Prognostic Value of the miR-17~92 Cluster in Chronic Lymphocytic Leukemia.

The aim of this study was to investigate the expression of miR-17∼92 cluster members in chronic lymphocytic leukemia (CLL) patients. Six microRNAs (miRNAs)-miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92a-1-very poorly characterized in CLL patients, were chosen for the study to consider their possible role as cancer biomarkers. It is currently unclear to which extent miR-17~92 expression is related to other routinely measured CLL markers, and whether the findings can be of any clinical significance. To achieve this goal, we report the expression levels of these miRNAs detected by RT-qPCR in purified CD19+ B lymphocytes of 107 CLL patients and correlate them with existing clinical data. The study provides new evidence regarding the heterogeneity of miR-17~92 cluster members' expression in CLL patients. Higher miR-17-5p expression was associated with unfavorable prognostic factors (i.e., 17p and 11q deletions, CD38 and ZAP-70 expression). On the other hand, miR-19a, miR-20a, and miR-92a-1 negatively correlated with these adverse factors. The presence of del(13q) as a sole aberration was associated with a significantly lower miR-17-5p as well as higher miR-19a-3p and miR-92a-1-5p expression compared to patients carrying unfavorable genetic aberrations. Particularly, miR-20a could be considered an independent favorable prognostic factor. In a multivariate analysis, high miR-20a expression remained an independent marker predicting long TTT (time to treatment) for CLL patients.

Reduced Percentage of CD14dimCD16+SLAN+ Monocytes Producing TNF and IL-12 as an Immunological Sign of CLL Progression.

Monocytes are one of the least studied immune cells with a potentially important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Nevertheless, data regarding the role of subpopulations of monocytes in the CLL microenvironment are still limited. For the very first time, this study presents an assessment of monocyte subsets divided according to SLAN and CD16 expression in CLL patients. The study involved 70 freshly diagnosed CLL patients and 35 healthy donors. Using flow cytometry, monocyte subpopulations were assessed among PBMCs. CD14+ monocytes can be divided into: "classical" (CD14+CD16-SLAN-), "intermediate" (CD14+CD16+SLAN-) and "non-classical" (CD14dimCD16+SLAN+). In our study, we noted an increased percentage of non-classical monocytes with intracellular expression of TNF and IL-12. On the other hand, among the intermediate monocytes, a significantly higher percentage of cells synthesizing anti-inflammatory IL-10 was detected. The percentage of CD14dimCD16+SLAN+ monocytes producing TNF and IL-12 decreased with the stage of CLL and inversely correlated with the expression of the prognostic factors ZAP-70 and CD38. Moreover, the percentage of CD14dimCD16+SLAN+ monocytes producing TNF and IL-12 was lower in CLL patients requiring treatment. This may indicate the beneficial effect of non-classical monocytes on the anti-tumor response.

An association of circulating Tregs and Th17 cells producing IL-21 and IL-22 with the ROMA in ovarian cancer patients.

The purpose of the present study was to assess the association of regulatory T cells (Tregs; CD4+ FOXP3+) and helper T lymphocytes (Th17) releasing interleukin (IL)-21 and IL-22, with the Risk of Ovarian Malignancy Algorithm (ROMA). Similar association was made with two additional tumour markers, human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125) from patients serum. The presence of Tregs and Th17 was determined both in the peripheral blood and in the tissue of epithelial ovarian tumors. Mononuclear cells obtained from patient's peripheral blood (PBMCs) and from ovarian tissue were isolated by density gradient centrifugation. As a control group patients who had undergone surgery for infertility without ovarian pathology were selected. The percentage of Tregs and Th17 releasing IL-21 or IL-22 cells from both peripheral blood and tumor tissue was measured by flow cytometry. No differences in demographic parameters like body mass index, age, or gravidity were observed among the studied groups. However, an increased concentration of marker HE4 and value of ROMA was identified in individuals with ovarian cancer when compared with women with cystadenomas. Furthermore, a negative correlation between the ROMA value in the serum and Tregs from the peripheral blood of patients with cystadenoma ovarian tumors was detected. The presented work documents, for the first time, the negative association between peripheral blood Tregs and ROMA evaluation based on the tumour markers present in the serum of women with ovarian cystadenoma. Such an effect might result from the negative impact of Tregs on the inflammation process and on tumorigenesis caused by the persistent inflammation.

Can Galectin-3 Be a Novel Biomarker in Chronic Lymphocytic Leukemia?

Galectin-3's (Gal-3) effect on the pathogenesis of chronic lymphocytic leukemia (CLL) has not yet been extensively studied. The present study aims to analyze the potential role of Gal-3 as a prognostic biomarker in CLL patients. The Gal-3 expression was evaluated in CLL cells with RT-qPCR and flow cytometry. Due to the unclear clinical significance of soluble Gal-3 in CLL, our goal was also to assess the prognostic value of Gal-3 plasma level. Because cell survival is significantly affected by the interaction between Gal-3 and proteins such as Bcl-2, the results of Gal-3 expression analysis were also compared with the expression of Bcl-2. The results were analyzed for known prognostic factors, clinical data, and endpoints such as time to first treatment and overall survival time. Our research confirmed that Gal-3 is detected in and on CLL cells. However, using Gal-3 as a potential biomarker in CLL is challenging due to the significant heterogeneity in its expression in CLL cells. Moreover, our results revealed that Gal-3 mRNA expression in leukemic B cells is associated with the expression of proliferation markers (Ki-67 and PCNA) as well as anti-apoptotic protein Bcl-2 and can play an important role in supporting CLL cells.

Prognostic Value of Tie2-Expressing Monocytes in Chronic Lymphocytic Leukemia Patients.

Tie2-expressing monocytes (TEMs) are associated with tumor progression and metastasis. This unique subset of monocytes has been identified as a potential prognostic marker in several solid tumors. However, TEMs remain poorly characterized in hematological cancers, including chronic lymphocytic leukemia (CLL). This study analyzed, for the first time, the clinical significance of TEM population in CLL patients. Flow cytometry analysis of TEMs (defined as CD14+CD16+Tie2+ cells) was performed at the time of diagnosis on peripheral blood mononuclear cells from 104 untreated CLL patients. Our results revealed an expansion of circulating TEM in CLL patients. These monocytes express high levels of VEGF and suppressive IL-10. A high percentage of TEM was associated closely with unfavorable prognostic markers (ZAP-70, CD38, 17p and 11q deletion, and IGHV mutational status). Moreover, increased percentages of circulating TEMs were significantly higher in patients not responding to the first-line therapy as compared to responding patients, suggesting its potential predictive value. High TEM percentage was also correlated with shorter overall survival (OS) and shorter time to treatment (TTT). Importantly, based on multivariate Cox regression analysis, TEM percentage was an independent predictor for TTT. Thus, we can suggest the adverse role of TEMs in CLL.

An association of iNKT+/CD3+/CD161+ lymphocytes in ovarian cancer tissue with CA125 serum concentration.

The purpose of this study was to investigate the association of iNKT (human invariant natural killer T) cells with the key marker of ovarian cancer (OC) - CA125 (cancer antigen125) in serum. The study reports the assessment of iNKT cells in peripheral blood and tissue of benign and borderline ovarian tumors (BOTs) and in the advanced-stage ovarian cancer. The study groups were as follows: 25 women with benign ovarian tumors, 11 women with BOTs, and 24 women with primary advanced-stage ovarian cancers. The control group consisted of 20 patients without the ovarian pathology. The rates of iNKT lymphocytes in the peripheral blood and tissue specimens were evaluated by a flow cytometry. Significant differences in the percentage of iNKT+/CD3+ of CD3+ lymphocytes, iNKT+/CD3+/CD161+ among CD3+ and iNKT+/CD3+/CD161+ among CD3+/iNKT+ between the control group and patients with ovarian tumors in the peripheral blood and tumor tissue were identified. Significant correlations were noticed between the proportion of lymphocytes iNKT+/CD3+/CD161+ among CD3+/iNKT cells in blood and in cancer tissue of both benign and malignant tumors. In the OC group, neither the ratio of iNKT cells in the blood (P = 0.07), nor the intra-tumor NKT-cell infiltration (P = 0.5) were independent prognostic factors for the follow-up. An increased rate of iNKT cells was detected in benign ovarian tumors compared to OCs. In patients with ovarian cancer, a higher rate of iNKT cells in tumor tissue was present related to that noted in the patient's blood. In addition, a correlation was discovered between the CA125 serum marker and NKT cells from the ovarian cancer tissue. This article has for the first time demonstrated a negative relationship between serum levels and NKT lymphocyte count from ovarian tissue. The inflammatory process in ovarian cancer tissue and the potential infiltration of endothelial immune cells, may result in a reduced number of NKT cells in the tumor microenvironment and increased circulation of the CA125 marker. Presented findings underscore new aspects of the iNKT cells involvement in the ovarian cancer development.

A Rare Oncologic Emergency: Spontaneous Tumor Lysis Syndrome in Metastatic Colon Adenocarcinoma.

Tumor lysis syndrome is an oncologic emergency that can present with variable symptoms and is truly a laboratory-based diagnosis without pathognomonic clinical findings. The classical teaching is to consider this diagnosis in cancer patients undergoing chemotherapy. We present the case of a 66-year-old female with newly diagnosed metastatic liver adenocarcinoma, not on chemotherapy, who was diagnosed with spontaneous tumor lysis syndrome. Cognizance of this syndrome and associated laboratory findings are paramount to diagnosis and rapid intervention.

Genetically Engineered Lung Cancer Cells for Analyzing Epithelial-Mesenchymal Transition.

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological process that also exerts important roles in disease progression. Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in vivo in the future.

RORγT is overexpressed in iNKT and γδ T cells during relapse in relapsing-remitting multiple sclerosis.

The aim of the current study is to evaluate IL-17 production and RORγT, and IL-23R expression by iNKT, Th17 and γδ T cells in the peripheral blood of relapsing-remitting multiple sclerosis patients. Samples of peripheral blood from 21 relapse patients and 12 remission patients, and 15 healthy volunteers were stained with monoclonal antibodies for flow cytometry analysis. No significant differences in iNKT, γδ T and Th17 percentages were noted. The significant overexpression of RORγT was observed in all three subpopulations - therefore, iNKT, γδ T and Th cells may be an important source of IL-17 shortly prior to the relapse.

Intracellular IL-4 and IFN-γ expression in iNKT cells from patients with chronic lymphocytic leukemia.

Malignant B cells in chronic lymphocytic leukemia serve an essential role in the whole immune response, so their interactions with other immune cells are more complex than observed in solid tumors. The latest study results indicate that the immune dysregulation in chronic lymphocytic leukemia (CLL) also affects a small population of invariant natural killer T cells (iNKT). Using peripheral blood iNKT cells obtained from patients with CLL, the objective of the present study was to assess the intracellular expression of typical cytokines involved in the Th1 (IFN-γ) and Th2 (IL-4) response pathways following stimulation with the iNKT-specific ligand α-galactosylceramide. iNKT cells from patients with CLL exhibited upregulated IL-4 and IFN-γ expression in comparison to those from HVs. No significant association between the ability of iNKT cells to produce IL-4 or IFN-γ and the expression of CD1d on leukemic B lymphocytes or monocytes was identified. However, the function of iNKT cells was compromised in patients with CLL by a strong Th2 bias (high IL-4 and low IFN-γ expression). The ratio of iNKT+IFN-γ+:iNKT+IL-4+ was significantly decreased in the CLL group when compared with HVs, and this decreased further as the disease progressed. This change may result in the promotion of leukemic B lymphocyte survival. Therefore, in the pathogenesis of CLL, Th2 bias may delay the antitumor response that relies on stimulation of the Th1 immune response.

CD1d expression is higher in chronic lymphocytic leukemia patients with unfavorable prognosis.

Through the analysis of CD1d expression by flow cytometry and qRT-PCR we showed lower CD1d molecule and CD1d mRNA expression in B cells of CLL patients than of healthy controls. The frequency of CD1d(+)/CD19(+) cells, CD1d staining intensity and CD1d transcript levels increased with the disease stage. CD1d expression was positively associated with ZAP-70 and CD38 expressions as well as with unfavourable cytogenetic changes. We established the relationship between high CD1d expression and shorter time to treatment and overall survival. We observed that CD1d expression in individual patients significantly changed over time. The percentage of CD1d(+)/CD19(+) cells inversely correlated with the percentage of iNKT cells.

Th17/IL-17A might play a protective role in chronic lymphocytic leukemia immunity.

Th17 cells, a recently discovered subset of T helper cells that secrete IL-17A, can affect the inflammation process autoimmune and cancer diseases development. The purpose of this study was to evaluate the role of Th17 cells and IL17A in biology of CLL. The study group included 294 untreated CLL patients in different clinical stages. Here, we show that higher Th17 and IL-17A values were associated with less advanced clinical stage of CLL. Th17 cells' percentages in PB were lower in patients who died due to CLL during follow-up due to CLL (as compared to surviving patients) and in patients responding to first-line therapy with fludarabine-based regimens (as compared to non-responders). IL-17A inversely correlated with the time from CLL diagnosis to the start of therapy and was lower in patients who required treatment during follow-up. Th-17 and IL-17A values were lower in patients with adverse prognostic factors (17p and 11q deletion, CD38 and ZAP-70 expression). CLL patients with detectable IL-17A mRNA in T cells were in Rai Stage 0 and negative for both ZAP-70 and CD38 expression. Th17 percentages positively correlated with iNKT and adversely with Treg cells. The results of this study suggest that Th17 may play a beneficial role in CLL immunity.