The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2015  |  Volume : 40  |  Issue : 4  |  Page : 159--165

TET2 expression in a cohort of Egyptian acute myeloid leukemia patients


Nahla A Hamed1, Nabil A El Halawani1, Heba S Kassem2, Mona W Ayad3, Enas A Dammag1,  
1 Internal Medicine Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Clinical Genetic Center, Faculty of Medicine, Alexandria University, Alexandria, Egypt
3 Clinical Pathology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Correspondence Address:
Mona W Ayad
Clinical Pathology Department, Alexandria Medical Faculty, Alexandria University, Alexandria
Egypt

Abstract

Ten-eleven translocation 2 gene (TET2) expression plays a crucial role in DNA methylation and hematopoietic stem cell functions. The prognostic relevance of the TET2 mutation in cytogenetically normal acute myeloid leukemia (AML) patients is still not well established. The aim of the present study was to determine the level of TET2 expression in AML patients, its prognostic significance, and its relation to cytogenetics. We studied TET2 gene expression by real-time PCR in 33 AML patients and 34 healthy controls matched for age and sex. The median age of AML patients at presentation was 40 years, with a female to male ratio of 1.06 : 1. A total of 87.9% were de-novo AML and 12.1% were chronic myeloid leukemia in blastic crisis. In all, 66.6% of cases had normal cytogenetics. Underexpression of the TET2 gene was present in 90.6% (0.3098 ± 0.3846) of the patients. TET2 expression was not affected by age (P = 0.609) or cytogenetic findings (P = 0.057). Yet, it was correlated inversely with pretreatment white blood cell count (rs = −0.366, P = 0.04). It also correlated with lower remission rates (P = 0.002) and relapse (P = 0.000). TET2 probably plays a role in the pathogenesis and progression of AML. This can play a role in targeted therapy in the future. Further studies are recommended to assess TET2 expression in response to hypomethylating agents to determine its predictive role in response to therapy using these agents.



How to cite this article:
Hamed NA, El Halawani NA, Kassem HS, Ayad MW, Dammag EA. TET2 expression in a cohort of Egyptian acute myeloid leukemia patients.Egypt J Haematol 2015;40:159-165


How to cite this URL:
Hamed NA, El Halawani NA, Kassem HS, Ayad MW, Dammag EA. TET2 expression in a cohort of Egyptian acute myeloid leukemia patients. Egypt J Haematol [serial online] 2015 [cited 2019 Dec 11 ];40:159-165
Available from: http://www.ehj.eg.net/text.asp?2015/40/4/159/170194


Full Text

 Introduction



Acute myeloid leukemia (AML) is a disease characterized by genetically heterogeneous somatic mutations in hematopoietic progenitor cells that disrupt cell cycle including cellular growth, proliferation, and differentiation [1],[2] .

Conventional cytogenetics is an important prognostic factor in AML. However, 40-50% of patients have a normal karyotype and are categorized as the intermediate-risk group [3] . Cytogenetically normal acute myeloid leukemia (CN-AML) represents the largest subgroup of adult primary AML [4] , and within this molecularly heterogeneous group, gene mutations are increasingly being used to assess prognosis and guide risk-adapted treatment [3] . AML has multiple pathophysiologic mechanisms that involve both genetics and epigenetics. Epigenetics are the modifications of nucleic acid bases without alterations in the primary DNA base sequence and are being responsible for a wide range of biological functions. The most common enzymatic modification is the methylation of the 5-position of cytosine [5] .

The ten-eleven translocation (TET) gene family is one of the epigenetics genes. The TET proteins TET1, TET2, and TET3 are a-ketoglutarate and Fe 2+ -dependent enzymes that can modify DNA methylation by converting 5-methylcystosine (5mC) into 5-hydroxymethylcystosine (5hmC) [6] . Furthermore, it can oxidize 5mC or 5hmC further and convert them into 5-formylcytosine (5fC) and/or 5-carboxylcytosine (5caC), leading to further complicated epigenetic regulation during development [7] .

Of all TET family members, TET2 is most frequently mutated in myeloid malignancies [8] . The TET2 gene is located on chromosome 4q24 [9] . Homozygous and heterozygous mutations in the TET2 gene are recurrent events in human hematopoietic malignancies. Most of these mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. Loss of TET2 protein could contribute toward gene-specific hypermethylation that is often observed in hematologic malignancies [9] . The reported mutation rate of TET2 is about 20%; it varies according to the different disease entities. It was reported that 6-7% of AML patients, mostly in the CN-AML subgroup, carry the TET2 mutation, but its prognostic relevance is still a controversial issue [5] .

The aim of the present study was to determine the level of TET2 gene expression in AML patients relevant to an age-matched and sex-matched control series and to study its prognostic significance and its relation to conventional cytogenetics in a cohort of Egyptian AML patients.

 Patients and methods



The current study included 67 participants; 33 adult AML patients were recruited from Alexandria Main University Hospital (Haematology Department) during the period from May to December 2013 (group A); 34 age-matched and sex-matched healthy individuals were recruited as a control group (group B). Patients younger than 16 years old, or patients with hepatic, renal failure or pregnant women were excluded from the study. Patients' median age was 40 years, with 51.5% women and 48.5% men.

Informed written consent forms were signed by patients according to the Declaration of Helsinki (1964) and the study was approved by the Alexandria Faculty of Medicine the Ethics Committee.

All patients were subjected to a thorough assessment of history, complete clinical examination, routine chemical investigations [10] , complete blood count, bone marrow examination, immunophenotyping (myeloperoxidase, CD13, CD33, CD14, CD11C, CD19, CD3, and CD117) [11] , and conventional cytogenetics [12] .

Conventional cytogenetics (karyotyping)

Preparation of cell culture: 0.5 ml of blood was added to 5 ml of growth medium RPMI, and 10% fetal calf serum and PHA were added. The cells were grown for 60-72 h at 37°C in a moist atmosphere containing 5% CO 2 with gentle inversion twice a day.The cell division was stopped at the metaphase: using prewarmed 37°C colcemid (final concentration 0.05 μg/ml).Hypotonic treatment of the red blood cell and white blood cell (WBC) was performed by KCl 0.075 mol/l 37°C.Fixing the cells: 5 ml of fixative solution (three parts of chilled absolute methanol was added to one part glacial acetic acid) was added to the centrifuge tube.Preparation of the chromosome slides: Five or six slides were laid next to each other on paper toweling with no separation between them. From a height of about 18 inches, two or three drops of fluid onto each side were dropped. The slides were allowed to dry thoroughly by placing them in the incubator (37°C) overnight. The slide was stained by immersion in fresh Giemsa stain [12] .Twenty metaphases were evaluated.

TET2 gene expression

Genomic RNA extraction was performed using the Qiagen QIAamp RNA mini kit (Hiden, Germany) (cat. No. 52304). QIAamp spin columns represent a technology for total RNA preparation that combines the selective binding properties of a silica-based membrane with the speed and convenience of microspin technology. A specialized high-salt buffering system allows RNA species longer than 200 bases to bind to the QIAamp membrane. The samples were run on agarose gel electrophoresis and RNA concentrations were obtained from nanodrop spectrophotometer readings at 260, 280, and 260/280 ratios [13] .

Real-time quantitative PCR assay

TaqMan-based RT-PCR technology was used. A relative quantification of TET2 gene expression was normalized to the endogenous housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GAPDH gene was used as the internal control and the quantification of relative expression was performed by real-time PCR using real-time cycler Rotor gene Q (Qiagen, Hilden, Germany), Hs_TET2_QF_1 QuantiFast Probe Assay Taqman FAM (NM_001127208, NM_017628, QF00281211), and the ready-to-use QuantiFast probe one-step RT-PCR assay (Qiagen). After the genomic DNA removal step, the PCR mixture contained 12.5 μl QuantiFast multiplex PCR master mix (HotStar Taq plus DNA polymerase, QuantiFast Probe, PCR Buffer and dNTP mix), 1.25 μl QuantiFast Primers and Probe assay for target gene (FAM*), 1.25 μl QuantiFast Primers and Probe assay for the housekeeping gene (MAX*), 0.25 μl QuantiFast RT Mix, 0.5 μl ROX dye solution, and RNase-free water and RNA (≤100 ng/reaction) in a total reaction volume of 25 μl. PCR was performed under the following conditions: 50°C for 20 min for reverse transcription of RNA, 95°C for 5 min, followed by 45 cycles of 95°C for 15 s, and 60 ° C for 30 s. A negative control was included in each experiment [14] .

The comparative cycle threshold (Ct ) method was used to determine the relative expression levels of TET2. The mean of the cycle number difference was calculated. Data were presented as 2−ΔΔCt , where Ct is the threshold cycle and ΔCt is the Ct value of target amplification minus that of reference amplification (ΔCt = GAPDH−TET2) [14] . Using the −ΔΔCt , a relative quantification in the means of fold change [fold change 2 (−ΔΔCt) 2 (−ΔΔCt) ] is measured using a reference of 1 for the control [15] .

Follow-up and treatment outcome

Response to induction therapy was assessed after two courses of chemotherapy. In accordance with standard criteria, complete remission (CR) was defined as less than 5% bone marrow blasts, an absolute neutrophil count of 1.0 × 10 9 /l or more, a platelet count of 100 × 10 9 /l or more, no blasts in the peripheral blood, and no extramedullary leukemia. Therapeutic failures were classified as either refractory disease or early death, which was death before treatment. Relapse was defined as more than 5% BM blasts unrelated to recovery from the preceding course of chemotherapy or new extramedullary leukemia in patients with previously documented CR.

Statistical analysis

Data were analyzed using SPSS v. 20 (IBM, NY, USA). Data were tested for normality using the Kolmogorov-Smirnov test and the Shapiro-Wilk test. Data were presented as range, mean ± SD. Qualitative data were tested using Pearson's χ2 -test and the Fisher exact test. The t-test was used to compare the means of parametric data. Nonparametric data were presented as median value and range. Mann-Whitney (U) test was used to compared the median value. Spearman bivariate correlation analysis was carried out to analyze correlation. P value less than 0.05 indicated statistical significance.

 Results



Clinical characteristics of the 33 AML patients studied: 29 study participants had de-novo AML (87.9%) and four patients had chronic myeloid leukemia in blastic crisis (12.1%). The median age of the patients was 40 years, with a range from 19 to 75 years. In all, 51.5% of the patients were women and 48.5% were men. At presentation, four patients (12.1%) presented with relapsing AML, whereas 29 patients (87.9%) had been newly diagnosed. FAB classification of the studied cohort was as follows: three (9.1%) patients with M0, 15 (45.5%) M1, three (9.1%) M2, one (3%) M3, five (15.2%) M4, three (9.1%) M5a, two (6.1%) M6, and one (3%) M7. Cytogenetic risk classification was categorized as follows: 9.7% as favorable risk, 70.9% as intermediate risk, and 19.4% as unfavorable risk. Two-third of the patients, 22/31 (66.6%), had normal karyotype. t(9;22) was found in four patients, complex karyotype in one patient (3.2%), hyperploidy in one patient (3%), t(8;21) in two patients (6.5%), and t(15;17) in one patient, whereas two cases failed to yield culture.

TET2 gene expression was assessed by relative comparison using ΔΔCt in comparison with a normal group where the normal individuals had one as the gene level of expression. Patients with AML had a mean of 0.3098 ± 0.3846. When categorizing patients as underexpressed and overexpressed, it was noted that 29/32 (90.4%) of patients showed underexpression of the TET2 gene and 3/32 (9.4%) of patients showed overexpression of TET2. One case failed PCR. The 3 patients who had overexpression of TET2 were De novo AML ([Table 1] and [Figure 1]).{Figure 1}{Table 1}

Patients' response to therapy: of the 33 patients enrolled, 25 (75.8%) were suitable for conventional chemotherapy in the form of a 3+7 regimen, whereas seven (21.2%) were managed palliatively (cytarabine alone, hydroxyurea) and only one (3%) died before treatment. Those who received treatment showed the following responses ([Table 2]).{Table 2}

There was a significant correlation between TET2 expression level and hematological parameters. It was directly proportional to platelet count and Hb concentration and inversely proportional to WBC counts. There was no significant correlation between TET2 expression and age of the patient or % bone marrow blast cells ([Table 3]).{Table 3}

Relation between cytogenetics and TET2 gene expression: no statistically significant difference was found between the level of TET2 gene expression and different karyotypes (F = 9.247; P = 0.057) ([Table 4]).{Table 4}

Mortality among cases

Of all AML patients enrolled, only 17 (51.5%) survived the follow-up period after two chemotherapy cycles (induction/first consolidation or induction/reinduction), whereas 16 patients (48.5%) died during this period; most of the deaths were because of sepsis 62.5% (10/16) and bleeding 37.5% (6/16). Bleeding sites leading to death were intracerebral hemorrhage and intra-alveolar hemorrhage.

Comparison between TET2 gene expression and therapeutic outcome

There was a statistically significant relation between the level of TET2 expression and failure to achieve CR (F = 10.089; P = 0.002), and relapse (P = 0.011). However, no significant relation was found between TET2 expression and mortality (χ2 = 2.921; P = 0.087), or refractoriness to therapy (χ2 = 0.002; P = 0.968) ([Table 5]).{Table 5}

 Discussion



TET2 is hypothesized to act as a tumor-suppressor gene at critical loci important for myelopoiesis and leukemogenesis. TET2 is frequently mutated in AML patients. Thus, it loses its role in the regulation of methylation and hence hypermethylation occurs. As a result, silencing of genes is needed for differentiation that occurs in AML [16] .

In the present study, TET2 gene underexpression was present in 90.8% of the patients, with a mean expression level of 0.3098 ± 0.3846 compared with an expression level of 1 in the control group. Underexpression of TET2 in AML was also reported by Solary et al. [9] and Chou et al. [17] . Also, Zhu et al. [18] found that TET2 gene expression levels from bone marrow mononuclear cells and CD34 + cells in CN-AML were significantly lower than those in healthy controls (P = 0.026 and 0.034, respectively).

TET2 deficiency confers the cells with a competitive advantage over wild-type cells and induces hematopoietic differentiation abnormalities, but is not directly responsible for full cellular transformation [19] .

In contrast to the few data available on TET2 gene expression, considerable data are available on mutations of TET2. Most TET2 (mut) are characterized by a weak gene-expression pattern [20] . Several studies have shown that TET2 abnormalities in myeloid malignancies are highly heterogeneous, consisting of insertions, nonsense/mis-sense mutations, and deletions. They may occur as homozygous or heterozygous alterations including loss of heterozygosity [21] because of homozygous deletion or uniparental disomy [22] . TET2 underexpression might be attributed to TET2 mutations such as frame shift or nonsense mutations (likely resulting in a truncated protein) [4] .

In the present study, about 10% of patients showed an overexpressed TET2 gene; all were de-novo AML patients. However, all patients with secondary AML showed an underexpressed TET2 gene. Similarly, reports on patients with AML have often focused on secondary AML arising from myeloproliferative neoplasm. Recently, Makishima et al. [23] reported that TET2 mutations were shown to be a late event in myelodysplastic syndrome (MDS), myelodysplastic/myeloproliferative (MDS/MPN), and MPN because they are rarely present in low-grade MDS and are frequent in secondary AML.

Pretreatment karyotype remains to be the most important predictor of outcome in adult AML. Patients with favorable cytogenetics have the best prognosis and those with unfavorable cytogenetics have the highest risk for induction failure and relapse and shortened survival [21] .

Most of our patients had a normal karyotype (22/31) (73.3%). There was no statistically significant relation between cytogenetic findings and the age (P = 0.653) or the sex of the patient (P = 0.609). Similarly, Preiss et al. [24] reported no statistical significance between age or sex and conventional cytogenetics in both de-novo and secondary AML.

In the present study, there was no statistically significant relation between the level of TET2 gene expression and karyotyping (P = 0.057). Similarly, Nibourel et al. [25] found no significant association between normal karyotypes and TET2 expression (P = 0.62), whereas Chou et al. [17] found a significant relationship between the intermediate-risk group (P < 0.001), normal karyotyping (P = 0.0064), and the TET2 mutation.

In the present study, a statistically significant positive correlation was found between TET2 gene expression level and Hb (P = 0.033) and platelets (P = 0.013), and a negative correlation with pretreatment WBCs counts (P = 0.04). Metzeler et al. [4] and Chou et al. [17] reported that TET2 mutations were associated with older age (P<0.001) and higher pretreatment WBCs (P = 0.04). However, Nibourel et al. [25] reported that there was no significant relation between TET2 and either age (P = 0.17), Hb (P = 0.45), WBCs (P = 0.14), or platelets (P = 0.85). They stated that TET2 gene mutations similar to those observed in myeloid and lymphoid malignancies also accumulate with age in otherwise healthy individuals with clonal hematopoiesis [20] . TET2 mutations were associated with deregulation of genes involved in stem-cell self-renewal, cell cycle control, and cytokine and growth factor signaling, which may help explain their adverse prognostic impact [4] .

When comparing therapy outcome with TET2 expression level, there was a statistically significant relation between low level of TET2 expression and failure to achieve CR (F = 10.089; P = 0.002) and relapse (χ2 = 12.789; P = 0.011), whereas no significant relation was found between TET2 expression and mortality (χ2 = 2.921; P = 0.087) or refractoriness to therapy (χ2 = 0.002; P = 0.968).

Similarly, Metzeler and colleagues found a statistically significant association between low gene expression levels and lower CR rate (P = 0.002) and relapse (P = 0.000), whereas it was not significant with mortality (P = 0.087) or refractory cases (P = 0.968). They reported that TET2-underexpression patients had shorter event-free survival (P<0.001) because of a lower CR rate (P = 0.007), and shorter disease-free survival (P = 0.003), and also had shorter overall survival (P = 0.001) [4] .

Also, Aslanyan and colleagues found that TET2 mutations were an independent marker of poor prognosis. Loss-of-function TET2 mutations predicted poor outcome; they speculated whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival Aslanyan et al. [27] .

Liu et al. [26] found that TET2 mutations have an adverse impact on prognosis and may help to justify risk-adapted therapeutic strategies for patients with AML.

Chou et al. [17] reported that the TET2 mutation is an unfavorable prognostic factor in patients with intermediate-risk cytogenetics, and its negative impact was further enhanced when the mutation was combined with FLT3-ITD, NPM1-wild, or unfavorable genotypes (other than NPM1+ /FLT3-ITD− or CEBPA+ ).

However, Nibourel et al. [25] did not find any prognostic impact of TET2 mutation in primary AML achieving CR [27] . Gaidzik et al. [20] found that TET2(mut) did not impact the response to therapy and survival. Mutations were mutually exclusive with IDH(mut), which supported recent data on a common mechanism of action that might obscure the impact of TET2(mut) compared against all other patients with AML.

Schlenk et al. [28] reported that epigenetics-modifying gene (DNMT3a, TET2, and IDH1/2) mutations are present, with no change between diagnosis and relapse samples, and may be used as a minimal residual disease marker. Indeed, these mutations are found at high frequency in conjunction with other mutations, supporting the hypothesis that genetically unstable preleukemic cells acquire numerous mutations and chromosomal abnormalities, resulting in a stepwise progression to the onset of leukemia [29] .

There are several unresolved issues related to the TET2 gene in primary AML. First, the association of TET2 mutations with other genetic alterations has not been fully addressed. Second, the prognostic significance of the TET2 mutation in AML is still controversial. Third, the stability of TET2 mutations during disease evolution in AML remains unknown [17] .

 Conclusion



TET2 low expression is a common finding in AML cases in Egypt. TET2 probably plays a role in the pathogenesis and progression of AML. This can play a role in targeted therapy in the future. Further studies on a large scale are recommended to assess types of TET2 mutations by sequencing and its expression in younger age, especially in the intermediate-risk group. Its relation to other gene mutations, response to hypomethylating agents and peripheral hematopoietic stem cell transplantation should also be assessed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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