Nitin Mathur

Hypoxia improves expansion potential of human cord blood–derived hematopoietic stem cells and marrow repopulation efficiency

Objectives:  In bone marrow, hematopoietic stem cells (HSCs) reside in the most hypoxic endosteum niche, whereas the proliferating progenitors are located near the relatively oxygen‐rich vascular region. High oxygen tension is potentially detrimental to HSCs. The objective of this investigation was to compare cellular, functional, and molecular responses of human umbilical cord blood (UCB)‐derived hematopoietic stem and progenitor cells in culture under hypoxic and normoxic conditions.

Evaluation of four methods for processing human cord blood and subsequent study of the expansion of progenitor stem cells isolated using the best method.

BACKGROUND AIMS The application of cord blood (CB)-derived hematopoietic cells in allogeneic transplantation has been restricted because of the unavailability of adequate cell numbers from one unit of CB and their delayed engraftment in recipients. The main purpose of this study was to develop an economic process for isolating nucleated cells from CB and expanding enriched CD133(+) cells. METHODS Four protocols for processing CB, using different combinations of density-gradient centrifugation, hydroxyethyl starch (HES) and NH4Cl treatment, were compared regarding the yields of CD45(+), CD34(+)/CD133(+) and colony-forming cells. CD133-enriched cells were expanded for 10 days in the presence of hematopoietic growth factor-supplemented medium. The performance of the culture was evaluated by analyzing colony-forming potential, fold-expansion of primitive hematopoietic stem cells (HSC) and lineage commitment by flow cytometry. RESULTS A two-step treatment of CB with HES followed by NH4Cl resulted in the highest recovery of CD45(+) (84.3%) and CD34(+) (69.1%) cells compared with that present in umbilical CB. A suspension culture of CD133-enriched cells resulted in an average 150-fold increase in nucleated cells, of which CD133(+), CD34(+) and CD34(+) CD38 cell expansions were 17.2+/-1.3, 40+/-4.6, and 6.9+/-1.1 fold, respectively. The total myeloid colony-forming cell count was almost 3800-fold higher than that present in the processed CB. CONCLUSIONS The highest yields of nucleated and progenitor stem cells were obtained with a two-step processing of CB. The CD133(+) cells obtained by this method are expected to yield enough hematopoietic progenitors for potential allogeneic transplantation.

Minimal residual disease evaluation in autologous stem cell transplantation recipients with multiple myeloma

The use of novel agents for the treatment of multiple myeloma (MM) has enabled attainment of deeper responses and the information that progression-free survival (PFS) and overall survival (OS) correlate with the depth of response has further created a need for the development of sensitive laboratory assays for the detection of minimal residual disease (MRD).[1,2] The serum protein electrophoresis (SPEP) has been in use for more than 30 years and although, still a gold standard for the diagnostic work-up, it has limited role in MRD detection due to its low sensitivity, false positivity due to presence of oligoclonal bands during the phases of bone marrow regeneration after chemotherapy or autologous stem cell transplantation (ASCT) and inability to monitor light-chain and nonsecretory MM.[3–5] The serum-free light-chain assay (SFLC) was initially propagated for the diagnosis and monitoring of nonsecretory and light-chain MM, but a recent study in patients with detectable M band has revealed its ability to predict a better PFS and OS for those who attained normal SFLC ratio (SFLCR) after treatment.[6] The same is reflected in the international response criteria for MM where the patients with normalization of SFLCR are accorded the status of being in stringent CR (sCR).[7] Multiparametric flow cytometry (MFC) has limited role at the diagnostic front in MM, but a number of studies have shown its prognostic utility in detecting MRD.[1,5,8,9] However, hemodilution of the bone marrow aspirate and the patchy nature of myeloma disease are major limitations resulting in false-negative MRD result using MFC.[10,11] Assessments of adequacy of the bone marrow aspirate on morphological assessment or the presence of normal plasma cells (PC) on MFC or estimating neoplastic plasma cell index are some of the remedial measures suggested to offset the false negativity of MFC in MRD detection in MM.[10,11] However, till date, there is no consensus on the method that is best suited for MRD detection in MM. We report, here, a comparison of the conventional CR (negative SPEP and IFX plus <5% PCs in BM) with sCR (CR plus normal SFLC ratio [SFLCR]) and iCR by MFC in MM patients undergoing ASCT and discuss the discrepant results. In this prospective observational study, 71 consecutive patients of MM who received ASCT after conditioning with high-dose melphalan were evaluated for residual disease using conventional laboratory methods of serum and urine protein electrophoresis plus immunofixation, SFLCR and MFC. The MFC was performed using a fivecolor a panel of antibodies: CD138 FITC, CD38 PE-Cy5, CD45 PE-Cy7, CD56 PE and CD19 PE-CF594 (BD Biosciences). A minimum of 500,000 events were acquired on a flow cytometer (FC-500, Beckman Coulter) and the data were analyzed using FCS-Express software version 4.0 (Denovo Software). Plasma cells were gated as events with bright expression of CD38, CD138 and dim expression of CD45. Minimal residual disease was defined as positive if a discrete population of phenotypically aberrant PC defined as >50 CD19-CD56þ events was identified in the 500,000-event free file (0.01% limit of detection). All the investigations were done as part of pretransplant work-up and at Dþ 100 post-transplant and at Dþ 180 except MFC which was done at first two time points only. Disease response was assessed as per International Myeloma Working Group (IMWG) response criteria for multiple myeloma;[7,11] stringent complete response (sCR) was defined as bone marrow PC <5% with negative serum and urine protein electrophoresis with

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia

BackgroundIn chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients.ResultsThe integration of DNA methylation profile (n = 14) with the gene expression profile (n = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients (n = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1, PMEPA1, SOX7, SPRY1, CDK6, TBX2, and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup (p < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p = 0.005) or PAX9 (RR = 1.87, p = 0.001). High expression of CRY1 (HR: 3.53, p < 0.001) or PAX9 (HR: 3.14, p < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172–9.272, p = 0.016) was also predictive of shorter overall survival in CLL.ConclusionsThe DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.

Immunoglobulin heavy chain variable region gene repertoire and B-cell receptor stereotypes in Indian patients with chronic lymphocytic leukemia

Abstract In chronic lymphocytic leukemia (CLL), the geographical bias in immunoglobulin heavy-chain variable (IGHV) gene usage lead us to analyze IGHV gene usage and B-cell receptor stereotypy in 195 patients from India. IGHV3, IGHV4, and IGHV1 families were the most frequently used. 20.5% sequences had stereotyped BCR and were clustered in 12 pre-defined and 6 novel subsets. Unmutated IGHV was significantly associated with reduced time to first treatment (p < 0.033) and poor overall survival (OS; p = 0.01). We observed a significant difference in OS between IGHV1, IGHV3, and IGHV4 family cases (p = 0.045) in early stage patients. Regarding subfamily usage, only IGHV1-69 expression was found to have statistically significant poor outcome (p = 0.017). Our results from the analysis of various molecular and clinical features suggest that the expression of specific IGHV gene influences the outcome in early stage CLL, and hence its assessment may be added to the clinical leukemia laboratory armamentarium.

Vascular endothelial growth factor expression in pediatric non-Hodgkin lymphoma: A prospective study

Serum vascular endothelial growth factor (VEGF) level is higher in children with nonHodgkin lymphoma (NHL) than healthy controls, but its prognostic significance is unknown [1]. VEGF expression has been evaluated in serum [2] and in tissue using immunohistochemistry [3] and polymerase chain reaction (PCR) [4]. After institute ethical approval, treatment-naïve patients of pediatric NHL 18 years were included from July 2012 to March 2014. Patients with early-stage nonlymphoblastic lymphomas were treated with short-course chemotherapy [5]; advanced-stage nonlymphoblastic lymphoma received NHL-BFM-90 protocol [6] and lymphoblastic lymphoma BFM-90 ALLlike protocol [7]. Survival was censored on 15 September 2015. Baseline serum VEGF-A immune reactivity was assessed using quantitative sandwich enzyme immunoassay (Quantikine Human VEGF-A Immunoassay; R&D Systems, Minneapolis, MN, USA). Cytoplasmic VEGF expression in tumor cells was assessed using immunohistochemistry with mouse monoclonal VEGF antibody (VEGF:Ab-3, Clone:JH121; Neomarkers, Fremont, CA, USA). Intensity was graded negative (0), weak, moderate, or intense. VEGF staining intensity 1+ in 10% tumor cells was considered positive. Polymerase chain reaction (PCR) was performed using gene-specific primer pair for VEGF-A and glyceraldehyde-3-phosphate dehydrogenase. There were 50 patient samples and 10 healthy ageand sex-matched controls. Median age of patients was 11 years (2–18 years). Stage III/IV disease was present in 74% patients; bone marrow was involved in 9 patients. Median VEGF level in patients was 648.2 pg/mL (16.98–3335.5 pg/mL). Median serum VEGF in patients was higher than controls (P = .03). Bone marrow involvement in lymphoma was associated with increased serum VEGF (P = .03) (Table 1). Immunohistochemistry for VEGF was performed in 27/50 tissue blocks. Positivity was observed in 17/27 (63%) patients (Supplemental Figure 1). No significant correlation was observed with VEGF positivity and baseline characteristics (Table 1). The number of patients with moderate or intense VEGF expression was 3/7 in stages I and II and 5/20 in stages III and IV (P = .37). Serum VEGF versus VEGF positivity (plotted as positive/negative) using Spearman’s correlation showed positive correlation (r = .44; P = .02).

Serial assessment of circulating T regulatory cells and T helper 17 cells in pediatric non-Hodgkin lymphoma: a prospective study

T regulatory cells (Tregs) (CD4 + CD25 + cells) have immunosuppressive function and constitute 5–10% of all peripheral CD4 + lymphocytes. T-helper 17 (TH17) cells are new effector T-Helper cells that play an important role in autoimmunity and protection from bacteria. TH17 cells and Tregs constitute two opposing immune responses that are critically involved in modulation of inflammation induced by either autoimmunity or bacterial infection [1]. Studies of Tregs in adult patients with non-Hodgkin lymphoma (NHL) have reported variable results with regard to prognosis [2]. There is no existing data to describe the relevance of Tregs and TH17 cells in pediatric NHL. In view of this gap in our knowledge, this study was planned with the aim to determine Tregs and TH17 cells at baseline and on follow-up in children with NHL and compare the same with healthy controls and baseline demographics. The secondary objective was to evaluate interrelationship of Tregs and TH17 cells, and also to evaluate their impact on outcome. Treatment naive pediatric patients ( 18 years) of NHL were included in this study from October 2012–March 2014. The study was approved by Institute Ethics Committee. Informed consent was obtained from guardians of patients and control subjects. Patients with early stage non-lymphoblastic lymphomas were treated with short course chemotherapy [3], advanced stage non-lymphoblastic lymphomas were treated with NHL BFM-90 protocol [4] and patients with lymphoblastic lymphoma patients were treated with BFM-90 ALL like protocol [5]. Five millilitres of peripheral blood was collected in anticoagulant coated vacutainers from patients at baseline (Sample-0) prior to initiation of treatment, post treatment (Sample-1) and at relapse/progression (Sample-R). Sample-1 was obtained at the following time-points: in lymphoblastic lymphoma at end of induction; early stage non-lymphoblastic lymphomas at end of treatment; and in non-lymphoblastic stage 3 and 4 lymphoma after two cycles of chemotherapy. For Sample-1, the samples were specifically collected after recovery of blood counts prior to administration of next cycle of chemotherapy. Sample-R was obtained at relapse or progression prior to initiation of salvage chemotherapy. Samples were also collected from ten healthy controls. All samples were processed within 6 h. Mononuclear cells (MNCs) were separated from peripheral blood using density gradient media (Ficoll Histopaque-1077, Sigma Aldrich) and stained with CD4 Phycoerythrin carboxy flourescein 5 (PECF594), CD25 phycoerythrin cyanin 7 (PE-Cy7), CD127 PE and FoxP3 Fluorescein isothiocyante (FITC) (BD Biosciences, San Jose, CA). Briefly, 2 10 peripheral blood MNCs were incubated with antibodies for surface staining followed by fixation and permeabilization for FoxP3 staining as per manufacturer’s recommendations (BD Biosciences). Minimum of 2 10 events were acquired on flowcytometer (FC500, Beckman Coulter) and analyzed using FCS expression ver 3.0 (denovo software). Using sequential gating strategy, Tregs were identified as CD4 + CD127 CD25 high cells expressing FoxP3. Treg events were the positive cells from total CD4 + cells of gated lymphocytes, while absolute numbers of Tregs per cubic millimeter were calculated from absolute lymphocyte counts. For TH17 cell, MNCs were incubated in Rosewell Park Memorial Institute medium and 5% CO2 at 37 C with leucocyte activation cocktail and golgi plug (BD Biosciences), followed by fluorochrome staining with CD3 (PC5), CD8 (FITC), IL-17 (PE) and anti-human IFN-g monoclonal antibody (PECy 7) after fixation and