TET2 (tet oncogene family member 2) on chromosome 4q24 was identified as a candidate tumor suppressor gene. Recurring submicroscopic deletions and copy-neutral loss of heterozygosity (CN-LOH) involving 4q in MDS patients detected by SNP microarray analyses suggested TET2 as an interesting candidate gene. Subsequent sequencing studies revealed TET2 mutations in 10–25% of patients with AML, MDS, and MPNs, while a mutation frequency of up to 42% was reported in CMML. Only a subset of studies evaluated both TET2 mutation status and TET2 copy number status, although this might be of pathophysiological and even of prognostic relevance if TET2 functions as a classical tumor suppressor gene. In the majority of studies copy number status was determined by SNP array analysis, although being expensive and time consuming. Here, in order to investigate TET2 deletions in a large cohort of patients an easy to perform FISH assay was developed applying BACs covering the TET2 gene (RP11-351K6 and RP11-16G16; BlueGnome, Cambridge, UK). This assay was validated on samples with TET2 deletions proven by SNP array analysis. With these FISH probes we analyzed 843 cases with myeloid malignancies (404 AML (323 de novo AML, 68 s-AML, 13 t-AML), 166 MDS, 201 CMML, and 72 MPN). Overall 50 (5.9%) cases with TET2 deletion were identified. These included 22 AML (5.0%), in detail 14 de novo AML (4.3%), 6 s-AML (8.8%), and 2 t-AML (15.4%) as well as 15 CMML (7.5%), 9 MDS (5.4%) and 4 MPN (5.6%). Patients with TET2 deletions showed the following karyotypes: normal: n=15, cytogenetically balanced rearrangements involving 4q24: n=3, 4q deletion as the sole abnormality: n=2, complex: n=25, other abnormalities: n=5. In 25/50 (50%) cases the TET2 deletion was cytogenetically cryptic. In patients with complex aberrant karyotype loss of 4q material was due to interstitial deletion in 7/25 cases and due to unbalanced rearrangements in 16/25 cases, while in 2/25 cases chromosomes 4 were normal in chromosome banding analysis. Furthermore, in patients with TET2 deletions mutation analyses was performed for mutations in TET2 (n=37 investigated), RUNX1 (n=13), NPM1 (n=18), JAK2V617F (n=18), CBL (n=36), NRAS (n=17), KRAS (n=36), FLT3-ITD (n=26), FLT3-TKD (n=8), IDH1 (n=9) and MLL-PTD (n=24). Mutations in TET2 were detected in 19/37 cases (51%), in RUNX1 in 1/13 (8%), in JAK2V617F in 6/18 (33.3%), in CBL in 2/36 (5.6%), in NRAS in 1/17 (6%), in KRAS in 1/36 (2.8%) and in NPM1 in 1/18 (6%) cases, whereas no mutations within the other genes analyzed were found. In the cohort of cases with TET2 deletion and concomitantly TET2 mutation (n=19) 10 had a normal karyotype (52.6%), 5 a complex karyotype (26.3%) and 4 had other abnormalities (21.1%). Importantly, in the cohort of CMML, in 10 of 14 cases (71.4%) both a TET2 deletion and TET2 mutation was detected. Overall, TET2 mutations were significantly more frequent in patients with cytogenetically cryptic TET2 deletion as compared to cytogenetically visible 4q deletions (68.2% vs. 26.7%, p=0.020). In addition FISH screening identified 2 cases (one CMML, one t-MDS) which showed a translocation involving the TET2 locus not leading to a deletion of the BAC signals but a separation suggesting a fusion with yet unidentified partner genes. In conclusion, FISH analyses identified TET2 deletions in 6% of myeloid malignancies. 50% of these deletions were submicroscopic and therefore not detectable by chromosome banding analysis. TET2 deletions were accompanied by TET2 mutations in 51% of respective cases. FISH is a reliable and efficient method to determine the copy number state of TET2. Still, the prognostic impact of TET2 deletions with and without additional TET2 mutations in the various myeloid malignancies has to be evaluated in future investigations.
Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Wille:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
Asterisk with author names denotes non-ASH members.