Irena Misiewicz-Krzeminska

Upregulation of Dicer is more frequent in monoclonal gammopathies of undetermined significance than in multiple myeloma patients and is associated with longer survival in symptomatic myeloma patients

Dicer and Drosha are key enzymes in the miRNA-processing pathway which is altered in many human cancers. We analyzed Dicer and Drosha expression levels by quantitative PCR in 151 patients with monoclonal gammopathies: 102 symptomatic myeloma patients, 23 smoldering myelomas and 26 monoclonal gammopathy of undetermined significance. We found that Dicer expression values were significantly higher in monoclonal gammopathy of undetermined significance than in smoldering myelomas and symptomatic myeloma (mean ± SD, 0.84±0.36 vs. 0.60±0.23 and 0.62±0.51; P<0.01). Moreover, the median progression-free survival was significantly longer in symptomatic myeloma patients with high expression of Dicer (not reached vs. 23.6 months; P=0.02). By contrast, no differences in the expression of Drosha among these groups of patients were observed. Our data suggest that Dicer expression may play an important role in the progression and prognosis of monoclonal gammopathies. (Clinicaltrials.gov identifier: NCT00461747 for MM patients under 65 years of age and NCT00443235 for MM patients over 65 years of age)

Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

Molecular Mechanisms of p53 Deregulation in Cancer: An Overview in Multiple Myeloma

The p53 pathway is inactivated in the majority of human cancers. Although this perturbation frequently occurs through the mutation or deletion of p53 itself, there are other mechanisms that can attenuate the pathway and contribute to tumorigenesis. For example, overexpression of important p53 negative regulators, such as murine double minute 2 (MDM2) or murine double minute 4 (MDM4), epigenetic deregulation, or even alterations in TP53 mRNA splicing. In this work, we will review the different mechanisms of p53 pathway inhibition in cancer with special focus on multiple myeloma (MM), the second most common hematological malignancy, with low incidence of p53 mutations/deletions but growing evidence of indirect p53 pathway deregulation. Translational implications for MM and cancer prognosis and treatment are also reviewed.

MicroRNA-223 is a novel negative regulator of HSP90B1 in CLL

BackgroundMicroRNAs are known to inhibit gene expression by binding to the 3′UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients.MethodsBy applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3′UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status, HSP90B1 expression and clinico-biological data were assessed.ResultsHSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome.ConclusionsHSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target.

PD71-08 ANDROGEN RECEPTOR SPLICE VARIANT 7 (AR-V7) IN PATIENTS WITH LOCAL ADVANCED, METASTATIC AND CRPCM: A NOVEL CAPILLARY NANO-INMUNOASSAY TECHNIQUE

INTRODUCTION AND OBJECTIVES: Docetaxel or cabazitaxel-based chemotherapy continues to have a critical role in the treatment of men with metastatic castration-resistant prostate cancer (mCRPC). However, responses are heterogeneous and resistance to therapy is a pressing clinical problem. With the goal of developing liquid biomarkers to aid in treatment selection, we sought to identify genes associated with resistance to chemotherapy using a circulating tumor cell (CTC)-based approach. METHODS: Whole blood (~5mL) was obtained from 25 patients with mCRPC starting docetaxel (n1⁄421) or cabazitaxel (n1⁄44). CTCs were isolated using anti-EpCAM-conjugated magnetic beads, and following cell lysis, mRNA was extracted followed by multiplex qRTPCR for 44 prostate cancer-related genes plus internal controls. Gene expression was normalized to controls, and samples were considered CTC-positive based on a previously established set of epithelial markers (EpCAM, EGFR, DSG2, KRT8, KRT18 and KRT19). The primary endpoint was PSA progression-free survival (PFS), with PSA progression defined as an increase of 25% or more above the nadir. Univariable Cox regression analyses were performed to assess for genes associated with PFS at false discovery rate (FDR) < 0.20. RESULTS: Among 25 patients with mCRPC, we identified 84% (21/25) with detectable CTCs. The median age of the cohort was 62 years (IQR 58-70). At a median (IQR) follow up of 5.4 (3.4-9.3) months, 47.6 % (10/21) of patients showed a PSA decrease of at least 30% following treatment initiation. 18/21 patients (85.7%) experienced PSA progression at a median of 2.8 months (IQR 1.7-4.8). In the Cox analysis, KLK2 (HR 2.54, 95%CI 1.24-5.21, p1⁄40.011), GAS6 (HR 3.50, 95% CI 1.30-9.42, p1⁄40.013), and BMP7 (HR 2.01, 95%CI 1.15-3.52; p1⁄40.014) were associated with shorter PFS. CONCLUSIONS: Wehave identified three genes associatedwith progression in mCRPC patients initiating chemotherapy. While these early results need further confirmation, they suggest that CTCs may be utilized to help guide precision-based treatment strategies in patients with mCRPC. Additionally, these results corroborate our recent in vitro and in vivo findings (Lee et al, J Cell Biochem, 2016) indicating that GAS6 protects prostate cancer cells from docetaxel-induced apoptosis.