TET2 Gene Mutations Research Articles

Relationship between DTA Mutations and Thromboembolism in Patients with Myeloproliferative Neoplasms

To analyze the DTA (DNMT3A, TET2, ASXL1) mutations in patients with myeloproliferative neoplasms (MPN), and preliminarily explore their correlation with thromboembolism. Clinical characteristics of 62 patients diagnosed de novo MPN at Central Hospital Affiliated to Shandong First Medical University from September 2016 to September 2022 were retrospectively analyzed. Next-generation sequencing was used to detect 35 MPN-related genes, and the DTA mutations in MPN patients and their relationship with thromboembolic events were analyzed. 75.8% (47/62) of the patients presented pathogenic non-driver mutations, and the mean number of pathogenic non-driver mutations per patient was 1.08. Among them, the most frequently mutated non-driver genes were TET2 (38.7%, 24/62), DNMT3A (9.7%, 6/62) and ASXL1 (6.5%, 4/62). The presence of DTA gene mutations was 50% (31/62) in the total MPN patients, and mainly accompanied by driver mutations. The mutation rate of DTA in patients aged ≥60 years was significantly higher than that in patients <60 years old (P =0.039). The incidence of thromboembolism in patients with DTA mutation was 58.1% (18/31), which was significantly higher than that in patients without DTA mutation (19.4%, 6/31) (P =0.002). The TET2 gene mutation rate in MPN patients with thromboembolism was 66.7% (16/24), which was significantly higher than that in patients without thromboembolism (21.1%, 8/38) (P =0.00). Patients with MPN have a higher incidence of DTA mutations, which are mainly accompanied by driver gene mutations. The incidence of thromboembolism in MPN patients with DTA mutations is higher than that in patients without DTA mutations. Especially, the elderly (≥60 years) essential thrombocythemia(ET) and polycythemia vera(PV) patients with TET2 mutation should be vigilant for thromboembolic events.

Mutation characteristics of angioimmunoblastic T-cell lymphoma: an analysis of 75 cases

Objective: To investigate the characteristics of gene mutations in angioimmunoblastic T-cell lymphoma (AITL). Methods: Seventy-five AITL cases diagnosed at the Department of Pathology, Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China from June 2021 to June 2023 were included. Their formalin-fixed and paraffin-embedded or fresh tissues were subject to targeted next generation sequencing (NGS). The sequencing data was collected, and the distribution and type of gene mutations were analyzed. Results: 492 potential driver mutations were identified in 74 out of the 84 genes. Targeted sequencing data for the 75 AITL patients showed that the genes with mutation frequencies of ≥10% were TET2 (89.3%), RHOA (57.3%), IDH2 (37.3%), DNMT3A (36.0%), KMT2C (21.3%), PLCG1 (12.0%), and KDM6B (10.7%). There were significant co-occurrence relationships between TET2 and RHOA, TET2 and IDH2, and RHOA and IDH2 gene mutations (P<0.05), respectively, while TET2 and KDM6B gene mutations were mutually exclusive (P<0.05). Conclusions: The study reveals the mutational characteristics of AITL patients using NGS technology, which would provide insights for molecular diagnosis and targeted therapy of AITL.

Identification Of Tet2 Gene Level Of Expression In Acute Leukemias

Objective: TET2 gene (4q24) is one of the most important regulatory factors of the methylation process in human genome. The homologous protein is directly involved in convertion of 5-methylcytosine into 5-hydroximethylcytosine (1). Loss of function mutations in TET2 gene are considered to be driver mutations (initiating mutations) for leukemogenesis. In some cases they can be found in early clonal cells of Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) or myelodisplastic syndromes (MDS) (2). The aim of this study is to determinate the frequency of TET2 mutations in Acute Leukemia (AL) patients. Materials and methods: This retrospective study enrolled 24 patients newly diagnosed with AML (19 patients) and ALL (5 pacients). By using Real-Time PCR we obtained a relative cuantification of TET2 gene level of expression taking ABL gene as reference. Outcomes: Low levels of TET2 gene expression were found in 6 patients (25%), 3 of them being diagnosed with AML and 3 with ALL. No cytogenetic anomalies were noticed. However, all these patients had blastocytosis and higher white blood cells count than pacients with normal levels of expression. There was one case with mutations in both TET2 gene and NPM1 gene and one case that associated a defective TET2 gene and an internal tandem duplication of FLT3-ITD. Conclusions: In our study, TET2 gene loss of function mutations occured on a specific background which involved some characteristic mutations for AL and some particular features of cell morphology. We aim to continue this study by investigating the long term effects of this epigenetic mutation in AL patients.

Genetic landscape of acute myeloid leukemias with leukocytosis

Background. Tumor cell proliferation in acute myeloid leukemia (AML) may manifest with high leukocyte counts. In our work, we evaluate the association of high leukocyte counts with individual mutations, as well as their total contribution to the development of leukocytosis in AML. The results obtained should improve our understanding of pathogenic mechanisms leading to the leukocytosis in AML.Aim. To study the genetic landscape of AML with leukocytosis.Materials and methods. The laboratory data of 214 AML patients admitted to the National Medical Research Center for Hematology (Moscow) from 2010 to 2022 were retrospectively examined. Real-time PCR, capillary electrophoresis and NGS (next generation sequencing) methods were used to detect mutations of FLT3, NPM1, CEBPA, IDH1/2, DNMT3A, TET2 genes, and CBFB::MYH11, RUNX1::RUNX1T1 chimeric gene transcripts.Results. Mutations of the FLT3 gene (odds ratio 5.45; p &lt; 0.0001), inv(16)/CBFB::MYH11 (odds ratio 10.03; p = 0.0009) are most associated with leukocyte counts higher than 30 × 109 / L in the debut of AML. Translocation t(8;21)/RUNX1::RUNX1T1 and adverse cytogenetic aberrations, such as -5/del(5q); -7 / del(7q); -17 / abn(17p), complex and monosomic karyotype were significantly associated with leukocyte counts lower than 30 × 109 / L at the time of disease manifestation (p &lt; 0.0001). In the group of patients with intermediate cytogenetic risk bearing only IDH1/2, DNMT3A, and TET2 gene mutations, leukocyte counts at AML debut were significantly lower, whereas the most pronounced leukocytosis was observed in patients with a combination of driver mutations with IDH1/2, DNMT3A, and TET2 gene mutations or FLT3, NPM1, and CEBPA gene mutations.Conclusion. In addition to the individual effect of certain genetic lesions and cytogenetic aberrations on the proliferative potential of tumor cells, there is a total contribution of various types of genetic events to the development of leukocytosis in AML. High leukocyte counts at the time of AML manifestation in patients with intermediate cytogenetic risk can serve as an indirect marker of the presence of a large number of genetic aberrations with a combination of IDH1/2, DNMT3A, and TET2 gene mutations or FLT3, NPM1, and CEBPA gene mutations.

Clinical Analysis of Patients with Blastic Plasmacytoid Dendritic Cell Neoplasm

To explore the clinical characteristics, treatment, and prognosis of patients with blastic plasmacytoid dendritic cell neoplasm(BPDCN). Clinical data of 5 patients diagnosed with BPDCN in Wuhan First Hospital and Wuhan Tongji Hospital from June 2016 to November 2021 were retrospectively analyzed. Among the 5 patients, 3 were male and 2 were female, with a median age of 28(10-52) years old. Four patients showed obvious skin damage at the initial diagnosis; the other one showed clinical manifestations of acute leukemia rather than obvious skin damage at the initial diagnosis, but infiltrated skin when the disease relapsed after treatment. Other infiltration sites of lesions included bone marrow (2/5), peripheral blood (2/5), lymph nodes (3/5), liver and spleen (2/5). All patients had no clinical manifestation of central nervous system infiltration. Tumor cell specific immune markers CD4, CD56, CD123 were all positive, and the median Ki-67 index was 70%. TET2, ASXL1 and NRAS gene mutations were found respectively in 3 patients by next-generation sequencing technique (NGS). ALL-like, AML-like and invasive NK/T cell lymphoma-like first-line induction chemotherapy regimens were used for the patients. One patient died of severe complications during the early stage of chemotherapy, 3 patients were evaluated as CR, and 1 patient was evaluated as PR. 2 patients were recurred and progressed after induction of chemotherapy, and one of them was evaluated as CR after re-treatment. One patient received autologous hematopoietic stem cell transplantation (auto-HSCT) and got long-term survival (OS 87 months). 3 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT), of which one died of transplantation related complications, and 2 cases survived. The median follow-up time of 4 patients with evaluable efficacy was 28.5(9-84) months, the median OS time was 31.5(10-87) months. BPDCN is a highly heterogeneous malignant tumor with a poor prognosis. HSCT, especially allo-HSCT can significantly improve the prognosis of BPDCN patients.

Molecular Genetics of Thrombotic Myeloproliferative Neoplasms: Implications in Precision Oncology.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include polycythaemia vera, essential thrombocythaemia, and primary myelofibrosis. Unlike monogenic disorders, a more complicated series of genetic mutations are believed to be responsible for MPN with various degrees of thromboembolic and bleeding complications. Thrombosis is one of the early manifestations in patients with MPN. To date, the driver genes responsible for MPN include JAK2, CALR, MPL, TET2, ASXL1, and MTHFR. Affords have been done to elucidate these mutations and the incidence of thromboembolic events. Several lines of evidence indicate that mutations in JAK2, MPL, TET2 and ASXL1 gene and polymorphisms in several clotting factors (GPIa, GPIIa, and GPIIIa) are associated with the occurrence and prevalence of thrombosis in MPN patients. Some polymorphisms within XRCC1, FBG, F2, F5, F7, F12, MMP9, HPA5, MTHFR, SDF-1, FAS, FASL, TERT, ACE, and TLR4 genes may also play a role in MPN manifestation. This review aims to provide an insightful overview on the genetic perspective of thrombotic complications in patients with MPN.

Gene Mutation and Clinical Characteristics of Patients with Acute Leukemia

To investigate the characteristics of gene mutation, clinical characteristics and significance in acute leukemia (AL) patients. The clinical data of 102 AL patients in Hebei General Hospital from September 2016 to September 2020 were collected and analyzed retrospectively, including the characteristics of gene mutation, age, peripheral blood cells, bone marrow blasts, leukemia subtypes and myeloperoxidase (MPO). The total gene mutation rate was 87.25% (89/102) in all 102 patients. A total of 275 gene mutations were detected, with an average of 2.70 gene mutations per patient. The most frequent mutations of 102 patients were as follows: CEBPA (6.91%), NPM1 and ASXL1(6.18%), TET2 (5.82%), DNMT3A (5.45%), IDH2 and FLT3-ITD (5.09%). Gene mutations often occurred simultaneously. CEBPA mutation occurred in 10 cases of M2 subtype, while TET2 mutation occurred in 9 cases of M2 subtype. Among the most common gene mutations in MPO low expression group, mutation rates of NPM1, DNMT3A, IDH2, SF related gene mutation and RUNX1 were significantly different than those in MPO high expression group (all P<0.05). Univariate analysis showed that age, NPM1, DNMT3A and FLT3-ITD had significant effects on leukocyte level. Logistic regression analysis showed that patients with positive NPM1 mutations may had higher leukocyte levels (p=0.038), and those with positive DNMT3A mutations may had higher platelet levels (p=0.042). The incidence of gene mutation in patients with AL is high, and it often occurs simultaneously. CEBPA and TET2 gene mutations are more common in M2 subtype. In patients with MPO low expression, the most common gene mutations are NPM1, DNMT3A and IDH2. AL patients with NPM1 gene mutation had higher white blood cell levels, while with DNMT3A gene mutation had higher platelet levels.

MPN-225 Next Generation Sequencing (NGS) in the Diagnosis, Prognosis and Features of the Disease in Ph-Negative Myeloproliferative Neoplasms.

Over the past decade NGS has expanded our understanding of the genomic landscape of hematological malignancies. New biomarkers discovered during NGS are now widely used in clinical practice to improve diagnosis, stratify patients into prognostic groups, and select targeted therapy for Ph-MPN patients. To assess possibilities of NGS in diagnosis and determination of prognostic features of disease course in Ph-MPN patients. The study included 36 patients from clinics in Moscow and St. Petersburg (17 men and 19 women), the median age was 55 (IQR 27-79) with diagnosed PV, ET, PMF, MDS, AML. A panel of 116 genes with an average reading depth of 200x or 1000x on MiSeq (Illumina) was used. NGS panel had a detection limit of 3% variant allele frequency. The clinical significance of mutations was determined using COSMIC and ClinVar. Genetic abnormalities were detected in all patients. On average, 10 mutations per one patient were revealed, including 4 somatic mutations in 92% (33/36) patients. In 70% (25/36), from 1 to 3 mutations with known clinical significance were found. In all patients without driver and somatic mutations (ASXL1, EZH2, etc.) and chromosomal aberrations, somatic mutations (both clinically significant and unknown clinical significance) were detected, that served as clonality confirmation. All patients were divided into two groups: 1) driver mutations, normal karyotype (16/36) (favorable prognosis) and 2) triple-negative mutational status (20/36) with additional patients having mutations in epigenetic regulators genes (4/36) (unfavorable prognosis). In the first group, NGS revealed additional mutations in 8/16 (50%) patients (ASXL1, EZH2, IDH1) and in 3/16 (19%) were noted mutations in TET2 gene. In an unfavorable group, additional mutations were also found in genes of epigenetic regulation (ASXL1, IDH1, SRSF2) and in signal transduction genes (RAS genes, PTPN11) and TET2. NGS data can confirm disease clonal nature, assess disease prognosis, and select target therapy for patients with Ph-negative myeloproliferative neoplasms. To get new information it is possible to expand the panel of sequenced genes, as well as study other genetic mechanisms underlying disease pathogenesis (e.g., changes in gene expression level, microRNAs, or transcription factors).

Prognostic factors of autologous hematopoietic stem cell transplantation in intermediate-risk acute myeloid leukemia patients with minimal residual disease negativity

To explore prognostic factors in intermediate-risk acute myeloid leukemia (AML) patients with minimal residual disease (MRD) negativity (MRD<0.1%,MRD-)receiving autologous hematopoietic stem cell transplantation (auto-HSCT).A total of 59 intermediate-risk AML patients with MRD-were treated with auto-HSCT from January 2015 to September 2021 at Affiliated People's Hospital of Ningbo University. The clinical data and laboratory results were collected retrospectively. Efficacy, clinical outcome and prognostic factors were analyzed. Univariate analysis was conducted by using log-rank test, the multivariate analysis by Cox proportional risk model.Among 59 patients, there were 27 males and 32 females with median age of 55 (31-69) years old.The median follow-up was 761(317-1 861)days. The 2-year overall survival (OS) rate and event-free survival (EFS) rate were 76.1%±11.4% and 73.4%±11.6% respectively.The univariate analysis showed that age older than 50 years, TET2 gene mutation (TET2+), achieving MRD negativity over 30 days (MRD30+) were unfavorable factors of OS (χ2=6.20, 33.20, 7.18;P=0.013,<0.001, 0.007). TET2+, WT1 gene mutation (WT1+), CD34+cells<2×106/kg, MRD30+were negative factors of EFS (χ2=17.29, 4.47, 3.94, 9.393;P<0.001, 0.035, 0.047, 0.002).Multivariate analysis showed that MRD30+, TET2+ were independent prognostic factors of OS and EFS (OS:HR=9.251, 25.839, P=0.036, 0.001;EFS:HR=5.851, 9.199, P=0.043, 0.002). Intermediate-risk AML patients with MRD30+or TET2+ have very poor prognosis after auto-HSCT. Alternative regimens should be investigated.

Analysis of Gene Mutation Characteristics and Prognosis of Elderly Patients with Acute Myeloid Leukemia

To investigate the characteristics of gene mutation in elderly patients with acute myeloid leukemia (AML) and its effect on prognosis. The clinical and laboratorial characteristics of 54 AML patients (≥60 years old) in Department of Hematology, Tangdu Hospital were analyzed retrospectively during April 2016 to October 2019. Thirty-four AML/myelodysplastic syndrome/myeloproliferative neoplasm related mutant genes were detected by second-generation sequencing technology, and their clinical characteristics, treatment effect, and influence on prognosis were analyzed. All the patients received DAC+CAG induction treatment, after 1-2 couses of treatment, 36 cases (66.7%) achieved complete response, with a total effective rate of 75.9%, and the median survival time was 17 months. The most frequent mutant genes were TET2 (33.3%), CEBPA (31.5%), DNMT3A (18.5%), ASXL1 (16.7%), NRAS (14.8%), RUNX1 (14.8%), FLT3-ITD (12.9%), TP53 (12.9%), NPM1 (12.9%), and IDH2 (12.9%). Among 7 patients with TP53 mutation, 6 cases obtained complete response after 1-2 courses of induction treatment, but there was no statistically significant difference in the effect on prognosis. Patients with FLT3-ITD and NRAS mutations had shorter overall survival time compared with who had no mutation (P=0.47, P=0.48). Multivariate analysis showed that FLT3-ITD and NRAS mutations were poor prognostic factors. The incidence of TET2 gene mutation is high in elderly AML patients. AML patients with TET2 and TP53 mutations may benefit from Decitabine-based chemotherapy. However, patients with FLT3-ITD and NRAS mutations have a short survival time, and may have a poor prognosis.

The prognostic value of the interaction between ASXL1 and TET2 gene mutations in patients with chronic myelomonocytic leukemia: a meta-analysis

ABSTRACT Purpose The prognostic role of TET2 and/or ASXL1 mutations which are common gene mutations in chronic myelomonocytic leukemia (CMML) remains controversial. Therefore, we conducted this meta-analysis to evaluate the prognostic efficacy of ASXL1 and TET2 mutations in CMML population. Methods PubMed, Cochrane and Embase for relevant research were employed to identify 16 studies. Overall survival rate (OS) with hazard ratios (HRs) was used for analysis, and each individual HR was applied to calculate the combined HR. Results The total HR of OS was 0.74, 95% CI = 0.61 - 0.91, P = 0.005, compared with CMML patients without TET2 mutations (TET2MT), and the total HR of OS was 1.56, 95% CI = 1.34 - 1.80, P = 0.000, compared with CMML patients without ASXL1 mutation (ASXL1WT), indicating that TET2MT and ASXL1WT were favorable for prognosis of CMML. According to whether the gene is mutated or not, the acute transformation rate of disease and mortality rate were further considered for assessment. Compared with the CMML patients with TET2MT and ASXL1WT, the HR of patients with in both TET2MT and ASXL1MT was 1.51 (95% CI = 1.14 - 1.99; P = 0.004), the HR of patients with neither TET2MT nor ASXL1MT was 1.49 (95%CI = 1.12 - 1.98; P = 0.007), and the HR of TET2WT and ASXL1MT patients was 1.88 (95%CI = 1.21 - 2.94; P = 0.005). Conclusion Presence of TET2MT and ASXL1WT genotype was the most beneficial for the survival of CMML patients.

Spectrum of Myeloid Mutations in Patients with Elevated Hemoglobin

▪Background: JAK2 V617F and exon 12 mutations are the characteristic driver mutations in polycythemia vera (PV), identified in more than 95% of patients. In addition, other genetic mutations have previously been described in JAK2-positive PV that appear to have prognostic significance (Tefferi et al., Blood 2016). The incidence of other driver mutations in unselected patients referred for elevated hemoglobin is less well studied. This study aims to characterize the genetic mutational landscape in a real-world population of patients referred for elevated hemoglobin using a targeted Next-Generation Sequencing (NGS)-based assay.Methods: We reviewed all patients referred for elevated hemoglobin levels (>160 g/L in females or >165 g/L in males) between 2018 and 2020 to hematology clinics at London Health Sciences Centre in Southwestern Ontario, Canada who underwent testing for genetic variants using the NGS-based Oncomine Myeloid Research Assay (ThermoFisher Scientific, MA, USA). This assay targets 40 key genes with diagnostic and prognostic implications in several myeloid malignancies (17 full genes and 23 genes with clinically relevant “hotspot” regions) and a panel of 29 fusion driver genes (>600 fusion partners). Patient demographics, laboratory data and final diagnosis were extracted from the electronic medical record. For all patients with genetic mutations, clinical diagnosis was confirmed by three independent reviewers.Results: A total of 529 patients underwent genetic testing for elevated hemoglobin levels: 389 (73.5%) were males (mean age 58; range 18-95) and 140 (26.5%) were female (mean age 60; range 24-85). JAK2 mutations were detected in 10.9% (58/529) of patients and a diagnosis of PV was confirmed. The majority of JAK2-mutated PV patients (n=57) were positive for JAK2 V617F, while one patient had an exon 12 mutation. Additional single myeloid mutations were detected in 34.5% (20/58) of JAK2-positive patients and involved the following genes: TET2 (11; 19%), DNMT3A (2; 3.4%), ASXL1 (2; 3.4%), SRSF2 (2; 3.4%), BCOR (1; 1.7%), TP53 (1; 1.7%) and ZRSR2 (1; 1.7%) (Figure 1A). JAK2 mutations were not detected in 89.0% (471/529) of our cohort. A diagnosis of PV was confirmed in 2 JAK2-negative patients based on clinical features and myeloid mutations were detected in both: SRSF2 and TET2 gene mutations in 1 patient and SRSF2, IDH2, ASXL1 gene mutations in the other patient. Three JAK2-negative patients tested positive for the BCR-ABL fusion and were diagnosed with chronic myeloid leukemia. The remaining 466 JAK2-negative patients were diagnosed with secondary erythrocytosis and myeloid mutations were found in 6% (28/466) of these cases. Mutations were detected in DNMT3A (12; 2.6%), TET2 (5; 1.1%), ASXL1 (5; 1.1%), TP53 (2; 0.4%), NF1 (2; 0.4%), KIT (1; 0.2%), U2AF1 (1; 0.2%) (Figure 1B). All patients with JAK2-negative secondary erythrocytosis had only one myeloid gene mutation detected.Conclusion: Additional myeloid mutations other than JAK2 mutations are frequently identified in patients referred for erythrocytosis, with the highest frequencies observed in the TET2, DNMT3A and ASXL1 genes. The spectrum of myeloid mutations and overall incidence in JAK2-negative patients with secondary erythrocytosis is similar to the reported incidence of Clonal Hematopoiesis of Indeterminate Potential (CHIP) (Jaiswal et al., NEJM 2014), and suggests that these may represent incidental age-related mutations. By contrast, among the JAK2-positive patients, 34.5% had at least one additional myeloid mutation supporting a pathogenic role in these patients with myeloproliferative neoplasms. While concomitant myeloid mutations in patients with PV are well-described, further research is required to elucidate the significance of variants identified in JAK2-negative patients classified as secondary erythrocytosis in order to determine whether these mutations contribute to clinical phenotype or represent background CHIP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combinatorial Inhibition of Focal Adhesion Kinase and BCL-2 in AML

Focal adhesion kinase (FAK), activated by integrins and cytokines, promotes cell growth, migration and metastasis. We previously reported that a selective FAK inhibitor, VS-4718, inhibited cell growth and induced cell death in leukemia cell lines in vitro, effectively reduced leukemia burden in peripheral blood and tissues including bone marrow, spleen, liver and lung, and significantly prolonged survival time in a human xenograft murine model (Carter BZ et al., MCT, 2017), 16(6):1133).Although BCL-2 inhibition by the BCL-2 inhibitor ABT-199 (Venetoclax) has demonstrated antileukemia activities, an acquired drug resistance frequently ensues. When combined with ABT-199, VS-4718 significantly potentiated ABT-199-induced apoptosis in AML cell lines and primary AML cells. VS-4718 treatment of Molm14 AML cells decreased levels of p-STAT5, MCL-1, and BCL-XL (Wang et al., ASH 2016) that play important roles in the acquired resistance to ABT-199.To further examine the effect of VS-4718 as a single agent and in combination with ABT-199 on primary AML cells in vivo, we generated patient-derived xenografts (PDX) from AML patient bone marrow cells expanded in NSGS (NOD-SCID IL2Rgnull-3/GM/SF, NSG-SGM3) mice. The xenograft used AML cells from a patient who had failed multiple chemotherapies and had NPM1, FLT3-ITD, TET2, DNMT3A, and WT1 gene mutations and a complex karyotype. The combination of VS-4718 and ABT-199 synergistically killed the PDX cells in vitro even under MSC co-culture conditions. Once PDX cells were engrafted into NSGS mice, animals were randomized into 4 groups (N=10/group) and treated with vehicle, VS-4718 (75 mg/kg, twice daily), ABT-199 (100 mg/kg, once per day), or the combination via oral gavage for 29 days. We observed that ABT-199 had a stronger anti-leukemic effect on peripheral blood than on tissue-resident cells, while VS-4718 was more effective in tissues. At the end of the treatments, VS-4718 significantly reduced circulating human CD45+ cells (P=0.0003), and ABT-199 alone (P=5.90E-07) and the ABT-199/VS-4718 combination (P=5.38E-07) were ever more effective. VS-4718 alone or in combination with ABT-199 reduced leukemia burden in spleen and liver and effectively reduced splenomegaly, while ABT-199 alone did not. VS-4718-treated mice survived significantly longer than the untreated controls (median survival 72 vs. 36 days, P=0.0002), while ABT-199 did not prolong survival (median survival 35 days). Also, the combination of VS-4718 with ABT-199 significantly prolonged survival (median survival 65.5 days, P=0.011 vs. control and P=0.0072 vs. ABT-199). No statistical difference in survival was found between VS-4718 and the combination treatment groups (P=0.33) (Fig. 1). All the mice had large spleen at death except the combination group: the 1st dead mouse had small and the 2nd to 4th ones had slightly enlarged spleen and only the last two had large spleen as others suggesting that additional reasons such as toxicity at the dose used may have contributed to the death of the 1st 4 mice in the combination group, hence shortened the median survival in this group.We determined expression of FAK and BCL-2 family proteins in cells from mouse spleens at the end of treatment and found that the combination of VS-4718 and ABT-199 decreased protein levels of FAK. Consistent with our in vitro results, the increased level of MCL-1 by ABT-199 was antagonized by the combination with VS-4718 in vivo suggesting the combination potentially be more effective.Conclusions: These results suggest a potential clinical benefit of combining a FAK inhibitor with Venetoclax for patients with AML. [Display omitted] DisclosuresPachter:Verastem, Inc.: Employment. Weaver:Verastem, Inc.: Employment. Carter:Daiichi Sankyo: Research Funding; novartis: Research Funding.

Prophylactic Modified Donor Lymphocyte Infusion after Allogeneic Peripheral Blood Stem Cell Transplantation for Very High-Risk Hematological Malignancies: Low Incidence of Relapse without Increasing the Risk of Graft-Versus-Host Disease

Introduction The prognosis of patients with very high-risk hematologic malignancies who have relapsed/refractory diseases or carrying very poor-risk gene mutations were dismal even after allogeneic hematopoietic stem cell transplantation. Donor lymphocyte infusion (DLI) is a promising prophylactic strategy for reducing relapse after transplant. However, DLI-associated graft-versus-host disease (GVHD) and non-relapse mortality (NRM) were still concerned, especially after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Therefore, prophylactic DLI should be safe with tolerable DLI-associated GVHD and NRM except for its anti-leukemia effect. To evaluate the safety and efficacy of prophylactic DLI for the very high-risk hematologic malignancies after allo-PBSCT, we performed a cohort study in 160 consecutive patients with hematological malignancies who received unmanipulated allo-PBSCT. DLI procedure was modified to use G-CSF mobilized peripheral progenitors instead of steady state lymphocytes and 4 to 6 weeks of immunosuppressant for prevention of DLI-associated GVHD. The outcomes of those very high-risk patients (group A: DLI group; group B: non-DLI group) and the patients without the very high-risk features (group C) who were not planned for prophylactic DLI were compared.Patients and Methods The underlying diseases consist of acute myeloid leukemia (n = 78), myelodysplastic syndrome (n = 22), acute lymphoblastic leukemia/lymphoma (n = 45), chronic myeloid leukemia (n = 7), non-Hodgkin's lymphoma (n = 7), and plasma cell leukemia (n = 1). Sixty five patients (41.2%) received allo-PBSCT from HLA-matched sibling donors, 7 patients (4.4%) from matched unrelated donors, and 87 patients (54.4%) from haploidentical donors (Table 1). The very high-risk features were defined as 1) disease in non-remission state at time of transplant; 2) acute leukemia achieving complete remission (CR1) with 3 or more cycles of induction chemotherapy; 3) leukemia in CR1/CR2 carrying TP53, TET2 or DNMT3A gene mutation. Patients in CR with FLT3/ITD or BCR/ABL T315I gene mutation were enrolled due to the unavailability of effective targeted drugs. Prophylactic modified DLI was planned at 30 to 60 days post-transplant for all very high-risk patients with the absence of GVHD. The infusion was G-CSF mobilized peripheral progenitors from cryopreserved cell aliquots of the original graft. The trough concentration of CSA was maintained at 180 to 250 ng/mL for at least 4 to 6 weeks post-DLI and then discontinued within 2 weeks when no GVHD occurred. All the patients were followed-up for a median of 11.1 (0.5 - 29.7) months after transplant.Results Prophylactic DLI was administered for 29 very high-risk patients (group A) at a median of 63 (25 - 176) days after transplant. The median numbers of mononuclear cells and CD3+ lymphocytes infused were 0.7 (0.1 - 1.4) × 108/kg and 18.5 (5.4 - 69.2) × 106/kg with a single dose. Nineteen patients (39.6%) with very high-risk features did not receive prophylactic DLI (group B) for the reasons of early relapse (n = 8), occurrence of GVHD (n = 8), and uncontrolled infections (n = 3). The cumulative incidence (CI) of grade II-IV acute GVHD at day 100 after transplant was 33.2% in group A, 38.0% in group B (p= 0.318), and 28.2% in group C (p= 0.746). The 2-year CI of chronic GVHD was 29.9% in group A, 15.8% in group B (p= 0.428), and 54.6% in group C (p= 0.255). The 2-year CI of relapse was 54.6% in group A, which was significantly lower than that in group B (68.4%, p= 0.019). The relapse-free survival in group A was superior to that in group B (37.3% vs . 24.6%, p = 0.032). In the very high-risk group of patients (group A and B), prophylactic DLI was correlated with a lower risk of relapse (p= 0.043, hazard ratio = 0.45). The 2-year CI of NRM in group A was not significantly different from that in group B (22.4% vs . 15.8%, p= 0.461) or group C (22.4% vs . 23.2%, p= 0.949). In addition, there was no significant difference in overall survival between group A and group B (48.6% vs . 35.1%, p = 0.134). The 6-month CI of CMV/EBV reactivations in group A was not significantly different from that in group B (p= 0.107 and p= 0.668) or group C (p= 0.130 and p= 0.596).Conclusions This study showed that the prophylactic DLI after unmanipulated allo-PBSCT was well tolerated and effective for the patients with very high-risk hematologic malignancies in decreasing relapse without increasing the risks of GVHD and NRM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Prevalence of TET2 Gene Mutations in Patients with BCR-ABL-Negative Myeloproliferative Neoplasms (MPN): A Systematic Review and Meta-Analysis.

Simple SummaryMany molecular biology techniques have been widely used to study the pathogenesis of different diseases, particularly haematologic malignancies which are generally caused by abnormalities in the genome. TET2 gene is one of the commonly found mutated genes in BCR-ABL-negative myeloproliferative neoplasms. However, the prevalence of TET2 gene mutations in the disease remains unclear. Therefore, this study aims to estimate the prevalence of TET2 gene mutations in myeloproliferative neoplasms. The findings may be helpful for future research, diagnoses and the identification of better therapeutic strategies to manage the diseases.Multiple recurrent somatic mutations have recently been identified in association with myeloproliferative neoplasms (MPN). This meta-analysis aims to assess the pooled prevalence of TET2 gene mutations among patients with MPN. Six databases (PubMed, Scopus, ScienceDirect, Google Scholar, Web of Science and Embase) were searched for relevant studies from inception till September 2020, without language restrictions. The eligibility criteria included BCR-ABL-negative MPN adults with TET2 gene mutations. A random-effects model was used to estimate the pooled prevalence with 95% confidence intervals (CIs). Subgroup analyses explored results among different continents and countries, WHO diagnostic criteria, screening methods and types of MF. Quality assessment was undertaken using the Joanna Briggs Institute critical appraisal tool. The study was registered with PROSPERO (CRD42020212223). Thirty-five studies were included (n = 5121, 47.1% female). Overall, the pooled prevalence of TET2 gene mutations in MPN patients was 15.5% (95% CI: 12.1–19.0%, I2 = 94%). Regional differences explained a substantial amount of heterogeneity. The prevalence of TET2 gene mutations among the three subtypes PV, ET and MF were 16.8%, 9.8% and 15.7%, respectively. The quality of the included studies was determined to be moderate–high among 83% of the included studies. Among patients with BCR-ABL-negative MPN, the overall prevalence of TET2 gene mutations was 15.5%.

WITHDRAWN: Molecular genetics of thrombotic myeloproliferative neoplasms: Implications in precision oncology

Classical BCR-ABL -negative myeloproliferative neoplasms (MPN) include polycythaemia vera, essential thrombocythaemia and primary myelofibrosis. Unlike monogenic disorders, a more complicated series of genetic mutations are believed to be responsible for MPN with various degrees of thromboembolic and bleeding complications. Thrombosis is one of the early manifestations in patients with MPN. To date, the driver genes responsible for MPN include JAK2, CALR, MPL, TET2, ASXL1 and MTHFR . Affords have been done to elucidate these mutations and the incidence of thromboembolic events. Several lines of evidence indicate that mutations in JAK2, MPL, CALR, TET2 and ASXL1 gene and polymorphisms in several clotting factors ( GPIa, GPIIa and GPIIIa ) are associated with the occurrence and prevalence of thrombosis in MPN patients. Some polymorphisms within XRCC1, FBG, F2, F5, F7, F12, MMP9, HPA5, MTHFR, SDF-1, FAS, FASL, TERT, ACE and TLR4 genes may also play a role in MPN manifestation. This review aims to provide an insightful overview on the genetic perspective of thrombotic complications in patients with MPN.

Iron Overload Impairs Bone Marrow Mesenchymal Stromal Cells from Higher-Risk MDS Patients by Regulating the ROS-Related Wnt/β-Catenin Pathway.

The bone marrow microenvironment plays important roles in the progression of the myelodysplastic syndrome (MDS). The higher incidence of ASXL1 and TET2 gene mutations in our iron overload (IO) MDS patients suggests that IO may be involved in the pathogenesis of MDS. The effects of IO damaging bone marrow mesenchymal stromal cells (MSCs) from higher-risk MDS patients were investigated. In our study, IO decreased the quantity and weakened the abilities of proliferation and differentiation of MSCs, and it inhibited the gene expressions of VEGFA, CXCL12, and TGF-β1 in MSCs regulating hematopoiesis. The increased level of reactive oxygen species (ROS) in MSCs caused by IO might be inducing apoptosis by activating caspase3 signals and involving in MDS progression by activating β-catenin signals. The damages of MSCs caused by IO could be partially reversed by an antioxidant or an iron chelator. Furthermore, the MSCs in IO MDS/AML patients had increased levels of ROS and apoptosis, and the expressions of caspase3 and β-catenin were increased even further. In conclusion, IO affects gene stability in higher-risk MDS patients and impairs MSCs by inducing ROS-related apoptosis and activating the Wnt/β-catenin signaling pathway, which could be partially reversed by an antioxidant or an iron chelator.

Analysis of Factors Influencing Overall Survival of MDS Patients Transplanted with HSCs

To analyze the effect of clinical features, routine laboratory examination and related gene mutation on the OS of patients with myelodysplastic syndrome (MDS) after hematopoietic stem cell transplantation (HSCT). 121 patients diagnosed as MDS and underwent hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University from October 2013 to August 2018 were selected. Basic information of the patients was collected, and blood cells, bone marrow blasts at initial diagnosis, chromosomal karyotypes and gene mutations of the patients were detected.The effect of different factors on overall survival (OS) was analyzed by statistical method. Kaplan-Meier univariate analysis shows that OS was significanly different among different age groups. The 3-year OS rate of patients aged 0-29 years was (83.3±7.7) %, the 3-year OS rate in patients aged 30-49 years was (58.1±7.7 %), and the 3-year OS rate of patients aged 50-69 years was (31.0±22.6) %, which was statistically different (P＜0.05) between different groups. There were also significant differences in OS among patients with different transplantation types. 3-year OS rate: HLA-matched sibling HSCT＞unrelated HLA-matched HSCT＞haploidentical HSCT＞micro HSCT. The OS rate of patients with bone marrow blasts≥10% seems lower than blasts＜10%, but there was no statistical difference.The 3-year OS rate of patients with chromosomal karyotype complex abnormality was (47.7±11.5) %, and that of patients without complex abnormality was (80±4.2) % which was statistical difference (P＜0.05). Patients with DNMT3A, NRAS, TP53 and GATA2 mutations had shorter OS time compared with patients without mutation of these genes, which shows statistically significant (P＜0.05). COX multivariate analysis showed that age, chromosome karyotype, DNMT3A, TET2, GATA2 and NRAS were the independent factors influencing OS of patients after HSCT, with statistically significant difference. age of patients, donor selection of HSCT, chromosome karyotype, DNMT3A, NRAS, TP53, GATA2 and TET2 gene mutations are all independent factors affecting the OS of patients after HSCT. Therefore, the assessment of the OS of MDS patients with transplantation requires comprehensive consideration.

Analysis of the Effect of TET2 Mutation and SNP on Clinical Characteristics and Prognosis of AML Patients

To evaluate the clinical significance of TET2 mutation(TET2mut) and SNP in adult acute myeloid leukemia (AML) and its effect on prognosis. A total of 24 genes, including TET2, FLT3-ITD and NPM1, were detected in 124 adult AML patients using second-generation sequencing technology, and their clinical characteristics and effect on prognosis of patients were analyzed. A total of 25 TET2 gene mutations were detected in 124 AML patients, the mutation rate was 20.2%, there were 75 cases of TET2 single nucleotide polymorphisms(SNP), accounting for 60.5%. There were 47 cases of SNPrs2454206(G>A), accounting for 37.9%. Compared with TET2 wild-type(TET2wt) patients, TET2mut patients were mostly elderly. TET2SNP was commonly seen in male, with statistically significant differences (P＜0.05). SNP rs2454206 had no significant correlation with sex and age (P＞0.05). However, the analysis found that the complete remission rate of 1 course and the total complete rate (CR) of patients with TET2AG/GG were both obviouslysuperior to those with TET2AA (P＜0.05). In the univariate analysis, the overall survival(OS) rate of the patients with TET2mut was lower than that of patients with TET2wt, and the event free survival (EFS) rate was higher than that of TET2wt patients, but the difference was not statistically significant (P＞0.05).The 1-year OS rate and EFS rate of the patients with TET2AA were significantly lower than those of the patients with TET2AG/GG (P＜0.05). Multivariate analysis showed that TET2AA was an independent risk factor for OS and EFS in AML patients. TET2 SNPrs2454206(G＞A) commonly appears in patients with acute myeloid leukemia, and these patients have the better response to chemotherapy and a better prognosis.

Gene mutational analysis by NGS and its clinical significance in patients with myelodysplastic syndrome and acute myeloid leukemia

BackgroundIn this study, we retrospectively summarized the differences of molecular gene mutations between MDS and AML patients, as well as the young and older age groups of MDS and AML patients. We also analyzed the response of newly diagnosed AML patients to standard DA or IA induction chemotherapy and the relationship between the chemotherapy outcome and the frequency of different gene mutation abnormalities.MethodsNGS assay covering 43 genes was studied in 93 de novo MDS and 325 non-M3 AML patients. Bone marrow samples from all patients underwent gene mutational analysis by NGS.ResultsAt least one non-synonymous gene mutation was detected in 279 AML patients (85.8%) and 85 MDS patients (91.4%). Contrary to 59 years and younger AML patients, there was a significantly higher incidence of gene mutation in 60 years and older AML patients (2.37 vs 1.94, p = 0.034). Gene mutation incidence in 60 years and older MDS patients increased, but no statistical significance was present (1.95 vs 1.64, p = 0.216). AML patients had a significantly higher gene mutation incidence compared with MDS-MLD patients (2.02 vs 1.63, p = 0.046). Gene mutation incidence was higher in patients with MDS-EB1/EB2 compared with patients with MDS-MLD but there was no statistical significance present (2.14 vs 1.63, p = 0.081). AML patients had significantly higher incidences of CEBPA, FLT3-ITD, DNMT3A, NPM1 and IDH1/2 gene mutations (p = 0.0043, 0.000, 0.030962, 0.002752, and 0.000628, respectively) and a lower incidence of TET2 and U2AF1 gene mutations (p = 0.000004 and 0.000, respectively) compared with MDS patients. Among the individual genes in different age groups, there were significantly higher incidences of RUNX1, IDH2, TP53 and SF3B1 gene mutations (p = 0.0478, 0.0028, 0.0024 and 0.005, respectively) as well as a trend of higher ASXL gene mutation (p = 0.057) in 60 years and older AML patients compared to 59 years and younger patients. There was no statistically significant difference in MDS patients with the different age groups and among the individual genes. Between AML patients and MDS patients among the different gene functional groups, AML patients had a significantly higher incidence of transcriptional deregulation (27.4% vs 15.1%, p = 0.014963), activated signalling (36.3% vs 10.8%, p = 0.000002) related gene mutations as well as a significantly lower incidence of RNA spliceosome (6.15% vs 60.1%, p = 0.000) related gene mutations. Furthermore, among the patients who received either IA or DA regimen for induction chemotherapy, patients with IA regimen had a significantly better CR rate than those with DA regimen (76.6% vs 57.1%, p = 0.0228).ConclusionsDifferent gene mutations had been found in majority of MDS and AML patients. MDS and AML patients had different gene mutation patterns. AML patients with fewer or no gene mutations had a better chance of achieving CR when treated with IA and DA regimen induction chemotherapy.