Maria Spiliotaki

Caveolin-1 regulates EGFR signaling in MCF-7 breast cancer cells and enhances gefitinib-induced tumor cell inhibition.

Caveolin-1, an essential protein constituent of caveolae, is involved in cell signalling through its association with various signalling molecules. Epidermal growth factor receptor (EGFR) interacts directly with caveolin-1 and this interaction functionally regulates EGFR tyrosine kinase activity. In this report we investigated the role of caveolin-1 overexpression on EGFR signalling in MCF-7 breast cancer cell line. We demonstrate here that although total EGFR expression levels are similar, EGFR phosphorylation is diminished in MCF-7/CAV1 cells compared to parental MCF-7 cells. In addition, MCF-7/CAV1 cells exhibit higher total and activated Akt levels. Moreover, MCF-7/CAV1 cells stimulated with EGF display higher EGFR and Akt phosphorylation associated with enhanced proliferative and motility rates, compared to MCF-7 cells. Pretreatment with gefitinib inhibits EGF-induced stimulation of both EGFR and downstream Akt and MAPK more efficiently in MCF-7/CAV1 than in MCF-7 cells. In accordance, EGF-induced proliferation and migration is more effectively suppressed with gefinitib in MCF-7/CAV1 compared to parental cells. In conclusion these results suggest that caveolin-1 overexpression in MCF-7 breast cancer cell line modulates EGFR activation levels and EGF-induced EGFR signalling. This is associated with enhanced antitumor effects of gefitinib suggesting a role for EGFR inhibition in caveolin-1 overexpressing breast cancers.

Targeting the insulin-like growth factor I receptor inhibits proliferation and VEGF production of non-small cell lung cancer cells and enhances paclitaxel-mediated anti-tumor effect.

The effects of AVE1642, a human monoclonal antibody against IGF-IR, were examined in NSCLC cell lines in order to characterize its anti-proliferative and anti-angiogenic activity as a single agent and in combination with chemotherapy. AVE1642 inhibited IGF-IR signaling and suppressed IGF-I-induced, serum-stimulated or autocrine-mediated proliferation of NSCLC cells in vitro. Furthermore, the combination of paclitaxel and AVE1642 resulted in a sequence-dependent increase in the inhibition of cell proliferation, compared to each agent alone, which was associated with a dose-dependent increase in phosphorylated IGF-IR and Akt. Moreover, inhibition of IGF-IR signaling by AVE1642 reduced IGF-I-induced VEGF production by NSCLC cells as well as the migratory capacity of HUVEC cells challenged with conditioned media from lung cancer cells previously exposed to IGF-I. The above results suggest that inhibition of IGF-IR signaling by AVE1642 enhances the efficacy of chemotherapy and modulates VEGF and angiogenesis in NSCLC. These effects may have important clinical implications in the treatment of NSCLC.

Abstract 2397: Evaluation of proliferation and apoptosis markers in circulating tumor cells (CTCs) of women with early breast cancer who are candidates for tumor dormancy

Background: In breast cancer (BC) recurrences frequently occur many years after removal of the primary tumor. These relapses are thought to originate from cells that disseminated from the primary tumor and underwent a period of inactivity, called tumor dormancy, followed by active growth. Clinical dormancy could be related to the presence of solitary dormant cells (cellular dormancy), and/or micrometastases, in which cell proliferation is counterbalanced by apoptosis (tumor mass dormancy). Clinical cancer dormancy is frequently observed in BC, whereas CTCs have been identified in clinically disease-free BC patients 7-22 years after surgery. In the present study, we aimed to detect and characterize CTCs of dormancy candidates with BC by analyzing the expression of proliferation and apoptosis markers. Patients and Methods: A total of 104 patients with early BC presenting no evidence of disease for at least 5 years after initial diagnosis were included. In addition, 36 patients relapsing at least 3 years after initial diagnosis were also included as controls. Cytospins were prepared from peripheral blood mononuclear cells (PBMCs), obtained from dormancy candidates and metastatic BC patients at the time of follow-up and prior to the initiation of and first-line therapy, respectively. Samples were stained with pancytokeratin antibody along with ki-67 as a proliferation marker and M-30 an antibody recognizing a neo-epitope expressed only after caspase cleavage of cytokeratin 18 during early apoptosis. A total of 106 PBMCs per patient were analyzed by the use of ARIOL system. Results: CTCs were detected in 40 (36%) out of 104 dormancy candidate patients with early BC. In 25 (63%) of these patients, all CTCs detected were negative for both ki-67 and M30, whereas in 8 (20%) ki-67 positive CTCs, and in 9 (23%) M-30 positive CTCs were detected. Two (5%) patients had CTCs expressing either ki-67 or M30. Among a total of 223 CTCs detected, 194 (87%), were both ki-67 and M-30 negative, 8 (3%) were ki-67 positive and 20 (9%) were M-30 positive. In the group of 36 patients with metastatic disease, CTCs were identified in 10 (28%). In 4 (40%) patients, ki-67 positive CTCs (p=0.01 compared to dormancy candidates) were detected, whereas, no M-30 positive CTCs were observed. Among of total 126 CTCs identified, 50 (40%) were ki-67 positive (p=0.01, compared to dormancy candidates). Conclusions: CTCs are often detected in dormancy candidates with BC. The great majority of these CTCs express neither proliferation, nor apoptotic markers, thus possibly representing dormant CTCs. On the contrary, in patients relapsing long after initial diagnosis, the proliferation index is increased in CTCs. The above findings suggest that proliferation marker expression in CTCs could be used for monitoring disease progression in dormancy candidates with BC. 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 2397. doi:1538-7445.AM2012-2397

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic 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.

Expression of insulin‐like growth factor‐1 receptor in circulating tumor cells of patients with breast cancer is associated with patient outcomes

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.

Abstract P2-02-16: The proliferation index of circulating tumor cells (CTCs) is not influenced by the administration of adjuvant chemotherapy in early breast cancer (BC) and seems to reflect Ki67 expression of the primary tumor

Background: The assessment of Ki67 in the primary tumor represents a prognostic marker with potential predictive implications in early BC. We evaluated Ki67 expression in CTCs from patients with early BC and assessed the effect of adjuvant chemotherapy as well as Ki67 expression in the primary tumor. Methods: Ki67 was evaluated in CTCs of 97 patients with early BC pre- and post- adjuvant chemotherapy by the use of immunofluorescence analysis. Cytospins of peripheral blood mononuclear cells were double stained with A45-B/B3 cytokeratin mouse antibody and a Ki67 rabbit antibody. Ki67 staining of the primary tumor was also performed for 13 CTC-positive patients. A proliferation index (PI) in CTCs was considered as the ratio of Ki67-positive CTCs/total CTCs. A PI of up to 14% was defined as 9low9 whereas a PI above 14% was defined as 9high9. Results: CTCs were detected in 26 (26.8%) patients before and/or after chemotherapy. Seven (27%) of 26 CTC-positive and 8 (11%) of 74 CTC-negative patients relapsed (p = 0.047). Ki67-positive CTCs were identified in 20 (76.9%) of 26 patients, whereas in 1 (3.9%) and 5 (19.2%) patients, exclusively Ki67-positive and Ki67-negative CTCs, respectively, were detected. Seven (33%) of 21 Ki67-positive patients relapsed in contrast to none among the exclusively Ki67-negative patients (p = 0.13). A total of 154 and 161 CTCs were detected pre- and post-chemotherapy, respectively; the PI in CTCs was 56% and 55%, respectively. In 8 patients with detectable CTCs at both time points, the PI was 65% and 49% pre- and post-chemotherapy, respectively. In 5 (62.5%) out of 8 patients, the PI remained high, in 2 (25%) increased and in 1 patient no Ki67–positive CTCs were detected post-chemotherapy. Seventeen patients were CTC-positive at baseline [HER2 positive, n=5; triple negative, n=1; hormone receptor positive, n=11]. A concordance in Ki67 staining between the primary tumor and CTCs was recorded in 10 (77%) out of 13 patients. Moreover, in 2 (67%) of 3 patients with exclusively Ki67-negative CTCs, low Ki67 expression was also observed in the primary tumor. Interestingly, 2 out of 5 patients with HER2 positive primary relapsed and both had high PI in their CTCs, whereas 2 out the 3 HER2 positive patients that did not relapse had low CTC proliferation index. Similarly, the triple negative patient had low PI in her CTCs and has not relapsed after 4 years of follow up. Conclusions: Adjuvant chemotherapy fails to decrease the proliferation index in CTCs. Ki67 expression in CTCs seems to reflect Ki67 expression in the primary tumor and could be predictive of patient outcome. Citation Format: Agelaki S, Spiliotaki M, Spanaki A, Kassiou L, Tzardi M, Koinis F, Kafousi M, Georgoulias V, Mavroudis D. The proliferation index of circulating tumor cells (CTCs) is not influenced by the administration of adjuvant chemotherapy in early breast cancer (BC) and seems to reflect Ki67 expression of the primary tumor. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-16.

Abstract 374: Expression of the autophagosomal marker LC3 on circulating tumor cells (CTCs) of patients with early and metastatic breast cancer (BC)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Autophagy is a protein and organelle degradation pathway involved in the regulation of cancer development and progression. Recent evidence suggests that autophagy serves as a cell survival mechanism under stressed conditions and it is considered to be involved in determining the response of cancer cells to anticancer therapy. Microtubule-associated protein 1 light chain 3 (LC3) has been identified as a marker for monitoring autophagy. CTCs, which have been linked to tumor progression in BC, may employ autophagy associated mechanisms to survive. Here, we investigated the expression of LC3B on CTCs isolated from patients with BC. Methods: The expression of LC3B was first assessed by immunofluorescence using MCF7 treated with tamoxifen as a model system. LC3B expression was then evaluated in 22 and 25 CTC positive patients with early and metastatic BC before the initiation of adjuvant and first-line chemotherapy, respectively. Metastatic patients were also assessed before the administration of the second treatment cycle. Cytospins of MCF7 cells and of peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 anti-cytokeratin (CK) antibody and an LC3B anti-rabbit antibody. LC3B density was measured using the automated fluorescent microscope ARIOL system. The cutoff value for high and low LC3B expression, was set at the median value of the integrated optical density (IOD). Results: Among 142 CTCs identified in patients with early BC, 55 (38%) presented high LC3B expression. A total of 176 CTCs were detected in metastatic patients before the initiation of first-line chemotherapy and 99 (59%) presented high LC3B expression (p = 0.0003 compared with early BC patients). After the first treatment cycle, 62 CTCs were detected with 37 (60%) of them demonstrating high LC3B expression. Nine metastatic patients remained CTC-positive both before and after the first cycle of chemotherapy. In these patients, the proportion of cells presenting high LC3B expression was 40% and 75%, pre- and post- chemotherapy, respectively (p = 0.0049). Conclusions: The autophagosome marker LC3B is expressed in CTCs from patients with BC. High LC3B expression in CTCs was more commonly observed in metastatic, whereas low LC3B expression prevailed in early disease. Moreover, LC3B expression on CTCs was significantly increased after the administration of chemotherapy. The above observations suggest that metastatic progression in breast cancer could be linked to LC3B expression. Moreover the evaluation of LC3B on CTCs merits further investigation regarding its association with chemotherapy efficacy. Citation Format: Maria Spiliotaki, Chrysa Koukorava, Alexios Matikas, Vassilis Georgoulias, Dimitrios Mavroudis, Sofia Agelaki. Expression of the autophagosomal marker LC3 on circulating tumor cells (CTCs) of patients with early and metastatic breast cancer (BC). [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 374. doi:10.1158/1538-7445.AM2015-374

Abstract P4-01-13: CTC enumeration and characterization has predictive and prognostic implications in patients with metastatic breast cancer treated with exemestane plus the mTOR inhibitor everolimus

Background: The utility of CTC enumeration in predicting patient (pt) outcome has been demonstrated in metastatic breast cancer (MBC) treated with chemotherapy or endocrine therapy. In this study we evaluated the clinical impact of CTC assessment in terms of both enumeration and characterization in breast cancer pts treated with exemestane plus everolimus. Patients and methods: Thirty-nine pts with hormone receptor (HR)-positive, HER2-negative MBC, received exemestane plus everolimus. CTC enumeration in peripheral blood (7.5 ml) was performed before treatment (n=39), post cycles 1 (n=39) and 3 (n=29), on disease re-evaluation and on relapse, whichever occurred first, using the CellSearch System. CTC characteristics were determined at the same time points by immunofluorescence (IF) analysis of PBMC cytospins (10 6 cells), triple stained with pancytokeratin (CK) antibody along with Ki67 and M30 as proliferation and apoptosis markers, respectively, using the Ariol System. Patients were assessed by CT scans and bone scan, every 3 months or as clinically indicated. Results: At the cut-off of ≥ 1 CTC, 25 of 39 (64%) pts had detectable CTCs at baseline, 12 (31%) of 39 post-1 st and 10 (34.5%) of 29 post-3 rd cycle. Ten (25.6%) pts remained CTC(+) and 12 (30.8%) CTC(-) both at baseline and post-1 st cycle; 15 (38.5%) CTC(+) pts turned to CTC(-) and 2 (5%) CTC(-) turned to (+). CTC positivity after the first cycle was associated with shorter median progression-free survival (PFS) compared to CTC(-) status (3.9 vs 8 mo, p=0.031). Shorter PFS was also recorded for pts that remained CTC(+) at both time points compared to all other (p=0.02). At the cut-offs of ≥ 2 and ≥ 5 CTCs, 16 (41%) and 9 (23%) pts were CTC(+) at baseline, respectively; post-1 st cycle, 7 (18%) and 4 (10%) pts were CTC(+) (at ≥ 2 and ≥ 5 CTCs, respectively). Post-3 rd cycle the positivity rate was 17% for both cut-offs and these pts had significantly shorter PFS compared to CTC(-) pts (3.7 vs 8.7 months, p=0.048). Efficacy assessment revealed partial response in 3 (7.7%) pts, stable disease in 27 (69.23%) and progressive disease (PD) in 8 (20.5%); 1 pt was non-evaluable for response. Among pts determined CTC(+) post-1 st cycle (cut-off ≥ 2 CTCs), 57% progressed compared to 13% of CTC(-) pts (p=0.02). In addition, at the post-3 rd cycle evaluation, pts with PD had significantly higher CTC counts compared to non-progressors (mean ± SEM; 10 ± 5.78/pt vs 1.62±0.83/pt, p=0.027). By the use of IF 43%, 44% and 40% of CTC(+) pts had proliferative [Ki67(+)/M30(-)] CTCs at baseline, post -1 st and -3 rd cycles, respectively (cut-off ≥ 1 CTC); 67%, 50% and 50% of those pts, respectively, experienced PD. Apoptotic [Ki67(-)/M30(+)] CTCs were detected in 14%, 22% and 60% of CTC(+) pts at baseline, post -1 st and -3 rd cycles, respectively; none of the pts with apoptotic CTCs experienced PD. Conclusions: CTC enumeration and characterization in terms of proliferation and apoptosis during the course of treatment has significant predictive and prognostic implications in patients with MBC receiving the combination of exemestane plus everolimus. Citation Format: Sofia Agelaki, Dimitris Mavroudis, Maria Spiliotaki, Eleni Politaki, Maria A Papadaki, Stella Apostolaki, Christos Nikolaou, Vassilis Georgoulias. CTC enumeration and characterization has predictive and prognostic implications in patients with metastatic breast cancer treated with exemestane plus the mTOR inhibitor everolimus [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-13.

Abstract P1-04-12: Prognostic value of CTC detection by RT-PCR and the CellSearch system before first-line chemotherapy in metastatic breast cancer

Background: CTCs have significant prognostic role in patients with breast cancer. Several techniques developed for CTC detection might help in monitoring treatment efficacy. The CellSearch System is the only clinically validated for use in patients with metastatic breast cancer (MBC). We have previously shown a concordance between RT-PCR for cytokeratin 19 (CK-19) and CellSearch System in the detection of CTCs in MBC. In this study we aimed to evaluate the prognostic value of CTC detection using these assays in the same cohort of patients. Methods: Blood was obtained from patients with MBC before the initiation of first-line chemotherapy. Different aliquots of the same blood sample were evaluated for the presence of CTCs by RT-PCR for CK-19 mRNA and by the CellSearch System. Disease progression and response to treatment were determined using standard clinical and imaging criteria. Results: In 142 patients with available clinical data, CTC evaluation was performed using both methods. Median age was 60.7 years (range, 23-82), 74.6% were post-menopausal and 16.9% were HER2-positive. CTCs were detected in 52.1% and 36.6% of patients by the CellSearch System (cut-offs ≥2 and ≥5 CTCs/7.5 ml, respectively) and in 38.7% by RT-PCR; 27.5% (cut-off ≥2) and 20.4% (cut-off ≥5) of patients were CTC-positive by both methods. Response rate was 40% in patients with CTCs detected by CellSearch (cut-off ≥2) vs 62% in CTC-negative patients; there was no difference in objective responses using the ≥5 cut-off, or using RT-PCR for CTC detection. Median PFS was 9.0 (cut-off ≥2) and 7.5 months (cut-off ≥5) for CTC-positive versus 20.0 (cut-off ≥2) and 18.7 months (cut-off ≥5) for CTC-negative patients using the CellSearch System (p = 0.0001). No significant difference in PFS was evident according to CTC detection by RT-PCR (10.0 vs 13.1 months, p = 0.253). Median survival was 24.7 vs 57.3 (cut-off ≥2) and 18.5 vs 53.7 (cut-off ≥5) months for CTC-positive vs CTC-negative patients using the CellSearch System (p = 0.0001); by RT-PCR, median survival was 30.3 and 50.1 months for CTC-positive and CTC-negative patients, respectively (p = 0.021). Conclusions: The detection of CTCs before first-line chemotherapy using either the CellSearch System or RT-PCR for CK-19 mRNA has significant prognostic value in patients with MBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-12.