ALBERT

Description: Because survival of pediatric acute lymphoblastic leukaemia (ALL) has improved in the last years, adverse effects acquire increasing importance. Avascular osteonecrosis (ON) is a long term complication of ALL treatment affecting the patients' quality of life. Its pathogenesis recognizes multiple factors (age, race, genetics, chemotherapies and steroids). The different roles of dexamethasone (DXM) versus prednisone (PDN) is still under discussion, as dosages and rate of administration. The prevalence of ON varies in different series (0.43-17.6%). It affects mostly the weight-bearing epiphyseal bone, usually knee and hip. Multiple joint involvement is common. ON treatment is not established: efficacious drugs are not available; surgery is strongly invasive for pediatric patients (pts); among physical treatments, the use of hyperbaric oxygen therapy (HBO) is uncommon. We report our experience in children treated consecutively from 2000 to 2015 for ALL at the Pediatric Hematology Clinic of Padua, who developed ON. HBO was used in some pts with variable efficacy, suggesting its possible role in early stages. Materials and methods Prevalence ofON was assessed retrospectively in 469 pts with ALL enrolled in AIEOP 2000 Protocol (P 2000, 327 pts) and AIEOP BFM ALL 2009 Protocol (P 2009, 142 pts); median age was 5 years (yrs, range 1-17). Girls were 53% and boys 47% (Table 1). ON was defined as persistent pain in one or more major joints (hip, knee, elbow, shoulder, ankle) and peculiar radiological findings (plain XR or MRI). Severity was graded radiologically by the ARCO (Association of Research Circulation Osseous) staging system; clinically by the CTCAE v3.0. Statistical analysis was done using z- and t-tests. Results 19/469 pts (4%) developed ON of CTCAE grade 2-4. Age at leukaemia diagnosis was over 10 yrs in 17/19 pts. ON always presented during maintenance or off therapy. Median follow up after ON development in P 2000 was 54 months (range 21-170) and 28.5 months (8-48) in P 2009. The most frequent sites were knee, hip and ankle. Multiple joint involvement was frequent (11/19, 58%); 14/19 (74%) had also functional limitation at diagnosis (grade 3-4). Nine ON pts (3 in P 2000, 6 in P 2009) were at high risk, receiving higher steroid dosages than low risk (p

Description: Introduction. Acute erythroid leukemia (AEL) is a high-risk leukemia subtype of poorly understood genetic basis. In integrated comparative genomic analysis of 159 AEL and 1509 non AEL myeloid tumors, we identified 5 age-related AEL subtypes with distinct genomic features and outcome: adult AEL with bi-allelic alterations in TP53 (31%), frequently co-occurring with alterations in DNMT3A,BCOR, EZH2, RB1, or NFIX; NPM1-mutated (12%); KMT2A-mutated/rearranged (11%), mostly co-mutated with STAG2; pediatric, NUP98-rearranged (5%) and adult, DDX41-mutated (3%). Thirty-eight percent of cases lacked an identifiable exclusive recurrent founding alteration but were enriched in mutations in ASXL1, TET2 and splicing factors. Methods. To explore the roles and cooperativity of the identified alterations in leukemogenesis we used CRISPR-Cas9 genome editing to induce combinations of loss-of-function mutations in 9 recurrently mutated genes in AEL (Tp53, Tet2, Dnmt3a, Asxl1, Ezh2, Stag2, Bcor, Ppm1d, Rb1 and Nfix). Based on patterns of mutation association, we generated 6 pools of lentiviral vectors (Table 1) with different combinations of single guide RNA (sgRNA) to induce multiplex genome editing in Cas9-eGFP-mouse lineage-negative hematopoietic stem cells (HSCs). Transduced cells were transplanted into lethally irradiated congenic mice. Tumors were characterized by morphology, immunophenotyping, and genomic analysis (sequencing of sites of editing, and exome, methylation and transcriptome sequencing). Ex vivo drug screening was performed to test sensitivity to 192 therapeutic agents, including conventional chemotherapeutic agents, and compounds targeting epigenetic regulators, protein kinases and cell cycle checkpoints. Results. In contrast to the uniform representation of guide RNAs observed in HSCs pre-transplant, tumors exhibited enrichment of specific sgRNAs with tumors of specific phenotype. We frequently observed bi-allelic editing of Tp53, Bcor, Tet2, Dnmt3a, Rb1 and Nfix in agreement with the presence of bi-allelic loss in patients. Concomitant editing and inactivation of Tp53/Bcor/Dnmt3a, or Tp53/Bcor/Rb1/Nfix promoted the development of acute erythroid leukemia (GATA1+/RUNX1+/GlyA+/-Ter119+/- and B220/CD19/PAX5/CD3/Mac1/Gr1-). Concomitant editing of Tp53/Bcor/Tet2 promoted the development of B-lineage ALL, and editing of Dnmt3a and Tet2 without Tp53 promoted T-cell ALL. Leukemic clones from primary tumors were serially transplantable across multiple different genetic backgrounds, with the same dominant clone present in all transplanted mice. Notably, mice that did not develop leukemia showed enrichment of different combinations of sgRNAs for Tet2, Dnmt3a and/or Asxl1, genes commonly mutated in clonal hematopoiesis of indeterminate potential, confirming that these mutations are alone not sufficient to induce leukemogenesis. Additional somatic mutations were acquired during clonal expansion of leukemic cells such as alterations of Notch1 and Ikzf1 in T-ALL; Setd2 at the serial passaging of T-ALL; Ptpn11, Kit (D816V), Kras (Q61L) and Lmo7 in the AEL models; and Sf3b3 in the B-ALL model (Fig 1A). Tumors with mutated Tp53 acquired aneuploidy whereas Tet2-mutated cells were genomically stable (Fig. 1B). Unsupervised hierarchical clustering of gene expression profiling identified 3 subgroups (Fig. 1C) associated with distinct genotypes and methylation profiles (Fig. 1D). Tet2-mutated tumors showed increase of hypermethylated sites and co-mutated Bcor/Dnmt3a leukemic cells showed loss of methylation. Eleven tumors representative of key AEL genotypes from the established models were selected to explore drug sensitivity. Response to individual drugs was associated with genotype with co-mutated Tet2/Dnmt3a T-ALL cells sensitive to bromodomain and histone methyltransferase inhibitors; co-mutated Bcor/Tp53/Dnmt3a or Bcor/Rb1 AEL cells to CDK9 inhibitor (LY2857785); Tp53 mutations alone were exclusively sensitive only to PARP inhibition (Talazoparib). In vivo mouse studies are ongoing to confirm ex vivo results. Conclusions: We successfully generated genetically defined models of AEL, demonstrated the role of Tp53 and Bcor mutations in driving the erythroid phenotype, and showed that sensitivity to different classes of compounds is genotype-dependent. These results provide the rational for testing new targeted agents in AEL. Disclosures Mullighan: Abbvie: Research Funding; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Description: Introduction We have evaluated Antithrombin (AT) values and Whole Blood Rotation Thromboelastometry (ROTEM) profiles in children affected by acute lymphoblastic leukemia (ALL), followed at the Clinic of Pediatric Hematology Oncology of Padua. L-Asparaginase, used in protocols for pediatric ALL, is an inhibitor of protein synthesis. Typical collateral effects are coagulation disturbances. Guidelines for identification of patients at risk of thrombosis and hemorrhages are not yet established. AT levels are the only reliable parameter for thrombotic risk. Very low levels of fibrinogen are not correctly detected by classical Clauss method; ROTEM test using FIBTEM is considered a valid tool to identify hypofibrinogenemia and hemorrhagic risk in surgical patients. The aim of the study was to analyze coagulation patterns during treatment with Pegilated L-Asparaginase (Peg-Asp). Methods Forty-two children (25 males, 17 females; 31 pB-ALL; 11 T-ALL; 23 at standard/medium risk; 19 at high risk) were consecutively diagnosed and treated with AIEOP BFM ALL 2009 protocol from June 2012 to March 2014. They received 3 (standard/medium risk) or 8 (high risk) doses of Peg-Asp (2500UI/mq/dose). ROTEM and AT determinations were performed at 5 fixed time-points before and after each Peg-Asp administration. Prothrombin time (PT), fibrinogen plasma levels and platelets counts were recorded. Maximum Clot Firmness (MCF; normal value 9-25 mm), measuring the maximum amplitude reached in FIBTEM thromboelastogram, assessed the specific role of fibrinogen in the whole blood clot formation following platelets inhibition by Cytocalasin D. Results: 798 AT and 706 FIBTEM tests were performed; details are reported in Table. We divided abnormal MCF in 3 grade levels. Only values of MCF 〉9 mm had a linear correlation with fibrinogen levels. Plasma fibrinogen values

Description: Background Multiparametric flow cytometry (MFC) is critical in the diagnosis and management of pediatric acute lymphoblastic leukemia (ALL), through immunophenotyping (IP), and minimal residual disease (MFC MRD) analysis. The aberrant expression of myeloid markers in B- and T-lineage ALL is a well-known phenomenon. It may be the result of an adaptive mechanism by lineage switch (SW), defined as any variation of blast IP over time. CD371 is a transmembrane glycoprotein usually expressed on normal myeloid cells and most of the myeloid blasts. Aberrant expression of CD371 was observed in DUX4-rearranged B cell precursor ALL (BCP ALL). Aims To investigate the clinical and biological features of CD371 positive (CD371+) pediatric BCP ALL, pointing out its potential implication in MFC MRD monitoring on Day 15. Materials and Methods From June 2014 to January 2017, 862 children with newly diagnosed t(9;22)(q34.1;11.2);BCR-ABL1 negative BCP ALL, were consecutively enrolled in the AIEOP BFM ALL 2009 study by AIEOP centers. Peripheral blood (PB) and bone marrow (BM) samples (SMPs) were processed and analyzed in the Laboratory of Diagnosis and Research of Pediatric Hematology-Oncology, University of Padua, Italy, according to standardized operating protocols designed by the AIEOP BFM Flow Network. At diagnosis, 9 combinations of 8 monoclonal antibodies (MoAbs) were used for IP. MFC MRD (Day 8 on PB, Day 15, 33, and 78 on BM) was performed with 2 combinations of 8 MoAbs from June 2014 to May 2016. Later, a dry 10 colors preformulated tube was adopted for MFC MRD monitoring. Results At diagnosis, CD371 expression was assessed in 823 of 862 (95.5%) SMPs by as many patients (pts; age: 1-17 years; male/female: 446/377). Of those, CD371 was positive in 75 of 823 SMPs (9.1%). CD371 positivity was associated with older age, euploidy, a more immature immunophenotype (B-I as per EGIL classification), and the aberrant expression of CD2 antigen, as well as at least one myeloid marker (Table). CD371+ SMPs showed a stronger expression of CD34, CD45, and CD58 antigens than CD371 negative SMPs (Table). We performed MFC MRD analysis on 207 SMPs of CD371+ BCP ALL (42 on Day 8, 72 on Day 15, 40 on Day 33, and 53 on Day 78). During the first 15 days of Induction therapy, a monocytoid population appeared in 76 SMPs [26 of 42 (61.9%) on Day 8 and 50 of 72 (69.4%) on Day 15]. It was characterized by a strong expression of CD34, CD58 and CD45, reduced expression of CD19, and high SSC. We interpreted that phenomenon as an SW to the myelomonocytic lineage, as previously described in a subtype of BCP ALL expressing CD2 at diagnosis. Myelomonocytic SW displayed 2 different patterns: (a) a single population of blasts with heterogeneous expression of CD19 (strong to dim/negative); (b) 2 distinct populations: the first one with the IP of diagnosis, the second one showing a downregulation of CD19 and CD34, an intensification of CD45, and an increase of SSC (Figure). At the same time points, a clear monocytoid population was visible on May-Grunwald-Giemsa stained smears. The comparison between MFC MRD and PCR MRD data showed a higher concordance when both the populations were included in the final amount of blasts on Day 15 (concordance rate: 89% vs. 82%). SW was transient and disappeared after Day 15, even though chemotherapy was always carried on as per therapeutic ALL protocol. CD371 antigen was an accurate marker of SW in our cohort [sensitivity = 0.93 (95% CI ± 0.06), specificity = 0.98 (95% CI ± 0.005), accuracy = 0.98]. Finally, CD371 positivity was associated with a worse response to Induction therapy, showing a higher proportion of pts enrolled in the high therapeutic risk group of the trial, most frequently due to a slow early response according to PCR MRD on Day 33 and 78 (Table). Conclusions We described a new subtype of pediatric BCP ALL, characterized by the aberrant expression of CD371 and a potential myelomonocytic SW during the first phase of Induction Therapy. Accurate identification of the lineage SW is mandatory to properly assess MFC MRD on Day 15 in these pts, avoiding an underestimation of blast cells. This is particularly important, considering that CD371+ BCP ALL was associated with a poor response to Induction therapy. Even in presence of a prevalent monocytoid population, chemotherapy should be carried on according to a therapeutic protocol for ALL. CD371 antigen should be part of IP diagnosis panel for ALL. A multicenter study of AIEOP BFM Flow Network centers is ongoing. Disclosures Brüggemann: Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy.

Description: Introduction. Chronic hypoxia, transfusional iron overload and chelation therapy are all contributing factors to the deterioration of renal function in thalassemia major (TM ) patients. Among chelators, deferasirox (DFX) is considered the most toxic drug for kidneys: increment of more than 30% of serum creatinine levels is used for dosage modulation or interruption. Recently tubular abnormalities have also been described. We have observed renal toxicity in nine children affected by TM and treated with DFX or deferiprone (DFP) and described the differences in glomerular or tubular function between the two oral chelators. Material and methods. Nine children, 4 males and 5 females, aged 3-17 years (median 7 y), were retrospectively evaluated. They were regularly transfused every 2-3 weeks, receiving a median of 260 g erythrocytes concentrates/kg/year. The median serum ferritin level at baseline before starting oral chelation was 1763 ug/L (range 1127-4198). Four patients (pts) received only one type of chelator (1 DFP and 3 DFX). Five pts received both DFX or DFP in different periods. Overall 8 DFX treatments (dosage 15-30 mg/kg, for 18-112 months, mean 40) and 6 DFP treatments (dosage 75-130 mg/kg for 14-54 months, mean 24) were administered. Three pts had previously received deferoxamine; 3/8 DFX pts and 1/6 DFP pts were naïf to chelation before first renal evaluation. The following renal indices were considered: − glomerular function: serum creatinine and cystatine C levels, estimated glomerular filtration rate (eGFR), according to modified Schwartz formula; urine albumin to creatinine ratio (ACR), urine protein excretion. − tubular function: glycosuria, urinary N-acetyl-beta-D-glucosamidase activity/u-creatinine (u-NAG/u-cr), (index of acute tubular damage); urine daily Beta2 microglobulin (B2MG) and Alfa1 microglobulin (A1MG) excretion (both indices of chronic tubular damage). Analyses were performed at different intervals, according to clinical conditions and patients' age, with different frequency among children. Creatinine impairment was defined as an increase over 30% of basal level. All indices were graded according to local laboratory range. Toxicity was considered significant when the tests were abnormal in at least 25% of evaluations. Results. Initial renal function was normal in all patients (median eGFR 209 ml/min/1.73 m2, range 157-295) and eGFR remained stable over time in all cases. A total of 5 pts with DFX and 3 pts with DFP showed an increase of creatinine over 30% of basal levels. Increase of ACR 〉 30 mg/g cr was seen in 6/8 pts (75%) in DFX and in 1/6 pts (17%) in DFP; 2 pts (1/8 in DFX and 1/6 in DFP) had macroalbuminuria (ACR 〉300 mg/g creatinine). No association was found between cumulative DFP dose and eGFR over time (Pearson coefficient, r = 0.06), while a moderate association was found with DFX (r = 0.5; p = 0.06). Symptomatic acute tubular toxicity was observed in 2 pts, with tubular acidosis, glycosuria and abnormal acute tubular indices (u-NAG/u-cr). A1MG and B2MG were also altered, suggesting a chronic injury substrate. Both pts were in DFX group (receiving a dose of 20 and 40 mg/kg/day, respectively) and had experienced a fast reduction in ferritin levels over a short period of time. No signs of concomitant infection were detected. u-NAG/u-cr was always abnormal in all children in both groups; mean values were 2.8 U/mmol for DFX and 2.4 U/mmol for DFP. B2MG was altered in 6/8 DFX pts and in 2/6 DFP pts (75% vs 33%). Median values were 3.0 mg/24 h for DFX and 0.5 mg/24 h for DFP, respectively. A1MG was altered in 4/8 DFX pts and 3/6 DFP pts, with median values of 164 mg/24 h for DFX and 55 mg/24 h for DFP, respectively. Conclusions. Patients withTM can experience abnormalities in renal function, especially as regards tubular damage. Acute tubular damage indices were altered in all chelated pts, whereas tests for chronic damage were abnormal more often in the DFX group than in DFP-treated pts. Albeit the significance of our findings needs further confirmation in a longer follow up, these results suggest that extensive renal evaluation is needed, in particular in children whose life expectancy is open and long-term prognosis for multiple organ function is an important issue. Disclosures No relevant conflicts of interest to declare.

Description: Background: Traumatic and bloody lumbar punctures (TLPs and BLPs, respectively) at the diagnosis of pediatric acute lymphoblastic leukemia (ALL) impair the interpretation of central nervous system status and are associated with worse outcomes. We previously analyzed risk factors for TLP and BLP in 958 patients with ALL treated at St. Jude Children's Research Hospital (St. Jude) between 1984 and 1998. With an incidence of 19% and 5% at diagnosis, respectively, TLPs and BLPs were associated with unmodifiable and modifiable risk factors (URFs and MRFs, respectively). URFs included age less than 1 year and black race. MRFs included a platelet count (PLT) of

Description: Introduction. Hypofibrinogenemia and its correlation with hemorrhagic risk is usually detected by serum fibrinogen level with Clauss test. Recently the role FIBTEM test of rotational thromboelastometry (ROTEM) has been evaluated for prevention and management of acute bleeding in intesive care and surgical settings. We applied it for monitoring fibrinogen deficiency for the first time in children affected by acute lymphoblastic leukemia (ALL) treated with Peg-asparaginase (PEG-Asp). Antithrombin levels (AT) were measured as parameter for thrombotic risk. This drug impairs protein synthesis and causes severe coagulative deficiencies, with both hemorrhagic and thrombotic complications. Guidelines for the management of coagulation abnormalities being missing, we tried to identify parameters for management and prevention. Methods. Eightytwo children (age 1-17 years, mean 6 year) affected by ALL treated with AIEOP BFM ALL 2009 protocol at our Institution were studied. Platelet count and coagulation tests (protrombin time PT/INR, activated partial tromboplastin time aPTT, AT, serum fibrinogen level (SF) at Clauss test and fibrinogen function at MCF) were performed before and after each PEG-Asp administration. We previously found linear correlation with SF levels only for MCF 〉 9 mm. We then tried to identify clinically significant deficiency of SF. Severe hypofibrinogenemia was considered when Clauss test showed SF levels 〈 1g/L. MCF limit for severe fuctional hypofibrinogenemia was considered