ALBERT

Description: Myeloid sarcoma (MS) is defined as a tumor mass consisting of myeloid blasts with or without maturation occurring at an anatomical site other than bone marrow (BM). MS may occur before, concurrently or after a characterized acute myeloid leukemia (AML). Cytogenetic abnormalities are found in 50% of the cases but molecular alterations are less well described and involved FLT3 and/or NPM1 mutations. Mutations in IDH1 and IDH2 genes are found in 15% to 20% of patients with AML but have never been described in MS. Mutated IDH enzymes produce in vast excess D-2-hydroxyglutarate (2-HG) in leukemic cells, which can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations. As availability of DNA sequencing techniques on paraffin samples are limited, molecular characterization of MS remained difficult. We asked whether in MS, serum 2-HG would predict the presence of IDH1/2 mutations at diagnosis, and could provide a biomarker for follow up. Tissue samples and serum samples from 8 patients with a MS diagnosis were analyzed. High quality genomic DNA was extracted from frozen MS samples using conventional phenol/chloroforme extraction procedures. Exon 4 of IDH1 and IDH2 genes (IDH1/R132 and IDH2/R140 and /R172 codons) was amplified by PCR using HotStar Taq polymeraze (Qiagen) and primers. Direct sequencing was performed using the Sanger method as previously described. In case of MS relapse or AML evolution, IDH1 and IDH2 genes were analyzed in the same way from frozen tissue sample or bone marrow sample. Serum samples at MS diagnosis were analyzed for total 2-HG, D-2-HG and L-2-HG by reverse-phase liquid chromatography coupled to mass spectrometry. In case of myeloid sarcoma with IDH1/2 mutation, 2-HG values were compared to 18F-FDG-PET results when available during remission phase and at relapse. Three patients (3/8; 37.5%) had an IDH2 R140Q mutation at diagnosis of MS localized to lymph node, soft tissue, skin or pharynx. At MS diagnosis, serum total 2-HG, D-2-HG and ratio D/L-2-HG were significantly higher in case of myeloid sarcoma with IDH2 R140Q mutation compared to patients with no IDH mutation (Table 1). Serum total 2-HG level ≥2µM or D-2-HG level ≥1.8µM or ratio D/L 2-HG 〉2.5 were significantly associated with the presence of IDH2 mutation (Fisher's exact test P≤0.02). Table 1. Myeloid sarcoma with IDH2 R140Q mutation (N=3) Myeloid sarcoma without IDH2 R140Q mutation (N=5) Median total 2-HG (µM) 4.1 (range: 3.1-30.1) 1.4 (range: 1-1.6) Median D-2-HG (µM) 3.7 (range: 2.3-28) 0.6 (range: 0.5-0.8) Median L-2-HG (µM) 0.8 (range: 0.4-2.1) 0.8 (range: 0.4-0.8) Median ratio D/L 2-HG 8.3 (range: 2.9-18.8) 1 (range: 0.7-1.7) All 3 patients with IDH2 R140Q mutated MS received intensive chemotherapy treatment and achieved complete remission (CR). Two patients relapsed: one experienced isolated extramedullary relapse (thigh muscle); one had a bone marrow relapse. IDH2 R140Q mutation was found at the site of relapse in both cases. When available, serum 2-HG values and 18F-fluorodeoxyglucose-positron-emission tomography (FDG-PET) were compared at different time points (at diagnosis, remission and relapse; Table 2). Table 2. Patient #1 Patient #2 Patient #3 FDG-PET at diagnosis (SUVmax) - 17 5.25 Serum 2-HG at diagnosis (µM) 4.1 3.1 30.1 FDG-PET in remission (SUVmax) 0 - 9.6 (N=4) 0 (N=4) - Serum 2-HG in remission (µM) 0.5 - 1.1 (N=4) 0.6 - 3.1 (N=4) 3.4 - 17.9 (N=3) FDG-PET at MS relapse/evolution to AML (SUVmax) 6.1 - 0 Serum 2-HG at MS relapse/evolution to AML (µM) 2.3 - 16.7 Time between diagnosis and MS relapse/evolution to AML (months) 30 - 9 Serum 2-HG values were in accordance with FDG-PET interpretations except in patient #1 who presented a transient hypermetabolic splenic nodule (SUVmax 9.6) without serum 2-HG increase. Patient #2 remained in CR but had recently increased 2-HG values without overt relapse. Patient #3 presented relapse as a refractory anemia with excess of blast without extra-medullary localization. FDG-PET didn't find any abnormality contrary to the persistent increased value of serum 2-HG (total 2-HG: 16.7µM). These data show that myeloid sarcoma can be associated with IDH2 R140Q mutation and suggest that 2-HG measurement in the serum predicts the presence of IDH1/2 mutations at diagnosis. During follow-up, serum 2-HG values could be representative of the disease status. Because of IDH inhibitors promising results in AML, 2-HG screening at MS diagnosis could be useful. Disclosures Ribrag: Celgene: Research Funding; Esai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. De Botton:Agios pharmaceuticals: Research Funding.

Description: Introduction: Mutations in IDH1 and IDH2 genes are found in 15% to 20% of patients with acute myeloid leukemia (AML). Mutated IDH enzymes produce in excess the D stereoisomer 2-hydroxyglutarate (2-HG) in leukemic cells, which can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations. Total serum 2-HG (D and L stereoisomers) 〉2µM and ratio D/L 2-HG 〉2.5 are strong predictors of the presence of IDH1/2 mutations at diagnosis (Janin et al; JCO 2014). We measured 2-HG levels in serial samples of 47 patients with IDH1/2 mutations during induction or first salvage therapy to determine whether 2HG can serve as a surrogate marker of treatment efficacy. Methods: Between 2007 and 2015, 47 AML patients with IDH1 or IDH2 mutation received intensive chemotherapy as induction therapy (n=42) or as first salvage regimen (n=5) in our center (Table 1). Genomic DNA was extracted from BM samples using conventional procedures. IDH1-(codon R132) and IDH2- targeted (codons R140 and R172) regions were amplified by PCR with primers containing Ion Torrent adapters and unique barcodes to generate libraries. Pooled amplicons libraries were clonally amplified on Ion Spheres using the Ion Xpress Template 200 Kit. All available serum samples (n=394) prior and during induction/salvage chemotherapy were analyzed for total 2-HG, D-2-HG and L-2-HG by reverse-phase liquid chromatography coupled to mass spectrometry. We studied the serum 2-HG values (D-2-HG and ratio D/L 2-HG) evolution during IC according to complete remission (CR) evaluated at the end of IC. When a patient had a missing value for 2-HG, the missing value was replaced by the last available observation (up to 7 days before the missing value). At each day we tested the difference between the dosage values of the responders and the non-responders with non-parametric Wilcoxon 2-sided tests. The analyses were done with SAS software (version 9.3; SAS Institute, Cary, NC). Table 1. Patients and treatment characteristics: AML at diagnosis / RR AML (N) 42 / 5 Median age (years) 58.2 (23.1 - 75.7) Sex ratio (M/F) 23/24 Conventional cytogenetic: - Normal karyotype - Trisomy 8 - Monosomy 7 39 (83%) 3 (6%) 2 (4%) Associated gene mutations: - NPM1- FLT3-ITD- FLT3-TKD 24 (51%) 10 (21%) 5 (11%) IDH mutation: - IDH1 R132 (N) - IDH2 R140 (N) - IDH2 R172 (N) 8 (17%) 32 (65%) 7 (15%) Intensive treatment: - "3+7" regimen - Intensified induction chemotherapy - High-dose cytarabine based chemotherapy 24 (51%) 19 (40%) 4 (8%) Results: Complete remission (CR) 1 was obtained in 35/42 patients (83%) and CR2 in 3/5 (60%) after IC. All (8/8) IDH1 -R132 mutated patients achieved CR1, 25/28 (89%) IDH2 -R140 mutated patients and 3/4 achieved CR1 and CR2 respectively; 2/6 (33%) IDH2 -R172 mutated patients and 0/1 achieved CR1 and CR2, respectively. Median serum 2-HG values before IC were as follow: total 2-HG 10.9 µM (range: 2.1-135.5); D-2-HG 10.7 µM (range: 1.7-132.6) and ratio D/L 36.9 (range: 3.9-131.2). Median number of serum 2-HG values per patient was 7 (range: 5-10). Serum D-2-HG and ratio D/L 2-HG evolution according to CR status after IC began to differ significantly at day 5 after IC (Wilcoxon test P=0.02, Figure 1). The difference between responders and non-responders increased overtime (Wilcoxon test P

Description: Background: Angioimmunoblastic T cell lymphoma (AITL) is one of the most frequent peripheral T cell lymphoma, and has a poor prognosis. Neoplastic T cells originate from T follicular helper cells, and are admixed among a prominent microenvironment, making their identification sometimes difficult. IDH2 mutations are present in 20-30% AITL patients, where they often co-exist with TET2, DNMT3A and RHOA mutations. They affect almost exclusively the codon R172 of IDH2, providing to the IDH2 enzyme a neo-activity that converts α ketoglutarate (αKG) to D 2-hydroxyglutarate (2HG). D-2HG, the dextrogyre form of 2HG, is an oncometabolite present at very low level under physiological condition, which inhibits many αKG dependent dioxygenases, including TET proteins and is involved in oncogenesis of various cancers such as gliomas or acute myeloid leukemias (AML). Preliminary data, based on small series, showed that increased level of 2HG was detectable in tumor and in serum of IDH2 mutated AITL. However, 2HG level, as well as D/L ratio, has not been evaluated as a surrogate marker of IDH2 mutation in AITL, at diagnosis or during the follow-up. Patients and Methods: Serum from 69 AITL patients, collected in REVAIL trial (NCT00169156) (N=48), RAIL trial (NCT01553786) (N=9) or TENOMIC collection (N=12) were included in this study. IDH2 mutations were assessed in formalin fixed paraffin embedded tumor tissue by deep next generation sequencing of exon 4, using PGM technology (N=63) or allele specific PCR (N=6). For the purpose of the study the cohort was enriched in mutated patients. Serum was collected at diagnosis and at the end of the frontline treatment in 6 patients, 5 of them being IDH2 mutated. D and L 2HG was measured in serum using a liquid tandem mass spectrometry method as previously described (Poinsignon et al. J Chromatogr B 2016) to determine total (D+L) 2HG and D/L 2HG ratio. Results: Twenty-four patients (35%) were IDH2 mutated. Median IDH2 variant allele frequency (VAF) was 7% (IQR, 4%-12.5%). Median total 2HG was 3.63 µM (IQR, 1.6-6.1) in mutated patients versus 1.17 µM (IQR, 0.85-1.68) in wild type patients (p

Description: Background: Busulfan (Bu) is the corner stone of hematopoietic stem-cell transplantation (HSCT) regimens with a narrow therapeutic window (TW). Graft rejection or toxicities are reported according to plasmatic exposure. In very young children, Bu exhibits large pharmacokinetic (PK) variability. Bu clearance was demonstrated to be non-linearly related to body weight (BW) and thus BW is used to optimally define the Bu dosage in children [Vassal et al., CCP 2006]. Search for additional data to confirm the TW and to describe clinical outcomes is still a topical question to better understand Pk/Pd relationship and to safely use Bu in young children and infants. Objective: To study the Pk/Pd relationship of Bu in pediatric recipients of bone marrow transplantation (BMT) receiving Bu-based conditioning regimens (CR). To correlate early toxicities (mainly hepatic veno-occlusive disease (VOD), non infectious pulmonary disease (niPD) and outcome i.e. overall survival (OS) at last follow up with type of CR, the underlying diseases, age at transplantation and Pk of busulfan Patients and Method: This multicenter prospective observational study included 307 pts transplanted between 2006 and 2013 from 14 French Pediatric BMT units; median age at transplantation was 18.4 months [1.3-289], with a median BW of 11.3 kg [3.4-82]. 100 pts were younger than 1 year, 71 pts 〈 9 kg and 171 pts 〈 16 kg. Patients were mostly affected by non-malignant diseases (primary immune deficiency (n=143), inherited metabolic disorders (n=50), hemoglobinopathies (n=20) while 78 patients suffered from malignant disease. 257 patients received allogenic HSCT (genoidentical donor n=79, matched unrelated donor n=33, mismatch unrelated donor n=18, haploidentical intra-familial donor n=59, other mismatched intra familial donor n=8 or unrelated cord blood n=51) and 41 autologous BMT. All patients received Bu-based conditioning regimen in combination with cyclophosphamide (BuCy n=119), fludarabine (BuFlu n=88), melphalan (BuMel n=36) or thiotepa (BuTTP n=3). 40 patients received BuFlu associated with a second alkylating agent (Mel, TTP or Cy), 12 patients received BuCy associated with a third agent (Mel or VP16). Serotherapy was given in 70% of the patients. Starting doses of Bu were given according to the European SmPC or EBMT-ESID recommendations. The median posology was 1 mg/kg 4x/day for 4 days [0.6-1.3 mg/kg]. PK was assessed on plasma samples with area under the curve (AUC) evaluation from the 1st(D1), 9th (D9) and 13th (D13) dose in 150 patients, D1 and D9 in 40 patients, D1 and D13 in 46 patients while 62 patients were monitored on D1 only (684 PK datasets). PK analysis was performed using Non linear Mixed Effect Modelling. A one-compartment PK model suitably fitted the concentrations vs time data. Median follow up is 27 months (0.33 to 96 months post BMT). Results: At D1, 67% of patients were within the therapeutic window (TW) [900-1500 µM.min] and 66% of patients reached the cumulative TW [14400-24000 µM.min]. Incidence of VOD and niPD were respectively 35% and 14%. Both toxicities correlated with type of CR and age

Description: Abstract 1811 Background: Busulfan (Bu) is a standard component of HSCT regimens and shows a narrow therapeutic range. In very young children, Bu exhibits large pharmacokinetic (PK) variability. Intravenous (IV) Bu clearance was demonstrated to be non-linearly related to body weight (BW) and thus BW is used to optimally define the IV Bu dosage in children [Vassal G. et al., CCP 2006]. Search for additional relevant covariates is still a topical question to further explain the variability in young children (Pts) and infants. Objective: to screen for the effect of heterogeneous patient characteristics on IV Bu PK disposition, and to evaluate the consistency of weight-based dosing strategy (as recommended in the European SmPC) in a large cohort of children, especially in patients with rare diseases and a BW less than 9 kg. Patients and Method: The prospective observational study included 115 patients (pts), mostly infants (median age: 13 months [0.3-148], median BW: 9 kg [3.5-58]). Main diagnoses were immunodeficiency (n=71, mainly SCID and lymphohistiocytosis) and inherited metabolic disorders (n=15). Additional data (90 pts, mostly malignant diseases) from two prospective clinical trials previously described [Nguyen L. et al., BMT 2004] [Vassal G. et al., CCP 2006] were pooled with the observational dataset. Altogether, 205 children aged from 10 days to 15 years (median of 30 months) and weighed from 3.5 to 62.5 kg (median of 11.0 kg) were analysed. IV Bu (Busilvex®) was given over 2 h q6h × 16 doses. For most of the pts (88%), the starting doses were given according to the European SmPC: 1.0 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 0.95 mg/kg and 0.8 mg/kg for pts 34 kg BW, respectively. PK was assessed on plasma samples from the 1st, 9th and 13th dose (536 PK datasets). PK analysis was performed using Non Linear Mixed Effect modelling with the NONMEM program. Results: A one-compartment PK model suitably fitted the concentrations vs time data. BW remained the main determinant of IV Bu PK disposition. Increase of IV Bu clearance with child's growth was observed to be faster in younger than in older children. For children 〈 9 kg, a doubling in BW is associated with a 2.4-fold increase in Bu clearance while a 1.7-fold increase in Bu clearance is observed in pts ≥ 9 kg for the same BW growth. To take into account this difference, the relationships between Clearance (CL) and BW was modelled using a different allometric exponent for children with a BW of 〈 9 kg and ≥ 9 kg. The predictive equations were: CL (L/h) = 2.18 × (BW/9)1.25 and CL(L/h) = 2.18 × (BW/9)0.76 in pts 〈 9 kg and ≥ 9 kg, respectively. The inter-individual variability decreased from 64% (covariate-free model) to 23% for Bu CL. No further significant impact was identified even when considering large fluctuations of the following covariates: creatinine clearance (up to 11 × Upper Normal Limit), bilirubin (up to 4 × UNL), ferritine (up to 43 × UNL), hepatic transaminases (up to 39 × UNL), alkaline phosphatases (up to 4 × UNL), gGT (up to 86 × UNL), total protein (0.5 to 1.4 × UNL) and lactate deshydrogenase (up to 3 × UNL). No PK difference was observed between patients under Benzodiazepine and those under Phenytoin prophylaxis, neither when comparing different groups of diagnoses. Similar performances for targeting an AUC range of 900 – 1500 μM.min were achieved when comparing Bu dosing either adjusted according to the model predictive equation or with the use of the European SmPC recommendations. The success rates were about 60% and 80% in pts 〈 9 kg and ≥ 9 kg respectively. Conclusion: BW is confirmed to be the only relevant predictor to optimally define the IV Bu dosing in children. The population PK model has established a higher allometric exponent for younger children (〈 9kg) in order to take into account the faster maturation of clearance in this weight group. This model is consistent with the dosing strategy recommended in the EMA registration. Disclosures: Daher Abdi: Pierre Fabre Médicament: Employment. Pétain:Pierre Fabre Médicament: Employment. Nguyen:Pierre Fabre Médicament: Employment.