SNP-a Karyotyping Provides a Clonal Molecular Marker and Is Associated with a High Incidence of Segmental Uniparental Disomy in Patients with CMML
Authors: McDevitt Michael , Dunbar Andrew Jeffrey , O’Keefe Christine , Makishima Hideki , Tiu Ramon V. , Gondek Lukasz , Karp Judith E. , Wang Xiao-Fei , Levis Mark J. , Maciejewski Jaroslaw 
Published: October 2019

Abstract
Chronic myelomonocytic leukemia (CMML) patients display poor prognosis with median survival of 20 months (range 10–60). The WHO classification recognizes CMML as an overlap syndrome with mixed clinical and histomorphological features of both myelodysplastic syndrome (MDS) and myeloproliferative disorders (MPD). The underlying molecular basis for the poor prognosis and clinical and pathological features including the dysplasia and monocytosis are currently unknown. A lack of clonal markers in the majority of patients impedes diagnosis and prognosis assignment, as well as treatment response monitoring. Individual cases are associated with rare (5;12) translocations, RAS mutations, and unbalanced chromosomal changes. Since chromosomal defects have a major impact on the diagnosis and prognosis of myeloid malignancies, it is likely that cytogenetic methods with higher resolution and an ability to detect uniparental disomy (UPD) could explain clinical heterogeneity and point to potential therapeutic targets in CMML. Our study included 101 cases including 70 cases of both CMML1 and 2 and CMML-derived AML. We also studied 31 M4/M5/monocytic subtype AML cases (8 FLT3+) to allow interpretation of the 17 CMML cases that were analyzed at the time of AML transformation. Both 250K and a more sensitive 6.0 SNP-A array were utilized. Copy number variants (CNV) were identified and excluded from analysis by comparison with simultaneous arrays with CD3+ normal T cells from the test individual, or when not available, by comparison of results with published and our extensive internal CNV databases, and experimentally determined size exclusion parameters from over 500 MDS and AML cases run on the same platform. In total, 67/70 patients had samples sent for metaphase cytogenetics (MC) and 45% of these cases had an abnormal karyotype. Expected lesions commonly associated with MDS/CMML, such as +8 (N=4) and −7/del(7q) (N=5) were observed. When SNP-A was applied to the entire cohort, defects found by MC were confirmed. Also, new chromosomal abnormalities were identified for a total detection rate of 55/70 (79%). When patients with CMML-1, CMML-2 vs CMML-derived AML were analyzed for gains, losses and UPD, several results are apparent. First, subcytogenetic loci on the chromosomes affected by large gains (e.g., 8 and 21) and losses (e.g., del7 or del5) were identified in additional patients. Second, cytogenetic amplification (amp) and deletion (del) regions seen by MC were confirmed by SNP-A. Examples of recurrent amplifications and deletions included amp21q21 (N=8), amp8p23.2–q24.3 (N=5), amp13q31.1(N=4), amp20p(N=3), del7q21.1(N=4), del12p12.2(N=4), del7q34(N=3), and del5q(N=3). We found a high prevalence of segmental UPD, not otherwise detectable by MC, occurring in 54 % of primary CMML/CMML-derived AML. Recurrent lesions were identified on chrs. 1(N=3), 2(N=3), 4(N=5), 6(N=3), 7(N=5), 11(N=7), 13(N=3), 14(N=3), and 21(N=4). Fifteen of seventy (21%) had UPD as a sole or isolated abnormality. In 6/7 of the UPD11 cases, this was present as a sole abnormality. In a separate abstract we present 12 UPD11q cases with MDS/MPD-U and sAML; in 7 of these cases inactivating c-Cbl mutations were found. All were associated with CMML cases associated with AML transformation. Overall survival was compared for patients with CMML (WHO 1 and 2), CMML into AML, and M4/M5 with either normal karyotype and normal SNP-A results (no amplifications, deletions, UPD), relative to those with detectable genomic lesions. Outcome was poor for all CMML groups however. In summary, SNP-A-based karyotyping complements MC and allows for precise definition of chromosomal aberrations in patients with CMML, including copy-neutral LOH/UPD. UPD is common in CMML and overlapping regions may point to potential causative genes. The identification of c-Cbl demonstrates the utility of this approach, identifying pathways to explore for therapeutic intervention. Overall, SNP-A reveals evidence for significant cytogenetic and subcytogenetic genomic instability in CMML, perhaps not surprisingly with the known adverse prognosis of this disorder. UPD 7 and UPD 21 abnormalities are particularly interesting based on the frequency of these lesions, and the known association of monosomy 7 with CMML. They may provide markers for diagnosis and response monitoring, as more novel therapeutics are being developed for this poor prognosis disorder.