ALBERT

Description: HLA incompatibility between the donor and recipient is the most critical factor governing the incidence of rejection and GVHD after conventional allogeneic stem cell transplantation. But the impact of HLA disparity on GVHD and graft rejection after RICT remains to be elucidated. We retrospectively analyzed the outcomes of 437 patients who underwent bone marrow (n=95) or peripheral blood stem cell RICT (n=342). The numbers of patients who received a graft from a HLA-matched (275 from siblings, 11 from family members, and 54 from unrelated donors), one-locus-mismatched, 2- or 3-loci-mismatched donor were 340, 65, and 32, respectively. The HLA-matched group included significantly higher population of patients who received cyclosporine alone for GVHD prophylaxis. The overall cumulative incidence of grade II-IV acute GVHD was 40% for all subjects. It was 38% (95% CI; 33%–43%) in recipients of HLA-matched donors, 43% (95% CI; 31%–54%) in those of one-locus-mismatched donors, and 54% (95% CI; 37%–68%) in those of 2–3-loci-mismatched donors. A Cox regression model adjusted for potential confounders including GVHD prophylaxis demonstrated that 2-3 loci-mismatch was identified as an independent risk factor of grade II-IV acute GVHD (Table). Use of antithymocyte globulin was identified as an independent better protective factor for GVHD (HR;0.66, p=.003). Cumulative incidence of rejection was significantly higher after one-locus mismatch RICT (Table) and the risk tended to increase in relation to an increase of HLA disparity. Malignant disease was identified as an independent prognostic factor for rejection. In patients with hematologic malignancies, overall survival (OS) of recipients of 2–3-loci-mismatched RICT at 1 year (38%, 95%CI; 21%–54%) was significantly worse than that after HLA-matched RICT (65%, 95%CI; 59%–70%). By contrast, there was no statistical difference in the incidence of grade II-IV acute GVHD and OS between HLA-matched RICT and one-locus-mismatched RICT. Multivariate analysis demonstrated 2–3-loci-mismatch (Table) and high-risk disease (HR; 2.3, p=.001) as independent risk factors for OS. Thus, HLA incompatibility between the donor and recipient is an important risk factor for rejection, acute GVHD and overall survival after RICT. Therefore RICT from a one-locus-mismatched donor may represent an effective alternative approach in patients lacking HLA-matched sibling donors. multivariate analysis n acute GVHD Rejection OS HR (95%CI) p HR (95%CI) p HR (95%CI) p match 340 1.0 1.0 1.0 1-mismatch 65 1.4 (0.9–2.2) 0.20 4.5 (1.1–17.9) 0.03 1.0 (0.6–1.6) 0.88 2–3-mismatch 32 2.2 (1.2–4.1) 0.02 7.0 (0.8–64.8) 0.08 3.3 (1.8–6.2)

Description: Background: The optimal therapy for relapsed acute promyelocytic leukemia (APL) after arsenic trioxide (As2O3)-induced remission is unclear. Hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality. Moreover, lasting remission is observed in many patients who are not candidates for HSCT, owing to advanced age or lack of donors, implying that HSCT is not mandatory for durable remission. We evaluated our results of an As2O3-based, non-HSCT regimen for patients with relapsed APL. Materials and methods: Forty-two consecutive patients (18 men, 24 women, median age: 35 years, 12–72) with relapsed (relapse 1, R1=39, R2=3) APL were treated with an-As2O3 based, non-HSCT regimen. The time from last complete remission (CR) was 22 (6–243) months (mo). Initial treatment was As2O3 (10 mg/day) either intravenously (n=16) or orally (n=28) until CR, followed by idarubicin consolidation (6 mg/m2/day x 9). Twenty-five patients received oral-As2O3 maintenance. Post-As2O3 relapses were treated with oral As2O3 + all-trans retinoic acid (ATRA, 45 mg/m2/day) until CR, followed by maintenance (two weeks of ATRA+As2O3 every 2 mo. for 2 years). Post-As2O3/ATRA relapses were treated with oral As2O3+ATRA+ascorbic acid (1g/day) until CR, followed by consolidation/maintenance with the same regimen (2 weeks every 2 mo. for 2 years). Part of the induction and all of the maintenance therapies were given in the outpatient clinic. Results: Forty-one patients (98%) achieved CR after initial As2O3 treatment. One 72-year old man with XYY syndrome, diabetes and mental retardation died of pneumonia. Thirteen relapses occurred at a median of 15 (6–22) mo. As2O3-maintenance significantly decreased further relapses (3/24 with versus 10/17 without As2O3-maintenance, p=0.003). Two relapses died of cerebral APL before further treatment could be administered. Of eleven patients treated with As2O3+ATRA, 10 achieved CR, 8 of whom have remained in remission (median follow-up: 33 mo.). Two post-As2O3/ATRA relapses achieved CR again with As2O3+ATRA+ascorbic acid, and have remained in remission after maintenance treatment with As2O3+ATRA+ascorbic acid. All patients in continuous remission (n=38) were PML/RARa negative on polymerase chain reaction (sensitivity 10−3 to 10−4). Conclusion: Our regimen resulted in a leukemia-free-survival of 89.3%. The results suggest that an oral and mainly outpatient As2O3-based, non-HSCT strategy is efficacious for relapsed APL. In terms of survival, costs, treatment side effects and patient tolerance, the results appear to be comparable to, if not more favorable than, other treatment options based on high dose chemotherapy, graft-versus-leukemia effect, or anti-myeloid antibody therapy.

Description: Background: Chronic lymphocytic leukemia (CLL) cells are weakly immunogenic, a property that may contribute to disease progression and inhibit the effectiveness of immunotherapies such as vaccines. Low surface expression of co-stimulatory molecules contributes to this poor immunogenicity. CLL cells express Toll-Like-Receptor-7 (TLR7), a powerful modulator of innate immunity. TLR7 agonists may be capable of enhancing the immunogenicity of CLL cells and thereby increasing T cell mediated killing of CLL. Methods: Circulating CLL cells were isolated directly from consenting patients by negative selection. TLR7 mRNA expression by CLL cells was demonstrated by RT-PCR. CLL cells were then incubated with S28690 (a TLR7 agonist), or with a negative control for 24–72h. Expression of the costimulatory molecules CD80, 83, 86, and 54 was determined by flow cytometry pre and post-incubation. Experiments were repeated in the presence of a NFkB inhibitor (dexamethasone), a p38 MAPK inhibitor, and a protein kinase C agonist (PDB). The effects of S28690 on phosphorylated-IkB and phosphorylated-STAT3 levels were measured by immunoblotting. The capacity of S28690-incubated CLL cells to stimulate T cell proliferation and killing was determined in mixed lymphocyte responses. Results: All tested CLL samples (n=20) expressed TLR7 mRNA, while Jurkat cells (T cell origin) did not. After incubation with S28690, CD80, 83, 86, and 54 surface expression increased on all CLL samples tested. The relative increase varied from 4 to 9-fold and was positively correlated with CD38 expression. NF-kB and p38 inhibitors decreased the effects of S28690 on co-stimulatory molecule expression while PDB amplified the effect. After incubation with S28690, IkB and STAT3 phosphorylation increased in CLL cells. S28690-incubated-CLL cells were able to stimulate moderate T cell proliferation, but did not increase T cell mediated killing of CLL cells. However, CLL cells incubated with both S28690 and PDB (a PKC agonist), exhibited much lower amounts of phosphorylated STAT3, triggered marked T cell proliferation, and stimulated T cell mediated killing of CLL cells. Conclusions: S28690 (a TLR7 agonist) causes increased expression of co-stimulatory molecules by CLL cells in vitro and transforms CLL cells into moderate stimulators of T cell proliferation. The effects of S28690 are synergistic with PKC agonists, potentially as a result of S28690-mediated NFkB activation and concurrent PKC-mediated inhibition of STAT3. These findings may find clinical application in immunotherapeutic approaches to CLL.

Description: We evaluated the toxicity-profile, engraftment potential, and efficacy of fludarabine-based nonmyeloablative allogeneic HCT in patients with a variety of nonmalignant hematological disorders. Twenty three patients (median age 29 years; range 11–52) with nonmalignant hematological disorders including ATG refractory SAA (n=13), severe paroxysmal nocturnal hemoglobinuria (PNH: n=9), and pure red cell aplasia (PRCA; n=1) were transplanted from 5/99 – 8/2004 at the NHLBI. The majority of patients had an extensive transfusion history including 11/23 who had HLA allo-antibodies and 4/23 with allo-antibodies to RBCs. Conditioning with fludarabine (25 mg/m2 x 5 days), ATG (40mg/kg x 4 days) and cyclophosphamide (60mg/kg x 2 days) was followed by infusion of an un-manipulated G-CSF mobilized allograft from an HLA matched sibling (n=18), parent (n=2), or single antigen mismatched sibling (n=3). GVHD prophylaxis consisted of cyclosporine (CSA) either alone (n=2) or combined with mycophenolate mofetil (n=10) or mini-dose methotrexate (n=11). Despite a high prevalence of pre-transplant allo-immunization, all patients achieved sustained donor engraftment in both myeloid and T-cell lineages. Myeloid recovery (neutrophils 〉500cells/uL) occurred at a median 14 days post transplant (range 8–18 days). Conversion from mixed to full donor myeloid and T-cell chimerism occurred in all patients by 110 days post-transplant. CMV reactivation occurred in 11/21 patients at risk (KM probability 52%) without any cases of CMV disease. Grade II–IV and III–IV acute GVHD was the major transplant complication occurring in 13/23 (KM probability 60%) and 8/23 (KM probability 38%) patients respectively. Fourteen of 21 evaluable patients developed chronic GVHD (limited in 11 cases), which resolved completely with low-dose alternate day steroids and/or CSA in all but 1 case. One patient who received an allograft from his HLA matched father died 16 months post-transplant from complications related to chronic GVHD. With a median follow up of 25 months (range 1–64 months), 20/21 patients evaluable more than 100 days post-transplant survive in complete remission with full donor chimerism in all lymphohematopoietic lineages (KM probability of long-term survival 92.8 %-see figure ). Conclusion: Fludarabine-based nonmyeloablative transplantation achieves excellent donor engraftment and long-term disease free survival in heavily transfused and allo-immunized patients with ATG refractory SAA and other nonmalignant hematological disorders associated with bone marrow failure.

Description: Introduction. B cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease as shown by differential expression of a variety of surface and cytoplasmic markers. In a search for markers that could define biological activity of different B-CLL subsets, we have studied the surface expression of the Toll-like receptor (TLR) family member CD180 in relation to other surface markers and mutation status of IgVh genes. Methods. Seventy eight B-CLL patients (68 untreated and 10 treated) and 15 age-matched controls were studied in three different clinics. CD19+ B cells were stained using indirect immuno fluorescence for CD180, surface IgM (sIgM), CD79b and CD38, analysed by flow cytometry and data expressed as the relative antibody binding sites (RBS)/cell for each marker. Monoclonal anti-CD5 antibodies were also used with anti CD180 to determine levels of expression of CD180 in control CD5+ cells. IgVh mutation was determined for 47 patients. Results B-CLL cells had variable levels of CD180 expression, but this was always less (1036 ± 935 RBS/cell) than that expressed by normal blood B cells (5548 ± 2271 RBS/cell) and was stable for up to 18 months. Significantly higher levels of CD180 were expressed by B-CLL cells with mutated (M) compared with those using unmutated (UM) IgVh genes. This was in contrast to the higher levels of expression of sIgM by B-CLL cells using UM than M IgVh genes (Figure). Conclusions. CD180 is expressed at higher levels on B-CLL cells using M than those using UM IgVh genes and is in contrast to the level of expression of sIgM which is higher on B-CLL cells using UM versus M genes. This differential expression of CD180 supports the notion that B-CLL cells using UM IgVh genes represent a population of cells actively responding to signals (perhaps to self antigens) via their surface IgM. Figure Figure

Description: BACKGROUND: Disease manifestations in myelofibrosis with myeloid metaplasia (MMM) that require therapy include anemia, thrombocytopenia, hepatosplenomegaly, and constitutional symptoms. We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone for the treatment of both anemia and thrombocytopenia in MMM (Blood2003;101:2534) and of etanercept (tumor necrosis factor-alpha antagonist) for alleviation of constitutional symptoms (Blood2002;99(6):2252). In the current study, we investigated the value of combining all three drugs (PET). METHODS: Study eligibility criteria included a histologically confirmed diagnosis of MMM and either severe anemia (hemoglobin 〈 10 g/dL) or symptomatic splenomegaly. Protocol treatment was initiated with 50 mg of oral thalidomide daily, etanercept 25 mg subcutaneously twice-a-week, and a three month prednisone taper starting at 0.5 mg/kg/day for the first month followed by a 50% reduction for the second months and a further 50% reduction for the third and final month of treatment with prednisone. Patients with a demonstrable clinical response after three months were able to remain on thalidomide and etanercept. RESULTS: To date 12 patients have been enrolled to the trial (median age 67 years, range, 54–77; 11 males). At enrollment, all patients were severely anemic (6 were red cell transfusion dependent), 6 were at least moderately thrombocytopenic (platelet count 〈 100 x 109//L), and 4 had severe constitutional symptoms. The responses to date included resolution of constitutional symptoms in all 4 pertinent patients, a 12–326% improvement in thrombocytopenia in 4 of 6 pertinent patients, and anemia response in 4 patients including one patient who became red cell transfusion independent. A more than 50% decrease in splenomegaly was documented in 3 patients. Only one patient experienced greater or equal to grade 3 toxicity (i.e. elevated bilirubin and infection). Follow-up bone marrow examination, when available, did not show post-treatment histological changes. CONCLUSIONS: The preliminary findings from the current ongoing phase II study of combination therapy with thalidomide-prednisone-etanercept in patients with MMM confirms the previously recognized value of etanercept in alleviating constitutional symptoms. However, at least preliminarily the PET regimen does not appear to be superior to thalidomide-prednisone combination in terms of therapeutic value for anemia, thrombocytopenia, or splenomegaly.

Description: Delayed administration of DLI to mixed hematopoietic chimeras following allogeneic bone marrow transplantation (alloBMT) leads to conversion from mixed to full donor chimerism without GVHD. However, in the clinical setting development of GVHD is still the most common complication. Donor T cells are the main mediators of GVHD in alloBMT. However, the precise role of host dendritic cells (DC) and their different subsets needs still to be elucidated. CD8a+ DCs seem to have a regulatory function and to reduce experimental GVHD. Recently, it has been shown that the ICSBP (Interferon Consensus Sequence Binding Protein) gene is essential for the development and activation of CD8a+ DCs. ICSBP-gene deficient mice (ICSBP KO) are characterized by several immune defects and systemic expansion of myeloid cells mimicking human CML and may be therefore an attractive in vivo model to evaluate the role of DCs in alloBMT. The aim of the study was to evaluate the 1. feasibility of inducing mixed hematopoietic chimerism following nonmyeloablative conditioning in ICSBP KO mice using fully MHC-mismatched BMT (Balb/c [H2Dd] to B6 x 129 [H2b]), and 2. susceptibility of ICSBP KO mice to the development of GVHD following administration of DLI. Nonmyeloablative conditioning consisted of in-vivo T cell depletion (anti-CD4-[GK1.5], anti-CD8-[2,43] mAb, d-5), fludarabine (30 mg/kg, d-4 to d-2), cyclophosphamid (200 mg/kg, d-1) and 3 Gy TBI (d0) followed by i.v. injection of 1,5 x 10E7 Balb/c bone marrow cells. Stable mixed chimerism was observed in ICSBP wildtype (wt) mice through week 35 post-BMT without signs of clinical GVHD. In contrast, we observed increasing donor CD4 and CD8 T cell chimerism over time in ICSBP KO mice suggesting a lack of donor T cell tolerance. Furthermore, chimerism was higher in granulocyte, B cells and monocyte compared to ICSBP wt mice. In a second experiment we were able to observe spontaneous conversion to full donor hematopoietic chimerism in 2 of 9 mice after nonmyeloablative BMT. In two separate experiments ICSBP wt or KO mice with mixed chimerism received (DLI) on day 35 post BMT. After injection of DLI all wt mice (n = 10) showed a rapid conversion to full donor chimerism and remained healthy until the end of the observation period without signs of GVHD. Despite myeloid hyperproliferation all ICSBP KO mice showed conversion to full donor chimerism. Furthermore, in ICSBP KO mice conversion to full donor chimerism was associated with development of severe GVHD (n = 9). In vitro studies using mixed lymphocyte reactions showed that splenic stimulator cells from ICSBP KO mice had a higher stimulatory capacity compared to wt stimulators (SI 1,5 versus 2,3). Using the same mixed lymphocyte culture (MLC) conditions, supernatants collected from BALB/c responders with ICSBP KO stimulators had higher levels of TNF-alpha and IFN-g compared to MLC using ICSBP wt stimulators. In conclusion, following alloBMT a defective development of host-specific tolerance and high susceptibility for GVHD in ICSBP KO mice is suggested. Further studies are warranted to delineate the precise underlying mechanism by addressing the potential contribution of defective regulatory host T cells and the lack of host CD8a+ DCs in ICSBP KO mice.

Description: Isolated chronic thrombocytopenia (CT) following allogeneic HCT is associated with an increased risk of transplant-related mortality and a reduction in long-term survival. Although the pathogenesis of CT is not entirely clear, it has been speculated that immune activation and cytokine dysregulation characteristic of chronic GVHD (CGVHD) play a similar role in the pathogenesis of this disorder. To better understand the impact of endogenous cytokine expression on platelets, we analyzed pro and anti-inflammatory cytokines in the serum of patients with CT following allogeneic HCT, comparing levels to healthy donors and to patients with normal platelet counts after allo-transplantation. Serum was obtained from 3 patient groups; Group 1- healthy donor controls (n=12): Group 2- post-transplant from allogeneic HCT patients with normal platelet counts (n=31): Group 3- post transplant from patients with CT (n=11). Using micro sphere-based Luminex flow cytometry, serum was analyzed for levels of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α as well as Th(1) cytokines IL1-b, IL-2, IL-12, IFN-g and Th(2) cytokines IL-4, IL-5, and IL-10. Results: Both post transplant cohorts (Group 2 and 3) had higher mean levels of GM-CSF (110 and 120 pg/ml), IFN-g (17 and 16 pg/ml), and IL-12 (318 and 202 pg/ml) compared to their respective non-transplant (Group-1) controls (GM-CSF 23 pg/ml: p

Description: 112 patients with senile acute myelocytic leukemia (AML), refractory or relapsed AML, and MDS-RAEBt entered this study to receive aclarubicin and low dose Arabinoside Cytosine (Ara-C) in combination with Granulocyte Colony Stimulating Factor (G-CSF) for evaluation of efficacy and tolerance of this CGA regimen. Low dose Ara-C was given at the dosage of 10mg/m2/12h, subcutaneously, D1-14 and aclarubicin 14mg/m2/d, intravenously, D1-4 (regimen A) or 7mg/m2 D1-8 (regimen B). Recombinant G-CSF was given at the dosage of 200μg/m2/d, subcutaneously, D1-14. We proved that overall response rate was 19/26 (73.1%) in senile AML, 48/62 (77.4%) in refractory AML, 12/18 (66.7%) in relapsed AML and 10/13 (76.9%) in MDS-RAEBt; and CR rate was at 8/26 (30.8%) in senile patients, 30/62 (48.4%) in refractory AML, 8/18 (44.4%) in relapsed AML and 5/13 (38.5%) in MDS-RAEBt, which were comparable in four groups of patients. 52 patients were followed up. Median progression free survival (PFS) and overall survival (OS) were 9.0±2.2 months and 11.0±1.6 months, respectively. The Kaplan-Meier estimated PFS and OS rate at 12 months were 40.73±8.15% and 42.85±8.23%, respectively. 1 year OS and PFS rate of the patients with CR were 60%±10.8% and 51.3%±13.7%, respectively, compared with the patients with PR, 17.7%±9.3% and 6.4%±6.1%, respectively. The toxic effects were very rare, mainly manifested as hematological changes, neutropenia and thrombocytopenia due to myelosuppression, which was around 70–80% exceeding NCI grade II. And non-hematological toxicities were not observed in this study. CAG regimen seems to be promising for the treatment of various categories of poor-prognosis AML or MDS-RAEBt, with acceptable toxicity.