Autologous hematopoietic stem cell transplantation (AHSCT) remains the most prevalent type of stem cell transplantation. In our study, we investigated the changes in circulating miRNAs in AHSCT recipients and their potential to predict early procedure-related complications. We collected serum samples from 77 patients, including 54 with multiple myeloma, at four key time points: before AHSCT, on the day of transplantation (day 0), and at days + 7 and + 14 post-transplantation. Through serum miRNA-seq analysis, we identified altered expression patterns and miRNAs associated with the AHSCT procedure. Validation using qPCR confirmed deviations in the levels of miRNAs at the beginning of the procedure in patients who subsequently developed bacteremia: hsa-miR-223-3p and hsa-miR-15b-5p exhibited decreased expression, while hsa-miR-126-5p had increased level. Then, a neural network model was constructed to use miRNA levels for the prediction of bacteremia. The model achieved an accuracy of 93.33% (95%CI: 68.05-99.83%), with a sensitivity of 100% (95%CI: 67.81-100.00%) and specificity of 90.91% (95%CI: 58.72-99.77%) in predicting bacteremia with mean of 6.5 ± 3.2 days before occurrence. In addition, we showed unique patterns of miRNA expression in patients experiencing platelet engraftment delay which involved the downregulation of hsa-let-7f-5p and upregulation of hsa-miR-96-5p; and neutrophil engraftment delay which was associated with decreased levels of hsa-miR-125a-5p and hsa-miR-15b-5p. Our findings highlight the significant alterations in serum miRNA levels during AHSCT and suggest the clinical utility of miRNA expression patterns as potential biomarkers that could be harnessed to improve patient outcomes, particularly by predicting the risk of bacteremia during AHSCT.

Despite advances in multiple myeloma (MM) treatment, drug resistance remains a clinical challenge. We aimed to develop a prognostic model for bortezomib resistance based on miRNA expression profiling. The study included 40 previously untreated MM patients receiving bortezomib-based regimens (20 treatment-sensitive, 20 resistant). Pretreatment venous blood samples were analyzed for miRNA expression. Differential expression analysis revealed upregulated miR-27b-3p (FC 1.45, p = 0.017) and let-7b-5p (FC 1.44, p = 0.025) in the resistant group. Univariate analysis identified let-7b-5p (OR 3.17, 95%CI: 1.19-11.4, p = 0.04) and miR-27b-3p (OR 4.73, 95%CI: 1.4-26.6, p = 0.036) as risk factors for resistance. The final multivariate model included miR-27b-3p (OR 23.1, 95% CI: 2.8-452, p = 0.015), let-7b-5p (OR 4.38, 95% CI: 1.28-22.2, p = 0.038), and miR-103a-3p (OR 15.3, 95% CI: 1.33-351, p = 0.049). These miRNAs may serve as biomarkers of treatment response in MM. However, external validation is necessary to confirm the clinical utility of our model.

BACKGROUND:Autologous hematopoietic stem cell transplantation (AHSCT) is currently the backbone of the treatment of multiple myeloma (MM) and relapsed and refractory lymphomas. Notably, infections contribute to over 25% of fatalities among AHSCT recipients within the initial 100 days following the procedure. In this study, we aimed to evaluate three selected miRNAs: hsa-miR-155-5p, hsa-miR-320c, and hsa-miR-361-3p, in identifying AHSCT recipients at high risk of infectious events up to 100 days post-transplantation after discharge. MATERIALS AND METHODS:The study group consisted of 58 patients (43 with MM, 15 with lymphoma) treated with AHSCT. Blood samples were collected from all patients at the same time point: on day +14 after transplantation. RESULTS:Fifteen patients (25.9%) experienced infectious complications after post-transplant discharge within the initial +100 days post-transplantation. The median time to infection onset was 44 days (interquartile range, 25-78). Four patients required hospitalization due to severe infection. High expression of hsa-miR-361-3p (fold change [FC], 1.79; P=0.0139) in the patients experiencing infectious complications and overexpression of hsa-miR-320c (FC, 2.14; P<0.0001) in patients requiring hospitalization were observed. In the multivariate model, both lymphoma diagnosis (odds ratio [OR], 6.88; 95% confidence interval [CI], 1.55-30.56; P=0.0112) and high expression of hsa-miR-361-3p (OR, 3.00; 95% CI, 1.40-6.41; P=0.0047) were independent factors associated with post-discharge infectious complications occurrence. Our model in 10-fold cross-validation preserved its diagnostic potential with an area under the receiver operating characteristic curve of 0.78 (95% CI, 0.64-0.92). CONCLUSION:Elevated serum hsa-miR-361-3p emerges as a promising biomarker for identifying patients at risk of infection during the early post-discharge period, potentially offering optimization of the prophylactic use of antimicrobial agents tailored to the specific risk profile of each AHSCT recipient.

Hepatic complications are an acknowledged cause of mortality and morbidity among patients undergoing hematopoietic stem cell transplantation. In this study, we aimed to evaluate the potential role in the prediction of liver injury of five selected microRNAs (miRNAs)—miR-122-5p, miR-122-3p, miR-15b-5p, miR-99b-5p, and miR-125a-5p—in the setting of autologous hematopoietic stem cell transplantation (ASCT). A total of 66 patients were included in the study: 50 patients (75.8%) with multiple myeloma (MM) and 16 (24.2%) with lymphoma. Blood samples were collected after the administration of the conditioning regimen, on the day of transplant (day 0). The expression levels of selected miRNAs were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the miRCURY LNA miRNA Custom PCR Panels (QIAGEN). In a multivariate logistic regression analysis adjusted for age, sex, and the administered conditioning regimen, two miRNAs, hsa-miR-122-5p (odds ratio, OR 2.10, 95% confidence interval, CI: 1.29–3.42, p = 0.0029) and hsa-miR-125a-5p (OR 0.27, 95% CI: 0.11–0.71, p = 0.0079), were independent for hepatic toxicity occurrence during the 14 days after transplant. Our model in 10-fold cross-validation preserved its diagnostic potential with a receiver operating characteristics area under the curve (ROC AUC) of 0.75, 95% CI: 0.63–0.88 and at optimal cut-off reached 72.0% sensitivity and 74.4% specificity. An elevated serum level of miR-122-5p and decreased level of miR-125a-5p on day 0 are independent risk factors for hepatotoxicity in ASCT recipients, showing promise in accurately predicting post-ASCT complications. Identifying patients susceptible to complications has the potential to reduce procedure costs and optimize the selection of inpatient or outpatient procedures.

Autologous hematopoietic stem cell transplantation (AHSCT) remains the most prevalent type of stem cell transplantation. In our study, we investigated the changes in circulating miRNAs in AHSCT recipients and their potential to predict early procedure-related complications. We collected serum samples from 77 patients, including 54 with multiple myeloma, at four key time points: before AHSCT, on the day of transplantation (day 0), and at days + 7 and + 14 post-transplantation. Through serum miRNA-seq analysis, we identified altered expression patterns and miRNAs associated with the AHSCT procedure. Validation using qPCR confirmed deviations in the levels of miRNAs at the beginning of the procedure in patients who subsequently developed bacteremia: hsa-miR-223-3p and hsa-miR-15b-5p exhibited decreased expression, while hsa-miR-126-5p had increased level. Then, a neural network model was constructed to use miRNA levels for the prediction of bacteremia. The model achieved an accuracy of 93.33

Infectious complications of autologous hematopoietic stem cell transplantation (AHSCT) are the most common adverse effects of the therapy, resulting in prolonged hospitalization and deterioration of patient well‐being. Identifying predictors of these complications is essential for improving patient outcomes and guiding clinical management. This study aimed to examine thrombospondin‐1 (THBS‐1) serum levels as a potential biomarker for predicting bacteremia in AHSCT recipients. Blood samples were collected from 30 patients undergoing BeEAM/BEAM (bendamustine/carmustine, etoposide, cytarabine, melphalan) conditioning regimen at subsequent time points during AHSCT. THBS‐1 levels were quantified using ELISA kits. Patients who developed bacteremia ( n = 11) during the AHSCT course had lower THBS‐1 concentration compared with those without ( n = 19) (22.88 ± 11.53 µg/mL vs. 15.24 ± 5.62 µg/mL, p = .0325). The ROC curve analysis revealed that THBS‐1 serum concentration at the first day of BeEAM/BEAM regimen had an area under the curve of 0.732 (95%CI: 0.5390.925, p = .0186) with an optimal cut‐off value of 16.5 µg/ml resulting in 82% Sensitivity and 53% Specificity for predicting bacteremia with a median of 11 days before its occurrence. Patients with lower THBS‐1 concentrations experienced febrile neutropenia significantly earlier, with a median difference of 5 days ( p = .0037). Patients with a low concentration of THBS‐1 had a higher risk of bacteremia and a shorter time to febrile neutropenia, indicating its potential value as a complications biomarker. Patients with lower serum THBS‐1 concentrations, indicating an increased risk, may be more suitable for an inpatient AHSCT procedure, where close monitoring and immediate intervention are accessible. image

Background: High-dose melphalan (MEL) followed by autologous hematopoietic stem cell transplantation (AHSCT) remains the treatment of choice for newly diagnosed multiple myeloma (MM) patients who are transplant-eligible. Despite variations in response, AHSCT outcomes are crucial in MM patients. Complete remission (CR) correlates with extended overall survival, while early relapse is linked to reduced survival, irrespective of cytogenetic risk. Identifying predictive biomarkers that might stratify patients according to their chance of achieving a response to AHSCT is essential for optimizing maintenance and consolidation therapy and improving patient outcomes. Aims: In our study, we investigated the changes in serum exosomal content in AHSCT recipients during the procedure and the potential differences in proteomic signatures depending on the response to AHSCT. Materials and methods: The study group consisted of 27 MM (12 women, 15 men) patients. The mean age was 60.5 ± 8.0 years. Nine patients (33.3%) had stage III, according to the Revised International Staging System (R-ISS). Most patients (18, 66.7%) received VCD (bortezomib, cyclophosphamide, dexamethasone) as an induction regimen. Most patients (18, 66.7%) received a MEL-200 mg/m2 conditioning regimen, 9 (33.3%) received a MEL-140. Patients received a median of 4.2 x 10 6 CD34+ cells/kg. Response assessment was available in 26 patients (96.4%). At +100 days after AHSCT, 12 patients (44.4%) achieved CR, four patients (14.8%) had a very good partial response, 6 (22.2%) had a partial response (PR), two patients (7.4%) had stable disease and two patients had disease progression. Blood samples were collected at four time points: before conditioning chemotherapy (T1), on day 0 (T2), day +7 (T3), and on day +14 after AHSCT (T4). Exosomes were isolated from 108 serum samples using exoEasy Maxi Kit (Qiagen). To perform protein identification, the proteins from exosome samples were trypsin digested. The obtained peptides were labelled using TMT10plex™ Isobaric Label Reagent Kit. After labelling, samples were subjected to LC-MS/MS analysis (RSLCnano liquid chromatography station Ultimate 3000, Thermo Scientific, mass spectrometer- Orbitrap Exploris 240, Thermo Scientific). All samples were analyzed in triplicates. Mass spectrometry data were searched against UP000005640_9606.fasta human database in Proteome Discoverer 2.4 (Thermo Scientific) software with Sequest algorithm. The average abundance of each protein was calculated. The heteroscedastic t-test was used to identify differentially expressed proteins (DEPs) according to clinical factors, and the paired t-test was used to compare protein abundances between study time points. The enrichment of functions and signaling pathways of the identified DEPs was determined using STRING database. Results: A total of 370 proteins with an average of 178.6 per sample were identified in exosomes derived from MM patients. Using the STRING database, the majority of proteins (207, 55.9%) were found to be localized in extracellular exosomes, confirming that isolated vesicles were exosomes. Proteins present in at least 50% of all samples were included for further analyses. When comparing time points with the application of correction for multiple comparisons, in T2 compared to T1, two downregulated DEPs were found. In T3 compared to T1, 21 DEPs were found, five up-regulated and 16 down-regulated. In T4 compared to T1, 21 DEPs were identified, seven were up-regulated, and 14 were down-regulated (Figure 1). In functional enrichment analysis, among the biological processes with the most involved DEPs between time points were response to stress, defense response, cell adhesion, regulation of stress response, and inflammatory response. In patients with suboptimal response to AHSCT (≤ PR), at T1 we found 13 DEPs, one up-regulated and 12 downregulated (Figure 2), that were highly enriched in genes related to platelet activation, innate immune system, platelet degranulation, post-translational protein phosphorylation and regulation of insulin-like growth factor. Conclusions: Our findings highlight the significant alterations in serum exosomal protein content during AHSCT and suggest its potential utility in risk-benefit stratification and optimizing treatment decisions in MM patients undergoing AHSCT.

Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM) microenvironment. Despite the progress made in treatment, some MM patients still die within the first year of diagnosis. Numerous studies investigating microRNA (miRNA) expression patterns suggest they may be good prognostic markers. The primary aim of this study was to analyze the expression of selected miRNAs in the serum of MM patients who were later treated with bortezomib-based regimens, and to determine their potential to predict early mortality. The study was conducted in 70 prospectively recruited patients with newly diagnosed MM admitted to the Department of Hematology of the Copernicus Memorial Hospital, Lodz (Poland) between 2017 and 2021. Among them, 17 patients experienced death within 12 months of diagnosis. The expression of 31 selected miRNAs was determined using a miRCURY LNA miRNA Custom PCR Panel. The obtained clinical data included patient characteristics on diagnosis, treatment regimen, response to treatment, and follow-up. Differential expression analysis found two miRNAs to be significantly downregulated in the early mortality group: hsa-miR-328-3p (fold change—FC: 0.72, p = 0.0342) and hsa-miR-409-3p (FC: 0.49, p = 0.0357). Univariate and multivariate logistic regression analyses were performed to assess the early mortality rate. The final model consisted of hsa-miR-409-3p, hsa-miR-328-3p, age, and R-ISS 3. It yielded an area under the curve (AUC) of 0.863 (95%CI: 0.761–0.965) with 88.2% sensitivity and 77.5% specificity. Further external validation of our model is needed to confirm its clinical value.

IntroductionAHSCT is the treatment of choice for newly diagnosed patients with transplant-eligible multiple myeloma (MM). However, considerable variability in response to autologous hematopoietic stem cell transplantation (AHSCT) results in only 50% of patients achieving complete response (CR) after AHSCT, which is directly associated with improved progression-free and overall survival (OS). In this study, we aimed to investigate the potential predictive role of selected serum miRNAs in MM patients who underwent AHSCT.Patients and methodsSerum expression level of 6 miRNAs: miR-221-3p, miR-15b-5p, miR-223-3p, miR-320c, miR-361-3p, and miR-150-5p was evaluated in 51 patients who underwent AHSCT. Blood samples were collected at two time points: before conditioning chemotherapy (T1) and fourteen days after transplant (+14) (T2).ResultsAll selected miRNAs significantly changed their expression level across the procedure- two were up-regulated after AHSCT: hsa-miR-320c (FC 1.42, p<0.0001) and hsa-miR-361-3p (FC 1.35, p=0.0168); four were down-regulated: hsa-miR-15b-5p (FC 0.53, p<0.0001), hsa-miR-221-3p (FC 0.78, p=0.0004), hsa-miR-223-3p (FC 0.74, p=0.0015) and hsa-miR-150-5p (FC 0.75, p=0.0080). Notably, before AHSCT, hsa-miR-223-3p was down-regulated in International Staging System (ISS) III patients (FC=0.76, p=0.0155), and hsa-miR-320c was up-regulated (FC=1.27, p=0.0470). These differences became non-significant after AHSCT. Eight (15.69%) patients achieved CR before AHSCT and 17 patients (33.33%) at +100 days after AHSCT. In multivariate logistic regression analysis, achievement of CR after induction and hsa-miR-223-3p at T1 were independent predictors of CR after AHSCT. In multivariate Cox regression analysis, hsa-miR-223-3p at T1 expression level was associated with prolonged OS (HR 0.06, 95%CI: 0.00 - 0.99, p=0.0488).ConclusionSerum expression of has-miR-223-3p is a predictor of CR and prolonged OS in MM patients undergoing AHSCT.

Multiple myeloma (MM) is characterized by the malignant proliferation of monoclonal plasma cells in the bone marrow with an elevation in monoclonal paraprotein, renal impairment, hypercalcemia, lytic bony lesions, and anemia. Immune cells and associated cytokines play a significant role in MM growth, progression, and dissemination. While some cytokines and their clinical significance are well described in MM biology, others remain relatively unknown. The present study examines the influence on progression-free survival (PFS) and overall survival (OS) by the serum levels of 27 selected cytokines in 61 newly diagnosed MM patients receiving first-line therapy with bortezomib-based regimens. The measurements were performed using a Bio-Rad Bio-Plex Pro Human Cytokine 27-Plex Assay and a MAGPIX Multiplex Reader, based on the Bio-Plex® 200 System (Bio-Rad). The following levels were determined: IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF. Most patients received a VCD chemotherapy regimen (bortezomib, cyclophosphamide, and dexamethasone). In the final multivariate model, IL-13 cytokine level (HR 0.1411, 95% CI: 0.0240–0.8291, p = 0.0302) and ASCT (HR 0.3722, 95% CI: 0.1826–0.7585, p = 0.0065) significantly impacted PFS. Furthermore, ASCT (HR 0.142, 95% CI: 0.046–0.438, p = 0.0007), presence of bone disease at diagnosis (HR 3.826, 95% CI: 1.471–9.949, p = 0.0059), and two cytokine levels—IL-1Ra (HR 1.017, 95% CI: 1.004–1.030, p = 0.0091) and IL-4 (HR 0.161, 95% CI: 0.037–0.698, p = 0.0147)—were independent predictors of OS. Three clusters of MM patients were identified with different cytokine profiles. In conclusion, serum pretreatment levels of IL-13 and IL-4 are predictors of better PFS and OS, respectively, whereas IL-1Ra pretreatment levels negatively impact OS in MM patients treated with bortezomib-based chemotherapy. Cytokine signature profile may have a potential influence on the outcome of patients treated with bortezomib.

While multiple myeloma (MM) treatment with proteasome inhibitors and other agents yields encouraging results, primary and secondary resistance remains an emerging problem. An important factor in such treatment resistance is the overexpression of several proteins. The present study comprehensively evaluates the expression of POMP, PSMB5, NRF2, XBP1, cMAF and MAFb proteins in plasma cells isolated from the bone marrow of 39 MM patients treated with bortezomib-based regimens using an enzyme-linked immunosorbent assay (ELISA). The proteins were selected on the basis of previous laboratory and clinical studies in bortezomib-treated MM patients. It was found that the expression of the investigated proteins did not significantly differ between bortezomib-sensitive and bortezomib-refractory patients. However, the expression of some proteins correlated with overall survival (OS); this was significantly shorter in patients with higher POMP expression (HR 2.8, 95% CI: 1.1–7.0, p = 0.0277) and longer in those with higher MAFB expression (HR 0.32, 95% CI: 0.13–0.80, p = 0.0147). Our results indicate that a high expression of POMP and MAFB in MM plasma cells may serve as predictors of OS in MM patients treated with bortezomib-based regimens. However, further studies are needed to determine the role of these factors in effective strategies for improving anti-myeloma therapy.

Proteasome inhibitors, like bortezomib, play a key role in the treatment of multiple myeloma (MM); however, most patients eventually relapse and eventually show multiple drug resistance, and the molecular mechanisms of this resistance remain unclear. The aim of our study is to assess the expression of previously described genes that may influence the resistance to bortezomib treatment at the mRNA level (ABCB1, CXCR4, MAF, MARCKS, POMP, PSMB5, RPL5, TXN, and XBP1) and prognosis of MM patients. mRNA expression was determined in 73 MM patients treated with bortezomib-based regimens (30 bortzomib-sensitive and 43 bortezomib-refractory patients) and 11 healthy controls. RPL5 was significantly down-regulated in multiple myeloma patients as compared with healthy controls. Moreover, POMP was significantly up-regulated in MM patients refractory to bortezomib-based treatment. In multivariate analysis, high expression of PSMB5 and CXCR and autologous stem cell transplantation were independent predictors of progression-free survival, and high expression of POMP and RPL5 was associated with shorter overall survival.

Abstract Introduction: Autologous hematopoietic stem cell transplantation (AHSCT) is an acknowledged and effective treatment method for hematopoietic system diseases. MicroRNAs were reported to impact the bone marrow niche microenvironment and regulate proliferation and survival of the hematopoietic stem cells in such a manner may also influence bone marrow convalescence after AHSCT. The project aimed to identify changes in the signature of miRNAs freely circulating in the serum during AHSCT related to chemotherapy-induced injury and further bone marrow recovery using next-generation sequencing. Patients and methods: Serum samples from 10 patients undergoing ASCT were collected. Blood samples were taken from each patient at four time points: (T1) before conditioning with high dose chemotherapy, (T2) on the day of AHSCT (day 0), on day +7 (T3), and on +14 day after AHSCT (T4). The myeloablative conditioning regimen for patients with MM was melphalan 200 mg/m 2, while in lymphoma patients, BEAM was used. Total RNA was extracted from 200 μl serum using miRNeasy Serum/Plasma Advanced Kit (QIAGEN) following manufacture instructions. Libraries were prepared from 5 μl of total RNA using QIAseq® miRNA Library Kit. The libraries were pooled in equimolar concentrations and sequenced on a NextSeq 550 System using a single-end read length of 75 nucleotides at an average of 10 million reads per sample (Illumina). In bioinformatics analysis, after adapter cut-off, filtration and mapping, miRNAs were counted based on mapping to reference miRbase 22 (tools: fastp, bowtie, samtools, picard). MicroRNAs were filtered to have at least 10 counts-per-million (CPM) of classified sequences in at least two samples. MiRNAs expression levels between time points were compared using paired t-test with Bonferroni correction. Results: The study group consisted patients with multiple myeloma (N=4), Hodgkin lymphoma (HL, N=3), and non-HL (N=3) aged 48±13 years. There was a significant decrease in the hematological parameters during ASCT with a nadir at T3, including hemoglobin (T1 vs. T3, p<0.0012), white blood cell count (p<0.0001), neutrophil count (p=0.0003), and platelet count (p<0.0001). Similarly, the decrease was observed in hsa-miR-223-3p (T1 vs. T3, p=0.048) and hsa-miR-18a-5p (T2 vs. T3, p=0.033) with a nadir at T3. On the other hand, an increase with a peak at T3 was observed in the expression of hsa-miR-320b (T1 vs. T3, p=0.007), hsa-miR-320c (T1 vs. T3, p=0.007), hsa-miR-320a-3p (T1 vs. T3, p=0.009), and hsa-miR-320d (T1 vs. T3, p=0.042). Interestingly, we have observed a gradual decrease across study timepoints in the expression of hsa-let-7f-5p, hsa-let-7i-5p, and hsa-miR-155-5p with a nadir at T4 (T1 vs. T4, p=0.004, p=0.01, and p=0.019, respectively). Similar changes were observed in the expression of hsa-miR-486-5p, but the statistically significant decline was only noted between T3 and T4 (p=0.024). Conversely, a gradual decrease was also seen in the expression of hsa-miR-96-5p, but there was a significant increment between T3 and T4 (p=0.036). Figure 1 presents the heatmaps for the miRNAs with significant expression changes and corresponding hematological parameters during AHSCT. Conclusion: Several significant changes in the miRNA expression profile were identified, both related to the chemotherapy-induced injury and subsequent bone marrow recovery. Figure 1 Figure 1. Disclosures Wierzbowska: Novartis: Consultancy; Abbvie: Consultancy; Jazz: Research Funding; Janssen: Consultancy; Astellas: Consultancy; Celgene/BMS: Consultancy.

Bortezomib is the first-in-class proteasome inhibitor, commonly used in the treatment of multiple myeloma (MM). The mechanisms underlying acquired bortezomib resistance in MM are poorly understood. Several cell-free miRNAs have been found to be aberrantly regulated in MM patients. The aim of this pilot study was to identify a blood-based miRNA signature that predicts bortezomib-based therapy efficacy in MM patients. Thirty MM patients treated with bortezomib-based regimens were studied, including 19 with refractory disease and 11 who were bortezomib sensitive. Serum miRNA expression patterns were identified with miRCURY LNA miRNA miRNome PCR Panels I+II (Exiqon/Qiagen). Univariate analysis found a total of 21 miRNAs to be differentially expressed in patients with MM according to bortezomib sensitivity. Multivariate logistic regression was created and allowed us to discriminate refractory from sensitive patients with a very high AUC of 0.95 (95%CI: 0.84–1.00); sensitivity, specificity and accuracy were estimated as 0.95, 0.91, and 0.93. The model used expression of 3 miRNAs: miR-215-5p, miR-181a-5p and miR-376c-3p. This study is the first to demonstrate that serum expression of several miRNAs differs between patients who are bortezomib refractory and those who are sensitive which may prove useful in studies aimed at overcoming drug resistance in MM treatment.

The aim of the study was to determine the levels of selected cytokines and chemokines in the serum of multiple myeloma (MM) patients treated with bortezomib-based regimens. A total of 71 MM patients were examined: 41 with primary refractory disease (17) or early relapse (28), and 30 who were bortezomib sensitive with no progression for at least six months. Patients who demonstrated CR or PR after bortezomib-based therapies longer than six months after treatment discontinuation were designated bortezomib sensitive. Serum cytokine levels were assayed with Bio-Rad Bio-Plex Pro Human Cytokine 27-Plex Assay on the MAGPIX Multiplex Reader and the Bio-Plex® 200 System (Bio-Rad). Higher levels of MIP-1 α and lower levels of MIP-1 β and IL-9 were associated with better responses to bortezomib-based treatment, and higher levels of IL-1ra and IL-8 were associated with bone involvement. MCP-1 was elevated in patients with hemoglobin<10 g/dl compared to those without anemia. The levels of IL-8, MIP-1 α , and TNF- α were significantly higher in patients with renal insufficiency. Only MIP-1 α was elevated in patients with hypercalcemia compared to patients with normal calcium levels. In conclusion, distinct cytokines are involved in the pathogenesis of MM and may play a prominent role in the prediction of treatment response. However, a single measurement of serum cytokines should be interpreted with caution and further studies are needed.

Chronic lymphocytic leukemia (CLL) treatment is improving; however, some patients do not respond to therapy. Due to the high heterogeneity in disease development, there is an urgent need for personalization of therapy. In the present study, the response of leukemic mononuclear cells to anticancer drugs used for CLL treatment (cladribine + mafosfamide; CM or CM combined with rituximab; RCM) was compared with the response to new cyclin-dependent kinase (CDK) inhibitors: BP14 and BP30. Viable apoptotic and necrotic cells were quantified by flow cytometry using propidium iodide and Yo-Pro stains. CDK inhibitors were studied in several doses to determine the reduction of necrosis and simultaneous increase of apoptosis in leukemic cell incubations with anticancer agents. The distinct cell response to applied doses/anticancer agents was observed. Results obtained in the current manuscript confirmed that modulation of doses is important. This was particularly indicated in results obtained at 24 h of cells incubation with anticancer agent. While an important time for analysis of anticancer response efficacy (monitoring of apoptosis induction potential) seems to be 48 h of cells exposition to anticancer agents. High variability in response to the drugs revealed that both the nature and the dose of the anticancer agents could be important in the final effect of the therapy. The present findings support the thesis that personalized medicine, before drug administration in the clinic, could be important to avoid the application of ineffective therapy.

OBJECTIVE:MicroRNAs engaged in angiogenesis and hematopoiesis can influence hematopoietic stem cells (HSCs) homing after transplantation by targeting bone marrow niche microenvironment. This study aimed to examine the kinetics of miRNA-15a, miRNA-16, miRNA-126, miRNA-146a, and miRNA-223 in autologous HSC transplantation settings.METHODS:The study comprised of 51 patients with hematological malignancies (42 multiple myeloma, 9 lymphoma). Samples were taken at four time points: before conditioning, after chemotherapy but prior to autologous HSC transplantation (day 0), on day +7, and +14 days after HSCT. The miRNA levels were evaluated by the real-time PCR method.RESULTS:A significant, steady decline of all tested microRNAs in the course of transplantation, as compared to the baseline, was found. The study revealed that higher levels of miRNA-15a, miRNA-16, miRNA-126, and miRNA-146a on day 0 correlated with longer time to engraftment. Additionally, a positive correlation between the levels of miRNA-15a, miRNA-146a, and miRNA-223 assessed on day +7 and the time to engraftment was observed.CONCLUSIONS:In conclusion, all investigated microRNAs changed significantly in the course of transplantation. Our results suggest that the miRNAs may participate in hematopoietic recovery in the early post-transplant period and influence engraftment efficiency after HSCT.

The Wnt/β-catenin signaling pathway is shown to play a significant role in the control of the survival, proliferation, and differentiation of hematopoietic cells. Studies have confirmed that aberrant activation of canonical Wnt signaling occurs in various forms of leukemia, and is crucial for chronic lymphocytic leukemia (CLL) pathogenesis. The aim of the study is to evaluate the influence of maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) on Wnt/β-catenin pathway gene expression in CLL (MEC-1) cells and to compare these findings with those obtained with fludarabine (FA). Microarray data analysis reveals seven of 19 Wnt/β-catenin pathway genes whose expression changes significantly during dendrimer and FA treatment: WNT10A, WNT6, and CDH1 among others. PPI-G4-M3 is already known to influence MEC-1 cell apoptosis and proliferation. The obtained results suggest that the reduction in cell survival under the influence of glycodendrimers and FA may be due to loss of Wnt signaling.

Patients with multiple myeloma (MM) are at increased risk of thrombosis. Growing evidence indicates that oxidative and nitrative modifications of proteins, including fibrinogen, may lead to changes in hemostasis. The study compares samples from patients with MM at diagnosis and healthy volunteers with regard to the oxidative/nitrative modifications of proteins, ROTEM and thrombin-catalyzed fibrin polymerization. The content of carbonyl groups in plasma proteins of patients with MM was significantly higher than in controls (2.981 vs. 1.807 nmol/mg of protein, p = 0.005), while no differences were seen in the concentrations of nitrated proteins. Maximum clot firmness readings were significantly higher in the samples of patients than in controls according to FIBTEM test (23.5 vs. 15 mm, p = 0.006). The lag time of the fibrin polymerization process and the velocity of clot lysis (V Lys) were found to be significantly higher in the group of MM patients than controls. In contrast, no marked differences were identified between studied groups in reference to maximal velocity of fibrin polymerization process (V max), maximal absorbance (A max) and plasmin amidolytic activity values. In conclusion, our study demonstrates that at the time of diagnosis, patients with MM demonstrated greater oxidative stress than healthy volunteers, which is reflected in a higher amount of carbonylated proteins. Some prothrombotic features found in ROTEM tests in MM patients were not confirmed by turbidimetry.

Abstract Introduction MicroRNAs are a class of small (19-25 nucleotides), endogenous RNA which play a significant role in regulation of gene expressions, cell proliferation, differentiation, apoptosis. These class of small molecules, engaged in angiogenesis and hematopoiesis can influence hematopoietic stem cells (HSC) homing after transplantation by targeting bone marrow niche microenvironment. The aim of the study was to evaluate expression of miRNA-15a, miRNA-16 (engaged in angiogenesis, apoptosis, proliferation and differentiation of HSC) miRNA-126, miRNA-146a (regulating homing of HSC after transplantion) and miRNA-223 (involved in apoptosis and granulopoiesis) in patients with lymphoproliferative malignancies prior to autologous hematopoietic stem transplantation (HSCT) and in early posttransplant period. Patients Fifty one patients were enrolled to the study (23 F, 28 M). The median (Me) age was 59 years. The investigated group consisted of 42 multiple myeloma (7 CR, 27 VGPR, 8 PR), 7 non-Hodgkin lymphoma (3 CR, 4 PR), and 2 Hodgkin lymphoma (2 PR). The blood (plasma samples) were collected on 4 time points: before chemotherapy - "BC", on the day of HSCT (after chemotherapy) - "0", seven days after HSCT "N" (nadir) - "N" and fourteen days after HSCT - "+14". Methods Total RNA was isolated using miRNeasy Serum/Plasma KIT (Qiagen, Hilden, Germany). MiRNA conversion to cDNA was performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). We used TaqMan MicroRNA Assays (Applied Biosystems): has-miR-15a-5p, has-miR-15a-5p, has-miR-146a-5p, has-miR-126-3p and has-miR-223-3p primers) to detect mature miRNA. Reverse transcription conditions were normalized using 5 nmol mirVana miRNA Mimic (cel-miR-39) as an endogenous control (Ambion, Austin, TX). The miRNA level was evaluated by Real Time PCR method. The Wilcoxon matched-pairs test was used to compare groups of dependent continuous variables: miRNA's RQ levels at two different time points. Spearman rank correlation coefficient (R) was used to compare independent variables: regeneration and miRNA's RQ level. Comparisons and correlations were considered significant if p<0.05. Results Kinetics of miRNA before conditioning treatment and after HSCT Our research revealed significant, steady decline of miRNA-15a, miRNA-16, miRNA-126, miRNA-146a and miRNA-223 at all time points as compared to baseline (BC). We observed increased miRNA-126 and miRNA-223 expression on "+14" day as compared to "N" day (Tab. 1). Correlation between miRNA expression and regeneration We observed that higher level of investigated miRNAs after chemotherapy resulted in longer time to engraftment. Our research revealed positive correlation between miRNA-15a, miRNA-16, miRNA-126 and miRNA146a levels on "0' day and the time to engraftment (R=0.36, p=0.009), (R=0.41, p=0.003), (R=0.30, p=0.03), (R=0.29, p=0.03) respectively. Additionally we found positive correlation between levels of miRNA-15a, miRNA-146a and miRNA-223 assessed on "N" day and time of regeneration (R=0.33, p=0.02), (R=0.34, p=0.02) and (R=0.30, p=0.04) respectively. No correlation between expression of all investigated miRNAs before chemotherapy and time to engraftment was found. Conclusions In conclusion, all investigated miRNAs changed significantly after conditioning treatment. Low expression of miRNA-15a and miRNA-16, as HSC proliferation and angiogenesis factors, play important role in regeneration process after chemotherapy and in early post-transplant period. Low expression of miRNA-223 on nadir day correlates with faster engraftment, which is probably related with miRNA-223 activity on different stages of hematopoietic cells development, especially myeloid precursors differentiation and maturation. Effective regeneration, correlated with decreased miRNA-126, miRNA-146a level after conditioning treatment, may be affected by well-known homing processes regulated by VCAM-1 and CXCR4/SDF-1 signaling. We suggest that investigated miRNAs expression in the early post-transplant period is significant for time to engraftment after HSCT. Disclosures Grzybowska-Izydorczyk: BMS: Honoraria; Novartis: Honoraria. Robak:AbbVie: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.