ALBERT

Description: Key Points Risk-adapted therapy and broad use of HSCT resulted in a significant improvement in outcome. AUTO- or ALLO-HSCT in high-risk patients resulted in a cumulative incidence of leukemia relapse superimposable to that of SR.

Description: Background. Disease-free survival (DFS) for children with ALL exceeds 80%. Nevertheless, the cure rate can be significantly lower in patients with specific chromosomal translocations, confering poor prognosis. The t(4;11) translocation can be detected in 1-3% of children with ALL and is associated with aggressive clinical course and high risk of treatment failure and relapse. Consequently, patients with t(4;11) may be offered allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission (CR), although the role of HSCT in this setting is still unclear. Patients and Methods. Data on children with t(4;11) positive ALL treated with allogeneic HSCT in AIEOP Centers from 1990 to 2013 were retrospectively collected, and the impact of patient- and treatment-related variables on the clinical outcome was evaluated. A total of 72 consecutive children with t(4;11) positive ALL were analyzed; 33 patients were males and 39 female. The median age at diagnosis was 11 months (range, 1.2 months - 15 years). 38 patients (53%) had an age at diagnosis 〈 12 months (defined as infants) and 22 were younger than 6 months at diagnosis. The median WBC count was 167x109/l. The majority of infants (30 patients) were enrolled into Interfant 99 and 06 protocols, while the 34 children older than 12 months of age were enrolled into AIEOP 95, 2000, R-2006 and 2009 protocols, respectively. 46 children (64%) were transplanted in first CR, 18 (25%) in second CR and the remaining 8 (11%) in a more advanced disease phase. 35 patients received HSCT from a matched unrelated donor (MUD), 28 from a matched family donor (MFD) and 9 from a partially matched family donor (PMFD). TBI was used in 40% of patients, while a busulfan-based conditioning regimen was used in 56% of cases. Results. 5-year overall survival (OS) and DFS were 54% (42-67) and 47% (35-60), respectively. Transplant-related mortality (TRM) and relapse incidence (RI) were 20% (13-33) and 32% (23-46), respectively. DFS by donor type was as follow: MFD 51%, MUD 52% and PMFD 22%, respectively (P = 0.04). TRM by donor type was 20% for MFD, 14% for MUD and 44% for PMFD, respectively (P = 0.03). DFS was 60% for children transplanted in 1st CR, 30% for patients transplanted in 2nd CR and 25% for those with more advanced disease (P = 0.002). DFS was 40% for children with an age at diagnosis 〈 6 months, 46% for those with an age at diagnosis between 6 and 12 months, 40% for the 12-24 months group and 57% for patients with an age at diagnosis 〉 24 months (P = N.S.). DFS was 61% for children receiving TBI and 39% for those treated with a chemo-based conditioning (P = 0.089). Conclusions. Our analysis suggests that HSCT in first remission is a valid therapeutic option for children with t(4;11) positive ALL. Patients transplanted in 1st CR, as well as those receiving a TBI-based conditioning regimen, had a better outcome. MFD and MUD transplants were associated with a similar DFS probability. Disclosures No relevant conflicts of interest to declare.

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 aims of this study were twofold: (1) to assess the marrow of patients with T-lineage acute lymphoblastic leukemia (T-ALL) for the presence of molecular residual disease (MRD) at different times after diagnosis and determine its value as a prognostic indicator; and (2) to compare the sensitivity, rapidity, and reliability of two methods for routine clinical detection of rearranged T-cell receptor (TCR). Marrow aspirates from 23 patients with T-ALL diagnosed consecutively from 1982 to 1994 at the Division of Pediatric Hematology and Oncology, University of Catania, Italy, were obtained at diagnosis, at the end of induction therapy (6 to 7 weeks after diagnosis), at consolidation and/or reinforced reinduction (12 to 15 weeks after diagnosis), at the beginning of maintenance therapy (34 to 40 weeks after diagnosis), and at the end of therapy (96 to 104 weeks after diagnosis). DNA from the patients' marrow was screened using the polymerase chain reaction (PCR) for the four most common TCR δ rearrangements in T-ALL (Vδ1Jδ1, Vδ2Jδ1, Vδ3Jδ1, and Dδ2Jδ1) and, when negative, further tested for the presence of other possible TCR δ and TCR γ rearrangements. After identification of junctional rearrangements involving V, D, and J segments by DNA sequencing, clone-specific oligonucleotide probes 5′ end-labeled either with fluorescein or with [γ-32P]ATP were used for heminested PCR or dot hybridization of PCR products of marrows from patients in clinical remission. For 17 patients with samples that were informative at the molecular level, the estimated relapse-free survival (RFS) at 5 years was 48.6% (±12%). The sensitivity and specificity for detection of MRD relating to the outcome were 100% and 88.9% for the heminested fluorescence PCR and 71.4% and 88.9% for Southern/dot blot hybridization, respectively. Predictive negative and positive values were 100% and 90.7% for heminested fluorescence PCR, respectively. The probability of RFS based on evidence of MRD as detected by heminested fluorescence PCR at the time of initiation of maintenance therapy was 100% and 0% for MRD-negative and MRD-positive patients, respectively. Thus, the presence of MRD at the beginning of maintenance therapy is a strong predictor of poor outcome, and the molecular detection of MRD at that time might represent the basis for a therapeutic decision about such patients. By contrast, the absence of MRD at any time after initiation of treatment strongly correlates with a favorable outcome. The heminested fluorescence PCR appears to be more accurate and more rapid than other previously used methods for the detection of residual leukemia.

Description: The aims of this study were twofold: (1) to assess the marrow of patients with T-lineage acute lymphoblastic leukemia (T-ALL) for the presence of molecular residual disease (MRD) at different times after diagnosis and determine its value as a prognostic indicator; and (2) to compare the sensitivity, rapidity, and reliability of two methods for routine clinical detection of rearranged T-cell receptor (TCR). Marrow aspirates from 23 patients with T-ALL diagnosed consecutively from 1982 to 1994 at the Division of Pediatric Hematology and Oncology, University of Catania, Italy, were obtained at diagnosis, at the end of induction therapy (6 to 7 weeks after diagnosis), at consolidation and/or reinforced reinduction (12 to 15 weeks after diagnosis), at the beginning of maintenance therapy (34 to 40 weeks after diagnosis), and at the end of therapy (96 to 104 weeks after diagnosis). DNA from the patients' marrow was screened using the polymerase chain reaction (PCR) for the four most common TCR δ rearrangements in T-ALL (Vδ1Jδ1, Vδ2Jδ1, Vδ3Jδ1, and Dδ2Jδ1) and, when negative, further tested for the presence of other possible TCR δ and TCR γ rearrangements. After identification of junctional rearrangements involving V, D, and J segments by DNA sequencing, clone-specific oligonucleotide probes 5′ end-labeled either with fluorescein or with [γ-32P]ATP were used for heminested PCR or dot hybridization of PCR products of marrows from patients in clinical remission. For 17 patients with samples that were informative at the molecular level, the estimated relapse-free survival (RFS) at 5 years was 48.6% (±12%). The sensitivity and specificity for detection of MRD relating to the outcome were 100% and 88.9% for the heminested fluorescence PCR and 71.4% and 88.9% for Southern/dot blot hybridization, respectively. Predictive negative and positive values were 100% and 90.7% for heminested fluorescence PCR, respectively. The probability of RFS based on evidence of MRD as detected by heminested fluorescence PCR at the time of initiation of maintenance therapy was 100% and 0% for MRD-negative and MRD-positive patients, respectively. Thus, the presence of MRD at the beginning of maintenance therapy is a strong predictor of poor outcome, and the molecular detection of MRD at that time might represent the basis for a therapeutic decision about such patients. By contrast, the absence of MRD at any time after initiation of treatment strongly correlates with a favorable outcome. The heminested fluorescence PCR appears to be more accurate and more rapid than other previously used methods for the detection of residual leukemia.

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: Background. Acute Myeloid Leukemia is a heterogeneous disease, characterized by the uncontrolled proliferation of hematopoietic precursor cells. AML accounts for 15% of acute leukemias in children, with a cure rate of 55-60% as overall survival (Pession A Blood 2013). Complete remission (CR) is achieved in 80-85% of children with AML. Current protocol defines CR by morphological evaluation (blasts 3, FDR adjusted p value -3, FDR adjusted p value

Description: The timing and developmental sequence of events for BCR-ABL1+ acute lymphoblastic leukemia (ALL), usually associated with IKAROS (IKZF1) deletions, are unknown. We assessed the status of BCR-ABL1 and IKZF1 genes in 2 pairs of monozygotic twins, one pair concordant, the other discordant for Philadelphia chromosome positive (Ph+) ALL. The twin pair concordant for ALL shared identical BCR-ABL1 genomic sequence indicative of monoclonal, in utero origin. One twin had IKZF1 deletion and died after transplantation. The other twin had hyperdiploidy, no IKZF1 deletion, and is still in remission 8 years after transplantation. In the twin pair discordant for ALL, neonatal blood spots from both twins harbored the same clonotypic BCR-ABL1 sequence. Low level BCR-ABL1+ cells were present in the healthy co-twin but lacked the IKZF1 deletion present in the other twin's leukemic cells. The twin with ALL relapsed and died after transplantation. The co-twin remains healthy and leukemia free. These data show that in childhood Ph+ ALL, BCR-ABL1 gene fusion can be a prenatal and possibly initiating genetic event. In the absence of additional, secondary changes, the leukemic clone remains clinically silent. IKZF1 is a secondary and probable postnatal mutation in these cases, and as a recurrent but alternative copy number change is associated with poor prognosis.

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.