Next-Generation Sequencing Technology Reveals a Characteristic Pattern of Molecular Mutations in 75% of Chronic Myelomonocytic Leukemia (CMML) by Detecting Frequent Alterations in TET2, RUNX1, CBL, and RAS.

Abstract 417

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Here, we analyzed 81 CMML cases (45 CMML-1, 36 CMML-2). In chromosome banding analysis 59/76 (77.6%) patients showed a normal karyotype (data not availabel in 5 cases). Recurrent chromosome aberrations were trisomy 8 (n=6; 7.9%), monosomy 7 (n=3; 3.9%), and loss of the Y-chromosome (n=5; 6.6%). Fluorescence in situ hybridization (FISH) detected the deletion of one allele of the TET2 gene in 4/71 cases (5.6%). Thus, the majority of cases can not be genetically characterized by these techniques. Therefore, we applied next-generation sequencing (NGS) technology to investigate 7 candidate genes, represented by 43 PCR-products, at known mutational hotspot regions, i.e. CBL (exons 8 and 9), JAK2 (exons 12 and 14), MPL (exon 10), NRAS (exons 2 and 3), and KRAS (exons 2 and 3). In addition, complete coding regions were analyzed for RUNX1 (beta isoform) and TET2. NGS was performed using 454 FLX amplicon chemistry (Roche Diagnostics Corporation, Branford, CT). The median number of base pairs sequenced per patient was 9.24 Mb. For each target gene a median of 911 reads was generated (coverage range: 736-fold to 1606-fold). This approach allowed a high-sensitive detection of molecular mutations, e.g. detecting the JAK2 V617F mutation down to 1.16% of reads. In total, 146 variances were detected by this comprehensive molecular mutation screening (GS Amplicon Variant Analyzer software version 2.0.01). In 80.4% of variances consistent results were obtained after confirming NGS mutations with melting curve analysis and conventional sequencing. In the remaining discrepant variances (19.6%) NGS deep-sequencing outperformed conventional methods due to the higher sensitivity of the platform. After excluding 19 polymorphisms or silent mutations 127 distinct mutations in 61/81 patients (75.3%) were detected: CBL: n=21 point mutations and one deletion (18 bp) found in 20 cases (24%); JAK2: n=8 mutations (V617F) found in 8 cases (9.8%); MPL: no mutations found; NRAS: n=23 mutations found in 18 cases (22.2%); KRAS: n=12 mutations found in 10 cases (12.3%); RUNX1: n=6 point mutations and one deletion (14 bp) found in 7 cases (8.6%); and TET2: n=49 point mutations and 6 deletions (2-19 bp; 5/6 out-of-frame) found in 41 cases (50.6%). Furthermore, in 21 TET2-mutated cases 11 mutations previously described in the literature were detectable, whereas 28 cases carried novel mutations (n=28). In the cohort of TET2-mutated cases 17/41 (41.3%) patients harbored TET2 abnormalities as sole aberration. Interestingly, CBL mutations were found to be significantly associated with TET2 mutations (Fisher's exact test, p=0.008). In 17 of 20 (85.0%) CBL-mutated cases TET2 abnormalities were concomitantly observed. In contrast, no significant associations were found between any of the point mutations or deletions and the karyotype. There were also no associations observed between molecular aberrations and the diagnostic categories CMML-1 and CMML-2. With respect to clinical data a trend for better outcome was seen for patients that carried either or both TET2 and CBL mutations (median OS 130.4 vs. 17.3 months, alive at 2 yrs: 72.0% vs. 43.9%; p=0.13). In conclusion, 75.3% of CMMLs harbored at least one molecular aberration. In median 2 mutations per case were observed. Compared to limited data from the literature we detected not only a higher frequency of CBL mutations, but also add data on novel TET2 mutations. In particular, comprehensive NGS screening here for the first time has demonstrated its strength to further genetically characterize and delineate prognostic groups within this type of hematological malignancy.