ALBERT

Description: Purpose: Determine the current pattern of use of non-ablative and reduced intensity conditioning regimens (NST) with volunteer unrelated donor progenitor cell transplantation (URD-T) in patients with malignant diseases, and identify potential prognostic factors for transplant outcomes. Patients and Methods: The National Marrow Donor Program (NMDP) database was queried to identify adult patients (greater than 18 years old) who had undergone an URD-T from 01/01/94 until 05/31/01, had a malignant disorder and received a conditioning regimen which fulfilled one of the following criteria: 500 cGy or less of total body irradiation, 9 mg/kg or less of total busulfan dose; 140 mg/m2 or less total melphalan dose and included a purine analog (fludarabine, cladribine, or pentostatin). A total of 293 patients were available for analysis of which 38% had acute leukemia or MDS, 18% had CML or another myeloproliferative disorder (MPD), 33% had CLL or lymphoma and 11% had a plasma cell disorder (PCD). The following transplant outcomes were analyzed: acute and chronic graft versus host disease (GVHD), non-relapse mortality (NRM), overall survival (OS) and event free survival (EFS). Results: The number of URD-T performed as NST have increased for all disease categories. From 1995 to 1999, 24 transplants were performed for acute leukemia (AML, ALL and MDS); 13 for MPD; 25 for lymphoproliferative disorders (CLL and lymphoma) and 1 for PCD. In the year 2000 alone these increased to 56 transplants for acute leukemia, 53 for CLL or lymphoma, 30 for MPD and 20 for PCD. When compared to 7015 NMDP recipients of ablative allografts performed during the same time period, recipients of NST were older (52 vs. 32 years), were more likely to have undergone transplant in a disease phase other than CR1, CR2 or first Chronic Phase (79% vs. 51%) and were more commonly treated for NHL and AML. NST patients had received a prior transplant in 63% of cases, and 35% had a Karnofsky performance score (KPS) of 〈 90 prior to transplant. The incidence of Grade II-IV acute GVHD for the whole group was 39% ± 5%. The 100 day and 1 year NRM rates were 20% ± 4% and 31% ± 5% respectively. Currently 83 patients are alive at a median of 985 days (range = 99–1825) with 77 patients free of progression. The 2 year OS and EFS rates are 33% ± 5% and 31% ± 5% respectively. On multivariate analysis favorable prognostic factors for OS were better HLA match, blood versus marrow stem cells and pre-transplant KPS ≥ 90%. Hazard Ratio 95% Confidence Intervals HLA Matching Match 1 Potential Match 4.701 3.097–7.136 Mismatch 2.128 1.509–3.001 Karnofsky Performance Scale 〈 90 1 ≥ 90 0.607 0.443–0.834 Stem Cell Source Peripheral Blood 1 Bone Marrow 1.602 1.151–2.229 Conclusions: URD-T with NST regimens are being performed more frequently, particularly in older patients. Although feasible and effective in a small fraction of patients, the procedure is still associated with significant morbidity and mortality. Factors relevant for outcomes after conventional myeloablative allografting (HLA match and performance status) remain important for outcomes after NST.

Description: High-dose therapy and autologous stem cell transplant (SCT) is an option for patients with AML most commonly performed in first complete remission (CR1) or CR2. Stem cell (SC) collection in CR1 typically follows consolidation. SC collection in CR2 is limited by the need to achieve a second remission prior to harvest, and the use of additional induction therapy that may result in marrow toxicity and SC depletion. In addition, a SC product collected in CR2 might be more likely to have leukemia cell contamination that could contribute to subsequent relapse. Prophylactic collection of SC from patients in CR1 who are not imminently going on to SCT may therefore be reasonable and theoretically could improve outcome of SCT in CR2. However, many patients never relapse, and for those who do, alternative options, and the need to achieve a 2nd CR, further limit the chance that these cells will be used. The morbidity and cost of SC collection, and the need for prolonged storage, call into question the practice of routine prophylactic SC harvest. To determine the utility of SC collection in CR1, we identified 67 patients with AML who had autologous SCs collected between 1995 and 2002. Charts were reviewed to assess whether the collection was prophylactic or for immediate use and we reviewed the timing and outcomes of transplant. 61/67 patients had SCs collected in CR1, 5 in CR2, 1 in CR3. 22 had collection for imminent therapeutic use and 45 for potential future use. Among the 22 patients whose cells were collected for planned SCT, cells were collected in CR1 in 17 cases and CR2 in 5 cases. 11 (50%) remain in CR a median of 58 mo (range 4–103) after SCT, and 11 died a median of 11 mo (4 – 28) after SCT. Causes of death were relapse (n=8), transplant related mortality (TRM) (n=1), TRM after allogeneic SCT (n=1), and unrelated causes (n=1). Of 17 patients transplanted in CR1, 8 (47%) remain in CR. Of the patients whose cells were both collected and used in CR2, 3/5 remain in remission 8, 53, and 103 mo after SCT and 2 died of relapse 10 and 24 mo after SCT. 5/45 patients whose stem cells were collected prophylactically in CR1 used these cells for SCT in CR2 a median of 16 months (15 – 28) after collection. 2/5 of these patients remain in CR 21 mo and 51 mo and 3 had died of relapsed disease 9–12 mo after SCT. Stem cells remain unused in 40 of these patients and 21 (53%) remain in CR a median of 39 mo (7–98 ) after collection. 15/21 remain in CR without further therapy. 3 patients are currently alive receiving therapy for relapse and therefore may use cells in the future. 6 patients are in CR after allogeneic SCT in CR1 (2), CR2 (3) or untreated relapse (1). 16/40 will never use stored cells. 12 died of disease a median of 10 mo (4–22) after prophylactic SC collection and 4 died from complications of allogeneic SCT in CR1 (3) or CR2 (1). In summary, of the 45 patients with SCs harvested prophylactically in AML CR1, 5/21 (23%) who required further therapy went on to use these cells with 2/5 long-term survivors. Another 24 have done well with initial therapy but remain at risk for relapse and may use SCs in the future. In our experience, prophylactic autologous SC harvest and storage from patients with AML in CR1 remains a reasonable option. Additionally, SCT for AML in CR has resulted in frequent long-term remission at our institution.

Description: Relapse remains the major limitation to successful autologous stem cell transplantation for Hodgkin’s lymphoma (HL) and is associated with limited long-term survival. There is now strong evidence that allogeneic donor cells can induce a direct and potent graft vs tumor reaction in patients with HL, ie, a Graft vs HL (GvHL) effect. Based on these findings, we evaluated the outcomes for 17 patients with incurable HL, all having relapsed after high dose therapy with autologous PBSC rescue, treated with non-myeloablative conditioning with allogeneic stem cell transplant (NMT). Patients were conditioned with fludarabine/cytoxan +/− Campath followed by allogeneic peripheral blood stem cells mobilized with G-CSF (12 from HLA-identical sibling donors, 3 from matched unrelated donors, 2 from single antigen mis-matched unrelated donors). Median recipient age was 37 (range 12–51). Median time from prior autotransplantation to relapse was 4.8 months (range 1–15m); median time from autotransplantation to NMT was 12m (range 5–53m), identifying this cohort as an extremely poor risk group. Conditioning in all patients included fludarabine 90–120 mg/m2/d combined with either ‘low dose’ cytoxan (900 mg/m2/d over 3 d) in 11 patients with sibling donors; or with ‘high dose cytoxan (3–4 gm/m2) in 6 pts with unrelated donors. 4 pts with unrelated donors, receiving high dose cytoxan with fludara also received 100 mg Campath 1-H divided over 5 days from D-9 to D-5. Post transplant GvHD prophylaxis consisted of cyclosporine (CSA) alone in 7 pts, CSA/methotrexate in 4 pts, and CSA/mycophenolate mofetil in 6 pts. Median MNC content of the graft was 7.5 x 108/kg (range 2.4–14); median CD34+ content was 4.1 x 106/kg (range .9–15.4). The median survival post allotransplant for all patients was 21.5 months (range 1–48.5m) and EFS was 8.5 months (range 1–41.5m) and was not influenced by graft source (sibling vs URD) or conditioning regimen. The 100 day TRM for all patients was 12%. The incidence of grade II-IV acute GvHD in 15 evaluable patients was 6/15 (40%). The incidence of chronic GvHD was 4/15 (27%) and was not correlated to a lower relapse rate among affected patients. Two patients were inevaluable for chronic GvHD and relapse secondary to early TRM. Of the 15 patients evaluable for response, 7 achieved CR (46%), 6 PR (40%), and 2 SD for an overall response rate of 86% in this chemoresistant cohort. Despite a high response rate, death from progressive disease occurred in the majority of patients. These data suggest a GVHL response can be induced, contributing to a response rate beyond what one would expect from salvage chemotherapy alone in this resistant cohort. However, the durability of response remains a limiting factor. Further studies designed to combine cytoreduction with allogeneic cellular therapy to enhance GVHL activity are warranted to improve outcomes after NMT for patients with poor-risk Hodgkin’s Lymphoma.

Description: CD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and more recently allograft tolerance in mice. Studies of human Treg have been hindered by the presence of CD25-dim conventional T cells that copurify during Treg isolation. We compared adult and cord blood sources for Treg, with the hypothesis that cord blood should lack significant numbers of memory cells or environmentally reactive T cells. We found that cord blood was a superior source for Treg isolation compared to adult blood. CD4+CD25+ cells were readily purified, and generated cell lines that consistently exhibited potent suppressor activity, with 〉95% suppression of allogeneic MLR (29/30 donors). The cells could markedly suppress an allo-MLR at a 1/16-1/32 suppressor/responder cell ratio. The cultured Treg cells blocked cytokine accumulation in MLR, with a less robust inhibition of chemokine production. Cultured Treg uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular CTLA4. Upon re-stimulation with anti-CD3/CD28 beads, the cultured Treg produced minimal cytokines (IL2, IFN-gamma, and IL10), and preferentially expressed TGF-beta latency associated protein on the cell surface, while conventional CD4+CD25− derived cell lines did not. Cytokine production however, could be largely restored by stimulation with PMA/ionomycin. Abundant FoxP3 mRNA was detected in fresh, cultured, and anti-CD3/CD28 bead restimulated CD4+CD25+ cells (approximately 2–4 fold less than cyclophillin A). Low amounts of FoxP3 mRNA were present in fresh CD25− cells, and they increased 32 fold on culture. However, FoxP3 protein, as assessed by western blot, was specifically expressed in the CD25+ derived cell lines, and was not detected in the CD25− derived cell lines. Restimulation with anti-CD3/CD28 beads led to increased expression of FoxP3 protein in CD25+ derived cell lines, but not in CD25− derived cell lines. Cord blood derived cultured suppressor cells were not cytolytic in chromium release assays, and mediated suppression of alloreactivity that was cell contact dependent and predominantly independent of IL10 and TGF-beta. Antibodies to GITR, GITR-L, OX40, OX40-L, CTLA4, and PD1 did not significantly affect suppression of the culture activated Treg cells. These results demonstrate virtually pure populations of potent suppressor cells can be cultured from cord blood, and these cell lines form an ideal model system for the evaluation of suppressor cell biology and mechanisms of action.

Description: Donor leukocyte infusions (DLI) can induce a potent GVT effect for some patients who relapse after allogeneic stem cell transplantation (SCT). However, GVT effects are most pronounced for patients with early phase CML and less effective for acute leukemia or other hematologic malignancies. One hypothesis for disease resistance suggests that donor T cells are not appropriately activated in-vivo to induce an anti-tumor response. Ex-vivo co-stimulation of T cells via CD3 and CD28 might overcome disease-induced anergy and augment GVT activity. To explore the possibilities for this approach, we performed a phase I trial of DLI followed by escalating doses of ex-vivo co-stimulated donor T cells (activated, or aDLI) for patients with relapse after allogeneic SCT (excluding CP-CML). Activated donor T cells are produced by co-stimulation and expansion by exposure to Dynal-magnetic beads coated with anti-CD3 (OKT3) and anti-CD28. 18 patients (11 male) with a median age of 45 (range 12–57) were treated for relapse after matched sibling SCT for ALL (7), AML (4), NHL (2), CLL (1), CML-BC (1), myeloma (1), Hodgkin’s disease (1) and lymphoblastic lymphoma (LL, 1). The median time from SCT to relapse was 11.5 mo (2–90) and relapse to DLI was 6 wks (2–31). All patients received standard unstimulated DLI (median 1.5 x 108 mononuclear cells/kg, range 0.9–3.5) followed 10 days later by ex-vivo co-stimulated donor T cells as aDLI. Patients with acute leukemia or BC-CML first received standard induction chemotherapy followed by DLI at the nadir. aDLI was dose escalated between every 3–4 patients from 1 x 106 CD3+ cells/kg to 1 x 108 CD3+ cells/kg in 5 dose levels. Ex-vivo co-stimulation resulted in a median 130-fold expansion of CD3+ cells (range 32–526) consisting of 56% CD4+ cells (range 30–84%) and 34% CD8+ cells (range 5–71%). The infusion of activated donor T cells was well tolerated with minor infusional toxicity at the highest dose level. Following aDLI, 7 patients developed acute GVHD (5 grade I-II skin only, and 2 grade III skin and liver). 2 patients have chronic GVHD limited to the oral mucosa and 2 have extensive chronic GVHD. No patient died of complications related to GVHD. 6/16 evaluable patients achieved CR (3/5 ALL, 1/4 AML, 1/1 CLL and 1/2 NHL) and 5 of 6 remain alive in CR a median of 25 mo after aDLI (range 8–43). 1 patient with ALL in CR relapsed 8 months after aDLI despite prior grade II aGVHD and died of disease progression. 1 patient with APML has residual disease detectable only by PCR 5 wks after aDLI and 4 other patients remain alive with disease (AML, ALL, HD, NHL) a median 4 mo after aDLI (range 3–5). 7 patients died of disease (3 ALL, CML-BC, MM, AML, LL) a median of 4 mo after aDLI (range 1–14). Functional assays show that immunity against pathogens such as EBV and CMV are retained after aDLI. These data demonstrate that adoptive transfer of co-stimulated allogeneic T cells is feasible, can induce a durable CR in a subset of patients, and does not result in excessive GVHD or other toxicity. In diseases where conventional DLI has been disappointing, response rates are impressive. Further studies to enhance GVT activity and tumor-specificity of aDLI are therefore warranted.

Description: Allogeneic hematopoietic stem cell transplantation (HSCT) can cure adults with hematologic malignancies, but results in significant morbidity and mortality. Graft-vs.-host disease (GVHD) is a major complication; attempts to reduce the risk of GVHD include selecting donors based on several characteristics, including parity, a criterion which has been controversial. This retrospective cohort study using data from the Center for International Blood and Marrow Transplant Research (CIBMTR) is the first multi-center analysis of the effects of donor and recipient parity on outcomes of HSCT in the modern transplant era. Methods: We studied patients at least 18 years old who received a non-T-cell-depleted, myeloablative HLA-identical sibling HSCT between 1995 and 1999, for acute lymphoblastic or myelogenous leukemia or chronic myelogenous leukemia. The study endpoints included acute and chronic GVHD, overall survival, and relapse. There were 2,626 patients who met inclusion criteria and had complete information on both donor and recipient pregnancy status. Donors and recipients were categorized as: males, nulliparous females, or parous (one or more pregnancies) females. Analysis: We compared all possible combinations of donor and recipient pregnancy status (9 groups in total); the reference group was male donor/male recipient pairs. Multivariate Cox proportional hazards regression was used to adjust for other prognostic factors. Because multiple groups were compared, significant p-values were considered to be less than or equal to 0.006. Results: After controlling for important patient-, disease-, and transplant-related covariates, the risk of chronic GVHD was significantly increased in parous female donor/male recipient pairs (HR 1.56, 95% CI 1.23 – 1.94, p 〈 0.0001). Neither donor nor recipient parity had an impact on overall survival, acute GVHD, or relapse risk. Conclusions: This multi-center retrospective registry study showed that parous female donors resulted in a significantly increased risk of chronic GVHD in male recipients, but without concomitant reduction in relapse. Thus, H-Y antigens may be important targets of GVHD, but not of a graft-versus-leukemia effect. As when selecting unrelated donors, avoidance of parous female donors, particularly for male patients, in HLA-identical sibling transplants is recommended when possible.

Description: Recombinant Erythropoietin (+/− G-CSF) is an effective therapy for the anaemia of selected patients with MDS. Validated response prediction models are available, but response rates are only 60% in the “high” predicted response group. Furthermore, half of the total cost of one year’s therapy for a cohort of patients selected for intermediate / high predicted response, is incurred within the initial 12-week therapeutic trial (Cassadeval et al, Blood 2004,104;321). Our hypothesis was that the erythroid response to a single bolus of EPO + G-CSF (Part 1) may predict for sustained response to a therapeutic trial (Part 2). 21 MDS patients (30x109/l was 100% predictive of subsequent response. In patients with erythroid response in Part 2, haemoglobin concentration at qw EPO either did not change compared to tiw dosing (P〉.05, n=5), or increased (P=.002, n=1). Serum ferritin, transferrin saturation, CHr (Bayer Advia) and serum transferrin receptor (TfR)concentrations were assayed weekly. Two patients became biochemically iron deficient during weeks 1–8, both of whom had baseline serum ferritin 100 mg/l. Serum non-transferrin bound iron concentration correlated closely with transferrin saturation both at baseline (n=21 patients), and on treatment (n=4 responders and 4 non-responders). In Part 2, neither ΔHb, nor ΔTfR at weeks 1 or 2 predicted response. No baseline erythroid parameters differed between responders and non-responders. New observations: 1. Absolute reticulocyte increment at Day 8 post s.c. bolus EPO/G-CSF predicts for therapeutic response in this small study, 2. Once weekly EPO is as effective as thrice weekly EPO in similar doses, 3. Functional iron deficiency may impair response in MDS patients with iron-limited erythropoiesis.

Description: The inhibitor of the apoptosis protein (IAP) survivin is expressed in proliferating cells such as fetal tissues and cancers. We previously reported that survivin is expressed and growth factor regulated in normal adult CD34+ cells. Herein, we examined survivin expression in CD34+ cells before and after cell cycle entry and demonstrate a role for survivin in cell cycle regulation and proliferation. Analysis of known human IAPs revealed that only survivin is cytokine regulated in CD34+ cells. Survivin expression is coincident with cell cycle progression. Up-regulation of survivin by thrombopoietin (Tpo), Flt3 ligand (FL), and stem cell factor (SCF) occurred in underphosphorylated-retinoblastoma protein (Rb)positive, Ki-67negative, and cyclin DnegativeCD34+ cells. Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) and multivariate flow cytometry demonstrated that Tpo, SCF, and FL increase survivin mRNA and protein in quiescent G0 CD34+cells without increasing Ki-67 expression, indicating that cytokine-stimulated up-regulation of survivin in CD34+cells occurs during G0, before cells enter G1. Selective inhibition of the PI3-kinase/AKT and mitogen-activated protein kinase (MAPKp42/44) pathways blocked survivin up-regulation by growth factors before arresting cell cycle. Retrovirus transduction of survivin-internal ribosome entry site–enhanced green fluorescent protein (survivin-IRES-EGFP) in primary mouse marrow cells increased granulocyte macrophage–colony-forming units (CFU-GM) by 1.7- to 6.2-fold and the proportion of CFU-GM in S phase, compared to vector control. An antisense survivin construct decreased total and S-phase CFU-GM. These studies provide further evidence that survivin up-regulation by growth factors is not a consequence of cell cycle progression and strongly suggest that survivin is an important early event for cell cycle entry by CD34+cells.

Description: Iron overload is a potentially life-threatening medical problem for patients with chronic transfusion-dependent anemias who cannot receive adequate iron chelation with existing therapies. ICL670 (deferasirox) is an investigational, tridentate, once-daily oral iron chelator that has demonstrated high iron binding potency and selectivity. The efficacy and safety of ICL670 is being investigated across a broad range of patient ages in a Phase II multicenter study in 7 countries in patients who are transfusion-dependent due to a variety of chronic anemias. The study population includes patients with various rare chronic anemias (n=98) and β-thalassemia patients who cannot be adequately treated with deferoxamine (n=86). The rare anemia category comprises 47 patients with myelodysplastic syndrome, 28 with Diamond-Blackfan anemia and 23 with other anemias of diverse etiologies. Between March and November 2003, 184 patients were enrolled in the following countries: Italy (57), US (36), France (20), UK (20), Canada (18), Germany (17), Belgium (16). Based on liver iron content (LIC) at baseline (2–3, 〉3–7, 〉7–14 and 〉14 mg Fe/g dw), patients were allocated to receive once daily oral ICL670 at doses of 5, 10, 20 or 30 mg/kg, respectively. Treatment was for one year initially, to be followed by an extension phase. LIC, the primary outcome measure, was assessed at baseline by liver biopsy or, when biopsy was contraindicated and also in some pediatric patients, non-invasively by magnetic susceptometry using a Superconducting QUantum Interference Device (SQUID). LIC will be reassessed after 12 months of therapy in each patient using the same methodology as at baseline. Liver biopsies are analyzed at a single center (Rennes, France) and 3 centers (Turin, Italy; Hamburg, Germany; Oakland, US) are performing SQUID assessments. At baseline, median (25–75th percentiles) LIC was 19.6 mg Fe/g dw (15.1–28.8) by biopsy and 9.1 (6.8–13.3) in those patients assessed by SQUID. Baseline patient demographics and disease characteristics (median values) are summarized in the table. ICL670 has been generally well tolerated. As of 30 April, 28 patients, predominantly with rare anemias, had discontinued the study due mainly to complications of the underlying disease. The key efficacy and safety data from the initial 12 months of therapy will be available for presentation in early December 2004. Disease group (by initial dose) Rare anemias (n = 98) β-thalassemia (n = 86) ≤ 10 mg/kg n = 16 20 mg/kg n = 29 30 mg/kg n = 53 ≤ 10 mg/kg n = 10 20 mg/kg n = 23 30 mg/kg n = 53 Age (yrs) median 56 39 49 21 23 25 No. of ≥ pts 2 -

Description: Desferrioxamine (DFO) and the hydroxypiridinone (HPO) deferiprone (CP20) chelate iron as well as other metals. These chelators are used clinically to treat iron overload, but they induce apoptosis in thymocytes. Thymocyte apoptosis is potentiated by zinc deficiency, suggesting that these iron chelators may induce apoptosis by depleting stores of zinc. Exposure of murine thymocytes to either DFO or deferiprone resulted in significant reductions in the labile intracellular zinc pool. Moreover, increasing intracellular zinc levels, by chronic zinc dietary supplementation to mice or in vitro loading with zinc, abrogated deferiprone-induced murine thymocyte apoptosis. Bidentate hydroxypyridinones such as deferiprone interact with intracellular zinc pools in a manner distinct from that of DFO, which is a hexadentate iron chelator. Whereas deferiprone acts synergistically with the zinc chelator NNNN-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to induce apoptosis, DFO does not. This difference is most likely due to the ability of HPOs but not DFO to “shuttle” zinc onto acceptors such as metallothioneins. By nature of its structure, DFO is larger than deferiprone and is thus less able to access some intracellular zinc pools. Additionally, metal complexes of DFO are more stable than those of HPOs and thus are less likely to donate zinc to other acceptors. The ability of deferiprone to preferentially access zinc pools was also demonstrated by inhibition of a zinc-containing enzyme phospholipase C, particularly when combined with TPEN. These findings suggest that bidentate iron chelators access intracellular zinc pools not available to DFO and that zinc chelation is a mechanism of apoptotic induction by such chelators in thymocytes.