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Digital polymerase chain reaction for detecting c-MYC copy number gain in tissue and cell-free plasma samples of colorectal cancer patients.

ABSTRACT: We focused on the utility of the droplet digital polymerase chain reaction (ddPCR) for detecting c-MYC gene copy number (GCN) gain in cell-free plasma and tumor tissue of colorectal cancer (CRC) patients. c-MYC GCN status was determined using dual-color silver in situ hybridization (SISH) and ddPCR in retrospective cohort 1 (192 CRC patients) and prospective cohort 2 (64 CRC patients). In cohort 1, c-MYC GCN gain was observed in 34 (17.5%) patients by SISH, and in 7 (3.6%) patients by ddPCR. c-MYC GCN by SISH significantly correlated with ddPCR results (ρ = 0.532, P < 0.001). Although 40 cases (20.7%) showed intratumoral genetic heterogeneity, it did not cause discordance in results obtained by the two methods. c-MYC GCN gain, by both SISH and ddPCR was independently correlated with worst prognosis (P = 0.002). In cohort 2, c-MYC GCN estimation in tissue by ddPCR was also significantly associated with results obtained by SISH (ρ = 0.349, P = 0.005), but correlated with plasma ddPCR with borderline significance (ρ = 0.246, P = 0.050). Additionally, detecting c-MYC GCN gain in plasma with ddPCR might have relatively low sensitivity but high specificity. Our study suggests that ddPCR can be a useful tool for detecting c-MYC GCN gain as a potential prognostic biomarker in CRC tissue samples; however, this will need further verification in plasma samples.

SUBMITTER: Lee KS

PROVIDER: S-EPMC6367402 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Digital polymerase chain reaction for detecting c-MYC copy number gain in tissue and cell-free plasma samples of colorectal cancer patients.

Lee Kyu Sang KS Nam Soo Kyung SK Seo Soo Hyun SH Park Kyoung Un KU Oh Heung-Kwon HK Kim Duck-Woo DW Kang Sung-Bum SB Kim Woo Ho WH Lee Hye Seung HS

Scientific reports 20190207 1

We focused on the utility of the droplet digital polymerase chain reaction (ddPCR) for detecting c-MYC gene copy number (GCN) gain in cell-free plasma and tumor tissue of colorectal cancer (CRC) patients. c-MYC GCN status was determined using dual-color silver in situ hybridization (SISH) and ddPCR in retrospective cohort 1 (192 CRC patients) and prospective cohort 2 (64 CRC patients). In cohort 1, c-MYC GCN gain was observed in 34 (17.5%) patients by SISH, and in 7 (3.6%) patients by ddPCR. c-M ...[more]

PMID: 30733532

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Project description:Background aimsVector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct.MethodsThe authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer-probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct.ResultsddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors' assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities.ConclusionsThe authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test-retest reliability, and that specific assays can be developed for detection of unique constructs.

Dataset Information

Digital polymerase chain reaction for detecting c-MYC copy number gain in tissue and cell-free plasma samples of colorectal cancer patients.

Publications

Digital polymerase chain reaction for detecting c-MYC copy number gain in tissue and cell-free plasma samples of colorectal cancer patients.

Similar Datasets

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