Poor prognostic implications of myelodysplasia-related mutations in both older and younger patients with de novo AML.

A set of myelodysplasia-related (MDS-R) gene mutations are incorporated into the 2022 European LeukemiaNet risk classification as adverse genetic factors for acute myeloid leukemia (AML) based on their poor prognostic impact on older patients. The impact of these mutations on younger patients (age < 60 years) remains elusive. In the study of 1213 patients with de novo non-M3 AML, we identified MDS-R mutations in 32.7% of the total cohort, 44.9% of older patients and 23.4% of younger patients. The patients with MDS-R mutations had a significantly lower complete remission rate in both younger and older age groups. With a median follow-up of 9.2 years, the MDS-R group experienced shorter overall survival (P = 0.034 for older and 0.035 for younger patients) and event-free survival (P = 0.004 for older and 0.042 for younger patients). Furthermore, patients with MDS-R mutations more frequently harbored measurable residual disease that was detectable using next generation sequencing at morphological CR than those without MDS-R mutations. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) might ameliorate the negative impact of MDS-R mutations. In summary, AML patients with MDS-R mutations have significantly poorer outcomes regardless of age. More intensive treatment, such as allo-HSCT and/or novel therapies, is warranted for AML patients with MDS-R mutations.

Investigations on the Wound Healing Potential of Tilapia Piscidin (TP)2-5 and TP2-6.

Wound healing is a highly orchestrated process involving many cell types, such as keratinocytes, fibroblasts and endothelial cells. This study aimed to evaluate the potential application of synthetic peptides derived from tilapia piscidin (TP)2, TP2-5 and TP2-6 in skin wound healing. The treatment of HaCaT keratinocytes with TP2-5 and TP2-6 did not cause cytotoxicity, but did enhance cell proliferation and migration, which could be attributed to the activation of epidermal growth factor receptor signaling. In CCD-966SK fibroblasts, although TP2-5 (31.25 µg/mL) and TP2-6 (125 µg/mL) showed cytotoxic effects, we observed the significant promotion of cell proliferation and migration at low concentrations. In addition, collagen I, collagen III, and keratinocyte growth factor were upregulated by the peptides. We further found that TP2-5 and TP2-6 showed pro-angiogenic properties, including the enhancement of human umbilical vein endothelial cell (HUVEC) migration and the promotion of neovascularization. In a murine model, wounds treated topically with TP2-5 and TP2-6 were reduced by day 2 post-injury and healed significantly faster than untreated wounds. Taken together, these findings demonstrate that both TP2-5 and TP2-6 have multifaceted effects when used as topical agents for accelerating wound healing.

Distinct clinico-biological features in AML patients with low allelic ratio FLT3-ITD: role of allogeneic stem cell transplantation in first remission.

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 < 0.5), clinical features, as well as genomic and transcriptomic profiles remain unclear, and evidence supporting allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) remains controversial. This study aimed to elucidate the genomic features, prognosis, and transplantation outcome of FLT3-ITDIow in AML patients with intermediate-risk cytogenetics. FLT3-ITDlow was associated with a negative enrichment of the leukemic stem cell signature, a marked enrichment of the RAS pathway, and with higher frequencies of RAS pathway mutations, different from those with FLT3-ITDhigh. Concurrent CEBPA double mutations were favorable prognostic factors, whereas MLL-PTD, and mutations in splicing factors were unfavorable prognostic factors in FLT3-ITDlow patients. Patients with FLT3-ITDlow had a shorter overall survival (OS) and event-free survival (EFS) than those with FLT3wt. Allo-HSCT in CR1 was associated with a significantly longer OS and EFS compared with postremission chemotherapy in patients with FLT3-ITDlow. In conclusion, FLT3-ITDlow is associated with different mutational and transcriptomic profiles compared with FLT3-ITDhigh. The presence of concomitant poor-risk mutations exert negative prognostic impacts in patients with FLT3-ITDlow, who markedly benefit from allo-HSCT in CR1.

Tilapia Piscidin 4 (TP4) Reprograms M1 Macrophages to M2 Phenotypes in Cell Models of Gardnerella vaginalis-Induced Vaginosis.

Gardnerella vaginalis is associated with bacterial vaginosis (BV). The virulence factors produced by G. vaginalis are known to stimulate vaginal mucosal immune response, which is largely driven by activated macrophages. While Tilapia piscidin 4 (TP4), an antimicrobial peptide isolated from Nile tilapia, is known to display a broad range of antibacterial functions, it is unclear whether TP4 can affect macrophage polarization in the context of BV. In this study, we used the culture supernatants from G. vaginalis to stimulate differentiation of THP-1 and RAW264.7 cells to an M1 phenotype. The treatment activated the NF-κB/STAT1 signaling pathway, induced reactive nitrogen and oxygen species, and upregulated inflammatory mediators. We then treated the induced M1 macrophages directly with a non-toxic dose of TP4 or co-cultured the M1 macrophages with TP4-treated vaginal epithelial VK2 cells. The results showed that TP4 could not only decrease pro-inflammatory mediators in the M1 macrophages, but it also enriched markers of M2 macrophages. Further, we found that direct treatment with TP4 switched M1 macrophages toward a resolving M2c phenotype via the MAPK/ERK pathway and IL-10-STAT3 signaling. Conversely, tissue repair M2a macrophages were induced by TP4-treated VK2 cells; TP4 upregulated TSG-6 in VK2 cells, which subsequently activated STAT6 and M2a-related gene expression in the macrophages. In conclusion, our results imply that TP4 may be able to attenuate the virulence of G. vaginalis by inducing resolving M2c and tissue repair M2a macrophage polarizations, suggesting a novel strategy for BV therapy.

Clinical implications of sequential MRD monitoring by NGS at 2 time points after chemotherapy in patients with AML.

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.

Incorporation of long non-coding RNA expression profile in the 2017 ELN risk classification can improve prognostic prediction of acute myeloid leukemia patients.

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 to 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. FUND: This work was supported Ministry of Science and Technology, and Ministry of Health and Welfare of Taiwan.

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.

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.

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

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.

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

Histone Methyltransferase G9a Drives Chemotherapy Resistance by Regulating the Glutamate-Cysteine Ligase Catalytic Subunit in Head and Neck Squamous Cell Carcinoma.

Transient chemotherapeutic response is a major obstacle to treating head and neck squamous cell carcinomas (HNSCC). Histone methyltransferase G9a has recently been shown to be abundantly expressed in HNSCC, and is required to maintain the malignant phenotype. In this study, we found that high G9a expression is significantly associated with poor chemotherapeutic response and disease-free survival in HNSCC patients. Similarly, G9a expression and enzymatic activity were elevated in cisplatin-resistant HNSCC cells. Genetic or pharmacologic inhibition of G9a sensitized the resistant cells to cisplatin, increasing cellular apoptosis. Mechanistic investigations indicated that G9a contributes to transcriptional activation of the glutamate-cysteine ligase catalytic subunit (GCLC), which results in upregulation of cellular glutathione (GSH) and drug resistance. In addition, we observed a significant positive correlation between G9a and GCLC expression in tumors of HNSCC patients. Taken together, our findings provide evidence that G9a protects HNSCC cells against chemotherapy by increasing the synthesis of GSH, and imply G9a as a promising target for overcoming cisplatin resistance in HNSCC. Mol Cancer Ther; 16(7); 1421-34. ©2017 AACR.

Distinct mutation profile and prognostic relevance in patients with hypoplastic myelodysplastic syndromes (h-MDS).

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematologic malignancies. Although most MDS patients have normal or increased BM cellularity (NH-MDS), some have hypocellular BM (h-MDS). The reports concerning the differences in genetic alterations between h-MDS and NH-MDS patients are limited. In this study, 369 MDS patients diagnosed according to the WHO 2008 criteria were recruited. h-MDS patients had lower PB white blood cell and blast counts, and lower BM blast percentages, than those with NH-MDS. h-MDS was closely associated with lower-risk MDS, defined by the International Prognostic Scoring System (IPSS) and revised IPSS (IPSS-R). IPSS-R could properly predict the prognosis in h-MDS (P<0.001) as in NH-MDS patients. The h-MDS patients had lower incidences of RUNX1, ASXL1, DNMT3A, EZH2 and TP53 mutations than NH-MDS patients. The cumulated incidence of acute leukemic transformation at 5 years was 19.3% for h-MDS and 40.4% for NH-MDS patients (P= 0.001). Further, the patients with h-MDS had longer overall survival (OS) than those with NH-MDS (P= 0.001), and BM hypocellularity remains an independent favorable prognostic factor for OS irrespective of age, IPSS-R, and gene mutations. Our findings provide evidence that h-MDS indeed represent a distinct clinico-biological subgroup of MDS and can predict better leukemia-free survival and OS.

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.

Clinical and prognostic implications of Roundabout 4 (robo4) in adult patients with acute myeloid leukemia.

BACKGROUND: Robo4 is involved in hematopoietic stem/progenitor cell homeostasis and essential for tumor angiogenesis. Expression of Robo4 was recently found in solid tumors and leukemia stem cells. However, the clinical implications of Robo4 expression in patients with acute myeloid leukemia (AML) remain unclear. METHODS: We investigated the clinical and prognostic relevance of mRNA expression of Robo4 in bone marrow (BM) mononuclear cells from 218 adult patients with de novo AML. We also performed immunohistochemical staining to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the cohort. RESULTS: Higher Robo4 expression was closely associated with lower white blood cell counts, expression of HLA-DR, CD13, CD34 and CD56 on leukemia cells, t(8;21) and ASXL1 mutation, but negatively correlated with t(15;17) and CEBPA mutation. Compared to patients with lower Robo4 expression, those with higher expression had significantly shorter disease-free survival (DFS) and overall survival (OS). This result was confirmed in an independent validation cohort. Furthermore, multivariate analyses showed that higher Robo4 expression was an independent poor prognostic factor for DFS and OS in total cohort and patients with intermediate-risk cytogenetics, irrespective of age, WBC count, karyotype, and mutation status of NPM1/FLT3-ITD, and CEBPA. CONCLUSIONS: BM Robo4 expression can serve as a new biomarker to predict clinical outcomes in AML patients and Robo4 may serve as a potential therapeutic target in patients with higher Robo4 expression.

GATA2 mutations in patients with acute myeloid leukemia-paired samples analyses show that the mutation is unstable during disease evolution.

Recently, mutations of the GATA binding protein 2 (GATA2) gene were identified in acute myeloid leukemia (AML) patients with CEBPA double mutations (CEBPA (double-mut)), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, 14 different missense GATA2 mutations, which were all clustered in the highly conserved N-terminal zinc finger 1 domain, were identified in 27.4, 6.7, and 1 % of patients with CEBPA (double-mut), CEBPA (single-mut), and CEBPA wild type, respectively. All but one patient with GATA2 mutation had concurrent CEBPA mutation. GATA2 mutations were closely associated with younger age, FAB M1 subtype, intermediate-risk cytogenetics, expression of HLA-DR, CD7, CD15, or CD34 on leukemic cells, and CEBPA mutation, but negatively associated with FAB M4 subtype, favorable-risk cytogenetics, and NPM1 mutation. Patients with GATA2 mutation had significantly better overall survival and relapse-free survival than those without GATA2 mutation. Sequential analysis showed that the original GATA2 mutations might be lost during disease progression in GATA2-mutated patients, while novel GATA2 mutations might be acquired at relapse in GATA2-wild patients. In conclusion, AML patients with GATA2 mutations had distinct clinic-biological features and a favorable prognosis. GATA2 mutations might be lost or acquired at disease progression, implying that it was a second hit in the leukemogenesis of AML, especially those with CEBPA mutation.

The N-terminal CEBPA mutant in acute myeloid leukemia impairs CXCR4 expression.

CXC chemokine receptor 4 (CXCR4) is an essential regulator for homing and maintenance of hematopoietic stem cells within the bone marrow niches. Analysis of clinical implications of bone marrow CXCR4 expression in patients with acute myeloid leukemia showed not only higher CXCR4 expression was an independent poor prognostic factor, irrespective of age, white blood cell counts, cytogenetics, and mutation status of NPM1/FLT3-ITD and CEBPA, but also showed CXCR4 expression was inversely associated with mutations of CEBPA, a gene encoding transcription factor C/EBPα. Patients with wild-type CEBPA had significantly higher CXCR4 expression than those with mutated CEBPA. We hypothesized that CEBPA might influence the expression of CXCR4. To test this hypothesis, we first examined endogenous CXCR4 expression in 293T and K562 cells over-expressing wild-type C/EBPα p42 and demonstrated that CXCR4 levels were increased in these cells, whilst the expression of the N-terminal mutant, C/EBPα p30, diminished CXCR4 transcription. We further showed p42 was bound to the CXCR4 promoter by the chromatin immunoprecipitation assays. Induction of p42 in the inducible K562-C/EBPα cell lines increased the chemotactic migration. Moreover, decreased expression of C/EBPα by RNA interference decreased levels of CXCR4 protein expression in U937 cells, thereby abrogating CXCR4-mediated chemotaxis. Our results provide, for the first time, evidence that C/EBPα indeed regulates the activation of CXCR4, which is critical for the homing and engraftment of acute myeloid leukemia cells, while p30 mutant impairs CXCR4 expression.

IPSS-R in 555 Taiwanese patients with primary MDS: Integration of monosomal karyotype can better risk-stratify the patients.

The revised International Prognostic Scoring System (IPSS-R) was recently developed to better assess the clinical outcome of adult patients with myelodysplastic syndrome (MDS). In this study, we aimed to investigate the prognostic impact of this new risk model on 555 MDS patients in Taiwan. Generally, the IPSS-R could discriminate MDS patients regarding risk of leukemia evolution and overall survival in our cohort and it further refined prognostic stratification in all IPSS risk categories. However, we could not find the inter-group difference between IPSS-R very low and low risk subgroups in both leukemia-free survival (LFS) and overall survival (OS). IPSS-R couldn't distinguish the prognosis between very good and good and between good and intermediate risk cytogenetic categories in OS, and between very good and good and between intermediate and poor cytogenetic-risk categories in LFS, either. On the other hand, incorporation of monosomal karyotype (MK) into IPSS-R could further stratify MDS patients with higher-risk IPSS-R (intermediate, high and very high risk) into four groups, rather than three groups, with different OS (P < 0.001). Intriguingly, patients receiving allogeneic hematopoietic stem cell transplantation had longer survival than those without in the IPSS-R high and very high, but not other risk groups. Similarly, patients treated with hypomethylating agents had better survival than those not in the IPSS-R very high risk group. In conclusion, IPSS-R can risk-stratify MDS patients in Taiwan but with some limitations, especially in very low risk category, and MK has additional prognostic value in discriminating MDS patients with higher-risk IPSS-R.