Dataset Information

Diagnosis and Treatment Monitoring of a Patient with Gastrointestinal Stromal Tumor by Next-Generation Sequencing and Droplet Digital Polymerase Chain Reaction Assay of a PDGFRA Mutation in Plasma-Derived Cell-Free Tumor DNA.

ABSTRACT: In patients with a suspected malignancy, standard-of care management currently includes histopathologic examination and analysis of tumor-specific molecular abnormalities. Herein, we present a 77-year-old patient with an abdominal mass suspected to be a gastrointestinal stromal tumor (GIST) but without the possibility to collect a tumor biopsy. Cell-free DNA extracted from a blood sample was analyzed for the presence of mutations in GIST-specific genes using next generation sequencing. Furthermore, liquid biopsies were used to monitor the levels of mutant DNA copies during treatment with a tumor-specific mutation droplet digital PCR assay that correlated with the clinical and radiological response. Blood-based testing is a good alternative for biopsy-based testing. However, it should only be applied when biopsies are not available or possible to obtain because overall, in only 50%-85% of the cell-free plasma samples is the known tumor mutation detected.

SUBMITTER: Boonstra PA

PROVIDER: S-EPMC6656511 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Diagnosis and Treatment Monitoring of a Patient with Gastrointestinal Stromal Tumor by Next-Generation Sequencing and Droplet Digital Polymerase Chain Reaction Assay of a PDGFRA Mutation in Plasma-Derived Cell-Free Tumor DNA.

Boonstra Pieter A PA Ter Elst Arja A Tibbesma Marco M Gietema Jourik A JA Schuuring Ed E Reyners Anna K L AKL

The oncologist 20190122 6

In patients with a suspected malignancy, standard-of care management currently includes histopathologic examination and analysis of tumor-specific molecular abnormalities. Herein, we present a 77-year-old patient with an abdominal mass suspected to be a gastrointestinal stromal tumor (GIST) but without the possibility to collect a tumor biopsy. Cell-free DNA extracted from a blood sample was analyzed for the presence of mutations in GIST-specific genes using next generation sequencing. Furthermo ...[more]

PMID: 30670599

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Project description:Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

Dataset Information

Diagnosis and Treatment Monitoring of a Patient with Gastrointestinal Stromal Tumor by Next-Generation Sequencing and Droplet Digital Polymerase Chain Reaction Assay of a PDGFRA Mutation in Plasma-Derived Cell-Free Tumor DNA.

Publications

Diagnosis and Treatment Monitoring of a Patient with Gastrointestinal Stromal Tumor by Next-Generation Sequencing and Droplet Digital Polymerase Chain Reaction Assay of a PDGFRA Mutation in Plasma-Derived Cell-Free Tumor DNA.

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