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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.

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

The SOX4 transcription factor is a key regulator of embryonic development, cell-fate decision, cellular differentiation and oncogenesis. Abnormal expression of SOX4 is related to malignant tumor transformation and cancer metastasis. However, no reports are available regarding the clinical significance of SOX4 in acute myeloid leukemia (AML) and the role of SOX4 in leukemogenesis. In the current study, we found that AML patients with low bone marrow (BM) SOX4 expression had higher remission rates and longer overall survival than those with high SOX4 expression, regardless of age, white blood cell count at diagnosis, karyotype profile and NPM1/FLT3-ITD status. To elucidate the role of SOX4 in leukemogenesis, we generated a transgenic zebrafish model that overexpressed human SOX4 in the myeloid lineage Tg(spi1-SOX4-EGFP). These transgenic zebrafish showed, at 5 months of age, increased myelopoiesis with dedifferentiation in kidney marrow. At 9 months of age, their kidney structure was significantly effaced and distorted by increased infiltration of myeloid progenitor cells. These results suggest that SOX4 is not only an independent prognostic factor of AML, but also an important molecular factor in leukemogenesis.

Suppressor of cytokine signaling 1 (SOCS1) protein, which encodes a member of signal transducers and activators of transcription-induced inhibitors, takes part in a negative regulation of cytokine signaling. The mechanism of SOCS1 in tumor carcinogenesis is complex and there have been no studies concerning the clinic-biologic implication of SOCS1 expression in acute myeloid leukemia (AML). Here, we first identified that higher bone marrow (BM) SOCS1 expression was closely associated with older age, FLT3-ITD, NPM1 and DNMT3A mutations, but negatively correlated with CEBPA mutation in patients with de novo AML. Compared to patients with lower SOCS1 expression, those with higher expression had lower complete remission rates and shorter overall survival. Further, higher expression of SOCS1 in the BM was an independent unfavorable prognostic factor irrespective of age, white blood cell, cytogenetics and gene mutations. Next, we generated zebrafish model overexpressing SOCS1 by spi1 promoter, which showed kidney marrow from adult SOCS1 zebrafish had increased myelopoiesis, myeloid progenitors and the kidney or spleen structure were effaced and distorted, mimicking leukemia phenotype. The SOCS1/FLT3-ITD double transgenic fish could further facilitate the leukemic process. The results indicate SOCS1 plays an important role in AML and its higher expression serves as a new biomarker to risk-stratify AML patients.

The TP53 mutation is frequently detected in acute myeloid leukemia (AML) patients with complex karyotype (CK), but the stability of this mutation during the clinical course remains unclear. In this study, TP53 mutations were identified in 7% of 500 patients with de novo AML and 58.8% of patients with CK. TP53 mutations were closely associated with older age, lower white blood cell (WBC) and platelet counts, FAB M6 subtype, unfavorable-risk cytogenetics and CK, but negatively associated with NPM1 mutation, FLT3/ITD and DNMT3A mutation. Multivariate analysis demonstrated that TP53 mutation was an independent poor prognostic factor for overall survival and disease-free survival among the total cohort and the subgroup of patients with CK. A scoring system incorporating TP53 mutation and nine other prognostic factors, including age, WBC counts, cytogenetics and gene mutations, into survival analysis proved to be very useful to stratify AML patients. Sequential study of 420 samples showed that TP53 mutations were stable during AML evolution, whereas the mutation was acquired only in 1 of the 126 TP53 wild-type patients when therapy-related AML originated from different clone emerged. In conclusion, TP53 mutations are associated with distinct clinic-biological features and poor prognosis in de novo AML patients and are rather stable during disease progression.

Recently, mutations of the additional sex comb-like 1 (ASXL1) gene were identified in patients with myelodysplastic syndrome (MDS), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, ASXL1 mutations were identified in 106 (22.7%) of the 466 patients with primary MDS based on the French-American-British (FAB) classification and 62 (17.1%) of the 362 patients based on the World Health Organization (WHO) classification. ASXL1 mutation was closely associated with trisomy 8 and mutations of RUNX1, EZH2, IDH, NRAS, JAK2, SETBP1 and SRSF2, but was negatively associated with SF3B1 mutation. Most ASXL1-mutated patients (85%) had concurrent other gene mutations at diagnosis. ASXL1 mutation was an independent poor prognostic factor for survival. Sequential studies showed that the original ASXL1 mutation remained unchanged at disease progression in all 32 ASXL1-mutated patients but were frequently accompanied with acquisition of mutations of other genes, including RUNX1, NRAS, KRAS, SF3B1, SETBP1 and chromosomal evolution. On the other side, among the 80 ASXL1-wild patients, only one acquired ASXL1 mutation at leukemia transformation. In conclusion, ASXL1 mutations in association with other genetic alterations may have a role in the development of MDS but contribute little to disease progression.