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The mechanism of tumor-selective replication of oncolytic measles virus (MV) is poorly understood. Using a stepwise model of cellular transformation, in which oncogenic hits were additively expressed in human bone marrow-derived mesenchymal stromal cells, we show that MV-induced oncolysis increased progressively with transformation. The type 1 interferon (IFN) response to MV infection was significantly reduced and delayed, in accordance with the level of transformation. Consistently, we observed delayed and reduced signal transducer and activator of transcription (STAT1) phosphorylation in the fully transformed cells. Pre-treatment with IFNβ restored resistance to MV-mediated oncolysis. Gene expression profiling to identify the genetic correlates of susceptibility to MV oncolysis revealed a dampened basal level of immune-related genes in the fully transformed cells compared to their normal counterparts. IFN-induced transmembrane protein 1 (IFITM1) was the foremost basally downregulated immune gene. Stable IFITM1 overexpression in MV-susceptible cells resulted in a 50% increase in cell viability and a significant reduction in viral replication at 24 h after MV infection. Overall, our data indicate that the basal reduction in functions of the type 1 IFN pathway is a major contributor to the oncolytic selectivity of MV. In particular, we have identified IFITM1 as a restriction factor for oncolytic MV, acting at early stages of infection.

The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance., How the bone marrow microenvironment evolves during induction chemotherapy to facilitate acute lymphoblastic leukaemia (ALL) survival remains unclear. Here the authors show that adipocytes emergent during ALL therapy aid the survival of ALL cells through a restrictive effect on the ALL proteome.

Lymphoblastic lymphoma (LBL) is a rare, malignant disorder of precursor T- or B- cells, which forms the lymphoma variant of acute lymphoblastic leukemia (ALL). Whilst morphologically and immunophen...

Patients with an ABL-class fusion have a high risk of relapse on standard chemotherapy but are sensitive to tyrosine kinase inhibitors (TKI). In UKALL2011, we screened patients with post-induction MRD ≥1% and positive patients (12%) received adjuvant TKI. As the intervention started during UKALL2011, not all eligible patients were screened prospectively. Retrospective screening of eligible patients allowed the outcome of equivalent ABL-class patients who did and did not receive a TKI in first remission to be compared. ABL-class patients who received a TKI in first remission had a reduced risk of relapse/refractory disease: 0% vs. 63% at four years (P = 0·009).

Introduction: Recent developments in management of patients with myeloma have resulted in longer survival. Availability of newer class of agents for treatment has prolonged survival, improved quality of life and achieved better disease control. Use of SCT, either autologous (AutoSCT) and to a lesser extent allogeneic (AlloSCT) still forms an important aspect of myeloma treatment pathway. Improved survivorship mandates evaluation of long term consequences and risk of SMN is one of the most important long-term consequences affecting overall outcome. Aim: Estimate the risk of SMN post SCT in patients with Myeloma. Methods and Materials: Analysis includes 779 patients who received SCT for myeloma from January 2002 to December 2019. Data was collected using case records, electronic patient records, transplant database and information from referring hospitals. Follow-up was updated to June 2020. Results: Analysis includes 779 patients with myeloma (M: 488, F:291; median age:59yr, range: 26-75) receiving AutoSCT (n=716) or AlloSCT (n=63). Conditioning for AutoSCT was high dose melphalan in majority (n=714, 99.7%). AlloSCT conditioning was RIC (n=40/63, 63.5%) or MAC (n=23/63, 36.5%). TBI was part of conditioning in 51/779 (6.5%). There was no difference in demographics between two groups. Results: Median follow-up was 46 mo (range: 0.2-220). Second malignancy was identified in 48 of 779 cases (6.0%). SMN developed in 6/63 (10%) AlloSCT and 42/716 (6%) AutoSCT patents (p=0.25). SMN types were haematological (n=21), lymphoma (n=6) and solid tumours (n=21). MDS was the only haematological SMN in this series. Secondary MDS developed in 20/21 of AutoSCT as compared to 1/63 in AlloSCT (p=0.01). Non-haematological SMN were breast (n=3), upper GI (n=3), lower GI (n=2), hepato-billiary (n=2), prostate (n=3), skin (n=5), and unknown primary (n=1). SMN incidence was higher with increasing follow-up (2002-2014, 8%; 2015-2017, 2.8%, 2018-2019, 0%, p=0.002). There was no association with development of SMN and gender, age at transplant, type of stem cell source or transplant conditioning. Incidence of SMN was significantly higher for all sites as compared to general population. Cumulative incidence of SMN was 5% at 5 yr, and 15% at 10 yr for AutoSCT patients. Conclusion: Myeloma patients are at increased risk of SMN and need monitoring for long term side effects. Development of MDS post AutoSCT is the commonest SMN post AutoSCT but there is increased incidence of solid tumours as well. Impact of new modalities of treatment needs long term monitoring. Disclosures Bloor: Novartis: Honoraria; Kite, a Gilead Company: Honoraria.

Use of Cryopreserved Allogeneic PBSC Results in Delayed Engraftment And Increased Incidence of Poor Graft Function Introduction: During COVID Pandemic, national and international transplant centres agreed to use cryopreserve the donor PBSC as a safer option to deliver allogeneic transplants. Published data suggests that use of cryopreserved allogeneic PBSC is safe and comparable to use of fresh PBSC but cryopreservation of stem cells may lead to cell loss and hence efficacy. During COVID pandemic, use of cryopreserved allogeneic PBSC was adopted as policy on 01/06/2020. This look back analysis evaluates the impact of change in policy. Aims: Evaluate Engraftment time, compare with historical data, blood component support, and use of growth factors Methods and Materials: Data was collected from health records (paper and electronic) and laboratory records. Transplant features and engraftment kinetics were analysed. Results: Group A June 2020 to November 2020, 19 patients [M: 13; F: 6; median age: 50yr (range: 23-69)] who received cryopreserved allogeneic PBSC were compared to 35 patients [M:24; F:11; median age: 59yr (range: 21-71)] receiving fresh allogeneic PBSC for engraftment kinetics. There were no differences between two groups regarding underlying diagnosis (p=0.31), sex mismatch, CMV mismatch, blood group mismatch, reduced intensity conditioning [RIC](p=0.28), type of donor (p=0.98) or use of Alemtuzumab (p=0.88). Median infused Cell dose in group A was 5.3 (3.4-7.16) and in group B 4.9 (1.03-6.85), [p=0.11]. Neutrophil engraftment was significantly faster with fresh PBSC as compared to cryopreserved PBSC (16d vs. 25d, p=0.0025) predominantly with MUD (18d vs. 27d, p=0.009) and RIC (16d vs. 25d, p=0.009). Platelet engraftment to 25 was faster with fresh PBSC (13d vs. 20d, p=0.021) with delayed engraftment in MUD (20d vs. 13d, p=0.006) and RIC (23d vs. 13d, p=0.039). Day to engraftment per unit CD34 was shorter with fresh PBSC for neutrophils (median: 3.2, range: 2.0-7.7 vs. 5.3, range: 2.5-16.7; p=0.006) and platelets (median: 2.4, range: 1.7-25 vs. 3.8, range: 2.2-25; p=0.001) but only for MUD. This suggests 35-40% less efficiency with use of cryopreserved PBSC. There was no difference in the need for transfusion support [RBCs (6 units vs. 3 units, p=0.32); platelets (5 pools vs. 7 pools, p=0.33)]. G-CSF use was higher with cryopreserved PBSC in RIC (54% vs. 20%, p=0.031). Two patients experienced TRM before day 90 (3.7%). At day 90, 17/52 (32.7%) had cytopenia in one lineage and 8/52 (16%) had cytopenia in more than one lineage. Delayed engraftment was observed in 10 of 33 patients (30.3%) transplanted in 2020 and the only significant association was use of cryopreserved PBSC (0% vs. 53%, p=0.001). There was no difference in the incidence of aGVHD, hepatic VOD, microangiopathy and bacterial infections. Numbers are not sufficient to make disease specific comparisons. Conclusion: Cryopreserved PBSC result in delayed neutrophil and platelet engraftment predominantly with MUDS and RIC. Incidence of delayed engraftment and poor graft function is higher. Per unit CD34 dose, cryopreserved PBSC are 30-40% less efficient to achieve engraftment. Delayed engraftment with cryopreserved PBSC especially in MUD raises the possibility that time from harvest to cryopreservation contributes to reduced efficacy. Based on these findings it was decided to infuse higher CD34 dose (6-7x10^6/kg as compared to usual 4-5x10^6/kg) for cryopreserved MUD PBSC. Disclosures Bloor: Kite, a Gilead Company: Honoraria; Novartis: Honoraria.

Background: Tisagenlecleucel was approved in Europe for relapsed/refractory acute lymphoblastic leukemia (ALL) in young patients in 2018. In England, a national CAR T cell panel (NCCP ALL) ensures equity of access and assesses eligibility using inclusion and exclusion criteria based on the ELIANA study. All UK cases are discussed. Eligible cases are allocated to one of 9 JACIE-FACT IEC-approved centres based on age-appropriate service, capacity, distance and patient preference. We systematically reviewed all cases from panel inception providing an opportunity to analyse complete real-world ALL outcomes from this national access programme for tisagenlecleucel on an intention-to-treat (ITT) basis. Methods: We included all patients discussed in the NCCP ALL from Nov 2018 up to July 16th 2020. Clinical data were retrospectively reported in a standardised dataset to allow central analysis of disease and toxicity-related outcomes. Survival outcomes were assessed as for other CAR studies, using Kaplan-Meier estimates of survival from infusion. These included overall survival (interval to death from any cause) and event-free survival as defined in the ELIANA study (interval to death, disease relapse, or treatment failure defined in turn as failure to respond by day 30, with patients receiving further therapy being censored). In order to report outcomes on an ITT basis, survival outcomes were also assessed from time of treatment allocation for all patients considered eligible for tisagenlecleucel. Further, a more comprehensive event-free survival measure encompassing interval to molecular or frank relapse, further therapy, B cell recovery, death or treatment failure was analysed. Median follow up was calculated using a reverse Kaplan-Meier method. Results: Figure 1 demonstrates all patients: 66 patients were screened, 60 patients were deemed eligible for therapy (the ITT cohort), 57 patients were harvested and 49 infused. A total of 3 patients were not harvested and 2 not infused because of progressive disease (n=4) and 1 manufacturing failure. Patient and disease characteristics are summarised in Table 1. The cohort comprised patients with advanced ALL, having been treated with a median of 2.5 therapy lines not including HSCT (range 1-6), and with 47.5% of the cohort having had a prior HSCT. The median follow-up from infusion was 9.9 months and from treatment allocation for the ITT cohort was 11.9 months. The median lead time from allocation to infusion was 2 months. The CR/CRi rate was 95% in the first 90 days, 78.9% were MRD-. On an ITT basis, CR/CRi rate was 84.8%. Median OS and EFS as defined in the ELIANA study were not reached. Overall survival at 6 and 12 months for infused patients was 97.6% and 86.1%, and for the ITT cohort was 90.4% and 78.3%. EFS for infused patients at 6 and 12 months were 74.8% and 68.2%, and for the ITT cohort were 78.5% and 60.9%. Of 49 patients infused, 14 (28.5%) received further therapy including 6 (8.2%) who received allogeneic SCT for relapse or B cell recovery. For patients relapsing following CR (n=10 infused) there were 4/10 CD19- relapses. A composite EFS including molecular or frank relapse, further therapy, B cell recovery, death or treatment failure was analysed. Using this more stringent definition from infusion, the 6, 12 month EFS were 47.6%, and 33.6% and from treatment allocation for the ITT cohort were 63% and 34.3% Toxicity outcomes are summarised in Table 2. Severe (grade ≥3) CRS, neurotoxicity, infection or cytopenia after day 30 post infusion occurred in 20.4%, 10.2% 27.1% and 54.2% respectively. 71.4% of the cohort developed hypogammaglobulinaemia, (Figure 3A). The 6 and 12 month probabilities of B cell depletion were 67.1% and 51.3% respectively. Conclusions: Whilst registry data provide outcomes of large cohorts receiving Tisagenlecleucel, standardised data collection on complete populations are lacking. The framework of a national access scheme provides a unique opportunity to study outcomes on an ITT basis. The CR and MRD negative CR rates, as well as conventional EFS and OS and severe toxicities noted in our cohort compare favourably to published registry reports (Grupp et al., 2019). We found a greater proportion of CD19+ compared to CD19- relapses than noted in the ELIANA study. Consideration of OS and EFS on an ITT basis are informative for clinicians when screening patients for eligibility. If selected for presentation at ASH 2020, updated data will be presented Disclosures Ghorashian: Amgen: Honoraria; Novartis: Honoraria; UCLB: Patents & Royalties. O'Reilly:Gilead: Honoraria; Novartis: Honoraria, Other: Travel support. Roddie:Celgene: Honoraria; Gilead: Honoraria; Novartis: Honoraria. Neill:Novartis: Other: Funded attendance at academic conferences; Celgene: Other: Funded attendance at academic conferences. Pagliuca:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Patel:Novartis: Honoraria, Other: travel support. Hough:Novartis: Other: Travel support.

The mechanism by which oncolytic measles virus (MV) kills cancer cells remains obscure. We previously showed that neutrophils are involved in MV-mediated tumor regressions and become activated, upon MV infection. In the present study, we attempted to enhance the neutrophil response toward MV-infected tumor targets by generating an oncolytic MV-expressing human granulocyte colony-stimulating factor (MVhGCSF). Evaluating the effects in two different models of B-cell malignancy, we showed that depletion of neutrophils abrogated the MV therapeutic effect in an in vivo Raji-but not Nalm-6 tumor model. Next, we compared MVhGCSF with the unmodified backbone virus MVNSe. MVhGCSF enhanced the oncolytic capacity of MV in the Raji model in vivo, whereas in the Nalm-6 model, the opposite was unexpectedly the case. This finding was recapitulated by exogenously administered hGCSF. MVhGCSF replicated within an MV-infectable CD46 transgenic mouse model with detectable serum levels of hGCSF but no toxicity. Our data suggest that a "one-size-fits-all" model of immune response to viral oncolysis is not appropriate, and each tumor target will need full characterization for the potential of both direct and indirect, innate immune responses to generate benefit.

The distinct nature of acute lymphoblastic leukemia (ALL) in adults, evidenced by inferior treatment outcome and different genetic landscape, mandates specific studies of disease-initiating mechanisms. In this study, we used NOD/LtSz-scid IL2Rγ null(c) (NSG) mouse xenotransplantation approaches to elucidate leukemia-initiating cell (LIC) biology in primary adult precursor B (pre-B) ALL to optimize disease modeling. In contrast with xenografting studies of pediatric ALL, we found that modification of the NSG host environment using preconditioning total body irradiation (TBI) was indispensable for efficient engraftment of adult non-t(4;11) pre-B ALL, whereas t(4;11) pre-B ALL was successfully reconstituted without this adaptation. Furthermore, TBI-based xenotransplantation of non-t(4;11) pre-B ALL enabled detection of a high frequency of LICs (

Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL.

Measles virus (MV) has been shown to be oncolytic in various tumor models and is being evaluated in various Phase I clinical trials. The mechanisms by which MV kills cancer cells still remain obscure. We aimed to enhance the therapeutic efficacy of MV by expressing the human granulocyte colony stimulating factor (hGCSF), as an additional transcription unit of MV. We showed that MVhGCSF is therapeutic in two different in-vivo subcutaneous model of B cell malignancy, Raji (Burkitt's lymphoma) and Nalm-6 (acute lymphoblastic leukemia). In the Raji model, MVhGCSF treatment generated a highly significantly superior (p=0.0001) anti tumor effect by comparison to MVNSe and the MVhGCSF treated mice survived significantly longer than MVNSe treated mice (p=0.0098). In the Nalm-6 model, both MVhGCSF and MVNSe resulted in complete regression of the tumors in both groups with no survival advantage to MVhGCSF therapy. We then determined therapeutic efficacy of MVhGCSF in disseminated Nalm-6 xenografts as our previous data showed that approx. 60% of mice have complete regression of the tumors with IV delivered MV, offering a greater potential to observe any therapeutic benefit to MVhGCSF. Nalm-6 tumor cells expressing luciferase were used, in order to enable in-vivo imaging of disease progression by imaging. Mice were treated with MVNSe, MVhGCSF, or UV-MV control and an additional control of hGCSF alone, using pegylated hGCSF. Mice treated with the controls alone succumbed to leukemia the most quickly; in the group treated with GCSF alone, the median survival was 50 days compared to 75 days in the UV-MV treated group (p=0.012). As expected, 75% of the mice treated with MVNSe responded well and were alive at the end of the experiment. Surprisingly, mice receiving treatment with MVhGCSF had a median survival of 78.5 days compared to the MVNSe treated cohort where the median was not reached by the end of the experiment (p=0.0149). At humane end point, the spleen sizes and cellularity were similar across all the groups. hGCSF was detected at comparable levels in the serum of the hGCSF and MVhGCSF treated cohorts. Flow cytometric analysis of CD11b, Ly6G6C, NK and Mac3 cells did not show any difference in the PMN, macrophage and NK cell percentages between the different groups. Finally, to be certain that the less good outcome of mice treated with MVhGCSF did not also result from toxicity of high GCSF levels, IfnarKO * CD46Ge mice were injected IV with either MVNSe or MVhGCSF. They were monitored for 35 days after which spleen size, differential cell count (NK, macrophage, PMN percentages) and serum hGCSF levels were determined. There was no difference between the groups. The mice treated with MVhGCSF showed 200-800pg/ml of hGCSF in the serum at Day 35, but there was no toxicity. Taken together these data in the disseminated Nalm-6 model, suggests that any adverse effect of expressing hGCSF as an additional transcription unit in tumor bearing mice relates solely to promotion of tumor growth and not to toxicity of GCSF production.Our data suggest that a “one-size fits all” model of immune response to viral oncolysis is not appropriate and each tumor target will need full characterization for the potential of both direct and indirect, innate immune responses to generate benefit.

Vaccine strain measles virus (MV) is oncolytic in numerous models of malignancy. The mechanism behind the selectivity of MV for transformed cells is poorly understood. To investigate further, an established step-wise model of cellular transformation was used; in which progressive oncogenic hits were stably and additively expressed in human bone marrow derived mesenchymal stromal cells following retroviral transfer of human telomerase reverse transcriptase (hTERT), human papilloma virus16 E6 and E7 (3H), SV40 small T antigen (4+V), finally, H-RAS (5H) (Funes at al, 2007). The most highly transformed cells (5H) were more permissive to oncolytic MV infection than any of the less transformed counterparts, with significantly greater viral titres. MV-induced cell-death increased progressively with progressive transformation. This was not explained by any differences in MV receptors CD46, SLAM or Nectin-4 expression. Investigation of anti-viral type 1 IFN response in this model 24 and 48 hours post MV infection (hpi) by ELISA demonstrated a robust induction of IFNβ (to a lesser extent IFNα) in hTERT cells, which was significantly and progressively reduced in 3H, 4+V and 5H according to level of transformation, suggesting that defective IFN pathway is a potential mechanism for the enhanced MV permissiveness observed in transformed cells. Examination of integrity of the RLR signalling pathway, which triggers IFNα/β production, revealed that expression levels of RIG-I, MDA5 and MAVS, determined by RQ-PCR at 6, 12, 24 and 48 hpi, were lowest in 5H and highest in hTERT cells, proposing a role for the RLR pathway in MV-mediated oncolysis. To gain insight into the differential effects of MV infection in transformed versus non-transformed cells, metabolic effects post MV infection were investigated. A significant increase in oxygen consumption rate (OCR) was observed 24 hpi, proportional to the stage of transformation, followed by a steep decline at 48 hpi. This increase was abrogated in the presence of fusion inhibitory peptide (FIP), implicating the role of syncytia formation, the cytopathic effect of MV infection, in MV-mediated oncolysis in vitro. A similar pattern was observed in ATP levels post MV infection; highest at 24hpi in 4+V and 5H and dropping off dramatically at 48hpi when cell viability is compromised. To further characterize the consistent increase in OCR and ATP levels upon MV infection of the more transformed cell lines, we sought to assess mitochondrial biogenesis using the fluorescent probes TMRM, Calcein AM and Hoechst. Preliminary results indicated significantly increasing numbers of mitochondria per cell at 24 hpi principally in 4+V (16%) and 5H (~29%) cells compared to their uninfected counterparts potentially explaining the observed high levels of OCR and ATP. Altogether, our data suggests that altered mitochondrial numbers and functions correlate with the degree of MV permissiveness and thus MV-induced cell death. Mitochondrial mass was also seen to be increased with MV infection. However, this effect was not specific to transformed cells. Metabolomics and further evaluations will be crucial to comprehensively assess differences in metabolism between cells in this model and to understand the role of bioenergetics in the differential levels of MV-mediated oncolysis.

Introduction: As the number of long-term survivors following HSCT is increasing, the long-term risks and associated morbidity has become important component of survivorship program. The known risk factors for developing cancer include use of chemotherapy agents, radiation exposure, immune dysfunction, previous malignancy in addition to other factors and as HSCT process involves all these factors, this single centre retrospective analysis was undertaken to evaluate the risk of developing SSC in the patients receiving transplant. Methods: From February 1982 to February 2016, 2231 patients received 2495 transplants (median age: 46yr., range: 14-76 yr.; M: 1586, F: 909) for haematological malignancies (Leuk: 744, lymphoma:767, myeloma:848, solid tumours/other:136). Donor was allogeneic (n=744) or autologous (n=1751) and conditioning was with (n=614) or without TBI (n=1881). Donor was sibling (n=375), matched unrelated (n=355), haploidentical relative (n=3) or umbilical cord blood (n=11). Source of stem cell was marrow (n=367), PBSC (n=2086), both (n=31) or cord blood (n=11). GVH prophylaxis included Campath or ATG in 369 cases. Of all the patients 1985 received single transplant, 231 had two, 13 had 3 and 2 had 4 HSCT procedures. Data was analysed as of 15/04/2016 using competing risk model with death as the competing event. Comparison of incidence to general population was performed by computing standardized incidence rates (SIR). Patients with second haematological malignancy were not included in this analysis. Results: Median follow-up was 5.3 years (range: 0-32 years). Patient follow-up was more than 10 years in 467 cases (19%), between 5 to 10 years in 430 (17%), 2 to 5 years in 607 (24%) and less than 1 year in 997 cases (40%). 36% patients were followed-up for more than 5 years. Second solid cancers developed in 116 patients with the incidence of 1% at 5yr (95% CI: 0.5-2.6), 3% at 10 yr (95% CI: 1.6-5.3), 6% at 15yr (95%CI: 3.6-8.8) and 10% (95% CI: 5.9-15.5) at 20 years. Median time to develop SSC from date of HSCT was 11 yr (range: 0.4-28.1 yr). Primary site for SSC included skin (n=37), breast (n=22), GI (n=15), GU (n=16), H&N (n=10), lung (n=6), CNS (n=4), Endocrine (n=4) & HPB (n=2). There was no difference with type of transplant i.e. auto or allograft. Autograft and allograft groups were analysed separately. In univariate analysis, allograft group showed higher cumulative incidence of SSC with use of PBSC (p Conclusion: This single centre analysis confirms that the risk of developing SSC increases with advancing age, use of RIC allograft, longer follow-up and leads to inferior survival. Since the year 2000, SSC are developing early after transplant and it needs to be evaluated if this is a trend seen at other centers and if so, is it related to increasing use of RIC, increasing number of elderly patients, severity of immune-suppression or higher incidence of GVHD. Disclosures Somervaille: Novartis: Consultancy, Honoraria; Imago Biosciences: Consultancy. Bloor:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; GSK: Consultancy, Speakers Bureau; Gilead: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Introduction: Community acquired SRVI increase hospital referrals, hospitalization and ICU admissions resulting in high morbidity and mortality during winter season. As there are no defined preventative or treatment measures for most of the SRVI, there is increasing burden on health service resources during SRVI season. This analysis was carried out to evaluate the SRVI incidence, risk factors, impact on mortality and changes in incidence trends over 9 year period in immunocompromised cancer patients. Methods: 2906 cancer patients (haematology: n=1098, 37.8%, lymphoma: n=643, 22.1%, other cancers: n= 1156, 40.1%) treated from January 2006 to January 2015 who had respiratory virus PCR were evaluated. Patients with haematological cancers included ALL (n=137), AML (n=338), Myeloma (n=396), CLL (n=131) or other cancers (n=96) [median age: 50 yr., 5-87, Male: 692, Female: 406). Common solid tumour diagnosis included cancer of Breast (n=280, 24.0%), GI tract (n=207, 17.8%), Lung (n=190, 16.3%), Genitourinary (n=180, 15.5%) or other sites (n=299, 25.7%), [Males: 461, Females: 695; median age: 55 yr., range: 6-89]. Patients with haematological malignancies were younger than patients with other cancers (median age: 50 yr. vs. 55 yr., p Results: In patients with malignancy, the season for ParaFlu, Rhinovirus, Metapneumovirus and FluA lasted longer than in general population (average: 2 months, started early and ended later). Incidence of RSV (6.2%, 4.9%, 1.6%, p=0.001), Adenovirus (1.3%, 1.7%, 0.33%, p=0.004), Rhinovirus (16.6%, 19.9%, 8.5%, p=0.001) and ParaFlu (7.4%, 6.3%, 2.6%, p=0.057) was higher in hematology and lymphoma patients. Incidence of PCP was higher in oncology patients (15.1%, 7.2%, 9.6%, p=0.001). Incidence of PCP was higher with increasing age (5.8% age< 50, 12.2% age>50 yr., p=0.001). Rhinovirus (18.7% age75 yr., p=0.001) and ParaFlu (8.1% age 25 yr., p=0.02) was higher in younger patients. Stem cell transplant increased risk of RSV (6.8% vs. 3.5%, p=0.001), Adenovirus (1.7% vs. 0.6%, p=0.001) and ParaFlu (8.1% vs. 0.23%, p=0.001) but risk of PCP (7.7% vs. 11.8%, p=0.0001) was lower. Risk of positive PCR for any respiratory virus was higher with increasing age, hematological cancers, and use of stem cell transplant. Surprisingly, diagnosis of CLL and Myeloma did not increase SIRV risk. Thirty-day mortality was higher in patients who had SRVI (p=0.041). Mortality was higher in patients with solid tumours (p Conclusion: This is one of the first reports that compares incidence of SRVI in patients with cancer to that in general population. The analysis of SRVI using PCR based diagnosis demonstrates that incidence of SRVI in cancer patients show different trends than in general population. SRVI season lasts longer and RSV, FluA and PCP contribute to 30-day mortality. Increasing PCP incidence in patients with solid tumours raises the questions about need to use of PCP prophylaxis in all these cases. Disclosures Somervaille: Novartis: Consultancy, Honoraria; Imago Biosciences: Consultancy. Bloor:Janssen: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Consultancy, Speakers Bureau; Gilead: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Introduction: Prevention of Graft versus host disease (GVHD) forms the mainstay of obstacle in the successful outcome of alloHSCT. Various strategies are employed but combination of calcineurin inhibitors with methotrexate or mycophenolate is the commonest preventative measure used. Cyclosporine is dosed according to trough (C0) levels and the optimum level is defined as between 200 and 300 ng/ml. Daily doses are adjusted to achieve the therapeutic levels. As there can be fluctuations in the daily levels, it was postulated that area under the curve (AUC) extrapolated from daily levels may be more useful to define the optimum cyclosporine exposure for prevention of GVHD. Hence this retrospective analysis was carried out to evaluate if cyclosporine AUC for first 14 days has correlation with the incidence of GVHD. Methods: 549 patients who received allograft and had cyclosporine C0 levels available for first 14 days were included in the analysis. The patient population included 353 males (64.3%) and 196 females (35.7%) with a median age of 44 yr. (range: 16-71). Underlying diagnosis included Ac. Leukaemia (n=319, 58.1%), Chr. Leukaemia (n=42, 7.7%), lymphoma (n=119, 21.7%), myeloma (n=57, 10.4%) or other (n=12, 2.2%).Donor was matched unrelated (n=330, 60.1%), sibling (n=204, 37.2%) or cord (n=15, 2.7%). Stem cell source was PBSC (n=454, 82.7%), BM (n=78, 14.2%), both (n=2, 0.4%) or cord blood (n=15, 2.7%). 415 patients had RIC (75.6%) and 134 had full intensity SCT (24.4%). Alemtuzumab or ATG was used in conditioning in 311 cases (56.6%). The target Cyclosporine AUC with a daily level of 200 was predicted as 2600, 2470 for cyclosporine level of 190 and 2730 for levels of 210. Results: The median AUC for the entire cohort was 3338 (range: 370-6390). Only 56 cases (10.2%) had AUC below 2600. Incidence of AGVHD was significantly higher with use of PBSC (53.7% vs. 21.7%, p Conclusion: This retrospective analysis had high proportion of cases receiving Alemtuzumab/ATG based GVHD prophylaxis and shows that even though there is no effect of cyclosporine AUC on the incidence of either AGVHD or CGVHD, patients receiving Alemtuzumab/ATG with sub-therapeutic cyclosporine AUC have poor OS. This raises the possibility that in this group of patients it is imperative to have target cyclosporine levels in first 14 days. The duration of target cyclosporine levels before tapering the doses needs to be established to improve OS without increasing the toxicity. Disclosures Somervaille: Imago Biosciences: Consultancy; Novartis: Consultancy, Honoraria. Bloor:Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria; GSK: Consultancy, Speakers Bureau.

Clinical trials of oncolytic attenuated measles virus (MV) are ongoing, but successful systemic delivery in immune individuals remains a major challenge. We demonstrated high-titer anti-MV antibody in 16 adults with acute lymphoblastic leukemia (ALL) following treatments including numerous immunosuppressive drugs. To resolve this challenge, human bone marrow-derived mesenchymal stromal cells (BM-MSCs) were used to efficiently deliver MV in a systemic xenograft model of precursor B-lineage-ALL. BM-MSCs were successfully loaded with MV ex vivo, and MV was amplified intracellularly, without toxicity. Live cell confocal imaging demonstrated a viral hand-off between BM-MSCs and ALL targets in the presence of antibody. In a murine model of disseminated ALL, successful MV treatment (judged by bioluminescence quantification and survival) was completely abrogated by passive immunization with high-titer human anti-MV antibody. Importantly, no such abrogation was seen in immunized mice receiving MV delivered by BM-MSCs. These data support the use of BM-MSCs as cellular carriers for MV in patients with ALL.

We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.

We have previously shown that PMNs play a role in MV-mediated oncolysis (Grote et al) and that oncolytic vaccine strain of MV (as opposed to wild type) stimulates release of numerous cytokines with potential anti-tumor effects (Zhang et al). Recent data from our laboratory indicated that normal healthy PMNs were capable of specific killing of 35-40% of infected Jurkat target cells. On the basis that MV-induced cell-cell fusion amplifies IFN-alpha (α)/beta (β) production in infected cells, we sought to determine whether fusion between infected target cells and normal human PMNs specifically mediates cytotoxicity. First, we showed that PMN-mediated MV killing completely disappeared in the presence of FIP (fusion inhibitory peptide). Next, we examined the role of cell-cell fusion on PMN degranulation, reactive oxygen species (ROS) production and IFN production. We carried out all experiments with and without FIP. Jurkat cells were infected with MVNSe or mock-infected and incubated with PMNs isolated from healthy donors at 8:1 E:T ratio. At 24 hours, the cells were collected. PMN degranulation was determined by increase in cell surface expression of CD66b, CD63 and CD35 and ROS generation was determined by flow cytometry. IFN α and β production in the supernatant was quantified by ELISA. During co-culture of infected target cells and PMNs, IFN α (3000-5000pg/ml) and β (70-90pg/ml) were produced. No IFN α and β was produced by infected or uninfected Jurkats without PMNs present, confirming PMNs as the source. This was highly significantly abrogated in the presence of FIP (α – 200-300pg/ml; β – 0-3pg/ml). PMN degranulation and ROS generation followed the same pattern with abrogation in the presence of FIP, although the differences were not statistically significant. We confirmed that the α or β IFN was not directly responsible for cytotoxicity, since addition of the same quantity of exogenous IFN directly to Jurkat cells did not lead to cell death. Hence, we sought downstream cytotoxic effectors and evaluated TRAIL, which we know from our previous work, can be released from pre-formed granules in response to MV-infection of PMNs. The conditions that generated high levels of IFN also produced significant levels of soluble TRAIL (800-1500pg/ml), which was also abrogated in the presence of FIP (190-650pg/ml). Our data suggests infected target cells stimulate PMNs to display a cytotoxic effector phenotype, directly killing infected target cells. High levels of type I IFNs, are generated which are not in themselves responsible for the cytotoxicity. None of this occurs without target cell-neutrophil fusion as it is abrogated by FIP and does not occur in infected target cells alone or infected PMNs alone. This is a novel mechanism of stimulation of innate immunity by an oncolytic virus.

Idiopathic myelofibrosis (IMF) is a clonal disorder resulting from the proliferation of aberrant hematopoietic stem cells. Conventional treatment is unsatisfactory, and with the exception of supportive blood transfusions, none of the standard therapies have been shown to confer a survival advantage. Allogeneic stem cell transplantation represents the only treatment modality with proven curative potential. Myeloablative conditioning regimens are associated with high transplant-related mortality, particularly in the elderly, making most patients with IMF ineligible for this treatment. Strategies using reduced intensity conditioning regimes have allowed application of allogeneic transplantation to a broader range of patients and a number of recent reports have demonstrated potential efficacy.

Although humanised models of ALL are well described they are largely based on paediatric disease. The distinct nature of ALL in adults - evidenced by inferior outcome and different genetic landscape - points to a need for targeted study of this disease. Key to this is development of in vivo models of adult ALL. In this study we sought to establish the optimal modelling system for adult pre-B ALL using NOD/LtSz-scid IL2Rγ nullc (NSG) mouse xenotransplantation approaches. Our aims were to define the conditions for sensitive assaying of adult ALL LIC and low frequency clones. Pre-conditioning irradiation has been shown to negatively influence pre-B ALL engraftment. Studies of paediatric ALL suggest this may be due to reduced homing of ALL cells to the bone marrow (BM) resulting from aberrant CXCR4 receptor desensitisation induced by SDF-1 which is upregulated after irradiation. To investigate the impact of irradiation on engraftment of adult ALL, 5 - 10 million viable cells were injected intravenously (IV) into 6-8 week mice either unconditioned or (+24 hrs) following irradiation (2.5Gy). After 6 months, BM leukaemic engraftment (>0.1% hCD45+/19+) was assessed. Engraftment occurred in only 2/17 (11.8%) samples from non-irradiated recipients but in 11/14 samples (78.6%) after pre-conditioning irradiation (p = 0.0003). Comparison of 8 samples assayed by both models demonstrated that pre-conditioning irradiation was a pre-requisite for successful engraftment in 7/8 cases. Contrasting this overall finding was t(4;11) ALL where engraftment was higher in non-irradiated models (2/3). To determine homing capacity in adult ALL, SDF-1 (0, 10ng, 50ng, 125ng + 1ug) induced directional migration was investigated. A dose dependant response was observed with maximal migration (37±15 fold) of adult pre-B ALL cells at high (125ng/ul) concentrations of SDF-1 (n=5, non t(4;11) ALL). Hence, in contrast to paediatric ALL, adult ALL tumours do not appear to exhibit significant CXCR4 desensitisation to high levels of SDF-1 which may explain the positive effect of irradiation on adult ALL engraftment. Notably, t(4;11) positive tumours were relatively insensitive to SDF-1 induced migration (max fold change ∼2.5). CXCR4 cell surface receptor analysis revealed a trend towards low expression in t(4;11) cases (8.7±3.3% blasts) vs non t(4;11) (41 ±12% blasts) which may account for the differential SDF-1 induced in vitro migration patterns. Further modifications to the NSG xenotransplantation system were investigated: direct intra bone marrow (IBM) vs IV injection, 2.5Gy vs 2.0Gy and IBM +/- 2.5Gy irradiation. An IBM injection route, 2.5Gy irradiation and IBM + irradiation were all associated with an overall faster engraftment kinetic and time to disease manifestation. However, overall engraftment potential was not affected by these factors. Hence, the optimal xenotransplantation assay for adult ALL was defined as IBM delivery in 2.5Gy irradiated mice. This system enabled quantification of at least 1:1000 adult ALL LICs as assessed by limiting dilution transplantation (cell dose:103-106 ). Finally, we used the optimised xenotransplantation system to study 9 MRD positive ALL BM samples (8.33 x 10-2 – 2.64-4 positivity by BCR/ABL or I/g TCR based PCR quantification). MRD positive BM samples (unfractionated) were injected at a cell dose of 8.7 x104 – 6 x106/mouse and, strikingly, in the absence of cellular enrichment, MRD induced leukaemic engraftment (83.6%) was achieved in 1/9 samples after an extended modelling phase of 7 months and importantly gave rise to overt leukemia associated morbidity. Hence we establish proof of principle, that MRD + ALL can be modelled in vivo. In conclusion we have established the parameters of a functional system for in vivo study of adult ALL. Our findings highlight the key role of irradiation in the homing of adult ALL cells and also in their retention and repopulation, possibly mediated by SDF-1/CXCR4 interaction. Importantly, we found distinct in vivo growth requirements not only between adult ALL subtypes but also compared to paediatric disease underlining the importance of age/leukaemia specific investigation. Our optimised system should facilitate the study of LIC and pre-clinical disease modelling in adult ALL. Crucially we establish a strategy for assaying arguably the most informative populations (MRD) for understanding disease resistance and evolution of relapse. Disclosures: No relevant conflicts of interest to declare.

Abstract 4300 Since the first allogeneic umbilical cord blood transplantation in 1988, there has been an increasing recognition and utilisation of cord blood as a source of haematopoietic stem cells. Donor cell leukaemia (DCL) following cord blood transplantation (CBT) remains a rarely reported phenomenon with only a handful of cases appearing in the literature to date. A plethora of possible mechanisms have been suggested for the development of DCL following CBT. In addition to the presence of undetected pre-leukaemic clones in the cord blood, chronic antigenic stimulation in an impaired tumour immune surveillance environment, perturbations of the host environment and premature ageing of the donor cells with associated chromosomal instability are among the proposed mechanisms for leukaemogenesis. Here we report a case of donor cell leukaemia in a 35-year-old female suffering from chronic myelogenous leukaemia. At 17 months following sex mismatched cord blood transplantation, donor cell derived BCR/ABL1 negative acute myeloid leukaemia (FAB M0) was diagnosed. In addition to the finding of monosomy 7, amplification of part of chromosome 21 containing the RUNX1, Down Syndrome Critical Region (DSCR) and ERG genes among others was identified by high-density array comparative genome hybridisation. The array findings were confirmed by qPCR and the RUNX1 amplification was mapped by FISH onto a marker chromosome, der(17)t(17;21)(p13;q?21). Importantly, these genetic changes were not found in the retrospectively tested cord blood cells using the same techniques. Whereas aberrations of chromosome 7 have been previously reported in cases of DCL post CBT, here we describe for the first time co-amplifications of several genes from the 21q22 chromosome region in conjunction with the finding of monosomy 7. RUNX1 amplifications are known to occur in acute leukaemia of both myeloid and lymphoid lineage. Gains of the 21q22 region with multiple copies of RUNX1 gene have been associated with therapy related AML and MDS, but thus far has not been reported in the context of DCL following CBT. Furthermore, the possibility that additional genomic imbalances may have contributed to the pathogenesis of DCL in this case is also raised. Co-amplification of genes from the DSCR at 21q22 in the presence of unmutated GATA1 and JAK3 genes may be relevant in this context, as located within this region are genes with powerful effects on differentiation (such as DYRK1A) and several genes whose over-expression has been linked with disturbed megakaryopoiesis and myeloproliferation including miR155, BACH1, RCAN1 (DSCR1), RUNX1 and ERG. In addition to the classical genomic imbalances such as monosomy 7 that typify the genetic makeup of secondary AML, this case highlights for the first time the potential cooperative role of oncogenic co-amplification in the pathogenesis of donor cell leukaemia. Disclosures: No relevant conflicts of interest to declare.