BACKGROUND:Graft-versus-host disease (GVHD) remains a barrier to long-term success of haploidentical donor (HID) allogeneic hematopoietic cell transplantation (HCT). Post-transplant cyclophosphamide (PTCy) is considered standard prophylaxis, while the addition of anti-thymocyte globulin (ATG) has been utilized by some centres to further reduce GVHD. METHODS:We conducted a retrospective, multicenter analysis using the Cell Therapy Transplant Canada (CTTC) registry to compare outcomes of ATG-PTCy versus PTCy-only GVHD prophylaxis among adults undergoing HID HCT for hematologic malignancies between 2016 and 2023. Endpoints included overall survival (OS), acute GVHD (aGVHD), chronic GVHD (cGVHD), GVHD-free/relapse-free survival (GRFS), relapse, and non-relapse mortality (NRM). Multivariable Cox regression and propensity score matched analyses accounted for patient-, disease-, and transplant-related factors. RESULTS:Of 385 patients (148 ATG-PTCy, 237 PTCy) were included. Two-year OS and cumulative incidence of relapse (CIR) were similar between groups: 61% vs. 64% (p = .29) and 19.6% vs. 19.4% (p = 1.0). ATG-PTCy significantly reduced moderate-severe cGVHD: 11% vs. 30% (HR: 2.28, p = .001), but two-year NRM was higher with ATG-PTCy: 27% vs. 18% (p = .04), likely related to increased infection risk. Propensity-score matched analysis revealed no difference in OS (p = .46), but lower moderate-severe cGVHD rates (p = .002), higher relapse (p=0.009) and similar NRM (p = .62) with ATG-PTCy. In AML patients (n = 196), relapse remained similar, but OS favored PTCy (OS at 2-year 71% vs. 50%, p < .001) due to excess NRM with ATG-PTCy. Multivariable analyses confirmed age and comorbidity burden as predictors of worse OS, while prophylaxis regimen independently influenced cGVHD risk. CONCLUSION:In this registry analysis, ATG-PTCy significantly reduced the incidence of moderate-to-severe cGVHD compared with PTCy alone; however, this benefit was offset by higher risks of relapse and NRM, ultimately yielding similar survival between the two regimens. These findings highlight the need for individualized prophylaxis strategies balancing GVHD prevention with infection-related risks.

BackgroundAllogeneic hematopoietic stem cell transplant (alloHSCT) is a mainstay of treatment for hematologic malignancies such as acute leukemias and aggressive lymphomas. Historically, fresh Hematopoietic Progenitor Cell (HPC) products have been preferred to cryopreserved products (cryo-HPC) due to concerns of loss of stem cell viability and number with the cryopreservation procedure.ObjectiveWe aimed to analyze the outcomes of patients who received cryo-HPCs during the COVID-19 pandemic and compare this against historical cohorts that received fresh HPC.Study DesignA retrospective chart review was conducted on all adult patients who received a peripheral blood alloHSCT in British Columbia, Canada between June 2017 and November 2021. Baseline characteristics, Kaplan-Meier (KM) Overall Survival (OS), engraftment, and incidences of acute and chronic graft versus host disease were compared between patients who received cryo-HPCs and fresh HPCs. Univariable analysis followed by multivariable analysis was performed using a backward stepwise selection procedure to generate predictors of OS, cumulative incidence of relapse (CIR), non-relapse mortality (NRM), and primary and secondary graft failure.Results383 patients were included in the analysis, with cryo-HPC representing 40%. Median viability was higher in the fresh-HPC group at 99.2% (IQR 98.3-99.5) versus cryo-HPCs at 97.0% (96.0, 98.6) (p<0.01). The 12-month actuarial survivals were 77% in the fresh HPC and 75% in the cryo-HPC groups (p=0.21). There were no differences between cryo-HPCs and fresh HPCs on univariable analysis of OS, CIR, or NRM. There was a shorter median time to platelet engraftment in patients receiving fresh HPC at 17 days (IQR 16, 20) versus cryo-HPC at 21 days (IQR 18, 29), p<0.001. There was a shorter median time to neutrophil engraftment in the fresh HPC group at 17 days (IQR 14, 20) versus 20 days (17, 23), p<0.001. Cryo-HPC accounted for 5 out of 6 cases of primary graft failure (p=0.04), and 3 out of five cases of secondary graft failure (p=0.39). There were no significant differences in acute GVHD between the fresh HPC and cryo-HPC groups (p=0.34). The incidence of moderate or severe chronic GVHD was 32% in the fresh-HPC group and 17% in the cryo-HPC group (p<0.001).In multivariable analysis, cryopreservation did not emerge as an independent predictor of OS, CIR, NRM, primary GF or secondary GF. However, viability <90% on arrival at our center was a significant predictor of OS (HR 5.3, 2.3-12.3, p<0.01), primary graft failure (OR 36.3, 5.4-210.2, p<0.01), and secondary graft failure (OR 18.4, 1.7-121.1, p<0.01).ConclusionPatients who received cryo-HPCs had similar OS and relapse rates to those who received fresh-HPCs but typically took 2-3 days longer to achieve engraftment of platelets or neutrophils and were associated increased primary graft failure. However, after accounting for multiple variables, cryopreservation was no longer a significant predictor of survival or engraftment while viability <90% emerged as an important predictor of OS, primary graft failure, and secondary graft failure. If confirmed, this suggests that viability on arrival at the infusion center may be a good quality control indicator used to identify HPC products that may warrant recollection if the risk of graft failure is sufficiently increased.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single- domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAbCARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. . Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

INTRODUCTION:Antithymocyte globulin (ATG) has been demonstrated to reduce the incidence of graft-versus-host disease (GVHD); however, it remains controversial whether these gains are offset by an increase in relapse. METHODS:We conducted a retrospective historical control study consisting of patients (n = 210) who underwent myeloablative allogeneic hematopoietic stem-cell transplantation (HSCT) from 2014 to 2020. RESULTS:The incidence of acute GVHD was lower in the ATG group (51.4%) than the non-ATG group (control) (70.0%, p = 0.010). The incidence of chronic GVHD was also lower in the ATG group at 1-year (36.4% vs. 62.9%, p < 0.001) and 2-year (40.0% vs. 65.7%, p < 0.001) post-HSCT. The mortality due to GVHD was higher in the control (18.5%) than the ATG group (4.3%; p = 0.024). The severe GVHD-relapse-free survival was higher in the ATG group (36.4%) than the control (12.9%; p < 0.001). Nevertheless, the 2-year overall survival was similar. CONCLUSION:Our results confirm the effectiveness of ATG in prevention of GVHD in the real-world setting and enhanced GVHD-free survival. An important result is the equalization of overall survival between the ATG and control groups at 1- and 2-year post-HSCT and implies that earlier GVHD-associated mortality may be offset by later relapse mortality producing similar overall survival over time.

Supplementary Figure 4. Activation of HUVECs by serum from patients with neurotoxicity.

Introduction: FLT3 internal tandem duplication (ITD) mutations have been historically associated with inferior outcomes in patients with acute myeloid leukemia (AML) and an intermediate risk status as per the European LeukemiaNet (ELN) 2022 guidelines. In 2017, the US FDA granted approval to midostaurin for frontline treatment and a year later, gilteritinib for use in relapsed/refractory (R/R) AML. These agents began to be widely used in our provincial leukemia program in early 2018 as per the label indications. In this study, we compared clinical outcomes for patients with FLT3-ITD mutated AML before and after this change in British Columbia. Methods: We identified patients in our program database diagnosed with FLT3-ITD mutated AML from January 1, 2010 to December 31, 2021. Only patients receiving intensive chemotherapy with cytarabine and anthracycline were included. Patients were divided into two eras: 2010-2017 (era 1) and 2018-2021 (era 2). The two eras were compared in terms of patient characteristics, genetics, rate of complete remission (CR) and CR with incomplete count recovery (CRi), receipt of allogeneic stem cell transplant (alloSCT) and the overall and relapse free survival (OS and RFS). OS was calculated from diagnosis to death from any cause and was not censored at the time of alloSCT. RFS was calculated from CR/CRi to relapse. Patients without an event during the study period were censored at the time of last follow-up. Categorical variables were compared using Chi-square and Fisher exact tests and continuous variable were compared using paired T tests. Kaplan-Meier survival method and log rank test were used to estimate and compare survival. Uni- and multivariate analyses were performed in a Cox proportional regression model and alloSCT was considered as a time dependent covariate. Results: 169 patients were included (era 1=101, era 2=68). The median duration of follow up of the entire cohort was 17 months (range 0.5-148.6 months) and follow-up of surviving patients (n=70) was longer in era 1 vs. era 2 (median 88 months vs. 32 months, p<0.001). The median age of the entire cohort was 60 years (range 26-79 years) and it was comparable in both eras. Most patients had normal cytogenetics (era 1: 76%; era 2: 69%). NPM1 was mutated in 69/101 (68%) and 38/68 (56%) patients in eras 1 and 2 respectively. Due to lack of genetic data for era 1, we did not risk stratify based on the recent ELN classification. The rate of CR/CRi was 58/101 (57%) vs. 40/68 (59%) after induction chemotherapy and 78/101 (77%) vs. 58/67 (87%) (p=0.09) at any time in eras 1 and 2 respectively. AlloSCT was performed in 58% and 72% of patients in the two cohorts. Midostaurin was used in 82% and gilteritinib was used in 26% of era 2 patients in the second era. OS was significantly longer in era 2 (median OS 73 months vs. 11 months, HR (death) 6.2, p=0.012) (Figure). The 2-year OS rate was higher in era 2 (57%) than in era 1 (39%). In patients achieving CR/CRi, median RFS was longer in era 2 (51 vs. 16 months, p=0.43), although this was not statistically significant. In univariate Cox regression analysis the following factors were associated with improved OS: use of midostaurin (HR 0.487, 95% CI 0.304-0.779, p=0.003), diagnosis in era 2 (HR 0.583, 95% CI 0.38-0.895, p=0.014) and receipt of alloSCT (HR 0.346,) 95% CI 0.219-0.545, p<0.001). Other parameters including age, sex, NPM1 status and cytogenetics were not significant. In multivariate analysis including era, midostaurin and alloSCT, only the receipt of alloSCT was associated with significantly improved OS (HR 0.362, 95% CI 0.229 - 0.572, p<0.001) whereas use of midostaurin (HR=0.465, CI 0.207-1.042, p=0.063) and era (HR 1.12, 95% CI 0.538-2.34, p=0.76) were not significant. Conclusions: Our results show thatOS for patients with FLT3-ITD AML has improved in patients diagnosed in a more recent treatment era. Patients diagnosed after 2018 were more frequently treated with AlloSCT and FLT3-inhibitors, which may have resulted in improvements in OS. Our findings support the assignment of FLT3-ITD AML to theintermediate risk category as per the current ELN guidelines. A limitation of this study is the differences in follow-up time between eras, which may influence the event rate between the groups.

Introduction: Lung involvement in chronic graft versus host disease (cGVHD) is an uncommon but potentially devastating complication with significant morbidity and mortality in patients who have received an allogeneic hematopoietic stem cell transplant (alloHSCT). Unfortunately, early detection remains difficult as early lung cGVHD tends to be asymptomatic or present with nonspecific symptoms such as cough or mild shortness of breath. In British Columbia (BC), Canada, routine pulmonary function tests (PFTs) have been employed in the screening of lung cGVHD in this population at an interval of every 3-4 months for the first 2-3 years after alloHSCT. However, it is unknown whether routine screening 1) improves detection of lung cGVHD, and 2) enables earlier intervention to lead to better outcomes. We aimed to assess the real-world outcomes of lung cGVHD screening and compared the characteristics and clinical results of patients who had lung cGVHD detected via routine PFT screening, versus those who were detected after development of symptoms. Methods: A retrospective chart review was conducted on all adult patients who underwent an alloHSCT at BC, Canada between June 2017 and January 2021. Patients were followed until August 2022. Patients were excluded if they had primary graft failure or if they died within 100 days after transplant. All types of pulmonary GVHD were included analysis and cases were established by the treating clinician in the clinical record. NIH consensus criteria were used to grade cGVHD severity. Statistical significance was calculated using the Chi square test for categorical variables and T-test for continuous variables. Results: Among 387 eligible consecutive patients who underwent alloHSCT, 189 (49%) developed cGVHD, with 33 (9%) cases of lung involvement. The mean time to diagnosis of lung cGVHD from the date of transplant was 13 ± 8 months. There were 16 cases of lung cGVHD (48%) that were initially detected by routine screening, while 17 cases (52%) were first detected due to clinical symptoms that developed in between screening visits or after the screening period ended (two patients developed symptoms at 27 months and 43 months respectively). Patient characteristics are presented on Table 1. Lung involvement was the first cGHVD sign in 17/33 (51%) of patients, with 5 (15%) detected by routine screening and 12 (36%) due to symptoms. At time of diagnosis of lung involvement, 12/33 (36%) of patients were already on systemic immunosuppression for cGVHD with 9 (56%) of these cases detected from screening and 3 (18%) from symptoms (p=0.02). All patients had a maximum overall cGVHD severity by NIH criteria of 2-3. Sixteen of 33 (48%) patients had a lung cGVHD score by NIH criteria of 2-3 at diagnosis, with a mean FEV1 of 54% ± 15, compared to 17 (52%) patients with a score of 1 and mean FEV1 of 78% ± 12. The NIH score at diagnosis was not significantly different between those picked up by routine screening versus symptoms (p=0.76). Eighteen of 33 (55%) patients had a maximum lung cGVHD score by NIH criteria of 2-3, with the mean lowest FEV1 of 52% ± 15, compared to 15 patients (45%) with a maximum lung cGVHD score of 1, with the mean lowest FEV1 of 77% ± 10. Similarly, the maximum NIH lung cGVHD score were not significantly different between those picked up by routine screening versus symptoms (p=0.08). However, patients picked up on screening were more likely to have significant clinical improvements in lung function (4/16 [25%] versus 0/17 [0%], p=0.03), and less likely to die from lung cGVHD (0/16 [0%] versus 5/17 [29%], p=0.02). Conclusions: Routine screening PFTs are effective for early diagnosis of asymptomatic lung cGVHD, and are associated with improved outcomes compared to patients who were diagnosed after symptoms developed. Since a significant number of these patients were not previously receiving immunosuppression for other cGVHD, this suggests a potential benefit in screening all patients every 3 months, regardless of prior GVHD status.

Supplementary Figure 1. Treatment of neurotoxicity

Supplementary methods, figure legends, and Supplementary Table 1. Lymphodepletion regimens prior to CAR-T cell infusion; Supplementary Table 2. Neurologic adverse event terms

Chimeric antigen receptor (CAR) T-cells are an emerging therapy for the treatment of relapsed/refractory B-cell malignancies. While CD19 CAR-T cells have been FDA-approved, CAR T-cells targeting CD22, as well as dual-targeting CD19/CD22 CAR T-cells, are currently being evaluated in clinical trials. This systematic review and meta-analysis aimed to evaluate the efficacy and safety of CD22-targeting CAR T-cell therapies. We searched MEDLINE, EMBASE, Web of Science, and the Cochrane Central Register of Controlled Trials from inception to March 3rd 2022 for full-length articles and conference abstracts of clinical trials employing CD22-targeting CAR T-cells in acute lymphocytic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). The primary outcome was best complete response (bCR). A DerSimonian and Laird random-effects model with arcsine transformation was used to pool outcome proportions. From 1068 references screened, 100 were included, representing 30 early phase studies with 637 patients, investigating CD22 or CD19/CD22 CAR T-cells. CD22 CAR T-cells had a bCR of 68% [95% CI, 53-81%] in ALL (n= 116), and 64% [95% CI, 46-81%] in NHL (n= 28) with 74% and 96% of patients having received anti-CD19 CAR T-cells previously in ALL and NHL studies respectively. CD19/CD22 CAR T-cells had a bCR rate of 90% [95% CI, 84-95%] in ALL (n= 297) and 47% [95% CI, 34-61%] in NHL (n= 137). The estimated incidence of total and severe (grade ≥3) CRS were 87% [95% CI, 80-92%] and 6% [95% CI, 3-9%] respectively. ICANS and severe ICANS had an estimated incidence of 16% [95% CI, 9-25%] and 3% [95% CI, 1-5%] respectively. Early phase trials of CD22 and CD19/CD22 CAR T-cells show high remission rates in ALL and NHL. Severe CRS or ICANS were (1)rare and dual-targeting did not increase toxicity. Variability in CAR construct, dose, and patient factors amongst studies limits comparisons, with long-term outcomes yet to be reported. Systematic review registration:https://www.crd.york.ac.uk/prospero, identifier CRD42020193027.

DISCUSSION AND CONCLUSION Chimeric antigen receptor (CAR)-T cells directed against B-cell maturation antigen (BCMA), have yielded impressive results in clinical trials for multiply relapsed/refractory multiple myeloma (MM). However, progression-free survival is short, demonstrating a need for improvements. While numerous advances have been made to improve this treatment modality, these have not always translated to superior clinical outcomes. A major limitation is that novel therapeutics are often not tested in a relevant in vivo model. These novel strategies have been largely tested only in immunodeficient models of MM. To better recapitulate the host-tumour-effector cell interactions, particularly in the TME, we sought to develop a system to study CAR-T cell activity in the Vk*MYC model of MM. Here, we report on the first known successful model of CAR-T cell activity in an immunocompetent model of MM. We sought to identify a BCMA CAR construct that is capable of binding to both human and mouse BCMA. A novel nanobody recognizing BCMA was developed by immunizing llamas with human BCMA and the resulting single-domain antibodies (sdAbs) were subsequently isolated and cloned. These sdAbs were then screened for binding to mouse BCMA. A binder capable of recognizing both human and mouse BCMA was identified and cloned into our murine CAR construct, with CD28 and CD3z signaling domains, herein referred to as sdBCMA2.CD28.CD3z. Murine CAR-T cells were then generated by transducing activated healthy murine splenocytes with a retroviral vector carrying the sdBCMA2.CD28.CD3z construct. These CAR-T cells were then characterized in vitro using standard cytotoxicity, proliferation, and cytokine release assays. To assess activity in an immunocompetent model, female C57BL/6J mice were injected intravenously with Vk12598 Vk*MYC cells. CAR-T cells were later administered intravenously. Mice were bled weekly to quantify M-protein disease burden by serum protein electrophoresis and for phenotypic characterization of circulating cells. When co-cultured with BCMA+ human MM cell lines and with Vk*MYC cell lines, murine sdBCMA2.CD28.CD3z CAR-T cells effectively lysed target cells, proliferated, and released inflammatory cytokines. To assess the in vivo activity of these CAR-T cells, we injected intravenously female C57BL/6J mice with Vk12598 cells, and then administered sdBCMA2.CD28.CD3z CAR-T cells intravenously. While there was some evidence of survival improvement, the median overall survival (mOS) of 35 days compared to 28 days, for CAR-T vs. Mock T-cells, respectively, the difference was non-significant, and the survival benefit was not durable. To address this, we incorporated lymphodepletion by total body irradiation (TBI) followed by CAR-T cell administration the next day. By incorporating lymphodepletion prior to CAR-T cell administration we were able to significantly reduce M-protein surrogates of disease burden, as well as achieve durable remissions in the TBI + CAR-T arm with an mOS not yet reached after experimental day 91, compared to an mOS of 42 days in the TBI + Mock T-cell arm. The addition of TBI also significantly improved the engraftment of CAR-T cells, as assessed by flow cytometry. The TBI + CAR-T arm also had significantly increased IL-6, IL-1b, IL-2, and TNF-a in the serum, compared to the TBI + Mock arm, at 7 days post administration of CAR-T cells. The durable remission achieved using the combination of our novel sdBCMA2.CD28.CD3z CAR-T cells with lymphodepletion is a pivotal advancement in modelling MM CAR-T cell activity in this clinically relevant model system. Our approach represents the first time that CAR-T cells were successfully used to treat the Vk12598 model of MM that faithfully recapitulates relapsed MM. The incorporation of lymphodepletion significantly improves CAR-T cell engraftment, in line with clinical CAR-T cell therapy paradigms. The cytokine profile observed is consistent with the cytokine elevations typically observed after CAR-T cell administration in patients. As our construct is capable of binding to both human and mouse BCMA, we are therefore able to fully characterize our lead assets in a relevant model system. Our strategy will enable further research to dissect host-tumour-effector cell interactions, to better inform the design and characterization of novel CAR-T cell therapies in MM.

Supplementary Figure 4. Activation of HUVECs by serum from patients with neurotoxicity.

Supplementary Figure 2. Serum cytokines after CD19 CAR-T cell immunotherapy

Background Chimeric antigen receptor T cell therapy (CAR-T) represents a promising and exciting new therapy for hematologic malignancies, where prognosis for relapsed/refractory patients remains poor. Encouraging results from clinical trials have often been tempered by heterogeneity in response to treatment among patients, as well as safety concerns including cytokine release syndrome. The identification of specific patient or treatment-specific factors underlying this heterogeneity may provide the key to the long-term sustainability of this complex and expensive therapy. An individual patient data meta-analysis (IPMDA) may provide potential explanations for the high degree of heterogeneity. Therefore, our objective is to perform a systematic review and IPDMA of CAR-T cell therapy in patients with hematologic malignancies to explore potential effect modifiers of CAR-T cell therapy. Methods and analysis We will search MEDLINE, Embase, and the Cochrane Central Register of Controlled Clinical Trials. Studies will be screened in duplicate at the abstract level, then at the full-text level by two independent reviewers. We will include any prospective clinical trial of CAR-T cell therapy in patients with hematologic malignancies. Our primary outcome is complete response, while secondary outcomes of interest include overall response, progression-free survival, overall survival, and safety. IPD will be collected from each included trial and, in the case of missing data, corresponding authors/study sponsors will be contacted. Standard aggregate meta-analyses will be performed, followed by the IPD meta-analysis using a one-stage approach. A modified Institute of Health Economics tool will be used to evaluate the risk of bias of included studies. Ethics and dissemination Identifying characteristics that may act as modifiers of CAR-T cell efficacy is of paramount importance and can help shape future clinical trials in the field. Results from this study will be submitted for publication in a peer-reviewed scientific journal, presented at relevant conferences and shared with relevant stakeholders.

Supplementary Figure 7. Blood-brain barrier disruption in severe neurotoxicity after CD19 CAR-T cell immunotherapy

Introduction Allogeneic stem cell transplantation (AlloHSCT) remains the only potentially curative treatment for myelofibrosis (MF). It confronts many challenges stemming from the older age of the typical patients and their age-related comorbidities, as well as disease-specific factors such as splenomegaly and hostile marrow microenvironment, which raise concerns for delayed engraftment and graft failure. Methods We performed a retrospective review of the 58 adult patients who received AlloHSCT for MF in British Columbia, Canada, in the 20-year period between January 2001 and December 2020. Data pertaining to patient demographics, disease characteristics, treatment/transplant details and clinical outcomes were gathered from the available paper and electronic records. Relapse-free survival (RFS) was defined as the time between the day of the transplant and the day of disease relapse or death from any cause. Overall survival (OS) was defined as the time between the day of the transplant and death from any cause. Patients who did not have an event during follow up were censored at the time of the last known follow up. Survival analysis was performed by Kaplan-Meier survival estimator and log-rank test, using Stata version 16.1 (Texas, USA). Results The median age of the cohort was 56 years (range 26-68), with 36% being 60 years old or older. The male to female ratio was 1.6:1. The median Karnofsky Performance Status index was 80 (range 70-100) and the median age-adjusted HCT-specific Comorbidity Index was 2 (range 0-5). The pre-transplant DIPPS plus risk category was: high in 13 patients (22%); intermediate-2 in 29 (50%); intermediate-1 in 15 (26%); and low in 1 (2%). 19/58 (33%) had a peripheral blast percentage greater than 2%. 51/58 (88%) had grade 3 fibrosis seen on the pre-AlloHSCT bone marrow biopsy. 27/57 (47%) patients were transfusion-dependent for pRBC and/or platelets. 28/54 (52%) evaluable patients had a normal karyotype and 6/54 (11%) a complex karyotype. Sixteen patients (28%) had a myeloid gene panel performed by PCR; the most commonly seen variants were ASXL1 (10 patients) and TET2 (5 patients). The median number of variants seen in a single patient was 3 (range 0-5). 42/58 (72%) patients received myeloablative conditioning before AlloHSCT. The donor type was: a volunteer unrelated donor in 54%; a matched sibling in 42%; and an alternative source (double cord or haploidentical donor) in 4%. Peripheral blood stem cells were used in 93% of the AlloHSCT. 24/58 (41%) patients received ruxolitinib pre-AlloHSCT (starting in June 2012) for a median duration of 10 months (range 1-44); 17/24 (71%) of the recipients had a clinical response to ruxolitinib but 3 had lost the response before their AlloHSCT. One patient had stopped ruxolitinib due to cytopenia. During a median follow up period of 41.7 months (range 0.5-260.7), 24/58 (41%) experienced acute graft-versus-host disease (GVHD) and 33/49 (67%) evaluable patients developed chronic GVHD. Graft failure was seen in 2 patients - one primary and the other secondary. MF relapse was seen in 13/58 (22%) after a median time of 1.1 years post-AlloHSCT (IQR 0.7-2.4). The transplant-related mortality (TRM) was 25% at 1 year and 28% at 3 years post-AlloHSCT. The most common causes of death were infection (7 patients), disease relapse (6 patients), and multiorgan failure (5 patients). Transfusion independence was achieved in 45/58 (78%) patients by day +100 and in 40/44 (90%) by 1-year post-AlloHSCT. 17/48 (35%) evaluable patients had persistent grade 3 fibrosis on their day+100 bone marrow biopsy. The estimated 1-year, 5-year and 10-year RFS rates were 66% (95% CI 52-76), 55% (95% CI 41-67) and 47% (95% CI 42-61) respectively. The estimated 1-year, 5-year and 10-year OS rates were 72% (95% CI 59-82), 63% (95% CI 49-74) and 54% (95% CI 37-68) respectively. There were no differences in RFS (p=0.40) and OS (p=0.23) between those who received ruxolitinib pre-transplant vs. those who did not. Conclusion In our cohort, approximately half the AlloHSCT recipients achieved long-term survival, many in remission (10-year OS 54%; 10-year RFS 47%). TRM was seen in 25% within the first year after AlloHSCT, with patients most commonly succumbing to infections. The graft failure rate was low at 3%. An increasing number of patients received pre-AlloHSCT ruxolitinib in the recent years but we were unable to demonstrate improved transplant outcomes with its use in this single centre cohort.

Supplementary Figure 5. Immunohistochemistry of brain in a patient with fatal neurotoxicity