- Open Access
Molecular dissection of a hyper-aggressive CBFB-MYH11/FLT3-ITD–positive acute myeloid leukemia
Journal of Translational Medicine volume 20, Article number: 311 (2022)
Acute Myeloid Leukaemia (AML) is a haematological malignancy showing a hypervariable landscape of clinical outcomes and phenotypic differences, explainable by heterogeneity at the cellular and molecular level. Among the most common genomic alterations, CBFB-MYH11 rearrangement and FLT3-ITD gene mutations, have opposite clinical significance and are unfrequently associated. We present here a Molecular Case Report in which these two events co-exist an ultra-aggressive phenotype resulting in death in 4 days from hospital admittance. Somatic and germline Whole Exome Sequencing analysis was performed to uncover other putative driver mutations, de-novo genomic structural events or germline clusters increasing cancer insurgence. Only three mutations in LTK, BCAS2 and LGAS9 were found, unlikely causative of the exhibited phenotype, prompting to additional investigation of the rare CBFB-MYH11/ FLT3-ITD scenario.
Acute Myeloid Leukaemia (AML) is a heterogeneous disease characterized by clonal expansion of undifferentiated myeloid precursors with variable phenotype and relevant molecular differences. This heterogeneity, coupled with the high relapse rate is responsible for the relatively low five-year overall survival (OS) for childhood AML, which lays at around 70%, compared to 90% for the more common acute lymphoblastic leukaemia [1, 2]. Indeed, over 30% of patients achieving complete remission (CR) after intensive chemotherapy develop relapses which are usually drug-resistant, with significant worsening of prognosis [1, 3].
Nowadays, the evolution of sequencing technologies allows for a genomic-based differential diagnosis for this form of cancer, with the identification of key mutations as new criteria to distinguish different forms of AML, granting more accurate prognostication , and the development of new, specific pharmacologic treatments . Nonetheless, our understanding of the synergies of concurrent genomic alterations is still incomplete. Most patients are screened for the presence of known genomic anomalies only, without accounting for the role of co-occurrences in driving development of resistance, and epigenetic reprogramming of cancer clones is not well established .
In this article, we present the unusual case of early death in a child with a FLT3-ITD positive CBFB-MYH11 rearranged AML, characterized by trio-filtered Whole Exome Sequencing (WES) analysis, and a comparison to available published data, to foster the identification of molecular determinants of disease aggressiveness in this case.
All samples were equally treated for gDNA extraction from PBMCs using QIAamp DNA Mini Kit (Qiagen). The quantity and quality of the DNA was assessed by the Qubit and by NanoDrop Spectrophotometer, respectively. DNA exome libraries for sequencing were generated using 150 ng of DNA according to the TruSeq DNA Exome kit (Illumina). Quality of the libraries has been verified with the Bioanalyzer (High Sensitivity DNA Kit); library quantity has been determined with qPCR. The samples have then been sequenced in paired-end mode, sequencing from each side 76 bp with NextSeq 500 (Illumina).
Somatic and Germline variants were called via the Dragen Somatic/Germline pipelines available on the Illumina Basespace Cloud (vv. 3.6.3). VCF files were downloaded, normalized and decomposed via the VT package (Tan et al., 2015) and intersected via custom R scripting. A final manual curation was performed to validate called somatic SNPs. Copy Number Variations were called via the CNVkit package (Talevich et al. 2014) batch algorithm. CNV baseline was established with all the available in-house data sequenced (N = 8) with the same library preparation kit in order to smooth out technical biases. Only CNVs with significant p value (< 1e−10) and a strong effect on deletion (CN ≤ 1) or amplification (CN ≥ 4) were considered. Structural Variations were computed with Manta (Chen et al. 2016), excluding events flagged as imprecise and other events that were exceeding the insert size length according to 75 bp read length. One section of our results is partially based upon data generated by the Therapeutically Applicable Research to Generate Effective Treatments initiative, phs000218. The data used for this analysis are available at https://portal.gdc.cancer.gov/projects.
A fourteen-year-old female, with no previous history of diseases, was hospitalized after referral from the general practitioner, presenting worsening nausea and fever, and ocular hyposphagma. Upon reaching the hospital (day 1) she developed dyspnoea and peripheral blood (PB) tests showed 364 × 109/L white blood cells (WBC, normal values: 4–12 × 109/L), prompting AML diagnosis. Cytarabine was started as continuous infusion, 150 mg IV every twelve hours, followed by Idarubicin, 10 mg, and dexamethasone, 6 mg from day 2. On the same day, the patient was moved to the paediatric intensive care unit for respiratory failure, eventually needing intubation by the end of the day. The patient, now showing hepatosplenomegaly and peripheral bleeding manifestations, was awake until sedation by administration of midazolam. On day 3, patient conditions further worsened, and transfusion support was started, while WBC showed only a moderate decrease (215 × 109/L). On day 4, after multiple organ failures (MOF), the patient died due to a mesencephalic haemorrhage.
Upon hospitalization, the patient was screened for genetic alterations commonly associated with AML, detecting both a FLT3-ITD mutation and CBFB-MYH11 rearrangement. She was negative for RUNX1/RUNX1T1, BCR-ABL1, MLL (KMT2A) rearrangements, FLT3-TKD and NPM1 mutations. Polymerase chain reaction (PCR) analysis of primary cells in accordance with European Leukemia Net (ELN) guidelines , confirmed the presence of a FLT3-ITD (Internal Tandem Duplication) with an Allelic Ratio (AR) of 0.15. Sequencing of the gene showed a 7 bp insertion in the juxta membrane domain of FLT3, associated with a 62 bp tandem duplication spanning to the TK Domain 1. Analysis of karyotype confirmed an inv(16). The association of FLT3-ITD mutations with inv(16) is a rare event in the AML landscape , and given the aggressive disease onset and the rapid fatal outcome, we decided to characterize in depth the genomic features of this AML case.
This study was approved by the Regina Elena Ethics Committee (CE 1353/20). Written informed consent was collected from the parents before blood sampling, on behalf of their child for the publication of any potentially identifiable data included in this article, and clinical data were collected from the patient chart and her parents.
Whole Exome Sequencing (WES) was performed on blast cells. In absence of a germline sample, PB Mononuclear Cells (PBMCs) samples were obtained from both parents to perform a family trio analysis of genetic variants, in order to achieve higher sensitivity and ease of data filtering compared to patient WES alone . The methods are presented visually in Fig. 1 and expanded in the supplementary section. After filtering variants shared with the parents, only two potentially relevant Single Nucleotide Variants (SNVs) were identified, presented in Table 1.
Analysis of gene Copy Number Variations (CNVs) revealed a focal shallow deletion (CN = 1) on the TUBB8B gene, and two shallow larger events on chr4 and chr17. Other two large deletions were discarded since overlapping the validated inversion on chr16. When considering significant amplifications with a strong signal (CN ≥ 4), only one event emerged, presented in Table 1. A further analysis on other genomic structural events such as translocations, insertions, and inversions, was performed: of the four alterations detected, one was excluded via further manual filtering (imprecise breakpoints). The three remaining insertions on chr3, chr9 and chr20 were not considered since they exceeded the software length threshold for insert size.
We then assessed the prevalence of concurrent FLT3-ITD and CBFB-MYH11 across several patient cohorts exploiting the cBioPortal interface. The overall population of double-mutant is comprising of 5 patients (0.5%) in the TARGET study and 2 patients (1%) in the TCGA study, in agreement with previously published data [11,12,13,14,15]. Interestingly, none of the mutations described in Table 1 could be observed in the somatic landscape of the double-mutated population. Furthermore, when querying for mutational events (SNPs, Indels, and CNVs) on LTK, BCAS2, LGASL2, and TUBB8B, the amount of somatic point mutations was none on said patients, while only 2 Copy Number Amplifications in BCAS2 and 1 in LTK were found. Taken together, these results could point out to a molecular background that acts in a sinergistic manner with the FLT3-ITD + CBFB-MYH11 combination.
We then sought to provide an overview of the prognostic value of all these variants on Overall Survival, while unfortunately such scarce numerosity of events prevents reaching statistical significance. Nonetheless, Overall Survival on all considered datasets suggests that these patients have worse prospects and a higher chance of relapse, especially in the paediatric population,
The median survival was 17 months on the 7 combined cases, against a median OS of 34.5 for FLT3-only, 59 for CBFB-MYH11 only and 47 for none of these events (Additional file 1: Fig. S1A).
Finally, blast median count was 61.5, 79.5, 73, 70 on the 4 groups, confirming the double mutant scenario not be the sole hallmark of hyperleukocytosis (Additional file 1: Fig. S1B).
To usher the era of precision medicine, a detailed understanding of the significance of genetic alterations is required. Indeed, not only their presence but also the interaction between the different genetic abnormalities, should be considered to explain the variability in response to treatment observed in patients.
The category of core binding factor (CBF) acute myeloid leukaemias (AMLs) includes AML with inv(16)(p13.1q22) and AML with t(8;21) (q22;q22,1), which translates into CBFβ/MYH11 and RUNX1/RUNX1T1 fusion genes, respectively . These abnormalities have an incidence of 10–15% in adults and 20% in childhood de novo AMLs, and they are considered prognostically favourable [7, 16, 17]. The presence of CBF-AML chimeric transcripts causes disruption of the CBF complex with consequent block of differentiation of myeloid blasts . However, several evidences suggest that the leukemogenesis process is more complex and could be supported by additional mutational events . In 2002, Kelly et al. proposed a two-hit model for CBF leukaemia in which the proliferative advantage was conferred by a mutation in a gene coding for a tyrosine kinase . The detection of an additional mutation in CBF-AMLs dates back to 2003, when Care et al. demonstrated that mutations of c-Kit or FLT3 genes could be found in 40% of AML with CBFβ/MYH11 cases . Otherwise, subsequent studies reported the co-existence of a mutually exclusive mutational process for activating tyrosine kinases pathway in up to 70% of CBF-AML cases [21, 22].
FLT3 mutations can be subdivided into internal tandem duplicates (ITD) that are in-frame duplications of variable size located within the juxatamembrane domain, and point mutations in the tyrosine kinase domain (TKD). FLT3-TKD mutations are observed in 11–13% of AMLs with CBFβ/MYH11, and they do not seem to affect prognosis [23,24,25], and in the paediatric population only anecdotal cases are reported . FLT3-ITD mutations are even more rare in CBFβ/MYH11 AMLs, with an incidence of 3 to 8%, and their prognostic role is negative. A mouse model study reported aggressive behaviour, low platelet counts and rapid peripheral dissemination of blasts . Moreover, a retrospective collection of cases described poorer outcome of adult patients, as compared to those with CBF-AMLs without FLT3-ITD mutations. In literature, no data is available on FLT3-ITD-positive CBF-AMLs in paediatric population . The presence of FLT3-ITD mutations confers to leukemic cells a proliferative and survival advantage by constitutive activation of downstream signalling events involving PI3K/AKT/mTOR, MEK/ERK and STAT5 pathways . Consequently, these mutations correlate with hyperleukocytosis and poor prognosis [30,31,32].
Our patient presented at onset with all the biological features described above, including hyperleukocytosis, which is a known independent prognostic factor for inferior outcome, poor response to therapy and aggressive course of the disease . It remains to be clarified whether a FLT3-ITD double mutation may have contributed to enhancing the unfavourable features known to be associated with a single mutation in this gene. Only another case of a 54-year-old patient with a de novo AML with FLT3-ITD double mutation is reported in the literature, with a particularly aggressive course and adverse outcome .
In conclusion, we reported the unique molecular profile of a paediatric patient affected by a CBFβ/MYH11 AML, carrier of two FLT3-ITD co-mutations. Even though the genetic features would classify this AML as favourable risk, according to the 2017 ELN risk-stratification , the course of the disease was unusually rapid and fatal. This could be partly explained by the high leukocyte count at presentation. Further studies are needed to assess the clinical significance of a FLT3-ITD double mutations and clarify their prognostic features in CBF-AMLs.
Availability of data and materials
List of annotated mutations are available upon request.
Acute Myeloid Leukemia
Single Nucleotide Variants
Copy Number Variation
Rasche M, Zimmermann M, Borschel L, et al. Successes and challenges in the treatment of pediatric acute myeloid leukemia: a retrospective analysis of the AML-BFM trials from 1987 to 2012. Leuk. 2018;32(10):2167–77.
Kuhlen M, Klusmann J-H, Hoell JI. Molecular approaches to treating pediatric leukemias. Front Pediatr. 2019;7:368.
Saultz JN, Garzon R. Clinical medicine acute myeloid leukemia: a concise review. Cancers. 2020;12(11):3225.
Sallman DA, Padron E. Integrating mutation variant allele frequency into clinical practice in myeloid malignancies. Hematol Oncol Stem Cell Ther. 2016;9(3):89–95.
Stanchina M, Soong D, Zheng-Lin B, Watts JM, Taylor J. Advances in acute myeloid leukemia: recently approved therapies and drugs in development. Cancers. 2020;12(11):3225.
Kantarjian H, Kadia T, DiNardo C, et al. Acute myeloid leukemia: current progress and future directions. Blood Cancer J. 2021;11(2):1–25.
Dohner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424–47.
Conneely SE, Rau RE. The genomics of acute myeloid leukemia in children. Cancer Metastasis Rev. 2020;39(1):189–209.
Kuhlen M, Taeubner J, Brozou T, et al. Family-based germline sequencing in children with cancer. Oncogene. 2019;38(9):1367–80.
Cerami E, Gao J, Dogrusoz U, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401–4.
Papaemmanuil E, Gerstung M, Bullinger L, et al. Genomic classification and prognosis in acute Myeloid Leukemia. N Engl J Med. 2016;374(23):2209–21. https://doi.org/10.1056/NEJMoa1516192.
Tyner JW, Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526–31.
Welch JS, Petti AA, Miller CA, et al. TP53 and Decitabine in Acute Myeloid Leukemia and Myelodysplastic Syndromes. N Engl J Med. 2016;375(21):2023–36.
Ley TJ, Miller C, Ding L, et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368(22):2059–74.
Duployez N, Marceau-Renaut A, Boissel N, et al. Comprehensive mutational profiling of core binding factor acute myeloid leukemia. Blood. 2016;127(20):2451–9.
Opatz S, Bamopoulos SA, Metzeler KH, et al. The clinical mutatome of core binding factor leukemia. Leukemia. 2020;34(6):1553–62.
Harrison CJ, Hills RK, Moorman AV, et al. Cytogenetics of childhood acute myeloid leukemia: United Kingdom medical research council treatment trials AML 10 and 12. J Clin Oncol Off J Am Soc Clin Oncol. 2010;28(16):2674–81.
Quan X, Deng J. Core binding factor acute myeloid leukemia: Advances in the heterogeneity of KIT, FLT3, and RAS mutations (Review). Mol Clin Oncol. 2020;13(2):95–100.
Kelly LM, Gilliland DG. Genetics of myeloid leukemias. Annu Rev Genomics Hum Genet. 2002;3:179–98.
Care RS, Valk PJM, Goodeve AC, et al. Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol. 2003;121(5):775–7.
Valk PJ, Bowen DT, Frew ME, et al. Second hit mutations in the RTK/RAS signaling pathway in acute myeloid leukemia with inv(16). Haematologica. 2004;89(1 SE-Letters to the Editor):106.
Boissel N, Leroy H, Brethon B, et al. Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia. 2006;20(6):965–70.
Kok CH, Brown AL, Perugini M, et al. The preferential occurrence of FLT3-TKD mutations in inv(16) AML and impact on survival outcome: a combined analysis of 1053 core-binding factor AML patients. Br J Haematol. 2013;160(4):557–9.
Mrózek K, Marcucci G, Paschka P, Bloomfield CD. Advances in molecular genetics and treatment of core-binding factor acute myeloid leukemia. Curr Opin Oncol. 2008;20(6):711–8.
Mead AJ, Linch DC, Hills RK, et al. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood. 2007;110(4):1262–70.
Shih L-Y, Liang D-C, Huang C-F, et al. Cooperating mutations of receptor tyrosine kinases and Ras genes in childhood core-binding factor acute myeloid leukemia and a comparative analysis on paired diagnosis and relapse samples. Leukemia. 2008;22(2):303–7.
Kim H-G, Kojima K, Swindle CS, et al. FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia. Blood. 2008;111(3):1567–74.
Kayser S, Kramer M, Martínez-Cuadrón D, et al. Characteristics and outcome of patients with core binding factor acute myeloid leukemia and FLT3-ITD: results from an international collaborative study. Haematologica. 2021;107:836–43.
Watanabe D, Nogami A, Okada K, et al. FLT3-ITD activates RSK1 to enhance proliferation and survival of AML cells by activating mTORC1 and eIF4B Cooperatively with PIM or PI3K and by inhibiting bad and BIM. Cancers. 2019;11(12):1827.
Best-Aguilera C, Rodrigo Gómez-Vázquez O, Elizabeth Guzmán-Hernández A, Monserrat R-S. Treatment of acute myeloid leukemia with the FLT3 gene mutation. Curr Oncol Rep. 2017;19(3):21.
Arrigoni P, Beretta C, Silvestri D, et al. FLT3 internal tandem duplication in childhood acute myeloid leukaemia: association with hyperleucocytosis in acute promyelocytic leukaemia. Br J Haematol. 2003;120(1):89–92.
Semary SF, Hammad M, Soliman S, et al. Outcome of childhood acute myeloid leukemia with FLT3-ITD mutation: the experience of children’s cancer hospital Egypt, 2007–17. Clin Lymphoma Myeloma Leuk. 2020;20(8):e529–41.
Stahl M, Shallis RM, Wei W, et al. Management of hyperleukocytosis and impact of leukapheresis among patients with acute myeloid leukemia (AML) on short- and long-term clinical outcomes: a large, retrospective, multicenter, international study. Leukemia. 2020;34(12):3149–60.
Alarbeed IF, Wafa A, Moassass F, et al. De novo adult acute myeloid leukemia with two new mutations in juxtatransmembrane domain of the FLT3 gene: a case report. J Med Case Rep. 2021;15(1):22.
Roll JD, Reuther GW. ALK-activating homologous mutations in LTK induce cellular transformation. PLoS ONE. 2012;7(2):e31733.
Salmerón-Hernández Á, Noriega-Reyes MY, Jordan A, Baranda-Avila N, Langley E. BCAS2 enhances carcinogenic effects of estrogen receptor alpha in breast cancer cells. Int J Mol Sci. 2019;20(4):966.
Ruvolo PP. Galectins as regulators of cell survival in the leukemia niche. Adv Biol Regul. 2019; 71: 41–54.
We thank the Nana Onlus association for its valuable support through all the phases of the study. We thank Tania Merlino for editorial assistance.
This work was supported by the Italian Ministry of Health (Ricerca Corrente 2021) and by the Nana Onlus association via a one-year fellowship and reagent support.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1: Figure S1A.
Kaplan–Meier curve showing Overall Survival among double mutant, inv16-only, FLT3-ITD only, and double negative cases in TCGA and TARGET studies. B: Blast count distribution in the 4 populations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Lo Iudice, G., De Bellis, E., Savi, A. et al. Molecular dissection of a hyper-aggressive CBFB-MYH11/FLT3-ITD–positive acute myeloid leukemia. J Transl Med 20, 311 (2022). https://doi.org/10.1186/s12967-022-03486-5