Concomitant targeting of FLT3 and SPHK1 exerts synergistic cytotoxicity in FLT3-ITD+ acute myeloid leukemia by inhibiting ß-catenin activity via the PP2A-GSK3ß axis.

BACKGROUND: Approximately 25-30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML. METHODS: We performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, ß-catenin, PP2A, and GSK3ß. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results. RESULTS: We demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates ß-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit ß-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3ß (GSK3ß) pathway. CONCLUSIONS: These findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.

Influence of genetic co-mutation on chemotherapeutic outcome in NPM1-mutated and FLT3-ITD wild-type AML patients.

BACKGROUND: Nucleophosmin 1 (NPM1) gene-mutated acute myeloid leukemia (NPM1mut AML) is classified as a subtype with a favorable prognosis. However, some patients fail to achieve a complete remission or relapse after intensified chemotherapy. Genetic abnormalities in concomitant mutations contribute to heterogeneous prognosis of NPM1mut AML patients. METHODS: In this study, 91 NPM1-mutated and FLT3-ITD wild-type (NPM1mut/FLT3-ITDwt) AML patients with intermediate-risk karyotype were enrolled to analyze the impact of common genetic co-mutations on chemotherapeutic outcome. RESULTS: Our data revealed that TET1/2 (52/91, 57.1%) was the most prevalent co-mutation in NPM1mut AML patients, followed by IDH1/2 (36/91, 39.6%), DNMT3A (35/91, 38.5%), myelodysplastic syndrome related genes (MDS-related genes) (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes) (35/91, 38.5%), FLT3-TKD (27/91, 29.7%) and GATA2 (13/91, 14.3%) mutations. Patients with TET1/2mut exhibited significantly worse relapse-free survival (RFS) (median, 28.7 vs. not reached (NR) months; p = 0.0382) compared to patients with TET1/2wt, while no significant difference was observed in overall survival (OS) (median, NR vs. NR; p = 0.3035). GATA2mut subtype was associated with inferior OS (median, 28 vs. NR months; p < 0.0010) and RFS (median, 24 vs. NR months; p = 0.0224) compared to GATA2wt. By multivariate analysis, GATA2mut and MDS-related genesmut were independently associated with worse survival. CONCLUSION: Mutations in TET1/2, GATA2 and MDS-related genes were found to significantly influence the chemotherapeutic outcome of patients with NPM1mut AML. The findings of our study have significant clinical implications for identifying patients who have an adverse response to frontline chemotherapy and provide a novel reference for further prognostic stratification of NPM1mut/FLT3-ITDwt AML patients.

Homoharringtonine may help improve the outcomes of venetoclax and azacitidine in AML1-ETO positive acute myeloid leukemia.

PURPOSE: T(8;21)(q22;q22.1)/AML1-ETO positive acute myeloid leukemia (AE-AML) is sensitive to conventional chemotherapy with a favorable prognosis. However, recent small case reports suggest the limited effectiveness of venetoclax (VEN) and hypomethylating agents (HMA) in treating AE-AML. The aim of this retrospective study was to evaluate the effectiveness of VEN plus AZA (VA) in AE-AML and explore whether adding homoharringtonine (HHT) to VA (VAH) could improve the response. METHODS: Patients who received VEN plus AZA and HHT (VAH) or VEN plus AZA (VA) regimens were included in this retrospective study. The endpoints of this study were to evaluate the rate of composite complete remission (CRc), measurable residual disease (MRD), event-free survival (EFS), overall survival (OS), and relapse between VAH and VA groups. RESULTS: A total of 32 AE-AML patients who underwent VA or VAH treatments (newly diagnosed with VA, ND-VA, n = 8; relapsed/refractory with VA, R/R-VA, n = 10; relapsed/refractory with VAH, R/R-VAH, n = 14) were included. The CR (complete remission) /CRi (CR with incomplete count recovery) rate of ND-VA, R/R-VA and R/R-VAH were 25%, 10%, and 64.3%, respectively. Measurable residual disease (MRD) negative was observed in 66.7% of R/R-VAH and none of VA-R/R patients. Co-occurring methylation mutations are associated with poor outcomes with VA but exhibit a more favorable response with VAH treatment. Additionally, patients with c-kit mutation presented inferior outcomes with both VEN-based regimens. All regimens were tolerated well by all patients. CONCLUSION: Our data confirmed the poor response of VA in AE-AML, whether used as frontline or salvage therapy. Adding HHT to VA may improve outcomes and enhance the efficacy of VEN in this population.

Impact of different CEBPA mutations on therapeutic outcome in acute myeloid leukemia.

Biallelic mutations of the CEBPA gene (CEBPAbi) are generally associated with favorable prognosis in patients with acute myeloid leukemia (AML). Monoallelic mutations of the CEBPA gene in carboxy-terminal DNA-binding region (CEBPAsmbZIP) and amino-terminal transactivation domains (CEBPAsmTAD) indicate distinct clinical characteristics and therapeutic outcomes. However, further investigation is required to fully understand these differences. In this retrospective study, we enrolled 77 AML patients with CEBPA mutations, including 53 with CEBPAbi, 12 with CEBPAsmbZIP and 12 with CEBPAsmTAD. The clinical characteristics of the three CEBPAmut groups presented significant differences in age, FAB classification, hemoglobin level and platelet count at diagnosis. The CEBPAsmTAD group exhibited shorter 2-year overall survival (OS) and relapse-free survival (RFS) compared to the CEBPAbi group and CEBPAsmbZIP group in AML patients. The most common co-mutations observed in CEBPAmut AML patients were TET2 and GATA2, which had no effect on prognosis. 2-year RFS of 27 CEBPAmut AML patients who underwent allo-HSCT was better than those who did not. MRD3 positive was identified as an influencing factor for 2-year OS and RFS. Allo-HSCT was found to improve the prognosis of CEPBAmut AML patients with positive MRD3 and adverse co-mutations.

Sorafenib plus triplet therapy with venetoclax, azacitidine and homoharringtonine for refractory/relapsed acute myeloid leukemia with FLT3-ITD: A multicenter phase 2 study.

BACKGROUND: Patients with relapsed or refractory acute myeloid leukemia (R/R AML) and FLT3-internal tandem duplication (FLT3-ITD) respond infrequently to salvage chemotherapy. OBJECTIVE: To investigate the efficacy of sorafenib plus triplet therapy with venetoclax, azacitidine, and homoharringtonine (VAH) as a salvage therapy in this population. METHODS: This multicenter, single-arm, phase 2 study was conducted at 12 hospitals across China. Eligible patients had R/R AML with FLT3-ITD (aged 18-65 years) who were treated with VAH. The primary endpoint was composite complete remission (CRc) after two cycles. Secondary outcomes included the overall response rate (ORR), safety, and survival. RESULTS: Between July 9, 2020, and March 19, 2022, 58 patients were assessed for eligibility, 51 of whom were enrolled. The median patient age was 47 years (interquartile range [IQR] 31-57). CRc was 76.5% with ORR of 82.4%. At a median follow-up of 17.7 months (IQR, 8.7-24.7), the median duration of CRc was not reached (NR), overall survival was 18.1 months (95% confidence interval [CI], 11.8-NR) and event-free survival was 11.4 months (95% CI, 5.6-NR). Grade 3 or 4 adverse events occurring in ≥10% of patients included neutropenia in 47 (92.2%), thrombocytopenia in 41 (80.4%), anemia in 35 (68.6%), febrile neutropenia in 29 (56.9%), pneumonia in 13 (25.5%), and sepsis in 6 (11.8%) patients. Treatment-related death occurred in two (3.9%) patients. CONCLUSIONS: The sorafenib plus VAH regimen was well tolerated and highly active against R/R AML with FLT3-ITD. This regimen may be a suitable therapeutic option for this population, but larger population trials are needed to be explored. TRIAL REGISTRATION: Clinical Trials Registry: NCT04424147.

Changing epidemiology, microbiology and mortality of bloodstream infections in patients with haematological malignancies before and during SARS-CoV-2 pandemic: a retrospective cohort study.

OBJECTIVE: This study was to explore the changes in bacterial bloodstream infection (BSI) in patients with haematological malignancies (HMs) before and during SARS-CoV-2 pandemic. DESIGN: Retrospective cohort study between 2018 and 2021. SETTING: The largest haematological centre in southern China. RESULTS: A total of 599 episodes of BSI occurring in 22 717 inpatients from January 2018 to December 2021 were analysed. The frequencies of the total, Gram-negative and Gram-positive BSI before and during the pandemic were 2.90% versus 2.35% (p=0.011), 2.49% versus 1.77% (p<0.001) and 0.27% versus 0.44% (p=0.027), respectively. The main isolates from Gram-negative or Gram-positive BSI and susceptibility profiles also changed. The 30-day mortality caused by BSI was lower during the pandemic (21.1% vs 14.3%, p=0.043). Multivariate analysis revealed that disease status, pulmonary infection and shock were independent predictors of 30-day mortality. CONCLUSION: Our data showed that the incidence of total and Gram-negative organisms BSI decreased, but Gram-positive BSI incidence increased in patients with HMs during the pandemic along with the changes of main isolates and susceptibility profiles. Although the 30-day mortality due to BSI was lower during the pandemic, the new infection prevention strategy should be considered for any future pandemics.

Influence on therapeutic outcome of platelet count at diagnosis in patients with de novo non-APL acute myeloid leukemia.

BACKGROUND: Platelet (PLT) count at diagnosis plays an important role in cancer development and progression in solid tumors. However, it remains controversial whether PLT count at diagnosis influences therapeutic outcome in patients with non-acute promyelocytic leukemia (APL) acute myeloid leukemia (AML). METHODS: This study analyzed the relationship between PLT count at diagnosis and genetic mutations in a cohort of 330 newly diagnosed non-APL AML patients. The impact of PLT count on complete remission, minimal residual disease status and relapse-free survival (RFS) were evaluated after chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT). RESULTS: Our studies showed that patients with DNMT3A mutations have a higher PLT count at diagnosis, while patients with CEBPA biallelic mutations or t(8;21)(q22; q22) translocation had lower PLT count at diagnosis. Furthermore, non-APL AML patients with high platelet count (> 65 × 109/L) at diagnosis had worse response to induction chemotherapy and RFS than those with low PLT count. In addition, allo-HSCT could not absolutely attenuated the negative impact of high PLT count on the survival of non-APL AML patients. CONCLUSION: PLT count at diagnosis has a predictive value for therapeutic outcome for non-APL AML patients.

Venetoclax Combined with Azacitidine and Homoharringtonine in Relapsed/Refractory AML: A Multicenter, Phase 2 Trial.

BACKGROUND: Relapsed or refractory acute myeloid leukemia (R/R AML) has a dismal prognosis. The aim of this study was to investigate the activity and tolerability of venetoclax combined with azacitidine plus homoharringtonine (VAH) regimen for R/R AML. METHODS: This phase 2 trial was done at ten hospitals in China. Eligible patients were R/R AML (aged 18-65 years) with an Eastern Cooperative Oncology Group performance status of 0-2. Patients received venetoclax (100 mg on day 1, 200 mg on day 2, and 400 mg on days 3-14) and azacitidine (75 mg/m2 on days 1-7) and homoharringtonine (1 mg/m2 on days 1-7). The primary endpoint was composite complete remission rate [CRc, complete response (CR) plus complete response with incomplete blood count recovery (CRi)] after 2 cycles of treatment. The secondary endpoints include safety and survival. RESULTS: Between May 27, 2020, and June 16, 2021, we enrolled 96 patients with R/R AML, including 37 primary refractory AML and 59 relapsed AML (16 relapsed after chemotherapy and 43 after allo-HSCT). The CRc rate was 70.8% (95% CI 60.8-79.2). In the patients with CRc, measurable residual disease (MRD)-negative was attained in 58.8% of CRc patients. Accordingly, overall response rate (ORR, CRc plus partial remission (PR)) was 78.1% (95% CI 68.6-85.4). At a median follow-up of 14.7 months (95% CI 6.6-22.8) for all patients, median overall survival (OS) was 22.1 months (95% CI 12.7-Not estimated), and event-free survival (EFS) was 14.3 months (95% CI 7.0-Not estimated). The 1-year OS was 61.5% (95% CI 51.0-70.4), and EFS was 51.0% (95% CI 40.7-60.5). The most common grade 3-4 adverse events were febrile neutropenia (37.4%), sepsis (11.4%), and pneumonia (21.9%). CONCLUSIONS: VAH is a promising and well-tolerated regimen in R/R AML, with high CRc and encouraging survival. Further randomized studies are needed to be explored. Trial registration clinicaltrials.gov identifier: NCT04424147.

Autophagy activation mediates resistance to FLT3 inhibitors in acute myeloid leukemia with FLT3-ITD mutation.

BACKGROUND: Autophagy plays a critical role in drug resistance in acute myeloid leukemia (AML), including the subtype with FLT3-ITD mutation. Yet how autophagy is activated and mediates resistance to FLT3 inhibitors in FLT3-ITD-positive AML remains unsure. METHODS: We detected the expression of autophagy markers in FLT3-ITD-positive leukemic cells after vs. before acquired resistance to FLT3 inhibitors; tested the stimulative effect of acquired D835Y mutation and bone marrow micro-environment (BME) on autophagy; explored the mechanism of autophagy mediating FLT3 inhibitor resistance. RESULTS: Sorafenib-resistant cells markedly overpresented autophagy markers in comparison with sorafenib-sensitive cells or the cells before sorafenib treatment. Both acquired D835Y mutation and BME activated cytoprotective autophagy to mediate FLT3 inhibitor resistance. Autophagy activation decreased the suppression efficacy of FLT3 inhibitors on FLT3 downstream signaling and then weakened their anti-leukemia effect. Inhibition of autophagy with CQ significantly enhanced the suppressive effect of FLT3 inhibitor on FLT3 downstream signaling, in the end overcame resistance to FLT3 inhibitors. CONCLUSIONS: Autophagy might be stimulated by acquired mutation or BME, and bypass activate FLT3 downstream signaling to mediate FLT3 inhibitor resistance in FLT3-ITD-positive AML. Targeting autophagy could be a promising strategy to overcome resistance.

Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia.

BACKGROUND: Epigenetic dysregulation plays important roles in leukemogenesis and the progression of acute myeloid leukemia (AML). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) reciprocally regulate the acetylation and deacetylation of nuclear histones. Aberrant activation of HDACs results in uncontrolled proliferation and blockade of differentiation, and HDAC inhibition has been investigated as epigenetic therapeutic strategy against AML. METHODS: Cell growth was assessed with CCK-8 assay, and apoptosis was evaluated by flow cytometry in AML cell lines and CD45 + and CD34 + CD38- cells from patient samples after staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). EZH2 was silenced with short hairpin RNA (shRNA) or overexpressed by lentiviral transfection. Changes in signaling pathways were detected by western blotting. The effect of chidamide or EZH2-specific shRNA (shEZH2) in combination with adriamycin was studied in vivo in leukemia-bearing nude mouse models. RESULTS: In this study, we investigated the antileukemia effects of HDAC inhibitor chidamide and its combinatorial activity with cytotoxic agent adriamycin in AML cells. We demonstrated that chidamide suppressed the levels of EZH2, H3K27me3 and DNMT3A, exerted potential antileukemia activity and increased the sensitivity to adriamycin through disruption of Smo/Gli-1 pathway and downstream signaling target p-AKT in AML cells and stem/progenitor cells. In addition to decreasing the levels of H3K27me3 and DNMT3A, inhibition of EZH2 either pharmacologically by chidamide or genetically by shEZH2 suppressed the activity of Smo/Gli-1 pathway and increased the antileukemia activity of adriamycin against AML in vitro and in vivo. CONCLUSIONS: Inhibition of EZH2 by chidamide has antileukemia activity and increases the chemosensitivity to adriamycin through Smo/Gli-1 pathway in AML cells (Fig. 5). These findings support the rational combination of HDAC inhibitors and chemotherapy for the treatment of AML.

Impact of FLT3-ITD allele ratio and ITD length on therapeutic outcome in cytogenetically normal AML patients without NPM1 mutation.

Mutations of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD) contribute to poor prognosis in cytogenetically normal acute myeloid leukemia (CN-AML). Chemotherapy has limited effect, while allogeneic hematopoietic stem cell transplantation (allo-HSCT) plus sorafenib maintenance is a promising protocol to improve their therapeutic outcome. However, the prognostic significance of FLT3-ITD mutant status remains controversial. To investigate this, we detected FLT3-ITD mutant ratio (high and low) and length (long and short) in enrolled 184 CN-AML patients without NPM1 mutation, and evaluated their impact on complete remission (CR), overall survival (OS), relapse-free survival (RFS) and relapse risk (RR) after chemotherapy or allo-HSCT plus sorafenib maintenance. Our studies showed that FLT3-ITD mutation had negative impact on chemotherapeutic response, OS and RFS in CN-AML patients. There was no significant difference in CR rate between high and low ratio, or long and short length. Increasing ITD mutant ratio and length were associated with decreasing OS, and long length had shorter RFS and higher RR than the short after chemotherapy. Allo-HSCT plus sorafenib maintenance was an effective strategy to improve RFS and decrease relapse probability in FLT3-ITD AML patients, and benefited to these regardless of mutant ratio, and those with long length instead of the short.

Suppressing Hedgehog signaling reverses drug resistance of refractory acute myeloid leukemia.

BACKGROUND: Hedgehog (Hh) signaling is involved in the pathogenesis of tumors. By performing gene chip analysis, we predicted that Hh signaling might regulate multiple downstream pathways in acute myeloid leukemia (AML). METHODS: In this study, the potential role of the Hh pathway in refractory AML, and the impact of Hh expression on clinical prognosis were examined. We also investigated the role of the Hh inhibitor NVP-LDE225 in reversing drug resistance of refractory primary AML cells in vitro and the roles of multiple drug-resistant HL60/Adriamycin-resistant cells in vitro and in vivo (in a xenograft mouse model). Finally, we explored the underlying mechanisms. RESULTS: Hh pathway was highly active in chemotherapy-resistant AML cells; by contrast, activation was less pronounced in chemosensitive cells and non-refractory primary cells. Strong activation of this pathway was associated with higher recurrence rates and poorer relapse-free and overall survival. NVP-LDE225 inhibited MRP1 protein expression, increased intracellular accumulation of Adriamycin, and reversed chemotherapeutic resistance. These effects were likely mediated through inhibition of the IGF-1R/Akt/MRP1 pathway. In the AML xenograft mouse model, NVP-LDE225 plus Adriamycin resulted in marked tumor regression. CONCLUSION: These findings suggest that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance and preventing refractory AML.

Gene mutation profile and risk stratification in AML1­ETO­positive acute myeloid leukemia based on next­generation sequencing.

Gene mutations play an important role in the development and progression of AML1­ETO­positive acute myeloid leukemia (AE­AML). Nevertheless, the gene mutation profile in this subtype of leukemia remains unclear. In addition, the clinical and prognostic effects of different mutant genes may be underestimated. In the present study, gene sequencing was conducted at diagnosis and relapse with next­generation sequencing (NGS) in 64 patients with newly diagnosed AE­AML, and 44/64 (68.8%) patients were found to present with a median of 2 (1­10) recurrent mutations at diagnosis and 6/11 (54.5%) cases were found to present with genetic alterations at relapse. c­KIT mutation was the most common in this cohort, with an incidence of 27/64 (42.2%) at diagnosis, followed by ASXL1 (n=10, 15.6%), MET (n=8, 12.5%), MLH1 (n=6, 9.4%), TET2 (n=5, 7.8%), and FBXW7, TP53 and DNMT3A (n=5, 7.8%). Survival analysis showed that c­KIT (exon 8, 17) but not exon 10 adversely affected survival. In addition, ASXL1 and TP53 were poor impact factors for recurrence­free survival (RFS) (P<0.05), and ASXL1, MET, FBXW7 and TP53 had a negative impact on overall survival (OS) (P<0.05). Multivariate analysis showed that c­KIT (exon 8, 17) [RFS: hazard ratio (HR) 3.36, 95% confidence interval (CI) 1.54­7.34, P=0.002; OS: HR 2.84, 95% CI 1.20­6.71, P=0.018] and ASXL1 mutations (RFS: HR 3.13, 95% CI 1.34­7.32, P=0.009; OS: HR 3.94, 95% CI 1.62­9.61, P=0.003) were independent adverse factors for survival. Further, co­mutation of these two genes showed even worse effect on disease outcome. Collectively, additional gene mutations play critical role in AE­AML. C­KIT and ASXL1 mutations are the two most common mutations in this subtype of leukemia. C­KIT (exon 8, 17) but not exon 10, and also the ASXL1 mutation poorly affect the disease outcome of this disease.

[Clinical characteristics of chronic myeloid leukemia with T315I mutation and the efficacy of ponatinib].

OBJECTIVE: To analyze the clinical features of chronic myeloid leukemia (CML) with T315 I mutation (CML-T315I) and compare the effectiveness of different treatments. METHODS: We retrospectively analyzed the clinical data and outcomes of 19 patients with CML-T315I receiving different treatments. The T315 I mutations in these patients were detected by examination of BCR-ABL kinase domain (KD) mutation by RTQ-PCR and Sanger sequencing. The relapse following the treatments, defined as hematological, cytogenetic and molecular biological recurrences, were analyzed in these patients. RESULTS: Of the 19 patients with CML-T315I, 14 (73.7%) were in CML-CP stage at the initial diagnosis, and 13 (81.2%) were high-risk patients based on the Sokal scores. All the 19 patients were treated with TKI after the initial diagnosis, and during the treatment, 15 (78.9%) patients were found to have additional chromosomal aberrations, and 10 (52.6%) had multiple mutations; 13 (68.4%) of the patients experienced disease progression (accelerated phase/blast crisis) before the detection of T315I mutation, with a median time of 40 months (5-120 months) from the initial diagnosis to the mutation detection. After detection of the mutation, 12 patients were treated with ponatinib and 7 were managed with the conventional chemotherapy regimen, and their overall survival rates at 3 years were 83.3% and 14.2%, respectively (P < 0.001). CONCLUSIONS: CML patients resistant to TKI are more likely to have T315I mutations, whose detection rate is significantly higher in the progressive phase than in the chronic phase. These patients often have additional chromosomal aberrations and multiple gene mutations with poor prognoses and a high recurrence rate even after hematopoietic stem cell transplantation. Long-term maintenance therapy with ponatinib may improve the prognosis and prolong the survival time of the patients.

Influence of initiation time and white blood cell count on the efficacy of cytotoxic agents in acute promyelocytic leukemia during induction treatment.

The present study retrospectively analyzed 96 newly diagnosed acute promyelocytic leukemia (APL) patients with low-intermediate mortality risk to identify the optimum timing to initiate cytotoxic chemotherapy following all-trans retinoic acid (ATRA) administration. Based on white blood cell (WBC) at chemotherapy initiation, the patients were divided into three groups: low WBC (WBC count ≤4×109/l), intermediate WBC (WBC count >4×109/l and <15×109/l) and high WBC group (WBC count ≥15×109/l). According to the period from ATRA commencement to chemotherapy, 96 patients were further divided into two groups: ≤3 days group (chemotherapy within 3 days of ATRA) and >3 days group (chemotherapy >3 days after ATRA). Clinical effects were compared by univariate analysis and multivariate analyses. The incidence rate of differentiation syndrome (DS; also termed retinoic acid syndrome) was 0.0, 11.1 and 40.0% in the low, intermediate and high WBC groups, respectively (P<0.001); complete remission (CR) rate was 90.5, 100.0 and 73.3%, respectively (P<0.001); and the rate of early mortality (defined as fatality during induction treatment) was 4.8, 0.0 and 26.7%, respectively (P<0.001). No differences were identified in clinicolaboratory parameters between the ≤3 days and >3 days groups, except in time to achieve CR (P=0.004) and rate of bleeding related to chemotherapy (P=0.009), both being higher in the >3 days group. Multivariate analyses indicated WBC count at chemotherapy was the only independent risk factor for the occurrence of DS [P=0.002; odds ratio (OR) =1.058, 95% confidence interval (CI) =1.021-1.095] and early mortality (P=0.036; OR =1.036, 95% CI =1.002-1.070). For newly diagnosed APL patients with low-intermediate risk, chemotherapy initiation should be recommended until WBC count rises to between 4×109/l and 15×109/l during induction treatment.

[Influence of FLT3-ITD Mutation and ITD Length on the Outcome on Patients with Acute Myeloid Leukemia].

OBJECTIVE: To explore the influence of FLT3-ITD mutation and ITD length on the overall survival (OS) and relapse free survival(RFS) in patients with non-M3 acute myeloid leukemia. METHODS: Clinical features and therapeutic effect were retrospectively analyzed in 75 AML patients with FLT3-ITD mutation and 76 FLT3-ITD- AML patients with a normal karotype from June 2011 to April 2016. Genomic DNA was amplified by PCR, and FLT3-ITD mutation length was analyzed by DNA sequencing in 40 patients. RESULTS: AML patients with FLT3-ITD mutation had higher WBC count and the ratio of BM blast cells at initial diagnosis was also higher than those in AML patients without FLT3-ITD mutation (95.13 vs 10.85)(P<0.01); 72% vs 59%(P<0.01). The CR rates in AML patients with FLT3-ITD mutation less than those in AML patients without FLT3-ITD mutation(70.42% vs 94.7%)(P<0.01). OS (P<0.01) and RFS (P<0.01) were significantly increased in patients with AML who received allo-HSCT as compared with the patients who received consolidation chemotherapy and similar to AML patients without FLT3-ITD mutation who received HSCT. Patients with maintenance sorafenib after HSCT had longer OS (P<0.05) and RFS (P<0.05) than controls. ITDs exceeding 60 bp in length were associated with decreasing OS as compared with shorter ITD in AML patients with FLT3-ITD mutation (P<0.05). OS and RFS were similar among the 2 groups receiving consolidation chemotherapy. Besides, the patients with allo-HSCT had shorter ITDs and longer OS than ITDs exceeding 60 bp (P<0.05) and similar to AML patients without FLT3-ITD mutation. CONCLUSION: AML patients with FLT3-ITD mutation has poorer outcome, among which the prognosis was worse in patients with ITD exceeding 60 bp, and the chemotherapy alone can not improve the prognosis of FLT3-ITD+. Allo-HSCT is an effective treatment for AML patients with FLT3-ITD mutation; Sorafenib appears to be an effective maintenance therapy after allo-HSCT in FLT3-ITD AML.

Amyloid precursor protein has clinical and prognostic significance in AML1-ETO-positive acute myeloid leukemia.

Amyloid precursor protein (APP) has been reported to be highly expressed in acute myeloid leukemia (AML)1-eight-twenty one (ETO)-positive AML. In the present study, the clinical and prognostic significance of APP expression was assessed in 65 patients with AML1-ETO-positive AML using reverse transcription-quantitative polymerase chain reaction. The patients were divided into an APP-high expression (APP-H) group (n=32) and an APP-low expression (APP-L) group (n=33) according to the cut-off value of APP relative expression, which was calculated by receiver operating characteristic curve analysis. It was observed that C-KIT mutations (14/32 vs. 3/33, P=0.009), white blood cell count (median, 23.2×109 vs. 12.4×109 cells/l; P=0.011) and bone marrow cellularity (median, 91.0 vs. 84.0%; P=0.039) and incidence of extramedullary leukemia (11/32 vs. 3/33, P=0.013) were all significantly increased in the APP-H group compared with the APP-L group. Furthermore, significantly lower rate of cumulative two-cycle complete remission (83.9 vs. 100%, P=0.016), major molecular remission following two courses of consolidation (34.5 vs. 71.4%, P=0.005), and poorer relapse-free survival (RFS) (33.5±5.2% vs. 76.3±6.9%, P<0.001) and overall survival (OS) (44.5±7.0% vs. 81.9±5.8%, P=0.002) were associated with APP overexpression. Multivariate analysis revealed that APP overexpression was a significant adverse factor affecting both RFS and OS. Taken together, these data suggest that APP may be correlated with C-KIT mutations and involved in leukemia cell proliferation, and its overexpression has an adverse effect on the prognosis in AML1-ETO-positive AML.

Disruption of Wnt/ß-Catenin Exerts Antileukemia Activity and Synergizes with FLT3 Inhibition in FLT3-Mutant Acute Myeloid Leukemia.

Purpose: Wnt/ß-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases ß-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3-mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/ß-catenin and FLT3 inhibition in FLT3-mutant AML.Experimental Design: Wnt/ß-catenin signaling was inhibited by the ß-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF.Results: We found significantly higher ß-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow-resident leukemic cells compared with circulating blasts. Disrupting Wnt/ß-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3-mutated AML cells and stem/progenitor cells in vitro The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/ß-catenin and FLT3 cooperatively decreased nuclear ß-catenin and the levels of c-Myc and other Wnt/ß-catenin and FLT3 signaling proteins. Importantly, ß-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells.Conclusions: Disrupting Wnt/ß-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3-mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3-mutated AML patients. Clin Cancer Res; 24(10); 2417-29. ©2018 AACR.

Analysis of early death in newly diagnosed acute promyelocytic leukemia patients.

The aim of this study was to identify risk factors for early death (ED) in acute promyelocitic leukemia (APL) patients.Clinical records of 49 APL patients who suffered ED were divided into 4 groups: death before treatment or within the first 3 days (immediate death; iED group), death during treatment at least 3 days after commencement (ED after treatment), low/intermediate risk, and high-risk groups.White blood cell (WBC) count, high-risk cases, prothrombin time (PT) prolongation, international society on thrombosis and hemostasis (ISTH) scores (P < .05), bleeding (P = .05), and death due to severe hemorrhage (P = .010) were higher in iED group than ED after treatment. And the time from onset to initial hospitalization or death was significantly shorter (P < .05) in iED patients. LDH level (P = .002), PT prolongation (P = .014), and incidence of grades 3 or 4 bleeding (P = .049) were higher in high-risk group than in ED and low/intermediate-risk groups, while the times from onset to the initial hospitalization or death were lower for ED patients in high-risk group (P = .037).We found that different types of EDs have different clinical features. A high WBC count contributes to the occurrence of more ED, which is usually not associated with delay of diagnosis and hospitalization. Current therapeutic strategies to reduce the incidence of ED in these cases are not adequate and will benefit from focused research attention.