Sofia Agelaki

Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer

The CD44(+)/CD24(-/low) and ALDH1(+) cell phenotypes are associated with stemness and enhanced tumorigenic potential in breast cancer. We assessed the expression of CD44, CD24 and ALDH1 on tumor cells circulating in the peripheral blood (CTCs) of patients with metastatic breast cancer using triple-marker immunofluorescence microscopy. Among a total of 1439 CTCs identified in 20 (66.7%) out of 30 patients, 35.2% had the stem-like/tumorigenic phenotype CD44(+)/CD24(-/low), whereas 17.7% of the CTCs analyzed in seven patients, were ALDH1(high)/CD24(-/low). In conclusion, we report the existence of a subpopulation of CTCs with putative stem cell progenitor phenotypes in patients with metastatic breast cancer.

Phosphorylated EGFR and PI3K/Akt signaling kinases are expressed in circulating tumor cells of breast cancer patients

IntroductionThe phosphoinositide-3 kinase (PI3K)/Akt pathway, operating downstream of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER)2, is implicated in cell migration and survival. EGFR and HER2 are expressed in circulating tumor cells, but the activation status of downstream signaling molecules has not yet been reported.MethodsTo investigate expression levels of EGFR, HER2, PI3K, and Akt in circulating tumor cells, we used peripheral blood mononuclear cells from 32 cytokeratin-19 mRNA-positive patients with early (n = 16) and metastatic (n = 16) breast cancer.Peripheral blood mononuclear cell cytospins were double stained with cytokeratin antibody along with one of the following: EGFR, phospho-EGFR, HER2, phospho-PI3K, or phospho-Akt antibodies.ResultsEGFR and HER2 were expressed in circulating tumor cells of 38% and 50% patients with early and 44% and 63% patients with metastatic disease, respectively. Interestingly, phospho-PI3K and phospho-Akt expression levels were similar at 88% (14 out of 16) and 81% (13 out of 16), respectively, in circulating tumor cells of patients with early and metastatic disease. Phospho-EGFR was observed in circulating tumor cells of two (33%) early and six (86%) metastatic EGFR-positive patients. Immunomagnetic separation of peripheral blood mononuclear cells, using EpCAM antibody, and subsequent double-staining experiments of circulating tumor cells showed that EGFR was co-expressed with HER2, phospho-Akt and phospho-PI3K kinases, indicating activation of the corresponding survival signaling pathway.ConclusionsOur findings demonstrate that circulating tumor cells express receptors and activated signaling kinases of the EGFR/HER2/PI3K/Akt pathway, which could be used as targets for their effective elimination.

Front-line bevacizumab in combination with oxaliplatin, leucovorin and 5-fluorouracil (FOLFOX) in patients with metastatic colorectal cancer: a multicenter phase II study.

PurposeTo evaluate the efficacy and the toxicity of front line FOLFOX4 combined with bevacizumab in patients with metastatsic CRC (mCRC).Patients and MethodsChemotherapy-naïve patients with mCRC, received bevacizumab (5 mg/kg every 2 weeks d1), oxaliplatin (85 mg/m2 on d1), leucovorin (200 mg/m2) on days 1 and 2 and 5-Fluorouracil (400 mg/m2 as i.v. bolus and 600 mg/m2 as 22 h i.v. continuous infusion on days 1 and 2) every 2 weeks.ResultsFifty three patients (46 with a PS 0–1) were enrolled. Complete and partial response was achieved in eight (15.1%) and 28 (52.8%) patients, respectively (ORR: 67.9%; 95% C.I.: 53.8%–92%); 11 (20.7%) patients had stable disease and six (11.3%) progressive disease. With a median follow up period of 13.5 months, time to tumor progression was 11 months while the median survival has not yet been reached; the probability of 1-, 2- and 3- year survival was 79.8%, 63.8% and 58.3%, respectively; Two patients relapsed during the follow up period. Eight (15%) patients underwent metastasectomy with R0 resections. Grade 3–4 neutropenia occurred in 15.1% of patients and one (1.9%) of them presented febrile neutropenia. Non-hematologic toxicity included grade 3 diarrhea (7.6%) and grade 2 and 3 neurotoxicity in 16.9 and 15.1% of patients, respectively. One (1.9%) patient presented pulmonary embolism and one (1.9%) cardiac ischaemia. There was one (1.9%) sudden death after the first cycle.ConclusionThe combination of FOLFOX4/bevacizumab appears to be highly effective, well tolerated and merits further evaluation in patients with mCRC.

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.

Pemetrexed versus erlotinib in pretreated patients with advanced non–small cell lung cancer: A Hellenic Oncology Research Group (HORG) randomized phase 3 study

In this superiority study, pemetrexed was compared with erlotinib in pre‐treated patients with metastatic non–small cell lung cancer (NSCLC).

Detection of cytokeratin-19 mRNA-positive cells in the peripheral blood and bone marrow of patients with operable breast cancer

Background:To compare detection rates and evaluate the clinical relevance of cytokeratin-19 (CK-19) mRNA-positive cells in the peripheral blood (circulating tumour cells, CTCs) and bone marrow (disseminated tumour cells; DTCs) of patients with early breast cancer.Methods:Paired samples of peripheral blood and bone marrow were obtained from 165 patients with stage I–II breast cancer before the initiation of adjuvant chemotherapy. In 84 patients, paired blood and bone marrow samples were also available after chemotherapy. The detection of CK-19 mRNA-positive CTCs and DTCs was assessed by real-time PCR.Results:CK-19 mRNA-positive CTCs and DTCs were detected in 55.2 and 57.6% of patients before chemotherapy, respectively. After chemotherapy, CTCs and DTCs were identified in 44 (52.4%) and 43 (51.2%) of the 84 patients, respectively. There was a 93.9% (McNemar; P=0.344) and 72.6% (McNemar; P=0.999) concordance between blood and bone marrow samples before and after chemotherapy, respectively. The detection of CK-19 mRNA-positive CTCs or DTCs before chemotherapy was associated with decreased overall survival (P=0.024 and P=0.015, respectively). In addition, their simultaneous detection was also associated with an increased incidence of disease-related death and decreased overall survival (P=0.016).Conclusions:The detection of CK-19 mRNA-positive CTCs using reverse transcription-PCR (RT–PCR) both before and after chemotherapy is correlated with the detection of CK-19 mRNA-positive DTCs in patients with early-stage breast cancer. The determination of the CTC status by RT–PCR conveys clinically relevant information that is not inferior to DTC status and, owing to the ease of sampling, warrants further evaluation as a tool for monitoring minimal residual disease.

Activation of FAK/PI3K/Rac1 Signaling Controls Actin Reorganization and Inhibits Cell Motility in Human Cancer Cells

We have recently identified a specific signaling pathway that regulates actin reorganization in malignant human breast and prostate epithelial cells associated with FAK, PI-3K and Rac1 activation. Here we report that this pathway operates in MCF7 cells upon activation of membrane androgen receptors (mAR). Stimulation of mAR by the non-permeable testosterone-BSA conjugate resulted in early actin reorganization documented by quantitative measurements of actin dynamics and morphological analysis of microfilament organization. This effect was regulated by early phosphorylation of FAK and subsequent PI-3K and Rac1 activation. The functional role of this pathway was further shown in A375 melanoma cells. Treatment with the opioid antagonist αs1 casomorphin resulted in rapid and potent actin remodeling in A375 cells, regulated by rapid activation of the FAK/PI-3K/Rac1 signaling. Pretreatment of both cell lines with the specific PI-3K inhibitor wortmannin blocked actin reorganization. Interestingly, wound healing assays revealed that testosterone-BSA and α s1 casomorphin significantly inhibited MCF7 and A375 cell motility respectively. These effects were abrogated through blockade of PI-3K signaling by wortmannin. The results presented here indicate that actin reorganization through FAK/PI3-K/Rac-1 activation operates in various human cancer cell systems supporting a functional role for FAK/PI-3K/Rac1/actin signaling in controlling cell motility.

Apoptotic Circulating Tumor Cells in Early and Metastatic Breast Cancer Patients

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.

Co-expression of putative stemness and epithelial-to-mesenchymal transition markers on single circulating tumour cells from patients with early and metastatic breast cancer

BackgroundThe detection of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with breast cancer predicts poor clinical outcome. Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) features display enhanced malignant and metastatic potential. A new methodology was developed in order to investigate the co-expression of a stemness and an EMT marker (ALDH1 and TWIST, respectively) on single CTCs of patients with early and metastatic breast cancer.MethodsTriple immunofluorescence using anti-pancytokeratin (A45-B/B3), anti-ALDH1 and anti-TWIST antibodies was performed in cytospins prepared from hepatocellular carcinoma HepG2 cells and SKBR-3, MCF-7 and MDA.MB.231 breast cancer cell lines. Evaluation of ALDH1 expression levels (high, low or absent) and TWIST subcellular localization (nuclear, cytoplasmic or absent) was performed using the ARIOL system. Cytospins prepared from peripheral blood of patients with early (n = 80) and metastatic (n = 50) breast cancer were analyzed for CTC detection (based on pan-cytokeratin expression and cytomorphological criteria) and characterized according to ALDH1 and TWIST.ResultsCTCs were detected in 13 (16%) and 25 (50%) patients with early and metastatic disease, respectively. High ALDH1 expression (ALDH1high) and nuclear TWIST localization (TWISTnuc) on CTCs was confirmed in more patients with metastatic than early breast cancer (80% vs. 30.8%, respectively; p = 0.009). In early disease, ALDH1low/neg CTCs (p = 0.006) and TWISTcyt/neg CTCs (p = 0.040) were mainly observed. Regarding co-expression of these markers, ALDH1high/TWISTnuc CTCs were more frequently evident in the metastatic setting (76% vs. 15.4% of patients, p = 0.001; 61.5% vs. 12.9% of total CTCs), whereas in early disease ALDH1low/neg/TWISTcyt/neg CTCs were mainly detected (61.5% vs. 20% of patients, p = 0.078; 41.9% vs. 7.7% of total CTCs).ConclusionsA new assay is provided for the evaluation of ALDH1 and TWIST co-expression at the single CTC-level in patients with breast cancer. A differential expression pattern for these markers was observed both in early and metastatic disease. CTCs expressing high ALDH1, along with nuclear TWIST were more frequently detected in patients with metastatic breast cancer, suggesting that these cells may prevail during disease progression.

Variable expression levels of keratin and vimentin reveal differential EMT status of circulating tumor cells and correlation with clinical characteristics and outcome of patients with metastatic breast cancer

BackgroundCTCs expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported. However, no information exists for keratin expression levels of CTCs in association with disease status, whereas assays for the characterization of transitional EMT phenotypes of CTCs in breast cancer are rather lacking. We investigated the correlation between keratin expression of CTCs and patients’ outcome and characterized the EMT status of CTCs via the establishment of a numerical “ratio” value of keratin and vimentin expression levels on a single cell basis.MethodsKeratin expression was evaluated in 1262 CTCs from 61 CTC-positive patients with metastatic breast cancer, using analysis of images obtained through the CellSearch System. For the determination of vimentin/keratin (vim/K) ratios, expression levels of keratin and vimentin were measured in cytospin preparations of luminal (MCF-7 and T47D) and basal (MDA.MB231 and Hs578T) breast cancer cell lines and 110 CTCs from 5 CTC-positive patients using triple immunofluorescence laser scanning microscopy and image analysis.ResultsMCF-7 and T47D displayed lower vim/K ratios compared to MDA.MB231 and Hs578T cells, while MCF-7 cells that had experimentally undergone EMT were characterized by varying intermediate vim/K ratios. CTCs were consisted of an heterogeneous population presenting variable vim/K values with 46% of them being in the range of luminal breast cancer cell lines. Keratin expression levels of CTCs detected by the CellSearch System correlated with triple negative (p = 0.039) and ER-negative (p = 0.025) breast cancer, and overall survival (p = 0.038).ConclusionsKeratin expression levels of CTCs correlate with tumor characteristics and clinical outcome. Moreover, CTCs display significant heterogeneity in terms of the degree of EMT phenotype that probably reflects differential invasive potential. The assessment of the vim/K ratios as a surrogate marker for the EMT status of CTCs merits further investigation as a prognostic tool in breast cancer.