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Due to the drastic and exponential growth of information systems and their use, the technology has taken a quantum leap. To ensure safe data transportation, different protection systems are used, such as intrusion detection systems, intrusion prevention systems, and firewalls. In this chapter, the proposed system has an anomaly-based cyber threats detection system using advanced machine learning algorithms. The anomaly-based detection system was used to analyze repackage signatures of malware which is not predefined. Machine learning with the use of previous datasets and algorithms to make the IDS intelligent. In this chapter, the authors use K-NN, which takes the similarity between new attack footprints and compare it with the older footprints in the dataset, and tell which has a higher resemblance. The major challenges of this IDS are minimization of false alarms and gaining high accuracy. The proposed IDS system is not only tested manually but is tested by automated utilities as well, and minimizing of overfitting and underfitting is also checked.

The expression of cancer germline antigens (CGAgs) is normally restricted to the testis but is also present in many types of malignant cells including plasma cells from patients with myeloma. Because T-cell immune responses to CGAg have been identified in patients with solid tumors, this may offer a novel target for immunotherapy in patients with myeloma. We have used 12 peptide epitopes from a range of CGAgs to screen for CGAg-specific T cells in blood from patients with multiple myeloma at various stages of their disease. T cells from 15 of 37 patients responded to one or more CGAg peptides and the magnitude of the CGAg-specific CD8+ T-cell response ranged between 0.0004% and 0.1% of the total CD8+ T-cell pool. Serial analyses showed that these immune responses were detectable in individual patients at multiple time points during the course of their disease. In patients undergoing treatment or in disease relapse, the magnitude of the CGAg-specific T-cell response was positively correlated with the level of paraprotein. Functional T cells specific for CGAgs are therefore present in a proportion of patients with multiple myeloma and offer the possibility of a novel approach for immunotherapy in this disease.

We report the outcomes after reduced-intensity conditioning allogeneic stem cell transplantation (RIT) for non-Hodgkin lymphoma (NHL) in 88 patients (low-grade NHL [LG-NHL], n = 41; high-grade NHL [HG-NHL], n = 37; mantle cell lymphoma [MCL], n = 10). Thirty-seven patients had previously received autografts, and 21 were in complete remission (CR) at transplantation. Conditioning therapy consisted of alemtuzumab, fludarabine, and melphalan. Sixty-five patients received peripheral blood stem cells (PBSCs) from HLA-identical siblings, and 23 received bone marrow (BM) from matched unrelated donors. Prophylaxis for graft-versus-host disease (GVHD) consisted of cyclosporin A. Grade III-IV acute GVHD developed in 4 patients, and chronic GVHD developed in 6 patients. With a median follow-up of 36 months (range, 18-60 months), the actuarial overall survival (OS) rates at 3 years were 34% for HG-NHL, 60% for MCL, and 73% for LG-NHL (P < .001). The 100-day and 3-year transplant-related mortality (TRM) rates for patients with LG-NHL were 2% and 11%, respectively, and were better (P = .01) than they were for patients with HG-NHL (27% and 38%, respectively). The actuarial current progression-free survival (PFS) rate at 3 years, including the rate for patients who achieved remission after donor lymphocyte infusion (DLI) for progression, was 65% for LG-NHL, 50% for MCL, and 34% for HG-NHL (P = .002). Twenty-one patients underwent DLI for matched related donor (MD)-persistent disease or relapse, and 15 underwent DLI for mixed hematopoietic chimerism. Patients who experienced relapses of LG-NHL and chronic lymphocytic leukemia (CLL) achieved excellent PFS with extremely low TRM and GVHD, even when matched related donors were unavailable. (Blood. 2004;104:3865-3871)

We report the outcomes of reduced-intensity allogeneic stem cell transplantation using BEAM-alemtuzumab conditioning (carmustine, etoposide, cytosine arabinoside, melphalan, and alemtuzumab 10 mg/d on days –5 to –1) in 6 United Kingdom transplant centers. Sixty-five patients with lymphoproliferative diseases underwent sibling (n = 57) or matched unrelated donor (n = 8) transplantation. Sustained donor engraftment occurred in 60 (97%) of 62 patients. Of the 56 patients undergoing chimerism studies, 35 (63%) had full donor chimerism. Overall, 73% were in complete remission (CR) after transplantation. At a median follow-up of 1.4 years (range, 0.1-5.6 years), 37 remain alive and in CR. Acute graft-versus-host disease (GVHD) occurred in 11 (17%) of 64, grades I-II only. Estimated 1-year transplantation-related mortality (TRM) was 8% for patients undergoing first transplantation but was significantly worse for those who had previously undergone autologous transplantation. Six patients relapsed (estimated 2-year relapse risk, 20%). Histologic diagnosis (mantle cell lymphoma and high-grade non-Hodgkin lymphoma) and age at transplantation (> 46 years) were significantly associated with higher relapse risk and worse event-free survival. Relapse did not occur in any patient who developed acute or chronic GVHD. This study demonstrates that reduced-intensity allogeneic stem cell transplantation for lymphoproliferative diseases using a BEAM-alemtuzumab preparative regimen is associated with sustained donor engraftment, a high response rate, minimal toxicity, and a low incidence of GVHD.

Melphalan is widely used as a preparative agent in patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (SCT). Although disease relapse is the major cause of death after a melphalan-conditioned autograft, the mechanism remains unclear. Melphalan produces a number of DNA adducts with the DNA interstrand crosslink (ICL) considered to be the critical cytotoxic lesion. By using a modification of the single-cell gel electrophoresis (Comet) assay, we have measured formation and repair of DNA ICL in plasma cells from melphalan- naive and melphalan-treated patients (ie, those who have relapsed after a melphalan-conditioned autologous SCT or oral melphalan therapy). Similar levels of dose-dependent DNA interstand crosslinking were observed in cells from both melphalan-naive and -treated patients. However, marked differences in ICL repair were observed: cells from naive patients showed no repair, whereas those from treated patients exhibited between 42% and 100% repair at 40 hours. In vitro sensitivity to melphalan in plasma cells was found to correlate with ICL repair. These findings suggest that ICL repair may be an important mechanism by which melphalan resistance emerges after autologous SCT or oral therapy. This mechanism may have implications for MM patients undergoing melphalan therapy.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8+ T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4+ cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4+ cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4+ T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3+ T cells in cGVHD.

A joint working group established by the Haemato-oncology subgroup of the British Committee for Standards in Haematology and the British Society for Bone Marrow Transplantation has reviewed the available literature and made recommendations for the supportive care and management of organ-specific complications of chronic graft-versus-host disease (cGvHD). This guideline includes recommendations for the specific therapy of skin, oral, liver, gut, lung, ocular and genital manifestations of cGvHD and for the supportive care of these patients, including vaccinations and prophylaxis against infection. The goal of treatment should be effective control of GvHD while minimizing the risk of toxicity and relapse.

A joint working group established by the Haemato-oncology subgroup of the British Committee for Standards in Haematology (BCSH) and the British Society for Bone Marrow Transplantation (BSBMT) has reviewed the available literature and made recommendations for the diagnosis and management of chronic graft-versus-host disease (GvHD). This guideline includes recommendations for the diagnosis and staging of chronic GvHD as well as primary treatment and options for patients with steroid-refractory disease. The goal of treatment should be the effective control of GvHD while minimizing the risk of toxicity and relapse.

A joint working group established by the Haemato-oncology subgroup of the British Committee for Standards in Haematology (BCSH) and the British Society for Bone Marrow Transplantation (BSBMT) has reviewed the available literature and made recommendations for the diagnosis and management of acute graft-versus-host disease. This guideline includes recommendations for the diagnosis and grading of acute graft-versus-host disease as well as primary treatment and options for patients with steroid-refractory disease. The goal of treatment should be effective control of graft-versus-host disease while minimizing risk of toxicity and relapse.

Herpes zoster infections are frequently observed after allogeneic bone marrow transplantation (alloBMT). In the majority of cases, the infection is restricted to specific dermatomes and responds to oral acyclovir, without visceral dissemination. We report the case of a 40-year-old male who developed dermatomal herpetic infection 8 months post alloBMT. The herpetic rash responded well to treatment with high-dose oral acyclovir. However, within a week of cessation of therapy, the patient re-presented with dermatomal zoster and meningoencephalitis. Although the cutaneous lesions resolved with intravenous acyclovir, clinical features of meningoencephalitis persisted, along with evidence of varicella zoster virus (VZV) DNA in cerebrospinal fluid (CSF). A satisfactory response to treatment was observed only after the addition of intravenous foscarnet to acyclovir. Based on our experience with this patient, we suggest that in a subset of alloBMT recipients, late dermatomal herpes zoster infections may respond only partially to treatment with standard oral acyclovir. The use of oral acyclovir preparations with higher bioavailability (valacyclovir) or intravenous acyclovir early on may prevent the considerable morbidity associated with disseminated zoster infection. Bone Marrow Transplantation (2000) 26, 795-796.

Introduction Up-front autologous stem cell transplantation (ASCT-1) post front line therapy with novel agents is standard of care in newly diagnosed multiple myeloma. The role for salvage transplantation (ASCT-2) in relapsed patients after retreatment remains unclear in the era of the novel agents. Majority of published studies include patients treated in pre-thalidomide era. Our retrospective study investigates the safety and efficacy of ASCT-2 in patients exclusively treated with novel drugs both at upfront and at relapse. Primary end point was non relapse mortality (NRM) at day 100. Secondary end points were progression free survival from ASCT-2 (PFS-2) and overall survival (OS) Patients and Methods Thirty-nine patients (21 female and 18 male) underwent ASCT-2 at 4 centres between 2008 and 2013. At initial presentation all received thalidomide based treatments pre ASCT-1. Therapy at progression was bortezomib based in 92% and thalidomide in 8%. Melphalan 200 mg/m2 was used as conditioning for 90% of patients, 140 mg/m2 in 10%. OS and PFS-2 were calculated from ASCT-2. Statistical analysis was carried out using IBM SPSS 19 for Windows. Results Median progression free survival (PFS-1) post ASCT-1 was 35 (10-90) months with 4 patients receiving thalidomide maintenance. Median age at ASCT-2 was 60 (37-68) years with a median stem cell dose of 2.7×106 (2-7) CD34 cells /kg body weight. All patients engrafted with median times to neutrophil (>0.5) and platelet (>20) engraftment of 12 days each with a day+100 and 1 year NRM of 0%. With a median follow up from ASCT-2 of 18 (3-52) months, the median PFS-2 was 18 (12-24) months and OS was 42 (33-50) months. PFS-1 of greater than 18 months was associated with prolonged PFS-2 (19 vs. 6 months, p=0.001, log rank), however there was no statistical difference observed for PFS-1 beyond 24 months. Similarly PFS-1 >18 months predicted for improved OS (39 vs. 14 months, p=0.007, log rank). Age at ASCT-2(>or 60 and with PFS-1 of >24 months had a median PFS-2 of 25months as compared to 14 months in patient age 24 months. Conclusion In the era of novel agents ASCT-2 can be safely delivered with 0% 1 year NRM. PFS-1 greater than 18 months gives better PFS-2 and OS suggesting a definite role of this therapy in a selected population Age greater than 60 years does not have adverse impact on either PFS or OS. Thus ASCT-2 should be considered in treatment strategies at disease progression and warrants further prospective studies Disclosures: No relevant conflicts of interest to declare.

Background Treatment of transformed follicular lymphoma (TFL) remains undefined and there is no consensus on the role of consolidation after induction chemotherapy. Outcomes for TFL in the pre-rituximab era were poor with a reported median overall survival (OS) of only 1.7 years1. This study investigates whether R-CHOP is an effective therapy for patients with TFL and which patient factors present at transformation help to predict outcome. Methods 1900 lymphoma diagnoses from January 2000 - June 2012 were retrospectively screened and identified 60 patients with transformed indolent lymphoma. 40 eligible patients with histologically confirmed TFL were identified for this study. Overall survival (OS) and progression-free survival (PFS) were the primary study end-points. Results The median follow up time for the cohort was 66 months (interquartile range (IQR) - 25-92 months) and the median age at diagnosis of TFL was 60 years (52-68 years). The majority (60%) of the cohort had high-grade (HG) transformation subsequent to diagnosis of follicular lymphoma (asynchronous TFL) with a median time to transformation of 48 months (24-78 mo). 32.5% of patients were simultaneously diagnosed with follicular lymphoma (FL) and HG disease (synchronous TFL). In a minority (3%), FL was diagnosed after HG disease. Univariate analysis revealed no significant relationship between outcome and patient age, sex or disease stage. However, patients with an elevated LDH (defined as >1.5x the upper limit of normal) at the time of diagnosis had a significantly reduced OS (5 mo vs 86 mo; p=0.003) and PFS (2.25 mo vs 56 mo; p=0.02). When compared with synchronous TFL, patients with asynchronous TFL had a reduced median survival (9 mo vs 67 mo; p=0.03) and PFS (7 mo vs 67 mo; p=0.003). When multivariate analysis is performed, elevated LDH and asynchronous TFL remain significant factors in determining PFS (p=0.021 and p=0.008 respectively). However, only elevated LDH remains a significant factor in determining OS (p=0.002). 25/40 patients were treated with R-CHOP for TFL, with the remaining 15 either treated palliatively: steroids and etoposide (n=8), radiotherapy (n=1); with CHOP (n=4) or another R-chemo (n=2; R-ESCHAP; R-CVP). All R-CHOP treated patients were rituximab naïve. Of the 25 patients treated with R-CHOP: 20 were treated with R-CHOP alone, 2 received R-CHOP and another chemotherapy, 1 had R-CHOP and autograft and 2 received R-CHOP and allograft. In our cohort, R-CHOP treated patients had a 5-year OS of 62%, a median OS of 86 months (31-120 months) and PFS of 56 months (14-86 months). This represents a marked improvement in outcome compared to historic data, which showed a 5-year OS of 33% for TFL patients treated with CHOP alone2. The patients treated with R-CHOP alone (without further chemotherapy, autograft or allograft) had a similar outcome with a median OS of 86 months (37-120 months) and median PFS of 56 months (11-86 months). Conclusion Our results demonstrate the excellent outcomes achieved with R-CHOP alone in rituximab naïve patients with transformed follicular lymphoma. Patients with TFL presenting with synchronous disease and non-elevated LDH have significantly improved outcomes. In such patients, we may consider reserving consolidation with autograft or allograft for the future. 1. Al-Tourah, A. J. et al. Population-based analysis of incidence and outcome of transformed non-Hodgkin’s lymphoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology26,5165–9 (2008). 2. Al-Tourah, A. J. et al. Addition of Rituximab to CHOP Chemotherapy Significantly Improves Survival of Patients with Transformed Lymphoma. Blood (ASH Annual Meeting Abstracts)110, (2007). Disclosures: Chaganti: Roche: Membership on an entity’s Board of Directors or advisory committees, Receipt of travel grant Other.

Abstract 460 Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic stem cell transplantation (alloSCT). Monitoring of EBV genomes in blood using quantitative PCR (EBV qPCR) coupled with pre-emptive administration of Rituximab in response to high-level EBV reactivation has emerged as a strategy to reduce mortality from PTLD. However, the effect of pre-transplant Rituximab therapy on the risk of EBV reactivation and survival post-alloSCT is unknown. This retrospective study examined 193 consecutive adult patients undergoing T cell depleted or cord blood alloSCT at University Hospital Birmingham, UK (UHB) and Nottingham University Hospital, UK (NUH) between May 2009 and April 2011. Median age at transplant was 54 years (range 16–73 years). Conditioning was reduced intensity in 84% and myeloablative in 16%. Stem cell source was matched unrelated donor in 70%, sibling in 24% and cord blood in 6%. T cell depletion was with in vivo Alemtuzumab in 89% and ATG in 6%. Patients were monitored by EBV qPCR whole blood assay, performed every 1–2 weeks post-transplant. EBV reactivation was defined as a single positive EBV qPCR result, whilst high-level EBV reactivation was defined according to institutional thresholds; 30,000 and 10,000 EBV genomes/ml for UHB and NUH respectively. All patients with high-level reactivation were pre-emptively treated with Rituximab. Median follow-up was 23 months (interquartile range [IQR] 18–30 months), with EBV qPCR testing for a median of 8 months (IQR 4–13 months) post-transplant. The cumulative incidence of EBV reactivation, adjusting for the competing risk of death, was 41% at 2 years post-transplant. Amongst those reactivating, the median time to EBV qPCR positivity was 120 days (IQR 77–198 days). High-level EBV reactivation was observed in 34/193 (18%) patients, accompanied by PTLD in 10 patients (4 biopsy-proven and 6 probable cases). Of patients developing high-level EBV reactivation, in 30/34 (88%) the interval from first EBV qPCR positivity to high-level reactivation was less than 4 weeks, with 15/34 (44%) exhibiting high-level reactivation at first qPCR positivity. In univariate analysis, significant predictors for EBV reactivation were older age (hazard ratio [HR] 1.02 per year; p=0.04), male sex (HR 1.75; p=0.03) and T depletion with ATG (HR 4.8; p Twenty-nine patients received Rituximab in the year preceding alloSCT, of whom 25 had NHL, 3 had chronic lymphocytic leukaemia (CLL) and 1 had acute lymphocytic leukaemia. Only one of these patients developed EBV reactivation by 12 months post-transplant - a patient with high-level reactivation associated with PTLD. Two other patients developed low-level EBV reactivation beyond 12 months. In univariate analysis, pre-transplant Rituximab was highly predictive for (lack of) EBV reactivation (HR 0.15, 95% confidence interval [CI] 0.05–0.48; p=0.0002; see figure). Applying a multivariate model including age, sex and ATG use, pre-transplant Rituximab remained highly predictive (HR 0.15, CI 0.05–0.47; p=0.0012). As expected, strong confounding between NHL and pre-transplant Rituximab made a model including both uninformative. There was no significant association between pre-transplant Rituximab and the risk of relapse, acute GvHD or CMV reactivation. Overall mortality was 50% at 2 years, with 4 deaths due to PTLD. There was no evidence of a link between EBV reactivation and survival (p=0.33). Pre-transplant Rituximab was associated with a significantly reduced risk of mortality (HR 0.49, CI 0.23–1.00; p=0.05) although the aforementioned confounding with NHL should be noted. We report the novel and clinically important finding that pre-transplant Rituximab is associated with a markedly reduced risk of EBV reactivation and a possible survival benefit after alloSCT. Our data make a strong case for prospectively evaluating the role of Rituximab in allograft conditioning. Disclosures: Off Label Use: Rituximab for prevention of Epstein-Barr virus reactivation after allogeneic stem cell transplantation. Fox:Roche: Honoraria.

The ability of reduced intensity conditioned (RIC) allografts to deliver long term disease free survival is dependent on the genesis of an immunologically mediated graftversus tumor (GVT) effect. Post-transplant immunosuppression plays an important role in limiting graft-versus-host disease (GVHD) but also modulates a GVT effect. Although cyclosporine A (CsA) is the most commonly utilised immunosuppressive agent in patients allografted using a RIC regimen the impact of CsA dose intensity on disease relapse and overall survival (OS) has not been studied. We have therefore measured CsA exposure in the first 21 days post-transplant and correlated individualised CsA area under the curve (AUC) values with OS and disease relapse in patients undergoing a RIC allograft. 132 patients with a diagnosis of acute myeloid leukemia (AML) (n=41), myelodysplasia (MDS) (n=17), Non-Hodgkin’s lymphoma (NHL)(n=51) or Hodgkin’s disease (n=23) were transplanted using an alemtuzumab containing RIC regimen. All patients with a myeloid malignancy (AML or MDS) were transplanted using a regimen consisting of fludarabine, melphalan and alemtuzumab (FMA). Patients with an underlying lymphoid disease (NHL or HD) were transplanted using FMA (n=31) or a regimen consisting of BCNU, etoposide, cytosine arabinoside, melphalan and alemtuzumab (BEAMA) (n=43). 39 patients had chemoresistant disease at the time of transplant. All patients received intravenous CsA at a loading dose of 5 mg/kg on day -1 followed by 2.5 mg/kg b.i.d. Patients were switched to oral CsA prior to discharge. Trough CsA levels were measured thrice weekly for the first three weeks after stem cell infusion and the dose of CsA adjusted to achieve levels in the region of 200–300 mg/l during this period. Trough levels obtained during the first 21 days post-transplant were used to calculate the CsA AUC for each patient. The median age of patients studied was 48 years (range 17–68). 71 patients were transplanted from HLA identical siblings and 61 from volunteer unrelated donors. The incidence of acute GVHD (Grades 2–4) was 34%. The median CsA AUC was 3682 mg.hr/l (range 2162- 8084 mg.hr/l). In univariate analysis the presence of chemoresistant disease at the time of transplant and a high CsA AUC were both associated with a decreased OS. In multivariate analyses chemoresistant disease (HR=2.60, 95% CI 1.44–4.65, p=0.002), increased age (HR=1.04, 95% CI 1.01–1.07, p=0.002) and linearly increasing CsA AUC were associated with an increased hazard of death (HR=1.1, 95%CI 1.02–1.24, p=0.02). The two year OS for patients with a CsA AUC less than 3682 mg.hr/l was 77% compared to 30% for patients with CsA of 3682 mg.hr/l and over (p

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.

Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality in patients undergoing allogeneic stem cell transplantation (SCT). This is particularly true in patients transplanted using volunteer unrelated donors (VUDs) where despite improvements in antiviral therapy CMV positive serostatus remains an adverse prognostic indicator. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones reduces the rate of viral reactivation but the complexity of this approach severely limits its clinical application. We have previously reported the purification of CMV-specific CD8+ cytotoxic T lymphocytes (CMV-CTL) from the blood of stem cell transplant donors using HLA-peptide tetramers and their subsequent transfer. Six patients undergoing stem cell transplantation from HLA identical sibling donors were studied. CMV-CTL were infused on a pre-emptive basis in patients who developed evidence of CMV viremia. All patients showed prompt and durable CMV-specific immune reconstitution demonstrating durable CMV-specific immune reconstitution. CMV-CTL constituted up to 21% of the patient lymphocyte CD8+ T cell compartment and were detectable up to 24 months in all patients. There was a reduced requirement for anti-viral agents in treated patients (33%) compared with a control group (82%) p=0.11. No secondary reactivation was seen in any patient in the treatment group. No infusion-related toxicity was seen in any treated patients. We have now extended these studies to recipients of VUD transplants in whom no CMV-CTL are detectable within the first 100 days of stem cell infusion. Tetramer-selected CMV-CTL have been adoptively transferred into six patients undergoing transplantation from an unrelated donor. CMV-CTL were given either for treatment of refractory CMV infection (n=5) or prophylactically 21 days post SCT (n=1). A cell dose of between 6x102/kg and 1.25x105/kg was administered with median purity of 95% of the infused T cell dose. No infusional toxicity or acute graft-versus-host disease was noted. CMV-CTL were not present in any patient prior to therapy. Within 7 days of adoptive transfer CMV-CTL were detectable in all patients comprising 0.6% – 20% of CD8+ T cell compartment. All patients demonstrated a reduction in the viral load and 5/6 cleared their viremia. In the patient who received prophylactic therapy durable expansion of CMV-CTL were observed up to 20% of CD8+ T cell pool. Taken together these data are consistent with the hypothesis that antigen-specific CMV specific CTL can be safely infused to recipients of VUD SCT and expand directly in vivo. Such an approach has considerable potential in the treatment or prophylaxis of CMV infection in patients undergoing VUD SCT.