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SummaryData from large case series of children with cerebral thrombotic events are pivotal to improve prevention, early recognition and treatment of these conditions. The Italian Registry of Pediatric Thrombosis (R. I. T. I.) was established in 2007 by a multidisciplinary team, aiming for a better understanding of neonatal and paediatric thrombotic events in Italy and providing a preliminary source of data for the future development of specific clinical trials and diagnostic-therapeutic protocols. We analysed data relative to the paediatric cerebral thrombotic events of the R. I. T. I. which occurred between January 2007 and June 2012. In the study period, 79 arterial ischaemic stroke (AIS) events (49 in males) and 91 cerebral sinovenous thrombosis (CSVT) events (65 in males) were enrolled in the R. I. T. I. Mean age at onset was 4.5 years in AIS, and 7.1 years in CSVT. Most common modes of presentation were hemiparesis, seizures and speech disturbances in AIS, and headache, seizures and lethargy in CSVT. Most common etiologies were underlying chronic diseases, vasculopathy and cardiopathy in AIS, and underlying chronic diseases and infection in CSVT. Time to diagnosis exceeded 24 hours in 46 % AIS and 59 % CSVT. Overall data from the Italian Registry are in substantial agreement with those from the literature, despite small differences. Among these, a longer time to diagnosis compared to other registries and case series poses the accent to the need of an earlier recognition of paediatric cerebrovascular events in Italy, in order to enable prompt and effective treatment strategies.

We analyzed the long-term outcome of 4865 patients treated in Studies 82, 87, 88, 91 and 95 for childhood acute lymphoblastic leukemia (ALL) of the Italian Association of Pediatric Hematology and Oncology (AIEOP). Treatment was characterized by progressive intensification of systemic therapy and reduction of cranial radiotherapy. A progressive improvement of results with reduction of isolated central nervous system relapse rate was obtained. Ten-year event-free survival increased from 53% in Study 82 to 72% in Study 95, whereas survival improved from 64 to 82%. Since 1991, all patients were treated according to Berlin-Frankfurt-Muenster (BFM) ALL treatment strategy. In Study 91, reduced treatment intensity (25%) yielded inferior results, but intensification of maintenance with high-dose (HD)-L-asparaginase (randomized) allowed to compensate for this disadvantage; in high-risk patients (HR, 15%), substitution of intensive polychemotherapy blocks for conventional BFM backbone failed to improve results. A marked improvement of results was obtained in HR patients when conventional BFM therapy was intensified with three polychemotherapy blocks and double delayed intensification (Study 95). The introduction of minimal residual disease monitoring and evaluation of common randomized questions by AIEOP and BFM groups in the protocol AIEOP-BFM-ALL 2000 are expected to further ameliorate treatment of children with ALL.

Background Acute B-cell leukemia (B-ALL) is a rare form of pediatric leukemia characterized by a very high-proliferation index, rapid clinical progression, and a high frequency of central nervous system (CNS) involvement. Commonly, it is treated in the clinical trials for Burkitt lymphoma, of which it represents the leukemic counterpart. Procedure Children with B-ALL diagnosed between 1988 and 1999 were enrolled in the AIEOP-8805 protocol. Treatment included six high-dose chemotherapy courses. No prophylactic CNS irradiation was administered. Results Sixty-five consecutive patients were enrolled in the study. L3 morphology was observed in 57 of 65 patients (88%). Twenty-five children (38%) had tumor mass in addition to massive bone marrow infiltration; 11 children (17%) had CNS disease at diagnosis. Sixty-two patients obtained complete morphological remission of which 13 suffered a relapse, including 3 with initial CNS involvement. Ten-year overall survival and event-free survival were 77% and 75%, respectively. Neither relevant long-term toxicity nor second malignancies were observed. Conclusions The AIEOP-8805 confirmed that short high-dose chemotherapy is highly effective for the treatment of B-ALL without significant long-term adverse sequelae. Therapy modifications to reduce relapse rate, such as the use of anti-CD20 monoclonal antibody and more effective CNS treatment, are being tested. Pediatr Blood Cancer 2011;56:544–550. © 2010 Wiley-Liss, Inc.

In spite of high cure rates and improved overall survival, 25% of pediatric patients with acute lymphoblastic leukemia (ALL) relapse after obtaining complete remission. Additionally a small proportion of patients are refractory and do not attain remission. Microarray expression analysis of matched diagnosis-relapse B-lineage ALL sample pairs identified DEFA1-3 as a potential marker of relapse. Here, validation of DEFA1-3 as a marker for therapy resistance is explored. DEFA1-3 expression was analysed by RQ-PCR in patient paired samples at diagnosis and relapse of 6 early-relapse (within 18 months) and 8 late-relapse (beyond 18 months) B-lineage ALL. Diagnostic samples of 19 patients with ALL who are in continuous complete remission (median time from diagnosis 47 month) and diagnostic samples of 5 refractory patients who had not achieved remission at day 35 of therapy were also analyzed. In addition, overexpression of alpha-defensin1-3 proteins in blast cells at relapse was analysed by flow cytometry. DEFA1-3 was overexpressed at relapse as compared to diagnosis in 12 of 14 samples. At diagnosis, the expression of DEFA1-3 was significantly higher in samples from refractory patients as compared to those of patients who are in CR and to those of patients who experienced late relapse. At diagnosis, patients who relapsed early after diagnosis could not be distinguished from refractory patients based on DEFA1-3 expression levels. Results suggest that high levels of DEFA1-3 mRNA and alpha-defensin1-3 protein expression are correlated with disease progression and failure of adequate response to conventional chemotherapy.

The outcome of children with acute lymphoblastic leukaemia (ALL) and early relapse remains unsatisfactory. In January 1995, the AIEOP (Associazione Italiana di Oncologia ed Ematologia Pediatrica) group opened a trial for children with ALL in first isolated or combined bone marrow relapse defined at high risk according to the length of first remission and the immunophenotype. The treatment plan included the combination of a single high-dose idarubicin and high-dose cytarabine as induction therapy followed by an intensive consolidation and stem cell transplant (SCT). In total, 100 children from 16 Italian centres were enrolled; 80 out of the 99 evaluable patients (81%) achieved second complete remission; eight (8%) died during induction and 11 (11%) failed to respond. A total of 42 out of the 80 responders (52.5%) received a SCT: 19 from an identical sibling, 11 from a matched unrelated donor and 12 from umbilical cord blood cells. The estimated 4-year overall survival and event-free survival were 25% and 21% respectively. Disease-free survival at 4 years was 25.8% for the 80 responders. At 4 years, 39 out of 100 children remain alive, with 27 of them free of leukaemia. This induction therapy has shown antileukaemic efficacy with acceptable toxicity; moreover, all responders proved eligible for intensive consolidation.

PURPOSE: To assess in a randomized study the therapeutic effect of the addition of high-dose l-asparaginase (HD ASP) in the context of a Berlin-Frankfurt-Münster (BFM)–based chemotherapy regimen for intermediate risk (IR) childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: From March 1991 to April 1995, a total of 705 patients, with 59% of the cohort of patients fewer than 15 years old, with newly diagnosed non-B ALL, enrolled onto the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-91 study, were assigned to the IR group. Patients in remission at the beginning of the reinduction phase were randomized either to the standard treatment (SD ASP arm) or the experimental treatment (HD ASP arm; weekly intramuscular administration of HD ASP 25,000 IU/m2 repeated for a total of 20 weeks). Most of the patients (90%) were treated with Erwinia chrysanthemi l-asparaginase product. RESULTS: Among the 610 patients randomized to the SD ASP arm (n = 322) or to the HD ASP arm (n = 288), relapse occurred at a median time of 24 months after randomization in 76 (24%) and in 64 children (22%), respectively. Most of the relapses occurred in the marrow (100 isolated, 21 combined). There was no significant difference between the disease-free survival in the two treatment arms (P = .64), with estimated values at 7 years from randomization of 72.4% (SE 3.1) v 75.7% (SE 2.6) in the SD ASP and HD ASP arms, respectively. CONCLUSION: No advantage was observed for IR ALL children treated with BFM-based intensive chemotherapy who received protracted E chrysanthemi HD ASP during reinduction and the early continuation phase.

BACKGROUND AND OBJECTIVES: Treatment of childhood standard-risk (SR) acute lymphoblastic leukemia (ALL) is generally successful. However, intensive chemotherapy regimens may be associated with severe treatment sequelae. Efforts are therefore being made to identify those patients in whom less intensive treatment would be equally successful but cause fewer long-term sequelae. The aim of this study was to evaluate the efficacy of treatment reduction in a subset of children with ALL at minimal risk of failure. DESIGN AND METHODS: The population of patients with SR ALL included children aged between 1 and 6 years with less than 20,000 WBC/mm3, non-T immunophenotype, DNA index between 1.16 and 1.6, absence of t(9;22) and t(4;11) clonal translocations, no extramedullary leukemia, good response to prednisone and complete remission (CR) at the end of induction therapy. A reduced-intensity, BFM-type treatment schedule (AIEOP-ALL 9501 protocol) was used. Induction therapy was based on vincristine, prednisone, L-asparaginase and intrathecal methotrexate only; high-dose-methotrexate (2 g/m2) was given x4. The BFM Protocol II was given as reinduction therapy; thus the total dose of anthracyclines was 120 mg/m2 and no epipodophyllotoxins or cranial irradiation were employed. RESULTS: Between May 1995 and December 1999, 123 patients were identified as having SR-ALL (7.8% of the ALL-95 population), of whom 102 received the SR protocol. After a median follow-up of 5.9 years, 11 patients in the SR protocol had relapsed, 1 had died in remission, and 1 had developed a second malignant neoplasm. The probabilities (standard errors) of survival and event-free survival (EFS) were, respectively, 97.0% (1.7) and 86.7% (3.5) at 5 years, and 95.3% (2.4) and 86.7% (3.5) at 7 years. INTERPRETATION AND CONCLUSIONS: Although most of the relapsed patients were rescued, the long-term EFS probability in this small, highly selected group of patients remains inferior to expectation. Thus, alternative selection criteria, such as treatment response measured by minimal residual disease, should be considered to address the issue of treatment reduction.

The aim of the study was to validate the impact of the single-nucleotide polymorphism rs2413739 (T > C) in the PACSIN2 gene on thiopurines pharmacological parameters and clinical response in an Italian cohort of pediatric patients with acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD). In ALL, PACSIN2 rs2413739 T allele was associated with a significant reduction of TPMT activity in erythrocytes (p = 0.0094, linear mixed-effect model, multivariate analysis considering TPMT genotype) and increased severe gastrointestinal toxicity during consolidation therapy (p = 0.049). A similar trend was present also for severe hematological toxicity during maintenance. In IBD, no significant effect of rs2413739 could be found on TPMT activity, howeverazathioprine effectiveness was reduced in patients carrying the T allele (linear mixed effect, p = 0.0058). In PBMC from healthy donors, a positive correlation between PACSIN2 and TPMT protein concentration could be detected (linear mixed effect, p = 0.045). These results support the role of PACSIN2 polymorphism on TPMT activity and mercaptopurine adverse effects in patients with ALL. Further evidence on PBMC and pediatric patients with IBD supports an association between PACSIN2 variants, TPMT activity, and thiopurines effects, even if more studies are needed since some of these effects may be tissue specific.

Purpose Between May 1995 and August 2000 the Associazione Italiana di Ematologia Oncologia Pediatrica conducted the ALL-95 study for risk-directed, Berlin-Frankfurt-Muenster (BFM) –oriented therapy of childhood acute lymphoblastic leukemia, aimed at exploring treatment reduction in standard-risk patients (SR) and intensification during continuation therapy in intermediate-risk patients (IR) as randomized questions and treatment intensification in high-risk patients (HR). The prognostic value of DNA index was explored in this setting. Patients and Methods A total of 1,744 patients were enrolled (115, SR; 1,385, IR; and 244, HR). SR patients (DNA index ≥ 1.16 and < 1.60; age, 1 to 5 years; and WBC < 20,000, non–T-immunophenotype, with no high-risk features) received a reduced induction therapy (no anthracyclines); IR patients were randomly assigned to receive or not receive vincristine and dexamethasone pulses during maintenance; HR therapy was based on a conventional BFM schedule intensified with three chemotherapy blocks followed by a double reinduction phase. Results The event-free survival and overall survival probabilities at 10 years for the entire group were 72.5% (SE, 1.3) and 83.6% (SE, 0.9); 85.0% (SE, 3.4) and 95.5% (SE, 2.0) in SR, 75.1% (SE, 1.5) and 87.5% (SE, 0.9) in IR, and 51.0% (SE, 3.2) and 57.2% (SE, 3.3) in HR patients, respectively. Patients with a favorable DNA index had superior EFS in both IR (83.8% [2.7%] v 73.9% [1.7%]) and in HR (67.8% [9.4%] and 49.6% [3.5%]). Of the six patients with DNA index less than 0.8, only one remained in remission. Conclusion Favorable DNA index was associated with a better prognosis in IR and HR patients defined by presenting clinical criteria and treatment with a BFM-oriented chemotherapy.

Asparagine levels in cerebrospinal fluid and serum asparaginase activity were monitored in children with acute lymphoblastic leukemia treated with pegylated-asparaginase. The drug was given intravenously at a dose of 2,500 IU/m2 on days 12 and 26. Serum and cerebrospinal fluid samples obtained on days 33 and 45 were analyzed centrally. Since physiological levels of asparagine in the cerebrospinal fluid of children and adolescents are 4-10 μmol/L, in this study asparagine depletion was considered complete when the concentration of asparagine was ≤0.2 μmol/L, i.e. below the lower limit of quantification of the assay used. Over 24 months 736 patients (AIEOP n=245, BFM n=491) and 903 cerebrospinal fluid samples (n=686 on day 33 and n=217 on day 45) were available for analysis. Data were analyzed separately for the AIEOP and BFM cohorts and yielded superimposable results. Independently of serum asparaginase activity levels, cerebrospinal fluid asparagine levels were significantly reduced during the investigated study phase but only 28% of analyzed samples showed complete asparagine depletion while relevant levels, ≥1 μmol/L, were still detectable in around 23% of them. Complete cerebrospinal fluid asparagine depletion was found in around 5-6% and 33-37% of samples at serum asparaginase activity levels

Purpose Delayed intensification (DI) is an integral part of treatment of childhood acute lymphoblastic leukemia (ALL), but it is associated with relevant toxicity. Therefore, standard-risk patients of trial AIEOP-BFM ALL 2000 (Combination Chemotherapy Based on Risk of Relapse in Treating Young Patients With ALL) were investigated with the specific aim to reduce treatment intensity. Patients and Methods Between July 2000 and July 2006, 1,164 patients (1 to 17 years of age) with standard-risk ALL (defined as the absence of high-risk cytogenetics and undetectable minimal residual disease on days 33 and 78) were randomly assigned to either experimental reduced-intensity DI (protocol III; P-III) or standard DI (protocol II; P-II). Cumulative drug doses of P-III were reduced by 30% for dexamethasone and 50% for vincristine, doxorubicin, and cyclophosphamide, which shortened the treatment duration from 49 to 29 days. The study aimed at noninferiority of reduced-intensity P-III; analyses were performed according to treatment given. Results For P-III and P-II, respectively, the 8-year rate of disease-free survival (± SE) was 89.2 ± 1.3% and 92.3 ± 1.2% ( P = .04); cumulative incidence of relapse, 8.7 ± 1.2% and 6.4 ± 1.1% ( P = .09); and overall survival, 96.1 ± 0.8% and 98.0 ± 0.6% ( P = .06). Patients with ETV6-RUNX1–positive ALL and patients 1 to 6 years of age performed equally well in both arms. The incidence of death during remission was comparable, which indicates equivalent toxicity. The 8-year cumulative incidence rate of secondary malignancies was 1.3 ± 0.5% and 0.6 ± 0.4% for P-III and P-II, respectively ( P = .37). Conclusion Although the criteria used for the standard-risk definition in this trial identified patients with exceptionally good prognosis, reduction of chemotherapy was not successful mainly because of an increased rate of relapse. The data suggest that treatment reduction is feasible in specific subgroups, which underlines the biologic heterogeneity of this cohort selected according to treatment response.

The outcome of high-risk (HR) Acute Lymphoblastic Leukemia (ALL) patients enrolled in AIEOP-BFM ALL 2000 study (NCT00613457) in Italy is described. Overall, 1999 Philadelphia negative ALL patients entered the study. HR criteria were: minimal residual disease (MRD) levels ≥10-3 at day 78 (HR-MRD), no complete remission (no-CR) at day 33, t(4;11) translocation, Prednisone Poor Response (PPR). Treatment (2 years) included protocol I, 3 polychemotherapy blocks, delayed intensification (protocol IIx2 or IIIx3), cranial radiotherapy, maintenance. 312 HR patients (15.6% of the total) had 5-year event-free survival (EFS) and overall survival (OS) of 58.9%(SE 2.8) and 68.9%(2.6). In hierarchical order, EFS was 45.9%(4.4) in 132 HR-MRD patients, 41.2%(11.9) in 17 patients no-CR at day 33, 36.4%(14.5) in 11 patients with t(4;11), 74.0%(3.6) in 152 HR patients only for PPR. No statistically significant difference was found for disease-free survival (DFS) in patients with very high risk features (HR-MRD, no-CR at day 33, t(4;11) translocation), given HSCT (n=66) or chemotherapy only (n=88), after adjusting for waiting time to hematopoietic stem cell transplantation (HSCT) (5.7 months). Patients at HR only for PPR have favorable outcome. High risk MRD is associated with poor outcome despite intensive treatment and/or HSCT and may qualify for innovative therapies. The study is registered at the US National Institutes of Health website http://clinicaltrials.gov as "Combination Chemotherapy Based on Risk of Relapse in Treating Young Patients With Acute Lymphoblastic Leukaemia" with the protocol identification number NCT00613457.

The Associazione Italiana di Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000) study has for the first time introduced standardized quantitative assessment of minimal residual disease (MRD) based on immunoglobulin and T-cell receptor gene rearrangements as polymerase chain reaction targets (PCR-MRD), at 2 time points (TPs), to stratify patients in a large prospective study. Patients with precursor B (pB) ALL (n = 3184) were considered MRD standard risk (MRD-SR) if MRD was already negative at day 33 (analyzed by 2 markers, with a sensitivity of at least 10-4); MRD high risk (MRD-HR) if 10-3 or more at day 78 and MRD intermediate risk (MRD-IR): others. MRD-SR patients were 42% (1348): 5-year event-free survival (EFS, standard error) is 92.3% (0.9). Fifty-two percent (1647) were MRD-IR: EFS 77.6% (1.3). Six percent of patients (189) were MRD-HR: EFS 50.1% (4.1; P < .001). PCR-MRD discriminated prognosis even on top of white blood cell count, age, early response to prednisone, and genotype. MRD response detected by sensitive quantitative PCR at 2 predefined TPs is highly predictive for relapse in childhood pB-ALL. The study is registered at http://clinicaltrials.gov: NCT00430118 for BFM and NCT00613457 for AIEOP. (Blood. 2010;115(16):3206-3214) © 2010 by The American Society of Hematology.

Induction therapy for childhood acute lymphoblastic leukemia (ALL) traditionally includes prednisone; yet, dexamethasone may have higher antileukemic potency, leading to fewer relapses and improved survival. After a 7-day prednisone prephase, 3720 patients enrolled on trial Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Munster (AIEOP-BFM) ALL 2000 were randomly selected to receive either dexamethasone (10 mg/m(2) per day) or prednisone (60 mg/m(2) per day) for 3 weeks plus tapering in induction. The 5-year cumulative incidence of relapse (± standard error) was 10.8 ± 0.7% in the dexamethasone and 15.6 ± 0.8% in the prednisone group (P < .0001), showing the largest effect on extramedullary relapses. The benefit of dexamethasone was partially counterbalanced by a significantly higher induction-related death rate (2.5% vs 0.9%, P = .00013), resulting in 5-year event-free survival rates of 83.9 ± 0.9% for dexamethasone and 80.8 ± 0.9% for prednisone (P = .024). No difference was seen in 5-year overall survival (OS) in the total cohort (dexamethasone, 90.3 ± 0.7%; prednisone, 90.5 ± 0.7%). Retrospective analyses of predefined subgroups revealed a significant survival benefit from dexamethasone only for patients with T-cell ALL and good response to the prednisone prephase (prednisone good-response [PGR]) (dexamethasone, 91.4 ± 2.4%; prednisone, 82.6 ± 3.2%; P = .036). In patients with precursor B-cell ALL and PGR, survival after relapse was found to be significantly worse if patients were previously assigned to the dexamethasone arm. We conclude that, for patients with PGR in the large subgroup of precursor B-cell ALL, dexamethasone especially reduced the incidence of better salvageable relapses, resulting in inferior survival after relapse. This explains the lack of benefit from dexamethasone in overall survival that we observed in the total cohort except in the subset of T-cell ALL patients with PGR. This trial was registered at www.clinicaltrials.gov (BFM: NCT00430118, AIEOP: NCT00613457).

This analysis compared the numbers of patients treated at Italian pediatric oncology group (Associazione Italiana Ematologia Oncologia Pediatrica [AIEOP]) centers with the numbers of cases predicted according to the population-based registry. It considered 32,431 patients registered in the AIEOP database (1989-2012). The ratio of observed (O) to expected (E) cases was 0.79 for children (0-14 years old) and 0.15 for adolescents (15-19 years old). The proportion of adolescents increased significantly over the years, however, from 0.05 in the earliest period to 0.10, 0.18, and then 0.28 in the latest period of observation, suggesting a greater efficacy of local/national programs dedicated to adolescents.

The role of all-trans retinoic acid (ATRA) in pediatric acute promyelocytic leukemia (APL) is the topic of several ongoing studies. The results of the Italian pediatric experience with the multicentric Gruppo Italiano per le Malattie Ematologiche dell'Adulto (GIMEMA)-Italian Pediatric Hematology and Oncology Group (AIEOP) "AIDA" (ATRA and idarubicin) trial are presented. Of the 983 patients with APL enrolled in this protocol between January 1993 and June 2000, 124 (13%) had younger than 18 years. Treatment consisted of ATRA and idarubicin induction followed by 3 polychemotherapy consolidation courses. Molecular response by reverse transcriptase-polymerase chain reaction (RT-PCR) was assessed after consolidation and patients who were PCR- were randomized for different maintenances. One hundred and seven children were eligible and evaluable for induction: 103 (96%) achieved a hematologically complete remission. Overt ATRA syndrome was observed in 2 patients and pseudotumor cerebri was observed in 10 patients. Ninety-four patients were evaluable for RT-PCR analysis at the end of consolidation: 91 (97%) proved PCR+ and 3 PCR-. The overall survival and event-free survival (EFS) are 89% (95% confidence interval [c.i.]: 83%-95%) and 76% (c.i.: 65%-85%), respectively, at more than 10 years. A white blood cell (WBC) count at diagnosis of greater than 10 x 10(9)/L had a significant impact on EFS (59% vs 83% at 10 years). These results highlight the efficacy and feasibility of the AIDA protocol in the pediatric APL population.

The toxicity and effi cacy of intrathecal liposomal cytarabine (LC) were evaluated in children with central nervous system (CNS) relapsed/refractory acute leukemia/lymphoma. Thirty patients (male:female ratio 21:9; median age 9.4 years) with CNS relapsed/ resistant disease were treated with intrathecal LC at dosages adjusted for age. Twenty-seven (90%) patients simultaneously received systemic chemotherapy, including concurrent highdose cytarabine or methotrexate in 21 (70%) cases. Of 28 patients evaluable for response, 25 patients (89%) achieved CNS complete remission and three (11%) partial remission. The median number of intrathecal LC administrations per patient was 4. The cerebrospinal fl uid was cleared after a median of 3 intrathecal LC administrations. Neurological toxicity ≥ grade 3 occurred in four (13%) patients. No permanent sequelae were observed. The median overall survival was 20.9 months and the 5-year probability of survival was 46%. These encouraging data suggest that intrathecal LC is well tolerated and eff ective in children with relapsed/refractory CNS leukemia/lymphoma.

Purpose Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Monitoring minimal residual disease (MRD) by using real-time quantitative polymerase chain reaction (RQ-PCR) provides information for patient stratification and individual risk-directed treatment. Cooperative studies have documented that measurement of blast clearance from the bone marrow during and after induction therapy identifies patient populations with different risk of relapse. We explored the possible contribution of measurements of MRD during the course of treatment. Patients and Methods We used RQ-PCR to detect MRD in 110 unselected patients treated in Italy in the International Collaborative Treatment Protocol for Children and Adolescents With Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000). The trial took place in AIEOP centers during postinduction chemotherapy. Results were categorized as negative, low positive (below the quantitative range [< 5 × 10−4]), or high positive (≥ 5 × 10−4). Patients with at least one low-positive or high-positive result were assigned to the corresponding subgroup. Results Patients who tested high positive, low positive, or negative had significantly different cumulative incidences of leukemia relapse: 83.3%, 34.8%, and 8.6%, respectively (P < .001). Two thirds of positive cases were identified within 4 months after induction-consolidation therapy, suggesting that this time frame may be most suitable for cost-effective MRD monitoring, particularly in patients who did not clear their disease at the end of consolidation. Conclusion These findings provide further insights into the dynamic of MRD and the ongoing effort to define molecular relapse in childhood ALL.

Approximately 10–15% of pediatric patients with acute lymphoblastic leukemia (ALL) have a T-cell phenotype. The prognosis of these patients has improved over the last years, owing to the use of more intensive treatment strategies. Characterization of molecular alterations with prognostic impact in T-ALL may be of great help for an early identification of patients at high risk (HR) of failure in whom more intensive treatments, including allogeneic hematopoietic stem cell transplantation, may be considered.

Venous thromboembolic events (VTEs) are frequent complications of childhood acute lymphoblastic leukemia (ALL) treatment. The aim of the study was to evaluate the rate of symptomatic VTEs in children with ALL and the predictive value of clinical and biological factors and routine monitoring of coagulation parameters in identifying children at a higher risk of this complication.Between September 2000 and July 2006, 2042 children (≥1 and younger than 18 y) with newly diagnosed ALL were enrolled in Italy in the AIEOP (Italian Association of Pediatric Hematology and Oncology)-BFM (Berlin-Frankfurt-Muenster) ALL 2000 trial. Patients with symptomatic VTEs (deep venous thromboses or cerebral venous thromboses) were identified after a careful review of clinical records. The impact of coagulation derangement at the onset of VTEs was evaluated by a nested case-control study.Forty-eight (2.4%) children presented with a VTE. The rate of VTEs was higher in male patients (P=0.001); patients randomized to receive dexamethasone tended to have a higher rate of VTE compared with those who received prednisone (P=0.10). The coagulation derangement at the onset of VTE was not associated with VTE occurrence. The prevalence of a factor V Leiden G1691A mutation and the prothrombin G20210A variant was higher in children with VTE than that expected in the general population.

Despite the success in treating the majority of children with newly diagnosed acute leukemia, children with relapsed or refractory disease are an exceptionally difficult group of patients to cure. We assessed the combination of fludarabine with cytarabine and granulocyte colony-stimulating factor (FLAG) and non-pegylated liposomal doxorubicin (Myocet) in children with either acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) refractory to first-line therapy or who had relapsed after risk-tailored chemotherapy. We treated 35 patients with FLAG-Myocet. The median age at treatment was 9 years and 7 months (range, 1 to 18 y). The 94% of ALL patients (16/17) and the 61% AML patients (11/18) achieved complete remission after FLAG-Myocet. A partial response was observed in the 17% of AML patients (3/18). Twenty-eight of 35 (80%) patients received hematopoetic stem cell transplantation in remission induced by FLAG-Myocet regimen. The ALL and AML overall survival at 3 years after FLAG-Myocet is 33% and 38%, respectively. The probability of ALL and AML event-free survival at 3 years after FLAG-Myocet is 33% and 40%, respectively. The probability of ALL and AML disease-free survival at 3 years after hematopoietic stem cell transplantation is 19% and 58%, respectively. Non-hematological toxicity was remarkably low, while almost all patients showed severe hematological toxicity. FLAG-Myocet is an efficient and a well-tolerated regimen that allows nearly all patients to undergo hematopoetic stem cell transplantation. FLAG-Myocet proved to be safe in terms of acute cardiac toxicity although particular care must be taken to reduce infectious complications due to severe myelosuppression. The promising results shown in our study need to be confirmed by larger and possibly randomized trials.

One hundred ninety-eight children and adolescents were entered in the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP)-ALL95 study for high-risk acute lymphoblastic leukemia (ALL). Inclusion criteria were poor response to initial prednisone/intrathecal methotrexate (prednisone-poor response [PPR]), resistance to induction therapy, translocation t(9;22), infants with the t(4;11), or CD10− ALL. The event-free survival (EFS) rate at 4 years was 56.5% (SE, 3.9%) for the entire group. The overall EFS rate in the current study was significantly better (P = .002) than that obtained in a comparable group of patients treated in the early 1990s in the AIEOP-ALL91 study. In particular, patients with PPR had a 4-year EFS of 61.1% (SE, 4.4%) versus 42.8% (SE, 5.4%) in the ALL 91 study (P = .008). Among PPR patients, those who were PPR-only (60.1%)—that is, they achieved CR and were negative for t(9;22) and t(4;11) translocations—had the best outcomes with this intensive treatment, even when additional adverse features (hyperleukocytosis, T phenotype) were present (4-year EFS, 70.1%; SE, 4.7%). We attribute this improvement to the replacement of 6 alternating blocks of non–cross-resistant drugs with an 8-drug reinduction regimen (Berlin-Frankfurt-Muenster [BFM] protocol II), repeated twice, in the context of a standard BFM-type intensive chemotherapy for high-risk ALL. This modified therapy may lead to high cure rates for patients defined as at high risk for intrinsic resistance to corticosteroids only.

// Chiara Palmi 1 , Angela M. Savino 1 , Daniela Silvestri 2, 3 , Ilaria Bronzini 4 , Gunnar Cario 5 , Maddalena Paganin 4 , Barbara Buldini 4 , Marta Galbiati 1 , Martina U. Muckenthaler 6 , Cristina Bugarin 1 , Pamela Della Mina 7 , Stefan Nagel 8 , Elena Barisone 9 , Fiorina Casale 10 , Franco Locatelli 11 , Luca Lo Nigro 12 , Concetta Micalizzi 13 , Rosanna Parasole 14 , Andrea Pession 15 , Maria C. Putti 4 , Nicola Santoro 16 , Anna M. Testi 17 , Ottavio Ziino 18 , Andreas E. Kulozik 6 , Martin Zimmermann 19 , Martin Schrappe 5 , Antonello Villa 7 , Giuseppe Gaipa 1 , Giuseppe Basso 4 , Andrea Biondi 3 , Maria G. Valsecchi 2 , Martin Stanulla 19 , Valentino Conter 3 , Geertruy te Kronnie 4 , Giovanni Cazzaniga 1 1 Centro Ricerca M. Tettamanti, Clinica Pediatrica, Universita di Milano Bicocca, Fondazione MBBM/Ospedale San Gerardo, Monza, Italy 2 Center of Biostatistics for Clinical Epidemiology, Department of Health Sciences, University of Milano-Bicocca, Milan, Italy 3 Clinica Pediatrica, Universita di Milano Bicocca, Fondazione MBBM/Ospedale San Gerardo, Monza, Italy 4 Laboratory of Onco-Hematology, Department SDB, Universita di Padova, Padova, Italy 5 Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 6 Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg and EMBL/Medical Faculty Molecular Medicine Partnership Unit, Heidelberg, Germany 7 Microscopy and Image Analysis Consortium, Universita di Milano-Bicocca, Monza, Italy 8 Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 9 Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, Regina Margherita Children’s Hospital, Turin, Italy 10 Pediatric Oncology Service, Pediatric Department of 2nd University of Naples, Naples, Italy 11 Department of Pediatric Hematology/Oncology, IRCCS Ospedale Bambino Gesu, Rome - University of Pavia, Pavia, Italy 12 Center of Pediatric Hematology Oncology, Azienda Ospedaliero-Universitaria “Policlinico Vittorio Emanuele”, Catania, Italy 13 Hematology/Oncology Unit, G. Gaslini Children’s Hospital, Genoa, Italy 14 Department of Pediatric Hemato-Oncology, Ospedale Pausilipon, Napoli, Italy 15 Department of Pediatrics, “Lalla Seragnoli” Hematology-Oncology Unit, University of Bologna, Bologna, Italy 16 Department of Pediatrics, Division of Pediatric Hematology-Oncology, University “A. Moro” of Bari, Bari, Italy 17 Division of Hematology, Department of Biotechnologies and Hematology, “Sapienza” University of Rome, Rome, Italy 18 Pediatric Hematology and Oncology Unit, A.R.N.A.S. Civico, Di Cristina and Benfratelli Hospital, Palermo, Italy 19 Department of Paediatric Haematology and Oncology, Hannover Medical School, Hannover, Germany Correspondence to: Giovanni Cazzaniga, email: gianni.cazzaniga@hsgerardo.org Andrea Biondi, email: abiondi.unimib@gmail.com Keywords: CRLF2, pediatric leukemia, T acute lymphoblastic leukemia, prognostic marker, high risk Received: May 20, 2016 Accepted: July 01, 2016 Published: July 15, 2016 ABSTRACT Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL. We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers. Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median ( CRLF2-high ); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated. Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib. In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway.