scholarly journals Post-Transplant Cyclophosphamide (PTCY) Vs Anti-Thymoglobulin (ATG) As Part of the Gvhd Prophylaxis for Fludarabine/Clofarabine/Busulfan Reduced Intensity Conditioning (RIC) in Allogeneic Stem Cell Transplantation (allo-SCT): Influence on Early Immune Reconstitution

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1955-1955
Author(s):  
Christelle Retiere ◽  
Catherine Willem ◽  
Thierry Guillaume ◽  
Henri Vié ◽  
Laetitia Gautrot-Rolland ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Conditioning Regimen ◽  
Immune Recovery ◽  
Post Transplant ◽  
Gvhd Prophylaxis ◽  
The Impact

Abstract Introduction: The influence of PTCY on early immune reconstitution after allo-SCT has been poorly studied so far, especially in comparison to standard use of ATG as GVHD prophylaxis. Patients and Methods: A prospective study was conducted at the CHU of Nantes with the aim to compare early immune recovery between patients receiving PTCY or ATG as GVHD prophylaxis after a RIC allo-SCT. Secondary objectives were to study the impact of 1 vs 2 days of PTCY (CY1 vs CY2) or ATG (A1 vs A2), and of fludarabine (Flu) vs clofarabine (Clo) as part of the RIC regimen. As such, 3 RIC regimens were considered in both groups: FluCY2 (Baltimore regimen, Luznic, BBMT 2008), FluCY1, CloCY2 (where Clo replaces Flu), on one hand, and FluB2A2, CloB2A2, CloB2A1 (Chevallier, Haematologica, 2014), on the other hand. FluCY2 and FluB2A2 are currently standard of care RIC regimens for patients with haplo-identical and matched donors, respectively. Five patients had to be included in each RIC subgroup, depending on the type of disease and donor (/): FB2A2 (lymphoid/matched); FluCY2 (lymphoid/haplo); CloB2A2 or CloB2A1 (myeloid/matched); CloCY2 (myeloid/haplo), FluCY1 (myeloid or lymphoid/matched). The source of graft was peripheral blood stem cells for all cases. Blood samples were collected before starting the conditioning regimen then 3 times per week from day +0 until day+30, and at days +60 and +90. The following cell subsets were studied: a/b and g/d CD3+, CD8+ and regulatory (Tregs) T cells, B and NK cells and monocytes. The median percentage (%) compared to total lymphocytes was considered for all lymphocytes subsets between days 0-30. Thereafter, median absolute numbers (AN)/mm3 were considered for samples collected at days +30, +60 and +90. The study was approved by the IRB of the CHU of Nantes and all patients provided informed consent. Results: Between August 2014 and May 2015, thirty patients were included, including 15 in both groups and 5 in each RIC subgroups. Median age was 61 years. There were more patients with active disease at transplant (47% vs 7%) and more haplo-identical donors (67% vs 0%) in the PTCY group. All patients engrafted and were alive at day +90. However, 1 PTCY patient with T-ALL relapsed before day+100. Within the first month post-transplant, PTCY group had a significantly higher median % of a/b T cells (69.1 vs 18.9, p<0.0001) and Tregs (3.46 vs 0.45, p<0.0001) while ATG group had higher median % of NK (23 vs 2.57, p<0.0001) and B-cells (0.88 vs 0.43, p=0.0002). Between day+30 and day+90, ATG group had significant higher median counts of a/b T cells at days +60 (1316 vs 79.6, p=0.0001) and +90 (795.8 vs 151.6, p=0.03); g/d T cells at day+60 (27.6 vs 1.26, p=0.002); CD8 T cells at day+60 (735 vs 29.6, p=0.008); NK cells at day+30 (203.7 vs 89, p=0.04) and monocytes at days +30 (455.5 vs 221.7, p=0.009) and +60 (832.5 vs 247.2, p=0.004). Compared to the standard FluCY2 regimen, although not significant, FluCY1 was associated with higher median %, between days 0-30 of g/d T cells (2.32 vs 0.8) and higher median AN of g/d T cells at days +30 (9.2 vs 1.02) and +60 (9.22 vs 1.05), of B cells at days +30 (0.4 vs 0.14) and +60 (1.6 vs 0.39) and of NK cells at day+30 (213.9 vs 82.7). Compared to the standard FB2A2 regimen, although not always significant, CloB2A1 was associated with higher median % between days 0-30 of Tregs (0.97 vs 0.25, p=0.002) and higher median AN of g/d T cells at day+30 (6.8 vs 2), B cells at days +30 (2.35 vs 0) and +60 (43.7 vs 0.19), NK cells at day +30 (288 vs 62.1) and Tregs at days +30 (4.3 vs 0.2), +60 (8.8 vs 1.14) and +90 (8.96 vs 3.45). Compared to the standard FB2A2 regimen, although not always significant, CloB2A2 was associated with higher median % between days 0-30 of a/b T cells (28.87 vs 3.78, p=0.01) and B cells (0.76 vs 0.5, p=0.02) and higher median AN of a/b T cells at days +30 (324.3 vs 125.8) and +90 (1594 vs 604.3), of g/d T cells at day +30 (7.35 vs 2), of CD8 + T cells at days +30 (209.7 vs 38.9) and +90 (1211.7 vs 504.6), of B cells at day +30 ( 1.41 vs 0), of NK cells at days +30 (303 vs 62.1), +60 (514.5 vs 225.7) and +90 (647 vs 102.8, p=0.03) and of monocytes at days +30 (688.9 vs 257.4, p=0.03) and +90 (2157.4 vs 611.2). Conclusion: Strong differences exist in term of early immune recovery when using PTCY or ATG as part of the GHVD prophylaxis for RIC allo-SCT. Dose or drug modifications within the standard RIC regimen in both groups may be envisaged to favor some cell population recoveries after allo-SCT in order to increase outcome in patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Post-Transplant Cyclophosphamide Ameliorates Lethal Thymic GVHD By Restoring Regulatory and Effector T Cell Homeostasis in Recipients with Prior PD-1 Blockade Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 479-479
Author(s):  
Shuntaro Ikegawa ◽  
Yusuke Meguri ◽  
Takumi Kondo ◽  
Hiroyuki Sugiura ◽  
Yasuhisa Sando ◽  
...  
Keyword(s):  
Overall Survival ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
T Cell Subsets ◽  
Post Transplant ◽  
Gvhd Prophylaxis ◽  
Donor T Cells ◽  
The Impact

Abstract Allogeneic HSCT has a curative potential for patients with hematological malignancies. However, graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after HSCT. Regulatory T cells (Tregs) are critical mediator for immune tolerance after HSCT and we recently reported that PD-1 plays an essential role for Treg survival (Asano et al, Blood 2017). Clinical studies suggested that PD-1 blockade prior to HSCT could be a risk of increasing severe GVHD. However, the mechanisms about GVHD induced by PD-1 blockade have largely unclear and there remains a paucity of data on appropriate GVHD prophylaxis for patients who undergo HSCT after PD-1 blockade. To address these issues, we investigated the impact of PD-1 expression on donor T cells on immune reconstitution with murine BMT models. First, lethally irradiated B6D2F1 mice were transplanted with 10 million of C57BL/6-background PD-1+/+ or PD-1-/- spleen cells with 5 million of bone marrow cells from normal C57BL/6, and GVHD scores and overall survival was monitored. Recipients receiving PD-1-/- graft developed severe GVHD resulting in a significant shorter survival than recipients receiving PD-1-/- graft (P<0.0001). We analyzed lymphocytes in spleen and thymus on day3, 7, and 14. We found that CD8 T cells in PD-1-/- group showed markedly higher Ki67 expression and CFSE-dilution until day3. Interestingly, PD-1-/- Tregs increased aggressively at day3 but it could not maintain until day14, while PD-1-/- CD8 T cells and conventional CD4 T cells (CD4 Tcons) continued to increase until day+14, resulting in the significant higher CD8/Treg ratio in PD-1-/- group (P<0.05, vs PD-1+/+ group). PD-1-/- Tregs showed significantly higher expression of Annexin V on day+7 and thymus CD4- and CD8- double-positive (DP) cells were in the extremely low levels in PD-1-/- group on day+14 (P<0.05, vs PD-1+/+ group). Thymic analysis showed that donor PD-1-/- graft-derived CD8 T cells infiltrated thymus in PD-1-/- group, suggesting reconstruction of thymic function was critically disturbed by severe GVHD. These data suggest that loss of PD-1 signaling resulted in unbalanced reconstitution of donor-derived T cell subsets as a consequence of continuous CTL expansion and increased Treg apoptosis. Next, to evaluate the impact of post-transplant cyclophosphamide (PTCy) on the abnormal reconstitution after PD-1 blockade, we administered 50mg/kg of Cy or control vehicle on day3. PTCy efficiently ameliorated GVHD in PD-1-/- group and extended overall survival by safely regulating the proliferation and apoptosis of T cell subsets. Of note, after PTCy, Tregs regained the ability of continuous proliferation in the first 2 weeks, resulting in well-balanced reconstitution of donor-derived T cell subsets. Thymic DP cells on day 14 was markedly increased in PD-1-/- group with PTCy intervention as compared to without PTCy, suggesting PTCy could rescue thymus from PD-1 blockade-related severe GVHD. Finally, to evaluate GVL activity, we performed BMT with co-infusion of P815L tumor cells on day0 and we confirmed that PTCy treatment for PD-1-/- recipients reduced the severity of GVHD with maintaining sufficient GVL effect. In summary, our data suggested three insights about the impact of PD-1 signaling on immune reconstitution. First, PD-1 inhibition influenced graft-derived T cells very differently within T cell subsets. PD-1-/- Tregs increased transiently but it was counterbalanced by accelerated apoptosis, while PD-1-/- CD4+Tcons and CD8 T cells continued the drastic expansion. Second, we found that PD-1-/- donor T cells developed severe GVHD in thymus. Few reports have concentrated on the impact of donor graft PD-1 expression to thymus after BMT and acute GVHD in thymus could lead late central immune disturbance. Third, PTCy successfully ameliorated GVHD induced by PD-1-/- donor T cells preserving GVL effect. Cell proliferation study implied that PD-1-/- graft-derived CD8 T cells might be more susceptible for PTCy because of the high-rate proliferation. In conclusion, PD-1-/- graft cause lethal thymic GVHD and PTCy successfully ameliorated it. The influence of PD-1 inhibition was different within T cell subtypes. PTCy might be appropriate GVHD prophylaxis strategy for patients who had prior usage of PD-1 blockade. Disclosures No relevant conflicts of interest to declare.

Induction of Alloantigen Specific CD4 T Cell Anergy by B7: CD28 Mediated Costimulatory Blockade Significantly Alters the Functional Program of Bystander CD8, Monocytes, NK and B Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3656-3656
Author(s):  
Gullu Gorgun ◽  
Fenglong Liu ◽  
Eleanor Howe ◽  
Patrick Philpot ◽  
Eva Guinan ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Functional Program ◽  
The Impact

Abstract Retention of antigen (Ag) specific immunity to pathogens and tumor cells in the context of adequate control of alloreactivity remains a challenge for allogeneic hematopoietic stem cell transplantation (HSCT). Global or subset depletion of T cells achieves control of alloreactivity at the cost of loss of non-allospecific repertoire. Induction of Ag specific anergy has been explored as a way to selectively impact alloreactivity. Anergy is induced when Ag is presented to CD4 T cells without CD28:B7 mediated costimulation. We and others have shown that CD4 anergy results from a positive signaling cascade, rendering Ag specific T cells unable to proliferate and produce cytokines on Ag specific rechallenge. When this concept was translated to a haploidentical HSCT clinical trial, all donor marrow harvest mononuclear cells were subjected to inhibition of CD28 mediated costimulation by CTLA-4-Ig, a fusion protein binding both B7.1 and B7.2 costimulatory molecules. In our ongoing clinical trial, unfractionated donor peripheral blood mononuclear cells (PBMC) are cultured with allostimulators in the presence of costimulatory blockade (CSB) provided by anti-B7.1 and anti-B7.2 humanized monoclonal antibodies. To understand the impact of inducing alloAg specific CD4 T cell anergy on bystander PBMC, we investigated the effects on CD8 T cells, monocytes, B and NK cells. Mimicking our ex vivo clinical anergization protocol, primary MLR using PBMC from fully HLA mismatched healthy donors (n=12) were performed in the presence or absence of CSB. CSB resulted in 73% mean inhibition of proliferation after 72 hrs of primary MLR. CD4 and CD8 T cells, monocytes, NK and B cells from these MLRs were isolated by negative selection as were control unmanipulated CD4 and CD8 T cells. From each population, RNA was extracted and global gene expression profiling performed using Affymetrix human 133plus2 chips. Unsupervised analysis was performed using DNA-Chip Analyzer and supervised analysis using Significance Analysis for Microarrays. While the frequency of alloreactive CD4 T cells in human PBMC is only 1–10%, we observed global gene expression variance (436 genes) between unstimulated CD4 cells vs. CD4 cells isolated from MLR in the presence or absence of CSB (P≤0.05). Even more surprising was the impact of CSB on bystander CD8, monocytes, NK and B cells. Between cells from MLR with or without CSB, there were 632 differentially expressed genes in CD8 T cells, 105 differentially expressed in NK cells, 85 differentially expressed in monocytes and 1781 in B cells (P≤0.05 for all populations tested). We observed not only expected alterations (e.g, in inflammatory cytokines and receptor mediated signaling) but also observed changes [e.g. in proteasome degradation (i.e. CD4, CD8, NK and B cells) and in expression of genes regulating cell motility (i.e. CD8 T and NK cells)]. These results demonstrate that induction of alloAg specific anergy within a small population of alloreactive CD4 T cells results in dramatic alterations of the genetic repertoire of bystander cells. Thus, blockade of the CD28:B7 costimulatory pathway not only inactivates alloreactive CD4 T cells but also alters the functional program of other immune cells that will repopulate the host post-HSCT.

B-cell and T-cell values in peripheral blood in Polish mixed-breed rabbits with addition of blood of meet breeds

2014 ◽  
Vol 17 (3) ◽  
pp. 421-426 ◽  
Author(s):  
B. Tokarz-Deptuła ◽  
P. Niedźwiedzka-Rystwej ◽  
B. Hukowska-Szematowicz ◽  
M. Adamiak ◽  
A. Trzeciak-Ryczek ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Laboratory Animals ◽  
Immunological Parameters ◽  
Four Seasons ◽  
Mixed Breed ◽  
The Impact

Abstract In Poland, rabbit is a highly valued animal, due to dietetic and flavour values of its meat, but above all, rabbits tend to be commonly used laboratory animals. The aim of the study was developing standards for counts of B-cells with CD19+ receptor, T-cells with CD5+ receptor, and their subpopulations, namely T-cells with CD4+, CD8+ and CD25+ receptor in the peripheral blood of mixed-breed Polish rabbits with addition of blood of meet breeds, including the assessment of the impact of four seasons of the year and animal sex on the values of the immunological parameters determined. The results showed that the counts of B- and T-cells and their subpopulations in peripheral blood remain within the following ranges: for CD19+ B-cells: 1.05 - 3.05%, for CD5+ T-cells: 34.00 - 43.07%, CD4+ T-cells: 23.52 - 33.23%, CD8+ T-cells: 12.55 - 17.30%, whereas for CD25+ T-cells: 0.72 - 2.81%. As it comes to the season of the year, it was observed that it principally affects the values of CD25+ T-cells, while in the case of rabbit sex, more changes were found in females.

Engineered immunostimulatory cells can convert PBMCs from chronic lymphocytic leukemia (CLL) patients into potent tumor killing immune cells.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 7517-7517
Author(s):  
Joshua W. Keegan ◽  
Frank Borriello ◽  
Stacey M. Fernandes ◽  
Jennifer R. Brown ◽  
James A. Lederer
Keyword(s):  
T Cells ◽  
B Cells ◽  
Tumor Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Killing Activity ◽  
Tumor Killing ◽  
Tumor Cell Killing ◽  
Potent Tumor

7517 Background: Alloplex Biotherapeutics has developed a cellular therapeutic that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand populations of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). This process leads to a 300-fold expansion of NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells that are called SUPLEXA cells, which will be cryopreserved and transferred back into patients as an autologous immune cell therapy for cancer. In this study, PBMCs from CLL patients were used to generate SUPLEXA cells as a first approach to comparatively profile SUPLEXA cells from cancer patients and normal healthy volunteers (NHVs). Methods: ENLIST cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million (M) PBMCs from 10 CLL patients or 2 NHVs were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Results: Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF) using a 47-marker antibody panel. CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all 10 CLL patients showed expansion of NK cells (17%), CD8 T cells (11%), and CD4 T cells (7.5%) that were similar in phenotype to SUPLEXA cells from NHVs showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells (60% ± 9.5%) was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and NHV patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell (MEL-14 melanoma cells expressing RFP) ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients (77.8% ± 2.6% at 2:1) and NHVs (81.5% ± 0.3% at 2:1) were nearly identical at all effector to target ratios. Conclusions: We demonstrate for the first time that PBMCs from CLL patients can be converted into SUPLEXA cells despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

scholarly journals Impact of Different T Cell Depletion Protocols on Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Amandine Pradier ◽  
Adrien Petitpas ◽  
Anne-Claire Mamez ◽  
Federica Giannotti ◽  
Sarah Morin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Cell Depletion ◽  
Unrelated Donor ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapeutic modality for a variety of hematological malignancies and congenital disorders. One of the major complications of the procedure is graft-versus-host-disease (GVHD) initiated by T cells co-administered with the graft. Removal of donor T cells from the graft is a widely employed and effective strategy to prevent GVHD, although its impact on post-transplant immune reconstitution might significantly affect anti-tumor and anti-infectious responses. Several approaches of T cell depletion (TCD) exist, including in vivo depletion using anti-thymocyte globulin (ATG) and/or post-transplant cyclophosphamide (PTCy) as well as in vitro manipulation of the graft. In this work, we analyzed the impact of different T cell depletion strategies on immune reconstitution after allogeneic HSCT. Methods We retrospectively analysed data from 168 patients transplanted between 2015 and 2019 at Geneva University Hospitals. In our center, several methods for TCD are being used, alone or in combination: 1) In vivo T cell depletion using ATG (ATG-Thymoglobulin 7.5 mg/kg or ATG-Fresenius 25 mg/kg); 2) in vitro partial T cell depletion (pTCD) of the graft obtained through in vitro incubation with alemtuzumab (Campath [Genzyme Corporation, Cambridge, MA]), washed before infusion and administered at day 0, followed on day +1 by an add-back of unmanipulated grafts containing about 100 × 106/kg donor T cells. The procedure is followed by donor lymphocyte infusions at incremental doses starting with 1 × 106 CD3/kg at 3 months to all patients who had received pTCD grafts with RIC in the absence of GVHD; 3) post-transplant cyclophosphamide (PTCy; 50 mg/kg) on days 3 and 4 post-HSCT. Absolute counts of CD3, CD4, CD8, CD19 and NK cells measured by flow cytometry during the first year after allogeneic HSCT were analyzed. Measures obtained from patients with mixed donor chimerism or after therapeutic DLI were excluded from the analysis. Cell numbers during time were compared using mixed-effects linear models depending on the TCD. Multivariable analysis was performed taking into account the impact of clinical factors differing between patients groups (patient's age, donor type and conditioning). Results ATG was administered to 77 (46%) patients, 15 (9%) patients received a pTCD graft and 26 (15%) patients received a combination of both ATG and pTCD graft. 24 (14%) patients were treated with PTCy and 26 (15%) patients received a T replete graft. 60% of patients had a reduced intensity conditioning (RIC). 48 (29%) patients received grafts from a sibling identical donor, 94 (56%) from a matched unrelated donor, 13 (8%) from mismatched unrelated donor and 13 (8%) received haploidentical grafts. TCD protocols had no significant impact on CD3 or CD8 T cell reconstitution during the first year post-HSCT (Figure 1). Conversely, CD4 T cells recovery was affected by the ATG/pTCD combination (coefficient ± SE: -67±28, p=0.019) when compared to the T cell replete group (Figure 1). Analysis of data censored for acute or chronic GVHD requiring treatment or relapse revealed a delay of CD4 T cell reconstitution in the ATG and/or pTCD treated groups on (ATG:-79±27, p=0.004; pTCD:-100±43, p=0.022; ATG/pTCD:-110±33, p&lt;0.001). Interestingly, pTCD alone or in combination with ATG resulted in a better reconstitution of NK cells compared to T replete group (pTCD: 152±45, p&lt;0.001; ATG/pTCD: 94±36, p=0.009; Figure 1). A similar effect of pTCD was also observed for B cells (pTCD: 170±48, p&lt;.001; ATG/pTCD: 127±38, p&lt;.001). The effect of pTCD on NK was confirmed when data were censored for GVHD and relapse (pTCD: 132±60, p=0.028; ATG/pTCD: 106±47, p=0.023) while only ATG/pTCD retained a significant impact on B cells (102±49, p=0.037). The use of PTCy did not affect T, NK or B cell reconstitution when compared to the T cell replete group. Conclusion Our results indicate that all TCD protocols with the only exception of PTCy are associated with a delayed recovery of CD4 T cells whereas pTCD of the graft, alone or in combination with ATG, significantly improves NK and B cell reconstitution. Figure 1 Disclosures No relevant conflicts of interest to declare.

Natural Killer Cell Immune Reconstitution Predicts Outcomes for Patients with Chronic Lymphocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3300-3300
Author(s):  
Don Benson ◽  
Leslie Andritsos ◽  
Mehdi Hamadani ◽  
Thomas Lin ◽  
Joseph Flynn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Nk Cell ◽  
Disease Status ◽  
Lymphocytic Leukemia ◽  
T Cell Subsets ◽  
Cell Recovery

Abstract Introduction: Chronic lymphocytic leukemia (CLL), the most common form of leukemia in the Western hemisphere, is associated with severe innate, adaptive and humoral immune dysregulation. CLL remains essentially incurable, with the potential exception of allogeneic stem cell transplantation (ASCT). Natural killer (NK) cells are CD56(+), CD3(−) large granular lymphocytes that comprise a key cellular subset of the innate immune system. Preliminary in vitro data suggest an NK cell versus CLL effect exists, similar to that observed in acute myeloid leukemia (AML) and other blood cancers. Novel immune therapies for CLL (e.g., rituximab, alemtuzumab) likely exert anti-tumor effect, in part, through NK cells, in fact. Although NK cells contribute to the graft-versus-tumor effect following ASCT for other blood cancers, little is known regarding the potential role NK cells may play in the clinical allogeneic transplant setting for CLL. Herein, we provide, to our knowledge, the first report regarding NK cell immune reconstitution following ASCT for CLL. Methods: 27 CLL patients underwent reduced intensity conditioning (RIC) with ASCT. Median age was 52 years (43–69), median number of prior therapies was 3 (2–11). 55% had chemotherapy-refractory disease, and 55% had “high-risk” cytogenetics by FISH (deletion 17p or 11q22-23 abnormality). 14 patients had sibling donors, 15 had volunteerunrelated donors. Conditioning regimens included Fludarabine/TBI/Alemtuzumab (n=8), Fludarabine/Busulfan with (n=9) or without ATG (n=6), and Fludarabine/Cyclophosphamide (n=4). GVHD prophylaxis consisted of tacrolimus/MMF (n=8) or tacrolimus/methotrexate (n=19). Patients underwent bone marrow assessment prior to day +75 following ASCT. Marrow was studied for engraftment, donor chimerism, and disease status as well as lymphoid immune reconstitution by percentage of total lymphocytes and absolute lymphocyte counts by multi-color flow cytometry. Results: NK cell immune reconstitution was predicted by disease status at transplantation. Patients in complete or partial remission at the time of ASCT had more robust NK cell recovery (mean = 45% of total lymphocytes +/− SEM 5%) as compared to patients entering transplant with refractory disease (16% +/− 1, p < 0.01). No differences were observed in CD4(+) or CD8(+) T cells and no lymphocyte subset recovery was associated with CD34(+) or CD3(+) cell dosage. Achieving complete donor chimerism by day +60 was associated with robust NK cell recovery (55% +/− 1 versus 7% +/−1, p = 0.02), recovery of CD4 and CD8 T cells was not associated with chimerism status, however. Patients who went onto exhibit a complete response to ASCT had greater early NK cell reconstitution (31% +/− 3) as compared to those who had no response (8% +/− 1, p = 0.01). No differences in T cell subsets were associated with response. Patients who ultimately achieved complete remission following transplant had a lower CLL:NK cell ratio in marrow (0.35 +/− 0.07) than those who did not (8.1 +/− 1, p = 0.01). However, differences in CLL:CD4(+) and CLL:CD8(+) T cells were not predictive of response. Trends to improvement in progression free survival and overall survival were observed for patients with NK cell reconstitution above the median for the group as compared to those below; no such trends were observed regarding T cell subsets. Greater NK cell reconstitution trended towards ultimate eradication of minimal residual disease following ASCT, but no such trends were observed for T cell subsets. Conclusions: Early NK cell recovery predicts survival following autologous and allogeneic SCT in a number of hematologic malignancies; however, little is known regarding this phenomenon in CLL. To our knowledge, these are the first findings to implicate a potentially important therapeutic role for early NK cell compartment recovery in CLL following ASCT. Further research into restoring and augmenting NK cell function following RIC/ASCT for CLL is warranted.

Anti-GvHD Effect of Antithymocyte Globulin Is Mediated by Antibodies Binding to T Cells and Regulatory NK Cells, and Less So or Not at All by Antibodies Binding to Other Mononuclear Cell Subsets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2346-2346
Author(s):  
Mette Hoegh-Petersen ◽  
Minaa Amin ◽  
Yiping Liu ◽  
Alejandra Ugarte-Torres ◽  
Tyler S Williamson ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Antithymocyte Globulin ◽  
Chronic Gvhd ◽  
Wilcoxon Test ◽  
Effector Memory

Abstract Abstract 2346 Introduction: Polyclonal rabbit-anti-human T cell globulin may decrease the likelihood of graft-vs-host disease (GVHD) without increasing the likelihood of relapse. We have recently shown that high levels of antithymocyte globulin (ATG) capable of binding to total lymphocytes are associated with a low likelihood of acute GVHD grade 2–4 (aGVHD) as well as chronic GVHD needing systemic therapy (cGVHD) but not increased likelihood of relapse (Podgorny PJ et al, BBMT 16:915, 2010). ATG is polyclonal, composed of antibodies for antigens expressed on multiple cell subsets, including T cells, B cells, NK cells, monocytes and dendritic cells. These cell subsets may play a role in the pathogenesis of GVHD. The anti-GVHD effect of ATG may be mediated through killing/inhibition of one or several of these cell subsets (eg, T cells) or their subsets (eg, naïve T cells as based on mouse experiments naïve T cells are thought to play a major role in the pathogenesis of GVHD). To better understand the mechanism of action of ATG on GVHD, we set out to determine levels of which ATG fraction (capable of binding to which cell subset) are associated with subsequent development of GVHD. Patients and Methods: A total of 121 patients were studied, whose myeloablative conditioning included 4.5 mg/kg ATG (Thymoglobulin). Serum was collected on day 7. Using flow cytometry, levels of the following ATG fractions were determined: capable of binding to 1. naïve B cells, 2. memory B cells, 3. naïve CD4 T cells, 4. central memory (CM) CD4 T cells, 5. effector memory (EM) CD4 T cells, 6. naïve CD8 T cells, 7. CM CD8 T cells, 8. EM CD8 T cells not expressing CD45RA (EMRA-), 9. EM CD8 T cells expressing CD45RA (EMRA+), 10. cytolytic (CD16+CD56+) NK cells, 11. regulatory (CD16-CD56high) NK cells, 12. CD16+CD56− NK cells, 13. monocytes and 14. dendritic cells/dendritic cell precursors (DCs). For each ATG fraction, levels in patients with versus without aGVHD or cGVHD were compared using Mann-Whitney-Wilcoxon test. For each fraction for which the levels appeared to be significantly different (p<0.05), we determined whether patients with high fraction level had a significantly lower likelihood of aGVHD or cGVHD than patients with low fraction level (high/low cutoff level was determined from ROC curve, using the point with maximum sum of sensitivity and specificity). This was done using log-binomial regression models, ie, multivariate analysis adjusting for recipient age (continuous), stem cell source (marrow or cord blood versus blood stem cells), donor type (HLA-matched sibling versus other), donor/recipient sex (M/M versus other) and days of follow up (continuous). Results: In univariate analyses, patients developing aGVHD had significantly lower levels of the following ATG fractions: binding to naïve CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients developing cGVHD had significantly lower levels of the following ATG fractions: capable of binding to naïve CD4 T cells, CM CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients who did vs did not develop relapse had similar levels of all ATG fractions. In multivariate analyses, high levels of the following ATG fractions were significantly associated with a low likelihood of aGVHD: capable of binding to naïve CD4 T cells (relative risk=.33, p=.001), EM CD4 T cells (RR=.30, p<.001), naïve CD8 T cells (RR=.33, p=.002) and regulatory NK cells (RR=.36, p=.001). High levels of the following ATG fractions were significantly associated with a low likelihood of cGVHD: capable of binding to naïve CD4 T cells (RR=.59, p=.028), CM CD4 T cells (RR=.49, p=.009), EM CD4 T cells (RR=.51, p=.006), naïve CD8 T cells (RR=.46, p=.005) and regulatory NK cells (RR=.55, p=.036). Conclusion: For both aGVHD and cGVHD, the anti-GVHD effect with relapse-neutral effect of ATG appears to be mediated by antibodies to antigens expressed on naïve T cells (both CD4 and CD8), EM CD4 T cells and regulatory NK cells, and to a lesser degree or not at all by antibodies binding to antigens expressed on B cells, cytolytic NK cells, monocytes or DCs. This is the first step towards identifying the antibody(ies) within ATG important for the anti-GVHD effect without impacting relapse. If such antibody(ies) is (are) found in the future, it should be explored whether such antibody(ies) alone or ATG enriched for such antibody(ies) could further decrease GVHD without impacting relapse. Disclosures: No relevant conflicts of interest to declare.

Influence of Graft Versus-Host Disease Prophylaxis Regimen On T-Cell Repertoire Diversity Following Reduced-Intensity HLA-Matched Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3054-3054 ◽  
Author(s):  
Rachel B. Salit ◽  
Frances T. Hakim ◽  
Michael R. Bishop ◽  
Thea M. Friedman ◽  
Robert Korngold ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Unrelated Donor ◽  
Matched Unrelated Donor ◽  
Cell Repertoire ◽  
Post Transplant ◽  
Gvhd Prophylaxis ◽  
Repertoire Diversity ◽  
Reduced Intensity

Abstract Abstract 3054 Background: A clearly superior graft-versus-host disease (GVHD) prophylaxis regimen has not been established for patients undergoing reduced intensity allogeneic hematopoetic stem cell transplantation (HSCT) from matched unrelated donors (URD). Encouraging results have been reported with both the combination of alemtuzumab and cyclosporine (AC) and the regimen of tacrolimus, methotrexate, and sirolimus (TMS) in the URD setting. These two regimens work by biologically distinct mechanisms and may have markedly different effects on immune reconstitution. T-cell receptor (TCR) spectratyping analysis, which provides information on antigen receptor diversity, is a valuable method for monitoring post-transplant immune reconstitution. As part of a randomized pilot study, we prospectively assessed the effects of AC vs. TMS on TCR Vb repertoire diversity in patients undergoing reduced intensity HLA-matched unrelated donor transplantation. Methods: Twenty patients (median age 53 yrs; range 24–70 yrs) with hematologic malignancies received reduced intensity conditioning (fludarabine 30 mg/m2/day and cyclophosphamide 1200 mg/m2/day IV Day -6 to -3) followed by a 10/10 HLA-matched unrelated donor T-cell replete mobilized peripheral blood allograft. Patients were randomized to receive either: AC (n=10): alemtuzumab 20 mg/day IV over 8 hours Days -8 to -4 and cyclosporine starting at Day -1 with a 10% per week taper starting at Day +100 or TMS (n=10): tacrolimus and sirolimus starting at Day -3 with a 33% taper at Day +63 and Day +119 and methotrexate 5 mg/m2 IV, Days +1, +3, +6, and +11. Blood samples were collected from the donor and patient at baseline and the patient at 1, 3, 6 and 12 months post-transplant for TCR spectratyping analysis. All comparisons are based on an exact Wilcoxon rank sum test; p values < 0.01 were significant because of multiple comparisons. Results: Patients on the AC arm had significantly fewer T-cells on Day +14 compared with the TMS arm (median CD3+ = 1 cells/μl vs 356 cells/μl; CD4+ = 0 cells/μl vs 243 cells/μl; CD8+ = 0 cells/μl vs. 59 cells/μl; each p<0.0001); there was less disparity at Day +28 (median CD3+ = 45 cells/μl vs. 398 cells/μl; CD4+ = 36 cells/μl vs. 218 cells/μl; CD8+= 5 cells/μl vs 152 cells/μl; each p 0.002). By Day +100, lymphocyte recovery was not appreciably different between the two arms (median CD3+ = 242 cells/μl vs. 445 cells/μl (p = 0.095): CD4+ = 106 cells/μl vs. 212 cells/μl (p=0.28); CD8+ = 72 cells/μl vs. 135 cells/μl (p = 0.03). NK-cell recovery was slightly less in the AC vs. TMS arm at Day +14 (median NK = 27 cells/μl vs. 70 cells/μl; p = 0.01) and at Day +28 (median NK = 29 cells/μl vs. 150 cells/μl; p=0.02). There was no difference by Day +100 (median NK = 124 cells/μl vs. 88 cells/μl; p=0.31). B-cell reconstitution was negligible in both arms through Day +100. Assessment of CD4+ TCR Vb repertoire diversity by spectratyping demonstrated significantly lower diversity in patients receiving AC at 1 (p = 0.0003), 3 (p = 0.0003) and 6 (p=0.003) months post transplant compared with patients receiving TMS. CD8+ TCR spectratyping similarly revealed significantly reduced diversity in the AC arm at 3 (p = 0.001) and at 6 months (p = 0.003), and a trend toward significance at 12 months (p = 0.07). On each of the 2 arms, 2 of 10 patients developed acute Grade II-IV GVHD. Of the 5 patients on the AC arm who were seropositive for CMV, all 5 reactivated CMV by PCR within the first 60 days and reactivated 2–5 times in the first year. In contrast, only 3 of 5 seropositive patients reactivated CMV on the TMS arm and only one reactivated in the first 60 days. Conclusions: Two factors may have contributed to the loss of repertoire diversity in the AC arm. First, the alemtuzumab regimen may have severely depleted the infused donor T-cells. Second, stimulation by reactivating virus may have induced expansion of CMV-specific memory and effector T-cells, resulting in a skewed and oligoclonal T-cell repertoire. Especially in CD8+ T-cells, CMV has been shown to produce significant oligoclonal expansion (including CD4+: CD8+ ratio inversion). The loss of T-cell numbers and repertoire may in turn have contributed to the prevalence of early CMV reactivation. Thus, despite the similarities in frequency of acute GVHD in this small sample, it appears that these two commonly used GVHD prophylaxis regimens have very different effects on post-transplant immune reconstitution in the first 6 months after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Prognostic Impact of Hematopoietic Recovery After Fludarabine, IV Busulfan and Antithymocyte Globulins (FB2 regimen) Reduced-Intensity Conditioning Regimen (RIC) Allogeneic Stem Cell Transplantation (allo-SCT)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4483-4483
Author(s):  
Amandine Lebourgeois ◽  
Marion Loirat ◽  
Benoit Tessoulin ◽  
Elsa Lestang ◽  
Pierre Peterlin ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Univariate Analysis ◽  
Conditioning Regimen ◽  
Prognostic Impact ◽  
Immune Recovery ◽  
Post Transplant ◽  
Factors Associated ◽  
Before And After ◽  
Circulating Lymphocytes ◽  
The Impact

Abstract Abstract 4483 Introduction: RIC regimens are increasingly used prior to allo-SCT. The FB2 regimen (Fludarabine 120–150 mg/m2 + IV Busulfan 6.4 mg/Kg + ATG Thymoglobuline 5mg/Kg) is currently the most widely used RIC regimen in many European centres. This retrospective analysis aimed to assess the hematopoietic and immune recovery in a homogeneously treated cohort of 53 patients (males: n=33; median age: 59 years (range: 22–70)) who received the FB2 regimen between January 2007 and October 2010 in our department. Patients and Methods: Diagnoses were as follow: AML n=23; ALL n=1; biphenotypic leukemia n=1; lymphoma n=16; myelodysplastic syndrome n=9; multiple myeloma n=3. Nineteen patients (36%) had received a prior autologous SCT. The majority of patients (n=40, 75.5%) were transplanted in complete remission. Thirty patients received a graft from a matched sibling donor (56.5%). All patients, but one (who received unmanipulated bone marrow) received G-CSF-mobilized PBSCs. GVHD prophylaxis consisted of cyclosporine (CsA) alone in patients transplanted with an HLA-identical sibling, and CsA+ mycophenolate mofetyl in other cases. None of the patients received G-CSF during aplasia following transplant while nine patients received erythropoietin before day+100. Results: Engraftment was achieved in 96% of patients (n=51). Median times for neutrophils (n=51) and platelets (n=22) recovery were 17 days (range: 0–39) and 10 days (range: 4–186), respectively. The majority of patients (n=31, 58%) did not receive platelet support during aplasia. The cumulative incidences of grade II-IV and grade III-IV acute GVHD were 30% and 15%, respectively, while overall incidence of chronic extensive GVHD was 33%. With a median follow-up of 19 months (range: 2–53), the 2-year OS, DFS, relapse incidence, and NRM were 63%, 59.5%, 35% and 6%, respectively. In univariate analysis, when regarding pre-transplant factors associated with outcome, the only factor correlated with a significantly higher 2-year OS and DFS was a higher total circulating lymphocytes count at transplant (> 730/mm3) (OS: 81.5% vs 43.2%, p=0.01; DFS: 73.2% vs 45.5%, p=0.03). Regarding post-transplant factors, we found that higher recovery of leukocytes (>5000/mm3) (2-year OS: 78% vs 46%, p=0.007; 2-year DFS: 70% vs 48%, p=0.08), neutrophils (>3230/mm3) (2-year OS: 76% vs 50%, p=0.02; 2-year DFS: 67.5% vs 52.0%, p=0.09), and monocytes (>590/mm3) (2-year OS: 80% vs 47%, p=0.004; 2-year DFS: 75% vs 42%, p=0.007) at day+30 post-transplant were the most significant factors associated with outcome. In multivariate analysis, the only independent factors associated with a significantly higher OS and DFS were a better immune status at transplant (lymphocytes count >730/mm3; HR 0.22; 95%CI: 0.08–0.63, p=0.005; and HR: 0.29; 95%CI: 0.12–0.71, p=0.006, respectively) and a higher monocytes count at day+30 post-transplant (>590/mm3) (HR: 0.24; 95%CI: 0.08–0.66, p=0.006; and HR: 0.28; 95%CI: 0.11– 0.68, p=0.005; respectively). Conclusion: These results suggest that hematopoietic status and recovery before and after FB2 RIC allo-SCT can be significant predictors of outcome. This paves the way for future studies aiming to closely monitor the kinetics of immune recovery after RIC allo-SCT and to evaluate the impact of growth factors and other immunostimulatory cytokines in the setting of RIC allo-SCT. Disclosures: No relevant conflicts of interest to declare.

Rituximab Administration before Allogeneic HSCT Is Associated with Accelerated T Cell Reconstitution after Transplantation: Kinetic Evidence for a Graft-Versus-Leukemia Effect

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3905-3905
Author(s):  
Sakura Hosoba ◽  
Christopher R. Flowers ◽  
Catherine J Wu ◽  
Jens R. Wrammert ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Subset ◽  
Post Transplant ◽  
T Cell Reconstitution ◽  
Relative Value ◽  
History Of

Abstract Introduction: Rituximab (R) administration results in depletion of blood B cells and suppression of B cell reconstitution for several months after, with suggestions that T cell reconstitution may also be impaired. We hypothesized that pre-transplant R would be associated with delayed B and T cell reconstitution after allo-HSCT compared with non-R-treated allo-HSCT recipients. Methods: We conducted a retrospective analysis of 360 patients who underwent allo-HSCT using BM or G-CSF mobilized PB. Recipients of cord blood, T cell depleted grafts and 2nd allo-HSCT were excluded. Analysis of lymphocyte subsets in at least one blood at 1, 3, 6, 12, and 24 months post-allo-HSCT was available for 255 eligible patients. Data on lymphocyte recovery was censored after DLI or post-transplant R therapy. Post-HSCT lymphocyte recovery in 217 patients who never received R (no-R) was compared to 38 patients who had received R before allo-HSCT (+R) including 12 CLL, 19 NHL, and 7 B-cell ALL patients. +R patients received a median of 9 doses of R with the last dose of R at a median of 45 days pre-transplant. Results: Mean lymphocyte numbers in the blood at 1, 3, 6, 12, and 24 months were B-cells: 55 ± 465/µL, 82 ± 159/µL, 150 ± 243/µL, 255 ± 345/µL, and 384 ± 369/µL (normal range 79-835); and T-cells: 65 ± 987/µL, 831 ± 667/µL, 1058 ± 788/µL, 1291 ± 985/µL, and 1477 ± 1222/µL (normal range 675-3085). Lymphocyte reconstitution kinetics did not vary significantly based upon the intensity of the conditioning regimen or related vs. unrelated donors allowing aggregation of patients in the +R and no-R groups (Figure). B cell reconstitution in the +R patients was higher at 1 month post-allo-HSCT (relative value of 143% p=0.008) and lower at 3 months post-transplant (19.2%, p=0.069) compared to no-R patients. Blood B cells in the +R group rebounded by the 6th month post-allo-HSCT and remained higher than the no-R group through the 24th month post-HSCT (197% at the 6th month, p=0.037). Higher levels of B-cells at 1 month in the +R group was due to higher blood B-cells at 1 month post-HSCT among 12 CLL patients compared with no-R patients (423%, p<0.001; Figure), while B-cell counts in the remaining +R patients (B-cell NHL and B-cell ALL) were lower than the no-R patients at both 1 and 3 months. Reconstitution of CD4+ and CD8+ T cells among +R patients were similar to no-R patients in the first month post-allo-HSCT and then rebounded to higher levels than the no-R group of patients (relative value 194%, p=0.077 at the 24th month for CD4+ T cell subset, and 224%, p=0.020 for CD8+ T cell subset; Figure). CLL patients had a striking increase in blood levels of donor-derived CD4+ and CD8+ T cells at 3 months post-transplant concomitant with the disappearance of blood B cells compared with no-R patients (relative value of 178% and 372%, p=0.018 and p=0.003, respectively; Figure). Long term T cell reconstitution remained higher for +R patients compared with no-R patients, even when CLL patients were excluded (relative value of 203%, p=0.005 at 24 months post-HSCT; Figure). Conclusions: We observed higher levels of blood B cells and T cells ³ 6 months post-allo-HSCT in +R patients compared with no-R patients. B cell recovery at 6 months post-transplant is consistent with clearance of residual plasma R given the 1-2 months half-life of R, and the median of 1.5 months between the last dose of R and allo-HSCT. The increased blood CD8+ T cells in the blood of CLL patients at 3 months post-allo-HSCT associated with clearance of the B-cells seen 1 month post-HSCT is consistent with a donor T cell-mediated GVL effect. Pre-transplant R therapy does not appear to have any long-term deleterious effect on immune reconstitution, indicating that post-allo-HSCT vaccination at ≥6 months may be efficacious. Figure: Kinetics of lymphocyte reconstitution after allo-HSCT varied by history of pre-transplant R administration and primary disease. Panels show mean counts of each lymphocyte subset at 1, 3, 6, 12 and 24 months post-allo-HSCT for: (1) B cell, (2) T cell, (3) CD4+ and (4) CD8+ T cells. Solid lines with triangle show no-R group; dashed lines with circles shows subgroups of CLL and NHL/ALL +R patients. Asterisks show p values from t-test of the comparison between CLL +R or the NHL/ALL +R patients with no-R patients. *p<0.05; ** p<0.01; *** p<0.001. Figure:. Kinetics of lymphocyte reconstitution after allo-HSCT varied by history of pre-transplant R administration and primary disease. Panels show mean counts of each lymphocyte subset at 1, 3, 6, 12 and 24 months post-allo-HSCT for: (1) B cell, (2) T cell, (3) CD4+ and (4) CD8+ T cells. Solid lines with triangle show no-R group; dashed lines with circles shows subgroups of CLL and NHL/ALL +R patients. Asterisks show p values from t-test of the comparison between CLL +R or the NHL/ALL +R patients with no-R patients. *p<0.05; ** p<0.01; *** p<0.001. Disclosures No relevant conflicts of interest to declare.

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