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Supplementary Table 1 from Nucleophosmin Mutations in De novo Acute Myeloid Leukemia: The Age-Dependent Incidences and the Stability during Disease Evolution

Nucleophosmin (NPM) mutations have been found in a significant proportion of adults with de novo acute myeloid leukemia (AML), especially in those of a normal karyotype. These results provide a basis for studies of the pathogenesis in this specific subgroup of AML. In this study, NPM mutations were analyzed in 173 Chinese patients of de novo AML, including adults and children. We found that NPM mutations were present in 19.1% of the overall population and 40.3% of those with a normal karyotype. Adults had a significantly higher incidence of NPM mutations than children [32 of 126 (25.4%) versus 1 of 47 (2.1%), P < 0.001]. NPM mutations were closely associated with normal karyotype (P < 0.001) and internal tandem duplication of FLT3 (P = 0.002), but negatively associated with CEBPA mutations (P = 0.032) and expression of CD34 (P < 0.001) and HLA-DR (P = 0.003). Serial analyses of NPM mutations showed the mutation disappeared at complete remission, but the same mutation reappeared at relapse, except for one who lost the mutation at the second relapse, when new cytogenetic abnormalities emerged. None acquired novel mutations during the follow-up period. In conclusion, NPM mutations occur in an age-dependent fashion. Moreover, the findings that NPM mutations are stable during disease evolution and closely associated with disease status make it a potential marker for monitoring minimal residual disease. (Cancer Res 2006; 66(6): 3310-6)

Myelodysplastic syndromes (MDS) are a group of clinically and genetically diverse diseases that impose patients with an increased risk of leukemic transformation. While MDS is a disease of the elderly, the interplay between aging and molecular profiles is not fully understood, especially in the Asian population. Thus, we compared the genetic landscape between younger and older patients in a cohort of 698 patients with primary MDS to advance our understanding of the distinct pathogenesis and different survival impacts of gene mutations in MDS according to age. We found that the average mutation number was higher in the older patients than younger ones. The younger patients had more WT1 and CBL mutations, but less mutated ASXL1, DNMT3A, TET2, SF3B1, SRSF2, STAG2, and TP53 than the older patients. In multivariable survival analysis, RUNX1 mutations with higher variant allele frequency (VAF) and U2AF1 and TP53 mutations were independent poor prognostic indicators in the younger patients, whereas DNMT3A and IDH2 mutations with higher VAF and TP53 mutations conferred inferior outcomes in the older patients. In conclusion, we demonstrated the distinct genetic landscape between younger and older patients with MDS and suggested that mutations impact survival in an age-depended manner.

The 2022 International Consensus Classification (ICC) recategorized myeloid neoplasms based on recent advances in the understanding of the biology of hematologic malignancies, in which myelodysplastic syndrome (MDS) with blasts of 10%-19% is classified as MDS/acute myeloid leukemia (AML), MDS with mutated SF3B1, irrespective of the number of ring sideroblasts, as MDS-SF3B1, and those with multi-hit TP53 mutations as MDS with mutated TP53. In the analysis of 716 patients with MDS diagnosed according to the 2016 WHO classification, we found that 75.3% of patients remained in the MDS group based on the ICC, while 24.7% of patients were reclassified to the MDS/AML group after the exclusion of 15 patients who were classified to the AML group. Patients with MDS/AML showed a distinct mutational landscape and had poorer outcomes, compared to those with MDS. In the MDS group, patients with MDS-SF3B1 had higher frequencies of DNMT3A and TET2 mutations than those with MDS, not otherwise specified, with single lineage dysplasia or multilineage dysplasia. Patients with mutated TP53 were associated with dismal outcomes, irrespective of the blast percentage. In conclusion, this study showed that the ICC facilitates efficient segregation and risk-stratification of MDS which can help guide the treatment choice of patients with the disease.

The mutant burden of FLT3-ITD modulates its prognostic impact on patients with acute myeloid leukemia (AML). However, for patients with low allelic ratio (AR) FLT3-ITD (FLT3-ITDlow, AR

Next-generation sequencing (NGS) has been applied to measurable/minimal residual disease (MRD) monitoring after induction chemotherapy in patients with acute myeloid leukemia (AML), but the optimal time point for the test remains unclear. In this study, we aimed to investigate the clinical significance of NGS MRD at 2 different time points. We performed targeted NGS of 54 genes in bone marrow cells serially obtained at diagnosis, first complete remission (first time point), and after the first consolidation chemotherapy (second time point) from 335 de novo AML patients. Excluding DNMT3A, TET2, and ASXL1 mutations, which are commonly present in individuals with clonal hematopoiesis of indeterminate potential, MRD could be detected in 46.4% of patients at the first time point (MRD1st), and 28.9% at the second time point (MRD2nd). The patients with detectable NGS MRD at either time point had a significantly higher cumulative incidence of relapse and shorter relapse-free survival and overall survival. In multivariate analysis, MRD1st and MRD2nd were both independent poor prognostic factors. However, the patients with positive MRD1st but negative MRD2nd had a similar good prognosis as those with negative MRD at both time points. The incorporation of multiparameter flow cytometry and NGS MRD revealed that the presence of NGS MRD predicted poorer prognosis among the patients without detectable MRD by multiparameter flow cytometry at the second time point but not the first time point. In conclusion, the presence of NGS MRD, especially after the first consolidation therapy, can help predict the clinical outcome of AML patients.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal myeloid malignancies. Though several recurrent mutations are closely correlated with clinical outcomes, data concerning the association between mutation variant allele frequencies (VAF) and prognosis are limited. In this study, we performed comprehensive VAF analyses of relevant myeloid-malignancy related mutations in 698 MDS patients and correlated the results with their prognosis. Mutation VAF in DNMT3A, TET2, ASXL1, EZH2, SETBP1, BCOR, SFSF2, ZRSR2, and TP53 mutations correlated with outcomes. In multivariable analysis, DNMT3A and ZRSR2 mutations with high VAF and mutant IDH2, CBL, U2AF1, and TP53 were independent poor prognostic factors for overall survival. A substantial portion of patients in each revised International Prognostic Scoring System (IPSS-R) risk group could be adjusted to different prognostic groups based on the integrated VAF and mutational profiles. Patients with these unfavorable mutations in each IPSS-R risk subgroup had survivals worse than other patients of the same risk but similar to those in the next higher-risk subgroup. Furthermore, patients harboring U2AF1 mutation might benefit from hypomethylating agents. This study demonstrated the critical role of VAF of mutations for risk stratification in MDS patients and may be incorporated in novel scoring systems.

Key Points CIBERSORTx analytical approach reveals M0, M2, and eosinophil fractions in the BM to be the most relevant prognostic factors in patients with MDS.High-risk immune cell scores correlate with NF-κB signaling, oxidative stress, and leukemic stem cell signature pathways., Visual Abstract, Aside from cell intrinsic factors such as genetic alterations, immune dysregulation in the bone marrow (BM) microenvironment plays a role in the development and progression of myelodysplastic syndromes (MDS). However, the prognostic implications of various immune cells in patients with MDS remain unclear. We adopted CIBERSORTx to estimate the relative fractions of 22 subtypes of immune cells in the BM of 316 patients with MDS and correlated the results with clinical outcomes. A lower fraction of unpolarized M0 macrophages and higher fractions of M2 macrophages and eosinophils were significantly associated with inferior survival. An immune cell scoring system (ICSS) was constructed based on the proportion of these 3 immune cells in the BM. The ICSS high-risk patients had higher BM blast counts, higher frequencies of poor-risk cytogenetics, and more NPM1, TP53, and WT1 mutations than intermediate- and low-risk patients. The ICSS could stratify patients with MDS into 3 risk groups with distinct leukemia-free survival and overall survival among the total cohort and in the subgroups of patients with lower and higher disease risk based on the revised International Prognostic Scoring System (IPSS-R). The prognostic significance of ICSS was also validated in another independent cohort. Multivariable analysis revealed that ICSS independently predicted prognosis, regardless of age, IPSS-R, and mutation status. Bioinformatic analysis demonstrated a significant correlation between high-risk ICSS and nuclear factor κB signaling, oxidative stress, and leukemic stem cell signature pathways. Further studies investigating the mechanistic insight into the crosstalk between stem cells and immune cells are warranted.

Aberrant alternative splicing (AS) is a hallmark of cancer development. However, there are limited data regarding its clinical implications in myelodysplastic syndrome (MDS). In this study, we performed an in-depth analysis of global AS in 176 primary MDS patients with 20 normal marrow transplant donors as reference. We found that 26.9% of the expressed genes genome-wide were aberrantly spliced in MDS patients compared with normal donors. These aberrant AS genes were related to pathways involved in cell proliferation, cell adhesion and protein degradation. A higher degree of global aberrant AS was associated with male gender and U2AF1 mutation, and predicted shorter overall survival and time to leukemic change. Moreover, it was an independent unfavorable prognostic factor irrespective of age, revised international prognostic scoring system (IPSS-R) risk, and mutations in SRSF2, ZRSR2, ASXL1, TP53, and EZH2. With LASSO-Cox regression method, we constructed a simple prognosis prediction model composed of 13 aberrant AS genes, and demonstrated that it could well stratify MDS patients into distinct risk groups. To our knowledge, this is the first report demonstrating significant prognostic impacts of aberrant splicing on MDS patients. Further prospective studies in larger cohorts are needed to confirm our observations.

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.

OBJECTIVES 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. METHODS A total of 693 newly diagnosed de novo non-M3 AML patients were consecutively enrolled. We compared relevant mutations in 20 genes between AML patients with or without HL and exposed their prognostic implications. RESULTS Hyperleukocytosis, defined as initial white blood cell counts above 50 000/μL, occurred in 28.9% of AML patients. HL patients had higher incidences of FLT3-ITD, NPM1, DNMT3A, CEBPA, and TET2 mutations. Multivariate analysis demonstrated that HL was an independent poor prognostic factor for overall survival and disease-free survival in total patients, those with intermediate-risk cytogenetics and normal karyotype irrespective of genetic alterations. Intriguingly, HL predicted poor survival in CEBPA double mutated, NPM1 + /FLT3-ITD- and NPM1-/FLT3-ITD- patients. Further, HL patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR) had a significantly longer overall survival and disease-free survival than those without allo-HSCT. CONCLUSIONS Hyperleukocytosis is an independent poor prognostic factor irrespective of cytogenetics and mutation status. Allo-HSCT in first CR seems to ameliorate the poor prognostic impact of HL.

Background DNMT3A 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. Results In 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. Conclusions DNMT3A 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. Electronic supplementary material The online version of this article (10.1186/s13148-018-0476-1) contains supplementary material, which is available to authorized users.

Homeodomain-only protein homeobox (HOPX) is the smallest homeodomain protein. It was regarded as a stem cell marker in several non-hematopoietic systems. While the prototypic homeobox genes such as the HOX family have been well characterized in acute myeloid leukemia (AML), the clinical and biological implications of HOPX in the disease remain unknown. Thus we analyzed HOPX and global gene expression patterns in 347 newly diagnosed de novo AML patients in our institute. We found that higher HOPX expression was closely associated with older age, higher platelet counts, lower white blood cell counts, lower lactate dehydrogenase levels, and mutations in RUNX1, IDH2, ASXL1, and DNMT3A, but negatively associated with acute promyelocytic leukemia, favorable karyotypes, CEBPA double mutations and NPM1 mutation. Patients with higher HOPX expression had a lower complete remission rate and shorter survival. The finding was validated in two independent cohorts. Multivariate analysis revealed that higher HOPX expression was an independent unfavorable prognostic factor irrespective of other known prognostic parameters and gene signatures derived from multiple cohorts. Gene set enrichment analysis showed higher HOPX expression was associated with both hematopoietic and leukemia stem cell signatures. While HOPX and HOX family genes showed concordant expression patterns in normal hematopoietic stem/progenitor cells, their expression patterns and associated clinical and biological features were distinctive in AML settings, demonstrating HOPX to be a unique homeobox gene. Therefore, HOPX is a distinctive homeobox gene with characteristic clinical and biological implications and its expression is a powerful predictor of prognosis in AML patients.

Table S1. Correlation of HOXB-AS3 expression with clinical characteristics and frequent somatic gene mutations in de novo AML patients. Table S2. Correlation of HOXB-AS3 expression with clinical characteristics in MDS patients. Table S3. Correlation of HOXB-AS3 expression with frequent gene mutations in MDS patients. Figure S1. Overview of Hoxb5os in mouse HOXB cluster. Figure S2. Alignment of mouse Hoxb5os and human HOXB-AS3. Figure S3. HOXB-AS3 promotes S phase entering in the cell cycle regulation. Figure S4. GSEA pathway analysis of the differentially expressed genes in HOXB-AS3 knockdown cells compared to the control cells. Figure S5. Cell cycle pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S6. DNA replication pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S7. RB pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S8. G1-S pathway from Wikipathway analysis of downstream pathways affected by downregulation of HOXB-AS3 in the myeloid cell lines. Figure S9. Quantitative PCR analysis of the expressions of indicated genes in TF-1 cells infected with lentivirus carrying pAS5.1w-Pbsd (control), or pAS5.1w-Pbsd-HOXB-AS3 (HOXB-AS3 overexpression). Figure S10. HOXB-AS3 expressions of AML patients and health donors. Figure S11. Survival analysis of AML patients stratified by the expressions of HOXB-AS3. Figure S12. HOXB-AS3 expression of non-APL AML patients and health donors. Figure S13. Survival analysis of non-APL AML patients stratified by the expressions of HOXB-AS3 in the NTUH AML cohort. Figure S14. HOXB-AS3 expressions of MDS patients and health donors. Figure S15. Overall survival of MDS patients stratified by the expressions of HOXB-AS3. Figure S16. Quantitative PCR analysis of the expressions of different variants in TF-1 and OCI/AML3 cell lines. (PDF 2900 kb)

Background: Expression of long non-coding RNAs (lncRNAs) has recently been recognized as a potential prognostic marker in acute myeloid leukemia (AML). However, it remains unclear whether incorporation of the lncRNAs expression in the 2017 European LeukemiaNet (ELN) risk classification can further improve the prognostic prediction. Methods: We enrolled 275 newly diagnosed non-M3 AML patients and randomly assigned them into the training (n=183) and validation cohorts (n=92). In the training cohort, we formulated a prognostic lncRNA scoring system composed of five lncRNAs with significant prognostic impact from the lncRNA expression profiling. Findings: Higher lncRNA scores were significantly associated with older age and adverse gene mutations. Further, the higher-score patients had shorter overall and disease-free survival than lower-score patients, which were also confirmed in both internal and external validation cohorts (TCGA database). The multivariate analyses revealed the lncRNA score was an independent prognosticator in AML, irrespective of the risk based on the 2017 ELN classification. Moreover, in the 2017 ELN intermediate-risk subgroup, lncRNA scoring system could well dichotomize the patients into two groups with distinct prognosis. Within the ELN intermediate-risk subgroup, we found that allogeneic hematopoietic stem cell transplantation could provide better outcome on patients with higher lncRNA scores. Through bioinformatics approach, we identified high lncRNA scores were correlated with leukemia/hematopoietic stem cell signatures. Interpretation: Incorporation of lncRNA scoring system in 2017 ELN classification can improve risk-stratification of AML patients and help clinical decision-making. Funding Statement: This work was supported Ministry of Science and Technology, and Ministry of Health and Welfare of Taiwan. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This study was approved by the Research Ethics Committee of the NTUH in accordance with the Declaration of Helsinki.

Introduction Presence of minimal residual disease (MRD) detected by multicolor flow cytometry (MCFC) or quantitative polymerase chain reaction has been recognized as an independent important prognosticator for patients with acute myeloid leukemia (AML). Next-generation sequencing (NGS) can simultaneously detect various mutations and be applied to the majority of patients with AML, but the clinical implication of its use in MRD monitoring remains to be clarified. Recently, it was shown that NGS MRD of mutants other than the common mutations occurring in clonal hematopoiesis of indeterminate potential, including the DTA (DNMT3A, TET2, and ASXL1) mutations, carry prognostic impacts on relapse rates and overall survival (OS) in AML patients. However, the proper time point for NGS MRD detection after treatment is still unclear. Our hypothesis is that the NGS MRD detected at different time points might have different clinical implications. In this regard, we aimed to explore the clinical implication of NGS MRD at different time points in AML patients after chemotherapy. Method We enrolled 306 de novo non-M3 and non-M6 AML patients who attained complete remission (CR) after standard induction chemotherapy and received 2-4 courses of post-remission chemotherapy with high-dose cytarabine with or without anthracycline. We analyzed bone marrow samples serially collected at diagnosis, first CR (1st time point for MRD analysis), and after the first consolidation chemotherapy (2nd time point). We used the TruSight myeloid panel (Illumina) to survey the 54 genes related to myeloid malignancies. Because of the sequencing sensitivity issue, we excluded CEBPA mutation and FLT3-ITD in the subsequent analyses. The median follow-up time was 92.0 months. Result At diagnosis, 91% of patients had at least one gene mutation with a median of 2.0 mutations (range 1-6) per patient; 49.4% had molecular gene mutations alone and 41.6% had both cytogenetic changes and molecular mutations. Mutations in NPM1, DNMT3A, NRAS and IDH2 were the most common mutations. According to the 2017 ELN recommendation, 49.3% of patients were in the favorable-risk group; 29.1%, the intermediate-risk group; and 21.6%, the unfavorable-risk group. Among the patients harboring at least one gene mutation at diagnosis, we randomly assigned them into the training (n=167) and validation cohort (n=111); the two cohorts had similar clinical features, and distribution of cytogenetic and molecular abnormalities. Based on the result from the analysis in the training cohort, we set 0.3% as the cut-off for MRD positivity because patients carried gene mutations lower than this limit had a similar outcome as those without detectable mutations. The allele frequencies of the mutants in MRD ranged from 0.3 to 50.5%. Excluding DTA mutations, 47.3% patients in the training cohort had MRD at 1st time point, and 26.9% at 2nd time point. The patients with positive NGS MRD had significantly higher relapse rate (P=0.042 for 1st MRD and P=0.035 for 2nd MRD), shorter disease-free survival (DFS, P=0.037 for 1st MRD and P=0.007 for 2nd MRD) and OS (P=0.015 for 1st MRD and P Conclusion NGS-based MRD monitoring can be applied to more than 90% of AML patients who have detectable mutations at diagnosis. The presence of NGS MRD after treatment can predict outcome of AML patients, especially after the first consolidation chemotherapy (2nd MRD). Positivity of 2nd MRD is an independent unfavorable prognostic factor for DFS and OS. Further prospective trials are warranted to validate these findings and to clarify the role of pre-emptive treatment. Disclosures Tsai: Celgene: Research Funding; Astellas, BMS, Celgene, Chugai, Johnson & Johnson, Kirin, Novartis, Pfizer, Roche, Takeda: Honoraria. Tien:Novartis: Other: Travel Grant. Hou:Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. Tien:Celgene: Honoraria; Novartis: Honoraria; Alexion: Honoraria; BMS: Honoraria; Roche: Research Funding; Pfizer: Honoraria; Roche: Honoraria; Celgene: Research Funding; Abbvie: Honoraria; Johnson &Johnson: Honoraria; Daiichi Sankyo: Honoraria.

Introduction One of the contributing factors to the relapse of acute myeloid leukemia (AML) is the presence of leukemia stem cells (LSCs). Interleukin 1 receptor accessory protein (IL1RAP) was reported to be one of the LSC markers. Most studies regarding clinical implications of IL1RAP expression in AML focused on small and selected patient groups. Besides, its correlation with other molecular alterations has not been reported yet in literature. In this study, we aimed to elucidate the relationship between bone marrow IL1RAP expression level and clinical and biological features in patients with de novo non-M3 AML. Furthermore, we would like to explore its prognostic impact and potential underlying mechanism. Method We enrolled 275 newly diagnosed de novo non-M3 AML patients. Among them, 187 (68%) patients received standard induction chemotherapy and 2-4 courses of high-dose cytarabine based post-remission therapy. Analyses of 54 gene mutations were performed by next generation sequencing. The global gene expressions were profiled with the Affymetrix GeneChip Human Transcriptome Array 2.0. Result We used the median as the cut-off value to define the higher and lower IL1RAP expression groups. The patients with higher IL1RAP expression had significantly higher white blood cell counts at diagnosis, higher peripheral blast counts, and higher lactate dehydrogenase levels. Higher IL1RAP expression was closely associated with t(8;21), favorable-risk cytogenetics based on the refined MRC classification, but inversely with unfavorable-risk cytogenetics. Compared with low-expression patients, the high-expression patients had significantly more FLT3/ITD and KIT mutations, but less mutations in U2AF1, TP53, or CEBPA. Among the 187 patients receiving standard intensive chemotherapy, those with lower IL1RAP expression had significantly longer overall survival (OS) than those with higher expression (P=0.047) after a median follow-up time of 91.1 months, but disease-free survival (DFS) was not significantly different between the two groups (P=0.311). Among the 77 patients who relapsed after first complete remission (CR), the second CR rate was similar between the two groups (P=0.649), but the second DFS was significantly longer in the low-expression patients than the high-expression patients (P=0.028) which was also reflected in a significantly longer survival after first relapse in the former group than the latter group (P=0.014). The prognostic impact of IL1RAP expression on OS could be externally validated in the TCGA cohort (P=0.038). Its prognostic implication remained significant in the subgroup of our cohort with intermediate-risk cytogenetics (P=0.006) and those with normal karyotype (P=0.025). In multivariate analysis incorporating age, transplantation status, 2017 ELN risk-stratification and IL1RAP expression as covariates, the higher IL1RAP expression was an independent poor prognostic factor for OS (HR=1.555, P=0.025). The Gene Set Enrichment Analysis revealed significant up-regulation of LSC related genes in the higher IL1RAP expressed patients (Figure 1 and 2). We further profiled genome-wide RNA expression with 70,523 probes to survey the potential molecular mechanisms underlying the IL1RAP expression signature. Totally, 313 differentially expressed genes were identified (>1.5-fold change and Student t-test P Conclusion Higher IL1RAP expression is associated with distinct clinical and genetic alterations. It is an independent prognostic factor for OS irrespective of the risk category based on the ELN classification. Transcription factors, such as GATA1 and GATA2, ABCB6, ELAVL1 and NFκB might be involved in the underlying mechanism. Further prospective large cohort is warrant to validate our findings. Disclosures Tien: Novartis: Other: Travel Grant. Hou:Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. Tien:Daiichi Sankyo: Honoraria; Roche: Honoraria; Abbvie: Honoraria; Alexion: Honoraria; Celgene: Honoraria; Johnson &Johnson: Honoraria; Novartis: Honoraria; Celgene: Research Funding; BMS: Honoraria; Pfizer: Honoraria; Roche: Research Funding.

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

Although clinical features, cytogenetics, and mutations are widely used to predict prognosis in patients with acute myeloid leukemia (AML), further refinement of risk stratification is necessary for optimal treatment, especially in cytogenetically normal (CN) patients. We sought to generate a simple gene expression signature as a predictor of clinical outcome through analyzing the mRNA arrays of 158 de novo CN AML patients. We compared the gene expression profiles of patients with poor response to induction chemotherapy with those who responded well. Forty-six genes expressed differentially between the two groups. Among them, expression of 11 genes was significantly associated with overall survival (OS) in univariate Cox regression analysis in 104 patients who received standard intensive chemotherapy. We integrated the z-transformed expression levels of these 11 genes to generate a risk scoring system. Higher risk scores were significantly associated with shorter OS (median 17.0 months vs. not reached, P < 0.001) in ours and another 3 validation cohorts. In addition, it was an independent unfavorable prognostic factor by multivariate analysis (HR 1.116, 95% CI 1.035~1.204, P = 0.004). In conclusion, we developed a simple mRNA expression signature for prognostication in CN-AML patients. This prognostic biomarker will help refine the treatment strategies for this group of patients.

Background Additional sex combs-like 1 (ASXL1) is frequently mutated in myeloid malignancies. Recent studies showed that hematopoietic-specific deletion of Asxl1 or overexpression of mutant ASXL1 resulted in myelodysplasia-like disease in mice. However, actual effects of a “physiological” dose of mutant ASXL1 remain unexplored. Methods We established a knock-in mouse model bearing the most frequent Asxl1 mutation and studied its pathophysiological effects on mouse hematopoietic system. Results Heterozygotes (Asxl1 tm/+) marrow cells had higher in vitro proliferation capacities as shown by more colonies in cobblestone-area forming assays and by serial re-plating assays. On the other hand, donor hematopoietic cells from Asxl1 tm/+ mice declined faster in recipients during transplantation assays, suggesting compromised long-term in vivo repopulation abilities. There were no obvious blood diseases in mutant mice throughout their life-span, indicating Asxl1 mutation alone was not sufficient for leukemogenesis. However, this mutation facilitated engraftment of bone marrow cell overexpressing MN1. Analyses of global gene expression profiles of ASXL1-mutated versus wild-type human leukemia cells as well as heterozygote versus wild-type mouse marrow precursor cells, with or without MN1 overexpression, highlighted the association of in vivo Asxl1 mutation to the expression of hypoxia, multipotent progenitors, hematopoietic stem cells, KRAS, and MEK gene sets. ChIP-Seq analysis revealed global patterns of Asxl1 mutation-modulated H3K27 tri-methylation in hematopoietic precursors. Conclusions We proposed the first Asxl1 mutation knock-in mouse model and showed mutated Asxl1 lowered the threshold of MN1-driven engraftment and exhibited distinct biological functions on physiological and malignant hematopoiesis, although it was insufficient to lead to blood malignancies. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0508-x) contains supplementary material, which is available to authorized users.

Supplementary methods, figures and table. (PDF 913 kb)

The SF3B1 mutation can be detected in patients with myelodysplastic syndrome (MDS), but the report regarding the association of this mutation with other genetic alterations and its stability during disease progression is limited. In this study, SF3B1 mutations were identified in 10% of total cohort of 479 MDS patients and 61.8% of 34 patients with refractory anemia with ring sideroblasts (RARS). SF3B1 mutations were closely associated with older age, higher platelet counts, lower lactate dehydrogenase levels, good-risk cytogenetics, and mutations of DNMT3A, but inversely related to ASXL1 mutations. Most SF3B1-mutated patients had concurrent other genetic alterations, including DNMT3A and RUNX1 mutations. There was no prognostic difference between patients with SF3B1 mutations and those without. Sequential studies in 417 samples from 142 patients demonstrated that all SF3B1-mutated patients retained the same mutations during disease evolution with the exception of two patients who lost the mutation after allogeneic hematopoietic stem cell transplantation, whereas none of the SF3B1-wild patients acquired a novel mutation during clinical follow-ups. In conclusion, the patients with SF3B1 mutations had distinct clinic-biologic features. SF3B1 mutations, accompanied with other genetic alterations, especially DNMT3A mutations, may play a role in the development of MDS, but have little role in disease progression. Am. J. Hematol. 89:E109–E115, 2014. © 2014 Wiley Periodicals, Inc.

Current information about clinical significance of IDH mutations in myelodysplastic syndromes (MDS), their association with other genetic alterations and the stability during disease progression is limited. In this study, IDH mutations were identified in 4.6% of 477 patients with MDS based on the FAB classification and in 2.2 % of 368 patients based on the 2008 WHO classification. IDH mutations were closely associated with older age, higher platelet counts, and mutations of DNMT3A (36.4% vs. 8.7%, P < 0.001), ASXL1 (47.6% vs. 22.0%, P = 0.007), and SRSF2 (45.5% vs. 11.8%, P < 0.001). IDH2 mutation was a poor prognostic factor for overall survival in patients with lower-risk MDS, based on international prognosis scoring system (IPSS), FAB classification, WHO classification, or revised IPSS (all P ≦ 0.001), but not in higher-risk groups. Sequential studies in 151 patients demonstrated that all IDH-mutated patients retained the same mutation during disease evolution while none of the IDH-wild patients acquired a novel mutation during follow-ups. In conclusion, IDH mutation is a useful biomarker for risk stratification of patients with lower-risk MDS. IDH mutations are stable during the clinical course. The mutation, in association with other genetic alterations, may play a role in the development, but not progression of MDS.

Introduction Recent advances in the discovery of the genomic landscape in AML prompts necessity to re-examine the 2017 European LeukemiaNet (ELN) recommendation. In this study we aimed to validate the usefulness of 2017 ELN risk stratification in a large Taiwan cohort with special focus on the prognostic relevance of FLT3-ITD allelic ratio and its interaction with other mutations. Methods We retrospectively included 1040 de novo non-M3 AML patients. AML was risk-stratified according to the 2017 ELN recommendation. 739 (71.1%) patients who received standard chemotherapy were included for survival analysis. Allogeneic hematopoietic stem cell transplantation (HSCT) was performed in 293 (39.6%) patients. Mutational analyses of fifteen genes, including CEBPA, NPM1, FLT3, RUNX1, ASXL1, TP53, splicing factors (SF), such as SRSF2, U2AF1, and SF3B1, as well as KIT, NRAS, KRAS, DNMT3A, TET2, and WT1 were performed. FLT3-ITD/wild allelic ratios were calculated as the ratio of the area under the curve by fragment analysis. High FLT3-ITD allelic ratio (FLT3-ITDhigh) was defined as ³ 0.5 and low allelic ratio (FLT3-ITDlow) defined as < 0.5. Results According to the 2017 ELN risk classification, favorable, intermediate and adverse categories comprised 34.6%, 29.2% and 36.2% patients, respectively. NPM1 mutations and FLT3-ITD, the most common mutations in this cohort, were detected in 217 (20.9%) and 216 (20.8%) patients, respectively, with a significant association between each other. The median value of the FLT3-ITD/wild ratio was 0.68 without difference between NPM1-mutated and NPM1-wild group. Of note, patients with FLT3-ITDhigh had higher WBC count and LDH level than those with FLT3-ITDlow. Overall, the CR rate and relapse rate were 74.2% and 54.7%, respectively and 5-year overall survival (OS) was 43.2±1.9%. The CR rate (92.3%) was higher in the 2017 ELN favorable risk group than in the intermediate (73.0%) and adverse groups (52.0%, P As to the prognostic impact of FLT3-ITD, we showed that FLT3-ITD patients had significantly lower CR rate, higher relapse rate, reduced DFS and OS than those without. There was a strikingly difference in treatment response between the low and high FLT3-ITD allelic ratio groups: CR rate (80.7% vs. 63.6%, P=0.0319), relapse rate (56.5% vs. 66.2%, P=0.329), DFS (14.2 vs. 4.6 months P=0.011) and OS (24.0 vs. 11.9 months, P=0.048). Interestingly, patients with FLT3-ITDhigh had a better OS if they received allogeneic HSCT than those who did not. Among the 2017 ELN favorable-risk category, we found that patients with mutated NPM1 and FLT3-ITDlow had significantly shorter OS (median, not reached vs. 31.6 months, P=0.003, Figure. 1A) and a trend of shorter DFS (median 14.9 months vs. 93.9 months, P=0.089, Figure. 1B) compared to other ELN favorable subgroups. To find the cause of the difference, we investigated the concurrent mutations in the patients with mutated NPM1 and FLT3-ITDlow. 46.2% of them had concurrent poor-risk mutations, such as ASXL1, RUNX1, TP53, WT1, TET2, DNMT3A, and SF mutations. Similarly, among the 2017 ELN intermediate-risk category, patients with mutated NPM1 and FLT3-ITDhigh had more unfavorable outcomes compared to those with wild-type NPM1 and without FLT3-ITD (DFS, median 3.7 vs. 11.6 months, P=0.028 and OS, median, 11.4 vs. 26.5 months, P=0.067). Presence of concurrent poor-risk mutations were also identified in 72.9% of these patients. Based on these findings, we postulated that concomitant poor-risk genetic alterations at least partially affected the prognosis of FLT3/ITD patients. In the cohort of FLT3-ITD patients, patients harboring poor-risk mutations had shorter DFS and OS than those without (P=0.028 and P=0.031, respectively). Further, co-occurrence of FLT3-ITDhigh and poor-risk mutations that predicted a worst outcome, seemed to define a highly adverse prognostic group. Conclusions We showed that ELN 2017 risk classification could well stratify AML patients in Taiwan. The prognostic relevance of FLT3-ITD may further depend on the presence or absence of co-occurring poor-risk genetic alterations, which seemed to add an adverse effect in patients with FLT3-ITD. These observations warrant confirmation in other prospective and large-scale studies. Disclosures Ko: Roche: Research Funding; GNT Biotech & Medicals Crop.: Research Funding; Abbevie: Research Funding; Mumdipharma Taiwan: Consultancy.

Conventionally, acute myeloid leukemia (AML) patients are categorized into good-, intermediate- and poor-risk groups according to cytogenetic changes. However, patients with intermediate-risk cytogenetics represent a largely heterogeneous population regarding treatment response and clinical outcome. In this study, we integrated cytogenetics and molecular mutations in the analysis of 318 patients with de novo non-M3 AML who received standard chemotherapy. According to the mutation status of eight genes, including NPM1, CEBPA, IDH2, RUNX1, WT1, ASXL1, DNMT3A and FLT3, that had prognostic significance, 229 patients with intermediate-risk cytogenetics could be refinedly stratified into three groups with distinct prognosis (P

Recurrent somatic mutation of SRSF2, one of the RNA splicing machinery genes, has been identified in a substantial proportion of patients with myelodysplastic syndrome (MDS). However, the clinical and biologic characteristics of MDS with this mutation remain to be addressed. In this study, 34 (14.6%) of the 233 MDS patients were found to have SRSF2 mutation. SRSF2 mutation was closely associated with male sex (P = .001) and older age (P < .001). It occurred concurrently with at least 1 additional mutation in 29 patients (85.3%) and was closely associated with RUNX1, IDH2, and ASXL1 mutations (P = .004, P < .001, and P < .001, respectively). Patients with SRSF2 mutation had an inferior overall survival (P = .010), especially in the lower risk patients. Further exploration showed that the prognostic impact of SRSF2 mutation might be attributed to its close association with old age. Sequential analyses in 173 samples from 66 patients showed that all SRSF2-mutated patients retained their original mutations, whereas none of the SRSF2-wild patients acquired a novel mutation during disease evolution. In conclusion, SRSF2 mutation is associated with distinct clinical and biologic features in MDS patients. It is stable during the clinical course and may play little role in disease progression.

DNMT3A mutations are associated with poor prognosis in acute myeloid leukemia (AML), but the stability of this mutation during the clinical course remains unclear. In the present study of 500 patients with de novo AML, DNMT3A mutations were identified in 14% of total patients and in 22.9% of AML patients with normal karyotype. DNMT3A mutations were positively associated with older age, higher WBC and platelet counts, intermediate-risk and normal cytogenetics, FLT3 internal tandem duplication, and NPM1, PTPN11, and IDH2 mutations, but were negatively associated with CEBPA mutations. Multivariate analysis demonstrated that the DNMT3A mutation was an independent poor prognostic factor for overall survival and relapse-free survival in total patients and also in normokaryotype group. A scoring system incorporating the DNMT3A mutation and 8 other prognostic factors, including age, WBC count, cytogenetics, and gene mutations, into survival analysis was very useful in stratifying AML patients into different prognostic groups (P < .001). Sequential study of 138 patients during the clinical course showed that DNMT3A mutations were stable during AML evolution. In conclusion, DNMT3A mutations are associated with distinct clinical and biologic features and poor prognosis in de novo AML patients. Furthermore, the DNMT3A mutation may be a potential biomarker for monitoring of minimal residual disease.

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.

The impact of WT1 mutations in acute myeloid leukemia (AML) is not completely settled. We aimed to determine the clinical implication of WT1 mutation in 470 de novo non-M3 AML patients and its stability during the clinical course. WT1 mutations were identified in 6.8% of total patients and 8.3% of younger patients with normal karyotype (CN-AML). The WT1 mutation was closely associated with younger age (P < .001), French-American-British M6 subtype (P = .006), and t(7;11)(p15;p15) (P = .003). Multivariate analysis demonstrated that the WT1 mutation was an independent poor prognostic factor for overall survival and relapse-free survival among total patients and the CN-AML group. A scoring system incorporating WT1 mutation, NPM1/FLT3-ITD, CEBPA mutations, and age into survival analysis proved to be very useful to stratify CN-AML patients into different prognostic groups (P < .001). Sequential analyses were performed on 133 patients. WT1 mutations disappeared at complete remission in all WT1-mutated patients studied. At relapse, 3 of the 16 WT1-mutated patients who had paired samples lost the mutation and 2 acquired additional mutations, whereas 3 of 110 WT1-wild patients acquired novel mutations. In conclusion, WT1 mutations are correlated with poor prognosis in AML patients. The mutation status may be changed in some patients during AML progression.