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Since being identified in China in December 2019, coronavirus disease 2019 (Covid‐19) has rapidly evolved into a global pandemic with over 4 million cases and more than 270,000 deaths.(1) Following the first reported cases in the United Kingdom (UK) in late January 2020, numbers have continued to rise with 223,060 cases and 32,065 deaths reported as of 11th May 2020.(2) Initial reports from China have indicated that Covid‐19 has an overall mortality rate of 1.4%. However, the prognosis varies widely between groups, with age over 60 years and underlying conditions including hypertension, diabetes, cardiovascular disease and cancer identified as risk factors for severe disease and death.(3) The initial reports from China show that patients with cancer are over‐represented among individuals who develop severe Covid‐19 after contracting the virus.(4) Patients with haematological malignancies are expected to be at increased risk of adverse outcomes from this viral infection, due being immunosuppressed as a consequence of the underlying cancer, and from the effects of therapy.

Background: Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. Methods: miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. Results: Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsamiR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. Conclusion: This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function. © 2016 Petty et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To date, studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation, block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens, we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique, unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia, was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33, CD34, CD123, CD11c and CD36, to deplete myeloblasts.

Preclinical animal models have largely ignored the immune-suppressive mechanisms that are important in human cancers. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in nonmalignant cells by cancer, we examined T-cell function in the chronic lymphocytic leukemia (CLL) Eμ- TCL1 transgenic mouse model. With development of leukemia, Eμ- TCL1 transgenic mice developed functional T-cell defects and alteration of gene and protein expression closely resembling changes seen in CLL human patients. Furthermore, infusion of CLL cells into young Eμ- TCL1 mice induced defects comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T-cell defects. Altered pathways involved genes regulating actin remodeling, and T cells exhibited dysfunctional immunological synapse formation and T-cell signaling, which was reversed by the immunomodulatory drug lenalidomide. These results further demonstrate the utility of this animal model of CLL and define a versatile model to investigate both the molecular mechanisms of cancer-induced immune suppression and immunotherapeutic repair strategies.

B-cell chronic lymphocytic leukemia (CLL) would appear to be an ideal target of T-cell-mediated responses against the cancer cell. The cancer arises in cells that can act as antigen-presenting cells (APCs), CLL cells express tumor antigens, and the cells can be a target of the allogeneic T cells in a graft-versus-leukemia effect. Despite these potential benefits, immune responses against CLL cells have been difficult to elicit. CLL induces immune defects in the host, the tumor cells are inefficient APCs, and therapies given to patients with CLL are themselves immunosuppressive. Successful vaccination approaches in this disease will require steps to overcome these difficulties, including steps to improve the immune defects in this disease, identification of the targets of the immune response to monitor immunologic responses, and improved presentation of antigen.

Although there have been no randomized trials comparing the outcome of stem cell transplantation (SCT) with standard chemotherapy for patients with chronic lymphocytic leukemia (CLL), increasingly, both autologous and allogeneic SCT approaches are being explored in this disease. Clinical trials have demonstrated that these approaches are feasible, but current data suggest that autologous transplantation is not curative and myeloablative SCT, although offering the potential for cure, is associated with high treatment-related mortality. There is a clear demonstration of a graft-versus-leukemia effect in CLL, with encouraging results seen after SCT with reduced-intensity conditioning. Because no other treatment modalities are currently capable of improving survival in this disease, the treatment of choice for younger patients with poor-risk CLL may well be SCT, but continued enrollment of appropriate patients into well-designed clinical trials is vital to compare advances in SCT with the advances occurring in chemoimmunotherapy in CLL.

Understanding how the immune system in patients with cancer interacts with malignant cells is critical for the development of successful immunotherapeutic strategies. We studied peripheral blood from newly diagnosed patients with acute myeloid leukemia (AML) to assess the impact of this disease on the patients' T cells. The absolute number of peripheral blood T cells is increased in AML compared with healthy controls. An increase in the absolute number of CD3+56+ cells was also noted. Gene expression profiling on T cells from AML patients compared with healthy donors demonstrated global differences in transcription suggesting aberrant T-cell activation patterns. These gene expression changes differ from those observed in chronic lymphocytic leukemia (CLL), indicating the heterogeneous means by which different tumors evade the host immune response. However, in common with CLL, differentially regulated genes involved in actin cytoskeletal formation were identified, and therefore the ability of T cells from AML patients to form immunologic synapses was assessed. Although AML T cells could form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signaling molecules to the synapse was significantly impaired. These findings identify T-cell dysfunction in AML that may contribute to the failure of a host immune response against leukemic blasts.

Cancer is associated with immune deficiency, but the biologic basis of this is poorly defined. Here we demonstrate that impaired actin polymerization results in CD4+ and CD8+ T cells from patients with chronic lymphocytic leukemia (CLL) exhibiting defective immunological synapse formation with APCs. Although this synapse dysfunction was in part a result of the CLL cells having poor APC function, defective actin polymerization was also identified in T cells from patients with CLL. We further demonstrate that, following contact with CLL cells, defects in immune synapse formation were induced in healthy allogeneic T cells. This required direct contact and was inhibited by blocking adhesion molecules on CLL B cells. In T cells from patients with CLL and in T cells from healthy individuals that had been in contact with CLL cells, recruitment of key regulatory proteins to the immune synapse was inhibited. Treatment of autologous T cells and CLL cells with the immunomodulating drug lenalidomide resulted in improved synapse formation. These results define what we believe to be a novel immune dysfunction in T cells from patients with CLL that has implications for both autologous and allogeneic immunotherapy approaches and identifies repair of immune synapse defects as an essential step in improving cancer immunotherapy approaches.

In HIV, the immune defects seen are due not only to a decrease in T-cell numbers, but also to qualitative impairment in T-cell function as well as decreased antigen-presenting cell (APC) function. These defects in cell-mediated immunity lead to increased level of infection, contributing to inability to clear the HIV virus, and an increased incidence of tumours. One of the major defects in HIV appears to be the failure of CD4 T cells to provide CD 154 (CD40 ligand)-mediated help, which is required for APC function. In lymphomas, activation through CD40 leads to increased APC activity and induction of immune responses against tumours. Such an effect may also be useful in HIV to increase response against the virus and improve immune surveillance of tumours.

Introduction IMiDs like lenalidomide have immunostimulatory effects and therefore the potential to reduce relapse after allogeneic haematopoietic cell transplant (AHCT) by increasing graft-versus-tumour (GvT) effects. However, early clinical experience using IMiDs after AHCT has been limited by induction of graft-versus-host disease (GvHD). Although lenalidomide has been shown to augment mitogen-stimulated T cell responses, the effects of this drug on T cell alloresponses that mediate both GvT and GvHD have not been well defined. Better understanding of the immune mechanisms involved would facilitate tracking and manipulation of lenalidomide-potentiated alloresponses and could reveal ways to use the drug to maximise GvT without excess GvHD. Therefore we used an HLA-mismatched in vitro model to analyse in depth the effects of lenalidomide on functional human T cell alloresponses. Materials and Methods We cocultured CFSE-labelled PBMC from healthy donors with irradiated allogeneic PBMC in the presence of 1μM lenalidomide, vehicle control or following pre-incubation with 1μM lenalidomide for 24 hours. Functional alloresponses were quantified after 7-9 days of allo-coculture by flow cytometry. In addition, allo-coculture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling. Results Addition of lenalidomide to allo-cocultures increased the total number of responder cells (p2-fold altered expression of >500 genes mostly associated with DNA synthesis and cellular proliferation when compared to non-proliferative CD8 cells. Lenalidomide-treated alloCD8 cells showed further increases in expression of many of these genes; however treatment also resulted in significant changes in expression of additional genes in alloCD8 cells compared to untreated alloCD8 cells (Fig 1B). These included >8 fold increases in expression of genes reported to potentiate T cell immune responses in other settings including PFKFB4,Pirin, and SOCS2 (part of the E3 ubiquitin ligase complex with cereblon), and >5 fold decreases in genes which can suppress T cell activation and memory differentiation including FAIM3 and PMCH. Conclusion We have shown for the first time that lenalidomide potentiates human alloresponses primarily by increasing alloproliferation of effector memory CD8 T cells. This likely results from altered expression of (i) multiple genes common to the intrinsic CD8 alloproliferative response and (ii) additional genes involved in the control of T cell activation and differentiation specific to lenalidomide-potentiated CD8 alloresponses. Furthermore treatment enhances the functional capacity of these cells by conferring greater polyfunctional effector potential. These findings could enable tracking of CD8 alloresponses induced by lenalidomide after AHCT and could inform novel clinical strategies for the use of the drug to augment GvT effects. Figure 1 Figure 1. Disclosures Gribben: Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Abstract 1945 Introduction: Identification of prognostic indicators which predict outcome of allogeneic stem cell transplantation (allo-SCT) is important for selection of patients who may benefit from the procedure. The European Group for Blood and Marrow Transplantation (EBMT) proposed a scoring system for CML which, after modification, also predicted outcomes for patients transplanted for other hematologic malignancies. We have recently shown that the level of C-reactive protein (CRP) measured shortly before transplantation is another important prognostic factor in patients undergoing myeloablative allo-SCT. Patients and Methods: In this study we tested the value of CRP together with other known prognostic factors in 187 consecutive patients who underwent reduced intensity HCT in a single institution from 1995 through 2010. Disease stage was assessed in accordance with EBMT criteria. Preconditioning serum CRP levels were measured at a median of 15 days before stem cell infusion using a standard latex immunoassay. The median age at HCT of the 116 (62%) males and 71 (38%) females was 49 (range 15–67) years. Fifty seven patients (31%) were transplanted in early stage, 34 (18%) in intermediate stage and 96 (51%) in late stage of their disease. The diagnoses included: AML (n = 36; 19%), Hodgkin and non-Hodgkin lymphoma (n = 42; 23%), CML in first chronic phase (n = 29; 16 %), advanced phase CML (n = 21; 11%), MDS (n = 15; 8%), myeloma (n = 16; 9%), ALL (n = 7; 4 %), myelofibrosis (n = 5; 3%), and other (n = 16; 9 %). Patients were transplanted after conditioning containing fludarabine in combination with busulfan (n = 81, 43 %), cyclophosphamide (n = 37, 20 %), and melphalan (n = 47, 25 %); and lomustine, cytarabine, cyclophosphamide and etoposide (LACE) conditioning (n = 21; 11 %). Donors were HLA matched (n = 116; 62 %) and 1 – 2 antigen mismatched (n = 8; 4 %) siblings, matched (n = 46; 25 %) and 1–2 antigen mismatched unrelated (n = 11; 6%), and HLA haploidentical family members (n = 6; 3 %). Results: In univariate analysis, factors associated with day 100 non-relapse mortality (NRM) were disease stage, preconditioning CRP level and donor and recipient HLA match; whereas disease stage, CRP level, HLA match and number of previous allogeneic stem cell transplantations were associated with overall survival. In multivariate analysis only two factors showed independent prognostic value: disease stage (early / intermediate versus late) and CRP level (above versus below 10 mg/L). A CRP level > 10 and late disease stage predicted for higher NRM (RR: 1.83, CI 1.1 – 3.1, P =.021) and (RR: 1.78, CI 1.0 – 3.0, P =.037), and inferior survival (RR: 1.61, CI 1.1 – 2.4, P =.016) and (RR: 1.58, CI 1.1 – 2.3, P =.023) respectively. The day 100 NRM was 31% (95% CI 22 – 44) for patients with elevated CRP, and 13 % (95% CI 8–21) for those with normal CRP (P =.002; figure) and 20 % (95% CI 15–27) for the whole cohort. The 5-year survival was 41 % (95% CI 27 – 56) for patients with normal CRP, 21% (95% CI 12 – 35) for those with elevated CRP (P =.004) and 34 % (95% CI 25 – 44) for the whole cohort. Conclusion: There is no obvious explanation for increased NRM and reduced survival in patients with elevated CRP before initiation of conditioning. Nevertheless our findings provide evidence that preconditioning level of CRP constitutes a key prognostic factor in allo-SCT and should be integrated into existing risk assessment tools when considering allo-SCT. Disclosures: No relevant conflicts of interest to declare.

Contrary to expectation, we have shown that the absolute number of T cells in the peripheral blood of patients presenting with AML is higher than that of healthy individuals (p=0.0006). Previous publications have demonstrated that in vitro the microenvironment in AML is immunosuppressive to healthy T cell function (Buggins et al. 2001). To analyze the molecular nature of T cell defects in patients with AML, we performed global gene expression profiling of peripheral blood CD4 and CD8 T cells from 10 AML patients compared to that of 21 healthy volunteers. The purity of the CD4 T cells was 86% in AML and 94% in healthy donors and 92% for AML and 87% in healthy donors for CD8 T cells. cRNA was hybridised on Affymetrix U133Plus2 chips using standard protocols. Samples were normalised using the MAS5 algorithm and subsequent analysis using Bioconductor software indicated marked differences between the gene expression profiles of CD4 and CD8 T cells from AML patients compared with normal individuals. Using a false discovery rate of 0.01 and fold change greater than 2, 1407 genes were found to be differentially regulated for CD4 and 779 genes for CD8. There was a large overlap in the two gene lists. The microarray data was validated using real-time quantitative PCR. Initial pathway analysis using Ingenuity software indicated many genes involved in cell proliferation, cell death and gene expression. Molecular defects in T cells from AML patients show marked differences from those we have already noted in T cells from patients with CLL. Only 2% of genes for CD4 and 6% of genes for CD8 are differentially regulated in T cells from both AML and CLL patients. These data suggest a different molecular basis for the T cell defects in these disease types. However, 2 genes downregulated in CD8 T cells in AML patients, ACTN1 and FILIP1 suggested impaired actin cytoskeletal activity may contribute to immune dysfunction in AML T cells as we have already demonstrated in CLL. We therefore tested the ability of AML CD4 and CD8 T cells to form intact immunological synapses (IS) with autologous AML blasts in the presence or absence of superantigen. F-actin was visualised with rhodamine phalloidin and recruitment of activated T cell receptor mediated signalling molecules was detected using an anti-phosphotyrosine antibody. Conjugate and synapse formation and phosphotyrosine signalling was assessed by confocal microscopy with at least 50 random conjugates analyzed. Unlike CLL B cells, AML blasts can act as antigen presenting cells (APCs) for conjugate and IS formation with healthy T cells. Again, in contrast to CLL, AML T cells are able to form conjugates with autologous tumour cells but demonstrate impaired IS formation and, in CD8 T cells, decreased phosphotyrosine signalling at the synapse site. This data indicates that T cells in patients presenting with AML are abnormal in terms of their gene expression profile and although they retain the ability to conjugate with autologous blasts, reduced polarisation of F-actin to form immune synapses is seen. We are currently investigating if these changes are induced directly by tumour cells and the functional consequences of the gene expression changes identified. The ability of AML blasts to function as APCs for IS formation is in keeping with the observed GVL effect in this disease. Understanding the nature of T cell defects in patients presenting with AML is fundamental before successful autologous immunotherapeutic strategies can be implemented.

Despite recent interest in the use of natural killer T (NKT) cells for cancer immunotherapy, there remains confusion over the phenotype and function of these cells. We observed a population of CD3+/CD56+ cells in the peripheral blood (PB) of patients with acute myeloid leukemia (AML) at diagnosis. We therefore performed more detailed multiparametric flow cytometric analysis on PB from previously untreated patients with AML (n=30), chronic lymphocytic leukemia (CLL) (n=12) and healthy normal volunteers (n=17) to characterize these cells in more detail. Since we were assessing rare cell populations, at least 200,000 events were always acquired from the mononuclear and live cell gates. In healthy individuals CD3+/CD56+ cells comprise 1% to 8.6% (median 2.74%) of PB T cells, but these cells are significantly increased both in percentage of T cells as well as in absolute numbers in the PB of patients with AML (p=0.0009) and CLL (p

Eight patients were referred from a private clinic between August 2003 and May 2004 for management of severe uncontrolled bleeding after late second trimester termination of pregnancy (TOP). TOP was perfomed at a mean gestation period of 21.4 weeks (median 21 weeks, 21–23 weeks) and was performed in 2 stages. Stage 1 comprised cervical dilatation, rupture of membrane, severing the cord and insertion of laminara. 24 hours later a dilatation and evacuation procedure was carried out. Mean age was 24.25 years (range 17–35). None of the women had any previous bleeding problems. Mean pre-operative Haemoglobin was 10.5 gm/dl (8.7 to 12.2 gm/dl). 1 patient had a preoperative clotting screen and this was normal. The pattern of bleeding was as follows: PV bleed 8/8, generalised bleed 2/8, haemodynamic failure 5/8. Median time to onset of bleeding after the second stage was 1.5 hours and median time to control bleeding was 9.5 hours. Details of coagulation screen and transfusion support are provided in tables 1 and 2. Factor VIIa was not approved for use in the hospital at that time. table 1 . coagulation parameters UPN nadir fibrinogen (1.8–4.5 g/l) hours to nadir from TOP hours to normal fibrinogen PT (9.6–11.6 secs) APTT (24–32 secs) platelets (150–400 × 109/l) 1 0.44 8 18 16.7 37.2 203 2 0.19 2 7 22.6 54.8 108 3 0.67 7 13 16.8 38 44 4 0.21 5 11 23.4 34.2 145 5 0.35 5 6 21.5 78 47 6 0.5 6 12 31 37.2 141 7 0.57 3 5 15.6 42.7 88 8 0.94 5 12.8 37.7 144 table 2 . blood product support UPN cryoppt (units) FFP(units) RBC(units) platelets(units) 1 12 2 2 2 20 4 14 1 3 15 4 9 4 40 4 12 5 20 5 17 2 6 10 3 8 7 18 4 6 1 8 2 2 Obstetric interventions included hysterectomy (1), balloon occlusion (2) ERPC (1), hysterotomy (1), cervical tear repair (2) and vaginal pack (2). Obstetric cause of haemorrhage was identified to be uterine atony (4), retained products (2), uterine injury (2) and no identifiable cause (2). All women recovered fully. Results: The profound coagulopathy in these women was characterised by a reduction in fibrinogen that was out of proportion to the anomalies in other clotting parameters and platelet counts. Treatment with cryoprecipitate reversed this reduction and controlled the haemorrhage although 5/8 patients required significant surgical intervention. Conclusions: The coagulation anomalies in these women are indicative of a defibrination syndrome that differs from DIC in the disproportionate reduction in fibrinogen level and the relatively rapid and complete resolution with blood product replacement and treating uterine pathology. Two stage TOPs in late second trimester is associated with significant morbidity.

Homoharringtonine is a plant alkaloid that inhibits the synthesis of proteins and results in apoptosis. In vitro homoharringtonine shows synergistic or additive effects with imatinib in CML cell lines and in blast cells derived from patients with advanced disease. We undertook a phase I/II study in patients who had achieved a sub-optimal response to imatinib alone to investigate whether the addition of semi-synthetic homoharringtonine (sHHT) (Myelostat®) would reduce the level of residual disease. Patients with CML who had achieved at least 35% Ph-negativity on imatinib were included. All patients were treated with imatinib at ≥ 400mg/day for at least 2 years; and achieved a plateau in BCR-ABL transcripts defined by measuring BCR-ABL transcripts on at least 4 occasions over a minimum period of 1 year with the latest value not lower than the previous minimum value. When sHHT was introduced, imatinib was continued at the previous dosage. sHHT was given subcutaneously at a dose of 1.25 mg/m2 twice daily for one day. Courses were repeated every 28 days. The dose of sHHT was escalated by adding one day of treatment every two courses (eg dose level 2: 1.25mg/m2 BD x 2 days) if the ANC was ≥2.5x109/l and the platelet count ≥200x109/l by day +28 of the previous course, or adding half a day if the ANC was ≥1.5x109/l and the platelet count ≥ 100x109/l. Patients were considered to have reached the maximal tolerable dose (MTD) when the sHHT could not be escalated further. Response was assessed by serial monitoring of peripheral blood levels of BCR-ABL transcripts assayed by RT-PCR at the start of each course. Results 10 patients have been enrolled so far. Patients received a median of six courses of sHHT (range 4–8). The MTD has been achieved in all cases (9 cases in account of myelosuppression and in one case in account of grade III asthenia). The median MDT was 2 days (range 1.5–3 days). The transcript level decline in all patients (table 1). In 7 patients the transcript levels declined by >0.5 log and in 5 patients by >1 log with respect the baseline value. In 2 patients transcripts became undetectable. The combination was relatively well tolerated with the principal toxicities being grade II asthenia (n=9), grade III astenia (n=1), grade III neutropenia (n=3) and grade III thrombocytopenia (n=1). We believe these preliminary clinical observations justify further study of the use of sHHT in patients on imatinib who fail to obtain low levels of minimal residual disease. Table 1 | n | Time from onset of imatinib to sHHT therapy (months) | Median R-PCR over the year preceding sHHT therapy | Minimal R-PCR during the year preceding sHHT therapy | R-PCR at screening for sHHT | R-PCR at last follow up | |:----------------------------------------------------------- | ---------------------------------------------------- | ------------------------------------------------- | ---------------------------------------------------- | --------------------------- | ----------------------- | | Transcript levels are expressed as a BCR-ABL/ABL percentage | | 1 | 27.2 | 7.3 | 5.4 | 16.9 | 0.6 | | 2 | 35.9 | 2.2 | 0.04 | 4.5 | 1.18 | | 3 | 34.1 | 0.012 | 0.01 | 0.028

Increasing numbers of allogeneic hematopoietic stem cell transplantation (allo-SCT) are being performed for patients who have failed a previous allogeneic or autologous SCT. We investigated whether the EBMT risk score could predict outcome after a subsequent allo-SCT. We analyzed prognostic factors in 124 consecutive patients who underwent a second transplantation using an allogeneic donor at our institution. Patients with either a first autologous (N = 64) or first allogeneic (N = 60) SCT were included. Age, disease stage, time interval from diagnosis to transplantation, donor type, and donor-recipient sex combination were used to establish a score from 0 to 7 points, from which 3 groups were identified. The 5-year survival probability decreased from 51.7% for risk scores 0-3 (low, n = 25), to 29.3% for risk score 4 (intermediate, n = 42), and only 10.4% for risk scores 5-7 (high, n = 57), P = .001. We propose that the EBMT risk score can identify patients most likely to benefit from a second transplantation.

Immunomodulatory drugs (IMiDs), such as lenalidomide provide a tool to enhance both direct anti-tumor and graft-versus-tumor effects after allogeneic haematopoietic stem-cell transplantation (AHCT). However, early clinical experience with IMiDs after AHCT using adult peripheral blood (APB) as a stem cell source has been limited by induction of graft-versus-host disease. Characterization of the mechanisms by which IMIDs can modulate alloresponses of T-cells and identification of differential effects on T-cells from disparate cell sources could facilitate more effective use of these drugs in the setting of AHCT. We therefore used in vitro modelling, multi-parameter flow cytometry and gene expression analysis to delineate the impact of the widely used IMiD lenalidomide on quantitative and qualitative alloresponses mediated by T-cells derived from APB and umbilical cord blood (UCB). We co-cultured carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMC) from healthy adult donors or donated UCB units with irradiated allogeneic PBMC in the presence of 1μM lenalidomide or vehicle control. In this model, cellular and supernatant concentrations of lenalidomide (determined by mass spectrometry) were similar to in vivo levels achieved at doses used as maintenance after AHSCT in published studies. Functional alloresponses were quantified after 7-9 days of allogeneic co-culture by flow cytometry. In addition, co-culture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling. We demonstrate that lenalidomide potentiates net alloproliferation of APB derived T-cells cells by selectively enhancing allospecific proliferation of CD8+ T-cells (median 58% (lenalidomide-treated) versus 43% (untreated), n=40, p In common with our findings in APB T-cells, lenalidomide also potentiated proliferation of alloreactive CD8+ T-cells from UCB (median 43% (lenalidomide-treated) vs 24% (untreated), n=17, p=0.0005) with similar polyfunctional effector memory differentiation, immunoregulatory gene expression changes and cellular ikaros depletion. Importantly, lenalidomide reduced allospecific proliferation of UCB CD4+ T-cells (median 58% (untreated) versus 41% (lenalidomide-treated), n=17, p Our findings show that lenalidomide has a qualitatively different impact on alloresponses of T-cells from different cell sources; alloresponses of APB T-cells are increased by lenalidomide via selective expansion of polyfunctional CD8+ effectors while lenalidomide limits alloresponses of UCB T-cells by reducing CD4+ effector expansion and increasing tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHCT. Disclosures No relevant conflicts of interest to declare.