Combination chemotherapy with ABVD is widely used as first line therapy for adult classical Hodgkin lymphoma (cHL). However, for patients in the Adolescent and Younger Adult (AYA) age group, there is no consensus regarding preferred upfront therapy and diverse approaches have been used. Given this, we sought to assess the suitability of ABVD for AYA patients by undertaking a comparative analysis of outcomes for AYA patients and other adults treated at a single institution employing an ABVD-based approach.

Autologous PBSC transplantation using high dose melphalan conditioning remains the mainstay of consolidation at the post-induction stage in patients with myeloma. However, trial-based data has generally had an age cut-off of 65 years and not included patients with significant renal impairment. We demonstrate that with dose adjusted for age and/or renal function, patients can safely undergo autograft and achieve the same PFS.

In this multicentre retrospective study we have studied the impact of T cell chimerism on the outcome of 133 patients undergoing an alemtuzumab based reduced intensity conditioning allograft (RIC). The median age of the patients was 50 years (range 42–55 years). 77 patients were transplanted using an HLA identical sibling donor while 56 patients received a fully matched volunteer unrelated donor graft. 64 patients had a lymphoid malignancy and 69 were transplanted for a myeloid malignancy. 38 patients (29%) relapsed with no significant difference in risk of relapse between patients developing full donor and mixed donor chimerism in the T-cell compartment on D+90 and D+180 post transplant. Day 90 full donor T cell chimerism correlated with an increased incidence of acute GVHD according to NIH criteria (p=0.0004) and the subsequent development of chronic GVHD. Consistent with previous observations, our results confirmed a correlation between the establishment of T cell full donor chimerism and acute GVHD in T deplete RIC allografts. However our study failed to identify any correlation between T cell chimerism and relapse risk and challenge the use of pre-emptive donor lymphocyte infusions (DLI) in patients with mixed T cell chimerism transplanted using an alemtuzumab based RIC regimen.

Abstract Abstract 3088 Background: High dose chemotherapy followed by autologous stem cell rescue is superior to salvage chemotherapy alone in relapsed Hodgkin's Lymphoma (HL) and Non-Hodgkin's lymphoma (NHL). A commonly used regimen is BEAM (BCNU, Etoposide, Cytarabine and Melphalan). Unfortunately, BCNU is associated with potentially lethal pulmonary interstitial toxicity, in a dose dependent manner. Therefore, it is often omitted in conditioning regimens for patients who have pre-existing impaired lung function. We aimed to determine the outcomes of patients given this EAM (Etoposide, Cytarabine, Melphalan) schedule in our centre. Method: At our unit, from 2000–2010, 338 patients had been transplanted with BEAM conditioning and 23 patients with an EAM regimen. BEAM chemotherapy involved cumulative doses of BCNU at 300 mg/m2, etoposide at 800 mg/m2, cytarabine at 1600 mg/m2 and melphalan at 140 mg/m2. The EAM regimen was the same, except for the omission of BCNU. We wanted to compare overall survival, event free survival and transplant related mortality between the two groups of patients. A matched-pair analysis based on age and sex on a 1:1 basis was conducted. Survival rates were estimated by the Kaplan-Meier method. Differences between the survival distributions were compared with the log-rank test. Results: 23 patients transplanted with an EAM regimen was matched with an equal number transplanted with a BEAM conditioning regimen. The median age was 53 in both groups. The median follow-up for patients alive was 3.2 years. Further baseline characteristics of the two groups are described in table 1. Other than a slight predominance of more advanced disease in the BEAM cohort, the two groups were evenly matched. Survival estimates were statistically significantly poorer for the EAM group compared to the BEAM group. The median overall survival for the EAM cohort was 29 months compared to 77 months (p=0.03) for the matched BEAM cohort. The median event free survival for the EAM cohort was 11 months compared to 63 months (p=0.02) for the BEAM cohort. The 100 day transplant related mortality was 8.7% for both cohorts. During the follow-up period 11 patients died of disease related causes in the EAM group compared to 6 in the BEAM group. Conclusion: Despite the prevalence of pre-existing lung disease in the EAM group of patients, the conditioning was reasonably well tolerated. Our analysis suggests that patient transplanted with an EAM regimen had an inferior survival outcome to patients treated with the standard BEAM regimen. This data provides evidence for the importance of BCNU in lymphoma control; consideration should be given to a reduction in BCNU dosage, rather than simple omission, for patients with borderline lung function results. Collaboration with other centres is warranted to determine if this experience with EAM is replicated elsewhere. Disclosures: No relevant conflicts of interest to declare.

Background Allogeneic stem cell transplantation is associated with a powerful ‘graft-versus-leukemia’ effect that is generally considered to result from an alloreactive T-cell immune response. However, disease remission can also be observed after syngeneic transplantation and we investigated whether a T-cell immune response to cancer-testis antigens can be detected in patients in the post-transplant period.Design and Methods The T-cell immune response against cancer-testis antigens was studied in a cohort of 41 patients who underwent allogeneic stem cell transplantation for the management of acute myeloid leukemia or multiple myeloma. The cytokine secretion assay was combined with magnetic selection to allow detection of an interferon-γ-secreting T-cell response to a panel of cancer-testis antigen peptides.Results A cancer-testis antigen-specific CD8+ T-cell immune response was observed in the peripheral blood of five patients with an average magnitude of 0.045% of the CD8+ T-cell repertoire. Four of these patients had undergone reduced intensity conditioning transplantation with alemtuzumab for the treatment of acute myeloid leukemia and three remain in long-term remission. T-cell immunity was focused against peptides derived from MAGE proteins and was markedly increased within the bone marrow.Conclusions Functional cancer-testis antigen-specific CD8+ T-cell immune responses develop in the early period following reduced intensity allogeneic stem cell transplantation and are preferentially localized to bone marrow. These immune responses are likely to contribute to the cellular basis of the graft-versus-leukemia effect.

Abstract Abstract 2361 Introduction: Reduced intensity allogeneic transplants represent a potentially curative therapy in older patients with haematological malignancies. However the upper age limit for transplantation using a sibling or unrelated donor is unclear and few studies have addressed this important issue. In the UK in vivo T cell depletion utilising alemtuzumab is commonly used as a strategy to reduce the risk of acute and chronic graft-versus-host disease (GVHD) but this manoeuvre impairs immune reconstitution and may pose a particular problem in older patients. Aim: We analysed the outcome of patients over the age of 60 after an alemtuzumab based reduced intensity allograft from five UK Transplant Centres with the aim of identifying factors determining long term outcome and also factors affecting the duration of inpatient admission during the first 100 days post transplant. A modified Charlson's comorbidity index score (Sorror modified HCT comorbidity index) was applied to analyse the transplant outcomes of these patients according to the presence of transplant co-morbidities. Patients and Methods: We have studied the outcome of 161 patients (89 male, 72 female) after an alemtuzumab conditioned reduced allograft allograft for a haematological malignancy. The median age of the patients was 62 years(range 60–72). 115 patients had a transplant for myeloid malignancies (87 acute myeloid leukaemia, 21 myelodysplastic syndrome, 2 chronic myeloid leukaemia, 5 myelofibrosis) and 46 for lymphoid malignancies (18 Non_Hodgkin's lymphoma, 14 chronic lymphocytic leukaemia, 4 T-prolymphocytic leukaemia, 7 multiple myeloma and 3 acute lymphoblastic leukaemia). 93 patients received an unrelated donor transplant and 68 had a sibling transplant. The great majority of patients were transplanted using a Fludarabine/Melphalan conditioning regimen(n=118) whilst 13 received a combination of fludarabine and busulphan, 21 BEAM (BCNU, cytarabine, etoposide, melphalan) and 9 a combination of BEAM and fludarabine. The median follow up was 19.1 months (range 2–94 months). Results: The one year overall survival for the whole group was 52% and the predicted two year OS 46%. The transplant related mortality (TRM) was 19% in the first 100 days post transplant and 23% in the first year post transplant. 40 patients (25%) relapsed. 25 patients (21%) developed Grade III-IV acute GvHD and 16 (10%) patients developed chronic extensive GvHD. There was a weakly negative correlation between Age and Bed Days(p:0.05843) but the correlation between high comorbidity index (3 or greater than 3) and increased bed days is significant(P:0.0013). Transplant outcomes were affected by Sorror modified comorbidity index for HCT.A score of 3 and above was significantly associated with decreased overall survival (P:0.001) and interestingly with decreased disease free survival (P:0.02). CMV status and stem cell dose did not have any impact on overall survival and disease free survival and CR1 at transplantation was showed a trend towards increased overall survival(P:0.05). Conclusions: Reduced intensity alemtuzumab based stem cell transplant can be delivered safely in patients above the age of 60. It appears that a Sorror comorbidity score of 3 and above has a negative impact on overall survival and disease free survival of these patients and significantly increases inpatient days.These observations require confirmation in a larger cohort of patients but suggest that the selection of patients above the age of 60 for a reduced intensity transplants will be facilitated by incorporation of the Sorror HCI comorbidity index into a transplant algorithm. Disclosures: No relevant conflicts of interest to declare.

The chemotherapy dose‐intensity in two adapted German BFM paediatric protocols (BFM 90 and NHL 86) was compared in contemporaneously treated adults <50 years with Burkitt lymphoma and B‐cell lymphoma unclassifiable, with features intermediate between diffuse large B‐cell lymphoma and Burkitt lymphoma (collectively referred to as BL). In BFM 90, primary prophylaxis with Granulocyte‐colony‐stimulating factor was used, postinduction treatment was started at granulocytes ≥0.5 × 10 9 /L (≥1.0 × 10 9 /L in NHL 86) with a higher mean methotrexate dose (2.9 g/m 2 /cycle, n = 23; 1.6 g/m 2 /cycle in NHL 86, n = 22, P < 0.001). Intervals between consecutive treatment‐cycles were shorter in BFM 90 ( P < 0.001) with no additional toxicity. However, the two‐year failure‐free survival with BFM 90 (82%) was similar to that achieved with NHL 86 (72%, P = 0.33). We conclude that BFM 90 enables safe intensification of therapy in young adults with BL compared to NHL 86, but registry‐based studies are required to further evaluate the antineoplastic effects and cost‐effectiveness of the two therapeutic approaches. Am. J. Hematol., 2010. © 2010 Wiley‐Liss, Inc.

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1HversusHA-1R) in theHMHA1gene. The HA-1Hpeptide is restricted by HLA-A2 and is immunogenic inHA-1R/RintoHA-1Htransplants, whileHA-1Rhas been suggested to be a “null allele” in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1Rvariant. To understand these findings, we determined the structure of an HLA-A2-HA-1Hcomplex to 1.3Å resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1H-specific T-cells bound HA-1Hpeptide with moderate affinity but failed to bind HA-1R, indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

The use of reduced intensity conditioning transplants gained momentum over the last 10 years enabling older patients with haematological malignancies to go through a potentially curative treatment option. This is based on the premise of graft versus leukaemia effect with low toxicity of conditioning therapy as older patients have other co morbidities. Full donor chimerism is the optimal desired result of transplantation. We have analysed here whether pretransplant characteristics could predict full donor chimerism on whole blood and T-cell fraction on day +90 and +180 and also the effect of the chimeric status on the transplant outcomes. This study included 70 patients with lymphoid (n=43) and myeloid (n=27) malignancies with a median age of 47 years (9–63). Median follow up was 22.3 months (range 2.2 – 49.6 months). Pretransplant baseline characteristics included age, sex, donor type, previous stem cell transplant, CD34 cell dose, CD3 cell dose, disease status at the time of transplant, alemtuzumab dose (50 vs. 100mg) and conditioning regimen. Multivariate logistic regression showed CD34 cell count was significant in predicting full donor T cell chimerism at 90 days (p=0.05). High CD3 cell dose (median 20.5 vs. 2.4; p=0.01) predicted full donor whole blood chimerism at 90 days. High CD34 cell count (median 6.0 vs. 5.0, p=0.02) and type of donor (Unrelated vs. sibling: 45% vs. 13%; p=0.003) were predictors for full donor chimerism at 180 days. Full donor T cell chimerism at 180 days was predicted by high CD34 cell count (median 6.0 v. 5.0, p=0.02) and type of donor (Unrelated vs. sibling: p=0.006). Donor type (unrelated vs. sibling; 68% vs. 33%: p=0.01), high CD3 cell dose (median 130 vs. 2, p=0.0001) and dose of alemtuzumab (high vs. low; 74% vs. 33%, p=0.006) were predictors for full whole blood chimerism at 180 days. Full donor and mixed T cell chimerism at 90 days did not impact on overall survival (OS), disease free survival (DFS), relapse free survival (RFS) and chronic graft versus host disease (GvHD). Full as compared to mixed donor chimerism at 90 days (74% vs.27%, p=0.001) was associated with acute GvHD. T cell mixed chimerism as compared to full at 180 days was a predictor for relapse (HR=7.6, p=0.02). Full donor chimerism at 180 days was a predictor for both acute (OR=3.3, p=0.04) and chronic GvHD (OR=4.6, p=0.02). Whole blood chimerism at 180 days did not impact OS, DFS, RFS and acute or chronic GvHD, but it appeared to show a trend for increased relapse (HR=2.52, p=0.05). Our study revealed CD3 & CD34 cell dose along with unrelated donor type were predictors for whole blood & T cell chimerism at days +90 and +180 in reduced intensity alemtuzumab conditioning transplantation. Our study also showed donor chimerism may be a predictor for GvHD. More studies are warranted to understand the predictors for donor chimerism. This will enable to establish optimal transplant strategies.

ATL-104 is a potent mitogen for epithelial cells of the gastrointestinal tract. In animal models, ATL-104 aids regeneration of the gastrointestinal tract after treatment with chemotherapeutic agents. The effect of ATL-104 on mucositis in patients requiring high-dose melphalan or BEAM before peripheral blood SCT (PBSCT) was investigated in a randomized, placebo-controlled, double-blind, two-part study. Patients were randomized to ATL-104 (50, 100 or 150 mg) or placebo once daily for 3 days before chemotherapy and 3 days after PBSCT. Part one of the study was a dose-escalation design; part two was a parallel group design using all three ATL-104 doses. Patients were followed up for 28 days post-treatment. Severity of signs and symptoms were assessed and used to calculate scores for standard toxicity rating scales (WHO, Western Consortium for Cancer Nursing Research (WCCNR)). Sixty-three patients were treated. Treatment with ATL-104 substantially reduced the median duration of severe oral mucositis (WHO grade 3 or 4) compared with placebo (median duration: ATL-104 groups 2 or 3 days, placebo 10.5 days). The effect of ATL-104 on the incidence of severe oral mucositis was inconclusive. Similar results were obtained using the WCCNR Scale. Adverse events (AEs) were predominantly mild or moderate in intensity. Gastrointestinal AE were most common.

Reactivation of CMV is a common complication following allogeneic haematopoietic SCT and is associated with significant morbidity and mortality. The relative importance of the CD4+ and CD8+ components of the CMV-specific immune response in protection from reactivation is unclear. The CMV-specific CD4+ and CD8+ immune response was measured at serial time points in 32 patients following allogeneic HSCT. Intracellular cytokine staining following CMV lysate stimulation and HLA-peptide tetramers were used to determine CMV-specific CD4+ and CD8+ responses, respectively. A deficient CMV-specific CD4+ T-cell immune response within the first 30–50 days post transplant was associated with high risk of viral reactivation. Patients with combined impairment of the CD4+ and CD8+ immune response within the first 100 days were susceptible to late viral reactivation. The frequency of CMV-specific CD4+ T cells correlated with CMV-specific CD8+ T cells, comprising 10% of the whole T-cell repertoire. Early CMV-specific CD4+ T-cell reconstitution was dominated by effector memory cells with normal levels of IL-2 resuming 6 months following transplantation. In summary, both CD4 and CD8 CMV-specific immune reconstitution is required for protection from recurrent activation. Measurement of the magnitude of the CMV-specific CD4+ immune response is useful in managing viral reactivation following HSCT.

Acute graft-versus-host disease (aGVHD) remains a serious complication following allogeneic stem-cell transplantation (SCT), and is mediated by infiltration of alloreactive donor T cells into recipient tissue. Chemokines and their receptors play a central role in controlling the recruitment of T cells into discrete tissue sites, and determine the clinical features of GVHD in murine models. In this study, we have analyzed the serum concentration of molecules that control leukocyte migration in serial samples from 34 patients following allogeneic SCT. The chemokine CXCL10 (IP-10) was significantly elevated (> 2-fold) in serum at the time of aGVHD. Because the ligand for CXCL10 is CXCR3, the number of CXCR3(+) T cells was determined in peripheral blood, but was not increased during episodes of GVHD. To investigate the role of chemokines in the recruitment of T cells to the anatomic site of GVHD, skin biopsies were stained for CXCL10 and CXCR3 expression. CXCL10 expression was observed in the basal keratinocytes of the epidermis in patients with GVHD together with positive staining for CXCR3 on cells in dermal infiltrates. These findings indicate that CXCL10 plays a central role in the pathogenesis of skin aGVHD by the recruitment of CXCR3(+) T cells to the sites of inflammation.

Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) is an uncommon but potentially fatal complication of allogeneic haematopoietic stem cell transplantation (SCT), usually presenting within 6 months post-transplant. Diagnosis of LPD may be difficult as the clinical picture can mimic sepsis or severe GvHD without obvious lymphadenopathy. High blood EBV load is a useful diagnostic feature but in isolation has limited positive predictive value for LPD. We report 2 patients in whom expansion of CD38+ activated B cells in peripheral blood was an important diagnostic feature of EBV-associated LPD developing early after T- deplete myeloablative allogeneic SCT. The first patient, a 39 year old female with relapsed acute lymphoblastic leukaemia, presented 4 months after an unrelated donor SCT with a febrile illness in association with EBV and adenovirus reactivation (5.7 x 105 and 2.6 x 106 copies/ml of blood respectively). There was no evidence of lymphadenopathy either clinically or on imaging. Following Cidofovir treatment there was a log reduction in the adenovirus load but EBV load increased coupled with a rising peripheral blood lymphocyte (PBL) count from 0.2 x 109/l to 3.2 x 109/l. The patient died soon after from multi-organ failure. The second patient, a 19 year old female with acute myeloid leukaemia, presented 2 months after a haplo-identical SCT with a similar clinical picture and EBV load of 2 x 107 copies. Her PBL count increased rapidly from 0.5 x 109/l to 11.3 x 109/l. Cell marker studies in both patients revealed B-lymphoproliferation with CD19+ B cells constituting 80% of PBL with a profound reduction in NK and T cell numbers. The B cells displayed an abnormal activated phenotype with strong CD38 expression and were CD20+, CD22+, CD79b+, CD5neg, CD10neg, and CD103neg. Concomitant CD23 expression was noted in a proportion of B cells and a minority expressed CD34. Clonal IgH gene rearrangement was noted in the second patient; furthermore, elispot assays demonstrated a complete absence of EBV-specific CD8 T cell immunity. This patient received 2 doses of Rituximab with a log reduction in EBV load, but succumbed to overwhelming sepsis-like syndrome. In both patients, a rising PBL count due to expansion of activated CD38+ B cells was an important manifestation of EBV-associated LPD in the absence of obvious lymphadenopathy or organomegaly. This response pattern is in sharp contrast to that seen in immunocompetent patients with infectious mononucleosis (IM) where lymphocytosis is due to proliferation of CD8+ T rather than B cells. Interestingly, our recent work on tonsils from IM patients has identified CD38 expression as a useful marker for B cells activated by EBV infection and it is likely that the abnormal B cells noted in the above two patients represent the same. Our experience emphasises the usefulness of peripheral blood cell marker studies in evaluating patients with suspected early onset PTLD following SCT. An expansion of CD38+ activated B cells and absence of T cell response can be useful diagnostic features which can aid in making treatment decisions in this difficult situation.