ALBERT

Description: Background. We previously reported data from 103 pts receiving PBSC from HLA-matched unrelated donors after conditioning with 2 Gy total body irradiation (TBI) plus 90 mg/m2 fludarabine. Postgrafting immunosuppression included MMF (administered from day 0 until day +40 with taper through day +96), and CSP (given from day -3 to day +100, with taper through day 180) (historical group). The incidences of grade III–IV acute and chronic extensive GVHD were 14% and 48%, respectively. Several studies have suggested that CSP could prevent activation-induced cell death of T-cells, and thus potentially delay the eradication of donor-versus-host alloreactive T-cells, preventing tolerance induction. Conversely, antimetabolite inhibitors, such as MMF, could delete alloreactive T-cells by induction of activation-induced cell death and apoptosis, and thus might favor tolerance induction Pts and Methods. Here, we investigated whether postgrafting immunosuppression with prolonged MMF (given at 15 mg/kg orally thrice a day from day 0 to day +30, then at 15 mg/kg orally twice a day until day 150, and then tapered at day 150 and discontinued at day 180) and truncated CSP (abruptly discontinued on day 80), would promote tolerance induction and reduce the incidence of GVHD (current protocol, n=71) after unrelated HCT with nonmyeloablative conditioning. Results. Sustained donor engraftment was achieved in 68 pts (96%) in the current protocol, versus in 98 pts (95%) in the historical group. Grades II, III and IV acute GVHD were seen in 36 (50.7%), 11 (15.5%) and 7 (9.9%) pts, respectively, in the current protocol, versus 39 (37.9%), 11 (10.7%) and 4 (3.9%) pts, respectively, in the historical group. The incidences of grade II–IV and grade III–IV acute GVHD were 75% and 23% in the current protocol, versus 52% (P=.05) and 14% (P=.14) in the historical group. The 1-yr incidence of chronic GVHD was 46% in the current protocol, versus 48% in the historical group. Finally, the 1-yr probabilities of relapse, nonrelapse mortality and progression-free survival were 24%, 36% and 40%, respectively, in the current protocol, versus 26% (P=.9), 18% (P=.005) and 56% (P=.04) in the historical group. The increased incidence of nonrelapse mortality in the current protocol was not due only to the increased incidence of grade II–IV acute GVHD, since nonrelapse mortality remained significantly higher in the current protocol than in the historical group after adjusting for occurrence of grade II and grade III–IV acute GVHD (P=.04). Evaluation of pretransplant comorbidities is ongoing. Conclusions. Postgrafting immunosuppression with prolonged MMF and truncated CSP failed to decrease the incidence of GVHD after nonmyeloablative conditioning with URD. Ongoing efforts are directed at reducing the risk of acute GVHD after nonmyeloablative conditioning for unrelated donors by replacing tacrolimus for CSP, and by adding sirolimus to MMF and CSP.

Description: Donor lymphocyte infusions (DLI) have radically changed the prognosis of patients relapsing after allogeneic hematopoietic stem cell transplant (SCT) for chronic myeloid leukemia (CML). Major obstacles to success with DLI are represented by leukemia resistance and by secondary GvHD (GvHD2). The best result of is when a patient treated with DLI achieve a durable molecular remission without experiencing GvHD2. It is unclear which factors may predict for such a favourable outcome when CML patients are treated with DLI. We retrospectively identified 500 patients (59% males, median age 39 years, range 4–64), treated with DLI for CML relapse (81 molecular [16%], 150 cytogenetic [30%], 211 hematological chronic [42%], and 58 hematological accelerated [12%]) at 68 EBMT centers before 2004 with adequate information collected on disease response, GvHD2 and survival after DLI. Donor was an HLA-identical sibling in 73%, unrelated in 27%. DLI started with a cell dose 1997), showed that chronic GvHD after transplant and prior to relapse (hazard ratio [HR]: 1.5, 95CI: 1.2–1.9, p

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: Introduction: Relapse of the malignant disease remains the major challenge after reduced intensity conditioning (RIC) HCT. Here we investigate the outcome of haematological relapse after low dose TBI-based conditioning regimens and the possibility to monitor minimal residual disease in patients with AML with the aim to prevent haematological relapse. Patients and methods: From 7/98 – 12/06, 156 consecutive pts [86 m/70 f; median age 61 (range 21–75) y] with CD34+ AML, n=138 and high-risk MDS, n=18 received RIC-HCT. Stage of disease at HCT was CR1, n= 99 (63%), CR2, n=18 (12%), and 〉CR2, n=39 (25%). HCT were performed from matched related donors (n=40) or matched unrelated donors (n=116) after 200cGy TBI ± fludarabine 30 mg/m2/day on 3 days followed by mycophenolate mofetil and cyclosporine. Chimerism was monitored at days (d) 28, 56, 84, and at 3 months interval thereafter in unsorted as well as flow-sorted CD3+ and CD34+ bone marrow cells by FISH for the XY chromosome in gender mismatched (100–300 IP) or PCR based analysis of polymorphic micro satellite regions in gender matched HCT. A decrease of 〉5% CD34+ donor chimerism (“CD34+ relapse”) was followed by a taper of immunosuppression. Haematological relapse was defined as increase of blasts to 〉5%. Results: Of the 156 patients, 141 pts engrafted as shown by donor T-cell chimerism. OS, RI, and NRM at 5 y were 38±0,05%, 47±0,06%, and 30±0,05% respectively. From the 141 patients, 48 had hematological relapse. Treatment of relapse included reduction of immunosuppression, low dose AraC and intensive chemotherapy ± mobilized buffy coat. CR was achieved in 50% of the patients. Reduction of immunosuppression and/or DLI (p=0,04), amount of overall donor chimerism at diagnosis (p=0,01), the interval from HCT to relapse (p=0,02), and the occurrence of GvHD (p=0,05) were factors associated with CR in the multivariate analysis. Survival of patients with haematological relapse, however, was poor (3,6±0.03% at 5a). All patients with haematological relapse within 100 d died despite therapy. Survival was independently influenced by the time of relapse after HCT (100 d; p=0.001), by the myeloblast count in the marrow (p=0.01) and by the type of relapse (haematological vs. “CD34 relapse” only). All patients with haematological relapse and available CD34+ donor chimerism (38/48 patients) had a decrease of CD34+ chimerism a median of 17 (range 0–181) d before the diagnosis of haematological relapse. Decrease of CD34+ chimerism without total chimerism decrease was observed in 12 additional patients (8,7%) a median of 149 (range 56–852) d after HCT. CD34+ donor chimerism amounted 84 (0–92)% before and returned to 100 (93–100)% after reduction of immunosuppression. Survival of these patients was 90±9% at 4 y. Conclusions: We conclude that relapse after low-dose TBI based conditioning has a poor outcome with survival of

Description: Introduction: Bortezomib is a novel proteasome inhibitor that has shown important clinical efficacy either as a single agent or in combination with other cytostatic agents in relapsed/refractory multiple myeloma (MM). The combinated treatment of bortezomib with bendamustine and prednisone (BPV) was assessed to determine the efficacy and toxicity of this regiment in patients with advanced MM. Methods: Between January 2005 and July 2007, 46 patients (median age 63; range 31–77 years) with relapsed or refractory MM (29 patients stage III a, 17 patients III b) were treated with bendamustine 60 (−80) mg/qm on day 1 and 2, bortezomib 1,3 mg/qm on day 1, 4, 8 and 11, and prednisone 100 mg on day 1, 2, 4, 8 and 11. Cycles were repeated every 21 days until maximum response or progressive disease. The time from first diagnosis ranged from 1 to 183 (median 36) months. The duration of the last remission before beginning the BPV-therapy was 6 (range 0–36) months. Previous therapy lines (median 2, range 1–6) included 18 × thalidomide, 10 × autologous PBSCT, and 9 × autologous/allogeneic PBSCT. 16 patients were refractory to the last treatment. 22 patients had preexistent severe thrombocytopenia, leukocytopenia or anemia (WHO grade 3 or 4). Response was assessed using EBMT criteria modified to include near complete remission (nCR) and very good partial remission (VGPR). Results: 36 patients (78%) responded after at least one cycle of chemotherapy with 2 CR, 5 nCR, 6 VGPR, 15 PR and 8 MR. 4 patients had stable disease and 6 patients had a progress. With a median follow up of 13 months, EFS, and OS at twelve months for patients without severe haematological toxicities due to previous treatments (n=24) were 46% and 79%, respectively. Outcome for these patients was significantly better compared to patients with severe haematological toxicities (grade 3 or 4, n=22) where EFS, and OS were 10% and 22%, respectively (p

Description: Conventional allogeneic hematopoietic cell transplantation (HCT) is effective treatment for AML, but its use has been restricted to younger patients in good medical condition. However, AML is increasingly common with advancing age, and older patients have more adverse prognostic features than younger patients. The vast majority of older patients who reach a CR with chemotherapy relapse within two years from diagnosis and die within three years. The situation is similar in patients with secondary AML or with AML in CR2, more advanced remission status or in partial remission, where median duration of disease-free survival (DFS) is generally

Description: In the last years, focus of regenerational studies has pointed on mesenchymal stem cells (MSC) and their ability to differentiate into several mesenchymal tissues. MSC have been shown to play a pivotal role in the microenvironment of bone marrow cells and in the modulation of immune response as they can suppress lymphocytic proliferation in vitro. Moreover, some animal studies have suggested they could favor the proliferation of malignant cell clones in solid tumor models. Their role in hematological malignancies, however, remains to be further elucidated and little is known about the influence of MSC in the development and maintenance of the malignant clone in chronic myeloid leukemia (CML). This disease is characterized by the presence of the Philadelphia (Ph) chromosome, a fusion product generated by the reciprocal translocation between chromosomes 9 and 22. Previous reports showed that hepatocytes precursors, found in the liver of CML patients carry the Ph translocation. Our intent was to elucidate whether MSC isolated from patients with CML in different stages of the disease originate from the malignant clone. To this purpose bone marrow aspirates of 11 patients with CML were obtained after informed consent. Five patients were analyzed at diagnosis, two after allogenic stem cell transplantation, three on treatment with the tyrosine kinase inhibitor imatinib and one on treatment with interferon alpha in combination with hydroxyurea. MSC were then generated as previously described. Briefly, cells were isolated by density gradient methods, resuspended in RPMI1640 medium containing 10% fetal bovine serum and plated in culture flasks to adhere. After 4–5 weeks of culture cells were collected and characterized by the expression of several surface markers in a fluorescence activated cell sorter (FACS). The presence of the Ph chromosome was assessed by both fluorescence in situ hybridization (FISH) and polymerase chain reaction (nested PCR). Moreover whole bone marrow was analyzed and results compared with those obtained in the MSC population. MSC showed a typical morphological pattern, growing to confluence after a few weeks of culture and appearing as an adherent, spindle shaped cell layer. In FACS they stained positive for SH2 and SH3 and did not express CD34, CD45 and CD14. MSC were then analyzed by FISH using probes for BCR-ABL. We could not detect the Ph translocation in any of the analyzed patients, though it was present at variuos levels in the remnant bone marrow cells. Results did not change, if expression of BCR-ABL was measured by high sensitivity RT-PCR. Our results showh that MSC of patients with CML are Philadelphia negative irrespective of the stage of disease and the treatment given, suggesting that these cells are not involved in the development of the malignancy. However, their interactions with leukemic cells as well as their role in the immune response against the tumor remains to be further characterized.

Description: NK-cells have been shown to play a pivotal role in haploidentical hematopoietic cell transplantation (HHCT) for engraftment, GvL effects and to combat infectious complications. Different strategies have been employed to hasten NK-cell recovery after HHCT. Here we compare the immune recovery of 17 patients after CD34 selected HHCT receiving additional adoptive CD3-depleted CD56-enriched NK cells 2 days after HHCT (adoptive NK-cells), with 18 patients receiving CD3/CD19 depleted grafts (CD3/CD19) for HHCT. Transplantations were performed at two different institutions with a median follow-up of 〉1 year. Conditioning consisted of 12 Gy TBI, thiotepa (10mg/kg), fludarabine (150 mg/m2) and OKT3 (day −4 to +2) in the group receiving CD34 selected grafts and adoptive NK-cell transfusions. All patients in the CD3/CD19 group received conditioning with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (day −5 to +14). No postgrafting immunosuppression was used in both groups. Seven out of the 17 patients in the adoptive NK-cell group received IL-2 activated NK cells. Median age was 37 years in the adoptive NK-cell group compared to 40 years in the CD3/CD19 group. Diagnoses in the adoptive NK-cell group included AML (n=10), ALL (n=3), CML (n=2), and Hodgkin’s disease (n=1) and MDS (n=1). Diagnoses in the CD3/CD19 group were AML (n=10), ALL (n=5), NHL (n=1), CML (n=1) and multiple myeloma (n=1). The grafts contained a median of 12.5x10E6 CD34+ cells/kg and 1.1×10E4 CD3+ cells/kg in the CD34 selected group versus 9.2×10E6 CD34+cells/kg and 2.3×10E4 CD3+cells/kg in the CD3/CD19 group. The number of transferred CD56+ cells was 8.3×10E6/kg in the adoptive NK-cell group and 7.2×10E7/kg cells in the CD3/CD19 group. Hematopoietic recovery was similar in both groups. Among the patients receiving adoptive NK-cells we observed a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells: patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant (p=

Description: We evaluated the utility of allogeneic HCT using nonmyeloablative conditioning as a potential treatment option for 79 patients (pts) with HL who were ineligible for high dose conventional allogeneic HCT and compared outcome based on donor cell source (MRD n=34, URD n=24, Haplo n=21). Conditioning consisted of 2 Gy TBI alone (n=15, MRD) or combined with either 90 mg/m2 (MRD n=19, all URD) or 150 mg/m2 (all Haplo) fludarabine. All haplo recipients also received cyclophosphamide pre (29 mg/kg) and post (50 or 100 mg/kg) HCT. GVHD prophylaxis consisted of mycophenolate mofetil and a calcineurin inhibitor. Pts were heavily pretreated with a median number of 5 (range, 2–10) prior regimens. Most pts failed prior autologous HCT (91%) and radiation therapy (86%). There were no graft rejections. The incidences of acute grades III–IV and extensive chronic GVHD were 15%/47% (MRD), 8%/60% (URD), and 10%/31% (Haplo). There were no statistically significant differences in the ability to discontinue immunosuppression based on donor cell sources. With a median follow up of 20 (range, 4–91) months for living pts; the 18 month overall survivals (OS), progression free survivals (PFS), and incidences of relapse/progressive disease (PD) were 47%, 20%, and 55% (MRD), 74%, 27%, and 65% (URD), and 71%, 60%, and 35% (Haplo), respectively. Nonrelapse mortalities (NRM) were significantly lower for URD (HR 0.16; p=0.03) and Haplo (HR 0.13; p=0.02) recipients compared to MRD recipients. There were also significantly decreased risks of relapse for Haplo recipients compared to MRD (HR 0.35; p=0.03) and URD (HR 0.36; p=0.02) recipients. These data suggest that salvage allogeneic HCT using nonmyeloablative conditioning provides anti-tumor activity in pts with very advanced disease; however, Rel/PD continue to be major problems regardless of stem cell source. Importantly, alternative donor sources are a viable option particularly for those pts who may have a limited window of opportunity to proceed to HCT. Pt Characteristics MRD (n=34) URD (n=24) Haplo (n=21) Median Age (yrs) 33 28 31 Disease Status at HCT CR 11 (32%) 5 (21%) 5 (24%) PR 16 (47%) 8 (33%) 5 (24%) Rel/Ref 7 (21%) 11 (46%) 11 (52%) Disease Bulk 〉 5cm 3 (9%) 3 (13%) 3 (14%) HCT-Comorbidity Index 〉2 21 (62%) 17 (74%) 13 (62%) Regimens 〉 5 16 (47%) 16 (67%) 12 (57%) Failed Prior Auto HCT 30 (88%) 23 (96%) 19 (90%) Median Time Diagnosis - Allo HCT (months) 33 31 32 Median Time Auto-Allo HCT (months) 16 19 17 Outcomes by donor type MRD (n=34) URD (n=24) Haplo (n=21) Median f/u living pts months (range) 15 (4–91) 26 (8–58) 15 (4–49) Acute GVHD grade II–IV, III–IV 53%, 15% 50%, 8% 43%, 10% 18 month extensive chronic GVHD 47% 60% 31% Day 200 NRM 18% 0% 0% 18 month OS 47% 74% 71% NRM 25% 8% 5% Rel/PD 55% 65% 35% PFS 20% 27% 60%

Description: Objectives: Patients with CML who achieve molecular remission (MR, defined as a RT-PCR negativity for BCR-ABL transcripts) after myeloablative stem cell transplantation (SCT) have a low risk of relapse, and the majority may be cured. The frequency of MR on imatinib varies greatly and the durability of these responses has not been reported. To investigate if MR after SCT and on imatinib are equally stable, we directly compared two cohorts of patients treated with imatinib or SCT, respectively, from the time of their first negative RT-PCR result. Patients and Methods: One hundred and forty-four CML patients in chronic (n=104) or accelerated phase (n=40) treated with standard dose imatinib were routinely monitored by conventional cytogenetics, quantitative RT-PCR (qPCR) and conventional nested PCR in case of negative qPCR results. Nineteen patients (13.2%) had at least 1 negative nested PCR. To assess the level of residual disease in patients with a single negative RT-PCR result, 10 replicate reactions were performed, each corresponding to 〉 106 white bone marrow cells. Thirty-six samples (median 3, range 1–4) from patients in MR on imatinib and 45 samples (median 2, range 1–3) from patients in MR after SCT were available. Twenty samples from healthy individuals were tested as controls. Results: The first negative result was noted after a median of 16.8 months (range 11.5–36.1) of imatinib therapy and 6.6 months (range 4.7–9.5) after SCT, respectively. The projected risk of molecular relapse at 12 months after the first negative RT-PCR result was 83% in patients on imatinib but only 20% in patients after SCT (P = 0.0001). Only two patients on imatinib remained in molecular remission at 13.8 and 16.6 months. While none of the patients with molecular relapse after allograft lost CCyR, one patient on imatinib progressed to cytogenetic relapse. The replicate assay was positive in 18/36 samples (50%) from patients on imatinib, 8/46 (17.4%) after allografting and 4/20 (20%) from healthy individuals. These differences were significant between patients on imatinib and after allografting (P = 0.003) and between patients on imatinib and healthy individuals (P = 0.005), but not between patients after allografting and healthy individuals (P = 0.9). Negativity by replicate testing was more stable in patients after allografting, although, even in these patients, positive replicate reactions continued to occur with longer follow-up. Conclusion: Imatinib-induced MR is usually not durable, in contrast to MR after transplant. Consistent with this, the level of residual disease in samples negative by single nested PCR is higher in patients on imatinib compared to patients after SCT. These results suggest that disease eradication with imatinib monotherapy may be rare. Patients on imatinib followed by PCR should be made aware of the fact that a single negative test does not have the same significance as in patients after SCT.