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Relapse following CD19-directed chimeric antigen receptor T-cells (CAR-T) for relapsed/refractory large B-cell lymphoma (r/r LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multi-antigen targeting and PD-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in r/r LBCL as inpatient or outpatient therapy (NCT03289455, https://clinicaltrials.gov/ct2/show/NCT03289455). Endpoints include toxicity (primary) and response rates (secondary). AUTO3 was manufactured for 62 patients using autologous leukapheresis, modified with a bicistronic transgene. 52 patients received AUTO3 (7/52,50x106; 45/52,150-450x106) and 48/52 received pembrolizumab. Median age was 59 years (range,27-83) and 46/52 had stage III/IV disease. Median follow-up was 21.6 months (range,15.1-51.3) at last data cut (Feb 28, 2022). AUTO3 was safe: grade 1-2 and grade 3 CRS affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), HLH affected 2 patients, and no Pembrolizumab-associated autoimmune sequalae were observed. On this basis, outpatient administration was tested in 20 patients, saving a median of 14 hospital days/patient. AUTO3 was effective: overall response rates were 66% (48.9%, CR; 17%, PR). For patients with CR, median DOR was not reached, with 54.4% (CI: 32.8, 71.7) projected to remain progression-free beyond 12 months after onset of remission. DOR for all responding patients was 8.3 months (95% CI: 3.0, NE) with 42.6% projected to remain progression-free beyond 12 months after onset of remission. Overall, AUTO3 +/- pembrolizumab for r/r LBCL was safe, lending itself to outpatient administration, and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion/persistence in vivo, and selection of CAR binders active at low antigen densities.

Publisher's Note: There is a Blood Commentary on this article in this issue.

The impact of Coronavirus disease 2019 (COVID-19) on outcomes in patients with cancer remains unclear. Acute Myeloid Leukemia (AML)/high-risk myelodysplasia (MDS) are common hematological malignancies resulting in profound immunosuppression, which is exacerbated by intensive and less-intensive chemotherapy. Importantly, venetoclax based regimens have been increasingly used during the pandemic as a strategy to reduce patient hospitalization however, there is little information concerning the impact of such regimens on COVID-19 infection rates. We therefore opened a prospective clinical study (PACE), at the start of the current pandemic in April 2020 to characterize the risk of COVID-19 infection in patients with AML/MDS-EB2 receiving intensive or non-intensive treatment, including patients treated with venetoclax-based regimens. The primary aim was to determine the incidence of COVID-19 in patients with AML /MDS-EB2 including both, prior to study entry and during treatment until 4 weeks after the last cycle of treatment. Secondary aims were to: characterize the presentation of COVID-19; define the severity and type of both non-COVID-19 and COVID-19 infections; and undertake an exploratory analysis to quantify the incidence of COVID-19 infection in patients receiving (less-intensive) venetoclax based regimens. All analysis conducted to date has been descriptive. 211/230 recruited patients had full treatment histories available, of whom 116 patients received intensive chemotherapy and 95 low intensity regimens. 48 patients received a venetoclax-based regimen. The median age of the non-intensive treatment arm was 72 years; (range 19.1-86.5) and of the intensive arm was 59 years (range 16.1-76.1). There were more cases of secondary AML and relapsed disease in the non-intensive arm as compared to the intensive arm. 25/226 evaluable patients tested positive for COVID-19 as defined by positive SARS-CoV2 PCR test, 10 with a prior diagnosis at study entry and 15 tested positive during the study. The incidence of COVID-19 infection for patients with AML/MDS-EB2 was 11.1% (90%CI: 7.8%-15.1%) (Table). A lower proportion of patients (n=6/91 6.6%) undergoing non-intensive treatment suffered COVID-19 as compared to those undergoing more intensive chemotherapy regimens (n=19/116, 16.4%). Specifically, only 3/48 (6.3%) patients undergoing a venetoclax regimen were infected with SARS-CoV2. The most common presenting symptoms of COVID-19 in this study, regardless of the intensity of chemotherapy, was fever and cough with 6/25 patients asymptomatic. The risk of death at 30 days following study entry in patients who had prior COVID-19 infection or who contracted COVID-19 during this period was 13.6%, compared to 3.9% in the overall cohort without COVID-19 infection. There was a lower incidence of non-COVID-19 related infections in patients receiving venetoclax-based regimens, n=43 infections in 24 (50.0%) of patients; with 313 infections in 94 (81%) of intensively treated patients. The overall occurrence of non-COVID-19 infection in the non-intensive arm was 87 infections in 50 (54.9%) patients. Our multi-center study provides real-world estimates for the incidence and presentation of COVID-19 infection in a cohort of patients with AML/MDS-EB2, and indicates a higher risk of death at 30 days in patients with prior COVID-19 infection prior to, or during treatment. Venetoclax based, and other non-intensive, regimens, increasingly implemented during the pandemic, to minimize patient exposure and reduce usage of hospital beds, appeared to be associated with a low incidence of COVID-19. Further follow-up will be required to understand the long-term impact of this strategy. Analysis of immune responses to COVID-19 infection and vaccination is on-going. Acknowledgments: This study was funded by Cure Leukaemia under the Trials Acceleration Program (TAP), and grants from BMS and Blood Cancer UK. Figure 1 Figure 1. Disclosures Loke: Novartis: Other: Travel; Janssen: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Daichi Sankyo: Other: Travel. Knapper: Pfizer: Consultancy, Speakers Bureau; Astellas: Ended employment in the past 24 months, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Khan: Abbvie: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Jazz: Honoraria; Gilead: Honoraria; Novartis: Honoraria. Dillon: Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Jazz: Other: Education events; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees. Culligan: AbbVie Ltd: Honoraria, Speakers Bureau; Celgene Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Jazz Pharma: Honoraria, Speakers Bureau; Takeda UK Ltd: Honoraria, Speakers Bureau. McMullin: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other: clinical trial support, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan: Research Funding, Speakers Bureau. Murthy: Abbvie: Other: support to attend educational conferences.. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) CD19 CAR-T therapies are licensed in the UK for relapsed/refractory large B-cell lymphoma (LBCL). Corticosteroids for CAR-T toxicity were administered to 28% and 10% of patients in ZUMA-1 and JULIET respectively, but real-world steroid use is reported to be much higher (Nastoupil et al, JCO 2020), with concerns that this may adversely impact on OS and PFS following CAR-T. Analysis of real-world datasets may facilitate the identification of modifiable risk factors for toxicity. Here, we report the UK experience of CAR-T toxicity and its management in 341 LBCL patients with a focus on predictors for corticosteroid use and the impact of steroids on OS/PFS post CAR-T therapy. Methods Data were collected in 10 UK centres from January 2019 to April 2021. CRS/ICANS were graded prospectively (ASTCT). Low (LG) and high-grade (HG) CRS/ICANS were classified as grade 1-2 and 3-5 respectively. Hyper-acute CRS was defined as onset within 24 hours of cells. Steroid cumulative dosing for CAR-T toxicity was calculated as dexamethasone equivalent up to day 30. Product selection was at the discretion of the treating physician. Toxicity management in the UK follows EBMT and ASCT guidance. Results A total of 341 patients received axi-cel (n=261) and tisa-cel (n=80) with a median follow-up of 14.8 (3.4-30) and 13.9 (range 7.5-27) months. Median age was 59 (range 18-80), 61% were male (M=207, F=134) and 79% received bridging therapy (BT). HG CRS, hyperacute CRS and HG ICANS with axi-cel (8.8%; 38%; 21% respectively) and with tisa-cel (7.5%; 26%; 5% respectively) were observed, with incidence of HG ICANS post axi-cel lower than published reports. Risk factors for HG CRS were age0 at infusion (0.001); and hyper-acute CRS was associated with stable (SD) or progressive disease (PD) after BT (p=0.036). On multivariate analysis (MVA), ECOG >0 (OR 3.4, 95% CI 1.3-9.1, p=0.015) and SD/PD post BT (OR 1.8, 95% CI 1.03-3.3, p=0.039) were predictive of HG CRS and hyper-acute CRS respectively. Risk factors for HG ICANS were age0 (p=0.009), SD/PD post BT (p=0.011), extranodal disease (p=0.02) and LDH>normal on Day 0 (p=0.02). By MVA, LDH>normal on Day 0 (OR 3.8, 95% CI 1.4-10.5, p=0.009) was predictive of HG ICANS. Steroids were used in 44% (n=115) of axi-cel treated patients, administered at day 6 (median; range 1-22), at a median dose of 165mg (range 10-2182) over a median of 8 days (range 1-86). Anakinra was given as an adjunctive agent for high-grade ICANS (n=26, 10%), CRS (n=10, 4%) or HLH (n=3, 1%) at a median of day 8 (range 5-43) for a median of 6 days (range 1-16). 18% of tisa-cel patients (n=14) received steroids, administered at day 4 (median; 2-26) at a median dose of 95mg (range 10-220) over a median of 4 days (range 1-8). Tocilizumab was used in 74% and 48% of axi-cel and tisa-cel patients respectively. Clinical factors associated with steroid use included ECOG>0 (OR 1.8, 95% CI 1.2-5.6, p=0.002), and the emergence of SD/PD post BT (OR 1.7, 95% CI 1.02-2.8, p=0.04). SD/PD post BT also predicted for higher total steroid dose (>median; OR 1.7, 95% CI 1.1-2.9, p=0.03), and prolonged (>1 week) administration (OR 1.8, 95% CI 1.1-2.9, p=0.03) (Table 1). The impact of clinical parameters including toxicity and steroids on PFS and OS post-CAR-T were assessed. Adjusted for age, sex, ECOG and LDH>normal pre-LD, HG ICANS (but not HG CRS) was associated with worse OS (HR 2.4, 95% CI 1.3-4.4, p=0.004). 100 day NRM was 3.5% for the whole cohort. Median OS was not reached. In contrast to other published series, early steroid use (HR 1.0, 95% CI 0.6-1.6, p=0.99), higher total doses (HR 1.2, 95% CI 0.7-1.9 p=0.6) and prolonged (>1 week) steroid exposure (HR 1.3, 95% CI 0.8-2.2, p=0.25) were not associated with worse OS. Early steroid use (HR 0.7, 95% CI 0.3-1.6, p=0.4), higher doses (HR 0.5, 95% CI 0.2-1.2, p=0.13) and prolonged exposure (HR 0.9, 95% CI 0.4-2.0, p=0.8) also had no significant impact on PFS. Conclusion Patients who are refractory to BT or with other markers of high disease burden at infusion are more likely to require steroids for CAR-T toxicity, with axi-cel associated with more HG ICANS and cumulative steroid exposure than tisa-cel. Despite this, steroid use for CAR-T toxicity did not negatively impact on PFS or OS. Rates of HG ICANS with axi-cel were comparatively lower in this UK dataset. Figure 1 Figure 1. Disclosures Sanderson: Kite, a Gilead Company: Honoraria; Novartis: Honoraria. Kuhnl: Kite: Honoraria; Novartis: Research Funding. Menne: Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Astra Zeneca: Research Funding; Jazz: Other: Travel grants; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Bayer: Other: Travel grants; Kyowa Kirin: Other: Travel grants; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Atara: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Honoraria for Lectures; Roche: Other: Honoraria for Lectures. Chaganti: Celgene-BMS: Consultancy, Honoraria, Other: Travel support; Takeda: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Atara Bio: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding; Incyte: Honoraria, Speakers Bureau. Nicholson: Novartis: Consultancy, Other: Conference fees; Kite, a Gilead Company: Other: Conference fees, Speakers Bureau; BMS/Celgene: Consultancy; Pfizer: Consultancy. Latif: Takeda UK: Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis,: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Kite: Consultancy, Honoraria, Speakers Bureau. Jones: Janssen: Consultancy; Kite/Gilead: Honoraria; Novartis: Honoraria. Sharplin: Kite Gilead: Honoraria; Novartis: Other: Travel Support. Jalowiec: Kite Pharma Gilead Sciences': Honoraria, Speakers Bureau. Roddie: Novartis: Consultancy; Celgene: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau. OffLabel Disclosure: Anakinra for CAR-T toxicity management

Allogeneic stem-cell transplantation is an important curative strategy in acute myeloid leukaemia (AML) consequent upon the development of a graft-versus-leukaemia (GVL) effect. Disease relapse is the major cause of transplant failure and novel therapeutic strategies are required in patients with measurable residual disease (MRD) post-transplant. Recognizing that full donor T cell chimerism serves as a biomarker of GVL and that mixed donor T cell chimerism reflects the presence of bi-directional tolerance, we report the first prospective correlation of the impact of post-transplant T cell chimerism and MRD in patients allografted for AML/MDS. Methods: 186 patients from FIGARO, a prospective randomized trial of reduced intensity conditioning regimens in AML/MDS, were alive and relapse free, at the first MRD timepoint and provided post-transplant bone marrow samples for flow cytometric MRD monitoring (day+42, month+3/+6/+9/+12) and peripheral blood samples for T cell chimerism analysis (3-monthly during the first year). Results were not available to clinicians. Flow cytometric MRD incorporating an unsupervised immunophenotypic analysis approach was used on 778 sequential samples. The prognostic significance of post-transplant MRD from each sampling timepoint was assessed by landmark analysis. Results: 29 (15.6%) patients were MRD+ at one or more timepoints in the first year after transplant. The presence of post-transplant MRD was associated with reduced OS (HR:2.19, p=0.0026) and RFS (HR:5.36, p Next, we examined the impact of T-cell chimerism, and post-transplant MRD on outcomes. In patients with mixed-donor T-cell chimerism (MDTC) at month+3 or +6, the presence of post-transplant MRD (preceding or at time of first MDTC) was associated with decreased OS (p=0.001) and RFS. Specifically, 2yr RFS in MRD+ patients was 23.1% (95%CI:5.6-47.5), compared to 63.6% (95%CI:44.9-77.5) in patients who were MRD negative (p=0.004) (Figure). In contrast, in 48 patients with full donor T-cell chimerism (FDTC) at months 3 and 6, only 5 patients were MRD+ post-transplant and their outcomes were equivalent to patients who were MRD negative (FDTC/MRD negative 2yr RFS: 80.9% (95% CI:65.3-90.0)). Downregulation of HLA class II molecules on AML blasts after transplant may result in immune evasion from GVL: we examined whether this had an additional impact on outcome in post-transplant MRD+ patients. Both RFS and OS were significantly lower in MRD+ patients when MRD+ blasts had down-regulated HLA-DR expression. In MRD+ patients, 2yr OS was 20.0% (95%CI:3.1-47.5) in those with HLA-DR down-regulation, versus 57.9% (95%CI 33.2-76.3) in those without (p=0.001). Conclusion We demonstrate that dynamic assessment of post-transplant MRD is an important predictor of outcome in patients allografted for AML/MDS, with additional prognostic value to pre-transplant MRD assessment. Post-transplant MRD and T-cell chimerism results are most informative when combined, underlining the importance of a GVL effect in AML/MDS. This suggests post-transplant strategies which result in FDTC may improve outcomes in patients allografted for AML/MDS. Our data also supports the clinical relevance of monitoring for leukemic immune escape as HLA-DR down-regulation identifies a post-transplant MRD positive subgroup at risk of almost inevitable relapse. Figure 1 Figure 1. Disclosures Loke: Novartis: Other: Travel; Janssen: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Daichi Sankyo: Other: Travel. Protheroe: Jazz Pharmaceuticals: Honoraria; Astellas: Honoraria; Kite Gilead: Honoraria. Dillon: Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Jazz: Other: Education events; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction The clinical manifestations of COVID-19 infection in recipients of allogeneic hematopoietic stem cell transplantation (HSCT) have been reported in multiple retrospective cohorts of patients, but there have been no prospective studies to date. Previous studies report that HSCT recipients are at higher risk, with cumulative incidence of death between 17-35%. Although an excessive pro-inflammatory viral response has been documented in the general population, its role in the immune incompetent HSCT setting has not been documented. We present a combined prospective and retrospective national study run through the UK IMPACT trial network to characterize the clinical and immunological features of COVID-19 infection in 96 adult and pediatric recipients of HSCT in the United Kingdom. Methods HSCT recipients of any age and transplanted for any indication, with an RT-PCR-proven COVID-19 infection, were eligible for this study. Patients within 72 hours of COVID-19 diagnosis, who had not received cytokine-targeted treatment, were recruited to a prospective cohort. All other patients were eligible for a retrospective cohort. Prospective patients provided blood samples within 72 hours of COVID-19 diagnosis, and again within 72 hours of clinical deterioration (defined as requirement for oxygen administration) if applicable. Follow-up data were collected on patients 30 and 100 days after COVID-19 diagnosis. Results 100 patients were recruited from 16 sites across the UK between May 2020-June 2021, comprising 12 in a prospective cohort and 88 recruited retrospectively. 96 patients were evaluable, as 4 proved ineligible post-registration. Patients were diagnosed with COVID-19 at a median of 11 months after HSCT. Patient/HSCT characteristics are shown Table 1. The most common symptoms associated with the onset of COVID-19 were fever in 8 prospective (73%) and 35 (41%) retrospective patients, followed by cough in 5 (45%) prospective and 35 (41%) retrospective patients and dyspnea in 4 (36%) prospective and 16 (19%) retrospective patients. 8 (73%) prospective and 40 (47%) retrospective patients were actively immunosuppressed at the time of COVID-19 infection. 16% of the patients had moderate/severe disease at baseline. At day 30 (±2 days) after COVID-19 diagnosis, 2 prospective and 8 retrospective patients continued to demonstrate SARS-CoV-2 positivity on respiratory PCR testing. The median time to viral clearance was 40 (IQR 17-78) days for the prospective and 34 (IQR 15-70) days for the retrospective cohort. Prolonged (more than 14 days) neutropenia was reported in 4 (5%) patients in the retrospective cohort, prolonged thrombocytopenia in 2 (18%) prospective and 11 (13%) retrospective patients. 1 retrospective patient developed secondary hemophagocytic lymphohistiocytosis, and graft rejection was reported in 1 (1%) retrospective patient, within 30 days of COVID-19 diagnosis. In the prospective cohort, 3 (27%) patients died, all by day 30, and all due to COVID-19. In the retrospective cohort, 13 (17%) patients died by day 30, rising to 18 (21%) by day 100, 61% of deaths were attributed to COVID-19. Lower baseline platelets (p=0.013, Mann-Whitney U test), lymphocytes (p=0.012), albumin (p=0.028), and higher baseline CRP (p=0.007), were seen in patients who died following COVID-19 diagnosis. Additionally, exploratory univariate logistic regression of the retrospective cohort found mortality at day 100 to be associated with increased age at diagnosis (OR 1.04, 95% CI 1.01-1.08, p=0.04), and no requirement compared with requirement for invasive ventilation (OR 0.02, 95% CI 0.00-0.16, p=0.001). The 11 prospective patients showed normal levels of interleukin (IL)-2, -4, -10, interferon gamma and tumor necrosis factor alpha at COVID-19 presentation. IL-6 was minimally raised (up to 127 pg/ml, nv Conclusion Our study confirms a significant mortality rate in patients affected by COVID-19 post HSCT and confirms age as well as requirement for invasive ventilation to be independent risk factors associated with death at day 100. Baseline laboratory data at disease presentation can identify patients at higher risk of COVID-19 related death. In the prospective cohort of our study, pathophysiology of the viral disease did not seem related to cytokine storm-mediated inflammation. Figure 1 Figure 1. Disclosures Protheroe: Jazz Pharmaceuticals: Honoraria; Astellas: Honoraria; Kite Gilead: Honoraria. Peggs: Autolus: Consultancy, Current equity holder in publicly-traded company. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nicholson: BMS/Celgene: Consultancy; Kite, a Gilead Company: Other: Conference fees, Speakers Bureau; Novartis: Consultancy, Other: Conference fees; Pfizer: Consultancy. Amrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties.

INTRODUCTION: Allogeneic stem cell transplantation (allo-SCT) is an important curative strategy in adults with high risk acute myeloid leukemia (AML) and myelodysplasia (MDS). Disease relapse represents the major cause of treatment failure and whilst retrospective analyses have identified that pre-transplant measurable residual disease (MRD) is an important predictor of transplant outcome this has never been examined prospectively. The advent of reduced intensity conditioning (RIC) regimens has substantially increased the number of older adults eligible for allo-SCT but the optimal RIC regimen in high risk AML remains unknown. Registry data have demonstrated improved outcomes using a sequential transplant regimen utilizing cytosine arabinoside (araC)/amsacrine (AMSA) cytoreduction followed by a fludarabine (Flu)/busulfan (Bu) based RIC regimen (FLAMSA-Bu). However, although the FLAMSA-Bu regimen is now widely used in adults with high risk AML and MDS its benefit has not been evaluated in a randomized trial. We report the results of a randomized trial evaluating the FLAMSA-Bu regimen compared with standard RIC regimens which also represents the first prospective evaluation of the impact of pre-transplant MRD levels on transplant outcome. PATIENTS AND METHODS: 244 patients (median age 59 yrs) with high risk AML (n=164) or high risk myelodysplasia (n=80) were randomized 1:1 to a control arm determined by investigator's choice of either Flu/B2/ATG (Flu, Bu 3.2 mg/kg x 2 days, ATG 2.5 mg/kg x 2 days); Flu/Mel/Alemtuzumab (A) (Flu, Mel 140 mg/m2, A 50 mg) or Flu/Bu2/A (Flu, Bu 3.2 mg/kg x 2 days, A 50 mg) versus an experimental arm of FLAMSA-Bu (Flu, araC 2g/m2 x 4 days, AMSA 100mg/m2 x 4 days, Bu -total dose 11.2 mg/kg). Patients over the age of 60 received an adjusted FLAMSA-Bu regimen utilising a reduced dose of araC (1mg/m2 x 4 days) and a total Bu dose of 6.4 mg/kg. Patients were transplanted using either an HLA identical sibling (n=49) or matched (10/10 or 9/10) unrelated donor (n=195). All patients received cyclosporine GVHD prophylaxis. 155 patients with AML were in CR1 or CR2 at the time of transplant and 9 had primary refractory disease. MRD was monitored by flow cytometry (applying different-from-normal analysis when no diagnostic/relapse leukemic aberrant immunophenotype was available). Pre-transplant MRD levels were measured up to four weeks prior to transplantation in 201 patients (MRD positive = 80 (40%), MRD negative = 94 (47%), inadequate sample = 27 (13%)). MRD results were not reported to clinicians. The primary outcome was overall survival. RESULTS: Baseline characteristics including CR1/CR2 status, adverse cytogenetics and MRD levels were similar between regimens. Median follow up was 35 months. Transplant outcomes were comparable between patients allografted in the control and FLAMSA-Bu arms. 2 yr overall survival (OS) and cumulative incidence of relapse (CIR) were 61% and 30% respectively in the control arm vs 62% and 26% for the FLAMSA-Bu arm. Transplant related mortality at 100 days was 3.0% in patients allografted using the control regimen vs 14% in patients allografted using the FLAMSA-Bu regimen and 17% vs 21% at 1 year. In the study cohort pre-transplant MRD positivity was associated with both an increased CIR compared to patients testing MRD negative (2 yr CIR 42% vs 19%, p=0.009) and decreased OS (2 yr OS 52% vs 71%, p=0.048). The FLAMSA-Bu regimen failed to improve OS or reduce CIR in either MRD positive or MRD negative patients. CONCLUSIONS: This trial, the largest randomized trial of RIC regimens in AML to date, did not detect any benefit of intensification of the conditioning regimen in adults with high risk AML or MDS. Specifically, the FLAMSA-Bu regimen was not associated with improved transplant outcome in patients who were MRD positive pre-transplant. These data include the first demonstration in a prospective analysis that the presence of pre-transplant MRD measured in real time is associated with reduced OS consequent upon an increased risk of disease relapse. Further randomized studies of novel conditioning regimens in adult AML, crucially with integrated MRD studies, are now required but these results support exploration of alternative strategies, such as pre or post-transplant pharmacological intervention, as the most promising strategy to reduce the risk of disease relapse post allograft. Disclosures Russell: Jazz: Consultancy, Honoraria, Speakers Bureau; DSI: Consultancy, Honoraria, Speakers Bureau; Pfizer Inc: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau. Freeman:Jazz Pharmaceuticals: Speakers Bureau. OffLabel Disclosure: We report data using the combination of fludarabine, busulphan, amsacrine and cytosine arabinsoide as a conditioning regimen in patients allografted for high risk acute myeloid leukemia

Karl S Peggs and Sarah J Albon contributed equally to the work and are joint first author Introduction Alemtuzumab reduces the incidence of GVHD after unrelated donor stem cell transplant (MUD SCT) but delays immune reconstitution resulting in high morbidity/mortality from viral infections. Previous studies have suggested that adoptive transfer of allodepleted donor T cells (ADTs) improves immunity after SCT but this has never been tested in a randomised study. We developed a methodology for selective immunomagnetic depletion of alloreactive T-cells upregulating CD25 and CD71 after activation with host dendritic cells (DC) and showed that ADTs retain anti-viral responses with minimal host alloreactivity (Samarasinghe et al Blood 2010). We have now tested whether ADTs can safely be used to improve immune reconstitution after MUD SCT for haematological malignancies in a randomised Phase II multi-centre clinical study; ICAT (NCT01827579). Methods Patients undergoing Alemtuzumab-based peripheral blood SCT from a 9/10 or 10/10 MUD for haematological malignancy were randomised 2:1 to receive either the ATIMP (ADTs) or standard of care. Two weeks prior to SCT, patients randomised to ATIMP underwent a leucapheresis from which DCs were generated. Irradiated patient-derived DCs were then co-cultured with peripheral blood mononuclear cells (PBMC) from an unstimulated leucapheresis/500ml blood draw from the donor to activate alloreactive T cells. Four days later, the co-culture was depleted of CD25+ and CD71+ fractions by immunomagnetic depletion on the CliniMACs, sampled for residual alloreactivity and sterility, and cryopreserved. Patients randomised to the ATIMP were scheduled to receive 3 escalating doses of ADTs (0.1x106/Kg at day 30, 0.3x106/Kg at day 60 and 1x106/Kg at day 90 post-SCT) until either there was >grade 1 aGVHD or they had normal circulating T cells (>700/µL). The primary end-point of the study was circulating CD3+ T cell count at 4 months post-SCT with one-sided 15% significance level. Acute/chronic GVHD were graded using the Seattle/NIH criteria respectively. Results Twenty one patients were treated, 13 on the ATIMP arm and 8 on the control arm. The median age was 53 years and 67% (14) were male. 12 were AML/Myelodysplasia, 5 NHL, 3 CLL/CML and 1 HL. The median follow-up time is 14 months. Five of 13 ATIMP patients received 1 dose of ADTs, 4/13 2 doses and 4/13 all 3 doses. The incidence of acute and chronic GVHD was comparable between the arms. Overall, 7/13 ATIMP patients developed significant (>Grade 2) acute GVHD compared to 4/8 of the control arm (p>0.99). 3/13 patients in the ATIMP arm and 2/8 patients in the control arm developed severe aGVHD (all Grade 3). Three of 13 ATIMP cohort patients developed chronic GVHD (1 mild, 1 moderate, 1 severe), compared to 3/8 (all mild) in the control cohort. At 4 months, the circulating CD3+ T cell count mean was 730/µL (range 10-4080) in the ATIMP group and 212.5/µL (range 10-500) in the control group (1-sided p=0.11). However, the data was not normally distributed (Wilcoxon 1-sided p=0.18). Three ATIMP patients had high CD3+ T cell count at 4 months (>1000/µL). At 6 months, the mean circulating CD3+ T cell count was 833.6/µL (range 20-2690) and 327.5/µL (range 10-860). At month 4, the mean PHA stimulation index in the ATIMP arm was 16.8 (range 0.67- 73.1) vs 3.8 (range 1.1-8.2) in the control group. At 4 and 6 months post-SCT, spectratyping analysis showed no evidence of a difference in Vβ diversity between the 2 arms in both CD4+ and CD8+ cells. The 1-year survival rate in the ATIMP cohort is 92% vs 88% in the control, and 1-year disease free survival rate 67% in the ATIMP cohort vs 70% in the control. Conclusions These data suggest that adoptive transfer of ADTs improves T cell reconstitution in some patients after MUD SCT and that the GVHD rates were similar between ATIMP and control groups. Figure 1: Kinetics of T cell recovery after transplant in ATIMP (blue) and Control (red) patients. Mean +/- SEM shown. Figure 1 Disclosures Peggs: Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees. Ghorashian:UCLB: Patents & Royalties: UCLB; Celgene: Honoraria; novartis: Honoraria. Amrolia:UCLB: Patents & Royalties.

MSI2 is highly expressed in human myeloid leukemia (AML) cell lines, and high expression of MSI2 mRNA is associated with decreased survival in AML, suggesting its use as a new prognostic marker. To test this, we measured MSI2 protein level by immunohistochemistry in 120 AML patients. Most cases (70%) showed some nuclear or cytoplasmic positivity, but the percentage of positive cells was low in most cases. Despite this, MSI2 protein expression was negatively associated with outcome, particularly for patients with good cytogenetic subgroup. For practical diagnostic purposes, the strongest significance of association was seen in cases with > 1% of cells showing strong MSI2 staining, these having a very poor outcome (P < .0001). Multivariate analysis with cytogenetic category, age, white cell count, and French-American-British subtype demonstrated that nuclear MSI2 levels were independently predictive of outcome (P = .0497). These results confirm the association of MSI2 expression with outcome in AML at the protein level and demonstrate the utility of MSI2 protein as a clinical prognostic biomarker. In addition, although positive at some level in most cases, its prognostic power derived from few positive cells, supporting its role in control of normal hematopoietic stem cell function and highlighting its role in disease progression.

Introduction: Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and non-relapse mortality in allogeneic haematopoietic stem cell transplant patients (Allo-HSCT). Extracorporeal Photopheresis (ECP) is now an established second-line treatment for steroid-refractory cGVHD. Here we report a large, multi-centre case series of 115 patients treated with ECP for cGVHD. To the best of our knowledge, this is the largest reported series of patients receiving fortnightly ECP for cGVHD. Method:A retrospective case note audit identified 115 consecutive Allo-HSCT patients who commenced fortnightly ECP for cGVHD between 2007 and 2016 at The Christie and Central Manchester NHS Foundation Trusts. cGVHD was classified and graded using the National Institutes of Health (NIH) consensus response criteria. Results: Median age when starting ECP was 49 years (range 18-75). 61% patients were male (n=70) and 39% Female (n=45). Underlying disease groups undergoing Allo-HSCT included Acute Leukaemia/Myelodysplastic Syndrome (n=69; 60%), Lymphoma (10%; n=12), Chronic leukaemia (13%; n=15) Myeloma (14%; n=16) and Aplastic Anaemia (3%; n=3). 24% of patients had myeloablative conditioning (n=27) and 76% had reduced-intensity conditioning (n=88). 61% (n=70) received stem cells from Voluntary Unrelated Donors (VUD) and 39% (n=45) Siblings (Sib). Patients received post Allo-HSCT GvHD prophylaxis with Calcineurin inhibitors, either alone, or in combination with Methotrexate and/or Mycophenolate Mofetil. Median time to stopping Calcineurin inhibitor post Allo-HSCT was 12 months (range 0-88). 14 patients received Donor Lymphocyte Infusions (DLI). Median time to development of GvHD post Allo-HSCT was 5 months (range 0-33). 94% developed cutaneous cGVHD (n=108), 32% oral cGVHD (n=37), 23% gut cGVHD(n=27), 24% liver cGVHD (n=28), 11% lung cGVHD (n=13) and 26% ocular cGVHD (n=30). 49% (n=57) patients had sclerodermoid disease. 37% (n=43) had one, 31% (n=36) two, 21% (n=24) three, 9% (n=10) four and 2% (n=2) five organ involvement. All 115 patients had been previously treated with immunosuppressive drugs - 109 Prednisolone; 73 Calcineurin Inhibitors; 60 Mycophenolate mofetil and other (ATG, n=1; Basiliximab, n=1; Methotrexate, n=6; Rituximab, n=10; and Thalidomide, n=18). Median duration of ECP treatment was 12 months (range 1-27 months). Response to ECP was assessed using NIH Criteria - 29% (n=33) Complete Response (CR), 19% (n=22) Partial Response (PR), 16% (n=18) Stable Disease (SD), 11% (n=13) Progressive Disease (PD), 17% (n=19) Death and 9% (n=10) Other. As a result of ECP treatment 26% (n=30) were able to stop steroid treatment while the remaining 74% were able to reduce their steroid dose. There was no overall difference in response between the two centres. Patients with cutaneous and oral cGVHD were more likely to achieve a PR or better (cutaneous p=0.03; oral p=0.05) but there was no difference with other organ involvement. The chance of response was not influenced by the number of organs involved. Median Overall Survival (OS) from the date of Allo-HSCT was 110 months (range 8-221). OS was not influenced by age, donor type, diagnostic group (Acute Leukaemia/Myelodysplastic Syndrome, Lymphoma, Chronic Leukaemia, Myeloma and Aplastic Anaemia), gender or the number and type of organs involved. The only exception was oral cGVHD which was associated with a lower OS (p=0.03). Patients with cGVHD achieving a CR/PR with ECP treatment, had a significantly better OS (40 months vs. median not reached, p Conclusion: ECP is an effective second line treatment for steroid-refractory and steroid-dependent cGVHD with a 50% response rate. This is not affected by the underlying diagnostic group or pattern of organ involvement, however patients with cutaneous and oral cGVHD seem to respond better. As response to ECP translates into better OS, this most likely reflects the reduction in immunosuppressive related complications. Therefore it is worth considering the use ECP treatment early in the management of cGVHD. Disclosures Routledge: Celgene: Honoraria; Jazz Pharmaceuticals: Honoraria; Gilead: Honoraria. Dignan:Jazz: Honoraria; Adienne: Speakers Bureau; Therakos/Mallinckrodt: Honoraria, Research Funding, Speakers Bureau; Gilead: Other: Cl for GvHD Study. Bloor:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria; GSK: Consultancy, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees. Tholouli:Johnson and Johnson: Speakers Bureau; MSD: Speakers Bureau; Celgene: Honoraria; Giles: Speakers Bureau; Amgen: Honoraria, Speakers Bureau.

Introduction: Alemtuzumab is used for in vivo T-cell depletion to reduce graft versus host disease in allogeneic Stem Cell Transplantation (SCT). Profound lymhotoxicity of this monocloncal antibody can potentially increase morbidity and mortality in SCT due to excessive viral infections and increased risk of graft rejection. We retrospectively analysed outcome of patients with myeloid disorders (acute myeloid leukaemia and myelodysplasia) who received in vivo T-cell depletion using Alemtuzumab based conditioning for allogeneic SCT over a period of 3 years in our centre. Methods: Patients were identified from department transplant data base. Data was collected for 73 consecutive patients over a period of three years using patient medical records, clinical work station and electronic patient records. The conditioning regimen included Fludarabine 30mg/m2 x5 (days -7 to -3), Alemtuzumab 10mgx5 (days -8 to -4) and Melphalan 140mg/m2 (day -2). Chimerism analysis was performed by polymerase chain reaction (PCR) to identify short tandem repeats within peripheral blood leucocytes and CD3 fraction. The quantification of donor chimerism was done by using gel photography system and LabWroks software. Viral testing was performed by PCR analysis. Results: Median age for the patients was 59 years (range 41-71). Median duration of follow up was 19 months (4-49 months). The majority of patients (67%) received SCT from a voluntary unrelated donor. Sixty four patients (88%) had AML whilst 9 had myelodysplasia. Sixty five (89%) patients were in morphological complete remission ( Conclusion: Our retrospective data show that Alemtuzumab based conditioning regimen in myeloid disorders have low incidence of GVHD, very low risk of graft rejection and comparable overall survival to those conditioning regimens which utilize in vivo T-cell depletion strategies other than Alemtuzumab. Moreover, a high incidence of viremia, in our cohort, did not translate into worse overall survival. Disclosures Saif: Novartis: Honoraria; Alexion: Honoraria. Dignan:Jazz pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Therakos: Honoraria, Speakers Bureau. Tholouli:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Introduction: The introduction of novel agentshas revolutionised the treatment of multiple myeloma, with improvements in survival of newly diagnosed and relapsed/refractory patients. However, despite these advances, the role for allogeneic haematopoietic stem cell transplantation (HSCT) in the treatment of these patients remains in question. Reduced-intensity conditioning (RIC) HSCT with Seattle regimen (30 mg/m2 fludarabine and 2 Gray total-body irradiation, TBI) for multiple myeloma has been reported to be an effective treatment with low upfront toxicity, and the potential for graft-versus-myeloma effect (Niederwieser D et al, Blood, 2003). Its use as part of tandem HSCT (autologous HSCT followed by RIC-HSCT) is associated with a treatment-related mortality (TRM) rate as low as 15% (Björkstrand B et al, J Clin Onc, 2011). However, data on survival following auto-HSCT/RIC-HSCT is inconsistent and no unifying consensus has been reached regarding the optimal timing of RIC-HSCT in the treatment algorithm. In this retrospective multi-centre study, we investigated the safety, tolerability and efficacy of RIC-HSCT in multiple myeloma patients in remission following previous auto-HSCT. Methods: A retrospective series was conducted for early-phase, high-risk myeloma patients who underwent RIC-HSCT following prior auto-HSCT in two tertiary transplant centres in Manchester in North West England, between November 2006 and July 2014. Results: Over the eight-year period, 42 myeloma patients (male n=29, female n=13) underwent Seattle-conditioned RIC-HSCT following prior auto-HSCT. 66.6% (n=28) were performed in tandem. Median age at RIC-HSCT was 52 years (range 41-65), and median β2-microglobulin at diagnosis was 3.1 mg/L (range 1.6-18.0). 16 patients had cytogenetics assessed at the time of diagnosis, of which loss of TP53 and translocation of chromosome 14 were most common. 37 patients (88.1%) had previously been exposed to immunomodulatory agents (IMiDs). 53.3% patients (n=24) were in first remission at RIC-HSCT, whilst 37.8% (n=17) were in second remission. 31.1% (n=14) of RIC-HSCTs were performed after second auto-HSCT following previous relapse and re-induction chemotherapy. 34 patients (55.9%) had achieved at least a very good partial response (VGPR) prior to RIC-HSCT. RIC-HSCT was performed a median of 20 months (range 10-89) from diagnosis. Two patients (4.8%) required re-transplantation for failed engraftment, but subsequently engrafted following in vivo T cell-depleting regimens. Mortality at early and interim time-points was low relative to registry and trial series, with TRM of 2.4% (n =1) and 9.5% (n=4) at day 100 and 365, respectively. 37 patients (82.2%) experienced chronic graft-versus-host disease (GVHD). Chronic skin GVHD was observed in 21 patients (grade 1-2: n=20; grade 3-4: n=1); chronic gastrointestinal GVHD in four patients (grade 1-2: n=1; grade 3-4: n=3); chronic hepatic GVHD in six patients (grade 1-2: n=4; grade 3-4: n=2); chronic oral GVHD in 17 patients (grade 1-2: n=17); and chronic ophthalmic GVHD in five patients (grade 1-2: n=5). There was an association between GVHD and enhanced disease control, as complete response rates increased from 26.5% to 51.5% by day 100 post-RIC-HSCT. However, 22 patients (48.9%) eventually relapsed with a median time to relapse of 6.5 months. 13 patients remain in CR one year post-RIC-HSCT. Relapse was treated with escalating donor lymphocyte infusions (DLI, n=13) or IMiDs (n=16). Response to lenalidomide at relapse was potentiated, despite the majority of patients having prior exposure to IMiDs. The median length of follow-up from diagnosis was 27 months (range 1-90). Median overall survival (OS) has not been reached; OS at two years was 69.2% (Figure 1). Conclusions: In this retrospective series we demonstrate the feasibility of Seattle-conditioned RIC-HSCT in multiple myeloma. We observed a very low early TRM of 9.5% and presence of allo-immune disease control (graft-versus-myeloma effect), thereby augmenting IMiD efficacy. Chronic GVHD was prevalent and relapse occurred in a significant proportion of patients, but salvage with DLI and IMiDs highlighted the efficacy of the graft-versus-myeloma effect elicited by RIC-HSCT. Figure 1. Survival outcomes after RIC-HSCT in myeloma. Median OS has not been reached, two-year OS was 69.2%. Figure 1. Survival outcomes after RIC-HSCT in myeloma. Median OS has not been reached, two-year OS was 69.2%. Disclosures Cavet: Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Speaker. Tholouli:Pfizer: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Speakers Bureau. Gibbs:Celgene: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Introduction: Qualitative and quantitative deficiencies in T-cell mediated immunity following allogeneic hematopoietic stem cell transplantation (HSCT) are associated with a high incidence of cytomegalovirus (CMV) infection, which remains an important cause of morbidity and cost, and contributes to mortality. Adoptive cellular therapy (ACT) can potentially expedite reconstitution of CMV-specific immunity and improve outcomes post HSCT. A number of uncontrolled studies have demonstrated proof of concept for such strategies. Previously we presented data from a randomized controlled trial assessing the effect of CMV-specific ACT (Cytovir CMV™) on immune reconstitution following unrelated donor HSCT (CMV~ASPECT). Now we report on a randomized controlled trial to evaluate the incidence of CMV reactivation and safety in a larger cohort of related donor HSCT patients treated with CMV-specific ACT. (CMV~IMPACT: ClinicalTrials.gov NCT 01077908). Methods: The study enrolled CMV-seropositive patients >16 years old, undergoing T-cell depleted (alemtuzumab) HSCT from a matched sibling CMV-seropositive donor, in 14 UK transplant centers. Patients and donors were selected using standard criteria. Patients were randomized into either ACT or control arms. CMV-specific cells were manufactured from a dedicated donor apheresis by direct selection at a central facility using either a StreptamerTM or gamma-capture technique (CliniMACS® Cytokine Capture System, IFN-gamma). Products were manufactured using Streptamers if the donor expressed a suitable HLA allele, or using gamma-capture if not. The 14 patients receiving the latter product are not included in this preliminary analysis. CMV surveillance was performed using quantitative PCR. All groups received standardized antiviral pre-emptive treatment. Cells were cryopreserved at a maximum dose of 5x104CD3+/kg and administered to the patient on day 27/28 post-transplant, regardless of CMV surveillance results (prophylactic administration). Patients with ≥ grade 2 acute GvHD or systemic steroid administration at baseline (D27/28) were withdrawn. Patients were assessed for CMV reactivation based on blinded retrospective assessment by the Chief Investigator. Patients were also assessed for evidence of GvHD and CMV-specific immune reconstitution. Results: As of August 2014, all but one patient had completed the study. There were no clinically apparent differences in serious adverse event (SAE) or acute GvHD events between the two arms. Notably, the number of patients experiencing >1 treatment episode was considerably lower than had been predicted in the control group (26% vs 60% predicted). There were fewer CMV reactivations in the ACT arm compared to the control arm (0.75 vs 1.0/patient), and fewer patients experiencing >1 treatment episode (15% vs 26%), although neither reached statistical significance. There was a trend toward reduced overall treatment duration in the ACT arm (Table 2, p=0.14). Preliminary data are summarized below. A complete and final analysis will be available at the time of the conference. Table1: Patient Disposition/Safety ACT Control Enrolled (withdrawn prior to baseline visit) Safety set Per protocol set (no ACT administered in 10 randomized patients) 40 (10) 30 20 35 (4) 31 31 Reasons for withdrawal · GvHD or steroids at baseline · Other 2 8 2 2 No. (%) with new onset acute GvHD (safety set; 100 day follow up) 2 (6.7) 1 (3.2) No. (%) with at least 1 SAE (safety set) 12 (40.0) 10 (32.3) Table2: Duration of CMV Treatment (days; per protocol set) ACT (n=20) Control (n=31) p Mean (stdv) 19.1 (27.8) 27.3 (31.3) 0.14 Median (min:max) 11 (0 : 114) 25 (0 : 133) Conclusions: In this randomized trial of CMV specific T-cells post-HSCT, adoptive cell therapy (Cytovir CMV™) was not associated with a significant increase in safety events, and preliminary analysis indicates Cytovir CMV administration may be associated with lower rates of CMV reactivation and shorter durations of viral treatment, though the lower than predicted reactivation rate in the control group reduced the power of the trial to demonstrate this at a statistically significant level. Disclosures Cobb: Cell Medica Ltd: Employment. Newton:Cell Medica Ltd: Employment. Thomas:Cell Medica Ltd: Employment. Moss:Cell Medica Ltd: Membership on an entity's Board of Directors or advisory committees.

Background Respiratory syncytial virus (RSV) is a common cause of respiratory viral infections and is associated with increased morbidity and mortality in patients undergoing haematopoietic stem cell transplantation (HSCT). Little is known about the economic burden associated with RSV infection. We sought to analyse the readmission rates and costs associated with RSV infection in a single UK centre. Patients and method We undertook a retrospective case analysis of 48 consecutive patients diagnosed with RSV infection post HSCT between December 2010 and April 2014. The frequency of infection was 35/227 (17%) in allogeneic HSCT and 13/180 (7%) in autologous HSCT recipients. Patient characteristics: male 28, median age 55 (range 23 to 71), diagnosis: acute leukaemia 15, multiple myeloma 16, myelodysplasia 6, aplastic anaemia 5, chronic leukaemia 3, other 3. In patients who had received an allogeneic transplant 22 had reduced intensity conditioning (RIC) regimen and 13 myeloablative conditioning. One patient received a cord blood transplant (CBT), 14 had a sibling allograft, whereas 20 had a volunteer unrelated donor (VUD) HSCT. In 20 patients, Alemtuzumab was part of the conditioning regimen. Twenty-seven patients were on immunosuppressive drugs (ciclosporin, mycophenolate mofetil, tacrolimus or corticosteroids) at time of diagnosis and 15 had active graft versus host disease (GVHD). At diagnosis, 37 patients were lymphopenic with lymphocytes below 1.5x10e9/L. Diagnosis was established by polymerase chain reaction (PCR) assay for respiratory viruses and was performed as part of routine screening and in patients with respiratory symptoms. At diagnosis, 26 patients presented with lower respiratory tract infection (LRTI) as defined by new infiltrate on chest X-ray (CXR), signs on auscultation or hypoxia, whereas 22 experienced upper RTI. A CXR was performed in 39 patients and revealed infective or inflammatory changes in 12 patients, all with LRTI signs. The primary indication for treatment was LRTI signs. Patients who were felt to be at high risk of progression to LRTI also received therapy. In total, 42 patients received Ribavirin: orally 16; aerosolised 17; aerosolised and orally combined 5; aerosolised and IV combined 4. Six patients received no treatment. The median duration of treatment was 7 days (range 5 to 47 days). Additional therapy was given: 13 patients received immunoglobulin replacement, 33 had concomitant antibiotic treatment and 11 also received antifungal treatment. Results Ribavirin was well tolerated with mild side effects associated with aerosolised administration: claustrophobia 2, headaches 1, nausea 1. Twenty patients were admitted for treatment of RSV infection, 6 of which required intensive care unit (ICU) admission including 4 who required invasive ventilation. Fifteen patients were treated as out-patients. Median length of ward stay was 10 days (range 4 to 55 days). ICU admission was associated with a median stay of 5 days (range 4 to 60 days). An additional 13 patients were diagnosed as in-patients and RSV was not felt to have extended their hospital stay. After a median follow-up period of 6.5 months (range 0-39): 33 patients are alive, 15 patients died. The causes of death were sepsis 3, relapsed disease 3, GVHD 2, gastrointestinal bleed 1, stroke 1. In 5 patients the cause of death was attributed to RSV infection, 4 having had previous ITU stay. No RSV-related deaths were recorded in the group treated with oral Ribavirin. Admission costs were calculated based on the 2014 hospital tariff. Median cost was £3,700 for ward stay (range £1,480 to 20,350) and ICU admission with a median cost of £7,340 (range £5,872 to 88,080). The median cost of aerosolised ribavirin (6 g/day 2014 pharmacy prices) was £800 (range £570 to 1,596). The median cost of oral ribavirin (1000 mg/day based on 70kg) was £98 (range £28 to 658) and for the IV formulation median cost was £7,920 (range 3,394 to 12,445). Conclusions RSV in post HSCT patients remains a challenge due to the high frequency of infection and high rates and costs of readmission. Oral ribavirin may be an option for lower risk patients and strict isolation of patients in waiting areas to prevent transmission could be a cost effective measure. Prompt initiation of treatment in high risk patients is essential due to increased morbidity and mortality associated with RSV- associated LRTI. Disclosures No relevant conflicts of interest to declare.

Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal growth and accumulation of tissue mast cells (MC) in various organ systems, including bone marrow (BM). Indolent and advanced forms of SM have been described. Whereas patients with ISM have a normal or near normal life-expectancy, patients with advanced SM, including those suffering from mast cell leukemia (MCL) have a poor prognosis. In these patients, neoplastic MC are usually resistant against conventional drugs and various targeted drugs. In rapidly progressive aggressive SM (ASM) and MCL, polychemotherapy followed by allogeneic hematopoietic stem cell transplantation (alloHCT) has been proposed. However, outcome of alloHCT in advanced SM is unknown, and it also remains uncertain whether clinically relevant graft-versus-SM (GVSM) effects may occur in these patients, as only sporadic case reports have been published. We performed a retrospective multi-center analysis to evaluate the outcome of alloHCT in patients with advanced SM. Fifty-four advanced SM patients receiving SCT in 32 transplantation centers in Europe and America were identified between 1990 and 2013. The median patient age was 45 years. Donors were: HLA identical siblings (31), unrelated donors (URD) (15), umbilical cord blood donors (UCB) (2), and haploidentical donors (1). In 5 patients, stem cell source was not defined (5). Thirty-four patients received myeloablative conditioning (MAC) and 18 received reduced intensity conditioning regimens (RIC). In 2 patients, conditioning regimen was not specified. Indications for alloHCT were SM with an associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) (n=32), MCL (n=13, including one with MCL-AHNMD), 8 with ASM and 1 with myelomastocytic leukemia (MML). The most prevalent AHNMD was acute myeloid leukemia (AML, n=16). With follow-up of 35-6180 (median 365) days, SM responses (defined as ≥50% decrease in BM mast cells ± decrease in serum tryptase ± regression of other organ manifestations) were observed in 39 patients (72%), including complete responses (CR) documented in 12 patients (22%). Eleven patients had stable disease, whereas 4 patients (7%) progressed immediately after alloHCT (primary resistance). In addition, 10 patients progressed (5 of them within 100 days) after an initial response. Progression was most frequently seen in MCL patients (n=6, 50%). In the AHNMD group, only 8 patients relapsed/progressed (25%). The overall survival (OS) and SM progression-free survival (PFS) at 1 year were 63% and 50% for all patients, 77% and 68% for SM-AHNMD, 63% and 50% for ASM, and 25% and 17% for MCL, respectively. The strongest predictive variable associated with inferior survival was a diagnosis of MCL. Other factors associated with poor outcome were: Karnofsky performance status ≤70%, ≥2 SM regimens given before alloHCT (e.g., steroids, cladribine, chemotherapy, tyrosine kinase inhibitor), donor source (alternative donors-UCB and haploidentical compared to sibling or URD), SM progression within the first 100 days, normal cytogenetics (compared to t(8;21) (q22;q22), and RIC (compared to MAC). The following variables were not associated with poor outcome: patient and donor age, recipient-donor sex match status, graft source (BM vs. peripheral stem cells), BM mast cell percentage at time of alloHCT, and CR status of AML or SM response at time of alloHCT. This largest multi-center analysis of results in advanced SM provides evidence for clinical efficacy of alloHCT, presumably because of a GVSM effect of alloHCT (achieving CR, and response to donor-lymphocyte infusions and RIC alloHCT). However, responses varied among different SM categories: while patients with SM-AHNMD enjoyed excellent outcomes, the OS for MCL patients in general, was poor. Nevertheless it is remarkable that 3 of 13 patients with MCL – an otherwise fatal disease with a median survival of Disclosures: Vercellotti: Sangart Inc.: Research Funding; Seattle Genetics: Research Funding. Akin:Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding.

Abstract 1441 Introduction: Hedgehog (Hh) signaling is essential in many developmental pathways and a growing body of evidence supports a role for aberrant activation of the Hh pathway in a variety of hematological malignancies including Acute Myeloid Leukemia (AML). Primary CD34+ myeloid blasts and cell lines preferentially demonstrate active Hh signaling suggesting that the Hh pathway may be involved in leukemic stem cell maintenance and survival (Bai et al. Leukemia. 2008;22(1):226-8). In addition, disruption of Hh signaling by targeted agents in this setting results in cell death and confers sensitivity to Ara-C in chemoresistant CD34+ leukemic cell lines (Kobune et al. Cancer Science. 2009;100(5):948-55). These findings provide a rationale for targeted disruption of the Hh pathway in AML and imply that the identification of Hh signaling in AML could correlate with disease that is refractory to standard induction therapy. Methods: To extend these previous findings, the presence of Hh signaling was assessed by the identification of nuclear GLI1 by immunofluorescence in tissue microarrays (TMA) prepared from a large cohort of primary human AMLs and the data correlated with clinical outcome. Presentation bone marrow trephine samples from patients diagnosed with AML between 1994 and 2005 were retrieved from the histopathology archives at Manchester Royal Infirmary. These biopsy samples were routinely processed, formalin-fixed, paraffin-embedded, and EDTA-decalcified. All material used for preparation of the TMA was residual diagnostic tissue, anonymized and consented for its use in research. Each TMA core contained at least 20% leukemic blasts and was representative of the whole biopsy sample. A specific and highly sensitive human GLI1 immunofluorescence assay was developed to visualize nuclear GLI1in four micron thick TMA sections. Human embryonic palatal mesenchymal (HEPM) cells stimulated with SHH and treated with the Hh antagonist saridegib (IPI-926) were used as staining controls to demonstrate assay specificity. To determine the cellular context of GLI1 staining in AML, a GLI1/CD34 double immunoflourescence assay was also developed. All samples were scanned in using the TissueGnostics TissueFAXS 2.0 system. The Cy5 channel was used to detect nuclear GLI1 and the Texas Red channel was used to detect CD34 when double staining was employed. A nuclear algorithm was developed to analyze the GLI1 staining using the TissueQuest version 2.0 software. Results: Of the 160 patient samples stained, 71 samples were evaluable after filtering on the basis of the presence of adequate tissue cores on the TMA and a full set of clinical follow-up data. All patients (36 male, 34 female, 1 unknown; age range 17 to 83 years at diagnosis with a median age of 53 years) received intensive chemotherapy according to standard UK MRC AML protocols and had follow-up data for up to 5124 days. Nuclear GLI1 as a function of total marrow cells showed a wide range of expression (0.0–32.0%) with a mean of 4.8% and a median of 3.1%. In a subset of cases with CD34 positive blasts, nuclear GLI1 co-expression could be readily identified as coexpressed in the CD34 positive cells. The degree of GLI1 expression did not correlate with AML subtype or cytogenetics. Correlation of GLI1 positivity with clinical features was performed and while no association with overall survival was observed in the total population, there was a significant decrease in overall survival in patients with secondary AML that had GLI1 levels above the median (HR = 0.18; p value = 0.0112). In addition, there was a significant correlation between GLI1 either above or below the median and remission status (i.e. complete remission, partial remission or refractory disease) with higher GLI1 levels observed in patients with refractory disease (p value = 0.045) and a more significant correlation with GLI1 values as a continuous variable and remission status (p value = 0.026). Conclusions: These data suggest that high levels of GLI1 predict for poor remission status in patients with AML and poor overall survival in patients with secondary AML. The presence of high levels of nuclear GLI1 in AMLs that are refractory to chemotherapy supports a role for Hh signaling in chemoresistance, particularly in patients with secondary AML, and provides a rationale for targeted inhibition of the Hh pathway in selected AML patients. Disclosures: Campbell: Infinity Pharmaceuticals, Inc.: Employment. Quigley:Infinity Pharmaceuticals, Inc.: Employment. Keilty:Infinity Pharmaceuticals, Inc.: Employment. Kelly:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. Read:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.

Abstract 1405 Despite improvements in the treatment of AML, high-risk disease including 8p11 myeloproliferative syndrome/stem cell leukemia remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. Based on the results of a genome-wide functional screen in AML using a retroviral library of short hairpin RNAs (shRNAs), we discovered that hepatocyte growth factor (HGF) is aberrantly expressed in about 40% of patients with AML, leading to activation of its receptor MET. Autocrine activation of MET is required for survival of AML cells, as genetic and pharmacologic inhibition of HGF or MET induces apoptosis and inhibits cell growth. However, AML cells treated for more than one week with the MET kinase inhibitor crizotinib develop resistance as a result of 13-fold upregulation of HGF expression, leading to restoration of MET pathway activity, and emphasizing autocrine HGF production as the basis for MET signaling addiction. Using simple thermodynamic models, we find that ligand-mediated receptor activation due to increased ligand expression effectively antagonizes the effects of targeted kinase inhibitors, causing complete restoration of aberrant signaling activity. Here we show that this mechanism of resistance can be overcome by combining MET kinase inhibition with depletion of HGF or targeting of pathways required for compensatory HGF upregulation. In the case of KG-1 cells derived from a patient with 8p11 myeloproliferative syndrome AML with chromosomal translocation mediated activation of FGFR1, we find that signaling downstream of the FGFR is required for the upregulation of HGF that occurs in response to MET kinase inhibition. Sustained MET inhibition due to the blockade of compensatory HGF upregulation leads to durable logarithmic cell kill as a result of combined treatment with the MET kinase inhibitor crizotinib and FGFR kinase inhibitor PD173074. In addition, we find that aberrant expression of HGF is highly correlated with expression of FGF2 (Pearson's correlation = 0.79, p < 0.001), with 10% of all patients exhibiting co-expression of HGF and FGF2. This co-expression leads to autocrine co-activation of MET and FGFR1, which can be effectively targeted using combined treatment with MET and FGFR kinase inhibitors. In all, these findings suggest a therapeutic strategy for patients with co-activation of MET and FGFR, including patients with 8p11 myeloproliferative syndrome AML, establish a paradigm of effective therapeutic targeting of aberrant HGF/MET signaling in AML, and emphasize the importance of targeting compensatory ligand-dependent receptor tyrosine kinase activation, as required for the development of rational combination therapy. Disclosures: Christensen: Pfizer: Employment. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding.

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.

Abstract 1042 Despite improvements in the treatment of acute myeloid leukemia (AML), high risk disease such as complex aberrant karyotype AML remains largely refractory to current therapy, and is mostly fatal. Identification of effective therapeutic targets by using candidate gene approaches has been limited by the number and variety of genetic defects associated with AML. Thus, we carried out a genome-wide functional screen in complex karyotype AML using a retroviral library of short hairpin RNAs (shRNAs), and discovered that shRNA-mediated depletion of hepatocyte growth factor (HGF) specifically inhibits growth of AML cells but not a panel of lymphoid cancer cells. HGF was to found to be aberrantly expressed in about 15% of patients with AML, including most patients with complex karyotype disease. In contrast to normal CD34+ cells that express MET (but not HGF), 5 of 7 cell lines derived from patients with complex karyotype AML exhibited aberrant expression of HGF that was associated with autocrine activation of its receptor MET. Depletion of HGF or MET using multiple independent shRNAs profoundly reduced proliferation and induced cell death in AML cells lines that express HGF but not those that lack HGF expression. Inhibition of MET using the tyrosine kinase inhibitor (SU11274) or HGF using neutralizing anti-HGF antibody (R&D Systems) also inhibited growth and induced apoptosis in AML cell lines dependent on HGF/MET activation but not those that lack HGF expression. Thus, aberrant HGF expression causes autocrine MET activation and oncogene dependence in a subset of patients with AML, confers sensitivity to HGF/MET inhibition, and provides a novel therapeutic target for this otherwise lethal disease. Disclosures: No relevant conflicts of interest to declare.

Microarray-based expression profiling has identified prognostic gene signatures for many cancers and validation is required in clinical samples. However, most clinical material is in the form of formalin fixed and paraffin embedded tissue (FFPET) in which gene expression analysis is problematic. We have developed a generic quantum dot (QD) based multiplexed in-situ hybridization (ISH) method enabling quantitative localization of multiple mRNA targets in FFPET. We expand on our previous work introducing a method for standardization of ISH signal, enabling comparative measurement of gene expression across multiple samples. This was applied to tissue microarrays (TMAs) using archived trephine biopsies from patients with acute myeloid leukaemia (AML) to identify prognostic genes. A total of 15 TMAs were prepared using FFPET samples from 240 patients with AML diagnosed and treated between 1994 and 2005 at Manchester Royal Infirmary (Manchester, UK). For the analysis, 192 patients were included as the remainder either died before, during or immediately after one course of chemotherapy or there was incomplete data collection. The median age was 52 years (range 17–77) and all patients received intensive chemotherapy according to standard UK MRC AML protocols. Three cores were taken from each sample for TMA preparation. A standard was prepared using a cell pellet obtained from whole blood white cells which was embedded, in triplicate, in each TMA. QD-ISH was performed for nine genes recognized to be of prognostic value in AML. Triplex QD-ISH using QD labeled anti-sense cDNA oligonucleotides was performed for the following targets: Bcl2, survivin and XIAP; DNMT1, DNMT3A and DNMT3B; HOXA4, HOXA9 and Meis1. Signal intensity for each gene was measured using spectral imaging. Scrambled sense cDNA oligonucleotides were used to measure the level of background staining for each gene in each core. Background noise was corrected for by dividing expression levels of anti-sense probes by that of the scrambled probe, for both samples and standards. This enabled direct comparison between TMAs as gene expression values of samples were normalized against the standard. The mean expression of each gene was calculated for each patient, divided into quartiles and correlated with clinical outcome data. Statistical analysis was performed using contingency tables, the chi-square test and Mann Whitney-U. Overall survival (OS) and disease free survival (DFS) were displayed using the Kaplan-Meier method and Cox regression was performed for univariate and multivariate analysis. The OS in this cohort of patients was 43% at 5 years with 80% achieving complete remission (CR) after induction chemotherapy. Patient age (

Quantum dots (QDs) are fluorescent semiconductor nanocrystals (2–10-nm core diameter) possessing the unique properties of extremely high fluorescence efficiency, lack of photobleaching and long fluorescence lifetime, making them an ideal tool for bioimaging. We have developed a novel technique for in situ hybridisation (ISH) using biotinylated oligonucleotides conjugated to streptavidin coated QD, and used them in this study to label bone marrow trephine samples. 50-mer long oligonucleotide probes were conjugated to QDs prior to ISH and conjugation efficiency was demonstrated by gel electrophopresis. ISH conditions and molar ratio of QDs to probe were optimised using a polyT probe. Images were captured using a CRI Nuance spectral imaging system and signal intensity was semi-quantitated using IPLab software. Specific oligonucleotide hybridisation was demonstrated using a probe for myeloperoxidase (MPO) in 40 bone marrow sections infiltrated by acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) as well as reactive bone marrow. In each case hybridisation signal was consistent with the distribution of MPO by standard immunohistochemistry - MPO was strongly expressed by myeloid blasts and absent in lymphoid blasts; in CML, most, but not all, cells were positive for MPO, in comparison to many fewer positive cells in reactive marrow. Duplex ISH was demonstrated using a probe for bcl-2 together with MPO in 5 bone marrow sections infiltrated by follicular lymphoma (FL). Strong hybridisation signal for bcl-2 was detected in all cells of the paratrabecular aggregates of FL but showed only scattered positivity in the remainder of the bone marrow. Conversely, MPO was absent in the paratrabecular aggregates and present in the myeloid cells in the remainder of the marrow. This pattern was present in both single and duplex ISH for MPO and bcl-2 in the marrow infiltrated by FL. Duplex ISH was performed both by sequential hybridisation with bcl-2 followed by MPO, and simultaneously with a mix of bcl-2 and MPO probes. As negative controls, scrambled oligonucleotide probes for the corresponding genes were used in each case and did not show hybridisation. In summary, we have developed a generic method for QD labelling and semi-quantitative detection of oligonucleotide ISH in routinely processed clinical tissue samples. Although, in this study we primarily used bone marrow trephine samples, this technique can be applied to any tissue. It has the potential to facilitate transfer of microarray-identified gene signatures to clinical research and diagnostics, whilst the ability of spectral imaging to resolve multiple signals offers the possibility of multiplexed probe detection.