Abstract
Background: Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease that can vary from a myelodysplastic (MD) predominant profile to a myeloproliferative (MP) one. CMML has a variable course, with a median overall survival of 20 months and 15-30% of progression to acute myeloid leukemia (AML). Cytogenetic abnormalities are present in 20-40% of cases and recurrent gene mutations have been reported in several genes. Patients with low risk cytogenetic features (normal karyotype and isolated -Y) account for approximately 80% of CMML patients and often fall into the low risk categories of CMML prognostic scores. Our hypothesis is that mutational study can contribute to diagnosis and prognostic stratification in this subset of patients.
Methods: A retrospective study was performed on 57 patients with CMML. Cases with normal karyotype (n=53), isolated -Y (n=2) and no metaphases (n=2) were selected. DNA was extracted from BM (n=52) and PB (n=5) samples at diagnosis. Targeted deep-sequencing was performed in a panel of 83 myeloid-related genes. Libraries were prepared with 1μg of genomic DNA using the KAPA Library Preparation Kit (Kapa Biosystems) and then enriched using the SeqCap EZ capture chemistry (Nimblegen, Roche). Libraries were sequenced with 150 bp paired-end reads on an Illumina MiSeq. Herein we present the results of 43 cases which were preliminarily analyzed using the Illumina MiSeq Reporter and Variant Studio softwares. High-probability oncogenic mutations were called by eliminating sequencing and mapping errors and known SNPs based on the available databases. An in-house bioinformatics pipeline will be designed to analyze the whole series of patients. A preliminar statistical analysis was performed with SPSS. Fisher's exact test was used to compare variables between patient subsets. Complete study, including the correlation of the sequencing findings with the clinical data, will be presented in the meeting.
Results: Median age at diagnosis was 70 years (range 48-87) and there was a 2:1 male predominance. Median follow up of patients was 23 months (range 1-116) during which 23% (11/43) of cases progressed to AML. Morphological WHO subtypes were CMML-1 in 36 (84%) cases and CMML-2 in 7 (16%). According to the FAB criteria 34 (79%) cases were classified as CMML-MD and 9 (21%) as CMML-MP. According to the CMML-specific scoring system (CPSS) 28/43 (65%) patients belonged to the low-risk category, 10/43 (23%) to the intermediate-1 and 4/43 (12%) to the intermediate-2. The mean depth of the targeted resequencing per base per sample was 810-fold. After excluding sequencing and mapping errors a mean of 293 single nucleotide variants (snv) and insertions/deletions (indels) were called per sample. After filtering non-silent variants and excluding known polymorphisms a mean of 6 variants per sample were called as high-probability somatic changes. Distribution of detected variants across the patients can be seen in Figure 1. Most frequently affected genes were TET2 (70%), ASXL1 (47%) and SRSF2 (35%); followed by RUNX1 (23%), NRAS (16%), CBL (12%), EZH2 (12%), SETBP1 (12%) and ZRSR2 (12%). Variants detected in 5-10% of patients included IDH2, CRBBP, SH2B3, UMODL1, DNMT3A, JAK2, PTPN11, SF3B1 and U2AF1 genes. Statistical analysis revealed that some variants correlated with CMML subtypes: SH2B3 (P=0.010) and STEBP1 (P=0.024) associated with CMML-2; JAK2 (0.007), NRAS (P=0.026) and EZH2 (P=0.05) associated with CMML-MP. Variants in NRAS also correlated with progression to AML (P=0.04) and patients in intermediate groups of CPSS associated with JAK2 (P=0.008) and EZH2 (P=0.011) variants.
Conclusions: Genetic profiling using targeted deep-sequencing is a highly promising approach for CMML diagnosis and varies according to the cytological subtypes. With the correlation of the results with the clinical data of patients, we expect to determine if targeted molecular profiling can contribute to prognostic stratification of patients with CMML and low risk cytogenetic features. For the moment, we have already found a correlation with progression to AML.
Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519; PI 11/02010); RTICC, FEDER (RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Josep Carreras, Obra Social "La Caixa" and Celgene Spain.
Distribution of the affected genes across the 43 studied patients with CMML
Sole:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.
