ALBERT

Description: Backgound : Sinusoidal obstruction syndrome [SOS, also known as hepatic veno-occlusive disease (VOD)] is frequent after HSCT and may have a mortality rate of up to 85%. Defibrotide has shown efficacy not only in the treatment of established SOS (Richardson et al. Blood 1998:92;737 and 2016:127;1656) but also in SOS prevention in children (prospective study: Corbacioglu et al. Lancet 2012:379;1301) as well as in adults (several retrospective studies). Patients and methods : Between 1999 and 2009, we gave defibrotide intravenously to 248 successive patients transplanted for hematological diseases starting at day -7 up to day +20 post-transplantation (dose range 800-2400 mg/d) in combination with heparin. We previously published the data of the 52 first patients compared to 52 historical controls who were transplanted just prior to the study period. We now expand the study with 196 additional patients treated successively with defibrotide prophylaxis while adding patients transplanted after 2010 when patients did not receive defibrotide as prophylaxis anymore (2011-2015) to the control group. The characteristics of patients are shown in Table 1. Results: Median follow-up for the study group was 10 (range 2-16) years and for the control group 2.7 (range 1-18) years. None of the 248 patients in the defibrotide group developed SOS (Baltimore criteria). The 100 day cumulative incidence (CI) of SOS was 0% in the defibrotide group as compared to 4.8% (95%CI 2.6-8%) in the control group, p=0.00046. The day 100 event free survival (EFS) where an event was defined as the 1st occurrence of one of the following: SOS, acute graft versus host disease (GvHD) ≥2, relapse or death, was not significantly different with 60% (95%CI 54%-66%) in the defibrotide group vs 53% (95%CI 47%-59%) in the controls, p=0.165, but the one year EFS was statistically different with 38% (95%CI 32%-44%) vs 28% (95%CI 22%-34%), p=0.00969. The 100 day CI of acute GVHD was not significantly different between the two groups [27% (95%CI 22%-33%) in the defibrotide group vs 29% (95%CI 24%-35%) in the control group, p= 0.707] while the 1 year acute GvHD CI was significantly reduced in the defibrotide group [31% (95%CI 25%-37%)] compared with the control group [42% (95%CI 36%-48%), p=0.026]. The one year overall survival (OS), relapse incidence (RI) and non-relapse mortality (NRM) were not statistically different being respectively 73% (95%CI 67%-78%) vs 65% (95%CI 59%-71%), p=0.0704, 32% (95%CI 27%-38%) vs 28% (95%CI 22%-34%), p=0.331 and 14% (95%CI 10%-18%) vs 19% (95%CI 14%-24%), p=0.148. Multivariate analysis, performed taking into account clinical factors known to influence the risk of SOS, confirmed the favorable impact of defibrotide on 100 day SOS CI [HR 7.5x10-7 (95%CI 1.8x10-7-3.2x10-6), p

Description: Self-renewal, pluripotency, and long-term reconstitution are defining characteristics of single hematopoietic stem cells.Pax5−/− precursor B cells apparently possess similar characteristics. Here, using serial transplantations, with in vitro recloning and growth of the bone marrow–homed donor cells occurring after all transplantations, we analyzed the extent of self-renewal and hematopoietic multipotency ofPax5−/− precursor B-cell clones. Moreover, telomere length and telomerase activity in these clones was analyzed at various time points. Thus far, 5 successive transplantations have been performed. Clones transplanted for the fifth time, which have proliferated for more than 150 cell divisions in vitro, still repopulate the bone marrow with precursor B cells and reconstitute these recipients with lymphoid and myeloid cells. During this extensive proliferation, Pax5−/− precursor B cells shorten their telomeres at 70 to 90 base pairs per division. Their telomerase activity remains at 3% of that of HEK293 cancer cells during all serial in vivo transplantations/in vitro expansions. Together, these data show thatPax5−/− precursor B-cell clones possess extensive in vivo self-renewal capacity, long-term reconstitution capacity, and hematopoietic multipotency, with their telomeres shortening at the normal rate.

Description: Non-HLA polymorphisms (NHP) influence risk of GVHD and outcome of allogeneic hematopoietic stem cell transplants (HSCT) however their influence on GvHD vs GvL remains to be defined. A cohort of 291 CML HLA matched sibling transplants with known clinical risk factors; eg stage of disease, gender mismatch (female donor/male recipient), patient age and time from diagnosis to transplant as defined by the EBMT risk score, were typed via SNPs or microsatellites for cytokines (IL-1Ra, IL-4, IL-6, IL-10, IFNγ, TNFα, TNFR 11), steroid hormone receptors (VDR and ERα) and NOD2/CARD15 mutations. TNFRII-196 allele R; IL-10 ATC/ACC; IL-1 Ra (allele 2) and IL-4T were significantly associated with survival using univariate analysis. Two clinical Cox proportional hazards models were generated for the statistical analysis and used as a basis for further development: (i) using the EBMT risk score as a single variable on an ordinal scale or (ii) using the individual clinical factors of the EBMT risk score as categorical variables. After step-wise variable selection using the significant genetic factors as candidates, the resulting multivariate models indicated that absence of TNFRII-196 R, i.e. down regulation of TNF in the recipient, absence of IL-10 ATC/ACC, i.e. intermediate IL-10 production in the donor and presence of IL-1Ra (allele 2) i.e. down regulation of IL-1 in the donor were associated with poor outcome. The addition of the genetic variables significantly improved the preferred model containing the EBMT risk score as a single variable. The Goodness of Fit of the models was assessed by Kaplan-Meier curves showing clinically relevant differences between good, intermediate and poor prognostic groups. The worst prognostic scores included the absence of ATA/ACC in the donor, evidenced by a steep change in survival probability. Relapse was associated with clinical factors; absence of female to male transplants; absence of bone marrow transplants and presence of T cell depletion but no significant association was found with genetic factors. This study suggests that distinct high risk patterns of NHP of patients and donors can be defined, which influences survival due to factors associated with an increased risk of GvHD without the potential benefit of increased GvL response. Data add to the clinical factors (eg age, sex, multiparity of the donor) where an unrelated donor might be the preferred choice compared to a high risk sibling donor.

Description: Using quantitative fluorescence in situ hybridization and flow cytometry, the telomere length of telomere repeat sequences after stem cell transplantation (SCT) were measured. The study included the telomeres of peripheral blood monocytes that should reflect the length of telomeres in stem cells and the telomeres of T lymphocytes that could shorten as a result of peripheral expansion. The loss of telomeres in monocytes and in memory T cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. In addition, the telomere length in the naive T cells that were produced by the thymus was comparable to the telomere length in the naive T cells of the donor. Compared to the total length of telomeres available, the loss of telomere repeats in leukocytes after SCT resembles the accelerated shortening seen in early childhood and remains, therefore, relatively insignificant.

Description: VOD of the liver is reported to occur in 10–30% of patients after allogeneic HSCT, with mortality rates up to 50%. We previously reported a pilot study of defibrotide (Prociclide® Crinos, Como, Italy) VOD prophylaxis showing a significantly lower rate of VOD as compared to historical controls. We now report the results of an extended study. Between 2000 and 2005, 157 consecutive patients were transplanted for hematological diseases. Median age was 43 y (range 5–61 y), M/F: 96/61; diagnosis was AML=42, ALL=19, CML=28, MDS/MPS=24, lymphoma=22, MM=12, AA=7, solid tumor=3. Donors were HLA-identical siblings (65%), unrelated (32%) mismatched relatives (3%). Stem cell source was peripheral blood (87%) and bone marrow (13%). The transplants were T-cell depleted by CAMPATH-1H in vitro, with (69%) or without (7%) T-cell add-back or left unmanipulated (24%). Conditioning was myeloablative with CyTBI (50%), BuCy (9%) other myeloablative (11%) or non-myeloablative (30%) using Fludara/Bu/ATG. GVHD prophylaxis was CSA with or without MTX or MMF. Nine pts received a second transplant for relapse or graft failure. Defibrotide was given intravenously 6 hourly starting prior to conditioning up to day +20 posttransplant (dose range 20–30 mg/kg) in addition to low dose heparin. The Baltimore criteria were used to score VOD. Results: No VOD was observed after 165 transplants. Defibrotide was well tolerated and no side effects were attributed to this medication. Median peak levels of bilirubin were 21 umol/l (range 9–286 umol/l), aPTT: 35 sec (range 24–83 sec) and fibrinogen: 7.1 g/l (range 3.1–9.5 g/l). Day 100 survival was 91±5%. With a median follow-up of 25 mths (range 3–80 mths), 1 year OS, TRM and RR were 74±7%, 13±5% and 29±8% respectively. The incidence of aGVHD 〉=II was 32±7 %. Conclusion: The results of this study suggest that defibrotide is an effective prophylaxis for VOD.

Description: Introduction: To decrease graft versus host disease (GvHD), the Geneva transplantation team has performed allogeneic hematopoietic stem cell (alloHSCT) with reduced intensity conditioning (RIC) and T cell depletion (TCD) to treat hematological malignancies for older or non fit for myeloablative conditioning patients. This is a new approach of engineering stem cell products that lowers the risk of GvHD while preserving graft versus leukemia (GvL) as much as possible. Patient and methods: We report a retrospective study of 73 patients who received alloHSCT with RIC and TCD between 2001-2013. The median age was 59 years (21-70), 60% were male. Disease at transplant time was acute leukaemia for 45%, Hodgkin lymphoma and non-Hodgkin lymphoma for 24%, myelodysplastic disorders for 13%, myeloproliferative disorders for 9,3 % and multiple myeloma for 8%. Source of stem cell was peripheral in 96% of the cases. 41% of the donors were matched related donor, 37% matched unrelated donor, 19% mismatched unrelated donor and 3% mistmatched related donor. The conditioning regimen consisted on fludarabine with busulfan or melphalan and ATG. Extensive T-cell depletion was done using Campath in the bag followed by washing procedures to remove free antibody. Fixed number of CD3+ T-cell addback was given on d+1 to preserve GvL with minimal residual disease (MRD) assessment and early donor lymphocyte infusions (DLI) given if MRD positive. Doses of DLI were preserved and frozen at the time of stem cell harvest. GvHD prophylaxis was with ciclosporine and mycophenolate mofetil. Results: With a median follow up of 5 (0.5-11) years, the 5-year overall survival (OS), disease free survival (DFS), current disease free survival, relapse rate and non relapse mortality (NRM) were 41.7% (95%CI 30.7-53.7%), 38.8% (95%CI 28.8-50.8%), 39,5% (95%CI 27.7-51.7%), 45.3% (95%CI 32.7-57.2%) and 15.8% (95%CI 8.3-25.4%) respectively. The main cause of death was relapse 38.7 % followed by GvHD 17% and infection 1.3%. In this cohort, the cumulative incidence (CI) of acute GvHD was 15.1% (95% CI: 8.0-24.3%) as well as for acute GvHD grade II-IV. CI of chronic GvHD was 14.7% (95%CI:7.2-23.6%) with extensive chronic GvHD CI being 5.9% (95% CI: 1.9-13.4%). Five patients received DLI for relapses, 27 for mixed chimerism and 8 for both causes. The average number of DLI was 2. Twenty-eight patients entered CR, 4 PR and 13 did not respond to DLI. In univariate analysis, two factors GvHD before DLI and GvHD after first DLI have a tendency for favorable impact on OS respectively p=0.093 and 0.071. For DFS, two factors are significant: disease risk index and GvHD after first DLI respectively p=0.013 and 0.044. For NRM disease risk index is the only factor which is statistically significant p=0.005. For relapse no factors were significant. Discussion: Our study showed a lower rate of acute and chronic GvHD as compared to other studies with unmanipulated stem cells. However, we describe a high rate of relapse incidence and relapse mortality. We have found in univariate analysis two factors statistically significant for DFS GvHD before and after first DLI. Our cohort is a heterogeneous group with different diseases at different stages, which can explain those results. It’s a monocentric study and small number of patient can be a limit for this work. Of note, since 2009 we have changed our strategy introducing a day +100 preemptive DLI infusion in the absence of GvHD, with escalading doses of lymphocytes every 8 weeks up to 5x 107 CD3/kg in the absence of GvHD to improve response. We don’t have enough patients and follow up to draw any conclusion regarding this new strategy. To improve the outcomes, the selection of patients who may receive partial T-cell depletion should be refined, avoiding transplanting patients with high risk of relapse with this strategy. To help decision making, the revised disease risk index as presented by Armand et al. (Blood 2014;123:3664) may be useful. Disclosures No relevant conflicts of interest to declare.