ALBERT

Description: Key Points Human BM-MSCs can be used to successfully deliver systemic oncolytic measles virotherapy to ALL tumor targets. This approach permits circumvention of preexisting anti-measles humoral immunity and enhanced therapeutic outcomes.

Description: Key Points Efforts to understand mechanisms of disease initiation in human adult pre-B ALL are hampered by lack of appropriate animal models. Optimized xenotransplant assays show that niche-based SDF-1/CXCR4 interaction is crucial for adult non-t(4;11) pre-B ALL leukemia initiation.

Description: Introduction:Thrombocytopenia is a common problem in hospitalized patients, which has a diverse etiology. One of the infrequent causes is heparin-induced thrombocytopenia (HIT), a complex immune disorder in which heparin leads to the production of IgG antibodies, targeting platelet factor 4 (PF4). HIT diagnosis is based on a decrease in the platelet count of more than 50% beginning 5 to 10 days after starting heparin, in association with platelet-activating HIT antibodies (screening test), positive functional tests (confirmatory tests), in patients with no alternative causes for thrombocytopenia, necrosis or thrombosis (Figure 1). Most patients admitted to our institution receive unfractionated or low molecular weight heparin for venous thromboembolism prophylaxis. The risk of HIT depends on the type of heparin the patient receives. Even though HIT is rare, we tend to have a high suspicion and low threshold to order laboratory workup for this condition, occasionally without using the 4T«s score (Table 1). The 4T«s score is a validated system with a very high negative predictive value (NPV) of up to 99%, when the score is ²3. The aim of this study is to determine if the 4T«s score has been used appropriately in our institution. It is a very useful tool with a high negative likelihood ratio and using it systematically may decrease unnecessary laboratory testing, consequently decreasing costs and potentially length of stay. Methods:This is a retrospective descriptive study from a single teaching community hospital. Between January and December of 2015, 57 HIT screening tests (PF4 ELISA) were ordered in our institution. We reviewed all of the patient charts to determine their 4T«s score. The patients were divided into low, moderate and high pretest probability based on their score (low probability if ²3, intermediate 4-5, and high if ³6). The data analysis was completed with SPSS software. Results:57 tests were ordered, 5 patient charts did not have enough information to calculate 4T«s score and were excluded. 52 charts were reviewed, 28 patients were male and 24 were female (53 and 46% respectively), and they were between 25 and 89 years of age. 7 (13.4%) did not receive heparin (during current hospitalization or within 100 days), 11 (21.1%) received therapeutic doses of unfractionated heparin, 11 (21.1%) received prophylactic doses of unfractionated heparin, and 23 (44.2%) received prophylactic low molecular weight heparin (enoxaparin). The 4T«s score was calculated for each patient; 40 were low risk, 10 intermediate risk and 2 high risk (76.9, 19.2, 3.8% respectively). All the patients had HIT screening antibodies ordered; only 10 (19.2%) were positive and from those only 1 (1.9%) was confirmed by the serotonin release assay. This confirmed patient had a 4T«s score of 6. Of the 7 tested patients who did not receive heparin, none had a positive screening test (all had 4T«s score of 1 or 2). 35 of the screening tests were ordered by internal medicine residents, 5 by surgical residents, 12 by attending physicians (67.3, 9.6 and 23% respectively). Previous studies validated that a 4T«s score of ²3 can predict negative results for the confirmatory tests. By using theMcNemar«stest for matched pairs, we attempted to determine a cut off for the 4T«s score that would predict a negativeHIT screeningtest. We found that a 4T«s score ²2 predicted a negativeHIT screeningtest, with aNPVof 75% (p=0.0018). Since our study included a small patient cohort and due to the possibility of laboratory work up not being ordered in patients with a low 4T«s score, we suspect theNPVis actually lower than our current finding. A prospective study with a larger cohort of patients would be necessary to confirm these findings. Conclusions:Only 12% of the patients had a 4T«s score ³4 that would warrant laboratory work up, therefore we concluded that PF4 ELISA screening tests have been over utilized in our institution, increasing false positive results, length of stay and cost of hospitalization in patients without a significant risk for HIT. This could be avoided by calculating the 4T«s score before obtaining any further laboratory tests. The 4T«s scoring system has been validated by multiple studies with large cohorts in the past. We will continue to work with our staff, especially the residents, to improve education on HIT and adequate diagnosis, based on current guidelines. Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: UKALL14 (NCT01085617) randomised 655 patients aged 25-65 years with B-precursor ALL, irrespective of Philadelphia chromosome (Ph) status or cell surface CD20 expression to determine if the addition of four doses of rituximab to standard induction chemotherapy (SOC+R) resulted in improved event free survival (EFS). Patients were recruited between Dec 2010 - Jul 2017 and the primary analysis population comprises 577 patients recruited after an April 2012 amendment in which the SOC therapy was altered. The trial was powered with an 84% chance to detect a 12% improvement in EFS (Hazard ratio (HR): 0.71). Secondary endpoints included complete remission (CR), OS, non-relapse mortality, levels of minimal residual disease (MRD) after induction and relationship of response to CD20 expression. SOC consisted of daunorubicin 30mg/m2, vincristine 1.4mg/m2, dexamethasone 4 day blocks of 10mg/m2 starting d1, 8, 15 and 22. Pegylated asparaginase 1000IU/m2 was added on d4 and 18 (d18 only if 〉40 years) for Ph- ALL and continuous daily imatinib 600mg for Ph+ ALL. Intrathecal MTX was given on d14. Rituximab 375mg/m2 was given on d3,10,17 and 24. Two patients did not start trial treatment, both were in the SOC+R arm. Analysis is by intention-to-treat. There were 288 patients in the SOC arm and 289 in the SOC+R arm, 273 (95.5%) of whom received all 4 doses of rituximab. The arms were well-balanced for risk characteristics. Of note, 63.9% (SOC) and 62.6% (SOC+R) were aged over 40 years at randomisation and 86 patients in each arm (29.9% and 29.8%, respectively) had Ph+ ALL. CR rate, 92.7% SOC and 94.8% SOC+R, did not differ between the arms. There was no difference in the MRD response between the arms, whether assessed as positive vs. negative or as a continuous variable; 121 (42.2%) of patients were MRD negative at induction completion in the SOC arm vs. 120 (41.8%) in the SOC+R arm. Likewise, the rate of severe/adverse events and non-relapse mortality did not differ between arms. At a median follow-up of 50.5 months (7 days - 83.6 months), 3 year EFS for SOC is 41.9% (95% CI: 35.8 - 48.0) versus SOC+R 48.7% (42.4 - 54.8), Hazard Ratio(HR) 0.88 (0.71 - 1.11), p=0.28, see figure1a. Pre-planned subgroup analyses by cytogenetic ("high risk" was defined as t(9;22), t(4;11), low hypodiploidy/near triploidy and complex karyotype) and other risk groups (age, presenting WBC) as well as by cell surface CD20 expression did not reveal any significant interactions. However, we did find that % blasts expressing CD20 was an independent poor prognostic factor; Youden's cut-off, to determine the best cut-off for a continuous variable, suggested a % CD20 expression of 11.7% as the optimal cut-off. EFS HR for 10-20% CD20 was 1.74 (0.98-3.10) and 〉20% was 2.20 (1.27 - 3.81), compared to

Description: Key Points CMV serostatus significantly influences chimerism levels after T-cell–depleted allogeneic transplantation. CMV-specific T cells are exclusively of recipient origin after R+/D− T-cell–depleted transplants and appear to provide protective immunity.