TET2 Gene Mutations Research Articles

Roles of TET2 and C-CBL Mutations In the Progression of De Novo Myelodysplastic Syndrome to Acute Myeloid Leukemia

AbstractAbstract 4019 Background.The molecular pathogenesis of myelodysplastic syndrome (MDS) and its role in the progression to secondary acute myeloid leukemia (sAML) remain to be further explored. Somatic TET2 and C-CBL mutations have recently been described in myeloid neoplasms including MDS and sAML. Most studies of TET2 or C-CBL mutations were carried out separately either at MDS or at sAML phases. There was also a discrepancy in the results of the impact of TET2 and C-CBL mutations on outcome of MDS patients. Aims.We aimed (1) to correlate the TET2 and C-CBL mutations with clinicohematological features and outcome, and (2) to determine the role of TET2 or C-CBL gene mutations in sAML derived from MDS. Materials and Methods.Bone marrow (BM) samples from 161 MDS patients (3 RCUD, 1 RARS, 36 RCMD, 118 RAEB, 1 MDS5q-, 2 MDS-U) at initial diagnosis were analyzed for both TET2 and C-CBL mutations; 49 of them had matched paired sAML BM samples available for comparative analysis. Mutational analysis was performed by DHPLC and/or direct sequencing of all RT-PCR or DNA-PCR products amplified with different primer pairs covering the whole coding sequences of TET2 and exons 7 to 9 of C-CBL. Results.The frequency of TET2 and C-CBL mutations in 161 MDS patients was 17.9 % and 1.9 %, respectively. Seven patients with polymorphism/germ line mutations or missense mutations outside of BOX 1 or 2 of TET2 gene were excluded. Of the 26 patients with 38 TET2 mutations, 14 had nonsense, 11 missense, 11 frameshift, 1 deletion, and 1 splice site mutations; 12 of them had double mutations. Three patients had C-CBL mutations (Y371S, F418S and G415S) at MDS phase. No patient had coexistence of TET2 and C-CBL mutations. TET2 mutations were significantly associated with increased risk of AML transformation (P= 0.037), whereas C-CBL mutations did not influence the risk of AML transformation (P=0.335). Patients carrying TET2 mutations had a trend of shorter time to sAML compared with those without TET2 mutations (estimated median time to sAML 8.7 months vs 15.0 months, P=0.067). Except for lower platelet count in patients with TET2 mutations (P=0.038), there were no significant differences in clinicohematological features including age, sex, hemoglobin level, white blood cell count, percentage of blasts in BM or peripheral blood, cytogenetics or IPSS (≤1.5 vs ≥2.0) between patients with and without TET2 or C-CBL mutations. No significant differences were found in overall survival with respect to mutation status of TET2 (P= 0.244) or C-CBL (P= 0.646). Of the 49 paired BM samples, 3 patients harboring C-CBL mutations at MDS phase retained the identical mutations and another 3 acquired C-CBL mutations (L370_Y371 ins L, L399V and C416W) during sAML evolution. There was an increased risk of acquiring C-CBL mutations during AML transformation (odds ratio=4.16, P=0.041), whereas TET2 mutation patterns remained unchanged and none acquired or lost TET2 mutations in sAML progression. Conclusions.Our results showed an increased risk of acquiring C-CBL mutations during sAML transformation. TET2 mutations played a role as an initial event in the development of MDS in a subset of patients and were associated with more frequent and rapid sAML evolution.Supported by grants NHRI-EX99-9711SI, NSC97-2314-B-182-011-MY3 and DOH99-TD-C-111-006. Disclosures:No relevant conflicts of interest to declare.

TET2 gene is not deleted in chronic myelomonocytic leukemia: a FISH retrospective study

We read with interest the paper TET2 gene mutation is a frequent and adverse event in chronic myelomonocytic leukemia , published in this Journal in December 2009. This study contributes significantly to the study of chronic myelomonocytic leukemia (CMML), providing important molecular and survival

Involvement of TET2 in Regulation of Epigenetic Silencing in MDS and Related Myeloid Malignancies.

Abstract 2908Poster Board II-884Genomic (chromosomal gain losses and UPD) and genetic damage (mutations) are hallmarks of MDS and related myeloid malignancies. In addition, aberrant hypermethylation of CpG islands, leading to epigenetic silencing of tumor suppressor and differentiation genes, appears to play an important pathogenic role in evolution to AML. While various mechanisms leading to chromosomal instability have been identified, the pathogenesis of epigenetic instability leading to aberrant methylation remains unexplored. The recent discovery of mutations in TET2 gene in MDS/MPN, sAML and especially CMML provided an intriguing possibility that dysfunction of this bona fide tumor suppressor gene may result in increased promoter methylation. This hypothesis is based on the description of the function of the highly related TET1 gene: the corresponding protein mediates hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which prevents the DNMT1-catalyzed maintenance methylation during cell division. Consequently, TET2 mutations could lead to epigenetic instability and accumulation of aberrantly hypermethylated CpG sites.Both homozygous and heterozygous TET2 mutations were found suggesting that the presence of WT allele or residual activity is not protective. A large proportion of these mutations results in introduction of frame shifts or stop codons resulting in loss of function whilst missense mutations may lead to decreased function. To clarify the function of TET2 and consequences of the mutations we transduced M5 THP-1 AML cell line with lentiviral vectors, containing partial TET2 cDNA (TET2+) and shRNA (TET2-), to achieve overexpression or knockdown of TET2 gene. Following selection, TET2+ cell line showed 3-fold TET2 mRNA overexpression while TET2- cells demonstrated 85% knockdown of TET2 gene as compared to control cells. Based on the putative function of TET2, we performed thin layer chromatography (TLC) comparing 5hmC DNA content in THP1, TET2+, TET2- and appropriate control cells. Parental THP1 cells revealed abundant level of 5hmC. Whilst a 1.5-fold increase of 5hmC content was found in TET2+ cells, it remained unchanged in TET2- cells. However, early results showed a 20% decrease in 5hmC in bone marrow of patients with myeloid malignancies and homozygous TET2 mutations. Illumina 27k methylation arrays were used to assess the effects of altered TET2 expression on methylation patterns. Although global methylation levels, as expressed by averaged β-values, did not significantly differ, the number of hypermethylated CpG sites, defined by individual β-values >0.9, was decreased by 25% (3756 vs. 2839) in TET2+ cells. No difference was seen in TET2- cells vs. controls. However, a number of promoter CpG in TET2- displayed >2-fold change in methylation level compared to TET2+, including MGMT, PCGF2, PTPRO and NOX1. Among differentially silenced promoter CpG we selected CDKN2B (p15) promoter as a marker for methylation activity. When p15 promoter controlling a luciferase reporter was transfected into TET2+ and control cells, a 2.4-fold increase of luciferase activity occurred, consistent with increased demethylating activity in cells expressing higher levels of TET2. Additionally, qPCR showed a 2.5-fold increase in p15 expression in TET2+ cells. Based on this result we also studied methylation status of p15 promoter in patients with WT (N=20) and mutant TET2 (N=27); patients with MDS and MDS/MPN who carried TET2 mutations presented consistent methylation of various promoters including p15 in 2/3 mutant cases vs. 1/3 WT cases.Whereas proliferation kinetics was not significantly altered by changes in TET2 levels, 72h treatment with decitabine (0.5 μM) resulted in a differential effect on TET2+ and TET2- cells. TET2+ cells were not significantly affected however survival of TET2- cells decreased by 30%. In addition, TET2+ cells showed a relative resistance to HDAC inhibitor Entinostat (1μM) with 26% inhibition of proliferation vs. 60% seen in TET2- cells measured after 72h by cell proliferation assay (MTS).In sum, our results suggest that TET2 gene may be involved in epigenetic regulation of promoter CpG that are relevant to proliferation/differentiation of myeloid cells and changes in the expression/activity of this gene may result in altered methylation and thereby epigenetic silencing patterns. Disclosures:No relevant conflicts of interest to declare.

Deep Sequencing the Tet2 Gene in 360 Patients with Myeloid Neoplasms Provides a Comprehensive and Quantitative Mutation Map and Reveals Low Level Mutant Clones.

Abstract 733Tet2 is a novel candidate tumor suppressor and mutations in this gene have been found in 24% of myeloid neoplasms following conventional Sanger sequencing. Using a massively parallel pyrosequencing methodology (Roche GS FLX) we sequenced the entire Tet2 gene coding region in 360 patients with myeloid malignancies (MDS 279; CMML 32; AML 10; MDS/MPD 10; 5q syndrome 8; others 21). We also utilized high density SNP6 arrays to screen for chromosomal aberrations including those at the Tet2 locus itself. Initially we found 106 mutations in Tet2 corresponding to 75 patients (21%), where the average sequence coverage was more than 100-fold for >90% of the gene. 25 patients had more than one mutant Tet2 clone. 83 mutations (in 17% of patients) were found in 30% to 95% of sequenced clones, where 50% or 100% could be indicative of heterozygous and homozygous mutations that would be found in all sample bone marrow cells respectively. The remaining 23 mutations were found in ≤30% of sequence clones, applying a lower threshold of 17% to avoid false positives, and were not reliably picked up by confirmatory Sanger sequencing. The 106 mutations were distributed as 44 nonsense or InsDel mutations throughout the gene, 59 nonsynonomous mutations within (23) and outside (36) the two known conserved domains, and 3 spice site mutations.Tet2 mutations were not correlated with survival outcome (probability of survival after 3 and 5 years from diagnosis 40% and 38% [p=0.3 log rank]for mutants vs non-mutants respectively). Patients with small (≤30%) or multiple mutant clones did not deviate from the overall survival trend. Furthermore, no difference in survival was seen when patients were analysed according to their WHO subgroups or IPSS scores. CMML featured as the only subgroup with a relatively high Tet2 mutation rate (53% of patients). Chromosomal aberrations linked to the Tet2 locus, including UPD or micro-deletion, were detected in 20-25% of patients, but this was not strongly correlated to detectable mutations in the Tet2 gene itself (24% coincidence), perhaps indicating that several forms of mutation or instability at this region are linked to disease. Interestingly, high percentage mutant clones which are indicative of homozygosity and thus loss of heterozygosity (LOH), were found as InsDel mutations in Tet2 for the majority (85%), suggesting a common defect in DNA replication or repair.Following up on additional smaller Tet2 mutant clones, we lowered our threshold for mutation detection and after further analysis and removal of experimental background, 42 additional candidate mutations of clone size between 8% and 17% were found. Taken as a whole, Tet2 gene mutations were found in 30% of patients but this may rise considerably if deeper sequencing screens for even smaller mutant clones yield further positive results.We therefore show via highly sensitive ‘Deep sequencing' on a relatively large patient cohort a comprehensive and highly quantitative mutation map across the Tet2 gene to allow increased prognostic comparisons. We also show that mutation figures rise significantly when smaller mutant clones are considered that cannot be detected via Sanger sequencing. In addition to this, if larger chromosomal aberrations such as UPD are added to the evolving number of mutant Tet2 patients, then Tet2 abnormalities may in fact be present in a majority of patients with myeloid neoplasms. Disclosures:Gattermann:Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.