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Despite enormous advances, management of multiple myeloma (MM) remains challenging. Multiple factors impact the decision to treat or which regimen to use at MM relapse/progression. Recent major randomized controlled trials (RCTs) showed widely varying progression-free survivals (PFS), ranging from a median of 4 months (MM-003) to 23.6 months (ASPIRE). Based on these RCTs, next-generation proteasome inhibitors (carfilzomib and ixazomib), next-generation immunomodulatory agent (pomalidomide), and monoclonal antibodies (elotuzumab and daratumumab) were approved for relapsed and refractory MM. Daratumumab, targeting CD38, has multiple mechanisms of action including modulation of the immunosuppressive bone marrow micro-environment. In addition to the remarkable single agent activity in refractory MM, daratumumab produced deep responses and superior PFS in MM when combined with lenalidomide/dexamethasone, or bortezomib/dexamethasone. Other anti-CD38 antibodies, such as isatuximab and MOR202, are undergoing assessment. Elotuzumab, targeting SLAMF7, yielded superior response rates and PFS when combined with lenalidomide/dexamethasone. New combinations of these next generation novel agents and/or antibodies are undergoing clinical trials. Venetoclax, an oral BH3 mimetic inhibiting BCL2, showed single agent activity in MM with t(11;14), and is being studied in combination with bortezomib/dexamethasone. Selinexor, an Exportin-1 inhibitor, yielded promising results in quad- or penta-refractory MM including patients resistant to daratumumab. Pembrolizumab, an anti-PD1 check-point inhibitor, is being tested in combination with lenalidomide/dexamethasone or pomalidomide/dexamethasone. Chimeric antigen receptor-T cells targeting B-cell maturation antigen have yielded deep responses in RRMM. Finally, salvage autologous stem cell transplantation (ASCT) remains an important treatment in MM relapsing/progressing after a first ASCT. Herein, the clinical trial data of these agents are summarized, cautious interpretation of RCTs highlighted, and algorithm for salvage treatment of relapse/refractory MM proposed.

CD34+ cell selection significantly improves GvHD-free survival in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, specific information regarding long-term prognosis and risk factors for late mortality after CD34+ cell-selected allo-HSCT is lacking. We conducted a single-center landmark analysis in 276 patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT for AML (n=164), ALL (n=33) or myelodysplastic syndrome (n=79). At 5 years’ follow-up after the 1-year landmark (range 0.03–13 years), estimated relapse-free survival (RFS) was 73% and overall survival (OS) 76%. The 5-year cumulative incidence of relapse and non-relapse mortality (NRM) were 11% and 16%, respectively. In multivariate analysis, Hematopoietic Cell Transplantation Comorbidity Index score⩾3 correlated with marginally worse RFS (hazard ratio (HR) 1.78, 95% confidence interval (CI) 0.97–3.28, P=0.06) and significantly worse OS (HR 2.53, 95% CI 1.26–5.08, P=0.004). Despite only 24% of patients with acute GvHD within 1 year, this also significantly correlated with worse RFS and OS, with increasing grades of acute GvHD associating with increasingly poorer survival on multivariate analysis (P<0.0001). Of 63 deaths after the landmark, GvHD accounted for 27% of deaths and was the most common cause of late mortality, followed by relapse and infection. Although prognosis is excellent for patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT, risks of late relapse and NRM persist, particularly due to GvHD.

Patients with acute myeloid leukemia (AML) who fail to achieve complete remission (CR) have a dismal prognosis. Although data suggest that durable remissions can be achieved in approximately 30% of patients with refractory or relapsed AML after allogeneic hematopoietic cell transplantation (HCT), only a small fraction of those patients are offered this therapeutic option. Importantly, patients with primary refractory AML have distinctly better outcomes following allogeneic HCT than those with refractory relapse. Access to suitable donors could be one of the main barriers in these situations. However, with recent developments in the field of allogeneic HCT, such as alternative donor sources, high-resolution HLA-typing, reduced intensity conditioning regimens and improvements in supportive care, this approach has the potential to offer long-term survival for patients with refractory and relapsed AML and should be considered as early after diagnosis as possible. Incorporating novel agents into the conditioning regimen or as post-transplant maintenance therapy could further improve outcomes and render older or medically infirm patients with refractory or relapsed AML eligible for allogeneic HCT. In this review, we summarize existing data on allogeneic HCT in patients with refractory or relapsed AML and explore novel approaches with the potential to improve outcomes in this patient population.

Calcineurin inhibitor (CNI)-sparing T-cell depleted (TCD) hematopoietic stem cell transplants (HSCTs) are presumed to be less nephrotoxic than conventional HSCTs. We evaluated incidence and risk factors for kidney failure and chronic kidney disease (CKD) in 231 TCD and 212 conventional HSCT recipients. Kidney failure required a median glomerular filtration rate (GFR) <60 ml/min/1.73 m2for ⩾100 days anytime after 180-days post-HSCT. Two-year cumulative incidence (CI) of kidney failure was 42% in the conventional versus 31% in the TCD group (P=0.005). TCD, age, acute kidney injury and number of toxic CNI levels all impacted on kidney failure, which was associated with increased all-cause mortality (hazard ratio 2.86 (95% CI: 1.88–4.36), P<0.001). Renal recovery occurred in 28% of kidney failure patients whereas the remaining patients were defined to have CKD. In those with baseline GFR>60 ml/min/1.73 m2, only exposure to nephrotoxic medications was associated with CKD (P=0.033). In the myeloablative-conditioning subgroup only total body irradiation was associated with CKD (P=0.013). Of all patients, five (1.13%) required dialysis. These results confirm an impact of TCD on kidney failure but not CKD for which other risk factors such as radiation or nephrotoxic drug exposure may have a role.

PR1, an HLA-A2-restricted peptide derived from both proteinase 3 and neutrophil elastase, is recognized on myeloid leukemia cells by cytotoxic T lymphocytes (CTLs) that preferentially kill leukemia and contribute to cytogenetic remission. To evaluate safety, immunogenicity and clinical activity of PR1 vaccination, a phase I/II trial was conducted. Sixty-six HLA-A2+ patients with acute myeloid leukemia (AML: 42), chronic myeloid leukemia (CML: 13) or myelodysplastic syndrome (MDS: 11) received three to six PR1 peptide vaccinations, administered subcutaneously every 3 weeks at dose levels of 0.25, 0.5 or 1.0 mg. Patients were randomized to the three dose levels after establishing the safety of the highest dose level. Primary end points were safety and immune response, assessed by doubling of PR1/HLA-A2 tetramer-specific CTL, and the secondary end point was clinical response. Immune responses were noted in 35 of 66 (53%) patients. Of the 53 evaluable patients with active disease, 12 (24%) had objective clinical responses (complete: 8; partial: 1 and hematological improvement: 3). PR1-specific immune response was seen in 9 of 25 clinical responders versus 3 of 28 clinical non-responders (P=0.03). In conclusion, PR1 peptide vaccine induces specific immunity that correlates with clinical responses, including molecular remission, in AML, CML and MDS patients.

Stem cell transplantation (SCT), an effective therapy for amyloid light chain (AL) amyloidosis patients, is associated with low treatment-related mortality (TRM) with appropriate patient selection and risk-adapted dosing of melphalan (RA-SCT). Consolidation after SCT increases hematologic complete response (CR) rates and may improve overall survival (OS) for patients with

The extant literature documents burden among caregivers of patients undergoing a hematopoietic stem cell transplantation (HSCT), but little is known about the burden of caregivers of patients receiving outpatient and homebound HSCTs. This scoping study sought to evaluate what is known about the burden of the increasing number of adult caregivers of patients receiving outpatient HSCTs and to create practice guidelines for how to best support this vulnerable group. Online databases were searched for studies that evaluated caregiver burden in adult caregivers of HSCT patients since 2010 (the publication date of the most recent systematic review on HSCT caregiver burden). Of the 1271 articles retrieved, 12 met the inclusion criteria, though none specifically examined outpatient or homebound caregivers. Overall, studies corroborated existing literature on the experience of significant burden among HSCT caregivers across the HSCT trajectory, and highlighted the emotional costs of outpatient transplants on caregivers and the need to identify caregivers at high risk for burden early in the transplant process. Future studies of outpatient caregivers should include a comprehensive assessment of burden and seek to identify points along the transplant trajectory at which caregivers are at particular risk for negative outcomes and when intervention is most appropriate.

Peer review is believed to be important in maintaining the quality and integrity of research in academic endeavors. Recently, the value of the current peer review process, which is more than 100 years old has come into question. In the field of hematopoietic cell transplantation (HCT), peer review was unable to prevent the publication of the largest and most notorious scientific fraud in our field. In order to assess how the HCT community views and how engaged it is with the peer review process, the American Society of Blood and Marrow Transplantation conducted a survey of all of its members in 2014. The survey was sent to all active members through multiple email communications in August and September 2014. Of a total of 1183 members, 149 responded. Almost all of the respondents had participated in the peer review process, with few respondents declining ever to review manuscripts. The most common cause for declining review requests was lack of time. Most respondents (68%) thought that the current peer review process was relatively fair and unbiased, whereas only 9% of the respondents stated that they did not believe in the peer review process. In conclusion, among the respondents of this survey most felt the peer review process to be valuable and fair, however, the lack of response suggests that further study into improving the peer review process in the field of HCT is warranted in the era of electronic publishing and communication.

Cyclophosphamide plus G-CSF (C+G-CSF) is one of the most widely used stem cell (SC) mobilization regimens for patients with multiple myeloma (MM). Plerixafor plus G-CSF (P+G-CSF) has demonstrated superior SC mobilization efficacy when compared with G-CSF alone and has been shown to rescue patients who fail mobilization with G-CSF or C+G-CSF. Despite the proven efficacy of P+G-CSF in upfront SC mobilization, its use has been limited, mostly due to concerns of high price of the drug. However, a comprehensive comparison of the efficacy and cost effectiveness of SC mobilization using C+G-CSF versus P+G-CSF is not available. In this study, we compared 111 patients receiving C+G-CSF to 112 patients receiving P+G-CSF. The use of P+G-CSF was associated with a higher success rate of SC collection defined as ⩾5 × 106CD34+ cells/kg (94 versus 83%, P=0.013) and less toxicities. Thirteen patients in the C+G-CSF arm were hospitalized owing to complications while none in the P+G-CSF group. C+G-CSF was associated with higher financial burden as assessed using institutional-specific costs and charges (P<0.001) as well as using Medicare reimbursement rates (P=0.27). Higher rate of hospitalization, increased need for salvage mobilization, and increased G-CSF use account for these differences.

Stem cell transplantation (SCT) has been used in the treatment of multiple myeloma (MM) for decades and has become a standard of care for newly diagnosed MM patients. However, several important questions remain regarding the optimal use of SCT, particularly in light of the many recent advances in the treatment of MM. Bortezomib-based therapy or, in some cases, lenalidomide-based therapy should be considered as an induction therapy in transplantation-eligible patients. Efforts to improve upon the efficacy and safety of standard transplantation regimens (that is, high-dose melphalan) are also underway. Most published studies on the use of tandem autologous SCT were conducted before the advent of novel agents, such as thalidomide, lenalidomide and bortezomib, making it difficult to establish the current role of tandem SCT. Allogeneic SCT continues to be evaluated in clinical trials, and may have an important role in the treatment of transplantation-eligible patients with suitable donors. Post-transplantation consolidation and maintenance therapy using novel agents should be considered to improve outcomes in patients who fail to achieve a complete response following SCT. Patients in remission should be advised that continued therapy has been shown to prolong remission, improve quality of life and extend survival. Additional data on the optimal approach to post-transplantation therapy are needed. New strategies in development aimed at improving patient selection, safety and efficacy of SCT are likely to improve future outcomes.

Donor lymphocyte transfusion has gained considerable interest as adoptive cellular immunotherapy for prevention or treatment of relapse after allogeneic stem cell transplantation. This study was designed to compare the yield of CD3⁺, CD3⁺4⁺, CD3⁺8⁺, CD19⁺, CD3⁻56⁺16⁺, and CD34⁺ cells contained in apheresis products from 61 consecutive non-cytokine treated, human leukocyte antigen (HLA)-matched donors for lymphocyte collection with the corresponding apheresis-derived cell yield from 112 consecutive, HLA-matched donors for blood stem cell collection who received recombinant human granulocyte colony stimulating factor (rhG-CSF, filgrastim) 6 μg/kg every 12 hours until cell collection was completed. Apheresis was started on day 4 or 5 of rhG-CSF treatment. The yield of lymphoid subsets was significantly different in the two sample groups, rhG-CSF treated product yields exceeding untreated product yields by a median of 2.1-fold (range: 1.3–2.6). However, the CD34⁺ cell yield in rhG-CSF-treated apheresis products exceeded untreated products by 26-fold. A single untreated apheresis procedure was usually sufficient to collect a target dose of 1 × 10⁸/kg CD3⁺ cells. Untreated apheresis products contained a median of 0.2 × 10⁶/kg CD34⁺ cells. A potential engraftment dose of ≥0.5 × 10⁶ CD34⁺ cells per kg of recipient body weight was contained in 16% of 57 untreated apheresis products. One single apheresis performed in a normal, untreated donor provides a sufficient amount of CD3⁺ cells for adoptive immunotherapy. Compared with that of an rhG-CSF stimulated apheresis product, the CD34⁺ cell count is usually, but not always, below the engraftment dose range. RhG-CSF treatment has little effect on the yield of lymphoid subsets collected by apheresis but is highly selective of the release of CD34⁺ cells. This report provides baseline data for studies that will show whether other cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF) and/or Flt-3 Ligand can immunomodulate allotransfusates in vivo to improve the graft-vs.-leukemia (GVL) effect after allogeneic stem cell transplantation, while lowering the incidence and severity of graft-vs.-host disease (GVHD). J. Clin. Apheresis. 16:82–87, 2001. © 2001 Wiley-Liss, Inc.

Donor lymphocyte transfusion has gained considerable interest as adoptive cellular immunotherapy for prevention or treatment of relapse after allogeneic stem cell transplantation. This study was designed to compare the yield of CD3+, CD3+4+, CD3+8+, CD19+, CD3−56+16+, and CD34+cells contained in apheresis products from 61 consecutive non‐cytokine treated, human leukocyte antigen (HLA)‐matched donors for lymphocyte collection with the corresponding apheresis‐derived cell yield from 112 consecutive, HLA‐matched donors for blood stem cell collection who received recombinant human granulocyte colony stimulating factor (rhG‐CSF, filgrastim) 6 μg/kg every 12 hours until cell collection was completed. Apheresis was started on day 4 or 5 of rhG‐CSF treatment. The yield of lymphoid subsets was significantly different in the two sample groups, rhG‐CSF treated product yields exceeding untreated product yields by a median of 2.1‐fold (range: 1.3–2.6). However, the CD34+cell yield in rhG‐CSF‐treated apheresis products exceeded untreated products by 26‐fold. A single untreated apheresis procedure was usually sufficient to collect a target dose of 1 × 108/kg CD3+cells. Untreated apheresis products contained a median of 0.2 × 106/kg CD34+cells. A potential engraftment dose of ≥0.5 × 106CD34+cells per kg of recipient body weight was contained in 16% of 57 untreated apheresis products. One single apheresis performed in a normal, untreated donor provides a sufficient amount of CD3+cells for adoptive immunotherapy. Compared with that of an rhG‐CSF stimulated apheresis product, the CD34+cell count is usually, but not always, below the engraftment dose range. RhG‐CSF treatment has little effect on the yield of lymphoid subsets collected by apheresis but is highly selective of the release of CD34+cells. This report provides baseline data for studies that will show whether other cytokines such as granulocyte macrophage colony stimulating factor (GM‐CSF) and/or Flt‐3 Ligand can immunomodulate allotransfusates in vivo to improve the graft‐vs.‐leukemia (GVL) effect after allogeneic stem cell transplantation, while lowering the incidence and severity of graft‐vs.‐host disease (GVHD). J. Clin. Apheresis. 16:82–87, 2001. © 2001 Wiley‐Liss, Inc.

Daclizumab, a humanized monoclonal IgG1 directed against the ? chain of the interleukin-2 receptor (IL-2R), is a competitive inhibitor of IL-2 on activated lymphocytes. To test the hypothesis that specific inhibition of activated lymphocytes in patients with ongoing acute graft-versus-host disease (GVHD) might ameliorate the process, we treated 43 patients with advanced or steroid-refractory GVHD with daclizumab. The first cohort of 24 patients was treated with daclizumab 1 mg/kg on days 1, 8, 15, 22, and 29. On day 43, the complete response (CR) rate was 29% (95% confidence interval [CI], 13%-51%). Survival on day 120 was 29% (95% CI, 13%-51%). A second cohort of 19 patients was treated with daclizumab 1 mg/kg on days 1, 4, 8, 15, and 22. For these patients, the CR rate on day 43 was 47% (95% CI, 24%-71%), and survival on day 120 was 53% (95% CI, 29%-76%). There were no infusion-related reactions and no serious side effects related to daclizumab. Following treatment, there was a reduction in serum concentrations of soluble IL-2R and peripheral blood CD3?+?25+ lymphocytes, but these changes were not predictive of response. Daclizumab has substantial activity for the treatment of acute GVHD, and the second regimen evaluated is recommended for a controlled study. (Blood, 2000; 95:83-89)

Peripheral blood stem cells (PBSCs) have been used rarely for allogeneic transplantation because of concerns regarding graft failure and graft-versus-host disease (GVHD). We evaluated the results of allogeneic PBSC transplantation (allo-PBSCT) in 9 patients with refractory leukemia or lymphoma receiving myeloablative therapy followed by allo-PBSCT from an HLA-identical sibling donor. Three patients had relapsed 11 to 21 months after allogeneic bone marrow transplantation (allo-BMT) and underwent allo-PBSCT using the same donor. Six patients received PBSCs as their initial allogeneic transplant. Filgrastim-mobilized PBSCs were collected from the donors in 3 to 4 aphereses and cryopreserved. The apheresis collections contained a median nucleated cell count of 16.5 x 10(8)/kg (range, 10.8 to 28.7 x 10(8), 10.7 x 10(6) CD34+ cells/kg (range, 7.5 to 22.5 x 10(6)), and 300.0 x 10(6) CD3+ cells/kg (range, 127.8 to 1,523.2 x 10(6)). The median recovery of CD34+ progenitor cells after freezing, thawing, and washing was 106.4% (range, 36.7% to 132.0%). All patients received filgrastim posttransplant through engraftment, and cyclosporine and methylprednisolone were used for GVHD prophylaxis. Neutrophil recovery to greater than 0.5 x 10(9)/L and greater than 1.0 x 10(9)/L occurred at a median of 9 (range, 8 to 10) and 9 days (range, 8 to 11) posttransplant, respectively, which was similar to historical controls after allo-BMT and granulocyte colony-stimulating factor therapy. Platelets recovered to greater than 20 x 10(9)/L and greater than 50 x 10(9)/L at a median of 12 (range, 8 to 25) and 15 days (range, 11 to 59), respectively, which was significantly more rapid than for the controls (P < .01). Donor cell engraftment was documented by cytogenetics, fluorescence in situ hybridization, and/or restriction fragment length polymorphisms with longest follow-up of 283 + days. Three patients developed grade 2 acute GVHD involving only the skin. Three of five evaluable patients show limited chronic GVHD. Cryopreserved, filgrastim-stimulated allogeneic PBSCs may be a suitable alternative to allogeneic marrow for transplantation with the advantage of more rapid platelet recovery. Acute GVHD was minimal despite the infusion of 1 log more CD3 cells than with marrow allografts. Further studies are required to assess long-term risks of chronic GVHD.

We collected peripheral blood mononuclear cells and bone marrow cells soon after recovery from conventional-dose chemotherapy-induced myelosuppression and transplanted these cells into advanced chronic myelogenous leukemia (CML) patients after treating these patients with 120 mg/kg cyclophosphamide, 750 mg/m2 VP-16, and 1,020 cGy of total body irradiation (TBI). Of the 10 late chronic-phase patients and the eight accelerated-phase CML patients evaluable posttransplant, 90% and 87%, respectively, remain alive posttransplant, whereas none of the three blast crisis CML patients given this therapy remain alive posttransplant. We measured the percentage of Philadelphia chromosome (Ph)-negative cells in the autologous cells collected after conventional-dose chemotherapy-induced myelosuppression before autologous transplant and in the marrow of these same CML patients after autologous transplantation of these cells into recipients treated with the cyclophosphamide, VP-16, and TBI. A direct correlation (correlation coefficient = 0.91) was observed between the level of Ph+ cells in the transplanted cells and the percentage of Ph+ marrow cells after transplant in 21 patients so transplanted. The data show that the chance of generating cytogenetic remissions post-transplant depends on the percentage of diploid cells in the preparations of autologous cells used for transplant and the stage of disease of the patients at the time of collection of the autologous cells.

Donor lymphocyte infusions can reinduce complete remission in the majority of patients with chronic myelogenous leukemia (CML) who relapse into chronic phase after allogeneic bone marrow transplantation (BMT). Such infusions are associated with a high incidence of graft- versus-host disease (GVHD) and marrow aplasia. BMT using selective depletion of CD8+ lymphocytes from donor cells reduces the incidence of GVHD without an increase in leukemia relapse. We hypothesized that infusion of CD8-depleted donor peripheral blood lymphocytes could also reinduce complete remissions with a lesser potential to produce symptomatic GVHD in patients with CML who relapsed after allogeneic BMT. Ten patients with Ph(+) CML who relapsed a median of 353 days after BMT (range, 82 to 1,096 days) received donor lymphocyte infusions depleted of CD8+ cells. Nine patients received a single infusion and 1 received two infusions. Four patients were treated while in chronic phase with clonal evolution, 2 during accelerated phase, 3 during blast crisis, and 1 in a cytogenetic relapse. A mean of 0.9 +/- 0.3 x 10(8) mononuclear cells/kg were infused, containing 0.6 +/- 0.4 x 10(6) CD3+CD8+ cells/kg. Six patients achieved hematologic and cytogenetic remission at 4, 8, 11, 15, 39, and 54 weeks after lymphocyte infusion. Two patients developed > or = grade II acute GVHD, and 1 patient developed mild chronic GVHD. We conclude that donor lymphocyte infusions depleted of CD8+ cells can induce remissions with a low rate of severe acute GVHD in patients with CML who relapse after allogeneic BMT, supporting the hypothesis that CD8+ lymphocytes are important effectors of GVHD, but may not be essential for the graft-versus- leukemia effect against this disease. Further controlled studies are required to confirm these preliminary observations.

The outcome of allogeneic haemopoietic transplants including the rate of immune complications for patients with chronic lymphocytic leukaemia (CLL) refractory to or relapsing after chemotherapy with fludarabine was analysed.
Fifteen patients with advanced CLL who received allogeneic transplantation were prospectively analysed. All patients had previously received chemotherapy with fludarabine for 3-15 courses; 12 were refractory. The median number of circulating CD4^plus; and CD8^plus; lymphocytes at the time of transplant was 0.49 x109 l and 0.23 x109 l, respectively. One patient was transplanted from a one HLA-antigen mismatched unrelated donor. Three others received a one or two antigen mismatched graft and 11 had HLA-identical sibling donors. Patients received cyclosporine or tacrolimus in addition to methotrexate or methylprednisolone for prophylaxis of acute graft-versus-host disease (aGVHD).
Fourteen patients engrafted; one patient had graft failure, but recovered after therapy with intravenous immunoglobulin. 13 (87%) achieved complete remission (CR). Nine (53%) remain alive and in CR with a median follow-up of 36 (range 3-60) months. None developed visceral graft-versus-host disease. These data compared favourably to published reports in other leukaemia patients and for patients with CLL who received a comparable immunosuppressive therapy but without prior fludarabine exposure.
This data indicates that allogeneic haemopoietic transplantation can induce durable remission in patients with CLL refractory to fludarabine and that it is reasonable to delay transplantation until failure of fludarabine therapy. It also suggests that prior exposure to fludarabine may decrease the incidence of severe aGVHD, possibly through its immunosuppressive effects. Further studies are warranted to evaluate this observation.

Thirty adults with leukemia or lymphoma undergoing marrow transplantation from HLA-compatible unrelated donors received tacrolimus (FK506), a new immunosuppressive macrolide lactone, and minidose methotrexate to prevent acute graft-versus-host disease (GVHD). The group had a median age of 36 years (range 21 to 49 years). Twenty-four patients had advanced disease, and 11 were resistant to conventional therapy. Tacrolimus was administered at 0.03 mg/kg/d intravenously (i.v.) by continuous infusion from day -2, converted to oral at four times the i.v. dose following engraftment, and continued through day 180 posttransplant. Methotrexate 5 mg/m2 was given i.v. on days 1, 3, 6, and 11. All patients engrafted. Grades 2–4 GVHD occurred in 34% (95% CI, 17% to 52%), and grades 3–4 GVHD in 17% (95% CI, 3% to 31%). Mild renal toxicity was common before day 100; 63% of patients had a doubling of creatinine, and 52% had a peak creatinine greater than 2 mg/dL, but only one patient was dialyzed. The median last i.v. dose of tacrolimus was 53% of the scheduled dose, and the median oral dose on day 100 was 41% of that scheduled. Overall survival at 1 year was 47% (95% CI, 27% to 66%). We conclude that tacrolimus can be combined safely with minidose methotrexate, and the combination has substantial activity in preventing acute GVHD after unrelated donor marrow transplantation.

Forty patients with multiple myeloma received thiotepa (750 mg/m2), busulfan (10 mg/kg), and cyclophosphamide (120 mg/kg) (TBC) followed by autologous bone marrow or blood stem cell support. Granulocyte-Colony stimulating factor (G-CSF) was administered to accelerate hematopoietic recovery. Sixty-five percent of all patients responded to this treatment. Eighty-eight percent of patients transplanted in partial remission had a further reduction of the myeloma and 53% achieved a complete remission. Forty-eight percent of patients with refractory myeloma responded. All responding patients transplanted during partial remission or with primary refractory myeloma remain free of progression for a period of 4 to 24 months post-transplant, but the remission duration of patients treated in refractory relapse was short (4 months). Five of 24 patients transplanted with marrow and none of 16 receiving blood stem cells died of treatment-related complications. Use of blood stem cells resulted in more rapid granulocyte and platelet recovery. We conclude that TBC is an effective, relatively well tolerated, preparative regimen for patients with multiple myeloma.

Following the publication of this article the authors noted that the MRD data under the Table 1 column “Remark” of Aspire should go to that of Pollux. The authors wish to apologize for any inconvenience caused. The corrected table is attached to this correction.

Vitamin D deficiency has long been understood to place patients at increased risk for reduced bone density and the development of pathological fractures. In addition to these classical manifestations, a wide body of emerging evidence suggests that Vitamin D deficiency may also increase the risk of illnesses associated with inflammation, including cardiovascular disease, infectious disease, autoimmune disease, and many common cancers. Our own in vitro data has confirmed that Vitamin D receptors are sharply upregulated on activated T cells and that physiologic calcitriol concentrations appear to have direct immunomodulatory functions (Joseph and Komanduri, unpublished). Vitamin D sufficiency is estimated by measuring 25-hydroxyvitamin D (25-OH D or calcidiol), and although there is no consensus on the optimal levels, many clinicians define sufficiency as >30 ng/ml, insufficiency as 20–30 ng/ml, and deficiency as <20 ng/ml. As one of the main sources of Vitamin D is through exposure to sunlight, allogeneic stem cell transplant (SCT) patients are at especially high risk of developing Vitamin D deficiency secondary to prolonged hospitalizations and standard recommendations to avoid sun exposure due to their increased risk of photosensitivity. To our knowledge, there are no large studies describing the incidence of Vitamin D deficiency in allogeneic SCT patients. We initially conducted a pilot study to assess Vitamin D levels in a group of allogeneic SCT recipients approximately assessed at approximately post-SCT day +100 (n=71) and found that only 18% of patients were sufficient, while the incidence of deficiency and insufficiency were to be 62%, and 20% respectively. To further elucidate if the deficiency was present pre-transplant or occurred post-transplant because of hospitalization and avoidance of sun exposure, we then assessed pre-transplant 25-hydroxyvitamin D levels in a larger group of allogeneic SCT recipients (n=108), and found that only 8% had normal baseline levels, while the vast majority were deficient (71%) or demonstrated insufficiency (21%). The baseline characteristics of this population are shown in the table below. Although this pilot study was not designed to assess the clinical significance of Vitamin D deficiency in the allogeneic transplant population, we believe that the unexpectedly high incidence of Vitamin D deficiency in SCT recipients deserves further study. Given the increasing epidemiologic and laboratory data implicating the Vitamin D axis in inflammation, we plan to assess traditional clinical outcomes associated with deficiency (including bone density and fracture rates) as well as immunological endpoints (GVHD and infection incidence) in future longitudinal studies. In conclusion, the incidence of Vitamin D deficiency in the allogeneic transplant setting is very high and additional studies are warranted to determine the clinical consequences associated with deficiency, and the potential therapeutic benefits of Vitamin D repletion in SCT recipients.

Vitamin D deficiency has long been understood to place patients at increased risk for reduced bone density and the development of pathological fractures. In addition to these classical manifestations, a wide body of emerging evidence suggests that Vitamin D deficiency may also increase the risk of illnesses associated with inflammation, including cardiovascular disease, infectious disease, autoimmune disease, and many common cancers. Our own in vitro data has confirmed that Vitamin D receptors are sharply upregulated on activated T cells and that physiologic calcitriol concentrations appear to have direct immunomodulatory functions (Joseph and Komanduri, unpublished). Vitamin D sufficiency is estimated by measuring 25-hydroxyvitamin D (25-OH D or calcidiol), and although there is no consensus on the optimal levels, many clinicians define sufficiency as >30 ng/ml, insufficiency as 20–30 ng/ml, and deficiency as <20 ng/ml. As one of the main sources of Vitamin D is through exposure to sunlight, allogeneic stem cell transplant (SCT) patients are at especially high risk of developing Vitamin D deficiency secondary to prolonged hospitalizations and standard recommendations to avoid sun exposure due to their increased risk of photosensitivity. To our knowledge, there are no large studies describing the incidence of Vitamin D deficiency in allogeneic SCT patients. We initially conducted a pilot study to assess Vitamin D levels in a group of allogeneic SCT recipients approximately assessed at approximately post-SCT day +100 (n=71) and found that only 18% of patients were sufficient, while the incidence of deficiency and insufficiency were to be 62%, and 20% respectively. To further elucidate if the deficiency was present pre-transplant or occurred post-transplant because of hospitalization and avoidance of sun exposure, we then assessed pre-transplant 25-hydroxyvitamin D levels in a larger group of allogeneic SCT recipients (n=108), and found that only 8% had normal baseline levels, while the vast majority were deficient (71%) or demonstrated insufficiency (21%). The baseline characteristics of this population are shown in the table below. Although this pilot study was not designed to assess the clinical significance of Vitamin D deficiency in the allogeneic transplant population, we believe that the unexpectedly high incidence of Vitamin D deficiency in SCT recipients deserves further study. Given the increasing epidemiologic and laboratory data implicating the Vitamin D axis in inflammation, we plan to assess traditional clinical outcomes associated with deficiency (including bone density and fracture rates) as well as immunological endpoints (GVHD and infection incidence) in future longitudinal studies. In conclusion, the incidence of Vitamin D deficiency in the allogeneic transplant setting is very high and additional studies are warranted to determine the clinical consequences associated with deficiency, and the potential therapeutic benefits of Vitamin D repletion in SCT recipients.

The use of CB for stem cell transplantation is increasing. A major disadvantage of CB is delayed and failed engraftment when compared to marrow or blood transplantation. Double CBT and ex-vivo expansion are both strategies being investigated in order to improve hematopoietic reconstitution. We compared these approaches with the aim of improving the clinical outcomes of CBT patients. 48 patients were randomized to receive either two unmanipulated (UNM) CB units (N=24) or one UNM unit and one unit from which all the cells were EXP ex-vivo (N=24). Most CB units were HLA 4/6 matches. Diagnoses were AML (N=16), ALL(N=13), NHL(N= 5), HD(N=7), CML (N=5), and CLL(N=2). Patients (Table 1) were heavily pre-treated, with a median of 3 (1–8) prior regimens including autotransplants in 18%. Preparative regimens included ATG and either fludara plus dose-adjusted busulfan(N=13; myeloid diseases), or melphalan and thiotepa (N=21; lymphoid malignancies/HD); patients not eligible for high-dose therapy received non-myeloablative fludara plus cyclophosphamide and 200 GyTBI (=11) or melphalan(n=3). GVHD prophylaxis was tacrolimus plus either 3 doses of 5 mg/m2 methotrexate or MMF (table 1). Ex-vivo expansion: the smallest unit was CD133-selected using the CliniMACS device (day -14). The T cell-containing CD133-negative fraction was frozen. The CD133+ fraction was cultured for 14 days in media containing SCF, G-CSF and TPO. On day 0, the 2nd UNM unit was infused, followed by the CD133-negative and the EXP fractions. Results. Infused median total nucleated cells (TNC)×108/Kg was 0.36 and 0.36, and median CD34×106/Kg was 0.16 and 0.13, respectively for EXP and UNM pts. Median TNC fold-expansion was 26 (0.44–275) and for CD34+ cells, 2.2 (0–18). There was a nonsignificant trend to more rapid engraftment in patients receiving EXP cells. Patients with >50-fold versus <50-fold TNC expansion engrafted in a median of 19 days(11–21) versus 23 (16–31), and achieved platelets>20K/ul at a median of 29 days(17–44) versus 42(29–56). 25 patients are alive [median follow up is 11.1 mo (2–33.6)]. 100-day and 1-year non-relapse mortality rates are 10% and 22%, respectively (table 2). Chimerism showed that 1 CB unit dominated in all patients. Among 19 evaluable EXP patients, in 63% the UNM unit provided 100% of the hematopoiesis from day +30; the EXP unit was predominant in 3 cases (for 12, 2 and 2 mo.) and present but not predominant in 4 (5–25%, for 12, 7, 3 and 3 mo). Conclusion: EXP CBT was safe. The range of fold-expansion was highly variable. Accrual continues focusing on strategies which improve CB expansion. Outcomes Expanded Unmanipulated P C.Incid.: cumulative incidence Time to ANC500 (median/95%CI) 20 days (15, 31) 23.5 (21, 34) 0.4 Proportion engrafting ANC500 82% (n=19) 79% (n=19) 1 Time to PLT20K (median/95%CI) 41 (32, NA) 54 (40, NA) 0.4 Proportion engrafting PLT 67% (n=16) 58% (n=14) 0.38 1-year survival 60% 40% 0.45 aGVHD gd II–IV/ III–IV 32% / 5% 41% / 9% NS C.Incid. cGVHD 47% (28–78) 31% (15–65) NS Expanded Unmanipulated P Complete remission at UCBT 42% 67% 0.15 Median weight 82 (20-113) 76 (51-144) 0.6 Median age 40.4 (4-66) 38.1 (9-65) 0.8 Ablative preparative regimen 67% 75% 0.8 GVHD prophylaxis with methotrexate 48% 48% NS

Acute (a)GVHD is a major cause of morbidity and mortality after UD or mismatched related (MRD) donor stem cell transplantation (HSCT). The nucleoside analog pentostatin (pentos) is a potent inhibitor of the enzyme adenosine deaminase that induces lymphocyte depletion, with low risk of myelosuppression. We have investigated the addition of pentos to tacrolimus (tacro) and MTX as GVHD prophylaxis.

The use of CB for stem cell transplantation is increasing. A major disadvantage of CB is delayed and failed engraftment when compared to marrow or blood transplantation. Double CBT and ex-vivo expansion are both strategies being investigated in order to improve hematopoietic reconstitution. We compared these approaches with the aim of improving the clinical outcomes of CBT patients. 48 patients were randomized to receive either two unmanipulated (UNM) CB units (N=24) or one UNM unit and one unit from which all the cells were EXP ex-vivo (N=24). Most CB units were HLA 4/6 matches. Diagnoses were AML (N=16), ALL(N=13), NHL(N= 5), HD(N=7), CML (N=5), and CLL(N=2). Patients (Table 1) were heavily pre-treated, with a median of 3 (1–8) prior regimens including autotransplants in 18%. Preparative regimens included ATG and either fludara plus dose-adjusted busulfan(N=13; myeloid diseases), or melphalan and thiotepa (N=21; lymphoid malignancies/HD); patients not eligible for high-dose therapy received non-myeloablative fludara plus cyclophosphamide and 200 GyTBI (=11) or melphalan(n=3). GVHD prophylaxis was tacrolimus plus either 3 doses of 5 mg/m2methotrexate or MMF (table 1). Ex-vivo expansion: the smallest unit was CD133-selected using the CliniMACS device (day -14). The T cell-containing CD133-negative fraction was frozen. The CD133+ fraction was cultured for 14 days in media containing SCF, G-CSF and TPO. On day 0, the 2nd UNM unit was infused, followed by the CD133-negative and the EXP fractions. Results. Infused median total nucleated cells (TNC)×108/Kg was 0.36 and 0.36, and median CD34×106/Kg was 0.16 and 0.13, respectively for EXP and UNM pts. Median TNC fold-expansion was 26 (0.44–275) and for CD34+ cells, 2.2 (0–18). There was a nonsignificant trend to more rapid engraftment in patients receiving EXP cells. Patients with >50-fold versus <50-fold TNC expansion engrafted in a median of 19 days(11–21) versus 23 (16–31), and achieved platelets>20K/ul at a median of 29 days(17–44) versus 42(29–56). 25 patients are alive [median follow up is 11.1 mo (2–33.6)]. 100-day and 1-year non-relapse mortality rates are 10% and 22%, respectively (table 2). Chimerism showed that 1 CB unit dominated in all patients. Among 19 evaluable EXP patients, in 63% the UNM unit provided 100% of the hematopoiesis from day +30; the EXP unit was predominant in 3 cases (for 12, 2 and 2 mo.) and present but not predominant in 4 (5–25%, for 12, 7, 3 and 3 mo).

Acute (a)GVHD is a major cause of morbidity and mortality after UD or mismatched related (MRD) donor stem cell transplantation (HSCT). The nucleoside analog pentostatin (pentos) is a potent inhibitor of the enzyme adenosine deaminase that induces lymphocyte depletion, with low risk of myelosuppression. We have investigated the addition of pentos to tacrolimus (tacro) and MTX as GVHD prophylaxis. Methods: This was an adaptive randomized, dose finding study that took into account toxicity and efficacy in a Bayesian adaptive design. Probability of assignment to the control group was fixed at 20%. Probability of assignment to the 4 dose groups was based on the probability that an arm’s success rate was greater than the control’s. Probabilities were recalculated after each observed patient outcome. Recipients of UD and MRD were eligible; all analysis is done by intention to treat. Success was patient being alive, engrafted, in complete remission (CR), without GVHD at study completion(100 days post HSCT). Grade III-IV aGVHD defined failure at any time, while gd. I-II did not constitute failure if absent on day 100. The study was designed to have a false positive rate of 0.05 and a power of 0.7 to detect a dose that had a 60% success rate for low-risk patients (HLA matched, in CR) and 45% for high-risk pts (mismatched, not in CR). High-resolution typing was prospectively available for 95% of donor-recipient pairs (HLA-A, B, C, DRB1 and DQB1 loci). Treatment plan: tacro from day-2; MTX 5 mg/m2 on days +1, +3, +6; day+11 was given only to controls. Pentos was given on days +8, +15, +22 and +30, in 4 ‘arms’: 0.5, 1, 1.5 and 2 mg/m2. Results: 147 pts, median age 49 yrs (18–72) were enrolled from 2001 to 2007. Diagnosis were AML/MDS (n=105), ALL(n=17), CML (n=18) and NHL (n=7); 48% of the pts were not in CR. Conditioning regimens (with ATG in 92% of the cases) were busulfan based (n=104), melphalan based(n=26), BEAM(n= 2), and CyTBI (n=15); 75% were ablative and 25%, reduced intensity. Stem cell source: bone marrow(n=120) and peripheral blood(n=27). Donors: UD(n=140) and MRD (n=7). Proportion of pts with donor-recipient HLA mismatches (HLA A, B, C, DRB1, DQB1) was 27%, 60%, 38%, 21% and 30%, respectively for the 5 study arms; median age was similar. 90% of the intended pentos doses were delivered. Time to engraftment was unafected. Incidence of toxicities (control vs study arms): renal(gd I-III) 34% vs 36%; TTP/HUS 6% vs 6%; early relapse 9% vs 5%; engraftment failure 3% vs 5.5%; delayed engraftment (>21 days): 6% vs 5%. Extensive cGVHD rate (control vs 1.5 mg/m2) was 52% vs 38%. Relapse rates are comparable. Conclusions: aGVHD rate may be reduced with pentostatin 1.5 mg/m2. A multicenter controlled study is planned in order to confirm these findings. Pentostatin dose control group n=37 0.5 mg/m² n=10 1 mg/m² n=29 1.5 mg/m² n=61 2 mg/m² n=10 gd II-IV aGVHD 57% 44% 40% 33% 50% gd III-IV aGVHD 20% 33% 18% 10% 10% Skin only aGVHD 45% 25% 50% 63% 67% Hepatic aGVHD 17% 50% 14% 2% 0 gd III-IV aGVHD n pts with <10/10 HLA match 1 in 9 2 in 5 5 in 11 0 in 11 0 in 3 CMV reactivation 63% 30% 48% 49% 50% Fungal infection 14% 10% 30% 16% 20% Not evaluable (n) 2 1 1 5 0

Disease relapse is the most frequent cause of treatment failure after HSCT in patients (pts) with refractory AML/MDS. AZA inhibits the enzyme DNA methyltransferase and leads to DNA hypomethylation. It may induce leukemic cell differentiation and increased immunogenicity, therefore potentially magnifying the graft-versus-leukemia effect. Lower doses are likely to be better tolerated after HSCT and to be as effective as larger doses in inducing hypomethylation. We hypothesized that AZA maintenance will result in lower relapse rates, and designed a phase I clinical trial to determine the safest dose and schedule combination. We also monitored global methylation post HSCT as a surrogate marker for changes in methylation status during AZA. Methods: Pts with AML or high-risk MDS not in 1st complete remission (CR), not candidates for ablative regimens were eligible. Conditioning regimen was gemtuzumab ozogamicin 2 mg/m2 (day -12), fludarabine 120mg/m2, and melphalan 140mg/m2. GVHD prophylaxis was tacrolimus/mini-methotrexate. ATG was administered to recipients of unrelated donor (UD) HSCT. We investigated 3 AZA doses: 8, 16, and 24 mg/m2 daily × 5 starting on day +42, and given for 1–4 28-day cycles (schedule). An outcome-adaptive method was used in order to determine both dose and schedule (number of cycles): pts were assigned to a dose/schedule combination chosen on the basis of the data (toxicity) from all pts treated previously in the trial. Patients in CR on transplant day +30, without gd III/IV GVHD, platelet >10,000/mm3 and ANC >500/mm3 were eligible to receive SQ AZA. The methylation status of long interspersed nuclear elements (LINE) was analyzed by pyrosequencing and used as a surrogate marker of global DNA methylation in mononuclear cells of 22 pts(55%) that received AZA for at least 1 cycle. Results: 40 patients were enrolled; median age was 57 years (22–72). Diagnoses were MDS with high IPSS(n=6) and AML (n=34). Disease status at HSCT: complete remission (CR), 10% (n=4); chemo-naive MDS, 2% (n=1); induction failure, 35% (n=14), 1st/2nd relapse, 42.5% (n=21). Patients had received a median of 5 courses of chemotherapy (0–23) prior to HSCT, and the median comorbidity Charlson score was 3 (0–8). Donors were related (n=23) or UD(n=17), and stem cell source was bone marrow(n=8) or peripheral blood(n=32). Median follow-up of alive pts is 11 mo (2.5–20; n=21). 49 cycles of AZA were delivered at 8 (n=7 pts), 16 (n=4) and 24 mg/m2 (n=12 pts). AZA-associated toxicities were grade I/II hematologic, nausea and fatigue. There was no increase in GVHD: gd II-IV aGVHD, and chronic GVHD rates were 34% and 30%. 11 pts have relapsed, 2 while on AZA (16 and 24 mg/m2). Day +30 and +100 non-relapse mortality was 5% and 12%. Mean LINE methylation results were as follows: baseline: 43.51% (±5; n=22); on cycle 1, 5th day of AZA: 24 mg/m2=43.58% (±5; n=9); 16 mg/m2: 36.93% (±6.5; n=4); 8 mg/m2: 42.86% (±5; n=3). There were trends towards lower levels of global methylation at 16mg/m2 in subsequent cycles. The trial design has reached the higher dose and the maximum number of cycles given the absence of AZA-related major toxicities. Conclusion: AZA at 24 mg/m2 is safe and can be administered for at least 4 cycles. The lack of toxicity and the methylation studies indicate that higher doses and longer periods of administration should be investigated.

Disease relapse is the most frequent cause of treatment failure after HSCT in patients (pts) with refractory AML/MDS. AZA inhibits the enzyme DNA methyltransferase and leads to DNA hypomethylation. It may induce leukemic cell differentiation and increased immunogenicity, therefore potentially magnifying the graft-versus-leukemia effect. Lower doses are likely to be better tolerated after HSCT and to be as effective as larger doses in inducing hypomethylation. We hypothesized that AZA maintenance will result in lower relapse rates, and designed a phase I clinical trial to determine the safest dose and schedule combination. We also monitored global methylation post HSCT as a surrogate marker for changes in methylation status during AZA.

We previously demonstrated early safety and efficacy of fixed-dose IV BuFlu. Here we compare IV BuFlu and IV BuCy2, with long follow-up in a large number of pts. Since pts were treated on consecutive, not randomized programs, we used pts receiving Flu-Melphalan (M) in a program spanning the time of the IV Bu-based studies to estimate/correct for this non-randomized “period” effect. Methods: 293 pts received Bu 130 mg/m2, Flu 40 mg/m2 (after April 2001; n=148); Bu 0.8 mg/kg × 16, Cy 60 mg/kg × 2 (before April 2001; n=67); Flu 30mg/m2 × 4, M 140–180 mg/m2 (FM; before and after April 2001; n=78). Patients received FM mainly based on higher age and coorbidities. Graft-vs-host dx (GVHD) prophylaxis was tacrolimus-based. Bayesian log-normal regression models were used to assess covariate- and treatment effects on survival, non-relapse mortality (NRM) and time to progressive disease (TPD). Covariates and survival model are shown in the table. The model accounts for the “period” effect, the effects of Bu-vs-FM, and Flu-vs-Cy within IV Bu pts. Patient and treatment characteristics were (BuFlu, BuCy2 and FM): median age 46 (19–66), 39 (13–64) and 54 (22–74) years. At HSCT 47%, 48% and 21% of the patients were in CR1-3. Donors were HLA-identical siblings in 53%, 79% and 41%. 30% of the Bu groups and 47% of FM had poor prognosis cytogenetics. Results: Median follow-up in months (BuFlu, BuCy2 and FM; alive pts): 30 (9–56; n=80; all but 2 pts followed for at least 1 yr), 69 (24–102; n=23) and 48 (17–89, n=25). 100-day NRM for pts in CR at HSCT was 2% and 9% after Bu-Flu and BuCy2, while 1 yr-NRM was 7% and 18%. BuFlu produced significantly better EFS (P=0.04) and overall NRM (P=0.02) than BuCy2, but TPD was similar (P=0.34). 2-yr actuarial survival (CR pts): 75% (BuFlu) vs. 50% (BuCy), P=0.01. Conclusion: After accounting for covariate and treatment period effects, there was a significant benefit to being treated with BuFlu compared to BuCy2 in spite of a higher median age and proportion of MUDs. The benefit was most evident for CR-pts due to a significant reduction in NRM, without loss of antileukemic activity due to exchanging Cy with Flu. Fitted Bayesian log-normal overall survival model Variable Mean SD Posterior 95% credible interval 2.5% 97.5%* Probability of beneficial effect *1=beneficial effect; 0=low likelihood of beneficial effect Cytogenetics −0.85 0.4 −1.652 −0.045 0.017 Disease status (CR vs other) 1.12 0.55 0.019 2.15 0.975 Donor (sibling vs other) 0.606 0.237 0.149 1.056 0.992 Age −0.022 0.010 −0.041 −0.003 0.017 Circulating blasts (0 vs >0) 0.951 0.268 0.454 1.463 1 Platelet count 0.002 0.001 0.000 0.004 0.967 Period effect −0.914 0.458 −1.795 0.041 0.029 IV Bu (vs FM) −0.513 0.481 −1.455 0.420 0.148 BuFlu (vs Bucy2) 1.476 0.544 0.406 2.555 0.995 Survival – patients in CR at transplant Survival – patients in CR at transplant

We previously demonstrated early safety and efficacy of fixed-dose IV BuFlu. Here we compare IV BuFlu and IV BuCy2, with long follow-up in a large number of pts. Since pts were treated on consecutive, not randomized programs, we used pts receiving Flu-Melphalan (M) in a program spanning the time of the IV Bu-based studies to estimate/correct for this non-randomized “period” effect.

Most patients (pts) transplanted with active myeloid leukemias will relapse after HSCT. Intensity of the conditioning regimen is an important component of disease control. We hypothesized that GO could safely increase the anti-disease activity of FM, and investigated this hypothesis in the trial reported here. Patients and Methods: Objective: to determine the dose of GO with the lowest toxicity (tox), and the highest response probabilities. Tox: grade 3–4 organ tox, engraftment failure or early death (ED; first 30 days post HSCT). Response was defined as no tox, engraftment, and remission (CR) on day +30. Trial was designed with GO doses of 4, 6 and 9 mg/m2 but given tox observed at 4mg/m2, doses 6 and 9 mg/m2 were not used and dose 2mg/m2 was added. Eligible were pts aged 12–75 years, not candidates for high-dose regimens or with high-risk CD33+ disease. Treatment: GO 2 or 4mg/m2 day -12, F 30mg/m2 (days -5 to -2), M 140mg/m2 (day -2); HSCT day zero. ATG was added in unrelated (UD) HSCT. Graft-versus-host disease (GVHD) prophylaxis: tacrolimus and mini-methotrexate (5mg/m2 day+1,+3,+6,+11). GO was given on day -12 to minimize probability of delaying engraftment. We treated 52 pts, median age 53 yrs (13–72), with AML (n=47), MDS (n=4) or blast crisis CML(n=1). Disease status at HSCT: CR (n=3),induction failure (n=15), first/second relapse (n=33), untreated MDS (n=1). 18 pts had a previous HSCT (allogeneic, n=11 or autologous, n=7). At study entry, median number of bone marrow blasts was 17% (0–95%); 33% of the pts had circulating blasts, 4 pts were FLT3 positive and 38% had poor prognosis cytogenetics. Median blast CD33 expression: 89% (22.5–99.6). Donors: related (n=33) or UD (n=19). Stem cell source was peripheral blood (n=43) or bone marrow (n=9). Results: Median day 30 donor cell chimerism was 100%. Median time to ANC> 500/mm3 was 13 days. Eight pts received GO at 4 mg/m2; 2 died early (due to pneumonia and renal failure) and 2 developed gd III gastro-intestinal and renal tox. 44 pts were then treated at 2 mg/m2: 3 pts died of regimen-related tox (2 ED due to pulmonary bleeding and sepsis, and 1 death within the first 100 days due to pneumonia, renal failure/TTP/HUS). 100-day treatment-related mortality (including 2 deaths due to aGVHD and 1 due to infection) was 15% (n=8). Tox included reversible gd III-IV bilirubin (n=2), transaminase elevation (n=5), moderate hepatic VOD (n=1) and gd I-II GI tract (n=39). CR rate was 90%; 1 pt failed to respond. Gd II-IV and III-IV aGVHD rates were 44% and 23%, respectively, and 58% developed cGVHD. Median follow-up is 9 months (2.5–36) for surviving pts (n=29); 17 pts have relapsed. Median event-free-survival (EFS) is 3.8 mos. Median EFS of a historic control group treated with FM140 (n=36) was 2.2 mos (Figure). Tox of GOFM is similar to that documented by our group with FM140 mg/m2 in a somewhat better prognosis population.(de Lima et al. Blood.2004; 104:865–72) Conclusion: GO 2mg/m2 can be safely added to FM, and may improve the anti-leukemic efficacy of the regimen. EFS - patients with active disease at HSCT EFS - patients with active disease at HSCT

Donor-recipient histocompatibility is a major determinant of outcome after UD HSCT, and matching pairs by high-resolution (Hi-res) HLA typing is likely to improve results. Methods: We studied 66 pts with myeloid leukemias in complete remission (CR) transplanted with UD bone marrow (n=58) or peripheral blood (n=8) from 01/02 to 12/04. Preparative regimens were ablative [Busulfan (Bu)/Cyclophosphamide (Cy)(n=13), Cy/TBI (n=2), Fludarabine (Flu)/Bu 130 mg/m2x4 doses (n=33)] and reduced intensity [Flu/Bu 130 mg/m2x2 doses+Gleevec (n=7), Flu/Melphalan 140mg/m2 (n=11)]. ATG was given in 61 cases. GVHD prophylaxis was tacrolimus and mini-methotrexate, with additional pentostatin in 24 pts. Hi-res typing of HLA-A, B, DRB1 and DQB1 loci was done prospectively; HLA-DPB1 and C loci were typed prospectively in 50% of cases and retrospectively in 50%. Hi-res typing was sequencing-based for HLA-A, B, DRB1, SSP-based for DRB3/4/5, DQB1 and DPB1; and SBT/SSOP-based for HLA-C. A Cox’s proportional hazards regression model was used to study overall (S), event-free (EFS) and aGVHD-free survival. Variables with a p-value <0.25 by univariate analysis were included in the multivariate model (MV). Variables were age, conditioning regimen, use of pentostatin, diagnosis, cytogenetics, SC source, ABO typing, infused total nucleated cell (TNC) dose, HLA matching and GVHD. Results: Median age was 45yrs (21–72). Diagnoses were MDS (n=5), AML (n=39), and CML (n=22). 21 pts were in 1st CR, 23 pts in 2nd or 3rd CR, 22 pts in 2nd or greater chronic phase (CP) CML. 45 pts were 10/10 HLA match (HLA-A, B, C, DRB1, DQB1), and 21 had one or more mismatches. All pts engrafted neutrophils at a median of 13 days; 32 pts (48%) had gd. II–IV aGVHD and 33 pts (57%) had cGVHD. With a median follow-up of 9 mo, 44 pts are alive; 11 pts have relapsed. There were no regimen-related deaths; 100-day mortality was 13% due to aGVHD (n=7), relapse (n=1) and infection (n=1). Median S and aGVHD-free survival in 10/10 pts have not been reached. Median EFS for 10/10 pts vs others is 34 vs.9.66 mos. MV for S: age ≥60yrs (P=0.02; HR 4.17 (95%CI 1.24–14), Flu-based regimen (P=0.0004; HR 0.14 (95%CI 0.05–0.41), use of pentostatin (P=0.02; HR 0.27 (95%CI 0.09–0.78) and grade II–IV aGVHD (P=0.02; HR 3.25 (95%CI 1.19–8.88). MV for EFS: age ≥60yrs (P=0.046; HR 3.06 (95%CI 1.02–9.18), Flu-based regimen (P=0.001; HR 0.21 (95%CI 0.08–0.54) and use of pentostatin (P=0.02; HR 0.34 (95%CI 0.13–0.85). MV for aGVHD-free survival: age ≥60yrs (P=0.039; HR 3.43 (95%CI 1.06–11.1), Flu-based regimen (P=0.0027; HR 0.09 (95%CI 0.02–0.43), use of pentostatin (P=0.003; HR 0.22 (95%CI 0.08–0.6) and CP CML (P=0.004; HR 0.06 (95%CI 0.01–0.41). S and EFS for 10/10 matched pts were improved with Flu-based regimen (p=0.01), and higher infused TNC dose (p=0.02). aGVHD-free survival was improved by using reduced intensity regimens (p=0.004) and worsened by presence of 2 DPB1 mismatches (1.18 mo vs not reached; HR=3.43; p=0.08). Conclusion: A combination of donor selection by high-resolution typing, less toxic preparative regimens and improved GVHD prophylaxis can optimize outcomes in this high-risk disease context. Figure Figure

RIC with FM has extended the use of HSCT to patients otherwise not eligible for this treatment. Longer follow-up and larger number of patients now allow for more robust evaluation of risk factors and outcomes. Herein are the results of such an evaluation. Patients and Methods: We evaluated outcomes of 112 patients with high-risk AML/MDS treated from August 1996 to December 2003 with FM (fludarabine 100–180 mg/m2 and melphalan 100–180 mg/m2) and unmanipulated HSCT. Eligibility included age >54 yrs. or comorbidity precluding an ablative preparative regimen. Disease status at HSCT was relapsed/refractory (n=43, 38.4%), primary induction failure (n=32, 28.6%), untreated (n=7, 6.3%) or complete remission (CR, n=30, 26.8). Cytogenetic risk was intermediate (n=59, 53%), high (n=47, 42%), low (n=3, 2.5%) or unknown (n=3, 2.5%). Donors were HLA matched related (MRD; n=59) or unrelated (UD; n=53). GVHD prophylaxis was tacrolimus based in all but one patient. Anti-thymocyte-globulin was added in 31 UD HSCT. Stem cell sources were bone marrow (n=56) or peripheral blood (n=57). Median age was 55 (range 22–74). Evaluated were the following variables and their influence on disease progression and overall survival: - age, donor type, duration of first CR, disease status at transplant (categorized as CR, No CR with (NoCR/CB) and without circulating blasts (NoCR/NoCB)), cytogenetics, acute and chronic GVHD (time dependent variables), and blood counts on day 30 (lymphocytes, monocytes and platelets). We used a Cox’s regression analysis. Results: Median time of follow up among survivors (n=43) was 28.4 mo (3.3–88.9). CR rate at day 30 post transplant was 87% (n=97), 8 patients died early and 7 did not respond. 25 (26%) of 97 patients progressed after day 30. All but 3 patients relapsed within the first year post HSCT, and only one relapsed more than 2 years after HSCT. In a landmark analysis, disease status at transplant was the only significant risk factor for progression among these 97 patients (HR of 3.7 for the NoCR/CB group compared to the CR group). 69 of 112 patients died with a median survival of 4.6 mo. Seven deaths (10% of all deaths) were observed more than 2 yrs. after HSCT, due to GVHD (n=3), infection (n=2), relapse (n=1) and unknown causes (n=1). Two-year OS and PFS was 44% and 69% respectively. Disease status at HSCT and grade II-IV aGVHD were the only significant predictors of OS on univariate and multivariate analysis. Blood counts on day 30 were associated with disease status at transplant, donor type and aGVHD. Their independent effect on outcome could not be evaluated given sample size. Conclusion: A significant portion of older patients with high-risk AML/MDS may achieve long-term PFS, but early relapses are the major cause of treatment failure in this context. Prognostic factors for event-free and overall survival Variables Multivariate analysis for disease progression CB=circulating blasts Disease status n Events (n) HR 95% CI p 2-yr PFS CR 30 6 1.0 57% (39–72) NoCR/NoCB 41 7 1.1 0.4–3.2 0.9 46% (30–60) NoCR/CB 26 12 3.7 1.4–9.8 0.001 22% (9–38) Multivariate analysis for overall survival (OS) Disease status HR 95% CI p 2-yr OS CR 30 12 1.0 66% (48–80) NoCR/NoCB 49 29 1.8 0.9–3.5 0.06 40% (26–53) NoCR/CB 34 28 2.8 1.4–3.5 0.002 23% (11–37) gd II-IV aGVHD 2.8 1.8–4.6 <0.001

Donor-recipient histocompatibility is a major determinant of outcome after UD HSCT, and matching pairs by high-resolution (Hi-res) HLA typing is likely to improve results.

Most patients (pts) transplanted with active myeloid leukemias will relapse after HSCT. Intensity of the conditioning regimen is an important component of disease control. We hypothesized that GO could safely increase the anti-disease activity of FM, and investigated this hypothesis in the trial reported here.

RIC with FM has extended the use of HSCT to patients otherwise not eligible for this treatment. Longer follow-up and larger number of patients now allow for more robust evaluation of risk factors and outcomes. Herein are the results of such an evaluation.

Cord blood (CB) is used to restore hematopoiesis in transplant patients lacking marrow donors. CB is associated with higher rates of delayed/failed engraftment. Peled et al developed an expansion technology using the copper chelator tetraethylenepentamine (TEPA), which enhanced the expansion of primitive CB populations when combined with early acting cytokines. A phase I clinical trial employing this technology was initiated. 10 patients with high-risk, heavily pre-treated hematologic malignancies (AML-2, ALL-5, HD-2, and NHL-1) have been enrolled with CB units that were cryopreserved in 2 fractions [20:80% (n=2), 40:60% (n=5) or 50:50% (n=3)]. 21days prior to infusion, AC133+ cells were isolated from the smaller (if unequal) or 50% CB fractions using the CliniMACS device and cultured for 21 days in media containing 10% FBS and SCF, FLT-3, IL6, TPO plus the copper chelator TEPA (Gamida). Patients then received myeloablative therapy with ATG and either fludara and busulfan (AML), or fludara, melphalan, thiotepa (ALL, HD, NHL) with infusion of the unmanipulated CB fraction on day 0, and the expanded fraction on day +1. GVHD prophylaxis was methotrex 5 mg/m2days 2, 4,7, and tacrolimus for 6 months. The median age was 21 (range 7–51) and weight 69 (range 31–156) kg. The CB units were matched at 4/6 (n=8) or 5/6 (n=2) HLA antigens. The pre-thaw total nucleated cell (TNC) dose of the CB units was a median of 2.5x107/kg with post-thaw TNC of 2.4 x107/kg. Following AC133-selection, the manipulated CB fractions were a median of 73 (range 38–95)% AC133+ with a median of 0.650 (range 0.16–2.7) x106TNCs, which were placed in culture. After 21 days of culture the expanded fraction had 69 (range 2–1638) x106TNCs representing a 207 (range 2–616) fold TNC expansion. Patients received a total (expanded plus unmanipulated) median of 1.8 (range 1.1–6.1) x107TNC/kg and 1.6x105(range 0.4–49.9) CD34+ cells/kg. Two patients have CB cultures in progress. Of the 8 patients transplanted, 1 had autologous recovery with relapse of AML on day 30 and death. Of the remaining patients, 7 were evaluable for neutrophil engraftment and 4 of them for platelet engraftment (2 too early for platelet evaluation and 1 early death). The median time to engraftment was 27 days for neutrophils (range 16–46) and 48 days for platelets (range 27–96). Preliminary analysis suggest a correlation between a shorter time to neutrophil engraftment and total TNC/kg infused (p=0.02), and a trend for CD34+ cells/kg infused (p=0.09). Three patients have developed grade ≤2 acute skin GVHD and one had chronic extensive GVHD of the skin and GI tract; all resolved with steroids. One patient (without GVHD) died of a systemic viral infection on day 56, despite adequate neutrophil recovery (not platelets). All of the remaining patients are all alive and free of malignancy at a median follow-up of 4 (range 1–16) months.

Cord blood (CB) is used to restore hematopoiesis in transplant patients lacking marrow donors. CB is associated with higher rates of delayed/failed engraftment. Peled et al developed an expansion technology using the copper chelator tetraethylenepentamine (TEPA), which enhanced the expansion of primitive CB populations when combined with early acting cytokines. A phase I clinical trial employing this technology was initiated. 10 patients with high-risk, heavily pre-treated hematologic malignancies (AML-2, ALL-5, HD-2, and NHL-1) have been enrolled with CB units that were cryopreserved in 2 fractions [20:80% (n=2), 40:60% (n=5) or 50:50% (n=3)]. 21days prior to infusion, AC133+ cells were isolated from the smaller (if unequal) or 50% CB fractions using the CliniMACS device and cultured for 21 days in media containing 10% FBS and SCF, FLT-3, IL6, TPO plus the copper chelator TEPA (Gamida). Patients then received myeloablative therapy with ATG and either fludara and busulfan (AML), or fludara, melphalan, thiotepa (ALL, HD, NHL) with infusion of the unmanipulated CB fraction on day 0, and the expanded fraction on day +1. GVHD prophylaxis was methotrex 5 mg/m2 days 2, 4,7, and tacrolimus for 6 months. The median age was 21 (range 7–51) and weight 69 (range 31–156) kg. The CB units were matched at 4/6 (n=8) or 5/6 (n=2) HLA antigens. The pre-thaw total nucleated cell (TNC) dose of the CB units was a median of 2.5x107/kg with post-thaw TNC of 2.4 x107/kg. Following AC133-selection, the manipulated CB fractions were a median of 73 (range 38–95)% AC133+ with a median of 0.650 (range 0.16–2.7) x106 TNCs, which were placed in culture. After 21 days of culture the expanded fraction had 69 (range 2–1638) x106 TNCs representing a 207 (range 2–616) fold TNC expansion. Patients received a total (expanded plus unmanipulated) median of 1.8 (range 1.1–6.1) x107 TNC/kg and 1.6x105 (range 0.4–49.9) CD34+ cells/kg. Two patients have CB cultures in progress. Of the 8 patients transplanted, 1 had autologous recovery with relapse of AML on day 30 and death. Of the remaining patients, 7 were evaluable for neutrophil engraftment and 4 of them for platelet engraftment (2 too early for platelet evaluation and 1 early death). The median time to engraftment was 27 days for neutrophils (range 16–46) and 48 days for platelets (range 27–96). Preliminary analysis suggest a correlation between a shorter time to neutrophil engraftment and total TNC/kg infused (p=0.02), and a trend for CD34+ cells/kg infused (p=0.09). Three patients have developed grade ≤2 acute skin GVHD and one had chronic extensive GVHD of the skin and GI tract; all resolved with steroids. One patient (without GVHD) died of a systemic viral infection on day 56, despite adequate neutrophil recovery (not platelets). All of the remaining patients are all alive and free of malignancy at a median follow-up of 4 (range 1–16) months. Conclusion: There was no toxicity associated with infusion of the TEPA-expanded CB cells. Additional data is necessary to determine the efficacy of this approach. Future directions include the expansion of the entire CB unit and removal of methotrex from the GVHD regimen to improve time to neutrophil engraftment, as well as comprehensive assessment of immune reconstitution.

GVHD remains a major obstacle to a successful unrelated (UD) or mismatched related (MRD) donor hematopoietic stem cell transplantation (HSCT). Pentostatin is a purine nucleoside analog that targets adenosine deaminase and leads to lymphocyte depletion, with low potential for myelosuppression. We are investigating the incorporation of pentostatin to our standard GVHD prophylaxis regimen with tacrolimus (tacro) and methotrexate (MTX). Methods: This is an adaptive randomized, dose finding study that takes into account toxicity and efficacy in a Baysean “play the winner” design. The “winner” dose moves to the phase II portion of the study. Probability of assignment to the control group was fixed at 20%. Recipients of UD and MRD are eligible; all analysis is done by intention to treat. Success was defined as being alive, engrafted, in complete remission(CR), without GVHD at study completion (100 days post HSCT). Development of grade III-IV acute (a) GVHD defined failure at any time, while grade I–II did not constitute failure if absent by day 100. This design has power 0.7 to detect a dose that has a success rate of 60% for low-risk patients (HLA matched, in CR) and 45% for high-risk patients (mismatched, not in CR). High-resolution typing was available for all donor-recipient pairs at HLA-DRB1 and -DQB1 loci, and to 83% of the pairs at HLA-A and -B loci; all patients had low-resolution -C typing. Treatment plan: tacro from day -2 (target level of 5–15 ng/ml) and MTX 5 mg/m2 on days +1, +3, +6 to all patients; day +11 was given only to the control group. Pentostatin was given on days +8, +15, +22 and +30, in treatment arms: 0.5 mg/m2, 1 mg/m2, 1.5 mg/m2, and 2 mg/m2. Results: 73 patients, median age 45 yrs (range 18–72) have been enrolled. Diagnosis were AML/MDS(n=48), ALL(n=8), CML (n=10) and NHL(n=7); 58% of the patients were not in CR at HSCT. Conditioning regimens were busulfan based(n=52), melphalan based(n=10), BEAM(n= 2), and CyTBI(n =9); 71% were ablative and 29%, reduced intensity. ATG was used in the regimen in 86% of the cases. Stem cell source: bone marrow (n=67) and peripheral blood(n=6). Donors: UD(n=67) and MRD(n=6). Proportion of patients with donor-recipient HLA mismatches was 24%, 20%, 33%, 21% and 40%, respectively for the 5 study arms; median age was similar. 85% of the intended pentostatin doses were delivered. Pentostatin did not delay engraftment. Incidence of toxicities (control vs. study arms): renal (all grade I/II)=47% vs 36%; TTP/HUS= 12% vs 9% (more severe among pentostatin patients); early relapse= 12% vs 5%; engraftment failure=6% vs 3%; delayed engraftment (>21 days): 0 vs. 5%. Probability that dose 1.5 mg/m2 is better than control is 0.9341. Pentostatin dose control group (n=17) 0.5 mg/m2 (n=10) 1 mg/m2 (n=12) 1.5 mg/m2 (n=24) 2 mg/m2 (n=10) gd II-IV aGVHD 47% 44% 63% 29% 50% gd III-IV aGVHD 20% 33% 27% 10% 10% CMV reactivation 41% 30% 33% 50% 50% bact/fungal infection 59% /12% 60% /10% 50% /18% 55% /17% 70% /10% Not evaluable n=2 n=1 n=1 n=4 n=0 Failure rate 47% 70% 33% 29% 40% Conclusions: This preliminary analysis indicates that aGVHD rate may be reduced with pentostatin 1.5 mg/m2, without interference with engraftment. Longer follow-up and larger number of patients will be needed to assess impact on survival.

Outcomes of patients with active AML/MDS remain poor after HSCT. Treatment failure is in part a result of poor disease control with the preparative regimen. Here we report toxicity and efficacy of the immunoconjugate GO incorporated to a reduced intensity regimen.

Outcomes of patients with active AML/MDS remain poor after HSCT. Treatment failure is in part a result of poor disease control with the preparative regimen. Here we report toxicity and efficacy of the immunoconjugate GO incorporated to a reduced intensity regimen. Methods: Objective: determine which dose of GO between doses 2 and 4mg/m2 has the lowest toxicity(tox) probability with the highest response probability when added to FM (statistical method of Thall, Estey, Sung (1999)). Tox was defined as grade 3–4 organ tox, engraftment failure or early death (first 30 days post HSCT). Response was defined as engraftment, no tox and remission (CR) on day +30. Trial was planned with dose levels 4, 6 and 9 mg/m2 but given tox observed at 4mg/m2, the other dose levels were not explored and dose 2mg/m2 was added. Eligibility: patients (pts) age 12–75 years, ineligible for high-dose regimens or with high-risk CD33+ AML/MDS. Donors: related or unrelated (MUD). Treatment plan: GO 2 or 4mg/m2 day -12, fludarabine 30mg/m2 (days -5 to -2), melphalan 140mg/m2 (day -2); HSCT day zero. ATG was added in MUD HSCT. Graft-versus-host disease (GVHD) prophylaxis: tacrolimus and mini-methotrexate (5mg/m2 day+1,+3,+6,+11). GO was given on day -12 to avoid delay in engraftment and to decrease the number of committed myeloid progenitors prior to FM. Here we also included pts. treated under a compassionate IND and performed a non-randomized comparison with historic pts. treated with FM140 mg/m2 without GO. Results: 29 pts. have been treated. Table shows baseline characteristics. GO dose 0 (n=49) 2 mg/m2 (n=21) 4 mg/m2 (n=8) P value Remission at BMT 26% 9% 12% 0.25 MUD 41% 28% 25% 0.52 Median age (years-range) 54 (21–75) 53 (13–72) 30 (23–67) 0.08 Peripheral blood stem cell transplant 47% 86% 100% 0.0005 High-risk cytogenetics 43% 33% 37% 0.83 Median follow-up is 10 months (range 2.5–36) for GOFM alive patients (n=18). CR rate: GO 2mg/m2 =90%; GO 4 mg/m2= 71%; control=71%. There was only 1 case of moderate, reversible hepatic VOD (GO 2mg/m2). Grade III-IV tox and early death rates were: GO 2 mg/m2, 19% and 5% (n=1 due to pulmonary hemorrhage); GO 4mg/m2, 25% and 25% (n=2, due to pneumonia and multi-organ failure); control, 21% and 12% (n=6). Acute gd. II-IV GVHD rate was 44% for GO treated patients. All evaluable patients had engrafted by day +30; 8 pts have relapsed. Comparison with historic controls: active disease and high-risk cytogenetics were significantly associated with worse event-free (EFS) and overall survival (unadjusted log-rank test). Cox proportional hazards model: risk of death or relapse was increased with older age (RR 1.02; P=0.06), MUD vs related donor (RR 1.91; P=0.03), active disease vs CR (RR 0.23; P=0.001), and having GO 4 mg/m2 or zero vs 2 mg/m2 (RR 0.46, P=0.04).Figure shows survival. Figure Figure Conclusions: The addition of GO at the 2 mg/m2 dose level is promising and merits evaluation in a larger, controlled trial.