Abstract: Background: Immune thrombocytopenia (ITP) is characterized by antibody-mediated platelet destruction and impaired platelet production. Residual long-lived autoreactive plasma cells may be a source of treatment resistance in autoimmune cytopenias. Antiplatelet-specific plasma cells have been detected in the spleen of the rituximab refractory ITP patients. These cells can also migrate and reside in bone marrow as long-lived plasma cells. Daratumumab, an anti-CD38 antibody, targets plasma cells and is approved for the treatment of multiple myeloma. Daratumumab has been successfully used to treat refractory autoimmune cytopenias in children and a few cases of post-transplant autoimmune cytopenias and refractory SLE in adults. We hypothesized that long-lived autoreactive plasma cells may be the source of treatment failure in some ITP patients. Based on that, we initiated a multicenter, open-label, dose-escalating phase II study with a safety run-in to evaluate the safety and efficacy of daratumumab in patients with ITP (NCT04703621). The first 3 patients were included in the safety run-in during Jan - May 2021. Study design and methods: Main inclusion criteria include age ≥18, primary ITP with a platelet count of ≤30X109/L, failure of corticosteroid therapy, and at least one second-line therapy including rituximab and/or TPO-RA. Main exclusion criteria include active bleeding, secondary ITP, concomitant autoimmune hemolytic anemia. Twenty-one patients will be included in the study. The safety run-in phase includes 3 patients who receive 4 weekly subcutaneous daratumumab injections followed by a 4-week observational period. Enrollment of the next patient in this phase occurs after the previous patient has completed treatment and an observational period. In cohort 1, 9 patients will receive 8 weekly injections. If the response rate is & lt;100% and no severe adverse events appear, the subsequent 9 patients will receive 8 weekly daratumumab injections followed by 2 injections administered every other week. Standard premedication before all daratumumab injections consists of antihistamine, corticosteroid (methylprednisolone 100 mg or equivalent before the 1 st daratumumab injection and 60 mg or equivalent before subsequent injections), and paracetamol. Rescue ITP medications are allowed during the first 8 weeks of the study. Steroid or TPO-RA (eltrombopag or romiplostim) dosing must remain stable during the 2 weeks preceding the inclusion. Dose escalation is not allowed during the study. The primary endpoint is a platelet count & gt;50x10 9/L in 2 measurements 12 weeks after treatment initiation for cohort 1, and 16 weeks for cohort 2, without rescue therapy after week 8. Safety will be assessed by the incidence and severity of adverse events. Secondary endpoints will include the number of weeks with platelet count & gt;50x10 9/L between the end of treatment and end of study without rescue therapy or dose increments of corticosteroids. Time to treatment failure (TTF) is defined as time to first platelet count & lt;30x10 9/L or administration of any platelet elevating therapy after achieving response. Exploratory endpoints include: role of anti-GPIIb/IIIa and Ib antibodies; serial characterization of various subsets of immunocompetent cells in the bone marrow and blood; measurement of HRQoL and fatigue Statistics: The primary outcome (treatment response) will be reported separately for each cohort and the entire study population, expressed by absolute numbers and rates with the corresponding 95% confidence interval. Daratumumab treatment will be considered "successful" if we observe a response rate of 30% or higher. Current enrollment status: As of July 10, 2021, 2 sites are open. Two patients have completed the safety run-in; one is close. One/two responded to treatment at week 12. Platelet response in all 3 patients is shown in Figure 1. No serious or grade 3 adverse events were reported. Cohort 1 will start in August 2021. Figure 1 Figure 1. Disclosures Tsykunova: Ablynx: Consultancy; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Sobi: Consultancy; Sanofi: Consultancy. Holme: bayer, Octapharma, Pfizer: Other: support to institution, Research Funding; Bayer, Novo Nordisk, Octapharma, Pfizer, Roche, Takeda, Sobi: Consultancy, Honoraria. Tran: Astra Zeneca: Consultancy; Novartis, Janssen, Abbvie, Takeda, CSL Bering: Consultancy. Tvedt: Ablynx,Alexion, Novartis: Membership on an entity's Board of Directors or advisory committees. Michel: Amgen,Novartis,UCB,Argenx,Rigel: Honoraria. Frederiksen: Novartis: Research Funding; Abbvie: Research Funding; Janssen Pharmaceuticals: Research Funding; Alexion: Research Funding; Gilead: Research Funding. Bussel: CSL: Other: DSMB; UCB: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Principia/Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; UptoDate: Honoraria; RallyBio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dova/Sobi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Momenta/Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kuter: Rubius: Current equity holder in publicly-traded company; Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, BioCryst, Bristol Myers Squibb (BMS), Caremark, CRICO, Daiichi Sankyo, Dova, Genzyme, Immunovant, Incyte, Kyowa-Kirin, Merck Sharp Dohme, Momenta, Novartis, Pfizer, Principia, Protalex, Protalix, Rigel: Consultancy, Other: grant support and consulting fees; Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, Bristol Myers Squibb (BMS), Immunovant, Kezar, Principia, Protalex, Rigel, Takeda (Bioverativ), UCB: Research Funding; Platelet Disorder Support Association: Membership on an entity's Board of Directors or advisory committees; Up-to-Date: Patents & Royalties: Up-To-Date. Ghanima: Amgen, Novartis, Pfizer, Bristol Myers Squibb, SOBI, Griffols, Sanofi: Honoraria; Bayer, BMS/Pfizer: Research Funding; Amgen, Novartis, Pfizer, Principia Biopharma Inc- a Sanofi Company, Sanofi, SOBI, Griffols, UCB, Argenx: Consultancy.

Abstract: Wilms tumor 1 (WT1) mutations have recently been identified in approximately 10% of adult acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) and are associated with poor outcome. Using array-based comparative genome hybridization in pediatric CN-AML samples, we detected a WT1 deletion in one sample. The other WT1 allele was mutated. This prompted us to further investigate the role of WT1 aberrations in childhood AML. Mutations were found in 35 of 298 (12%) diagnostic pediatric AML samples. In 19 of 35 (54%) samples, more than one WT1 aberration was found: 15 samples had 2 different mutations, 2 had a homozygous mutation, and 2 had a mutation plus a WT1 deletion. WT1 mutations clustered significantly in the CN-AML subgroup (22%; P 〈 .001) and were associated with FLT3/ITD (43 vs 17%; P 〈 .001). WT1 mutations conferred an independent poor prognostic significance (WT1 mutated vs wild-type patients: 5-year probability of overall survival [pOS] 35% vs 66%, P = .002; probability of event-free survival 22% vs 46%, P 〈 .001; and cumulative incidence of relapse or regression 70% vs 44%, P 〈 .001). Patients with both a WT1 mutation and a FLT3/ITD had a dismal prognosis (5-year pOS 21%). WT1 mutations occur at a significant rate in childhood AML and are a novel independent poor prognostic marker.

Abstract: TEL-AML1 fusion transcripts are found in 25% of children with B-cell precursor ALL (BCP-ALL). From June 1993 to December 1999, 1195 children with BCP-ALL were included in the FRALLE 93 protocol. Out of these, 792 were evaluated for TEL-AML1 transcript expression. There is no difference in terms of initial features, DFS, EFS, survival between evaluated (792) and non evaluated (403) patients. Out of the 792 pts, 191 (24%) expressed TEL-AML1 transcripts at diagnosis. To assess the potential prognostic value of TEL-AML1 transcripts quantification, we have retrospectively analysed follow up marrow samples using Europe Against Cancer procedures for real time quantitative RT-PCR assay, on ABI PRISM 7700 (2 reference labs) and Light Cycler apparatus (1 reference lab). Out of the 191 TEL-AML1+ve pts, 83 were evaluated for MRD at different time points after induction therapy (median = D41 (34–55) (53 evaluable pts), at D111 (62–158) (62 pts), at D216 (159–325) (33 pts) and at D838 (365–1287) (49 pts). According to normalized Ct values, samples were attributed to 4 MRD level ranging from 0 to 3 and defined as follows: 0: Ct 〉 40 ; 1 : 36 〈 Ct≤40 ; 2 : 33 〈 Ct≤36 ; 3 : Ct≤33, corresponding respectively to undetectable MRD ; MRD 〈 10-4 ; 10-4≤MRD 〈 10-3 ; MRD≥ 10-3, with respect to dilution of REH cDNA. Distribution of pts according to MRD level at different time points after induction treatment are summarized in the following table. Seventeen relapses have occurred at a median time of 41 months (17–73)(bone marrow: 7, BM + other: 5, testis: 3, CNS: 2). A level 2 positivity at the end of induction was associated with an increased risk of relapse of 3.31(95%CI:1.02 – 10.76, p =.047) while level 3 positivity was associated with a relative risk of 9.52 (95%CI: 2.91 – 31.08, p =.0002). Positivity at D111 was associated with an increased risk of relapse of 8.6 (2.0 – 38.5, p = 0.0042), whatever the level. Combination of data obtained at D41 and D111 allows to distinguish 3 subsets of pts with decreasing relapse-free survival: from 97.5% (95%CI: 85–100%) in pts with no positivity at D111 whatever the D41 result, to 75% (95%CI: 58–92%) in pts with MRD +ve at D111 with low level at D41 and 42% (95%CI: 14–69%) in pts with MRD +ve at D111 with level 2 or 3 at D41 (p 〈 .0001). No other prognostic factor was found (age, sex, WBC, D8 steroid response, D21 bone marrow response) which renders the MRD profile unique in this matter. Conclusion: RQ-PCR-based MRD detection is a powerful prognostic tool in TEL-AML1+ve leukemia. Combination of two time points allows a relevant stratification of pts according to the risk of relapse, compatible with clinical decision making towards intensification or deescalation in the setting of controlled trials FU time point Number of pts in MRD classes (number of relapses) 0 1 2 3 Not evaluated D41 27 (3) 11 (2) 10 (4) 5 (4) 30 (4) D111 40 (2) 14 (6) 7 (2) 1 (1) 21 (6) D216 29 (2) 2 (1) 1 (0) 1 (1) 50 (13) D838 47 (8) 1 (0) 1 (1) 0 34 (8)

Abstract: Abstract 1508 Background. The long term prognosis of ETV6/RUNX1 -positive acute lymphoblastic leukemia (ALL) remains to be evaluated with regard to the frequency of late relapses and the possible existence of a preleukemic stem cell. We performed a retrospective study based on a long-term follow up of the FRALLE 93 ALL relapses to address the issue of the outcome of ETV6/RUNX1 -positive ones. Methods. 1395 patients aged 0 to 20 years with untreated ALL (except L3) were included between 01-Jan-1993 and 31-Dec-1999. From 1995, children were systematically screened for four fusion transcripts (ETV6-RUNX1, BCR-ABL, E2A-PBX1, MLL-AF4). The FRALLE 93 study population was stratified into three groups (low-risk [LR], intermediate-risk [IR] , and high-risk [HR]) based on the following prognostic factors: age, white-cell count at diagnosis, haemoglobin level, immunophenotype, karyotype, and response to steroids. Patients received an initial treatment comprised of a prednisone prophase and a triple-drug intrathecal injection. Induction treatment then included prednisone, vincristine, L-asparaginase, daunorubicin (except for the LR group), and one or two more triple-drug intrathecal injections (TIT). The main treatment features of the SR and IR protocol were induction, consolidation, delayed intensification, and maintenance (total treatment duration of 26 and 38 months for girls and boys respectively). Treatment of the HR patients consisted of induction, consolidation, two delayed intensifications, and maintenance with a total treatment time of 2 years. Depending on subgroups, CNS-directed therapy included intrathecal injections +/− high-dose methotrexate +/− cranial irradiation. Following factors influencing survival after first relapse were analyzed: age, leukocytosis, gender, duration of first remission (CR1), risk groups defined in the REZ-BFM 95/96 study, sites of relapse and post CR2 consolidation treatment (AlloSCT or not). Results. ETV6/RUNX1 status was defined for 724 patients B lineage ALL. Overall, 162 of the 713 children who reached CR1 (45 % of boys) relapsed, including 43 with t(12;21). Cumulative incidence of relapses did not differ between ETV6/RUNX1 -positive and negative ALL (p=0.94), with a 5-year estimate at 19.4% and 19.9%, respectively nor according to gender and type of relapse. Nevertheless, 11 out of 26 relapses in the ETV6/RUNX1 -positive ALL males (43%) were testicular (4 testis isolated) versus 16 out of 70 (23%) in ETV6/RUNX1 -negative ALL cases (p= 0.04). Thirty three (77%) had been stratified as LR (n=6) or IR (n=27) group and 32 displayed good early response at initial diagnosis. Thirty five (81.4%) patients were classified as S1/S2 and 8 (18.6%) as S3/S4. All but three received second line salvage therapy (37/40 were included in the COOPRALL 97) and 16 underwent an AlloSCT (6 S3/S4). Based on univariate analyses, the overall survival of ETV6-RUNX1 -positive ALL after relapse was significantly affected by the duration of the first remission with a OS that was significantly improved when relapse occurred after 36 months (5-year OS: 80.6+/−7.9% versus 34.7+/− 12.3, p=0.002). Female gender was also associated with a poor survival (p= 0.015), whereas the site of relapse (p= 0.13), age at initial diagnosis (p= 0.81), leukocytosis (p=0.42), and consolidation strategy (p=0.18) had no affect on survival. In multivariate Cox-regression analysis, only the duration of first remission remained associated with the outcome (Figure 1). Conclusions. We found a high rate of testicular relapse without any increase of other extramedullary sites and an excellent outcome for ETV6/RUNX1 -positive leukemia relapses occurring over 36 months post-diagnosis. These findings highlight the interest of a primary treatment able to cross the testicular barrier. They also support the hypothesis that ETV6/RUNX1 “late relapses” are due to a novel leukemic clone, sensitive to a novel cycle of ALL chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 1605 Poster Board I-631 CCAAT/enhancer binding protein alpha (C/EBPá) function is frequently disrupted in acute myeloid leukemia (AML). This can be caused by different mechanisms, including mutations in CEBPA, the gene encoding for C/EBPá. Recently, promoter hypermethylation resulting in CEBPA silencing has been described. CEBPA mutations are associated with a favorable outcome in adult AML. Recent studies however suggested that the favorable outcome is uniquely associated with CEBPA double-mutated AML (CEBPAdoublemut) (presence of 〉 1 CEBPA mutation), and not with CEBPA single-mutated AML (CEBPAsinglemut). In pediatric AML, data on outcome of CEBPA-mutated AML is limited to one study, showing favorable outcome for CEBPAdoublemut as well as CEBPAsinglemut, which is in contrast with the adult data. So far, data on CEBPA hypermethylation in pediatric AML is lacking. We therefore studied a large pediatric AML cohort (n=252) to characterize CEBPA mutations by sequencing the entire coding region, and CEBPA promoter hypermethylation by methylation-specific PCR (MSP). Survival analyses were performed in 185 patients with de novo AML (excluding t(15;17) and secondary AML) treated on uniform DCOG and BFM protocols. Furthermore, we generated gene expression profiles using the Affymetrix HGU133 plus 2.0 microarrays to compare subgroups with CEBPA aberrations. Thirty four CEBPA mutations were identified in 20/252 diagnostic samples (7.9%). In 14 cases double mutations were present, which combined an N-terminal frame shift mutation with an in-frame mutation in the bZIP region (n=13) or with a frame shift-causing insertion before bZIP (n=1). In 6 cases a single mutation was present; i.e. in-frame bZIP mutation (n=4), or frame shift mutation respectively in the TAD2 domain (n=1) or before the bZIP domain (n=1). CEBPAdoublemut were only present in children above 3 years of age and in FAB M1/2 subtypes, in contrast to CEBPAsinglemut, which presented also in children 〈 3 years of age (1/6) and in other FAB subtypes (3/6). CEBPAdoublemut and CEBPAsinglemut were both exclusively found in cases with a normal karyotype (57% and 33%, respectively) and in cases with ‘other’ karyotypes (defined as 'other than t(8;21), inv(16), t(15;17) and MLL-rearrangements'), in 36% and 50%, respectively. However, in both subgroups additional molecular aberrations, i.e. in RAS, FLT3/ITD and WT1, were equally distributed. CEBPAdoublemut patients (n=10) had a significantly better overall survival compared with CEBPAsinglemut (n=5) (5-years pOS 79±13% vs. 25±22%, p=0.04; pEFs 58±16% vs. 30±24%, p=0.16). Furthermore, they showed a trend for favorable outcome compared with CEBPA wild-type AML patients, after excluding CBF-AML cases (n=120; pOS 79±13% vs. 47±5%, p=0.07; pEFS 58±16% vs. 34±4%; p=0.06). Their survival was comparable to the CBF-AML subgroup (n=50) (pOS 91±4%, pEFS 61±8%). Multivariate analysis, including age, WBC, CBF-AML, NPM1 mutations and FLT3/ITD, showed that CEBPAdoublemut were an independent favorable prognostic factor for pOS (HR 0.23; p=0.04) and pEFS (HR 0.32; p=0.03). CEBPA promoter hypermethylation was detected in 3/237 cases, which resulted in CEBPA silencing. Using an unsupervised clustering analysis as previously published by Valk et al. (NEJM 2004) of our de novo AML cases (n=237), CEBPA-mutated cases predominantly aggregated in 1 cluster with the CEBPAsil cases, revealing a common underlying gene expression profile. Two additional cases with silenced CEBPA (CEBPAsil) were identified in this cluster, resulting in 5/237 (2.1%) CEBPAsil cases. All CEBPAsil showed T-lymphoid characteristics, (e.g. high expression of CD7 and high LCK expression). However, NOTCH1 mutations were not found. Three of 5 patients relapsed within 1 year of diagnosis, but the other 2 are in continuous complete remission for 4.8 and 8.5 yrs. In conclusion, CEBPAdoublemut were identified as an independent predictor of good clinical outcome. Hence, if these results could be confirmed in a prospective serie, CEBPAdoublemut may be used for further refinement of risk-group stratification. Of interest, the other cases with CEBPA aberrations, i.e. CEBPAsinglemut and CEBPAsil, seem to predict for poor outcome, in line with data presented in adults. The subgroup with CEBPAsil due to hypermethylation may potentially benefit from the use of demethylating agents. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 1628 Poster Board I-654 Since the cloning of the t(12 ;21) in 1995 the prognosis of children with ETV6-RUNX1(+)ALL seems to further increase in the current era. From december 2000 to july 2008, 1461 children and adolescents aged from 1 to 20 years with B cell lineage ALL have been treated according the FRALLE protocols: one for standard-risk (SR) ALL (age 1-9 y, WBC 〈 50 G/L, no extra medullary involvement) F2000-A; the other one FRALLE 2000-B for high and very high risk ALL (HR) (all other pts). The two protocols mainly differ by a larger use of dexamethasone and a reduced use of anthracyclins in F2000A and the use of HDMTX and a double delayed intensification in FRALLE 2000B. ETV6-RUNX1 presence has been assessed by RT-PCR in 1392 pts (i.e. 96%) and found positive in 321 pts, i.e. 23%. Initial features include a median age of 4.2 y (1.2-15.6), a sex ratio (M/F) of 1.2, a median WBC of 10 G/L(1-293). Both peripheral blood D8 response to prednisone (PRED) and D21 marrow response to chemotherapy were evaluable in 316 pts: 94% of the pts have a rapid early response at D8 and D21. Only 3% of the pts were qualified as D8 poor PRED responders and 3% slow marrow responders at D21. The D35-42 CR rate is 100%. End of induction (EOI) minimal residual disease (MRD) using Ig-TCR methods is known in 259 out of 321 pts (81%) pts. Only 4 pts (2%) had a very high (≥10-2) EOI-MRD. Five year EFS, DFS and OS are 95±2%, 95±2%, 98±1%, respectively. The 5y EFS is 96±2% for the 252 SR pts and 90±7% for the 69 HR pts, p=0.30. The 5y EFS is significantly better for children with ETV6-RUNX1(+)ALL compared to those with ETV6-RUNX1(-) B-lineage ALL: 95±2% vs 84±2%, respectively (p=.001). Nine relapses have been reported in the bone marrow (4) the testis (4), the CNS (1) after a median time of 44 m (25-68), i.e. significantly longer than in the ETV6-RUNX1(-) cases (28m (1-92), p=.01). More testis relapses are observed in boys with ETV6-RUNX1(+)ALL (4 out 7 relapses vs 2 out of 62 in boys with ETV6-RUNX1(-) ALL). These results represent a significant progress compared to the previous protocol F 93 (191 children) both in terms of EFS and overall survival (5y EFS: 95±2% vs 78±3%, p=.001; 5 y OS: 98±1% vs 92±2%, p=.001). Conclusion an excellent prognosis of children with t(12;21)/ETV6-RUNX1 positive acute lymphoblastic leukemia is now observed in the FRALLE 2000 protocol. The question of a cautious de-escalation in this subgroup will be envisaged in the next protocol. Disclosures No relevant conflicts of interest to declare.

Abstract: In an array-CGH screening study of cytogenetically normal AML (CN-AML), we detected a cryptic 11p13-deletion including the WT1 gene in one childhood AML sample. The remaining WT1 allele in this sample carried a nonsense mutation. WT1 gene mutations have recently been identified in approximately 10% of adult CN-AML. Of interest, WT1 mutations were found to be a new independent poor prognostic factor in adult CN-AML (Virappane et al. JCO2008, Paschka et al. JCO2008). WT1 mutations have also been reported in childhood AML; however, their clinical relevance in childhood AML is not known. In this study, we investigated the frequency, clinical characteristics and prognostic value of WT1 mutations (exons 7–10) in a large, well-characterized cohort of childhood AML samples (n=298). Additionally, a subset of these samples was screened for mutations in exons 1–6 (n=68), and for micro-deletions in the WT1 gene (n=24). Survival analysis was restricted to the subset of patients with de novo AML who were treated using uniform DCOG and BFM treatment protocols (n=232). Fifty-three pathogenic WT1 mutations were detected in 35/298 (12%) samples taken at diagnosis. Mutations were mainly located in exon 7 (n=43), but also in exons 1 (n=2), 2 (n=1), 3 (n=2), 8 (n=1) and 9 (n=4). Predominantly frame-shift mutations were found (n=41), next to nonsense mutations (n=6) and missense mutations (n=6); the former two resulting in a truncated WT1 protein. In 19/35 (54%) of the WT1-mutated samples, we detected more than one WT1 aberration. This included either a different WT1 mutation (n=15), a homozygous WT1 mutation (n=2), or a deletion of the other WT1 allele (n=2). WT1 mutations clustered significantly in the CN-AML subgroup (21/94=22%; p & lt;0.001). NPM1 and WT1 mutations were mutually exclusive, but WT1-mutated samples were more likely to carry FLT3/ITD (43% vs. 17%; p & lt;0.001) and CEBPα mutations (26% vs. 9%; p=0.007). Mutations in patients below the age of 3 years were only found sporadically (1/60=2%). The highest frequency was found in the age category 3–10 years (17/76=18%), and decreased above the age of 10 years (17/128=12%; p=0.008). WT1-mutated AML was correlated with a higher white blood cell count at diagnosis (WBC) (57.2×109/l vs. 34.1×109/l; p=0.007); no correlation was found with sex or FAB-classification. WT1-mutated AML patients had a significantly worse outcome when compared with patients with WT1 wild-type AML (5-year overall survival (pOS) 35% vs. 66%; p=0.002; 5-year event-free survival (pEFS) 22% vs. 46%; p & lt;0.001; and 5-year cumulative incidence of relapses (CIR) 70% vs. 44%, respectively; pGray & lt;0.001). Moreover, using multivariate analysis including age, WBC, cytogenetics, FLT3/ITD and stem cell transplantation, WT1 mutations were identified as an independent poor prognostic factor for pOS (HR1.79; p=0.04), pEFS (HR2.05; p=0.005) and relapse-free survival (pRFS) (HR2.44; p=0.001). We identified patients carrying both a WT1 mutation as well as a FLT3/ITD as a very poor prognostic subgroup (5-year pOS 21%). The mutational hotspots in the WT1 gene were located within areas of primer-probe combinations used for WT1-based minimal residual disease (MRD) detection. Furthermore, in 4/28 (14%) wild-type diagnostic-relapse pairs a mutation was gained at relapse, which may also effect MRD detection. In conclusion, WT1 mutations are present in 12% of childhood AML at diagnosis and in 22% of patients with CN-AML, and are a novel independent poor prognostic marker in childhood AML. Furthermore, their presence may have implications for current WT1-based MRD detection.

Abstract: From January 2000 to July 2006, 580 BCR-ABL negative patients with HR/VHR-ALL (200 T-ALL and 380 BCP-ALL (age≥10 or WBC≥50 or CNS+ or MLL-R) were included in the FRALLE 2000-BT trials. Induction regimen is prednisone (PRED) prephase + IT MTX, VCR, L-Aspa, DNR 120mg/m2 cumulated dose or DNR 160mg/m2 + cyclophosphamide 1g/m2 (T-ALL and D21 M2M3 marrow or MLL-R BCP-ALL). MRD at EOI is quantitatively determined by DNA-based PCR for Ig/TCR rearrangements either competitive PCR with GeneScan analysis or RQ-PCR with clone specific primers (sensitivity ranges 0.5×10−3–10−3, and 10−3–10−5, respectively). MRD status at EOI are available for 425 out of 552 CR (77%). MRD results are reported as negative, positive & lt;10−3, highly positive (10−3≤MRD & lt;10−2), very highly positive (≥10−2). Patients with EOI MRD ≥10−2 were to receive an intensified treatment. Results: 30% (89/293) of HR/VHR BCP-ALL and 34% (45/132) T-ALL have detectable MRD at EOI, NS. A high or very high MRD is encountered in 16% (47/293) of BCP-ALL and 21% (27/132) of T-ALL (NS). Surprisingly 57% (20/35) of the pts with BCP-ALL and D8 poor PRED response (PPR) and 71% (36/51) of the pts with D8 PPR T-ALL have a negative or slightly positive MRD ( & lt;10−3) at EOI, NS. 30% (7/23) of the pts with D21 M2M3 BCP-ALL and 46% (6/13) of the pts with D21 M2M3 T-ALL have also a negative or slightly positive MRD ( & lt;10−3) at EOI. By contrast, 11% of good early responders (D8 good PRED response and D21 M1) with BCP-ALL or T-ALL (27/245 and 11/74, respectively) have a high or very high positive MRD. Overall, 20 out of 449 (4.5%) HR/VHR pts in CR have received an intensified treatment due to a very high MRD only. 3y DFS for pts with a highly positive or very highly positive MRD are 65±7% (BCP-ALL) and 64±9% (T-ALL), NS. 3y DFS for pts with a negative or slightly positive MRD ( & lt;10−3) are 94±2% (BCP-ALL) and 87±4% (T-ALL), p=.04. There is a statistically significant difference in term of DFS between pts with MRD≥10−3 and MRD & lt; 10−3, p=.001 and p =.05 for BCP- and T-ALL, respectively. Conclusions: the incidence of high and very high MRD at EOI is identical in children with HR/VHR-BCP- and T-ALL. High and very high MRD levels are found in good early responders defined by morphology. Conversely, excellent molecular responses can be found in bad early responders. High or very high MRD values are associated to a comparable prognosis between BCP- and T-ALLs.

Abstract: In pediatric T-ALL, oncogenetic markers, MRD, and WBC count are independent predictors of outcome. These factors should be used together for individual treatment stratification.

Abstract: From Dec 2000 to Dec 2003, 390 children with SR-BCP-ALL (age: 1–9, WBC & lt;50 G/L, CNS-, no MLL-R, no BCR-ABL) were included in the FRALLE 2000 -A protocol. Induction regimen is: prednisone prephase for 7 days (60 mg/m2/d) +IT MTX, dexamethasone 6 mg/m2 (D8–D28), vincristine 1.5mg/m2(D8, D15, D22, D29), L-asparaginase 6000 U/m2 (9 infusions). Good marrow responders at D21(M1 pts) are randomized to receive or not daunorubicin (DNR) 40 mg /m2 at D22 and D29. D21 M2/M3 pts are not randomized and given DNR. MRD at EOI is determined by DNA-based PCR for Ig/TCR rearrangements. Two methods are used for quantification (competitive PCR with GeneScan analysis -sensitivity: 0.5x 10-3-, RQ-PCR with clone-specific probes- sensitivity range: 10-3-10-5). EOI MRD data are evaluable for 343 pts with D21 M1 response (DNR+= 169/ DNR−= 173/ not randomized = 1) and for 20 pts M2M3 ; two pts died during induction and thus are NE. MFU of these 365pts is 22m (3–39). Median age is 4.1y(1.1–9.9), median WBC is 7.47 G/L (.9–47). MRD results are classified for analysis either in three categories (negative, weakly +ve if & lt; 10−3, highly positive if & gt; 10−3, or according to the exact level of positivity. Results: 1) 15% (51/336 pts) of the SR-BCP ALL have a detectable MRD at EOI 2) As expected, whatever the threshold, pts with D21 M2/M3 marrow are more likely to have a detectable MRD (p=.0017). But 43/316 M1 pts (14%) have a highly +ve (n=19;6%) or weakly +ve MRD (n=24 ; 8%). Surprisingly, only 1 out 20 M2M3 pts had a MRD & gt; 10−2 while it is the case for 8 out 316 M1 pts (p=NS). If only pts receiving DNR are considered (DNR+ M1 pts and all M2/M3 pts), again pts with D21 M2/M3 marrow are more likely to have a detectable MRD (p=.0015). 3) If we compare the MRD levels in the 2 arms (DNR+ve or neg) in the 315/343 M1 pts evaluable for MRD: 136 pts in each arm had no detectable MRD; 16 and 8 have a weak positivity in the DNR− and DNR+ arm respectively while 10 and 9 have a weak positivity in the DNR− and DNR+ arm respectively: p= .29). If the exact level is considered, this absence of difference remains at all levels considered. Conclusions: 15% of the SR-BCP ALL have a detectable MRD at EOI after a three or four-drug induction. D21 M2/M3 pts are more likely than M1 pts to have a detectable MRD at EOI but 14% of the D21 M1 pts have a high ( & gt; 10−3) or very high MRD ( & gt; 10−2) which confirms the added value of MRD detection to classical morphology. The MRD level at EOI is not different in SR-BCP-ALL after a three or four-drug induction regimen. This is of paramount importance since EOI MRD is a surrogate marker for probability of relapse.