Abstract
The purpose of this study was to assess the effect of preoperative chemoradiotherapy (CRT) for operable gastric cancer on lymphocyte subpopulations counts. A total of 35 patients with ...adenocarcinoma of the stomach were investigated. The preoperative CRT regime consisted of radiotherapy and chemotherapy. The total dose of 45 Gy was administered in 25 fractions, with 5 fractions per week for 5 weeks. Analysis of lymphocyte subpopulations in peripheral blood was carried out before and during treatment (5 analyses for each patients) using a BD Multitest IMK Kit (Becton Dickinson). The data were analyzed with ANOVA. Before and after CRT, the mean values (MVs) of the subtype counts (SD) were as follow: lymphocytes, 1,788 cells/µl (±694) and 239 cells/µl (±131); CD3+CD4+ cells, 738 cells/µl (353) and 135 cells/µl (55); CD3+CD8+ cells, 562 cells/µl (±398) and 56 cells/µl (±41); CD3-CD19+ cells, 150 cells/µl (±112) and 2 cells/µl (±2); and CD16+CD56+ cells, 329 cells/µl (±155) and 28 cells/µl (±36). Every week, we have observed the gradual depletion of all subset of lymphocytes and those differences were statistically significant (p<0.0000). Before CRT, the MV of the CD4+/CD8+ ratio was 1.58 (SD±0.97 ). This ratio has gradually increased during CRT. At the end of treatment, the MV of the CD4+/CD8+ ratio was 2,96 (SD±2,04), p<0.0000. Preoperative chemoradiotherapy induces depletion of all lymphocyte subpopulations.
•The 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.•TGFβ 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.
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.
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.
The results has shown different composition of analyzed cytokines (IL-1 β, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-β1, IFN-γ and TNF-α) 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-β1 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.
Abstract Non-cytotoxic innate lymphoid cells (ILCs) have been added to the list of immune cells that may contribute to the tumor microenvironment. Elevated levels of total ILCs and their subgroups ...have been reported in peripheral blood and tissue samples from patients with solid tumors, but their frequency in non-Hodgkin lymphomas, particularly diffuse large B-cell lymphoma (DLBCL), has not been clearly established. This study examined frequency and subset distribution in newly diagnosed DLBCL patients (nodal and extra-nodal) and compared it with blood specimens from healthy donors. The percentage of total ILCs (Lin − CD127+) was assessed by flow cytometry, as well as the four ILC subsets, defined as ILC1 (Lin − CD127 + cKit − CRTH2−), ILC2 (Lin − CD127 + cKit+/- CRTH2+), ILCp NCR- (Lin − CD127 + cKit + CRTH2- NKp46-) and NCR + ILC3 (Lin − CD127 + cKit + NKp46+). In the studied group of patients ( n = 54), significantly lower levels of circulating total ILCs, ILC1, and ILCp NCR- were observed compared to the control group ( n = 43). Similarly, there was a statistically significant decrease in the median frequency of NKp46 + ILC3 cells in lymphoma patients. Analysis of the ILC2 subpopulation showed no significant differences. The correlation of the distribution of individual subpopulations of ILCs with the stage and location of the tumor was also demonstrated. Our results suggest that circulating ILCs are activated and differentiated and/or differentially recruited to the lymph nodes or tumor microenvironment where they may be involved in antitumor defense. However, our observations require confirmation in functional studies.
High-dose therapy with autologous stem cell support (autologous stem cell transplantation, autoSCT) remains the standard of care for eligible patients with multiple myeloma (MM). Planned tandem ...autoSCT which can also be considered for all transplant candidates requires collection of at least 5 x 10^6 CD34+ cells/kg. Randomized trials comparing chemomobilization with use of cyclophosphamide (CY) + G-CSF to G-CSF alone did not demonstrate clear advantage of addition of CY to growth factor. In recent years, intermediate-dose cytosine arabinoside (ID-AraC) + G-CSF has been proposed, showing high mobilization potential, as demonstrated in retrospective analysis (Giebel et al, Bone Marrow Transplant 2013, 48: 915-21). The goal of this prospective, randomized trial (ClinicalTrials.gov identifier: NCT01908621) was to compare the efficacy of ID-AraC + G-CSF with G-CSF alone in patients with MM referred for double autoSCT.
The inclusion criteria were set as follows
1) the diagnosis of MM, 2) age 18-65 years, 3) at least partial remission achieved after one or more lines of therapy including six or more cycles containing components like thalidomide, lenalidomide , bortezomib, or melphalan, 4) planned tandem autoSCT procedure.
The proportion of patients with stem cell yield of at least 5 × 10^6 CD34+ cells/kg was the primary study end-point.
Mobilization regimens were as follows
1) ID-AraC arm - AraC 2 x 0.4 g/m2 on days 1,2 (total 1.6 g/m2) + filgrastim 10 ug/kg/day starting from day 5; 2) G-CSF arm - filgrastim 10 μg/kg/day for five consecutive days. Leukaphereses were started when the level of circulating CD34+ cells in peripheral blood (PB) reached at least 10/uL and were continued for the maximum period of three days or until reaching the target CD34+ cell yield. Leukaphereses were performed using Spectra Optia Apheresis System, Mononuclear Cell Collection Procedure (Therumo BCT Inc., Lakewood, CO, USA), processing 2 total blood volumes.
Between March 2013 and March 2016, 44 and 46 patients were randomly assigned to ID-AraC and G-CSF study arm, respectively. Patients in the ID-AraC arm were younger (median age 56 years, range (33-65) for ID-AraC and 60 years (37-65) for G-CSF, p=0.04). The groups did not differ in terms of MM remission status at mobilization (p=0.3), the number of preceding lines of chemotherapy (p=0.5) and the frequency of preceding radiotherapy (p=0.4).
The level of CD34+ cells in PB required to start leukaphereses was achieved in all patients in the ID-AraC arm and in 44 (96%) of them in the G-CSF arm, p=0.2.
In the ID-AraC group, 43 patients (98%) collected at least 5 x 10^6 CD34+ cells/kg compared to 32 patients (70%) in the G-CSF group (p=0.0003). In a multivariate model adjusted for age, the use of G-CSF alone was independently associated with increased risk of mobilization failure (Odds ratio = 17.5; 95% CI = 2.1-144.9; p=0.007).
The median peak number of circulating CD34+ cells was 346 (11-1044)/µL vs. 40 (1-326)/µL (p<0.000001), while the median number of collected CD34+ cells was 20.2 (2.9-59.4) x10^6/kg vs. 5.9 (0-11) x10^6/kg, respectively (p<0.000001). A single apheresis was sufficient to achieve the threshold number of harvested CD34+ cells in 37 of cases (86%) after ID-AraC+G-CSF compared to 13 (41%) after G-CSF alone (p=0.00008). The median day of the first apheresis was 13 (range, 12-15; SD=0.7) after ID-AraC.
Mobilization with ID-AraC + G-CSF is associated with significantly higher efficacy than G-CSF alone. In the studied group it allowed for collection of CD34+ cell number adequate for tandem autoSCT in almost all MM patients, usually with a single leukapheresis. This study provides the first evidence coming from prospective, randomized trial for the advantage of chemomobilization over G-CSF monotherapy in terms of proportion of patients achieving CD34+ yield sufficient for autoSCT.
No relevant conflicts of interest to declare.
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.
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.
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.
Abstract 3018
Cryopreservation of autologous peripheral blood progenitor cells (PBPCs) in 10% DMSO is a routine in most transplantation centers. During ASH 2011 Meeting, we presented the results of ...in vitro research, concerning the optimization of DMSO concentrations for recovery and clonogeneic potential of PBPCs after thawing. We concluded, that reduction of DMSO concentration from 10% to 7.5% may have favorable impact for cell clonogeneicity. Accordingly, we implemented new cryoprotective mixture (7.5% instead of 10% DMSO) into clinical practice. The purpose of this study was to clinically evaluate the changed protocol.
In our department, between Jan 2012-Aug 2012, 56 patients were transplanted with autologous PBPCs cryopreserved in 7.5% DMSO solution (median of age: 57 years, range: 21–66). We compared the data concerning hematopoietic engraftment and the frequency of side effects with historical control – 52 subsequent patients treated with transplantation of PBPSc cells cryopreserved in 10% DMSO (median of age: 57 years, range: 21–66) in a preceding period. Both study groups did not differ significantly with regard to the diagnosis (mostly lymphoproliferative disorders) or disease status at transplantation. As well, the number of transplanted CD34+ cells was comparable: median 6.5′106/kg (range 1.5–24.7) for 7.5% DMSO and 7.5′106/kg (2.1–24.6) for 10% DMSO group, p=0.68. All received G-CSF (filgrastim) starting on day +7 after transplantation.
The volume of infused DMSO was significantly lower in patients who obtained PBPBc cryopreserved in 7.5% DMSO (median 22.5 ml, range 7.5–45) than 10% DMSO (median: 30 ml, range, 10–160); p=0.02. The time to leukocyte recovery >1′109/L was faster for 7.5% DMSO (median: 11 days, range: 9–12) than in 10% DMSO (median 11 days, range: 10–13), p=0.03. Similarly, reconstitution of neutrophils >0.5′109/L was faster for 7.5% DMSO group: median 11 days (range 9–13 days) vs. 11 days (range 10–13 days), respectively; p = 0.04. We didn’t observe significant difference with regard to platelet recovery >50′109/L (median 12 days, range: 0–21 days for 7.5% DMSO vs. median 12.5, range: 0–19 days for 10% DMSO). Hospital stay since HSCT was shorter in case of 7.5% group (median: 14 days, range: 11–21) than 10% group (median: 15 days, range: 13–25); p=0.04. Number of RBC and platelets transfusions as well as transfusion-related complications did not differ between the groups. Adverse events after transplantation were mild and transient, usually grade 1 nausea, and occurred in 20 (38%) patients in 10% DMSO group compared to 24 (43%) in 7.5% DMSO group (p=0.7).
The analysis of newly implemented cryopreservation protocol suggest that reduction of the DMSO concentration from 10% to 7.5% is associated with faster leukocyte and neutrophil recovery as well as shorter hospital stay. These findings require verification in a prospective, randomized trial. Display omitted
No relevant conflicts of interest to declare.
Abstract 4131
High-dose therapy followed by autologous peripheral blood stem cell transplantation (autoPBSCT) is widely applied in the treatment of lymphoid malignancies. However, a significant ...proportion of patients fail to mobilize sufficient number of stem cells after commonly used protocols.
In this study we evaluated efficacy of first-line mobilization based on cytosine arabinoside (AraC) 1600 mg/m2 + G-CSF (filgrastim) 5–10 ug/kg. Results of 70 subsequent patients (diagnosis multiple myeloma, MM-39, Hodgkin’s lymphoma, HD-9, non-Hodgkin lymphoma, NHL-22) were compared with 45 individuals mobilized with cyclophosphamide (CTX) (4 g/m2) + G-CSF in a preceding period (MM-25; HD-4; NHL-16). In respective groups, 39 (56%) and 30 (67%) patients were classified as “predicted” poor mobilizers, defined as the presence of at least one risk factor: age >60 years, thrombocytopenia, preceding treatment with melphalan, fludarabine, preceding radiotherapy on pelvis, >8 courses of chemotherapy for MM, >3 courses containing cisplatin or carboplatin for lymphomas, preceding high-dose treatment with autoHSCT. In addition, we analyzed efficacy of AraC+G-CSF in 14 patients who had failed previous chemo-mobilization, mostly with CTX+G-CSF.
All but three patients (96%) treated with AraC reached >15 CD34+ cells/uL in peripheral blood, compared to 67% in the CTX+G-CSF cohort (p<0.0001). Median peak level was 127 (3–780) CD34+ cells/uL vs. 33 (1–240), respectively (p<0.0001). After AraC, 68 (97%) patients collected >=2×10e6 CD34+ cells/kg and 58 (84%) collected >=5×10e6 CD34+ cells/kg with the median 14.9 (2.3–54.6), which was achieved with a single leukapheresis in 92% patients. Using CTX, 67% patients collected sufficient number of stem cells (p<0.0001) and 76% required 2 or more leukaphareses (p<0.0001). AraC+G-CSF was effective in 37 (97%) predicted poor mobilizers compared with 16 (64%) in the CTX+G-CSF group (p=0.0006). The toxicity related to both regimens was comparable. Among patients who proceeded to autoPBSCT the time to neutrophil recovery after transplantation was the same for AraC and CTX cohorts (median 12 days), while platelet engraftment was faster for patients mobilized with AraC (13 days vs. 14 days, p=0.06).
All 14 patients who had failed previous mobilization collected >=2×10e6 CD34+ cells/kg after AraC+G-CSF, with the median 9.3 (2.5–25.9). It was achieved with a single apheresis in 10 (71%) cases.
Mobilization based on intermediate doses of AraC+G-CSF is highly effective allowing adequate CD34+ harvest in almost all patients with lymphoproliferative diseases, including predicted and proven poor mobilizers. The level of mobilized CD34+ cells is four times higher compared to protocol based on CTX.
No relevant conflicts of interest to declare.
PDF
