ALBERT

Description: By reducing treatment intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become feasible for elderly patients. Different reduced-intensity conditioning (RIC) regimens are available, but there is little consensus about the optimal preparative regimen to use, in particular with regard to the outcomes counterbalancing the aim of feasibility and tolerability with higher rates of relapse. Here, we retrospectively evaluate the outcome of sequential therapy employing RIC with fludarabine 30 mg/m2, cytarabine 2g/m2 and amsacrine 100 mg/m2 for 4 days (FLAMSA; Schmid C et al. JCO 2005) followed by busulfan 10 x 0.8 mg/kg (FLAMSA-Bu) compared to RIC utilizing fludarabine 5 x 30 mg/m2, carmustine (BCNU) 2 x 150 mg/m2 and melphalan 110 mg/m2 (FBM; Marks R et al. Blood 2008) in elderly patients treated consecutively at our institution between July 2005 and October 2012. We analyzed the course of 114 patients (pts) with acute myeloid leukemia (AML; n=99) or myelodysplasia (MDS; n=15) aged ≥ 59 years with 59 pts aged ≥ 66 years who were treated with either FLAMSA-Bu (n=66; n=24 ≥ 66 years) or FBM (n=48; n=35 ≥ 66 years). All patients received sero-therapy with anti-thymocyteglobuline (ATG). Median patient age was 66 years for the entire cohort (68 years FBM; 64 years FLAMSA-Bu). 36 patients (75%) of the FBM and 42 patients (63 %) of the FLAMSA-Bu group suffered from high risk disease defined as relapsed or refractory AML or refractory anemia with excess of blasts in transformation (RAEB-T). The hematopoietic cell transplantation comorbidity index (HCT-CI) was higher for the patients of the FBM group than for the FLAMSA-Bu group with 26 (54 %) versus (vs) 24 patients (36 %) scoring ≥ 2 (p 0.085). Graft source after conditioning with FBM/FLAMSA-Bu was bone marrow (1/2), G-CSF mobilized peripheral blood stem cells (40/62) and double-umbilical cord-blood (7/1). In 23 pts (20 %) HLA-matched related and in 91 pts (80 %) HLA-matched unrelated donor transplantation was performed. Engraftment failure was observed in 1 patient after FLAMSA-Bu, while engraftment was achieved in all evaluable patients of the FBM group in a median of 23 days vs 18 days after FLAMSA-Bu (p 0.003), while 7 pts with double-umbilical cord-blood transplantation where included in the FBM group vs 1 pt in the FLAMSA-Bu group. Non-hematological treatment-related acute toxicity ≥ CTC III (gastrointestinal, hepatic, cardiovascular, renal, centralnervous system) occurred in 12/48 pts (25 %) after FBM and in 18/66 pts (27 %) after FLAMSA-Bu. Incidence of severe acute (III-IV) and chronic GvHD was 22.9 %/16.6 % for FBM vs 18.2 %/19.7 % for FLAMSA-Bu, respectively. After conditioning with FBM 2/48 pts vs 9/66 pts after FLAMSA-Bu were diagnosed with a secondary malignancy (p 0.08). Non-relapse mortality (NRM) after 12 months was 26.8 % for FBM versus 25.2 % for the FLAMSA-Bu group. Incidence of relapse after FBM vs FLAMSA-Bu conditioning was 22.9 % vs 15.2 % after 1 year and 31.3 % vs 16.7 % after 2 years. Occurrence of relapse was significantly related to an incomplete or mixed chimerism (donor cells ≤ 95 % in peripheral blood and/or bone marrow) at day +30 (p 0.001). After a median follow up of 31.4 months (range 4.4-97.5) estimated overall survival (OS) and relapse-free survival (RFS) after 2 years was 55.4 % and 51.4 % for the FBM vs 58 % and 56.7 % for the FLAMSA-Bu group, respectively. Analyzing different subgroups, FBM conditioning might be favorable for pts aged ≥ 66 years when suffering from high risk AML (n=26): Within this group 1-year OS after FBM vs FLAMSA-Bu was 71.4 % vs 66.7 % (p 0.58) and 1-year RFS was 71.4 % vs 58.3 % (p 0.59), respectively. Notably, for pts at highest risk (aged ≥ 66 years and suffering from secondary or therapy-related AML; n=24) the benefit of FBM conditioning becomes more pronounced: 1-year OS after FBM vs FLAMSA-Bu was 62.5 % vs 37.5 % (p 0.26) and 1-year RFS 54.2 % vs 37.5 % (p 0.17). Both conditioning regimens are feasible, and provide similar rates of acute toxicity, NRM and GvHD. There might be evidence for a benefit of conditioning with FBM for the subgroup of “the oldest patients at highest risk”. Taking into account that there is an increasing group of ‘medically fit’ elderly patients in the field of allogeneic transplantation, prospective clinical trials are needed to investigate different conditioning regimens considering their special requirements. Disclosures No relevant conflicts of interest to declare.

Description: Chronic GvHD (cGvHD) is associated with endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in skin biopsies as well as increase of von Willebrand factor in plasma (BC Biedermann, Lancet 2002). Moreover an increased quantity of CD45RO positive lymphocytes has been observed in bone marrow biopsies (bmb) in CML pts with acute GvHD (aGvHD) grade III and lV, who were transplanted from unrelated donors (J Thiele, Bone marrow transplantation 2001). We analysed factors of T-cell activation and microvessels in bmb of pts with cGvHD to evaluate the function of the bone marrow in cGvHD. Methods: In an associated protocol of a prospective phase II study in 35 pts for the therapy of bone loss following HSCT with a bisphosphonate one bmb was taken prior of entry into the study and embedded in paraffin after decalcification. The interval between HSCT and time of biopsy was median 477 +/−998 days. For conventional semiquantitative evaluation of marrow and spongiosa a Giemsa staining was used. For immunological quantitative evaluation the following monoclonal antibodies were used with the APAAP method: CD34 and von Willebrand factor (VWF) for endothels of microvessels and CD45RO and CD8 for T-lymphocytes subsets. The pts were divided in 5 groups: neither aGvHD nor cGvHD; no cGvHD but aGvHD before entry; cGvHD limited; cGvHD extensive inactive; cGvHD extensive active, needing immunosuppression. Results: Conventionally analysed no pt had evidence of vasculitis. 1 pt of group 4 had one small immunoreactive aggregate of lymphocytes another pt of group 5 had a diffuse infiltration with lymphocytes. 32 pts had no or very small reduction of spongiosa and 3 had severe reduction of spongiosa 〈 15%. The significant immunological results were: Number/mm2 1. no GVHD (n=6) 2. aGvHD before (n=4) 3. cGvHD limited (n=5) 4. cGvHD ext. inactive (n=8) 5. cGvHD ext. active (n=12) p (group No) CD34+ vessels 12,6 10,4 8,5 20,7 27,9 〈 0,001 (1,2,3 vs 4,5) V. Willebrand 6,5 13,2 18,6 17,6 18,9 0,005 (1,2 vs 3,4,5) CD8+ 5,8 4,0 6,8 10,2 25,3 0,034 (1,2,3 vs 4,5) The number of CD34 as well as the number of vWF positive microvessels increased with the grade of involvement and activity of cGvHD (11,1 vs 25,3 respectively 8,7 vs 18,1). Additionally the content of CD8 positive lymphocytes increased with the grade and activity of disease. In contrast to reported skin biopsies, bone marrow capillaries were not destroyed by cytotoxic lymphocytes with increased activity. The bone marrow seems to show a different immunological reaction in contrast to pts with cGvHD of whom the target organs are skin, liver or gut. This was not observed on conventional biopsies. Further studies will be directed to the pattern of microvessels with regard to hematopoietic function and relapse.

Description: Hematopoiesis of the host is a primary target organ of the graft-versus-host reaction. However histological analyses of the bone marrow are rarely reported. Here we report histological changes in the bone marrow of patients (pts) with and without chronic graft-versus-host disease (cGvHD). Bone marrow biopsies were obtained between 101 days and 4623 days (median:419 days) after transplantation as part of a controlled prospective phase ll study of patients with osteopenia/osteoporosis after allogeneic hematopoietic stem cell transplantation (HCT). Previously we reported an increased density of microvessels using an antibody against v. Willebrand factor (vW) (Hill W. et al Blood110, abstract No 1963; 2007). Here we report additional immunohistological and immunocytological findings in marrow and blood. We analyzed the number of CD34+ and vW+ microvessels as well as CD8+ suppressor/cytotoxic T-cells/mm² (T-S) in sequential biopsies of pts with (n=9) or without (n=6) cGvHD after median 2 years apart. Biopsies of 3 pts without HCT and without lymphoma involvement served as controls. Simultaneously lymphocyte subpopulations were evaluated in peripheral blood samples of pts with (n=16) or without (n=8) cGvHD. The pts were divided in 5 groups: neither aGvHD nor cGvHD; no cGvHD but acute GvHD before entry; cGvHD limited; cGvHD extensive without immunosuppression; cGvHD extensive with immunosuppression. Results: In the first biopsies the content of CD34+, vW+ microvessels and T-S cells were significantly higher in pts with cGvHD (group 3–5) than in those without cGvHD (group 1–2) (21,3 vs 8,2 p=0,03; 22,0 vs 9,2 p=0,002 respectively 106,2 vs 32,1 p=0,04). In the second biopsies these parameters were also increased in cGvHD: CD34+ (18,3 vs 11,2 p=0,02), vW+ (17,3 vs 9,0 p=0,08) microvessels and T-S cells (63,2 vs 37,8 p=0,27). The increased density of CD34+ and vW+ microvessels correlated with the number of T-S cells (p=0,05). As compared to normal controls we observed a significantly higher content of vW+ microvessels in all groups of transplanted pts (16,9 vs 4,2 p=0,03). In pts with cGvHD (group 3–5) CD34+ and vW+ microvessels were further increased (p=0,02 respectively p=0,002). At the time of the first biopsy the absolute T-S cell content in peripheral blood was moderately increased in group 5 (1124/ul) and minimally increased in group 2 (993/ul) (normal 270 – 880), whereas the overall T cell (CD3) content was normal in all groups. The percentage of activated T-S (HLA-DR+) cells was increased in all groups of transplanted pts (61,8% vs normal =33%; p=0,05). After two years T-S cells content was reduced in pts under immunosuppressive therapy (group 5) (1415 vs 900/ul; p=0.000) but remained increased over the norm. In group 4 T-S cell content was increased over the norm (800 vs 920/ul; p=0,043). In conclusion, sequential immunohistology and immunocytology analyses on bone marrow biopsies and peripheral blood provide evidence for the existence of a chronic graft-versus-host reaction of the bone marrow in pts with cGvHD. This is characterized by an increased content of CD34+ and vW+ microvessels and an increased content of T-S cells at least initially. However this reaction does not lead to a generalized hematopoietic insufficiency.

Description: Decreased bone mineral density (BMD) has been found to be a relatively common complication following haematopoietic cell transplantation (HCT). Only some studies exist about therapy with antiresorptive substances such as bisphosphonates. These studies analyse either short time therapy or only the use for female patients (pts). The aim of this study was to test the effect and tolerance of zoledronic acid in long time survival pts after HCT. Methods: 40 pts with osteoporosis (WHO T-score

Description: Abstract 4540 Microangiopathy (MA) and graft-versus-host disease (GvHD) are serious complication after allogeneic hematopoietic cell transplantation (HCT). MA is comparable to a slight form of thrombotic thrombocytopenic purpura (TTP), characterized as de novo thrombocytopenia, hemolysis, evidence of fragmentocytes in the blood, hypertension, cerebral confusion and uremic syndrome. Conglomerates of platelets and uncleaved multimere of vWillebrand-Factor (vWf)) are the pathophysiological correlate and result in multiple occlusions of microvessels. Up to now the exact pathomechanism for chronic GvHD (cGvHD) is unknown but oftenly associated with decreasing platelets counts. In patients (pts) with cGvHD we previously described an increased number of vWf +and CD34+ microvessels and of CD8+ T-cells in the bone marrow (Hill,W Blood 112 abstract No 1166, 2008). The aim of this study is to screen bone marrow biopsies of pts after HCT for histological changes of vascular quality regarding to MA and correlate these findings with the development of cGvHD. Bone marrow biopsies of 36 pts (25 male, 11 female, 45 years median, range 18–64) after HCT (day 105 to 4623) were screened for overall number of vWf+ vessels, the number of sinus with a thick wall of vWf + deposit with a size 〉 15 × 8μ (plaquesinus) and the number of vWf+ deposit without evidence of sinus (plaques) as well as the number of megakaryocytes between Oct 2001 and Jul 2006. Morphological findings were correlated with GvHD or non-GvHD and with MA by the well known grading (aMA) at the time of acute GvHD (aGvHD) and with as a modificated grading (cMA) at the time of simultaneous cGvHD. Results: 26 of 36 pts had cGvHD (6 with limited and 20 with extensive disease). 22 had aGvHD before and 4 did not. 8 of the 36 pts had neither aGvHD nor cGvHD, 2 pts suffered from aGvHD. Regarding to aMA a moderate grade was found in 14 pts, a mild form was detected in 15 pts, and 7 pts did not have any signs of aMA. The levels of maximal elevation of lactatdehydrogenase (LDH) and maximal decrease of platelets (each p value 〈 0,02) were as followed: absent grade LDH mean 248 U/L and platelets decrease mean 36 %; mild grade LDH 299 U/L and platelets 52 % and moderate grade LDH 505 U/L and platelets 70%. Additional parameters (each p

Description: Acute graft-versus-host disease (aGVHD) is a major obstacle of allogeneic stem cell transplantation (SCT). We compared a cohort of 58 patients with HLA-haploidentical transplants (haplo) and a contemporary group of 229 patients with HLA-identical transplants (id) for the manifestation of aGVHD. Haplo-patients were given unmodified marrow (bm) and CD6- depleted mobilized blood cells (mbc) 6 days after marrow transplantation. The combination of cyclosporin A (CSA) and a short course of methotrexate (sMTX) was given post-grafting. Standard intensity conditioning was given to 34 and reduced intensity conditioning to 24 patients. Id-patients were given bm in 140, mbc in 79 and the combination of bm and mbc in 6 cases. Post grafting immunosuppression consisted of CSA either alone (N=10) or in combination with sMTX (N=158), and/or mycophenolate mofetil (MMF) (N=57). Conditioning was of reduced intensity in 50 patients and standard in 175 patients. Haplo-patients were younger in age (34 vs. 44 yrs.), more frequently male and in a more advanced stage of their disease. Manifestations of aGVHD, microangiopathy characterized by schistocytes and elevated LDH and virus infections were evaluated. Haplo-patients had more severe aGVHD of the skin than id-patients (IBMTR index B – D: 53% vs. 37%; p

Description: Allogeneic stem cell transplantation is limited to patients with a histocompatible donor, but for patients with advanced acute leukemia and high-grade lymphoma HLA-haploidentical transplantation may be considered. Rejection of the transplant and graft-versus-host disease are major obstacles and T-cell depletion eliminates the graft-versus-leukemia effect and causes prolonged immune deficiency. Here we studied the use of CD6-depleted G-CSF mobilized blood cells (mbc) 6 days after transplantation of unmodified marrow in order to suppress host-versus-graft and graft-versus-host reactions (GVHD) retaining a graft-versus-leukemia effect and the capacity to reconstitute the immune system. CD6-depleted mbc contain a large proportion of NK and NK-T cells. 63 patients with advanced disease (AML 32, ALL 15, NHL 11, CLL 2, CML 2, SAA 1) were transplanted with marrow from family donors sharing one HLA-haplotype and differing in 0 – 4 HLA-antigens of the second haplotype. Conditioning consisted of total body irradiation (TBI), antithymocyte globulin (ATG) and cyclophosphamide (CY), post-grafting immunosuppression of cyclosporin A (CSA) and a short course of methotrexate (sMTX). A transfusion of donor leukocytes was given prior to CY. Complete engraftment was observed in 34 evaluable patients given 12 Gy TBI. The dose of TBI could be reduced to 4 Gy without rejection in 25 evaluable patients. GVHD was severe (grade III and IV) in 12 of 48 evaluable patients. An improved method of CD6-depletion was administered to mbc in 9 patients and severe GVHD did not develop. GVHD responded to corticosteroids in most patients. 15 patients survive disease free up to 6 years (median 784 days). Recurrent infections including PTLD were the major cause of transplant-related mortality. Absolute counts of lymphocytes, CD4 and CD8 subpopulations were not different from those of a contemporary group of 46 patients in advanced disease given HLA-identical sibling transplants. However naïve CD4 cells and TRECs were low. Rejection, GVHD and GVL have been controlled by this regimen, but immune reconstitution remains a problem that may be solved by early discontinuation of immunosuppression in this regimen.

Description: Allogeneic stem cell transplantation produces a strong graft-versus-leukemia effect which may be enhanced by HLA-mismatches and immune recognition as a result of pregnancy. Therefore we compared the results of HLA-haploidentical transplantation (N=113) to those of HLA-identical sibling (N=195) transplantation performed in the same time. Cases were matched by disease category (acute myeloid leukemia-myelodysplastic syndromes, AML/MDS, acute lymphoid leukemia ALL and lymphoma-chronic lymphocytic leukemia NHL/CLL) and stage of the disease (early, intermediate and advanced). HLA-haploidentical transplants were performed with unmodified bone marrow followed by CD6-depleted mobilized blood stem cells on day 6.; patients with leukemia were in more advanced disease in leukemia, they were younger and more often male. In multivariate analysis survival was influenced by HLA-mismatch, stage of the disease and age of the patient, but conditioning treatment and CMV-seropositivity had no significant effect. Transplant related mortality was influenced by the age of the patient and HLA-match, to a lesser extent by the disease category and the stage, whereas CMV-seropositivity and conditioning had no influence. Remission duration was dependent on the stage of the disease, the donor recipient gender combination and the conditioning treatment in univariate analysis; in multivariate analysis only CMV-seropositivity had a poor prognostic impact. In comparable disease stages the relapse rate was not different in HLA-haploidentical from HLA-identical transplantation as was the rate of acute GVHD. In contrast the rate of chronic GVHD was lower in HLA-haploidentical transplantation. In ALL, relapse rate and remission duration was inferior with non-myeloablative conditioning as compared to myeloablative total body irradiation. In HLA-haploidentical transplantation the response rate of leukemia was better in patients homozygous for the cross reactive HLA-C group given a transplant from a heterozygous donor suggestive of NK activity. This GVL activity was not associated with GVH activity. Similarly maternal donors were superior to paternal donors and female donors in male recipients better than other gender combinations for the control of leukemia without increased GVHD. Non-inherited maternal antigen (NIMA) and inherited paternal antigen (IPA) in the graft-vs-host direction had a weak influence on the relapse rate, but no influence on GVHD. On an observational basis several sons and daughters transplanted with stem cells from the mother had a strong GVL effect without any signs of GVHD. However two have been lost due to untreatable pulmonary complications. Donors with cytotoxic antibodies have been excluded from donation, but cellular reactivity will have to be assessed in more detail. Recent progress in controlling EBV-associated disease by selection of donor T cells without expansion will improve HLA-haploidentical transplantation and show the way to selecting donor with the appropriate immune repertoire including reactivity to leukemia.

Description: Allogeneic stem cell transplantation from HLA-haploidentical family members has been studied in 80 patients for the treatment of hematological malignancies in advanced stages. The protocol involves unmodified marrow on day 0 and CD6-depleted G-CSF-mobilized blood cells on day 6. Engraftment was seen in patients treated with myeloablative (12 Gy) and non-myeloablative (4 Gy) doses of total body irradiation. Graft-versus-host disease (GVHD) was mild or absent in 53% and severe GVHD did not develop after changing to a more effective method of depletion of CD6-positive cells. Acute GVHD resolved more often and chronic GVHD was less severe than in HLA-identical sibling transplantation. Here we evaluated survival and relapse risk. Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) was the diagnosis in 41 patients, acute lymphoid leukemia (ALL) in 19 patients, chronic myelogenous leukemia (CML) in 2, chronic lymphocytic leukemia (CLL) in 3, high grade lymphoma (NHL) in 14 and severe aplastic anemia (SAA) in one patient. The patient’s age ranged from 17 to 62 years (median 36.5 yrs), for 65 patients a relative of first degree and for 15 patients a second degree relative was the donor. The major cause of death was recurrence of the disease (29 pts.), 7 patients died early and 22 pts. died of complications (infections 12, GVHD 3, pulmonary compl. 2, PTLD 3, cerebr. hemorrhage 1, liver tox. 1). Survival was significantly better in male patients with female donors (2 yr survival 36% vs 13%; p=0.001), patients transplanted with cells from their mother rather than from the father (2 yr survival 55% vs 8%; p=0.02) and patients transplanted in an earlier phase (2yr survival 50% vs 15%; p=0.048). Patients transplanted for ALL (2 yr. survival 31%) fared better than patients with AML/MDS (2yr survival 23%)and patients with NHL (2 yr. survival 12.5%) (p=0.08). Age, number and type of HLA-mismatches, as well as the depletion method had no effect on survival and on relapse risk. The favorable effect of the female gender of the donor on survival was still significant in multivariate analysis after adjustment for the stage of the disease and the diagnosis (p=0.01). The possibility of non-inherited maternal antigens (NIMA) inducing tolerance was tested in siblings sharing the paternal haplotype. There was a weak effect in the host-versus-graft direction (p=0.06), but no effect in the graft-versus-host direction. The risk of relapse was lowest in patients transplanted with stem cells from their mother as compared to those transplanted from their father (p=0.002). Female gender of the donor was favorable even in patients without GVHD. In summary female donors are preferable to male donors for control of leukemia and survival.