Acute lymphoblastic leukemia (ALL) is the most prevalent type of cancer occurring in children. ALL is characterized by structural and numeric genomic aberrations that strongly correlate with prognosis and clinical outcome. Usually, a combination of cyto- and molecular genetic methods (karyotyping, array-CGH, FISH, RT-PCR, RNA-Seq) is needed to identify all aberrations relevant for risk stratification. We investigated the feasibility of optical genome mapping (OGM), a DNA-based method, to detect these aberrations in an all-in-one approach. As proof of principle, twelve pediatric ALL samples were analyzed by OGM, and results were validated by comparing OGM data to results obtained from routine diagnostics. All genomic aberrations including translocations (e.g., dic(9;12)), aneuploidies (e.g., high hyperdiploidy) and copy number variations (e.g., IKZF1, PAX5) known from other techniques were also detected by OGM. Moreover, OGM was superior to well-established techniques for resolution of the more complex structure of a translocation t(12;21) and had a higher sensitivity for detection of copy number alterations. Importantly, a new and unknown gene fusion of JAK2 and NPAT due to a translocation t(9;11) was detected. We demonstrate the feasibility of OGM to detect well-established as well as new putative prognostic markers in an all-in-one approach in ALL. We hope that these limited results will be confirmed with testing of more samples in the future.

• Publikační typ
• časopisecké články MeSH

Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL including refractory relapse cases confirmed strong anti-leukemia activity with IC50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.

• MeSH
• akutní lymfatická leukemie farmakoterapie   patologie   MeSH
• anthelmintika farmakologie   terapeutické užití   MeSH
• apoptóza účinky léků   MeSH
• chemorezistence MeSH
• lidé MeSH
• myši SCID MeSH
• nádorové buňky kultivované MeSH
• nádorové mikroprostředí účinky léků   MeSH
• přehodnocení terapeutických indikací léčivého přípravku * MeSH
• protinádorové látky farmakologie   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• akutní lymfatická leukemie * diagnóza   epidemiologie   genetika   MeSH
• lidé MeSH
• mutace MeSH
• pre-B-buněčná leukemie * diagnóza   epidemiologie   genetika   MeSH
• prognóza MeSH
• transkripční faktor Zeb2 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH

• Publikační typ
• časopisecké články MeSH

Asparaginase-associated pancreatitis (AAP) frequently affects children treated for acute lymphoblastic leukemia (ALL) causing severe acute and persisting complications. Known risk factors such as asparaginase dosing, older age and single nucleotide polymorphisms (SNPs) have insufficient odds ratios to allow personalized asparaginase therapy. In this study, we explored machine learning strategies for prediction of individual AAP risk. We integrated information on age, sex, and SNPs based on Illumina Omni2.5exome-8 arrays of patients with childhood ALL (N=1564, 244 with AAP 1.0 to 17.9 yo) from 10 international ALL consortia into machine learning models including regression, random forest, AdaBoost and artificial neural networks. A model with only age and sex had area under the receiver operating characteristic curve (ROC-AUC) of 0.62. Inclusion of 6 pancreatitis candidate gene SNPs or 4 validated pancreatitis SNPs boosted ROC-AUC somewhat (0.67) while 30 SNPs, identified through our AAP genome-wide association study cohort, boosted performance (0.80). Most predictive features included rs10273639 (PRSS1-PRSS2), rs10436957 (CTRC), rs13228878 (PRSS1/PRSS2), rs1505495 (GALNTL6), rs4655107 (EPHB2) and age (1 to 7 y). Second AAP following asparaginase re-exposure was predicted with ROC-AUC: 0.65. The machine learning models assist individual-level risk assessment of AAP for future prevention trials, and may legitimize asparaginase re-exposure when AAP risk is predicted to be low.

• MeSH
• akutní lymfatická leukemie * farmakoterapie   genetika   MeSH
• asparaginasa * škodlivé účinky   MeSH
• celogenomová asociační studie MeSH
• dítě MeSH
• lidé MeSH
• pankreatitida * chemicky indukované   genetika   MeSH
• protinádorové látky * škodlivé účinky   MeSH
• strojové učení MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Survival for childhood acute lymphoblastic leukaemia surpasses 90% with contemporary therapy; however, patients remain burdened by the severe toxic effects of treatment, including asparaginase-associated pancreatitis. To investigate the risk of complications and risk of re-exposing patients with asparaginase-associated pancreatitis to asparaginase, 18 acute lymphoblastic leukaemia trial groups merged data for this observational study. METHODS: Patient files from 26 trials run by 18 trial groups were reviewed on children (aged 1·0-17·9 years) diagnosed with t(9;22)-negative acute lymphoblastic leukaemia between June 1, 1996, and Jan 1, 2016, who within 50 days of asparaginase exposure developed asparaginase-associated pancreatitis. Asparaginase-associated pancreatitis was defined by at least two criteria: abdominal pain, pancreatic enzymes at least three times the upper limit of normal (ULN), and imaging compatible with pancreatitis. Patients without sufficient data for diagnostic criteria were excluded. Primary outcomes were defined as acute and persisting complications of asparaginase-associated pancreatitis and risk of re-exposing patients who suffered an episode of asparaginase-associated pancreatitis to asparaginase. Data were collected from Feb 2, 2015, to June 30, 2016, and analysed and stored in a common database at Rigshospitalet, Copenhagen, Denmark. FINDINGS: Of 465 patients with asparaginase-associated pancreatitis, 33 (8%) of 424 with available data needed mechanical ventilation, 109 (26%) of 422 developed pseudocysts, acute insulin therapy was needed in 81 (21%) of 393, and seven (2%) of 458 patients died. Risk of assisted mechanical ventilation, need for insulin, pseudocysts, or death was associated with older age (median age for patients with complications 10·5 years [IQR 6·4-13·8] vs without complications 6·1 years [IQR 3·6-12·2], p<0·0001), and having one or more affected vital signs (fever, hypotension, tachycardia, or tachypnoea; 96 [44%] of 217 patients with affected vital signs vs 11 [24%] of 46 patients without affected vital signs, p=0·02). 1 year after diagnosis of asparaginase-associated pancreatitis, 31 (11%) of 275 patients still needed insulin or had recurrent abdominal pain or both. Both the risk of persisting need for insulin therapy and recurrent abdominal pain were associated with having had pseudocysts (odds ratio [OR] 9·48 [95% CI 3·01-35·49], p=0·0002 for insulin therapy; OR 11·79 [4·30-37·98], p<0·0001 for recurrent abdominal pain). Within 8 years of asparaginase-associated pancreatitis, risk of abdominal symptoms dropped from 8% (26 of 312) to 0% (0 of 35) but the need for insulin therapy remained constant (9%, three of 35). 96 patients were re-exposed to asparaginase, including 59 after a severe asparaginase-associated pancreatitis (abdominal pain or pancreatic enzymes at least three times the ULN or both lasting longer than 72 h). 44 (46%) patients developed a second asparaginase-associated pancreatitis, 22 (52%) of 43 being severe. Risk of persisting need for insulin or abdominal pain after having had two versus one asparaginase-associated pancreatitis did not differ (three [7%] of 42 vs 28 [12%] of 233, p=0·51). Risk of a second asparaginase-associated pancreatitis was not associated with any baseline patient characteristics. INTERPRETATION: Since the risk of a second asparaginase-associated pancreatitis was not associated with severity of the first asparaginase-associated pancreatitis and a second asparaginase-associated pancreatitis did not involve an increased risk of complications, asparaginase re-exposure should be determined mainly by the anticipated need for asparaginase for antileukaemic efficacy. A study of the genetic risk factors identifying patients in whom asparaginase exposure should be restricted is needed. FUNDING: The Danish Childhood Cancer Foundation and The Danish Cancer Society (R150-A10181).

• MeSH
• akutní lymfatická leukemie komplikace   farmakoterapie   MeSH
• asparaginasa škodlivé účinky   MeSH
• dítě MeSH
• kohortové studie MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• pankreatitida chemicky indukované   epidemiologie   terapie   MeSH
• předškolní dítě MeSH
• protinádorové látky škodlivé účinky   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH

A better description of the leukemia cell surface proteome (surfaceome) is a prerequisite for the development of diagnostic and therapeutic tools. Insights into the complexity of the surfaceome have been limited by the lack of suitable methodologies. We combined a leukemia xenograft model with the discovery-driven chemoproteomic Cell Surface Capture technology to explore the B-cell precursor acute lymphoblastic leukemia (BCP-ALL) surfaceome; 713 cell surface proteins, including 181 CD proteins, were detected through combined analysis of 19 BCP-ALL cases. Diagnostic immunophenotypes were recapitulated in each case, and subtype specific markers were detected. To identify new leukemia-associated markers, we filtered the surfaceome data set against gene expression information from sorted, normal hematopoietic cells. Nine candidate markers (CD18, CD63, CD31, CD97, CD102, CD157, CD217, CD305, and CD317) were validated by flow cytometry in patient samples at diagnosis and during chemotherapy. CD97, CD157, CD63, and CD305 accounted for the most informative differences between normal and malignant cells. The ALL surfaceome constitutes a valuable resource to assist the functional exploration of surface markers in normal and malignant lymphopoiesis. This unbiased approach will also contribute to the development of strategies that rely on complex information for multidimensional flow cytometry data analysis to improve its diagnostic applications.

• MeSH
• akutní lymfatická leukemie imunologie   metabolismus   MeSH
• CD antigeny analýza   MeSH
• imunofenotypizace MeSH
• lidé MeSH
• membránové proteiny * analýza   metabolismus   MeSH
• myši MeSH
• nádorové biomarkery * analýza   MeSH
• proteom * analýza   metabolismus   MeSH
• průtoková cytometrie MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

Most relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.

• MeSH
• akutní lymfatická leukemie * farmakoterapie   MeSH
• amidohydrolasy terapeutické užití   MeSH
• B-lymfocyty MeSH
• chemorezistence * genetika   MeSH
• dítě MeSH
• GPI-vázané proteiny MeSH
• hematopoetické kmenové buňky MeSH
• lidé MeSH
• molekuly buněčné adheze MeSH
• prospektivní studie MeSH
• protokoly protinádorové kombinované chemoterapie MeSH
• retrospektivní studie MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Recently, several studies have demonstrated a negative prognostic impact of Ikaros (IKZF1) gene alterations in acute lymphoblastic leukemia (ALL). However, controversies still exist regarding the impact of IKZF1 in current treatment protocols. PROCEDURE: We simultaneously detected IKZF1 gene deletions by multiplex ligation-dependent probe amplification and gene expression of IKZF1 isoforms in 206 children with BCR/ABL-negative ALL treated with ALL IC-BFM 2002 protocol, in which risk stratification was not based on minimal residual disease (MRD), and validated the results on a cohort of 189 patients treated with MRD-directed ALL-BFM 2000 protocol. RESULTS: Deletion of IKZF1 was present in 14 of 206 (7%) ALL IC patients. Interestingly, gene expression did not completely correlate with the deletion status in either cohort. Deletions were not always reflected in the gene expression of dominant-negative isoforms, and conversely, 7 of 395 (2%) non-deleted cases overexpressed dominant-negative isoform Ik6. IKZF1 deletions significantly affected event-free survival (EFS) of the ALL IC cohort (41 ± 14% vs. 86 ± 3%, P < 0.0001). Regarding IKZF1 isoforms, only Ik6 overexpression had negative prognostic impact (EFS 50 ± 16% vs. 85 ± 3%, P = 0.003). In multivariate analysis, which included ALL IC risk criteria, flow-cytometric MRD and IKZF1 alterations, day 15 MRD and IKZF1 deletion status displayed an independent prognostic impact. CONCLUSIONS: We show that MRD-directed treatment diminishes prognostic impact of IKZF1 alterations. However, IKZF1 status alone or combined with day 15 flow cytometry can significantly improve risk stratification within BFM protocols at centers that do not perform antigen-receptor-based MRD monitoring.

• MeSH
• akutní lymfatická leukemie genetika   mortalita   patologie   MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• dítě MeSH
• lidé MeSH
• předškolní dítě MeSH
• přežití bez známek nemoci MeSH
• prognóza MeSH
• průtoková cytometrie MeSH
• reziduální nádor genetika   patologie   MeSH
• rizikové faktory MeSH
• transkripční faktor Ikaros genetika   MeSH
• transkriptom MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH