Marketa Zaliova

Hsa-mir-125b-2 is highly expressed in childhood ETV6/RUNX1 (TEL/AML1) leukemias and confers survival advantage to growth inhibitory signals independent of p53

MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose-dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic function of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT–PCR in four types of childhood acute lymphoblastic leukemia (ALL) characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly, high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies showed that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together, these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53-independent manner.

IKZF1 status as a prognostic feature in BCR-ABL1–positive childhood ALL

Childhood BCR-ABL1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has an unfavorable outcome and shows high frequency of IKZF1 deletions. The prognostic value of IKZF1 deletions was evaluated in 2 cohorts of BCR-ABL1-positive BCP-ALL patients, before tyrosine kinase inhibitors (pre-TKI) and after introduction of imatinib (in the European Study for Philadelphia-Acute Lymphoblastic Leukemia [EsPhALL]). In 126/191 (66%) cases an IKZF1 deletion was detected. In the pre-TKI cohort, IKZF1-deleted patients had an unfavorable outcome compared with wild-type patients (4-year disease-free survival [DFS] of 30.0 ± 6.8% vs 57.5 ± 9.4%; P = .01). In the EsPhALL cohort, the IKZF1 deletions were associated with an unfavorable prognosis in patients stratified in the good-risk arm based on early clinical response (4-year DFS of 51.9 ± 8.8% for IKZF1-deleted vs 78.6 ± 13.9% for IKZF1 wild-type; P = .03), even when treated with imatinib (4-year DFS of 55.5 ± 9.5% for IKZF1-deleted vs 75.0 ± 21.7% for IKZF1 wild-type; P = .05). In conclusion, the highly unfavorable outcome for childhood BCR-ABL1-positive BCP-ALL with IKZF1 deletions, irrespective of imatinib exposure, underscores the need for alternative therapies. In contrast, good-risk patients with IKZF1 wild-type responded remarkably well to imatinib-containing regimens, providing a rationale to potentially avoid hematopoietic stem-cell transplantation in this subset of patients.

Quantification of fusion transcript reveals a subgroup with distinct biological properties and predicts relapse in BCR/ABL-positive ALL: implications for residual disease monitoring

Minimal residual disease (MRD) monitoring is an essential tool for risk group stratification in current treatment protocols for childhood acute lymphoblastic leukaemia (ALL). Although quantitative detection of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is currently considered to be the standard method, leukaemia fusion genes provide other possible targets for MRD follow-up, as already demonstrated in TEL/AML1-positive ALLs. We analysed and compared MRD levels quantified by BCR/ABL transcript detection and by the standard Ig/TCR-based method in 218 bone marrow specimens from 17 children with BCR/ABL-positive ALL. We found only a limited overall correlation of MRD levels as assessed by the two methods (correlation coefficient R2=0.64). The correlation varied among patients from excellent (R2=0.99) to very poor (R2=0.17). Despite identical sensitivity of the approaches, 20% of the samples were negative by the Ig/TCR approach whereas positive by the BCR/ABL method. We show that multilineage involvement is at least partly responsible for the discrepancy. Moreover, our data demonstrate that BCR/ABL monitoring enables better and earlier prediction of relapse compared to the standard Ig/TCR methodology. We conclude that BCR/ABL-based MRD monitoring of childhood ALL is a clinically relevant tool and should be performed in parallel with the standard Ig/TCR follow-up.

ERG deletion is associated with CD2 and attenuates the negative impact of IKZF1 deletion in childhood acute lymphoblastic leukemia

ERG deletion is associated with CD2 and attenuates the negative impact of IKZF1 deletion in childhood acute lymphoblastic leukemia

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options

TCF3-HLF−positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF−positive and treatment-responsive TCF3-PBX1−positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Acute leukemias with ETV6/ABL1 (TEL/ABL) fusion: poor prognosis and prenatal origin.

The ETV6/ABL1 (TEL/ABL) fusion gene is a rare aberration in malignant disorders. Only 19 cases of ETV6/ABL1‐positive hematological malignancy have been published, diagnosed with chronic myeloid leukemia, other types of chronic myeloproliferative neoplasm, acute myeloid leukemia or acute lymphoblastic leukemia (ALL). This study reports three new cases (aged 8 months, 5 years, and 33 years) of ALL with the ETV6/ABL1 fusion found by screening 392 newly diagnosed ALL patients (335 children and 57 adults). A thorough review of the literature and an analysis of all published data, including the three new cases, suggest poor prognosis of ETV6/ABL1‐positive acute leukemias. The course of the disease in the two pediatric patients is characterized by minimal residual disease monitoring, using quantification of both the ETV6/ABL1 transcript and immunoreceptor gene rearrangements. Eosinophilia could not be confirmed as a hallmark of the ETV6/ABL1‐positive disease. Studies of neonatal blood spots demonstrated that, in the child diagnosed at five years, the ETV6/ABL1 fusion initiating the ALL originated prenatally.

CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage

Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, ‘monocytoids’ share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14negCD19posCD34pos B lymphoblasts and CD14posCD19negCD34neg ‘monocytoids’ were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.

Characterization of leukemias with ETV6-ABL1 fusion

To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with tyrosine kinase inhibitors should be considered for patients with this fusion.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: An international collaborative study

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4–7 and exons 1–8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case–control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2–7 (P=0.03), DEL 2–8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4–7 and DEL 1–8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

The Identification of (ETV6)/RUNX1-Regulated Genes in Lymphopoiesis Using Histone Deacetylase Inhibitors in ETV6/RUNX1-Positive Lymphoid Leukemic Cells

Purpose: Chimeric transcription factor ETV6/RUNX1 (TEL/AML1) is believed to cause pathologic block in lymphoid cell development via interaction with corepressor complex and histone deacetylase. We wanted to show the regulatory effect of ETV6/RUNX1 and its reversibility by histone deacetylase inhibitors (HDACi), as well as to identify potential ETV6/RUNX1-regulated genes. Experimental Design: We used luciferase assay to show the interaction of ETV6/RUNX1 protein, ETV6/RUNX1-regulated gene, and HDACi. To identify ETV6/RUNX1-regulated genes, we used expression profiling and HDACi in lymphoid cells. Next, using the flow cytometry and quantitative reverse transcription-PCR, we measured differentiation changes in gene and protein expression after HDACi treatment. Results: Luciferase assay showed repression of granzyme B expression by ETV6/RUNX1 protein and the reversibility of this effect by HDACi. Proving this regulatory role of ETV6/RUNX1, we identified, using complex statistical analysis, 25 genes that are potentially regulated by ETV6/RUNX1 protein. In four selected genes with known role in the cell cycle regulation (JunD, ACK1, PDGFRB, and TCF4), we confirmed expression changes after HDACi by quantitative analysis. After HDACi treatment, ETV6/RUNX1-positive cells showed immunophenotype changes resembling differentiation process compared with other leukemic cells (BCR/ABL, ETV6/PDGFRB positive). Moreover, ETV6/RUNX1-positive leukemic cells accumulated in G1-G0 phase after HDACi whereas other B-lineage leukemic cell lines showed rather unspecific changes including induction of apoptosis and decreased proliferation. Conclusions: Presented data support the hypothesis that HDACi affect ETV6/RUNX1-positive cells via direct interaction with ETV6/RUNX1 protein and that treatment with HDACi may release aberrant transcription activity caused by ETV6/RUNX1 chimeric transcription factor.