Cheng-Hong Tsai

Genetic alterations and their clinical implications in older patients with acute myeloid leukemia

A number of patient-specific and leukemia-associated factors are related to the poor outcome in older patients with acute myeloid leukemia (AML). However, comprehensive studies regarding the impact of genetic alterations in this group of patients are limited. In this study, we compared relevant mutations in 21 genes between AML patients aged 60 years or older and those younger and exposed their prognostic implications. Compared with the younger patients, the elderly had significantly higher incidences of PTPN11, NPM1, RUNX1, ASXL1, TET2, DNMT3A and TP53 mutations but a lower frequency of WT1 mutations. The older patients more frequently harbored one or more adverse genetic alterations. Multivariate analysis showed that DNMT3A and TP53 mutations were independent poor prognostic factors among the elderly, while NPM1 mutation in the absence of FLT3/ITD was an independent favorable prognostic factor. Furthermore, the status of mutations could well stratify older patients with intermediate-risk cytogenetics into three risk groups. In conclusion, older AML patients showed distinct genetic alterations from the younger group. Integration of cytogenetics and molecular mutations can better risk-stratify older AML patients. Development of novel therapies is needed to improve the outcome of older patients with poor prognosis under current treatment modalities.

Splicing factor mutations predict poor prognosis in patients with de novo acute myeloid leukemia.

Mutations in splicing factor (SF) genes are frequently detected in myelodysplastic syndrome, but the prognostic relevance of these genes mutations in acute myeloid leukemia (AML) remains unclear. In this study, we investigated mutations of three SF genes, SF3B1, U2AF1 and SRSF2, by Sanger sequencing in 500 patients with de novo AML and analysed their clinical relevance. SF mutations were identified in 10.8% of total cohort and 13.2% of those with intermediate-risk cytogenetics. SF mutations were closely associated with RUNX1, ASXL1, IDH2 and TET2 mutations. SF-mutated AML patients had a significantly lower complete remission rate and shorter disease-free survival (DFS) and overall survival (OS) than those without the mutation. Multivariate analysis demonstrated that SFmutation was an independent poor prognostic factor for DFS and OS. A scoring system incorporating SF mutation and ten other prognostic factors was proved very useful to risk-stratify AML patients. Sequential study of paired samples showed that SF mutations were stable during AML evolution. In conclusion, SF mutations are associated with distinct clinic-biological features and poor prognosis in de novo AML patients and are rather stable during disease progression. These mutations may be potential targets for novel treatment and biomarkers for disease monitoring in AML.

Incorporation of mutations in five genes in the revised International Prognostic Scoring System can improve risk stratification in the patients with myelodysplastic syndrome

Gene mutations have not yet been included in the 2016 WHO classification and revised International Prognostic Scoring System (IPSS-R), which are now widely utilized to discriminate myelodysplastic syndrome (MDS) patients regarding risk of leukemia evolution and overall survival (OS). In this study, we aimed to investigate whether integration of gene mutations with other risk factors could further improve the stratification of MDS patients. Mutational analyses of 25 genes relevant to myeloid malignancies in 426 primary MDS patients showed that mutations of CBL, IDH2, ASXL1, DNMT3A, and TP53 were independently associated with shorter survival. Patients within each IPSS-R or 2016 WHO classification-defined risk group could be stratified into two risk subgroups based on the mutational status of these five genes; patients with these poor-risk mutations had an OS shorter than others in the same risk group, but similar to those with the next higher risk category. A scoring system incorporating age, IPSS-R and five poor-risk mutations could divide the MDS patients into four risk groups (P < 0.001 for both OS and leukemia-free survival). In conclusion, integration of gene mutations in current IPSS-R improves the prognostication of MDS patients and may help identify high-risk patients for more aggressive treatment in IPSS-R lower risk group.

Mayo Alliance Prognostic Model for Myelodysplastic Syndromes: Integration of Genetic and Clinical Information

Objective: To develop a new risk model for primary myelodysplastic syndromes (MDS) that integrates information on mutations, karyotype, and clinical variables. Patients and Methods: Patients with World Health Organization–defined primary MDS seen at Mayo Clinic (MC) from December 28, 1994, through December 19, 2017, constituted the core study group. The National Taiwan University Hospital (NTUH) provided the validation cohort. Model performance, compared with the revised International Prognostic Scoring System, was assessed by Akaike information criterion and area under the curve estimates. Results: The study group consisted of 685 molecularly annotated patients from MC (357) and NTUH (328). Multivariate analysis of the MC cohort identified monosomal karyotype (hazard ratio [HR], 5.2; 95% CI, 3.1‐8.6), “non‐MK abnormalities other than single/double del(5q)” (HR, 1.8; 95% CI, 1.3‐2.6), RUNX1 (HR, 2.0; 95% CI, 1.2‐3.1) and ASXL1 (HR, 1.7; 95% CI, 1.2‐2.3) mutations, absence of SF3B1 mutations (HR, 1.6; 95% CI, 1.1‐2.4), age greater than 70 years (HR, 2.2; 95% CI, 1.6‐3.1), hemoglobin level less than 8 g/dL in women or less than 9 g/dL in men (HR, 2.3; 95% CI, 1.7‐3.1), platelet count less than 75 × 109/L (HR, 1.5; 95% CI, 1.1‐2.1), and 10% or more bone marrow blasts (HR, 1.7; 95% CI, 1.1‐2.8) as predictors of inferior overall survival. Based on HR‐weighted risk scores, a 4‐tiered Mayo alliance prognostic model for MDS was devised: low (89 patients), intermediate‐1 (104), intermediate‐2 (95), and high (69); respective median survivals (5‐year overall survival rates) were 85 (73%), 42 (34%), 22 (7%), and 9 months (0%). The Mayo alliance model was subsequently validated by using the external NTUH cohort and, compared with the revised International Prognostic Scoring System, displayed favorable Akaike information criterion (1865 vs 1943) and area under the curve (0.87 vs 0.76) values. Conclusion: We propose a simple and contemporary risk model for MDS that is based on a limited set of genetic and clinical variables.

Prognostic impacts and dynamic changes of cohesin complex gene mutations in de novo acute myeloid leukemia

Prognostic impacts and dynamic changes of cohesin complex gene mutations in de novo acute myeloid leukemia Cheng-Hong Tsai , Hsin-An Hou, Jih-Luh Tang, Yuan-Yeh Kuo , Yu-Chiao Chiu , Chien-Chin Lin, Chieh-Yu Liu, Mei-Hsuan Tseng, Tzung-Yi Lin, Ming-Chih Liu, Chia-Wen Liu, Liang-In Lin, Ming Yao, Chi-Cheng Li, Shang-Yi Huang, Bor-Sheng Ko, Szu-Chun Hsu, Chien-Ting Lin, Shang-Ju Wu, Chien-Yuan Chen, Woei Tsay, Eric Y. Chuang, Wen-Chien Chou and Hwei-Fang Tien

Concomitant WT1 mutations predict poor prognosis in acute myeloid leukemia patients with double mutant CEBPA

Acute myeloid leukemia (AML) with double mutant CCAAT/enhancer binding protein α (CEBPA) is a new entity in the 2016 World Health Organization (WHO) classification with unique biologic features and prognostic implications. The incidence of CEBPA ranges from 7.5% to 11% in AML. CEBPA AML patients, when treated with standard chemotherapy, achieve a high complete remission (CR) rate. However, relapse occurs in 40% of patients who attain CR. This has raised the clinically relevant question whether concomitant genetic alterations influence the prognosis of CEBPA patients. Apart from GATA2, the prognostic impact of other concomitant gene mutations is largely unsettled because limited patient numbers preclude informative analyses. Given that AML is a heterogeneous disease, risk-adapted treatment may not only improve the prognosis, but also reduce toxicity from the therapy. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first CR is not beneficial for cytogenetically normal AML (CN-AML) patients with CEBPA . If any concomitant mutations adversely affect the clinical outcome of CEBPA patients, it will be interesting to know whether allo-HSCT should be performed for these patients. As yet, there is no data to answer this question. In this study, the aim was to identify additional mutations in CEBPA AML patients that conferred prognostic significance. Furthermore, we investigated the role of allo-HSCT in CEBPA patients with concurrent adverserisk mutations. Mutation analyses in CEBPA and 19 other relevant genes, including FLT3-ITD, FLT3-TKD, NRAS, KRAS, KIT, PTPN11, RUNX1, GATA2, MLL/PTD, ASXL1, IDH1, IDH2, TET2, DNMT3A, SF3B1, SRSF2, U2AF1, NPM1, WT1, and TP53 were performed by Sanger sequencing for patients (n=500) diagnosed from 1994 to 2007. For patients (n=256) diagnosed after 2008, Ion Torrent next generation sequencing (NGS) (Thermo Fisher Scientific, MA, USA) was performed. The WT1 mutations detected by NGS were all confirmed by Sanger sequencing. We identified 102 (13.5%) CEBPA-mutated patients from 756 patients with newly diagnosed de novo AML (Online Supplementary Table S1); 33 (4.4%) had CEBPA single mutation (CEBPA) and 69 (9.1%), CEBPA. Sixtynine CEBPA patients were found to have 109 distinct mutations (Figure 1A, Online Supplementary Table S2). All patients had a combination of one N-terminal and one C-terminal mutation. Most (53 of 56, 94.6%) of the N-terminal mutations were frame-shift mutations, while most (42 of 53, 79.2%) of the C-terminal mutations were in-frame mutations with internal tandem duplications clustered in the junction between the basic region and the leucine zipper. CEBPA patients were significantly younger and had higher hemoglobin levels at diagnosis than CEBPA and CEBPA wild-type patients. All except one CEBPA patient had intermediate-risk cytogenetics (P<0.0001) (Figure 1A). The most frequent intermediate-risk cytogenetic change was del(9) (n=4, 5.8%), and CNAML occurred in 81.2% of CEBPA patients (n=56). Fifty (72.5%) of the CEBPA patients had additional genetic alterations (Online Supplementary Table S3). Among them, 29 (58%) had one, 17 (34%) had two, 3 (6%) had three and 1 (2%) had four changes. The most common concurrent molecular event in CEBPA patients was GATA2 mutation (33.8%), followed by FLT3-ITD (14.5%), NRAS (14.5%), TET2 (13.2%), and WT1 (11.8%) mutations. GATA2was more frequently mutated in CEBPA patients than in CEBPA wild-type patients (33.8% vs. 2.8%, P<0.0001). In contrast, CEBPA patients less frequently harbored NPM1, ASXL1, IDH2, DNMT3A and RUNX1 mutations (Figure 1B). Survival analyses were restricted to 530 patients, including 62 CEBPA patients and 468 others (22 with CEBPA and 446 CEBPA wild-type), who received standard intensive chemotherapy. The CR rate was 90.2% for CEBPA patients and 72.2% for others (P=0.003). In multivariate analysis, CEBPA was an independent favorable prognostic factor for OS and DFS (RR 0.420, 95% CI 0.246-0.718, P=0.002 and RR 0.544, 95% CI 0.351-0.842, P=0.006, respectively, Online Supplementary Table S4). Of the 56 CEBPA patients who achieved first CR, 10 received allo-HSCT and 46 had postremission chemotherapy alone. The reasons for frontline alloHSCT were persistent residual leukemia cells in 4 patients, concurrent FLT3-ITD in 3 patients, initial hyperleukocytosis in 2 patients and complex cytogenetics in 1 patient. Intriguingly, the relapse rate was 45.7% in the postremission chemotherapy group and 0% in the alloHSCT group (P=0.009). DFS was significantly better in

Hyperleukocytosis is associated with distinct genetic alterations and is an independent poor-risk factor in de novo acute myeloid leukemia patients

Acute myeloid leukemia (AML) with hyperleukocytosis (HL) is intuitively thought as a unique group with dismal prognosis. However, comprehensive studies regarding the genetic landscape and clinical outcome in this group of patients are limited.

Clinically validated machine learning algorithm for detecting residual diseases with multicolor flow cytometry analysis in acute myeloid leukemia and myelodysplastic syndrome

Background Multicolor flow cytometry (MFC) analysis is widely used to identify minimal residual disease (MRD) after treatment for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, current manual interpretation suffers from drawbacks of time consuming and interpreter idiosyncrasy. Artificial intelligence (AI), with the expertise in assisting repetitive or complex analysis, represents a potential solution for these drawbacks. Methods From 2009 to 2016, 5333 MFC data from 1742 AML or MDS patients were collected. The 287 MFC data at post-induction were selected as the outcome set for clinical outcome validation. The rest were 4:1 randomized into the training set (n = 4039) and the validation set (n = 1007). AI algorithm learned a multi-dimensional MFC phenotype from the training set and input it to support vector machine (SVM) classifier after Gaussian mixture model (GMM) modeling, and the performance was evaluated in The validation set. Findings Promising accuracies (84·6% to 92·4%) and AUCs (0·921–0·950) were achieved by the developed algorithms. Interestingly, the algorithm from even one testing tube achieved similar performance. The clinical significance was validated in the outcome set, and normal MFC interpreted by the AI predicted better progression-free survival (10·9 vs 4·9 months, p < 0·0001) and overall survival (13·6 vs 6·5 months, p < 0·0001) for AML. Interpretation Through large-scaled clinical validation, we showed that AI algorithms can produce efficient and clinically-relevant MFC analysis. This approach also possesses a great advantage of the ability to integrate other clinical tests. Fund This work was supported by the Ministry of Science and Technology (107-2634-F-007-006 and 103–2314-B-002-185-MY2) of Taiwan.

Dynamics of DNMT3A mutation and prognostic relevance in patients with primary myelodysplastic syndrome

BackgroundDNMT3A gene mutation has been associated with poor prognosis in acute myeloid leukemia, but its clinical implications in myelodysplastic syndrome (MDS) and dynamic changes during disease progression remain controversial.ResultsIn this study, DNMT3A mutation was identified in 7.9% of 469 de novo MDS patients. DNMT3A-mutated patients had higher platelet counts at diagnosis, and patients with ring sideroblasts had the highest incidence of DNMT3A mutations, whereas those with multilineage dysplasia had the lowest incidence. Thirty-one (83.8%) of 37 DNMT3A-mutated patients had additional molecular abnormalities at diagnosis, and DNMT3A mutation was highly associated with mutations of IDH2 and SF3B1. Patients with DNMT3A mutations had a higher risk of leukemia transformation and shorter overall survival. Further, DNMT3A mutation was an independent poor prognostic factor irrespective of age, IPSS-R, and genetic alterations. The sequential study demonstrated that the original DNMT3A mutations were retained during follow-ups unless allogeneic hematopoietic stem cell transplantation was performed, while DNMT3A mutation was rarely acquired during disease progression.ConclusionsDNMT3A mutation predicts unfavorable outcomes in MDS and was stable during disease evolutions. It may thus be a potential biomarker to predict prognosis and monitor the treatment response.

GATA2 zinc finger 1 mutations are associated with distinct clinico-biological features and outcomes different from GATA2 zinc finger 2 mutations in adult acute myeloid leukemia

Mutations of the GATA binding protein 2 (GATA2) gene in myeloid malignancies usually cluster in the zinc finger 1 (ZF1) and the ZF2 domains. Mutations in different locations of GATA2 may have distinct impact on clinico-biological features and outcomes in AML patients, but little is known in this aspect. In this study, we explored GATA2 mutations in 693 de novo non-M3 AML patients and identified 44 GATA2 mutations in 43 (6.2%) patients, including 31 in ZF1, 10 in ZF2, and three outside the two domains. Different from GATA2 ZF2 mutations, ZF1 mutations were closely associated with French-American-British (FAB) M1 subtype, CEBPA double mutations (CEBPAdouble-mut), but inversely correlated with FAB M4 subtype, NPM1 mutations, and FLT3-ITD. ZF1-mutated AML patients had a significantly longer overall survival (OS) than GATA2-wild patients and ZF2-mutated patients in total cohort as well as in those with intermediate-risk cytogenetics and normal karyotype. ZF1 mutations also predicted better disease-free survival and a trend of better OS in CEBPAdouble-mut patients. Sequential analysis showed GATA2 mutations could be acquired at relapse. In conclusion, GATA2 ZF1 mutations are associated with distinct clinico-biological features and predict better prognosis, different from ZF2 mutations, in AML patients.