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The spectrum of CBL-mutated JMML
Introduction
Material and studies
We received bone marrow and/or peripheral blood from all patients, collected at the time of initial diagnosis or shortly there- after. Mononuclear cells were isolated using a Ficoll method and DNA was extracted using the Gentra Puregene kit (Qiagen, Germany). For identification of genomic mutations, DNA was extracted from bone marrow or peripheral blood mononuclear cells. DNA from peripheral blood was used for DNA methylation analysis. The germline status of CBL mutations was determined using material from either a buccal swab, cord blood or after sort- ing CD3+ T cells from peripheral blood. A somatic mutation was defined as a mutation that was identified in leukemia tissue (peripheral blood or bone marrow), but had a minimum variant allele fraction (VAF) of <5% in any germline tissue (epithelial cells from buccal swab, cord blood, or T cells).
Next-generation sequencing of juvenile myelomonocytic leukemia-associated mutations
Genomic DNA samples were sequenced using a custom ampli- con-based sequencing approach (Paragon Genomics, Hayward, CA, USA) targeting 26 genes that are known to be recurrently mutated in JMML18 (Online Supplementary Table S1 and Online Supplementary Methods). A VAF of 0.05 (=5%) at diagnosis was required for reporting.
Targeted MethylSeq library preparation, sequencing and hierarchical clustering
Genomic DNA (300 ng) was bisulfite converted and 100 ng of converted single-strand DNA were used as the input for a custom 3000 CpG loci targeted MethylSeq assay (Tecan). Single-index library pools were sequenced on the Illumina NovaSeq with paired-end 150 base-pair (bp) mode averaging 20x106 reads per library preparation. MethylSeq hierarchical clustering and classifi- cation were performed according to the international consensus definition12 as described in the Online Supplementary Methods.
Statistical analyses
Spontaneous resolution of disease was defined as normalization of monocyte counts in the peripheral blood and reduction of spleen to normal size without any JMML-directed therapy. Persistent disease was defined as not achieving complete remis- sion according to the International Working Group definition19 with or without therapy excluding HSCT. Overall survival was defined as time from initial diagnosis to death from any cause and was estimated using the Kaplan-Meier method. The median fol- low-up time was estimated using the reverse Kaplan-Meier method. Differences in clinical features between patients with germline CBL mutations and those with somatic-only CBL muta- tions were tested for significance using the Fisher exact test for cat- egorical variables and the Mann-Whitney U test for continuous variables. The level of statistical significance for all tests was a P- value less than 0.05. All calculations were performed using Microsoft Excel (version 16.16.3), GraphPad Prism software (ver- sion 8.0) and R (version 3.4.1).
Results
Patients’ characteristics
The cohort consisted of 33 patients with a median age at diagnosis of 1.1 years (range, 1 month - 25 years). The median follow-up in this study was 3.5 years (range, 0.2- 12.1 years). The patients’ characteristics at diagnosis are shown in Table 1. Additional clinical information for each patient can be found in Online Supplementary Table S2.
Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive disease of young children that presents with fea- tures of both myelodysplasia and myeloproliferation. To date, hematopoietic stem cell transplantation (HSCT) is the only curative therapy and results in long-term survival of about 50-60% of patients.1,2 The mutational landscape of JMML has been well described in several large studies.3-6 Nearly all patients have initiating mutations in genes that activate the Ras pathway, most commonly, PTPN11, NRAS, KRAS, NF1 or CBL. Secondary mutations are found in approximately 30% of patients at diagnosis and are associ- ated with inferior survival.
CBL mutations were first described in JMML patients in 2009 by our group and others7-10 and account for approxi- mately 15% of all JMML cases.3,11,12 Germline tissue includ- ing cord blood and buccal samples were heterozygous for mutations, while leukemia cells were homozygous, indicat- ing that loss of heterozygosity was required for leukemoge- nesis. The majority of these children presented with syn- dromic features including facial dysmorphia, growth retar- dation, cryptorchidism and autoimmune phenomena, specifically vasculitis – a condition that is now called “CBL- syndrome”. Patients with CBL-syndrome are at risk of developing JMML. Surprisingly, several of these patients with confirmed JMML experienced spontaneous resolution of their leukemia. Based on this observation and in stark contrast to other forms of JMML, CBL-syndrome JMML has been thought to be associated with relatively good progno- sis. Treatment has evolved accordingly for these patients, who undergo a period of close observation of blood counts and assessment of splenomegaly, with HSCT utilized only after more conventional therapies have failed.13
In overlapping adult myelodysplastic syndromes/myelo- proliferative neoplasms, somatically acquired CBL muta- tions occur in about 10-15% of cases and are associated with a poor prognosis. Several studies in chronic myelomonocytic leukemia (CMML), a disease of adults with many similarities to JMML, have shown that CBL mutations are associated with inferior survival.14-16
In our experience, we have observed that the spectrum of clinical courses of patients with CBL-mutated JMML is wide, ranging from spontaneous resolution to aggressive disease with transformation to acute myeloid leukemia. This observation prompted us to perform a systematic study of clinical presentations and molecular features to identify potential factors that can identify patients who may be observed and those who need immediate therapy.
Methods
Patients
We collected samples and clinical data from 33 patients diag- nosed with JMML according to international diagnostic criteria17 who were found to have a clonal CBL mutation. The patients were diagnosed between 2006 and 2019 in 27 different centers across the USA and Canada. Treatment decisions were at the dis- cretion of the treating physicians and were independent of this study. Written consent and specimens were obtained from all patients at the time of routine clinical assessments. The study design was reviewed and approved by the institutional review board of University of California San Francisco, in accordance with the Declaration of Helsinki.
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