RESEARCH LETTER TO THE EDITOR: KMT2A-CBL rearrangements in acute leukemias: clinical characteristics and genetic breakpoints lie Caye-Eude,4 Marion Strullu,5 Arthur Sterin,6 Oscar Molina,2 Patrice Chevallier,7 Alex Bataller,1,2,* Francesca Guijarro,3,* Aure Marketa Zaliova, Jan Zuna, Pablo Mozas, Laura Magnano,1 Nathalie Grardel,9 Pascale Cornillet-Lefebvre,10 Jen-Fen Fu,11 8 8 1 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/24/5617/1855916/advancesadv2021005736.pdf by guest on 08 February 2023 Lee-Yung Shih,12 Temenuzhka Boneva,13 Elisabeth Nacheva,13 Silvia Be a,3,14 M opez-Guerra,3,14 Clara Bueno,2,14,15 onica L 2,14-16 Pablo Men endez, Jordi Esteve, Patrizia Larghero, Claus Meyer, and Rolf Marschalek17 1,2 17 17 1 Hematology Department, Hospital Clınic de Barcelona, Institut d’Investigacions Biom ediques August Pi i Sunyer (IDIBAPS); 2Josep Carreras Leukemia Research Institute, School of Medicine, 3Hematopathology Section, Hospital Clınic de Barcelona, University of Barcelona, Barcelona, Spain; 4Department of Genetics and Molecular Genetics; 5 Department of Pediatric Hematology, AP-HP, Robert Debr e Hospital, Paris, France; 6Department of Pediatric Hematology, Immunology and Oncology, APHM, La Timone Children Hospital, Marseille, France; 7Hematology Department, CHU Hotel-Dieu, Nantes, France; 8CLIP, Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University Prague and University Hospital Motol, Prague, Czech Republic; 9Department of Hematology, University Hospital Claude Huriez, Lille, France; 10 Laboratoire d'H ematologie, H^ e, Reims, France; 11Department of Medical Research, Chang Gung Memorial Hospital-Linkou, Taoyuan, Taiwan; 12Division of opital Robert Debr Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University, Taipei, Taiwan; 13HSL Analytics LLP OncoGenomics, London, United Kingdom and UCL Cancer Institute, UK; 14Centro de Investigaci on Biom edica en Red de Cancer, 15RICORS-TERAV, ISCIII, Madrid, Spain; 16Instituci o Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; and 17Institute of Pharmaceutical Biology/Diagnostic Center of Acute Leukemia, Goethe University, Frankfurt/Main, Germany KMT2A (MLL) abnormalities are common in leukemias of various lineages. Specifically, KMT2A chromo- somal rearrangements are present in about 5% to 15% of all adolescents and young adults diagnosed with acute lymphoblastic leukemia (ALL) and in 3% of all adult patients with acute myeloid leukemia (AML).1,2 Thus far, more than 100 KMT2A partner genes have been described, AFF1 (AF4) and MLLT3 (AF9) being the most common ones, mainly associated with ALL and AML, respectively.3 Although the KMT2A recombinome has been largely studied in B-cell ALL (B-ALL), its characterization in T-cell ALL (T-ALL) is still limited, with KMT2A rearrangements accounting for about 2% to 5% of all T-ALL cases.4 In T-ALL, in turn, the most frequent partner genes involved in KMT2A rearrangements are MLLT1 (ENL) and AFDN (AF6).3,4 CBL (also known as c-CBL, casitas B-lineage leukemia) encodes for a ubiquitin-ligase (E3s) involved in the negative regulation of signaling mediated by multiple tyrosine kinase receptors. This protein is a mem- ber of the Cbl family of proto-oncogenes, which has 3 members (CBL, CBLB, and CBLC) and is ubiqui- tously expressed in the cytoplasm of many tissues.5 The protein domain structure of Cbl is presented in Figure 1A. At its N-terminal portion, there is a tyrosine kinase–binding (TKB) domain, an EF hand, and a Src homology 2 domain (SH2). Its main function is to bind multiple cytoplasmatic substrates, including tyro- sine kinase proteins. The zinc-binding Really Interesting New Gene (RING) finger domain, which regulates the E3 ubiquitin ligase activity of c-Cbl, brings in an E2 ubiquitin-conjugating enzyme and therefore ubiquity- lates the substrate bound to the TKB domain. Adjacent to the RING domain, there is a proline-rich region involved in protein/protein interactions via inducible association with SH2 and SH3. At the C-terminal por- tion, there is a ubiquitin associated/leucine zipper domain (UBA/LZ), crucial for Cbl homodimerization. Several mutations in CBL have been described in various hematologic disorders, mostly in myeloid malignancies. The frequency of CBL mutations has been as high as 15% and 4% of adult myelo- dysplastic/myeloproliferative diseases and myeloproliferative neoplasms, respectively. In AML, its frequency is lower: approximately 1% to 2%.6 In juvenile myelomonocytic leukemia (JMML), CBL mutations are also common, with a frequency of 17%.7 In ALL, although rare, a few reports have described CBL mutations in both T- and B-cell lineages, especially in KMT2A-rearranged leuke- mias.8,9 CBL mutations consist mostly of point mutations clustering in exons 8 and 9, involving the linker and RING domain6 (Figure 1A). The proposed oncogenic mechanism of CBL mutations is Submitted 12 July 2021; accepted 13 September 2021; prepublished online on © 2021 by The American Society of Hematology. Licensed under Creative Blood Advances First Edition 12 October 2021; final version published online 28 Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC- December 2021. DOI 10.1182/bloodadvances.2021005736. ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other *A.B. and F.G. contributed equally to this study. rights reserved. Requests for data sharing may be submitted to Alex Bataller (abataller@clinic.cat). 28 DECEMBER 2021 • VOLUME 5, NUMBER 24 5617 A P7 P5, P6, P4, P3 and P1 P2 Linker TKB domain Mutation hotspot B KMT2A gene Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/24/5617/1855916/advancesadv2021005736.pdf by guest on 08 February 2023 P2 P5 P1 P3 P6 P7 P4 0 00 00 00 00 00 00 00 00 41 .5 .0 .5 .0 .5 .0 .5 .5 .3 45 46 46 47 47 48 48 89 90 P7 CBL gene P5 P6 P4 P3 P1 P2 0 26 0 00 00 72 0 00 00 73 0 00 00 75 0 75 0 76 0 76 0 77 0 77 0 00 78 0 00 00 00 93 0 00 94 0 94 0 00 95 0 00 04 50 0 0 0 0 0 0 0 0 0 0 0 0 0 .0 .5 .5 .0 .5 .0 .5 .0 .5 .0 .5 .0 .5 .0 .5 .0 .5 .0 .0 .5 .0 .5 .0 .5 .0 .5 .0 26 71 72 73 74 74 78 79 92 92 93 95 96 Figure 1. CBL structure and KMT2A-CBL rearrangements. (A) Structure of the CBL protein and its domains: 4-helix bundle (4H), EF-hand-calcium binding (EF), SRC homology 2 (SH2), RING finger (RF), proline-rich domain (PR), and ubiquitin-associated domain (UBA). Phosphorylation sites (P) and predicted protein breakpoints for each patient are also represented. (B) Schematic representation of KMT2A and CBL genes with the breakpoint for each patient. based on intracellular signaling deregulation because of the loss maintains the reading frame. This mechanism could also explain of CBL inhibitory function.10 Interestingly, in CBL-mutated mye- the KMT2A-CBL rearrangement of patient 7, but this hypothesis loid neoplasms, a loss of the germline CBL allele has been dem- could not be verified because of the lack of material. This study onstrated, mostly because of uniparental disomy.11 has been approved by the Ethics Committee of Hospital Clınic de Barcelona. Code:HCB/2021/0010 and was conducted in accor- To our knowledge, only 4 reports of hematologic neoplasms harbor- dance with the Declaration of Helsinki. ing a KMT2A-CBL gene fusion (3 of them under the diagnosis of AML) have been published.3,12,13 International collaborative efforts, Despite the limited number of patients, some biological insights such as the MLL recombinome database, are crucial to identify and about this rare rearrangement can be extracted from our findings. collect these rare rearrangements. Along with the previously pub- We report for the first time that KMT2A-CBL rearrangements are lished cases, we have now identified 3 new cases with the same present not only in AML and B-ALL but also in T-ALL. Moreover, 2 rearrangement but in distinct hematologic malignancies (Table 1). It of these cases were secondary neoplasms (JMML and polycythemia has been described that KMT2A breakpoints mostly localize within vera; both received prior chemotherapeutic agents), which is also exon 9 and intron 11, with an association of intron 9 breakpoints common in KMT2A-rearranged leukemias. CBL is located in chro- with AML or older patients and intron 11 with ALL and younger mosome 11q23, at a distance of 680 kb from KMT2A, suggesting patients.3 In our cohort, all patients had their breakpoint within the that KMT2A-CBL rearrangements are necessarily caused either by previous described limits, 4 of them clustering in intron 9, although an interstitial deletion or by a translocation event occurring between establishing associations between age or diagnosis with this finding both homologous chromosomes 11. Split-signal FISH analysis can seems far-fetched. CBL breakpoints also cluster in intron 9 (Figure 1). reveal these types of rearrangements, whereas conventional cytoge- The KMT2A-CBL fusions of patients 2 and 7 are per se out of netics usually fails to detect them. Subsequently, different diagnostic frame. However, the reverse transcriptase-polymerase chain reac- techniques can be used to identify CBL as the KMT2A fusion part- tion (RT-PCR) of case 2 revealed a cryptic exon composed of the ner, although their implementation in the clinical routine might be dif- truncated exon 10 of KMT2A and additional filler DNA that ficult. Targeted next-generation sequencing (NGS) is a feasible 5618 RESEARCH LETTER 28 DECEMBER 2021 • VOLUME 5, NUMBER 24 Table 1. Patients with KMT2A-CBL rearrangement Patient 1 (Marseille) Patient 2 (Reims) Patient 3 (Barcelona) Patient 43 (Prague) Patient 512 (Taiwan) Patient 63 (Nantes) Patient 713 (London) Sex, age (y) Male, 9 Male, 11 Male, 18 Male, 3 Female, 28 Female, 59 Female, 18 Medical history JMML - KRAS mut 6MP None None Development delay None Polycythemia vera JAK21 None & CB-AlloSCT (Bu/Cy) Hydroxyurea and pipobroman Hematologic T-ALL (EGIL T2) T-ALL (EGIL T2) T-ALL (EGIL T2) B-ALL (EGIL B1) AML (FAB M1) sAML (FAB M5) AML diagnosis White blood count (109 /L) 1.1 5.39 12.32 65.5 0.8 37.5 1.3 Hemoglobin (g/L) 90 97 83 107 44 107 74 Platelets (109 /L) 109 69 24 71 15 45 55 Blast count peripheral 0/97 33/99 85/99 80/93 47/96 65/83 blood/bone marrow (%) Flow cytometry cCD31, sCD32, cCD31, sCD32, CD711, cCD31, sCD31 dim, CD342, TdT1, CD191, CD341, CD131, CD331 CD342, CD131, CD341, cMPO1, TdT1, CD991, CD211, CD51/2 (13%), CD342, TdT2, CD991 CD79a1, CD101, CD71 CD331, CD41 CD331, CD151, CD71, CD21, CD51 CD341dim, CD3811, CD131 dim, CD1a2, CD711, dim, CD381 CD381, CD561 CD381 Cytogenetics Pseudodiploid and 47,XY,?del(5)(q31),18, 47, XY, der(7),119[20] NA 50,XX,122,13 4546,XX,-5,add(6)(p21), 46,XX[20] monosomic with del(9)(q13q34),del(11)(q21q23), mar [5]/49,XX, 219, -9,1i(9)(p10), 3 clonal anomalies: del(12)(p11)[cp6]/46,XY[28] 122,13mar add(11)(q23),-18,1mar[7], del(2q),-16,r [11]/46,XX,inv(9)[4] 4749,XX, 12,-5,-9,i(9q10),add(11)(q23), ?113,-18,119,120,inc[9] KMT2A FISH KMT2A 3' loss of signal KMT2A 3' loss of signal KMT2A 3' loss of signal KMT2A 3' loss of signal — KMT2A 3' loss of signal KMT2A 3' loss of signal 28 DECEMBER 2021 • VOLUME 5, NUMBER 24 NGS KRAS p.G13C (VAF 32%) IL7R p.V253_A254insVA No mutations detected KRAS p.A18D (VAF 44%) CBL p.L380P (VAF 70%) — RUNX1 p.G372S ASXL1 p.G646Wfs12 (VAF 26%) (VAF 13%) (VAF 50%) STAT3 p.N481I (VAF 36%) KMT2A p.N*2278fs (VAF 82%) NOTCH1 p.P2514fs (VAF 16%) NOTCH1 p.A1700D (VAF 10%) NOTCH1 p.L1678P (VAF 36%) ZFHX4 p.R2676Q (VAF 28%) Method for RNAseq and targeted NGS RNAseq and Targeted NGS Targeted NGS RNAseq and LDI-PCR cDNA panhandle PCR Validated RNAseq and LDI-PCR Chromosomal KMT2A-CBL by RT-PCR 1 cloning microarray analysis detection and sequencing of the KMT2A fusion product. Breakpoint KMT2A intron 9 KMT2A exon 10 KMT2A intron 9 KMT2A intron 10 KMT2A intron 9 KMT2A intron 9 KMT2A intron 10 CBL intron 9 CBL intron 15 CBL intron 9 CBL intron 9 CBL intron 9 CBL intron 9 CBL exon 2 Clinical outcome Induction Chemotherapy Induction chemotherapy Induction chemotherapy AEIOP/BFM 2000 ALL Induction chemotherapy Induction chemotherapy Induction (CALL-T protocol) – CR MRD1 (CAALL-F01 protocol) (PETHEMA 2019) – PR protocol CR MRD- CR CR chemotherapy CR Consolidation 1 Intensification – NR Re-induction (FLAG-IDA) – Relapse after 8 y Consolidation – CR Consolidation CR Loss of follow-up CR MRD-CB-AlloSCT Consolidation 1 Intensification CR MRD- IntReALL SR 2010 protocol Relapse 2 y after diagnosis RD-AlloSCT Alive, CR 1 y after diagnosis CR MRD- Consolidation – CR MRD- CR-MRD1 Reinduction Relapse 11 mo after diagnosis RD-AlloSCT MUD-AlloSCT Blinatumomab 3 2 CR Azacytidine: No response Alive, CR 3 y after diagnosis Alive, CR 6 mo after MUD-AlloSCT MUD-AlloSCT Deceased because of diagnosis Alive, CR 13 y Deceased due to alloSCT disease progression after diagnosis complications In cytogenetic studies, number of metaphases studied is represented in brackets. 6MP, mercaptopurine; AlloSCT, allogeneic stem cell transplantation; CB, cord blood; CR: complete response; EGIL, European Group for the Immunological Classification of Leukemia; FAB, French-American-British classification; LDI-PCR, long-distance-inverse PCR; MRD, minimal residual disease; MUD, matched unrelated donor; NR, no response; PR, partial response; RD, related donor; VAF, variant allele frequency. RESEARCH LETTER 5619 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/24/5617/1855916/advancesadv2021005736.pdf by guest on 08 February 2023 alternative to detect this and other rare rearrangements included in Spain; email: abataller@clinic.cat and Francesca Guijarro, the panel. Hematopathology Section, Hospital Clınic de Barcelona, Carrer Villarroel 170, 08036 Barcelona, Spain; email: fguijarro@clinic.cat. In terms of the leukemogenic effect of KMT2A-CBL rearrangements, to date, there are no specific models that can clearly explain its mechanism. CBL is a cytoplasmatic protein so its effect in the leuke- References mic cell might be different from that of nuclear factors like AFF1 or MLLT3.14 In fact, it has been hypothesized that rearrangements of KMT2A with cytoplasmatic proteins could trigger their oncogenic 1. Malard F, Mohty M. Acute lymphoblastic leukaemia. Lancet. 2020; effects through dimerization of the chimeric KMT2A protein, enhanc- 395(10230):1146-1162. ing a specific gene expression program.15 This is supported by the 2. Papaemmanuil E, Gerstung M, Bullinger L, et al. Genomic retention of the UBA domain responsible for the homodimerization of classification and prognosis in acute myeloid leukemia. N Engl J CBL in all the KMT2A-CBL cases reported in the present study. Med. 2016;374(23):2209-2221. Downloaded from http://ashpublications.org/bloodadvances/article-pdf/5/24/5617/1855916/advancesadv2021005736.pdf by guest on 08 February 2023 Thus, dimerization of KMT2A-CBL through the UBA domains could 3. Meyer C, Burmeister T, Gr€ oger D, et al. The MLL recombinome of be an important leukemogenic mechanism to be explored. Another acute leukemias in 2017. Leukemia. 2018;32(2):273-284. alternative explanation is that the translocated protein domains might 4. T€ urkmen S, Timmermann B, Bartels G, et al. Involvement of the MLL exert novel functions in the wrong cellular compartments. In addition, gene in adult T-lymphoblastic leukemia. Genes Chromosom Cancer. the resulting truncated CBL protein most likely represents a genetic 2012;51(12):1114-1124. “loss-of-function” situation, as already observed in hematological 5. Rao N, Dodge I, Band H. The Cbl family of ubiquitin ligases: critical neoplasms with CBL inactivating mutations.10 negative regulators of tyrosine kinase signaling in the immune To conclude, KMT2A-CBL rearrangements are rare events identi- system. J Leukoc Biol. 2002;71(5):753-763. fied in leukemias of various lineages. Conventional cytogenetics may 6. Schnittger S, Bacher U, Alpermann T, et al. Use of CBL exon 8 and miss such rearrangements but the increasingly widespread use of 9 mutations in diagnosis of myeloproliferative neoplasms and targeted sequencing techniques in the diagnostic work-up will prob- myelodysplastic/myeloproliferative disorders: an analysis of 636 cases. Haematologica. 2012;97(12):1890-1894. ably increase the number of cases identified. International collabora- tive efforts are necessary to improve the characterization of the 7. Loh ML, Sakai DS, Flotho C, et al. Mutations in CBL occur KMT2A-CBL rearrangement and investigate its prognostic impact. frequently in juvenile myelomonocytic leukemia. Blood. 2009; 114(9):1859-1863. Ultimately, functional experiments of KMT2A-CBL gain-of-function models and CD341 hematopoietic progenitor cells may provide fun- 8. Saito Y, Aoki Y, Muramatsu H, et al. Casitas B-cell lymphoma muta- damental knowledge about the leukemogenic potential and the cell- tion in childhood T-cell acute lymphoblastic leukemia. Leuk Res. 2012;36(8):1009-1015. of-origin of KMT2A-CBL leukemias. 9. Shiba N, Park MJ, Taki T, et al. CBL mutations in infant acute Acknowledgments: The authors thank Centres de Recerca de lymphoblastic leukaemia. Br J Haematol. 2012;156(5): Catalunya/Generalitat de Catalunya and Fundaci o Josep 672-674. Carreras-Obra Social la Caixa for institutional support. 10. Katzav S, Schmitz ML. Mutations of c-Cbl in myeloid malignancies. Contribution: A.B., F.G., M.S., A.S., O.M., P.C., M.Z., J.Z., Oncotarget. 2015;6(13):10689-10696. P.M., L.M., N.G., P.C.-L., J.-F.F., L.-Y.S., T.B., E.N., S.B., and 11. Sanada M, Suzuki T, Shih L-Y, et al. Gain-of-function of mutated M.L.-G. provided clinical and biological information of all C-CBL tumour suppressor in myeloid neoplasms. Nature. 2009; cases; A.C.-E., C.B., P.M., P.L., C.M., and R.M. provided 460(7257):904-908. genetic data; and A.B. and F.G. collected the data and wrote 12. Fu JF, Hsu JJ, Tang TC, Shih LY. Identification of CBL, a proto- the manuscript along with C.B., P.M., J.E., P.L., C.M., and R.M. oncogene at 11q23.3, as a novel MLL fusion partner in a patient All authors reviewed the final manuscript. with de novo acute myeloid leukemia. Genes Chromosom Cancer. 2003;37(2):214-219. Conflict-of-interest disclosure: The authors declare no compet- 13. Boneva T, Nacheva E. KMT2A-CBL fusion gene resulting ing financial interests. from del(11)(q23.3q23.3) identified by chromosome microarray analysis-second report in AML. Atlas Genet ORCID profiles: A.B., 0000-0002-6085-2745; F.G., 0000- Cytogenet Oncol Haematol. 2020;24(6):262-264. 0001-6376-0929; O.M., 0000-0001-7585-4519; M.Z., 0000- 0002-1639-7124; P.M., 0000-0001-9528-4971; N.G., 0000- 14. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev 0001-6573-1312; L-Y.S., 0000-0003-1866-7922; S.B., 0000- Cancer. 2007;7(11):823-833. 0001-7192-2385; R.M., 0000-0003-4870-3445. 15. So CW, Lin M, Ayton PM, Chen EH, Cleary ML. Dimerization Correspondence: Alex Bataller, Department of Hematology, Hos- contributes to oncogenic activation of MLL chimeras in acute pital Clınic de Barcelona, Carrer Villarroel 170, 08036 Barcelona, leukemias. Cancer Cell. 2003;4(2):99-110. 5620 RESEARCH LETTER 28 DECEMBER 2021 • VOLUME 5, NUMBER 24