Martha Zavridou

Prognostic significance of PD-L1 expression on circulating tumor cells in patients with head and neck squamous cell carcinoma

Background Successful application of programmed death 1 (PD1) checkpoint inhibitors in the clinic may ultimately benefit from appropriate patient selection based upon predictive biomarkers. Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while predictive biomarkers for response to PD1 checkpoint inhibitors are lacking. We sought to assess whether overexpression of PD-L1 in CTCs could be detected at baseline and at different timepoints during treatment in a prospective cohort of head and neck squamous cell carcinoma (HNSCC) patients and used to predict clinical outcome after treatment with curative intent. Patients and methods We developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA expression in EpCAM(+) CTCs. In a prospective cohort of 113 locally advanced HNSCC patients treated with curative intent we evaluated PD-L1 expression in the EpCAM(+) CTC fraction at baseline, after 2 cycles of induction chemotherapy (week 6) and at the end of concurrent chemoradiotherapy (week 15). Results PD-L1 overexpression was found in 24/94 (25.5%) patients at baseline, 8/34 (23.5%) after induction chemotherapy and 12/54 (22.2%) patients at the end of treatment. Patients with CTCs overexpressing PD-L1 at end of treatment had shorter progression-free survival (P = 0.001) and overall survival (P < 0.001). Multivariate analysis revealed that PD-L1 overexpression at end of treatment was independent prognostic factor for progression-free survival and overall survival. The absence of PD-L1 overexpression at the end of treatment was strongly associated with complete response with an odds ratio = 16.00 (95% CI = 2.76-92.72, P = 0.002). Conclusions We demonstrate that detection of CTCs overexpressing PD-L1 is feasible and may provide important prognostic information in HNSCC. Our results suggest that adjuvant PD1 inhibitors deserve evaluation in HNSCC patients in whom PD-L1(+) CTCs are detected at the end of curative treatment.

Direct Comparison of Metastasis-Related miRNAs Expression Levels in Circulating Tumor Cells, Corresponding Plasma, and Primary Tumors of Breast Cancer Patients

BACKGROUND Circulating tumor cells (CTCs) and microRNAs (miRNAs) are important in liquid biopsies in which peripheral blood is used to characterize the evolution of solid tumors. We evaluated the expression levels of miR-21, miR-146a, miR-200c, and miR-210 in CTCs of breast cancer patients with verified metastasis and compared their expression levels in corresponding plasma and primary tumors. METHODS Expression levels of the miRNAs were quantified by quantitative reverse transcription PCR (RT-qPCR) in (a) 89 primary breast tumors and 30 noncancerous breast tissues and (b) CTCs and corresponding plasma of 55 patients with metastatic breast cancer and 20 healthy donors. For 30 of these patients, CTCs, corresponding plasma, and primary tumor tissues were available. RESULTS In formalin-fixed, paraffin-embedded tissues, these miRNAs were differentially expressed between primary breast tumors and noncancerous breast tissues. miR-21 (P < 0.001) and miR-146a (P = 0.001) were overexpressed, whereas miR-200c (P = 0.004) and miR-210 (P = 0.002) were underexpressed. In multivariate analysis, miR-146a overexpression was significantly [hazard ratio 2.969 (1.231-7.157), P = 0.015] associated with progression-free survival. In peripheral blood, all miRNAs studied were overexpressed in both CTC and corresponding plasma. There was a significant association between miR-21 expression levels in CTCs and plasma for 36 of 55 samples (P = 0.008). In plasma, ROC curve analysis revealed that miR-21, miR-146a, and miR-210 could discriminate patients from healthy individuals. CONCLUSIONS Metastasis-related miRNAs are overexpressed in CTCs and corresponding plasma; miR-21 expression levels highly correlate in CTCs and plasma; and miR-21, miR-146a, and miR-210 are valuable plasma biomarkers for discriminating patients from healthy individuals.

miRNA-21 as a novel therapeutic target in lung cancer

Lung cancer is a leading cause of cancer death, and late diagnosis is one of the most important reasons for the high mortality rate. microRNAs (miRNAs) are key players in gene regulation and therefore in tumorigenesis. As far as lung carcinogenesis is concerned, miRNAs open novel fields in biomarker research, in diagnosis, and in therapy. In this review we focus on miR-21 in lung cancer and especially on how miR-21 is involved 1) as a biomarker in response or resistance to therapy or 2) as a therapeutic target.

MicroRNA signatures as clinical biomarkers in lung cancer

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Current Biomarker Findings 2015:5 35–45 Current Biomarker Findings Dovepress

Evaluation of Preanalytical Conditions and Implementation of Quality Control Steps for Reliable Gene Expression and DNA Methylation Analyses in Liquid Biopsies

BACKGROUND 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.

LIQUID BIOPSY IN OVARIAN CANCER: THE POTENTIAL OF CIRCULATING miRNAs AND EXOSOMES

&NA; Ovarian cancer still remains the most lethal female cancer, since in most cases it is diagnosed at an advanced stage. Usually after completion of primary treatment chemoresistance occurs, and recurrent disease is finally observed. Liquid biopsy, based on minimally invasive and serial blood tests, has the advantage of following tumor evolution in real time, offering novel insights on precision medicine. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating cell‐free microRNAs (cfmiRNAs) and circulating exosomes represent the major components of liquid biopsy analysis. Liquid biopsy has been already implemented in ovarian cancer, and most studies so far are mainly focused on CTCs and ctDNA. This review is mainly focused on the clinical potential of circulating miRNAs and exosomes as a source of liquid biopsy biomarkers in ovarian cancer diagnosis, prognosis, and response to treatment.

Abstract 1718: RT-qPCR gene expression analysis of CTCs isolated through an epitope-independent enrichment microfluidic device in patients with head and neck squamous cell carcinoma

Background: Molecular characterization of circulating tumor cells (CTCs) is very challenging since these cells are rare, and the amount of available sample for their analysis is very limited. Moreover, CTC are highly heterogeneous and enrichment technologies based on EpCAM expression present the risk of missing EpCAM-negative CTCs. The Parsortix system (ANGLE plc, UK), is a novel microfluidic technology platform designed for marker-independent capture of CTCs. In this study we used for the first time the Parsortix system to isolate CTCs from patients with Head and Neck Squamous Cell carcinoma (HNSCC), and proceeded to downstream molecular characterization through RT-qPCR gene expression analysis. Methods: Peripheral blood samples (10 mL) from head and neck squamous cell carcinoma (HNSCC) patients (n=19) and healthy donors used as a control group (n=10) were used for the isolation of CTCs using the Parsortix device. Enriched CTCs were harvested in Trizol reagent, followed by extraction of total RNA and cDNA synthesis. RT-qPCR was performed in the LightCycler (Roche) for the following gene targets: PD-L1, VIM, TWIST, EGFR, and B2M (used as a reference gene). The expression levels of PD-L1, VIM, TWIST and EGFR were normalized using the 2 -ΔΔCt approach in respect to the expression of B2M. Results: All samples analyzed were of excellent RNA quality as this was evaluated by B2M expression. According to our results, PD-L1 overexpression was detected in 5/19(26.3%) samples, VIM was overexpressed in 3/19 (15.7%) and TWIST-1 in 1/19 (5.3%) sample, while EGFR expression was not detected in any patient (0/19, 0%). These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Conclusions: This preliminary study is showing for the first time that RT-qPCR can be successfully used for the molecular characterization of CTCs isolated by the label-free Parsortix microfluidic device in HNSCC. Overexpression of individualized immunotherapy important biomarkers such as PD-L1 in CTCs of HNSCC patients could be of significant clinical importance for the selection and follow up of these patients. Citation Format: Martha Zavridou, Areti Strati, George Koutsodontis, Amanda Psyrri, Evi Lianidou. RT-qPCR gene expression analysis of CTCs isolated through an epitope-independent enrichment microfluidic device in patients with head and neck squamous cell carcinoma [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 1718. doi:10.1158/1538-7445.AM2017-1718

Abstract 501: PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer

Background: Programmed cell Death receptor Ligand 1 (PD-L1) is a very promising biomarker for the selection of patients for cancer immunotherapy. We recently developed a highly sensitive, specific and robust RT-qPCR assay for PD-L1 mRNA. The aim of the present study was to study the expression of PD-L1 in CTCs from breast cancer patients with verified metastasis. Methods: We quantified the expression of PD-L1 mRNA transcripts in EpCAM-positive CTCs, by using our recently developed RT-qPCR assay, based on the following procedure: i) immunomagnetic enrichment of EpCAM-positive CTCs from 20mL of peripheral blood, ii) total RNA isolation iii) cDNA synthesis and iv) RT-qPCR for PD-L1. PD-L1 expression in respect to the expression of B2M as a reference gene, was normalized using the 2-ΔΔCt approach. Peripheral blood samples (20mL) were obtained from 22 breast cancer patients with verified metastasis and 14 healthy donors. Results: According to our results, 11/22 (50%) of metastasis-verified breast cancer patients were found to be positive for PD-L1 overexpression in CTCs. These are preliminary results and these percentages may change, since the number of samples that we are analyzing is continuously increasing. Our results are in concordance with a recent study by Mazel et al (Mol Oncol 2015), that by using the CellSearch(®) system found PD-L1((+)) CTCs in 11/16(68.8%) patients with metastatic breast cancer. Conclusion: This is the first time that a quantitative RT-qPCR molecular assay is used for the evaluation of PD-L1expression levels in EpCAM-positive CTCs in metastatic breast cancer patients. The assay is closed-tube, quantitative, highly specific and sensitive, and high-throughput. We are currently evaluating the assay in a large number of clinical samples. Citation Format: Martha Zavridou, Areti Strati, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PD-L1 expressing circulating tumor cells (CTCs) in patients with breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 501.