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  • 1
    In: Journal of Separation Science, Wiley, Vol. 41, No. 13 ( 2018-07), p. 2819-2827
    Abstract: Malignant transformation in gliomas is frequently supplemented by somatic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes. It has recently emerged that mutations in these genes are associated with prolonged survival and should be used as prognostic factor in management of brain cancer patients. There are several approaches in use for the detection of isocitrate dehydrogenase 1 and 2 mutations; however, these often exhibit shortcomings such as convoluted protocols with long processing time, complex (and costly) dedicated fluorescent probes, and/or demand on amounts of input DNA. Therefore, a simple and rapid method would be highly desired. Here, we present development and validation of simple and reliable isocitrate dehydrogenase 1 and 2 mutation detection assay using denaturing capillary electrophoresis. The detection sensitivity in terms of the limiting mutated allele fraction detectable estimated from a series of dilution runs was 2.9%. The method was validated by comparing to results obtained by a widely accepted detection technique, the multiplex ligation‐dependent probe amplification, on a set of 85 brain tumors. The concordance of both methods was 100%, but denaturing capillary electrophoresis assay required fivefold lower input of DNA (1 versus 5 μL of DNA at concentrations typically between 10 and 30 ng/μL).
    Type of Medium: Online Resource
    ISSN: 1615-9306 , 1615-9314
    URL: Issue
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    Language: English
    Publisher: Wiley
    Publication Date: 2018
    detail.hit.zdb_id: 2047990-6
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  • 2
    In: Journal of Separation Science, Wiley, Vol. 33, No. 15 ( 2010-08), p. 2349-2355
    Abstract: The presence of activating mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene has been attributed to a positive response to biological therapy of lung cancer by small‐molecular tyrosine kinase inhibitors, gefitinib and erlotinib. Among the two most significant mutation types are deletions in exon 19 and a single point substitution in exon 21 (termed L858R). The exon 19 deletions can readily be examined by fragment analysis, due to the characteristic length difference between the normal and mutated PCR product. Analysis of the L858R point mutation, however, presents a greater challenge. The current paper is aimed at developing a sensitive, yet simple, low‐cost mutation detection assay directed at the L858R mutation using a method based on CE of heteroduplexes under partial denaturing conditions. We perform optimization of separation conditions on different commercial instruments including ones equipped with 8, 16 and 96 capillaries. We present normalized migration reproducibility in the range from 1 (8 and 16) to 5% (96) RSD. A reliable distinction of the R836R silent polymorphism from a potential presence of the L858R mutation is also demonstrated. In its implementation, the presented assay is just another application running on a conventional CE platform without the need of dedicated instrumentation.
    Type of Medium: Online Resource
    ISSN: 1615-9306 , 1615-9314
    URL: Issue
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    Language: English
    Publisher: Wiley
    Publication Date: 2010
    detail.hit.zdb_id: 2047990-6
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  • 3
    In: Lung Cancer, Elsevier BV, Vol. 77 ( 2012-6), p. S24-S25
    Type of Medium: Online Resource
    ISSN: 0169-5002
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2012
    detail.hit.zdb_id: 2025812-4
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  • 4
    In: Pathology & Oncology Research, Springer Science and Business Media LLC, Vol. 26, No. 1 ( 2020-1), p. 379-385
    Type of Medium: Online Resource
    ISSN: 1219-4956 , 1532-2807
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2020
    detail.hit.zdb_id: 2002501-4
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  • 5
    In: Gastroenterology, Elsevier BV, Vol. 156, No. 6 ( 2019-05), p. S-489-
    Type of Medium: Online Resource
    ISSN: 0016-5085
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    Language: English
    Publisher: Elsevier BV
    Publication Date: 2019
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  • 6
    In: Gastroenterology, Elsevier BV, Vol. 146, No. 5 ( 2014-05), p. S-864-S-865
    Type of Medium: Online Resource
    ISSN: 0016-5085
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    Language: English
    Publisher: Elsevier BV
    Publication Date: 2014
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  • 7
    Online Resource
    Online Resource
    American Society of Clinical Oncology (ASCO) ; 2017
    In:  Journal of Clinical Oncology Vol. 35, No. 15_suppl ( 2017-05-20), p. e23040-e23040
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 35, No. 15_suppl ( 2017-05-20), p. e23040-e23040
    Abstract: e23040 Background: The detection of circulating tumor DNA (ctDNA), referred to as “liquid biopsy” is becoming a preferred option for some patients with lung adenocarcinomas. The key for a suitable technology is high sensitivity enabling to detect small fractions of mutated alleles, the method has to be solid against false-positives. The aim of this work was to compare our method based on capillary electrophoresis under denaturing conditions (DCE) to FDA and CE IVD approved commercial kit - cobas EGFR Mutation Test v2. Methods: A total of 44 plasma samples were acquired from patients with clinicaly confirmed stage IV lung adenocarcinoma. There were samples from patients prior to receiving therapy (with a known tissue EGFR genotype) as well as several ones showing progression on a previously administered antiEGFR-therapy (with no tissue info available at the time of plasma sampling). DNA was isolated from multiple 2 mL aliquots of each plasma. The first aliquot was run on cobas z480 real-time instrument using unmodified manufacturer's protocol. Another 2ml aliquot was run by DCE assay (accredited method under ISO 15189:2012), which is based on a PCR followed by a fragment analysis on an ABI capillary analyzer (Sanger sequencer). Results: The mutual concordance was 84.1% (37/44). cobas assay identified 11 EGFR-positive plasma samples (6 x Ex19del, 3 x L858R, 1x T790M combined with Ex19del and 1x T790M combined with L858R). DCE identified 8 EGFR-positive samples (6 x Ex19del, 1 x L858R, 1xT790M combined with L858R). DCE missed 5 of cobas-positive samples (2x Ex19del, 2x L858R and 1x T790M) while in turn identifying 2x Ex19del plasma samples that were concordant with tissue, but missed by cobas. Overall concordance with tissue was 73.3% (22/30) for cobas, 70.0% (21/30) for DCE assay and 80.0% (24/30) for both combined. Conclusions: DCE approach is reliable for low levels of mutations in plasma of lung cancer patients and comparable (and complementary) to cobas. Its advantage is simplicity, low cost and a universal, straighforward adaptability to virtually any mutation at any site allowing to detect oncogenic as well as tumor supressor mutations in ctDNA. Supported by grant 17-30748A.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
    RVK:
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    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2017
    detail.hit.zdb_id: 2005181-5
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  • 8
    Online Resource
    Online Resource
    American Society of Clinical Oncology (ASCO) ; 2015
    In:  Journal of Clinical Oncology Vol. 33, No. 3_suppl ( 2015-01-20), p. 642-642
    In: Journal of Clinical Oncology, American Society of Clinical Oncology (ASCO), Vol. 33, No. 3_suppl ( 2015-01-20), p. 642-642
    Abstract: 642 Background: Patients with advanced stages of colorectal cancer have elevated levels of circulating-tumor DNA (ctDNA) in peripheral blood. Its presence can facilitate acquisition of DNA material for tumor molecular profiling without the invasive procedures. In this work we present utility of ctDNA for the determination of tumor phenotype within the basic division into the mutator phenotype (microsatellite instability, MSI and chromosomal instability, CIN) or methylator phenotype (CpG island methylator phenotype, CIMP). Methods: Tumor biopsy tissue and 5mL of peripheral blood samples were prospectively collected from 148 patients with colorectal tumors. In addition, plasma was extracted from peripheral blood immediately upon collection. Samples were then transported to the molecular laboratory where DNA and ctDNA were isolated from tissue and plasma, by a modified spin-column ctDNA extraction. Somatic mutations in tumor suppressors denoting CIN phenotype were detected by denaturing capillary electrophoresis (DCE), microsatellite instability was detected by MSI Analysis System, Version 1.2 (Promega corporation, Madison, WI) and DNA methylation (CIMP phenotype) was obtained by multiplex ligation-dependent probe amplification (MRC Holland, NL). Results: The samples included 75 adenomas and 70 carcinomas. The detected rates in tissues were: 4% for MSI, 32% for CIN and 7% for CIMP in adenomas and 10% for MSI, 36% for CIN and 32% for CIMP in carcinomas. In adenomas, none of the detected molecular types could be confirmed in ctDNA. In carcinomas, the concordance between tissue and ctDNA was 25% for MSI, 23% for CIN and 60% for CIMP. The success rates were related to the ctDNA concentration yields with a minimum requirement of ~0.2ng/uL. Conclusions: ctDNA may serve as alternative source for molecular classification of colorectal tumors (MSI, CIN and CIMP). A low concordance between tissue and ctDNA was found for CIN and MSI in comparison to 60% of CIMP-phenotypes found in both tissue as well as ctDNA. The results are predominantly dictated by disease stage and the corresponding levels of ctDNA. Supported by a Czech Ministry of health grant no. 14383.
    Type of Medium: Online Resource
    ISSN: 0732-183X , 1527-7755
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    Language: English
    Publisher: American Society of Clinical Oncology (ASCO)
    Publication Date: 2015
    detail.hit.zdb_id: 2005181-5
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  • 9
    Online Resource
    Online Resource
    American Association for Cancer Research (AACR) ; 2019
    In:  Cancer Research Vol. 79, No. 13_Supplement ( 2019-07-01), p. 412-412
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 79, No. 13_Supplement ( 2019-07-01), p. 412-412
    Abstract: INTRODUCTION: Patients in advanced stages of non-small cell lung carcinomas (NSCLC), who and unsuited for targeted biological therapy because of lack of actionable molecular predictors are frequently treated by anti-angiogenic therapy. The effectiveness of such therapy is primarily relying on imaging techniques including CT or hybrid PET/CT evaluating a combination of morphological factors (dimensions and volume) and, more recently, also functional parameters including the metabolic activity, tumor vascularization etc. The ability to track the course of the disease at the same time all of these parameters is prerequisite to enable timely monitoring and therapy change in case of an early detection of resistance. This study was aimed at utility of circulating-tumor DNA (ctDNA) as an tool for monitoring of therapy response. and assessment of phenotypic parameters of the tumor. PATIENTS AND METHODS: A total of 50 patients with confirmed Stage IV lung adenocarcinomas showing negativity on ALK, BRAF, EGFR, RET, ROS1 and MET predictors were prospectively enrolled into the study. Patients were treated under a standard protocol by a combination of paclitaxes/carboplatin/bevacizumab and followed by dual PET/CT. For each patient cytology tissue sample was subjected to test for a panel of somatic mutations. The found mutations were subsequently detected in ctDNA in plasma extracted from peripheral blood collected prior to therapy start and then in 1-month intervals during the therapy. The occurrence and quantity of ctDNA were correlated with the objective therapy response (RECIST) and to the functional imaging parameters of metabolic activity and vascularization. RESULTS: ctDNA was initially positive in 29 patients. ctDNA levels closely reflected the response to the therapy with complete or partial remission expressed as reduction or absence of ctDNA, while disease stabilization or progression signaled by persisting or increasing ctDNA levels. There was a borderline correlation between ctDNA and vascularization evaluated by dual PET/CT (p=0.055). CONCLUSION: In a subset of patients ctDNA can readily serve as a surrogate marker for semi-continuous monitoring of the effect of anti-angiogenic therapy. Work supported by Czech Ministry of Health project AZV 17-30748A. Citation Format: Marek Minarik, Martin Svaton, Barbora Belsanova, Anastasiya Semyakina, Jan Baxa, Ondrej Fiala, Milos Pesek, Lucie Benesova. Application of a serial liquid biopsy ctDNA assay for monitoring efficacy of anti-angiogenic lung cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 412.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    RVK:
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2019
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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  • 10
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 80, No. 16_Supplement ( 2020-08-15), p. 724-724
    Abstract: Introduction: Liquid biopsy based on detection of circulating tumor DNA (circulating-tumor DNA, ctDNA) is finding new applications in clinical management of solid cancers. From the initial use as an alternative to classical tissue biopsy for investigation of molecuar profiles in prediction or resistance detection of targeted therapies the approach is now applied to monitoring of minimal residual disease (MRD). Patients with metastatic colorectal cancer undergoing liver resection are subject to standard post-operative surveillance based mainly on periodic CT or PET/CT imaging (complemented eventually by MR imaging). Some reports have previously demonstrated benefit from performing a ctDNA detection complimentary to standard imaging to improve follow-up efficiency. Methods: In this report we demonstrate use of our oncoMonitor™ ctDNA technology, based on detection of somatic mutations derived from tumor in patient plasma. Unlike various approaches targeting oncogenic hotspot mutants, this technology can trace virtually any somatic mutation found in any gene (e.g. a tumor supressor). The relatively simple approach is based on rapid scanning of 100 to 140bp target amplicons for presence of mutations revealed based on differential melting by high-resolution denaturing capillary electrophoresis (DCE). The detection sensitivity is 0.1% MAF. Patients: In our concordance study during which a total of 41 patients with metatatic colorectal cancer were followed for 3 years from metastasectomy. oncoMonitor™ tast was applied to evaluate ctDNA along with standard CT or PET/CT imaging and tumor markers (CEA/CA19-9). Results: A total of 23 recurrences were clinically confirmed during surveillance. All recurrences were detected by oncoMonitor™ (23/23, 100%), while at the same time only 17 instances were detected by CT or PET/CT imaging (17/23, 73.9%) and 16 by tumor markers CEA/CA19-9 (16/23, 69.6%). There were 2 patients after R0 resection with persistent ctDNA positivity who had recurrence within 6 months from surgery. Conclusions: oncoMonitor™ technology is suitable for quick and sensitive detection of ctDNA in plasma of patients with metatatic colorectal cancer after liver resection. Evaluation of ctDNA presence improves efficiency of post-operative surveillance. Supported by Czech Ministry of Health project no. 17-31909A. Citation Format: Marek Minarik, Barbora Belsanova, Renata Ptackova, Tereza Halkova, Filip Pazdirek, Jiri Pudil, Miroslav Levy, Jaromir Simsa, Jiri Hoch, Miroslav Ryska, Lubos Petruzelka, Lucie Benesova. Application of oncoMonitor™ ctDNA tracking technology for monitoring of therapy and early detection of recurrence in metastatic colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 724.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    RVK:
    RVK:
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2020
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
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