Fedor Kryukov

Circulating serum microRNAs as novel diagnostic and prognostic biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance

Multiple myeloma still remains incurable in the majority of cases prompting a further search for new and better prognostic markers. Emerging evidence has suggested that circulating microRNAs can serve as minimally invasive biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance. In this study, a global analysis of serum microRNAs by TaqMan Low Density Arrays was performed, followed by quantitative real-time PCR. The analyses revealed five deregulated microRNAs: miR-744, miR-130a, miR-34a, let-7d and let-7e in monoclonal gammopathy of undetermined significance, newly diagnosed and relapsed multiple myeloma when compared to healthy donors. Multivariate logistical regression analysis showed that a combination of miR-34a and let-7e can distinguish multiple myeloma from healthy donors with a sensitivity of 80.6% and a specificity of 86.7%, and monoclonal gammopathy of undetermined significance from healthy donors with a sensitivity of 91.1% and a specificity of 96.7%. Furthermore, lower levels of miR-744 and let-7e were associated with shorter overall survival and remission of myeloma patients. One-year mortality rates for miR-744 and let-7e were 41.9% and 34.6% for the ‘low’ expression and 3.3% and 3.9% for the ‘high’ expression groups, respectively. Median time of remission for both miR-744 and let-7e was approximately 11 months for the ‘low’ expression and approximately 47 months for the ‘high’ expression groups of myeloma patients These data demonstrate that expression patterns of circulating microRNAs are altered in multiple myeloma and monoclonal gammopathy of undetermined significance and miR-744 with let-7e are associated with survival of myeloma patients.

Plasma cell leukemia: from biology to treatment

Plasma cell leukemia (PCL) is a very aggressive and rare form of malignant monoclonal gammopathy characterized by the presence of plasmocytes in peripheral blood. It is classified as primary PCL occuring ‘de novo’, or as secondary PCL in patients with relapsed/refractory multiple myeloma. Primary PCL is a distinct clinicopathological entity from myeloma with different cytogenetic abnormalities and molecular findings, which are usually found only in advanced multiple myeloma. The clinical course is aggressive with short remissions and reduced overall survival. The diagnostic criteria are based on the percentage (>20%) and absolute number (2 × 109/L) of plasma cells in peripheral blood. After establishing diagnosis, induction therapy should begin promptly which is aimed to rapid disease control and to minimize the risk of early death. Intensive chemotherapy regimens and bortezomib‐based regimens, followed by high‐dose therapy with autologous stem cell transplantation, are recommended. Allogeneic transplantation can be considered in younger patients. This article reviews recent knowledge of this hematological malignancy that is associated with a very poor prognosis.

Clinical implication of centrosome amplification and expression of centrosomal functional genes in multiple myeloma.

BackgroundMultiple myeloma (MM) is a low proliferative tumor of postgerminal center plasma cell (PC). Centrosome amplification (CA) is supposed to be one of the mechanisms leading to chromosomal instability. Also, CA is associated with deregulation of cell cycle, mitosis, DNA repair and proliferation. The aim of our study was to evaluate the prognostic significance and possible role of CA in pathogenesis and analysis of mitotic genes as mitotic disruption markers.Design and methodsA total of 173 patients were evaluated for this study. CD138+ cells were separated by MACS. Immunofluorescent labeling of centrin was used for evaluation of centrosome amplification in PCs. Interphase FISH with cytoplasmic immunoglobulin light chain staining (cIg FISH) and qRT-PCR were performed on PCs.ResultsBased on the immunofluorescent staining results, all patients were divided into two groups: CA positive (38.2%) and CA negative (61.8%). Among the newly diagnosed patients, worse overall survival was indicated in the CA negative group (44/74) in comparison to the CA positive group (30/74) (P = 0.019).Gene expression was significantly down-regulated in the CA positive group in comparison to CA negative in the following genes: AURKB, PLK4, TUBG1 (P < 0.05). Gene expression was significantly down-regulated in newly diagnosed in comparison to relapsed patients in the following genes: AURKA, AURKB, CCNB1, CCNB2, CETN2, HMMR, PLK4, PCNT, and TACC3 (P < 0.05).ConclusionsOur findings indicate better prognosis for CA positive newly diagnosed patients. Considering revealed clinical and gene expression heterogeneity between CA negative and CA positive patients, there is a possibility to characterize centrosome amplification as a notable event in multiple myeloma pathogenesis.

Centrosome amplification as a possible marker of mitotic disruptions and cellular carcinogenesis in multiple myeloma.

Centrosome amplification (CA) as a potential marker of mitotic disruptions in multiple myeloma (MM) was investigated in two populations of B-cell lineage: B-cells and plasma cells (PCs). Using immunofluorescent staining, it was shown that CA in B-cells is present in 3.2+/-2.5% in healthy donors versus 9.9+/-7.9% in MM patients (p<0.0001). Based on the calculated threshold value of CA in B-cells, 37% (14/38) of MM patients were positive. There was no significant correlation between CA-positive MM cases (based on PC samples evaluation) and the occurrence of cytogenetic abnormalities in PCs, including del(13)(q14), del(17)(p13), gain(1)(q21) and hyperdiploidy.

Bone marrow plasma cell separation - validation of separation algorithm

Monoclonal gammopathy is characterized by the presence of monoclonal immunoglobulin which is produced by a specifi c clone of terminally differentiated long-living plasma cells (PC) in the bone marrow. More detailed understanding of this clone at the genomic and proteomic level as well as its comparison with the population of normal PC is possible only after the clone is separated from other BM cells.

Flow cytometry in immunoglobulin light chain amyloidosis: Short review.

Flow cytometry (FCM) has found its application in clinical diagnosis and evaluation of monoclonal gammopathies (MG). Although, research has been mainly focused on multiple myeloma (MM), nowadays FCM becomes to be potential tool in the field of AL amyloidosis. Clonal plasma cells identification and specific phenotype profile detection is important for diagnosis, monitoring and prognosis of AL amyloidosis. Therefore, FCM could be a perspective method for study not only MM but also AL amyloidosis. This review provides an overview and possibilities of FCM application in AL amyloidosis.

Combination of serum microRNA‐320a and microRNA‐320b as a marker for Waldenström macroglobulinemia

IgM monoclonal gammopathies are a group of diseases characterized by increased level of IgM immunoglobulin produced by one clone of B cells. These diseases range trom benign (monoclonal gammopathy ot undetermined significance, MGUS) to malignant, such as Waldenstrom macroglobulinemia (WM) or to a lesser extent multiple myeloma (MM) [1,2]. The criteria that differentiate WM from IgM-MGUS are based on the extent of bone marrow (BM) involvement, amount of serum concentration of the M-proicin, presence or absence of symptomatic disease or more recently, MYD88 (L265P) or CXCR4 mutations [3-6]. Despite that, new criteria for the differential diagnosis between these conditions are still needed, circulating microRNAs (miRNAs) being one of them. Circulating miRNAs are present in different body fluids; they reflect physiological or pathologi cal conditions and can be used tor patient classification [7,8]. Thus, we aimed to investigate the ability of serum miRNAs to distinguish WM from IgM-MGUS as well as IgM-MM patients and healthy donors (HD).

Molecular heterogeneity and centrosome-associated genes in multiple myeloma.

Abstract In multiple myeloma (MM), biologic complexity originates from complex oncogenic processes involving somatic acquisition of myriad mutations coupled with genetic variability within the host. This pathogenically determined molecular heterogeneity predetermines clinical intricacy. In this study, we performed gene expression profiling (GEP) focusing on centrosome-related genes to determine the molecular heterogeneity for centrosome-associated genes in patients with MM. We identified the gene pattern with an impact on myeloma pathogenesis. According to expression tendency, three subgroups of patients were established. The revealed molecular signature is related to overall survival as well as to clinical parameters and the International Staging System. Associations with integral clinical parameters allow us to proclaim the impact of the revealed functional gene set in MM genesis. We believe that future investigation of this molecular heterogeneity will help to refine the broad prognoses offered by present-day established systems and even sub-stratify them.

In vitro activation of cytotoxic T-lymphocytes by hTERT-pulsed dendritic cells

Multiple myeloma has been considered a weakly immunogenic malignancy that can cause profound defects in the immune system. An important issue for the immunotherapy of myeloma is the identification of appropriate tumor-associated antigens (TAAs). Recently, hTERT (human telomerase reverse transcriptase) was detected on a majority of human malignancies. In the studies reported here, we studied antigen-specific and HLA-A2-restricted cytotoxic activity against an ARH77 myeloma cell line in vitro. An HLA-A2-specific hTERT-derived nonapeptide (540ILAKFLHWL548) was used as a TAA. Myeloma-specific cytotoxic activity of hTERT-reactive cytotoxic lymphocytes (CTLs) was established by repeated stimulation of the CTLs via dendritic cells loaded with hTERT-derived nonapeptide. These studies were able to demonstrate that hTERT-reactive T-lymphocytes can be identified and expanded using relatively simple in vitro techniques consisting of antigen-specific stimulation, immunomagnetic sorting, and then induction of rapid expansion.

Circulating Serum MicroRNA-130a as a Novel Putative Marker of Extramedullary Myeloma.

Poor outcome of extramedullary disease in multiple myeloma patients and lack of outcome predictors prompt continued search for new markers of the disease. In this report, we show circulating microRNA distinguishing multiple myeloma patients with extramedullary disease from myeloma patients without such manifestation and from healthy donors. MicroRNA-130a was identified by TaqMan Low Density Arrays and verified by quantitative PCR on 144 serum samples (59 multiple myeloma, 55 myeloma with extramedullary disease, 30 healthy donors) in test and validation cohorts as being down-regulated in myeloma patients with extramedullary disease. Circulating microRNA-130a distinguished myeloma patients with extramedullary disease from healthy donors with specificity of 90.0% and sensitivity of 77.1%, patients with extramedullary disease from newly diagnosed multiple myeloma patients with specificity of 77.1% and sensitivity of 34.3% in the test cohort and with specificity of 91.7% and sensitivity of 30.0% in the validation cohort of patients. Circulating microRNA-130a in patients with extramedullary myeloma was associated with bone marrow plasma cells infiltration. Further, microRNA-130a was decreased in bone marrow plasma cells obtained from patients with extramedullary myeloma in comparison to bone marrow plasma cells of myeloma patients without such manifestation, but it was increased in tumor site plasma cells of patients with extramedullary disease compared to bone marrow plasma cells of such patients (p<0.0001). Together, our data suggest connection between lower level of microRNA-130a and extramedullary disease and prompt further work to evaluate this miRNA as a marker of extramedullary disease in multiple myeloma.