ALBERT

Description: Backgound : Sinusoidal obstruction syndrome [SOS, also known as hepatic veno-occlusive disease (VOD)] is frequent after HSCT and may have a mortality rate of up to 85%. Defibrotide has shown efficacy not only in the treatment of established SOS (Richardson et al. Blood 1998:92;737 and 2016:127;1656) but also in SOS prevention in children (prospective study: Corbacioglu et al. Lancet 2012:379;1301) as well as in adults (several retrospective studies). Patients and methods : Between 1999 and 2009, we gave defibrotide intravenously to 248 successive patients transplanted for hematological diseases starting at day -7 up to day +20 post-transplantation (dose range 800-2400 mg/d) in combination with heparin. We previously published the data of the 52 first patients compared to 52 historical controls who were transplanted just prior to the study period. We now expand the study with 196 additional patients treated successively with defibrotide prophylaxis while adding patients transplanted after 2010 when patients did not receive defibrotide as prophylaxis anymore (2011-2015) to the control group. The characteristics of patients are shown in Table 1. Results: Median follow-up for the study group was 10 (range 2-16) years and for the control group 2.7 (range 1-18) years. None of the 248 patients in the defibrotide group developed SOS (Baltimore criteria). The 100 day cumulative incidence (CI) of SOS was 0% in the defibrotide group as compared to 4.8% (95%CI 2.6-8%) in the control group, p=0.00046. The day 100 event free survival (EFS) where an event was defined as the 1st occurrence of one of the following: SOS, acute graft versus host disease (GvHD) ≥2, relapse or death, was not significantly different with 60% (95%CI 54%-66%) in the defibrotide group vs 53% (95%CI 47%-59%) in the controls, p=0.165, but the one year EFS was statistically different with 38% (95%CI 32%-44%) vs 28% (95%CI 22%-34%), p=0.00969. The 100 day CI of acute GVHD was not significantly different between the two groups [27% (95%CI 22%-33%) in the defibrotide group vs 29% (95%CI 24%-35%) in the control group, p= 0.707] while the 1 year acute GvHD CI was significantly reduced in the defibrotide group [31% (95%CI 25%-37%)] compared with the control group [42% (95%CI 36%-48%), p=0.026]. The one year overall survival (OS), relapse incidence (RI) and non-relapse mortality (NRM) were not statistically different being respectively 73% (95%CI 67%-78%) vs 65% (95%CI 59%-71%), p=0.0704, 32% (95%CI 27%-38%) vs 28% (95%CI 22%-34%), p=0.331 and 14% (95%CI 10%-18%) vs 19% (95%CI 14%-24%), p=0.148. Multivariate analysis, performed taking into account clinical factors known to influence the risk of SOS, confirmed the favorable impact of defibrotide on 100 day SOS CI [HR 7.5x10-7 (95%CI 1.8x10-7-3.2x10-6), p

Description: Introduction Myeloproliferative Neoplasm (MPN) unclassifiable (MPN-U), is a heterogeneous disease that presents with an MPN-type clinical/ histological phenotype yet fails to meet diagnostic criteria for other MPN entities. Incidence is

Description: Abstract 3265 Poster Board III-1 In utero transplantation of large animals (sheep and goats) with human hematopoietic cells has proven valuable in distinguishing subsets of human cells with short- and long-term repopulating activity. Transplantation of secondary and tertiary fetal sheep with cells regenerated in primary sheep has also demonstrated the ability of human hematopoietic stem cells to retain and execute their self-renewal potential in a xenogeneic setting. We now describe the novel extension of this approach to the generation of a BCR-ABL gene transfer-based in vivo model of human myeloproliferative disease. Lin-CD34+ human cord blood (CB) cells were transduced with BCR-ABL retrovirus (MSCV-BCR-ABL-IRES-GFP) and the cells were then injected IP into pre-immune fetal goats at 45–55 days of gestation. This induced a high frequency of abortion among the injected fetuses: 79% (n=22) when 〉5×104BCR-ABL- transduced CB cells were injected as compared to 64% (n=9) when control (MIG)-transduced cells were injected. Six goats transplanted with 2×104 BCR-ABL-transduced cells were born alive and 3 weeks after birth, 3 of these were sacrificed so that their tissues could be analyzed. Interestingly, in the goats injected with BCR-ABL-transduced human cells, only GFP+(BCR-ABL+) human cells were detected and these were found in multiple hematopoietic and non-hematopoietic tissues, including peripheral blood, bone marrow (BM), liver, kidney, lung, heart, and skeletal and smooth muscle (1–49%) by fluorescence microscopy and confocal laser scanning microscopy, FISH and FACS. Immunohistochemical analysis allowed positive cells to also be detected in frozen sections of liver tissue. Continued follow-up of the other recipients of transduced cells showed that the 3 injected with BCR-ABL-transduced cells all developed features of early chronic phase human chronic myeloid leukemia (CML), as evidenced by a 3- to 5-fold elevation in their WBC count (up to 2.5×1010/L as compared to 5–8×109/L in the recipients of MIG-transduced cells, P2.5-fold higher levels in the BCR-ABL chimeric goats as compared to both control chimeric goats and normal human CB. These over-expressed genes are from several functional categories, including tyrosine kinases and other proteins with phosphorylation activities, cell cycle control, cell adhesion, homing and differentiation, transcription, nucleotide binding and ion transport. Several were confirmed by quantitative RT-PCR. These results demonstrate long-term engraftment, but slow expansion of primitive human hematopoietic cells transduced with BCR-ABL fusion gene and transplanted in utero in a large animal model. This novel xenotransplant goat model should be useful for characterizing the early (pre-symptomatic) phase of human CML and for assessing new therapies with potential long-term benefits. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: In a recent French FRALLE versus LALA comparison, we have demonstrated a large benefit in outcome when adolescents and young adults were treated in a pediatric rather than an adult ALL protocol (Boissel JCO 2003). Similar provocative results have been observed in three other pediatric versus adult ALL studies (Stock ASH 2000, de Bont Leukemia 2004, Testi ASH 2004). One explanation may be the larger amounts of steroids, vincristine (VCR), and L-asparaginase (L-aspa) administered in pediatric patients. The GRAALL-2003 study was thus designed to offer a pediatric approach in adults with Ph-negative ALL until 60 years of age. Methods: Treatment included a 5-drug induction, high dose-intensity consolidation blocks, delayed intensification, and 2-year maintenance. The comparison with the former LALA-94 protocol showed a 8.6-fold, 3.7-fold, and 16-fold increase in cumulative doses of prednisone, VCR, and L-aspa, respectively. One difference with childhood ALL therapy remained indication of allogeneic SCT in first CR which was offered to all patients with high-risk factor and a donor. In addition, induction was reinforced with a hyper-cyclophosphamide sequence (HyperC) in case of poor early response (cortico- and/or chemoresistant ALL). In the present report, 212 GRAALL-2003 patients with Philadelphia-negative ALL aged 15–55 years with a median follow-up of 18 months were compared to 712 patients previously treated in the LALA-94 trial. Results: Cohorts were comparable in terms of prognostic factors. CR rate was significantly higher in GRAALL patients (93 vs 88%, P=0.02) due to a reduction in resistant disease (0.5 vs 8%, P

Description: A high proportion of Ph-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients still undergo relapses despite the use of Tyrosine Kinase Inhibitors (TKIs) in addition to chemotherapy as frontline therapy. In this leukemia subtype, the role of BCR-ABL1 kinase domain (KD) mutations as a driver of resistance to TKIs has already been documented by previous studies and such mutations have been reported in up to 80% of the patients at relapse. Next-generation sequencing (NGS) has been proposed to characterize these mutations with a higher sensitivity than Sanger. We report here a prospective study aiming at detecting potentially resistant cell populations by NGS in Ph+ ALL patients enrolled in the GRAAPH 2014-trial. Between March 2016 and February 2019, 156 patients aged 18 to 59 years with newly diagnosed Ph+ and/or BCR-ABL1 positive ALL have been included in the GRAAPH 2014 trial (NCT02619630). BCR-ABL1 isoforms were E1A2 69%, B2A2/B3A2 29%, atypical 2%. After a prephase of steroid, treatment consisted of 4 blocks of chemotherapy + nilotinib. 118 patients (76%) underwent allogeneic or autologous stem cell transplantation (SCT). 22 medullary relapses were recorded within a median time of 9 months (range, 2 - 35). Blood and marrow samples harvested at diagnosis, after each treatment block, before and 3 months after SCT, and at relapse, were sequenced if BCR-ABL1/ABL1 ratio were above 0.001. Mutated BCR-ABL1 transcripts were detected by sequencing the KD of BCR-ABL1 transcripts by NGS with a limit of detection (LOD) of 0.03. T315I allele specific oligonucleotide (ASO) droplet digital RT-PCR (ddRT-PCR) with a LOD of 0.0005 was also performed at diagnosis on a subset of 63 patients, including 5 who have subsequently developed a T315I clone. NGS. At diagnosis, no KD mutation was found by NGS in pretreatment samples of 137 patients. During follow-up (FU), only 12 mutations were found by NGS in 7 out of the 88 patients tested (81, 45, 30, 20, 19, 9 after block 1, 2, 3, 4, before and 3 months after SCT, respectively). Mutations were T315I (N=6), Y253H (N=1), E255K (N=2), E255V (N=1), Q252H (N=1), Y253F (N=1). At relapse, 16 mutations were identified by NGS in 12 patients out of the 17 tested (71%). Mutations were T315I (N=7), Y253H (N=n=3), F359V (N=2), E255K (N=1), E255V (N=1), Q252H (N=1), Y253F (N=1). More than 1 mutated clone were present in 2 patients (E255V+T315I+F359V and Y253H+F359V), and a compound mutation was found in 1 patient (Q252H/Y253F). Out of the 7 patients found mutated during FU, 5 have relapsed with a rapid expansion (1 to 3 months) of the mutated clone. One patient harboring a sub-clonal (10%) E255K at MRD1 has relapsed 9 months later without any detectable mutation. One patient identified with 3 mutated clones (E255K 10%, E255V 10%, T315I 80%) underwent SCT and has not relapsed so far. We failed to anticipate expansion of any mutated clone in the 7 remaining patients found mutated at relapse. T315I ASO ddRT-PCR on diagnostic samples. Low-level T315I mutated BCR-ABL1 transcripts (0.00051 to 0.0013) were detected in 14 out of 63 patients (24%) tested. Only one has expanded a T315I clone later on. In the context of the GRAAPH 2014 trial, 71% of the 17 relapses tested so far were associated with BCR-ABL1 KD mutations. Expansion of the mutated clone could have been characterized before the onset of hematological relapses in only 5 out of 12 patients (42%). Unfortunately in these cases, lags between first detection and relapse were very short (1 to 3 months). On the contrary, occurrences of relapses associated with expansion of KD-mutated clones could not have been anticipated in 58%. All mutations identified, including T315I, F359V, E255K/V and Y253F/H, Q252H/Y253F are known for conferring resistance to nilotinib. NGS is a valuable method for KD mutation detection in Ph+ ALL. It allows a quantitative characterization of KD mutations at relapse. However in our hands and in the context of an intensive therapy combining chemotherapy, nilotinib and SCT, its enhanced sensitivity as compared to Sanger (3% vs 20%) does not translate into the capacity of anticipating expansion of KD-mutated clones. Moreover, in this study, NGS did not detect any mutation in pre-therapeutic samples while T315I mutated BCR-ABL1 transcripts were found at low-level in 24% of these samples by ddRT-PCR. However it should be emphasized that when detected, low-level T315I mutated sub-clones present at diagnosis failed to expand in most instances. Disclosures Cayuela: Incyte: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Chalandon:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Incyte Biosciences: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rousselot:Pfizer: Research Funding. Thomas:PFIZER: Honoraria; ABBVIE: Honoraria; INCYTE: Honoraria; DAICHI: Honoraria. Huguet:Amgen: Honoraria; Servier: Honoraria; Jazz Pharmaceuticals: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte Biosciences: Honoraria; Novartis: Honoraria. Chevallier:Incyte: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria; Daiichi Sankyo: Honoraria. Boissel:NOVARTIS: Consultancy. Vey:Novartis: Consultancy, Honoraria; Janssen: Honoraria. Berthon:PFIZER: Other: DISCLOSURE BOARD; JAZZPHARMACEUTICAL: Other: DISCLOSURE BOARD; CELGEN: Other: DISCLOSURE BOARD.

Description: PAX5 is a transcription factor central to B-cell differentiation, frequently mutated in pediatric B-ALL (38.9% in 192 B-ALL, Nature,2007;446:758). PAX5 mutations consist in whole or partial gene deletions or amplifications, fusion gene or point mutations. Adult B-ALL differs in many regards from pediatric B-ALL as demonstrated by differences in prognosis and genetic abnormalities (ETV6-RUNX1 almost absent from adult; BCR-ABL1 rare in children). We screened the occurrence of PAX5 mutations in a series of 119 adult patients with B-lineage ALL prospectively treated in the GRAALL-2003 (pediatric-like protocol for BCR-ABL1 negative patients) or GRAAPH-2003 (imatinib-containing protocol for BCR-ABL1 positive patients), with a median follow-up of 667 days. PAX5 copy number was evaluated using quantitative PCR of each exon performed in hexaplicate (10 exons). PAX5 point mutations of the paired and transactivation domains (exons 2, 3, 7, 8 and 9) were evaluated by sequence analysis. PAX5 rearrangement was evaluated by caryotype and FISH analysis. Globally, PAX5 mutations were identified in 35 cases (30%). Isolated complete deletion of 1 allele was found in 13 cases (11%). Hypomorphic PAX5 alleles were detected in 22 cases (19%) consisting either of partial deletion (13 cases), partial amplification (1), point mutations (7 cases, frequently associated with complete deletion of the remaining allele in 5 cases) or fusion gene (1 PAX5-ELN). In addition, 2 cases of whole PAX5 amplification were found (2%). The remaining 82 cases had two normal PAX5 alleles (68%). BCR-ABL1 fusion gene was significantly associated with PAX5 mutations (Pearson Chi2, p=0.045) occurring in 40% of patients with PAX5 mutations and 18.3% without. PAX5 mutations were rare in the early stages of B-ALL as estimated by the immunological EGIL classification (only 2/35 PAX5 mutant cases [5.7%] vs. 16/82 [19.5%] in normal PAX5 B-ALL cases blocked at the B-I stage). Frequencies of CD10+ and CD20+ blasts were significantly higher in PAX5 mutant cases (Mann-Whitney, p=0.022 and p=0.038, respectively). As PAX5 is essential to the immunoglobulin heavy chain DHJH to VHDHJH transition, we analyzed IGH rearrangement. No difference was detected regarding the frequency of IGH rearrangement (78% in normal vs. 74% in mutants) or the rearranged VH. Survival was not significantly different between the two groups of patients (log rank, p=0.45). In conclusion, PAX5 mutations were a frequent event in adult B-ALL, associated with BCR-ABL1 fusion gene but were not associated with a significant clinical evolution.

Description: Purpose: The use of rituximab, a chimeric monoclonal antibody to CD20, has led to significant improvement in the treatment of B-cell non-Hodgkin's lymphoma and mature B-cell ALL. CD20 is expressed in 30 to 50% of adult BCP-ALL patients. Although some single arm studies suggested that adding rituximab to chemotherapy could improve the outcome of these patients, no randomized study has been reported so far. Methods: To evaluate the potential benefit of adding rituximab, we conducted a multicenter randomized trial comparing the pediatric-inspired GRAALL protocol to the same regimen plus rituximab, in patients aged 18-59 years old with newly diagnosed CD20-positive Ph-negative BCP-ALL enrolled in the GRAALL 2005 trial. CD20 positivity was defined as expression of CD20 in more than 20% of leukemia blasts. Rituximab (375 mg/m2) was given during induction (day 1 and 7), salvage reinduction when needed (day 1 and 7), consolidation blocks (6 infusions), late intensification (day 1 and 7) and first year of maintenance (6 infusions) for a total of 16 to 18 infusions. Allogeneic stem cell transplantation (SCT) was offered in first complete remission (CR) to patients with one or more conventional high-risk criteria and a donor. The primary study objective was event-free survival (EFS). A study sample size of 220 patients was estimated in order to detect a 20% gain in EFS at 2 years (two-sided test, power 85%, type 1 error 5%). A sensitivity analysis was performed after censoring patients allografted in first CR at transplant time. This trial was registered at http://www.clinicaltrials.gov as #NCT00327678. Results: From 2005 to 2014, 220 patients from 56 centers were randomized. Eleven patients had non-eligibility criteria (n=5 Ph+ ALL; n=3 CD20-negative ALL; n=1 HIV infection) or withdrew their consent (n=2) and were accordingly excluded from this modified ITT analysis that dealt with 209 patients (105 in the rituximab arm and 104 in the control arm). Median age was 40 years. Both randomization arms were well balanced for pretreatment characteristics including age, ECOG status, WBC, and central nervous system (CNS) involvement (6% of the whole cohort). After induction ± salvage reinduction, CR rate was 92% and 91% in rituximab and control arm, respectively. In patients who reached CR after first induction and were evaluated for Ig/TCR minimal residual disease level (MRD), the rates of patients with MRD

Description: Patients undergoing treatment for acute lymphoblastic leukemia (ALL) are at risk for thrombosis, in part due to the use of L-asparaginase (L-ASP). Antithrombin (AT) replacement has been suggested to prevent VTE and thus might increase exposure to ASP. We report herein the results of the prophylactic replacement strategy in the pediatric-inspired prospective GRAALL-2005 study. Between 2006 and 2014, 784 adult patients with newly diagnosed Philadelphia-negative ALL were included. The incidence rate of VTE was 16% with 69% of them occurring during induction therapy. Most patients received AT supplementation (87%). After excluding patients who did not receive L-ASP or developed thrombosis before L-ASP, AT supplementation did not have a significant impact on VTE (8% versus 14%, OR: 0.6, p=0.1). Fibrinogen concentrates administration was associated with an increased risk of VTE (17% versus 9%, OR 2.2, p=0.02) whereas transfusion of fresh-frozen plasma had no effect. Heparin prophylaxis was associated with an increased risk of VTE (13% versus 7%, OR 1.9, p=0.04). Prophylactic measures were not associated with an increased risk of grade 3-4 bleeding complications. The rate of VTE recurrence after L-ASP reintroduction was 3% (1/34). In ALL patients receiving L-ASP therapy, the use of fibrinogen concentrates may increase the risk of thrombosis and should be restricted to rare patients with hypofibrinogenemia-induced hemorrhage. Patients developed VTE despite extensive AT supplementation which advocates for additional prophylactic measures. While this large descriptive study was not powered to demonstrate the efficacy of these prophylactic measures, it provides important insight to guide future trial design. NCT00327678.

Description: Background: After alloSCT, oral valganciclovir (VALGCV) is a promising alternative to IV GCV against CMV but its pharmacokinetics (PK) have not been studied. Methods: We investigated the PK of GCV after VALGCV in a randomized, crossover phase II-study of 48 patients (pts) after alloSCT. Pts who had 〉400 copies/ml of CMV DNA in plasma measured by quantitative PCR were randomized to receive a fixed dose of VALGCV 900 mg BID (adjusted for renal function) for 7 days, followed by IV GCV as 1-h-infusion at 5mg/kg every 12 h for another 7 days, or to receive the inverse sequence of study drug administration. The PK of GCV were assessed on days 4 and 11. Safety monitoring was done until day 84 after alloSCT. Results: 28 pts were fully assessable for PK analyses. Among the 22 pts without intestinal graft-versus-host disease (GVHD), the mean±SD AUC 0–12 (mg/L*h) was 53.8±18.0 on VALGCV vs 39.5±13.9 on IV GCV (mean difference 14.3 [95% CI 7.6 to 20.9]); Cmax (mg/L) was 8.8±2.4 vs 10.3±2.1; tmax was 2.7±0.8 vs 1.1±0.6; and t1/2 was 4.2±1.1 vs 3.4±0.8. Among the 6 pts with intestinal GVHD, the mean±SD AUC 0–12 (mg/L*h) was 46.6±24.9 on VALGCV vs 35.3±12.8 on IV GCV (mean difference 11.3 [95% CI −13.4 to 35.9]); Cmax (mg/L) was 7.1±3.6 vs 11.1±3.1; tmax (h) was 2.7±0.8 vs 1.2±0.4; and t1/2 (h) was 5.6±2.0 vs 3.3±0.7. The bioavailability of GCV after VALGCV was 53%. Using a VALGCV fixed oral dose (1.800mg) for preemptive therapy in pts with low body weight led to a sharp increase of the VALGCV / IV GCV ratio (i.e. 50kg = 1.9). With a limited number of pts with low body weight included in this study no severe GCV associated toxicity was seen in these pts. Non-fatal CMV pneumonia developed in 2 pts during follow-up after antiviral therapy, and servere neutropenia 〈 500/μl occurred in 3 pts. Clearance of CMV DNA was equally effective in both arms. Conclusions: In alloSCT, exposure to GCV after preemptive therapy using VALGCV is higher compared to that with IV GCV in patients with and without intestinal GVHD. In addition to patients with renal dysfunction patients with low body weight 〈 60kg and normal renal function should be treated very carefully to avoid over-exposure to GCV. Although no severe toxicity was demonstrated in this pk-study a further study to address the safety and efficacy of VALGCV in alloSCT is needed.