Advertisement

Invariant phenotype and molecular association of biallelic TET2 mutant myeloid neoplasia

Hassan Awada, Yasunobu Nagata, Abhinav Goyal, Mohammad F. Asad, Bhumika Patel, Cassandra M. Hirsch, Teodora Kuzmanovic, Yihong Guan, Bartlomiej P. Przychodzen, Mai Aly, Vera Adema, Wenyi Shen, Louis Williams, Aziz Nazha, Mohamed E. Abazeed, Mikkael A. Sekeres, Tomas Radivoyevitch, Torsten Haferlach, Babal K. Jha, Valeria Visconte and Jaroslaw P. Maciejewski

Key Points

  • Biallelic TET2 gene inactivation is frequently observed in myeloid neoplasia.

  • It might represent an auxiliary assessment tool to identify specific morphologic subentities of myeloid neoplasia.

Abstract

Somatic TET2 mutations (TET2MT) are frequent in myeloid neoplasia (MN), particularly chronic myelomonocytic leukemia (CMML). TET2MT includes mostly loss-of-function/hypomorphic hits. Impaired TET2 activity skews differentiation of hematopoietic stem cells toward proliferating myeloid precursors. This study was prompted by the observation of frequent biallelic TET2 gene inactivations (biTET2i) in CMML. We speculated that biTET2i might be associated with distinct clinicohematological features. We analyzed TET2MT in 1045 patients with MN. Of 82 biTET2i cases, 66 were biTET2MT, 13 were hemizygous TET2MT, and 3 were homozygous TET2MT (uniparental disomy); the remaining patients (denoted biTET2 hereafter) were either monoallelic TET2MT (n = 96) or wild-type TET2 (n = 823). Truncation mutations were found in 83% of biTET2i vs 65% of biTET2 cases (P = .02). TET2 hits were founder lesions in 72% of biTET2i vs 38% of biTET2 cases (P < .0001). In biTET2i, significantly concurrent hits included SRSF2MT (33%; P < .0001) and KRAS/NRASMT (16%; P = .03) as compared with biTET2. When the first TET2 hit was ancestral in biTET2i, the most common subsequent hits affected a second TET2MT, followed by SRSF2MT, ASXL1MT, RASMT, and DNMT3AMT. BiTET2i patients without any monocytosis showed an absence of SRSF2MT. BiTET2i patients were older and had monocytosis, CMML, normal karyotypes, and lower-risk disease compared with biTET2 patients. Hence, while a second TET2 hit occurred frequently, biTET2i did not portend faster progression but rather determined monocytic differentiation, consistent with its prevalence in CMML. Additionally, biTET2i showed lower odds of cytopenias and marrow blasts (≥5%) and higher odds of myeloid dysplasia and marrow hypercellularity. Thus, biTET2i might represent an auxiliary assessment tool in MN.

Introduction

Increasingly, genomic data are being used to classify myeloid neoplasia (MN). Examples include BCR/ABL in chronic myeloid leukemia (CML)1; t(8;21), inv(16), t(15;17), or MLL in acute myeloid leukemia (AML)2-4; PDGFRA/B translocations in chronic myelomonocytic leukemia (CMML), and, in hereditary cases, germline mutations in CEBPA,5 RUNX1,6,7 ETV6,8,9 DDX41,10 GATA2,11 TP53,12 etc. Such genetic alterations are beginning to supersede the use of morphologies in diagnoses, particularly when the pathomorphologies are less pronounced. For instance, ring sideroblasts are linked to SF3B1 mutations13,14 or refractory anemia with ring sideroblasts and thrombocytosis with a combination of SF3B1 with either CALR, JAK2, or cMPL mutations.13,15,16

Some mutations are common and thus unlikely to be molecular markers of specific morphologic subentities. Examples include mutations in TET2, ASXL1, and DNMT3A.17,18 TET2 is located on the long arm of chromosome 4 (4q24), a region susceptible to microdeletions, copy-neutral losses of heterozygosity, and rare translocations that also result in protein loss of function, producing TET2. TET2 is an Fe2+-dependent dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) to derepress silenced genes. TET2 mutations (TET2MT) impair TET2’s ability to carry out this reaction. This decreases 5hmC DNA levels, which in turn skews differentiation toward monocytic progenitors. Indeed, engineered tet2−/− mice have earlier onsets of myeloproliferative neoplasm (MPN) disease than tet2+/− mice.19,20 The role and function of TET2 has been studied in normal and malignant hematopoiesis.21-24 The contribution of TET2MT to clinicohematological features has, however, been controversial, possibly due to small-scale studies and combinatorial diversity of cooccurring lesions. Large studies accounting for clonal architecture and association between molecular and clinical features will be helpful to clarify the consequences of TET2MT on disease phenotypes.

Here, we report the clinical course of patients with biallelic TET2 inactivation (biTET2i) in the context of MN. Compared with corresponding monoallelic mutations, these events might be associated with gene-dose–dependent greater intensity phenotypes and clinical outcomes and perhaps exaggerated morphologic features associated with increased risk to leukemia progression. We have comprehensively dissected the clonal nature of TET2MT in 4930 patients with MN, of whom 40% harbored biTET2MT.25 We thus investigated possible associations between such abnormalities and clinical features and outcomes. We provide evidence supportive of the notion that biTET2i cases belong to a qualitatively distinct morphologic subentity of MN.

Material and methods

Patients

Peripheral blood and bone marrow (BM) samples from patients with MN were collected after receiving written informed consent according to the protocols approved by the Institutional Review Board of Cleveland Clinic in accordance with the Declaration of Helsinki. A total of 1045 patients were initially screened and enrolled in this study. Clinical parameters (age, sex, peripheral blood, BM counts, diagnosis, and overall survival) were obtained from medical records. Diagnosis was assigned based on the 2008 World Health Organization (WHO) classification criteria.26 Genomic and germline DNA obtained from CD3+ lymphocytes was subjected to molecular screening for the coding regions of TET2 and other gene mutations. Samples that yielded low sequencing quality due to low depth were excluded from the study. Cases in which no TET2MT was found by gene sequencing were investigated for possible deletions and microdeletions at chromosome 4q/24 by reviewing metaphase cytogenetics and results of single-nucleotide polymorphism (SNP) array, respectively. The ones with 4q deletion/microdeletion involving TET2 locus were considered monoallelic TET2MT (monoTET2MT), while those with absent aberrations were considered wild-type (TET2WT) (supplemental Figure 1A). All TET2MT were also screened for uniparental disomy (UPD) at chromosome 4q/24 by the SNP-array (SNP-A) method.

Next-generation sequencing

Whole-exome sequencing libraries were prepared according to the Nextera Rapid Capture Exome protocol (Illumina, San Diego, CA) and subjected to massive parallel sequencing using HiSeq 2000. Average coverage of samples subjected to whole-exome sequencing and targeted deep sequencing was 115× and 250×, respectively. Variants with a variant allele frequency (VAF) >5% were included. Multiamplicon targeted deep sequencing included a panel of 36 genes commonly mutated in myelodysplastic syndrome (MDS)18,27,28 and other myeloid malignancies (supplemental Table 1). Paired-end libraries were subjected to deep sequencing on MiSeq sequencers according to Illumina protocols. Variants were extracted using the GATK3.3 pipeline. TET2MT were called somatic when absent or at very low frequencies in germline CD3+ lymphocytes. Alterations found in both the myeloid and lymphoid cells with an equal VAF were considered germline and excluded from the study. Previously, usage of T cells as germline29,30 resulted in similar frequencies of TET2MT compared with skin or buccal swab specimens.18,31 For original data, please contact H.A. (awadah{at}ccf.org).

SNP-A–based karyotyping

SNP-A karyotyping for confirming metaphase cytogenetics and detecting copy-number–neutral loss of heterozygosity was performed as previously described.32,33 Briefly, Affymetrix 250K and 6.0 SNP-As were used to evaluate cop-number alterations and copy-number natural loss of heterozygosity. Using our internal database and a publicly available database (http://dgv.tcag.ca/dgv/app/home), the screening algorithm validated each lesion as somatic. Nonsomatic lesions were excluded from further analysis. Affected genomic positions in each lesion were visualized and extracted using CNAG (v3.0) or Genotyping Console (Affymetrix, Santa Clara, CA).34,35 Metaphase cytogenetic requires cellular proliferation, and its sensitivity and resolution depend on the proportion of clonal cells in the sample and size of the lesion, respectively. SNP-A does not depend on the presence of dividing cells and is able to detect copy-number variations and UPD with a high resolution. For this purpose, we included this method to further investigate for 4q/24 cryptic chromosomal lesions not identified by metaphase cytogenetic in our cohort.36

Conventional cytogenetics

Metaphase cytogenetics was performed on BM aspirates. The median number of metaphases analyzed was 20. Chromosomal preparation was performed on G-banded metaphase cells using standard techniques, and karyotypes were described in 86% (862/1001) of patients according to the International System for Human Cytogenetic Nomenclature.37

Ancestral/dominant-codominant and secondary mutation estimation

VAFs were used to categorize first-hit TET2MT into ancestral (dominant or codominant) mutations vs subclonal secondary mutations. A mutation with the highest VAF that is at least 5% more than the second highest VAF in each sample was defined as an ancestral/dominant mutation; those with <5% difference from the highest VAF were defined as ancestral/codominant, while VAFs with a >5% difference from the highest VAF were considered subclonal/secondary mutations (supplemental Figure 2A-F). Mutations in selected genes (commonly mutated in myeloid neoplasms) were assessed for differences in biTET2i vs biTET2- cases.

Statistical analyses

Fisher’s exact test was used to compare proportions. All P values were 2 sided; those <.05 were considered statistically significant. Kaplan-Meier methods were used to plot survival probabilities, and log-rank tests were used to compare such curves. Univariate and multivariate Cox model analyses were also performed. All statistical computations were performed using R 3.5.1 (www.r-project.org).

Results

Identification of biTET2i in myeloid neoplasms

We analyzed configurations of TET2MT using VAFs (see Materials and methods) in 1045 patients with MN. Patients with TET2MT (n = 200) were classified into heterozygous biallelic TET2MT (biTET2MT; ≥2 TET2MT with VAF sum of >55%; n = 66), biclonal TET2MT (≥2 TET2MT with VAF sum of <45%; n = 11), undetermined (either biallelic or biclonal TET2MT, as their VAF sum lays between 45% and 55%; n = 33), hemizygous TET2MT (single TET2MT with VAF >55% in the presence TET2 locus alteration on chromosome 4q24; n = 13), homozygous TET2MT (homozygous mutation at 4q24 with UPD detected by SNP analysis; n = 3), and monoTET2MT (single TET2MT with VAF <45% and normal cytogenetics; n = 74; Figure 1A). Biclonal TET2MT and undetermined cases were ambivalent and thus filtered out of the study (n = 44), resulting in a cohort of 1001 MN patients. BiTET2MT, hemizygous TET2MT, and homozygous TET2MT have all inactivation (impairment) of both parental copies of TET2 and therefore were grouped in as biTET2i cases (n = 82) (Figure 1D). The remainder of the population, with either monoTET2MT (n = 96; 74 cases with single TET2MT and normal metaphase cytogenetic/SNP-A screening; 22 cases with 4q/TET2 locus deletion in absence of TET2MT) or wild-type configuration (TET2WT, n = 823; normal metaphase cytogenetic/SNP-A screening and absent TET2MT), were considered negative for biTET2i (biTET2; n = 919). Patients were also divided into those with CMML and without CMML (−) according to the presence of WHO-defined CMML hallmark clinical features.1 The CMML (−) cohort (n = 885) was then subgrouped according to the presence of monocytosis. Monocytosis was present in 56% (n = 497) of these cases. TET2MT configuration and respective number of patients is summarized in (supplemental Figure 1A).

Figure 1.

TET2 gene mutation classification, type, and clinical characteristics. (A) Scatterplot of the VAFs of patients with TET2MT. The VAF of first-hit TET2MT was plotted on the x-axis and that of the second-hit TET2MT, if present, on the y-axis. Patients were categorized into 5 groups as explained in the text. The red oval corresponds to biTET2MT cases, the gray bar to undetermined cases, the blue oval to biclonal TET2MT cases, the light green oval to monoTET2MT cases, and the yellow oval to hemihomozygous TET2MT (UPD) cases. (B) Percentages of different types of TET2MT in biTET2i cases and significance of truncating mutations vs monoTET2MT. Fisher’s exact test was used for analysis. (C) Bar graphs showing the distribution of biTET2i and monoTET2MT cases per diagnosis and cytogenetics. The bar columns indicate percentages. (D) Pie chart showing the percentage of cases per configuration. (E) Pie charts of biTET2i, monoTET2MT, and TET2WT respectively representing the percentage of cases per classification. CMML (+) indicates cases with CMML at the time of presentation, CMML (−) indicates no CMML diagnosis, monocytosis (+) indicates the presence of monocytosis, and monocytosis (−) indicates the absence of monocytosis.

Somatic TET2MT was found in 156 out of 1001 of cases (16%), of which 53% were biTET2i. A total of 83% of biTET2i cases were truncating (frameshift deletion/insertion and nonsense), while 27% of somatic alterations were missense (Figure 1B). A comparison of the VAF ratio of the first and second TET2MT per type of mutation and succession in biTET2MT is presented in supplemental Figure 1D. The majority of monoTET2MT patients (77%) harbored a TET2MT with a VAF <45%; the minority had 4q/24 aberrations (locus deletion) (23%) (supplemental Figure 1B). MonoTET2MT cases included 65% truncating and 35% missense mutations (supplemental Figure 1C). Truncating mutations were significantly more common in biTET2i cases than monoTET2MT cases (odds ratio [OR], 2.7; P = .02; Figure 1B).

Clinical phenotypes of cases with biTET2i

Clinical analysis revealed that biTET2i, in comparison with biTET2, was associated with older age (91% ≥60 years vs 74%, P = .0004; Table 1). Among biTET2i cases, 27% were classified as MDS, 50% as MDS/MPN, and 23% as AML (10% pAML and 13% sAML). BiTET2i was enriched in patients with CMML1/2 (44%; P < .0001), predominantly in lower-risk cases (62% vs 47% in biTET2; P = .003) and more commonly had normal metaphase cytogenetics (65%; P = .0007; Figure 1C). We also assessed phenotype/genotype association of biTET2i (Table 2). In biTET2 cases, leukopenia (81%; P < .0001), neutropenia (52%; P = .008), pancytopenia (27%; P = .008), and increased marrow blast percentages (blasts ≥5% in 33%; P = .01) were more prevalent than in biTET2i cases, which in return cosegregated with monocytosis (84%; P < .0001; Table 2; Figure 1E), marrow hypercellularity (cellularity ≥70% in 67%; P < .0001), and more pronounced myeloid dysplasia (68%; P = .0003).

Table 1.

Comparison of baseline characteristics in biTET2i vs biTET2 cases

Table 2.

Comparison of clinical characteristics in biTET2i vs biTET2 cases

We next compared biTET2i cases to those with monoTET2MT to evaluate the consequence of a second TET2 inactivation on disease features (supplemental Tables 2 and 3). Biallelic inactivation of TET2 was more likely to occur with MDS/MPN (P = .001), particularly the CMML1/2 subtype (P < .0001; Figure 1C,E), and with normal cytogenetics (P = .003). In addition, it was correlated with a lower odds of leukopenia (P = .002) and ring sideroblasts (≥15%; P = .02) and a higher likelihood of monocytosis (P = .003) and marrow hypercellularity (P = .02) (Figure 1E).

Because we observed a highly significant (P < .0001) relationship between biTET2i and CMML diagnosis and/or monocytosis, we focused on patients without obvious CMML (monocytosis; absence of BCR/ABL1, PDGFRA/B, or 11q23; presence of <20% blasts; and myeloid dysplasia) and compared biTET2i and biTET2 for the association with monocytosis and myeloid dysplasia (supplemental Figure 1E). Increased monocyte counts among CMML (−) was also significantly overrepresented in biTET2i cases (72%; P = .03) compared with biTET2 cases (55%), as was myeloid dysplasia (72% vs 46%; P = .0001).

Clonal substructure of cases with biTET2i

The rank of TET2MT within the clonal hierarchy can be determined according to VAF methodology (see Materials and methods). We first defined each TET2MT as ancestral vs secondary and then identified other hits in relation to TET2 status. Due to resolution limits of the VAF approach and the difficulty of distinguishing subclonal from ancestral hits, we applied an arbitrary cutoff of 5% between VAFs to discriminate ancestral first hits (dominant and codominant) from subsequent “secondary” hits (supplemental Figure 2A-F). A summary of clonal hierarchy of somatic mutations, cytogenetic findings, and diagnoses in all biTET2i cases is presented in Figure 2A. Seventy-two percent of first TET2 hits in biTET2i were founder (dominant/codominant) lesions (P < .0001), while only 28% were secondary to another antecedent somatic mutations (Figure 2B). In monoTET2MT cases, only 38% TET2 hits were dominant. In biTET2i, when the first TET2MT was subclonal, the preceding founder clone was most likely characterized by BCOR/BCORL1MT, PRC2-familyMT, ZRSR2MT, ASXL1MT, and others (Figure 2C). When the first TET2 hit was ancestral, the most common secondary mutation affected TET2, followed in frequency by SRSF2MT, ASXL1MT, RASMT, and DNMT3AMT (Figure 2D-E).

Figure 2.

Clonal architecture and hierarchy of TET2MTin biTET2i. (A) Plot showing dominant, codominant, and secondary mutations in the 82 biTET2i patients. Mutated gene names, cytogenetics, and diagnosis are color coded as indicated. For this presentation, only genes mutated ≥3 times among the biTET2i population are represented. Each column represents 1 patient, and each row corresponds to 1 gene or family of genes. (B) Pie chart displaying the percentage of first-hit TET2 occurring as dominant (ancestral), codominant (ancestral), and subclonal (secondary) in the biTET2i population. (C-E) The bar graphs show the percentages of the corresponding dominant genes to the secondary/subclonal first-hit TET2 gene (C), secondary clones to the dominant first-hit TET2 gene (D), and codominant genes to the codominant first-hit TET2 gene (E). (F) Frequency (in percentage) of mutations in selected genes in the population. Ten genes that are frequently mutated in myeloid neoplasms were selected. Columns are color coded per TET2MT configuration (TET2WT, monoTET2MT, and biTET2i).

When we investigated associations between concurrent mutations and TET2MT configuration, ASXL1MT (25%), TP53MT (16%), and CBLMT (7%) were more frequent in monoTET2MT cases; SRSF2MT (33%), KRAS/NRASMT (16%), RUNX1MT (16%), and ZRSR2MT (6%) were more frequent in biTET2i cases; and DNMT3AMT (13%) and U2AF1MT (9%) were more common in the TET2WT population (Figure 2F). When compared with patients with and without biTET2i (Figure 3A), a significant co-occurrence with SRSF2MT (P < .0001) and KRAS/NRASMT (P = .03) in biTET2i and TP53MT (P = .03) in biTET2 was noted. SRSF2MT was also found to be significantly associated with biTET2i when compared with monoTET2MT (P = .02) (supplemental Figure 3). In contrast, in biTET2i without monocytosis (16%; n = 13), SRSF2MT was absent and KRAS/NRASMT was only detected in 8% (n = 1) of the cases.

Figure 3.

Significance of concurrent gene mutations in biTET2iand correlation per disease subtype. (A) Forest plot showing the OR of associated gene mutations in biTET2i vs biTET2 cases. As indicated, red squares correspond to significant cases, while red stars correspond to highly significant cases. Fisher’s exact test was used to test significance. (B) Frequency (in percentage) of mutations in selected genes per disease subtype in biTET2i vs biTET2. Significance was tested via Fisher’s exact test.

In CMML, among biTET2i cases, SRSF2MT was the most commonly found co-occurring lesion (53%; P = .005), followed by KRAS/NRASMT (28%), ASXL1MT (28%), and RUNX1MT (22%). Investigation for the incidence of a secondary/subclonal ASXL1MT among CMML with preexisting biTET2i (20%) vs CMML with monoTET2MT (72%) tended to be significant (P = .05). In biTET2 cases, ASXL1MT (31%), SRSF2MT (25%), KRAS/NRASMT (23%), and RUNX1MT (18%) were seen in high frequencies. In MDS/MPN (excluding CMML), SRSF2MT was present in 40% and TP53MT in 20% of biTET2i cases, respectively, while DNMT3AMT occurred in 14% and ASXL1MT in 11% of biTET2 cases. Similar to what we observed in CMML, patients with MDS/MPN carrying biTET2i showed a striking concordance with SRSF2MT (P = .05). In MDS/sAML, ASXL1MT (24%) and SRSF2MT (12%) were most commonly found together with biTET2i, while ASXL1MT (13%), DNMT3AMT (12%), and TP53MT (12%) clustered with biTET2 cases. Finally, in pAML, DNMT3AMT (63%), SRSF2MT (25%), and RUNX1MT (25%) were the most frequent molecular events in biTET2i cases, while DNMT3AMT (22%) and KRAS/NRASMT (18%) were seen in biTET2 cases (Figure 3B).

We then grouped biTET2i cases into those with and without diagnosis of CMML (supplemental Figure 4A) and found that biTET2i cases were strongly associated with SRSF2MT (P = .0009) and KRAS/NRASMT (P = .01) in CMML. Among biTET2i cases with SRSF2MT and those with KRAS/NRASMT, CMML was diagnosed in 70% (P = .001) and 77% (P = .01), respectively (supplemental Figure 4B), which is higher than what was seen in the biTET2i population (44%; Figure 1E). We then investigated the overall impact of biTET2i by comparing the effect of biTET2i vs biTET2 in relation to CMML diagnosis, the presence and absence of monocytosis, and the concomitant presence of SRSF2MT or KRAS/NRASMT. No differences in survival outcomes could be attributed to the presence of biTET2i as a sole factor or in combination with others (supplemental Figure 5A-J).

Independent features associated with biTET2i

When univariate analyses were conducted in the biTET2i vs TET2WT population (Figure 4A), biTET2i was associated with older age, lower risk, MDS/MPN, CMML, normal cytogenetics, monocytosis, marrow hypercellularity, myeloid dysplasia, and the presence of SRSF2MT and NRAS/KRASMT. In contrast, TET2WT status correlated with MDS, leukopenia, neutropenia, pancytopenia, elevated BM blasts, and TP53MT. We then conducted a multivariate Cox regression analysis (Table 3). For biTET2i vs TET2WT, older age (≥60 years; OR, 4.2; P = .002), CMML (OR, 3.4; P = .03), monocytosis (OR, 2.1; P = .05), myeloid dysplasia (OR, 1.8; P = .04), marrow hypercellularity (OR, 2.4; P = .005), and SRSF2MT (OR, 2.2; P = .02) were independent features associated with biTET2i. In biTET2i vs monoTET2MT, CMML (OR, 6.7; P = .02), truncating TET2MT (OR, 3.5; P = .02), and ancestral TET2MT (OR, 5.5; P = .0002) were independently associated with biTET2i. When we compared the cohort of biTET2i vs biTET2, CMML (OR, 6.9; P = .02), truncating TET2MT (OR, 3.5; P = .02) and ancestral TET2MT (OR, 5.5; P = .0002) were found to be independent prognostic factors in biTET2i, while elevated marrow blasts (OR, 0.2; P = .02) were more common in biTET2. Finally, for monoTET2MT vs TET2WT, elderly (age ≥60 years; OR, 3.3; P = .002), TP53MT (OR, 2.5; P = .01), and SRSF2MT (OR, 2.1; P = .03) were found to be distinct for monoTET2MT.

Figure 4.

Univariate analysis for baseline, clinical, and genomic features in biTET2ivs wild-type. Univariate analysis showing the significant results for baseline (older age, lower risk, MDS, MDS/MPN, CMML, and normal cytogenetics), clinical (leukopenia, neutropenia, monocytosis, pancytopenia, BM blasts, BM hypercellularity, and myeloid dysplasia), and genomic (KRAS/NRASMT, SRSF2MT, and TP53MT) features in biTET2i vs TET2WT.

Table 3.

Multivariate analysis showing significant independent results in biTET2i vs TET2WT, biTET2i vs monoTET2MT, biTET2i vs biTET2, and monoTET2MT vs TET2WT

Discussion

Objective molecular tools are complementing morphological methods in clinical practice. Morphology remains, however, the golden standard for identifying strong associations between phenotype and genotype in MN. We report here on phenotypical and morphological characteristics of cases harboring biallelic TET2 defects.

To date, only a few studies have investigated the clinical consequences of biTET2i in MN. We used a well-characterized cohort of patients with biTET2i. Our hypothesis was that biTET2i associates with a group of pathomorphological features that independently define a distinct MN subtype. To test our idea, we first identified biTET2i cases among a cohort of 1045 patients with MN and then studied correlations between mutational configuration and clinicohematological morphology in comparison with biTET2 cases.

While our finding that most TET2MT cases are truncating (frameshift or nonsense changes) agrees with previous studies,38-40 we further show that a second TET2 hit in biTET2MT cases significantly increases the chances of accumulating more truncating changes in those already harboring a TET2MT. The prevalence of biTET2i among older patients demonstrated in this investigation is also in agreement with patterns observed in other studies.25 This can be explained by the effect of aging on the accumulation of TET2MT in clonal hematopoiesis of indeterminate potential and subsequent subclonal hits over time.41,42

TET2MT is detected in a large fraction of myeloid disorders38,43 but predominantly in CMML.44 Here, we show that the MDS/MPN CMML subtype significantly correlated with biTET2i events (Figures 1C,E and 4). This relationship is modified by mutations in additional genes, such as SRSF2MT in cases of CMML,44,45 that are found to correlate strongly with biTET2i even when CMML is absent. This observation was further demonstrated by comparing biTET2i with monoTET2MT, which showed a significant accumulation of MDS/MPN, CMML subtype, monocytosis, and SRSF2MT solely resulting from the clonal succession following the second TET2 hit. When criteria for CMML diagnosis46 were not clinically fulfilled, biTET2i remained invariably associated with monocytosis and myeloid dysplasia, both hallmarks of CMML proliferative and dysplastic features.43,47

Along with studies suggesting that TET2MT has a neutral impact on the rate of progression to AML,25,48 we also identified that biTET2i tended to occur in lower-risk patients with a lower likelihood of leukemic transformation, unless additional deleterious events co-occur. Given that most TET2 hits were ancestral to subsequent secondary mutations (second TET2MT, SRSF2MT, ASXL1MT, RASMT, and DNMT3AMT), we concluded that these founder lesions represent a leukemogenic predisposition (mutator phenotype) rather than driving leukemia. BiTET2i correlated with normal karyotype, as did the second TET2 hit in those harboring TET2MT, implying a significantly lower likelihood of cytogenetic abnormalities and an association with lower-risk disease.

Other notable features of biTET2i included, in addition to monocytosis and SRSF2MT, significantly higher leukocyte and neutrophil counts and less pancytopenia. When reviewing BM morphology, in addition to myeloid dysplasia, we observed low percentages of marrow blasts despite prominent hypercellularity. Moreover, lower odds of leukopenia and ring sideroblasts and an increased marrow cellularity were strongly correlated with a second hit in TET2 in comparisons of biTET2i and monoTET2MT.

Inactivating TET2MT leads to low 5hmC levels. 5hmC is the first oxidative product of the TET demethylation pathway marking tissue and cell-specific genes. A decrease in 5hmC levels is associated with malignant phenotypes and poor survival outcomes.49 5hmC is highly localized at binding sites of the p300/CREB binding protein, and in vitro studies have shown that upon TET2 acetylation, 5hmC levels increase significantly. As previously reported, TET2MT displayed low levels of 5hmC showing hypomethylation compared with healthy subjects at the majority of differentially methylated CpG sites. The greater degree of deficiency by the impairment of both TET2 alleles, the more the hydroxylation function is affected.22 Consequently, more methylation of CpG sites would be expected. As a result, the previously described expansion of the stem cell compartment and differentiation that skewed toward monocytic differentiation should be expected to be more pronounced. In our study, biTET2i did not lead to a strong deleterious phenotype, suggesting that the acetylation mechanism might have been stabilized TET2 protein.

Given a more pronounced phenotype and genotype, it is important to speculate why biTET2i does not result in more serious clinical consequences. It is possible that the impairment of TET2 via inactivating mutations might be compensated by other TET enzymes (eg, TET1) or by decreased posttranslational modification (eg, acetylation) leading to a higher fraction of catalytically active protein. TET2 activity is regulated by acetylation, which increases TET2 stability (by protecting TET2 from ubiquitination and proteasome degradation) and promotes the cooperation with DNA methyltransferase 1 (DNMT1).44 Acetylation might represent a regulatory mechanism of TET2 protein. TET2 is acetylated by p300/CREB binding protein at key lysine residues (K110 and K111) located in the N terminus of the protein, and this acetylation can be switched by HDAC1/2 and SIRT1/2 deacetylases. The N terminus of TET2 seems to contain high levels of enzymatic activity and positive regulatory feedback, possibly because the acetylation of lysine residues is a natural mechanism increasing TET2 activity.50

In conclusion, our collective observations demonstrate that biTET2i is a frequent event in MN. Furthermore, the presence of biTET2i contributes additional information to the genetic complexity of MN and thus might represent a putative assessment tool of certain morphologic MN subentities associated with invariant phenotypic and molecular characteristics. The increasing collection of omics data and their correlation with clinical factors will further shed the light on the nature of biTET2i in MN.

Acknowledgments

This work was supported by the National Institutes of Health, National Heart, Lung, and Blood Institute (grants R01HL118281, R01HL123904, R01HL132071, and R35HL135795) and the Edward P. Evans Foundation (J.P.M.).

Authorship

Contribution: H.A. designed the study, collected, analyzed, and interpreted clinical and molecular data, and wrote the manuscript; Y.N. collected data and provided important feedback to the manuscript; A.G., M.F.A., B.P., and Y.G. collected and analyzed data; C.M.H. and B.P.P., performed and analyzed DNA sequencing data and edited the manuscript; T.K., M.A., V.A., W.S., and L.W. collected clinical data and edited the manuscript; A.N., M.E.A., M.A.S., T.H., and B.K.J. provided clinical specimens and important insights on the manuscript; T.R. provided statistical advice and important editing in the manuscript; V.V. provided important editing and wrote the manuscript; J.P.M. designed the study, conceptualized and sponsored the overall research, and wrote the manuscript; and all authors read and approved the final manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Jaroslaw P. Maciejewski, Department of Translational Hematology and Oncology Research, Lerner Research Institute, 9620 Carnegie Ave, N Building, NE6-250, Cleveland, OH 44106; e-mail: maciejj{at}ccf.org.

Footnotes

  • The full-text version of this article contains a data supplement.

  • Submitted August 1, 2018.
  • Accepted December 12, 2018.

References

  1. 1.
  2. 2.
  3. 3.
  4. 4.
  5. 5.
  6. 6.
  7. 7.
  8. 8.
  9. 9.
  10. 10.
  11. 11.
  12. 12.
  13. 13.
  14. 14.
  15. 15.
  16. 16.
  17. 17.
  18. 18.
  19. 19.
  20. 20.
  21. 21.
  22. 22.
  23. 23.
  24. 24.
  25. 25.
  26. 26.
  27. 27.
  28. 28.
  29. 29.
  30. 30.
  31. 31.
  32. 32.
  33. 33.
  34. 34.
  35. 35.
  36. 36.
  37. 37.
  38. 38.
  39. 39.
  40. 40.
  41. 41.
  42. 42.
  43. 43.
  44. 44.
  45. 45.
  46. 46.
  47. 47.
  48. 48.
  49. 49.
  50. 50.
View Abstract