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Background Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). Methods In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. Results All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. Conclusions In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

Background: Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis represents a liquid biopsy approach for real-time monitoring of tumor evolution. DNA methylation is considered to be an early event in the process of cancer development and progression. The aim of the present study was to evaluate whether detection of DNA methylation of selected tumour suppressor genes in size-based CTC fraction and matched ctDNA could provide prognostic information in early stage NSCLC. Methods: The methylation status of five selected gene promoters (APC, RASSFIA1, FOXA1, SLFN11, SHOX2) was examined by highly specific and sensitive real time methylation specific PCR assays in: a) a training group of 35 primary tumours and their corresponding adjacent non-cancerous tissues of early stage NSCLC patients, b) a validation group of 22 primary FFPEs tumor tissues and 42 peripheral blood samples of early stage NSCLC patients that gDNA was isolated from FFPEs, sized-based CTCs and plasma, and c) a control group of healthy blood donors (n=12). Results: All five gene promoters tested were highly methylated in the training group of primary tumours; methylation of SHOX2 promoter in the primary tissues was associated with unfavorable outcome. RASSFIA and APC were found methylated in plasma-cfDNA samples at 14.3% and 11.9% respectively, whereas in the corresponding size-based CTC-enriched fractions SLFN11 and APC promoters were methylated in 7.1%. The incidence of relapse was higher in patients with i) promoter methylation of APC and SLFN11 in plasma-cfDNA (P=0.037 and P=0.042 respectively) and ii) at least one gene promoter was methylated in the sized-based CTC fraction or plasma-cfDNA. Conclusions: Although in liquid biopsy components methylation of these gene promoters was significantly lower than in the primary tumors, the combination of DNA methylation analysis in sized-based CTC fraction and plasma-cfDNA revealed clinical relevance. Additional studies are required to validate our findings in a large cohort of early stage NSCLC patients. Citation Format: Athina N. Markou, Dora Londra, Victoria Tserpeli, John Kollias, Emilia Tsaroucha, Ioannis Pateras, K Potaris, Ioannis Vamvakaris, Athanasios Kotsakis, Vasilis Georgoulias, Evi Lianidou. Combined DNA methylation analysis in size based CTC fraction and matched plasma-cfDNA provides prognostic information in early stage NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3384.

Background In metastatic breast cancer (MBC) the molecular characterization of Circulating Tumor Cells (CTCs) provides a unique tool to understand metastasis-biology and therapy-resistance. We evaluated the prognostic significance of gene expression in EpCAM(+) CTCs in 46 MBC patients based on a long follow-up. Methods We selected a panel consisting of stem cell markers (CD24, CD44, ALDH1), the mesenchymal marker TWIST1, receptors (ESR1, PGR, HER2, EGFR) and the epithelial marker CK-19. Singleplex RT-qPCR was used for TWIST1 and CK-19 and multiplex RT-qPCR for a) CD24, CD44, ALDH1, HPRT and b) ESR1, PR, HER2, EGFR. A group of 19 healthy donors (HD) was used as control. Results Univariate (p=0.001) and multivariate analysis (p=0.002) revealed the prognostic value of combined gene expression of CK-19(+), CD44high/CD24-/low, ALDH1high/CD24-/low and HER2 over-expression for overall survival (OS). The Kaplan–Meier estimates of OS were significantly different in patients positive for CK-19 (p = 0.028), CD44high/CD24-/low (p = 0.002), ALDH1high/CD24-/low (p = 0.007) and HER2-positive (p = 0.022). Conclusions Our results indicate that combined gene expression analysis in EpCAM(+) CTCs provides prognostic information in MBC but need to be further confirmed in a prospective study, including a larger and well-defined patient cohort.

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.

Real-world evidence regarding the prevalence of epidermal growth factor receptor (EGFR) mutation-positive status (M+) and the clinicopathological characteristics associated with the presence of EGFR mutations in advanced non-small cell lung cancer (NSCLC) is scarce, especially among Caucasian populations. The present study aimed to bridge this gap, as well as to record treatment patterns and outcomes in routine-care settings.REASON (NCT01153399) was a prospective study of patients with stage IIIB/IV NSCLC and known EGFR mutation status. Clinicopathological, treatment characteristics and clinical outcomes were recorded and correlated with EGFR mutation testing results.Of 575 enrolled patients, EGFR mutations were detected in 15.7% of them. Male gender (p=0.008) and smoking (p0.001), but not adenocarcinoma, were associated with EGFR M+ status. In the EGFR M+ subpopulation (n=88), absence of bone and/or brain metastasis and presence of exon 19 EGFR M+ status at diagnosis were independently associated with longer progression-free survival (PFS) (p=0.011 and p=0.040, respectively).In our population, males and smokers had decreased odds of harboring an EGFR mutation, while adenocarcinoma histology was not a significant predictor of EGFR M+ status. EGFR M+ patients with bone and/or brain metastases at diagnosis or mutations other than exon 19 deletions were at increased risk for earlier disease progression.

Introduction To assess the smoking behaviour of Lung Cancer (LC) and Colorectal (CRC) patients at the time of diagnosis and one year after diagnosis. Material and Methods The study took place in the island of Crete, Greece. Data (LC patients=4,421; CRC patients=3,609) were derived from the Cancer Registry of Crete (1992-2010). Participants with I-III stage primary cancer and confirmed diagnosis (histologically/cytologically) were included. Patients with unknown stage were excluded after testing for potential statistical bias. Kruskal-Wallis (one-way analyses of variance) and logistic regression models estimated the risk of not quitting smoking after diagnosis. All tests were two-tailed (a=0.05). Results Overall, 75.1% of LC and 51.4% of CRC patients were current smokers at the time of diagnosis. One year after diagnosis 49.5%% of the LC and 38.1% of the CRC patients were still smoking. Males, public insurance, high number of pack/years, not receiving chemotherapy/radiotherapy, not undergoing surgery, higher number of co-morbidities, advanced cancer stage (III) and alcohol consumption were significant predictors of not quitting smoking one year after diagnosis (p

Introduction Τo provide insights on Lung Cancer (LC) and the associated risk factors before-after the economic crisis in Crete, Greece. To assess the smoking habits of LC patients during the austerity period. Material and Methods The study was conducted in Crete, Greece. Data (5,057 LC cases) were obtained from the Cancer Registry of Crete (CRC). Age-Standardized Incidence and Mortality Rates (ASIR, ASMR/100,000/year), adjusted Charlson’s Comorbidity Index (CCI%), deprivation index (HPI-2) and exposure to Outdoor Air Pollution (OAP) were estimated. The analysis was performed for two time periods (Period A: 1992-2008; Period B: 2009-2013). Results ASIR presented a significant increase during the economic crisis, while even higher increase was observed in ASMR (Period A: ASMR=30.5/100,000/year; Period B: ASMR=43.8/100,000/year; p

Introduction To identify the optimum geographic locations of establishing smoking cessation services for cancer patients in Crete. Material and Methods Data (1992-2013) for selected tobacco induced cancers (lung, oral cavity and pharynx, nasal cavity and paranasal sinus, larynx, esophagus, stomach, pancreas, kidney, liver, bladder, uterus, cervix, colon/rectum, ovary and leukemia) were obtained from the Cancer Registry of Crete (CRC). Age-Adjusted Incidence Rates (AAIR) and Smoking Attributable Fraction (SAF%) were estimated. Smoking rates were estimated by age group and other selected socio-economic and clinical variables. All rates were mapped and analyzed in the ArcGIS 10.3.1. The Getis ord statistic, K-Means (a=0.05) and multi-criteria model builder were performed. Results The AAIR for tobacco-related cancers was 160 new cases/100,000/year (AAIRmales=222.1/100,000/year; AAIRfemales=98.7/100,000/year). Larynx (SAF=71.4%), esophagus (SAF=42.4%), lung (SAF=41.9%), oral cavity (SAF=38.7%) and bladder (SAF=36.5%) presented the highest SAFs in all the municipalities of Crete. Significant variations were observed in the geographical distribution of all estimated rates (Pvalue

INTRODUCTION Detection of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in the peripheral blood of patients with solid tumors has been widely studied for the early detection of metastatic spread. We evaluated whether there was an association between the origin of cfDNA and CTCs. We investigated whether SRY (sex determining region Y)-box 17 (SOX17) promoter methylation in CTCs was associated with the methylation pattern of this gene in matched cfDNA isolated from plasma of patients with breast cancer. METHODS We examined SOX17 methylation in 79 primary breast tumors, in 114 paired samples of DNA isolated from CTCs and cfDNA, and in 60 healthy individuals. Isolated DNA was modified by sodium bisulfite and subjected to methylation specific PCR. RESULTS The SOX17 promoter was methylated in 68 (86.0%) of 79 of primary breast tumors. In CTCs, SOX17 was methylated in 19 (34.5%) of 55 patients with early breast cancer, 27 (45.8%) of 59 patients with metastatic cancer, and 1 (4.3%) of 23 healthy individuals, whereas in matched cfDNA SOX17 was methylated in 19 (34.5%) of 55, 24 (40.7%) of 59, and 1 (2.0%) of 49 of these same groups, respectively. There was a significant correlation between SOX17 methylation in cfDNA and CTCs in patients with early breast cancer (P = 0.008), but not in patients with verified metastasis (P = 0.283). CONCLUSIONS The SOX17 promoter is highly methylated in primary breast tumors, in CTCs isolated from patients with breast cancer, and in corresponding cfDNA samples. Our findings indicate a direct connection between the presence of CTCs and cfDNA in patients with operable breast cancer, after surgical removal of the primary tumor.

Myeloid-derived suppressor cells (MDSC) present in the human peripheral blood, represent a heterogeneous population of cells with monocytic and granulocytic features. To provide guidelines for reliable assessments of the frequency and function of MDSC, we compared fresh vs. cryopreserved peripheral blood mononuclear cell (PBMC) samples obtained from normal controls and patients with cancer. PBMC were obtained from 4 healthy donors and 21 patients with cancer. They were stained with labeled antibodies, and the frequency of DR⁻/LIN⁻/CD11b+, DR⁻/LIN⁻/CD15+, DR⁻/LIN⁻/CD33+ and DR(-/low)/CD14+ cells was determined by flow cytometry before and after cryopreservation. CFSE-based suppressor assays were used to test inhibitory functions of MDSC. Arginase I expression and reactive oxygen species (ROS) upregulation in MDSC subsets were evaluated by flow cytometry. The DR(-/low)/CD14+ and DR⁻/LIN⁻/CD11b+ subsets of MDSC were found to be more resistant to the cryopreservation/thawing procedure compared to the DR⁻/LIN⁻/CD15+ and DR⁻/LIN⁻/CD33+ subsets. The frequency of the latter two MDSC subsets was significantly reduced after cryopreservation. All but DR⁻/LIN⁻/CD15+ cells inhibited proliferation of autologous CSFE-labeled CD4+ cells but lost suppressor activity after cryopreservation. Only DR⁻/LIN-/CD15+ cells were positive for Arginase I, but lost its expression after cryopreservation. Only fresh DR⁻/LIN⁻/CD11b+ and DR⁻/LIN⁻/CD15+ cells produced ROS after in vitro stimulation. Studies of human MDSC should be performed in fresh blood samples. If samples have to be cryopreserved, monitoring of CD11b+ and CD14+ MDSC subsets provides the most reliable results. Arginase I expression or stimulated ROS production assessed by flow cytometry are useful markers for MDSC subsets only in fresh samples.

BACKGROUND Circulating tumor cells (CTCs) are associated with prognosis in a variety of human cancers and have been proposed as a liquid biopsy for follow-up examinations. We show that tumor suppressor and metastasis suppressor genes are epigenetically silenced in CTCs isolated from peripheral blood of breast cancer patients. METHODS We obtained peripheral blood from 56 patients with operable breast cancer, 27 patients with verified metastasis, and 23 healthy individuals. We tested DNA extracted from the EpCAM-positive immunomagnetically selected CTC fraction for the presence of methylated and unmethylated CST6, BRMS1, and SOX17 promoter sequences by methylation-specific PCR (MSP). All samples were checked for KRT19 (keratin 19, formerly CK-19) expression by reverse-transcription quantitative PCR. RESULTS In CTCs of patients with operable breast cancer, promoter methylation of CST6 was observed in 17.9%, BRMS1 in 32.1%, and SOX17 in 53.6% of patients. In CTCs of patients with verified metastasis, promoter methylation of CST6 was observed in 37.0%, BRMS1 in 44.4%, and SOX17 in 74.1%. In healthy individuals, promoter methylation of CST6 was observed in 4.3%, BRMS1 in 8.7%, and SOX17 in 4.3%. DNA methylation of these genes for both operable and metastatic breast cancer was significantly different from that of the control population. CONCLUSIONS DNA methylation of tumor suppressor and metastasis suppressor genes is a hallmark of CTCs and confirms their heterogeneity. Our findings add a new dimension to the molecular characterization of CTCs and may underlie the acquisition of malignant properties, including their stem-like phenotype.

Background: Patients with EGFRmt non-small cell lung cancer (NSCLC) develop resistance to first (erlotinib/ gefitinib) and second (afatinib) generation EGFR tyrosine kinase inhibitors (EGFR TKIs). Circulating tumor cells (CTCs) may be used as a potential biological material to monitor genetic alterations during the clinical course of the disease. We used the ParsortixTM (ANGLE) system, a label-independent microfluidic-based technology platform, to enrich CTCs from whole blood of NSCLC patients to evaluate the presence of HER-2 and c-MET amplification using fluorescence in situ hybridization (FISH). Materials and methods: Peripheral blood (PB) (15mL) from 42 patients with EGFRmt and resistant to 1st and 2nd generation EGFR TKIs NSCLC was obtained before, during and after treatment with osimertinib. CTCs were isolated using the ParsortixTM system and were further analyzed for HER-2 and c-MET amplification by FISH. Results: PB samples were obtained before (n=42), after one cycle (n=12) and at PD (n=7) during treatment with Osimertinib. One patient had HER-2 amplified CTCs whereas in total, 5 patients had marginally HER-2-positive CTCs; HER-2-positive CTCs were observed in 1/42 baseline sample but in none post-1st cycle and PD samples. Similarly, c-MET amplification was detected in 3/42 patients at baseline whereas in total 4 patients had marginally c-MET-positive CTCs. Treatment with Osimertinib resulted in a decrease of the number of c-MET amplified CTCs in these patients. In all c-MET and HER-2 amplified CTCs there was no HER-2 or c-MET amplification in the primary tumor. c-MET amplification in the primary tumor was detected only in one patient, however this patient had no detectable CTCs. Conclusions: Our preliminary results indicate that in selected patients with EGFRmt NSCLC, detection of HER-2 or c-MET amplified CTCs may be correlated with tumor resistance to EGFR TKIs. The ParsortixTM system can be successfully combined with downstream FISH analysis for the molecular characterization of CTCs in NSCLC patients. More samples are currently being analyzed in order to reveal the implication of HER-2 and c-MET amplification in resistance to therapy with first, second and third generation EGFR TKIs. Citation Format: Aliki Ntzifa, Martha Zavridou, Pavlos Pollakis, Georgia Bardi, Vasilis Georgoulias, Athanasios Kotsakis, Evi Lianidou. Combination of a label independent CTC enrichment platform with fluorescence in situ hybridization for the molecular characterization of CTCs in non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4576.

AIMS: Molecular characterization of Circulating Tumor Cells (CTCs) and analysis of circulating tumor DNA (ctDNA) in cancer patients holds promise as an extremely powerful and reliable non-invasive clinical tool for the individual molecular profiling of each patient in real time. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is implicated in human cancers, and somatic mutations in the p110α catalytic subunit of PI3K, are very frequent and play a crucial role in response to molecular targeted therapies in breast cancer. In this study, we analyzed PIK3CA hotspot mutations (1633 G>A, 3140 A>G) in CTCs and corresponding ctDNA of early and metastatic breast cancer patients. We also examined whether there is a correlation between the presence of PIK3CA mutations in CTCs and ctDNA. METHODS: We used our highly sensitive methodology for the detection of PIK3CA hotspot mutations in exons 9 (1633 G>A) and 20 (3140 A>G), based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis (HRMA) (Markou et al, CCR 2014). We analyzed PIK3CA hotspot mutations in the EpCAM-positive CTCs fraction and the corresponding plasma samples of: i) a group of 19 patients with operable breast cancer, ii) a group of 40 breast cancer patients with verified metastasis and iii) 30 healthy female volunteers. All ctDNA samples (extracted from 2 ml plasma) were examined for their DNA quality; to verify DNA quality, primers specific for the wild type in exactly the same PIK3CA gene region for exon 9 were used to assess for hotspots mutations. The mutation status of PIK3CA gene in ctDNA samples was detected by the developed methodology exactly as previously described. All samples were analyzed in triplicate. RESULTS: The assay is highly sensitive as it can detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons (9 and 20) and highly specific (0/30 healthy donors). PIK3CA hotspot mutations were identified in ctDNA in 12/40 (30%) of metastasis-verified breast cancer patients and 4/19 (21%) of operable breast cancer patients. In metastasis-verified breast cancer patients, the concordance between EpCAM- positive CTCs and ctDNA for 1633 G>A mutation was 72.5%, whereas the corresponding concordance for the 3140 A>G mutation was 97.5%. In operable breast cancer patients, the concordance between EpCAM-positive CTCs and ctDNA for the 1633 G>A mutation was 80%, whereas the corresponding concordance for the 3140 A>G mutation was 100%. CONCLUSIONS: Detection of PIK3CA hotspot mutations in EpCAM-positive CTCs and corresponding ctDNA has shown a correlation both in metastasis-verified and operable breast cancer patients. We will further evaluate our findings in a large cohort of patients before and after treatment, to evaluate response to molecular targeted therapies in breast cancer. Citation Format: Elena Tzanikou, Athina Markou, Nikos Malamos, Vasilis Georgoulias, Evi S. Lianidou. PIK3CA mutational status in circulating tumor cells (CTCs) and corresponding circulating tumor DNA in breast cancer patients. [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 497.

Introduction: In the early stages of cancer, the chance to detect rare CTCs is increasing by increasing the sample volume. The aim of our study was to evaluate the diagnostic sensitivity of a novel clinical device for the in-vivo isolation of EpCAM-positive CTCs (CellCollectorTM, GILUPI, GmBH), by using highly sensitive RT-qPCR molecular assays. Patients and methods: 29 breast cancer patients without overt metastases before the beginning of adjuvant chemotherapy (M0), 26 breast cancer patients with overt metastases before starting of therapy (M1) and 12/26 of them before the second cycle of therapy (M2), as well as 18 healthy donors participated in the study. After in-vivo isolation, total RNA was extracted from captured cells, lysed in Trizol, followed by cDNA synthesis. RT-qPCR was used for the molecular characterization of captured cells, for: CK-19, HER-2, TWIST1, VEGF, ER, PR, EGFR, CD44, CD24, and ALDH1, while B2M was used as a reference gene. Peripheral blood was also collected for CTC analysis by the FDA cleared CellSearchTM system. In addition, immunofluorescence staining of cytospins was performed and screened for CTCs using the ARIOL system, using ER, HER2, CK (8, 18, 19) and CD45 for CTC identification. Results: Results are shown in Table 1. At least one gene was expressed in 10(34.5%) of M0, 15(57.7%) of M1 and 4(33.3%) of M2 patient groups, but in none of healthy donors 0/18(0%). CellSearchTM gave positive results in 5(17.2%) of M0, 10(38.5%) of M1 and 0(0%) of M2. Immunofluorescence (Ariol system) was positive for ER, HER2, CK (8, 18, 19) in 5/15(33.3%) M0, in 4/12(33.3%) M1 and in 1/7(14.3%) M2 groups. Table 1.Gene expression in CTCHealthy N = 18M0 N = 29M1 N = 26M2 N = 12CK-190 (0%)6(20.7%)6 (23.1%)2 (16.7%)HER20 (0%)2 (6.9%)0 (0%)0 (0%)ER0 (0%)2 (6.9%)0 (0%)0 (0%)PR0 (0%)0 (0%)0 (0%)0 (0%)EGFR0 (0%)0 (0%)0 (0%)0 (0%)TWIST10 (0%)1 (3.4%)0 (0%)2 (16.7%)VEGF0 (0%)3 (10.3%)5 (19.2%)1 (8.3%)CD44+/CD24−,0 (0%)4 (13.8%)3 (11.5%)1 (8.3%)ALDH1high/CD24−,0 (0%)2 (6.9%)8 (30.8%)1(8.3%) Conclusions: In-vivo isolation of CTC is minimally invasive, and in combination with high specific and sensitive RT-qPCR assays for CTC detection and molecular characterization seems promising. Comparison studies with the CellSearch and immunofluorescence have shown poor agreement. These results should be validated in large patient cohorts, and in respect to the clinical outcome. Citation Format: Areti D. Strati, Martha Zavridou, Galateia Kallergi, Eleni Politaki, Tobias Gorges, Andra Kuske, Anna-Lena Bohnen, George Koutsodontis, Amanda Psyrri, Klaus Lucke, Vasilis Georgoulias, Klaus Pantel, Evi Lianidou. Molecular characterization of in vivo isolated EpCAM-positive circulating tumor cells in 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 503.

AIMS: DNA methylation is an epigenetic alteration which plays a decisive role in the regulation of signal translation processes. In our lab, we have demonstrated for the first time the epigenetic silencing of tumor and metastasis suppressor genes in CTCs through their promoter methylation. Estrogen receptor (ER) is an important prognostic biomarker and is predictive of response to endocrine therapy in breast cancer. In this study, we evaluated for the first time ESR1 methylation in CTCs of breast cancer patients. METHODS: We developed and validated a novel highly sensitive and specific qMSP assay for ESR1 methylation using commercially available DNA methylation controls and the MDA-MB-231 cell line. We further examined its performance in EpCAM-positive immune-magnetically isolated CTC fractions, followed by DNA isolation and sodium bisulfite (SB) treatment from: a) 74 operable, b) 48 metastasis- verified breast cancer patients and c) 30 healthy donors (control group). RESULTS: The developed assay is highly specific and sensitive since it can detect 0.1% methylated ESR1 sequences in the presence of 99.9% un-methylated. ESR1 was found to be methylated in 16/74 (21.6%) operable breast cancer patients, in 10/48 (20.8%) patients with verified metastasis, but only in 1/30 (3.3%) healthy donors (EpCAM-positive CTC fraction). CONCLUSIONS: The EpCAM-positive CTC fraction was found to be methylated for ESR1 in about 20% of patients with breast cancer. We will further evaluate these findings in respect to the clinical outcome of these patients, since the epigenetic silencing of ESR1 could be of important clinical significance especially for its impact on the efficacy of treatment. Citation Format: Sofia Mastoraki, Areti Strati, Maria Chimonidou, Nikos S. Malamos, Vasilis Georgoulias, Evi S. Lianidou. ESR1 methylation in circulating tumor cells of 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 499.

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.

Background CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation. Methods We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable. Results The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays. Conclusions The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.

Background Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited. Methods We quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer. Results RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT α+β+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT α+β+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch. Conclusions Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.

BACKGROUND Molecular characterization of circulating tumor cells (CTCs) is crucial to identify novel diagnostic and therapeutic targets for individualized therapies. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in CTCs. METHODS mRNA isolated from immunomagnetically enriched CTCs was subjected to multiplex PCR for KRT19 (keratin 19; also known as CK19), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2], SCGB2A2 (secretoglobin, family 2A, member 2; also known as MGB1, mammaglobin A), MAGEA3 (melanoma antigen family A, 3), TWIST-1 [twist homolog 1 (Drosophila)], and HMBS (hydroxymethylbilane synthase; also known as PBGD). Biotinylated amplicons were hybridized against fluorescent microspheres carrying gene-specific capture probes and incubated with streptavidin–phycoerythrin. We quantified the captured labeled amplicons and decoded the beads by Luminex flow cytometry. The assay was validated for limit of detection, specificity, and comparison with reverse-transcription quantitative PCR (RT-qPCR), and its clinical performance was evaluated in 64 patients with operable breast cancer, 20 patients with metastasis, and 17 healthy individuals. RESULTS The assay was specific for each gene in complex multiplexed formats and could detect the expression of each gene at the level of a single SK-BR-3 cell. The assay produced results comparable to those for RT-qPCR for each gene. None of the genes tested was detected in the CTC fraction of healthy donors. We detected KRT19, ERBB2, MAGEA3, SCGB2A2, and TWIST1 in 26.6%, 12.5%, 18.7%, 10.9%, and 31.2% of operable breast cancer patients, respectively, and detected the corresponding genes in 65%, 20%, 30%, 20%, and 20% of patients with verified metastasis, respectively. CONCLUSIONS The expression of 6 genes in CTCs can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis.

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

Introduction: The aim of our study was to evaluate the prognostic significance of our previously developed EpCAM independent RT-qPCR assay for CK-19 mRNA (Stathopoulou et al, Int J Cancer 2006) and validate its analytical and clinical performance in early breast cancer. Methods: Quality control on analytical sensitivity and specificity, linearity, intra- and inter-assay reproducibility, and stability of the external standard used for the preparation of the calibration curves was performed. Reproducibility of the assay between our labs was evaluated by analyzing 26 cDNAs. The prognostic significance of the assay in respect to DFI and OS was evaluated by analysing peripheral blood of 179 patients with stage I/II breast cancer postoperatively, before the administration of adjuvant chemotherapy. Results: The limit of detection (LOD) is 3 copies/reaction and limit of Quantitation (LOQ) 10 copies/reaction. The linear range of the calibration curve was 10-105copies/reaction and the intra- and inter-assay reproducibility are shown below: CK19 transcriptsReproducibility of the assay (n = 3)copies/reactionIntra-assay RSD (%)Inter-assay RSD (%)101.973.151020.932.241030.450.861040.122.581050.11.52 The inter-lab reproducibility of the assay was evaluated by analyzing 26 cDNA samples in both labs and there was a 100% concordance. During the follow up period (8 years) 32/179 (17.9%) patients relapsed and 18/179 (10%) patients died from the disease. 45/179 (25.1%) samples were found positive for CK-19 and 134/179 (74.9%) negative. In the group of CK-19 positive patients 15/45(33.3%) relapsed and 9/45(20.0%) died while in the group of CK-19 negative patients 17(12.7%) patients relapsed and 9(6.7%) died. Kaplan-Meier analysis showed that CK-19 mRNA positivity was significantly associated with DFI (P = 0.014) and OS (P = 0.051). Conclusions: This EpCAM independent assay can be used for a high-throughput detection of CTCs in peripheral blood and has prognostic significance in early breast cancer. Citation Format: Areti Strati, Athina Markou, Aliki Stathopoulou, Stella Apostolaki, Dimitris Mavroudis, Vasilis Georgoulias, Evi S. Lianidou. Analytical and clinical validation of an EpCAM-independent assay for CTC detection in peripheral blood of early breast cancer patients based on Cytokeratin-19 (CK-19) RT-qPCR. [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 373. doi:10.1158/1538-7445.AM2015-373

Introduction: The discovery of novel non-invasive tumor biomarkers for early diagnosis, prognosis and therapy monitoring remains an undeniable imperative necessity. Circulating miRNAs have recently attracted a lot of interest as novel promising tumor biomarkers. In the present study, we investigated the expression levels of three cancer-associated miRNAs in plasma and evaluated their prognostic significance in breast cancer. Materials and Methods: We examined the expression levels of miR-21, miR-146a and miR-210 in plasma samples of 134 breast cancer patients (57 patients with verified metastasis and 77 patients with operable breast cancer) and 20 healthy individuals. Circulating miRNAs were isolated from 200μL of plasma using mirVana PARIS kit and in each sample 25fmol of the synthetic cel-miR-39 were spiked as exogenous control. For the quantification of plasma miRNAs, a stem-loop cDNA approach and TaqMan real time PCR (LightCycler 2, Roche Applied Science) were used. The normalization procedure was based on a combination of cel-miR-39 as exogenous control and hsa-miR-191 as endogenous control. Relative expression of circulating miRNAs in plasma was evaluated as previously described (Sourvinou et al., J. of Mol. Diagn, 2013). Results: Our results indicate that the expression levels of all three circulating miRNAs are significantly increased in plasma both in operable and metastasis verified breast cancer patients in comparison to those of healthy individuals (p Conclusions: Our results indicate that quantification of circulating miR-21 and miR-146a in plasma are promising prognostic biomarkers in breast cancer and should be further evaluated in a large number of patients. Citation Format: Ioanna S. Sourvinou, Athina Markou, Nikolaos Malamos, Vasilis Georgoulias, Εvi S. Lianidou. Circulating miR-21, miR-146a and miR-210 levels in plasma are associated with clinical outcome in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4786. doi:10.1158/1538-7445.AM2014-4786

Background: Breast-cancer metastasis suppressor 1 (BRMS1) gene encodes for a predominantly nuclear protein that differentially regulates the expression of multiple genes, leading to suppression of metastasis without blocking orthotopic tumor growth. The aim of the present study was to evaluate for the first time the prognostic significance of BRMS1 promoter methylation in cell free DNA (cfDNA) circulating in plasma of non-small cell lung cancer (NSCLC) patients. Towards this goal, we examined the methylation status of BRMS1 promoter in NSCLC tissues, matched adjacent non-cancerous tissues and corresponding cfDNA as well as in an independent cohort of patients with advanced NSCLC and healthy individuals. Methods: BRMS1 promoter methylation was examined in 57 NSCLC tumors and adjacent non-cancerous tissues, in cfDNA isolated from 48 corresponding plasma samples, in cfDNA isolated from plasma of 74 patients with advanced NSCLC and 24 healthy individuals. Results: BRMS1 promoter was highly methylated both in operable NSCLC primary tissues (59.6%) and corresponding cfDNA (47.9%) but not in cfDNA from healthy individuals ( 0%), while it was also highly methylated in cfDNA from advanced NSCLC patients (63.5%). In operable NSCLC, Kaplan-Meier estimates were significantly different in favor of patients with non-methylated BRMS1 promoter in cfDNA, concerning both DFI (p=0.048) and OS (p=0.007). In advanced NSCLC, OS was significantly different in favour of patients with non-methylated BRMS1 promoter in their cfDNA (p=0.003). Multivariate analysis confirmed that BRMS1 promoter methylation has a statistical significant influence both on operable NSCLC patients' DFI time and OS and on advanced NSCLC patients' PFS and OS. Conclusions: BRMS1 promoter methylation in cfDNA isolated from plasma of NSCLC patients provides important prognostic information and merits to be further evaluated as a circulating tumor biomarker. Citation Format: Ioanna Balkouranidou, Maria Chimonidou, Georgia Milaki, Emily Tsaroucha, Stelios Kakolyris, Danny Welch, Vasilis Georgoulias, Evi Lianidou. Breast cancer metastasis suppressor-1 promoter methylation in cell free DNA provides prognostic information in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3826. doi:10.1158/1538-7445.AM2014-3826

Background: Circulating tumor cells (CTCs) are associated with prognosis and can be used as a liquid biopsy for repeated follow up examinations. The subpopulation of CTCs that successfully establish distant metastases share EMT and stemness features. These cells have been found to be associated with resistance to anti-cancer therapies and treatment failure. Patients and methods: Peripheral blood (20 mL in EDTA) was obtained and after density gradient centrifugation, immunomagnetic Ber-EP4 coated capture beads were used to enrich for epithelial cells from 102 patients with early breast cancer and 23 patients with verified metastasis. Messenger RNA was isolated from enriched epithelial cells using oligo (dT)25 coated magnetic beads. After cDNA synthesis the expression of CK-19, MAGE-A3, HER-2, PBGD and CD24, CD44, ALDH1, HPRT was tested, using two different quadruplex RT-qPCR assays. Moreover we performed RT-qPCR for the expression of TWIST1, hMammaglobin and hTERT α+β+. Results: The presence of circulating tumor cells was evaluated using CK-19, MAGE-A3, HER-2, hTERT α+β+, and mammaglobin gene transcripts. In 102 operable breast cancer patients 31 patients (30.4%) were found positive for the presence of CTCs. CTC biomarkers were significantly correlated with lymph nodes (P=0.032). In the CTC+ group, 10 patients (32.3%) were positive for TWIST1 and 16 (51.6%) were positive for CD44+/CD24-/low and ALDH1+ cell phenotype. We found a significant correlation between HER2 negative tumors with TWIST1 expression (P=0.027) and Stem Cell phenotype (P=0.012). The detection of both EMT and Stem Cell characteristics was associated with shorter disease-free survival (P=0.024). In the CTC positive group of patients with verified metastasis (39.1%) 1 patient (11.1%) was positive for TWIST1 and 4 (44.4%) were positive for CD44+/CD24-/low and ALDH1+ cell phenotype. The detection of CK19+, Stem Cell and Stem Cell and/or EMT phenotype was associated with shorter overall-free survival (P=0.036, P=0.001 and P=0.002). Discussion: CTCs in breast cancer patients share both stem cell and/or EMT phenotype. The detection and molecular characterization of CTCs with an EMT or stem cell-like characteristics could be a powerful diagnostic tool for the determination of prognosis and designing new therapeutic drugs for the elimination of minimal residual disease. Citation Format: Areti D. Strati, Vasilis Georgoulias, Evi Lianidou. Prognostic significance of stem cell and EMT phenotype in Circulating Tumor Cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1465. doi:10.1158/1538-7445.AM2013-1465

Introduction: Breast Cancer Metastasis Suppressor-1 (BRMS1) encodes a predominantly nuclear protein that differentially regulates expression of multiple genes, leading to suppression of metastasis without affecting orthotopic tumor growth. We examined the clinical relevance of BRMS1 promoter methylation in early breast cancer. Methods: We analyzed 118 formalin-fixed paraffin embedded samples: 5 pairs of breast tumors and adjacent non-cancerous tissues, 14 non-cancerous tissues, 10 benign fibroadenomas, and 84 primary breast tumors. Peripheral blood mononuclear cells were isolated from 39/84 of these patients and were fixed in cytospins. BRMS1 methylation status was investigated in all FFPE and cytospin stained circulating tumor cells using methylation specific PCR. BRMS1 expression was examined using double-immunofluorescence in isolated PBMC cytospins using anti-BRMS1 and pancytokeratin A45-B/B3 antibodies. Results: BRMS1 promoter methylation was observed in 0/19 noncancerous breast tissues, 0/10 benign fibroadenomas and 31/84(37.8%) breast tumors. During the follow-up period 27/84(32.1%) patients relapsed of which 19/84(22.6%) patients died as a consequence of disease progression. BRMS1 methylation was detected in 15/27(55.6%) patients that relapsed and in 10/19(52.6%) of patients that died. BRMS1 promoter methylation was associated with reduced disease-free interval (P=0.009) while a trend towards a reduced overall survival (OS) was also observed (P=0.071). Expression of BRMS1 on CTC was very heterogeneous: CTC with high BRMS1 expression were found in 7/13(53.8%) CTC positive samples while exclusively CTC with high BRMS1 were found in only 3 patients (23.0%). CTC with low BRMS1 expression were found in 3 patients. CTC negative for BRMS1 were found in 8/13(61.5%) patients. Exclusively CTC with low or negative BRMS1-expression were identified in 6/13(46.1%) patients. 4/13(30.8%) patients had both high and low or negative BRMS1 expression in their CTC. Conclusions: BRMS1 promoter methylation provides important prognostic information for disease free survival in early breast cancer patients. BRMS1 promoter methylation and protein expression were evaluated in primary tumors and corresponding CTC for the first time. BRMS1 expression in CTC was heterogeneous in individual patient samples. CTC expressing epithelial markers but no BRMS1 seem to define a worse prognosis group of patients. These results need to be further evaluated and validated in a larger cohort of breast cancer patients. Citation Format: Maria Chimonidou, Galatea Kallergi, Vasilis Georgoulias, Danny Welch, Evi Lianidou. Prognostic significance of BRMS1 promoter methylation in operable breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-283. doi:10.1158/1538-7445.AM2013-LB-283

Introduction: Molecular characterization of Circulating Tumor Cells (CTC) is a hot topic nowadays. SOX17 plays a very critical role in the regulation of development and stem/precursor cell function, at least partly through repression of Wnt pathway activity. Silencing of SOX17 due to its promoter methylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer. We have recently shown that the promoter of this gene is methylated in CTC (Chimonidou et al, Clin Chem 2011). The aim of the present study was to evaluate whether SOX17 promoter methylation in CTC, is associated with the methylation pattern of this gene in matched cell free DNA isolated from plasma of breast cancer patients. Patients and methods: We firstly developed a highly sensitive and specific Methylation Specific Real-time PCR method for SOX17. The methylation status of SOX17 was examined in paired specimens of DNA isolated from CTC and cell free DNA (cfDNA) circulating in plasma of the same individuals. 58 patients with operable breast cancer, 21 patients with verified metastasis, and 12 healthy individuals used as controls were enrolled in our study. For CTC analysis, DNA was extracted from EpCAM positive immunomagnetically isolated CTC fraction, while cfDNA was isolated from 200μL of plasma from the same individuals. DNA extracted from isolated CTC and cfDNA was modified by a Sodium Bisulfite (SB) conversion reaction and subjected to methylation specific real time PCR. Results: In CTC, promoter methylation of SOX17 was observed in 22/58 (37.9%) of patients with operable breast cancer, 12/21 (57.1%) of patients with verified metastasis and 1/12 (8.3%) of healthy individuals. In the matched plasma samples, promoter methylation of SOX17 was observed in 23/58 (39.6%) of patients with operable breast cancer, 11/21 (52.4%) of patients with verified metastasis and 1/12 (8.3%) in healthy individuals. By comparing these results we found that promoter methylation of SOX17 in CTC was significantly correlated with promoter methylation of SOX17 in cell free DNA isolated from plasma of the same patients (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2382. doi:1538-7445.AM2012-2382

Backround: Breast Cancer Metastasis Suppressor gene-1 (BRMS1) suppresses breast cancer metastasis in multiple experimental models by reducing solitary cell survival and inhibiting growth initiation. Loss of BRMS1 expression has been shown to predict reduced disease-free survival in subsets of breast cancer patients. The BRMS1 promoter region contains a promoter-associated CpG island (CGI) that encompasses the transcriptional start site.The aim of our study was to investigate the methylation status of BRMS1 in breast tumors (FFPES) of operable breast cancer patients and evaluate its prognostic significance. Patients and methods: Using methylation-specific PCR (MSP), we examined BRMS1 promoter methylation in 116 fixed paraffin-embedded tissues (FFPEs): 5 pairs of breast tumors and their surrounding non-cancerous tissues, 4 non-cancerous tissues, 10 benign breast tumors (fibroadenomas), 10 normal breast tissues and 82 breast tumors. All breast tumors were isolated from patients with operable breast cancer. Results: Methylation of BRMS1 promoter was observed in 0/19 (0%) noncancerous breast tissues, in 0/10 (0%) benign breast tumors (fibroadenomas) and in 31/82 (37.8 %) breast cancer tumors. During the follow-up period 22/82 (26.8%) patients relapsed and 20 (24.4%) patients died. BRMS1 methylation was detected in 13/22 (59.1%) patients that relapsed and in 12/20 (60.0%) of patients who died. Disease-free interval (DFI) and overall survival (OS) were significantly associated with BRMS1 promoter methylation (P=0.007). Conclusion: We show for the first time that BRMS1 promoter methylation status in FFPEs from operable breast cancer patients provides important prognostic information and merits to be further evaluated and validated in a larger cohort of patients. This finding is consistent with other studies that showed significant down-regulation of BRMS1 in some breast tumors, especially in metastatic disease, because of epigenetic silencing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3425. doi:1538-7445.AM2012-3425

Introduction: Somatic mutations in the gene encoding for the phosphatidyl-inositol 3-kinase (PI3K) catalytic subunit, PIK3CA, have been discovered in many different human cancers. We have recently developed a high-resolution melting curve analysis method for the detection of the three hotspot mutations of PIK3CA gene (Vorkas et al, J of Mol Diagn 2010). The aim of our study was to evaluate this method for the presence of PIK3CA mutations in cell-free DNA (cfDNA) circulating in plasma of breast cancer patients and healthy individuals. Patients and methods: cfDNA was isolated from plasma (200μL) of 30 patients with operable breast cancer, 45 patients with metastatic breast cancer and 12 healthy donors. After DNA extraction, real-time PCR was performed in triplicate for all samples in the LightCycler (Roche), in the presence of LC-Green Plus saturating dye (Idaho Technology Inc.). High-resolution melting curves were obtained with HR-1 High Resolution Melter (Idaho Technology Inc.). Results: We identified 15 cfDNA samples mutated out of 75 tested (20%). 3/75 (4%) cfDNA samples were mutated within the exon 9 amplified region, and 12/75 (16%) cfDNA samples were mutated within exon 20 (31.6%). In operable breast cancer 1/30 (3.3%) cfDNA sample was mutated in exon 9 and 3/30 (10%) cfDNA samples were mutated in exon 20, while in metastatic breast cancer 2/45 (4.4%) cfDNA samples wer mutated in exon 9 and 9/45 (20%) cfDNA samples were mutated in exon 20. PIK3CA mutations were not detected in any of the 12 cfDNA samples from healthy donors, used as negative controls. Conclusions: Using High-Resolution Melting curve Analysis (HRMA) PIK3CA gene mutations were detected in cfDNA circulating in plasma of patients with operable and metastatic breast cancer, but not in plasma of healthy individuals. The prognostic significance of this finding will be evaluated when a longer follow up period will be completed for these patients and a larger number of clinical samples will be analyzed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2088. doi:1538-7445.AM2012-2088

Introduction: Cystatin M or E/M (encoded by the CST6 gene) is an endogenous inhibitor of lysosomal cysteine proteases that functions to protect cells against uncontrolled proteolysis. CST6 gene is featuring a CpG island within the promoter and first exon, and is frequently methylated in cancer. We have recently shown that detection of CST6 methylation is of prognostic significance in operable breast cancer patients (Kioulafa et al, Int J Cancer, 2009). Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) enables highly sensitive, labor- and cost-efficient single locus methylation studies on the basis of DNA high-resolution melting technology. The aim of our study was to develop a novel closed-tube MS-HRMA methodology for the investigation of CST6 methylation status that is based on the combination of PCR and high-resolution melting analysis. Materials and methods: The study material consisted of a total of 117 breast formalin fixed paraffin-embedded tissues (FFPEs): 90 paraffin-embedded breast carcinomas from patients with operable breast cancer, 18 non cancerous breast tissues and 9 fibroadenomas, all obtained from the Department of Medical Oncology, University Hospital of Heraklion Crete. 1μg of extracted DNA was modified with sodium bisulfite, in order to convert all unmethylated, but not methylated-cytosines to uracil. In each sodium bisulfite conversion reaction, dH2O and MCF-7 were included as a negative and positive control, respectively. Our methodology was first optimized and evaluated by using certain mixtures of fully methylated to unmethylated bisulfite converted genomic DNA, as controls. We compared our results with Methylation-Specific PCR (MSP) for the same samples. Results: The developed methodology can detect the presence of methylation within the studied region, and additionally provides a semi-quantitative estimation of CST6 methylation level in terms of percentage. By using MS-HRMA for CST6 methylation we observed 42/90 (47.7%) of breast cancer FFPEs samples analyzed to be methylated at various methylation levels. In non cancerous tissues CST6 methylation was detected at very low levels in 2/18 (11.1%), while in benign breast tumors (fibroadenomas) CST6 methylation was observed in 1/9 (11.1%). Our results for MS-HRMA when compared to MSP for the same samples show a strong correlation (P Conclusions: The developed MS-HRMA assay for CST6 promoter methylation is highly sensitive, cost-effective, easy-to-perform, and can be used as a screening test prior to DNA sequencing for the exclusion of clearly unmethylated samples. MS-HRMA gives comparable results to MSP in less time, while its main advavantage is the ability to additionally provide an estimation of the level of methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 81. doi:10.1158/1538-7445.AM2011-81

Introduction: Circulating tumor cells (CTCs) represent an important biological link in the spread of breast cancer from primary to metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. The aim of our work was to develop a quantitative real-time multiplex PCR assay for CK-19, MAGE-A3, HER-2 and PBGD and validate its performance in CTCs of early and metastatic breast cancer patients. Materials and Methods: A tetraplex quantitative real time assay for CK-19, MAGE-A3, HER-2 and PBGD was developed based on the in-silico design of primers and four hybridization probe pairs with different fluorescence emission spectra. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. Specificity and sensitivity experiments were performed using the SKBR-3 cancer cell line as a positive control. The method was applied in 73 patients with early breast cancer before the administration of adjuvant chemotherapy, 27 patients with verified metastasis and 17 female healthy volunteers. Peripheral blood (20 mL in EDTA) was obtained and after density gradient centrifugation, immunomagnetic Ber-EP4 coated capture beads were used to enrich for epithelial cells, keeping for each sample two fractions: the CTC and corresponding PBMC fraction. Messenger RNA was isolated from enriched epithelial cells using oligo (dT)25 coated magnetic beads. After cDNA synthesis the expression of CK-19, MAGE-A3, HER-2 and PBGD was tested, in both fractions. Results: The analytical performance of the method was evaluated in SKBR-3 tumor cell line in respect to analytical sensitivity and specificity. Cross reaction studies, performed for each gene target in the presence of all other targets have shown a very high specificity for each analyte. RNA quality in all samples was evaluated by PBGD gene expression. We found 30/73 (41%) patients with early breast cancer positive for CK-19, 14/73 (19,2%) for MAGE-A3 and 11/73 positive for HER-2 (15,1%). In patients with verified metastasis we found 14/27 patients positive for CK-19 (51.8%) 4/27 for MAGE-A3 (14,8%), 5/27 patients positive for HER-2 (18,5%). In healthy individuals we found 1/17 positive for CK-19 (5,9%) while no samples were found positive for MAGE-A3 and HER-2 (0%). Conclusions: We report for the first time a highly specific, reproducible and sensitive quantitative multiplex real-time PCR assay for the simultaneous detection of CK-19, MAGE-A3, HER-2 and PBGD. The expression of these genes in CTCs will be further examined in a larger number of patients and results will be correlated with their clinical outcome. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 840.

Background Several researchers have claimed that classification of tumours on the basis of HER-2/neu overexpression or amplification may define a subset of breast cancer in which the net effect of a risk factor could be rather more obvious and its impact on breast cancer development more clear. We decided to investigate, in a group of patients from a geographical area with a low incidence of breast cancer, whether HER-2/neu positive tumours are correlated with established or suspected risk factors for breast cancer and thus to identify distinct subgroups of high risk women. Methods This study analysed data from patients who attended the Breast Unit at the University Hospital of Heraklion, Crete, Greece between 1996 and 2002. 384 women with primary invasive breast cancer were compared with 566 screened women who were referred to the Unit and had not developed breast neoplasm by the time the data were analysed. Risk factor data were obtained from each subject by personal interviews using a structured questionnaire. The detection and scoring of the HER-2/neu protein, estrogen and progesterone receptor expression were performed using immunochemistry. Odds ratios and 95% confidence intervals were determined by chi-square test and logistic regression analysis. Case-case odds ratios were calculated in order to measure the risk heterogeneity between HER-2/neu+ and HER-2/neu-tumours. Separate analyses were performed for premenopausal and postmenopausal women and according to estrogen receptor status. Results In multivariate analysis without HER-2/neu stratification, an increased breast cancer risk was associated with only four of the factors examined: use of oral contraceptives (OR = 4.40, 95%C.I: 1.46–13.28), use of HRT (OR = 7.34, 95%C.I: 2.03–26.53), an age at first full pregnancy more than 23 years (OR = 1.91, 95%C.I: 1.29–2.83) and body mass index more than 29 kg/m2 (OR = 3.13, 95%C.I: 2.02–4.84). Additionally, a history of abortion or miscarriage (OR = 0.56, 95%C.I: 0.38–0.82) was correlated with a decreased risk of breast cancer. In the case to case comparison only BMI >29 kg/m2 revealed a relative connection that was stronger with positive than with negative HER-2/neu tumours (ratio of OR's = 2.23, 95%C.I: 1.20–4.15, p = 0.011). This may indicate evidence of heterogeneity of a rather significant degree for this factor. In the ER negative group an age at first full pregnancy >23 years and a BMI >29 kg/m2 were associated with an increased risk in both HER-2/neu groups, but the association was significantly stronger for the latter factor in the positive HER-2/neu tumours (ratio of OR's = 2.46, 95%CI: 0.97–6.21). Conclusions Our study did not confirm that the established or putative hormonal breast cancer risk factors differ regarding their relations with HER-2/neu+ versus HER-2/neu-breast tumours, with the exception of increased BMI. Further innovative studies with larger sample sizes are needed to examine how the status of these potentially modifiable breast cancer risk factors interacts with biological markers such as HER-2/neu oncoprotein.