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Busulfan (Bu) combined with cyclophosphamide (Cy) is commonly used as a myeloablative conditioning regimen for allogeneic hematopoietic cell transplantation (allo-HCT). There is inter-individual variability of Bu pharmacokinetics (PK) and hence in toxicity and efficacy. The introduction of therapeutic drug monitoring (TDM) of Bu has decreased toxicity of the regimen. Hepatic metabolism of Bu is mediated through Glutathione-S-Transferases (GSTs), mainly GSTA1. Patients with GSTA1*A variants are considered normal metabolizers and GSTA1*B corresponds to poor metabolism, defined by nucleotide changes at −52 or −69 locus in GSTA1 promoter region. The aim of the study was to explore the correlation between GSTA1 polymorphisms and Bu-PK in 60 adult patients receiving an allo-HCT in the BuCyBu clinical study (ClinicalTrials.gov I, ID NCT01779882) comparing the sequence BuCy to CyBu. DNA samples prior to conditioning were genotyped for candidate variants at −52 (rs3957356) and −69 (rs3957357) loci in the GSTA1 promoter. Thirty-three % of patients were GSTA1*A*A, 49% GSTA1*A*B and 18% GSTA1*B*B. In GSTA1*A*A patients, median Bu-AUC was 3.6 ± 0.7 mg*h/L, in GSTA1*A*B 4.5 ± 1.6 and in GSTA1*B*B 4.9 ± 1.4 (AUC 35% higher than GSTA1*A*A, p = 0.03), with a similar significant correlation with Bu-clearance (p = 0.04). The correlation between GSTA1 polymorphism and AUC remained significant in multivariate linear regression analysis. There was a trend for lower non-relapse mortality (NRM) in patients with low AUC. We could not demonstrate a correlation between GSTA1 polymorphisms and NRM, acute graft-versus-host disease (aGvHD) in this small cohort, but there is a trend of higher aGvHD incidence in GSTA1*B*B patients.

In busulfan-based conditioning regimen for hematopoietic stem cell transplantation in children, accurate a priori determination of the first dose is important because of its narrow therapeutic window. Sickle cell disease (SCD) influences pharmacokinetics of the commonly used drugs by affecting organs responsible for drug metabolism and elimination. This pharmacokinetics study assesses the influence of SCD on the metabolic pathway of busulfan that is mainly metabolized in the liver. In this retrospective cross-sectional case-control study, 16 patients with SCD were matched to 50 patients without SCD on known busulfan clearance's covariates (glutathione-S-transferase alpha1 polymorphisms, age, weight). Clearance of the first dose of busulfan was not significantly different independently of genetic or anthropometric factors in patients with or without SCD.

Sinusoidal obstruction syndrome (SOS) is a well-recognized and potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT). SOS arises from endothelial cell damage and hepatocellular injury mostly due to the transplantation conditioning regimens but also to other patient, disease, and treatment-related factors. Understanding risk factors associated with the development of SOS is critical for early initiation of treatment or prophylaxis. The knowledge about genetic contribution is limited; few studies investigated so far selected a set of genes. To get more comprehensive insight in the genetic component, we performed an exome-wide association study using genetic variants derived from whole-exome sequencing. The analyses were performed in a discovery cohort composed of 87 pediatric patients undergoing HSCT following a busulfan-containing conditioning regimen. Eight lead single-nucleotide polymorphisms (SNPs) were identified after correction for multiple testing and subsequently analyzed in a validation cohort (n = 182). Three SNPs were successfully replicated, including rs17146905 ( P = .001), rs16931326 ( P = .04), and rs2289971 ( P = .03), located respectively in the UGT2B10, BHLHE22, and KIAA1715 genes. UGT2B10 and KIAA1715 were retained in a multivariable model while controlling for nongenetic covariates and previously identified risk variants in the GSTA1 promoter. The modulation of associations by conditioning regimens was noted; KIAA1715 was dependent on the intensity of the conditioning regimen, whereas the effect of UGT2B10 was equally applicable to all of them. Combined effect of associated loci was also observed ( P = .00006) with a genotype-related SOS risk of 9.8. To our knowledge, this is the first study addressing the genetic component of SOS at an exome-wide level and identifying novel genetic variations conferring a higher risk of SOS, which might be useful for personalized prevention and treatment strategies. (C) 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

The most frequent complication of allogeneic hematopoietic stem cell transplantation is acute Graft versus Host Disease (aGVHD). Proliferation and differentiation of donor T cells initiate inflammatory response affecting the skin, liver, and gastrointestinal tract. Besides recipient–donor HLA disparities, disease type, and the conditioning regimen, variability in the non-HLA genotype have an impact on aGVHD onset, and genetic variability of key cytokines and chemokines was associated with increased risk of aGVHD. To get further insight into the recipient genetic component of aGVHD grades 2–4 in pediatric patients, we performed an exome-wide association study in a discovery cohort (n = 87). Nine loci sustained correction for multiple testing and were analyzed in a validation group (n = 168). Significant associations were replicated for ERC1 rs1046473, PLEK rs3816281, NOP9 rs2332320 and SPRED1 rs11634702 variants through the interaction with non-genetic factors. The ERC1 variant was significant among patients that received the transplant from HLA-matched related individuals (p = 0.03), bone marrow stem cells recipients (p = 0.007), and serotherapy-negative patients (p = 0.004). NOP9, PLEK, and SPRED1 effects were modulated by stem cell source, and serotherapy (p < 0.05). Furthermore, ERC1 and PLEK SNPs correlated with aGVHD 3-4 independently of non-genetic covariates (p = 0.02 and p = 0.003). This study provides additional insight into the genetic component of moderate to severe aGVHD.

Purpose This study aimed to retrospectively evaluate the genetic association of null variants of glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with hematological malignancies (HMs) undergoing busulfan (BU)- containing allogeneic hematopoietic stem cell transplantation (HSCT) and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST gene-edited cell models. Methods GSTM1- and GSTT1-null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1-null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell death studies were conducted in GSTs- null and non-null LCLs and CRISPR–Cas9 gene-edited THP1 leukemia cell lines. Results Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76–15.42; p = 1.9 × 10–5]). BU-induced cell death preferentially in THP1GSTM1(non−null) and LCLsGSTM1(non−null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. Conclusion The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation.

Hematopoietic stem cell transplantation (HSCT) is currently the standard of care for many malignant and nonmalignant blood diseases. As several treatment-emerging acute toxicities are expected, optimal supportive measurements critically affect HSCT outcomes. The paucity of good clinical studies in supportive practices gives rise to the establishment of heterogeneous guidelines across the different centers, which hampers direct clinical comparison in multicentric studies. Aiming to harmonize the supportive care provided during the pediatric HSCT in Europe, the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) promoted dedicated workshops during the years 2017 and 2018. The present paper describes the resulting consensus on the management of sinusoidal obstructive syndrome, mucositis, enteral and parenteral nutrition, iron overload, and emesis during HSCT.

Background: Relapse is the major cause of treatment failure in children with hematological malignancies (HMs) undergoing busulfan (BU)- based allogeneic hematopoietic stem cell transplantation (HSCT). Glutathione S-transferases (GSTs) isoforms that participate in BU detoxification and protect cells against stress and cell death may be linked to post-HSCT outcomes. This study aimed to retrospectively evaluate the genetic association of null variants of Glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with HMs undergoing BU- containing allogeneic HSCT and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized and tumor lymphoblastoid cell lines (LCLs).Methods: GSTM1- and GSTT1- null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1- null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell-death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines. Results: Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76 - 15.42; p= 1.9 x 10-5]). BU induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. Conclusion: The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation. Trial registration: ClinicalTrials.gov identifier: NCT01257854, Registered February 2008 – retrospectively registered.

BackgroundRelapse is the major cause of treatment failure in children with hematological malignancies (HMs) undergoing busulfan (BU)- based allogeneic hematopoietic stem cell transplantation (HSCT). Glutathione S-transferases (GSTs) isoforms that participate in BU detoxification and protect cells against stress and cell death may be linked to post-HSCT outcomes. This study aimed to retrospectively evaluate the genetic association of null variants of Glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with HMs undergoing BU- containing allogeneic HSCT and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST-gene edited cell models.MethodsGSTM1- and GSTT1- null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1- null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell-death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines. ResultsCarrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76 - 15.42; p= 1.9 x 10-5]). BU induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. ConclusionThe clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation. Trial registrationClinicalTrials.gov identifier: NCT01257854, Registered February 2008 – retrospectively registered.

BackgroundChildhood cancer and its treatment may lead to many acute and chronic health complications. Related impairment in quality of life, excess in deaths, and accumulated health care costs are relevant. There is a wide inter-individual variability in the type and severity of health complications. Genetic variations are suggested to contribute to individual susceptibility. So far, only few genetic variants have been used to risk-stratify treatment and follow-up care. This study platform aims to identify germline genetic variants associated with acute and late complications of childhood cancer.MethodsThe Genetic Risks for Childhood Cancer Complications Switzerland (GECCOS) study is a nationwide cohort study. It includes patients and survivors who were diagnosed with childhood cancers or Langerhans cell histiocytosis before age 21 years, were registered in the Swiss Childhood Cancer Registry (SCCR) since 1976 and have consented to the Pediatric Biobank for Research in Hematology and Oncology (BaHOP), Geneva, host of the Germline DNA Biobank Switzerland for Childhood Cancer and Blood Disorders (BISKIDS). BISKIDS is a national biobank for the collection of germline DNA in childhood cancer patients and survivors.GECCOS uses demographic and clinical data from the SCCR and the associated Swiss Childhood Cancer Survivor Study (SCCSS), which contains health-related data of survivors. Phenotypic data consist of objective measurements, health conditions diagnosed by physicians, second primary neoplasms, self-reported and health-related information from participants. Germline genetic samples and sequencing data have been collected in BISKIDS. We will perform gene panel sequencing, whole-exome sequencing, or whole-genome sequencing depending on the research questions. We will perform association analyses to identify genetic variants associated with specified health conditions. We will use clustering and machine-learning techniques and assess multiple health conditions in different models.DiscussionGECCOS will serve as an overarching platform to enable genotype-phenotype association analyses on complications associated with childhood cancer and its treatments. Knowledge of germline genetic variants associated with childhood cancer-associated health conditions will help to further individualize cancer treatment and follow-up care, potentially resulting in improved efficacy and reduced side effects, for personalized cancer care.Trial registrationClinicaltrials.gov: NCT04702321

Acute Graft versus Host Disease (aGvHD) grades 2–4 occurs in 15–60% of pediatric patients undergoing allogeneic haematopoietic stem-cell transplantation (allo-HSCT). The collateral damage to normal tissue by conditioning regimens administered prior to allo-HSCT serve as an initial trigger for aGvHD. DNA-repair mechanisms may play an important role in mitigating this initial damage, and so the variants in corresponding DNA-repair protein-coding genes via affecting their quantity and/or function. We explored 51 variants within 17 DNA-repair genes for their association with aGvHD grades 2–4 in 60 pediatric patients. The cumulative incidence of aGvHD 2–4 was 12% (n = 7) in the exploratory cohort. MGMT rs10764881 (G>A) and EXO rs9350 (c.2270C>T) variants were associated with aGvHD 2–4 [Odds ratios = 14.8 (0 events out of 40 in rs10764881 GG group) and 11.5 (95% CI: 2.3–191.8), respectively, multiple testing corrected p ≤ 0.001]. Upon evaluation in an extended cohort (n = 182) with an incidence of aGvHD 2–4 of 22% (n = 40), only MGMT rs10764881 (G>A) remained significant (adjusted HR = 2.05 [95% CI: 1.06–3.94]; p = 0.03) in the presence of other clinical risk factors. Higher MGMT expression was seen in GG carriers for rs10764881 and was associated with higher IC50 of Busulfan in lymphoblastoid cells. MGMT rs10764881 carrier status could predict aGvHD occurrence in pediatric patients undergoing allo-HSCT.

GSTA1 encodes a member of a family of enzymes that function to add glutathione to target electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress. GSTA1 has several functional SNPs within its promoter region that are responsible for a change in its expression by altering promoter function. This study aims to investigate distributions of GSTA1 promoter haplotypes across different human populations and to assess their impact on the expression of GSTA1. PHASE 2.1.1 was used to infer haplotypes and diplotypes of six GSTA1 promoter SNPs on 2501 individuals from 26 populations classified by the 1000 Genomes Project into five super-populations that included Africa (N = 660), America (N = 347), East Asia (N = 504), Europe (N = 502), and South Asia (N = 488). We used pairwise FST analysis to compare sub-populations and luciferase reporter assay (LRA) to evaluate the impact of each SNP on activation of transcription and interaction with other SNPs. The distributions of GSTA1 promoter haplotypes and diplotypes were significantly different among the different human populations. Three new promoter haplotypes were found in the African super-population. LRA demonstrated that SNPs at -52 and -69 has the most impact on GSTA1 expression, however other SNPs have a significant impact on transcriptional activity. Based on LRA, a new model of cis-elements interaction is presented. Due to the significant differences in GSTA1 diplotype population frequencies, future pharmacogenomics or disease-related studies would benefit from the inclusion of the complete GSTA1 promoter haplotype based on the newly proposed metabolic grouping derived from the LRA results.

Aims The aim of this study is to develop a population pharmacokinetic (PopPK) model for intravenous busulfan in children that incorporates variants of GSTA1, gene coding for the main enzyme in busulfan metabolism. Methods Busulfan concentration-time data was collected from 112 children and adolescents (median 5.4 years old, range: 0.1-20) who received intravenous busulfan during the conditioning regimen prior to stem cell transplantation. Weight, sex, baseline disease (malignant vs. non-malignant), age, conditioning regimen and GSTA1 diplotypes were evaluated as covariates of pharmacokinetic parameters by using nonlinear mixed effects analysis. The ability to achieve the target AUC24h (3600-6000 μM min-1 ) was assessed by estimating the first dose based on the present PopPK model and by comparing the results with other available models in children. Results A one-compartment model with first-order elimination best described the data. Allometric scaling of weight and a factor of busulfan metabolism maturation were included in the base model. GSTA1 diplotypes were found to be a significant covariate of busulfan clearance, which was 7% faster in rapid metabolizers and 12% slower in poor metabolizers, in comparison with normal ones. Busulfan doses calculated using the parameters of the proposed PopPK model were estimated to achieve the target AUC in 85.2% of the cases (95% CI 78.7-91.7%). Conclusion This is the first PopPK for busulfan that successfully incorporated GSTA1 genotype in a paediatric population. Its use may contribute to better prediction of busulfan exposure in children and adolescents since the first dose, by tailoring the dose according to the individual metabolic capacity.

Busulfan (Bu) is a key component of conditioning regimens used before hematopoietic stem cell transplantation (SCT) in children. Different predictive methods have been used to calculate the first dose of Bu. To evaluate the necessity of further improvements, we retrospectively analyzed the currently available weight- and age-based guidelines to calculate the first doses in 101 children who underwent allogenic SCT in CHU Sainte-Justine, Montreal, after an intravenous Bu-containing conditioning regimen according to genetic and clinical factors. The measured areas under the curve (AUCs) were within target (900 to 1500 µM/min) in 38.7% of patients after the administration of the first dose calculated based on age and weight, as locally recommended. GSTA1 diplotypes linked to poor Bu metabolism (G3) and fludarabine-containing regimens were the only factors associated with AUC within target (OR, 4.7 [95% CI, 1.1 to 19.8, P = .04]; and OR, 9.9 [95% CI, 1.6 to 61.7, P = .01], respectively). From the 11 methods selected for dose calculation, the percentage of AUCs within the target varied between 16% and 74%. In some models G3 was associated with AUCs within the therapeutic and the toxic range, whereas rapid metabolizers (G1) were correlated with subtherapeutic AUCs when different methods were used. These associations were confirmed by clearance-prediction analysis, in which GSTA1 diplotypes consistently influenced the prediction errors of the methods. These findings suggest that these factors should be considered in Bu dose prediction in addition to the anthropometric data from patients. Furthermore, our data indicated that GSTA1 diplotypes was a factor that should be included in future population pharmacokinetic models, including similar conditioning regiments, to improve the prediction of Bu exposure after its initial dose.

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for several malignant and non-malignant diseases at the cost of serious treatment-related toxicities (TRTs). Recent research on extending the benefits of HSCT to more patients and indications has focused on limiting TRTs and improving immunological effects following proper mobilization and engraftment. Increasing numbers of studies report associations between HSCT outcomes and the expression or the manipulation of G protein-coupled receptors (GPCRs). This large family of cell surface receptors is involved in various human diseases. With ever-better knowledge of their crystal structures and signaling dynamics, GPCRs are already the targets for one third of the current therapeutic arsenal. The present paper assesses the current status of animal and human research on GPCRs in the context of selected HSCT outcomes via a systematized survey and analysis of the literature.

// Marc Ansari 1,2 , Patricia Huezo-Diaz Curtis 1,2,* , Chakradhara Rao S. Uppugunduri 1,2,* , Mohammed Aziz Rezgui 3 , Tiago Nava 1,3,5,6 , Vid Mlakar 1,2 , Laurence Lesne 1,2 , Yves Theoret 3,4,5 , Yves Chalandon 7 , Lee L. Dupuis 8 , Tao Schechter 8 , Imke H. Bartelink 9,17 , Jaap J. Boelens 9 , Robbert Bredius 10 , Jean-Hugues Dalle 11 , Saba Azarnoush 11 , Petr Sedlacek 12 , Victor Lewis 13 , Martin Champagne 14 , Christina Peters 15 , Henrique Bittencourt 3,4,5,16 and Maja Krajinovic 3 - 5,16,18 1 Department of Pediatrics, CANSEARCH Research Laboratory, Faculty of Medicine, Geneva, Switzerland 2 Department of Pediatrics, Onco-Hematology Unit, Geneva University Hospital, Geneva, Switzerland 3 Charles-Bruneau Cancer Center, CHU Sainte-Justine Research Center, Montreal, Quebec, Canada 4 Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada 5 Clinical Pharmacology Unit, CHU Sainte-Justine, Montreal, Quebec, Canada 6 Faculty of Medicine, Federal University of Rio Grande do Sul, Porto Alegre, Brazil 7 Department of Medical Specialties, Division of Hematology, Geneva University Hospital, Geneva, Switzerland 8 Department of Haematology/Oncology, Blood and Marrow Transplant Unit, The Hospital for Sick Children, Toronto, Ontario, Canada 9 Pediatric Blood and Marrow Transplantation Program, University Medical Center, Utrecht, The Netherlands 10 Department of Pediatrics, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands 11 Pediatric Hematology Department, Robert Debre Hospital, Assistance Publique, Hopitaux de Paris, Paris, France 12 Department of Pediatric Hematology and Oncology Teaching Hospital, 2nd Medical School, Charles University, Prague, Czech Republic 13 Department of Pediatrics, Alberta Children’s Hospital, Calgary, Alberta, Canada 14 Department of Hematology, Hospital Verdun, Montreal, Quebec, Canada 15 Department of Pediatrics, Stem Cell Transplantation Unit, St Anna Children’s Hospital, Vienna, Austria 16 Department of Pediatrics, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada 17 Department of Medicine, The University of California San Francisco, San Francisco, CA, USA 18 On Behalf of the Pediatric Disease Working Party of the European Society for Blood and Marrow Transplantation, Leiden, The Netherlands * These authors have contributed equally to this work Correspondence to: Patricia Huezo-Diaz Curtis, email: // Keywords : busulfan, pharmacokinetics, pharmacogenetics, toxicity, hematopoietic stem cell transplantion Received : July 18, 2017 Accepted : July 23, 2017 Published : August 27, 2017 Abstract Busulfan (BU) dose adjustment following therapeutic drug monitoring contributes to better outcome of hematopoietic stem cell transplantation (HSCT). Further improvement could be achieved through genotype-guided BU dose adjustments. To investigate this aspect, polymorphism within glutathione S transferase genes were assessed. Particularly, promoter haplotypes of the glutathione S transferase A1 ( GSTA1 ) were evaluated in vitro, with reporter gene assays and clinically, in a pediatric multi-center study (N =138) through association with BU pharmacokinetics (PK) and clinical outcomes. Promoter activity significantly differed between the GSTA1 haplotypes (p

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BackgroundBusulfan (Bu) is widely used as a component of myeloablative conditioning regimen before hematopoietic stem cell transplantation (HSCT) in children. Obtaining the ratio of Bu to its metabolite sulfolane i.e. metabolic ratio (MR) may serve as an indicator of Bu GSH conjugating capacity of an individual.ObjectiveTo evaluate the utility of Bu MR to predict EFS in children undergoing allogeneic HSCT.MethodsTwo different cohorts with children receiving Bu in four times daily (QID, n=44) and once daily doses (QD, n=13) at St. Justine’s Hospital, Montreal were studied. Bu and Su levels were measured on day 3 of the conditioning regimen at the end of infusion (dose 9 in QID or dose 3 in QD dosing). EFS was defined from the time of transplant until death, relapse, or rejection, whichever occurred first. A receiver-operator characteristic curve (ROC) of Bu MRs was analyzed in relation to EFS. Cutoff values were defined based on the Youden´s J statistic.ResultsTwenty-two males and 22 females aged from 0.1 to 19.9 years (mean±SD: 7.2 ± 5.7) from Bu QID cohort had the mean MR of 5.9 (SD: 3.2). A cut off value of 4.9 in MR was chosen in ROC analysis in this cohort, with better sensitivity (71%) and specificity (70%) for EFS prediction (p=0.01, AUC= 0.7 (95% CI= 0.6–0.8). In QD cohort nine females, and four males aged between 0.4 and 15.8 years (6.7±5.1) had the mean MR of 29.3 (SD: 16.6). In ROC analysis, a cut off value of 25.06 was chosen with better sensitivity (100%) and specificity (100%) for EFS prediction (p=0.003; AUC=1.0).ConclusionThe Bu MR on day 3 above 4.973 and 25.06 were associated with worse EFS in children undergoing HSCT and received Bu in QID and QD dosing schedules, respectively.Disclosure(s)Nothing to disclose

BackgroundHaematopoietic stem cell transplantation (HSCT) is the only current curative treatment for Sickle Cell Disease (SCD), with potential life-threatening consequences. Busulfan is an alkylating agent used in HSCT conditioning regimen. Because of its narrow therapeutic window, determining the optimal first dose a priori remains a challenge.Busulfan is metabolized in the liver by conjugation with glutathione, which is catalyzed by Glutathione-S-Transferases (GSTs). GSTA1 is a known determinant of Busulfan clearance1 2 (just like age and weight) suggesting that those characteristics should be known a priori to adjust the first dose of Busulfan.Haemoglobinopathies (SCD and thalassemia) are not associated with changes in Busulfan clearance in a recent study.3 However, SCD is known to alter pharmacokinetics of other drugs.4 5 As it leads namely to liver dysfunction6 it may affect busulfan pharmacokinetics independently from genetic or anthropometric factors.Our aim is to compare the clearance of the first dose of Bu between patients with and without SCD, considering other constitutional factors.MethodsPatients with SCD were paired to patients without SCD on known Busulfan clearance’s covariates including GSTA1 group, age and frequency of administration.Data were collected retrospectively from the HSCT Unit database at Sainte-Justine Hospital and also used in previous studies.1 2 Weight adjusted clearance was compared between the two paired groups using a mixed procedure on SAS software.ResultsAmong the 129 patients included, 16 had SCD. Each patient was matched with up to 4 controls (total of 50 controls). Mean weight adjusted clearance was 3.04 ml/min/kg [SD:0,18] in patients with SCD versus 3.11 ml/min/kg [SD:0,14] in controls (difference 0.07 ml/min/kg F=0,14 p>F=0.714).ConclusionsThe diagnosis of SCD did not reveal to influence independently the clearance of the first dose of Bu. Consequently, no dose tailoring is needed in those patients only by the fact of being affected by SCD.ReferencesAnsari M, Curtis PH-D, Uppugunduri CRS, Rezgui MA, Nava T, Mlakar V, et al. GSTA1 diplotypes affect busulfan clearance and toxicity in children undergoing allogeneic hematopoietic stem cell transplantation: a multicenter study. Oncotarget 2017 Oct 31;8(53).Nava T, Kassir N, Rezgui MA, Uppugunduri CRS, Curtis PH-D, Duval M, et al. Incorporation of GSTA1 genetic variations into a population pharmacokinetic model for IV busulfan in paediatric hematopoietic stem cell transplantation. British Journal of Clinical Pharmacology. 84(7):1494–504.McCune JS, Baker KS, Blough DK, Gamis A, Bemer MJ, Kelton-Rehkopf MC, et al. Variation in prescribing patterns and therapeutic drug monitoring of intravenous busulfan in pediatric hematopoietic cell transplant recipients. Journal of Clinical Pharmacology. 2013 Mar;53(3):264–75.Maksoud E, Koehl B, Kaguelidou F, et al. Population Pharmacokinetics of Cefotaxime and Dosage Recommendations in Children with Sickle Cell Disease. Antimicrob Agents Chemother 2018 Apr;62(4).Dampier CD, Setty BN, Logan J, Ioli JG, Dean R. Intravenous morphine pharmacokinetics in pediatric patients with sickle cell disease. J Pediatr 1995 Mar;126(3):461–7.Gremse DA, Fillingim E, Hoff CJ, Wells DJ, Boerth RC. Hepatic function as assessed by lidocaine metabolism in sickle cell disease. J Pediatr 1998 Jun;132(6):989–93.Disclosure(s)Nothing to disclose

Introduction: Acute Graft versus Host Disease (aGvHD) occurs in 20-50% of pediatric patients who undergo allogeneic stem cell transplantation. Despite advances in HLA-typing methods and post-transplant immune suppression, it remains a significant cause of mortality and morbidity. Conditioning regimens include alkylating agents that exert their effects through their ability to directly or indirectly damage DNA. Unfortunately normal cells are also damaged due to their rapid proliferation cycles, increasing the probability of treatment-related toxicities. The most vulnerable targets among these damaged tissues are the skin, the intestinal epithelium, and the liver: In this study, our aim was to find biomarkers for aGvHD by focussing on how inter-individual differences in DNA repair mechanisms due to genetic variants in genes encoding DNA repair proteins might affect toxicity. Methods: The study included 115 children that had undergone allo-HSCT at four different centers. All patients received a Busulfan(Bu)/Cyclophosphamide(Cy) conditioning regimen. Pharmacokinetic-guided dose adjustment was performed for Bu to obtain a concentration at the steady state (Css) between 600 and 900 ng/ml. Patients received 16 doses of Bu followed by, i.v. Cy (200mg/kg total dose, 80% of patients) or CyVP16 (120mg/kg total dose). Cyclosporine was given as GvHD prophylaxis, and Methotrexate (MTX) or steroids were added to bone marrow and cord blood transplantation, respectively. ATG was given to 75% of patients. HLA matching was as follows: MRD = 37%; MUD = 21%; MMRD = 4%; MMUD = 38%. Acute GvHD was diagnosed according to the 1994 consensus conference up to day 180 post HSCT. Peripheral blood was collected and the DNA was extracted prior to transplant. Fifty-one single nucleotide polymorphisms (SNPs) within seventeen DNA repair genes were chosen for investigation. Cumulative incidence analysis of aGvHD 2-4 was performed using Kaplan-Meier analysis and log-rank test. Multivariate Cox regression was performed to estimate the impact of genotype on clinical outcome in the presence of other covariates. Results: The most significant finding came from one SNP (rs10764881, G>A) located in the promoter of the MGMT gene. Patients with rs10764881 GG genotypes had a lower risk for aGvHD 2-4 incidence (Figure 1). This variant was not associated with any other treatment related toxicities nor relapses. Multivariate analysis including MGMT rs10764881 with known aGvHD risk covariates did not influence the model. In addition, from the expression analysis that we performed on 24 lymphoblastoid cell lines rs10764881 GG carriers showed 1.5 fold higher expression compared to AA or AG carriers (Figure 2). We were able to confirm experimentally with luciferase reporter constructs the impact of this variant on promoter function. Plasmids, which included the promoter sequence with rs10764881 demonstrated higher luciferase protein levels, compared to plasmids with a promoter sequence excluding rs10764881 (p= 0.01), suggesting the presence of an enhancement element close to the variant region (Figure 3). Electrophoretic mobility shift assays confirmed the presence of a Glucocorticoid Receptor Element (GRE) near to this variant. To understand whether the enhancing effects are related to corticosteroids, keratinocytes transfected with the gene reporter plasmids were stimulated with dexamethasone and luciferase expression was examined as previously. Exposing the cells with dexamethasone significantly increased protein expression of luciferase in both plasmids compared to their non-treated plasmid construct. The highest response was seen from the plasmid containing variant rs10764881 G (Figure 3). However, clinically we did not find clear differences between steroid and non-steroids recipients against rs10764881 with regards to aGvHD vulnerability (Figure 4). Conclusions: We hypothesize that the reason why variant rs10764881 GG might have a protective effect against aGvHD is due to its higher expression levels, resulting in more efficient DNA repair, in turn diminishing the immune response, reducing inflammation and hence causing less aGvHD. Thus aGvHD rs10764881 GG could potentially be a biomarker for aGvHD protection. The effects of steroids on MGMT expression needs to be addressed in future studies. Figure 1 Figure 1. Disclosures Bader: Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding. Bittencourt: Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel Grant; Amgen Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy.

Introduction: Busulfan (Bu) is a component of several conditioning regimens before hematopoietic stem cell transplant (HSCT). Bu has a narrow therapeutic window. High exposure was associated to higher rates of GVHD, VOD and mortality and low exposure to relapse or graft failure. Pharmacokinetic (PK) studies demonstrated high intra- and inter-patient variability. Several factors such as age, gender and weight have been associated to this variability (Vassal 1993, McCune 2009, Lee 2012, Veal 2012). The contribution of GSTA1 polymorphisms has been also documented. Haplotype *A2 was associated to higher clearance and less SCT-related complications, whereas haplotype *B (-69T) was associated to more frequent VOD occurrence (Ansari 2013). This study aims to analyse the influence of GSTA1 genotypes/haplotypes on Bu clearance. Methods: Eighty-nine patients underwent a SCT after Bu-based conditioning with therapeutic drug monitoring (TDM) from October 2000 to August 2010 at our center CHU Sainte Justine, Montreal. The median age was 8.2 (IQR 1.6 to 13.9) years, 50.6% were males, most with malignancies (64.4%). Most patients received BuCy (77%) as conditioning regimen. Bu four times daily dose was given as previously described (Ansari 2014) and ulterior doses were calculated depending on the TDM. Bu AUC, steady-state concentration (Css) and Clearance were assessed after the first dose in all patients. New PK studies could be performed between sixth and ninth dose. Adjusted doses were calculated assuming a linear correlation between dose and desired AUC. Bu clearancewas expressed in base to an allometric representation of patients' size accounting for age, body weight and body composition: Normal Fat Mass (NFM). The NFM of 62Kg corresponds to an allometrically-scaled body weight of 70kg. Then, clearance was normalized by 62Kg NFM as described previously (McCune 2014). GSTA1 genotypes was classified as carriers of *A/*B haplotypes and their subtypes (Ansari 2013). Continuous data was expressed as median and interquartile range (IQR). A linear mixed model was performed to evaluate the association of normalized clearance to GSTA1 genotypes/haplotypes. Subjects were considered as random effects of repeated measures. Results: Two hundreds and eighty PK measurements were performed (from one to 5 times per patient during the Bu exposure time). Median of Bu prescribed doses during the conditioning was 0.97mg/Kg. A TDM-based change of dose was possible from the 6th dose (30 hours after Bu initial dose). A dose change was made in 69.3% of patients. Median change was +32% (IQR 0 to +48.3%) of the initial dose. Eight percent of doses had to be decreased. Overall normalized clearance was 16.8 l/h/62 Kg NFM (IQR 13.7 to 19.4). Eighty-six percent were genotyped by GSTA1. In a model including age, gender and NFM normalized dose, clearance was significantly associated with GSTA1 haplotypes (p Conclusion: We have demonstrated that GSTA1 *B1a haplotype is an independent factor related to diminished Bu clearance. We suggest that GSTA1 genotyping should be included in the work-up before Bu-based conditioning regimens. Adjusted doses depending on *B1a status would limit the exposure to Bu in patients with lower rates of elimination and higher risk of HSCT-related complications, ensuring a more individualized and safer conditioning. However, it lacks Bu first dose prediction models accounting for genetics components such as GSTA1 haplotypes. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Busulfan (Bu) is a component of conditioning before stem cell transplant (SCT). Bu has a narrow therapeutic window. High exposure was associated to higher rates of GVHD, VOD and mortality and low exposure to relapse or graft failure. Pharmacokinetic (PK) studies demonstrated high intra- and inter-patient variability. Several factors such as age, gender, weight and genetic components have been associated to this variability (McCune 2009, Vassal 1993, Ansari 2013). Certain models and nomograms have been used to predict Bu first dose, in theory precluding need of further adjustments. Recent comparison of 12 different models showed acceptable performances but high coefficients of variation (CV) and failure to reach target AUC in at least 24% of cases (Zao 2015). The present study aims to evaluate the role of GSTA1 haplotypes on different methods' performances in predicting of Bu first dose. Methods: A hundred and ten patients underwent a SCT after Bu based conditioning from October 2000 to October 2011 and 104 had PK data. Median of age was 6.9 (IQR 2 to 13.7) years, 55.5% were males and most had malignancies (69%). Most received Bucy (78.2%) as conditioning regimen. Four-time daily dose of Bu was calculated as previously described (Ansari 2014). Bu AUC, steady-state concentration (Css) and Clearance (Cl) were assessed for all patients after the first dose. The same parameters were predicted based on 11 other methods previously described (Zao 2015) to calculate the initial dose of Bu assuming a linear relationship between predicted doses and AUC. For each model that predicted individualized clearances, a mean prediction error (MPE) was calculated as follows: MPE=(Clpredicted-Clobserved)/Clobserved. Target AUC was from 900μM.min through 1500μM.min. Two haplotypes, found previously to influence Cl were included in the analysis: haplotype *B1a (-1142G, -631G, -513C, -69T) and *A2 (-1142C, -631G, -513T, -69C) in heterozygous and homozygous state (Ansari 2013). X2 test was used to compare methods' performances and observed values. Continuous data was expressed as median and interquartile range (IQR). Multivariate linear model was used to evaluate the association of Cl MPE and the GSTA1 haplotypes. Results: Initial doses of Bu, calculated by the different methods, varied from 0.47 to 1.6mg/kg. Predicted AUCs varied from 392 to 2,968μ.min with CV ranging from 21.5 to 37.1%. Ability to predict the target AUC was from 28.8% to 76%, with 0 to 20% of measures above the range. Bartelink's (p Conclusion: Bartelink's and McCune's models seemed to be adequate methods to calculate the initial dose of Bu and demonstrated better performances than the method used in our center. For the clearance-predictor methods, although McCune's seemed to be the most reliable model for most patients, it had limited performance in GSTA1 haplotypes previously associated to extreme clearances. It suggests that genetic factors such as GSTA1 genotyping should be included in a future model design to better predict the initial dose of Bu. Disclosures No relevant conflicts of interest to declare.