Risk Of Extensive Chronic GVHD Research Articles

Donor Genotypes For Interleukin-17A Gene Single Nucleotide Polymorphisms (SNPs) Allow Anticipation Of Complications After HLA-Identical Allogeneic Stem Cell Transplantation (allo-SCT)

Introduction Graft versus host disease (GvHD) is the main cause of morbimortality after allogeneic stem cell transplantation (allo-SCT). Several single-nucleotide polymorphisms (SNPs) in in the promoter region of cytokine genes have shown to alter their expression and are therefore associated with donor-recipient alloreactivity and, ultimately, with SCT outcome. Interleukin 17 (IL-17) is secreted by CD4+ T-cells and has been implicated in the pathogenesis of various autoimmune diseases but its importance in SCT is not well-known. Objective To analyse the influence of IL-17A SNP genotypes on the risk and severity of GvHD and other complications after HLA-identical allo-SCT. Patients and Methods Genomic DNA obtained from peripheral blood samples belonging to 546 patients and their HLA-identical sibling donors (Table 1) included in the DNA Bank of the Spanish Group for Hematopoietic Stem Cell Transplantation (GETH). Genotyping of the polymorphisms of interest, rs8193036 (-737C>T), rs2275913 (-197G>A), rs3819024 (-444A>G), rs4711998 (-877A>G), were performed by multiplex primer extension followed by mass spectrometry (MALDI-TOF; Sequenom MassArray). Results Genotype frequencies are shown in Table 2 and the association between IL-17A genotypes and complications after allo-SCT are shown in Table 3. Patients transplanted from donors harboring genotype CC for the SNP rs8193036 show increased risk of grade III-IV acute GvHD (7/26 vs 47/397, p=0.035) and of grade II-IV acute GvHD (13/26 vs 133/409, p=0.048). Patients transplanted from donors harboring allele A in the SNP rs4711998 show increased risk of extensive chronic GvHD (53/161 vs 43/177, p=0.045). Relapse rate was not related with IL-17A SNP genotypes. Finally a higher risk of toxicity-related mortality (TRM) was observed in patients transplanted from donors harboring allele A for SNP rs2275913 (78/293 vs 46/227, p=0.048), donors harboring allele G for SNP rs3819024 (78/279 vs 46/242, p=0.011) and donors harboring allele A for SNP rs4711998 (68/250 vs 55/229, p=0.044). Conclusions IL-17A SNP genotyping might be useful to anticipate complications after sibling HLA-identical allo-SCT and, therefore, to improve the clinical management of transplanted patients. This results further support the idea of a genetic predisposition to certain complications after allo-SCT. Paper presented on behalf of the GvHD/Immunotherapy committee of the Spanish Group for Hematopoietic Transplantation (GETH). Disclosures: No relevant conflicts of interest to declare.

Antithymocyte Globulins (ATG) As Part Of The Myeloablative Conditioning (MAC) Regimen Can Reduce The Risk Of Severe Graft-Vs.-Host Disease (GVHD) After Allogeneic Stem Cell Transplantation (allo-SCT) From Matched-Unrelated Donors (MUD) [A SFGM-TC Study

Antithymocyte Globulins (ATG) As Part Of The Myeloablative Conditioning (MAC) Regimen Can Reduce The Risk Of Severe Graft-Vs.-Host Disease (GVHD) After Allogeneic Stem Cell Transplantation (allo-SCT) From Matched-Unrelated Donors (MUD) [A SFGM-TC Study

Antithymocyte Globulins (ATG) as Part of the Myeloablative Conditioning (MAC) Regimen Can Reduce the Risk of Severe Graft-Vs.-Host Disease (GVHD) after Allogeneic Stem Cell Transplantation (allo-SCT) from Matched-Unrelated Donors (MUD).

The use of ATG in the setting of standard MAC allo-SCT is still controversial. Some studies, however, suggested a beneficial effect of ATG in preventing acute GVHD. Here, we report the results of a large multicenter retrospective study analyzing the effect of ATG, incorporated within the MAC regimen for MUD-transplants in leukemic patients compared to patients not receiving ATG. The purpose of the study was to compare the incidence and severity of acute and chronic GVHD as well as non-relapse mortality (NRM), leukemiafree and overall survivals (LFS, OS). The study included 171 adult patients with acute leukemia and MDS (73% standard risk and 27% more advanced disease) reported to the registry of the SFGM-TC between 1998 and 2004, and for whom detailed allelic HLA typing (4 digits) for the recipient and the donor was available. Patients' characteristics were as follow: median age: 33 (range, 15–67), 57% male recipients, 35% female donors, 44% AML, 43% ALL, 11% MDS and 2% unclassified leukemia. The stem cell source was bone marrow in 72.5% of patients, while PBSCs were used in 27.5% of cases. 81% of patients were transplanted from 10/10 allelic MUD, and 19% from a MUD with at least one allelic difference. A high dose TBI-based MAC regimen was used in 84% of cases, while 16% received a high-dose chemotherapy containing MAC regimen. 85% of the patients received the classical CsA and short course methotrexate GVHD prophylaxis regimen. In this series, 120 patients (70%) did not receive ATG (“no-ATG” group), while 51 patients received ATG (“ATG” group; Thymoglobuline*-Genzyme in all cases; total ATG dose: ≤5 mg/Kg, n=13; >5 and <10 mg/Kg, n=17; ≥10 mg/Kg, n=21) as part of the MAC regimen. Except for a significantly higher number of allelic differences between recipient and donor (33% vs. 13%; P=0.002), the “no-ATG” and “ATG” groups were strictly comparable as for patients, disease and transplant characteristics. 95% of patients had a sustained engraftment at a median of 20 (range, 9–41) days after allo-SCT with no significant differences between the 2 groups. With a median followup of 30.3 (range, 2.6–68.1) months after allo-SCT, grade 0–1 and 2–4 acute GVHD occurred in 74 (46%) and 88 patients (54%) respectively, with grade 3–4 acute GVHD being significantly lower in the “ATG” group as compared to the “no-ATG” group (18% vs. 32%; P=0.04). In this series, 142 patients (83%) were evaluable for chronic GVHD. Limited and extensive chronic GVHD were observed in 22 and 25% of assessable patients respectively, with extensive chronic GVHD being significantly lower in the “ATG” group as compared to the “no-ATG” group (5% vs. 33%; P=0.001). Interestingly, patients from the “ATG” group had a higher incidence of limited chronic GVHD (33% vs. 18%; P=0.06). The use of ATG was not associated with a higher risk of infections: infection-related mortality was comparable between both groups (23% vs. 27%, P=NS). Also, NRM was comparable between both groups (30% vs. 29%; P=NS). In multivariate analysis including all relevant risk factors tested in the univariate analysis, we found that an HLA allelic mismatch and the non-use of ATG were associated with an increased risk of severe grade 3–4 acute GVHD (RR=2.80, 95%CI, 1.5–5.3), P=0.001; and RR=2.4, 95%CI, 1.1–5.0, P=0.02 respectively). Similarly, multivariate analysis showed that the absence of use of ATG was the unique and strongest parameter associated with an increased risk of extensive chronic GVHD (RR=6.9; 95%CI, 1.7–29.0, P=0.008). Finally, LFS and OS at 2 years were not significantly different between the “no-ATG” and “ATG” group (48.8% vs. 41.3%, P=NS; and 53.6% vs. 54.3%, P=NS; respectively) Despite its retrospective nature, these results strongly indicate a global long-term beneficial effect of ATG when used as part of the MAC regimen prior to allo-SCT from MUD (especially in the HLA mismatch setting). Though prospective studies are needed to assess the optimal ATG dosing and administration schedule, such protective effect of ATG against severe acute and chronic GVHD, can be likely achieved without an increased risk of infections or leukemia recurrence.

Higher Doses of Transplanted T and B Cells Are Associated with Greater Incidence of Extensive Chronic GVHD after PBSC Transplantation from HLA-Identical Sibling Donors.

Background. We investigated the impact of graft composition on GVHD in 1520 allogeneic transplant recipients given PBSC.Methods. Graft composition was determined by FACS analyses and expressed as cells/kg of recipient weight. Cell types were defined as follows: CD3+ T cell (CD3+CD56−), CD4+ T cell (CD3+CD4+), CD8+ T cell (CD3+CD8+), natural killer (NK) cell (CD3−CD56+), NK/T cell (CD3+CD56+), B cell (CD20+), monocyte (CD14+), and CD34+ cells (CD34+CD14−). Cell doses were modeled as continuous linear variables or by quartiles. Cox (for chronic) and logistic (for acute) regression models were adjusted for conditioning regimen intensity, pt age, female donor to male recipient vs. other gender combination, and disease risk.Results. The median age among all pts was 52 (45 among myeloablative, 57 among nonmyeloablative); 27% of pts were male transplanted from a female donor; 66% of pts had high-risk disease; 49% were transplanted from an alternative donor (47% unrelated, 2% mismatched sibling); 51% received a nonmyeloablative conditioning. Table 1 shows graft composition and GVHD incidences according to donor type and conditioning intensity. There were no statistically significant associations between cell dose and acute GVHD, although there was a suggestion that the association of NK/T cell was dependent on conditioning. In particular, increasing NK/T cells were associated with an increased probability of grades 2–4 GVHD among myeloablative pts (p=.03) but not among nonmyeloablative pts. There were no statistically significant associations between cell dose and chronic GVHD, but several cell types showed evidence of an interaction between donor and cell dose. In particular, increasing B cells and CD3+, CD4+, and CD8+ T cells were associated with an increased risk of chronic GVHD among pts with an HLA-identical donor (p=.006, p=.0003, p=.0002, p=.02, respectively) while the associations among alternative donors were in the opposite direction and were not statistically significant (p=.68, p=.26, p=.44, and p=.15, respectively). Lack of correlation in the alternative donor group may reflect differences in T-cell responses to the greater minor histocompatability divergence, or changes in T-cell functionality associated with the dose of G-CSF (higher in sibling donors) used for mobilization. Increasing CD34+ cells was not statistically associated with chronic GVHD in either myeloablative (negative association, p=.10) or nonmyeloablative (positive association, p=.11) pts.Conclusions. Higher numbers of transplanted T and B cells were associated with an increased risk of extensive chronic GVHD in pts given grafts from HLA-identical siblings. Increasing CD34+ cells were not associated with a statistically significant increase in chronic GVHD. Further analyses looking at other clinical outcomes such as survival are forthcoming.Table 1Graft composition and GVHD according to conditioning intensity and donor typeNonmyeloablative conditioning and HLA-id siblingMyeloablative conditioning and HLA-id siblingNonmyeloablative conditioning and alternative donorMyeloablative conditioning and alternative donorMedian # of T cell transplanted (× 108/Kg)3.42.52.52.8Median # of CD34+ cell transplanted (× 106/Kg)8.67.56.97.5Incidence of grade II–IV acute GVHD (%)47666181Incidence of extensive chronic GVHD (%)40544149

Unrelated donor or partially matched related donor peripheral stem cell transplant with CD34+ selection and CD3+ addback for pediatric patients with leukemias

Unmodified peripheral stem cell transplants are associated with an increased risk of extensive chronic GVHD. T depletion may reduce this risk, but the risk of graft failure or relapse may increase. To decrease the risks of both extensive chronic GVHD and graft failure, we added back a defined dose of CD3+ cells to CD34+ selected PSCs. Twenty-four patients were evaluable for outcome analysis. Donors were unrelated (23) or related (1). Conditioning was thiotepa, cyclophosphamide, and total body irradiation. Cyclosporine was used post transplant. Following CD34+ selection, a total of 5 x 10(5)/kg CD3+ cells were infused. Donors were matched for 12 patients. The median CD34+ dose infused was 7.1 x 10(6)/kg. Engraftment occurred in all patients at a median of 14 days (10-19). Twelve patients are alive in remission 15-34 months (median, 25) post PSCT. GVHD occurred in 17 patients, but was >grade II in only 2. Chronic GVHD occurred in 61.5% of evaluable patients, but was limited to skin and perioral cavity. Two patients relapsed, and 10 patients died of non-relapse causes. This study demonstrates that PSCT with CD34+ selection and a defined dose of CD3+ results in prompt engraftment and may limit development of extensive chronic GVHD.

Unrelated Donor (URD) or Partially Matched Related Donor (PMRD) Peripheral Stem Cell Transplant (PSCT) with CD34+ Selection and CD3+ Addback for Pediatric Patients with Leukemias.

PSCT is becoming increasingly more common, as large doses of PSCs may result in faster engraftment than bone marrow, and high PSC doses may be advantageous in decreasing graft rejection. PSCs contain several logs more T cells than bone marrow, and most studies have shown an increased risk of extensive chronic GVHD with unmodified PSCs. However, purified CD34+ cells may be associated with increased graft failure (Bornhauser, et al. Br J Haematol 118:1095, 2002) or relapse. To decrease the risks of both chronic GVHD and graft failure, and maintain potential graft vs. leukemia effect, we used CD34+ selection with a defined dose of CD3+ cells in the product at the time of infusion for URD or PMRD PSCT. We report on the first 23 patients (pts) on this IRB approved study. Conditioning was thiotepa 10mg/kg, cyclophosphamide 120 mg/kg and TBI 12 Gy/6 fractions. Lung shielding after 8 Gy and methylprednisolone 1 mg/kg at day+8 were initiated when it became apparent that engraftment syndrome was being observed in the first 10 patients. GVHD prophylaxis was cyclosporine, followed by oral tacrolimus, which was weaned by 120 days in the absence of GVHD. CD34+ selection was accomplished with the Isolex 300i device and resulted in an average T cell depletion of 4.2 logs. The positive fraction had an average purity of 94%, and T cells obtained from the negative fraction were added to the positively selected product at the time of infusion to achieve the defined T-cell dose of 5 x 105/kg CD3+. The remaining portion of the negative fraction was cryopreserved in multiple aliquots for DLI, if needed. There were 12 males; median age was10 yrs (range, 3–22). Diagnoses included: ALL: 6, AML/MDS:12, CML: 3, JMML: 2. URDs were used for 22 patients, and related donor for 1. This was the second transplant for one pt with MDS. Donors were matched by high resolution typing for13 pts; mismatches included A for 5 pts, B and C loci for 3 pts, DQB1 for 2 pts. The median CD34+ dose infused was 6.6 x 106/kg (1.3–12.4). Engraftment occurred in all pts, with ANC>500 at a median of 14 days (10–19), and platelets >20K in 15 pts at a median of 22 days (13–33). Eleven patients (48%) are alive in remission 7–25 months post SCT (med, 18). Grades I-II GVHD occurred in 15 pts, grade III in 1, and grade IV in association with HHV6 in 1 pt. Limited chronic GVHD occurred in 8 of 12 evaluable pts (67%), and has resolved in all but one. Two pts with AML relapsed at 3 and 18 months. Ten pts died at d 32–85 from CMV (2), ARDS (5), HHV6/infection (2), bacterial sepsis(1). Both pts who developed CMV pneumonitis were seropositive, with seronegative donors; one developed pneumonitis at day +20, with negative antigenemia. Pts with CML were negative for bcr-abl by PCR <6 months post SCT and have remained negative without GVHD. There was a trend toward higher mortality with mismatched donors (RR 2.6, p=0.06). This study demonstrates that PSC engineering with CD34+ positive selection with a defined dose of CD3+ cells results in prompt engraftment. With this approach, the risk of extensive chronic GVHD with URD or PMRD PSCs may be reduced.

MCMV Infection Lowers the Threshold for the Development of Clinical GvHD after Allogeneic Bone Marrow Transplantation.

CMV infection is reported to increase the incidence and severity of chronic GvHD and clinical data have shown that preemptive antiviral therapy decreased the risk of extensive chronic GvHD. Using mouse model of Allogeneic BMT, we investigated the mechanism for the association of MCMV infection and GvHD. We hypothesize that MCMV infection leads to generalized immune activation and increases the number of donor derived allo-reactive T cells and GvHD activity.Methods: A parenteral to F1 mouse BMT model was used to study anti-CMV immunity and GvHD. Low dose splenocytes (3x106) from MCMV immunized C57BL/6 donors (H-2b, Thy1.2+, CD45.1+) were transplanted with 5x106 T cell depleted bone marrow (TCD BM) from congeneic mice (H-2b, Thy1.1+, CD45.2+) into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C, H-2b/d, Thy1.2+, CD45.2+). Previous work has established this as a dose that protects against CMV without immediate lethality from GvHD. Non-GvHD control mice received a dose of Amotosalen treated splenocytes (10x106) and 5x106 TCD BM that protects against CMV without GvHD. Recipient mice were infected i.p. with a supralethal dose (2.5x104 pfu) of MCMV 7 days post transplant. Clinical GvHD was monitored twice weekly by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. T cell chimerism in recipient spleen and thymus, and MCMV peptide specific tetramer+CD8+ T cells were determined by flow cytometry. Liver and lung viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers.Results: During the acute phase of MCMV infection (day 3 to 14 post infection), recipient mice that received 3x106 untreated donor splenocytes developed GvHD characterized by weight loss and higher mortality than the non-GvHD control mice. Although both GvHD+ and control mice effectively cleared the virus from their liver, delayed viral clearance from the lung was found in non-GvHD recipients. Viral clearance was associated with expansion of donor spleen-derived MCMV peptide specific tetramer+ CD8+ T cells. The kinetics of donor T-cell expansion varied significantly between the two treatment groups, with GvHD+ recipients showed rapid early expansion of donor derived T-cells followed by the development of GvHD with subsequent lymphopenia. Recipients of Amotosalen-treated splenocytes had more gradual expansion of total and 400-fold expansion of antigen specific T-cells with sustained lymphoid reconstitution. GvHD+ recipients of untreated splenocytes had complete chimerism comprised of >80% of CD8+ donor T cells whereas non-GvHD controls had significant expansion of host derived T cells following MCMV infection and lacked allo-reactive of donor- spleen-derived T cells. Thymic function was inhibited among animals that developed GvHD and preserved among control mice throughout the infectious phase. Very delayed CMV infection (on day 60 after transplant) in mice with established chronic GvHD exacerbated GvHD and was associated with delayed lung viral clearance.Conclusion: After CMV infection there is extensive expansion of allo-reactive T cells in GvHD+ mice with associated damage to the microenvironment in the spleen and thymus. Amelioration of the immuno-suppressive effect of CMV infection (in clinical transplantation) will likely require more effective prophylaxis and treatment of GvHD in allotransplant recipients.