Magdalena Glowala-Kosinska

Association of circulating regulatory T cell number with the incidence and prognosis of diffuse large B‐cell lymphoma

Regulatory T (Treg) cells are essential for maintaining immune tolerance. High Treg frequencies have been reported in peripheral blood and tissue samples of patients with solid tumors while their role in lymphomas, including diffuse large B‐cell lymphoma (DLBCL) has not been clearly established. In this study, we analyzed the circulating Treg numbers in 27 patients with newly diagnosed DLBCL and 17 healthy individuals. Tregs were detected by flow cytometry based on CD4+CD25highFoxP3+ phenotype. In addition, the expression of CD45RA, HLA‐DR, CD62L, CD39, and CTLA4 was analyzed. The number of circulating Treg cells was lower in patients with DLBCL than in healthy controls: median 23 (range, 4–107)/μL vs. 41 (19–104)/μL (P = 0.04). In particular, the number of Tregs expressing CD45RA (naïve Tregs), HLA‐DR (marker of activation), and CD62L (L‐selectin) was decreased in the DLBCL group. Lower (below median) number of circulating Tregs was associated with reduced chance of achieving complete remission (29% vs. 69%, P = 0.05) and reduced probability of even‐free survival (24% vs. 84% at 1 yr, P = 0.0004), independently on the International Prognostic Index. We conclude that low number of circulating Tregs may be associated with poor prognosis in patients with DLBCL. However, our observations require confirmation in larger patient population.

A faster reconstitution of hematopoiesis after autologous transplantation of hematopoietic cells cryopreserved in 7.5% dimethyl sulfoxide if compared to 10% dimethyl sulfoxide containing medium ☆

Our previous in vitro studies proved a higher clonogenic potential of peripheral blood progenitor cells cryopreserved in 7.5% dimethyl sulfoxide (Me2SO) than in 10% Me2SO containing medium. Based on this findings 7.5% Me2SO cryopreservation medium was introduced to our protocol and both the hematopoietic recovery and infusion-related toxicity were compared with that obtained with standard 10% Me2SO containing solution. Two cohorts of consecutive patients treated with autologous hematopoietic stem cell transplantation were included in the analysis: 56 patients with PBPCs cryopreserved in 7.5% Me2SO solution and 52 patients who obtained cells cryopreserved in 10% Me2SO. Both study groups did not differ significantly with regard to age, diagnosis, and the number of transplanted CD34(+) cells. The time to leukocyte recovery was shorter for patients in the 7.5% Me2SO treated group than in the 10% one. Reconstitution of platelets and the frequency of adverse events did not differ in both groups. Reduction of Me2SO concentration from 10% to 7.5% in cryoprotective mixture has a beneficial impact on leukocyte recovery. These findings require verification in a prospective, randomized trial.

Very high efficacy of intermediate-dose cytarabine in combination with G-CSF as a second-line mobilization of hematopoietic stem cells

Autologous hematopoietic stem cell transplantation (autoHSCT) is used widely in the treatment of patients with lymphoid malignancies. Currently, 99 % of such procedures are performed using peripheral blood as a source of stem cells [1]. The generally accepted minimal level of CD34 cells required for rapid neutrophil and platelet recovery after autoHSCT is 2 9 10/kg. However, some data indicate that higher numbers are associated with less need for blood product transfusions and administration of antibiotics [2–4]. Mobilization regimens are based either on the use of granulocyte-colony stimulating factor (G-CSF) alone or G-CSF in combination with chemotherapy, most frequently cyclophosphamide 1.5–7 g/m or lymphoma-specific salvage regimens [5]. Unfortunately, a significant proportion of patients fail to mobilize sufficient number of CD34 cells, thus requiring additional attempts [6]. New mobilization strategies are being explored, including the use of plerixafor, CXCR4 inhibitor, in combination with G-CSF, with or without chemotherapy. This agent enabled sufficient CD34 cell harvest in 64.8–81.6 % of proven or predicted poor mobilizers [7, 8]. Unfortunately, such treatment is very expensive, which limits its worldwide application. Hence, the development of new strategies is still warranted. Although current studies focus on small molecules interfering with stem cell–stroma interactions, traditional chemotherapy-based salvage mobilization regimens have not been sufficiently explored. In our center, between November 2010 and September 2011, 14 patients who had failed chemotherapy-based mobilization were treated with salvage regimen including intermediate-dose cytarabine (ID-AraC) in combination with G-CSF. Most of the patients were referred for transplantation due to multiple myeloma (n = 8) or lymphoma (n = 5), while the remaining one had ovarian cancer. Patients had previously been treated with a median of 2 (range 1–5) lines of chemotherapy and most of them had received irradiation. Previous mobilization was usually based on cyclophosphamide plus G-CSF (Table 1). Six patients received AraC 400 mg/m (2 h infusions) every 12 h for three consecutive days (total dose, 2400 mg/m), while the following eight patients were treated with AraC for 2 days (total dose, 1600 mg/m). G-CSF 5–10 lg/kg was started on day 5 and continued daily until last leukapheresis. Leukaphereses were started once the peripheral blood CD34 cell count exceeded 15/lL and were performed using the Spectra-Optia Apheresis System (CaridianBCT Inc, Lakewood, CO, USA). The target number of CD34 cells collected was 5 9 10/kg for multiple myeloma (all planned for tandem autoHSCT) and 2 9 10/kg for the remaining patients. The maximum number of peripheral blood CD34 cells/lL after AraC ? G-CSF was 86 (17–312) and was significantly higher compared to the first-line mobilization: 8 (0–35) (Mann–Whitney U test, P = 0.002). No difference could be found according to the AraC dose: 80 (17–174) for 1.6 g/m versus 88 (24–312) for 2.4 g/m, P = 0.85. All patients collected required number of CD34 cells, which was achieved with a single apheresis in 10/14 (71.5 %) patients (Table 1). Although all patients experienced profound cytopenia and most required T. Kruzel M. Sadus-Wojciechowska J. Najda T. Czerw M. Glowala-Kosinska J. Holowiecki S. Giebel (&) Department of Bone Marrow Transplantation, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15 Street, 44-101 Gliwice, Poland e-mail: sgiebel@io.gliwice.pl

Evaluation of proinflammatory and immunosuppressive cytokines in blood and bone marrow of healthy hematopoietic stem cell donors

Introduction Cytokine composition of bone marrow microenvironment in comparison to blood is poorly explored. The goal of this study was to investigate the levels of cytokines present in peripheral blood and bone marrow of healthy hematopoietic stem cells donors. The data obtained on this subject with addition to cytometric analysis can provide new insight into the hematopoietic stem cells microenvironment. Methodology Study consisted of cytokine concentration analysis performed by ELISA tests of peripheral blood of healthy peripheral blood stem cells donors and bone marrow of healthy bone marrow donors. Additionally we have tested the expression of CD47 and CD274 proteins on the surface of hematopoietic stem cells by the flow cytometry analysis. Results The results has shown different composition of analyzed cytokines (IL‐1 &bgr;, IL‐2, IL‐4, IL‐6, IL‐10, IL‐17A, TGF‐&bgr;1, IFN‐&ggr; and TNF‐&agr;) present in bone marrow and blood of stem cells donors. The hematopoietic stem cells in peripheral blood are subjected to higher levels of proinflammatory cytokines whilst the lower level of those cytokines in bone marrow with a very high level of TGF‐&bgr;1 which possibly creates a more immunosuppressive environment. The IL‐10 level was significantly higher in peripheral blood of PBSC donors after the administration of mobilizing factor (G‐CSF). The percentage of CD47+HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors. HighlightsThe first study comparing cytokine concentration, expression of CD47 and CD274 immunosuppressive proteins on HSCs in bone marrow and blood of hematopoietic stem cells donors.Concentration of proinflammatory cytokines is higher in blood than in bone marrow.TGF&bgr; is present at very high concentrations in bone marrow.High level of IL‐10 was detected in peripheral blood of G‐CSF stimulated donors.CD47 protein expressions is higher in bone marrow than in peripheral blood.

The impact of circulating regulatory T cells number and their subpopulations on risk of lymph node metastasis

Background Regulatory T cells (Tregs) suppress the induction of immune response to cancer cells. An increased number of Tregs have been observed in many solid tumors. The aim of this study was to evaluate whether the number and percentage of circulating Tregs and their subpopulations differ in patients with gastric cancer (GC) and normal controls (NC). The relationship between Tregs subpopulations and clinical factors was analyzed.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Łomianki/Kiełpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Reduction of DMSO concentration in cryopreservation mixture from 10% to 7.5% and 5% has no impact on engraftment after autologous peripheral blood stem cell transplantation: results of a prospective, randomized study

The procedure of autologous peripheral blood stem cell transplantation (autoPBSCT) requires cryopreservation of cells in a mixture containing dimethyl sulfoxide (DMSO). DMSO is necessary to secure cell viability, however, its infusion may be toxic to stem cell recipient. The aim of this study was to prospectively evaluate the impact of DMSO concentration on engraftment after autoPBSCT.One-hundred-fifty patients were randomly assigned to one of three study arms; their leukapheresis products were cryopreserved in 10%, 7.5% or 5% DMSO. The study groups did not differ with regard to the diagnosis (mainly lymphomas and multiple myeloma), age, conditioning regimen, and the number of transplanted hematopoietic stem cells. 143 patients were treated with autoPBSCT. The frequency of adverse effects during and shortly after infusion was the lowest in 5% DMSO arm (p = 0.02 compared to 10% DMSO). 4 patients died due to infection before the engraftment. The median time to leukocyte and neutrophil recovery was 10 days in all study groups (p = 0.36 and p = 0.2). As well, the median day of platelet recovery was the same for all DMSO concentrations and equaled 15 days (p = 0.61).In view of these results, 5% DMSO mixture may be considered a new standard in cryopreservation of hematopoietic stem cells.

Increased Efficacy of Stem Cell Chemomobilization with Intermediate-Dose Cytarabine Plus Granulocyte Colony-Stimulating Factor (G-CSF) Compared with G-CSF Alone in Patients with Multiple Myeloma: Results of a Randomized Trial

Mobilization of hematopoietic stem cells for patients with multiple myeloma (MM) may be done using either steady-state granulocyte colony-stimulating factor (G-CSF) or a combination of chemotherapy with G-CSF. The goal of this randomized, open-label, phase 3 trial was to compare the efficacy of chemomobilization using intermediate-dose cytarabine (ID-AraC) plus G-CSF with G-CSF alone in patients with MM referred for tandem autologous stem cell transplantation (autoSCT). The percentage of patients with stem cell yield of at least 5 × 106 CD34+ cells/kg was the primary endpoint. Ninety patients were enrolled, including 44 assigned to the ID-AraC arm and 46 in the G-CSF arm. The threshold number of CD34+ cells was reached in 43 patients (98%) in the ID-AraC arm and in 32 patients (70%) in the G-CSF arm (P = .0003). The median number of collected CD34+ cells was 20.2 × 106 cells/kg in the ID-AraC arm versus 5.9 × 106 cells/kg in the G-CSF arm (P < .000001). A single apheresis was sufficient to achieve the required number of harvested CD34+ cells in 37 patients (86%) in the ID-AraC arm and in 13 patients (41%) in the G-CSF arm (P = .00008). The times to both neutrophil and platelet recovery after autoSCT were significantly shorter in the patients mobilized with ID-AraC. This study provides the first evidence of the advantage of chemomobilization over G-CSF monotherapy in terms of efficacy. ID-AraC with G-CSF should be the preferred chemomobilization protocol for patients with MM scheduled to undergo tandem autoSCT.

Immunological properties of bone marrow microenvironment 1 year after allogeneic hematopoietic stem cell transplantation

Regeneration of the bone marrow microenvironment after transplantation of allogeneic hematopoietic stem cells is poorly explored. The goal of our study was to investigate this process focusing on immunologic factors: concentrations of selected cytokines, expression of immunosuppressive proteins CD47 and CD274 on hematopoietic stem cells, and frequency of T regulatory lymphocytes (Tregs). Bone marrow samples were collected before transplantation, on the day of transplantation, and at the 1-year follow-up. As a control group, we used bone marrow from healthy donors. Prior to the conditioning, the percentage of Tregs and concentration of interleukin-10 were higher in the bone marrow of patients than in healthy donors. The conditioning regimen resulted in increased concentrations of interferon-γ and expression of CD274 on hematopoietic stem cells. Twenty-eight days after transplantation, level of Tregs, expression of CD47, and concentration of interleukin-10 and latency-associated peptide 1 were increased compared with the period before conditioning. Starting from day 100 after transplantation, the microenvironment tended to normalize; the level of Tregs and concentrations of most cytokines were similar to values in the bone marrow of healthy donors.