ALBERT

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: 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: In this study, we tried to select a very small subset of children with acute lymphoblastic leukemia (ALL) at minimal risk of treatment failure - identified to not only by early response in vivo, one of the strongest predictors in the I-BFM-SG experience, but also by age, blood count and in particular high DNA content - which we treated with a reduced-intensity BFM schedule. The AIEOP-ALL 9501 study enrolled patients with standard-risk (SR) ALL, defined as:

Description: Abstract 1432 Poster Board I-455 Whether a stochastic or hierarchical model best reflects the identity of leukemia initiating cell is still an open debate. In the case of acute lymphoblastic leukemia (ALL) little is known about the identity and the origin of leukemic stem cells (LSCs). In this study we focused on infant ALL carrying the t(4;11)/MLL-AF4, the most frequent and aggressive form of leukemia occurring within the first year of life. Using fluorescence-activated cell sorting (FACS) combined with fluorescence in situ hybridization (FISH) analysis, in vivo repopulating assay and clonal analysis, we investigated whether different BM subsets, sorted from infant ALL diagnostic samples on the bases of their CD34/CD19/NG2 phenotype, have similar or distinct LSC properties. Our studies demonstrate that in t(4;11)/MLL-AF4 infant ALL the 34+19- hematopoietic stem/progenitor cell pool is intact (MLL-AF4 negative by FISH), and that the LSC is not CD19-, as CD19- cells lacked repopulating ability (CD34-) or gave rise exclusively to normal multilineage reconstitution (CD34+). In contrast, CD19+ LSCs rapidly engraft and establish leukemia in NOD/SCID mice, with features recapitulating the human disease, and possess self-renewal potential in serial transplantation, regardless of CD34 and NG2 expression. However, although multiple leukemic subsets possess some “stemness”, we did observe differences in the kinetics and immunophenotypes of engraftment between phenotypically distinct LSC subpopulations, suggesting that in t(4;11)/MLL-AF4 infant ALL the LSCs are diverse but distinct. These findings might reconcile conflicting results with regard to previously proposed stochastic versus hierarchical models for LSCs. By designing patient-specific primers and probes on the junctional regions, we analyzed by RQ-PCR the clonal contribution to the leukemic grafts and found that, as in infant patients, ALL grafts in mice were oligoclonal, as several independent clones were able to sustain engraftment in recipient mice. However, among the engrafting clones, we observed different behaviours: either major or minor clones in the patient dominate in the graft; conversely some other clones which were predominant in the diagnostic sample failed to sustain leukemia in vivo. Specifically we observed that dominant clones in primary recipients reconstituted with leukemia can either persist over time or extinguish (or become dormant) in serial passages; while other clones, which were quiescent in primary recipents, never-the-less persisted, reactivated and became dominating in secondary recipients. In conclusion we provided evidences that LSC in infant ALL with t(4;11) is restricted to the CD19+ fraction and, among this, multiple phenotypically distinct BM subsets can initiate leukemia in NOD/SCID mice, with different kinetics and immunophenotypes of engraftment. Moreover, our clonal analysis results point out a further heterogeneity within purified LSC subsets and implicate the co-existence and competition between multiple clones. Disclosures No relevant conflicts of interest to declare.

Description: Slow early response indicates poor prognosis in childhood ALL. We aimed to evaluate if post-induction MRD levels had different prognostic impact in precursor B-cell (pB) or T-cell ALL. From 07/2000 to 06/2006, 4730 pts with ALL were enrolled in trial AIEOP-BFM ALL 2000. MRD levels were centrally measured by real-time quantitative polymerase chain reaction using the identification of clone-specific T-cell receptor and immunoglobulin gene rearrangements. MRD study time-points (TP) were treatment day 33 (TP1, end of induction) and day 78 (TP2, after consolidation). To define MRD negativity, two markers with a sensitivity of at least 10−4 were required. Patients were treated with BFM induction (protocol I-A), consolidation (I-B), extra-compartment/intensified consolidation (HD-MTX in non-high-risk patients, pulses in high-risk patients), reinduction, and maintenance. MRD analysis at one or two time points suceeded in 3707 pts; the immunophenotype was available from 3636 pts. MRD levels and corresponding estimated 5-year event-free survival (5y-pEFS) comparing pB- and T-ALL are shown in Table 1 (3yrs median follow-up). MRD response in T-ALL was slower than in pB-ALL resulting in a higher percentage of pts with high MRD load in T-ALL. In pB-ALL as well as T-ALL, high MRD levels at TP2 were well predictive to identify pts with poor prognosis. For prediction of good prognostic subgroups, TP1 was more appropriate identifying a subgroup with excellent 5y-pEFS of 〉90% in case of MRD negativity. Specificity of TP1 was poor in T-ALL if the pB-ALL criteria of MRD negativity were applied. If MRD low positive and MRD negative T-ALL pts were combined, the discrimination was as good as in pB-ALL. The optimal choice of MRD evaluation time points depends on biological factors and treatment, and is most relevant for MRD-based risk stratification. Table 1 pB-ALL T-ALL n % 5y-pEFS % (SE) n % 5y-pEFS % (SE) all 3177 100% 82.3 (1.0) 459 100% 77.2 (2.2) MRD TP1     neg 1399 44.1 92.5 (1.0) 75 16.4 94.3 (2.8)     10E-4/−5 1122 35.4 81.9 (1.7) 116 25.4 91.2 (2.8)     10E-3 393 12.4 66.4 (3.5) 110 24.1 75.3 (4.6)     ≥10E-2 256 8.1 53.2 (4.3) 156 34.1 59.8 (4.5) MRD TP2     neg 2464 77.6 87.7 (1.0) 220 47.9 91.9 (2.0)     10E-4/−5 523 16.5 68.9 (2.9) 143 31.2 76.6 (3.9)     10E-3 107 3.4 56.3 (6.5) 58 12.6 50.2 (8.1)     ≥10E-2 82 2.6 38.0 (7.3) 38 8.3 33.2 (8.3)

Description: Progress in the treatment of acute lymphoblastic leukaemia (ALL) has led to better survival rates; however, children have had a greater benefit from improved treatment modalities than adolescent who show an overall lower event-free survival (EFS) compared to younger patients. Some differences in the clinical and biologic characteristics of adolescents compared to childhood ALL may partly account for the different outcome, but adolescents treated on pediatric ALL trials seem to have a significantly better EFS than those treated on adult trials. We retrospectively compared the results obtained in a series of 245 patients ranging in age from 14 to 18 years diagnosed and enrolled in specific Italian children and adult ALL trials, between 4/1996 and 10/2003. One hundred and fifty patients, from 30 pediatric centers, underwent the childhood AIEOP ALL 95 and 2000 protocols; the other 95, from 28 adult centers, were enrolled in the GIMEMA ALL 0496 and 2000 protocols. The AIEOP 95 and 2000 trials are BFM-like protocols with a 7 drug induction followed by risk-modulated post-remission therapy that includes high-dose MTX and reinduction for low and intermediate groups, and intensive blocks (high-dose MTX and cytarabine) for high-risk patients. Standard maintenance therapy is administered up to a total of 2 years. Cranial radiotherapy is limited to high-risk patients. Stem cell transplantation is planned for very high-risk patients. The GIMEMA regimens are instead based on an induction with high-dose anthracyclines (cumulative dose 550 mg/m2), high-dose cytarabine as consolidation and do not include high-dose MTX and the reinduction phase. Standard maintenance with vincristine + daunorubicin/cyclophosphamide pulses is given for 2 years. Cranial radiotherapy is administered to all patients. The main patients characteristics at diagnosis, in the two groups under examination, were comparable except for age: median age was 15 and 16 years, respectively in the AIEOP and GIMEMA trials.Poor risk cytogenetic translocations and T-immunophenotype were equally dinstributed. Adolescents in the AIEOP protocols had a higher CR rate (94% vs 89%) and a lower relapse rate (17% vs 45%) compared to the adolescents enrolled in the GIMEMA trials. The 2-year overall survival rate was 80% in the AIEOP protocols and 71% in the GIMEMA trials. Detailed results according to the different clinical and biologic features of the adolescents analyzed will be presented. The results of our comparative study indicate that adolescents enrolled in pediatric trials have a more favourable clinical outcome.

Description: Abstract 2944 Poster Board II-920 Introduction: The chromosomal translocation t(8;14)(q24;q32) represents a specific tumor marker in Burkitt's lymphoma (BL). This chromosomal aberration involves the MYC oncogene on chromosome 8 and the immunoglobulin heavy-chain (IgH) locus on chromosome 14. We have previously demonstrated that this genetic abnormality can be used as a marker of Minimal Disseminated Disease (MDD) in BL (Mussolin et al, JCO, 2007). The aim of the study was to assess of the prevalence of MDD in BL at diagnosis in children enrolled in the AIEOP LNH-97 clinical protocol and the evaluation of its impact on prognosis. Patients and Methods: We established a simplified long-distance PCR (LD-PCR) assay which can amplify up to 15-20 Kb DNA sequence making it possible to detect the t(8;14) at the genomic level with the sensitivity of 10-4. The assay was based on 4 separate PCR reactions in which one primer complementary to the first exon of the MYC gene is used with one of four primers for the IgH locus (1 for the JH region and 1 for each of the 3 constant regions). Results: LD-PCR was applied to prospectively study 124 BL biopsies and detected a specific PCR product in 88 of them (71%). Of the 88 positive BL patients we studied both the tumor and the bone marrow (BM) at diagnosis in 76: BM was positive by LD-PCR in 25 patients (33%), whereas only 10 (13%) were positive at the standard morphological and/or immunophenotypical analyses. Most of the MDD positive patients (88%) belonged to the R4 Risk Group according to BFM definition (stage III or stage IV according to St. Jude staging classification and LDH≥1000 U/l). The 3-year progression-free survival (PFS) was 68% (SE 10%) in MDD positive R4 patients compared with 96% (SE 4%) in MDD negative R4 patients (p= 0.02), whereas there was no difference in PFS between children with morphological involvement of BM at diagnosis versus those who had negative BM (PFS=62.5% (SE 17%) vs. PFS= 87% (SE 6%), respectively, p= 0.09). By multivariate analysis (including MDD, gender, LDH, CNS involvement) MDD was predictive of higher risk of failure (Hazard Ratio: 8.4 , p= 0.04). Conclusions: We demonstrated that MDD identifies a poor prognosis subgroup among high risk Burkitt's lymphoma patients. We suggest that a more effective risk-adapted therapy, possibly including anti-CD20 monoclonal antibody, should be considered in these patients. Disclosures: No relevant conflicts of interest to declare.

Description: We compared the clinical and biologic features and treatment results of patients with DS-ALL (n=120, 1.9%), compared to those without DS (n=6237), enrolled in the AIEOP trials between 1988 and October 2004. DS patients had significantly more often the following characteristics: female gender (55.0% vs 44.9%, p=0.027), age 10 years or older (28.3 vs 17.7%, p=0.014), high risk by NCI criteria (41.7 vs. 33%, p=0.045); on the contrary significantly less often they belonged to high-risk group according to current stratification (10.8 vs. 20.3%, p=0.017) or had T-lineage immunophenotype (0.8 vs 11.8%, p=100K/mm3: 6.6 vs. 10.8%). TEL/AML1 rearrangement was found in only 1 of 44 tested (2.2%). Of the 120 DS patients, 5 died during induction (4.2 vs 1.2% in non-DS), 1 was resistant (0.8 vs 1.4%). Leukemia relapse occurred in 31.6% of DS patients, compared with 24.9% of non-DS: bone marrow 22.5 vs 15.5%, CNS isolated 4.2 vs 3.6%, testis 0.8 vs 1.6%, BM combined 3.3 vs. 3.6%, other site 0.8 vs 0.6%. Death in complete remission occurred in 4.2 vs 2.0%. No second malignancy was reported. Overall, 59.2% of DS patients remained in first remission, compared with 70.2% of non-DS patients. This translated into a probability of EFS (SE) at 10 years of 56.2% (4.8) versus 67.7% (0.6) and a survival of 60.8% (5.0) versus 77.2% (0.6). 57 DS patients were diagnosed

Description: Death early (DE) during the induction phase of therapy is a rare but dramatic event in children with acute myeloid leukemia (AML). Recent reports emphasize that DE in patients with leukemia is related to aggressive disease, signaled by hyperleukocytosis, and to the early administration of intensive chemotherapy. Many efforts have been made to better characterize subgroups of patients with AML by defining biologic markers associated with aggressive disease. Even if little is known about the molecular mechanisms that regulate blast migration into tissues, adhesion receptors are likely to play an important role in this process. We have attempted to identify the molecular characteristics of an aggressive subset of pediatric patients with AML through a prospective evaluation of the CD56+ neural cell adhesion molecule (NCAM) and CD94 expression. For this purpose we developed an RT-PCR semi-quantitative methodology, using specific primers from NCAM, CD94 and β-actin as an internal quality and quantity control. We analyzed a total population of 44 children with AML, distributed as follow: 5 with M3v (group A), 9 with APL (group B) and 30 with non-APL AML (group C). We found a dramatic correlation in group A between high expression of NK-related genes and occurrence of early death. In groups B and C, primarily used as controls, we found a strong correlations between a high level of NCAM-CD94 expression and two cases with APL, three with FAB M5, two with FAB M2 AML, one case with FAB M4 and a child with dendritic cell leukemia (DCL). We primarily found that 9 out of 13 cases presenting with a high level of NCAM-CD94 expression died of disease-related events during the early days of induction. Our data strongly suggest that doses and timing of chemotherapy induction in children with AML need to be modulated because of the high risk of hemorrhagic events. These appear to be related to the presence of high concentration of adhesion molecules secondary to acute blast lysis and to the associated severe coagulopathy. We know of no pharmacological correctives for this phenomenon. It would be important to develop specifically designed molecules in order to disrupt the interactions with adhesion molecules, and thus to improve the clinical course of both children and adults with AML during the early days of induction.

Description: Immunoglobulin (Ig) and T-cell Receptor (TcR) gene rearrangements are used as patient-specific PCR targets for MRD detection in ALL. However, oligoclonality was reported in childhood ALL, and in those cases, the uncertainty of which clone is going to emerge at relapse may give rise to false negative MRD results. In particular, oligoclonality is a peculiar characteristic of Infant ALL (less than 1 year of age at diagnosis). So far, the prognostic relevance of MRD monitoring in Infant ALL has not been defined, and the assessment of oligoclonality and stability of PCR markers may have a profound impact in MRD monitoring. We successfully studied by PCR the frequency and stability of the currently used rearrangements of the Ig Heavy (IgH), Ig Kappa deleting element (IgK-Kde), TcR delta (TcRD), and TcR gamma (TcRG) gene rearrangements in 42/43 Infant ALL patients prospectively enrolled in Italy in the Interfant-99 International ALL protocol. Pro-B, common and pre-B ALL were 29 (67%), 8 (19%), and 6 (14%), respectively. Sixty-three percent of cases were prednisone good responders (PGR), 70% were MLL-rearranged, 44% were aged less than 6 months, and 21% of cases had more than 300.000 wbc at diagnosis. Overall, rearrangements of the IgH, IgK, TcRD, and TcRG genes were found in 91, 21, 40, and 21% of Infant ALL, respectively. The pattern of Ig/TcR rearrangements in immunophenotypic subgroups of Infant ALLs has been compared to the one of older children prospectively enrolled in Italy into the AIEOP-BFM ALL2000 protocol (n=649). While the overall frequency of IgH and TcRD are similar in the two age groups, IgK and TcRG are less rearranged in Infant cases, mainly due to the low percentage in the pro-B group (10% versus 41% for IgK VK-Kde, and 17% vs 36% for TcRG). In particular, none of the 29 pro-B cases showed rearrangement of the IgK intron. Sixteen Infant relapsed so far (37%), and 12 of them were successfully analyzed for Ig/TcR rearrangement at diagnosis and relapse. Ten were early (less than 18 months from diagnosis), and 2 were late relapses. None of the patients showed an identical Ig/TcR pattern at diagnosis and relapse; 7 (58%) had at least one, and 1 had at least 2 PCR targets preserved, while 4 cases (33%) presented completely different markers at relapse. This distribution was independent on age at diagnosis, site and time of relapse. Interestingly, we observed an increase of complete-IgH (from 10 alleles in 7 patients to 23 alleles in 11) and TcRG (from 4 alleles in 2 patients to14 alleles in 8) rearrangements at relapse. The stability of Ig/TcR markers was as it follows: IgH, 6/26; IgK, 3/5; TcRD, 1/9 and TcRG, 0/4. In conclusion, the oligoclonality feature of Infant ALL may potentially hamper the MRD predictivity in Infant ALL. An appropriate identification and selection of Ig/TcR MRD-PCR targets in Infant ALL is a crucial premise for obtaining clinically relevant MRD data and for preventing false-negative MRD results. Ig Kappa may be a preferential marker for MRD studies in Infant ALL, although its use is limited by the low frequency of the rearrangement. The leukemia-specific MLL genomic breakpoint may potentially overcome these limitations and improve the MRD analysis.