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  1. Article ; Online: Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR.

    de Kock, Remco / Baselmans, Mieke / Scharnhorst, Volkher / Deiman, Birgit

    European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology

    2020  Volume 40, Issue 4, Page(s) 807–813

    Abstract: The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 ... ...

    Abstract The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.
    MeSH term(s) Autoantigens/genetics ; COVID-19/diagnosis ; COVID-19 Nucleic Acid Testing/methods ; Coronavirus Envelope Proteins/genetics ; Coronavirus Nucleocapsid Proteins/genetics ; Coronavirus RNA-Dependent RNA Polymerase/genetics ; DNA/analysis ; Genes, Essential ; Glucuronidase/genetics ; Humans ; Multiplex Polymerase Chain Reaction/methods ; Phosphoproteins/genetics ; RNA, Messenger/analysis ; RNA, Viral/analysis ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Ribonuclease P/genetics ; SARS-CoV-2 ; Sensitivity and Specificity
    Chemical Substances Autoantigens ; Coronavirus Envelope Proteins ; Coronavirus Nucleocapsid Proteins ; Phosphoproteins ; RNA, Messenger ; RNA, Viral ; RPP30 protein, human ; envelope protein, SARS-CoV-2 ; nucleocapsid phosphoprotein, SARS-CoV-2 ; DNA (9007-49-2) ; Coronavirus RNA-Dependent RNA Polymerase (EC 2.7.7.48) ; NSP12 protein, SARS-CoV-2 (EC 2.7.7.48) ; Ribonuclease P (EC 3.1.26.5) ; Glucuronidase (EC 3.2.1.31)
    Keywords covid19
    Language English
    Publishing date 2020-10-26
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 603155-9
    ISSN 1435-4373 ; 0934-9723 ; 0722-2211
    ISSN (online) 1435-4373
    ISSN 0934-9723 ; 0722-2211
    DOI 10.1007/s10096-020-04076-3
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  2. Article ; Online: Optimized (Pre) Analytical Conditions and Workflow for Droplet Digital PCR Analysis of Cell-Free DNA from Patients with Suspected Lung Carcinoma.

    de Kock, Remco / Deiman, Birgit / Kraaijvanger, Raisa / Scharnhorst, Volkher

    The Journal of molecular diagnostics : JMD

    2019  Volume 21, Issue 5, Page(s) 895–902

    Abstract: For patients with suspected lung carcinoma, the analysis of circulating tumor DNA, obtained by liquid biopsy, has the potential to support cancer diagnosis and guide targeted therapy. To ensure sensitive and reproducible detection of circulating tumor ... ...

    Abstract For patients with suspected lung carcinoma, the analysis of circulating tumor DNA, obtained by liquid biopsy, has the potential to support cancer diagnosis and guide targeted therapy. To ensure sensitive and reproducible detection of circulating tumor DNA in routine clinical practice, a standardized (pre) analytical workflow is required. Plasma was obtained from patients and healthy volunteers. Using the QIAmp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), six different procedures for the isolation of cell-free DNA (cfDNA) were compared. cfDNA was analyzed by droplet digital PCR (ddPCR) for KRAS G12/13 mutations and for EGFR Ex19Del, L858R, and L861Q mutations using an in-house EGFR multiplex assay. A new isolation procedure that yields extracts with significantly higher cfDNA concentrations than described previously was selected (P < 0.001). EGFR and KRAS assay sensitivity of at least 0.2% fractional abundance was guaranteed for approximately 76% of patient samples in one run. A flowchart that includes validity criteria for a standardized analytical workflow of ddPCR analysis was designed. An improved protocol for cfDNA isolation enables a higher cfDNA input for ddPCR. The use of sensitive KRAS and EGFR multiplex assays and accompanying validity criteria allows for controlled and efficient testing of patient samples at lower costs. Using the suggested workflow, a guaranteed, reliable, and sensitive analysis of cfDNA can be performed using ddPCR in routine clinical practice.
    MeSH term(s) Biomarkers, Tumor/blood ; Biomarkers, Tumor/genetics ; Cell-Free Nucleic Acids/analysis ; Cell-Free Nucleic Acids/genetics ; Circulating Tumor DNA/analysis ; Circulating Tumor DNA/genetics ; Humans ; Liquid Biopsy ; Lung Neoplasms/blood ; Lung Neoplasms/diagnosis ; Lung Neoplasms/genetics ; Mutation ; Polymerase Chain Reaction ; Specimen Handling/standards ; Workflow
    Chemical Substances Biomarkers, Tumor ; Cell-Free Nucleic Acids ; Circulating Tumor DNA
    Language English
    Publishing date 2019-06-21
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2000060-1
    ISSN 1943-7811 ; 1525-1578
    ISSN (online) 1943-7811
    ISSN 1525-1578
    DOI 10.1016/j.jmoldx.2019.05.003
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  3. Article: Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR

    de Kock, Remco / Baselmans, Mieke / Scharnhorst, Volkher / Deiman, Birgit

    Eur. j. clin. microbiol. infect. dis

    Abstract: The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 ... ...

    Abstract The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.
    Keywords covid19
    Publisher WHO
    Document type Article
    Note WHO #Covidence: #891909
    Database COVID19

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  4. Article ; Online: Therapy Monitoring of EGFR-Positive Non-Small-Cell Lung Cancer Patients Using ddPCR Multiplex Assays.

    de Kock, Remco / van den Borne, Ben / Youssef-El Soud, Maggy / Belderbos, Huub / Brunsveld, Luc / Scharnhorst, Volkher / Deiman, Birgit

    The Journal of molecular diagnostics : JMD

    2021  Volume 23, Issue 4, Page(s) 495–505

    Abstract: The detection of EGFR-sensitizing and EGFR-resistance mutations in advanced non-small-cell lung cancer patients is important for the selection and monitoring of EGFR tyrosine-kinase inhibitor therapy. Droplet digital PCR (ddPCR) multiplex assays allow ... ...

    Abstract The detection of EGFR-sensitizing and EGFR-resistance mutations in advanced non-small-cell lung cancer patients is important for the selection and monitoring of EGFR tyrosine-kinase inhibitor therapy. Droplet digital PCR (ddPCR) multiplex assays allow for sensitive and simultaneous detection of multiple mutations in cell-free DNA (cfDNA) with a minimum of extract needed and at lower cost. Patients were screened for the EGFR tyrosine-kinase inhibitor-sensitizing mutations Ex19Del, L858R, L861Q, G719S, and S768I using a novel ddPCR pentaplex assay. Patients who tested positive subsequently were monitored during treatment for the EGFR-sensitizing mutation and two EGFR-resistance mutations, T790M and C797S, using a ddPCR monitor triplex assay. The ddPCR multiplex assays enabled reliable detection of each mutation with a fractional abundance of at least 0.1%. For six patients, longitudinal data were analyzed and the ddPCR results provided a good reflection of the course of the disease and radiologic response. This study confirms that ddPCR on cfDNA supports the diagnosis and therapy selection, and shows that ddPCR multiplex assays on cfDNA could be a valuable additional diagnostic tool for therapy monitoring of non-small-cell lung cancer patients.
    MeSH term(s) Carcinoma, Non-Small-Cell Lung/drug therapy ; Carcinoma, Non-Small-Cell Lung/epidemiology ; Carcinoma, Non-Small-Cell Lung/genetics ; Carcinoma, Non-Small-Cell Lung/pathology ; Cell-Free Nucleic Acids/blood ; Cell-Free Nucleic Acids/genetics ; Drug Resistance, Neoplasm/genetics ; ErbB Receptors/antagonists & inhibitors ; ErbB Receptors/genetics ; Follow-Up Studies ; Humans ; Liquid Biopsy/methods ; Longitudinal Studies ; Lung Neoplasms/drug therapy ; Lung Neoplasms/epidemiology ; Lung Neoplasms/genetics ; Lung Neoplasms/pathology ; Molecular Targeted Therapy/methods ; Multiplex Polymerase Chain Reaction/methods ; Mutation ; Netherlands/epidemiology ; Protein Kinase Inhibitors/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Reproducibility of Results ; Treatment Outcome
    Chemical Substances Cell-Free Nucleic Acids ; Protein Kinase Inhibitors ; EGFR protein, human (EC 2.7.10.1) ; ErbB Receptors (EC 2.7.10.1)
    Language English
    Publishing date 2021-01-22
    Publishing country United States
    Document type Clinical Trial ; Journal Article ; Multicenter Study ; Research Support, Non-U.S. Gov't
    ZDB-ID 2000060-1
    ISSN 1943-7811 ; 1525-1578
    ISSN (online) 1943-7811
    ISSN 1525-1578
    DOI 10.1016/j.jmoldx.2021.01.003
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  5. Article ; Online: Sensitive cell-free tumor DNA analysis in supernatant pleural effusions supports therapy selection and disease monitoring of lung cancer patients.

    de Kock, Remco / Knoops, Chantal / Baselmans, Mieke / Borne, Ben van den / Brunsveld, Luc / Scharnhorst, Volkher / Deiman, Birgit

    Cancer treatment and research communications

    2021  Volume 29, Page(s) 100449

    Abstract: Supernatant pleural effusions (PE) have shown to be a valuable source for the detection of driver mutations in circulating tumor DNA (ctDNA). In this prospective study, the clinical value of ctDNA analysis in supernatant PE to support therapy selection ... ...

    Abstract Supernatant pleural effusions (PE) have shown to be a valuable source for the detection of driver mutations in circulating tumor DNA (ctDNA). In this prospective study, the clinical value of ctDNA analysis in supernatant PE to support therapy selection and disease monitoring in lung cancer patients is assessed. Paired PE and plasma samples were collected from lung cancer patients before initiation of therapy (N = 2) and from EGFR positive patients during therapy (N = 3). Supernatant PE and plasma were tested for mutations in EGFR, KRAS and BRAF by droplet digital PCR. In PE of two patients with suspected lung cancer, a KRAS mutation was detected with a 5- and 8-fold higher fractional abundance (FA) compared to plasma. For three patients with progressive disease during therapy, both the EGFR L858R and T790M mutations were detected in PE. However, in plasma only for two of these patients the L858R mutation was detected with a 46- and 14- fold lower FA, and only for one patient the T790M mutation was detected with a 8-fold lower FA. For one patient, longitudinal ctDNA analysis in PE revealed the T790M and L858R mutations already two months prior to detection of progressive disease by CT-scan. In this study, a higher ctDNA concentration and FA was obtained from PE compared to the corresponding blood samples, which enables more sensitive mutation analysis. Thus, PE is a valuable liquid biopsy, complementing plasma, for ctDNA analysis to support therapy selection and disease monitoring in lung cancer patients.
    MeSH term(s) Circulating Tumor DNA/blood ; Female ; Genotyping Techniques/methods ; Humans ; Lung Neoplasms/pathology ; Male ; Pleural Effusion/pathology
    Chemical Substances Circulating Tumor DNA
    Language English
    Publishing date 2021-08-25
    Publishing country England
    Document type Journal Article
    ISSN 2468-2942
    ISSN (online) 2468-2942
    DOI 10.1016/j.ctarc.2021.100449
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  6. Article: Up-front mutation detection in circulating tumor DNA by droplet digital PCR has added diagnostic value in lung cancer.

    Visser, Esther / de Kock, Remco / Genet, Sylvia / Borne, Ben van den / Soud, Maggy Youssef-El / Belderbos, Huub / Stege, Gerben / de Saegher, Marleen / 't Westeinde, Susan van / Broeren, Maarten / Eduati, Federica / Deiman, Birgit / Scharnhorst, Volkher

    Translational oncology

    2022  Volume 27, Page(s) 101589

    Abstract: Identification of actionable mutations in advanced stage non-squamous non-small-cell lung cancer (NSCLC) patients is recommended by guidelines as it enables treatment with targeted therapies. In current practice, mutations are identified by next- ... ...

    Abstract Identification of actionable mutations in advanced stage non-squamous non-small-cell lung cancer (NSCLC) patients is recommended by guidelines as it enables treatment with targeted therapies. In current practice, mutations are identified by next-generation sequencing of tumor DNA (tDNA-NGS), which requires tissue biopsies of sufficient quality. Alternatively, circulating tumor DNA (ctDNA) could be used for mutation analysis. This prospective, multicenter study establishes the diagnostic value of ctDNA analysis by droplet digital PCR (ctDNA-ddPCR) in patients with primary lung cancer. CtDNA from 458 primary lung cancer patients was analyzed using a panel of multiplex ddPCRs for EGFR (Ex19Del, G719S, L858R, L861Q and S768I), KRAS G12/G13 and BRAF V600 mutations. For 142 of 175 advanced stage non-squamous NSCLC patients tDNA-NGS results were available to compare to ctDNA-ddPCR. tDNA-NGS identified 98 mutations, of which ctDNA-ddPCR found 53 mutations (54%), including 32 of 45 (71%) targetable driver mutations. In 2 of these 142 patients, a mutation was found by ctDNA-ddPCR only. In 33 advanced stage patients lacking tDNA-NGS results, ctDNA-ddPCR detected 15 additional mutations, of which 7 targetable. Overall, ctDNA-ddPCR detected 70 mutations and tDNA-NGS 98 mutations in 175 advanced NSCLC patients. Using an up-front ctDNA-ddPCR strategy, followed by tDNA-NGS only if ctDNA-ddPCR analysis is negative, increases the number of mutations found from 98 to 115 (17%). At the same time, up-front ctDNA-ddPCR reduces tDNA-NGS analyses by 40%, decreasing the need to perform (additional) biopsies.
    Language English
    Publishing date 2022-11-19
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2443840-6
    ISSN 1936-5233
    ISSN 1936-5233
    DOI 10.1016/j.tranon.2022.101589
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  7. Article ; Online: Circulating biomarkers for monitoring therapy response and detection of disease progression in lung cancer patients.

    de Kock, Remco / Borne, Ben van den / Soud, Maggy Youssef-El / Belderbos, Huub / Stege, Gerben / de Saegher, Marleen / van Dongen-Schrover, Claartje / Genet, Sylvia / Brunsveld, Luc / Scharnhorst, Volkher / Deiman, Birgit

    Cancer treatment and research communications

    2021  Volume 28, Page(s) 100410

    Abstract: Liquid biopsies have become of interest as minimally invasive ways to monitor treatment response in lung cancer patients. Circulating tumor DNA (ctDNA) and protein biomarkers are evaluated for their added value in monitoring therapy response and early ... ...

    Abstract Liquid biopsies have become of interest as minimally invasive ways to monitor treatment response in lung cancer patients. Circulating tumor DNA (ctDNA) and protein biomarkers are evaluated for their added value in monitoring therapy response and early detection of disease progression. Plasma and serum samples of non-small cell or small cell lung cancer patients were analyzed for driver mutations in ctDNA (EGFR, KRAS or BRAF) using droplet digital PCR and protein biomarkers (CA125, CEA, CA15.3, Cyfra 21-1, HE4, NSE, proGRP and SCCA) using electrochemiluminescence immunoassays. Biomarker concentration changes were compared with the outcome of CT-scans during therapy. The median difference of the concentration of ctDNA, CA125 and Cyfra21-1 was significantly lower in patients with partial response (PR) compared to patients with progressive disease (PD) on the first evaluation CT-scan (P<0.001, P=0.042 and P=0.020, respectively). A substantial agreement between ctDNA or CA125 response and radiographic response was observed (k=0.692 and k=0.792, respectively). The median difference of the concentration of ctDNA and Cyfra21-1 was also significantly lower in PR patients compared to PD patients at the last CT-scan during therapy (P<0.001 and P=0.026, respectively). An almost perfect agreement between ctDNA and radiographic response (k=0.827) and a moderate agreement between Cyfra21-1 response and radiographic response was observed (k=0.553). Serial testing of the concentration of ctDNA, Cyfra21-1, and possibly CA125 could be a useful added tool for monitoring therapy response and early detection of disease progression in lung cancer patients.
    MeSH term(s) Aged ; Biomarkers, Tumor/metabolism ; Disease Progression ; Female ; Humans ; Liquid Biopsy/methods ; Lung Neoplasms/therapy ; Male ; Middle Aged ; Prospective Studies
    Chemical Substances Biomarkers, Tumor
    Language English
    Publishing date 2021-06-01
    Publishing country England
    Document type Journal Article ; Multicenter Study
    ISSN 2468-2942
    ISSN (online) 2468-2942
    DOI 10.1016/j.ctarc.2021.100410
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Liquid biopsy-based decision support algorithms for diagnosis and subtyping of lung cancer.

    Visser, Esther / Genet, Sylvia A A M / de Kock, Remco P P A / van den Borne, Ben E E M / Youssef-El Soud, Maggy / Belderbos, Huub N A / Stege, Gerben / de Saegher, Marleen E A / van 't Westeinde, Susan C / Brunsveld, Luc / Broeren, Maarten A C / van de Kerkhof, Daan / Deiman, Birgit A L M / Eduati, Federica / Scharnhorst, Volkher

    Lung cancer (Amsterdam, Netherlands)

    2023  Volume 178, Page(s) 28–36

    Abstract: Objectives: Pathologic subtyping of tissue biopsies is the gold standard for the diagnosis of lung cancer (LC), which could be complicated in cases of e.g. inconclusive tissue biopsies or unreachable tumors. The diagnosis of LC could be supported in a ... ...

    Abstract Objectives: Pathologic subtyping of tissue biopsies is the gold standard for the diagnosis of lung cancer (LC), which could be complicated in cases of e.g. inconclusive tissue biopsies or unreachable tumors. The diagnosis of LC could be supported in a minimally invasive manner using protein tumor markers (TMs) and circulating tumor DNA (ctDNA) measured in liquid biopsies (LBx). This study evaluates the performance of LBx-based decision-support algorithms for the diagnosis of LC and subtyping into small- and non-small-cell lung cancer (SCLC and NSCLC) aiming to directly impact clinical practice.
    Materials and methods: In this multicenter prospective study (NL9146), eight protein TMs (CA125, CA15.3, CEA, CYFRA 21-1, HE4, NSE, proGRP and SCCA) and ctDNA mutations in EGFR, KRAS and BRAF were analyzed in blood of 1096 patients suspected of LC. The performance of individual and combined TMs to identify LC, NSCLC or SCLC was established by evaluating logistic regression models at pre-specified positive predictive values (PPV) of ≥95% or ≥98%. The most informative protein TMs included in the multi-parametric models were selected by recursive feature elimination.
    Results: Single TMs could identify LC, NSCLC and SCLC patients with 46%, 25% and 40% sensitivity, respectively, at pre-specified PPVs. Multi-parametric models combining TMs and ctDNA significantly improved sensitivities to 65%, 67% and 50%, respectively.
    Conclusion: In patients suspected of LC, the LBx-based decision-support algorithms allowed identification of about two-thirds of all LC and NSCLC patients and half of SCLC patients. These models therefore show clinical value and may support LC diagnostics, especially in patients for whom pathologic subtyping is impossible or incomplete.
    MeSH term(s) Humans ; Lung Neoplasms/diagnosis ; Lung Neoplasms/genetics ; Carcinoma, Non-Small-Cell Lung/diagnosis ; Carcinoma, Non-Small-Cell Lung/genetics ; Carcinoma, Non-Small-Cell Lung/pathology ; Prospective Studies ; Biomarkers, Tumor ; Phosphopyruvate Hydratase ; Liquid Biopsy
    Chemical Substances antigen CYFRA21.1 ; Biomarkers, Tumor ; Phosphopyruvate Hydratase (EC 4.2.1.11)
    Language English
    Publishing date 2023-02-01
    Publishing country Ireland
    Document type Multicenter Study ; Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 632771-0
    ISSN 1872-8332 ; 0169-5002
    ISSN (online) 1872-8332
    ISSN 0169-5002
    DOI 10.1016/j.lungcan.2023.01.014
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