ALBERT

Description: Although usually confined to the bone marrow, extramedullary (EM) involvement has been reported in up to 15%–20% of patients with myeloma at diagnosis and develops in an additional 15% during the course of the disease. To date clinical observations on EM myeloma are based on small series of relapsed patients and results suggest that the disease course is often aggressive associated with both early progression and short survival. The purpose of this study was to compare the presenting clinical/laboratory features and outcome of newly presenting myeloma patients with and without extramedullary manifestations and to determine the optimum treatment for this group of patients. EM involvement was defined as the presence of extramedullary plasmacytomas clinically or radiologically; patients with solitary or multiple bony plasmacytomas were excluded. Among 459 newly-diagnosed symptomatic myeloma patients prospectively recorded in the Royal Marsden database, 75 (16.3%) had EM involvement. The main sites of involvement were paravertebral (56%), chest wall (15%) and subcutaneous (15%). The presence of EM disease was associated with higher haemoglobin, albumin, lower β2-M level, and consequently a more favorable ISS staging at diagnosis (p

Description: Allogeneic haematopoietic stem cell transplantation remains the only curative option for many patients with acute lymphoblastic leukaemia (ALL). The optimal timing for transplant, and donor selection in this context, as well as the differing outcomes in paediatric and adult patients, remain areas of study. We reviewed the results in a single institution treating both children and adults, over a 16-year period from 1991–2007, to address a number of these issues. The study included 196 patients. The median age of the patients was 16 years (range: 2–60). The donor was a sibling in 99, unrelated (UD) in 93 and mismatched relative in 4. The transplant was performed in CR1 (71), CR2 (83), 〉CR2 (24) or with persistent disease (16). 16% of patients were Philadelphia chromosome positive and 22% had T-cell ALL. 8% of patients had received a prior autograft. The majority of patients had conditioning with a TBI-containing regimen. Most of the patients with an UD had in-vivo T-cell depletion (TCD) with Alemtuzumab (78%), while sibling donor transplants were T-cell replete. The median follow-up was 3.9 years (0.8–14.9 years). The overall survival in the whole group was 48% and 42% at 5 and 10 years respectively. The relapse risk and non-relapse mortality (NRM) were 22% and 29% at 3 years and 25% and 30% at 5 years. Acute GvHD was present in 64% of patients (grade II–IV in 29%). We were able to show a significant improvement in outcome dependent on the time period over which the transplant was performed. The 3-year survival was 33%, 51% and 57% in those transplanted from 1991–1995, 1996–2003 and 2004–2007 respectively (p=0.03). This appeared to be largely due to a decrease in the NRM in more recent years (3 year: 40%, 32% and 18% for these groups, p=0.04). Interestingly, there was no significant difference observed in OS, NRM or relapse risk based on the use of either a sibling or UD. Whilst there was no significant difference in outcome dependent on whether the transplant was performed in CR1/CR2 or beyond CR2 in children, transplants beyond CR2 or with residual disease were associated with a worse outcome in adults (p=0.01). Survival was significantly worse in patients who had received a previous autograft (p=0.0001). Although only a small number of patients (10) received reduced intensity conditioning for their transplant, the OS was not significantly different to those receiving myeloablative conditioning (2 years: 45% vs 53%, p=0.4). We did observe a significant difference in outcome in adult (〉16) compared to paediatric patients (16 and younger), 5 year OS: 32% and 62% respectively, p=0.0004. The NRM was significantly higher in adult patients (3 year: 37% compared to 21% in children, p=0.01), however there was no significant difference in disease relapse (3 year: 28% in adults compared to 24% in children). In adult patients only one factor impacted significantly on OS in multivariate analysis: a transplant undertaken in 2004–2007 compared to earlier years resulted in a significant survival benefit (HR: 0.2, 95% CI 0.1; 0.7, p=0.009). In paediatric patients two factors impacted on OS in multivariate analysis: transplants performed after 1995 compared to earlier years resulted in a significant survival benefit (HR: 0.2, 95% CI 0.05; 0.8, p=0.024), and there was a significantly worse survival in patients with a T cell phenotype (HR: 2.7, 95% CI 1.0; 6.7, p=0.031). In conclusion, we report outcomes in a large group of patients transplanted for ALL in a single institution. We show an improvement in patient survival and NRM over time, particularly in adult patients, despite the inclusion of older patients and RIC regimens in recent years. It appears that unrelated and sibling donors can be used to achieve a similar long-term outcome.

Description: Introduction: PTCL is a rare and heterogeneous group of non-Hodgkin lymphoma (NHL) comprising ~10% of cases. CHOP is frequently used first-line, but with the exception of ALK+ anaplastic large-cell lymphoma (ALCL), long term outcomes are historically poor with reported 5-yr overall survival (OS) rates of 36%. We retrospectively evaluated the outcomes following first-line chemotherapy for patients with PTCL treated at the Royal Marsden (RM) and Christie (CH) hospitals over a 10-year period. Methods: All eligible patients with PTCL aged ≥18 years and treated at the RM and CH between 1st January 2002 and 31st January 2012 were included. The study was approved by our institutional review boards. Patients were identified from hospital databases and included if they had received at least 1 cycle of first-line chemotherapy. Precursor T-cell malignancies, mycosis fungoides and adult T-cell leukaemia/lymphoma were excluded, as was cutaneous T-cell lymphoma not requiring combination chemotherapy. Clinical data were collated from electronic patient records and the diagnosis of PTCL was confirmed in all cases by an expert haematopathologist. Response was assessed using the IWG 1999 criteria. OS and progression free survival (PFS) were calculated from date of start of 1st line treatment and analysed using Kaplan Meier methods and Cox regression model. The impact of clinical factors on survival was assessed using Cox regression analysis. Results: A total of 143 (RM n=69, CH n=74)patients were evaluable and the median follow-up was 63.4 months. The median age at diagnosis was 59 yrs (range 18-89 yrs). PTCL subtypes were: PTCL not otherwise specified (NOS) (n=48), angioimmunoblastic T-cell lymphoma (AITL) (n=37), ALCL ALK- (n=24), ALCL ALK+ (n=14) and other (n=20). First-line chemotherapy included CHOP (n=97), GEM-P (gemcitabine, cisplatin and methylprednisolone) (n=16), other gemcitabine containing regimen (n=7), asparaginase (n=2) or other (n=21). OS by PTCL subtype is shown in Figure 1. Response was evaluable for 125/143 patients. Overall response (ORR) to first-line chemotherapy was 81.4% with complete response (CR) seen in 42.4%. For the entire cohort (n=143) 5-yr PFS was 20.6% and 5-yr OS was 39.6%. For CHOP treated patients ORR was 80.5% with CR in 43.7%, 5-yr PFS was 25.5% and 5-yr OS was 41.2%. ORR with GEM-P was 78.6% with CR in 50%, 5-yr PFS was 13.6% and 5-yr OS was 39.1%. No statistically significant difference between CHOP and GEM-P was seen in terms of response, OS or PFS. Autologous stem cell transplantation (autoSCT) was performed post first-line induction in 15% (n=22). For patients in CR post induction (CR1) (n=41), we compared survival for those treated with (n=12) and without (n=29) subsequent autoSCT. AutoSCT in CR1 was associated with a trend towards better PFS (HR 0.36, 95%CI 0.13-1.02; p=0.056) but not OS (HR 0.72, 95% CI 0.21-2.47; p=0.599). Uni- (UVA) and multivariate analyses (MVA) were performed to determine the impact of the following on OS and PFS: age (≤60 vs 〉 60yrs), gender, stage (I-III vs IV), performance status (PS, 0-1 vs 2), B symptoms (present vs absent), ethnicity (white vs other), LDH (normal vs elevated), IPI (low vs intermediate vs high), PTCL subtype, number of extranodal sites (0-1 vs 〉1), chemotherapy (CHOP vs gemcitabine based vs other), CR post induction (present vs absent) and autoSCT (performed vs not). Factors with a p-value of

Description: Abstract 3125 The median progression free survival (PFS) for younger, fitter patients with multiple myeloma is between 3 and 4 years, however some patients respond very poorly and relapse within a year of autologous transplantation. These patients then go on to receive relapse chemotherapy often with novel agents and, surprisingly, some have a longer remission with second line therapy. The prognostic factors which describe such patients are not well understood, and identifying and treating these patients remains a challenge. To characterise these patients further and describe prognostic markers, we have utilised 2 datasets (1081 patients) comprising 619 patients from the Myeloma IX trial and 462 patients from the Royal Marsden database (RMH). All patients were initially treated with high dose melphalan and peripheral blood stem cell support. Patients were then divided into two groups: early relapse (12 months after transplant or disease free at last follow up). The Myeloma IX trial consisted of 147 patients (23.7%) in the early relapse group compared to 472 patients (76.3%) in the late relapse group, with a median PFS1 of 7.6 vs. 27.8 months (P

Description: The WHO classification (2008) defines "myeloid/lymphoid neoplasm with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1" as a rare subtype of myeloid neoplasms. Whilst patients with PDGFRA or PDGFRB rearrangements respond very well to imatinib, the optimal therapy for patients with FGFR1 rearrangements, which we refer to as FGFR1 fusion gene positive MLN-eo (FGFR1+ MLN-eo), remains to be defined. Encouraging in-vitro data using inhibitors of the FGFR1 tyrosine kinase prompted the implementation of ponatinib, which inhibits FGFR1, into therapeutical strategies. In a recent report, the clinical activity of ponatinib was reported in a single patient who concomitantly received high-dose chemotherapy and allogeneic stem cell transplantation (ASCT, Khodadoust et al, Leukemia 2015). We sought to evaluate efficacy of ponatinib in seven consecutive FGFR1+ MLN-eo patients. Median age was 52 years (range, 48-74) with a male predominance (n=5). Median observation time after diagnosis was 10 months (range, 5-36). All patients presented with left-shifted leukocytosis but only three patients [all with t(8;13)] had eosinophilia of 〉0.5 x 109/l. Bone marrow biopsy revealed a hypercellular marrow consistent with myeloproliferative neoplasm in all patients. Five patients presented with concomitantly diagnosed lymphoid neoplasms, i.e. T-lymphoblastic lymphoma (T-LBL, n=3), biclonal accelerated phase (n=1) or lymphoid blast phase of MPN/B-cell acute lymphoblastic leukemia (B-ALL, n=1). Cytogenetic analysis revealed a reciprocal translocation with involvement of chromosome band 8p11 in all patients [t(8;13)(p11;q12), n=3; t(8;22)(p11;q11), n=2; t(1;8;22)(?;p11;q11), n=1; t(6;8)(q27;p11), n=1]. On molecular level, RT-PCR identified the associated fusion genes ZMYM2-FGFR1 (n=3), BCR-FGFR1 (n=3), and FGFR1OP-FGFR1 (n=1), respectively. In one patient with T-LBL, the FGFR1 rearrangement was revealed by FISH analysis in 80% of lymph node cells indicating an origin of both MPN and T-LBL from the same progenitor/stem cell (myeloid/lymphoid stem cell neoplasm) and T-LBL as a feature of extramedullary lymphoid blast phase. All patients were initially treated with chemotherapy-based regimens including hydroxyurea (n=4) and/or high-dose chemotherapy (n=3), the latter exclusively in patients with concomitant aggressive lymphoid neoplasms. Lack of complete response, e.g. persisting features of MPN, relapse or progression led to the off-label use of ponatinib at a dose of 30mg/day (n=2) or 45mg/day (n=5). Median duration of treatment was 8 weeks (range, 2-52). A temporary partial hematologic response (control of peripheral blood cell count) was observed in 6 of 7 patients. One patient did not respond at all and died within a few weeks while on ponatinib due to progressive disease. Three of the 6 responders had cytogenetic analysis at a median of 3 months after the start of ponatinib. One patient with t(8;13) achieved a partial cytogenetic response (50% of metaphases positive after 3 months of treatment); in all other patients no cytogenetic response was observed. Four patients underwent ASCT and are in complete molecular remission and alive after a median time of 19 months (range, 8-36) after diagnosis and 13 months (range, 4-29) after ASCT. For one patient with BCR-FGFR1-positive MLN-eo without concomitant lymphoid disease ASCT is planned. One patient is on supportive care. Conclusion: Unexpectedly, response to standard dose ponatinib in FGFR1+ MLN-eo has been poor. There was either progressive disease or no evidence for sustained hematologic or cytogenetic response. However, there was also no evidence for a sustained complete remission on intensive chemotherapy in patients with full myeloid/lymphoid phenotype. Hence, ASCT currently remains the only option to achieve long-term remission and possibly cure in FGFR1+ MLN-eo. Disclosures Off Label Use: ponatinib, used as FGFR1 inhibitor. Bommer:Alexion Pharmaceuticals: Honoraria. Cross:Ariad: Consultancy, Honoraria, Research Funding; Qiagen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

Description: Abstract 2601 Poster Board II-577 Minimal residual Disease (MRD) monitoring is known to be of importance in guiding management in patients with acute myeloid leukaemia (AML) treated with chemotherapy. Many subtypes of AML do not, however, have a molecular marker available for MRD monitoring and in this setting the use of multiparametric flow cytometry (MFC) has been evaluated. Few studies have investigated the impact of MRD in the setting of allogeneic transplantation for AML. We speculate that, as with chemotherapy, the outcome in MRD positive patients will be inferior to MRD negative patients due to an increase in disease relapse. In order to investigate this hypothesis we studied 74 patients who underwent allogeneic transplantation for AML in a single centre from 2004–2008. The median age was 47.1 years (range: 20.8–70.1). The overall survival at 2 years was 48% with a median follow-up of 1.96 years. Conditioning was myeloablative in 37 patients and reduced intensity in 37 patients. 46 patients had T cell depletion as part of the conditioning. 48 patients had an unrelated donor and in 26 a sibling donor was used. MRD analysis by MFC (sensitivity 0.04%)in the bone marrow was performed pre-transplant and on day 28 and 100 post-transplant. Using conventional criteria, 47 patients were in CR1, 15 in CR2, 4 in CR 〉 2 and 8 patients were not in CR at the time of the transplant. Survival at 2 years was 58%, 50%, 25% and 12,5% respectively (p=0.001). We examined the impact of MRD by MFC in patients who were in CR at transplant. Survival at 2 years in patients who were MRD negative (MRD-) pre-transplant was 70% compared to 29% in those who were MRD positive (MRD+) (p=0.003). In multivariate analysis, the most significant factor affecting outcome was being MRD+ pre-transplant (RR of death of 3.04, p=0.011; 95% CI 1.2–7.1). Interestingly, there was no significant difference in survival seen between patients who were MRD+ and those who were not in CR (by conventional tests) pre-transplant (p=0.305). The MRD analysis at day 28 was not predictive of outcome. However, in patients alive and without relapse at day 100, MRD analysis was predictive of outcome (2 year: MRD-70% vs 27% MRD+; p=0.045). In patients who were MRD- at both time-points, survival at 2 years was 81% compared to 42% in those who were MRD+ at one or both time-point (p=0.040). The incidence of disease relapse was 32% at 2 years and non-relapse mortality (NRM) was 8% at day 100 and 22% at 1 year. Interestingly, while the pre-transplant stage was predictive of relapse (2 years: those in CR 28% (no difference for CR1 or 〉CR1) compared to those not in CR 63%, p=0.004), the presence of MRD positivity was not; neither pre-transplant (p=0.47) nor at day 100 (p=0.29). Conversely, the NRM was significantly higher in those who were MRD+ pre-transplant, but not at day 100. The NRM at day 100 was 4% in MRD- patients and 15% in MRD+ patients (p=0.01). In multivariate analysis this conferred a RR of NRM of 2.12 (p=0.014; 95% CI 1.1–3.8) to MRD+ patients. We conclude that the presence of MRD detected by flow cytometry pre- and day 100 post-transplant is predictive of a worse patient outcome. Interestingly this appears to be predominantly due to an increase in NRM rather than an increase in disease relapse. We speculate that differences in the duration and/or intensity of GVHD prophylaxis may be implicated in this finding and we are currently investigating this hypothesis. A better understanding of these factors will allow us to tailor treatment based on MRD status in an intelligent fashion. Disclosures: No relevant conflicts of interest to declare.

Description: Empirical antifungal therapy is the standard treatment in allogeneic transplant patients who have persistent febrile neutropenia. This approach can be associated with increased cost, toxicity and breakthrough infections. There are limited reports to date of strategies for the early diagnosis of invasive fungal infection (IFI). These include either invasive investigations or serum testing. (Oshima K et al, J Antimicrobial Chemother2007 Aug; 60(2): 350–5, Maertens J et al, Clin Infect Dis2005; 41:1242–50). To our knowledge, there are no reports to date of treatment strategies based only on high resolution computerised tomography (HRCT) scans. We used a CT-diagnosis based treatment strategy for early invasive aspergillosis in 99 consecutive patients undergoing allogeneic transplantation over a two year time period. A retrospective review of the electronic patient record, notes and drug charts was undertaken in all patients receiving an allogeneic transplant in our unit from 1st January 2006 to 31st December 2007. The study protocol was approved by the Royal Marsden Hospital audit committee. Patients received primary antifungal prophylaxis with itraconazole or secondary prophylaxis with voriconazole from day + 1. Patients had a HRCT scan performed if they had antibiotic resistant fever for 〉 72 hours. Parenteral antifungal treatment with caspofungin was commenced in patients with a positive HRCT result. Cavitation, air crescent sign and halo sign were classified as major changes. Nodules and new infiltrates including consolidation and effusions were classified as minor changes. Serum testing was not used due to the possibility of false positive results with tazobactampiperacillin antimicrobials and low sensitivity in patients receiving mould active azoles as prophylaxis. Neutropenic fever developed in 89/99 patients (90%). Fifty-four percent (53/99) of patients developed antibiotic resistant fever for 〉 72 hours and would have received parenteral antifungal treatment if an empiric strategy had been used. The HRCT-based strategy reduced the use of parenteral antifungal treatment to 17% of patients (17/99). Four of these patients had engrafted (absolute neutrophil count 〉0.5x109 cells/L for 3 days) at time of commencing caspofungin following a period of persistent neutropenic fever. Fifteen patients had a positive HRCT scan and 2 were treated empirically until a HRCT could be performed. The remaining 36/53 patients (68%) did not receive antifungal treatment although they would have met criteria for standard empirical therapy. Our nonempiric strategy reduced the use of parenteral antifungal treatment from 54% to 17% of allogeneic transplant patients. These findings represent a 68% reduction in use of parenteral antifungal agents Caspofungin (70mg once daily IV on day 1 and 50mg once daily IV thereafter) was given for a median of 13 days (3–34 days) and 11 patients responded to treatment. Six patients required second line antifungal therapy. Only one patient died from IFI after 100 days of follow-up. No patients had to stop caspofungin due to toxicity. Three of the 36 patients who did not receive initial antifungal treatment went on to receive empiric caspofungin within 100 days. One of these 3 patients died of enterococcal septicaemia. The other 2 patients recovered. None of this group had probable or proven IFI. During the extended follow-up period of 3–27 months (median 13 months) only 3/99 patients (3%) died of IFI. These results suggest that this non-empiric strategy of parenteral antifungal treatment based on HRCT scanning is feasible and may help to reduce toxicity, cost and breakthrough infections associated with the use of antifungal agents. Caspofungin may be a useful first line agent in this setting. A randomised controlled trial is warranted to further evaluate these results.

Description: T-cell prolymphocytic leukaemia (T-PLL) is a rare aggressive post thymic lymphoid disorder with distinctive clinical and pathological features. It is resistant to conventional chemotherapy and has a short median survival of 7 months. Intravenous (IV) alemtuzumab is an effective and well tolerated therapy for this disease, inducing remission in 76% of patients in the relapsed and refractory setting and increasing median survival to 24 months in responders. The aim of this study was to determine the efficacy of subcutaneous (SC) administration of alemtuzumab in previously untreated T-PLL patients. After an initial week of dose escalation, patients were treated with a dose of 30 mg 3 times weekly. Patients could be switched to the IV route if they had local side effects or showed a lack of response. Nine patients were enrolled. Male: female ratio was 2:1; median age was 61 years. All cases were centrally reviewed and confirmed to have a diagnosis of T-PLL. Eight had an immunophenotype typical of T-PLL (CD2, CD3, CD4, CD5 and CD 7 positive). One patient was unusually CD7 negative and also did not have an abnormality of chromosome 14. Seven had a complex karyotype and 8 had chromosome 14 abnormalities. Patients were treated on trial for a median of 7 weeks (range 3–13 weeks); 55% required a change to IV treatment due to a lack of response. This resulted in an increase in overall response (OR) from 33% (1 complete response [CR], 2 partial response [PR]) to 44 %.(2 CR, 2 PR). Five patients required addition of pentostatin to alemtuzumab at a dose of 4 mg/m2 IV weekly to augment response. This increased OR to 77% (1 additional CR and 2 PR). 5 patients had haemopoietic progenitor cell transplants (HPCT) as consolidation; 1 was autologous and 4 were allogeneic. Median overall survival was 20 months (range 1–26 months) and disease free survival was 12 months (range 0–20 months). The treatment was well tolerated; however 2 patients had skin reactions to the SC injections. One patient required treatment for CMV reactivation and 2 patients had grade 4 haematologic toxicity which was related to disease and not treatment. Four patients remain alive and well in CR with a follow up of between 12 and 23 months, of whom 3 had allogeneic HPCT. The study was terminated early as a data safety monitoring review felt that SC treatment was not efficacious and patients should not be offered SC treatment. This pilot study suggests that the SC route of administration of alemtuzumab is not as effective as IV in the treatment of T-PLL; our series of 16 patients receiving first line therapy with IV alemtuzumab has shown an OR of 94% with 88% CR. It is therefore advisable that IV treatment should be used. Pentostatin is an effective agent to augment response. Allogeneic HPCT should be used as consolidation therapy when possible.

Description: Veno-occlusive disease (VOD) is a common complication following the intensive conditioning regimens used in stem cell transplantation (SCT). The reported incidence is between 10–60% and the clinical spectrum varies from mild, reversible disease to a severe disorder with a mortality rate approaching 100% by day 100 post SCT (McDonald et al. Ann Intern Med1993, 118, 255–267). Defibrotide has recently been shown to be effective in the treatment of severe VOD in several series (Chopra et al. Br J Haematol2000, 111, 1122–1129; Richardson et al. Blood2002, 100, 4337–4343). Only one report to date details the use of prophylactic defibrotide in the allogeneic SCT population. This study showed a significant reduction in VOD in patients treated with prophylactic defibrotide and heparin (Chalandon et al. Biol Blood Marrow Transplant2004, 10, 347–354). We report a retrospective review of the incidence of VOD in 58 adult patients who received defibrotide prophylaxis during allogeneic SCT from May 2004 to December 2005. Heparin was not used routinely. Patients received 5mg/kg of defibroide twice daily IV from day + 1 to day + 21. All patients were assessed daily by clinical examination including weight and abdominal girth and laboratory tests including liver function tests. The Baltimore criteria were used to diagnose VOD. If VOD was suspected then US of the liver with a doppler test was performed. No patients met the Baltimore criteria for VOD and no patients died of suspected VOD within 100 days of SCT. The transplant related mortality was 5/58 (8.6%) of patients. Three patients died of bronchopnemonia, one of E.Coli septicaemia and one of multi-organ failure secondary to infection. VOD was not felt to have contributed to the deaths of any of these patients. The dose of defibrotide was increased to 10mg/kg four times daily IV in 3 patients in whom VOD formed part of the differential diagnosis for deranged liver function tests but who did not meet the Baltimore criteria. None of these patients had a positive US scan. One of these patients had a liver biopsy suggestive of mild VOD but also showed features consistent with GVHD and viral infection. The liver function of the other two patients improved after hepatotoxic drugs were stopped. All of these patients were alive at 100 days post transplant with no features of VOD. Patients tolerated defibrotide well and the drug did not have to be discontinued in any patient. There were no cases of haemorrhagic complications attibutable to defibrotide. It seems unlikely that differences in clinical characteristics accounted for the low incidence of VOD as many of our patients had several high risk factors. Sixty-seven % (39/58) had received a transplant from an unrelated donor, 18/58 (31%) had received a previous transplant and 28/58 (48%) had received either cyclophosphomide or busulphan as part of their conditioning regimen. This review suggests that the use of prophylactic defibrotide may reduce the incidence of VOD following allogeneic SCT. Defibrotide may be effective without concurrent heparin but a randomised controlled trial would be useful to further investigate this issue.

Description: Abstract 1912 The response to donor leukocyte infusions (DLI) can be improved by the use of pre-DLI lymphoreduction. The mechanism underlying this is poorly explained. In order to investigate this further we examined the T cell subsets in patients entered into a clinical trial testing the utility of pre-DLI lymphoreduction using fludarabine. Inclusion criteria were: 1. mixed chimerism (MC) in whole blood (〈 95% donor), 2. previous reduced intensity transplant for a haematological malignancy or failure to respond to a previous dose of DLI and 3. a lymphocyte count of 〉1.0 × 109/l. Patients consenting to the trial received three doses of oral fludarabine as an outpatient at 25mg/m2 on day −7, −6 and −5. DLI was given on day 0. The starting dose of DLI for patients with MC alone was 5 × 105 CD3/kg (unrelated donor) or 1 × 106 CD3/kg (sibling donor). Higher doses were used in those with evidence of both disease and MC. Samples were collected at D-7, D0, D+7, D+28, D+60 and D90 for T cell subset analysis. Samples were analysed for a combination of markers for CD4, CD8 and T regs using multicolour flow cytometry. CD4 and CD8 were analysed for subset: central memory (CM 62L+RA-), effector memory (EM 62L-RA-), naïve (N 62L+RA+) and effectors (EFF 62L-RA+). Tregs were characterised as CD4+25hi127loFOXP3+. The study was a phase II pilot study with 18 patients required. At the end of the trial, 17 patients were eligible for analysis (1 early death due to leukaemia relapse). The disease categories were: AML (7), ALL (1), MDS (4), T-PLL (1), HD (2), DLBCL (1) and MCL (1). Only 2 patients had co-existing evidence of disease (morphological relapse of AML, 2% positive by immunophenotyping in T-PLL). The median age was 55 years (range: 22–66). There were 10 males and 7 females. 12 patients had HLA-matched sibling donors and 5 had 10/10 matched unrelated donors (UD). The majority of patients (13) had conditioning with fludarabine, melphalan and alemtuzumab, with cyclosporine post transplant. Each received a single dose of DLI on the trial. The median time to DLI post-transplant was 7.4 months (range: 4–11.7). The median percentage of donor chimerism pre-DLI was 85% (range: 18–93). The median lymphocyte count pre-DLI was 1.3 (range: 1.0–3.8). Of the 17 eligible patients, 11 (65%) responded to a single dose of DLI (CR=9 (〉95%), PR=2 (chimerism 94% donor at study end)). Five patients developed GvHD – grade 1 (n=2), grade 2 (n=1), grade III/IV (n=2). GVHD resolved completely in all cases. 3 patients reactivated a virus (CMV, 2 × EBV), although not all required treatment. No pre-DLI clinical factor had a significant impact on DLI responsiveness including: disease type, age, gender, donor type, product source (sibling, UD), pre-DLI chimerism, pre-DLI lymphocyte count, time to DLI or source of DLI (mobilised or non-mobilised cells). Post DLI GvHD did not have a significant association with response. There was a significant decrease in the lymphocyte count, as well as the absolute numbers of CD8 (unpaired t test, p