ALBERT

Description: Background and aim: The AIEOP-BFM ALL 2009 trial (https://www.clinicaltrials.gov/NCT01117441) is an international collaborative treatment protocol for children and adolescents with acute lymphoblastic leukemia (ALL) wherein PEG-ASP (Oncaspar®, Servier) was used as the front-line ASP product. Since HSR represent the most relevant limitation to the completion of ASP treatment schedules in the context of any study protocol, we evaluated the cumulative incidence of HSR following PEG-ASP treatment for better understanding the impact of this product on the HSR phenomenon in the context of the treatment strategy outlined in the AIEOP-BFM ALL 2009 study protocol. Patients and Methods: Children with newly diagnosed ALL were enrolled in the AIEOP-BFM ALL 2009 protocol and stratified as Standard (SR), Intermediate (IR) or High-Risk (HR) according to their presenting and response criteria. A first exposure to PEG-ASP, administered I.V. at 2,500 IU/sqm throughout the whole protocol, was scheduled in all patients during induction (phase IA, 2 doses, on day 12 and 26), whilst second or further exposures were scheduled as follows: a. in SR and MR patients during the reintensification (protocol II, 1 dose); and b. in HR patients during each of the three intensive blocks and of the three protocols III (1 dose in each of these phases). In addition, MR patients were randomized (RMR) at the start of reintensification (protocol II) to receive 0 (Standard Arm, SA) vs 9 (Experimental Arm, EA) additional biweekly PEG-ASP doses throughout protocol II and continuation therapy. Therefore, all the SR patients and the MR patients randomized to SA overall received 3 PEG-ASP doses with an 18-week-long interval between doses #1+#2 (induction, first exposure) and dose #3 (reintensification, second exposure) whilst the MR patients randomized to the EA overall received 12 PEG-ASP doses with an 18-week-long interval between doses #1+#2 (induction, first exposure) and doses #3 to #12 (reintensification and continuation therapy, second exposure). HR patients were randomized (RHR) at the start of the consolidation phase (protocol IB) to receive 0 (SA) vs 4 (EA) additional weekly PEG-ASP doses. Therefore, HR patients randomized to the EA received overall 12 PEG-ASP doses (first exposure: #1+#2 in IA; further exposures: #3 to #6 in IB, #7 to #9 in each of the 3 blocks and #10 to #12 in each of the protocols III) without relevant time-intervals among the various doses, whilst HR patients randomized to the SA received overall 8 doses with an 8-week-long interval between the doses #1+#2 (IA, first exposure) and the second and further exposures (3 blocks and 3 protocols III, doses #3 to #8). HSR were recorded by treating physicians and collected in the trial database and cumulative incidence (CI-HSR) was estimated adjusting for competing risks (death for any reasons and leuekemia recurrence). Results: Between June 01, 2010 and February 28, 2017, 6136 patients were eligible and evaluable in the study and are here reported. An HSR occurred in 468 patients, with a CI-HSR of 7.6% (SE 0.3). In particular, 121 (2.0%) occurred in IA, 20 (0.3%) in IB, 140 (2.3%) in Protocol II, 183 (3.0%) in blocks, 4 (0.07%) in Protocols III and maintenance. According to the stratification group, CI-HSR was 4.9%(0.5) in SR, 4.9% (0.4) in MR and 16.8% (1.0) in HR groups. In the RMR study, the CI-HSR was 3.1% (0.6) vs 4.3% (0.8) in the SA vs the EA, respectively (p-value=0.26), whilst in the RHR study the CI-HSR was 18.3% (1.9) vs 6.1% (1.3) in the SA vs the EA, respectively (p-value

Description: Abstract 1578 Poster Board I-604 Background T-ALL accounts for about 15% of pediatric ALL and still represents a clinical challenge, because more than 20% of children experience a recurrent disease which has a dismal prognosis. Characterization of molecular alterations with prognostic impact may be useful for an early identification of patients at high risk of failure in whom more intensive treatments, including hematopoietic stem cell transplantation (HSCT) may be considered. CALM-AF10 results from a recurring t(10;11)(p13;q14-21) chromosomal translocation and is the most frequent fusion transcript in both adult and pediatric patients with T-ALL. Its presence has been associated with a poor prognosis (Asnafi V et al. Blood 2003; van Grotel M et al Haematologica 2006). The aim of the present study was i) to define the incidence of CALM-AF10 among homogeneously treated children with T-ALL and ii) to evaluate the outcome of these patients. Materials and Methods We studied 201 patients with T-ALL, diagnosed and enrolled between 9/2000 and 12/2007 in the ALL-2000 protocol and the subsequent modified 2000 study (ALL-R-2006) of the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP) which consist of an intensive chemotherapy strategy based on BFM-ALL schedules. Patients were mainly stratified according to prednisone response evaluation (good response: blast count at day 8 less than 1000/mmc) and detection of minimal residual disease (MRD) performed at day 33 (Timepoint 1) and at day 78 (Timepoint 2). When both TPs were negative children were considered to be at Standard Risk (SR); patients with TP1 and/or TP2 positive and TP2 '10-3 were considered to be at Intermediate Risk (MR); children with TP2 positive ≥10-3 belong to the high risk (HR) group. Patients with prednisone poor response and MRD-HR were considered eligible for HSCT from a sibling donor in first complete remission. Event free survival (EFS) and overall survival (OS) estimates with 95% CIs were calculated through the Kaplan-Meier method and differences compared with the log-rank test. RT-PCR reactions for detection of CALM-AF10 were performed as previously reported (Asnafi V et al Blood 2003) Results Ten patients resulted not eligible and were excluded from analysis. Among the 191 evaluable children with T-ALL, 14 (7,3%) were positive for CALM-AF10. Twelve (85%) of these patients were males. Median age was 8 years (range 2 – 13). Immunophenotyping showed thymic/intermediate features in 6 cases, mature in 5, early in 1, biclonal in 1 and unknown in 1, respectively. Eight cases showed a poor prednisone (PDN) response. Based on a randomized study performed in induction in the frame of the ALL-2000 protocol on the efficacy of PDN vs dexametasone (DXM) 8 children were treated with PDN and 3 with DXM The remaining 3 cases, belonging to the ALL R-2006 protocol, were treated with DXM (n=2) and PDN (n=1). MRD-based stratification allowed the allocation of 3, 8 and 3 patients in the SR, MR and HR, respectively. Four cases relapsed (3 in the central nervous system and 1 in the bone marrow). EFS at 5 years of the 14 CALM-AF10 positive T-ALL children versus the 177 who were negative was 70.1% vs 63.9%, respectively (p-value log-rank=0.61). Small numbers did not allow to fully evaluate the impact of different variables such as initial steroid treatment (PDN vs DXM) or the MRD-based risk-group assignment Conclusions This study performed in a vast cohort of children with T-ALL shows that CALM-AF10 is found in about 7% of children with T-ALL and that does not predict a poor outcome when an intensive chemotherapy strategy is employed. Disclosures No relevant conflicts of interest to declare.

Description: The definition “very-high risk” (VHR) in the setting of childhood acute lymphoblastic leukaemia (ALL) is usually adopted to identify children eligible for transplantation in first complete remission (CR1). The endpoint of this study is to assess whether, how many, and how VHR ALL children achieving CR1 and not transplanted in CR1 can be rescued in case of relapse. Eligiblity criteria to transplantation in AIEOP-BFM ALL 2000 study slightly changed overtime and differed between the BFM (Berlin Frankfurt Muenster) and AIEOP (Associazione Italiana di Ematologia ed Oncologia Pediatrica) Groups.. For this analysis VHR ALL was defined by one or more of the following criteria: induction failure (IF), high levels (≥ 1x10−3) of minimal residual disease at day 78 (VHR-MRD), clonal abnormality t(4;11), prednisone poor response associated (PPR+) with one or more of the followings: hyperleukocytosis, T-immunophenotype, ≥ 25% marrow blasts at day 15, pro-B immunophenotype. Patients carrying clonal abnormality t(9;22) were not included here since allocated in a specific study protocol since 2004. Out of 571 VHR patients, distributed among the four criteria (17%, 45%, 3%, 35%, as hierarchically listed), 249 (43%) were transplanted in CR1 (80%, 48%, 47%, 25% in each category) and 322 (57%) were not. Of the 322 patients treated with chemotherapy [5-year-EFS 53.5% (SE 3.1), 5-year-survival 66.6% (SE 2.9)], 19 died in CR1, 6 presented with a second malignancy, and 113 (35%) relapsed at a median of 15 months after diagnosis, mostly in the bone marrow (81%: 66% isolated, 15% combined) or in an extramedullary site (19%, 15% in the central nervous system) at a median time of 9 ms after diagnosis. The 3-year-survival after relapse was only 26.0% (SE 5.1) for the 113 patients overall, 38.7% (SE 7.8) for the 55 who underwent transplantation in CR2 and 16.2% (SE 5.3) for the 58 who were not transplanted in CR2. Interestingly the 3-year-survival after relapse by VHR criteria was only 7.1% (SE 9.3) in the IF, 28.6% (SE 7.1) in the VHR-MRD and 33.1% (SE 6.7) in the PPR+ subgroups. In conclusion very few VHR-ALL children could be rescued after relapse, particularly among those carrying most unfavourable criteria at the onset (IF and VHR MRD), confirming that transplantation should be performed in CR1. Transplantation in CR2 was feasible in only half of the patients and presumably their outcome was influenced by MRD response after relapse; this aspect is under investigation. New strategies are needed for this dismal subset of patients.

Description: Early measurement of blast clearance is a relevant prognostic indicator in childhood acute lymphoblastic leukemia (ALL). To this purpose we measured, by four-colour flowcytometry (FC), the percentage of blast cells in bone marrow samples from Italian patients enrolled in the multicentre AIEOP-BFM ALL 2000 trial. Samples were collected on day 15 (after 14 days of steroids, and one dose of IT-MTX, vincristine, daunorubicine, asparaginase) and shipped overnight to the reference laboratory. The data were compared to PCR-MRD performed, by study design, on day +33 and +78 BM samples. We report the results of patients enrolled between December 2000 and October 2004. The 561 patients studied were not different from the remaining ones (with no available material) including their cumulative incidence of relapse (SE): 17.3% (1.9) vs. 18.1% (1.5) in 850 patients not studied. According to the results of FC-MRD, 5 groups were defined: negative (blast count

Description: Between May 1995 and August 2000 the Associazione Italiana di Ematologia Oncologia Pediatrica (AIEOP) conducted the ALL-95 study for risk-directed, BFM-oriented therapy of childhood acute lymphoblastic leukemia (ALL), aimed at exploring treatment reduction in standard risk (SR) and intensification during continuation therapy in intermediate risk (IR) as randomized questions and treatment intensification in high risk (HR). The prognostic value of DNA index was explored in this setting. 1,744 patients were enrolled, 115 SR, 1,385 IR, and 244 HR risk. SR patients (DNA index ≥1.16 and

Description: Introduction: In the last two decades, treatment to prevent CNS relapses in childhood ALL has been characterized by a progressive replacement of cranial radiotherapy (CRT) with HD-MTX and/or protracted IT chemotherapy. AIEOP has already reported that HD-MTX (5 g/sqm x 4), associated with protracted use of IT MTX led to a 6-year isolated CNS relapse rate of 0.8% in a relatively small fraction of intermediate risk (IR) ALL children (30% of the total) treated in the BFM-oriented AIEOP ALL 88 study (J Clin Oncol, 13; 10: 2497–2502, 1995). Aim: To evaluate if adequate CNS relapse prevention is obtained also by using HD-MTX at lower doses (2 gr/sqm x 4 instead of 5 gr/sqm x 4) and in a larger proportion of patients (up to 80% of the overall population) when associated to protracted IT chemotherapy. Patients and methods: Eligibility: children with newly diagnosed non-T ALL with no HR features [i.e. no poor-prednisone response and/or no t(9;22) or t(4;11) clonal translocations and/or no CR after protocol IA] and no CNS or testicular involvement at the onset. Chemotherapy was based on a traditional BFM back-bone (protocols IA+B, M, II, continuation phase). CNS relapse preventive therapy consisted of one IT MTX at diagnosis; TIT x 4 in protocol IA+IB; HD-MTX (2 g/sqm x 4) + TIT x 4 in protocol M; TIT x 2 in protocol II; during the continuation phase, TIT x 6 in the IR group and TIT x 8 in the standard risk (SR) group. Results: From 4/95 to 8/2000 1745 patients were recruited in the study AIEOP ALL 95; of these, 1441 (82.6 % of the overall ALL population) were SR (n=115) or IR (n=1326) and fulfilled the eligibility criteria for the present study. Among these 1441 patients, the following events have been observed: 264 relapses, 8 deaths in induction, 13 deaths in complete remission and 3 secondary neoplasms. Among relapses, 198 were isolated in the bone marrow, 15 isolated in the CNS, 15 isolated in the testes, 12 in the CNS+bone marrow, 10 in the testis+bone marrow, 9 in other sites +bone marrow, 5 in other isolated extramedullary sites. With a median follow-up of 5.5 years, the 6-year event-free survival (EFS) of the 1441 patients was 78.6% (SE 1.2) with an isolated CNS relapse rate of 1% (SE 0.3); when also the combined relapses involving the CNS were counted, the CNS relapse rate was 2%. Conclusions: These data confirm and extend our previous findings, suggesting that, in the context of an intensive chemotherapy program, prevention of CNS relapse may be effectively obtained in non-HR ALL children using HD-MTX at 2 gr/sqm x 4, associated with protracted IT chemotherapy, thus permitting to avoid the use of CRT.

Description: Abstract 319 Aim: The Italian Association of Pediatric Haematology and Oncology (AIEOP) patients, diagnosed in the period September 2000-July 2006, were treated in the context of the AIEOP-BFM ALL 2000 Study. Some differences in high risk (HR) treatment and hematopoietic stem cell transplantation (HSCT) indications justify separate reporting of results obtained by AIEOP and BFM respectively. We report here the AIEOP experience. Patients and methods: Overall, 1999 AIEOP Ph negative Acute Lymphoblastic Leukemia (ALL) patients were eligible to the AIEOP-BFM ALL 2000 Study. High Risk (HR) criteria were: t(4;11) translocation, Prednisone Poor Response (PPR), no complete remission (CR) at day 33, high minimal residual disease (MRD) levels (≥10-3) at day 78 (HR-MRD). Treatment consisted of protocol I (patients were randomised to receive either dexametasone or prednisone in induction), 3 HR polychemotherapy blocks, a randomized comparison between delayed intensification based on protocol II repeated twice or protocol III repeated thrice, cranial radiotherapy (CRT), maintenance therapy for a total of 2 years of treatment. Results: 311 patients were classified as being at HR (15.6% of the total ALL population) and had an overall event-free survival (EFS) and Survival of 58.7%(standard error 2.9) and 70.1%(2.7), respectively. For the 204 patients randomized to different steroids in protocol I, we observed a 5-year EFS of 62.7%(5.0) and 62.3%(4.8) and a 5-year Survival of 72.7%(4.7) and 72.8%(4.3) for dexamethasone and prednisone arm, respectively. The 5-year EFS was 44.4%(4.5) in 132 patients at HR for MRD, 36.4%(14.5) for the 11 patients at HR for t(4;11), 41.2%(11.9) for the 17 patients at HR for no CR at day 33, 74.6%(3.7) for the 151 patients at HR only for PPR. Patients at HR with

Description: Abstract 3518 Introduction. In the AIEOP-BFM ALL 2000 study, the MRD-based risk groups stratification allowed to achieve more than 80% cure rate. However, relapse is still the most frequent adverse event, occurring mainly in the largest subgroup of non-high risk (HR) patients. This emphasizes the need for new prognostic markers for upfront identification of patients with a high risk of relapse. Recently, new genomic abnormalities of the CRLF2 and Ikaros (IKZF1) genes have been reported in the subset of BCP-ALL patients without known chromosomal aberrations. We recently analyzed the poor prognostic impact of P2RY8-CRLF2 fusion in a representative cohort of 464 non-DS Ph- BCP-ALL patients enrolled in Italy into AIEOP-BFM ALL2000 study from February 2003 to July 2005. Aim. In order to estimate the incidence and prognostic value of IKZF1 deletions, alone or in combination with CRLF2 alterations, we initiated the screening of the full cohort of patients. A pilot test of a subset of 154 non-DS, Ph-, BCP-ALL patients treated in Italy according to the AIEOP-BFM ALL 2000 protocol from February 2003 to June 2004 has been completed so far. Methods. Ig/TcR PCR-MRD and RT-PCR screening for known chromosomal translocation were routinely performed; P2RY8-CRLF2 fusion was detected by RT-PCR. Multiplex Ligation-dependent Probe Amplification (MLPA) (Salsa MLPA P335-A3 ALL-IKZF1 kit; MRC-Holland, Amsterdam, NL) was performed to identify IKZF1 deletions together with deletions in additional B-cell development genes. Patients positive for IKZF1 deletions were further analyzed by Salsa MLPA P202-A1 IKZF1 kit to confirm and better define the extension of the IKZF1 gene deletion. Results. IKZF1 deletions were detected in 22/154 cases (14.3%), in keeping with incidence data reported in the literature. In 9 cases (5.8%) the deletion was intra-genic, involving only some exons of the IKZF1 gene, while in 13 cases (8.4%) the deletion was encompassing the whole IKZF1 gene. Three out of 16 patients (18.8%) carried both IKZF1 and CRLF2 deletions. Overall, the 5-year Cumulative Incidence of Relapse (median follow-up: 5.8 years) was 23±9% for patients carrying IKZF1 deletions and 12.2±2.9% for patients who did not (p=0.22). Interestingly, out of 13 cases with complete deletion of IKZF1, only one case (1/13, 7.7%) relapsed, vs 4/9 cases (44.4%) positive for IKZF1 intra-gene deletion. Conclusions. IKZF1 intra-gene deletion, but not whole gene deletion, showed a tendency to be associated with poor prognosis in childhood BCP-ALL treated according to the AIEOP-BFM ALL 2000 protocol. Whether these results will be confirmed at the completeness of this study, the assessment of IKZF1 status might serve as a new stratification marker. It will be of particular interest to verify whether different IKZF1 deletions within non-HR patients by MRD will have a prognostic impact. Disclosures: No relevant conflicts of interest to declare.

Description: Background. The ‘early T-cell precursor’ (ETP) subtype of T-ALL comprises up to 15% of T-ALL and has been reported to be associated with high risk of relapse. In addition to properties of T cell development, gene expression profile and immunophenotype of ETP-ALL show stem cell and early myeloid features. Consistently, this leukemia subgroup shows lower frequencies of prototypical T-ALL lesions and a higher prevalence of mutations typically associated with AML, including RAS and FLT3 mutations. In particular, FLT3-ITD was identified in up to 35% of adult ETP-ALL but data on its prevalence in pediatric ETP-ALL are lacking. In agreement with its stem-cell signature, ETPs frequently lack Immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, the most used and sensitive targets for MRD monitoring. As a consequence, alternative markers are required to extend the application of molecular MRD to most ETP-ALL patients. Aim. We explored the prevalence of FLT3-ITD mutation in a large series of pediatric ETP-ALL enrolled in two consecutive protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP), and we evaluated the potential use of FLT3-ITD as an alternative DNA marker for MRD monitoring. Methods. Out of 439 T-ALL patients enrolled in Italy into the AIEOP-BFM ALL2000 and AIEOP ALLR2006 consecutive protocols, 145/168 Early-T ALL (TI/II) patients were screened for FLT3-ITD occurrence. Among Early-T patients, 34 were defined as ETP according to immunophenotype, and 31 of them were screened for FLT3-ITD. Twenty-two ETP patients enrolled in Italy into the ongoing AIEOP-BFM ALL2009 were also screened, together with T-ALL cases without IG/TR molecular markers only for the technical validation of the method. PCR screening and RQ-PCR for FLT3-ITD were performed as previously reported (Nakao, Leukemia 1996;10:1911; Beretta, Leukemia 2004;18:1441). Parallel MRD analysis for IG/TR on the same samples, and flow cytometry-MRD were performed by standard procedures. EuroMRD guidelines were applied for performance and interpretation of RQ-PCR. Results. Among ALL2000/R2006 and ALL2009 ETP cases, 4/31 (12.9%) and 3/22 (13.6%) were FLT3-ITD positive, respectively; 5/7 were PPR, and all 7 were stratified as high risk. For ALL2000/R2006 patients, IG/TR MRD monitoring was feasible in 2 cases, and both were MRD-HR; 3/4 cases are alive in CCR, and one died after HSCT. Overall, the FLT3-ITD marker was detected in 12 T-ALL cases; only 4 of them had valuable IG/TR markers, while 8/12 (66%) did not present a suitable IG/TR MRD marker. FLT3-ITD MRD monitoring was performed on 11/12 FLT3-ITD positive T-ALL cases. Mean length of the ITD was 44 nucleotides (nts) (range 24-71), with a mean of 7 randomly inserted nts (range 1-26). Standard curves performed by 10-fold dilutions in DNA from PB Healthy Donor, showed a quantitative range of at least 5.0E-04 in all cases and 1.0E-04 in 5/11. Sensitivity of the assay was at least 1.0E-04 in all tested cases, and 1.0E-05 in 7/11. A comparison between IG/TR and FLT3-ITD was feasible in 3 out of 4 cases (1 is ongoing); all 3 cases were monitored by 2 IG/TR markers. At day15 and day33 of the Induction therapy, when MRD was very high (10-1 to 10-3 range), the IG/TR and FLT3-ITD were fully comparable, with less than 2 times difference. At day78 (after IB Induction block) 1 case was fully negative for both markers, 1 was slightly positive by FLT3-ITD (although not quantifiable and at the limit of the sensitivity) and negative for both IG/TR. The latter case was highly positive for both IG/TR (5.0E-03) but low positive (

Description: Key Points Intensive BFM therapy is effective for HR childhood ALL if low MRD levels are achieved at the end of the induction/consolidation phase. Childhood ALL with high MRD levels at the end of induction/consolidation phase has a poor prognosis despite intensive BFM therapy or HSCT.