Chronic myelomonocytic leukemia (CMML) is a chronic clonal disease characterized by myelodysplastic (MDS) and myeloproliferative (MPN) features.1 It is defined by the occurrence of peripheral blood (PB) monocytosis (>1 × 109/l) without BCR-ABL1 fusion oncogene or PDGFRA or PDGFRB rearrangements. Cases are further subclassified in CMML-1 (<10% blasts in the bone marrow (BM)) and CMML-2 (10–19% blasts), accounting for 80 and 20% of cases, respectively. The genetic landscape is highly heterogeneous. Most frequently mutated genes are TET2 (59–60% of cases), SRSF2 (50–51%), ASXL1 (40–45%), RAS (30%), CBL (15–21%) and SETBP1 (6–15%), whereas TP53 mutations are rare (1%).2, 3 Majority of patients (up to 70%) show normal karyotype (NK), whereas clonal cytogenetic abnormalities have been identified in almost 30% of cases.4 Most frequent abnormalities are trisomy 8 (23%), loss of the Y chromosome (20%), monosomy 7 or 7q deletion (14%), complex karyotype (10%), trisomy 21 (8%), abnormalities of chromosome 3 (8%) and 20q deletion (8%). On the basis of the cytogenetic findings, the Spanish group defined three risk classes, that is, low (–Y and NK), intermediate (karyotype not classified as low or high risk) and high risk (+8 or monosomy 7).4 Balanced rearrangements have been described rarely.5
However, chromosome banding analysis (CBA) shows some limits related to the necessity of in vitro replication of the abnormal clone.6 Moreover, spatial resolution is limited to 10 MB size. Therefore, submicroscopic cytogenetic aberrations cannot be detected. Genomic arrays do not rely on proliferating cells and provide a very high spatial resolution, up to few Kb. Furthermore, arrays can detect not only copy number abnormalities (CNAs), that is, losses and gains, but also copy neutral loss of heterozygosity (CN-LOH).7 However, at least 15% of cells should harbor the genomic abnormality to allow a reliable detection. On the basis of these premises, we aimed to evaluate genomic abnormalities in CMML patients with normal CBA through the application of genomic array.
One hundred and forty patients have been analyzed. Diagnosis has been performed on morphological evaluation of PB and BM smears according to the WHO classification (2016).1 Cytogenetic and genomic array evaluation was performed on BM, and gene sequencing has been performed only on BM in 137 cases and BM/PB in three cases. Samples were referred to MLL Munich Leukemia Laboratory between October 2005 and February 2015. Morphology and cytogenetic evaluation as well as survival information were available for every patient. Median follow-up was 2.4 years. Cases with normal CBA were further studied with genomic array (SurePrint G3 ISCA CGH+SNP (4 × 180 K), Agilent, Waldbronn, Germany) and data were analyzed using Nexus Copy Number Software v. 6.1 (Biodiscovery, Inc., El Segundo, CA, USA) according to manufacturer’s recommendations. Amplicon-based next generation sequencing or polymerase chain reaction were applied to detect TET2, SRSF2, ASXL1, RUNX1, CBL, SETBP1, KRAS, NRAS, JAK2, EZH2, KIT, U2AF1, SF3B1, DNMT3A, SMC1A and CSF3R mutations in all patients. Library preparation was performed with Access Array Technology (Fluidigm, South San Francisco, CA, USA) and sequencing with MiSeq Instrument (Illumina, San Diego, CA, USA). Part of patients was the validation set published by Itzykson et al.8 and were also included in Meggendorfer et al.2, 3, 9 All patients gave written informed consent for use of data for scientific evaluations. The study was approved by the Internal Review Board and adhered to the tenets of the Declaration of Helsinki. Dichotomous variables were compared between different groups using χ2-test and continuous variables by Student’s t-test. Significance was set at P<0.05. Reported P-values are two-sided. Kaplan–Meier method was applied for overall survival (OS) curves and results were analyzed applying the two-sided log-rank test. Cox regression analysis was performed for multivariate analysis, including parameters significant (P<0.05) at univariate analysis.
Median age was 74 years and 95 patients out of 140 (68%) were male. Mean white blood cell (WBC) count was 17 × 109/l (±20 s.d.) with a mean percentage of monocytes of 29% (±12). The mean absolute monocyte count (AMC) was 5.2 × 109/l (±6.1). Thirty-one patients (22%) showed CMML-2 morphology. Ninety-five out of one hundred and forty patients (71%) showed a NK by CBA, whereas the remaining 41 patients (29%) showed CBA abnormalities. These two groups did not differ for age, gender, WBC, AMC, hemoglobin and CMML subtype. However, the group with abnormalities showed lower platelet count (mean 186 vs 131 × 109/l, P=0.004). In NK subgroup, mutations were identified at the following frequencies: TET2 (76/99, 77%), SRSF2 (55/99, 56%), ASXL1 (47/99, 48%), RUNX1 (20/99, 20%), CBL (14/99, 14%), SETBP1 (8/99, 8%), KRAS (12/99, 12%), NRAS (10/99, 10%), JAK2 (10/99, 10%), EZH2 (5/99, 5%), KIT (5/99, 5%), U2AF1 (5/99, 5%), SF3B1 (4/99, 4%), DNMT3A (2/99, 2%) and SMC1A (2/99, 2%). No mutations were detected on CSF3R gene. TET2 mutations were more frequent in NK subset (77 vs 56%, P=0.024), whereas SETBP1 mutations were more frequent in abnormal CBA subset (8 vs 22%, P=0.04). No statistical differences were evident regarding remaining genes.
Applying genomic array in all 99 cases with NK, we detected 50 CNA/CN-LOH in 40/99 patients (40%). These encompassed 28 CNA (22 deletions and 6 gains) in 24 patients and 22 CN-LOH in 20 patients (Figure 1a). The median CNA size was 0.79 MB (range 0.07–159). Only in one case, the abnormality was >10 MB, that is, a case with monosomy 7 not detected by CBA, probably due to insufficient in vitro proliferation of aberrant clone. The median CN-LOH size was 66.6 MB (range 10.14–135.44). In 19 cases, CN-LOH ranged to telomere and in three cases, it was interstitial. Majority of patients had one aberration (31/40, 78%), eight patients showed two aberrations and one patient showed three aberrations (all gains) (further details in the right panel of Figure 1a).
Except for mutual exclusion between NRAS mutations and CNA/CN-LOH, no other association with recurrently mutated genes was evident. Moreover, the number of additional mutations did not differ between normal and abnormal genomic array subgroups.
Most frequently recurrent aberration was the CN-LOH of chromosome 4q22.3q32.2 (97 791 611–190 803 979) in six patients involving TET2 gene (five harbored TET2 mutations and one patient showed SRSF2 mutation), followed by 11q13.5q25 CN-LOH (76 150 205–134 195 978) involving CBL gene (all of them showing CBL mutation) in four patients and 17q22q24.1 CN-LOH (53 404 930–63 868 162) in three patients, involving MPO gene. Moreover, 4q24 deletion (105 479 140–106 215 692), also involving TET2 gene, was found in three patients, all showing TET2 mutation. Two patients showed 7q21.3q36.3 CN-LOH (7 368 205–159 118 566) and other two patients showed 7q22.1q22.1 deletion (101 262 074–101 951 902) encompassing the CUX1 gene, a tumor suppressor gene frequently deleted in myeloid neoplasms.10 Two further patients showed Xp22.2p22.2 deletion (15 748 556–16 686 778) involving the ZRSR2 gene. The aforementioned findings are in line with the study by Tiu et al.,11 reporting a rate of aberrations of 37% in 55 CMML patients with normal CBA and Gondek et al.,12 indicating a rate of CN-LOH, among 24 unselected MDS/MPN patients, of 35%. Furthermore, we confirmed the recurrence of 11q, 7q, 17q CN-LOH.11
No specific molecular profile related to recurrent abnormalities, except for CBL mutation (n=14) that was strongly related to 11q CN-LOH (10 out of 95 patients without 11q CN-LOH, 11% vs 4 out of 4 with 11q CN-LOH, 100%, P=0.001). From the 14 patients with mutated CBL, four patients (28.6%) showed 11q CN-LOH, whereas only 2 of the 10 patients without 11q CN-LOH (20%) had two CBL mutations. This is in line with data stating that 11q CN-LOH strictly relates to CBL mutations.13 Among the 76 patients with mutated TET2, eight patients showed 4q CNA/CN-LOH. Five of these eight patients with CN-LOH showed TET2 homozygosity and three patients with deletions showed TET2 hemizygous status, whereas, among the 69 patients without 4q aberrations, 48 (70%) showed two TET2 mutations, corroborating the finding that TET2 acts as tumor suppressor gene.14 Noteworthy, only one patient did show neither any gene mutation nor any genomic array aberration.
Overall survival analysis showed a negative impact of ASXL1 (median 5.6 vs 1.7 years, P=0.008), EZH2 (4.3 years vs 4 months, P=0.03), RUNX1 (6.6 vs 1.5 years, P=0.014) and NRAS (4.3 vs 1.4 years, P=0.015) mutations (Figure 1b). However, at Cox multivariate analysis, only ASXL1 mutation retained statistical significance (hazard ratio 2.1; 95% confidence interval 1.02–4.4, P=0.04), in line with previous observations.8 On the other hand, no differences in OS were detected between normal and abnormal genomic array subgroups, also considering numbers of aberrations or recurrent lesions (Figure 1c). Recently, Palomo et al.15 investigated the impact of genomic array in 117 low-risk cytogenetic CMML (NK and loss of chromosome Y) and 11 cases with failed karyotyping. Even though they found aberrations at higher frequency compared with our series (i.e., 67%), they did not find any correlation with OS, regarding either the occurrence of any aberrations or their number, confirming our results.
In conclusion, genomic array analysis can detect aberrations in 40% of CMML patients with NK at CBA, enriching the genomic scenario (Figure 2). Recurrent genomic array aberrations were detected at 4q, 7q, 17q, 11q and Xp. However, the prognostic role of molecular genetic markers, in particular, ASXL1 mutations, overcomes the prognostic significance of any genomic aberration in terms of OS.
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We would like to thank all physicians for sending samples to MLL laboratory and for providing clinical information. In addition, we would like to thank all the coworkers at the MLL for the great work in performing technical evaluation.
CV performed statistical analysis and wrote the manuscript; CH was responsible for cytogenetics and genomic array; TH was responsible for cytomorphologic analysis; MZ performed cytogenetics and genomic array; NN provided bioinformatic support; WK contributed to statistical analyses; MM investigated molecular mutations and was involved in the collection of clinical data as well as manuscript preparation. All authors read and contributed to the final version of the manuscript.
CH, WK, and TH declare equity ownership of MLL Munich Leukemia Laboratory. NN, MZ and MM are employed by MLL Munich Leukemia Laboratory. CV declares no conflict of interest.
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Vetro, C., Haferlach, C., Haferlach, T. et al. Aberrations identified by genomic arrays in normal karyotype CMML can be detected in 40% of patients, but do not add prognostic information to molecular mutations. Leukemia 30, 2235–2238 (2016). https://doi.org/10.1038/leu.2016.158