Supplementary Figure 1. Expansion of adaptive NK cells from C1/C2 donors depends on original imprint. Supplemental Figure 2. Selective expansion of NKG2C NKG2A- NK cells depends on stimulation with HLA-E expressing feeder cells. Supplementary Figure 3. Expression of granzymes and perforin before and after in vitro expansion of adaptive NK cells. Supplementary Figure 4. Adaptive NK cells recognize MDS and AML blasts. Supplementary Figure 5. Sorting of adaptive NK cells for functional studies.

Background: Childhood cancer survivors treated before 1992, when blood donor screening for hepatitis C virus (HCV) infection was introduced, are at risk of transfusion-transmitted HCV infection. A national HCV screening campaign targeting blood transfusion recipients was launched in Sweden in 2007-2010. The aims of this study were to, among adult childhood cancer survivors in Stockholm County, investigate the prevalence of HCV infection, the natural course of infection, treatment outcome and anti-HCV testing frequency before, during and after the screening campaign and finally to actively screen the untested ones.Material and Methods: This was a combined retrospective register based and prospective screening study of adult childhood cancer survivors (n=686) treated for malignancy in Stockholm before 1992. In the first part, we investigated the prevalence of HCV infection and previous anti-HCV testing, and in the second part, we actively traced and HCV-screened the remaining untested cohort living in Stockholm. Analysis of previous documented anti-HCV tests in medical records, laboratory records, and the national communicable disease registry was performed. In the second part, 231 presumably untested individuals were contacted by mail and offered an anti-HCV test. The natural course of HCV infection and treatment outcome was analyzed for those found to be chronically infected.Results: In total, 235 patients were tested and 11 were HCV-RNA positive. The overall prevalence of chronic HCV infection among the tested childhood cancer survivors was thus 4.7% (95% CI = 2.6-8.2%), which is almost 10 times higher than the national prevalence of 0.5%. Only 12% of the Stockholm cohort were tested during the screening campaign in 2007-2010, while the test uptake using active tracing screening within this study was 40% (p<.001).Conclusion: With today's effective treatment options, active tracing and HCV screening of childhood cancer survivors are recommended.

Acute leukaemias comprise approximately 32% of all childhood cancers (National Cancer Institute, 2018). Up to 20% of paediatric patients with acute myeloid leukaemia (AML) have mutations in FLT3, conferring a poor prognosis (Balgobind et al, 2011; Zwaan et al, 2015). Approximately 80% of infants with acute lymphoblastic leukaemia (ALL) have gene rearrangements in KMT2A (KMT2A-R ALL), often in combination with overexpression of mutation-negative FLT3 (Stam et al, 2007). This combination of KMT2A-R ALL and FLT3 overexpression is associated with particularly poor long-term outcomes (Stam et al, 2007; Chillon et al, 2012). Preclinical and clinical work suggests that targeted inhibition of FLT3 may be a promising strategy for paediatric acute leukaemias (Annesley & Brown, 2014). Midostaurin (PKC412), a multikinase inhibitor with targets that include FLT3, was approved in adults with newly diagnosed FLT3-mutated AML (Stone et al, 2017). Midostaurin has demonstrated antileukaemic activity in preclinical models of FLT3-mutated AML and FLT3-overexpressing KMT2A-R ALL (Weisberg et al, 2002; Armstrong et al, 2003). This dose-escalation and dose-expansion study evaluated the safety and efficacy of single-agent midostaurin in paediatric patients with relapsed or refractory (R/R) FLT3-mutated AML or KMT2A-R ALL (NCT00866281). Patients had documented diagnosis of FLT3-mutated AML or KMT2A-R ALL according to local laboratory assessment. FLT3 mutations were confirmed at a central laboratory but were not required for inclusion of patients with KMT2A-R ALL. The primary objective was to determine the maximum tolerated dose (MTD) or recommended dose for expansion (RDE) of oral midostaurin solution, developed for paediatric use, in each age group using a single 3-parameter Bayesian logistic regression model (Appendix S1). A dose-limiting toxicity (DLT) was defined as a nonhaematological grade 3/4 adverse event (AE), drug-related abnormal laboratory value, or AE leading to study discontinuation within 14 days of starting treatment occurring in the dose-determining set. Midostaurin was administered to children in 2 age groups: younger (≥3 months to ≤2 years) and older (>2 years to ≤18 years) (Figure S1). From September 2009 to October 2014, 22 patients were enrolled (9 FLT3-mutant AML, 13 KMT2A-R ALL). The study was terminated early due to slow accrual (Appendix S1). Baseline characteristics are presented in Table 1 and Table SI. All 9 patients with AML were in the older age group. Of 13 patients with KMT2A-R ALL, 11 were younger and 2 older. According to the central laboratory analysis of FLT3-mutation status, not all patients with AML had a FLT3 mutation (6 FLT3-internal tandem duplication, 1 FLT3-mutation–negative [FLT3-mut−], and 2 missing/nonevaluable) and only 1 patient with KMT2A-R ALL had a FLT3 mutation (FLT3-tyrosine kinase domain; 11 FLT3-mut−; 1 missing/nonevaluable) (Table SII). Potential reasons for discrepancy between the local and central laboratory analyses are discussed in the Supplementary Appendix. High levels of phosphorylated FLT3 were not detected in any patient sample (AML or KMT2A-R ALL) at baseline (Table SIII). Of 22 patients, 7 (3 with FLT3-mutated AML and 4 with KMT2A-R ALL per local assessment) received the starting dose (30 mg/m2 twice daily [bid]) and 15 (6 with FLT3-mutated AML and 9 with KMT2A-R ALL per local assessment) received the maximum dose (60 mg/m2 bid). All patients discontinued treatment (Figure S2). No DLTs or significant safety concerns were observed among the 6 patients who received the 30 mg/m2 bid dose; therefore, dose escalation proceeded to 60 mg/m2 bid. Among 11 patients in the dose-determining set who received the 60 mg/m2 bid dose, a DLT was reported in 1 patient (grade 3 alanine aminotransferase elevation). One patient with FLT3-mutated AML in the 60 mg/m2 bid cohort completed the per-protocol follow-up; the remaining 21 patients died. Median duration of exposure to midostaurin for all patients was 16 days. Because the study design did not allow dosing beyond 60 mg/m2 bid, a true MTD could not be determined and the 60 mg/m2 bid dose was chosen as the RDE (Table SIV). Most patients (95·5%) experienced ≥1 AE on treatment or during the 28-day follow-up period (Table SV). Overall, 77·3% of patients experienced grade 3/4 AEs, with more AEs occurring in the 60 mg/m2 cohort than in the 30 mg/m2 cohort. AEs suspected to be study treatment–related were reported in 72·7% of patients. Efficacy was evaluated in all 22 patients. The rate of best clinical response (assessed per modified Cheson criteria; Appendix S1) to single-agent midostaurin was 55·5% (95% confidence interval [CI], 21·2–86·3%) and 23·1% (95% CI, 5·0–53·8%) in paediatric patients with R/R FLT3-mutated AML and KMT2A-R ALL, respectively (Fig 1). One patient with FLT3-mutated AML achieved a complete remission with incomplete count recovery on day 14; she discontinued midostaurin therapy on day 64 but remained in remission until her last response assessment (day 67), received a haematopoietic stem cell transplant (day 76), and was alive on day 960. In patients with FLT3-mutated AML and KMT2A-R ALL, median time to response was 14 (range, 8–22) days and 8 (range, 3–8 ) days, respectively; median overall survival (95% CI) was 3·7 (2·7–8·3) months and 1·4 (1·0–2·9) months, respectively (Figure S3). This study was the first to evaluate the safety, efficacy, and pharmacokinetics (Appendix S1) of oral midostaurin solution in children. The RDE was set at 60 mg/m2 bid for the 2 age groups. Single-agent midostaurin showed limited clinical activity in these children with heavily pretreated FLT3-mutated AML or KMT2A-R ALL. Despite preclinical data suggesting that midostaurin sensitivity in infant KMT2A-R ALL overexpressing FLT3 was similar to that in FLT3-mutated AML (Stam et al, 2007), single-agent midostaurin showed no clinical activity in children with R/R KMT2A-R ALL, possibly because overexpression of FLT3 alone is not sufficient to drive the disease or because FLT3 was not inhibited enough at the doses tested to elicit responses. Future studies should aim to understand the kinetics of FLT3 mutations between diagnosis and relapse, as they present an important prognostic factor for patients with AML. The unmet need in paediatric patients with R/R FLT3-mutant AML or KMT2A-R ALL is high. Because midostaurin plus chemotherapy demonstrated clinical benefit in adults with FLT3-mutated AML and single-agent midostaurin was generally well tolerated in paediatric patients, midostaurin will be evaluated in combination with chemotherapy in paediatric patients with FLT3-mutated AML. This study was supported by funding from Novartis Pharmaceuticals Corporation. We thank the patients enrolled in this trial and their families for their participation. We also acknowledge all of the investigators at the study sites that participated in the trial: Erasmus MC–Sophia Children's Hospital, Rotterdam, the Netherlands; Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden; Hôpital Universitaire Robert-Debré, Paris, France; IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy; Fondazione MBBM, Azienda Ospedaliera San Gerardo, Monza, Italy; Paediatric Onco-Haematology, Stem Cell Transplantation and Cellular Therapy Division, A.O.U. Città della Salute e della Scienza di Torino, Ospedale Infantile Regina Margherita, Torino, Italy; Seattle Children's Hospital, Seattle, WA; Haematology Unit, IRCCS Instituto Giannina Gaslini, Genova, Italy; and Princess Máxima Centre for Paediatric Oncology, Utrecht, the Netherlands. We would also like to thank Katherine Mills-Luján, PhD, CMPP, Amarallys Cintron, PhD, and Pamela Tuttle, PhD, CMPP, of ArticulateScience LLC for medical editorial assistance with this manuscript, which was funded by Novartis Pharmaceuticals Corporation. This study was designed by the sponsor, Novartis Pharmaceuticals Corporation, and the study steering committee, CMZ, ML, and RP. CMZ, SS, BB, ML, CR, RWS, FF, PAH, C Dufour and RP enrolled patients. EB oversaw the conduct of the study. C Dutreix performed the pharmacokinetic analyses. Data were collected and analysed by the sponsor in conjunction with the authors, who had full access to the data. CMZ and RP wrote the manuscript, with medical writing support from ArticulateScience LLC. All authors contributed to draft revisions and approved the final version of the manuscript. CMZ is a consultant for and has received funding from Novartis Pharmaceuticals Corporation. C Dufour is a consultant for Pfizer and has participated in advisory committees for Novartis Pharmaceuticals Corporation. PAH is a current employee of and has equity ownership in Seattle Genetics. EB and C Dutreix are current or former employees of Novartis Pharmaceuticals Corporation. SS, BB, ML, CR, RWS, FF and RP have no competing interests. Medical editorial assistance with this manuscript was funded by Novartis Pharmaceuticals Corporation. Appendix S1. Supplementary Methods; Supplementary Results; Supplementary Discussion Table SI. Expanded Patient Baseline Characteristics Table SII.FLT3 Mutation Status per Central Laboratory Assessment Table SIII. FLT3 Expression and Phosphorylation Analysis Table SIV. Posterior Probabilities of Dose-Limiting Toxicity by Age Strata and Dose Table SV. Most Common Adverse Events (occurring in ≥10% of patients) With Single-Agent Midostaurin in Paediatric Patients With Relapsed or Refractory Acute Leukaemia Table SVI. Bioavailability and Safety of Midostaurin Oral Solution and Capsule Formulations in Healthy Volunteers in Study A2108 Table SVII. Modified Cheson Criteria for Response Fig S1. Trial Design Fig S2. Patient Disposition Fig S3. Overall Survival Fig S4. Pharmacokinetics of Midostaurin and Its Metabolites Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract Manipulation of human natural killer (NK) cell repertoires promises more effective strategies for NK cell–based cancer immunotherapy. A subset of highly differentiated NK cells, termed adaptive NK cells, expands naturally in vivo in response to human cytomegalovirus (HCMV) infection, carries unique repertoires of inhibitory killer cell immunoglobulin-like receptors (KIR), and displays strong cytotoxicity against tumor cells. Here, we established a robust and scalable protocol for ex vivo generation and expansion of adaptive NK cells for cell therapy against pediatric acute lymphoblastic leukemia (ALL). Culture of polyclonal NK cells together with feeder cells expressing HLA-E, the ligand for the activating NKG2C receptor, led to selective expansion of adaptive NK cells with enhanced alloreactivity against HLA-mismatched targets. The ex vivo expanded adaptive NK cells gradually obtained a more differentiated phenotype and were specific and highly efficient killers of allogeneic pediatric T- and precursor B-cell acute lymphoblastic leukemia (ALL) blasts, previously shown to be refractory to killing by autologous NK cells and the NK-cell line NK92 currently in clinical testing. Selective expansion of NK cells that express one single inhibitory KIR for self-HLA class I would allow exploitation of the full potential of NK-cell alloreactivity in cancer immunotherapy. In summary, our data suggest that adaptive NK cells may hold utility for therapy of refractory ALL, either as a bridge to transplant or for patients that lack stem cell donors. Cancer Immunol Res; 5(8); 654–65. ©2017 AACR.

Introduction Outcomes for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) have improved, but the prognosis for pts with R/R disease remains poor. Childhood AML and MLL-rearranged ALL (MLL r-ALL) often overexpress or have activating mutations in the FLT3 tyrosine kinase (TK). Midostaurin (PKC412) is an orally available TK inhibitor that targets FLT3, KIT, and other kinases. Data in adults for single-agent midostaurin, including from studies CPKC412A2104 and CPKC412A2106, showed that 100 mg bid (≈ 60 mg/m2 bid) was adequately tolerated, elicited leukemic blast reduction, and inhibited FLT3 autophosphorylation. Here, we present the safety and preliminary clinical activity of midostaurin in children with R/R acute leukemia.

Ferrata Storti Foundation EUROPEAN HEMATOLOGY ASSOCIATION Haematologica 2016 Volume 101(1):68-76 ARTICLE Acute Lymphoblastic Leukemia doi:10.3324/haematol.2015.131680

Genomic characterization of pediatric acute lymphoblastic leukemia (ALL) has identified distinct patterns of genes and pathways altered in patients with well-defined genetic aberrations. To extend the spectrum of known somatic variants in ALL, we performed whole genome and transcriptome sequencing of three B-cell precursor patients, of which one carried the t(12;21)ETV6-RUNX1 translocation and two lacked a known primary genetic aberration, and one T-ALL patient. We found that each patient had a unique genome, with a combination of well-known and previously undetected genomic aberrations. By targeted sequencing in 168 patients, we identified KMT2D and KIF1B as novel putative driver genes. We also identified a putative regulatory non-coding variant that coincided with overexpression of the growth factor MDK. Our results contribute to an increased understanding of the biological mechanisms that lead to ALL and suggest that regulatory variants may be more important for cancer development than recognized to date. The heterogeneity of the genetic aberrations in ALL renders whole genome sequencing particularly well suited for analysis of somatic variants in both research and diagnostic applications.

Relapse is the main reason for treatment failure in childhood acute lymphoblastic leukemia. Despite improvements in the up-front therapy, survival after relapse is still relatively poor, especially for high-risk relapses. The aims of this study were to assess outcomes following acute lymphoblastic leukemia relapse after common initial Nordic Society of Paediatric Haematology and Oncology protocol treatment; to validate currently used risk stratifications, and identify additional prognostic factors for overall survival. Altogether, 516 of 2735 patients (18.9%) relapsed between 1992 and 2011 and were included in the study. There were no statistically significant differences in outcome between the up-front protocols or between the relapse protocols used, but an improvement over time was observed. The 5-year overall survival for patients relapsing in the period 2002-2011 was 57.5±3.4%, but 44.7±3.2% (P<0.001) if relapse occurred in the period 1992-2001. Factors independently predicting mortality after relapse included short duration of first remission, bone marrow involvement, age ten years or over, unfavorable cytogenetics, and Down syndrome. T-cell immunophenotype was not an independent prognostic factor unless in combination with hyperleukocytosis at diagnosis. The outcome for early combined pre-B relapses was unexpectedly poor (5-year overall survival 38.0±10.6%), which supports the notion that these patients need further risk adjustment. Although survival outcomes have improved over time, the development of novel approaches is urgently needed to increase survival in relapsed childhood acute lymphoblastic leukemia.

Background Therapy directed at the central nervous system (CNS) is an essential part of the treatment for childhood acute lymphoblastic leukemia (ALL). The current evaluation of CNS involvement based on cytomorphological examination of the cerebrospinal fluid (CSF) alone is not as sensitive with low cell counts as flow cytometric immunophenotyping (FCI) of the CSF. However, the importance of low CSF blasts counts at diagnosis is uncertain. We sought to determine the significance of FCI in relation to conventional morphological examination. Procedure We retrospectively compared FCI of the CSF with cytomorphology at diagnosis or relapse of childhood ALL. All patients were diagnosed 2000–2012 in Stockholm or Umeå, Sweden. Clinical data were collected from medical records and the Nordic leukemia registry. Treatment assignment was based on morphological examination only. Results The cohort was comprised of 214 patients with ALL. CSF involvement was detected by both methods in 20 patients, in 17 by FCI alone, and in one patient by cytomorphology alone. The relapse rate was higher for patients with negative cytology but positive FCI compared to those without CNS involvement using both methods. The difference was especially marked in the current protocol. However, none of the patients with negative CSF cytology but positive FCI had a CNS relapse. Conclusions FCI of the CSF increased the detection rate of CNS involvement of ALL approximately two times compared to cytomorphology. Patients with low‐level CNS involvement may benefit from additional intensified systemic or CNS‐directed therapy, but larger studies are needed. Pediatr Blood Cancer 2015;62:951–956. © 2014 Wiley Periodicals, Inc.

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children, with close to 200 cases per year in the Nordic countries. Despite recent advances in modern chemotherapies, 20% of the ALL patients experience a relapse. ALL has traditionally been stratified into subtypes with different risk classification and therapy using large genomic aberrations such as translocations and aneuploidies. In recent years technological advances have enabled the detection of smaller genetic variants, such as point mutations and small insertions/deletions. This thesis focuses on the detection of these smaller variants and their potential impact for ALL.The present work includes four studies. In the first study we investigated the effects of whole genome amplification and non-indexed pooling strategies to maximize the output of targeted sequencing. We found that whole genome amplified DNA is equivalent to genomic DNA when screening for point mutations in targeted sequencing data. We were able to accurately detect variants in non-indexed pools with up to ten samples. The second study describes further work on non-indexed pools where we pooled samples in an overlapping scheme and identified carriers of rare variants. The third study describes the whole genome and RNA sequencing of four patients with ALL and validated the results in a cohort of 168 additional ALL patients. In the whole genome sequenced patients we found somatic mutations in both known cancer driver-genes (KRAS and NOTCH1) and in putative driver-genes (KMT2D and KIF1B) after analysis of the additional ALL patients. We validated point mutations genome-wide and observed a large number of C>A mutations in one patient, in contrast to C>T mutations that are more common in cancer in general. The fourth study analyzed the same cohort as the third study and expanded the target to 872 genes linked to cancer, ALL or epigenetic regulation recorded in the literature. We found distinctive differences between BCP-ALL and T-ALL both in number and types of mutations. In addition we observed an association between mutations in the Notch signaling pathway and relapse.These results will have an impact on future studies of cancer, and add another piece to the genetic puzzle of ALL.

BackgroundWhen offered participation in clinical trials, families of children with cancer face a delicate balance between cure and toxicity. Since parents and children may perceive this balance differently, this paper explores whether adolescent patients have different enrollment patterns compared to younger children in trials with different toxicity profiles.ProcedureAge-dependent participation rates in three consecutive, randomized childhood leukemia trials conducted by the Nordic Society of Paediatric Haematology and Oncology were evaluated. The ALL2000 dexamethasone/vincristine (Dx/VCR) trial tested treatment intensifications to improve cure, and the back-to-back ALL2008 6-mercaptopurine (6MP) and ALL2008 PEG-asparaginase (ASP) trials tested treatment intensifications (6MP) and toxicity reduction without compromising survival (ASP). Patient randomization and toxicity data were prospectively registered by the treating physicians.ResultsParents of young children favored treatment intensifications (Dx/VCR: 12% refusal; 6MP: 14%; ASP: 21%), whereas parents of adolescents favored treatment reductions (Dx/VCR: 52% refusal; 6MP: 30%; ASP: 8%). Adolescents were more likely to refuse intensification trials than young children (adjusted ORs 6.3; P < 0.01 [Dx/VCR] and 2.1; P = 0.04 [6MP]). Adolescents were less likely to refuse the ASP trial, with varying effect size depending on the length of the preceding consolidation treatment (adjusted OR for median consolidation length 0.15; P = 0.01). Younger children participated more frequently in only 6MP than in only ASP (14% vs. 5%), and adolescents vice versa (2% vs. 17%; P = 0.001).ConclusionsParents' and adolescents' divergent inclinations toward intensified or reduced therapy emphasize the necessity of actively involving adolescents in the informed consent process, which should also address motives for trial participation.

BackgroundPrevious studies have indicated that patients with thiopurine methyltransferase (TPMT) low activity (TPMTLA) have reduced risk of relapse but increased risk of second malignant neoplasm (SMN) compared to patients with TPMT wild-type (TPMTWT) when treated with 6MP maintenance therapy starting doses of 75mg/m(2)/day. To reduce SMN risk, 6MP starting doses were reduced to 50mg/m(2)/day for patients with TPMT heterozygosity in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL2000 protocol.ProcedureWe explored the pattern of SMN and relapse in the NOPHO ALL2000 protocol (n=674) and NOPHO ALL92 protocol (n=601) in relation to TPMT pheno- and/or genotype.ResultsThe overall risk of any event did not differ significantly between the two protocols. However, in event pattern analyses considering only the patients with TPMTLA who experienced relapse or SMN, the risk of SMN versus leukemia relapse was significantly lower in the ALL2000 cohort for patients with a 6MP starting dose <75mg/m(2)/day when compared to the patients in ALL92 (relapse (n=11) and SMN (n=0) in ALL2000 versus relapse (n=5) and SMN (n=4) in ALL92, P=0.03). Furthermore, the 8-year cumulative incidence of relapse for patients with TPMTLA was significantly higher in the ALL2000 compared to the ALL92 cohort (19.7% (11.6-33.3%) vs. 6.7% (2.9-15.5%), P=0.03).ConclusionThis study indicates that reducing 6MP starting dose for patients with TPMTLA may reduce SMN risk but lead to a relapse risk similar to that of patients with TPMTWT. Pediatr Blood Cancer 2014;61:797-802. (c) 2014 Wiley Periodicals, Inc.

Children with acute lymphoblastic leukemia (ALL) have several risk factors for deep venous thromboses (DVTs) such as central venous catheters and asparaginase (ASP), related antithrombin (AT) deficiency. After introduction of a new standard and intermediate-risk ALL treatment protocol with prolonged continuous ASP treatment, two symptomatic DVTs in 10 patients were observed at the Children's Hospital, Helsinki, Finland. To prevent further thrombotic complications yet ensuring continuous exposure to ASP, an AT substitution strategy was adopted in Helsinki. The same ALL treatment protocol is used without AT substitution in the other Nordic countries. In this retrospective study, we describe the effect of prolonged ASP treatment on AT and fibrinogen levels in children without AT substitution in Stockholm, Sweden (n = 39) and the AT substitution in children with AT activity below 0.55 kIU/l in Helsinki (n = 36, intervention group). The intervention group is compared with children treated similarly earlier in Helsinki without AT substitution (n = 10). The median lowest AT activity during the ASP treatment without AT substitution was 0.55 kIU/l. Fibrinogen level of 1.0 g/l or less was found in 14% of all routine samples during the ASP treatment. In the intervention group, 23 (64%) received AT concentrate. Two (20%) children had symptomatic DVT before initiation of the AT substitution and two (6%) thereafter. We conclude that most children are exposed to low AT activity during ASP treatment predisposing to thrombosis. The effect of prophylactic AT substitution remains unclear.

Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)–deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (-9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

BackgroundIn children, T-cell acute lymphoblastic leukemia (T-ALL) has inferior prognosis compared with B-cell precursor ALL. In order to improve survival, individualized treatment strategies and thus risk stratification algorithms are warranted, ideally already at the time of diagnosis.ProcedureWe analyzed the frequency and prognostic implication of mutations in NOTCH1 and FBXW7 in 79 cases of Swedish childhood T-ALL treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL-1992 and ALL-2000 protocols. In a subgroup of patients, we also investigated the functional relevance of NOTCH1 mutations measured as expression of the HES1, MYB, and MYC genes.ResultsForty-seven of the cases (59%) displayed mutations in NOTCH1 and/or FBXW7. There was no difference in overall (P=0.14) or event-free survival (EFS) (P=0.10) in patients with T-ALL with mutation(s) in NOTCH1/FBXW7 compared with patients with T-ALL without mutations in any of these genes. T-ALL carrying NOTCH1 mutations had increased HES1 and MYB mRNA expression (HES1 9.21.9 (mean +/- SEM), MYB 8.7 +/- 0.8 (mean +/- SEM)) compared to T-ALL with wild-type NOTCH1 (HES1 1.8 +/- 0.7, MYB 5.1 +/- 1.2, P=0.02 and 0.008, respectively). In cases of T-ALL with high HES1 expression, improved overall (P=0.02) and EFS (P=0.028) was seen.ConclusionsIncreased NOTCH activity, reflected by increased HES1 expression, is associated with improved outcome in pediatric T-ALL, but its role as a diagnostic tool or a therapeutic target in future clinical treatment protocols remains to be elucidated. Pediatr Blood Cancer 2014;61:424-430. (c) 2013 Wiley Periodicals, Inc.

BACKGROUND:Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.RESULTS:We surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared with control blood cells, the methylomes of ALL cells shared 9,406 predominantly hypermethylated CpG sites, independent of cytogenetic background. Second, each cytogenetic subtype of ALL displayed a unique set of hyper- and hypomethylated CpG sites. The CpG sites that constituted these two signatures differed in their functional genomic enrichment to regions with marks of active or repressed chromatin. Third, we identified subtype-specific differential methylation in promoter and enhancer regions that were strongly correlated with gene expression. Fourth, a set of 6,612 CpG sites was predominantly hypermethylated in ALL cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status.CONCLUSIONS:Our results suggest an important biological role for DNA methylation in the differences between ALL subtypes and in their clinical outcome after treatment.