Array CGH Identifies Copy Number Changes In 10% Of 520 MDS Patients With Normal Karyotype: Deletions Encompass The Genes TET2, DNMT3A, ETV6, NF1, RUNX1, and STAG2 and Are Associated With Shorter Survival

In MDS, cytogenetic aberrations play an important role for classification and prognostication. The original IPSS and the revised IPSS classifiers have clearly demonstrated the prognostic impact of distinct cytogenetic abnormalities. The vast majority of chromosome aberrations in MDS are gains or losses of chromosomal material while balanced rearrangements are rare. However, more than 50% of MDS and even more in low risk MDS harbor a normal karyotype. Chromosome banding analysis can only detect gains and losses of more than 10 Mb size due to its limited resolution and is dependent on proliferation of the MDS clone in vitro to obtain metaphases. Array CGH has a considerably higher resolution and does not rely on proliferating cells.

In this study we addressed the question whether MDS with normal karyotype harbor cytogenetically cryptic gains and losses.

520 MDS patients with normal karyotype were analyzed by array CGH (Human CGH 12x270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). For all patients cytomorphology and chromosome banding analysis had been performed in our laboratory. The cohort comprised the following MDS subtypes: RA (n=22), RARS (n=43), RARS-T (n=27), RCMD (n=124), RCMD-RS (n=111), RAEB-1 (n=104), and RAEB-2 (n=89). Median age was 72.2 years (range: 8.9-90.1 years). Subsequently, recurrently deleted regions detected by array CGH were validated using interphase-FISH.

In 52/520 (10.0%) patients copy number changes were identified by array CGH. Only eight cases (1.5%) harbored large copy number alterations >10 Mb in size, as such generally detectable by chromosome banding analysis. These copy number alterations were confirmed by interphase-FISH. They were missed by chromosome banding analysis due to small clone size (n=2), insufficient in vitro proliferation (n=3) or poor chromosome morphology (n=3). In the other 44 patients with submicroscopic copy number alterations 18 gains and 32 losses were detected. The sizes ranged from 193,879 bp to 1,690,880 bp (median: 960,176 bp) in gained regions and 135,309 bp to 3,468,165 bp (median: 850,803 bp) in lost regions. Recurrently deleted regions as confirmed by interphase-FISH encompassed the genes TET2 (4q24; n=9), DNMT3A (2p23; n=3), ETV6 (12p13; n=2), NF1 (17q11; n=2), RUNX1 (21q22; n=2), and STAG2 (Xq25, deleted in 2 female patients). No recurrent submicroscopic gain was detected. In addition, we performed survival analysis and compared the outcome of patients with normal karyotype also proven by array CGH (n=462) to patients with aberrant karyotype as demonstrated by array CGH (n=52). No differences in overall survival were observed. However, overall survival in 35 patients harboring deletions detected solely by array CGH was significantly shorter compared to all others (median OS: 62.1 vs 42.4 months, p=0.023).

1. Array CGH detected copy number changes in 10.0% of MDS patients with cytogenetically normal karyotype as investigated by the gold standard method, i.e. chromosome banding analysis. 2. Most of these alterations were submicroscopic deletions encompassing the genes TET2, ETV6, DNMT3A, NF1, RUNX1, and STAG2. 3. Interphase-FISH for these loci can reliably pick up these alterations and is an option to be easily performed in routine diagnostics in MDS with normal karyotype. 4. Patients harboring deletions detected solely by array-CGH showed worse prognosis.

Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Raitner:MLL Munich Leukemia Laboratory: Employment. Holzwarth:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.