IKZF1 Deletions with COBL Breakpoints Are Not Driven by RAG-Mediated Recombination Events in Acute Lymphoblastic Leukemia

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in both childhood and adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Previously, we revealed that COBL is a hotspot for breakpoints in leukemia and could promote IKZF1 deletions. Through an international collaboration, we provide a detailed genetic and clinical picture of B-ALL with COBL rearrangements (COBL-r). Patients with B-ALL and IKZF1 deletion (n = 133) were included. IKZF1 ∆1-8 were associated with large alterations within chromosome 7: monosomy 7 (18%), isochromosome 7q (10%), 7p loss (19%), and interstitial deletions (53%). The latter included COBL-r, which were found in 12% of the IKZF1 ∆1-8 cohort. Patients with COBL-r are mostly classified as intermediate cytogenetic risk and frequently harbor ETV6, PAX5, CDKN2A/B deletions. Overall, 56% of breakpoints were located within COBL intron 5. Cryptic recombination signal sequence motifs were broadly distributed within the sequence of COBL, and no enrichment for the breakpoint cluster region was found. In summary, a diverse spectrum of alterations characterizes ΔIKZF1 and they also include deletion breakpoints within COBL. We confirmed that COBL is a hotspot associated with ΔIKZF1, but these rearrangements are not driven by RAG-mediated recombination

Gain-of-function mutations in interleukin-7 receptor-α (IL7R) in childhood acute lymphoblastic leukemias

IL7R-activating mutations identified in B-ALL and T-ALL patient leukemic cells facilitate cytokine-independent growth

The Bone Marrow Niche in B-Cell Acute Lymphoblastic Leukemia: The Role of Microenvironment from Pre-Leukemia to Overt Leukemia

Genetic lesions predisposing to pediatric B-cell acute lymphoblastic leukemia (B-ALL) arise in utero, generating a clinically silent pre-leukemic phase. We here reviewed the role of the surrounding bone marrow (BM) microenvironment in the persistence and transformation of pre-leukemic clones into fully leukemic cells. In this context, inflammation has been highlighted as a crucial microenvironmental stimulus able to promote genetic instability, leading to the disease manifestation. Moreover, we focused on the cross-talk between the bulk of leukemic cells with the surrounding microenvironment, which creates a “corrupted” BM malignant niche, unfavorable for healthy hematopoietic precursors. In detail, several cell subsets, including stromal, endothelial cells, osteoblasts and immune cells, composing the peculiar leukemic niche, can actively interact with B-ALL blasts. Through deregulated molecular pathways they are able to influence leukemia development, survival, chemoresistance, migratory and invasive properties. The concept that the pre-leukemic and leukemic cell survival and evolution are strictly dependent both on genetic lesions and on the external signals coming from the microenvironment paves the way to a new idea of dual targeting therapeutic strategy

2009

Design and Methods Patients The study cohort was constituted by 410 non-Down syndrome, non-T, Philadelphia chromosome-negative, B-cell precursor ALL patients consecutively enrolled in the AIEOP-BFM ALL2000 study in AIEOP Centers from February 2003 to July 2005, who were included in the previous study on CRLF2 alterations and for whom DNA was still available. 19 P2RY8-CRLF2 rearrangement was tested by reverse transcriptase polymerase chain reaction analysis in 372 (90.7%) patients. The project was approved by the AIEOP ALL Scientific Committee. Risk group definitions and treatment outlines have already been reported DNA copy number variations IKZF1 deletions, together with deletions in other genes (CDKN2A/B, PAX5, ETV6, BTG1, RB1 and EBF1) were investigated by multiplex ligation-dependent probe amplification (MLPA) using the Salsa MLPA P335-A3 ALL-IKZF1 kit (MRC-Holland, Amsterdam, the Netherlands) according to the manufacturer's instructions. Samples from pediatric ALL patients in complete remission were used as wild-type controls. Statistical analysis Event-free survival time was calculated from the date of diagnosis to the date of an event, which was resistance, relapse, death or second neoplasm, whichever occurred first. Patients were censored at last follow-up if no events occurred. Event-free survival was estimated according to Kaplan-Meier, and compared using the log-rank test. The cumulative incidence of relapse at 5 years was estimated by adjusting for competing risks of other events and compared using Gray's test. Results IKZF1 deletions at diagnosis IKZF1 deletions were detected in 54/410 cases (13.2%), in keeping with incidence data reported in the literature. The clinical characteristics of the patients are presented in Using the MLPA technique we further analyzed the presence of copy number variations of other genes frequently deleted in B-cell precursor ALL and known to be involved in lymphoid development (PAX5, ETV6, EBF1) or in cell cycle regulation (CDKN2A/B, BTG1, RB1). 10,20-22 We did not find statistically significant differences in the incidence of these genetic alterations in children positive or negative for IKZF1 deletions (Online Prognostic impact of IKZF1 deletions Compared to patients without a deletion of IKZF1, those with a deletion of part or all of this gene had an inferior event-free survival [70.2% (6.2) versus 85.2% (1.9) at 5 years, P=0.007] and a significantly higher cumulative incidence of relapse [24.2% (5.9) versus 13.1% (1.8) at 5 years, P= 0.049] ( These data are in accordance with those from other studies reported in the literature, in particular with those recently published by Dorge et al. who analyzed ALL patients enrolled in the AIEOP-BFM ALL2000 study in Germany [event-free survival 69% (5) versus 85% (1), P=<0.001 and cumulative incidence of relapse 21% (4) versus 10% © F e r r a t a S t o r t i F o u n d a t i o n N o c o m m e r c i a l u s e deleted patients. These events contributed to the statistical significance of the difference in the event-free survival. In both Cox model analyses, the P2RY8-CRLF2 aberration and risk group were significantly associated with outcome. Of note, when individually analyzed, the IKFZ1 deletion had a statistically significant effect on event-free survival and relapse, in keeping with results in We further analyzed the prognostic value of IKZF1 deletions within the subgroups according to protocol stratification. IKZF1 deletions were less frequent within the standard-risk group, being found in 8 out 117 standard-risk patients (6.8%), 42 out of 264 intermediate-risk patients (15.9%) and 4 out of 29 high-risk patients (13.8%) ( Discussion Previous studies Indeed, overall event-free survival for patients with IKZF1 deletions, after excluding the confounding effects of Down syndrome, T-immunophenotype and Philadelphia chromosome-positive patients, was around 70% at 5 years also in our experience. The three patients with IKZF1 deletions who were at high risk and relapsed had poor response to treatment (high minimal residual disease levels) and accordingly were all eligible for transplantation, thus identification of IKZF1 deletions would not have contributed to a better stratification. In the intermediate risk group, with a 5-year event-free survival of 70%, treatment intensification could be justified to improve results. In our context, the recent AIEOP-BFM ALL 2009 study, with a more intensive use of L-asparaginase, might already provide a benefit that reduces the impact of an IKZF1 deletion. This is especially true if we consider that in our study cohort the difference in the cumulative incidence of relapse was not so marked, being approximately 7% in the intermediate risk subgroup and 11% overall. This, as well as the lower number of events in the multivariate analysis, may explain why the presence of IKZF1 deletions is an independent prognostic factor for event-free survival but not for the hazard of relapse alone. In conclusion, based on our data, the suitability of IKZF1 deletions as an additional stratification marker for Philadelphia chromosome-negative, B-cell precursor ALL patients remains questionable, at least until new target therapy becomes available. (NoE-2011-261474). Acknowledgments The authors would like to thank Simona Songia, Lilia Corral, Eugenia Mella, Tiziana Villa (Monza), Elena Seganfreddo and Katia Polato (Padova) for AIEOP minimal residual disease monitoring and all medical doctors of the AIEOP centers. This work was supported by grants from: Fondazione Tettamanti (Monza), Fondazione Città della Speranza (Padova), Associazione Italiana Ricerca sul Cancro (AIRC) (to GB, AB, MGV, GteK and GC), MIUR (to GB and AB), Fondazione Cariplo (to AB, GC and GteK), and CARIPARO project of excellence (to GteK). This work was (partly) funded by the European Commission (FP7) under the contract ENCCA Authorship and Disclosure

Inhibition of inflammatory signaling in Pax5 mutant cells mitigates B-cell leukemogenesis

© The Author(s) 2020.PAX5 is one of the most frequently mutated genes in B-cell acute lymphoblastic leukemia (B-ALL), and children with inherited preleukemic PAX5 mutations are at a higher risk of developing the disease. Abnormal profiles of inflammatory markers have been detected in neonatal blood spot samples of children who later developed B-ALL. However, how inflammatory signals contribute to B-ALL development is unclear. Here, we demonstrate that Pax5 heterozygosis, in the presence of infections, results in the enhanced production of the inflammatory cytokine interleukin-6 (IL-6), which appears to act in an autocrine fashion to promote leukemia growth. Furthermore, in vivo genetic downregulation of IL-6 in these Pax5 heterozygous mice retards B-cell leukemogenesis, and in vivo pharmacologic inhibition of IL-6 with a neutralizing antibody in Pax5 mutant mice with B-ALL clears leukemic cells. Additionally, this novel IL–6 signaling paradigm identified in mice was also substantiated in humans. Altogether, our studies establish aberrant IL6 expression caused by Pax5 loss as a hallmark of Pax5-dependent B-ALL and the IL6 as a therapeutic vulnerability for B-ALL characterized by PAX5 loss.We would also like to thank all members of our groups for useful suggestions and for their critical reading of the manuscript. Research at G.C.’s laboratory was supported by Italian Association for Cancer Research (grant IG-17593 to GC) and Fondazione Cariplo (grant 2018-0339 to CP). Research at CC’s laboratory was partially supported by FEDER, EU, MINECO (SAF2017-83061-R), the “Fundación Ramón Areces,” a Research Contract with the “Fundación Síndrome de Wolf-Hirschhorn o 4p-”, and institutional grants from the “Fundación Ramón Areces” and “Banco de Santander” to the CBMSO. Research in the CVD group is partially supported by FEDER, “Miguel Servet” Grant (CPII19/00024—AES 2017-2020) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad), “Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III” (PI17/00167). Research in the ISG group is partially supported by FEDER and by SAF2015-64420-R MINECO/FEDER, UE, RTI2018-093314-B-I00 MCIU/AEI/FEDER, UE, by Junta de Castilla y León (UIC-017, CSI001U16, and CSI234P18), and by the German Jose Carreras Foundation (DJCLS R13/26; DJCLS 07R/2019). CVD, and ISG have been supported by the German Federal Office for Radiation Protection (BfS)-Germany (FKZ: 3618S32274). M.R.O., and ISG have been supported by the Fundacion Unoentrecienmil (CUNINA project). Research in the A.O. group is partially supported by "Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III" - FEDER-Ministerio de Economía y Competitividad (PI19/01183). AC-G and M.I.-H. are supported by FSE-Conserjería de Educación de la Junta de Castilla y León 2019 and 2020 (ESF- European Social Fund) fellowship, respectively. J.R.-G. is supported by a scholarship from University of Salamanca co-financed by Banco Santander and ESF