Areti Strati

Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

IntroductionThe detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.MethodsThe expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGF2), HIF-1α and phosphorylated-focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and confocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1α or pFAK antibodies, respectively.ResultspFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1α was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1α and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. Vascular endothelial growth mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1α and VEGF2.ConclusionsThe expression of pFAK, HIF-1α, VEGF and VEGF2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.

Molecular Characterization of Circulating Tumor Cells in Breast Cancer by a Liquid Bead Array Hybridization Assay

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.

DNA Methylation of Tumor Suppressor and Metastasis Suppressor Genes in Circulating Tumor Cells

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.

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

BackgroundCirculating 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.MethodsWe 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.ResultsRT-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.ConclusionsMolecular 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.

SOX17 Promoter Methylation in Circulating Tumor Cells and Matched Cell-Free DNA Isolated from Plasma of Patients with Breast Cancer

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.

Circulating tumor cells as promising novel biomarkers in solid cancers

Abstract The presence of circulating tumor cells (CTCs) in peripheral blood can serve as a “liquid biopsy” approach and has thus emerged lately as one of the hottest fields in cancer research. CTCs can be isolated from blood in a non-invasive approach, and can be used to follow patients over time since these cells can provide significant information for a better understanding of tumor biology and tumor cell dissemination. CTC molecular characterization offers the unique potential to better understand the biology of metastasis and resistance to established therapies, and analysis of these cells presents a promising field for both advanced and early-stage patients. CTC detection, enumeration, and molecular characterization are very challenging since CTCs are rare, and the amount of available sample is very limited. Since detection of CTCs has been shown to be of considerable utility in the clinical management of patients with solid cancers, various analytical systems for their isolation and detection have been developed. New areas of research are directed towards developing novel assays for single-CTC isolation and molecular characterization. The clinical significance of CTCs has been evaluated in many types of solid cancers, and the CTC enumeration test in metastatic breast, colorectal, and prostate cancer was cleared by the FDA almost a decade ago. This review is mainly focused on the clinical potential of CTCs as novel biomarkers in 10 different types of solid cancers: breast, ovarian, prostate, lung, colorectal, hepatocellular carcinoma, pancreatic, head and neck, bladder cancer and melanoma.

Molecular characterization of circulating tumor cells in breast cancer: challenges and promises for individualized cancer treatment

Blood testing using Circulating Tumor Cells (CTCs) has emerged as one of the hottest fields in cancer diagnosis. Research on CTCs present nowadays a challenge, as these cells are well defined targets for understanding tumour biology and improving cancer treatment. The presence of tumor cells in patient’s bone marrow or peripheral blood is an early indicator of metastasis and may signal tumor spread sooner than clinical symptoms appear and imaging results confirm a poor prognosis. CTC enumeration can serve as a “liquid biopsy” and an early marker to assess response to systemic therapy. Definition of biomarkers based on comprehensive characterization of CTCs has a strong potential to be translated to individualized targeted treatments and spare breast cancer patients unnecessary and ineffective therapies but also to reduce the costs for the health system and to downsize the extent and length of clinical studies. In this review, we briefly summarize recent studies on the molecular characterization of circulating tumor cells in breast cancer and discuss challenges and promises of CTCs for individualized cancer treatment.

CST6 promoter methylation in circulating cell-free DNA of breast cancer patients

OBJECTIVES We have recently shown that detection of CST6 promoter methylation in primary breast tumors can provide important prognostic information in patients with operable breast cancer and that CST6 promoter is also methylated in Circulating Tumor Cells (CTC). In this study we evaluated the presence of CST6 promoter methylation in cell-free DNA (cfDNA) circulating in plasma of breast cancer patients. DESIGN AND METHODS Our study material consisted of: a) a pilot testing group of 27 patients with stage I-III operable breast cancer, 46 patients with verified metastasis and 37 healthy donors and b) an independent cohort of 123 consecutive stage I-III operable breast cancer patients. Methylated and unmethylated CST6 promoter sequences were detected by using methylation-specific PCR (MSP). CST6 immunohistochemical detection was performed in 20 corresponding primary tumor tissues. RESULTS In the pilot testing group, CST6 promoter was methylated in 8/27 (29.6%) operable breast cancer patients, in 6/46 (13.0%) patients with verified metastasis but none of 37 healthy individuals (0%). In the independent cohort, 49/123 (39.8%) operable breast cancer patients were found positive. During the follow up period, 25/123 (20.3%) patients relapsed and 9/123 (7.3%) died. CST6 was methylated in cfDNA of 13/25 (52%) patients that relapsed and in 3/9 (33.3%) patients that died. CONCLUSIONS CST6 promoter is highly methylated in cfDNA of breast cancer patients, but not in healthy individuals. CST6 promoter methylation in cfDNA, should be prospectively validated as a novel plasma tumor biomarker for breast cancer in a large cohort of breast cancer patients.

Comparison of three molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer

IntroductionComparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice.MethodsWe compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin.ResultsIn early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%.ConclusionsAll CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies.

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.