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PDF file - 46K, Characterization of apoptotic and proliferative CTCs prior to and after adjuvant treatment.

The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (polycaspase kit: 8.1% vs. 47.4% of the total CTC number; P = 0.0001; M30-antibody: 32.1% vs. 76.63%; P = 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics. Mol Cancer Ther; 12(9); 1886–95. ©2013 AACR.

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PDF 111KB, Oligonucleotide sequences of BRMS1 primers used in this study.

Supplementary Figures S1-S2 from Prognostic Value of the Molecular Detection of Circulating Tumor Cells Using a Multimarker Reverse Transcription-PCR Assay for Cytokeratin 19, Mammaglobin A, and HER2 in Early Breast Cancer

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. There are few targeted therapies for these patients, leading to an unmet need for new biomarkers. The present study aimed to investigate the expression of PD-L1, CTLA-4, GLU, and VIM in CTCs of TNBC patients. Ninety-five patients were enrolled in this study: sixty-four TNBC and thirty-one luminal. Of these patients, 60 were in the early stage, while 35 had metastatic disease. Protein expression was identified by immunofluorescence staining experiments and VyCAP analysis. All the examined proteins were upregulated in TNBC patients. The expression of the GLU+VIM+CK+ phenotype was higher (50%) in metastatic TNBC compared to early TNBC patients (17%) (p = 0.005). Among all the BC patients, a significant correlation was found between PD-L1+CD45−CK+ and CTLA-4+CD45−CK+ phenotypes (Spearman test, p = 0.024), implying an important role of dual inhibition in BC. Finally, the phenotypes GLU+VIM+CK+ and PD-L1+CD45−CK+ were associated with shorter OS in TNBC patients (OS: log-rank p = 0.048, HR = 2.9, OS: log-rank p < 0.001, HR = 8.7, respectively). Thus, PD-L1, CTLA-4, GLU, and VIM constitute significant biomarkers in TNBC associated with patients’ outcome, providing new therapeutic targets for this difficult breast cancer subtype.

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.

Detection of CTCs represents a poor prognostic factor in patients with early and metastatic breast cancer (mBC) and treatment with everolimus–exemestane (E/E) is an established effective treatment in hormone receptor-positive/HER2-negative mBC patients. The effect of E/E on CTCs in mBC patients was prospectively investigated. CTCs from 50 pre-treated patients with mBC receiving E/E were analyzed using the CellSearch (CS) platform and triple immunofluorescence (IF) staining for cytokeratin, M30 and Ki67 expression to assess their proliferative and apoptotic status. CTCs (by CS) were detected in 64% of patients before treatment and E/E administration resulted in their decreased prevalence [(n = 18; 36%, p = 0.004) and (n = 7; 19.4%, p = 0.019) post-1st and post-3rd treatment cycle, respectively] whereas it was significantly increased at disease progression (PD: 61%) compared to post-1st and post-3rd cycle (p = 0.049 and p = 0.021, respectively). Ki67-positive CTCs were detected in 60%, 60%, 17% and 50% of patients before treatment, post-1st, post-3rd cycle and at PD, respectively, while the opposite was observed for M30-positive CTCs (0% at baseline, 10% after the 1st cycle, 50% after the 3rd cycle and 0% at PD). The detection of even ≥ 1 CTC/5 ml after one cycle was associated with decreased PFS (3.3 vs 9.0 months, p = 0.025) whereas the detection of even ≥ 2 CTCs at PD was associated with decreased OS (32.4 vs 19.5 months; p = 0.009). The combination of E/E resulted in early elimination of proliferating CTCs in mBC patients and this effect was associated with a favorable clinical outcome.

BRCA1 and PARP are involved in DNA damage repair pathways. BRCA1 mutations have been linked to higher likelihood of triple negative breast cancer (TNBC). The aim of the study was to determine PARP-1 expression and BRCA1 mutations in circulating tumor cells (CTCs) of BC patients. Fifty patients were enrolled: 23 luminal and 27 TNBC. PARP expression in CTCs was identified by immunofluorescence. Genotyping was performed by PCR-Sanger sequencing in the same samples. PARP-1 expression was higher in luminal (61%) and early BC (54%), compared to TNBC (41%) and metastatic (33%) patients. In addition, PARP-1 distribution was mostly cytoplasmic in luminal patients (p = 0.024), whereas it was mostly nuclear in TNBC patients. In cytokeratin (CK)-positive patients, those with the CK+PARP+ phenotype had longer overall survival (OS, log-rank p = 0.046). Overall, nine mutations were detected; M1 and M2 were completely new and M4, M7 and M8 were characterized as pathogenic. M7 and M8 were predominantly found in metastatic TNBC patients (p = 0.014 and p = 0.002). Thus, PARP-1 expression and increased mutagenic burden in TNBC patients’ CTCs, could be used as an indicator to stratify patients regarding therapeutic approaches.

Introduction: Liquid biopsy is a useful tool for monitoring treatment outcome in solid tumors, including lung cancer. The relevance of monitoring CTCs and plasma ctDNA as predictors of clinical outcome was assessed in EGFR-mutant NSCLC patients treated with osimertinib. Methods: Forty-seven EGFR-mutant NSCLC patients who had progressed on prior first- or second-generation EGFR inhibitors were enrolled in the study and treated with osimertinib, irrespective of the presence of the T790M mutation in the primary tumor or the plasma. Peripheral blood was collected at baseline (n = 47), post-Cycle 1 (n = 47), and at the end of treatment (EOT; n = 39). CTCs were evaluated in 32 patients at the same time points (n = 32, n = 27, and n = 21, respectively) and phenotypic characterization was performed using triple immunofluorescence staining (CK/VIM/CD45). Results: Osimertinib resulted in an ORR of 34% (2 CR) and a DCR of 76.6%. The median PFS and OS values were 7.5 (range, 0.8–52.8) and 15.1 (range, 2.1–52.8) months, respectively. ctDNA was detected in 61.7%, 27.7%, and 61.5% of patients at baseline, post-Cycle 1, and EOT, respectively. CTCs (CK+/CD45-) were detected in 68.8%, 48.1%, and 61.9% of patients at the three time points, respectively. CTCs expressing both epithelial and mesenchymal markers (CK+/VIM+/CD45-) were detected in 56.3% and 29.6% of patients at baseline and post-Cycle 1, respectively. The detection of ctDNA at baseline and post-Cycle 1 was associated with shorter PFS and OS, whereas the ctDNA clearance post-Cycle 1 resulted in a significantly longer PFS and OS. Multivariate analysis revealed that male sex and the detection of ctDNA at baseline were independent predictors of shorter PFS (HR: 2.6, 95% C.I.: 1.2–5.5, p = 0.015 and HR: 3.0, 95% C.I.: 1.3–6.9; p = 0.009, respectively). Conclusions: The decrease in both CTCs and ctDNA occurring early during osimertinib treatment is predictive of better outcome, implying that liquid biopsy monitoring may be a valuable tool for the assessment of treatment efficacy.

Recent studies revealed that the histone demethylase KDM2B regulates the epithelial markers E-Cadherin and ZO-1, the RhoA/B/C-small-GTPases and actin cytoskeleton organization, in DU-145 prostate- and HCT-116 colon-tumor cells. Here we addressed the role of KDM2B in the activation of Focal Adhesion Kinase (FAK)-signaling and its involvement in regulating tumor cell motility. We used RT-PCR for gene transcriptional analysis, Western blotting for the assessment of protein expression and activity and wound-healing assay for the study of cell migration. KDM2B overexpression or silencing controls the activity of FAK in DU-145 prostate- and HCT-116 colon-tumor cells without affecting gene transcription and protein expression of this kinase. Upon KDM2B overexpression in DU-145 cells, significantly enhanced migration was observed, which was abolished in cells pretreated by the specific phosphoinositide-3 kinase (PI3 K) inhibitor LY294002, implying involvement of FAK/PI3 K signaling in the migration process. In line with this, the p85-PI3 K-subunit was downregulated upon knockdown of KDM2B in DU-145 cells, while the opposite effect became evident in KDM2B-overexpressing cells. These results revealed a novel functional role of KDM2B in regulating the activation of the FAK/PI3 K signaling in prostate cancer cells that participates in the control of cell motility.

Detection and characterization of circulating tumor cells (CTCs) with an epithelial-to-mesenchymal transition (EMT) phenotype is very important, as it can contribute to the identification of high-risk for relapse and death patients. However, most methods underestimate CTC numbers, owing to their dependence on epithelial markers. In the current study, we evaluated the EMT phenotype in CTCs isolated from breast cancer (BC) patients, using the CellSearch system. Spiking experiments for the evaluation of the specificity and sensitivity of our method were performed using HeLa cells. Sixty-five breast cancer (BC) patients (47 early and 18 metastatic) were enrolled in the study. Vimentin is a mesenchymal marker that indicates tumoral cells acquiring invasive and malignant properties. We studied vimentin (VIM) expression using the extra channel of the CellSearch system and an anti-vimentin antibody conjugated with FITC. In our present results, we reported the percentage of circulating tumor cells that expressed vimentin in early and in metastatic breast cancer patients. Interestingly, the incidence of cells with a CK-VIM+CD45- phenotype was detected in both settings. These cells were detected in 31.4% of CK-negative (11/35) and 82.3% of CK-positive (10/12) early BC patients. The corresponding numbers for metastatic disease were 15.4% (2/13) and 100% (5/5), respectively. Our results suggest that in CTC-negative patients, potentially undetectable tumor cells could be identified using the FDA-approved CellSearch system, based on the (CK-VIM+CD45-)-phenotype, offering additional information regarding metastatic dissemination in cancer patients. Further experiments evaluating more biomarkers are necessary to elucidate the mechanisms that regulate tumorigenesis and metastasis.

In the present study, we evaluated the expression of JUNB and CXCR4 in circulating tumor cells (CTCs) of lung cancer patients and investigated whether these proteins have prognostic clinical relevance. Peripheral blood from 30 patients with non-small-cell lung cancer (NSCLC) was filtered using ISET membranes, and cytospins from 37 patients with small-cell lung cancer (SCLC) were analyzed using confocal and VyCAP microscopy. Both JUNB and CXCR4 were expressed in the vast majority of lung cancer patients. Interestingly, the phenotypic patterns differed between NSCLC and SCLC patients; the (CK+/JUNB+/CXCR4+) phenotype was present in 50% of NSCLC vs. 71% of SCLC patients. Similarly, the (CK+/JUNB+/CXCR4–) was present in 44% vs. 71%, the (CK+/JUNB–/CXCR4+) in 6% vs. 71%, and the (CK+/JUNB–/CXCR4–) phenotype in 38% vs. 84%. In NSCLC, the presence of ≥1 CTCs with the (CK+/JUNB+/CXCR4+) phenotype was associated with worse progression-free survival (PFS) (p = 0.007, HR = 5.21) while ≥2 with poorer overall survival (OS) (p < 0.001, HR = 2.16). In extensive stage SCLC patients, the presence of ≥4 CXCR4-positive CTCs was associated with shorter OS (p = 0.041, HR = 5.01). Consequently, JUNB and CXCR4 were expressed in CTCs from lung cancer patients, and associated with patients’ survival, underlying their key role in tumor progression.

Introduction : Despite advances in diagnostic and therapeutic techniques breast cancer is still associated with significant morbidity and mortality. CTCs play a crucial role in the metastatic process, which is the main cause of death in BC patients.Areas covered : This review discusses the prognostic and predictive value of CTCs and their prospective in management of BC patients.Expert opinion : The analysis of CTCs through improved technologies offers a new insight into the metastatic cascade. Assessment of the number and molecular profile of CTCs holds great promises for disease monitoring and therapeutic decisions. However, more research is needed until they can be used in therapeutic decisions in clinical practice.

Background: Artesunate (AS), besides being widely used as an antimalarial agent, has been also suggested to exert anti-tumor activity in a plethora of cancer types, including breast and lung cancer. Recently, AS has been shown to inhibit the expression of JunB, which is associated with metastatic progression. The aim of this study is to examine the effect of AS on the viability of both adherent and non-adherent (resembling circulating tumor cells; CTCs) breast and lung cancer cell cultures. Methods: Prevention of cell attachment and establishment of the CTCs’ model was achieved via polyHEMA [poly(2-hydroxyethyl methacrylate)] treatment. Viability of adherent and non-adherent MDA-MB-231 (triple negative breast cancer) and H1299 (non-small-cell lung cancer) cells was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay, 48 h following treatment with different concentrations of AS (3 μM to 100 μΜ) in serum starvation conditions. Cell migration was examined using the wound healing assay. Results: Optimization involving the duration (20 min vs. overnight) and concentration (10 mg/ml) of polyHEMA coating, as well as the duration of AS treatment (24 h vs. 48 h) were performed. 5-fluorouracil was used as a positive control. AS decreased viability of both cell lines in a concentration-dependent manner with comparable IC50, whether being adherent (pIC50 = 4.6 ± 0.1 and 5.1 ± 0.1 for MDA-MB-231 and H1299, respectively; n = 8) or non-adherent (pIC50 = 4.5 ± 0.1 and 4.9 ± 0.1 for MDA-MB-231 and H1299, respectively; n =8). AS (10 μΜ, 48 h) inhibited proliferation of both adherent MDA-MB-231 (34% ± 5, n = 8) and H1299 (57% ± 4, n = 8) cells. Interestingly, the effect of AS on MDA-MB-231 and H1299 non-adherent cultures was less prominent (15% ± 6 and 26% ± 6, P Conclusions: Evidently, non-adherent, free-floating cells seem to respond differently to AS compared to their attached counterparts. These results suggest that antitumor agents might exert different effects in CTCs compared to adherent tumor cells. Acknowledgements: This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2ΕΔΚ-01562). Citation Format: Evangelia Pantazaka, Dafni Graikioti, Constantinos M. Athanassopoulos, Galatea Kallergi. Non-adherent breast and non-small-cell lung cancer cell cultures as a promising CTCs’ model for evaluation of the anti-tumor effects of artesunate [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 1119.

Simple Summary Lung cancer is the most frequent malignancy in the world. Most lung cancer patients are diagnosed at an advanced stage. To make matters worse, the survival of patients is very poor. Circulating tumor cells (CTCs), albeit rare, have been portrayed as essential players in the progression of lung cancer. It is definitely not easy being a CTC. First, they escape from the primary tumor, then they travel in the bloodstream, have to survive really harsh conditions, and finally, they form metastases. The adoption of epithelial-to-mesenchymal transition as well as cancer stem cell features has been suggested to allow CTCs to survive and metastasize. This review will focus on how these features can be used to estimate the prognosis of lung cancer patients. Abstract Lung cancer is the leading cause of cancer-related mortality globally. Among the types of lung cancer, non-small-cell lung cancer (NSCLC) is more common, while small-cell lung cancer (SCLC) is less frequent yet more aggressive. Circulating tumor cells (CTCs), albeit rare, have been portrayed as essential players in the progression of lung cancer. CTCs are considered to adopt an epithelial-to-mesenchymal transition (EMT) phenotype and characteristics of cancer stem cells (CSCs). This EMT (or partial) phenotype affords these cells the ability to escape from the primary tumor, travel into the bloodstream, and survive extremely adverse conditions, before colonizing distant foci. Acquisition of CSC features, such as self-renewal, differentiation, and migratory potential, further reflect CTCs’ invasive potential. CSCs have been identified in lung cancer, and expression of EMT markers has previously been correlated with poor clinical outcomes. Thus far, a vast majority of studies have concentrated on CTC detection and enumeration as a prognostic tools of patients’ survival or for monitoring treatment efficacy. In this review, we highlight EMT and CSC markers in CTCs and focus on the clinical significance of these phenotypes in the progression of both non-small- and small-cell lung cancer.

Detection of CTCs represents a poor prognostic factor in patients with early and metastatic breast cancer (mBC) and treatment with everolimus-exemestane (E/E) is an established effective treatment in hormone receptor-positive/HER2-negative mBC patients. The effect of E/E on CTCs in mBC patients was prospectively investigated.CTCs from 50 pre-treated patients with mBC receiving E/E were analyzed using the CellSearch (CS) platform and triple immunofluorescence (IF) staining for cytokeratin, M30 and Ki67 expression to assess their proliferative and apoptotic status.CTCs (by CS) were detected in 64% of patients before treatment and E/E administration resulted in their decreased prevalence [(n = 18; 36%, p = 0.004) and (n = 7; 19.4%, p = 0.019) post-1st and post-3rd treatment cycle, respectively] whereas it was significantly increased at disease progression (PD: 61%) compared to post-1st and post-3rd cycle (p = 0.049 and p = 0.021, respectively). Ki67-positive CTCs were detected in 60%, 60%, 17% and 50% of patients before treatment, post-1st, post-3rd cycle and at PD, respectively, while the opposite was observed for M30-positive CTCs (0% at baseline, 10% after the 1st cycle, 50% after the 3rd cycle and 0% at PD). The detection of even ≥ 1 CTC/5 ml after one cycle was associated with decreased PFS (3.3 vs 9.0 months, p = 0.025) whereas the detection of even ≥ 2 CTCs at PD was associated with decreased OS (32.4 vs 19.5 months; p = 0.009).The combination of E/E resulted in early elimination of proliferating CTCs in mBC patients and this effect was associated with a favorable clinical outcome.

Upregulation of Vimentin (VIM), alpha-Tubulin (TUB) and Detyrosinated tubulin (GLU) in circulating tumor cells (CTCs) derived from breast cancer patients is related to poor prognosis. In the current study we evaluated for the first time, these cytoskeletal proteins in sixty Non-Small Cell Lung Cancer (NSCLC) patients’ CTCs (33 treatment-naïve and 27 pre-treated). Samples were isolated using the ISET platform and stained with a pancytokeratin (CK)/CD45/TUB, CK/GLU/VIM and CK/programmed death ligand 1 (PD-L1) combination of antibodies. Subsequently, slides were analyzed using confocal laser scanning microscopy. CTCs were detected in 86.7% of the patients. CTCs with TUB expression were identified in 65.4% (34/52) of the CK (+)-patients. GLU, VIM and PD-L1 were also evaluated. The frequency of the observed phenotypes was as follow: (CK+/GLU−/VIM−): 35.2%, (CK+/GLU+/VIM+): 63.0%, (CK+/GLU+/VIM−): 16.7%, (CK+/GLU−/VIM+): 72.2%, (CK+/PD-L1−): 75% and (CK+/PD-L1+): 55%. The OS was significantly decreased in patients with high GLU (3.8 vs. 7.9 months; p = 0.018) and/or high VIM (3.2 vs. 7.1 months; p = 0.029) expression in their CTCs. PD-L1 was also related to OS (3.4 vs. 7.21 months; p = 0.035). Moreover, TUB-high and TUB-low expression in CTCs inversely influenced patients’ OS as independent prognostic factors (p = 0.041 and p = 0.009). The current study revealed that TUB, GLU, VIM and PD-L1 were overexpressed in CTCs from NSCLC patients. Furthermore, the presence of GLU, VIM-positive and PD-L1 in CTCs is potentially related to patients’ outcomes.

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

In patients with breast cancer, markers of aggressiveness such as dysregulation of the insulin-like growth factor receptor (IGF1R) system and E-cadherin loss are commonly observed. Reduced IGF1R expression is correlated with decreased E-cadherin levels and increased cell motility. We assessed IGF1R and E-cadherin expression in circulating tumor cells (CTCs) in patients with breast cancer. Peripheral blood mononuclear cells of early (n = 87)- and metastatic (n = 126)-stage breast cancer patients (obtained prior to adjuvant and first-line chemotherapy) were evaluated using double immunofluorescence (IF) staining for cytokeratin (CK) and IGF1R. Triple IF using CK, IGF1R, and E-cadherin antibodies was performed in selected CTC(+) patients. IGF1R(+) CTCs were more frequently observed in early disease than in metastatic disease (86% vs 68% of CTCs, P = 0.04) stage, whereas IGF1R(-) CTCs were more common in metastatic than in early disease (32% vs 14% of CTCs, P = 0.002). 100% of CTC(+) patients with early disease, compared to 79% of those with metastatic disease, harbored IGF1R(+) CTCs (P = 0.007). Patients with early disease and exclusively IGF1R(+) CTCs had longer disease-free (P = 0.02) and overall survival (P = 0.001) compared to patients with both IGF1R(+) and IGF1R(-) CTC populations. 67% of early-stage CTC(+) patients evaluated had exclusively IGF1R(+)/E-cadherin(+) CTCs, 33% also had IGF1R(-)/E-cadherin(-) CTCs, and none had exclusively IGF1R(-)/E-cadherin(-) CTCs compared to 17%, 75%, and 8% of metastatic patients, respectively (P = 0.027). Similarly, in paired samples of patients with early disease that progressed to metastatic disease, the proportion of IGF1R(+)/E-cadherin(+) CTCs was reduced and IGF1R(-)/E-cadherin(-) CTCs were increased in the metastatic stage compared to early disease stage. IGF1R(+) CTCs are commonly detected in breast cancer, and their frequency decreases in the metastatic disease stage. IGF1R(+)/E-cadherin(+) CTCs also decrease in metastatic patients. IGF1R(+) CTCs are associated with favorable outcomes in early disease stage, suggesting that IGF1R expression is correlated with reduced metastatic potential in breast cancer.

Background: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

Background Circulating tumor cells (CTCs) are important for metastatic dissemination of cancer. They can provide useful information, regarding biological features and tumor heterogeneity; however, their detection and characterization are difficult due to their limited number in the bloodstream and their mesenchymal characteristics. Therefore, new biomarkers are needed to address these questions. Methods Bioinformatics functional enrichment analysis revealed a subgroup of 24 genes, potentially overexpressed in CTCs. Among these genes, the chemokine receptor CXCR4 plays a central role. After prioritization according to the CXCR4 corresponding pathways, five molecules (JUNB, YWHAB, TYROBP, NFYA, and PRDX1) were selected for further analysis in biological samples. The SKBR3, MDA-MB231, and MCF7 cell lines, as well as PBMCs from normal (n = 10) blood donors, were used as controls to define the expression pattern of all the examined molecules. Consequently, 100 previously untreated metastatic breast cancer (mBC) patients (n = 100) were analyzed using the following combinations of antibodies: CK (cytokeratin)/CXCR4/JUNB, CK/NFYA/ΥWHΑΒ (14-3-3), and CK/TYROBP/PRDX1. A threshold value for every molecule was considered the mean expression in normal PBMCs. Results Quantification of CXCR4 revealed overexpression of the receptor in SKBR3 and in CTCs, following the subsequent scale (SKBR3>CTCs>Hela>MCF7>MDA-MB231). JUNB was also overexpressed in CTCs (SKBR3>CTCs>MCF7>MDA-MB231>Hela). According to the defined threshold for each molecule, CXCR4-positive CTCs were identified in 90% of the patients with detectable tumor cells in their blood. In addition, 65%, 75%, 14.3%, and 12.5% of the patients harbored JUNB-, TYROBP-, NFYA-, and PRDX-positive CTCs, respectively. Conversely, none of the patients revealed YWHAB-positive CTCs. Interestingly, JUNB expression in CTCs was phenotypically and statistically enhanced compared to patients’ blood cells (p = 0.002) providing a possible new biomarker for CTCs. Furthermore, the detection of JUNB-positive CTCs in patients was associated with poorer PFS (p = 0.015) and OS (p = 0.002). Moreover, JUNB staining of 11 primary and 4 metastatic tumors from the same cohort of patients revealed a dramatic increase of JUNB expression in metastasis. Conclusions CXCR4, JUNB, and TYROBP were overexpressed in CTCs, but only the expression of JUNB was associated with poor prognosis, providing a new biomarker and a potential therapeutic target for the elimination of CTCs. Electronic supplementary material The online version of this article (10.1186/s13058-019-1166-4) contains supplementary material, which is available to authorized users.

Introduction: Vimentin (VIM) expression in CTCs is a poor prognostic factor for breast cancer patients. Furthermore, upregulation of alpha Tubulin (TUB) and Detyrosynated tubulin (GLU) in CTCs is related to patients' outcome. In the current study, we evaluated TUB, GLU and VIM in NSCLC cell lines and in patients' CTCs. Methods: Four NSCLC cell lines were used for the evaluation of our method. In addition, 60 patients with metastatic NSCLC before the initiation of any line of treatment, were enrolled in this study. Thirty-three patients' samples were taken prior to the 1st line treatment and 27 patients were enrolled before the initiation of ≥2 lines of treatment. CTCs were isolated using ISET platform and stained with pancytokeratin (CK)/CD45/TUB and CK/GLU/VIM antibodies. Samples were analyzed using confocal laser scanning microscopy. Results: In patients' samples, CK(+)/CD45(-) cells were observed in 90% (54 out of 60 patients). CTCs with high expression of TUB were detected in 62.96% (34/54) of the patients. Among the total isolated CTCs/patient, 30.57 % belonged to the (CK+TUB+CD45) phenotype. The rest of the CTCs presented with either low (31.2%) or no expression (24.8%) of TUB. Interestingly, 4.15% of the CTCs (only in advanced disease) belonged to (CK-TUB+CD45-) phenotype. The proportion of TUB low or negative CTCs was statistically higher in treatment-naïve compared to pre-treated patients (p= 0.004 and p=0.007, respectively) whereas the proportion of TUB high CTCs was increased in pre-treated compared to treatment-naïve patients (85.72% vs 64.52%), implying that these CTCs persist after chemotherapy. Further analysis revealed that patients with high TUB expression in their CTCs experienced shorter OS compared to patients with CTCs with low or no TUB expression (7 vs 14 months, respectively; p=0.023). GLU and VIM were also evaluated in the same cohort of patients. The frequency of the observed phenotypes were as follow: (CK-GLU-VIM-): 35.19% (19/54 patients), (CK+GLU+VIM+): 62.96% (34/54), (CK+GLU+VIM-): 16.67% (9/54) and (CK+GLU-VIM+): 72.22%. The last one was the most abundant subclone in treatment naïve patients 81.25% (26/32). Furthermore the most abundant phenotypes in advanced disease were the (CK+GLU-VIM-) 59.09% (13/22), (CK+GLU-VIM+) 59.09% (13/22) and (CK+ GLU+ VIM+): 54.55% (12/22). The expression of VIM in advanced patients was also related to poor OS (1 vs 10 months, p=0.010). Conclusions: In agreement to our previous results in breast cancer patients, current study also revealed that alpha-Tubulin, GLU and Vimentin are overexpressed in CTCs from NSCLC patients. In addition, the presence of TUB and/or VIM-positive CTCs is potentially related to poor patients' outcome. Citation Format: Galatea Kallergi, Spyridoula D. Katsarou, Anastasia Voumvouraki, Christos Stournaras, Stuart S. Martin, Vassilis Georgoulias. Alpha tubulin and EMT-related molecules expressed in circulating tumor cells (CTCs) isolated from non-small cell lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 780.

Background: Since tumor cells may escape from immune surveillance through the programmed cell death 1 (PD-1)/programmed death ligand (PD-L)1 axis, this study was designed in order to evaluate whether there is a correlation between the levels of PD-1+ and PD-L1+-expressing immune cells (ICs) and circulating tumor cells (CTCs) in patients with non-small cell lung cancer (NSCLC). Patients and methods: Peripheral blood was obtained from 37 chemotherapy-naïve patients with metastatic NSCLC before treatment. PD-1 and PD-L1 expression was evaluated (1) on ICs with anti-tumor function (CD4+ and CD8+ T-cells, B-cells, monocytes/dendritic cells) using flow cytometry, (2) on CTCs by immunofluorescence and (3) on cells from tumor tissues by immunohistochemistry. The levels of PD-1+ and PD-L1+-expressing ICs were correlated with progression-free survival (PFS). Results: The presence of PD-1+ CD8+ cells, with reduced interferon (IFN)-γ expression, but not other ICs, were positively correlated with PD-L1+ CTCs (p < 0.04). Increased percentages of PD-1+ CD8+ T-cells, were associated with a worse response to treatment (p = 0.032) and shorter PFS (p = 0.023) which, in multivariate analysis, was revealed as an independent predictor for decreased PFS [hazard ratio (HR): 4.1, p = 0.0007]. Conclusion: The results of the current study, for first time, provide evidence for a possible interaction between ICs and CTCs in NSCLC patients via the PD-1/PD-L1 axis and strongly support that the levels of PD-1+ CD8+ in these patients may be of clinical relevance.

Background: Circulating tumor cells (CTCs) could escape from the immune system through the programmed death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis leading to the development of metastasis. The current study investigated the expression of PD-1/PD-L1 on CTCs isolated from non-small cell lung cancer (NSCLC) patients treated with chemotherapy. Patients and methods: CTCs were isolated from 30 chemo-naïve stage IV NSCLC patients before and after front-line chemotherapy using the ISET filtration platform. CTCs were detected by Giemsa and immunofluorescence (IF) staining. Samples were analyzed with the ARIOL system. Results: Giemsa staining revealed that 28 (93.3%) out of 30 and 9 (81.8%) out of 11 patients had detectable CTCs at baseline and after the third chemotherapy cycle, respectively. Cytokeratin (CK)+/CD45- CTCs by IF could be detected in 17 of 30 (56.7%) patients at baseline and in 8 of 11 (72.7%) after the third chemotherapy cycle. Spearman analysis revealed a significant correlation ( p = 0.001) between Giemsa-positive and IF-positive (CK+/CD45-) CTCs. At baseline, PD-1 and PD-L1 expression was observed in 53% and in 47% CK-positive patients, respectively. After the third treatment cycle the corresponding numbers were 13% and 63% respectively. Median progression-free survival (PFS) was significantly shorter in patients with >3 PD-1(+) CTCs at baseline compared with those with 1 Giemsa-positive tumor cells ( p = 0.025). Conclusion: PD-1(+) and PD-L1(+) CTCs could be detected before and after front-line chemotherapy in patients with metastatic NSCLC. The presence of high PD-1(+) CTC numbers before treatment is associated with a poor patient clinical outcome.

Background To evaluate the phenotypic heterogeneity of circulating tumor cells (CTCs) based on the expression of proliferative, apoptotic and Epithelial-to-Mesenchymal Transmission (EMT) markers during front-line treatment in patients with small cell lung cancer (SCLC) and to evaluate their clinical relevance. Methods CTCs from 108 chemotherapy-naive patients with SCLC were analyzed by double immunofluorescence staining using anti-Ki67, anti-M30, anti-Vimentin along with anti-CKs antibodies. In 83 patients CTCs were also enumerated using the CellSearch. Results Sequential samples were available from 76 and 48 patients after one-treatment cycle and on disease progression (PD), respectively, for immunofluorescence and from 50 and 36 patients after one-cycle and on PD, respectively, for CellSearch. At baseline, 60.2% of the patients had detectable CTCs by either method. Both proliferative (CK67+) and non-proliferative (Ki67-), apoptotic (M30+) and non-apoptotic (M30-) as well as EMT (Vim+) CTCs were present in the same patient. Among 22 patients without detectable CTCs by CellSearch, CK+/Ki67+ and CK+/Vim+ CTCs could be detected in 6 (27.3%) and 6 (27.3%) patients, respectively. One-chemotherapy cycle reduced both the incidence of detection (p

Background: The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with breast cancer (BC) has been associated with poor disease outcome. Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) properties are considered to display enhanced metastatic potential. The presence of these features in CTCs could be an important determinant of increased metastatic behaviour. A new methodology was developed to investigate the co-expression of the stemness marker ALDH1 and the EMT marker TWIST on CTCs from patients with early and metastatic breast cancer, at the single-cell level. Patients and Methods : PBMC’ cytospins were prepared from PB obtained from 80 patients with early and 50 with metastatic breast cancer. Triple immunofluorescence staining was performed using anti-pancytokeratin (A45-B/B3), anti-ALDH1 and anti-TWIST antibodies. Classification of ALDH1 expression levels (high or low) and TWIST subcellular localization (nuclear or cytoplasmic) was performed in HepG2 control cells and three representative BC cell lines, using the ARIOL system. A total of 500.000 PBMCs per patient were subsequently analyzed. Results : CTCs were detected in 13 out of 80 (16%) and in 25 out of 50 (50%) patients with early and metastatic BC, respectively. Although co-expression of ALDH1 and TWIST was observed on almost all CTCs in both patient groups, CTCs expressing the phenotype ALDH1high/TWISTnuclear were more frequently detected among patients with metastatic compared to early disease (76% vs. 15% of patients, p = 0.0004). Moreover, a higher proportion of ALDH1high/TWISTnuclear CTCs was confirmed in the metastatic setting (62% vs. 13% of CTCs, p Conclusions : A new assay is provided for the evaluation of ALDH1 and TWIST co-expression at the single-CTC level. CTCs bearing the ALDH1high/TWISTnuclear phenotype were more commonly detected in metastatic compared to early BC, suggesting that this phenotype prevails during disease progression. The characterization of CTCs according to these markers could be used to improve risk stratification in BC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-08.

// Sofia Agelaki 1, 2 , Melina Dragolia 3 , Harris Markonanolaki 3 , Saad Alkahtani 4, 5 , Christos Stournaras 5 , Vassilis Georgoulias 3 , Galatea Kallergi 3, 5 1 Laboratory of Translational Oncology, School of Medicine, University of Crete, Voutes, Heraklion, Greece 2 Department of Medical Oncology, University General Hospital of Heraklion, Voutes, Heraklion, Greece 3 Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Voutes, Heraklion, Greece 4 Department of Zoology, Science College, King Saud University, Riyadh, Saudi Arabia 5 Department of Biochemistry, University of Crete Medical School, Voutes, Heraklion, Greece Correspondence to: Galatea Kallergi, email: kalergi@med.uoc.gr Keywords: CTCs, HER2, estrogen receptor, progesteron receptor, EGFR Received: August 01, 2016 Accepted: November 22, 2016 Published: December 24, 2016 ABSTRACT Introduction: Patients with triple negative breast cancer (TNBC), are considered as a poor prognosis group for whom no targeted therapies are currently available. The aim of the present study was to phenotypically characterize their CTCs in order to explore potential therapeutic targets. Methods: PBMC’s cytospins were prepared from 45 early (before and after adjuvant chemotherapy), 10 metastatic TNBC and 21 hormone receptor (HR) -positive patients. The expression of Cytokeratins (CK), ER, PR, EGFR and HER2 on CTCs was assessed using immunofluoresence staining and ARIOL analysis. Results: In early stage TNBC, ER, PR, HER2 and EGFR expressing-CTCs were detected in 24.4%, 24.4%, 20% and 40% of patients before the initiation of adjuvant chemotherapy, and in 17.8%, 13.3% 6.7% and 51.1% respectively after the completion of adjuvant treatment. Triple staining experiments revealed distinct subpopulations of CTC expressed HR, and ErbB family receptors. In patients with metastatic disease, the frequency of HER2+ CTCs was significantly increased compared to adjuvant setting (60% vs 20%, p=0.014). The presence of CK + PR - CTCs, before adjuvant treatment was associated with reduced OS (p=0.032) and DFI (p=0.04). Furthermore, the frequency of ER-, PR- and HER2+ CTCs was higher in HR(+) than in TNBC tumors (57.1%, p=0.006; 52.4%, p=0.021 and 52.38%, p=0.009, respectively). Conclusions: The CTCs in patients with early TNBC are phenotypically heterogeneous based on the expression of HR, EGFR and HER2 both before and after the completion of adjuvant chemotherapy whereas the presence of HER2 + CTCs prevails during disease evolution. These findings could be of clinical relevance in terms of CTC targeting.

Introduction: The presence of Disseminated (DTCs) and Circulating Tumor Cells (CTCs) in the bone marrow (BM) and the blood of breast cancer (BC) patients are poor prognostic factors. We have recently shown that CTCs derived from metastatic BC patients, overexpressed CXCR4 and JUNB and this expression was related to clinical outcome. In this study we applied the same methodology to BM of non-metastatic BC patients at primary diagnosis, before the onset of therapy. Methods: Bilateral BM (10 ml) aspirations were assessed for the detection of DTCs by a Ficoll density gradient centrifugation and subsequent centrifugation of the BM cells onto glass slides [1x106 mononuclear cells (MNCs) per slide]. Triple staining experiments with panytokeratin/CXCR4/JUNB antibodies were performed. An expression pattern of the examined proteins was created, using three different BC cell lines (SKBR3, MDA-MB231, and MCF7). Consequently, 10 BM aspirations from BC patients were analyzed. Mean intensity for each examined molecule was calculated automatically with the ARIOL system. Results: Quantification of CXCR4 among BC cell lines revealed that CXCR4 expression followed the subsequent hierarchy SKBR3>MCF7>MDA-MB231 with higher expression in SKBR3 and lower in MDA-MB 231 cells, respectively. Accordingly, the expression pattern of JUNB in cell lines was SKBR3>MCF7>MDA-MB231. Consequently, we analyzed 10 BM samples from primary BC patients. The (CXCR4+JUNB+CK+) phenotype was observed in 90% (9/10) of the samples. In addition, the (CXCR4-JUNB+CK+) phenotype was detected in 20% (2/10) of the patients. Interestingly, the (CXCR4+JUNB-CK+) phenotype was not observed in any of the patient's samples. Quantification of CXCR4 expression revealed that the mean intensity in DTCs (19.1±2) was statistically higher than in MCF7 (13±0.51; p=0.006) and MDA-MB231 (10.22±0.7; p= 0.0002) cell lines. It was also higher compared to normal donors' PBMCs (10.6±1.01; p=0.0004). In addition, the mean intensity of JUNB was statistically increased in DTCs (17.1±1.66) compared to MCF7 (8.9±0.56; p=0.00001) and MDA-MB231 (6.6±0.15; p= 0.00001) cells, respectively. Furthermore, it was increased compared to normal donors' PBMCs (5.2±0.15; p=0.0002). In two patients, we had available samples after treatment. In one patient, DTCs were eliminated after neoadjuvant therapy, for the other patient, DTCs were reduced from 10 to two DTCs. However, these cells were double positive for CXCR4 and JUNB. Conclusion: CXCR4 and JUNB were overexpressed in DTCs derived from primary BC patients, in agreement to previous findings in CTCs, implying an upregulation of the CXCR4 pathway in these cells. Quantification of this expression potentially defines a subgroup of patients with high expression of CXCR4 and JUNB that could benefit from targeted therapies. Citation Format: Galatea Kallergi, Oliver Hoffmann, Nefeli Zacharopoulou, Christos Stournaras, Vassilis Georgoulias, Sabine Kasmir-Bauer. Overexpression of CXCR4 and JUNB in disseminated tumor cells (DTCs) isolated from breast cancer patients before neoadjuvant treatment [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 5186.

Introduction: Circulating tumor cells (CTCs) are considered as a “liquid biopsy” that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers, including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab. Methods: CTCs were isolated based on their size using the ISET platform from 27 patients before treatment, after 1 cycle and 3 cycles. CTCs were detected with Giemsa staining and immunofluorescence (IF) experiments, using either pancytokeratin (A45-B/B3) (CK7)/PD-1/CD45 or (A45-B/B3)(CK7)/PD-L1/CD45 combination of antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC cell lines: H460, H1299, HCC827 and SKMES in normal blood were used to evaluate the detection method. Results: Giemsa evaluation in Nivolumab-treated patients at baseline (25 evaluable samples), after the 1st (9 evaluable samples) and the 3rd (8 evaluable patients) cycle of treatment showed that CTCs could be detected in 48% (12/25), 33.3% (3/9) and 50% (4/8) of patients, respectively. IF could also reveal the presence of CK-positive cells in 44.4% (12/27), 22% (2/9) and 75% (6/8) patients, respectively. PD-1 (+) CTCs were detected in 33.3% (4/12) of patients at baseline, in, 0% after the 1 and 16.7% (1/6) of patients after the 3rd cycle. The same percentages were identified for PD-L1 expression in the same cohort of patients. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Multivariate analysis revealed that the presence of CK-positive cells is an independent prognostic factor for OS (p=0.028) Conclusion: Nivolumab reduced the number of PD-1- and PD-L1-expressing CTCs in advanced NSCLC patients. Furthermore the expression of both markers at baseline is associated with the clinical outcome. Citation Format: Galatea Kallergi, Anastasios Koutsopoulos, Eleni Lagoudaki, Despoina Agouraki, Eleni-Kiriaki Vetsika, Anastasia Voumvouraki, Stuart S. Martin, Filippos Koinis, Christos Stournaras, Vassilis Georgoulias, Athanasios Kotsakis. Detection of PD-L1 and PD-1 positive circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [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 1587.