ALBERT

Description: Management of acute myeloid leukemia (AML) in India remains a challenge. A major constraint is the cost of therapy. In a predominantly self paying system the majority of patients will not have the resources to manage a subsequent relapse. Hence, the choice of consolidation therapy has to be carefully considered to balance cost and efficacy. An allogeneic SCT (alloSCT) with a reduced intensity conditioning regimen (RIC) in first remission (CR1) is an attractive option to fulfill these requirements of relatively low cost without compromising efficacy. The need for consolidation chemotherapy prior to offering a RIC alloSCT for AML CR1 remains controversial. To evaluate these aspects we undertook a retrospective analysis of patients with AML CR1 who received a RIC alloSCT from multiple centers in India. Conventional criteria were used for definition of conditioning regimens to be considered RIC (CIBMTR). Data from 8 centers in India was collected between 2005 and 2013. A total of 138 patients fulfilled the criteria of AML CR1 having received an alloSCT with a RIC regimen. The median age was 34 years (range: 2 – 63) and 60% were males. The median time from diagnosis of AML to transplant was 99 days (range: 41 – 504). 123 (89%) were HLA matched related donors, 3 (2.1%) were MUD transplants and the rest were HLA mismatched related donors. The majority by cytogenetics (n=115) were intermediate risk (76%) followed by high risk (23%). 70 (51%) received chemotherapy consolidation prior to transplant, 61 (44%) did not and data was not available in 7 (5%). 68% of those that received consolidation received intermediate or low dose cytosine based regimens. 129 (94%) were CMV serology positive pre-transplant. Fludarabine with melphalan (140mg/m2) (128{93%}) was the most commonly used regimen and cyclosporine with short course low dose methotrexate (126{91%}) the most commonly used GVHD prophylaxis regime. All patients received a PBSC graft with a median CD34 cell dose of 9.1x106/kg (range: 1.3 – 43). With the exception of one, all patients engrafted. The median time to ANC 〉500/mm3 was 13 days (range: 7 – 22) and platelet count of 〉20,000/mm3 was 15 days (range: 0-33). Of those that engrafted, 97% achieved complete chimerism at one month post transplant (data not available in 4). Post transplant CMV reactivation was seen in 32% and a fungal infection (possible, probable or definitive) in 13%. Acute GVHD Grade 2-4 was seen in 29% and of patients evaluated 62% had chronic GVHD, the majority of these being limited (61%). The 100 day treatment related mortality (TRM) was 7.5% and the one year TRM was 25.6%. At a median follow up of 24 months the 5 year EFS and OS was 64.0±5.07 (Figure 1A) and 71.1±4.0 respectively. The 5 year cumulative incidence of relapse was 21.8% (Figure 1A). The baseline characteristics as mentioned above were not significantly different between the group that received consolidation and the group that did not. The use of consolidation therapy prior to alloSCT did not have a significant impact on EFS or OS (Figure 1B). On univariate analysis the factors that adversely impacted EFS were mismatched non sibling family donor (RR 8.1; P-value 0.001), CMV reactivation (RR 2.6; P-value 0.001), fungal infection post transplant (RR 6.8; P-value 0.000) and acute GVHD (RR 2.1; P-value 0.02). On a forward stepwise multivariate analysis adjusting for these and other conventional risk factors only CMV reactivation (RR 2.0; 95% CI 1.03-3.87; P-value 0.042) and fungal infection (RR 7.1; 95%CI 3.154-16.12; P-value 0.000) retained their adverse impact. There was no correlation between CMV reactivation and relapse of disease post transplant. The mean costs of induction chemotherapy for these patients was US$ 9239±3596 (n=74), for consolidation chemotherapy it was 5007±3490 (n=21) and for alloSCT it was 18138±13826 (n=118; costing up to 1 year post transplant). Induction chemotherapy followed by HLA matched RIC alloSCT is likely to be a cost effective and affordable treatment option for young adults with AML in CR1in an Indian context. With an average gross net income in India of US$3500/year (http://indiabudget.nic.in) the limitation still remains the cost of treatment and number of centers that can offer this therapy. Figure 1 Figure 1. Disclosures Srivastava: Octapharma: Consultancy, Other. Off Label Use: Bortezomib in the treatment of acute promyelocytic leukemia.

Description: Abstract 1941 Busulfan in combination with cyclophosphamide (Cy) is the commonly used conditioning regimen for hematopoietic stem cell transplantation (HSCT) for various hematological diseases. Cy, a prodrug, undergoes hepatic biotransformation to 4-hydroxy cyclophosphamide (4-HCy) and subsequently to its active metabolite, phosphoramide mustard (PM) and carboxyethyl PM (CEPM), a nontoxic oxidation product of HCY/aldophosphamide. Though toxic complications like hemorrhagic cystitis (HC) and hepatic sinusoidal obstruction syndrome (SOS) have been associated with metabolites of Cy such as acrolein and CEPM, respectively, there is no data correlating pharmacokinetics (PK) of Cy, HCy or CEPM with toxicity and outcome of HSCT for ß thalassemia major. Aim of the present study was to evaluate Cy, 4-HCy and CEPM PK and the influence of these PK parameters on clinical outcome in patients with ß thalassemia major undergoing HSCT. Between January 2001 to June 2009, out of the 168 HSCT for thalassemia major conditioned with Bu/Cy regimen (including 8 second transplants), 90 patients for whom PK samples were collected were included in this study. Cy was administered at 50 mg/kg/day for 4 days (days −5 to −2) following 4 days of busulfan (days −9 to −6). Peripheral blood samples were collected during Cy infusion at various time interval and plasma samples were stored for Cy, HCy and CEPM PK analysis. Levels of Cy and 4-HCy were measured by high performance liquid chromatography, and CEPM, by a modified LCMSMS method. The population PK estimates were determined using non-linear mixed effects modeling analysis performed with Monolix (version 3.1, www.monolix.org). Specifically, a compartmental model which included two compartments each for Cy, HCy and CEPM was used to describe the data. Clinical outcome endpoints including graft rejection, event free survival (EFS), overall survival (OS), transplant related mortality (TRM), SOS, and HC were evaluated using standard criteria. The influence of Cy and metabolite PK on clinical outcome endpoints were compared using logistic regression analysis. Age range of the patients was 2 to 24 years. Four patients belonged to Lucarelli risk class I, 49 class II and 37 class III. Based on risk stratification that we have defined using liver size and age (high risk: age 〉7 years and liver size 〉5cm; and the rest as low risk; Mathews et al, 2007), 39 were in low risk, 40 intermediate and 11 were high risk. Overall incidence of OS, EFS, rejection, TRM, SOS, HC in this cohort was 77, 70, 14, 10, 18 and 31% respectively. It should be noted that this does not completely represent the outcome of HSCT in thalassemia during this period, as only patients with available Cy PK analysis were included for analysis. The high risk patients had significantly reduced OS (RR 2.59; p=0.04), EFS (RR 2.23; p=0.058), increased risk of TRM (RR: 3.56; p=0.03) and HC (RR: 3.19; p=0.036) compared to others, while this was not significantly different with respect to Lucarelli class except for increased incidence of HC in Lucarelli class III patients (RR: 2.6; p=0.04). Upon univariate analysis, there was significantly increased Cy AUC (1887 ng*h/ml; range: 679–8546; vs. 1544, range: 662–4434; p= 0.028) in patients who developed HC compared to those who did not. There was significantly decreased HCy AUC (median 5.172 ug*L/h, range: 0.795–6.457; vs. 6.224; 2.536–12.003 p=0.007) in patients who died compared to those who are alive; similar but more significant association was seen with EFS as well. There was decreased CEPM Cl/F in those who rejected the grafts (0.013 vs. 0.028 L/h/Kg; p=0.016), while it was significantly increased in patients who developed SOS (median 0.029; vs. 0.013, range: L/h/Kg; p=0.05). Upon forward stepwise multivariate analysis including all the Cy and metabolite PK parameters, only HCy AUC significantly influenced EFS and OS in these patients. We show here for the first time that Cy and metabolite PK influences HSCT outcome in a uniform cohort of patients with thalassemia major. However, due to the complex metabolic pattern of Cy, and the association of metabolite PK instead of the levels of the parent compound with outcome, the possibility of targeted dose adjustment of Cy to improve HSCT outcome may be more challenging than targeted dose adjustment of other drugs used in HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 518 Targeted dosing of busulfan (Bu) has been shown to improve outcome of allogeneic HSCT (aHSCT) in patients with leukemia. There is limited data on correlation of Bu PK with outcome in patients with thalassemia major (TM)) undergoing aHSCT. We have previously shown that first dose trough level of Bu (Cmin1) predicts graft rejection (Chandy et al, BMT 2005), and Bu Css is significantly lower in patients with hepatic veno-occlusive disease (HVOD) (Srivastava et al, Blood 2004). The aim of the present study was to evaluate the correlations of Bu PK with outcome of HSCT in a larger cohort of patients and to evaluate the pharmacogenetic basis for the differences. We retrospectively analyzed oral Bu PK after 1st and 13th doses of Bu in 255 patients out of the 291 thalassemic patients who underwent aHSCT from matched related donors between 1991 till February 2010 at our centre. All patients received busulfan (at a dose of 14 or 16 mg/kg/day or 600mg/m2/day) in combination with Cy (at a dose of 160mg/kg for those 〉15 years or 200mg/kg for all others) as part of the conditioning regimen. Bu levels were measured by HPLC as previously described (Poonkuzhali et al, 1999). We also analyzed GSTA1*1B, GSTM1 and GSTT1 deletion polymorphisms in these patients. Based on Lucarelli's risk stratification, 18/291 patients belonged to class I, 121/291 were class II and 151/291 were class III. The class III patients were further risk stratified into class 3 high risk and low risk based on age and liver size (high risk: age 〉7 years and liver size 〉5cm; and the rest as low risk; Mathews et al, 2007). None of the Bu PK parameters were significantly different between Lucarelli classes as well as between class III high and low risk patients. For the entire group, EFS was 77%, OS 81%, NRM (non rejection mortality)15% and graft rejection 8.6%. Class III patients had a significantly lower EFS (p=0.0007) and OS (p=0.0051) compared to class I and II. Bu Cmin1 (p=0.007), but not Bu Css1 was significantly lower in those who rejected their graft compared to those who did not. On quartile analysis, patients with Cmin1 156ng/ml (RR= 9.8; p=0.0001). Those with Bu Css1 in the lowest quartile also had significant risk of rejection (14/57 with Css1 490 ng/ml; RR= 3.8, p=0.027) but the correlation was not as strong as that with Cmin1. Upon multivariate analysis of all the variables that were significantly influencing aHSCT outcome in univariate analysis, only Lucarelli class III high risk (p=0.034), SGOT level (p=0.036) and Bu Cmin1 (p=0.0001) were significantly influencing graft rejection. In addition, GSTA1*1B homozygous variant genotype was significantly associated with higher Bu Cmin1 (p=0.008) and Css1 (p=0.009). Since Bu Cmin1 was significantly influenced by GSTA1*1B genotype, we compared the combined risk of Cmin1

Description: Abstract 546 The basis for 10–15% of patients with severe hemophilia having clinically mild disease is not fully understood. We have described two levels of heterogeneity that is clinically obvious – first in the number of bleeds and second is in the amount of joint damage with similar number of bleeds. We hypothesized that differences in inflammatory responses determine the latter phenomenon. A total 114 patients with hemophilia A (n=95) and hemophilia B (n=19) were studied. All these patients were on minimal on-demand treatment (50-150 IU/kg/year). Patients were evaluated for the frequency and site of hemorrhage. Their WFH clinical and radiological (Pettersson) joint scores were documented. Disease causing mutations in factor 8 or factor 9 genes was genotyped. Functional polymorphisms in the inflammatory cytokine genes (tumor necrosis factor alpha; transforming growth factor (TGF) beta; interleukin (IL) 10; IL 6; IL 1beta; IL 1 beta receptor antagonist; tumor necrosis factor beta), immunoregulatory cytokine genes (interferon gamma(IFNg); HLA B27; FC gamma receptor) were genotyped. Patients with extremes of joint damage marked by their clinical and radiological scores, were classified as having severe (n=10, upper 10 percentile, ≥21.8 clinical and ≥36.1 radiological scores) or mild (n=10, lower 10 percentile, ≤2.6 clinical and ≤2.9 radiological scores) disease. The hemostasis parameters, factor VIII/or IX levels and tests of global hemostasis as measured by clot curve analysis (MAX2) were comparable between these groups. The number of reported bleeds between the severe group and mild group of patients within the classification of clinical score (19.7 Vs 12 bleeds, p=0.08) or radiological scores (14.2 Vs 13.4 bleeds, p=0.544) was not significantly different. This reflects our clinical observations that even among those who bleed frequently, the degree of synovial reaction and the extent of damage to joint cartilage can vary. Of the polymorphisms studied, the IL-10 -819T allele (60 Vs 22%, p=0.009) and -592C (60 Vs 22%, p=0.009) allele predicting higher levels of this anti-inflammatory cytokine, and IFNg 874A allele (60 Vs 0%, p= 0.011) predicting lower levels of pro-inflammatory cytokine were associated with a mild phenotype in patients classified using radiological scores. Interestingly, the IFNg 874A allele (60 Vs 0%, p= 0.011) and the inheritance of moderate disease causing mutation (missense mutation, 88 Vs 28%, p=0.041) was also associated with mild phenotype by clinical score classification. This data suggests that a cytokine mileu in target joint comprising of lower levels of IFNg that results in reduced co-stimulation of pro-inflammatory cytokines and higher levels of anti-inflammatory IL-10 may reduce the risk of developing severe arthropathy. This phenomenon needs to be evaluated in larger number of patients receiving on-demand treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Azathioprine (AZA) has been used as a steroid sparing agent in allogeneic BMT program at the Princess Margaret Cancer Centre, Toronto, Canada for last two decades especially for cGVHD treatment. A previous clinical trial (Sullivan, Blood 1998) compared prednisone (PRD) alone vs PRD plus AZA for the treatment of extensive chronic GVHD (cGVHD) suggesting that PRD alone showed a better survival than PRD+AZA. However, the NIH consensus criteria (NCC, 2005) for cGVHD and new statistic endpoint to evaluate efficacy of cGVHD, failure free survival (FFS), have been recently introduced and increasingly used. Therefore, we conducted retrospective study attempted to evaluate the efficacy of PRD+AZA regimen compared to PRD alone regimen with respect to failure free survival (FFS) as well as overall survival (OS), non-relapse mortality (NRM)and relapse incidence. In order to adjust for the risk factors which affect the choice of treatment between different treatment options, propensity score matching (PSM) analysis was adopted in the present study. Methods: The patients diagnosed with late onset acute GVHD was excluded. A total of 240 patients were included in the analysis, transplanted at the Princess Margret Cancer Center between 2009 and 2013, diagnosed with cGVHD by NCC, and treated with PRD+AZA (n=98) or PRD alone (n=142) as first line treatment. Failure free survival (FFS), OS, NRM and relapse were compared between the 2 groups. A case-control study was performed with well-balanced pairs of PRD+AZA vs PRD patients. For the PSM analysis, propensity score (PS) was calculated. Clinical variables included in PS calculation were global score (GS) by NCC, subtype of cGVHD (classical vs overlapping), age, gender, duration from HCT to cGVHD initial treatment, performance status (PS), progressive type onset (PTO) of cGVHD, thrombocytopenia (TP) and each organ involvement of cGVHD per skin, gastrointestinal tract, liver, lung and musculoskeletal system. A total of 74 case-control pairs were selected within 0.1 of a difference in propensity score. RESULTS: With a follow-up of 43. 6 months, the 2-year FFS, OS, NRM and relapse incidence was 24.7 %, 75.6 %, 16.6% and 7.7%, respectively. The median FFS was 7.9 months (95% CI, 6.1-9.6 months). PRD+AZA group showed a longer FFS duration compared to PRD group (13.2 vs 5.6 months, p

Description: Acute myeloid leukemia (AML) is a clinically and biologically complex and heterogeneous hematopoietic neoplasm. Recent advances in acute myeloid leukemia (AML) biology have lead to prognosticate and predict treatment outcome in AML based on molecular markers. Mutations in NPM1, CEBPA are considered good prognosis and High BAALC, ERG & MN1 expression associate with worse outcome in AML patients treated with standard chemotherapy. Although many efforts have been made to identify genetic mutations and modulated gene expression levels that can be used to predict outcomes in patients with AML, the association between these prognostic markers has not been evaluated. We have reported previously that the NPM1 mutated patients have significantly high dCK and hENT1 gene expression (involved in cytarabine metabolism) and low ABCG2 and ABCB1transporter expression (Abraham et al, ASH abstracts; Nov 2011; 118: 3481 and Nov 2012; 120: 143), suggesting that the good prognostic nature of this mutation is possibly due to the better metabolism and transport of the chemotherapeutic drugs used in induction therapy.  We extended this study to look for association between NPM1/FLT3 mutation status and the RNA expression of other good or poor prognostic markers in patients with AML. We prospectively included 274 adult patients with AML in this study. The median age was 42 years (range 16-74y). AML was diagnosed according to the FAB and WHO classifications. There were 238 patients with de novo AML; Secondary AML -6; Therapy related AML- 2 and Relapsed AML-28. Bone marrow cytogenetics and immunophenotyping analysis was available for all patients at diagnosis and/or relapse. Diagnostic bone marrow MNCs were isolated by ficoll- density gradient centrifugation and stored in trizol reagent for RNA expression and mutation detection. RT-PCR was used to screen AML-ETO and Inv 16, and the expression of BAALC, ERG1, MN1, CXCR4 and WT1were analyzed using RQ-PCR. NPM1-c, FLT3 ITD and TKD were screened using DNA PCR followed by gene-scan, sequencing or RFLP methods. The basic demographics and the frequency of the markers are listed in Table 1. When analyzed separately in normal karyotype AML (NK-AML), the frequencies of the mutations were: NPM1: 52.2%; FLT3-ITD: 24%; TKD: 4.3%. When the RNA expression of BAALC, WT1, ERG1, CXCR4 and MN1 was compared in patients with NPM1 or FLT3 mutations, we noticed that patients with NPM1 had significantly low expression of BAALC, MN1 and ERG1 while those with FLT3 mutations (ITD or TKD) had higher expression of these genes (Figure1). There was no significant association with CXCR4 or WT1 expression and these mutations. When analyzed separately in the normal karyotype AML, these associations were still significant. In addition, the relapsed patients had significantly higher expression of BAALC, MN1, and ERG1 RNA compared to de-novoAML cases (data not shown). To conclude, we show that NPM1 or FLT3 mutations acquire the prognostic significance due to several factors including BAALC, ERG1 and MN1expression levels in addition to drug metabolizing enzymes’ and drug transporter expression. These factors must be taken into consideration when attempting to personalize chemotherapy in AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3486 Poster Board III-423 Haemophilia B (HB), an X linked inherited disorder is caused by heterogeneous mutations in the F9 gene. Approximately 3% of hemophilia B patients have major deletions in the F9 gene. Gross and small deletions in the F9 gene in HB affected males are easily detected by PCR but detecting the carrier state of females in the family is challenging due to the presence of the normal allele. Different methods like linkage analysis, real time PCR and MLPA have been used to assess the carrier status in this situation. Linkage analysis is limited by the availability of informative markers and adequate number of family members. Real time PCR involves standardisation and preparation of calibration curves for each run. Although MLPA is a better alternative, it can be time consuming and involves multiple steps. We have therefore developed a fluorescent PCR based gene dosage approach which is simple, rapid and cost-effective for determining the carrier status of females in families with deletions in the F9 gene. 200ng of DNA extracted by standard protocols was used for amplification with primers designed to amplify a 160bp product from exon h in the F9 gene. One of the primers was fluorescently labelled. Amplification was carried out using these primers for 20 cycles only and the amplified product was subjected to capillary electrophoresis on an ABI 310 genetic analyser. A 230 bp fragment in the albumin gene was used as the control. Analysis was done using Genescan and Genotyper software. The ratio between the peak heights of the exon h in the F9 and control genes in the patient samples were normalised to a normal control. Five families with deletional HB were analysed (in toto deletion-1; Ex g-h – 1; Ex g-poly A-1; Ex h-poly A-2). The ratios in the probands and the family members are presented in the table. Out of eight females analysed, 6 were carriers and 2 were normal. The mean ratio in the carriers was 0.49±0.08 and 0.75±0.05 in the normal. Deletions are not uncommon in HB and deletions involving the exon g and h constitute a major group. Among 212 families with HB assessed at our centre, we have identified large deletions in 8 families (3.7%). It is interesting to note that all except one of these deletional mutations involved exon h. This method confirmed the presence of these deletions in the males and helped us to identify the carrier status of the females in the family. Identification of carrier status of females with deletions in F9 gene by gene dosage Subject ID Peak height of Exh in F9 Peak height of albumin Normalised Ratio Interpretation HB5 284 442 0.59 Carrier HB6 305 489 0.57 Carrier HB22 188 372 0.47 Carrier HB63 85 165 0.48 Carrier HB129 247 295 0.78 Normal HB238 94 326 0.4 Carrier HB280 372 679 0.77 Normal HB384 202 670 0.4 Carrier Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2024 Introduction: Follicular lymphoma (FL) remains an incurable disease despite the use of immunochemotherapy or autologous stem cell transplant (ASCT). Allogeneic stem cell transplantation (Allo-SCT) remains a viable treatment option in FL because of its associated graft versus lymphoma effect and potential to achieve long term disease control. There is limited data in literature regarding long term outcome of follicular lymphoma post allo-SCT. Patients and Methods: We identified a total of 52 patients with chemosensitive FL underwent allo-SCT at our centre between 1989 and 2009. 89% were in a chemosensitive remission at time of transplantation. The conditioning regimens included BUCY in 48 (92%) patients. GVHD prophylaxis was cyclosporine and methotrexate until 2009 and subsequently cyclosporine and mycophenolate mofetil. Patients undergoing matched unrelated donor (MUD) transplant received alemtuzumab. Results: There were 30 (58%) males and 22 (42%) females. The median age at BMT was 45 years (range:24–64) and the median number of prior chemotherapy regimens was 3 (range: 1–6). Fifteen (29%) of patients had only received

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: Abstract 3349 Poster Board III-237 Busulfan in combination with cyclophosphamide is the commonly used conditioning regimen for hematopoietic stem cell transplantation (HSCT). We have studied the pharmacokinetics (PK) of oral busulfan in children with beta thalassaemia major undergoing BMT and observed up to 12 fold variation in PK. Since PK parameters of Bu have been shown to be influencing BMT outcome and regimen related toxicity, we attempted to understand factors influencing Bu PK using a population PK based model. The aim of this study was to explore the effect of several demographic, biological, and pharmacogenetic covariates on the disposition of busulfan in children with beta thalassaemia major undergoing hematopoietic stem cell transplant. One hundred and fifty children with beta thalassaemia major received 16 mg/kg/day of busulfan as part of the pre-BMT conditioning regimen. Plasma busulfan concentrations were measured after first and 13th doses using HPLC based method with UV detection. Non-linear mixed effects modeling analysis was performed with Monolix (version 2.4, www.monolix.org) to evaluate the effect of age, body weight, sex, Lucarelli class, serum ferritin levels, pre-transplant liver function and ten polymorphisms corresponding to GSTA1, GSTM1, GSTP1, GSTT1, CYP2B6, CYP2C9, CYP2C19, CYP3A4 (pharmacogenetic data was available for 133 of 150 patients). A one-compartment pharmacokinetic model with first-order oral absorption was used to describe the data. The pharmacokinetic parameters estimated included apparent clearance and volume (CL/F (L/hr or L/hr/kg), V/F (L or L/kg)), and the absorption parameter (ka (1/hr)). The bioavailability, F, was not identifiable since only oral drug was used. The distribution of the parameters was assumed log-normal. The main covariate which explained the largest portion of the inter-individual variation in busulfan kinetic parameters (45%, 22%, and 15% of clearance (CL), volume of distribution (V), and absorption rate constant (ka), respectively) was body weight. The next most significant covariate was GSTA1 promoter polymorphism. In particular, clearance decreased 124% and 148% and the oral absorption increased 16% and 15% in GSTA1 heterozygous and homozygous patients, respectively, compared to wild type. GSTA1 explained an additional 17% and 1% of inter-individual variation in the CL and ka, respectively, compared to the weight normalized model. By combining these two covariates, the interindividual variability on busulfan CL/F decreased from 45% to17%. We have developed a population pharmacokinetic model of oral busulfan in children with beta thalassaemia major undergoing BMT which explains the inter-individual variation in Bu PK, considering demographic and biological covariates. Disclosures: No relevant conflicts of interest to declare.