Publications by authors named "Marketa Zaliova"

53 Publications

Novel class of ZNF384 aberrations in acute leukemia.

Blood Adv 2021 Sep 16. Epub 2021 Sep 16.

CLIP, University Hospital Motol, Czech Republic.

Fusion of the ZNF384 gene as the 3' partner to several different 5' partner genes occurs recurrently in B-cell precursor acute lymphoblastic and mixed phenotype B/myeloid leukemia. These canonical fusions (ZNF384r) contain the complete ZNF384 coding sequence and are associated with a specific gene expression signature. Cases with this signature, but without canonical ZNF384 fusions (ZNF384r-like cases), have been described previously. Though some have been shown to harbor ZNF362 fusions, the primary aberrations remain unknown in a major proportion. We studied three patients with the ZNF384r signature and unknown primary genetic background and identified a previously unknown class of genetic aberration affecting the last exon of ZNF384 and resulting in disruption of the C-terminal portion of the ZNF384 protein. Importantly, in two cases, the ZNF384 aberration - indel - was missed during the bioinformatic analysis but revealed by the manual, targeted reanalysis. Two cases with the novel aberrations had a mixed (B/myeloid) immunophenotype commonly associated with canonical ZNF384 fusions. In conclusion, we present leukemia cases with a novel class of ZNF384 aberrations that phenocopy leukemia with ZNF384r. Therefore, we show that part of the so-called ZNF384r-like cases represent the same genetic subtype as leukemia with canonical ZNF384 fusions.
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http://dx.doi.org/10.1182/bloodadvances.2021005318DOI Listing
September 2021

The Clinical Utility of Optical Genome Mapping for the Assessment of Genomic Aberrations in Acute Lymphoblastic Leukemia.

Cancers (Basel) 2021 Aug 30;13(17). Epub 2021 Aug 30.

Department of Human Genetics, Hannover Medical School, 30625 Hannover, Germany.

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., , ) 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 and 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.
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http://dx.doi.org/10.3390/cancers13174388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8431583PMC
August 2021

Pharmacogenetics of the Central Nervous System-Toxicity and Relapse Affecting the CNS in Pediatric Acute Lymphoblastic Leukemia.

Cancers (Basel) 2021 May 12;13(10). Epub 2021 May 12.

2nd Department of Pediatrics, Semmelweis University, H-1094 Budapest, Hungary.

Despite improving cure rates in childhood acute lymphoblastic leukemia (ALL), therapeutic side effects and relapse are ongoing challenges. These can also affect the central nervous system (CNS). Our aim was to identify germline gene polymorphisms that influence the risk of CNS events. Sixty single nucleotide polymorphisms (SNPs) in 20 genes were genotyped in a Hungarian non-matched ALL cohort of 36 cases with chemotherapy related acute toxic encephalopathy (ATE) and 544 controls. Five significant SNPs were further analyzed in an extended Austrian-Czech-NOPHO cohort ( = 107 cases, = 211 controls) but none of the associations could be validated. Overall populations including all nations' matched cohorts for ATE ( = 426) with seizure subgroup ( = 133) and posterior reversible encephalopathy syndrome (PRES, = 251) were analyzed, as well. We found that patients with rs1045642, rs1128503 or rs2032582 TT genotypes were more prone to have seizures but those with rs1045642 TT developed PRES less frequently. The same SNPs were also examined in relation to ALL relapse on a case-control matched cohort of 320 patients from all groups. Those with rs1128503 CC or rs2032582 GG genotypes showed higher incidence of CNS relapse. Our results suggest that blood-brain-barrier drug transporter gene-polymorphisms might have an inverse association with seizures and CNS relapse.
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http://dx.doi.org/10.3390/cancers13102333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151239PMC
May 2021

Functional reconstruction of human AML reveals stem cell origin and vulnerability of treatment-resistant MLL-rearranged leukemia.

Sci Transl Med 2021 02;13(582)

Leukaemia and Stem Cell Biology Group, School of Cancer and Pharmaceutical Sciences, King's College, London SE5 9NU, UK.

Chemoresistance remains the major challenge for successful treatment of acute myeloid leukemia (AML). Although recent mouse studies suggest that treatment response of genetically and immunophenotypically indistinguishable AML can be influenced by their different cells of origin, corresponding evidence in human disease is still largely lacking. By combining prospective disease modeling using highly purified human hematopoietic stem or progenitor cells with retrospective deconvolution study of leukemia stem cells (LSCs) from primary patient samples, we identified human hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs) as two distinctive origins of human AML driven by Mixed Lineage Leukemia (MLL) gene fusions (MLL-AML). Despite LSCs from either MLL-rearranged HSCs or MLL-rearranged CMPs having a mature CD34/CD38 immunophenotype in both a humanized mouse model and primary patient samples, the resulting AML cells exhibited contrasting responses to chemotherapy. HSC-derived MLL-AML was highly resistant to chemotherapy and expressed elevated amounts of the multispecific anion transporter ABCC3. Inhibition of ABCC3 by shRNA-mediated knockdown or with small-molecule inhibitor fidaxomicin, currently used for diarrhea associated with infection, effectively resensitized HSC-derived MLL-AML toward standard chemotherapeutic drugs. This study not only functionally established two distinctive origins of human LSCs for MLL-AML and their role in mediating chemoresistance but also identified a potential therapeutic avenue for stem cell-associated treatment resistance by repurposing a well-tolerated antidiarrhea drug already used in the clinic.
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http://dx.doi.org/10.1126/scitranslmed.abc4822DOI Listing
February 2021

Outcomes of paediatric patients with B-cell acute lymphocytic leukaemia with ABL-class fusion in the pre-tyrosine-kinase inhibitor era: a multicentre, retrospective, cohort study.

Lancet Haematol 2021 Jan 22;8(1):e55-e66. Epub 2020 Dec 22.

Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands. Electronic address:

Background: ABL-class fusion genes other than BCR-ABL1 have been identified in approximately 3% of children with newly diagnosed acute lymphocytic leukaemia, and studies suggest that leukaemic cells carrying ABL-class fusions can be targeted successfully by tyrosine-kinase inhibitors. We aimed to establish the baseline characteristics and outcomes of paediatric patients with ABL-class fusion B-cell acute lymphocytic leukaemia in the pre-tyrosine-kinase inhibitor era.

Methods: This multicentre, retrospective, cohort study included paediatric patients (aged 1-18 years) with newly diagnosed ABL-class fusion (ABL1 fusion-positive, ABL2 fusion-positive, CSF1R fusion-positive, and PDGFRB fusion-positive) B-cell acute lymphocytic leukaemia enrolled in clinical trials of multidrug chemotherapy done between Oct 3, 2000, and Aug 28, 2018, in which tyrosine-kinase inhibitors had not been given as a first-line treatment. Patients from 14 European, North American, and Asia-Pacific study groups of the Ponte di Legno group were included. No patients were excluded, and patients were followed up by individual study groups. Through the Ponte di Legno group, we collected data on the baseline characteristics of patients, including IKZF1, PAX5, and CDKN2A/B deletion status, and whether haematopoietic stem cell transplantation (HSCT) had been done, as well as treatment outcomes, including complete remission, no response, relapse, early death, and treatment-related mortality, response to prednisone, and minimal residual disease (MRD) at end of induction therapy. 5-year event-free survival and 5-year overall survival were estimated by use of Kaplan-Meier methods, and the 5-year cumulative incidence of relapse was calculated by use of a competing risk model.

Findings: We identified 122 paediatric patients with newly diagnosed ABL-class fusion B-cell acute lymphocytic leukaemia (77 from European study groups, 25 from North American study groups, and 20 from Asia-Pacific study groups). 64 (52%) of 122 patients were PDGFRB fusion-positive, 40 (33%) were ABL1 fusion-positive, ten (8%) were CSF1R fusion-positive, and eight (7%) were ABL2 fusion-positive. In all 122 patients, 5-year event-free survival was 59·1% (95% CI 50·5-69·1), 5-year overall survival was 76·1% (68·6-84·5), and the 5-year cumulative incidence of relapse was 31·0% (95% CI 22·4-40·1). MRD at the end of induction therapy was high (≥10 cells) in 61 (66%) of 93 patients, and most prevalent in patients with ABL2 fusions (six [86%] of 7 patients) and PDGFRB fusion-positive B-cell acute lymphocytic leukaemia (43 [88%] of 49 patients). MRD at the end of induction therapy of 10 cells or more was predictive of an unfavourable outcome (hazard ratio of event-free survival in patients with a MRD of ≥10vs those with a MRD of <10 3·33 [95% CI 1·46-7·56], p=0·0039). Of the 36 (30%) of 119 patients who relapsed, 25 (69%) relapsed within 3 years of diagnosis. The 5-year cumulative incidence of relapse in 41 patients who underwent HSCT (17·8% [95% CI 7·7-31·3]) was lower than in the 43 patients who did not undergo HSCT (45·1% [28·4-60·5], p=0·013), but event-free survival and overall survival did not differ between these two groups.

Interpretation: Children with ABL-class fusion B-cell acute lymphocytic leukaemia have poor outcomes when treated with regimens that do not contain a tyrosine-kinase inhibitor, despite the use of high-risk chemotherapy regimens and frequent HSCT upon first remission. Our findings provide a reference for evaluating the potential benefit of first-line tyrosine-kinase inhibitor treatment in patients with ABL-class fusion B-cell acute lymphocytic leukaemia.

Funding: The Oncode Institute, Pediatric Cancer Foundation Rotterdam, Dutch Cancer Society, Kika Foundation, Deutsche Krebshilfe, Blood Cancer UK, Associazione Italiana per la Ricerca sul Cancro, Cancer Australia, National Cancer Institute, National Institute of Health, and St Baldrick's Foundation.
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http://dx.doi.org/10.1016/S2352-3026(20)30353-7DOI Listing
January 2021

Rapidly progressing acute myeloid leukemia with KAT6A-LEUTX fusion in a newborn.

Pediatr Blood Cancer 2020 10 11;67(10):e28663. Epub 2020 Aug 11.

Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic.

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http://dx.doi.org/10.1002/pbc.28663DOI Listing
October 2020

DUX4r, ZNF384r and PAX5-P80R mutated B-cell precursor acute lymphoblastic leukemia frequently undergo monocytic switch.

Haematologica 2021 08 1;106(8):2066-2075. Epub 2021 Aug 1.

CLIP-Dpt.of Paediatric Haematology/Oncology, Charles University, Prague, Czech Republic.

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.
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http://dx.doi.org/10.3324/haematol.2020.250423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8327733PMC
August 2021

Repurposing anthelmintic agents to eradicate resistant leukemia.

Blood Cancer J 2020 06 26;10(6):72. Epub 2020 Jun 26.

Department of Oncology and Children's Research Center, Children's Hospital Zurich, Lengghalde 5, Balgrist Campus AG, 8008, Zurich, Switzerland.

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 IC 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.
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http://dx.doi.org/10.1038/s41408-020-0339-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320149PMC
June 2020

Metabolic profile of leukemia cells influences treatment efficacy of L-asparaginase.

BMC Cancer 2020 Jun 5;20(1):526. Epub 2020 Jun 5.

CLIP - Childhood Leukaemia Investigation Prague, Prague, Czech Republic.

Background: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs.

Methods: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence.

Results: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us.

Conclusions: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.
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http://dx.doi.org/10.1186/s12885-020-07020-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275298PMC
June 2020

13q12.2 deletions in acute lymphoblastic leukemia lead to upregulation of FLT3 through enhancer hijacking.

Blood 2020 08;136(8):946-956

Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden.

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene in 13q12.2 are among the most common driver events in acute leukemia, leading to increased cell proliferation and survival through activation of the phosphatidylinositol 3-kinase/AKT-, RAS/MAPK-, and STAT5-signaling pathways. In this study, we examine the pathogenetic impact of somatic hemizygous 13q12.2 microdeletions in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) using 5 different patient cohorts (in total including 1418 cases). The 13q12.2 deletions occur immediately 5' of FLT3 and involve the PAN3 locus. By detailed analysis of the 13q12.2 segment, we show that the deletions lead to loss of a topologically associating domain border and an enhancer of FLT3. This results in increased cis interactions between the FLT3 promoter and another enhancer located distally to the deletion breakpoints, with subsequent allele-specific upregulation of FLT3 expression, expected to lead to ligand-independent activation of the receptor and downstream signaling. The 13q12.2 deletions are highly enriched in the high-hyperdiploid BCP ALL subtype (frequency 3.9% vs 0.5% in other BCP ALL) and in cases that subsequently relapsed. Taken together, our study describes a novel mechanism of FLT3 involvement in leukemogenesis by upregulation via chromatin remodeling and enhancer hijacking. These data further emphasize the role of FLT3 as a driver gene in BCP ALL.
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http://dx.doi.org/10.1182/blood.2019004684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498303PMC
August 2020

Novel SAMD9 Mutation in a Patient With Immunodeficiency, Neutropenia, Impaired Anti-CMV Response, and Severe Gastrointestinal Involvement.

Front Immunol 2019 18;10:2194. Epub 2019 Sep 18.

Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czechia.

Mutations in the Sterile alpha motif domain containing 9 () gene have been described in patients with severe multisystem disorder, MIRAGE syndrome, but also in patients with bone marrow (BM) failure in the absence of other systemic symptoms. The role of hematopoietic stem cell transplantation (HSCT) in the management of the disease is still unclear. Here, we present a patient with a novel mutation in (c.2471 G>A, p.R824Q), manifesting with prominent gastrointestinal tract involvement and immunodeficiency, but without any sign of adrenal insufficiency typical for MIRAGE syndrome. He suffered from severe CMV (cytomegalovirus) infection at 3 months of age, with a delayed development of T lymphocyte functional response against CMV, profound T cell activation, significantly reduced B lymphocyte counts and impaired lymphocyte proliferative response. Cultured T cells displayed slightly lower calcium flux and decreased survival. At the age of 6 months, he developed severe neutropenia requiring G-CSF administration, and despite only mild morphological and immunophenotypical disturbances in the BM, 78% of the BM cells showed monosomy 7 at the age of 18 months. Surprisingly, T cell proliferation after CD3 stimulation and apoptosis of the cells normalized during the follow-up, possibly reflecting the gradual development of monosomy 7. Among other prominent symptoms, he had difficulty swallowing, requiring percutaneous endoscopic gastrostomy (PEG), frequent gastrointestinal infections, and perianal erosions. He suffered from repeated infections and periodic recurring fevers with the elevation of inflammatory markers. At 26 months of age, he underwent HSCT that significantly improved hematological and immunological laboratory parameters. Nevertheless, he continued to suffer from other conditions, and subsequently, he died at day 440 post-transplant due to sepsis. Pathogenicity of this novel mutation was confirmed experimentally. Expression of mutant caused a significant decrease in proliferation and increase in cell death of the transfected cells. We describe a novel mutation in a patient with prominent gastrointestinal and immunological symptoms but without adrenal hypoplasia. Thus, SAMD9 mutations should be considered as cause of enteropathy in pediatric patients. The insufficient therapeutic outcome of transplantation further questions the role of HSCT in the management of patients with mutations and multisystem involvement.
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http://dx.doi.org/10.3389/fimmu.2019.02194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759462PMC
November 2020

Relapses and treatment-related events contributed equally to poor prognosis in children with ABL-class fusion positive B-cell acute lymphoblastic leukemia treated according to AIEOP-BFM protocols.

Haematologica 2020 07 10;105(7):1887-1894. Epub 2019 Oct 10.

Pediatric Hematology-Oncology Department, Ruth Rappaport Children's Hospital, Rambam Health Care Campus, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

ABL-class fusions other than characterize around 2-3% of precursor B-cell acute lymphoblastic leukemia. Case series indicated that patients suffering from these subtypes have a dismal outcome and may benefit from the introduction of tyrosine kinase inhibitors. We analyzed clinical characteristics and outcome of 46 ABL-class fusion positive cases other than treated according to AIEOP-BFM (Associazione Italiana di Ematologia-Oncologia Pediatrica-Berlin-Frankfurt-Münster) ALL 2000 and 2009 protocols; 13 of them received a tyrosine kinase inhibitor (TKI) during different phases of treatment. ABL-class fusion positive cases had a poor early treatment response: minimal residual disease levels of ≥5×10 were observed in 71.4% of patients after induction treatment and in 51.2% after consolidation phase. For the entire cohort of 46 cases, the 5-year probability of event-free survival was 49.1+8.9% and that of overall survival 69.6+7.8%; the cumulative incidence of relapse was 25.6+8.2% and treatment-related mortality (TRM) 20.8+6.8%. One out of 13 cases with TKI added to chemotherapy relapsed while eight of 33 cases without TKI treatment suffered from relapse, including six in 17 patients who had not received hematopoietic stem cell transplantation. Stem cell transplantation seems to be effective in preventing relapses (only three relapses in 25 patients), but was associated with a very high TRM (6 patients). These data indicate a major need for an early identification of ABL-class fusion positive acute lymphoblastic leukemia cases and to establish a properly designed, controlled study aimed at investigating the use of TKI, the appropriate chemotherapy backbone and the role of hematopoietic stem cell transplantation. (Registered at: .
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http://dx.doi.org/10.3324/haematol.2019.231720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327633PMC
July 2020

Molecular Basis of Cisplatin Resistance in Testicular Germ Cell Tumors.

Cancers (Basel) 2019 Sep 6;11(9). Epub 2019 Sep 6.

Department of Pediatric Hematology and Oncology, CLIP, 2nd Faculty of Medicine, Charles University and University Hospital Motol, 150 00 Prague, Czech Republic.

The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients' samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients' samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., , in one patient, and in another one). Among all primary tumor samples, the most commonly mutated gene was , affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.
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http://dx.doi.org/10.3390/cancers11091316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769617PMC
September 2019

Genomic landscape of pediatric B-other acute lymphoblastic leukemia in a consecutive European cohort.

Haematologica 2019 07 10;104(7):1396-1406. Epub 2019 Jan 10.

CLIP - Childhood Leukaemia Investigation Prague

Novel biological subtypes and clinically important genetic aberrations (druggable lesions, prognostic factors) have been described in B-other acute lymphoblastic leukemia (ALL) during the last decade; however, due to a lack of studies on unselected cohorts, their population frequency and mutual associations still have to be established. We studied 110 consecutively diagnosed and uniformly treated childhood B-other patients using single nucleotide polymorphism arrays and whole exome/transcriptome sequencing. The frequency of -rearranged, -like, -rearranged, -like, iAMP21 and -rearranged subtypes was 27%, 15%, 5%, 5%, 4%, and 2%, respectively; 43% of cases were not classified into any of these subtypes (B-rest). We found worse early response to treatment in -rearranged leukemia and a strong association of -rearranged leukemia with B-myeloid immunophenotype. Of the druggable lesions, JAK/STAT-class and RAS/RAF/MAPK-class aberrations were found in 21% and 43% of patients, respectively; an ABL-class aberration was found in one patient. A recently described negative prognostic factor, , was found in 14% of patients and was enriched in (but not exclusive for) -like subtype. fusions (including 4 novel), intragenic amplifications and P80R mutations were mutually exclusive and only occurred in the B-rest subset, altogether accounting for 20% of the B-other group. P80R was associated with a specific gene expression signature, potentially defining a novel leukemia subtype. Our study shows unbiased European population-based frequencies of novel ALL subtypes, recurrent (cyto)genetic aberrations and their mutual associations. This study also strengthens and widens the current knowledge of B-other ALL and provides an objective basis for optimization of current genetic diagnostics.
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http://dx.doi.org/10.3324/haematol.2018.204974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601078PMC
July 2019

deletions in childhood acute lymphoblastic leukemia with rearrangements are mostly polyclonal, prognostically relevant and their detection rate strongly depends on screening method sensitivity.

Haematologica 2019 07 10;104(7):1407-1416. Epub 2019 Jan 10.

CLIP - Childhood Leukaemia Investigation Prague.

-deletions occur recurrently in acute lymphoblastic leukemia, especially in the -rearranged subtype. The -deletion was shown to positively impact prognosis of patients with -deletion and its presence precludes assignment into group, a novel high-risk category on AIEOP-BFM ALL trials. We analyzed the impact of different methods on -deletion detection rate, evaluated -deletion as a potential marker for -rearranged leukemia, studied its associations with molecular and clinical characteristics within this leukemia subtype, and analyzed its clonality. Using single-nucleotide-polymorphism array, genomic polymerase chain reaction (PCR) and amplicon-sequencing we found -deletion in 34% (16 of 47), 66% (33 of 50) and 78% (39 of 50) of -rearranged leukemia, respectively. False negativity of -deletion by single-nucleotide-polymorphism array caused misclassification in 5 patients. No -deletion was found outside the -rearranged cases. Within -rearranged leukemia, the -deletion was associated with higher total number of copy-number aberrations, and, importantly, the -deletion positivity by PCR was associated with better outcome [5-year event-free survival (EFS), -deletion-positive 93% -deletion-negative 68%, =0.022; 5-year overall survival (OS), -deletion-positive 97% -deletion-negative 75%, =0.029]. Ultra-deep amplicon-sequencing revealed distinct co-existing -deletions in 22 of 24 patients. In conclusion, our data demonstrate inadequate sensitivity of single-nucleotide-polymorphism array for -deletion detection, unacceptable for proper classification. Even using more sensitive methods (PCR/amplicon-sequencing) for its detection, -deletion is absent in 22-34% of -rearranged leukemia and does not represent an adequately sensitive marker of this leukemia subtype. Importantly, the -deletion potentially stratifies the -rearranged leukemia into biologically/clinically distinct subsets. Frequent polyclonal pattern of -deletions shows that late origin of this lesion is more common than has been previously described.
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http://dx.doi.org/10.3324/haematol.2018.204487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601096PMC
July 2019

New biological and genetic classification and therapeutically relevant categories in childhood B-cell precursor acute lymphoblastic leukemia.

F1000Res 2018 28;7. Epub 2018 Sep 28.

Department of Pediatric Hematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic.

Traditionally, genetic abnormalities detected by conventional karyotyping, fluorescence hybridization, and polymerase chain reaction divided childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) into well-established genetic subtypes. This genetic classification has been prognostically relevant and thus used for the risk stratification of therapy. Recently, the introduction of genome-wide approaches, including massive parallel sequencing methods (whole-genome, -exome, and -transcriptome sequencing), enabled extensive genomic studies which, together with gene expression profiling, largely expanded our understanding of leukemia pathogenesis and its heterogeneity. Novel BCP-ALL subtypes have been described. Exact identification of recurrent genetic alterations and their combinations facilitates more precise risk stratification of patients. Discovery of targetable lesions in subsets of patients enables the introduction of new treatment modalities into clinical practice and stimulates the transfer of modern methods from research laboratories to routine practice.
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http://dx.doi.org/10.12688/f1000research.16074.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173109PMC
March 2019

Two novel fusion genes, AIF1L-ETV6 and ABL1-AIF1L, result together with ETV6-ABL1 from a single chromosomal rearrangement in acute lymphoblastic leukemia with prenatal origin.

Genes Chromosomes Cancer 2018 09 30;57(9):471-477. Epub 2018 Jul 30.

CLIP - Childhood Leukaemia Investigation Prague, Prague, Czech Republic.

Fusion genes resulting from chromosomal rearrangements represent a hallmark of childhood acute lymphoblastic leukemia (ALL). Unlike more common fusion genes generated via simple reciprocal chromosomal translocations, formation of the ETV6-ABL1 fusion gene requires 3 DNA breaks and usually results from an interchromosomal insertion. We report a child with ALL in which a single interchromosomal insertion led to the formation of ETV6-ABL1 and 2 novel fusion genes: AIF1L-ETV6 and ABL1-AIF1L. We demonstrate the prenatal origin of this complex chromosomal rearrangement, which apparently initiated the leukemogenic process, by successful backtracking of the ETV6-ABL1 fusion into the patient's archived neonatal blood. We cloned coding sequences of AIF1L-ETV6 and ABL1-AIF1L in-frame fusion transcripts from the patient's leukemic blasts and we show that the chimeric protein containing the DNA binding domain of ETV6 is expressed from the AIF1L-ETV6 transcript and localized in both the cytoplasm and nucleus of transfected HEK293T cells. Transcriptomic and genomic profiling of the diagnostic bone marrow sample revealed Ph-like gene expression signature and loss of the IKZF1 and CDKN2A/B genes, the typical genetic lesions accompanying ETV6-ABL1-positive ALL. The prenatal origin of the rearrangement confirms that ETV6-ABL1 is not sufficient to cause overt leukemia, even when combined with the 2 novel fusions. We did not find the AIF1L-ETV6 and ABL1-AIF1L fusions in other ETV6-ABL1-positive ALL. Nevertheless, functional studies would be needed to establish the biological role of AIF1L-ETV6 and ABL1-AIF1L and to determine whether they contribute to leukemogenesis and/or to the final leukemia phenotype.
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http://dx.doi.org/10.1002/gcc.6DOI Listing
September 2018

International cooperative study identifies treatment strategy in childhood ambiguous lineage leukemia.

Blood 2018 07 2;132(3):264-276. Epub 2018 May 2.

National Program for Antineoplastic Drugs for Children (PINDA), Chilean National Pediatric Oncology Group, Hospital Roberto del Rio/Universidad de Chile, Santiago, Chile.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Münster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19 leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19 ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19 and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.
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http://dx.doi.org/10.1182/blood-2017-12-821363DOI Listing
July 2018

Relapsed acute lymphoblastic leukemia-specific mutations in NT5C2 cluster into hotspots driving intersubunit stimulation.

Leukemia 2018 06 25;32(6):1393-1403. Epub 2018 Feb 25.

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6, 166 10, Czech Republic.

Activating mutations in NT5C2, a gene encoding cytosolic purine 5'-nucleotidase (cN-II), confer chemoresistance in relapsed acute lymphoblastic leukemia. Here we show that all mutants became independent of allosteric effects of ATP and thus constitutively active. Structural mapping of mutations described in patients demonstrates that 90% of leukemia-specific allelles directly affect two regulatory hotspots within the cN-II molecule-the helix A region: residues 355-365, and the intersubunit interface: helix B (232-242) and flexible interhelical loop L (400-418). Furthermore, analysis of hetero-oligomeric complexes combining wild-type (WT) and mutant subunits showed that the activation is transmitted from the mutated to the WT subunit. This intersubunit interaction forms structural basis of hyperactive NT5C2 in drug-resistant leukemia in which heterozygous NT5C2 mutation gave rise to hetero-tetramer mutant and WT proteins. This enabled us to define criteria to aid the prediction of NT5C2 drug resistance mutations in leukemia.
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http://dx.doi.org/10.1038/s41375-018-0073-5DOI Listing
June 2018

IKZF1 Defines a New Minimal Residual Disease-Dependent Very-Poor Prognostic Profile in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

J Clin Oncol 2018 04 2;36(12):1240-1249. Epub 2018 Mar 2.

Martin Stanulla, Elif Dagdan, Stefanie V. Junk, Laura Hinze, Norman Klein, Christian P. Kratz, Petra Dörge, Doris Steinemann, and Martin Zimmermann, Hannover Medical School; Petra Dörge, German Center for Infection Research, Hannover; Anja Möricke, Kirsten Bleckmann, Denis Schewe, Gunnar Cario, and Martin Schrappe, University Hospital Schleswig-Holstein; Britt-Sabina Petersen and Andre Franke, Kiel University, Kiel; Cornelia Eckert and Stefanie Groeneveld-Krentz, Charité University Hospital; Wolf-Dieter Ludwig, HELIOS-Clinic Berlin-Buch, Berlin; Rolf Koehler, Claus R. Bartram, Andreas Kulozik, and Martina U. Muckenthaler, University of Heidelberg, Heidelberg; Arndt Borkhardt, Heinrich-Heine University, Düsseldorf, Germany; Marketa Zaliova, Charles University and University Hospital Motol, Prague, Czech Republic; Chiara Palmi, Giovanni Cazzaniga, and Andrea Biondi, Azienda Ospedaliera San Gerardo; Maria Grazia Valsecchi, University of Milano-Bicocca, Monza; Geertruy te Kronnie and Giuseppe Basso, University of Padova, Padua, Italy; Jean-Pierre Bourquin and Beat Bornhauser, University Children's Hospital Zurich, Zurich, Switzerland; Shai Izraeli, Sheba Medical Center Tel-Hashomer and Tel Aviv University, Tel Aviv, Israel; Oskar A. Haas and Renate Panzer-Grümayer, St Anna Kinderkrebsforschung and Medical University Vienna, Vienna, Austria; Hélène Cavé, Robert Debré Hospital and Paris-Diderot University, Paris, France; and Richard S. Houlston, The Institute of Cancer Research, London, United Kingdom.

Purpose Somatic deletions that affect the lymphoid transcription factor-coding gene IKZF1 have previously been reported as independently associated with a poor prognosis in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). We have now refined the prognostic strength of IKZF1 deletions by analyzing the effect of co-occurring deletions. Patients and Methods The analysis involved 991 patients with BCP ALL treated in the Associazione Italiana Ematologia ed Oncologia Pediatrica-Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 trial with complete information for copy number alterations of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, Xp22.33/Yp11.31 (PAR1 region; CRLF2, CSF2RA, and IL3RA), and ERG; replication of findings involved 417 patients from the same trial. Results IKZF1 deletions that co-occurred with deletions in CDKN2A, CDKN2B, PAX5, or PAR1 in the absence of ERG deletion conferred the worst outcome and, consequently, were grouped as IKZF1. The IKZF1 group comprised 6% of patients with BCP ALL, with a 5-year event-free survival of 53 ± 6% compared with 79 ± 5% in patients with IKZF1 deletion who did not fulfill the IKZF1 definition and 87 ± 1% in patients who lacked an IKZF1 deletion ( P ≤ .001). Respective 5-year cumulative relapse incidence rates were 44 ± 6%, 11 ± 4%, and 10 ± 1% ( P ≤ .001). Results were confirmed in the replication cohort, and multivariable analyses demonstrated independence of IKZF1. The IKZF1 prognostic effect differed dramatically in analyses stratified by minimal residual disease (MRD) levels after induction treatment: 5-year event-free survival for MRD standard-risk IKZF1 patients was 94 ± 5% versus 40 ± 10% in MRD intermediate- and 30 ± 14% in high-risk IKZF1 patients ( P ≤ .001). Corresponding 5-year cumulative incidence of relapse rates were 6 ± 6%, 60 ± 10%, and 60 ± 17% ( P ≤ .001). Conclusion IKZF1 describes a new MRD-dependent very-poor prognostic profile in BCP ALL. Because current AIEOP-BFM treatment is largely ineffective for MRD-positive IKZF1 patients, new experimental treatment approaches will be evaluated in our upcoming trial AIEOP-BFM ALL 2017.
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http://dx.doi.org/10.1200/JCO.2017.74.3617DOI Listing
April 2018
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