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  • 1
    Online Resource
    Online Resource
    Informa UK Limited ; 2016
    In:  Lung Cancer: Targets and Therapy
    In: Lung Cancer: Targets and Therapy, Informa UK Limited
    Type of Medium: Online Resource
    ISSN: 1179-2728
    Language: English
    Publisher: Informa UK Limited
    Publication Date: 2016
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  • 2
    Online Resource
    Online Resource
    Informa UK Limited ; 2015
    In:  Current Biomarker Findings
    In: Current Biomarker Findings, Informa UK Limited
    Type of Medium: Online Resource
    ISSN: 2230-2492
    Language: English
    Publisher: Informa UK Limited
    Publication Date: 2015
    detail.hit.zdb_id: 2695566-0
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  • 3
    Online Resource
    Online Resource
    Elsevier BV ; 2019
    In:  Translational Research Vol. 205 ( 2019-03), p. 77-91
    In: Translational Research, Elsevier BV, Vol. 205 ( 2019-03), p. 77-91
    Type of Medium: Online Resource
    ISSN: 1931-5244
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2019
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  • 4
    In: Clinical Chemistry, Oxford University Press (OUP), Vol. 67, No. 4 ( 2021-03-31), p. 642-652
    Abstract: PD-L1, an immune checkpoint protein, is an important biomarker for monitoring cancer patients during the administration of cancer immunotherapy. Droplet digital PCR (ddPCR), is a highly sensitive and accurate tool for the quantification of cancer biomarkers in liquid biopsy. We report the development and analytical validation of a novel duplex RT-ddPCR assay for the simultaneous quantification of PD-L1 and hypoxanthine phosphoribosyltransferase (HPRT) (used as reference gene) transcripts in circulating tumor cells (CTCs). Methods RT-ddPCR experimental conditions were first optimized and the assay was analytically validated using synthetic standards and the BB49 and SCC47 cancer cell lines. The developed assay was further applied in 71 peripheral blood (PB) samples from head and neck squamous cell carcinoma (HNSCC) patients and 20 PB samples from healthy donors. PD-L1 and HPRT transcripts were quantified in cDNAs derived from CTCs isolated by a size-dependent microfluidic device. The developed RT-ddPCR assay was directly compared to RT-qPCR using 71 identical patient cDNA samples. Results Analytical sensitivity was 0.64 copies/μL, while estimation of intra- and interassay variation revealed a high reproducibility (within-run CV%:4.7–23%; between-run CV%:13%). Using the developed RT-ddPCR assay 33/71(46.5%) HNSCC patients’ samples were found positive for PD-L1 expression in CTCs, while by using RT-qPCR fewer samples (23/71, 32.4%) were positive (concordance: 55/71, 77.5%). Conclusions The developed RT-ddPCR assay for PD-L1 in CTCs is highly sensitive, specific, and reproducible; additionally, it offers improved diagnostic sensitivity over RT-qPCR. The clinical utility of the assay should be prospectively evaluated for the real-time monitoring of CTCs of cancer patients under immunotherapy.
    Type of Medium: Online Resource
    ISSN: 0009-9147 , 1530-8561
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2021
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  • 5
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 75, No. 15_Supplement ( 2015-08-01), p. 5187-5187
    Abstract: Introduction: CTC play a critical role in the metastatic spread of carcinomas and their detection is associated with prognosis in many human cancers while their enumeration has been cleared by the FDA for follow up of breast, colon, and prostate cancer patients. However, it is quite clear now that simple enumeration of CTC is not enough and that CTC molecular characterization is absolutely necessary, since it can play a crucial role in understanding the biology of metastasis and in selecting patients for targeted therapy. In the present study, we investigated for the first time the expression levels of five metastasis-related miRNAs in the EpCAM-positive CTC fraction, in corresponding plasma and paired FFPEs samples of metastatic breast cancer (MBC) patients. Materials and Methods: We studied the expression of miR-21, miR-31, miR-146a, miR-200c and miR-210 expression in the EpCAM-positive CTC fraction and corresponding plasma samples and FFPEs tissues of 22 breast cancer patients with verified metastasis and 20 healthy individuals. For CTC analysis 20mL peripheral blood were used, RNA was extracted from the EpCAM positive CTC fraction, while circulating miRNAs were isolated from 200μL of corresponding plasma samples. miRNA quantitative analysis in both EpCAM-positive fractions and in corresponding plasma was performed by a stem-loop cDNA approach and RT-qPCR in the LightCycler 2.0 (Roche, Germany) Results: We first evaluated the differences in the expression levels of these five metastasis-related miRNAs between primary breast cancer tissues (FFPEs) and non-camcerous breast tissues (mammoplasties). The expression of all miRNAs was significantly different in primary tumors in respect to cancer-free breast tissues. Based on this finding we further studied the expression levels of these miRNAs in the EpCAM-positive CTC fraction and plasma samples of metastatic breast cancer in respect to healthy donors and found that they were also significantly different. There was a concordance only between miR-21 expression in EpCAM positive CTC fraction and FFPEs for 16/22 (72.7%) of patients (P = 0.026, Pearson's χ2 test), but not for the other miRNAs tested. The expression levels of all miRNAs in the EpCAM positive CTC fraction and circulating miRNAs were not associated with any of the clinical and pathological characteristics of the patients. We found only a correlation between circulating miR-210 expression levels in plasma and expression of ER (P = 0.043) and progesterone receptors (P = 0.006). Conclusion: This is the first study to evaluate the expression levels of specific miRNAs in the EpCAM positive CTC fraction in relation to corresponding circulating miRNAs in plasma and paired primary tissues. Our results need further verification in larger patient cohorts. Citation Format: Athina Markou, Martha Zavridou, Ioanna Sourvinou, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. Expression of metastasis-related miRNAs in EpCAM-positive CTC fraction in corresponding plasma and FFPEs of breast cancer patients with verified metastasis. [abstract] . In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5187. doi:10.1158/1538-7445.AM2015-5187
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    RVK:
    RVK:
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2015
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  • 6
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 79, No. 13_Supplement ( 2019-07-01), p. 2290-2290
    Abstract: INTRODUCTION: Androgen-receptor splice variant 7 (AR-V7)is a highly promising liquid biopsy predictive biomarker indicating primary or acquired resistance to novel androgen receptor signaling inhibitors in metastatic castration resistant prostate cancer (mCRPC). We present for the first time the expression pattern of AR-FL, AR-V7, and AR-567es at a quantitative level in circulating tumor cells (CTCs) and paired plasma-derived exosomes in mCRPC. METHODS: We first developed and analytically validated a novel multiplex RT-qPCR assay for AR full length (AR-FL), AR-V7, AR-567es and AR-total. We then quantified the expression levels of AR-splice variants, CK-19 (epithelial marker) and B2M (reference gene) in EpCAM+ CTCs and paired plasma-derived exosomes isolated from peripheral blood (20mL) of 62 mCRPC patients and 10 healthy donors. RESULTS: In CTCs AR-FL was detected in 57/62(92%),AR-V7 in 32/62(52.0%),AR-567es in15/62(24.2%) and AR-total in 56/62(90.0%). In paired plasma-derived exosomes, AR-FL was detected in 45/62(72.5%), AR-V7 in 4/62(6.5%), AR-567 in 2/62(3.2%) and AR total in 47/62(75.8%).CK-19 expression was detected in 18/62(29.0%) of CTCs and in 28/62(45.2%) of exosomes. In all cases AR splice variants were expressed at higher levels in CTCs than in paired exosomes, while AR-V7 was detected at higher percentages than AR-567es. CONCLUSIONS: Our results reveal for the first time a remarkable heterogeneity on the expression levels of AR-FL, AR-V7 and AR-567es in EpCAM+ CTCs and paired exosomes between individual mCRPC patients. The clinical significance of this finding will be further investigated in large patient cohorts in respect to therapy response. Citation Format: Areti D. Strati, Martha Zavridou, Evangelos Bournakis, Sophia Mastoraki, Evi S. Lianidou. Expression pattern of androgen receptor (AR), splice variant 7 (AR-V7) and splice variant 567 (AR-567) in circulating tumor cells and paired plasma-derived exosomes in metastatic castration resistant prostate cancer [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 2290.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    RVK:
    RVK:
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2019
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  • 7
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 79, No. 13_Supplement ( 2019-07-01), p. 2241-2241
    Abstract: Background: Circulating tumor cells (CTCs) heterogeneity is highly affecting the efficiency of their isolation and thus the reliability of downstream analysis. Especially in Head and Neck Squamous Cell Carcinoma (HNSCC) epithelial mesenchymal transition (EMT) is highly affecting CTC isolation and downstream analysis. We directly compared two different approaches used for CTC isolation, a label-independent size-based microfluidic-based system versus an EpCAM-based positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in HNSCC. Methods: Peripheral blood (PB) in EDTA (20mL) was collected from 50 HNSCC patients and 18 healthy donors (HD). A size-based microfluidic device (Parsortix, ANGLE) and an EpCAM-based positive immune-magnetic isolation procedure were applied in parallel, using 10mL PB in each case. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M. Real time methylation specific PCR (MSP) was used to study the methylation status of SOX17, RASSF1A and MLL3 genes in DNAs isolated from the same enriched CTCs. Results: In identical blood draws, the label-free size-based CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-based CTC enrichment, since a significantly higher percentage of identical PB samples was found positive for all genes tested both at the gene expression and DNA methylation level, while the specificity was not affected. Conclusions: In HNSCC CTC molecular characterization at the gene expression and DNA methylation level should be based on a label-free size-based isolation system. Citation Format: Martha Zavridou, Sophia Mastoraki, Areti D. Strati, George Koutsodontis, Apostolos Klinakis, Amanda Psyrri, Evi S. Lianidou. Molecular characterization of circulating tumor cells in head and neck squamous cell carcinoma: Direct comparison of a label-independent size-based microfluidic device with EpCAM-based CTC enrichment [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 2241.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    RVK:
    RVK:
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2019
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  • 8
    In: Clinical Chemistry, Oxford University Press (OUP), Vol. 64, No. 10 ( 2018-10-01), p. 1522-1533
    Abstract: Liquid biopsy provides important information for the prognosis and treatment of cancer patients. In this study, we evaluated the effects of preanalytical conditions on gene expression and DNA methylation analyses in liquid biopsies. METHODS We tested the stability of circulating tumor cell (CTC) messenger RNA by spiking MCF-7 cells in healthy donor peripheral blood (PB) drawn into 6 collection-tube types with various storage conditions. CTCs were enriched based on epithelial cell adhesion molecule positivity, and RNA was isolated followed by cDNA synthesis. Gene expression was quantified using RT-quantitative PCR for CK19 and B2M. We evaluated the stability of DNA methylation in plasma under different storage conditions by spiking DNA isolated from MCF-7 cells in healthy donor plasma. Two commercially available sodium bisulfite (SB)-conversion kits were compared, in combination with whole genome amplification (WGA), to evaluate the stability of SB-converted DNA. SB-converted DNA samples were analyzed by real-time methylation-specific PCR (MSP) for ACTB, SOX17, and BRMS1. Quality control was assessed using Levey–Jennings graphs. RESULTS RNA-based analysis in CTCs is severely impeded by the preservatives used in many PB collection tubes (except for EDTA), as well as by time to analysis. Plasma and SB-converted DNA samples are stable and can be used safely for MSP when kept at −80 °C. Downstream WGA of SB-converted DNA compensated for the limited amount of available sample in liquid biopsies. CONCLUSIONS Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic.
    Type of Medium: Online Resource
    ISSN: 0009-9147 , 1530-8561
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2018
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  • 9
    In: Clinical Chemistry, Oxford University Press (OUP), Vol. 68, No. 10 ( 2022-10-06), p. 1323-1335
    Abstract: Gene expression in circulating tumor cells (CTCs) can be used as a predictive liquid biopsy test in metastatic castration-resistant prostate cancer (mCRPC). We developed a novel 6-plex reverse transcription droplet digital PCR (RT-ddPCR) assay for the absolute quantification of 4 prostate cancer biomarkers, a reference gene, and a synthetic DNA external control (DNA-EC) in CTCs isolated from mCRPC patients. Methods A novel 6-plex RT-ddPCR assay was developed for the simultaneous absolute quantification of AR-FL, AR-V7, PSA, and PSMA, HPRT (used as a reference gene), and a synthetic DNA-EC that was included for quality control. The assay was optimized and analytically validated using DNA synthetic standards for each transcript as positive controls. Epithelial cellular adhesion molecule (EpCAM)-positive CTC fractions isolated from 90 mCRPC patients and 11 healthy male donors were analyzed, and results were directly compared with reverse transcription quantitative PCR (RT-qPCR) for all markers in all samples. Results Linear dynamic range, limit of detection, limit of quantification, intra- and interassay precision, and analytical specificity were determined for each marker. Application of the assay in EpCAM-positive CTC showed positivity for AR-FL (71/90; 78.9%), AR-V7 (28/90; 31.1%), PSA (41/90; 45.6%), PSMA (38/90; 42.2%), and HPRT (90/90; 100%); DNA-EC concentration was constant across all samples. Direct comparison with RT-qPCR for the same markers in the same samples revealed RT-ddPCR to have superior diagnostic sensitivity. Conclusions Our 6-plex RT-ddPCR assay was highly sensitive, specific, and reproducible, and enabled simultaneous and absolute quantification of 5 gene transcripts in minute amounts of CTC-derived cDNA. Application of this assay in clinical samples gave diagnostic sensitivity and specificity comparable to, or better than, RT-qPCR.
    Type of Medium: Online Resource
    ISSN: 0009-9147 , 1530-8561
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2022
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  • 10
    In: Cancers, MDPI AG, Vol. 12, No. 5 ( 2020-05-08), p. 1188-
    Abstract: PIM-1 is an oncogene involved in cell cycle progression, cell growth, cell survival and therapy resistance, activated in many types of cancer, and is now considered as a very promising target for cancer therapy. We report for the first time that PIM-1 is overexpressed in circulating tumor cells (CTCs) from metastatic castration-resistant prostate cancer patients (mCRPC). We first developed and validated a highly sensitive RT-qPCR assay for quantification of PIM-1 transcripts. We further applied this assay to study PIM-1 expression in EpCAM(+) CTC fraction isolated from 64 peripheral blood samples of 50 mCRPC patients. CTC enumeration in all samples was performed using the FDA-cleared CellSearch® system. PIM-1 overexpression was detected in 24/64 (37.5%) cases, while in 20/24 (83.3%) cases that were positive for PIM-1 expression, at least one CTC/7.5 mL PB was detected in the CellSearch®. Our data indicate that PIM-1 overexpression is observed at high frequency in CTCs from mCRPC patients and this finding, in combination with androgen receptor splice variant 7 (AR-V7) expression in CTCs, suggest its potential role as a very promising target for cancer therapy. We strongly believe that PIM-1 overexpression in EpCAM(+) CTC fraction merits to be further evaluated and validated as a non-invasive circulating tumor biomarker in a large and well-defined patient cohort with mCRPC.
    Type of Medium: Online Resource
    ISSN: 2072-6694
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2527080-1
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