Characterization of CMML with Normal Karyotype in Comparison to CMML with Aberrant Karyotype

Background: CMML is a myelodysplastic/myeloproliferative neoplasm with distinct morphological and genetic features. Based on differences in blast count CMML is divided into CMML-1 (<1% blasts in the peripheral blood (pB) and <10% in the bone marrow (BM)) and CMML-2 (5-19% blasts in pB, 10-19% in BM or presence of Auer rods). Clonal cytogenetic abnormalities are detected in only 20-40% of patients by chromosome banding analysis (CBA) while >90% of patients harbor at least one molecular mutation. The most frequent cytogenetic abnormalities include abnormalities of chromosome 7, trisomy 8 and complex karyotype. The genes most frequently mutated in CMML are TET2, ASXL1 and SRSF2.

Aims: 1. Evaluate the frequency of submicroscopic gains and losses of chromosomal material as well as copy neutral loss of heterozygosity (CN-LOH) in CMML with normal karyotype in chromosome banding analysis (CBA). 2. Analyze the association of these lesions with molecular mutations and impact on survival.

Patients and Methods: 69 patients with CMML-1 and 31 with CMML-2 and normal karyotype by CBA were evaluated by array CGH (SurePrint G3 ISCA CGH+SNP, Agilent, Waldbronn, Germany). 32 patients were female, 68 male, median age was 75 years (range: 50-89 years). These were compared to 41 cases with aberrant karyotype by CBA. Patients were screened for mutations (mut) in ASXL1, CBL, DNMT3A, EZH2, JAK2V617F, KITD816, KRAS, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, and U2AF1.

Results: In 35 cases (35%) with normal karyotype by CBA 46 abnormalities were detected by array CGH (CGHpos). These were 6 gains, 17 losses and 23 CN-LOH. No recurrent gain was observed, while recurrent losses of 4q24 (n=2, including TET2) and of 13q14 (n=2) were identified. CN-LOH was recurrently observed on 4q (n=6, including TET2), 11q (n=5, including CBL), 17q (n=4) and 7q (n=2). Mutations were identified at the following frequencies: TET2: 77% (74/96), SRSF2: 56% (54/97), ASXL1: 48% (46/96), RUNX1: 20% (20/98), CBL: 15% (15/97), KRAS: 12% (12/97), JAK2V617F: 10% (10/98). The following genes were mutated in <10%: NRAS, SETBP1, EZH2, U2AF1, KITD816, SF3B1, DNMT3A. 85 patients were analysed for all mutations. In median 3 mutations were identified per patient (range 0-6), while only in 1 patient no mutation was detected. 4/5 (80%) cases with 11q CN-LOH harbored a CBLmut and 7/8 (88%) cases with CN-LOH 4q or 4q24 deletion harbored a TET2mut, indicating that these two gene mutations might contribute in homozygous manner to pathogenesis.

NRASmut were significantly less frequent in CMML CGHpos compared to CGHneg (0% vs 14.3%, p=0.024). Mutations in ASXL1 and RUNX1 frequently occurred together: 35% of ASXL1mut cases also carried a RUNX1mut as compared to 8% of ASXL1 wild-type cases (p=0.002). All 7 SETBP1mut cases also carried an ASXL1mut (p=0.04)

Patients with CGHneg (n=65) and CGHpos (n=35) were compared to 41 cases with aberrant karyotype by CBA. While TET2mut were detected at comparable frequencies in CGHneg and CGHpos patients (80% and 71%) they were significantly less frequent in CMML with aberrant karyotype (54%, p=0.021). On the other hand SETBP1mut were more frequent in CMML with aberrant karyotype as compared to CGHpos and CGHneg (21%, 7%, 9%, p=0.08). A distinct mutation profile was identified in 9 patients with monosomy 7 who showed ASXL1mut in 78%, SETBP1mut in 75%, CBLmut in 33% and TET2mut in only 22%. In CMML-2 RUNX1mut were more frequent than in CMML-1 (33% vs 12%. p=0.008).

No differences in overall survival (OS) were observed between patients with CGHneg, CGHpos and aberrant karyotype. However, Cox regression analyses revealed a negative impact on OS for ASXL1mut (relative risk (RR): 2.4, p=0.027), RUNX1mut (RR: 2.5, p=0.025) and CMML-2 (RR: 2.2, p=0.02).

Conclusions:  1. 35% of CMML cases with normal karyotype based on chromosome banding analysis harbor abnormalities detectable by array CGH. 2. Prognosis in CMML is determined by the molecular mutation profile, cytogenetic abnormalities play a minor role. 3. Mutations in ASXL1 and RUNX1 are associated with a negative impact on survival. 4. The poor prognosis described for monosomy 7 seems to be due to a high frequency of ASXL1 und SETBP1 mutations. 5. Inferior outcome in CMML-2 might be due to a higher frequency of RUNX1 mutations. 6. In CMML a molecular work up including screening for mutations in ASXL1 and RUNX1 provides more relevant prognostic information than chromosome banding analysis and/or array CGH.