failure (LRF) rate in human papilloma virus (HPV)-negative oropharyngeal squamous cell carcinoma (OPSCC) remains disappointingly high and toxicity is substantial. Response prediction prior to or early during treatment would provide opportunities for personalised treatment. Currently, there are no accurate predictive models available for correct OPSCC patient selection.

The prognostic and predictive value of ESR1 fusion gene transcripts in primary breast cancer.

Background: In breast cancer (BC), recurrent fusion genes of estrogen receptor alpha (ESR1) and AKAP12, ARMT1 and CCDC170 have been reported. In these gene fusions the ligand binding domain of ESR1 has been replaced by the transactivation domain of the fusion partner constitutively activating the receptor. As a result, these gene fusions can drive tumor growth hormone independently as been shown in preclinical models, but the clinical value of these fusions have not been reported.

Liquid biopsy in esophageal cancer: a case report of false-positive circulating tumor DNA detection due to clonal hematopoiesis.

Circulating tumor DNA (ctDNA) analysis is a promising non-invasive technique for active surveillance after chemoradiotherapy for locally advanced resectable esophageal carcinoma. In other malignancies false-positive results in ctDNA analysis have been reported due to clonal hematopoiesis. In this case, we present a 66-year-old male who had adenocarcinoma of the gastroesophageal junction for which he received neoadjuvant chemoradiotherapy and underwent a transhiatal esophagectomy.

Optimizing Nanopore sequencing-based detection of structural variants enables individualized circulating tumor DNA-based disease monitoring in cancer patients.

Here, we describe a novel approach for rapid discovery of a set of tumor-specific genomic structural variants (SVs), based on a combination of low coverage cancer genome sequencing using Oxford Nanopore with an SV calling and filtering pipeline. We applied the method to tumor samples of high-grade ovarian and prostate cancer patients and validated on average ten somatic SVs per patient with breakpoint-spanning PCR mini-amplicons. These SVs could be quantified in ctDNA samples of patients with metastatic prostate cancer using a digital PCR assay.

Detection of oncogenic mutations in paired circulating tumor DNA and circulating tumor cells in patients with hepatocellular carcinoma.

Background And Aims: Circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients.

Methods: Twenty-six mostly advanced HCC patients were enrolled.

Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer.

Background: Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA).

Methods: For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture.

Optimization of Pancreatic Juice Collection: A First Step Toward Biomarker Discovery and Early Detection of Pancreatic Cancer.

Introduction: Imaging-based surveillance programs fail to detect pancreatic ductal adenocarcinoma at a curable stage, creating an urgent need for diagnostic biomarkers.

Methods: Secretin-stimulated pancreatic juice (PJ) was collected from the duodenal lumen during endoscopic ultrasound. The yield of biomarkers and organoids was compared for 2 collection techniques (endoscope suction channel vs catheter-based) and 3 periods (0-4 vs 4-8 vs 8-15 minutes).

Comparison of variant allele frequency and number of mutant molecules as units of measurement for circulating tumor DNA.

Quantification of tumor-specific variants (TSVs) in cell-free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement.

Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

The aim of this study was to determine an optimal workflow to detect mutations in baseline and longitudinal serum cell free DNA (cfDNA) from high-grade serous ovarian carcinomas (HGSOC) patients and to define whether mutations are suitable as biomarker for disease. was investigated in tissue and archived serum from 20 HGSOC patients by a next-generation sequencing (NGS) workflow alone or combined with digital PCR (dPCR). AmpliSeq™-focused NGS panels and customized dPCR assays were used for tissue DNA and longitudinal cfDNAs, and Oncomine NGS panel with molecular barcoding was used for baseline cfDNAs.

Whole exome sequencing of cell-free DNA - A systematic review and Bayesian individual patient data meta-analysis.

Molecular profiling of tumor derived cell free DNA (cfDNA) is gaining ground as a prognostic and predictive biomarker. However to what extent cfDNA reflects the full metastatic landscape as currently determined by tumor tissue analysis remains controversial. Though technically challenging, whole exome sequencing (WES) of cfDNA enables thorough evaluation of somatic alterations.

High ctDNA molecule numbers relate with poor outcome in advanced ER+, HER2- postmenopausal breast cancer patients treated with everolimus and exemestane.

We determined whether progression-free survival (PFS) in metastatic breast cancer (MBC) patients receiving everolimus plus exemestane (EVE/EXE) varies depending on circulating tumour DNA (ctDNA) characteristics. Baseline plasma cell-free DNA (cfDNA) from 164 postmenopausal women with ER-positive, HER2-negative MBC refractory to a nonsteroidal aromatase inhibitor and treated with standard EVE/EXE (Everolimus Biomarker Study, Eudract 2013-004120-11) was characterised for 10 relevant breast cancer genes by next-generation sequencing with molecular barcoding. ctDNA molecule numbers, number of mutations and specific variants were related with PFS and overall survival (OS).

Plasma Cell-Free DNA Testing of Patients With EGFR Mutant Non-Small-Cell Lung Cancer: Droplet Digital PCR Versus Next-Generation Sequencing Compared With Tissue-Based Results.

Purpose: To compare the results of plasma cell-free DNA (cfDNA) droplet digital PCR (ddPCR) and next-generation sequencing (NGS) on detection of epidermal growth factor receptor () primary activating mutations and p.T790M with results of tissue analysis in patients with mutated non-small-cell lung cancer.

Methods: All patients with EGFR mutated non-small cell lung cancer for which a pathology and a plasma specimen were available upon progression between November 2016 and July 2018 were selected.

RAS and BRAF mutations in cell-free DNA are predictive for outcome of cetuximab monotherapy in patients with tissue-tested RAS wild-type advanced colorectal cancer.

In metastatic colorectal cancer, RAS and BRAF mutations cause resistance to anti-EGFR therapies, such as cetuximab. Heterogeneity in RAS and BRAF mutations might explain nonresponse in a subset of patients receiving cetuximab. Analyzing mutations in plasma-derived circulating tumor DNA (ctDNA) could provide a more comprehensive overview of the mutational landscape as compared to analyses of primary and/or metastatic tumor tissue.

High-throughput isolation of circulating tumor DNA: a comparison of automated platforms.

The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high-throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current 'gold standard' QA to facilitate high-throughput processing of samples in prospective trials.

An Optimized Workflow to Evaluate Estrogen Receptor Gene Mutations in Small Amounts of Cell-Free DNA.

The detection of mutated genes in cell-free DNA (cfDNA) in plasma has emerged as an important minimally invasive way to obtain detailed information regarding tumor biology. Reliable determination of circulating tumor-derived DNA, often present at a low quantity amidst an excess of normal DNA in plasma, would be of added value for screening and monitoring of cancer patients and for hypothesis-generating studies in valuable retrospective cohorts. Our aim was to establish a workflow to simultaneously assess four hotspot estrogen receptor mutations (mESR1) in cfDNA isolated from only 200 μL of plasma by means of uniplex or multiplex pre-amplification combined with digital PCR.