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Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for most children with juvenile myelomonocytic leukemia (JMML). Novel therapies controlling the disorder prior to HSCT are needed. We conducted a phase 2, multicenter, open-label study to evaluate the safety and antileukemic activity of azacitidine monotherapy prior to HSCT in newly diagnosed JMML patients. Eighteen patients enrolled from September 2015 to November 2017 were treated with azacitidine (75 mg/m2) administered IV once daily on days 1 to 7 of a 28-day cycle. The primary end point was the number of patients with clinical complete remission (cCR) or clinical partial remission (cPR) after 3 cycles of therapy. Pharmacokinetics, genome-wide DNA-methylation levels, and variant allele frequencies of leukemia-specific index mutations were also analyzed. Sixteen patients completed 3 cycles and 5 patients completed 6 cycles. After 3 cycles, 11 patients (61%) were in cPR and 7 (39%) had progressive disease. Six of 16 patients (38%) who needed platelet transfusions were transfusion-free after 3 cycles. All 7 patients with intermediate- or low-methylation signatures in genome-wide DNA-methylation studies achieved cPR. Seventeen patients received HSCT; 14 (82%) were leukemia-free at a median follow-up of 23.8 months (range, 7.0-39.3 months) after HSCT. Azacitidine was well tolerated and plasma concentration-–time profiles were similar to observed profiles in adults. In conclusion, azacitidine monotherapy is a suitable option for children with newly diagnosed JMML. Although long-term safety and efficacy remain to be fully elucidated in this population, these data demonstrate that azacitidine provides valuable clinical benefit to JMML patients prior to HSCT. This trial was registered at www.clinicaltrials.gov as #NCT02447666.

Acute promyelocytic leukaemia (APL) is a rare subtype ofchildhood acute myeloid leukaemia (AML), accounting for5–12% of all AML cases. Treatment with all-trans-retinoicacid (ATRA) and anthracyclines yields complete remission(CR) rates >90% and 10-year event-free survival (EFS) ratesaround 80%.1–3The recent front-line arsenic trioxide(ATO)-based regimens have further improved the APL out-come by reducing toxicity and preventing relapse.4–6Relapsed APL patients are a rare and heterogeneous group,whose prognosis depends on front-line treatment and timeelapsing between diagnosis and recurrence. Currently, ATOis the treatment of choice for relapsed APL. Due to the rarityof relapsed childhood APL, 17–27% in ATRA- andchemotherapy-1,3,7–9and 4% in ATO-containing regimens,10it will be difficult, in the future, to design future prospectivecomparative trials for these patients. In an attempt to designtherapeutic guidelines, recommendations on management ofrelapsed/refractory paediatric APL have been publishedrecently

The aim of this study was to investigate the association of neuroimaging, clinical, and laboratory findings in children with different underlying diseases who developed posterior reversible encephalopathy syndrome (PRES). All consecutive pediatric patients referred to our institute between 2000 and 2017 were retrospectively evaluated for newly diagnosed PRES. Clinical data, medical history, and therapies administered at the time of PRES and laboratory findings were reviewed as the magnetic resonance imaging (MRI) obtained at PRES presentation and during follow-up. Statistics included Mann–Whitney U, Chi-square, and Fisher's exact tests. A total of 39 pediatric patients (25 males, median age = 8.8 years) with a confirmed diagnosis of PRES were included. The patients were divided into four groups based on the underlying disease: (1) patients with hemato-oncological diseases not transplanted (n = 15, 38.5%), (2) who underwent hematopoietic stem cell transplantation (HSCT; n = 12, 30.8%), (3) affected by renal (n = 8, 20.5%), and (4) by autoimmune diseases (n = 4, 10.2%). Hemato-oncological patients and those undergoing HSCT presented a higher incidence of involvement of deep gray matter structures and hemorrhagic lesions when compared with patients with renal and autoimmune disorders (p = 0.036 and p = 0.036, respectively). No differences emerged among different groups of patients regarding presenting PRES symptoms, arterial blood pressure, laboratory findings, and extent of MRI abnormalities and these parameters did not show association with outcome.In conclusion, in pediatric patients with different diseases who develop PRES, involvement of deep gray matter structures and hemorrhagic lesions are more common in hemato-oncological patients and those undergoing HSCT. Clinical and radiologic outcome is favorable independently of clinical data and extent of MRI abnormalities.

Importance: Blinatumomab is a CD3/CD19-directed bispecific T-cell engager molecule with efficacy in children with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Objective: To evaluate event-free survival in children with high-risk first-relapse B-ALL after a third consolidation course with blinatumomab vs consolidation chemotherapy before allogeneic hematopoietic stem cell transplant. Design, Setting, and Participants: In this randomized phase 3 clinical trial, patients were enrolled November 2015 to July 2019 (data cutoff, July 17, 2019). Investigators at 47 centers in 13 countries enrolled children older than 28 days and younger than 18 years with high-risk first-relapse B-ALL in morphologic complete remission (M1 marrow

Background Anti-N-methyl- d -aspartate receptor encephalitis is a central nervous system inflammatory autoimmune disease affecting adults and children. The use of first- and second-line immunotherapies is supported. Recent reports suggest the efficacy of bortezomib in severe anti-N-methyl- d -aspartate encephalitis in adult patients not responsive to second-line treatment; there are no data about pediatric patients. patient description We describe an eight-year-old child with anti-N-methyl- d -aspartate encephalitis not responsive to first- and second-line treatments who experienced marked clinical improvement after bortezomib administration. Discussion Bortezomib is a selective and reversible inhibitor of the 26S proteasome, which is used to treat oncologic and rare autoimmune disorders in pediatric patients. As observed in adult patients, bortezomib administration induced anti-N-methyl- d -aspartate antibody titer decline and clinical improvement with an acceptable risk profile. Conclusion This is the first report of the use of bortezomib in children with anti-N-methyl- d -aspartate encephalitis; it could be a useful therapeutic option in children with refractory anti-N-methyl- d -aspartate encephalitis.

Key Points Immunological dysregulation may underlie unusual autoimmune diseases, which also deserve to be investigated from a genetic point of view.

Introduction Advances in paediatric oncology led to the increase in long-term survival, revealing the burden of therapy-related long-term side effects. We evaluated overall and cause-specific mortality in a large cohort of Italian childhood cancer survivors (CCSs) and adolescent cancer survivors identified through the off-therapy registry. Materials and methods CCSs alive 5 years after cancer diagnosis occurring between 1960 and 1999 were eligible; the last follow-up was between 2011 and 2014. Outcomes were reported as standardised mortality ratios (SMRs) and absolute excess risks (AERs). Results Among 12,214 CCSs, 1113 (9.1%) deaths occurred. Survival at 35 years since diagnosis was 87% (95% confidence interval [CI]: 86–88) and at 45 years was 81% (95% CI: 77–84). CCSs had an 11-fold increased risk of death (SMR 95% CI: 10.7–12), corresponding to an AER of 48 (95% CI: 45–51). Mortality decreased by 60% for survivors treated most recently (1990–1999). The most frequent causes of death were recurrence of the original cancer (56%), a subsequent neoplasm (19%) and cardiovascular diseases (5.8%). Among those who survived at least 15 years after diagnosis, a secondary malignancy was the leading cause of death. Conclusions This study confirms the impact of recent advances in anticancer therapy in reducing mortality, mainly attributable to recurrence but also to other causes. However, overall mortality continues to be higher than in the general population. A long-term follow-up is needed to prevent late mortality due to secondary neoplasms and non-neoplastic causes in CCSs.

Autoimmune lymphoproliferative syndrome (ALPS) is a congenital disorder that results in an apoptosis impairment of lymphocytes, leading to chronic lymphoproliferation and autoimmunity, mainly autoimmune cytopenias. FAS gene defects are often responsible for the disease, the phenotype of which can vary from asymptomatic/mild forms to severe disease. More rarely, defects are associated to other genes involved in apoptosis pathway, such as CASP10. Few data are available on CASP10-mutated patients. To date, two CASP10 mutations have been recognized as pathogenic (I406L and L258F) and others have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l). In this study, we evaluated apoptosis function in patients with an ALPS/ALPS-like phenotype carrying CASP10 variants. Molecular findings were obtained by next generation sequencing analysis of genes involved in immune dysregulation syndromes. Functional studies were performed after inducing apoptosis by FAS-ligand/TRIAL stimulation and analysing cell death and the function of CASP10, CASP8 and PARP proteins. We identified 6 patients with an ALPS (n = 2) or ALPS-like (n = 4) phenotype, carrying I406L (n = 1),V410l (n = 2),Y446C (n = 1) heterozygous CASP10 variants or the L522l polymorphisms (n = 2) associated with another polymorphic homozygote variant on CASP8 or a compound heterozygous mutation on TNFRSF13C. Apoptosis was impaired in all patients showing that such variants may play a role in the development of clinical phenotype.

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

OBJECTIVE: To develop and validate a diagnostic score that assists in discriminating primary hemophagocytic lymphohistiocytosis (pHLH) from macrophage activation syndrome (MAS) related to systemic juvenile idiopathic arthritis. STUDY DESIGN: The clinical, laboratory, and histopathologic features of 362 patients with MAS and 258 patients with pHLH were collected in a multinational collaborative study. Eighty percent of the population was assessed to develop the score and the remaining 20% constituted the validation sample. Variables that entered the best fitted model of logistic regression were assigned a score, based on their statistical weight. The MAS/HLH (MH) score was made up with the individual scores of selected variables. The cutoff in the MH score that discriminated pHLH from MAS best was calculated by means of receiver operating characteristic curve analysis. Score performance was examined in both developmental and validation samples. RESULTS: Six variables composed the MH score: age at onset, neutrophil count, fibrinogen, splenomegaly, platelet count, and hemoglobin. The MH score ranged from 0 to 123, and its median value was 97 (1st-3rd quartile 75-123) and 12 (1st-3rd quartile 11-34) in pHLH and MAS, respectively. The probability of a diagnosis of pHLH ranged from 99% for a score of =123. A cutoff value of =60 revealed the best performance in discriminating pHLH from MAS. CONCLUSION: The MH score is a powerful tool that may aid practitioners to identify patients who are more likely to have pHLH and, thus, could be prioritized for functional and genetic testing.

To the editor: Reinduction therapy (also known as delayed intensification, DI) is an essential part of childhood acute lymphoblastic leukemia (ALL) treatment.[1][1],[2][2] In Berlin-Frankfurt-Munster (BFM) studies, it includes same/similar drugs as those employed in induction therapy. The Children

Febrile neutropenia (FN) represents a life-threatening complication in hematological malignancies. Its etiology is most often due to infections even though FN of other origins, such as tumor-related fever and non-infectious inflammation, should rapidly be ruled out. Initially, C-reactive protein and, more recently, procalcitonin (PCT) have been proposed as useful biomarkers for differential diagnosis. PCT was shown to be a good biomarker of bacterial infections and their clinical outcomes. Definition of standard cut-offs and design of PCT-guided treatment protocols remain however to be defined. Areas covered: In this review, highlights on the current clinical use of PCT and its potential role as a diagnostic tool have been discussed by a panel of physicians from different areas of expertise. We provide current clinical evidence that PCT has been shown to be a reliable biomarker to differentiate fever of bacterial origin from other causes. Moreover, the Authors convened to a round-table to discuss their 'real-life experience' and offer their recommendations by a Delphi survey. Expert commentary: PCT has an important clinical role in FN. Issues such as the validation of a specific decision algorithm that includes PCT to monitor antibiotic choice and treatment duration will be addressed in prospective studies.

To the editor: Essential thrombocythemia (ET) is a myeloproliferative neoplasm (MPN) that mainly affects middle-aged patients. Although pediatric cases occur, they are rare, and their molecular features considerably differ from the adult counterparts: JAK2 V617F mutation occurs in only 25% of cases

Before 2000, allogeneic hematopoietic stem cell transplantation (HSCT) was the only potentially curative treatment for children with chronic myeloid leukemia (CML) in chronic phase (CP). The availability of imatinib (IM) and thereafter of second-generation (2G) tyrosine kinase inhibitors (TKIs) has extended therapeutic options for childhood CML. Experience with IM in managing children and adolescents with CP-CML resistant or intolerant to interferon treatment treated prompted us to outline guidelines with the aim of offering a uniform management approach for this category of patients (pts). IM was scheduled in newly diagnosed pts with CML in CP, including those with an HLA identical sibling (HLA+). Cytogenetics on bone marrow (BM) and molecular analysis on peripheral blood (PB) and on BM were suggested at planned time-points. These guidelines have been shared with both pediatric referral centers (P Centers) and reference center for adults and children (AP Centers) in Italy. The guidelines, summarized in Figure 1, have been updated over the years (yrs), with the contribution of colleagues managing adult CML. Fifty-seven CP-CML pts with a median age of 11.4 yrs, diagnosed between March 2001 and June 2015, were managed at 9 P centers (34 pts) and at 4 AP Centers (23 pts). All pts, including 16 with an HLA+, started IM that was discontinued early in 2 pts due to toxicity. HSCT from an HLA+ donor was performed in 2 pts in CP. A bone marrow aspirate at 3, 6, 9, 12, 18, 24 months was carried out in 85.5%, 98%, 83.5%, 100%, 95% and 94% of pts, respectively. Rates of molecular assessments on BM at 3, 6, 9, 12, 18 and 24 months were 73%, 78%, 92%, 84%, 86% and 100% of pts, respectively, significantly higher at 6 and 9 months in pts followed at P centers compared to those followed at AP centers. Cytogenetic evaluation rates at the same time points were 69%, 80%, 81%, 82% 73% and 75%, respectively, significantly higher in pts followed at AP centers compared to those followed at P centers. Rates of molecular assessments on PB at 3, 6, 9, 12, 18 and 24 months were 50%, 54%, 57%, 60%, 64% and 69% of pts, respectively, higher in pts followed at AP centers compared to those followed at P centers. Four pts (1 with HLA+) resistant to IM were rescued with 2G TKI and 3 of them then underwent a HSCT. Fifty pts (92.5%), including 13 HLA+ pts, achieved a cytogenetic or molecular (MR). Seven of the 50 responders (14%) underwent a HSCT from an HLA+ (6 pts) or from a matched unrelated donor (MUD), all in P centers before 2009. Seven HLA+ pts continued IM: in 3 it was given intermittently and thereafter discontinued in 1. Among the 36 responders without a HLA-identical sibling (HLA-), 10 pts (persistent MR, 2 pts; late side effects, 5 pts; poor compliance, 3pts) started intermittent IM, that was discontinued in 3 of them with deep MR. Overall, 18/36 (50%) pts failed IM: 2 had late toxicity and 16 (44%) developed disease recurrence (5 on intermittent IM and 1 pt after IM discontinuation). BCR-ABL1 mutation analysis was carried out in 11 pts: BCR-ABL1E255K combined with BCR-ABL1F359Vmutations were found in 1 pt and BCR-ABL135INS in 5 pts. Eleven pts were rescued with 2G TKIs (Dasatinib, 5 pts; Nilotinib, 6 pts), 5 pts restarted continuous IM and 2pts underwent a HSCT from a MUD. All 57 pts are alive at a median follow-up of 72 months (range, 12-184): 15 pts (26%) after a HSCT (HLA+, 9 pts; MUD, 6), 4 pts (7%) are treatment-free (1 pt after nilotinib), 27 pts (47.5%) are still receiving IM (continuous, 22 pts; intermittent, 5 pts), 10 pts (17.5%) are on treatment with 2G TKIs (dasatinib, 5 pts; nilotinib, 5 pts) and 1 pt with BCR-ABL1E255K and BCR-ABL1F359Vmutations is on third-line treatment with ponatinib. Our experience shows that pediatric guidelines are an important management tool for children and adolescents with CP-CML. Over the years, exchange of experience for monitoring and treatment of pediatric CP-CML with colleagues managing adult pts has contributed to update management strategies for this particular category of pts. Furthermore, exchange of knowledge and debate concerning some clinical situations within our network has allowed to have a progressively uniform approach for children and adolescents followed in these 13 Italian centers. Taking into account the rarity of the condition, shared pediatric guidelines and exchanges of experience and knowledge with adult hematologists will further contribute to an optimal management of childhood CML. Disclosures Biondi: BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board. Saglio:Ariad: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy. Foà:BMS: Consultancy; Ariad: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy; Gilead: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

POI is a relevant late complication after HSCT and occurring more frequently after MAC than after RIC regimens. Reports on the frequency of POI after RIC in a large pediatric and adolescent population are lacking. In this study, we describe a girl affected by CML diagnosed at the age of 15 yr and treated with oncarbide and interferon followed by imatinib and dasatinib. She had two pregnancies shortly after RIC performed according to the CML-SCT I-BFM protocol including TT, FLU, and MEL. Hypergonadotropic hypogonadism occurred four months after HSCT; menstruations resumed regularly six months after HSCT. Eight and 20 months after HSCT, the patient became pregnant and then delivered, respectively, two babies at term by cesarean section. Both newborns had no neonatal complications. Donor chimerism at time of two pregnancies and five yr after transplantation demonstrated complete donor engraftment. These findings suggest that I-BFM CML-SCT protocol could be a promising treatment option for adolescents or young adults with CML eligible for HSCT.

Imatinib mesylate (IM) is used for the management of childhood chronic myeloid leukaemia (CML). The most effective dosage of IM and its long-term efficacy in children are not well defined. The purpose of this multicentre study is to report on the long-term results of high-dose IM (340 mg/m2 /d) in CML patients in chronic phase (CP-CML) aged

Background Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening, heterogeneous, hyperinflammmatory disorder. Prompt identification of inherited forms resulting from mutation in genes involved in cellular cytotoxicity can be crucial. X-linked lymphoproliferative disease 1 (XLP1), due to mutations in SH2D1A (Xq25) encoding signaling lymphocyte activation molecule–associated protein (SAP), may present with HLH. Defective SAP induces paradoxical inhibitory function of the 2B4 coreceptor and impaired natural killer (NK) (and T) cell response against EBV-infected cells. Objective To characterize a cohort of patients with HLH and XLP1 for SAP expression and 2B4 function in lymphocytes, proposing a rapid diagnostic screening to direct mutation analysis. Methods We set up rapid assays for 2B4 function (degranulation or 51 Cr-release) to be combined with intracellular SAP expression in peripheral blood NK cells. We studied 12 patients with confirmed mutation in SH2D1A and some family members. Results The combined phenotypic/functional assays allowed efficient and complete diagnostic evaluation of all patients with XLP1, thus directing mutation analysis and treatment. Nine cases were SAP − , 2 expressed SAP with mean relative fluorescence intensity values below the range of healthy controls (SAP dull ), and 1, carrying the R55L mutation, was SAP + . NK cells from all patients showed inhibitory 2B4 function and defective killing of B-EBV cells. Carriers with SH2D1A mutations abolishing SAP expression and low percentage of SAP + cells showed neutral 2B4 function at the polyclonal NK cell level. Three novel SH2D1A mutations have been identified. Conclusions Study of SAP expression is specific but may have insufficient sensitivity for screening XLP1 as a single tool. Combination with 2B4 functional assay allows identification of all cases.

Imatinib (IM) is an established first-line treatment for children with chronic myeloid leukemia (CML). However, the most effective dosage of IM and overall management of newly-diagnosed childhood CML in chronic phase (CP) are not well defined. This study was designed to evaluate (a) the response to IM at a dosage equivalent in terms of drug exposure to the 600 mg daily utilized in adults and (b) the long-term outcome in newly-diagnosed children and adolescents with CML. Patients aged Between December 2002 and February 2014, 41 CML patients in CP (females: 13, males: 28; age 24 months. IM was started in all patients, including 16 with an HLA-matched sibling. The dosage of IM was modulated according to the occurrence of >2 WHO side-effects or response, mainly during the first 6 months of treatment. Forty patients are evaluable for treatment results. Median administered dosage of IM was 309 mg/m2/day, higher in males than in females (326 mg/m2vs 245 mg/m2, p .015) and in those younger than 10 years (314 mg/m2vs 262 mg/m2). Twenty-four patients (60%) experienced isolated or combined side effects: hematologic toxicity (medullary hypoplasia [n=1], neutropenia [n=7] and/or thrombocytopenia [n=6], anemia [n=1]) and/or extra-hematologic toxicity (arthralgia/myalgia [n=8], nausea [n=1], vomiting [n=1], diarrhea [n=1], abdominal pain [n=2], hepatitis [n=1], skin rash [n=2]. Persistent >3 WHO adverse events led to IM discontinuation in 6 patients (15%). At 3 months of IM treatment, hematologic response and PCyR rates were 91% and 54%, respectively; BCR-ABL1 transcript levels 7years successfully discontinued IM and 2 patients resumed continuous IM because of an increased BCR-ABL transcript. IM was interrupted in 8/33 (24%) responder patients, 4 of them in BCR-ABL1 IS In our experience, IM at a daily dose of 340 mg/m2 is effective in newly-diagnosed CML children with responses rates higher than those reported in children treated with IM at lower dosage. Considering the long-term follow-up, high-dose IM allowed to safely discontinue treatment in some patients with a deep MR; furthermore, it did not worsen the outcome both in patients submitted to a SCT and in those with disease progression or side-effects. Disclosures Saglio: BMS: Consultancy, Fees for occasional speeches Other; Novartis: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; Pfizer: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; ARIAD: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other.

Acute lymphoblastic leukemia (ALL) is the major pediatric cancer. At diagnosis, the developmental timing of mutations contributing critically to clonal diversification and selection can be buried in the leukemia's covert natural history. Concordance of ALL in monozygotic, monochorionic twins is a consequence of intraplacental spread of an initiated preleukemic clone. Studying monozygotic twins with ALL provides a unique means of uncovering the timeline of mutations contributing to clonal evolution, pre- and postnatally. We sequenced the whole genomes of leukemic cells from two twin pairs with ALL to comprehensively characterize acquired somatic mutations in ALL, elucidating the developmental timing of all genetic lesions. Shared, prenatal, coding-region single-nucleotide variants were limited to the putative initiating lesions. All other nonsynonymous single-nucleotide variants were distinct between tumors and, therefore, secondary and postnatal. These changes occurred in a background of noncoding mutational changes that were almost entirely discordant in twin pairs and likely passenger mutations acquired during leukemic cell proliferation.

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.

Natural history of acute lymphoblastic leukemia in neurofibromatosis type 1 monozygotic twins

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59-100%) and a specificity of 87% (CI 70-96%). Positive and negative predictive values were 71% (CI 42-92%) and 96% (CI 82-100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria. © 2013 Macmillan Publishers Limited. All rights reserved.

Imatinib (IM) is an established first-line treatment for children with chronic myeloid leukemia (CML). However, the most effective dosage and duration of IM treatment are not well defined. This study was designed to evaluate the response to high-dose IM and long-term outcome in pediatric CML patients, previously untreated or resistant to IFN. Patients aged From March 2001 to February 2013, 45 CML patients in CP (18 females, 27 males; median age: 119/12 years) were recorded from 9 Italian pediatric centers. Eight patients had previously received IFN. IM was started in all patients, including 16 with an HLA-matched sibling. The dosage was modulated according to hematologic toxicity and/or appearance of WHO >2 side- effects, mostly during the first 6 months of treatment (median administered dose: 309 mg/m2/day). Hematologic toxicity (medullary hypoplasia[n=1], neutropenia [n=11] and/or thrombocytopenia [n=6], anemia [n=1]) was observed in14/44 evaluable patients (32%); 13 patients (29.5%) experienced isolated or combined side effects: arthralgia/myalgia (n=10), nausea (n=1), vomiting (n=1), diarrhea (n=1), hepatitis (n=1), edema (n=1). After 3 months of IM treatment, 7/25 of tested patients (28%) obtained complete cytogenetic response (CCyR). Overall, 34/36 evaluable patients (94%) obtained a CCyR at a median time of 6 months. A molecular response (12 months received IM at the same daily dosage for 3 weeks a month (intermittent therapy). IM given without interruption was resumed in 3 of these 9 patients because of an increased BCR-ABL transcript. The growth rate showed a delay in height, which recovered over time, in 6 children who received IM prior to puberty. Overall, IM was stopped in 22/44 evaluable patients (50%) because of various reasons: stem cell transplant (SCT) in 8 patients (3 in CP, 1 in CCyR, 3 in MMR, 1 in CMR); hematologic (n=2) or extra-hematologic toxicity (n=2) (WHO grading >3) during the first 6 months in 4; recurrent disease in 6 (3 increased BCR-ABL transcript, 2 cytogenetic relapse,1 blast crisis [BC]); no response in 1; CMR ( 88 months in 2 and pancreatitis in 1 patient in CMR for 75 months. Twelve patients underwent a SCT after a median time of 8 months: 8 from an identical siblings (5 responders to IM [1 CMR, 3 MMR, 1 CCyR]), 3 MUD (2 in CCyR and 1 in MMR) and 1 cord blood in CCyR after chemotherapy for CB. Three patients, transplanted from an identical sibling, had disease recurrence after 24 (molecular relapse), 36 (cytogenetic relapse) and 83 (BC) months, respectively. At the last follow-up, all patients are alive (CMR= 25; MMR=14; CCyR=3; minor CyR=2; too early=1) at a median of 52 months (range: 3-146). Of 42 patients evaluable for treatment, 23 are receiving IM at a dosage of 340 mg/m2 (4 intermittent IM); 11 are in CMR without any treatment (8 after SCT; 3 at 32, 33 and 50 months after IM discontinuation); 4 are in treatment with dasatinib, 3 with nilotinib and 1 with IFN. In our experience, higher dose IM is an effective treatment for childhood CP CML, associated with sustained responses. Moreover, IM can be safely discontinued in pediatric CML patients with a deep CMR lasting more than 7 years. For patients candidates to a SCT, IM provides a safe bridging option to transplant, at no increased risk. Since patients may lose their response, a close and regular monitoring should be performed, mostly for those who stop IM, as SCT and new TK inhibitors may be successfully employed in patients failing IM. Disclosures: Saglio: Celgene: Consultancy, Honoraria; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Abstract 3586 Introduction: Acute megakaryoblastic leukemia (AMKL) is a biologically heterogeneous variant of acute myeloid leukemia (AML), known as AML-M7 according to the FAB classification. The incidence of AML-M7 among pediatric AML in previously published studies ranged between 7 and 15% of the whole number. Differently from children with Down Syndrome (DS)-AMKL who have an excellent outcome with specifically designed, low-intensity chemotherapy, some studies have reported that the outcome of children with non-DS-AMKL is poor (Ribeiro et al, 1993; Athale et al, 2001). We analyzed the clinical/biological characteristics and outcome of children with non-DS-AMKL treated with the AIEOP AML 2002/01 Protocol. Patients and Methods: Between 12/2002 and 06/2011, 44 patients (pts) were included in the analysis. They were assigned to the high-risk (HR) group. Treatment was administered as follows: pts were given 2 courses of 7-day induction therapy (idarubicin, cytarabine and etoposide); if they obtained first complete remission (CR1), they proceeded to receive 2 consolidation courses based on high-dose cytarabine (HD-Ara-c), combined with either etoposide during the first course (AVE 3+4) or mitoxantrone during the second course (HAM 3+2). At the end of consolidation, pts with AMKL in CR1 were eligible to be treated with allogeneic (ALLO) HSCT from a HLA-identical relative, if available, or from alternative donors, namely unrelated donors or HLA-mismatched relatives. We analyzed CR, early death (ED) and induction failure (IF) rate of pts with AMKL; moreover, we calculated the cumulative incidence of relapse and the probability of both overall survival (OS) and event-free survival (EFS). Results: AMKL was diagnosed in 44 of 482 (9.1%) children and adolescents below 18 years of age without DS enrolled in the AIEOP AML 2002/01 protocol. Twenty-one pts were males and 23 females. Median age at diagnosis was 1.6 years (range 0.3–15), with 12/44 (27%) pts aged < 1 year. The median count of white blood cells (WBC) at diagnosis was 18,910×103/ml (range 2,490–59,000). Involvement of central nervous system (CNS) at diagnosis was documented in 5/44 (11%), whereas extramedullary disease in sites other than CNS was found in 3/44 pts (7%). Cytogenetic results were available in 34/44 (77%) pts; they showed t(1;22)(p13;q13) in 3/34 (9%) pts, either as sole abnormality or together with other numerical and structural changes and 11q23 rearrangements in 4/34 (12%). Complex karyotype was present in 5/34 (15%) pts. Three out of 44 pts (7%) showed FLT3-ITD mutation. The induction treatment outcome of pts with AMKL was as follows: the CR rate was 86%, while ED and IF rates were 5% and 9%, respectively. The cumulative incidence of leukemia recurrence was 27% [standard error (SE) 8.7]. Thirty out of 44 pts with AMKL (68%) received an ALLO HSCT in first CR. With a median follow up of 57 months (range 3–130), the 8-year OS and EFS of AMKL were 58.4% (SE 7.9) and 55.6% (SE 7.7), respectively, these values being similar to those of children with other variants of AML. Conclusions: Although AMKL is generally considered an AML variant characterized by a poor prognosis, our data indicate that an intensive treatment strategy including a wide use of HSCT in first CR results in a favorable outcome, more than 50% of pts being alive and disease-free. The main cause of treatment failure remains leukemia recurrence; once relapsed, children with AMKL have a dismal probability of being rescued by second-line treatment. Overall, our results compare favorably with previously published data on AMKL, including those recently reported by Rubnitz et al (Lancet Oncol 2010;11:543–52). Disclosures: No relevant conflicts of interest to declare.

Clofarabine is a promising new chemotherapeutic agent that is active in the treatment of pediatric acute leukemia. Forty children (16 with acute myeloid leukemia [AML], 24 with acute lymphoblastic leukemia [ALL]), aged 1-20 years (median 7.6 years) with relapsed or refractory ALL or AML were treated because of resistance to first-line treatment (n =5), or for first (n =22), second (n =11) or third relapse (n =2). They received clofarabine (40 mg/m(2)/day) associated with etoposide (100 mg/m(2)/day) and cyclophosphamide (440 mg/m(2)/day) administered as one or two induction cycles (5 days of chemotherapy) in an attempt to reach complete remission (CR) or CR without platelet recovery (CRp). This was followed by 1-3 consolidation cycles (4 days of chemotherapy) for a maximum of four cycles. Seven (44%) out of 16 and 10 (42%) out of 24 evaluable children with AML and ALL, respectively, responded to treatment. The most common adverse events were infections and gastrointestinal and hepatic toxicity. Thirteen (76%) out of 17 responders underwent hematopoietic stem cell transplant. The 24-month overall survival was 25%, while it was 59% among patients who responded to the first induction cycle. Our study suggests that this drug regimen is well tolerated and can be effective in heavily pretreated pediatric patients with relapsed or refractory acute leukemia.

Abstract 2414 Introduction: Neurofibromatosis 1 (NF1) is an autosomal dominant disease, caused by mutation of neurofibromin (NF1) gene, located in the 17q11.2 chromosomal region. Children affected by NF1 have an increased risk to develop tumors, including leukemia. It has been suggested that NF1 aberrancy is a tumor predisposing factor, and that secondary events in somatic cells gives rise to the formation of neoplasm. However, the clonal evolution from NF1 mutation to tumor development has never been elucidated. Monozygotic twins constitute an ideal tool to understand the clonal evolution of leukemia in the context of a common genetic background. Patients: We performed the genomic characterization of a pair of monozygotic twins with diagnosis of NF1, who developed concordant B-cell precursor ALL at the age of 6 (Twin 1, T1) and 6.5 years (T2). The diagnostic and relapse samples showed common and unique lesions, allowing us to hypothesize a model for leukemia clonal evolution. At diagnosis, T1 had common ALL with 45,XX,-7,del(9)(p12),del(10)(q23)[8]/46,XX[12] whereas T2 had common ALL with 45,XX,-7,del(10)(q22)[14]. Both were treated according to AIEOP-BFM ALL 2000 protocol; T1 was classified as MRD high risk (HR), and T2 as MRD Intermediate risk (IR). T1 underwent BMT 8 months after diagnosis, and achieved clinical remission, still persisting, whilst T2 relapsed 13 months after diagnosis with 47,XX,del(9)(p21),+21[11]/46,XX[5]; she entered AIEOP REC 2003 protocol and, after achieving second complete remission and negativity for MRD, underwent allogeneic HLA-compatible BMT; she is in clinical remission 3.5 years after BMT. Results: Genome-wide copy number alteration (CNA) analysis by Cytogenetics Whole Genome 2.7M Arrays (Affymetrix) indicated that T1 and T2 shared a copy number neutral Loss Of Heterozygosity (LOH) of 17q arm, were the NF1 gene is located. At diagnosis, both had chromosome 7 monosomy but showed completely different Ig/TCR rearrangements. Three additional clonal rearrangements were found in T2 relapse sample. By backtracking the rearrangements, the relapse clone was detected in about 1% of T2 diagnostic cells, but not in T1 diagnosis sample. Several twin-specific abnormalities were detected: both had a deletion involving the q arm of chromosome 10, with different extent: del(10)(q23.33) in T1 and del(10)(q23.1) in T2. Moreover, T1 carried hemizygous deletions on chromosome 9 and a focal loss of 6q15 locus, containing the B-cell specific transcriptional regulator BACH2. Genomic analysis of T2 revealed a gain in the telomeric region of chromosome 2 and heterozygous losses in 14q32.13 and 15q21.3 regions, involving BX247990 (human full-length cDNA clone of B cells) and TCF12 (transcription factor 12) genes, respectively. The T2 relapse showed LOH 17q and del(15)(q21.3), as found in her diagnostic sample, and several additional CNAs, including the heterozygous deletions of IKZF1 (7p12.2), ETV6 (12p13.2) and C20orf94 (20p12.2) genes, trisomy of chromosome 21 and LOH of chromosome 20. Discussion: This is the first study that describes the natural history of events in NF1 patients (with 17q LOH) who developed ALL. The unique feature common to both twins in diagnosis and relapse samples was 17q LOH, that probably arose prenatally in a common hematopoietic progenitor (before somatic recombination) of one twin and spread to the other twin through intraplacental circulation. After birth, an independent clonal evolution is sustained by different Ig/TCR rearrangements as well as specific and different oncogenetic lesions. This model support a preleukemic state followed by at least-two-step mechanism for progression to leukemia. The absence of monosomy 7 at relapse indicates an independent and postnatal acquisition of this lesion; and that relapse in T2 occurs from a pre-leukemic clone before monosomy 7, which further evolved through acquisition of additional abnormailities, commom in BCP-ALL (i.e. ETV6 and Ikaros deletions). The role of BACH2 and TCF12 gene deletions in NF1 and ALL must be further explored. BACH2 is a human B-cell specific transcriptional repressor already described as tumor suppressor gene, involved in IgH@ translocations. TCF12 (also named HEB) is a basic helix-loop-helix transcription factor involved in B and T cell commitment. The altered expression of these genes could contribute to the differentiation arrest and the uncontrolled proliferation of leukemic blasts. Disclosures: No relevant conflicts of interest to declare.

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 Conclusions: These data show that AIEOP-BFM ALL 2000 HR therapy is very effective for patients defined at HR for PPR. These patients could thus be considered for some treatment reduction, i.e. sparing most toxic therapeutic elements such as CRT. MRD identifies patients at HR (8.7% of the analysed population, 42% of HR patients), who have a poor outcome despite receiving intensive BFM treatment, including HSCT, and who may thus be eligible for innovative therapies. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 17 Introduction: The prognosis of paediatric patients (pts) with Acute Myeloid Leukaemia (AML) has significantly improved over time, the published current probability of event-free survival (EFS) being in the order of 40–50%. We report the results of the AIEOP LAM 2002/01 Protocol, a multicentric prospective study conducted by Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP). Treatment was stratified according to the risk group at which the pt was assigned and the study was designed to evaluate if a large use of haematopoietic stem cell transplantation (HSCT) in high-risk (HR) pts was able to increase the 5-year EFS above 50%. Patients and Methods: Between 12/2002 and 12/2007, 217 children with de novo AML other than promyelocitic leukaemia were enrolled; 205 of them are evaluable. Children with Down syndrome were excluded. Pts with isolated AML1-ETO fusion transcript or anomalies of CBF-b achieving complete remission (CR) after the first induction course were considered to be at Standard Risk (SR), whereas all the others were assigned to the HR group. After 2 courses of 7-day induction therapy (idarubicin, cytarabine and etoposide: ICE 3+5+7), pts achieving CR received 2 consolidation courses, including high-dose cytarabine (HD-Ara-c), combined with either etoposide in the first course (AVE 3+4) or mitoxantrone in the second course (HAM 3+2). After consolidation, SR pts received a further cycle of 3-day HD-Ara-c, while HR children underwent either allogeneic (ALLO) or autologous (AUTO) HSCT depending on the availability of an HLA-identical relative. Children Results: 34 (16%) and 171 (84%) pts were assigned to SR and HR groups, respectively. Overall, 82% of 205 pts achieved CR after induction, the early death and induction failure rate being 4,3% and 11,7%, respectively. Pts in CR Relapsed in 35%, died in 5%. With a median follow-up of 43 months (range 4-86), the 5-year probability of overall survival (OS), EFS, and disease-free survival (DFS) for the whole cohort were 70%, 55%, and 63%, respectively. In the SR group OS, EFS and DFS were 97%, 78% and 78%, respectively, while in the HR group were 64%, 50% and 60%, respectively. Notably, EFS for the 25 children with M7-AML was 54%; 24 infants had an EFS of 62%. WBC count at diagnosis did not influence EFS. In the HR group, the EFS of pts who did or did not have FLT-3 internal tandem duplication was 31% and 51%, respectively (p Conclusions: AIEOP LAM 2002/01 study resulted in an EFS superior to 50%. The early-death rate was particularly low. The risk stratification adopted allowed to identify 2 groups with distinct prognosis (Fig1) and deserving post-remissional treatment of different intensity. The EFS of M7-AML and infant pts compares favourably with previously published results. Notably, among pts given ALLO-HSCT, the outcome was not influenced by the type of donor employed. Disclosures: No relevant conflicts of interest to declare.

While Essential Thrombocytemia (ET) is a typical chronic myeloproliferative disease (MPD) in adults of median/advanced age, it is rarely seen in children. Literature is scarce on this issue and mostly focused on case reports. Current diagnostic criteria in adults are those of WHO2008; new biological markers show a growing clinical relevance. In contrast, we suggested from individual Institutions’ experience that these criteria are questionable for children. Therapeutic options in pediatric age are far from established, as demonstrated by the heterogeneous treatments reported in the literature. We thus analyzed a large series of thrombocytemic children, diagnosed and followed in the Units of Italian Association of Pediatric Hematology and Oncology (AIEOP), to evaluate their clinical and biological features in comparison with adult counterparts. 90 children with sporadic ET were reported. Children with familiar history of MPD were excluded. Diagnosis was performed initially according to the PVSG criteria and since 2001 to the WHO criteria. All patients underwent bone marrow aspirate; biopsy was performed only in one third of children. Main clinical data, age at diagnosis, length of follow up (f-up), possible treatment and complications were recorded for all cases. Since 2002, most patients were analyzed for significant biological features, either retrospectively or at diagnosis. V617FJAK2mutation was analyzed by allele specific PCR on DNA extracted from granulocytes. Erythroid colonies (EEC) were obtained with and without addition of erythropoietin. Clonality was studied on female patients according to HUMARA method. The mutations of thrombopoietin (TPO) and its receptor (c-MPL) genes were performed by sequencing methods. Prevalence of females (62 girls and 28 boys) was seen as in adult ET. Median age at diagnosis was 6.75 years (range 1 mo-17 yrs); median f-up was 5 years (range 2–11 yrs). Platelets counts at diagnosis were 611–4020 × 109/l (median 1260); haemoglobin was normal, leukocytosis was frequent. Splenomegaly was present in 21% of cases. In 57 patients thrombocytemia was a casual finding. Headache was the most frequent complaint(25%). 10 patients presented minor symptoms. Major problems were seen at diagnosis in 2 infants (deep venous thrombosis with Budd-Chiari syndrome) and during f-up in 2 more children (deep venous thrombosis and splenic infarction with venous thrombosis); no major hemorrhagic events were recorded. One girl had autoimmune peripheral neuropathy. Treatment was heterogeneous and often modified during f-up: 28 children did not receive any treatment; 29 cases used low dose ASA alone or in association. Cytoreduction was obtained by hydroxyurea (29 cases), interferon alfa (17cases) and anagrelide (26 cases), aiming at platelets level 400–600 × 109/l. Treatment was stopped for toxicity in 1 case of IFN and 3 cases of Anagrelide. The V617F JAK2 mutation, present in about 50% of adult ET, was found in 16 out of 65 children (24%). Of 28 informative female patients, 17 (60%) had clonal myelopoiesis. Spontaneous EEC grew in 15 out of 32 cases (47%). We could not find any mutation of TPO or cMPL genes in sporadic patients. In conclusion, this represents the largest reported series of pediatric ET. Differences from adult cases are shown: most cases have little or no clinical symptoms; the incidence of thrombotic events is low. The primitive proliferative nature of these cases is not always confirmed: V617FJAK2 mutation is less frequent than in adults and its correlation with the clinical severity is not firm. This heterogeneity requires the definition of specific criteria for diagnosis and treatment of ET in children. Main features of pediatric ET Features Symptoms Treatment patients 90 None 52 (55%) None 28 M/F 28/62 Budd Chiari 2 ASA 29 Age at Dx 6.75 y (1m–17y) Deep Venous Thrombosis 2 Anagrelide 26 Follow-up 5 y (2–11) Haedache 22 (25%) a-IFN 17 Plts × 109/L 1260 (611–4020) Paresthesia 3 OHU 29 WBC × 109/L 11.75 Nosebleed 2 JAK2 V617F 16/65 (24%) Abdominal pain 4 EEC pos 17/28 (60%) Splenomegaly 19 (21%) Monoclonality 15/32 (47%) Peripheral neuropathy 1 MPL/TPO mutation none

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.

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

The anti-CD33 antibody calicheamicinconjugate gemtuzumab ozogamicin (GO) was used to treat 16 patients with acute promyelocytic leukemia (APL) who had relapsed at the molecular level. Of these patients, 8 were experiencing a first, 5 a second, 2 a third, and 1 a fourth relapse. GO was administered at 6 mg/m2 for 2 doses, and patients achieving a new molecular remission (MR) (ie, negativity of the reverse transcriptase-polymerase chain reaction [RT-PCR] test for PML/RARalpha) received a third dose. MR was obtained in 9 (91%) of 11 patients tested after 2 doses and in 13 (100%) of 13 patients tested after the third dose. Of the 3 remaining patients, 1 achieved MR after one GO administration and received no further therapy owing to hepatic toxicity, and 2 showed disease progression during treatment. Quantitative RT-PCR studies showed that responding patients experienced a dramatic decline (at least 2 logs) of the PML/RARalpha transcript after the first GO dose. Of 14 responders, 7 remained in sustained MR for a median of 15 months (range, 7-31 months) while 7 experienced relapse at 3 to 15 months. GO was administered again in 2 patients with relapse, and both obtained a new MR. These data indicate that GO is highly effective as a single treatment for patients with molecularly relapsed APL including those with very advanced disease.