Showing papers by "Evi Lianidou published in 2017"
TL;DR: It is demonstrated that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC, and suggest that adjuvant PD1 inhibitors deserve evaluation in H NSCC patients in whom PD- L1(+) C TCs are detected at the end of curative treatment.
141 citations
TL;DR: ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen and should be further evaluated as a potential liquid biopsy-based biomarker.
Abstract: Purpose: Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of circulating tumor cells (CTCs) and plasma-circulating tumor DNA (ctDNA). ESR1 epigenetic silencing potentially affects response to endocrine treatment. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA. We evaluated ESR1 methylation in CTCs and paired plasma ctDNA as a potential biomarker for response to everolimus/exemestane treatment.Experimental Design: A highly sensitive and specific real-time MSP assay for ESR1 methylation was developed and validated in (i) 65 primary breast tumors formalin-fixed paraffin-embedded (FFPE), (ii) EpCAM+ CTC fractions (122 patients and 30 healthy donors; HD), (iii) plasma ctDNA (108 patients and 30HD), and (iv) in CTCs (CellSearch) and in paired plasma ctDNA for 58 patients with breast cancer. ESR1 methylation status was investigated in CTCs isolated from serial peripheral blood samples of 19 patients with ER+/HER2- advanced breast cancer receiving everolimus/exemestane.Results:ESR1 methylation was detected in: (i) 25/65 (38.5%) FFPEs, (ii) EpCAM+ CTC fractions: 26/112 (23.3%) patients and 1/30 (3.3%) HD, and (iii) plasma ctDNA: 8/108 (7.4%) patients and 1/30 (3.3%) HD. ESR1 methylation was highly concordant in 58 paired DNA samples, isolated from CTCs (CellSearch) and corresponding plasma. In serial peripheral blood samples of patients treated with everolimus/exemestane, ESR1 methylation was observed in 10/36 (27.8%) CTC-positive samples, and was associated with lack of response to treatment (P = 0.023, Fisher exact test).Conclusions: We report for the first time the detection of ESR1 methylation in CTCs and a high concordance with paired plasma ctDNA. ESR1 methylation in CTCs was associated with lack of response to everolimus/exemestane regimen. ESR1 methylation should be further evaluated as a potential liquid biopsy-based biomarker. Clin Cancer Res; 24(6); 1500-10. ©2017 AACR.
117 citations
TL;DR: It is reported for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients and real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation.
Abstract: // Lydia Giannopoulou 1 , Issam Chebouti 2 , Kitty Pavlakis 3 , Sabine Kasimir-Bauer 2 , Evi S. Lianidou 1 1 Analysis of Circulating Tumor Cells Laboratory, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, University Campus, Athens, 15771, Greece 2 Department of Gynecology and Obstetrics, University Hospital of Essen, University of Duisburg-Essen, Essen, D-45122, Germany 3 Pathology Department, IASO Women’s Hospital, Marousi, Athens, 15123, Greece Correspondence to: Evi S. Lianidou, email: lianidou@chem.uoa.gr Keywords: circulating tumor DNA, RASSF1A, high-grade serous ovarian cancer, methylation specific PCR, high resolution melting analysis Received: April 10, 2016 Accepted: November 11, 2016 Published: February 10, 2017 ABSTRACT The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients.
51 citations
TL;DR: Current knowledge regarding CTCs and ctDNA in HNSCC is reviewed and open questions are addressed to address open questions in this fast-evolving field of research.
47 citations
TL;DR: DNA methylation analysis of SOX17 in CTCs and matched ctDNA provides significant prognostic value and was associated with CK-19 expression but was not reflecting the status of primary tumours in breast cancer.
Abstract: // Maria Chimonidou 1 , Areti Strati 1 , Nikos Malamos 2 , Sophia Kouneli 2 , Vassilis Georgoulias 3 and Evi Lianidou 1 1 Analysis of Circulating Tumour Cells Laboratory, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece 2 Department of Pathology, Oncology Unit, Helena Venizelou Hospital, Athens, Greece 3 Laboratory of Tumour Cell Biology, Medical School, University of Crete, Heraklion, Greece Correspondence to: Evi Lianidou, email: lianidou@chem.uoa.gr Keywords: liquid biopsy, circulating tumour cells, circulating tumour DNA, breast cancer, methylation specific PCR Received: March 25, 2016 Accepted: March 29, 2017 Published: June 27, 2017 ABSTRACT Circulating Tumour Cells (CTCs) and circulating tumour DNA (ctDNA) represent a non-invasive liquid biopsy approach for the follow-up and therapy management of cancer patients. We evaluated whether DNA methylation status in CTCs and ctDNA is comparable and whether it reflects the status of primary tumours. We compared the methylation status of three genes, SOX17, CST6 and BRMS1 in primary tumours, corresponding CTCs and ctDNA in 153 breast cancer patients and healthy individuals, by using real time methylation specific PCR. We report a clear association between the EpCAM-positive CTC-fraction and ctDNA for SOX17 promoter methylation both for patients with early ( P = 0.001) and metastatic breast cancer ( P = 0.046) but not for CST6 and BRMS1. In early breast cancer, SOX17 promoter methylation in the EpCAM-positive CTC-fraction was associated with CK-19 mRNA expression ( P = 0.006) and worse overall survival (OS) ( P = 0.044). In the metastatic setting SOX17 promoter methylation in ctDNA was highly correlated with CK-19 ( P = 0.04) and worse OS ( Ρ = 0.016). SOX17 methylation status in CTCs and ctDNA was comparable and was associated with CK-19 expression but was not reflecting the status of primary tumours in breast cancer. DNA methylation analysis of SOX17 in CTCs and matched ctDNA provides significant prognostic value.
32 citations
TL;DR: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC, and differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies.
Abstract: Background We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.
32 citations
TL;DR: It is felt that microfluidics-based exosome isolation techniques, if cost-effective, could be implemented in the clinic and/or resource-scarce settings and proposed a road map for future developments.
Abstract: Interindividual variability is yet to be fully characterized, and for this, optimum patient stratification and companion diagnostics are still lacking. Especially when complex disease phenotypes and/or polygenic diseases are considered, patient monitoring and disease management become rather challenging, while acquired resistance to therapy and/or toxicity events are among the unmet needs in the clinic. No doubt, biomarkers are of great importance to disease management and tailor-made theranostics. Microfluidics has gathered great attention lately, mostly due to its low-invasive nature compared to tissue biopsies. Low invasiveness becomes greatly advantageous for microfluidics practices as the latter mirror cell biology revolutionizing cancer diagnostics and management. Recent advances in microfluidics hold the promise of robust clinical diagnostics after they have demonstrated effective exosome separation. We feel that microfluidics-based exosome isolation techniques, if cost-effective, could be implemented in the clinic and/or resource-scarce settings. This article (a) discusses exosomes, (b) comments on the first microfluidic advances in the field of cancer theranostics, (c) presents such advances in exosomes as complementary to liquid biopsies with an emphasis on circulating tumor cells, and (d) proposes a road map for future developments.
27 citations
TL;DR: Using low-dose computed tomography screening, a 20% reduction in lung cancer mortality has been more recently reported and a special focus on molecular markers as an alternative approach to different imaging and cytology-based strategies is focused on.
Abstract: Lung cancer kills millions of people each year in the world and is the main cause of cancer death in men and the second cause in women (1). Surprisingly, a decade ago, lung cancer was not so frequently diagnosed compared to breast and prostate cancer, explained by the fact that these cancers can be detected at a very early stage. For lung cancer, the five-year survival rate was as low as 15%. However, when lung cancer can be detected at an early stage, the survival rate increases dramatically (2). Using low-dose computed tomography (CT) screening, a 20% reduction in lung cancer mortality has been more recently reported (3). Thus, during this last decade, many efforts have been made to develop innovative technologies to detect lung cancer very early, with a special focus on molecular markers as an alternative approach to different imaging and cytology-based strategies.
3 citations
01 Jan 2017
TL;DR: In the present chapter, latest developments on histone and DNA methylation based biomarkers in Lung cancer are described.
Abstract: Oncogenesis is driven by the accumulation of genetic and epigenetic alterations that result in dysregulation of key oncogenes, tumor suppressor genes, and DNA repair/housekeeping genes. One of the major clinical needs is the discovery and clinical validation of new molecular biomarkers using non-or minimally invasive procedures to assist early diagnosis, prognosis and prediction of response to treatment. Histone methylation has profound effects on nuclear functions such as transcriptional regulation, maintenance of genome integrity and epigenetic inheritance. On the other hand, aberrant DNA methylation can be detected in several biological fluids of patients and could be served as a potential tumor biomarker. In the present chapter we describe latest developments on histone and DNA methylation based biomarkers in Lung cancer.
3 citations
TL;DR: It is reported for the first time that ESR1 methylation can be detected in cfDNA of high-grade serous ovarian cancer patients, but not in healthy women.
Abstract: Introduction: ESR1 methylation is frequently observed in many types of solid malignancies, but few studies attempted to investigate ESR1 methylation status in ovarian cancer so far. We examined for the first time ESR1 methylation in plasma samples of high-grade serous (HGSC) ovarian cancer patients, using a novel real-time MSP assay. Materials and methods: We first developed and validated a highly specific and sensitive real-time MSP assay for ESR1 methylation. A commercially available 100% methylated standard was used as positive control in all experiments. We then evaluated ESR1 methylation in plasma (2mL) of 59 pre-surgery, mainly advanced stage HGSC patients. A control group of 51 plasma samples from healthy women was recruited for the evaluation of specificity of our assay. All cfDNA samples isolated from plasma were subjected to sodium bisulfite (SB) treatment and were then analyzed by real-time MSP for ESR1 methylation. For the normalization of the results, we used ACTB as a reference gene. The real-time PCR assay for ACTB was designed to amplify specifically SB treated DNA. Results: The real-time MSP assay is highly sensitive, as it detects down to 0.1% of ESR1 methylation in the presence of 99.9% non-methylated sequences. ESR1 methylation was detected in 18/59 (30.5%) high-grade serous ovarian patients, but only in 1/51 (2.0%) of healthy women. All results were obtained after normalization with ACTB as a reference gene. Conclusion: We report for the first time that ESR1 methylation can be detected in cfDNA of high-grade serous ovarian cancer patients, but not in healthy women. Further investigation is required to determine the clinical significance of ESR1 methylation status in cfDNA of HGSC patients. We also aim to evaluate our findings in a larger group of patients. Citation Format: Lydia Giannopoulou, Sophia Mastoraki, Areti Strati, Isaam Chebouti, Sabine Kasimir-Bauer, Evi S. Lianidou. ESR1 methylation in plasma cfDNA of patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5691. doi:10.1158/1538-7445.AM2017-5691
1 citations
TL;DR: Tzanikou et al. as mentioned in this paper developed a highly sensitive and specific methodology for the detection of ERS1 D538G hotspot mutation, based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis.
Abstract: AIMS: Molecular characterization of CTCs and ctDNA analysis holds promise as an extremely powerful tool for the molecular profiling of cancer patients in real time. Estrogen receptor alpha (ERα) is expressed in approximately 70% of all breast cancers and endocrine therapy represents a major treatment modality in ERα-positive disease. Recently, somatic mutations in the ERα gene (ESR1) were linked to acquired resistance to endocrine therapies in breast cancer. In this study, we analyzed the most frequent ERS1 mutation (D538G) in CTCs (DNA samples isolated from CellSearch cartridges), corresponding ctDNA from early and metastatic breast cancer patients and healthy donors. METHODS: We first developed a highly sensitive and specific methodology for the detection of ESR1 D538G hotspot mutation, based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis. We analyzed DNAs isolated from CTCs (CellSearch) and the corresponding ctDNA before and/or after therapy in: a) 25 patients with ER+ operable breast cancer, b) 11 patients with ER+ metastatic breast cancer, c) 13 patients with ER- early breast cancer, d) 5 patients with ER- metastatic breast cancer and e) 80 healthy female volunteers. In all cases ctDNA (extracted from 2 ml plasma) and DNA from CTCs were first examined for their DNA quality before analysis. RESULTS: The assay is highly sensitive (analytical sensitivity: 0.05%) and specific (0/80 healthy donors). ERS1 D538G hotspot mutation was identified in ctDNA in 4/18 (22.2%) of ER+ metastasis-verified and in 5/33 (15.2%) of ER+ early breast cancer. In CTCs, ERS1 D538G mutation was identified in 6/18 (33.3%) of ER+ metastasis-verified and 5/33 (15.2%) of ER+ early breast cancer. In ER-pos metastasis-verified breast cancer, the concordance for D538G mutation between CTCs and ctDNA was 10/18 (55.6%), whereas the corresponding concordance for ER+ operable breast cancer was 25/33 (75.8%). Moreover, ERS1 D538G hotspot mutation was identified in ctDNA in 2/9 (22.2%) of ER- metastasis-verified breast cancer and 2/16 (12.5%) of ER- early breast cancer. ERS1 D538G hotspot mutation was identified in CTCs in 3/16 (18.8%) of ER- operable breast cancer, whereas none of ER- metastasis-verified breast cancer patients (0/9) were positive. In ER- metastasis-verified breast cancer patients, the concordance between CTCs and ctDNA for D538G mutation was 7/9(77.8%), whereas the corresponding concordance for ER- early breast cancer was 15/16 (93.8%). CONCLUSIONS: We developed and validated an ultrasensitive and highly specific methodology for the detection of ERS1 D538G hotspot mutation. This mutation was detected not only in the ER+ group, but also in the ER- group of breast cancer patients. We will further evaluate our findings in a large cohort of patients before and after treatment, to evaluate response to endocrine therapies in breast cancer. Citation Format: Eleni Tzanikou, Athina Markou, Eleni Politaki, Giorgos Koytsodontis, Amanda Psyrri, Vassileios Georgoulias, Evi Lianidou. Detection of ESR1 D538G mutation in circulating tumor cells (CTCs) and paired circulating tumor DNA (ctDNA) samples of breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2017-1725
01 Jan 2017
TL;DR: Analysis of CTCs at the gene expression, DNA methylation, and DNA mutation level holds considerable promise for an individualized treatment approach for patients with metastatic breast cancer.
Abstract: Molecular characterization of CTCs holds considerable promise for the identification of therapeutic targets and resistance mechanisms and for real-time monitoring of the efficacy of systemic therapies in patients with metastatic breast cancer. A variety of state-of-the-art systems are available for CTC isolation, detection, and molecular characterization. Analysis of CTCs at the gene expression, DNA methylation, and DNA mutation level holds considerable promise for an individualized treatment approach for patients with metastatic breast cancer.