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CD40L generates immune responses in leukemia-bearing mice, an effect that is potentiated by IL-2. We studied the feasibility, safety, and immunologic efficacy of an IL-2– and CD40L-expressing recipient-derived tumor vaccine consisting of leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 (hIL-2) and hCD40L. Ten patients (including 7 children) with high-risk acute myeloid (n = 4) or lymphoblastic (n = 6) leukemia in cytologic remission (after allogeneic stem cell transplantation [n = 9] or chemotherapy alone [n = 1]) received up to 6 subcutaneous injections of the IL-2/CD40L vaccine. None of the patients were receiving immunosuppressive drugs. No severe adverse reactions were noted. Immunization produced a 10- to 890-fold increase in the frequencies of major histocompatibility complex (MHC)–restricted T cells reactive against recipient-derived blasts. These leukemia-reactive T cells included both T-cytotoxic/T-helper 1 (Th1) and Th2 subclasses, as determined from their production of granzyme B, interferon-γ, and interleukin-5. Two patients produced systemic IgG antibodies that bound to their blasts. Eight patients remained disease free for 27 to 62 months after treatment (5-year overall survival, 90%). Thus, even in heavily treated patients, including recipients of allogeneic stem cell transplants, recipient-derived antileukemia vaccines can induce immune responses reactive against leukemic blasts. This approach may be worthy of further study, particularly in patients with a high risk of relapse.

Purpose: Human CD40 ligand activates the malignant B-cell chronic lymphocytic leukemia cells and enhances their capacity to present tumor antigens. Human interleukin-2 further potentiates the immunogenicity of human CD40 ligand in preclinical murine models. Experimental Design: We prepared autologous B-cell chronic lymphocytic leukemia cells that expressed both human CD40 ligand (>90% positive) and human interleukin-2 (median secretion, 1,822 pg/mL/106 cells; range, 174-3,604 pg). Nine patients were enrolled in a phase I trial, receiving three to eight s.c. vaccinations. Results: Vaccinations were administered without evidence of significant local or systemic toxicity. A B-cell chronic lymphocytic leukemia–specific T-cell response was detected in seven patients. The mean frequencies of IFN-γ, granzyme-B, and IL-5 spot-forming cells were 1/1,230, 1/1,450, and 1/4,500, respectively, representing a 43- to 164-fold increase over the frequency before vaccine administration. Three patients produced leukemia-specific immunoglobulins. Three patients had >50% reduction in the size of affected lymph nodes. Nonetheless, the antitumor immune responses were observed only transiently once immunization ceased. High levels of circulating CD4+/CD25+/LAG-3+/FoxP-3+ immunoregulatory T cells were present before, during and after treatment and in vitro removal of these cells increased the antileukemic T-cell reactivity. Conclusions: These results suggest that immune responses to B-cell chronic lymphocytic leukemia can be obtained with human CD40 ligand/human interleukin-2–expressing s.c. vaccines but that these responses are transient. High levels of circulating regulatory T cells are present, and it will be of interest to see if their removal in vivo augments and prolongs the antitumor immune response.

Allogeneic hemopoietic stem cell transplantation (HSCT) is the only curative option for many patients with hematological malignancies. Since many of these patients lack HLA-identical sibling donors and are older or have comorbidity, a fully ablative HSCT is not feasible and an alternative approach is required. We studied 22 consecutive patients who could not have myeloablative conditioning because of comorbidity or age - 21/22 being over the age of 50 years (median 58 years range 20-70 years). A conditioning regimen consisting of fludarabine, total body radiation 450 cGy and alemtuzumab (CD52 mAb) was used for 15 patients. A second group of seven patients received CD45 monoclonal antibodies in addition. Unrelated donor stem cells were HLA matched (15 patients - 68%) or one locus mismatched (seven patients - 32%). In all, 16 patients had high-risk disease, including 12 with active malignancy at the time of transplant. With a median follow-up of 715 (216-1470) days, nonrelapse mortality, actuarial event-free and overall survival is 27, 45 and 45%, respectively. Hence the outcome of reduced intensity HSCT with lymphodepleting antibodies in older patients with intermediate/high-risk hematological malignancies appears comparable to that obtained with fully ablative transplantation in younger patients, even when these older recipients lack HLA-identical sibling donors.

Epstein Barr virus (EBV)+ Hodgkin's disease (HD) expresses clearly identified tumor antigens derived from the virus and could, in principle, be a target for adoptive immunotherapy with viral antigen–specific T cells. However, like most tumor-associated antigens in immunocompetent hosts, these potential targets are only weakly immunogenic, consisting primarily of the latent membrane protein (LMP)1 and LMP2 antigens. Moreover, Hodgkin tumors possess a range of tumor evasion strategies. Therefore, the likely value of immunotherapy with EBV-specific cytotoxic effector cells has been questioned. We have now used a combination of gene marking, tetramer, and functional analyses to track the fate and assess the activity of EBV cytotoxic T lymphocyte (CTL) lines administered to 14 patients treated for relapsed EBV+ HD. Gene marking studies showed that infused effector cells could further expand by several logs in vivo, contribute to the memory pool (persisting up to 12 mo), and traffic to tumor sites. Tetramer and functional analyses showed that T cells reactive with the tumor-associated antigen LMP2 were present in the infused lines, expanded in peripheral blood after infusion, and also entered tumor. Viral load decreased, demonstrating the biologic activity of the infused CTLs. Clinically, EBV CTLs were well tolerated, could control type B symptoms (fever, night sweats, and weight loss), and had antitumor activity. After CTL infusion, five patients were in complete remission at up to 40 mo, two of whom had clearly measurable tumor at the time of treatment. One additional patient had a partial response, and five had stable disease. The performance and fate of these human tumor antigen–specific T cells in vivo suggests that they might be of value for the treatment of EBV+ Hodgkin lymphoma.

Multiple sclerosis is thought to involve aberrant immune responses to myelin autoantigens. Haematopoietic stem-cell transplantation (HSCT) is in clinical trials for progressive multiple sclerosis based on the rationale that it destroys aberrant immune system, while recapitulation of lymphocyte ontogeny might alter the immune system and slow down disease progression. This study was undertaken to analyse characteristics of the T-cell receptor (TCR) repertoire, serum cytokine profile and the T-cell responses to myelin basic protein (MBP) in the reconstituted immune system in progressive multiple sclerosis. The study revealed that, following autologous HSCT, the T-cell immunity recovered in two distinctive phases. The first phase was characterized by limited T-cell immunity as a result of selective expansion of pre-existing T cells commonly expressing the TCR beta chain variable region (TCR BV) 20 and increased serum cytokine production during the first several months. The second phase of T-cell reconstitution coincided with increased thymic T-cell output 9-12 months after HSCT. T cells reconstituted from stem-cell grafts had the distinctive properties of comprehensive T-cell immunity and a broad TCR repertoire. T cells recognizing MBP were initially depleted by immunoablation and rapidly expanded from the reconstituted T-cell repertoire in 12 months. The reconstituted MBP-reactive T cells exhibited a broader epitope recognition repertoire while maintaining the same skewed reactivity pattern compared with that seen at baseline. The findings have important implications in the understanding of the role of HSCT as a potential treatment for multiple sclerosis.

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion characterized by dyspnea, hypoxemia, hypotension, fever, and bilateral pulmonary infiltrates. Although the frequency is estimated at 1/1,120 to 1/5,000 transfusions, few cases have been reported after hematopoietic stem cell transplant. We report a case occurring in an allogeneic transplant recipient who developed acute respiratory distress and bilateral pulmonary infiltrates 2 hr after a platelet transfusion due to the presence of anti granulocyte antibody HNA-3a in the product. As there is a wide differential diagnosis for pulmonary infiltrates developing post transplant, TRALI may be under-recognized and should be considered in this setting.

Systemic effects of oral budesonide in hematopoietic transplant: implications of drug interaction with azoles

Antithymocyte globulin (ATG) is accepted as a treatment option for steroid-refractory acute graft-versus-host disease (GVHD). We conducted an international survey to determine how steroid refractoriness is defined and how ATG is used in clinical practice. Responses were received from 153 centers in 36 countries. The most common threshold steroid dose to define steroid refractoriness was 2 mg/kg/day (67% of respondents), and the median duration of treatment before failure was declared varied from 3 to 5.5 days, depending on whether failure was defined as 'progressed', 'not improved' or 'not resolved'. The threshold corticosteroid dose was significantly higher in pediatric centers than in adult or combined programs (P = 0.003). ATG was used routinely for treatment of steroid-refractory GVHD by 67% of the respondents. Horse ATG was used more frequently than rabbit ATG overall (50% vs 24%, P < 0.001), and predominance of horse ATG was most evident in the western hemisphere, in small- to medium-sized centers, and in pediatric centers. A wide variety of dose schedules for both drugs was reported. We conclude that there is some degree of variation in the definition of steroid refractoriness, especially between pediatric and nonpediatric programs, and no consensus has emerged in identifying the optimal ATG dose schedule in this setting.

1.0 Checklist for Patient Eligibility and Necessary Information 2.0 Objectives 3.0 Background and Rationale 4.0 Study Design 5.0 Patient Eligibility 6.0 Treatment Plan 7.0 Patient Evaluation 8.0 Evaluation during Study 9.0 Statistical Considerations 10.0 Study Interpretation 11.0 Off Study Criteria 12.0 Records to Be Kept 13.0 Reporting Requirements 14.0 Informed Consent 15.0 References Appendix I

Predictors of severe reaction to polyvalent pneumococcal vaccine (PPV) are not established. In the absence of prospective studies, revaccination within 5 years and high titer of protective antibody appear to increase the risk of a local reaction that manifests as self-limiting pain, localized erythema, and edema. A more severe systemic reaction, manifesting as fever, arthralgia, and extensive edema involving the entire upper extremity, is rare, and we report a case in a patient with myeloproliferative disease.

Successful mobilization and transplantation of filgrastim mobilized hematopoietic stem cells in sickle cell-hemoglobin C disease

Matched related donor (MRD) bone marrow transplantation is the treatment of choice for pediatric patients with severe aplastic anemia; however, only 25% of patients will have an HLA-identical sibling. Alternative donor transplants may be an option for these patients, but such therapies have been associated with greater incidences of graft failures and graft-versus-host disease (GVHD). We retrospectively analyzed 32 pediatric patients who have undergone either bone marrow or peripheral blood stem cell transplant for severe aplastic anemia at our institution from April 1997 to April 2005. These patients had a total of 34 transplants. One patient had a MRD transplant followed by a matched unrelated donor (MUD) transplant eight years later, while another patient had a HLA-mismatched unrelated donor (MMUD) transplant followed by a transplant from a haplo-identical parent. Of the remaining 30 patients, 12 received MRD transplants, whereas 18 patients received alternative donor transplants - 11 MUD, 3 haplo-identical donors, and 4 MMUD. The median age at transplant was 9 years (range 1.5 to 18.4 yrs). All patients who received alternative donor transplants had previously failed therapy, including antithymocyte globulin (ATG) and cyclosporine. For MRD transplants, the conditioning regimen most often utilized cyclophosphamide 50 mg/kg x 4 days and ATG 30 mg/kg x 3 days. For alternative donor transplants, the conditioning regimen most often utilized cyclophosphamide 50 mg/kg x 4 days, Campath 3–10 mg x 4 days (dependent upon patient’s weight) or ATG 30 mg/kg x 3 days, and TBI (single fraction 200 cGy for MUD; two fractions 200 cGy for MMUD). Alternative donor recipients who received ATG in their preparative regimen were transplanted between December 1997 and March 2001 (n=9), whereas patients who received Campath were treated between November 2001 and April 2005 (n=11). GVHD prophylaxis was either FK506 or cyclosporine +/− mini-methotrexate. The overall survival for MRD patients was 91.7% versus 80% for alternative donor patients at a median follow-up of 47 months (range 3 to 100 months). Of the 32 patients, there were 5 deaths: pulmonary failure with extensive, chronic GVHD (n=1); poor graft function with infection (n=1); and infection (n=3). For patients receiving alternative donor transplants, the overall survival for the Campath group was 81.8% vs. 77.8% in the ATG group. None of the Campath patients developed extensive, chronic GVHD compared to 3/9 ATG patients. In conclusion, alternative donor transplantation using Campath or ATG in the preparatory regimens can establish donor engraftment and offers a curative therapy for pediatric severe aplastic anemia patients with survival similar to that of patients receiving matched sibling transplants. Although follow-up is shorter, Campath may be associated with a reduced incidence of extensive, chronic GVHD and further investigation is warranted.

We report the case of a 34-yearold woman with recurrent pure red cell aplasia and evidence of hepatitis Band C infection. Review of the English literature identified 19 prior cases in which pure red cell aplasia was associated with hepatitis. This case is the first in which serologic evidence of hepatitis C infection was documented. This patient also had porphyria cutanea tarda and marked hepatic siderosis but no active hepatitis or cirrhosis. Treatment with cyclophosphamide and prednisone produced complete remission of the pure red cell aplasia. Erythroid colony formation (colony-forming unit-erythroid and erythroid burst-forming unit) was reduced in cultures of bone marrow obtained during relapse but was normal in remission marrow. However, addition of the patient serum, whether collected during relapse or remission, inhibited erythroid colony formation by her bone marrow. These observations, and the known extrahepatic immunologic manifestations of hepatitis C infection, suggest that the pure red cell aplasia occurred because of an autoimmune mechanism provoked by the infection.

Viral infections remain a major cause of morbidity and mortality after allogeneic HSCT. We and others have demonstrated that the adoptive transfer of virus-specific T cells (VSTs) specific for EBV, CMV and Adv antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional problematic viruses has been limited by competition between viral antigens and time-consuming/laborious manufacturing. We therefore developed a simplified 10-day system for generating a single preparation of VSTs with activity against 12 antigens from 5 viruses (EBV, CMV, Adv, BK, HHV6) that commonly cause post-transplant morbidity and mortality. We report our initial clinical results using these pentavalent pVSTs. With NHLBI-PACT support, we prepared 35 clinical-grade pVSTs from PBMCs (3x107 cells/sample) that we exposed to overlapping peptide libraries spanning immunogenic Adv (Hexon, Penton), CMV (pp65, IE1), EBV (LMP2, EBNA1, BZLF1), BK (Large T, VP1) and HHV-6 (U11, U14, U90) antigens. Exposure was followed by a 9-11 day expansion phase in a G-Rex device in the presence of IL4+7, producing a mean of 374x106 T cells (range 99-713x106). These lines were polyclonal, comprising both CD4+ (57±5%) and CD8+ (35±5%) cells and retained expression of the memory markers CD45RO+CD62L+ (58±8%). Their specificity was dependent on the prior viral exposure of the cell donor; 32/35 lines had activity against Adv (Hexon: 446±153; Penton:317±108 SFC/2x105), 20/35 against CMV (IE1: 337±141; pp65 1059±479), 26/35 against EBV (LMP2: 175±87; EBNA1: 116±44; BZLF1: 129±88), 18/35 against BK (Large T: 130±67; VP1: 231±104) and 21/35 against HHV-6 (U90: 66±50; U11: 36±18; U14: 82±21). None of the lines reacted against recipient PHA blasts (mean Cr51release of 1% at a 20:1 E:T ratio). We have administered pVSTs to 10 allogeneic HSCT recipients in a dose escalation study; 4 on DL1 (5x106/m2), 4 on DL2 (1x107/m2) and 2 on DL3 (2x107/m2). There were no immediate infusional toxicities, and no de novo acute GvHD, demonstrating the in vivo safety of these pVSTs even after a single exposure to viral antigens in vitro. Three patients received the cells as viral prophylaxis (days 38-43 post-HSCT) and all remain well and virus-infection free at up to 3 months post-treatment. The other 7 patients received the cells as treatment for one or more active infections between days 59-139 post-HSCT. Based on viral load measurements by day 42 post-infusion, the pVSTs were successful in controlling active CMV (1 complete (CR) and 1 partial response (PR)), EBV (2 CRs, including a case of frank PTLD); Adv (1 CR); HHV6 (1 CR); and BK (3 CR, 1 PR, 1NR) infections. Of note, 3 of our BK virus responders had tissue disease with severe hemorrhagic cystitis and all had marked improvement or disappearance of hematuria following infusion. One subsequently had an episode of transient but severe bladder pain in association with inflammation seen on cytoscopy coincident with a 6 log fall in urine BK viral load. Our only non-responder was a patient with BK infection whose line lacked activity for this virus, likely reflecting the serostatus of the donor. In addition, 3 patients subsequently reactivated other viruses than those for which they were initially treated, but all cleared these infections by week 12, without requiring additional cell infusions (CMV: 1CR; EBV: 1CR; BK: 1CR; HHV6: 1CR). Finally, 1 patient received pVSTs under a single patient protocol as an emergency treatment for widespread and bulky rituximab-resistant EBV-PTLD. Post pVST there was an immediate decline in her EBV viral load with complete and sustained resolution of PTLD, coincident with an increase in circulating EBV-specific T cells. However, the profound anti-tumor activity mediated by the rapidly-expanding EBV-directed T cells also produced a transient systemic inflammatory response syndrome, which was controlled with steroids and anti-TNFR antibody, with no long term adverse effects. Thus, infusion of pVSTs as prophylaxis or treatment has been safe and is associated with the appearance of virus-reactive T cells in peripheral blood that have been able to control infection with all 5 targeted viruses. We are currently exploring the extension of this platform to include additional clinically relevant viruses and are planning to assess the activity of these cells in the 3rd party setting for broader implementation. Disclosures: Off Label Use: Virus-specific CTLs manufactured under an investigator-initiated IND. Vera:Wilson Wolf Corporation: Consultancy, Research Funding.

Background Bleeding is a common presentation of light chain AL amyloidosis mainly attributable to vascular fragility and impaired vasoconstriction also known as Amyloid Angiopathy. It has also been shown that acquired coagulation factor deficiencies such as Factor X can exacerbate this situation and can be the leading cause of mortality in these patients. We performed a retrospective review of Bleeding disorders in patients with AL amyloid from the Amyloid Database at The Houston Methodist Hospital between 2006 and 2013. Methods We performed a retrospective review extracting information from the Tumor Registry and the Medical Electronic Records by querying for the diagnosis of Amyloidosis. We obtained baseline demographics, laboratory data, diagnostic workup, treatment plan including bone marrow, Lung and Heart transplant, clinically documented bleeding events and overall outcomes. Results We reviewed 411 patients who met the inclusion criteria. At the time of review, Amyloid typing was available in 95 patients out of which 85 were Al Amyloid. The baseline demographics included 213 males and 198 females, whose median age was 67; 238 were Caucasians while 77 were African Americans, 34 Hispanic, 12 Asian and 49 reportedly as others. Since the primary end point was review of bleeding, we sorted the groups based on the events into bleeders requiring ICU admission, bleeders without ICU admission and the non-bleeders. As shown in Figure 1, bleeders with ICU admission were 16 patients with a median PT of 19.77, median INR 1.6 and a low median factor X assay of 45% (range 70-120); Median BNP was 1285, serum creatinine 1.5, platelets 133, and Fibrinogen 356. From this group only 1 patient received a Heart and Lung Transplant. In the group of bleeders not requiring ICU admission there were 50 patients with a median PT of 16.2, median INR 1.128 and a low median factor X assay of 76%. Median BNP was 795 serum creatinine 1.37, platelets 126, and Fibrinogen 403.5. In this cohort 7 received bone marrow transplants, 3 received both Heart and Bone Marrow Transplants. In the group of nonbleeders, we had a total of 340 patients with a median PT of 15.7, median INR 1.2 and a low median factor X assay of 66. Median BNP was 240, serum creatinine 1.3, platelets 173, and Fibrinogen 409. In this cohort 16 received Bone Marrow transplant, 1 received lung Transplant and 10 received Heart Transplant. Conclusion Even though Amyloid Angiopathy is a major cause of bleeding, acquired factor X deficiency could potentially be reversible. There are reports of no correlation of diminished Factor X activity and severity of bleeding. Our data indicates that patients with very low factor X deficiency have a high risk of bleeding potentially requiring critical care support. Further statistical review of this data set will be presented at the American Society of Hematology, New Orleans 2013. Disclosures: No relevant conflicts of interest to declare.

Donor lymphocyte infusions (DLI) following hematopoietic stem cell transplantation may reduce or control opportunistic infections and leukemia/lymphoma relapse, but the associated graft versus host disease (GvHD) limits the clinical success of this procedure. Since T cell immunotherapy may be a safer alternative to DLI we have now used a single T cell platform that mediates both antileukemic and antiviral activity. Autologous T cells modified to express CD19-specific chimeric antigen receptors (CD19.CAR) have had clinical activity against CD19-expressing malignancies, but it is unknown if similarly modified allogeneic T cells will be equally effective. Allogeneic virus specific T cells (VSTs) directed to cytomegalovirus (CMV), adenovirus (Adv), and Epstein Barr virus (EBV) have been shown to be safe and effective in preventing and treating life-threatening viral infections post HSCT. Therefore, we sought to determine whether allogeneic VSTs could be engineered to express CD19.CAR and would retain the safety and effectiveness of unmodified VSTs whilst gaining anti-tumor activity. VSTs were expanded ex vivo using antigen presenting cells engineered to express adenovirus and cytomegalovirus (using an Ad5f35 adenoviral vector expressing the CMV pp65 gene), and Epstein Barr virus (using EBV-infected lymphoblastoid cell lines) antigens. After 3 stimulations, the VST’s were modified to express CD19.CAR.28ζ using a retroviral vector encoding the CAR-CD19 receptor coupled to the CD28 co-stimulatory molecule and the T cell receptor zeta (ζ) chain. Nine CD19.CAR-modified virus specific T cell (CD19.CAR-VSTs) products were generated for infusion. All VST lines recognized at least one viral antigen as determined by Elispot or chromium release assays and 20% to 48% of cells expressed the CD19.CAR. All lines killed CD19-expressing cells in vitro. We treated nine patients with these CD19.CAR-VSTs, 3 months to 13 years after HSCT. Six patients received CD19.CAR-VSTs for relapsed disease and 3 patients received the T cells as adjuvant therapy to prevent viral infection and relapse after HSCT. Safety. There were no infusion-related toxicities. One patient presented with gastrointestinal symptoms following infusion subsequently determined to be unrelated to the T cells. Persistence. VSTs persisted a median of 8 weeks in the peripheral blood and up to 9 weeks at disease sites. In three patients (#1, #3 and #5), CD19.CAR signals were detectable in the bone marrow or the lymph nodes (44.8, 25.85, and 32 copies/1000 ng DNA) even when no signal was measurable in peripheral blood, indicating preferential accumulation of the infused T cells at the disease site. Anti-Tumor Activity. During the period of CD19.CAR-VST persistence, objective anti-tumor activity was evident in 2/6 patients with relapsed disease (patient # 1 had detectable blasts in the peripheral blood which disappeared within 1-2 weeks following infusion, patient # 2 had 16% circulating CLL cells which decreased within 2 weeks of T cell infusion) but disease recurred after 3 and 2 months, respectively. The two patients who received cells while in remission remain disease-free >3 and >9 months later. Anti-Viral Activity. In two patients with EBV reactivation, donor CD19.CAR-VSTs expanded concomitant with an increase in virus-specific T cell responses, and decreased viral load. A third patient had a rise in adenovirus specific VSTs during an episode of adenovirus associated diarrhea. Although the infection was controlled, there was no concomitant rise in CD19-CAR expressing T cells in this patient. No other patient had viral disease. In conclusion, allogeneic CD19.CAR-VSTs administered after allogeneic HSCT are safe and can exert both anti-tumor and anti-viral activity in the absence of GvHD. Earlier administration of CD19.CAR-VSTs after HSCT, when the host is lymphodepleted and the incidence of viral infection is higher, may allow these cells to better capture the potential advantages of native TCR stimulation (and associated co-stimulation) for expansion and persistence, and thereby produce a higher frequency of sustained tumor responses. Alternatively, intentional stimulation of the native TCRs by viral vaccines may produce equal benefit, with greater predictability. Disclosures: Savoldo: Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

Abstract 956 The tumor cells of EBV-associated Hodgkin's Lymphoma (HL) and of some non-Hodgkins lymphoma (NHL) express the subdominant EBV antigens LMP1 and LMP2 (Type II viral latency), and these may serve as target antigens for immunotherapy. We hypothesized that cytotoxic T lymphocytes (CTLs) enriched for effector cells specifically targeting LMP antigens would have efficacy in patients with EBV+ lymphoma. We prepared antigen presenting cells (dendritic cells for the initial stimulation, EBV- lymphoblastoid cell lines (LCLs) for the subsequent) that expressed transgenic and inactive LMP1 (ΔLMP1) and/or LMP2, following transduction with the adenoviral vectors Ad5f35LMP2 (n=16) or Ad5f35ΔLMP1-I-LMP2 (n=27). All LMP-CTL lines were polyclonal as measured by flow cytometry CD4+ (mean 17+/−18%; range 1–92%) and CD8+ (mean 74 +/− 25%; range 1–99%) T-cells. Analysis of memory markers revealed mixed populations of CD45RA- CD62L- T-cells (mean 45+/−15%; range 31–63%) and CD45RA- CD62L+ T-cells (mean 34+/−5%; range 28–41%). CTL lines had specificity for CD4+ and CD8+ restricted LMP2 epitopes alone (median 1 epitope; range 0–7) or both LMP1 and LMP2 epitopes (median 3 epitopes; range 0–9) per CTL line, as determined using overlapping LMP1 and LMP2 peptide pools to stimulate the T cells in γ-IFN ELISPOT assays. 43 patients with EBV+ HL (n= 18) and NHL (n=25) have been treated and received between 4×107/m2 and 3.2×108/m2 T cells; 16 received LMP2 CTLs and 27 were given LMP1/2 CTLs. No immediate toxicity was observed. Within 1 to 4 weeks after CTL infusion, the numbers of LMP-specific T-cells in peripheral blood rose 2 to 70 fold, and elevated levels persisted for up to 3 months. Lymph node biopsies from 3 patients taken 3–6 months post CTL showed selective accumulation of LMP-specific T-cells in lymph nodes compared to peripheral blood. 21/22 high-risk and/or multiply relapsed patients who received LMP-CTL as adjuvant treatment remain in remission for a median of 2.5 years after CTL with a PFS of 80% at 3 years. 21 patients had detectable disease at the time of CTL infusion. Following treatment, 4 had progressive disease by 8 weeks, 1 is too early and 16 had clinical responses. The median duration of the clinical responses is 1.5 years with 1 stable disease, 3 partial responses and 13 complete responses. The 3 year progression free survival for patients treated with disease is 60%. We compared outcomes between patients who received LMP2 CTL alone versus LMP1 and 2-specific CTL. For high-risk patients treated with CTLs as adjuvant, the 3 year PFS is 70% for LMP2 CTL recipients versus 100% for recipients of LMP1/2-CTLs (p=0.06). The 3 year PFS in relapsed patients receiving LMP2-CTL was 50% versus 62% for recipients of LMP1/2 CTL (p=0.3). In conclusion, immunotherapy with CTLs targeting LMP antigens is well tolerated in patients with EBV+ lymphoma. Infused LMP-CTL accumulate at tumor sites and have induced complete and sustained clinical responses in 13/20 patients with relapsed disease. We are now implementing a simpler LMP-specific CTL expansion protocol to enable a definitive efficacy study. Disclosures: Pro: Allos Therapeutics: Honoraria.

Abstract 4048 Introduction: Chemotherapy + GCSF mobilization and GCSF alone are the most common mobilization regimens for autologous stem cell transplant (ASCT). We examined the benefits and limitations of both regimens in terms of mobilization success, predictability and costs. Methods: A retrospective, multi-center chart review was conducted of multiple myeloma (MM) and lymphoma patients mobilized between January 1, 2006 and December 31, 2007 for ASCT. Patients were excluded if they were mobilized with plerixafor (Mozobil®) or enrolled in a clinical trial of mobilization regimens. Data collected included demographics, disease and treatment history, mobilization regimen, blood counts, aphaeresis, remobilization, cells transplanted, time to engraftment and resource use. Stem cell collection practices and related clinical outcomes were analyzed separately for patients that were mobilized with chemotherapy + GCSF vs. GCSF alone. Resource use was evaluated using US unit cost data. Results: Data were collected for 227 consecutive patients from 11 centers (143 patients that received a chemotherapy + GCSF mobilization regimen and 84 patients who received GCSF alone). Total cells collected were significantly higher in the chemotherapy + GCSF mobilization group compared to GSF alone (18.6 × 106/kg vs.7.0 × 106/kg, p Even with fewer aphaeresis days and re-mobilizations, the cost total cost of mobilization, remobilization and aphaeresis with chemotherapy + GCSF was higher than GCSF alone ($19,614 vs. 16,852; p=0.003). Higher chemotherapy + GCSF costs were driven by the costs of chemotherapy, and extra days of GCSF. Chemotherapy + GCSF was also harder to predict in terms of timing the first day of aphaeresis. In patients receiving GCSF alone, the majority (95%) of first day aphaeresis occurred between Monday and Wednesday with the remaining 5% occurring on a Thursday or Friday. In contrast, chemotherapy + GCSF mobilizations resulted in 74.1% of first day aphaeresis occurring between Monday and Wednesday, 12.6% on Thursday and Friday and a further 13.3% occurring on a Saturday or Sunday. Discussion: Significant differences in mobilization success, costs and predictability were observed between patients that received chemotherapy + GCSF and those that received GCSF alone. While a chemotherapy + GCSF mobilization regimen may provide better mobilization and collection, there is a trade-off in terms of predictability, time to collection, costs and rate of febrile neutropenia. However, as chemotherapy + GCSF may be used for the treatment of the underlying malignancy; its use may not be completely comparable to GCSF mobilization alone. Disclosures: Grima: Cornerstone Research Group Inc.: Employment, Equity Ownership. Holmberg:Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Research Funding; Otsuka: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Celgene: Research Funding. Fung:Genzyme: Consultancy, Honoraria, Speakers Bureau. Brown:Cornerstone Research: Employment. Horwitz:Genzyme: Honoraria, Research Funding. Bernard:Cornerstone Research: Employment, Equity Ownership. Shaughnessy:Genzyme: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millenium: Honoraria, Speakers Bureau; Otsuka: Honoraria, Speakers Bureau.

Abstract 1925 After mobilization, the timing of aphaeresis can be highly variable, often depending on rise in peripheral blood stem cell levels. Some centers that conduct autologous stem cell transplant (ASCT) do not conduct aphaeresis for stem cell collection on weekends. We examined stem cell collection practices and related outcomes in centers that do and do not conduct weekend aphaeresis. Methods: A retrospective, multi-center chart review was conducted of multiple myeloma (MM) and lymphoma patients mobilized between January 1, 2006 and December 31, 2007 for ASCT. Patients were excluded if they were mobilized with plerixafor (Mozobil®) or were enrolled in a clinical trial of mobilization regimens. Data collected included demographics, disease and treatment history, mobilization regimen, blood counts, aphaeresis, remobilization, cells transplanted, time to engraftment, and resource use. Stem cell collection practices and related clinical outcomes were analyzed separately for sites that did and did not conduct weekend aphaeresis. Results: Data were collected for 292 consecutive patients in the different centers (91 patients from 4 sites that performed weekday only aphaeresis and 201 patients from 7 sites who performed aphaeresis on weekends and weekdays). Weekday only aphaeresis sites were more likely to conduct GCSF alone mobilization compared to weekend sites (42% vs. 21%, p Discussion: Significant differences in the management of patients were observed between sites that conduct and don't conduct weekend aphaeresis. Weekday only aphaeresis sites used a more regimented collection schedule with most collections starting on a Monday, potentially missing the “peak” day of cell collection. Sites that conducted weekend aphaeresis used a more cell-count based approach and were more likely to monitor peripheral blood stem cell counts. Collection success appears to be superior with the cell-count based approach that includes greater use of chemo-mobilization, cell count monitoring and weekend collection. Disclosures: Chao: Genzyme: Research Funding. Bernard:Cornerstone Research: Employment, Equity Ownership. Fung:Genzyme: Consultancy, Honoraria, Speakers Bureau. Grima:Cornerstone Research Group Inc.: Employment, Equity Ownership. Holmberg:Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Research Funding; Otsuka: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Celgene: Research Funding. Brown:Cornerstone Research: Employment. Horwitz:Genzyme: Honoraria, Research Funding. Shaughnessy:Genzyme: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millenium: Honoraria, Speakers Bureau; Otsuka: Honoraria, Speakers Bureau. Tricot:Otsuka: I have an ongoing clinical study supported by Otsuka.

Abstract 4489 Dominant cardiac involvement by primary systemic amyloidosis (AL) precludes effective AL treatment and is associated with short survival (median survival for stage-III AL= 3–4 months). Three patients who presented with severe cardiac dysfunction as their major AL manifestation underwent orthotopic heart transplantation (OHT) followed by autologous hematopoietic stem cell transplantation (ASCT). The clinicopathological characteristics of these patients form the basis of this report. Age at AL presentation was 63, 62 and 44 years in 2 females and 1 male. The time from initial symptoms of AL to diagnosis was 12, 24 and 12 months. Diagnosis of cardiac AL was established via endomyocardial biopsy, Congo red staining and immunohistochemistry. Standard work-up for plasma cell dyscrasia included; serum and urine electrophoresis, immunofixation, measurement of serum-free light-chain ratio and bone marrow biopsy. All patients had stage III AL based on cardiac troponin I and NT-pro-BNP. All had end stage heart failure with New York Heart Association class 4 symptoms and developed cardiogenic shock requiring intra-aortic balloon pump support as a bridge to OHT. All were considered unsuitable for ASCT. Initial treatment was with lenalidomide, dexamethasone in two patients while the third received melphalan and dexamethasone. Table -1 shows the timeline of diagnosis and dual transplants.Table-1:Orthotropic cardiac transplantation (OHT) followed by ASCTAge/SexTime to AL diagnosis (months)Organ involvement (months)Time* to OHT (months)Time to ASCT s/p OHT (months)Survival (months)63/F12Heart, Kidney, PN71330+62/F24Heart, Kidney, PN51626+44/M12Heart, Kidney, tongue41314+*Time to OHT from onset of heart failure symptoms, PN= peripheral nerve, F = female, M= male, s/p= status post ASCT was performed 13, 16 and 13 months after OHT. All 3 patients were on tacrolimus and prednisone at the time of stem cell mobilization and hematopoietic transplant; two patients were also receiving mycophenolate mofetil and valganciclovir. We collected 6.1 and 6.2 × 106/kg CD-34+ cells in 2 days with filgrastim (5 ug/kg, twice, daily) and plerixafor (16 mg/kg based on day- 4 CD-34+ counts) in two subjects. The third initially failed to mobilize but 4.3×106/kg CD-34+ cells were subsequently obtained when she (patient #2) was remobilized with filgrastim and plerixafor 4 weeks after stopping mycophenolate mofetil. The creatinine clearance at the time of high-dose chemotherapy was 36, 30 and 43 ml/minute. All 3 patients received renal adjusted dose of melphalan at 140mg/m2. Mycophenolate mofetil and Valganciclovir were withheld during neutropenia until engraftment. Time to engraftment for neutrophils (ANC of >500/uL x3 days) was 13, 11 and 14 days. Platelets engraftment (untransfused >20 × 109 for 3 consecutive days) occurred in 14, 13 and 16 days. No patients received post-transplant filgrastim. Patients were hospitalized for 17, 18 and 18 days. Renal function remained stable during ASCT and non-hematological toxicity was limited to grade I-II apart from one grade III oral mucositis and colitis. One patient achieved hematologic complete remission (patient#2) while 2 patients had partial response following ASCT. Post OHT and ASCT all patients are alive and well at follow-up of 14, 26 and 30 months from OHT. OHT should be considered in select patients with dominant cardiac involvement and advanced heart failure to allow subsequent ASCT and improve survival. Disclosures: No relevant conflicts of interest to declare.

Cancer precursor/progenitor cells may initiate and sustain the growth of tumors, but evidence for their existence in human disease is indirect, relying on their in vitro properties and animal models. More directly, specific elimination of these rare cells from cancer patients should produce a delayed but progressive disappearance of differentiated malignant progeny. Here, we describe selective eradication of a putative precursor population in a patient with B-cell chronic lymphocytic leukemia, followed 6 months later by a progressive loss of mature tumor cells without further treatment. This outcome supports the presence of a rare population of precursor/progenitor cells in human malignancies, and suggests benefit from their removal.

B-chronic lymphocytic leukemia (B-CLL) cells express tumor associated antigens that may generate a T cell mediated immune response, but present these antigens poorly. Moreover, patients with B-CLL often have poor immune function due to the disease or its treatment. We have shown that expression of transgenic CD40L increases the immunogenecity of human B-CLL cells ex vivo and in vivo, and that this effect can be potentiated by co-expression of transgenic IL2. Previous studies described outcomes when adenoviral vectors were used to obtain gene transfer, but because of the complexities and expense of manufacture of viral vectors, and their lingering safety concerns, we determined whether it was possible to use electroporation (with the MaxCyte device) as a physical means of transferring CD40L and IL2 plasmids to produce vaccines with similar biological properties in vitro and in vivo. Table 1 compares the phenotype of the vaccines using each vector. Table 1. Comparision of immunogenic characteristics and viability of the adenoviral and plasmid vaccines Type of Vaccine CD40L (%) CD80 (%) CD86 (%) IL-2 (pg/ml/10e6 cells) Viability (%) IL2 CD40L All the values are given as mean ± SE. * P< 0.01, Paired Student’s t test. Adenoviral Pre 0.2 ± 0.01 2.6 ± 2.4 7.5 ± 3.9 Post 66.1 ± 5.5* 50.2 ± 7.8* 69.5 ±11* 253.5 ± 82.6 93.6 94.2 Plasmid Pre 1.3 ± 0.85 11.5 ± 6.2 19.7 ± 6.8 Post 55.5 ± 5.1* 19.2 ± 9.3 26.4 ± 9.7 4806.6 ±1398.9 84.4 88.4 Vaccines made by both approaches met the release criteria for CD40L and IL2 expression (CD40L ≥20% and IL-2 ≥ 150 pg/ml/1x10e6 cells ), but expression of IL2 was higher in the plasmid vaccines, expression of CD40L was equivalent in each and expression of the additional co-stimulatory molecules CD80 and CD86 (induced after CD40 activation by transgenic CD40L) was higher in the adenoviral vaccines. Fourteen patients were given adenoviral-vaccines and nine the plasmid transduced cells. Each of these patients received up to 18 s.c. injections of IL-2 secreting and CD40L expressing tumor cells. Both types of vaccine were well tolerated. Table 2 shows the results of culturing patient T cells with autologous B-CLL tumor cells. Table 2. Comparision of anti-B-CLL T cell responses induced by adenoviral and plasmid vaccines Type of Vaccine Pre-vaccine After 3rd vaccine After 6th vaccine All the values were are given as mean ± SE. *P After 3 and 6 injections, both the adenoviral and plasmid vaccines had induced a rise in spot forming cells (SFC) for IL5, a cytokine associated with Th2 cells, but the rise was greatest in the recipients of the electroporated plasmid vaccine. By contrast, only the adenoviral vaccine induced a rise in SFC that produced IFN-γ, a cytokine associated with Th1 cells. Studies using MHC class I and II blocking antibodies showed that the IL5 and IFN-γ responses to both types of vaccine were mediated by HLA restricted T lymphocytes. The 1-year progression-free survival rates (PFS) for adenoviral vaccine group and plasmid vector group were 43% and 22% respectively. Figure 1 shows 1-year PFS rates for each group. Hence electroporation provides a more rapid and simpler means of preparing IL2/CD40L expressing B-CLL vaccines, but the cells express higher levels of IL2 and lower levels of “secondary” co-stimulator molecules than adenoviral vaccines, and produce an anti-tumor immune response of different polarity. Currently, we are evaluating electroporation of mRNA encoded CD40L which appears to augment upregulation of additional costimulatory molecules. Figure Figure

EBV-associated Hodgkin’s Lymphoma (HL) and some non-Hodgkins lymphoma (NHL) have type II viral latency expressing the subdominant EBV antigens EBNA1, LMP1 and LMP2. These antigens may serve as targets for immunotherapy approaches and in previous studies, we used polyclonal EBV-specific CTL in patients with relapsed EBV +ve HL obtaining 2 complete and 1 partial response in 11 patients. Analyses of EBV-CTL lines showed that small populations of T cells reactive against the tumor-associated antigen LMP2 were present in the majority of the infused lines, with some expansion in the peripheral blood following infusion. We therefore hypothesized that CTL enriched for effector cells specifically targeting LMP antigens would have greater efficacy in these patients. LMP-CTL were generated using dendritic cells for initial stimulations then EBV-transformed lymphoblastoid cell lines (LCL) both of which had been genetically modified to overexpress either LMP2 alone or inactive LMP1 (ΔLMP1) and LMP2 by transduction with an Ad5f35LMP2 (n=16) or Ad5f35ΔLMP1-I-LMP2 (n=14) vector respectively. All LMP-CTL lines were polyclonal comprising CD4+ (mean 17±18%; range 1–92%) and CD8+ (mean 74 ± 25%; range 1–99%) T-cells. Flow cytometric analysis of memory markers revealed mixed populations of CD45RA- CD62L- T-cells (45±15%; range 31–63%) and CD45RA- CD62L+ T-cells (34±5%; range 28–41%). The CTL lines had specificity for CD4+ and CD8+ restricted LMP2 epitopes alone (n=19; mean 1; range 0–7) or both LMP1 and LMP2 epitopes (n=13; mean 2; range 0–6) per CTL line, as determined using overlapping LMP1 and LMP2 peptide pools in ELISPOT assays. Twenty-four patients with EBV+ Hodgkin’s Lymphoma and non-Hodgkin Lymphoma have been treated on dose escalation studies. 16 with LMP2 CTLs and 8 with LMP1/2 CTLs. No immediate toxicity was observed. After CTL infusion, increased numbers of LMP-specific T cells were detected in the blood of 15/22 evaluable patients, (range 2 to 70 fold) persisting for up to 3 months. Additionally, two patients had lymph node biopsies 3–6 months post CTL, which showed selective accumulation of LMP2-multimer positive cells in lymph nodes. 12/13 high-risk and/or multiply relapsed patients who received LMP-CTL as adjuvant treatment after chemotherapy remain in remission for a median of 2years (range >3months to >5years) after CTL. 11 patients had detectable disease at the time of CTL, 2 of these had progressive disease by 8 weeks and 9 had clinical responses. The median duration of the clinical responses is 1 year with one stable disease (>12months), one partial response (36 months) and 7 complete responses (range 9 months to >4.5 years). One of the complete responders was biopsied 7 weeks after receiving CTL, which were predominantly CD4+ (92%). Increased CD4+ T cells were seen compared to pre-CTL biopsy specimens and imaging studies confirmed remission. In conclusion, immunotherapy with CTL targeting LMP antigens is well tolerated in patients with EBV+ lymphoma and infused LMP-CTL can accumulate at tumor sites and induce complete and sustained clinical responses.

We agree that diagnosis of central nervous system (CNS) GVHD may be controversial and that the diagnosis currently remains one of exclusion. As we stated,1 the possibility of post transplant progressive multifocal leukoencephalopathy (PML)2 was indeed considered in both patients. The diagnosis of 'possible PML' has to be entertained in the presence of the typical clinical and radiologic picture of PML in the absence of demonstrable JC virus (JCV) infection. In the immunocompromised host, JCV DNA detection in the cerebrospinal fluid (CSF) has a sensitivity of 72–92% and specificity of 92–100%.3 As one-third of patients may not yield JCV DNA in CSF, brain biopsy with immunohistochemical studies along with tissue JCV DNA amplification by PCR remains the gold standard for diagnosis of PML. We did indeed seek the characteristic pathological changes of PML, namely, JCV-infected glial cells, bizarre asytrocytes, lipid-laden macrophages and demyelination, but they were lacking in our cases. Hence the absence of characteristic pathologic changes, the negative JCV studies (negative immunohistochemical stain in case no. 1 and negative CSF PCR in case no. 2), the rapid response to steroids and the exclusion of other etiologies lead one to consider the possibility of CNS GVHD. While PML is indeed a worthy candidate for consideration in cases of this type, it would be unwise to force this label on all 'white matter disease of uncertain origin' in the immunocompromised host. Continued reporting of similar cases may help delineate whether brain tissues truly are targets for the development of GVHD.

7112 Background: We investigated the safety and outcome after adding Rituximab to our standard high-dose chemotherapy conditioning regimen with BEAM in patients receiving autologous hematopoietic stem cell transplantation. Methods: Between November 2002 and May 2006, 25 consecutive patients with relapsed high-risk NHL (diffuse large B cell = 15, transformed follicular =10) were enrolled. The high-dose chemotherapy regimen consisted of BEAM with rituximab administered at 375 mg/m2 IV on days −6, +14, +21 and +28. Rituximab associated delayed neutropenia was defined as ANC of < 500 occurring at least 4 weeks after the last dose of rituximab. B cell and T cell reconstitution data was measured using phenotypic analysis for CD3, CD4, CD8, CD56, CD19 and CD20. Results: The median age was 55 years (range 22–69 years). The disease status at transplant included advanced disease (≥CR-2) in 15 while 10 had primary refractory disease. The previous initial and or salvage chemotherapy included Rituximab in all 25 patients. A median of 4.47 ×10 6/kg CD 34 + cells were infused resulting in neutrophil and platelet engraftment at 11 (10–23) and 21 (14–56) days respectively. The median time to T cell reconstitution (CD3 >1,000/uL) was 32 days, B cell recovery was delayed beyond 1 year with median of 72 CD19+ve cells/uL at 1 year. 24 (96%) patients achieved complete remission after transplant and 1 had a partial response. There was no transplant related mortality. Nine patients relapsed at a median of 4 months (2–23 months). 6 patients died at a median of 15 months from transplant (relapsed lymphoma =4 and secondary MDS=2). At a median follow-up of 24 months, the disease free survival and overall survival is 52% and 75% respectively. Rituximab associated delayed neutropenia occurred in 3 (12%) patients at a median of 39 (31–64) days from the last dose of rituximab and resolved at a median of 14 days (7–38) with or without filgrastim support. There were no infectious complications associated with neutropenia or delayed B cell recovery. Conclusion: Incorporation of rituximab into a high dose chemotherapy conditioning regimen is safe. Delayed B cell reconstitution and Rituximab associated delayed neutropenia are common but not associated with significant infectious complications. No significant financial relationships to disclose.

A patient with Philadelphia-positive (Ph+) chronic myelogenous leukemia (CML) was diagnosed with cutaneous sarcoidosis after treatment with interferon-alpha (IFN-alpha). Following IFN-alpha dose reduction, the skin lesions disappeared. Few cases of sarcoidosis associated with IFN treatment have been reported, and only in one patient with pre-existing CML. Our patient was unique in that (1) the sarcoidosis was induced by the IFN-alpha treatment alone, (2) it developed de novo, and (3) it was confined to the skin.

CMV, Adenovirus and EBV are major viral pathogens causing morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. Previous studies have shown that prophylactic adoptive immunotherapy with donor-derived Cytotoxic T Lymphocytes (CTL) directed against EBV and CMV can effectively prevent the clinical manifestations of these viruses. We have extended these studies by generating CTL from normal donor PBMC that can restore cellular immunity to CMV, EBV and adenovirus simultaneously. We have developed a protocol utilizing an initial round of stimulation with autologous mononuclear cells transduced with a recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber carrying a transgene for the immunodominant CMV antigen, pp65 (Ad5f35pp65). This is followed by 2 rounds of weekly stimulation with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector using MOIs of 10 and 100 respectively. After 3 rounds of stimulation, 9 CTL cultures contained a mean of 83% (range 8.4–98.99%) CD8+ve and a mean of 19.6% (range 2.2–91.6%) CD4+ve cells. In chromium release and/or IFNg ELISPOT assays, all CTL lines showed specific activity against CMV and EBV targets. 8/9 lines also showed specific lysis against adenovirus targets. Further, using MHC-peptide multimers we have been able to demonstrate the simultaneous presence of CD8+ve cells recognizing peptide epitopes from CMV pp65 (range 2.32–21%) and Adenovirus hexon (1.07–8.08%) in the CTL cultures. So far we have treated 6 patients in this phase I CMV prophylaxis study, 3 on dose level 1 (1x10e7/m2) and 3 on dose level 2 (5x10e7/m2). Patients received 1 infusion of virus-specific CTL from 54–120 days post transplant. We observed up to a 28-fold increase in CMV pentamer positive CD8+ve cells post CTL. At last follow-up (7–35 weeks post CTL infusion) all patients are CMV and EBV negative. 2 patients were transiently positive for CMV by PCR 4–9 weeks post CTL but both were negative 7 days later without anti-viral therapy, with a corresponding rise in CMV-specific CTL detected in the peripheral blood. 2 patients were culture positive for adenovirus in their stool pre CTL therapy. One of these patients was infected with adenovirus species from subgroups A, C and D. In both patients, we observed a 2-log reduction of adenovirus copies/g stool within 2–3 weeks post CTL infusion at which time their symptoms (fever, loose stools) resolved. In summary, we have developed a protocol for the efficient generation of multi-virus specific CTL: infusion of small numbers of these cells increased virus-specific CD8+ve T cells in the peripheral blood post CTL infusion. Further, reduction in adenovirus load in stool suggests efficacy of adenovirus specific CTL in vivo. However, expansion of virus-specific CTL in vivo may require presence of antigen. We will therefore complete this prophylaxis study and then proceed to using virus-specific CTL for the treatment of CMV and adenovirus disease post transplant.

EBV-associated Hodgkin’s Disease (HD) and some non-Hodgkins lymphoma (NHL) show type II latency expressing the subdominant EBV antigens EBNA1, LMP1 and LMP2, which may serve as targets for immunotherapy approaches. In previous studies, we used polyclonal EBV-specific CTL in patients with relapsed EBV +ve HD and saw 2 complete and 1 partial response in 11 patients. Analyses of EBV-CTL lines showed that small populations of T cells reactive against the tumor-associated antigen LMP2 were present in the majority of the infused lines, with some expansion in the peripheral blood following infusion. We therefore hypothesized that CTL specifically targeting LMP2 might have greater efficacy in these patients. LMP2-CTL were generated from 14 patients using Dendritic Cells for initial stimulations then Lymphoblastoid Cell Lines (LCL) both of which had been genetically modified to overexpress LMP2 by transduction with an Ad5f35LMP2 vector. Polyclonal LMP2-CTL lines recognized 1–7 (median 2) LMP2 epitopes, as determined using pentamers and overlapping LMP2 peptide pools in ELISPOT assays. A mean of 22.8% (5–42.1%) of CD8+ T cells bound HLA-restricted LMP2 pentamers, compared to a mean of 0.11% (0.01–0.24%) of LMP2-pentamer positive CD8+ T cells found in CTL generated with genetically unmodified LCL from the same patients. So far, 11 patients have been treated on this dose escalation study - 6 patients have been treated on dose level (DL)1 (2 doses of CTL at 2x107/m2/dose given 2 wks apart in the outpatient clinic), 4 patients on DL2 (2x107/m2 and 1x108/m2) and 1 patient on DL3 (1x108/m2 and 2x108/m2). No immediate toxicity was observed. After CTL infusion, an increase in the frequency of EBV +/- LMP2-specific T cells could be detected in the blood in 8/10 evaluated patients (range 2–17.6 fold). Five of 6 patients who received LMP2-CTL as adjuvant therapy post stem cell transplant or chemotherapy remain in remission up to 22mths post LMP2-CTL. 1 patient presented with progressive disease 8 wks post CTL therapy. Five patients had detectable disease at the time of CTL therapy of whom 1 had progressive disease 8 wks post CTL and 4 had clinical responses (1 very good partial response and 3 clinical or radiologic complete responses). One of these 3 patients was evaluated 7 wks after receiving CTLs, which were predominantly CD4+ve (91.6%). Biopsies showed minimal residual NHL cells with increased CD4+ve T cells compared to pre-CTL biopsy specimens. Imaging studies performed 1 wk later were negative for NHL. This patient received 2 extra doses of CTL (given 8 wks apart) and re-evaluations showed CR on PET and CT scans. Two other patients had stable disease 8 wks post LMP2-CTL. Both patients received 2 further doses of LMP2-CTL. One patient is without evidence of disease 12 months post CTL. The other patient had a complete radiological response. This patient had a supraclavicular lymph node resection, which showed selective accumulation of LMP2-tetramer +ve T cells (0.3% compared to 0.01% in the peripheral blood) with few residual tumor cells. Immunotherapy with autologous LMP2-CTL is therefore well tolerated in patients with relapsed EBV+ve HD/NHL and infused LMP2-CTL cells can accumulate at tumor sites and induce clinical responses.

6650 Alemtuzumab appears to be of promise when incorporated as a lymphocyte-depleting agent in reduced intensity conditioning (RIC) stem cell transplants (SCT). However, there is a high incidence o...

CMV and Adenovirus are major viral pathogens causing morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. Previous studies have shown that prophylactic adoptive immunotherapy with donor-derived Cytotoxic T Lymphocytes (CTL) directed against EBV and CMV can effectively prevent the clinical manifestations of these viruses. We have extended these studies by generating CTL from PBMC from CMV seropositive normal donors that should restore cellular immunity to both CMV and adenovirus simultaneously using gene transfer to modify antigen-presenting cells. We have generated a clinical grade recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber (Ad5f35pp65) carrying a transgene for the immunodominant CMV antigen, pp65. We have developed a protocol utilizing an initial round of stimulation with autologous mononuclear cells transduced with Ad5f35pp65, followed by 2 rounds of weekly stimulation with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector using MOIs of 10 and 100 respectively. After 3 rounds of stimulation, four CTL cultures contained a mean of 96% (range 93–99%) CD8+ve and a mean of 4% (range 1.3–7%) CD4+ve cells. All CTL lines showed significant cytotoxicity in chromium release assays against CMV targets and 3/4 lines also showed specific lysis against adenovirus targets. Further, using MHC-peptide multimers we have been able to demonstrate the simultaneous presence of CD8+ve cells recognizing peptide epitopes from CMV pp65 (range 2.32–21%) and Adenovirus hexon (1.07–8.08%) in the CTL cultures. So far we have treated 3 patients in this phase I CMV prophylaxis study. All patients received 1 infusion of virus-specific CTL at a dose of 110e7/m2 from day 30 post transplant. We observed up to a 28-fold increase in CMV pentamer positive CD8+ve T cells post CTL. At last follow-up (2–10 weeks) all patients remain CMV negative. One patient was culture positive for adenovirus in her stool pre CTL therapy. Within 2 weeks of CTL infusion we observed a 2-log reduction of adenovirus copies/gram stool at which time the patient's symptoms (fever, loose stools) resolved. In summary, we have developed a protocol for the efficient generation using genetically modified monocytes and LCL to expand CTL ex vivo with specificity for CMV and adenovirus. CMV-pentamer specific CD8+ve T cells increase in the peripheral blood post CTL infusion despite infusing relatively low numbers of cells. Further, reduction in adenovirus load in stool suggests efficacy of adenovirus specific CTL in vivo. However, expansion of virus-specific CTL in vivo may require presence of antigen. We will therefore complete this prophylaxis study and then proceed to using virus-specific CTL for the treatment of CMV and adenovirus infections post transplant.

Background : IThe CD52 monoclonal antibody Alemtuzumab appears to be of great promise when incorporated as a lymphocyte depleting agent in RIC stem cell transplants. However the degree of functional immunosuppression produced by this antibody in this application has not yet been well characterized. As a surrogate functional marker, we have studied the incidence and pattern of CMV infection following RIC transplantation regimens with a regimen incorporating this antibody. Methods : We analyzed CMV infections and risk factors in 49 patients who underwent allogeneic stem cell transplantation with a RIC regimen consisting of Fludarabine (120mg/m2), total body irradiation (450 cGy), antithymocyte globulin (40mg/kg), (n=11) or Alemtuzumab (40mg), (n=38) with or without CD45 monoclonal antibodies for various malignant disorders. Donors were HLA identical (related, n=20; unrelated, n=20) or one antigen mismatched (n=9). Ganciclovir was used as prophylaxis starting at engraftment or on recovery from cytopenias. CMV antigenemia and polymerase chain reaction based assays were performed on peripheral blood twice a week till day 100 and then on each clinic visit. Results : The cumulative incidence of CMV reactivation at 1 year post-transplant with Alemtuzumab, in patients at risk, was 60% (95% CI, 45%–78%). Median time to reactivation of 24 days (range, 5– 95 days). Fifty seven percent (12/21) of the reactivations occurred before day 30. Recurrence of CMV reactivation occurred in 38% (8/21) of patients with a median number of recurrences of 2 (range, 1–6) and they extended beyond day 100. Only one patient developed CMV colitis and fully recovered with treatment. There was no CMV related mortality. The overall non-relapse mortality was 16% and overall survival in CMV reactivators and non-reactivators was not statistically different. Risk factors for CMV reactivation analyzed were patients age, sex, acute graft vs host disease, Alemtuzumab use, Ganciclovir use before day 30 and absolute lymphocyte count at day 30. In univariate analysis, use of Alemtuzumab (P= 0.001) and starting Ganciclovir after day 30 (P= 0.002) were significantly associated with CMV reactivation but in multivariate analysis only starting Ganciclovir after day 30 was statistically significant, P= 0.009 (95% CI, 1.5%–17%). Conclusions : RIC stem cell transplantation using Alemtuzumab is associated with a particularly high incidence of early CMV reactivation, and is also associated with recurrent reactivation extending beyond day 100. Although the incidence of CMV disease was low, the high frequency of viral reactivation with Alemtuzumab is likely an indicator of profound post transplant immune suppression, suggesting that despite the “reduced intensity” of the conditioning, these patients require early, comprehensive and prolonged pre-emptive/prophylactic therapy for opportunistic infections.

Significant reduction in renal function secondary to protein deposition in the renal tubules is a common occurrence among patients with plasma cell dyscrasias such as Multiple Myeloma (MM) and Amyloidosis undergoing HSCT. Conditioning chemotherapy and uric acid release following cellular lysis may worsen already compromised renal function, leading to acute renal failure. Rasburicase, a recombinant urate oxidase enzyme, has been approved by the U.S. Food and Drug Administration for the treatment and prevention of hyperuricemia associated with tumor lysis syndrome in pediatric patients with leukemia, lymphoma, and solid tumor malignancies undergoing chemotherapy. We hypothesized that prompt treatment of hyperuricemia with rasburicase may prevent uric acid nephropathy and improve renal function, obviating the need for dialysis and improving the eventual outcome. Six adults, four with MM and two with amyloidosis, with rising uric acid levels after high dose Melphalan (200mg/m2) were given a single dose of rasburicase (0.15 mg/kg). At baseline, all patients had rising serum creatinine [mean: 2.1 mg/dL (range: 0.9–4.5 mg/dL)], rising BUN [mean: 27.6 mg/dL (range: 1.3–70 mg/dL)], and declining creatinine clearance (CrCL) [mean: 50.5 ml/min (range: 16–75 ml/min)]. After one dose of rasburicase, renal function significantly improved according to CrCL at 48 hours [mean: 55.2 ml/min (range: 17–82 ml/min)] and continued to improve at five days [mean: 68.2 ml/min (range: 18–109 mg/dL)]. Baseline uric acid was elevated [mean: 8.61 mg/dL (range: 7–9.9 mg/dL)] and improved 24 hours after treatment with rasburicase [mean: 0.2 mg/dL (range: 0–0.6 mg/dL)]. None of the patients required renal replacement therapy. There were no serious adverse events associated with rasburicase therapy. We therefore conclude that a single dose of rasburicase is promising in improving deteriorating renal function post high dose chemotherapy in patients with compromised renal function; further controlled studies are warranted.

Despite improvements in methods for the early diagnosis of CMV infection, 5% of HSCT patients receiving preemptive therapy develop CMV disease. In addition, the use of highly immunosuppressive therapies prior to and after transplant and the use of matched unrelated (MUD) and mismatched related (MRD) donors have increased the incidence of CMV infection and disease. We designed a prospective trial examining the safety and efficacy of valganciclovir for the prophylaxis of early CMV infection. Methods: CMV seropositive patients or CMV seronegative patients receiving CMV seropositive stem cell products with an estimated creatinine clearance of ≥ 50 mL/min, platelet count ≥ 50,000/μL, and WBC count ≥ 1,000/μL are eligible. Patients receive valganciclovir 900 mg daily Mondays through Fridays starting 21 to 35 days after transplant and continuing through Day 100, with dosage adjustments for myelosuppression and/or reduced creatinine clearance. Patients are monitored with weekly CMV PCR analysis at a central lab according to the Roche assay. Results: Twenty four (24) out of 50 planned subjects have been enrolled to date (MUD 9, MRD 15). All were CMV seropositive at the time of transplant. Stem cell source included peripheral blood = 21, bone marrow = 3. A total of 7 patients required corticosteroids for treatment of graft versus host disease. Three patients developed myeloid toxicity related to valganciclovir (absolute neutrophil count < 1,000/μL = 1, platelets < 50,000/μL = 2). All three (3) patients recovered their counts and restarted valganciclovir with dose adjustments. A total of 11 patients required dose adjustments due to myelosuppression (3), creatinine clearance (4), weight (1), institutional decision (3). Twelve (12) patients have not completed therapy due to: progressive disease or death before day +100 = 4, CMV infection =2, too early for analysis = 6. CMV infection occurred in 2/24 and CMV disease occurred in 0/24. The two (2) patients with positive CMV-PCR developed infection one (Day 40) and two (Day 36) weeks after starting valganciclovir. These two (2) patients were successfully treated with IV ganciclovir. Ten (10) patients have been followed between day +100 and 6 months post transplant. Three (3) of these 10 patients continued valganciclovir off study and had no evidence of CMV infection. Two (2) of the 10 patients who were not receiving valganciclovir developed CMV infection but were successfully treated with valganciclovir. Survival is 80% (16/20) at day +100 and 82% (9/11) at 6 months post transplant. Conclusions: We conclude that valganciclovir at the dose used in this study is well tolerated with minimal reversible myelosuppression. These preliminary findings suggest that prophylaxis with oral valganciclovir appears to be a promising strategy to prevent CMV infection and disease.

Recent studies have shown that flow cytometry immunophenotyping is a promising tool aiding in the diagnosis of myelodysplastic syndromes (MDS). However, the majority of these studies apply only qualitative pattern analysis in their interpretation of immunophenotypic data. The goal of this study was to analyze immunophenotyping results quantitatively, investigating the potential of this approach for providing additional information useful diagnosing MDS. Using flow cytometry immunophenotyping, we studied 56 bone marrow specimens from 13 patients with well-defined MDS by morphologic, clinical, and/or cytogenetic findings (5 RA, 3 RARS, 2 RAEB grade 1, 2 RAEB grade 2 and 1 secondary to chemotherapy), 15 cytopenic patients (controls, age-matched with MDS patients) with non-MDS/non-clonal hematologic disorders receiving marrow evaluation for other reason (ITP, fever of unknown origin, or lymphoma staging), 8 patients with AML transformed from MDS (t-AML), 6 patients with de novo AML, and 7 patients (14 specimens) with regenerating marrow after stem cell transplantation. These samples were analyzed qualitatively as reported in the literature as well as quantitatively for percentages of T-cells (CD3+), B-cells (CD20+), NK cells (CD3−/CD56+), granulocytes (moderate CD45 intensity and high side scatter characteristics), monocytes (CD14+/CD11c+), blasts (defined by dim CD45 and low side scatter, CD34+ or CD117+), erythroid precursors (CD71+/CD45−) and plasma cells (bright CD38), CD4/CD8 ratio, percentages of granulocyte subsets (CD10+, CD10−, CD36+/CD64+, CD36−/CD64+, CD11b−/CD16−, CD11b+/CD16− or CD11b+/CD16+ granulocytes per total granulocytes), percentage of CD56+ monocytes, and percentages of erythorid precursors subset (glycophorin A+ or glycophorin A- erythroid precursors per total erythroid precursors). In agreement with previous studies, qualitative analysis of these data demonstrated abnormal patterns of expression in myeloid and erythroid lineages in patients with MDS and t-AML. However, these patterns are also observed in age-matched controls and patients with de novo AML or with regenerating marrow. The quantitative analysis showed significantly increased T-cells and a significantly decreased granulocyte subset of CD11b+/CD16− in MDS patients when compared to age-matched controls (9.0 +/− 6.7% vs. 4.4 +/− 3.0 %, p = 0.023 and 28.1 +/− 14.9% vs. 44.5 +/−12.9%, p = 0.004, student t-test). There were also trends for increased NK cells and CD4:CD8 ratios and decreased total granulocytes in MDS patients as compared to the age-matched controls (p = 0.097, 0.094 and 0.059, respectively, student t-test). Other immunophenotypic parameters demonstrated no significant differences between these two groups. Furthermore, the changes observed in MDS patients were also seen in patients with t-AML. Patients with de novo AML or regenerating marrow post stem cell transplantation showed a quantitative immunophenotypic pattern between that of MDS patients and age-matched controls. These findings suggest that quantitative analysis of flow cytometry immunophenotyping data can aid in the diagnosis in MDS as well as the identification of AML arising from background MDS. The latter is clinically significant since these patients carry worse prognosis than those with de novo AML and may require novel therapies.

Autologous stem cell transplant (ASCT) requires therapy with very highly emetogenic chemotherapy regimens. Nausea and emesis are a major issue for patients undergoing ASCT. Aprepitant, a selective substance-P/neurokinin 1 (NK1) receptor antagonist, has been approved by the U.S. Food and Drug Administration for treatment of acute and delayed CINV in combination with a corticosteroid and 5-HT3 receptor antagonist. Due to little patient data in this area, a retrospective chart review was performed which evaluated 41 ASCT patients that had received standard therapy (control group, n=25) compared to aprepitant in addition to standard therapy (aprepitant group, n=16). Patients who underwent an ASCT and were greater than 18 years of age were eligible for inclusion in the study. Patients who underwent an allogeneic stem cell transplant were excluded. All data was collected from the hospital’s computerized nurse charting system. Episodes of nausea and emesis were collected from nursing notes while data for rescue medications was collected from the charted medications. Conditioning regimens given during the study included high dose (HD) melphalan, BEAM (carmustine, etoposide, cytarabine, and melphalan) or R-BEAM (rituximab, carmustine, etoposide, cytarabine, and melphalan). All patients evaluated in the aprepitant group had received aprepitant 125 mg orally once daily on the first day of chemotherapy administration and 80 mg orally once daily the following 2 days in the HD melphalan group (Day -1 through Day 0) and the following 5 days in the BEAM group (Day-6 through Day -1). Although aprepitant dosing was consistent, dosing was determined by the individual physician and prescribed on a patient-specific basis. The primary endpoint was the number of episodes of nausea and emesis after chemotherapy, while the secondary endpoint was the number of rescue medications that were used for nausea and emesis. For the primary endpoint of the study, the mean number of nausea episodes per patient was reduced in the aprepitant group compared to the control group in the acute phase (1.7 versus 2.2, p=0.51) and delayed phase (5.3 versus 5.6, p=0.83). Emetic episodes per patient were decreased in the aprepitant group compared to the control group in both the acute (0.06 versus 0.9, p=0.21) and delayed phases (0.9 versus 2.4, p=0.17). Use of rescue medication per patient was decreased in the aprepitant group compared to the control group in the acute phase (2.3 versus 3.6, p=0.40) but not in the delayed phases (8.1 versus 8.1, p=0.99). None of the results of this study were statistically significant, although the reduced number of emetic episodes with aprepitant may be clinically significant. The results of this study warrant consideration for a prospective evaluation of this therapy in ASCT patients to greater clarify the use of aprepitant in these patients.

EBV-associated Hodgkin’s Disease (HD) and non-Hodgkins Lymphoma (NHL) show type II latency. They express the subdominant EBV antigens EBNA1, LMP1 and LMP2, which may serve as targets for immunotherapy approaches. In our previous studies, we used polyclonal EBV-specific CTL (EBV-CTL) in patients with relapsed EBV +ve HD and saw 2 complete and 1 partial response in 11 patients. Tetramer and functional analyses of EBV-CTL lines showed that small populations of T cells reactive against the tumor-associated antigen LMP2 were present in the majority of the infused lines, with some expansion in the peripheral blood following infusion. We therefore hypothesized that CTL specifically targeting type II viral latency antigens might have greater efficacy in these patients. LMP2-CTL could be generated from normal donors using dendritic cells (DC) genetically modified with a recombinant adenovirus encoding LMP2a (Ad5LMP2a) to direct the CTL response to LMP2. However, this approach required the generation of large numbers of DC to expand LMP2-CTL and was not practical in these heavily pretreated patients. We therefore modified the LMP2-CTL generation protocol to use DC for the initial stimulation, followed by stimulation with LCL that had been genetically modified to overexpress LMP2a by transduction with an Ad5f35LMP2a vector. This approach allowed us to specifically expand LMP2-CTL from patients to the numbers required for clinical use. We have generated LMP2 specific CTL lines in 10 patients with EBV+ve HD or NHL. LMP2 peptide tetramers were used for analysis of the polyclonal LMP2-CTL lines where HLA-restricted tetramers were available, and the lines recognized 2–6 (median 4) LMP2 epitopes, as determined by ELISPOT assay. A mean of 22.8% (5–42.1%) of CD8+ T cells bound these LMP2 tetramers, compared to a mean of 0.11% (0.01–0.24%) of LMP2-tetramer positive CD8+ T cells found in CTL generated with genetically unmodified LCL from the same patients. So far, 6 patients have been treated, initially receiving 2 doses of 2x107 CTL/m2 two weeks apart. All patients were off other therapies for at least 1 month prior to receiving CTL. No immediate toxicity was observed. In patients with identified LMP2-epitopes, measurement of IFNγ secretion by CD8+ T cells after stimulation with appropriate LMP2-peptides in ELISPOT assays showed a 4–25-fold increase in spot forming cells after infusions. In contrast, the frequency of CMV and superantigen-specific T cells did not increase. Four patients without radiological evidence of disease who received CTL as adjuvant therapy post stem cell transplant or chemotherapy remain well up to 9 months post CTL. Two patients with measurable disease at the time of CTL infusion had stable disease 8 weeks post CTL. They received 2 further doses of LMP2-CTL at 2x107/m2/dose. Re-evaluation was performed 8 weeks after the additional CTL infusions and one patient continues with stable disease. In the second patient, radiological review now revealed no evidence of disease, and a supraclavicular lymph node resection showed selective accumulation of LMP2-tetramer positive cells (0.3% compared to 0.01% in the peripheral blood) with scanty residual tumor cells. Immunotherapy with autologous LMP2-CTL is therefore well tolerated in patients with relapsed EBV+ve HD/NHL and infused LMP2-CTL cells can localize to the tumor and induce a clinical response.

Patients with preexisting coronary artery disease (CAD) have traditionally not undergone stem cell transplantation because of the perceived cardiac risk of the procedure. With the advent of reduced intensity stem cell transplantation and increased numbers of elderly patients, the question of whether or not it is safe to transplant patients with CAD is increasingly pertinent. We reviewed the outcomes of eight transplants performed in patients with clinically significant CAD. We also assessed the contribution of anthracycline/cyclophosphamide use and mediastinal radiation to cardiac toxicity. Clinically significant CAD was defined as that associated with myocardial infarction or that requiring percutaneous intervention/coronary artery bypass grafting (see chart for individual details). All patients were clinically stable at the time of transplant although two had experienced transient angina within three months. For all patients, baseline echocardiogram demonstrated ejection fractions greater than 50%, and baseline EKG demonstrated sinus rhythm although Q waves, T wave inversions, diffuse T wave abnormalities, and first-degree AV block were present. One fully ablative allogeneic, three subablative allogeneic, and four autologous transplants were performed. Conditioning regimens are presented below. All transplants used peripheral blood stem cells. Transplants were performed for multiple myeloma, ALL, AML, and CML. None of the patients were exposed to mediastinal radiation or cardiac toxic doses of anthracyclines or cyclophosphamide. Median survival after transplant was 496.9 days (range 58 – 1367 days). Two patients have died; one from disease progression and the other from pneumonia related complications. One patient was retransplanted on day 721. No ischemic events have been reported on follow up which has included myocardial perfusion scanning in three transplants. We conclude that cardiac risk is minimal and acceptable in patients with stable CAD who undergo transplant. Baseline Cardiac History and Transplantation Characteristics Cardiac History Transplant Conditioning Follow up 1v stent Autologous Melphalan 200 mg/m2 Retransplanted day 721, no ischemic events, normal myocardial perfusion scan 7v CABG Autologous Melphalan 200 mg/m2 Alive day 579, no ischemic events 3v CABG Autologous Melphalan 200 mg/m2 Alive day 1367, no ischemic events 1v stent Autologous Melphalan 200 mg/m2 Alive day 877, no ischemic events 1v stent Haploidentical 4/6 TBI 450 cGy (single dose), campath 40 mg, fludarabine 120 mg/m2 Alive day 58, no ischemic events, normal myocardial perfusion scan 1v stent Matched related Busulfan 6.4 mg/kg, fludarabine 120 mg/m2 Alive day 86, no ischemic events, normal myocardial perfusion scan 3v CABG, 1v angioplasty Matched related TBI 450 cGy (single dose), campath 30 mg, anti-CD45 antibody 1.6 mg/kg, fludarabine 120 mg/m2 Dead day 68 from disease progression Small vessel disease with myocardial infarction Mismatched related 5/6 TBI 800 cGy, cyclophosphamide 120 mg/kg Dead day 219 from pulmonary complications

6627 Background: Infection is a frequent cause of morbidity and mortality following allogeneic stem cell transplantation. It is still not clear whether RIC regimens have an impact on incidence and severity of infections. Methods: We analyzed infectious complications and outcome in 53 patients with median age of 55 years (range, 16–75 years) who underwent allogeneic stem cell transplantation with a RIC regimen consisting of Fludarabine, total body irradiation, antithymocyte globulin or Campath with or without CD45 monoclonal antibodies. Sixty eight percent (36/53) of patients received Campath based regimen. Donors were HLA identical (24 related and 17 unrelated) or one antigen mismatched (3 related, 9 unrelated). Infectious prophylaxis was Levofloxacin, fluconazole, Ganciclovir and Bactrim. Results: There were 122 episodes of fever or other clinical infections, 54 were before day +30, 46 between day +30 and day +100, and 22 after day +100. Bacteria were isolated in 42% (5 gram negative, 46 gram positive), ...