ALBERT

Description: Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

Description: In patients with acute promyelocytic leukemia (APL), in spite of recent advances, early treatment related mortality (TRM) still remains a challenge and is largely related to the associated coagulopathy. It is well recognized that conventional coagulation parameters used in the clinic do not accurately predict the risk of adverse outcomes like major bleeding or thrombosis. There is limited data on the clinical utility of whole blood viscoelastic assays and extended coagulation parameters for evaluation of coagulopathy in patients with APL. Consecutive patients with PML-RARA positive APL were enrolled. Blood samples were collected prior to infusion of blood products on day 1 and again on day 4 for thromboelastometry done on whole blood and activated with recombinant tissue factor at a final dilution of 1:50,000 (ROTEM analyzer, Tem Innovations GmbH, Basel) and an extended panel of coagulation parameters done by immunoassays (TAT complex, protein C, ATIII, PIC complex, tPAIC, d-dimer and thrombomodulin). Platelet count and conventional coagulation parameters, which were prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and thrombin time (TT), were measured daily for the first 14 days (more frequently as clinically indicated). Patients received platelet transfusions to target a platelet count of 30x109/L (in the absence of bleeding). Fresh frozen plasma and cryoprecipitate transfusions were done to target a normal PT and aPTT and maintain a fibrinogen level above 140 mg%. Major bleeding, thrombosis and death during induction therapy were the outcome variables. Of the 61 patients enrolled, 11 were excluded (3 did not continue treatment at our hospital, 2 died within 48 hours of admission, and 6 took discharge against medical advice before completing induction). Of the remaining 50 cases that were evaluated, 40 were newly diagnosed and 10 were relapsed. The median age was 34.5 years (range 9-68). 25 were males (50 %). The WBC count was above 10 x 109/L in 20 patients (40%). The median platelet count was 28 x 109/L (range 4-339). There were 13 (26%) deaths in induction. Six patients had a major bleeding event. Five patients had a thrombotic event. Of the evaluated conventional coagulation parameters, extended coagulation parameters, platelet counts and ROTEM parameters at diagnosis (ROTEM data not available at diagnosis in 2 cases) only the ROTEM parameter of maximum clot firmness (MCF) as a continuous variable was significantly associated with death (p=0.012). On univariate cox regression analysis with death as the outcome, MCF≤ 30mm was a poor prognostic marker (hazard ratio of 13.24, 95%CI 1.71-102.19; p =0.013). On multivariate cox regression analysis including high WBC count (〉10 x 109/L), low platelet count (〈 40 x 109/L) and history of relapse, MCF ≤30mm was an independent poor prognostic variable (HR: 11.89; 95% CI 1.43 - 98.75; p 0.022). Four of the 6 major bleeding events, four of five thrombotic events and all deaths related to coagulopathy occurred in patients with MCF ≤30mm. The descriptive statistics of patients with MCF 〉30mm (n=21) vs those with MCF ≤ 30mm (n=27) are shown in table 1. Figure 1 illustrates the type of discordance one can see between conventional coagulation parameters and ROTEM values. This study suggests that the MCF value from a ROTEM assay at diagnosis is probably a better measure of the hemostatic defect at diagnosis in APL. Patients with APL with low MCF on ROTEM at diagnosis are at an increased risk of death during induction therapy despite an adequate blood product replacement strategy which was based on conventional coagulation parameters. In these patients, there is a need to develop novel and possibly a more intensive replacement strategy. Table 1. Comparison of baseline characteristics and clinical outcomes. Variables MCF ≤30 (n = 27)N (%) /Median (range) / Mean±SD MCF 〉 30 (n=21)N (%) /Median (range) / Mean±SD p value Age (years) 40(12-68) 27(9-48) 0.006 WBC 〉10 x x109/L 17(62.96) 2(9.52) 0.003 Platelet count (x109/L) 17(4-89) 45(10-339) 0.001 PT in seconds 14.5(11-87.5) 11.8(10.1-14.8) 0.004 aPTT in seconds 28(23.2-77.9) 30.2(22-53.6) 0.104 Fibrinogen in mg% 134.8(24.7-393) 172.2(14.7-549.5) 0.066 Major bleeding 4(14.8) 1(4.8) 0.368 Thrombosis 4(14.8) 1(4.8) 0.369 Death 12(44.4) 1(4.8) 0.003 Disclosures No relevant conflicts of interest to declare.

Description: Graft rejection (GR) following an allo-SCT occurs in 10-20% of patients with β thalassemia major (TM). The reported clinical outcome following second transplants have been associated with a high incidence of graft rejection, treatment related mortality, and graft versus host disease [Haematologica 2009; 94(9)]. There is limited data on the clinical profile and long term outcome of patients who have had a graft rejection. We undertook a retrospective analysis of patients who had a graft failure post allogeneic SCT for TM at our center. From October, 1991 to June 2016, 506 HLA matched related transplants for TM were done at our center. Of these 55 (11%) patients had a graft failure. An additional 7 patients with graft failure following an allo-SCT done at other centers were referred to us for a second transplant. Of the 62 patients with graft failure 32 (52.4%) were primary graft failures (PGF; 15 with aplasia and 17 with autologous recovery) while 30 (47.6%) were secondary graft failures (SGF; 5 with aplasia and 25 with autologous recovery). The median age of the patients who had graft failure was 8 years (range:1-19) and there were 38 (60.3%) males. On conventional risk stratification 40 (63.5%) were Class III. Eighteen (54.5%) cases with PGF and 16 (53.3%) with SGF did not receive a second transplant. From Oct 2009, at our center, a reduced toxicity myeloablative (MAC) treosulfan based conditioning regimen with a PBSC graft was offered to all high risk patients and for second transplants at the treating physician's discretion. Twenty nine (46%) of the patients with GR underwent a second allo-SCT. With the exception of one patient (first allo-SCT with an unrelated cord blood product) the donor for the second transplant was the same as the first transplant. Conditioning regimen for second SCT was busulfan based MAC in 7 (24%), treosulfan based MAC in 12 (41.3%) and the remaining received non-myeloablative conditioning regimens (fludarabine based, low dose TBI, OKT3, Cy-OKT3) often in view of pancytopenia and perceived inability to tolerate a MAC. All patients receiving a treosulfan based regimen had a PBSC graft. A BM graft was used in 7 (41%) of the remaining cases. None of the patients conditioned with a treosulfan based regimen had a graft rejection though one patient died of Grade IV acute GVHD. Of the remaining 17 patients 10 died following a second GR, 3 died of regimen related toxicity. Four are alive of which one has recurrent TM and the rest are well and transfusion independent at 55, 80 and 204 months from second transplant (all busulfan based MAC). The baseline characteristics and clinical outcomes of patients who received a treosulfan based MAC regimen versus the rest is summarized in table 1 and figure 1. On a univariate analysis a non-treosulfan based conditioning regimen and time from GR to second transplant of

Description: Among transplant related complications, graft versus host disease (GvHD) significantly affects survival among patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT). There is limited data on GvHD and its impact on outcomes of aHSCT in patients with thalassemia major (TM). We have reviewed the incidence and outcome of GVHD among patients with TM who underwent aHSCT at our center. All patients with TM undergoing their first aHSCT between January 2007 and December 2017 were included in this analysis. Till 2009, all patients received conditioning with busulfan (16mg/kg over 4 days) with cyclophosphamide (200mg/kg over 4 days). From 2010, most patients received treosulfan (42 G/m2 over 3 days) with thiotepa (8mg/Kg for one day) and fludarabine (160mg/m2over 4 days) based conditioning regimen. All patients receiving busulfan conditioning received bone marrow (BM) as the graft while most patients receiving treosulfan conditioning received mobilized peripheral blood stem cells (PBSC). GvHD prophylaxis was with short-course methotrexate (10mg/m2 on day +1, and 7mg/m2 on days 3, 6 and 11) with cyclosporine. Thymoglobulin was added for matched unrelated donors (MUD). GvHD was prospectively recorded and graded according to the Glucksberg classification. Between January 2007 and December 2017, 363 first transplants were done for patients with TM with HLA identical donors. There were 12(3.3%) class 1, 105(28.9%) class 2 and 246(67.8%) class 3 (Pesaro risk stratification), with 115(46.7%) of the latter being high risk (Vellore risk stratification - BBMT, 2007; 13: 889). The median age was 8 years (range: 1-25) with a male predominance (60%). 331 (91.2%) patients had matched related donors (MRD) and 32 (8.8%) had MUDs. Donor gender was mismatched in 207 (57%) of which 129 (35.5%) were female to male transplants. The graft was obtained from the bone marrow in 137 (37.7%) of whom 53 (38.7%) were class III, and from mobilized peripheral blood in 226 (62.3%) of whom 193 (85.4% were class III. 149 (41%) patients developed GvHD - acute GvHD (aGvHD) in 115 (31.7%) and chronic GvHD (cGvHD) in 80 (22%). aGvHD was grade I in 32 patients (27.8%), grade II in 36patients (31.3%), grade III in 16 patients (13.9%) and grade IV in 25 patients (21.7%), while 6 patients (5.2%) had features of overlap GvHD only (oral lichen planus). First line treatment was with steroids in all patients with grade II and above aGvHD (n=83) with 43 (51.8%) of them responding adequately. There were 37 patients (44.5%) who required various second line agents for aGvHD with 20 (24%) receiving more than one immunosuppressive agent. 20 patients (24%) with persistent aGvHD went on to develop cGvHD. Out of the total of 80 patients with cGvHD, 13 (16.3%) had limited and 67 (83.7%) had extensive cGvHD. 26 patients (32.5%) developed de novo cGvHD, 8 (10%) of them after donor lymphocyte infusion (DLI) for potential rejection. The other 46 patients (57.5%) had chronic overlap GvHD following previous aGvHD. Among the different variables evaluated for association with aGvHD (patient/donor age, gender mismatch, MUD vs MRD), none were significant. Among those with MRD, aGVHd occurred in 36/135 patients (26.7%) of patients receiving BM grafts compared to 65/196 patients (33.2%) who received PBSC grafts (p=ns). cGvHD occurred in 23/106 patients (21.7%) in those receiving BM grafts vs 52/171 patients (30.4%) receiving PBSC (p=ns). 30 patients (8.3%) persisted to have cGvHD at last follow-up but only 20 (5.2%) required treatment. Mortality of the whole cohort was 66 (18.2%), out of which 32 (8.8%) were related to GvHD - 24 (6.6%) due to aGvHD and 8 (2.2%) due to cGvHD. At a median follow up of 41 months (range: 0-148), the 5-year and 10-year overall survival (OS) was 81.1±2.1% each for the whole cohort. The 5-year OS of those with grade 2-4 aGvHD was significantly lower than those with grade 0/1 aGvHD (65.7±5.3% vs 85.7±2.2%, p=0.000) [figure 1]. The 5 year OS of those with cGvHD was 88.9% ± 3.8% as compared to those without cGVHD was 96.9% ± 1.2% (P=0.009) [figure 2]. There was no significant difference in OS among those with limited and extensive cGvHD (90.9±3.6% vs 88.4±4.2% (p=ns). Our data shows that, as expected, severe aGvHD and extensive cGvHD significantly lowers survival in patients with TM undergoing aHSCT. However, PBSC graft did not result in higher acute or chronic GVHD compared to BM. Disclosures No relevant conflicts of interest to declare.

Description: Multicenter large collaborative research groups have been the cornerstone for advances that have been made in multiple disciplines in medicine. These collaborative groups are specifically useful in situation where no single center based dataset is large enough to effectively address biological and clinical relevant questions that could advance the field. Most such collaborative groups exist in the developed countries and have contributed significantly to the development of the current standards of care in leukemia. The challenges in the developing countries or low middle income countries (LMIC) are distinctly different and often algorithms that have evolved in the developed world may not be applicable, relevant or accessible in the LMIC. It is imperative that these challenges be addressed through large multicenter studies that are located within the LMIC and appropriate local data driven solutions be implemented in response. The 'Hematological Cancer Consortium' is a collaborative group from India currently compromising twelve institutions spread across the country that have come together to collaborate in the field of leukemia. As an initial exercise, to establish denominators a retrospective data analysis was undertaken (Indian acute leukemia research database [INwARD]). Here we present the retrospective analysis of the acute myeloid leukemia (AML) data. Retrospective data from January 2013 to December 2017 was collected from 10 large tertiary centers from across the country (in one center data was available only from January 2017). A central online data capture and management system was in place which was independent of all the participating centers (Clinical Data Management Center [CDMC], Vellore, which is compliant with standard ICH-GCP regulations). In this initial phase some centers contributed data offline to the data management center. A total of 3848 were confirmed to have had a diagnosis of AML in this period of which it was noted that 1766 (46%) received definitive treatment (Fig 1 a). The median age of the patients was 40 years (range: 0-89) and there were 59% males. The age distribution of patients by each decade is illustrated in Fig 1b. 399 (10.4%) were ≤ 18 years). A sample for karyotyping was sent in 2609 (68%) however of these an evaluable karyotype was noted in only 1477 (57%) (Fig 1c), the reasons for lack of evaluable metaphases was not clear. A FLT3 and NPM1 mutation status was evaluated in 1338 (35%) and 1401 (36%) respectively. Of the evaluated patients 20.6% and 21.9% had FLT3-ITD and NPM1 mutated respectively (Fig 1d). Of the 1766 patients that were treated 858 (48.6%) received a conventional 7/3 induction, 170 (9.6%) received hypo-methylating agents while the rest received various abbreviated dose regimens and a small proportion (2.8%) received high dose cytosine based regimens as induction therapy. Antifungal prophylaxis was used by 82% of patients that received therapy. Of those that received induction therapy there were 18% induction deaths and 12.9% subsequently received an allogeneic SCT as part of their consolidation therapy (Fig 1a). The 5 year KM estimate for overall and event free survival for the patient that received treatment was 56.2±2.6% and 33.8±2.4% respectively. The data illustrates significant challenges and opportunities with the management of AML in India. A significant proportion of cases do not receive definitive therapy nor do they have conventional tests such as karyotyping or molecular tests done as part of the baseline diagnostic tests, various social and financial constraints could contribute to these and these need to be evaluated in more detail. Strategies to increase access to care and laboratory facilities along with an effort to reduce early induction deaths need relatively urgent attention. The relatively young age of the cohort and large number of cases would allow us to address relevant biological and clinically challenges effectively, in the future, in this cooperative setting. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Allogeneic stem cell transplantation (SCT) is the best form of therapy for a young patient (〈 50 years) with severe aplastic anaemia. In developing countries, there is a big time interval between diagnosis and SCT leading to increased transfusions and increased risk of infections, both of which adversely affects transplant outcome. This retrospective analysis is aimed at studying the outcomes of SCT among Indian patients with aplastic anaemia. Methodology: The Indian Stem cell transplant registry (ISCTR) is a group of transplant physicians representing about 30 active transplant centres in India. This retrospective analysis was done on data reported on 634 patients by 20 centres who reported outcomes of SCT for aplastic anaemia. Data was collected from individual medical records and databases. Analysis was done using SPSS software version 16.0 Results: Six hundred and thirty four patients [445 males and 189 females] with a median age of 21 years (range: 2 - 65) underwent allogeneic SCT between 1990 and March 2015. There were 209 children (age 〈 15 years). The median time from diagnosis to SCT was 5 months (range: 1 - 120) while the median number of transfusions was 20 (range: 1 - 150). All donors were HLA identical sibling or family donors; matched unrelated and haplo-identical donor transplants were excluded from this analysis. Conditioning regimen was Cyclophosphamide based (Cy/ Cy+ ATG/ Cy+ TBI/TLI) in 78 patients (12.3%) while majority received Fludarabine with Cyclophosphamide (n = 481; 75.8%) and 75 received other conditioning regimens (Flu/TBI, Flu/Bu, Bu/Cy etc). Graft source was bone marrow [BM] in 124 (19.5%) and peripheral blood stem cells in 510 patients (80.5%). Graft versus host disease (GVHD) prophylaxis predominantly consisted of Cyclosporine and methotrexate in 543 patients (85.6%). Engraftment was seen in 572 patients (90.4%) while 19 (2.9%) had primary graft failure and 43 (6.7%) expired prior to engraftment due to infection or bleeding. The median time to neutrophil engraftment was 13 days (range: 8 - 21) while platelet engraftment occurred at 13 days (range: 5 - 37). Grade II - IV acute GVHD occurred in 29.3% while grade III-IV was seen in 14.1%. Chronic GVHD was seen in 41% of evaluable patients which was limited in most patients. At a median follow up of 43 months (range: 1 - 264), 431 patients are alive. The 5 yr OS for the entire group is 66.3 + 2.0%. The OS was higher in children compared to adults (73.4 + 3.3% vs 62.8 + 2.5%; p = 0.006), better for Flu/Cy compared to Cy based conditioning (69.8 + 2.2% vs 57.8 + 5.6%; p = 0.002) and better for PBSC compared to BM (68.9 + 2.2% vs 56.1 + 4.8%; p = 0.020). There has been significant improvement in outcomes over the past 15 years [3 yr OS of 41.9 + 1.3% for 1984-1995, 40.9 + 1.2% for 1996-2000, 70.6 + 5.0% for 2001 -2005, 70.3 + 3.1% for 2006-2010 and 68.8 + 3.0% from 2011 onwards]. Conclusion: Outcomes of patients with aplastic anaemia are improving and patients have a 70% chance of getting cured with a HLA identical sibling donor transplant. The use of PBSC as graft source is not associated with inferior outcomes. Disclosures No relevant conflicts of interest to declare.

Description: A toxicity reduced conditioning regimen containing Treosulfan (Treo), fludarabine (Flu), thiotepa for high risk Thal Major (TM) has been used since 2009 at our centre that has significantly improved transplant outcomes of these patients compared to the historical cohort of patients receiving busulfan/ cyclophosphamide based myeloablative regimen (Mathews et al, 2013). Limited knowledge is available on the pharmacokinetics (PK), pharmacogenetics (PG) and pharmacodynamics of fludarabine and treosulfan, especially in non-malignant hematological disorders like TM. We describe here the PK of Flu and Treo in patients with TM undergoing HSCT, the factors influencing the inter-individual variability in PK and the role of these factors on HSCT outcome. Seventy one patients diagnosed with TM undergoing HSCT with Flu/Treo based conditioning regimen between January 2012 and January 2015 were included (Table: Patient demographics). Selected functional polymorphisms in the NT5E, DCK, hENT1 and GST genes that are involved in fludarabine or treosulfan metabolism were screened. All patients received Flu 40mg/m2/day x 4 days as an 1hr infusion on days 1 and 4 and Treo as 14g/m2/day x 3 days at the rate 5g/hr. Plasma was separated from the peripheral blood collected at predetermined time points after the infusion of Flu and Treo PK analysis. Plasma Flu was analyzed using a LC-MS/MS method and the concentration was expressed as mMole/ml while Treo was analyzed using a HPLC-RI method and concentration was expressed as mg/L. Flu and Treo PK was estimated using nonlinear mixed effects modeling via Monolix 4.3.3. The covariates tested for both PK were: age, sex, body weight, BSA, ferritin, and polymorphisms in NT5E, hENT1, dCK and GST genes. The PK parameters AUC, CL, V and k were estimated on day 1 for Treo and on day 1 and day 4 for Flu (Table). The influence of Flu and Treo PK and PG on graft rejection, early transplant related mortality (TRM) & chimerism status was estimated using logistic regression analysis. Wide inter-individual variation in Flu and Treo PK was noted (7 and 9 fold Vs 5 and 8 fold respectively for Day 1 & 4 Flu AUC & Cl; 33 & 31 fold variation in Treo AUC and Cl) (Table). Flu CL was significantly higher on day 4 compared to day1 (Figure A). The variation in Flu PK was explained by genetic variants in NT5E and dCK. Patients having variant genotype for the SNPs in NT5E (rs2295890) and dCK (rs11544786) showed significantly lower plasma Flu clearance compared to those with wild type genotype (p=0.006 & p=0.05 respectively) (Figure B). This is consistent with our previous report in patients with aplastic anemia undergoing HSCT (Mohanan et al. 2014; Blood: 124 (21)). None of the genetic variants in the GST genes explained the variation in Treo PK. Day21 mortality was seen in 6/71 patients (8.5%) and graft rejection in 3/66 evaluable patients (4.5%). Analysis of the influence of PK and PG variables on transplant outcome showed significantly high first dose Flu AUC to be associated with D21 mortality upon Univariate analysis (median 42.5, range 32.1-63.7 compared to 31.8, range 15.2-111 mMole*h/mL, in those with and without TRM respectively; p=0.043); none of these parameters were significantly associated with graft rejection or mixed chimerism. There was no association between Treo PK parameters and graft rejection or TRM. The influence of Flu and Treo PK on regimen related toxicity is yet to be evaluated. The lack of the influence of PK on transplant outcome could be due to lower incidence of rejection and TRM in this cohort. Further analysis in a larger cohort of patients will be done once we enroll more patients for PK analysis. Our results demonstrate that Flu PK is influenced by genetic variants in NT5E and dCK, the enzymes involved in Flu biotransformation. The relationship between high-plasma Flu exposure and TRM and given the fact that multiple factors influence TRM, we can extrapolate that the plasma Flu AUC may be a surrogate marker of overall preparative regimen intensity as reported previously (Long-Boyle et al, Bone Marrow Transplant, 2011). The lack of association of genetic variants in GST genes in explaining the inter-patient variability in treosulfan exposure suggests the involvement of other drug metabolizing genes on treosulfan PK. We are currently evaluating the role of genetic variants in a large panel of drug metabolizing genes on explaining this inter-individual variability in Treo PK. Disclosures No relevant conflicts of interest to declare.

Description: The focus of ATO resistance in acute promyelocytic leukemia (APL) has centered on mutations in PML-RARA gene (Blood 2011, NEJM 2014). However such mutations are rare and cannot explain the majority of relapses seen in the clinic. To evaluate the mechanisms of ATO resistance, we generated ATO resistant NB4 sub clone NB4-EVAsR1 (A216V - VAF-91.7%) in our laboratory. We also had another ATO resistant cell line (UF1) which does not have the A216V mutation. In an expression array we noted that redox signaling, AMPK signaling and energy metabolism pathways were significantly dysregulated in the ATO resistant cell lines compared to naïve NB4 cells. Towards validating the microarray data and to characterize the ATO resistant cell lines we measured the basal levels of reactive oxygen species (ROS), glutathione(GSH), mitochondrial membrane potential (MMP), glucose uptake and their sensitivity to glycolytic inhibitor 2-Deoxy glucose (2-DG) in comparison to naïve NB4 cells. We observed that resistant cell lines have significantly lower ROS, MMP, glucose uptake (Fig 1a) and increased GSH. We also observed that the resistant cell lines were significantly less susceptible to treatment with 2-DG in comparison to naïve NB4 cells (Fig 1b) suggesting that resistant cell lines were less dependent on glycolysis. ATO has been reported to directly inhibit the glycolytic pathway, this effect is believed to contribute to its cytotoxic effect (PNAS 2015). However, we did not observe any cytotoxic synergy between ATO and 2-DG on naïve NB4 cells and neither did this combination restore sensitivity to ATO in the resistant cell lines (Fig 1b). Next we assessed the sensitivity of these resistant cell lines to oxidative phosphorylation (OXPHOS) inhibitors. We used an uncoupler (FCCP at 10uM) of OXPHOS which promotes uncoupled respiration by deregulating the proton gradient which drives ATP synthesis via ATP synthase. We observed that the FCCP treatment alone did not reduced the viability of naïve NB4 cells. Similarly, viability of ATO resistant cell lines also did not reduce significantly suggesting the ability of these cells to uncouple their metabolic pathway from OXPHOS to glycolysis when inhibited. However, when FCCP was combined with ATO it significantly restored the sensitivity of the resistant cell lines to ATO (Fig 1c). The same combination did not have any additive effect on naïve NB4 cells. The combination not only restored the sensitivity of the ATO resistant cell lines but also sensitized the conventionally ATO resistant cell lines such U937 (Fig 1c) and THP1. In spite of the profound effect on leukemic cells we also observed a significant bystander effect on the normal peripheral blood mononuclear cells (Fig 1c). The data suggests that the sensitivity of these resistant cell lines could be potentially restored by combining ATO with an OXPHOS uncoupler. A number of molecules that are FDA approved and used in the clinic also have OXPHOS uncoupling activity and could potentially be evaluated for their synergistic activity with ATO in leukemia. This data also draws attention to possible severe systemic off-target toxicity of such combinations which may be inadvertently used in the clinic. Disclosures No relevant conflicts of interest to declare.

Description: There are approximately 10 therapeutic agents listed as second line therapy for steroid refractory or relapsed ITP but there are no clear guidelines for timing & order of their use or that of splenectomy. At our center, we have used dapsone or azathioprine as second line therapy prior to splenectomy in such patients who do not have critical bleeding. This study describes the outcome of that approach. Three hundred patients fulfilling ITP International Working Group definition of steroid non-response & relapse ITP (Rodeghiero et al Blood 2009) were included. Response to treatment was assessed using American Society of Hematology 2011 guidelines (Neunert et al Blood 2011). This analysis was approved by the institutional ethics committee. Patients received either dapsone (n = 170) at 1-2 mg/kg/day or azathioprine (n = 130) at 1-3 mg/kg/day in escalating doses based on physician preferences. Additional intermittent corticosteroids were given in 36 patients. Data was retrieved from individual medical records and electronic database. Statistical analysis was performed using SPSS version 16.0 Results: Baseline characteristics of the patients & outcome are described in Table 1. After 3 months of therapy, overall response was 58.6% while it was 58.8% for the dapsone group and 58.5% for azathioprine group. The number of patients achieving complete & partial response was 46% & 12.7% for overall group, 45.9% & 13% for dapsone group & 46.2% & 12.3% for azathioprine group. The median duration of response was 35 months (2-74 months) and was significantly longer with azathioprine - 60 months (2-60 months) compared to 27 months (5-74 months) with dapsone (p=0.015). Therapy was well tolerated with 4 patients discontinuing dapsone (methemoglobinemia, dapsone syndrome) and 2 discontinuing azathioprine (cytopenia). There were no deaths. At a median follow up of 33 months (24-42 months), 67 (38%) of the 176 responders have relapsed. These included 49 patients that relapsed while on therapy and 18 that relapsed after cessation of therapy. Relapses were significantly more common with dapsone (40.3%) than azathioprine (20.4%) (p=0.002). The median time to relapse while on therapy was 14 months for both agents. Any response to second line therapy less than complete response (p=0.030) & steroid nonresponsive ITP (p=0.042) were significantly associated with increased risk of relapse on therapy. Overall, 59.6% patients responding to dapsone and & 79.5% of patients responding to azathioprine continue to remain in remission both on & off therapy. Patients who switched to dapsone at the time of relapse showed significantly better response rate - 54% than azathioprine - 22.2% (p

Description: Allogeneic stem cell transplantation (HSCT) is the only curative option for patients with thalassemia major (BTM). HSCT in BTM is associated with increased risk of graft rejection. Some patients do not achieve full donor chimerism after transplant despite hematopoietic engraftment. Early post transplant mixed chimerism (MC) is a known predictor of secondary graft rejection. Data is limited on the role of donor lymphocyte infusion (DLI) to prevent secondary graft rejection following detection of early MC in BTM. We report our experience with DLI in BTM patients who had developed progressive worsening early MC after HSCT. All patients with BTM who underwent HSCT at our centre between September 1994 and December 2013 were included in the analysis. During this period, 406 patients underwent HSCT for BTM. Median age was 8 years (range: 1 - 25) and 247 were males (60.8 %). Conditioning regimen was busulphan based in 274 (67.5%) or treosulfan based in 132 (32.5%). The graft source was BM in 305 (75.1%) and PBSC in 101 (24.9%) etc. Median CD34 stem cell dose was 9.24x106/kg (range: 2.1- 33). GVHD prophylaxis consisted of CSA and short course MTX. Patients who engrafted underwent chimerism analysis of peripheral blood at day 28 and between days 60-90. Subsequently chimerism analysis was done at the discretion of the treating physician. Chimerism analysis was done by qualitative and quantitative methods up to 2007 and by quantitative method from 2007. MC was defined by 〉5% recipient cells at any time point post transplant. The severity of MC was determined as per previously published criteria (level I= 〈 10%, level II = 10-25 %, level III = 〉25 %). In patients who developed MC, immunosuppression was tapered and stopped and chimerism monitored. Cyclosporine was tapered by approximately 30% every 2 weeks until there was stable MC or complete donor chimerism. If progressive loss of donor chimerism leading to level II or III MC occured on two consecutive occasions with a concurrent drop in hemoglobin despite stopping immunosuppression and in the absence of GVHD, DLI was given. Patients should have had at least 10% donor chimerism in order to have a DLI. There were 109 (41.6 %, out of 262 patients in whom chimerism was done) patients who developed MC. Of these, 35 (32.1%) developed progressively worsening MC and drop in hemoglobin despite tapering of immunosuppression (91% of these were within day 60). Of these 35 patients, 23 (65.7%) received DLI while 12 developed anemia requiring transfusion (4continued to be on blood transfusion and 8 underwent a second transplant). Among 23 patients who received DLI, 9 (39.1%) showed response (rise in Hb with CC or stable MC) while 14 (60.9%) failed to show any response. The median time to show response was 28 (8-192) days and the median time to peak response was 44 (23-442) days. The 14 patients who did not show any response developed anemia requiring transfusion (11 continued to be on blood transfusion while 3 underwent a second transplant). While 80% of those with level II chimerism responded to DLI, only 31.2% of those with level III chimerism showed a response (summarized in Table 1). DLI was well tolerated by the majority of the patients. However, 7 (30.4%) patients developed cytopenia. While 6 of these had level III chimerism at the time of DLI, 1 had level II chimerism. Two patients developed Grade IV cytopenia. Grade 2 acute GVHD developed in 3 (13%; liver=2, gut=1) which responded to immunosuppressive treatment. Chronic GVHD developed in 2 (8.7%: extensive=1). There was no mortality related to DLI. DLI helped close to 40% patients achieve either complete donor chimerism or stable MC who otherwise might have required a second stem cell transplant with minimal risk. There was a trend to suggest that early administration of DLI at level II MC was superior to DLI administered at level III. Table 1. Factors affecting the response to DLI n(%) Effect of DLI Response(CC+Stable MC) No response p Chimerism level I 0 0 0 II 5 4(80%) 1 III 16 5(31.2%) 11 0.07 MC 2 0 2 Lucarelli class I 1 1(100%) 0 II 8 3(37.5%) 5 III 14 5(35.7% ) 9 0.4 Conditioning Bu-Cy-ATG/ALG 12 4(33.3%) 8 Thio-Treo-Flu 11 5(45.5%) 6 0.7 Graft BM 17 5(29.4%) 12 PB 6 4(66.7%) 2 0.1 Time of 1st DLI 180 days 5 2(40%) 3 1.0 Total 23 9(39.1%) 14 Disclosures No relevant conflicts of interest to declare.