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Children with Down syndrome have an augmented risk for B-cell acute lymphoblastic leukemia (DS-ALL), which is associated with lower survival than in non-DS-ALL. It is known that cytogenetic abnormalities common in childhood ALL are less frequent in DS-ALL, while other genetic aberrancies (ie, CRLF2 overexpression and IKZF1 deletions) are increased. A possible cause for the lower survival of DS-ALL that we herewith evaluated for the first time was the incidence and prognostic value of the Philadelphia-like (Ph-like) profile and the IKZF1plus pattern. These features have been associated with poor outcome in non-DS ALL and therefore introduced in current therapeutic protocols. Forty-six out of 70 DS-ALL patients treated in Italy from 2000 to 2014 displayed Ph-like signature, mostly characterized by CRLF2 (n = 33) and IKZF1 (n = 16) alterations; only 2 cases were positive for ABL-class or PAX5-fusion genes. Moreover, in an Italian and German joint cohort of 134 DS-ALL patients, we observed 18% patients positive for IKZF1plus feature. Ph-like signature and IKZF1 deletion were associated with poor outcome (cumulative incidence of relapse: 27.7 ± 6.8% versus 13 ± 7%; P = 0.04 and 35.2 ± 8.6% versus 17 ± 3.9%; P = 0.007, respectively), which further worsens when IKZF1 deletion was co-occurring with P2RY8::CRLF2, qualifying for the IKZF1plus definition (13/15 patients had an event of relapse or treatment-related death). Notably, ex vivo drug screening revealed sensitivity of IKZF1plus blasts for drugs active against Ph-like ALL such as Birinapant and histone deacetylase inhibitors. We provided data in a large setting of a rare condition (DS-ALL) supporting that these patients, not associated with other high-risk features, need tailored therapeutic strategies.

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

Recently, we defined “CML-like” subtype of BCR::ABL1-positive acute lymphoblastic leukemia (ALL), resembling lymphoid blast crisis of chronic myeloid leukemia (CML). Here we retrospectively analyzed prognostic relevance of minimal residual disease (MRD) and other features in 147 children with BCR::ABL1-positive ALL (diagnosed I/2000–IV/2021, treated according to EsPhALL (n = 133) or other (n = 14) protocols), using DNA-based monitoring of BCR::ABL1 genomic breakpoint and clonal immunoglobulin/T-cell receptor gene rearrangements. Although overall prognosis of CML-like (n = 48) and typical ALL (n = 99) was similar (5-year-EFS 60% and 49%, respectively; 5-year-OS 75% and 73%, respectively), typical ALL presented more relapses while CML-like patients more often died in the first remission. Prognostic role of MRD was significant in the typical ALL (p = 0.0005 in multivariate analysis for EFS). In contrast, in CML-like patients MRD was not significant (p values > 0.2) and inapplicable for therapy adjustment. Moreover, in the typical ALL, risk-prediction could be further improved by considering initial hyperleukocytosis. Early distinguishing typical BCR::ABL1-positive ALL and CML-like patients is essential to enable optimal treatment approach in upcoming protocols. For the typical ALL, tyrosine-kinase inhibitors and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, no relevant prognostic feature applicable for therapy tailoring was found so far.

ABL-class fusions other than BCR-ABL1 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 BCR-ABL1 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 ≥5x10-4 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: clinicaltrials.gov identifier: NTC00430118, NCT00613457, NCT01117441).

ABL-class fusions other than BCR-ABL1 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 BCR-ABL1 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 ≥5x10-4 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: clinicaltrials.gov identifier: NTC00430118, NCT00613457, NCT01117441).

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.

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 IKZF1plus. The IKZF1plus group comprised 6% of patients with BCP ALL, with a 5-year event-free survival of 53 6 6% compared with 7965% in patients with IKZF1 deletion who did not fulfill the IKZF1plus 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 IKZF1plus. The IKZF1plus 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 IKZF1plus patients was 94 6 5% versus 40 6 10% in MRD intermediate- and 30 ± 14% in high-risk IKZF1plus patients (P ≤.001). Corresponding 5-year cumulative incidence of relapse rates were 6 ± 6%, 60 ± 10%, and 60 ± 17% (P ≤.001). Conclusion IKZF1plus describes a new MRD-dependent very-poor prognostic profile in BCP ALL. Because current AIEOP-BF

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 tyros

Item does not contain fulltext, 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.

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.

__Background:__ Although distinct patterns of homeobox (HOX) gene expression have been described in defined cytogenetic and molecular subsets of patients with acute myeloid leukemia (AML), it is unknown whether these patterns are the direct result of transcriptional alterations or rather represent the differentiation stage of the leukemic cell. __Method:__ To address this question, we used qPCR to analyze mRNA expression of HOXA and HOXB genes in bone marrow (BM) samples of 46 patients with AML and sorted subpopulations of healthy BM cells. These various stages of myeloid differentiation represent matched counterparts of morphological subgroups of AML. To further study the transcriptional alterations of HOX genes in hematopoiesis, we also analyzed gene expression of epigenetic modifiers in the subpopluations of healthy BM and leukemic cells. __Results:__ Unsupervised hierarchical clustering divided the AMLs into five clusters characterized by the presence of prevalent molecular genetic aberrations. Notably, the impact of genotype on HOX gene expression was significantly more pronounced than that of the differentiation stage of the blasts. This driving role of molecular aberrations was best exemplified by the repressive effect of the PML-RARa fusion gene on HOX gene expression, regardless of the presence of the FLT3/ITD mutation. Furthermore, HOX gene expression was positively correlated with mRNA levels of histone demethylases (JMJD3 and UTX) and negatively correlated with gene expression of DNA methyltranferases. No such relationships were observed in subpopulations of healthy BM cells. __Conclusion:__ Our results demonstrate that specific molecular genetic aberrations, rather than differentiation per se, underlie the observed differences in HOX gene expression in AML. Moreover, the observed correlations between epigenetic modifiers and HOX ex pression that are specific to malignant hematopoiesis, suggest their potential causal relationships.

Acute lymphoblastic leukemia (ALL) accounts for ∼25% of pediatric malignancies. Of interest, the incidence of ALL is observed ∼20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5' region of SLX4IP. We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in ∼30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1-rearranged ALL (both overall P < 0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected (P = 0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared with girls.

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.