Hypomethylation Agent Research Articles

Venetoclax Combined with Hypomethylation Agents As Induction Therapy for Newly Diagnosed Acute Myeloid Leukemia - Priliminary Results from a Real World Study in China

Introduction: Venetoclax in combination with hypomethylation agents (HMAs) is a first-line induction regimen recommended by NCCN guidelines for patients aged older than 75 years or unfit for intensive chemotherapy. Since December, 2020, venetoclax has been approved in China for the treatment of AML patients who are older or unfit for intensive chemotherapy, and is gradually being used in an increasing numbers of hospitals in China. However, the efficacy and safety of venetoclax plus HMAs for the treatment of newly diagnosed adult AML patients, especially AML patients fit for intensive chemotherapy. Since January 2022, we have initiated a multicenter, prospective, observational study (No.2022219) to investigate the efficacy and safety of venetoxlax-based AML induction therapy in a real world setting. Here, we report the primary results of this study. Methods: A total of 25 sites in 19 provinces or municipalities across China participated in this study. The study population consisted of adult patients with newly diagnosed acute myeloid leukemia who had received venetoclax and HMA induction therapy and were available for evaluation. Diagnosis was based on the 2016 World Health Organization criteria. Risk stratification was performed according to the 2017 ELN recommendations. Clinical data were retrospectively collected from the electronic healthcare system and reviewed by the principal investigator of the lead site (The First Affiliated Hospital of Soochow University). The primary endpoint was composite complete remission (CRc, including complete remission, CR, and complete remission with incomplete hematologic recovery, CRi). Statistical analyses were performed using SPSS version 26.0 (Illinois, USA) and R4.0.4 (R Foundation for Statistical Computing, Vienna, Austria). Comparisons of categorical variables between groups were performed using the chi-square test or Fisher's exact test, and the Mann-Whitney U test and the Kruskal-Wallis test were used for continuous variables. P-values less than 0.05 were considered statistically significant. Results: Clinical data were collected from a total of 418 patients with newly diagnosed AML between January, 2022 to February, 2023. The minimum dose of venetoclax were 14 days. The baseline characteristics of all patients were shown in Table 1. The median age of all the patients were 64 (53-70 years). The was no difference in the gender distribution of patients (male to female ratio = 1.25:1). 231/418 (55.3%) patients had an ECOG score of ≥ 2 at the time of initiation of the induction treatment. 327 (80.9%) patients were considered unfit for intensive chemotherapy. 350/418 (83.7%) patients received azacitidine and venetoclax, and 12.3% of the patients received decitabine and venetoclax. Five patients were treated with specific targeted drugs along with decitabine and azacitidine, one patient was treated with flumatinib, two patients were treated with gititinib, and one patient was treated with sorafenib. A total of 384 (91.9%) de novo AML and 34 (8.2%) secondary AML patients were included in this study. According to the 2017 ELN criteria, 60 (14.4%), 114 (27.3%), and 244 (58.4%) patients were grouped to favorable, intermediate, and adverse risk groups, respectively. The overall response rate (ORR) for all patients was 77.5% (324/418). The composite complete remission (CRc) rate was 80% (48/60), 86% (98/114) and 73% (178/244) in the favorable, intermediate and adverse-risk group respectively (Figure 1A). There was no difference in CRc rates in patients with de novo AML or secondary AML(79% vs 65%, P=0.0699) (Figure 1B). Treatment responses varied among different sites (Figure 1C). The median amounts for platelet infusions and red blood cell infusions were 6.0 U (3.0-11.0 U) and 4.0 U (1.0-8.0 U). The median time of hospitalization during the induction treatment was 31 days (26-37 days). The median cost of induction treatment were $14,755 ($8,654- 22,991). Conclusion: In this study, venetoclax in combination with a hypomethylation agent showed comparable responses across all genetic subgroups in this real-world study of China，which was compared to the results of clinical studies conducted primarily in Western countries. Currently, we have collected 800 samples in this study. We will analyze and report the short- and long-term efficacy of venetoclax plus hypomethylating agent for the treatment of newly diagnosed AML in the near future.

Incidence of Pneumonia, Sepsis and Febrile Neutropenia in Transplant-Ineligible Patients with Newly Diagnosed Acute Myeloid Leukemia Treated with Venetoclax Based Combination Therapy: A Systematic Review and Meta-Analysis of Phase III Randomized Controlled Trials

Introduction Prognosis remains dismal for older patients with acute myeloid leukemia (AML) even after hypomethylation agent or low dose cytarabine. Venetoclax is a highly selective oral BCL-2 inhibitor which induces apoptosis in BCL-2 dependent leukemic cells. In recent years, phase III RCTs have shown that the addition of venetoclax to standard treatment in older patients with newly diagnosed AML (ND AML) significantly improved efficacy with notable toxicities. We conducted a combined analysis of randomized controlled trials (RCT) to determine the risks of pneumonia, sepsis and febrile neutropenia in transplant-ineligible patients with ND AML treated with venetoclax based combination therapy. Methods We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 30 th, 2023. Phase III RCTs utilizing venetoclax in transplant-ineligible patients with ND AML that mention febrile neutropenia, sepsis and pneumonia as adverse effects were incorporated in the analysis. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Heterogeneity was assessed with Cochran's Q- statistic. Fixed effects model was applied. Results A total of 637 transplant-ineligible patients with ND AML from 2 phase III RCTs (VIALE-A and VIALE-V) were eligible. VIALE-A and VIALE-V compared azacitidine + venetoclax vs azacitidine + placebo, and venetoclax + low dose cytarabine vs placebo + low dose cytarabine respectively. The randomization ratio was 2:1 in both studies. The incidence of any-grade pneumonia was 22.1% in study group vs 23.6% in control group (RR, 0.94; 95% CI: 0.70 - 1.27; P=0.69). High-grade pneumonia rate was nearly 2.9% lower in the study arm compared to control arm, but RR was not statistically significant at 0.86 (95% CI: 0.62 - 1.21; P = 0.39). High-grade sepsis was reported in the study arm (5.6%) versus 7.5% in control arm with RR of 0.75 (95% CI: 0.41- 1.38; P = 0.35). High-grade febrile neutropenia as hematologic adverse events was noted in 163 (38.4%) of patients in study group compared to 47 (22.2%) in control group with RR of 1.73 (95% CI: 1.31- 2.29; P = 0.0001). Febrile neutropenia as serious adverse events was reported in 107 (25.1%%) of patients in study group compared to 27 (12.7%) in control group with RR of 1.98 (95% CI: 1.34- 2.93; P = 0.0006). Conclusions Our meta-analysis showed that venetoclax based anti-leukemic therapy contributed to higher incidence of high-grade febrile neutropenia as hematologic and serious adverse events in transplant-ineligible patients with ND AML, with RR of 1.73 and 1.98. However, the risks of high-grade sepsis and pneumonia were found to be statistically not significant between the two groups. Timely intervention with proper supportive care is required.

Li-Fraumeni Syndrome: The Two Faces of a Coin in Myeloid Malignancies

Background Li-Fraumeni Syndrome (LFS) is a rare, autosomal dominant genetic disorder caused by germline mutations in the TP53 tumor suppressor gene. It is characterized by a predisposition to a wide range of cancers, including hematological malignancies. Hematological malignancies account for approximately 3-4% of all LFS-related cancers of which are acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Myelodysplastic syndromes (MDS) and lymphomas. Method and Results We retrospectively analyzed our clinical data for patients with hematological malignances from the period of Jan 2016-Jan 2022 at the national center for cancer care and research (NCCCR), the only tertiary hematology center in the country. We report on two cases with Li Fraumeni syndrome with acute leukemia and their variable spectrum of the disease manifestations and prognosis. Case 1: 16 years old male known case of rheumatic heart disease presented with double malignancy; left mandible osteosarcoma and lymphoblastic leukemia/ lymphoma concurrently in 2020. For his Leukemia he received BFM*2009 protocol and after remission he underwent surgical resection and received radiotherapy for the osteosarcoma. Patient has family history of breast cancer in his maternal aunt and his paternal great aunt. Germline Genetic testing from buccal swab revealed a pathogenic variant in TP53 gene confirming Li Fraumeni syndrome which is most likely De novo as it was not detected in both parents and his 3 siblings. In 2022 patient presented with pancytopenia and therapy related AML/MDS with complex Karyotype. He was started on salvage therapy and was planned for allogenic stem cell transplant (SCT) from his HLA matched brother, however, unfortunately, patient didn't achieve complete remission thus could not receive the SCT and he passed away one year later in 2023. Case 2: 60-year-old female known case of hypertension who was diagnosed with right breast cancer in 2011 for which she underwent surgery, chemotherapy, and radiotherapy. A year later, patient was diagnosed with left upper lobe squamous cell lung carcinoma, and underwent left upper lobectomy, chemotherapy and radiotherapy. Patient was in remission for 6 years, until 2018 when she presented with pancytopenia and therapy related AML with complex monosomic Karyotype. Due to patient's age and comorbidities, she was unfit for intensive therapy, thus she received hypomethylation agent (Azacitidine) till disease progression and was referred to genetic counseling. Patient has a family history of unknown cancer in her paternal half-sister at the age of 77 and an unknown cancer in her paternal cousin. Genetic testing reveled a mosaic pathogenic variant in TP53 gene present in 17% of the 1137 next generation sequencing reads. Shortly later, patient passed away without confirmatory fibroblast testing. Conclusion: Genetic testing indications for Li-Fraumeni syndrome present challenges as patients with full mutationin TP53 often but not always present with the full spectrum of the disease, whereas patients with mosaic profilepresent with a later onset of the disease and might not be identified early. In both scenarios, patients can be miss-identified and exposed to radiotherapy thus testing criteria warrants further evaluation. Acute Leukemia in Li-Fraumeni syndrome is associated with poor prognosis and usually presents in the context of therapy related myeloid malignancies . Identification of patients with Li-Fraumeni syndrome early on is crucial to avoid exposures to radiotherapy whenever possible thus reducing the risk of subsequent malignancies and the tragic outcomes of the disease. There are unmet clinical needs in Li-Fraumeni syndrome diagnostic and therapeutic approaches which needs further attention.

Azacitidine Combined with Novel Flavonoid Derivative GL-V9 Demonstrated Synergistic Anti-Leukemia Effect in Acute Myeloid Leukemia By Targeting DDIT4/mTOR Signaling

Background Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of malignant progenitor cells and impaired cell differentiation. GL-V9, a nature compound derived from wogonin, has shown superior anti-tumor potential by inhibiting cancer cell growth. Azacitidine (AZA), a DNA hypomethylation agent, has demonstrated therapeutic efficacy against AML. In this study, we investigated the anti-leukemia effect and potential mechanism of AZA combined with GL-V9 in AML cells, aiming to provide evidence for future clinical treatment. Methods We employed Cell Counting Kit-8 (CCK-8) to assess cell viability, Annexin-V/PI staining followed by flow cytometry analysis to measure apoptosis. CalcuSyn analysis was used to evaluate the synergistic effect. RNA-seq was performed in U937 cells and differentially expressed genes (DEGs) were identified and subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. RT-qPCR and Western Blot were used to examine gene expression. Results Our results demonstrated significant cell proliferation arrest in U937 and MV4-11 cells treated with the combination of AZA and GL-V9, compared to the single drug ( Fig. 1A&B). CalcuSyn analysis showed a strong synergistic effect for the combination ( Fig. 1A&B). Similar results were also found in primary AML cells derived from 2 AML patients [patient 1 exhibited t(6;11) (q27;q23) chromosomal translocation, while Patient 2 presented with secondary AML, transformed from CMML (Chronic Myelomonocytic Leukemia) and featured ASXL1 and SRSF2 mutations] ( Fig. 1C&D). Moreover, AZA plus GL-V9 induced a substantial increase in cell apoptosis in U937 and MV4-11 cells compared to the single drug ( Fig. 1E&F). Consistently, the AZA+GL-V9 combination led to a notable increase in the protein level of Bax, BAD, BIM, and a decrease in BCL2 ( Fig. 1G). These data indicated the combination of AZA with GL-V9 has synergistic anti-leukemia effects in AML. To understand the underlying mechanisms of this synergy, we performed RNA-seq analysis in U937 cells, identifying 1385 and 586 DEGs (|log 2FC|≥2.0, P<0.05) upon AZA or GL-V9 treatment, respectively ( Fig. 2A). The mTOR signaling pathway exhibits prominent enrichment in the KEGG analysis of the overlapping DEGs between AZA and GL-V9 ( Fig. 2B). DDIT4, a negative regulator of mTOR emerged as one of the top DEGs in response to both AZA and GL-V9 treatment. Importantly, the AZA+GL-V9 treatment exhibited a higher expression level of DDIT4 compared to either drug alone and suppressed the phosphorylation of mTOR ( Fig. 2C). Additionally, the DDIT4 expression level was significantly decreased in AML patients, which derived from a public database (TCGA-AML, GSE13159)] ( Fig. 2D) and Zhongda Hospital (Nanjing, China) compared to the healthy controls ( Fig. 2E). To be noticed, high DDIT4 expression was associated with extended overall survival (P=0.021) ( Fig. 2F) and relapse-free survival (P=0.015) ( Fig. 2G), indicating DDIT4 may act as a tumor suppressor in AML. These findings suggested that the combination may exert the anti-leukemia effect by targeting the DDIT4/mTOR signaling pathway, as summarized in Fig. 2H. Conclusions Our study provides novel evidence of the synergistic effect on cell growth arrest and apoptosis treated with a new nature compound derived from wogonin combined with AZA in AML. Furthermore, we identified the mechanism underlying the synergy through targeting of DDIT4/mTOR signaling. These results offer preliminary support for the potential application of this combination therapy in treating AML patients.

Abstract A08: Targeting HSP90 epichaperome in TP53 mutant AML

Abstract The Bcl-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, has improved outcomes for many patients with AML and is now considered standard of care for patients who are older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months on this regimen and only 2.5 months post relapse. Molecular analysis demonstrates that mutations in TP53 and oncogenic kinases are key determinants of lower response rates and early relapse. Preclinical studies also show that increased kinase signaling in AML stem/progenitor cells in TP53 mutant AML. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. HSP90-associated-epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, HSP90 epichaperome inhibition has the potential of targeting TP53 mutant AML. In contrast to other HSP90 ATP inhibitors, PU-H71 (zelavespib) is a competitive inhibitor specific for the ATP binding site of HSP90 epichaperomes. We here investigate the therapeutic potentials of targeting HSP90 epitherachorme with PU-H71 in TP53 mutated AML. Western blot analysis found increased HSP90 and several signaling proteins in TP53 knockout and mutant Molm13 cells generated by CRISPR/cas-9 or by exposing to idasanutlin, compared to the isogeneic wild-type controls. Using a fluorochrome-labelled PU-H71 and flow cytometry, we demonstrate the presence of HSP90 epichaperomes in AML cells and AML stem/progenitor cells with TP53 mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively kills AML cells and AML stem/progenitor cells with various TP53 mutations, and prolongs survival in TP53-mutant AML xenograft mice with minimal effects on normal CD34+ bone marrow cells and hematopoiesis. PU-H71 increased Bim expression and enhanced VEN activity in AML cells and AML stem/progenitor cells with TP53 mutations. Importantly, in a mixture of TP53 wild-type/R248W Molm13 cells (1000:1), nutlin3a selectively killed TP53 wild-type but enriched TP53 mutant Molm13 cells; VEN treatment favored the outgrowth of TP53-mutant cells, while PU-H71 effectively killed TP53 wild-type and mutant cells. Furthermore, PU-H71 exhibited anti-leukemia activity against both TP53 WT and mutant AML cells, which was further enhanced by VEN in vivo in a xenograft model of mixed TP53 WT and mutated Molm13 cells (10:1). Our data support that the HSP90 epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring mutant TP53. Inhibition of HSP90 by PU-H71 targets AML cells/stem/progenitor cells enhances VEN activity and prevents outgrowth of VEN-resistant TP53 mutant AML cells. This concept warrants clinical evaluations. Citation Format: Bing Carter, Po Yee Mak, Wenjing Tao, Lauren B Ostermann, Yuki Nishida, Steffen Boettcher, Michael Andreeff. Targeting HSP90 epichaperome in TP53 mutant AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A08.

Epichaperome Inhibition Targets Kinase and TP53 Mutant Therapy Resistant AML

The BCL-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, improves outcomes for many patients with AML and is now considered standard of care for patients older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months; 2.5 months after relapse. Mutations of FLT3, PTPN11, and RAS, leading to constitutive activation of cell survival signaling, and mutations of TP53 have been identified as key determinants of low response rates, early relapse and death. Mutant TP53 cells are also characterized by activation of signaling kinases. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. Epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, epichaperome inhibition has the potential of eliminating resistant subclones before they become dominant and cause relapse. In contrast to other HSP90 purine analog inhibitors, PU-H71(zelavespib) is a competitive inhibitor specific for the conformationally changed ATP binding site of HSP90 when incorporated into epichaperomes. We here investigate the therapeutic potential of targeting epichaperomes with PU-H71 in TP53 and oncogenic kinase mutant AML. Western blot analysis showed increased HSP90 and several signaling proteins such as STAT3 and c-Myc in TP53-knockout and mutant Molm13 cells generated by CRISPR/cas-9, compared to isogeneic wild-type (WT) controls. Using fluorochrome-labelled PU-H71 and flow cytometry, we demonstrated the presence of epichaperome in AML and AML stem/progenitor cells with TP53 and/or RAS mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively killed AML cells, including those with acquired resistance to BH3 mimetics and those with various FLT3 mutants. PU-H71 targets AML cells and stem/progenitor cells with TP53 mutations, and prolonged survival of TP53-mutant AML xenograft mice with minimal effects on mouse bone marrow LSK cells and hematopoiesis. PU-H71 increased the expression of pro-apoptotic BIM and the efficacy of VEN in AML cells and AML stem/progenitor cells with TP53 and/or RAS mutations (combination index < 1). Importantly, in a 1,000: 1 mixture of TP53-WT and TP53-R248W mutant isogenic Molm13 cells, MDM2 inhibitor nutlin3a selectively killed TP53-WT but enriched TP53-mutant Molm13 cells, as expected, and VEN treatment also favored the outgrowth of TP53-mutant cells. However, PU-H71 effectively killed TP53-WT and mutant cells (Fig. 1): 10 million TP53-WT cells were reduced to 335 cells, and 10,000 TP53-mutant cells to 26. The activity of PU-H71 against both TP53-WT and mutant AML cells was further enhanced by VEN in vivo in a xenograft model of mixed (10:1) TP53-WT and mutant Molm13 cells. Our data support the concept that the epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring TP53 and/or oncogenic kinase mutations. Inhibition of epichaperomes by PU-H71 targets AML cells/stem/progenitor cells, enhances VEN activity, and prevents the outgrowth of nutlin- or VEN-resistant TP53-mutant AML cells. This concept has entered clinical evaluation in MPN-derived secondary AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Synergistic Anti-Tumor Effect on Novel Combination of Selinexor with Azacitidine in AML By Targeting XPO1/eIF4E/C-MYC Signaling

Background: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with high mortality and poor outcome. Highly effective therapy options are always needed for AML. Selinexor (KPT-330) is an oral bioactive selective CRM1 (XPO1) inhibitor, and the clinical trial showed the combination of KPT-330 over standard induction therapy benefits much for AML patients. Azacitidine (AZA), a DNA hypomethylation agent, can induce cell apoptosis and exhibit potential therapeutic efficacy against AML. However, it has not yet determined the anti-tumor effect of KPT-330 with AZA in AML. Here we examined the anti-leukemia effect and potential mechanism of KPT-330 combined with AZA in AML cells to provide evidence for further clinical trials. Methods: A total of 64 AML patients’ BM samples and 64 normal control from healthy volunteers were obtained from Zhongda Hospital Southeast University, in which the 2 patients’ samples were further treated with indicated drugs in vitro. Cell Counting Kit-8 (CCK-8) was used for cell viability assay, Annexin-V/PI staining following flow cytometry analysis for apoptosis, and qPCR and Western Blot for gene expression in AML cell lines and patient samples. CalcuSyn analysis was performed for a synergistic effect. RNA-seq was performed in U937 cells treated with 0.7uM AZA for 48 hrs, and the significantly differentially expressed genes (DEGs) were identified and gene set enrichment analysis (GSEA) was performed. Results: Cell proliferation arrest and apoptosis were significantly observed in U937 and MV4-11 cells treated with the combination of KPT-330 with AZA compared to single drug control (data not shown). Then, we further explored the synergistic effect of the combination in patients’ samples. Results showed that cell growth arrest and apoptosis significantly increased in primary samples from two de novo AML patients compared to the single drug controls, and CalcuSyn analysis showed a synergistic effect of the combination on cell growth arrest (Fig. 1A &1B). To understand the mechanisms underlying the synergy, RNA-seq analysis was performed in U937 cells treated by 0.7uM AZA and 4655 DEGs were identified. XPO1, eIF4E, and MYC are in the top DEGs upon AZA treatment, and GSEA analysis indicated MYC_TARGETS and apoptosis pathway are enriched in DEGs (Fig. 1C &1D ). Moreover, the mRNA level of XPO1 and eIF4E was increased in our AML patient cohort compared with healthy controls (Fig. 1E), and the high expression of XPO1 or elF4E are associated with poor outcome (Fig. 1F). XPO1 is the key component of nuclear-exporting machinery; elF4E, the cargo of XPO1 is translation initiation factor and plays a critical role in translational machinery, which regulates the C-MYC translation. It is also reported that AZA regulates the transcription of C-MYC by affecting methylation. We observed a combination of KPT-330 with AZA significantly down-regulated the mRNA and protein level of oncogene eIF4E and C-MYC in two of the patients’ samples compared to single drug controls (Fig. 1G &1H ). Also, C-MYC knock-down was not only significantly increased the apoptosis in U937 cells compared to scramble shRNA (shCTL) control, but also significantly enhanced the combination-mediated apoptosis compared to that of single drug control in U937 cells (Fig. 1I). these data indicated that XPO1/eIF4E/C-MYC signaling plays critical roles in the combination-mediated anti-leukemia, and the mechanistic model is summarized in Fig.1J. Conclusions: Our data for the first time showed the synergistic effect of a novel combination of KPT-330 with AZA on cell growth arrest and apoptosis in AML patients and also identified the underlying mechanism by targeting XPO1/eIF4E/C-MYC signaling. Our results also highlighted the feasibility of the clinical trial for the combination in the therapy of AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Efficacy of Azacitidine Plus Hag in Acute Myeloid Leukemia: A Multi-Center, Phase 2 Clinical Trial

Background Homoharringtonine (HHT) is a plant cytotoxic alkaloid derived from the trees of the genus Cephalotaxus. HAG (HHT, low-dose cytarabine, and G-CSF), as a priming regimen, is utilized to treat acute myeloid leukemia (AML). Azacitidine (Aza), a DNA hypomethylation agent, has been a "backbone" for new combinational therapies. However, so far, it has not been well determined the efficacy and safety of adding Aza to HAG regimen. Methods This is a multi-center, single arm, phase 2 clinical trial done in 17 clinical institutions across China between Aug 2019 and Dec 2021 (ClinicalTrials.gov: NCT04248595). Induction therapy consisted of Aza (75mg/m2/d on days 1-7) was given in combination with the HAG regimen (Fig. 1A). Primary endpoints were complete remission (CR) or complete remission with incomplete hematologic recovery (CRi). Secondary endpoints were overall survival (OS), relapse free survival (RFS), and adverse events (AEs). Mutation screening was conducted through next generation sequencing (NGS) by targeting 58 frequently mutated genes. Results A total of 112 patients, including 72 newly diagnosed (ND) (56 de novo, 16 secondary AML [sAML]), and 40 relapsed/refractory (R/R) AML were enrolled. The CR/CRi was achieved in 79.2% (57/72) in the ND and 40.0% (16/40) in R/R AML, respectively (Fig. 1B). CR/CRi were achieved in 80.4% of de novo AML (45/56) and 75.0% of sAML (12/16), respectively. In addition, the median OS and RFS of ND AML patients were 22.8m (95%CI, 12.6 to not reached) and not reached, respectively (Fig. 1C&1D), which were significantly longer than the R/R group (median OS, 10.8m [95%CI, 9.5 to 12.7], P=0.0056; median RFS 5.21m [95%CI, 4.21 to 9.64], P=0.0051) (Fig. 1C&1D). Furthermore, 94.7% (18/19) of patients with favorable-risk reached CR/CRi (Fig. 1E), with median OS and RFS were both not reached (Fig. 1F&1G). 83.9% (26/31) of patients with intermediate-risk reached CR/CRi (Fig. 1E), and the median OS and median RFS were 22.8m (95%CI, 10.5 to not reached) (Fig. 1F) and 16.9m (95%CI, 4.64 to not reached) (Fig. 1G), respectively. Whereas in those with poor-risk, 59.1% (13/22) reached CR/CRi (Fig. 1E), with the median OS 17.3m (95%CI, 3.79 to not reached) (Fig. 1F) and median RFS not reached (Fig. 1G). In this study, infection was the most common non-hematological adverse event, presenting 58.0% (65/112). Common non-hematological AEs of grade 3 or higher includes: infection (38/112, 33.9%), hemorrhage (11/112, 9.82%), fatigue (6/112, 5.36%), hypokalemia (4/112, 3.57%), cardiac arrhythmia (2/112, 1.79%), and fever (2/112, 1.79%). The median duration of neutropenia and thrombocytopenia were 11 d (IQR, 7-19d) and 16d (IQR, 11-25d) among CR/CRi patients. No patients discontinued the induction therapy due to hematological or non-hematological toxicities. The early deaths within 4 weeks of the induction treatment occurred in 1.79% (2/112). Moreover, we detected 228 mutants involving 33 genes in 103 patients at the enrollment. The most frequently mutated genes were DNMT3A (25/103), TET2 (20/103), and NPM1 (18/103) (Fig. 1H), and mostly involved in DNA methylation (DNMT3A, IDH1/2, TET2; 48/103, 46.6%), followed by signal transducers (FLT3, NRAS, KRAS, KIT, PTPN11; 36/103, 35.0%), and transcription regulation (WT1, RUNX1, CEBPA, SETBP1, GATA2; 32/103, 28.2%). Upon Aza+HAG treatment, we observed the high CR/CRi rate in ND patients with mutated BCOR (100%,7/7), KIT (100%,3/3), IDH1 (100%,7/7), NPM1 (93.3%,14/15), ASXL1 (88.9%,8/9), DNMT3A (80. 0%,16/20), RUNX1 (75.0%,6/8), FLT3 (71.4%,5/7), and TET2 (71.4%,10/14) (Fig. 1I). We further observed the clearance of mutated genes after the induction therapy and found the variant allele fraction (VAF) of mutants was dramatically reduced among the CR/CRi patients. Specifically, VAF change of FLT3 mutant from 3 of 4 available patients reduced to <0.01%, with the other one, decreased by 91.9% (Fig. 1J); 4 out of 6 patients with IDH1 mutant reduced to <0.01%, with the other two, decreased 67.6% and 35.9%, respectively (Fig. 1K); 5 out of 6 patients with ASXL1 mutant reduced to <0.01%, with the other one, decreased by 33.4% (Fig. 1L); and 10 out of 14 patients with NPM1 mutant reduced to <0.01%, respectively (Fig. 1M). Conclusion This study demonstrated that the Aza+HAG regimen is a cost-effective first-line therapy with high efficacy and well tolerance for elderly/unfit AML, especially ND AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Azacitidine Combined with Homoharringtonine, Idarubicin/Daunorubicin, Cytarabine for Previously Untreated Patients with Acute Myeloid Leukemia: A Single-Center, Phase 2 Study

Background Previous studies have reported that a combination of Homoharringtonine (HHT), a plant cytotoxic alkaloid derived from the trees of the genus Cephalotaxus, plus intensive chemotherapy (IC) improved the clinical responses of acute myeloid leukemia (AML). Azacitidine (Aza), a DNA hypomethylation agent, has been a "backbone" for novel combinational therapies. In this trial, we assessed the efficacy and safety of adding Aza to HIA/HDA regimen in previously untreated young or eligible for IC patients with AML. Methods This is a single-center, phase 2 trial (ClinicalTrials.gov: NCT04248595). Aza (75mg/m2/d on days1-5) administered in combination with HIA/HDA [HHT 2mg/m2/d on days 4-8, idarubicin 6mg/m2/d or daunorubicin 40 mg/m2/d on days 4-6, and cytarabine 100mg/m2/d on days 4-10] regimen for patients age<60 or eligible for IC (Fig.1A) The primary endpoint was the composite CR. Secondary endpoints were overall survival (OS), relapse-free survival (RFS), and adverse events (AEs). A leukemia targeted-exome-seq panel including 58 genes and leukemia 47 fusion gene panel were used for screening. Results Between Aug, 2019 to May, 2022, a total of 20 de novo AML patients were enrolled. The CR/CRi was achieved in 95.0% (19/20) (Fig. 1B). 94.7% (18/19) reached CR/CRi after the 1st cycle of induction therapy and the median time was 25.0d (IQR, 21.0 to 27.0). The median follow-up was 628 days (range, 107 to 905) and the median OS and RFS were not reached (Fig. 1C& 1D). The estimated 2-year OS and RFS were 87.4% (95%CI, 58.1% to 96.7%) (Fig. 1C) and 82.0% (95%CI, 53.1% to 94.0%), respectively (Fig. 1D). No survival difference was observed between the favorable and intermediate versus poor-risk groups in both OS and RFS (Fig. 1E& 1F). Meanwhile, the estimated 2-year OS and RFS possibility of the favorable and intermediate-risk group was 90.9% (95%CI, 50.8% to 98.7%) and 80.0% (95%CI, 20.4% to 96.9%), respectively (Fig. 1E& 1F). While the estimated 2-year OS and RFS possibility of the poor-risk group was 85.7% (95%CI, 33.4% to 97.9%), and 75.0% (95%CI, 12.8% to 96.1%), respectively (Fig. 1E& 1F). We detected 30 mutants in 17 of 58 leukemia driver genes at the timepoint of enrollment. The median mutation number/patient was 1.50 (range, 0-4), and 17 of 20 (85.0%) patients had more than one mutation (≥1) (Fig. 1G). The most frequently mutated genes were FLT3 (ITD/TKD) (20.0%), KIT (15.0%), NRAS (15.0%), and DNMT3A (15.0%) (Fig. 1G). More than half of the patients (11/20) harbored the mutated genes involved in signal transducers (FLT3, KRAS,NRAS, KIT, PTPN11, CSF3R; 11/20, 55.0%), and all these patients achieved CR/CRi (11/11, 100%) after induction therapy (Fig 1G). It is worth noticing that, we observed a high mutant's clearance with VAF (variant allele frequency) dramatical reduction (to <0.01%) (Fig. 1H) after the induction therapy, indicating that mutations involved in signal transducers were rapidly and significantly associated with high clinical response. We also identified 2 patients with MLL-AF9 and 4 patients with CBF-β-MYH11. Both patients with MLL-AF9 reached complete molecular remission (<0.1%) after induction. 3 of 4 patients with CBF-β -MYH11 reached complete molecular remission (<0.1%), and the expression level of the CBF-β -MYH11 fusion gene in 1 patient decreased over 1000-fold at the molecular level (512.16% to 0.28%) after 1 cycle of induction. The MRD negative was 84.2% (16/19) in patients who reached CR/CRi after the 1st cycle of induction therapy (Fig. 1I, 1J). Also, MRD were negative in all 3 patients with favorable risk, 77.8% with intermediate-risk, and 87.5% with poor-risk (Fig. 1K). Infection (grade 1-4) was the most common non-hematological AE (65.0%). Other AEs of grade 3 or higher included fatigue (5.0%), decreased appetite (5.0%), and hepatic dysfunction (5.0%). The most common grade 3-4 hematological AEs were neutropenia and thrombocytopenia. The median duration of neutropenia and thrombocytopenia were 12d (IQR, 11 to 15) and 14d (IQR, 12 to 16), respectively. No patients discontinued the induction treatment and no patient died within 8 weeks of the treatment. Conclusion This trial demonstrated that the adding of Aza to HIA/HDA regimen is an effective first-line therapy for previously untreated young or fit AML patients, with high efficacy and well tolerance. This study provides preliminary results for the clinical application of this novel combination therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDM2 antagonist improves therapeutic activity of azacitidine in myelodysplastic syndromes and chronic myelomonocytic leukemia

Failure of hypomethylation agent (HMA) treatments is an important issue in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Recent studies indicated that function of wildtype TP53 positively impacts outcome of HMA treatments. We investigated the combination of the HMA azacitidine (AZA) with DS-3032b and DS-5272, novel antagonists of the TP53 negative regulator MDM2, in cellular and animal models of MDS and CMML. In TP53 wildtype myeloid cell line, combinational effects of DS-3032b or DS-5272 with AZA were observed. In Tet2-knockout mouse model of MDS and CMML, DS-5272 and AZA combination ameliorated disease-like phenotype. RNA-Seq analysis in mouse bone marrow hematopoietic stem and progenitors indicated that DS-5272 and AZA combination caused down-regulation of leukemia stem cell marker genes and activation of pathways of TP53 function and stability. These findings demonstrate that combining an MDM2 antagonist with AZA has potential to improve AZA treatment in TP53 wildtype MDS and CMML.

Palbociclib promotes the antitumor activity of Venetoclax plus Azacitidine against acute myeloid leukemia

Around 70 % of patients diagnosed with acute myeloid leukemia (AML) survive less than 5 years due to drug resistance and disease relapse. Consequently, improved clinical treatments are urgently needed. Some but not all AML patients benefit from the combination of the BCL-2 inhibitor Venetoclax with the hypomethylation agent Azacitidine. Here we investigated the utility of employing the cyclin dependent kinase (CDK6) inhibitor Palbociclib to improve the efficacy of Venetoclax/Azacitidine combination therapy. Our analysis of publicly available RNA sequencing datasets showed CDK6 was highly expressed in the major acute forms of leukemia including AML. Consistently, using qPCR and flow cytometry we found that CDK6 was overexpressed in bone marrow mononuclear cells from AML patients compared to healthy controls. Subsequent in vitro testing of Palbociclib, Venetoclax and Azacitidine, alone and in combination against CDK6-overexpressing AML cells lines THP-1 and KG-1 and primary AML cells showed that the Palbociclib/Venetoclax/Azacitidine combination improved treatment efficacy compared to Venetoclax/Azacitidine treatment alone. Additional investigations in a subcutaneous KG-1 mouse model showed similarly the three-drug combination produced the most significant reductions in tumor load together with the least amount of spleen infiltration. We established Palbociclib functioned in combination with Venetoclax/Azacitidine by increasing the rates of apoptosis in AML cells. Further investigations revealed that Palbociclib does not affect BCL-2 activity but downregulated the anti-apoptotic proteins MCL-1 and BCL-XL, making AML cells more sensitive to Venetoclax/Azacitidine treatment. Our results propose that the Palbociclib/Venetoclax/Azacitidine regimen warrants further preclinical research for clinical application in AML patients.

Variant Allele Frequency Status in Elderly Patients with Acute Myeloid Leukemia Can be Early Predictors of Responsiveness to Decitabine Treatment

As interest in elderly Acute Myeloid Leukemia (AML) patients increases, American society of hematology (ASH) 2020 guidelines for treating newly diagnosed AML in older adults suggested diverse treatment options. The guidelines suggest using monotherapy over combination of hypomethylation agent (HMAs) with other agents in newly diagnosed AML in older adults due to similar efficacy and the potential for more toxicity. HMAs alone is still used widely as an alternative treatment for patients who cannot use venetoclax due to the high cost and poor performance score. If there are early predictors of responsiveness to Decitabine mono therapy, it will be helpful to decide whether to combine Novel agents. This retrospective cohort study from a single institution aimed to evaluate the prognostic significance of Variant allele frequency (VAF) changes in elderly patients after 4 th cycle of decitabine. Total 123 patients with elderly AML were eligible. 57 patients performed follow-up bone marrow biopsy and 49 patients were available of follow up targeted NGS samples from biopsy after 4th cycle of decitabine. To clarify the immortal timed bias, landmark analyses were performed with patients (n=84) who remained at least the median time to perform follow-up bone marrow biopsy after 4th cycle of decitabine treatment. 24 patients (54.5%, 24 of 44) showed more than 50% decrease of VAF after 4 th cycle of decitabine (figure 1a). DMNT3A, TET2, IDH1, IDH2, and SETBP1 and SMC1A showed less than 50% of the decreases of VAF. Patients with DNA methylation genes showed significantly reduced VAF less than 50% (figure 1b). A significant difference of ∆VAF was observed depending on CR status (p=0.021). The survival outcome of patients who showed more than 50% decrease of initial VAF after 4th cycle of decitabine was significantly better than that that with less than 50% decrease of VAF(1-year OS VAF decrease ≥ 50% (n=23), 75.0%; VAF decrease < 50% (n=20), 38.5%; no mutation (n=12), 45.5%; not available of follow up targeted NGS sample (n=29), 16.6%; p < 0.001, figure 2a). Mutations in DNMT3A, TET2, and ASXL1 (DTA genes) were detected in samples from 19 patients at diagnosis. After the exclusion of DTA mutations, the survival outcome improved prognostic risk stratification power of NGS-based MRD assessment in AML. The survival outcome of patients who showed more than 50% decrease of initial VAF after 4th cycle of decitabine was significantly better than that that with less than 50% decrease of VAF(1-year OS VAF decrease ≥ 50% (n=24), 75.0%; VAF decrease < 50% (n=19), 35.1%; no mutation (n=12), 50.1%; not available of follow up targeted NGS sample (n=29), 16.6%; p<0.001, figure 2b). In conclusion, more than 50% decrease of VAF was important negative prognostic factors by improving overall response rate and OS. In case of patients with older adults who received decitabine treatment, if follow up BM biopsy after 4 th cycles of decitabine treatment showed more than 50% reduction of VAF, it may suggest to maintain decitabine treatment. However, if VAF is reduced by less than 50% in follow up BM biopsy, the residual disease burden is considered for the selection of combination treatment to improve survival outcome. [Display omitted] DisclosuresKim: Bristol-Meier Squibb: Research Funding; Paladin: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Concentration-Dependent Decitabine Effects on Primary NK Cells Viability, Phenotype, and Function in the Absence of Obvious NK Cells Proliferation-Original Article.

Acute myeloid leukemia (AML) cells can evade innate immune killing by modulating natural killer (NK) cells receptors and their cognate ligands in tumor cells, thus it may be possible to restore proper expression of immune receptors or ligands with immune sensitive drugs. Decitabine, as a hypomethylation agent, was approved for the treatment of AML and myelodysplastic syndrome. While clinical responses were contributed by epigenetic effects and the induction of cancer cell apoptosis, decitabine also has immune-mediated anti-tumor effects. After exposure to various concentration of decitabine for 24 h, the primary NK cells (AML-NK cells) cytotoxicity and receptor expression (NKG2D and NKp46) displayed parabola-shaped response, while U-shaped response was seen in cytokine release (IFN-γ and IL-10), and these effects were regulated by ERK and STAT3 phosphorylation level. Furthermore, AML-NK cells function displayed different response when the competitive MEK and STAT3 inhibitors applied respectively. Thus, we could conclude that the different dose of decitabine makes various effects on AML-NK cells function and receptors expression.

Hypomethylation of Decitabine Improved Outcomes of Hematopoietic Stem Cell Transplantation in Children with Juvenile Myelomonocytic Leukemia

BackgroundJuvenile myelomonocytic leukemia (JMML) is useally poor respond to chemotherapy, and approximately 50% of patients relapse after hematopoietic stem cell transplantation (HSCT). Recent studies have highlighted the importance of epigenetic aberrations in JMML and proved that some JMML stem cells were associated with hapermethylation. Hence,we desiged the current study to investigate whether low dose Decitabine could improve outcomes of JMML-HSCT.Patients and methodFourteen patients received HSCT combined with Decitabine between December 2014 and July 2017. Of them, 4 patients with NF-1 mutation, 4 with PTPN11 mutation, 1 with Nras somatic mutation , 1 with monosomy 7, 1 with multiple mutation (PTPN11+Kras), 3 with uncertain mutation. The median age at diagnosis was 35 months (range: 1-71 months).13/14 patients received 2~4 course mild chemotherapy (one patient, case 6, received only single course Decitabine therapy) before HSCT. Three patients received HSCT from HLA matched unrelated donors (MUD), and eleven patients received the complementary transplantation (CT, see blood 2016 128:1235), i.e. unrelated cord blood (UCB) was given at 6 day after haploidentical peripheral blood stem cell transplantation (PBSCT) using high dose cyclophosphamide (Cy) post-transplant (PTCy).Conditioning regimen composed of Cy, Busulfan (Bu)/Thiotepa (TT), fludarabine (Flu) ,and ATG-F in the MUD-HSCT, and Cy, Bu, Flu, TT and Cyrabine in the CT. Patients received a fixed dose of 8×108/kg mononuclear cells (MNC) in the MUD-HSCT, and a median dose of 48.5×108/kg (range, 34~88×108/kg) mobilized peripheral blood MNCs and a median dose of 8.22×107/kg (range, 4.7~9.74×107/kg) UCB nucleated cells in the CT, respectively.GVHD prophylaxis were consist of PTCy, mycophenolate mofetil (MMF) and tacrolimus in the CT, and ATG,Cs A and MMF in the MUD-HSCT.Decitabine was administrated for 2~3 courses (20mg/m2.d×5 day for each course with 4-week interval) before HSCT to reduce load of leukemia cells and for 2~4 courses (5~10mg/m2.day×5day for each course with the interval of 4~6 weeks) after HSCT to overcome immune-escape of leukemia cells.Results:The median follow-up time was 5 months (range, 1.5-39 months). Full donor cells engrafted in all patients (donor cell engraftment in case 6 occurred in a salvaged transplant from another haplo-donor after primary failure of first CT). Both overall survival and disease-free survival were 93% in total. In the CT, haplo-cells and UCB-cells engrafted finally in 5 and 6 patients, respectively. The median times to neutrophil more than 0.5x109/L and platelet more than 20 x109/L were 31 day (range,18~71 day) and 17 day (range, 12~35 day), and 25 day (range,12~105 day) and 12 day (range,10~30 day) post-transplant, respectively, in the CT and the MUD- HSCT. None of the patients developed relapse.The cumulative incidences of grades Ⅱ-Ⅳ acute GVHD (aGVHD) was 35.7% (5/14 patients). Case 6 had grade III aGVHD. A case died from gut grade IV aGVHD 50 days after the CT. Chronic GVHD occurred in 2 patients, and no chronic GVHD more than grade II (NIH criterion) occurred in all patients. The most common complication associated HSCT was infection. The incidences of CMV and EBV antigenmia were 43% (6/14) and 7.1%(1/14), respectively. Recoverable serious pancytopenia occurred in two patients with Decitabine therapy post-HSCT.Conclusion:The combination of hypomethylation agent with HSCT showed encouraging results in JMML-HSCT. It is impressed that no relapse occurred in all patients despite short follow-up time. A large-cohort study with extending follow-up time should be developed in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Investigating postharvest chilling injury in tomato (Solanum lycopersicum L.) fruit using magnetic resonance imaging and 5-azacytidine, a hypomethylation agent

Author(s): Albornoz, K; Chen, B; McCarthy, M; Zhang, L; Cantwell, M; Beckles, DM | Abstract: Tomato, like most species from tropical and subtropical regions, exhibits postharvest chilling injury (PCI) when stored at low temperatures. Because of its economic importance and the functional genomics tools available, we used tomato to investigate aspects of fruit PCI development. We asked two questions: First, are there spatial-temporal differences in the development of PCI that can be detected by magnetic resonance imaging (MRI)? Here, the aim was to use a non-invasive method to study PCI progression in vivo. At mature green and breaker, the pericarp, locular tissue and columella produced distinct D-values while in contrast, there was no such differentiation in riper fruit. Although the pericarp is where most PCI symptoms are visible, this tissue showed less dynamism upon cold exposure, compared to the inner tissues as detected by MRI. This suggests the occurrence of distinct, independently modulated mechanisms contributing to the development of PCI-symptomatology. Collectively our data showed that the MRI could detect fruit ripening, its attenuation by cold, and fruit tissue-specific responses to chilling stress. The second question we asked was if epigenetic modification of the tomato genome or transcriptome influences PCI response. We examined PCI severity in fruit injected with a demethylating agent, 5- azacytidine (AZA). Two tomato genotypes exposed to varying severities of cold-stress were studied. Results suggested that AZA was able to moderate PCI in 'Micro-Tom' after 3 weeks at 2.5°C, while different patterns were observed in 'Sun Cherry' across various cold treatments. The effects of AZA on PCI were complex, multilayered and highly context-dependent.

Decitabine: A Historical Review of the Development Process of an Epigenetic Drug

Decitabine (5-aza-2p-deoxycytidine) is a hypomethylation agent with a double-action mechanism, these are the reactivation of silenced genes; exhibiting differentiation at low doses and showing cytotoxicity at high doses. Decitabine was used as a classic anticancer drug in the original studies in the 1980s, 1500 to 2500 mg/m2 per cycle was the maximum clinically tolerated dose. The dosage was reassessed after a better understanding of epigenetics in cancer and the role of decitabine in epigenetic (hypomethylation) therapy was obtained, in about 1/20th of the previous doses (i.e., 'optimal biological' doses modulating hypomethylation). It has been found that decitabine (100 to 150 mg / m2 per cycle) can be used in patients with myelodysplastic syndromes (MDS) and other myeloid tumors, with manageable side effects. Combination therapies that amplify the epigenetic effect of decitabine will most likely improve the patient responses and allow it to be used in the treatment of other malignancies.

Complementary Transplantation with Unrelated Cord Blood Following Haploidentical Stem Cells for Juvenile Myelomonocytic Leukemia:Double-Centers Study from China

Background At present, allogeneic-HSCT is still the only proven potentially curative strategy for Juvenile myelomonocytic leukemia(JMML) children, being only able to cure about 50% of patients by either a relative donnor or an unrelated volunteer. Relapse represented the main causes of treatment failure. The data of HLA-haploidentical transplantation is poorly reported so far. But we have reported an encouraging result in the treatment of leukemia and Thalassemia with complementary transplantation(CT), i.e. unrelated umbilical cord blood(UCB) following haploidentical stem cells transplantation(see blood 2016 128:1235). The evidence of aberrant DNA methylation in JMML provided a rationale for the use of the DNA hypomethylating agent 5-azacytidine or Decitabine(DAC). Recent studies have indicated hypomethylating agent maybe reduce the disease burden in the pretransplant window with minimal toxicity and play an immunomodulatory role after transplantation. Therefore, we developed a novel CT strategy combinated with hypomethylation agent for children with JMML. Patients and method 48 patients received CT combined with DAC from December 2014 to April 2019 in two independent HSCT-centers. Of them, 44 patients had molecular genetics mutation(including NF1, PTPN11,Kras,Nras),and 6 patients had cytogenetic abnormalities with monosomy 7. The median age at diagnosis was 22 (1 to 90) ms. (Table.1). 47 of 48 patients received 2~6 courses of DAC therapy (20mg/m2/d ×5 days for each course with 4-weeks interval) and 1~2 courses of mild-moderate cytotoxic chemotherapy (according to the"A-triple-V"regimen from Korea or reduced-intensity FlAG regimen) as "Bridging therapy" in order to reduce the tumor burden prior to HSCT. There was only one patient who was accepted one course of DAC therapy prior to HSCT. In addition, low dose DAC was also administrated 2~6 courses (10mg/m2/day for 3 or 5 days for each course with the interval of 4~6 weeks) for all patients in order to overcome immune-escape of leukemia cells post HSCT. None of the patients underwent splenectomy. Conditioning regimen included DAC ,Cyclophosphamide(Cy) ,Busulfan,Fludarabine and Thiotepa (or replaced by Busulfan). GVHD prophylaxis consisted of Cy on day+3,+4, MMF and Tacrolimus from day+6. Noteworthily, UCB was infused on day+6 (Fig.1). Human leukocyte antigen of UCB-donor/recipient pairs had less than 3/10 loci incompatibility.The median number of infusion mononuclear cells from haploid-donor was 37.8×108/kg, and which of infusion nucleated cells from UCB was 8.8×107/kg(Table.1). Results The median and mean follow-up time was 13.5 and 16.2 (3 to 63)ms ,respectively. Full donor cells engrafted in all patients (donor cell engraftment in one case without any cytotoxic chemotherapy before HSCT, who was occurred in a salvaged HSCT from another haplo-donor and UCB-donor after primary failure of first CT ). 5-years OS, disease-free survival(DFS) and transplant-related mortality were 90.1%,86.9% and 4.4%, respectively(Fig.2). Haplo-cells and UCB-cells engrafted finally in 26 and 22 patients, respectively. The median times to neutrophil more than 0.5×109/L and platelet more than 20×109/L were 29 days and 24 days post-HSCT, respectively (Table.2). Platelets and neutrophi recovered earlier in patients with haplo-engrafted than those with UCB-engrafted (p=0.000)(Table.3). Five patients developed relapse after HSCT, who had PTPN11 mutation. 4 of them were Haplo-cells engraftment. The median time to relapse after HSCT was 5.5 (1.5 to 12 ) months. There ware no significant difference in the OS and DFS between UCB-engrafted group and Haplo-engrafted group(p=0.612 and p=0.227,respectively)(Fig.3).The cumulative incidences of grades Ⅱ-Ⅳ aGVHD was 38% (18 pts).The number of patients with grade III and Ⅳ aGVHD were 7 in total. Chronic GVHD occurred in 7 patients, and no chronic GVHD more than grade II (NIH criterion) occurred in all patients(Table.2).The most common complication associated CT was infection. The reactivation incidences of CMV, EBV and human herpes virus 6 were 27% ,13% and 5.0%, respectively. Recoverable serious pancytopenia occurred in four patients with DAC therapy post-HSCT. Summary CT have significantly improved the therapeutic effect in JMML-HSCT.Hypomethylation agent may play an important role in CT. Neither cord blood-engraftment nor haploid-engraftment demonstrated a clear superiority for CT. Disclosures No relevant conflicts of interest to declare.

Precision Medicine Treatment in Older AML: Results of Beat AML Master Trial

Precision Medicine Treatment in Older AML: Results of Beat AML Master Trial

A Double Priming Induction Regimen for Acute Myeloid Leukemia with Inferior PS Among Resource Limited Areas

The treatment options for patients with acute myeloid leukemia (AML) under inferior performance status (i.e. senior patients, MDS transformed AML, and patients with proven invasive fungal disease) are limited. The conventional “3+7” (idarubicin plus cytarabine) induction for those patients can be either too toxic, which leads to higher mortality rate, or requires prolonged recovery time thus raise medical cost. And the delay of consolidation may compromise outcome thus cause early relapse in such patients.Giving the rationale from the designing art of CPX-351, the prolonged IV time requirement for cytarabine, as well as mini-transplantation for AML treatment, at least in part, reflects the philosophy of a longer exposure to the chemo agent can be a more effective treatment approach for leukemia. On the other hand, the D-CAG protocol, which indicated an encouraging result for elderly patients with AML, indicated effectiveness of the strategy with reduced intensity. However, the cyto-toxic effect of decitabine with standard dose (20mg/m2) could still lead to severe treatment adverse events (AEs) thus raise the need of optimization of D-CAG regimen for patients with inferior PS.When considering the low dose hypo-methylation agent (HMA) can trigger the innate immunity response, the unique effect of homoharringtonine, as well as the effectiveness of CAG, we designed this DHCAG protocol following the principle of “longer exposure, lower intensity preceded by priming” and observed an unexpected excellent outcome with high CR rate, low induction failure and treatment mortality, as well as a higher cost effective value for patients with AML, when compared to “3+7” protocol, whom under inferior PS.From March 2016 - January 2018, we initiated this pilot study and investigated the safety and efficiency of this DHCAG protocol in patients with AML under poor PS. We enrolled 25 patients and administer the regimen as followings: i) G-CSF: 5μg/kg used when WBC <20×10^9/L at day 0-14 subcutaneous injection; ii) Decitabine: 6mg/m2 at day 0, 3, 6, 9, 12 iv drip; iii) Aclarubicin: 6mg/m2 at day 1-8 iv drip; iv) Homoharringtonine: 1mg/m2 at day 9-14 iv drip ( at day 9, if WBC >1*10^9/L then increase the dosage of homoharringtonine to 2mg/m2); iv) Cytarabine:10mg/m2 q12h at day 1-14 subcutaneous injection (if WBC >20*10^9/L then increase to 100mg/m2 by 24h CIV, or 50mg/m2 q12h by subcutaneous injection).The primary end point was complete hematologic remission, defined as a bone marrow blast cells ≤5%, Neutrophils in peripheral blood ≥1.0X109 /L, hemoglobin≥90g/L, and platelets ≥100X109/L and no any evidence of extramedullary leukemic infiltration; Secondary end points included numbers of adverse events, length of hospital stay, medical costs, and quality of life as measured with the use of the Functional Assessment of Cancer Therapy - General (FACT-G) questionnaire. Among the 25 patients in the study, 23 completed the induction therapy. And 21/25 patients had a hematologic complete remission after a median time of 19 days. Once complete remission had occurred, all 21 patients received post-remission treatment for another 5 cycles of DHCAG. Median follow-up was 14 months (range, 8 to 19) by June 2018. Interestingly, regardless of grade 3-4 myelo-suppression occurred all of our patients during induction, eight patients did not experienced grade 3-4 myelo-suppression during the following cycles. The median hospital stay is 22 days. The median of total medical costs for induction was $9,815 (range, $5,053 to $16,336) vs $14,705 for “3+7” induction protocol (history control. Data unpublished). Patients resumed their usual lifestyle during post-remission therapy, and their quality of life was rated as nearly normal on the FACT-G questionnaire. At the time of the last follow-up, seven patients had had a hematologic relapse. The results of our pilot study, in which we tested a priming based, low dose and longer exposure in 25 patients with AML under poor PS, showed that the treatment was safe, effective, and economical. A prospective multicenter, randomized trial comparing DHCAG with IA is now under way in China. DisclosuresNo relevant conflicts of interest to declare.

A Novel Therapy Protocol Improves Outcomes of Hematopoietic Stem Cell Transplantation in Juvenile Myelomonocytic Leukemia Patients Using Hypomethylation of Decitabine

BackgroundJuvenile myelomonocytic leukemia(JMML) has usually poor response to chemotherapy, and approximately 50% of patients relapse after hematopoietic stem cell transplantation (HSCT). Recent studies have highlighted the importance of epigenetic aberrations in JMML and proved that some JMML stem cells were associated with hypermethylation. Hence, we desiged the current study to investigate whether low dose Decitabine could improve outcomes of JMML-HSCT.We have reported the preliminary results of low-dose decitabine in the treatment of children with JMML in 2017 ASH as a poster(see blood 2017 130:3232). Then, we will report our latest study.Patients and method27 patients received HSCT combined with Decitabine between December 2014 and July 2018. Of them,6 patients with NF-1 mutation,11 with PTPN11 mutation, 2 with Kras somatic mutation ,1 with Nras somatic mutation, 3 with multiple mutation (PTPN11+NF-1),2 with monosomy 7,and 3 with uncertain mutation. The median age at diagnosis was 24 months (range: 1-72 months).26/27 patients received 1~4 course mild chemotherapy(one patient,case 6, received only single course Decitabine therapy)before HSCT.3 patients received HSCT from HLA matched unrelated donors(MUD),and 24 patients received the complementary transplantation(CT), i.e. unrelated cord blood(UCB) was given at day 6 after haploidentical peripheral blood stem cell transplantation(PBSCT) using high dose cyclophosphamide(Cy) post-transplant (PTCy), (see blood 2016 128:1235).Conditioning regimen was composed of Cy, Busulfan (Bu)/ Thiotepa (TT), Fludarabine (Flu) and ATG-F in the MUD-HSCT, and Cy, Bu/TT, Flu and Cytarabine in the CT. Patients received a fixed dose of 8×108/kg mononuclear cells(MNC) in the MUD-HSCT, and a median dose of 45.5×108/kg (range, 26.8~88×108/kg) mobilized peripheral blood MNCs and a median dose of 8.9×107/kg (range, 4.0~12.8×107/kg) UCB nucleated cells in the CT, respectively.GVHD prophylaxis consisted of PTCy, Mycophenolate Mofetil (MMF) and Tacrolimus in the CT, and Thymoglobuline, CsA and MMF in the MUD-HSCT.Decitabine was administrated for 2~4 courses (20mg/m2.d×5 day for each course with 4-week interval) before HSCT to reduce load of leukemia cells and for 2~4 courses (5~10mg/m2.day×5day for each course with the interval of 4~6 weeks) after HSCT to overcome immune-escape of leukemia cells.Results:The median follow-up time was 13months (range, 2-51 months). Full donor cells were engrafted in all patients (donor cell engraftment in case 6 occurred in a salvaged transplant from another haplo-donor after primary failure of first CT).The Overall survival(OS) and Disease-free survival(DFS) was 89.4% and 87.3% respectively. In the CT, haplo-cells and UCB-cells were engrafted in 10 and 14 patients, respectively.The median time to neutrophil more than 0.5x109/L was 31days (range,12~71 days) and 17 days (range, 12~35 days)post-transplant, and to platelet more than 20 x109/L was 22 days (range,9~105 days) and 12 days (range,10~30 days) post-transplant, respectively, in the CT and the MUD- HSCT. All the 3 patients with relapse were haploid-engrated. Two of the three patients with relapse had underwent secondary CT. One of them was Disease-free survival ,and the other died of viral encephalitis(HHV-6) after secondary CT.The cumulative incidence of grades Ⅱ-Ⅳ acute GVHD (aGVHD) was 25.9% (7/27 patients). Case 6 had grade III aGVHD. A case died of grade IV aGVHD(gut) 50 days after the CT. Chronic GVHD(cGVHD) occurred in 5 patients, and no cGVHD more than grade II (NIH criterion) occurred in all patients. The most common complication associated with HSCT was infection. The cumulative incidences of infection plus reactivation of CMV,EBV and HHV-6 were 30% (7/27),3.7%(1/27) and 11.1%(3/27), respectively. Recoverable serious pancytopenia occurred in 3 patients with Decitabine therapy post-HSCT.Conclusion:The combination of hypomethylation agent with HSCT still showed satisfactory results in JMML-HSCT when the follow-up time has been extended for one year. A large-cohort study with extending follow-up time should be developed continuously in the future and the interim results of five-year follow-up time will be reported. DisclosuresNo relevant conflicts of interest to declare.