Paulina Gronesova

Matrix metalloproteinase 1 and circulating tumor cells in early breast cancer

BackgroundMatrix metalloproteinases (MMPs) are involved in cancer invasion and metastasis. Circulating tumor cells (CTCs) play role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. The aim of this study was to assess correlation between CTCs and tumor MMP1 in BC.MethodsStudy included 149 primary BC patients treated by surgery from March 2012 to March 2013. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSepTM selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by qRT-PCR. Patient samples with higher epithelial and/or mesenchymal gene transcripts than those of healthy donors (n = 60) were considered as CTC positive. Expression of MMP1 in surgical specimens was evaluated by immunohistochemistry.ResultsCTCs were detected in 24.2% patients. CTCs exhibiting only epithelial markers were present in 8.7% patients, whereas CTCs with epithelial-mesenchymal transition (EMT) markers (CTC_EMT) were observed in 13.4% of patients and CTCs co-expressing both markers were detected in 2.0% patients. Patients with CTC_EMT in peripheral blood had significantly increased expression of MMP1 in tumor cells (p = 0.02) and tumor associated stroma (p = 0.05) than those of patients without CTC_EMT. In multivariate analysis, CTC_EMT and tumor grade were independently associated with MMP1 expression in cancer cells, while CTC_EMT and Ki67 were independently associated with MMP1 expression in cancer associated stroma.ConclusionOur data suggest link between MMP1 and CTCs with EMT phenotype and support role of MMPs and EMT in tumor dissemination.

Using nested RT-PCR analyses to determine the prevalence of avian influenza viruses in passerines in western Slovakia, during summer 2007

The prevalence of avian influenza virus (AIV), together with the distribution of different AIV subtypes, was studied in migratory waterfowl and terrestrial birds caught in western Slovakia during summer 2007. Both oropharyngeal and cloacal swabs were collected. Screening of samples revealed that 18% of oropharyngeal and 18% of cloacal samples were positive for AIV. Samples from both the oropharynx and cloaca were positive in only 6.6% of cases. A total of 10 different subtypes of haemagglutinin (H2, H3, H4, H6, H7, H9, H10, H11, H12, and H13) and 4 different subtypes of neuraminidase (N1, N2, N3, and N5) were detected in 32 samples from this location. The most abundant subtypes of HA in the samples were H12 and H9 (25% each), followed by H11 and H10 (15% each), and H13 (9%). There were 3 cases where different AIV infections were detected in oropharyngeal and cloacal samples originating from the same bird (H13N1 and H12N5; H13N3 and H9N5; H10N2 and H9N5 in the oropharynx and cloaca, respectively).

Prevalence of avian influenza viruses, Borrelia garinii, Mycobacterium avium, and Mycobacterium avium subsp. paratuberculosis in waterfowl and terrestrial birds in Slovakia, 2006

The prevalence of Borrelia, Mycobacteria and avian influenza virus (AIV) infections, together with the distribution of different AIV subtypes, was studied in migratory waterfowl and terrestrial birds trapped in three localities in Slovakia during 2006. Samples obtained from waterfowl captured in the Senianske Ponds area of Eastern Slovakia showed the highest diversity of AIV isolates. A total of 13 different subtypes were detected in 19 samples from this location (H1N2, H2N2, H3N2, H6N6, H7N6, H9N2, H9N5, H9N6, H10N5, H10N6, H12N6, H13N6, and H16N6). H3N5 virus was detected in 50% of passerines testing positive for AIV in the Parizske Wetlands, with H7N2, H9N2, H9N5, H12N1, and H13N2 infections also recorded at this locality. H9N5 virus predominated in passerines captured at Trnava Ponds, with isolates H1N6, H6N5, H7N2, H7N6, H10N3, and H10N6 also detected at this location. There were five cases where different AIV infections were detected in oropharyngeal and cloacal samples originating from the same bird (H13N6 and H1N2; H10N5 and H12N6; H9N5 and H6N5; H10N6 and H7N6; and H9N2 and H3N5 in the oropharynx and cloaca, respectively). Between 21% and 52% of captured birds tested positive for Borrelia burgdorferi sensu lato, with the proportion infected depending on bird species and locality. Samples were characterized by polymerase chain reaction-restriction fragment length polymorphism analysis and identified as Borrelia garinii species (either B/B′ or R/R′ pattern). Mycobacteria were detected in 42% and 26% of waders captured at Senianske Ponds and marsh-dwelling passerines captured in the Parizske Wetlands, respectively. Interestingly, forest-dwelling passerine species caught in the Trnava Ponds region were tested negative for Mycobacteria.

Relationship between circulating tumor cells, blood coagulation, and urokinase-plasminogen-activator system in early breast cancer patients.

Cancer is a risk factor for venous thromboembolism (VTE) and plasma d‐dimer (DD) and tissue factor (TF) are established VTE associated markers. Circulating tumor cells (CTCs) are associated with the risk of VTE in metastatic breast cancer. This study aimed to correlate CTCs, blood coagulation and the urokinase plasminogen activator (uPA) system in primary breast cancer (PBC) patients. This prospective study included 116 PBC patients treated by primary surgery. CTCs were detected by quantitative RT‐PCR assay for expression of epithelial (CK19) or epithelial‐mesenchymal transition (EMT) genes (TWIST1, SNAIL1, SLUG, ZEB1, FOXC2). Plasma DD, TF, uPA system proteins were detected by enzyme‐linked immunosorbent assays, while expressions of uPA system in surgical specimens were evaluated by immunohistochemistry. CTCs were detected in 27.6% patients. Patients with CTCs had a significantly higher mean plasma DD (ng/mL) than those of patients without CTCs (632.4 versus 365.4, p = 0.000004). There was no association between plasma TF and CTCs. Epithelial CTCs exhibit higher expression of uPA system genes compared to EMT_CTCs. Patients with CTCs had higher plasma uPA proteins than those of patients without CTCs; there was no correlation between tissue expression of uPA system, CTCs, DD or TF levels. In multivariate analysis CTCs and patients age were independent factors associated with plasma DD. We found association between plasma DD and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. CTCs could be directly involved in coagulation activation or increased CTCs could be marker of aggressive disease and increased VTE risk.

Modulation of cisplatin sensitivity in human ovarian carcinoma A2780 and SKOV3 cell lines by sulforaphane

Cisplatin resistance is one of the major obstacles in the treatment of ovarian cancer. In an effort to look for new possibilities of how to overcome this difficulty, we studied the mechanisms of the interactions between sulforaphane (SFN) and cisplatin (cisPt) in combined treatment of human ovarian carcinoma A2780 and SKOV3 cell lines. Synergy (A2780) and antagonism (SKOV3) found in MTT assay was confirmed by apoptosis. While SFN significantly potentiated cisPt-induced DNA damage in A2780 cells, it protected SKOV3 cells against cisPt-crosslinking. We revealed a less efficient Nrf-2 pathway inducibility by SFN in A2780 compared to SKOV3 cells, when activation of the Nrf-2 pathway incites its protectivity against cisPt. Thus, different activation of the Nrf-2 pathway may explain the dual effects of SFN.

A novel 3D mesenchymal stem cell model of the multiple myeloma bone marrow niche: biologic and clinical applications.

Specific niches within the tumor bone marrow (BM) microenvironment afford a sanctuary for multiple myeloma (MM) clones due to stromal cell-tumor cell interactions, which confer survival advantage and drug resistance. Defining the sequelae of tumor cell interactions within the MM niches on an individualized basis may provide the rationale for personalized therapies. To mimic the MM niche, we here describe a new 3D co-culture ex-vivo model in which primary MM patient BM cells are co-cultured with mesenchymal stem cells (MSC) in a hydrogel 3D system. In the 3D model, MSC with conserved phenotype (CD73+CD90+CD105+) formed compact clusters with active fibrous connections, and retained lineage differentiation capacity. Extracellular matrix molecules, integrins, and niche related molecules including N-cadherin and CXCL12 are expressed in 3D MSC model. Furthermore, activation of osteogenesis (MMP13, SPP1, ADAMTS4, and MGP genes) and osteoblastogenic differentiation was confirmed in 3D MSC model. Co-culture of patient-derived BM mononuclear cells with either autologous or allogeneic MSC in 3D model increased proliferation of MM cells, CXCR4 expression, and SP cells. We carried out immune profiling to show that distribution of immune cell subsets was similar in 3D and 2D MSC model systems. Importantly, resistance to novel agents (IMiDs, bortezomib, carfilzomib) and conventional agents (doxorubicin, dexamethasone, melphalan) was observed in 3D MSC system, reflective of clinical resistance. This 3D MSC model may therefore allow for studies of MM pathogenesis and drug resistance within the BM niche. Importantly, ongoing prospective trials are evaluating its utility to inform personalized targeted and immune therapy in MM.

Expression of SOCS1 and CXCL12 Proteins in Primary Breast Cancer Are Associated with Presence of Circulating Tumor Cells in Peripheral Blood.

Circulating tumor cells (CTCs) are independent prognostic factors in the primary and metastatic breast cancer patients and play crucial role in hematogenous tumor dissemination. The aim of this study was to correlate the presence of CTCs in peripheral blood with the expression of proteins in tumor tissue that have a putative role in regulation of cell growth and metastatic potential. This prospective study included 203 primary breast cancer patients treated by definitive surgery. CTCs were detected by quantitative real-time PCR for the expression of epithelial (CK19) or epithelial-to-mesenchymal transition–inducing transcription factor genes (TWIST1, SNAIL1, SLUG, and ZEB1). Expression of APC, ADAM23, CXCL12, E-cadherin, RASSF1, SYK, TIMP3, BRMS1, and SOCS1 proteins in primary breast tumor tissue was evaluated by immunohistochemistry. CTCs with epithelial markers were found in 17 (9.2%) patients. Their occurrence was associated with inhibition of SOCS1 expression (odds ratio [OR] = 0.07; 95% confidence interval [CI], 0.03-0.13; P < .001). CTCs with positive epithelial-to-mesenchymal transition markers were detected in 30 (15.8%) patients; however, no association with analyzed protein expressions was found. Overall, CTCs were detected in 44 (22.9%) patients. Presence of any CTC marker was significantly associated with positive CXCL12 expression (OR = 3.08; 95% CI, 1.15-8.26; P = .025) and lack of SOCS1 expression (OR = 0.10; 95% CI, 0.04-0.25; P < .001) in patient’s tumor tissues. As both CXCL12 and SOCS1 proteins are involved in cytokine signaling, our results provide support for the hypothesis that aberrant signaling cross talk between cytokine and chemokine responses could have an important role in hematogenous dissemination of tumor cells in breast cancer.

Sulforaphane-induced apoptosis involves the type 1 IP3 receptor.

In this study we show that anti-tumor effect of sulforaphane (SFN) is partially realized through the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). This effect was verified in vitro on three different stable cell lines and also in vivo on the model of nude mice with developed tumors. Early response (6 hours) of A2780 ovarian carcinoma cells to SFN treatment involves generation of mitochondrial ROS and increased transcription of NRF2 and its downstream regulated genes including heme oxygenase 1, NAD(P)H:quinine oxidoreductase 1, and KLF9. Prolonged SFN treatment (24 hours) upregulated expression of NRF2 and IP3R1. SFN induces a time-dependent phosphorylation wave of HSP27. Use of IP3R inhibitor Xestospongin C (Xest) attenuates both SFN-induced apoptosis and the level of NRF2 protein expression. In addition, Xest partially attenuates anti-tumor effect of SFN in vivo. SFN-induced apoptosis is completely inhibited by silencing of IP3R1 gene but only partially blocked by silencing of NRF2; silencing of IP3R2 and IP3R3 had no effect on these cells. Xest inhibitor does not significantly modify SFN-induced increase in the rapid activity of ARE and AP1 responsive elements. We found that Xest effectively reverses the SFN-dependent increase of nuclear content and decrease of reticular calcium content. In addition, immunofluorescent staining with IP3R1 antibody revealed that SFN treatment induces translocation of IP3R1 to the nucleus. Our results clearly show that IP3R1 is involved in SFN-induced apoptosis through the depletion of reticular calcium and modulation of transcription factors through nuclear calcium up-regulation.

Plasma Cytokines Correlated With Disease Characteristics, Progression-Free Survival, and Overall Survival in Testicular Germ-Cell Tumor Patients

Micro‐Abstract We performed a translational study and found a correlation among overall survival, progression‐free survival, and circulating cytokines in metastatic testicular germ‐cell tumor patients. Association between baseline clinicopathologic features and plasma cytokines was also assessed. Plasma cytokines could be a potential biomarker for identification of high‐risk patients. Background: Cytokines are the communicators of immune system and are involved in all immune responses. The aim of this study was to assess the correlation among plasma cytokines, patient and tumor characteristics, and clinical outcome in chemonaive testicular germ‐cell tumor (TGCT) patients. Patients and Methods: This study included 92 metastatic chemotherapy‐naive TGCT patients treated with platinum‐based chemotherapy from July 2010 to March 2014. Plasma was isolated before first administration of chemotherapy, and the concentration of 51 plasma cytokines were analyzed using multiplex bead arrays. Results: At a median follow‐up of 33.2 months (range, 0.1‐54.8 months), 10.9% of patients experienced disease progression, and 7.6% died. Several cytokines were associated with different baseline clinicopathologic features. Elevated plasma levels of interferon (IFN)‐&agr;2, interleukin (IL)‐2R&agr;, IL‐16, hepatocyte growth factor (HGF), and monocyte chemotactic protein (MCP)‐3 were significantly associated with worse progression‐free survival and overall survival (OS). Moreover, elevated levels of stem‐cell growth factor (SCGF)‐&bgr; were also associated with worse OS. Patients with elevated levels of all 6 cytokines experienced significantly worse outcomes compared to patients who had fewer than 6 cytokines elevated (hazard ratio = 12.06; 95% confidence interval, 7.39‐19.49; P = .002 for progression‐free survival, and hazard ratio = 39.65; 95% confidence interval, 25.03‐62.18; P < .00001 for OS, respectively). Results were independent of International Germ Cell Cancer Collaborative Group criteria. Conclusion: We found a correlation among progression free‐survival, OS, and circulating cytokines in TGCT. This suggests the existence an association between plasma cytokines and baseline clinicopathologic features in TGCT. Plasma cytokines could be used for identification of high‐risk patients who are candidates for new therapeutic approaches.

Prognostic value of serum carbonic anhydrase IX in testicular germ cell tumor patients

Despite the fact that testicular germ cell tumors (TGCTs) are one of the most chemosensitive solid tumors, a small proportion of patients fail to be cured following cisplatin-based first line chemotherapy. Upregulation of carbonic anhydrase IX (CA IX) in various solid tumors is associated with poor outcome. The current prospective study investigated the prognostic value of serum CA IX level in TGCTs. In total, 83 patients (16 non-metastatic following orchiectomy with no evidence of disease, 57 metastatic chemotherapy-naïve and 10 metastatic relapsed chemotherapy-pretreated) starting adjuvant and/or new line of chemotherapy and 35 healthy controls were enrolled in the study. Serum CA IX values were determined using an enzyme-linked immunosorbent assay, and intratumoral CA IX was analyzed by immunohistochemistry. Metastatic chemotherapy-naïve patients had significantly higher mean CA IX serum levels than healthy controls (490.6 vs. 249.6 pg/ml, P=0.005), while there was no difference in serum CA IX levels in non-metastatic or relapsed TGCT patients compared with healthy controls. There was no significant difference in the mean serum CA IX levels between different groups of patients and between the first and second cycle of chemotherapy, nor association with patients/tumor characteristics. Serum CA IX was not prognostic for progression-free survival [hazard ratio (HR)=0.81, P=0.730] or overall survival (HR=0.64, P=0.480). However, there was a significant association between intratumoral CA IX expression and serum CA IX concentration (rho=0.51, P=0.040). These results suggest that serum CA IX level correlates with tumor CA IX expression in TGCT patients, but fails to exhibit either a prognostic value or an association with patients/tumor characteristics.