Decitabine Research Articles

Venetoclax+Azacytidine Followed By Modified BuCy Conditioning Regimen for High Risk or Refractory/Relapsed Acute Lymphoblastic Leukemia and High Risk Myelodysplastic Syndromes

Background:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only potential curable treatment for myelodysplastic syndromes (MDS) and acute lymphoblastic leukemia (ALL). However, for patients with high-risk or refractory relapsed (R/R) B-ALL with allo-HSCT, the three-year overall survival (OS) rate is only 5-10%, more worse in T-ALL. It is widely known that MDS patients in the high-risk group do not benefit from induction chemotherapy, the five-year OS rate is 23-39% when they received allo-HSCT. Nevertheless, relapse remains a significant cause of treatment failure after allo-HSCT, highlighting the urgent need for improved conditioning regimens that can improve prognosis and reduce relapse rates. Azacytidine (AZA) combined with Venetoclax (VEN), showed a synergistic anti-tumor activity against several hematological malignancies, Therefore, we explored AZA+VEN (VA) combined with modified BuCy(mBuCy) (semustine, cytarabine, busulfan, and cyclophosphamide) conditioning regimen and assessed the safety and effectiveness. Aims ：The aim of this study is to assess the safety, and effectiveness of the VA treatment regimen followed by mBuCy as conditioning regimen for high-risk or R/R ALL(NCT05809167) and high-risk MDS(NCT03256071) . Methods:Patients diagnosed with high-risk or R/R ALL or high-risk MDS (IPSS-R score), undergoing VA+mBuCy as conditioning regimen, and consolidation treatment with demethylating drugs every three months after transplantation, for a total of 8-12 cycles. Results:Patients diagnosed with high-risk or R/R ALL(n=11) or high-risk MDS (IPSS-R score)(n=8) were enrolled between January 13, 2022, and June 9, 2023. The ALL cohort comprised of 5 Ph-negative B-ALL, 2 Ph-positive B-ALL, 3 mixed phenotype leukemias (MPAL), and one T-lymphoblastic lymphoma/leukemia (T-ALL/LBL). There were 7 males and 4 females, with a median age of 33 years (17-54 years) (Table 1). Out of the eleven patients, four had previously undergone Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) therapy, and one patient had received Blinatumomab. One patient experienced relapse, another patient was refractory, and 3 patients had extramedullary infiltration. All patients achieved morphological complete remission prior to transplantation, and 10 patients (90.9%) achieved a MRD negative remission. Additionally, all the transplantations successfully completed, with a median time to absolute neutrophil counts (ANC) recovery of 12 days (11-15 days) and platelet (PLT) recovery of 16 days (15-21 days). The median follow-up time was 6.4 months (2.4 -13.6 months), no patient experienced relapse or death, the OS and DFS are both 100%. There were no grade 3 or higher adverse events (AEs), hepatic veno-occlusive disease (VOD),and the transplant-related mortality (TRM). Four patients who developed acute graft-versus-host disease (aGVHD) post-transplantation, all cases involved grade I/II skin aGVHD, and one of them involved grade III GI aGVHD, and no chronic graft-versus-host disease (cGVHD) were observed during the follow-up period. By the endpoint of follow-up, Cytomegalovirus (CMV) activation was 45.4% (5/11) and Epstein-Barr virus (EBV) activation was 27.3% (3/11). The MDS cohort consisted of 7 cases of MDS-EB-I, 1 case of MDS-EB-II. There were 5 males and 3 females, with a median age of 42 years (23-57 years)(Table 2). Two patients received chemotherapy before transplantation, and the other six patients directly received allo-HSCT. In MDS cohort, the median recovery time for ANC was 12 days (11-13 days) and PLT was 17.5 days (16-20 days). The median follow-up was 14.3 months (1.2-18.3 months), no patient had relapsed or died, both OS and DFS are 100%. There were no grade 3 or higher AEs, and TRM. One patient developed VOD. Three patients developed aGVHD after transplantation, all involving degree I/II skin aGVHD, and there was one case of cGVHD during the follow-up period, where the patient developed a mild pulmonary cGVHD. By the endpoint of the follow-up, the rate of CMV activation was 37.5% (3/8) and EBV activation was 25% (2/8). Conclusion:The VA combined with the mBuCy proves to be a effective and safe conditioning regimen for high-risk ALL and MDS patients. However, more patients are needed for follow-up and validation.

A Phase 1 Study of Adding Pitavastatin to Venetoclax-Based Therapy in AML and CLL/SLL

Statins (HMG-CoA-reductase inhibitors) are drugs used widely for the treatment of high cholesterol and are generally well-tolerated. There has been increasing interest in repurposing statins for oncology based on the recognition that many cancer cells are “addicted” to the mevalonate biosynthetic pathway (Iannelli et al, 2017; Juarez and Fruman 2021). We have shown that statins increase expression of the pro-apoptotic BCL2 family member PUMA (Lee at al, 2018). We have strong pre-clinical evidence that statins improve responses to the BCL2 inhibitor venetoclax (VEN) in cell line models and ex vivo primary patient specimens from patients with both chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) (Lee et al, 2018). Additionally, retrospective review of patients enrolled into the early clinical trials of VEN in CLL revealed improved responses to VEN (complete remission [CR] and progression-free survival) in the patients who were concurrently on a statin medication (Lee et al, 2018). These retrospective analyses may underestimate the potential of statins for leukemia therapy, as the statin drugs most commonly prescribed for hypercholesterolemia are not pharmacologically optimized for oncology (Abdullah et al., 2018). We developed a phase 1 study of adding pitavastatin (PIT) to VEN-based therapy in patients with AML and CLL/small lymphocytic lymphoma (SLL) (NCT04512105). AML patients were eligible if receiving induction therapy with azacitidine (AZA) and VEN per standard of care. CLL/SLL patients could receive either VEN with obinutuzumab (O) or rituximab. A 3+3 design was employed. Dose level 1 was PIT 2 mg daily, and dose level 2 (DL2) was 4 mg daily. These doses are consistent with the doses used for the treatment of hypercholesterolemia. Those enrolling at DL2 had a creatinine clearance of 60 mL/min or higher based on the prescribing information for pitavastatin. Patients already on statins for the treatment for hypercholesterolemia were allowed; these patients stopped their prescribed statin for 72 hours prior to initiation of PIT. PIT started after patients completed VEN ramp-up and had been on a stable VEN dose for at least 5 days. Primary objectives were assessing safety and tolerability and determining the recommended phase 2 dose of PIT in combination with VEN-based therapies. Secondary objectives included CR rates. Exploratory objectives included performing BH3 profiling on pretreatment and post-treatment blood samples to assess whether add-on treatment with PIT increases apoptotic priming, as seen in preclinical models (Lee et al, 2018). 14 patients signed informed consent. 6 were deemed ineligible. 2 withdrew consent prior to starting PIT therapy. 6 patients received PIT on study. 2 patients had AML and received AZA+VEN. 2 patients had CLL, and 2 patients had SLL. The CLL/SLL patients all received VEN+O. Grade 1 myalgia was reported in one subject on the 4mg PIT dose. 1 patient experienced a grade 1 increase in AST on the 4mg dose. Another patient had grade 1 elevation in bilirubin and alkaline phosphatase at the 4 mg dose. Grade 3-4 toxicities included leukopenia, neutropenia (1 case of febrile neutropenia, grade 3), anemia, and thrombocytopenia. Cycle 1 was defined as the dose-limiting toxicity (DLT) period, and no hematologic toxicities occurred during the DLT period. 1 patient had grade 3 pancreatitis, but this was felt to be unrelated to therapy or disease (also outside the DLT period). No grade 5 toxicities occurred while on study therapy. Toxicities were overall felt to be similar to patients receiving VEN-based therapy in standard clinical practice. All evaluable patients (n=5) achieved clinical CR. 1 patient with SLL is awaiting re-staging at the time of this abstract. Correlative studies are ongoing. In 2 patient peripheral blood samples analyzed, the % blasts (for an AML sample) and % CD5+ B-cells (for a CLL sample) were reduced 24 hours after PIT dosing compared to prior to any treatment and before PIT/after VEN ramp-up. Based on the tolerability of PIT and encouraging clinical outcomes, a phase 2 study is being planned to better assess the efficacy of adding PIT to VEN-based therapies in AML and CLL/SLL.

Venetoclax Exposure Reduction in Venetoclax-Hypomethylating Agents Combination Is Feasible and Has the Same Efficacy in Patients with Acute Myeloblastic Leukemia. Experience of a Single Center

BACKGROUND The combination of venetoclax (VEN) and azacitidine (AZA) is the actual standard of care for patients with acute myeloid leukemia (AML) not eligible for intensive treatment ( Di Nardo et al. N Engl J Med 2020; 383:617-629). Despite being considered a low-intensity treatment, dose adjustments are frequently needed in this group of patients due to prolonged myelosuppression and increased incidence of infection. Reducing the duration of VEN administration might improve the safety profile, although its efficacy is still unknown in this setting. AIM We present the experience of the Institut Català d'Oncologia-Hospital Duran i Reynals in patients with AML treated with VEN in combination with hypomethylating agents (HMA) to evaluate the efficacy of VEN exposure reduction in patients who require VEN adjustment due to myelotoxicity. METHODS We retrospectively analyzed 68 patients with AML treated with VEN+HMA as a first-line or as salvage therapy between May 2019 and April 2023. We specifically evaluated patients in complete remission (CR) after 1 to 3 cycles of VEN combination who require VEN scheme shortening to 7 (low dose) or 14-21 (intermediate dose) days every 28 days cycle due to myelotoxicity. Response to treatment was defined according to ELN2022 criteria. RESULTS Median age at diagnosis was 75 years (range 33-85 years). Forty-seven (69%) were male. Twenty-eight (41.2%) were classified according to the 2022 ELN classification as an adverse risk, 33 (48.5%) as intermediate risk and 7 (10.3%) as a favorable risk group. The median number of cycles was 7 (range 1-23). ORR of the whole series was 76.5%. Forty-six (67.6%) patients achieved a composite complete response (CRc) (CR, CR with partial hematologic recovery, and CR with incomplete hematologic recovery). Among the 46 responding patients, seven of them (15.2%) discontinued treatment because of patient's decision after achieving CR and they were not considered for further analysis. The remaining 39 patients reached the best response in the first 2 cycles. Twenty-eight (71.8%) patients received VEN+HMA as first-line treatment and 11 (28.2%), as salvage therapy. No significant differences were found between these two groups regarding sex, age or ELN2022 risk. Nine out of these 39 patients (23.1%) received VEN for 28 days every 28 days cycle (VEN28 group), 21 (53.8%) patients received VEN for 14 or 21 per cycle (VEN14-21 group), and 9 (23.1%) patients received VEN for 7 days per cycle (VEN7 group). With a median follow-up of 18.8 months (range 14.6-23.1), the median OS of the VEN14-21 group was 15.9 months but it was not reached for the VEN7 group. Regarding EFS, the median EFS of the VEN14-21 group was 11.4 months though it was not reached for the VEN7 group. No statistically significant differences were found comparing OS and EFS between the VEN14-21 group and the VEN7 group nor were both of them with the VEN28 group (Figure 1). CONCLUSION Shortening VEN exposure to 14-21 or 7 days per cycle with concomitant HMA administration seems to induce a similar response rate and is likely to have comparable efficacy to the 28-day cycle. Among responders, VEN dose reduction seems to be associated with favorable outcomes. Prospective clinical trials that further evaluate the efficacy and safety of shortened VEN administration are required.

N/KRAS-Mutant AML LSCs Originate from Committed Myelomonocytic Progenitors and Drive Clinical Resistance to Venetoclax

Driver mutations in acute myeloid leukemia (AML) often exhibit distinct temporal acquisition patterns, but the biological basis for this, if any, remains unknown. RAS mutations occur invariably late in AML, upon progression or relapsed/refractory disease, in contrast to their function as early drivers in solid tumors. We developed synthetic leukemogenesis models in human induced pluripotent stem cell (iPSC)- and primary cord blood (CB)-derived human hematopoietic stem/progenitor cells (HSPCs) by introducing the prototypical NRAS G12D mutation alone or with other mutational combinations, using CRISPR, and engraftment of a transplantable leukemia into NSG mice as the readout. RAS mutations alone were not sufficient for leukemogenesis, but required specific cooperating mutations. Subsequently, by creating temporally controlled engineered models, we found that RAS mutations are obligatory late events that can only drive leukemic transformation when acquired after, but not before, cooperating mutations. We provide the mechanistic explanation for this in a requirement for mutant RAS to specifically transform committed granulocyte-monocyte progenitors (GMPs) harboring previously acquired driver mutations, through sorting and transplantation and multi-omics experiments. In contrast, acquisition of RAS mutations in earlier progenitors (common myeloid progenitors, CMP), with or without cooperating mutations, blocked the emergence of GMPs and abolished engraftment or gave rise to a myeloproliferative neoplasm. These results indicate that advanced RAS-mutant (MT) leukemic clones have a different cell type of origin from the ancestral clone. Next, using single-cell transcriptomics in cells isolated from xenografts of two AML-iPSC lines generated from an AML patient with a subclonal KRAS G12D mutation, one capturing the RAS WT major clone and one the KRAS G12D subclone, we show that KRAS G12D leukemia stem cells (LSCs) give rise to leukemia with a more monocytic immunophenotype, while the ancestral clone within the same patient generates more leukemic cells with primitive and neutrophil markers. These results were corroborated by Genotyping of Transcriptomes (GoT) in another AML patient with subclonal NRAS G12D mutation. Recent studies have linked monocytic AML and, independently, RAS pathway mutations to poor responses to Venetoclax (VEN)-containing regiments. In view of the causal link we found between RAS mutations and monocytic disease, we evaluated the associations between monocytic AML, RAS mutations and response to VEN. We reanalyzed data from a cohort of 118 older/unfit newly diagnosed AML patients treated on a prospective clinical trial with VEN and decitabine (DEC) (NCT03404193). All outcomes were comparable between monocytic and non-monocytic groups, including overall response rate, duration of response (DOR) and overall survival. On the other hand, patients with N/KRAS mutations had significantly shorter DOR. These results support the presence of N/KRAS mutations and not the monocytic stage as predictors of inferior responses to VEN regimens. To definitely test this, we treated defined cell types generated from RAS WT and RAS MT iPSC lines - CD34+ LSC-like and CD11b+/CD68+/CD14+ monocytic cells - with VEN. In agreement with previous studies, monocytes were resistant and RAS WT LSCs were sensitive to VEN. However, strikingly RAS MT LSCs were VEN-resistant. Consistent with these, single-cell transcriptomics in patient-derived AML-iPSC cells in vivo, GoT in primary AML cells and bulk transcriptomics in synthetic iPSC-AML cells showed downregulation of BCL2 and upregulation of MCL1 in the RAS MT LSCs. This study provides the first example of a mechanistic explanation of why the timing of mutational acquisition in AML is so strongly biased. It shows for the first time that interaction between an oncogenic driver and the non-genetic developmental hematopoietic hierarchy imposes a specific LSC cell-of-origin restriction and in turn shapes the hierarchical structure of the resulting AML and critically determines therapeutic outcomes in patients. Importantly, our results have direct implications for clinical practice, as they support that RAS MT LSCs drive clinical resistance to VEN and that treatment with VEN in patients with RAS mutations can accelerate disease progression by selection of RAS MT LSCs (Figure).

Phenotypic and Molecular Landscape of Bone Marrow CD8+ T Cells in Myeloid Neoplasms Predicts Response to Treatment with Hypomethylating Agents

Background and aims Myeloid malignancies encompass a diverse group of conditions, including myelodysplastic neoplasms (MDS) and chronic myelomonocytic leukemia (CMML). There is evidence suggesting the role of bone marrow (BM) environment and immune cells in the development and progression of MDS and CMML. Azacytidine (AZA) remains the backbone therapy for higher-risk MDS and non-proliferative CMML. To date, molecular predictors of response to AZA are not yet well-defined. Herein, we sought to investigate the immune landscape of BM samples from patients with lower-risk MDS (LR-MDS; i.e., IPSS-R <3.5), CMML, higher-risk MDS (HR-MDS; i.e., IPSS-R ≥3.5), and acute myeloid leukemia (AML). Methods Samples were collected before treatment initiation. Mass cytometry (CyTOF) was performed on isolated BM mononuclear cells from 48 patients, using the Maxpar® Direct™ Immune Profiling Assay. Flow cytometry was performed on a secondary cohort of 56 patients. scRNA-sequencing was performed on sorted BM CD8+ T cells from patients with HR-MDS (n=4) and AML (n=5), using a 10x Genomics pipeline. Mann-Whitney U test and the Kruskal-Wallis test were used as appropriate. Kaplan-Meier analysis was utilized for survival analysis, and the log-rank test was employed to assess the significance. The level of significance was established at P < 0.05. Results The immunophenotypic analysis using CyTOF demonstrated increased expression of CXCR3 on cytotoxic CD8+ T-cells in CMML and AML samples compared to samples from patients with MDS. Increased frequency of the CD57 +CXCR3 +CD8 + T-cell subset was further observed in patients with HR-MDS and AML that were non-responders to AZA, compared to responders, as observed by both CyTOF and flow cytometry. After determining the optimal cut-off for the frequency of CD8 +CD57 +CXCR3 + T cells at 29%, we observed that HR-MDS and AML patients (Figure 1A) with a frequency of CD57 +CXCR3 +CD8 + of <29% had better overall survival (OS).Multivariate analysisshowed that a frequency of CD57 +CXCR3 +CD8 + of >29% was independently associated with decreased survival in patients with TET2 mutations (HR=2.603; 1.275-5.317). scRNA-seq analysis of isolated BM CD8 + T cells was further performed to characterize the molecular signature that favors response to AZA, which resulted in the identification of several cell clusters based on gene expression. The cluster characterized by the expression of genes associated with cytotoxicity showed significant differences between responders and non-responders. Specifically, within the cytotoxic cluster, genes associated with IFN-related pathways were upregulated in cells from responders to AZA. In contrast, genes upregulated in non-responders displayed an enrichment for the TGF-β signaling pathway, which is known to be associated with the suppression of the killing capacity of T cells (Figure 1B). The aforementioned molecular signatures were further associated with a higher cytotoxic molecular signature in the cytotoxic cell cluster from the group of responders. Conclusion In this study, we focused on BM CD8 + T cells in samples from patients with myeloid neoplasias and performed deep phenotypic analysis using CyTOF coupled with scRNA, in order to characterize the BM immune environment. We demonstrate for the first time that BM CD8 + T cells show different phenotypic and transcriptomic characteristics in responders to AZA. Specifically, we show that the frequency of the effector CD57 +CXCR3 +CD8 + subset is decreased in responders before treatment initiation, and this is associated with better survival. Despite the decreased proportion of this cytotoxic cell population in responders, there are specific signatures associated with effector T cell functionality that can explain the improved outcomes in these patients. These findings underscore the significance of the crosstalk between leukemic and immune cells in the progression of the disease and the effect of treatment. Figure legend A. Increased (%) CD57 +CXCR3 + correlates significantly with worse survival of HR-MDS and AML patients. Kaplan Meier curves for Overall Survival in HR-MDS and AML patients respectively. B. Gene signature derived from the scRNAseq analysis. Interferon-stimulated gene (ISG) signature is increased in the cytotoxic cell cluster in responders and and TGF-β signature in non-responders.

Embryonic exposure to decitabine induces multiple neural tube defects in developing zebrafish.

Neural tube defects are severe congenital disorders of the central nervous system that originate during embryonic development when the neural tube fails to close completely. It affects one to two infants per 1000 births. The aetiology is multifactorial with contributions from both genetic and environmental factors. Dysregulated epigenetic mechanisms, in particular the abnormal genome-wide methylation during embryogenesis, have been linked to developmental abnormalities including neural tube defects. The current study investigated the influence of decitabine (DCT), a DNA methylation inhibitor, on embryonic development in zebrafish, with a focus on neural tube formation. The developing zebrafish embryos were exposed to graded concentrations of decitabine (from 13.69 μM to 1 mM) before the onset of neurulation. The developmental process was monitored at regular time intervals post fertilization. At 120 h post fertilization, the developing embryos were inspected individually to determine the incidence and severity of neural tube defects. Using alizarin red staining, the cranial and caudal neural tube morphology was examined in formaldehyde fixed larvae. Anomalies in neural tube and somite development, as well as a delay in hatching, were discovered at an early stage of development. As development continued, neural tube defects became increasingly evident, and there was a concentration-dependent rise in the prevalence and severity of various neural tube defects. 90% of growing embryos in the group exposed to decitabine 1 mM had multiple neural tube malformations, and 10% had isolated neural tube defects. With several abnormalities, the caudal region of the neural tube was seriously compromised. The histopathological studies supported the malformations in neural tube. Our study revealed the harmful impact of decitabine on the development of the neural tube in growing zebrafish. Moreover, these findings support the hypothesis that the hypomethylation during embryonic development causes neural tube defects.

Measurable Residual Disease Assessment in Patients with Acute Myeloid Leukemia Aged ≥60 Years Treated with a 10-Day Decitabine Schedule Versus Intensive Chemotherapy in the AML21 Study (NCT02172872)

Introduction Several studies have demonstrated that patients with acute myeloid leukemia (AML) who test measurable residual disease (MRD) positive during or after treatment have a higher probability of relapse and a shorter duration of survival, as compared to those who are persistently MRD negative. Indeed, a status of MRD positivity reflects a condition of resistant disease, existing below the threshold of the morphologic evaluation of complete remission (CR). The bulk of our knowledge in terms of MRD assessment has been generated in the setting of intensive chemotherapy; much less is known for older patients receiving less intensive treatments, such as decitabine (DEC) using a 10-day schedule. Methods The AML21 study was a randomized open-label phase 3 clinical trial conducted by the EORTC Leukemia Group, GIMEMA, and GMDS-SG. Patients with a newly diagnosed AML, aged ≥60 years and eligible for intensive chemotherapy were randomized 1:1 to receive DEC (20mg/m2 a day) using a 10-day schedule or intensive chemotherapy regimen consisting of daunorubicin 60 mg/m2 x 3 days and cytarabine 200 mg/m2 x 7 days (3+7), followed by 1-4 additional chemotherapy cycles. Patients who had an HLA-matched donor and at least stable disease were encouraged to undergo HSCT after >1 treatment cycle. Patients from the DEC arm not receiving HSCT could continue DEC treatment. A subset of patients treated in GIMEMA sites underwent a prospectively planned MRD evaluation post cycle 3 (DEC arm)/post consolidation (3+7 arm), and at 6, 12 and 18 months after the randomization, in case they were in a first CR and had not been allografted. MRD was studied by multiparameter flow cytometry. Based on several retrospective validations in the context of former EORTC/GIMEMA protocols, the threshold for discriminating MRD negative from MRD positive cases was set at 3.5x10-4 (0.035%) residual leukemic cells, upon full blood count recovery. Results In total, 130 out of 154 patients treated in GIMEMA centers provided consent for participation in translational research, had leukemia associated immunophenotype suitable for MRD monitoring, and initiated the protocol treatment, DEC (N=66) or 3+7 (N=64). Median age was 68 years (range 60-78), 33% of patients were ≥70 years old, 54% were male, and 31% and 30% had good and adverse European LeukemiaNet 2017 risk profile, respectively. At the MRD evaluation post cycle 3/consolidation, 9 out of 66 (14%) patients from the DEC and 12 out of 64 (19%) patients from the 3+7 arm had documented MRD negativity. The number of patients who either had documented MRD negativity or had undergone allografting and were alive and progression-free was 12 (18%) and 17 (27%) at 6 months, 14 (21%) and 13 (20%) at 12 months, and 14 (21%) and 7 (11%) at 18 months in the DEC and 3+7 arms, respectively. The inspection of individual profiles of patients who underwent the first MRD evaluation (and were at remission at that time) indicated that all MRD-positive patients from the DEC arm who were not allografted eventually relapsed, which often occurred just after the discontinuation of DEC maintenance. Conclusions 10-day DEC treatment can induce MRD-negativity. However, early MRD-negativity appeared more difficult to achieve with 10-day DEC than with 3+7. Among patients in whom 10-day DEC did not induce MRD-negativity, allografting or DEC maintenance appeared necessary to avoid a disease relapse.

Multi-Omics Reveals Azacitidine Partially Restores Hematopoiesis-Supportive Functions in Mesenchymal Stromal Cells from Patients with Chronic Myelomonocytic Leukemia

Background: Being the most standard first-line treatment option for chronic myelomonocytic leukemia (CMML), the mechanism by which hypomethylating agents exert therapeutic effects has not yet been elucidated. While it was previously believed that HMAs act primarily through inhibiting DNA methyltransferases and restoring the expression of tumour suppressor genes, recent findings suggested its therapeutic effects might involve remodelling the bone marrow mesenchymal niche. Bone marrow mesenchymal stromal cells (MSCs) were hypothesized to play a role in this process. Nevertheless, the lack of evaluations on MSC amenability to HMAs has hindered a complete understanding of the post-HMA treatment mechanisms in patients with CMML. Aims: The present study aimed to investigate the effects of azacitidine (AZA), the most commonly used HMA, on CMML patient-derived MSCs and to determine its associated clinical significance. The specific aims were to understand the AZA-induced modifications on the DNA methylome/transcriptome and, thus, to evaluate the molecular/functional impacts of AZA treatment on hematopoiesis-supportive functions in MSCs. At last, we attempted to evaluate the association between in vivo/ex vivo sensitivity of MSCs to AZA and post-HMA efficiencies in CMML patients. Methods: High-throughput multi-omic methods including DNA Methylation Profiling (Infinium HumanMethylation850K BeadChip, Illumina Inc), RNA-sequencing (RNA-seq), and exome sequencing were utilized to assess the regulatory effects of the treatment of AZA (10 μM for 48 hours) on the DNA methylome and transcriptome of CMML patient-derived MSCs. Molecular and functional investigations including DNA damage assessment assay and long-term culture initiating cell (LTC-IC) assay, were performed to validate the sensitivity of CMML patient-derived MSCs (Figure 1A). At last, using univariate survival analysis, we assessed the association between the sensitivity of patient-derived MSCs to AZA and the efficacy of HMA-treated patients. Results: AZA treatment induced global DNA demethylation in both CMML-MSCs and healthy MSCs, with CMML-MSCs showing a more complex and bidirectional response pattern to AZA than healthy MSCs. The combined multi-omic analysis identified 1395 “AZA-sensitive genes” as potential regulatory targets in CMML-MSCs (66.4% up-regulated and 37.5% down-regulated). These genes were associated with hematopoiesis support, cell adhesion, and immune activation. Functional assays confirmed that AZA restored the expression of hematopoiesis-supportive cytokines and improved the hematopoiesis-supportive abilities of CMML-MSCs. At last, the higher sensitivity of MSCs correlated with a significant superior progression-free survival (PFS; 21.8 months vs. 16.2 months, P=0.03) and higher overall response rate (77.8% vs. 22.2%) in CMML patients treated with at least 2 cycles of HMAs (Figure 1B). Conclusion: The study provides evidence that patient-derived MSCs are sensitive to AZA treatment at the DNA methylation level, suggesting a modulable bone marrow mesenchymal niche during HMA treatment. AZA treatment rejuvenated functionally defected CMML-MSCs and restored their ability to support normal hematopoiesis in vitro. The sensitivity of CMML-MSCs to HMA correlated with clinical outcomes, with patients having AZA-sensitive MSCs showing superior PFS and higher clinical response rates after HMA treatment. These findings support the theory that AZA may switch the dysfunctional MSCs from a “leukemic-supportive” phenotype to a “hematopoiesis-supportive” phenotype. However, further investigations are required to understand whether this recovery of the hematopoietic niche directly leads to the inhibition of malignant blast cells. Patient-derived xenograft models are recommended to further verify the potential therapeutic effects of HMA-associated modulations on the hematopoietic niche.

Preliminary Results of a Phase 2 Study of IMM01 Combined with Azacitidine (AZA) As the First-Line Treatment in Adult Patients with Chronic Myelomonocytic Leukemia (CMML)

Background: Patients with Chronic Myelomonocytic Leukemia (CMML) have poor prognosis and an inherent risk for transformation to acute myeloid leukemia. IMM01 is a recombinant signal regulatory protein α (SIRPα) IgG1 fusion protein that exerts anti-tumor activity via blocking “Don't eat me” signal and activating the “Eat me” signal to induce strong antibody-dependent cellular phagocytosis (ADCP). Furthermore, IMM01 activated macrophages could process and present tumor antigen to T cells and elicit long-last tumor-specific T cell immune response. Methods : This is an open-label, multi-center, phase 2 study (NCT05140811) that evaluated safety and efficacy of IMM01 in combination with AZA as the first-line treatment in patients with newly diagnosed CMML. We enrolled patients aged 18 years or older with the diagnosis of CMML, including subtypes CMML-1 and 2, not eligible for stem cell transplant. Patients with treatment-naive CMML were administered intravenous IMM01 at a dosage of 2.0mg/kg/week and subcutaneous AZA at a dosage of 75 mg/m 2 on D1-7 per 28-day cycle. Safety was assessed by CTCAE V5.0 and efficacy by IWG 2006 (MDS) criteria. Results : Between August 4, 2022 and July 7, 2023, 24 patients were enrolled: median age was 62 years, 15 (62.5%) males, 18(75.0%) ECOG≥1. Additionally, 1(4.2%), 7(29.2%) and 16(66.7%) patients were intermediate risk(IR)-1, IR-2 and high risk(HR) by CPSS-mol classification, respectively. According to World Health Organization (WHO) classification of CMML, patients enrolled included CMML-1(50.0%) and CMML-2(50.0%). Majority of patients had poor baseline hematologic conditions with a median Hb of 69.5 (32, 132) g/L and a median PLT of 73.5 (5, 667)×10 9/L. By the data cut-off date of July 7, 2023, the median follow-up for the study was 4 months (95%CI:3.3-4.8). Among the 16 efficacy evaluable patients who received initial treatment ≥3 months, overall response rate (ORR) was 87.5% (14/16), including 25.0% (4/16) complete response (CR), 18.8% (3/16) marrow CR (mCR) with hematologic improvement (HI), 6.3% (1/16) HI and 37.5% (6/16) mCR alone. Among the 9 efficacy evaluable patients who received initial treatment ≥4 months, ORR was 88.9% (8/9), including 44.4% (4/9) CR, 11.1% (1/9) mCR with HI and 33.3% (3/9) mCR alone. The CR rate of IMM01 combined with AZA is increasing with longer treatment duration. The median duration of response (DoR) was not reached. NGS analysis of bone marrow collected from responders were included in this study. The most frequent treatment related adverse events (TRAEs) (≥20%) were lymphopenia (70.8%), leukopenia (70.8%), neutropenia (70.8%), thrombocytopenia (58.3%), infusion related reaction (33.3%), anemia (25.0%), pyrexia (25.0%), pneumonia (25.0%), constipation (20.8%), nausea (20.8%), vomiting (20.8%) and leukocytosis (20.8%). The most common ≥Grade 3 TRAEs (≥10%) included lymphopenia (66.7%), neutropenia (54.2%), leukopenia (54.2%), thrombocytopenia (54.2%), anemia (20.8%) and pneumonia (16.7%), all of which were commonly reported in AZA monotherapy studies for treatment naive MDS/CMML patients in China. The study is ongoing. Conclusions: Preliminary data showed that IMM01 (without a low-dose priming) combined with AZA were well tolerated. The combo, when compared to the historical data of AZA alone, showed exciting efficacy result for patients with treatment-naive CMML1-2.

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.

Beat AML S8 Group 2: Gilteritinib (GILT) in Combination with Decitabine (DEC) and Venetoclax (VEN) in Untreated FLT3 Mutated Acute Myeloid Leukemia (AML) Patients Age ≥60 with High and Low Variant Allele Frequency (VAF)

Background: GILT is a potent oral selective FLT3 kinase inhibitor ( FLT3i) approved for the treatment of patients with relapsed/refractory FLT3 mutated ( FLT3m) AML. However, the optimal combination in older, newly diagnosed (ND) FLT3m AML patients is unknown. GILT in combination with azacitidine (AZA) had higher initial response rates, but did not improve overall survival (OS), compared to placebo in the LACEWING trial (Wang et al. Blood 2021). GILT followed by decitabine (DEC) was also tested and found to be safe, but the efficacy did not meet pre-determined criteria (NCT03013998, S8 Group 1). In the interim, VEN + AZA/DEC emerged as the new standard of care for older/unfit patients with AML (DiNardo et al. NEJM 2020). Thus, the Beat AML S8 study was amended to assess the safety and efficacy of GILT in combination with DEC and VEN in newly diagnosed FLT3m AML patients aged ≥60 years (NCT03013998, S8 Group 2). Methods: The S8 Group 2 sub-study was part of the multicenter (15 sites) Beat AML Master Trial. Key eligibility criteria included ND FLT3m AML patients aged ≥60 years who are not able to receive intensive induction chemotherapy, and Eastern Cooperative Oncology Group (ECOG) performance status 0-2. A conventional 3+3 design was used to determine the recommended phase 2 dose (RP2D) of GILT+DEC+VEN. Patients were treated with GILT at three dose levels (DL). During induction, treatment consisted of GILT 120mg/day on days 1-7 then 80mg/day on days 8-28 for dose level 1 (DL1), or on days 8-21 (DL-1), or on days 8-14 (DL-2). All patients received DEC at 20mg/m 2 on days 8-12, and VEN at 400mg/day (or at 100mg/day if concomitant antifungal) on days 8-28. Patients who achieved a complete remission (CR) or CR with hematologic improvement (CRh) received GILT 80mg/day on days 1-15 (DL1) or days 1-7 (DL-1, DL-2), DEC on days 1-5 (All DLs), and VEN on days 1-15 (DL1, DL-1) or days 1-10 (DL-2) for an additional 12 consolidation cycles. The primary end point was to determine the tolerability and toxicity of GILT in combination with DEC and VEN. Secondary end point was the composite CR (CRc: CR/CRh) rate. Response was assessed using modified 2017 European LeukemiaNet (ELN) AML criteria. Results: Between 10/22/2020 and 6/13/2022, 19 patients were consented, of whom 18 started treatment and were included in the analysis. The median age was 73 and baseline patient characteristics are shown in Table 1. The first 2 patients treated at DL1 both experienced hematologic dose-limiting toxicities (DLTs) with persistently low absolute neutrophil count (ANC). Therefore, the GILT dose was de-escalated to DL-1, where 8 patients were enrolled and treated. Among the 6 evaluable patients on DL-1, DLTs occurred in 2 patients; one had hematologic DLT and the other had failure to thrive. This led to dose de-escalation to DL-2. In DL-2, 2/8 patients experienced hematologic DLTs. The protocol was then amended to include DL-3 and DL-4, but the trial was closed by the sponsor due to priority transition and enrollment was halted so further dose reductions were not evaluated. Adverse events (AEs) were mostly hematologic, and 11 patients had treatment-related adverse events (TAEs), 10 of them grade 3 or higher. Median (range) time on treatment was 6 (1-25) cycles. Most common reason for treatment discontinuation were adverse event (3, 21.4%), recurrence (2, 14.3%), stem cell transplant (2, 14.3%), and death (2, 14.3%) Nine patients achieved CR, 2 patients achieved CRh and 1 patient achieved complete remission with incomplete hematologic recovery (CRi). The CRc rate (CR/CRh) was 61.1% (11/18, 95% CI: 38.6%-83.6%). After a median follow up of 19.7 months, the median overall survival and duration of response were not reached. One-year overall survival (OS) was 71.8% (95% CI: 44.9-87.2). Conclusion: Triplet therapy of GILT+DEC+VEN inND FLT3m AML patients ≥60 years old induced a high response rate (CRc rate 61.1%), and the median OS was not reached at time of this report (median follow-up 19.7 months). The typical dose of GILT 120mg/day with DEC+VEN was associated with significant hematology toxicities, which required multiple dose level reductions. At the reduced dose (GILT 80mg/day on days 1-7), the combination therapy was generally safe and well tolerated. A Phase 2 clinical trial sponsored by Astellas of GILT+AZA+VEN as frontline treatment for ND older patients with FLT3m AML is now enrolling (NCT05520567). Funding: this study was funded by Astellas Pharma Inc.

AK117 (anti-CD47 monoclonal antibody) in Combination with Azacitidine for Newly Diagnosed Higher Risk Myelodysplastic Syndrome (HR-MDS): AK117-103 Phase 1b Results

Background: CD47, an immune checkpoint overexpressed by tumor cells, plays a crucial role in evading the antitumor immune response. AK117, an IgG4 monoclonal antibody (mAb) targeting CD47, has emerged as a potential best-in-class therapy with high affinity binding to CD47 and without inducing hemagglutination effects. By blocking CD47, AK117 effectively triggers macrophage-mediated phagocytosis of hematological malignancy cells, and in combination with azacitidine (AZA), it synergistically enhances the activation of “eat me” signals. In this study, we present the Phase 1b data from the AK117-103 study conducted in patients with newly diagnosed higher risk myelodysplastic syndrome (HR-MDS). Methods: Eligible patients with newly diagnosed intermediate/high/very high-risk MDS, determined by the Revised International Prognostic Scoring System (IPSS-R) prognostic risk score >3, were enrolled to receive combination therapy with AK117 and AZA. AK117 was administered intravenously (IV) following various dosing regimens (20-30 mg/kg once weekly [QW], or 20-45 mg/kg every 2 weeks [Q2W], or 30 mg/kg every 4 weeks [Q4W]), whereas AZA was administered subcutaneously (SC) at the standard dosage (75 mg/m 2, Day 1-7, Q4W). The primary endpoints of this study were safety/tolerability and the complete remission (CR) rate based on the International Working Group (IWG) 2006 response criteria. Results: As of June 9, 2023, 72 patients were enrolled (median age: 66 years, 69.4% males). Most patients exhibited abnormal hematologic conditions at baseline, including decreased hemoglobin, neutrophil count, and platelet count. Specifically, 69.4% of patients had Grade ≥3 anemia, 61.1% had Grade ≥3 neutrophil count decrease, and 47.2% had Grade ≥3 platelet count decrease. For the recommended phase 2 dose (RP2D) group, treated with a combination of AK117 30 mg/kg Q2W and AZA, 30 patients were enrolled with a median age of 67 years, 80.0% of whom were males. According to IPSS-R risk assessment, 33.3%, 23.3%, and 43.3% of patients had intermediate, high, and very high risk, respectively. 30.0% of patients had poor-risk cytogenetics, with 23.0% of those being complex. After a median follow-up of 6.6 months (ranging from 0.4-17.2 months), anemia (a primary adverse event associated with CD47 blocking antibodies) occurred in 30.6% (22/72) of the patients, of which Grade ≥ 3 anemia accounted for 22.2% (16/72). Other commonly observed treatment-emergent adverse events (TEAEs) (≥30%) included decreased neutrophil count (77.8%), decreased white blood cell count (72.2%), decreased platelet count (69.4%), pyrexia (58.3%), decreased lymphocyte count (41.7%), constipation (41.7%), and vomiting (31.9%). Three patients (4.2%) discontinued treatment due to TEAEs. The efficacy of AK117 was compared at different dosages, and ultimately, 30mg/kg Q2W was chosen as the RP2D. As of June 21, 2023, among 27 evaluable patients, the CR rate was 48.1%. 8 patients achieved marrow CR (5 also with hematologic improvement [HI]), and 2 patients achieved HI alone. Among 24 evaluable patients with ≥ 3 months follow-up, the CR rate further escalated to 54.2%. Of 13 evaluable patients who initially required red blood cells (RBC) transfusions, 8(61.5%) became independent of RBC transfusion. Conclusions: AK117 in combination with AZA was well tolerated with low incidence of anemia and demonstrated promising efficacy in patients with newly diagnosed HR-MDS.

A Phase 1b Study Evaluating the Safety and Efficacy of AK117 (anti-CD47 monoclonal antibody) in Combination with Azacitidine in Patients with Treatment-Naïve Acute Myeloid Leukemia

Background: AK117 is a humanized IgG4 monoclonal antibody that specifically targets CD47, a “don't eat me” signal excessively expressed on tumor cells such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). CD47 blockade effectively stimulates macrophage-mediated phagocytosis of AML cells, and when combined with azacitidine (AZA), it synergistically enhances the activation of “eat me” signals. Here, we report the data from a Phase 1b study of AK117 plus AZA in patients with treatment-naïve AML. Methods: Patients newly diagnosed with AML who were deemed unfit for intensive chemotherapy were included in the study to receive combination therapy with AK117 and AZA. AK117 was administered intravenously (IV) following various dosing regimens (10-20 mg/kg once weekly [QW], or 30-45 mg/kg every 2 weeks [Q2W]), whereas AZA was administered subcutaneously (SC) at the standard dosage (75 mg/m 2, Day 1-7, every 4 weeks [Q4W]). The primary endpoints of the study were safety/tolerability, and composite complete remission (CCR) defined as the combination of complete remission (CR) and complete remission with incomplete hematologic recovery (CRi), based on the 2017 European Leukemia Net (ELN) response criteria. Results: As of June 9, 2023, 40 patients were enrolled (median age: 63 years, 42.5% males, 72.5% with ECOG≥1). Most patients had abnormal hematologic conditions at baseline, including decreased hemoglobin, neutrophil count, and platelet count. Specifically, 80.0% of patients had Grade ≥3 anemia, 62.5% had Grade ≥3 neutrophil count decrease, and 50.0% had Grade ≥3 platelet count decrease. For the recommended phase 2 dose (RP2D) group, treated with a combination of AK117 30 mg/kg Q2W and AZA, 22 patients were enrolled (median age: 63 years, 31.8% males, 72.7% with ECOG≥1). After a median follow-up of 6.7 months (ranging from 0.2-20.7 months), anemia (a primary adverse event associated with CD47 blocking antibodies) occurred in 32.5% (13/40) of the patients. Other commonly observed treatment-emergent adverse events (TEAEs) (≥30%) included decreased white blood cell count (70.0%), decreased lymphocyte count (62.5%), decreased platelet count (55.0%), decreased neutrophil count (52.5%), pyrexia (52.5%), constipation (40.0%), and vomiting (37.5%). The most frequently reported Grade ≥3 TEAEs (≥20%) were decreased white blood cell count (52.5%), decreased platelet count (47.5%), decreased neutrophil count (45.0%), decreased lymphocyte count (25.0%), and anemia (25.0%). One patient (2.5%) discontinued treatment due to TEAE. The efficacy of AK117 was compared at different dosages, and ultimately, 30mg/kg Q2W was chosen as the RP2D. Out of 20 evaluable patients, 11 (55%) achieved a CCR, including 9 (45%) with a CR and 2 (10%) with a CRi. Stable disease (SD) was reported in 35% of the patients. The median time to response (TTR) was 1.31 months, while the median time to CR (TTCR) was 1.84 months. The median duration of response (DoR) and duration of CR (DoCR) were not reached. Conclusions: The combination of AK117 and AZA exhibited a manageable safety profile with a low incidence of anemia, and demonstrated promising efficacy as a first-line treatment in AML patients who were unable to undergo intensive chemotherapy.

Suppression of KRAS and CBL Mutations and Hematological Improvement By Lenzilumab and Azacitidine Treatment in Proliferative Chronic Myelomonocytic Leukemia

Introduction: Mutations in RAS-pathway genes ( NRAS, KRAS, CBL) are amongst the most common somatic mutations in cancer and historically resistant to most therapies. Targeted therapies that impact the RAS-pathway constitute a major unmet need in oncology. The proliferative form of chronic myelomonocytic leukemia (CMML) is commonly associated with RAS-pathway mutations and has a high propensity to develop into acute myeloid leukemia. In pre-clinical models, Lenzilumab (LENZ; Humanigen, Inc., Short Hills, NJ), a proprietary Humaneered ® first-in-class monoclonal antibody with best-in-class off-rate and affinity that neutralizes GM-CSF, resulted in a reduction of colony numbers and viability of CMML cells, with the greatest sensitivity in cells possessing RAS-pathway ( NRAS/ KRAS/ CBL) mutations, suggesting benefit in the targeted treatment of CMML. The PREcision Approach to CHronic Myelomonocytic Leukemia (PREACH-M) trial assesses the efficacy of LENZ, in addition to azacytidine (AZA), in CMML subjects with RAS-pathway mutations and high dose sodium ascorbate (ASC) and AZA in CMML subjects without RAS-pathway mutations. Interim data from 11 subjects with RAS-pathway ( NRAS/ KRAS/ CBL) mutations in PREACH-M receiving LENZ/AZA demonstrate reductions in circulating GM-CSF and CRP with 8 subjects achieving complete response or optimal marrow response. This report describes improvements in variant allele frequencies (VAF) for RAS-pathway mutations and hematologic improvements associated with LENZ/AZA treatment in CMML. Methods: PREACH-M is a Phase 2/3 non-randomized, open-label trial in 72 subjects, aged at least 18 years, with newly diagnosed CMML based on the WHO 2016 criteria; and RAS-pathway mutations at a variant allele frequency ≥3%. Subjects received 24 cycles (every 28 days) of AZA (SC; 75 mg/m 2 for 7 days) and LENZ (IV; 552 mg; d1 & d15 of cycle 1 and d1 only for all subsequent cycles). Subjects without RAS-pathway mutations received the same AZA regimen and sodium ascorbate (IV; 30g for 7 days (15g for 1st dose only; 30g thereafter if no evidence of tumor lysis syndrome); PO; 1.1g on all other days). VAF was determined from bone marrow mononuclear cells using a 41-myeloid panel using Illumina Hi-Seq with a depth of 1000x performed on bone marrow aspirates obtained at baseline and Day 1 of treatment cycles 4, 7, and 12. Results: As of July 2023, 11 subjects completed at least 3 months of LENZ/AZA treatments and follow-up sequencing data were available for 10 subjects. Five of 10 subjects showed >10% decrease in VAF for at least one mutation detected at baseline, including 5 decreased alleles in the RAS-pathway ( KRAS, CBL) out of 22 total VAF mutation responses. CBL mutations showed the largest change with a decrease in >50% VAF in three subjects. Decreases in RAS-pathway clones by LENZ/AZA were durable at 6 months. No decreases in VAF for any mutations were observed in 2 evaluable subjects following ASC/AZA treatment. Following 3 months LENZ/AZA treatment, blood monocyte count improved from a baseline of 11.0 ± 6.0 x 10 9/L to 2.0 ± 1.0 x 10 9/L (p=0.030). Blast differential improved from 10.5 ± 5.0% to 4.0 ± 3.5% (p=0.038). Platelet count increased from 90.0 ± 50.0 x 10 9/L to 150.0 ± 60.0 x 10 9/L (p=0.010). Statistical improvements remained durable for at least 6 (n=6) and 12 (n=3) months. Hemoglobin concentration increased 100.0 ± 22.0 g/L to 115.0 ± 10.0 g/L at 3 months (p=NS, n=11) and progressively increased to 122.0 ± 10.0 g/L at 12 months (p=0.024, n=3). Spleen length decreased from 15.0 ± 4.0 cm to 13.0 ± 4.0 cm (p=0.03) and decreased further to 11.0 ± 2.0 cm in subjects who received 12 months of treatment (n=3). No evidence of disease progression or relapse was observed in any subject. Conclusion: In 11 evaluable subjects with proliferative CMML and RAS-pathway mutations, GM-CSF neutralization with LENZ in addition to AZA standard of care, resulted in significant decreases in the proportion of KRAS and CBL mutant leukemic cells, accompanied by clinically significant hematologic improvements and a reduction in splenomegaly. Lenzilumab may have efficacy in preventing outgrowth of RAS-pathway mutations, specifically KRAS and CBL, in the context of CMML and other myeloid malignancies.

Preliminary Results of a Phase 2 Study of IMM01 Combined with Azacitidine (AZA) As the First-Line Treatment in Adult Patients with Higher Risk Myelodysplastic Syndromes (MDS)

Background: Patients diagnosed with higher-risk myelodysplastic syndrome (MDS) have poor prognosis. IMM01 is a recombinant signal regulatory protein α (SIRPα) IgG1 fusion protein that exerts anti-tumor activities via blocking “Don't eat me” signal and activating the “Eat me” signal to induce strong antibody-dependent cellular phagocytosis (ADCP). Furthermore, IMM01 activated macrophages could process and present tumor antigen to T cells and elicit long-last tumor-specific T cell immune response. Methods: This is an open-label, multi-center, phase 2 study (NCT05140811) that evaluated safety and efficacy of IMM01 in combination with AZA as the first-line treatment for patients with untreated higher-risk MDS. Enrolled patients were aged ≥18 years with intermediate to very high risk MDS (IPSS-R >3.5) who were not eligible for stem cell transplant or intensive chemotherapy. Patients with treatment-naive MDS were administered intravenous IMM01 at a dosage of 2.0mg/kg/week and subcutaneous AZA at a dosage of 75 mg/m 2 on D1-7 per 28-day cycle. Adverse events (AEs) were reported according to CTCAE v5.0. Efficacy was assessed by IWG 2006 (MDS) criteria. Results: A total of 54 patients were enrolled from June 29, 2022 to June 11, 2023. The median age was 64 (30-83) years, with 39 (72.2%) being male, and 52 (96.3%) having an ECOG of ≥1. Based on risk classification per IPSS-R, 13 patients (24.1%) were intermediate risk (IR), 25 (46.3%) high risk (HR), and 16 (29.6%) very high risk (vHR). At baseline, the median levels of blood counts were 69 (35-95)g/L for hemoglobin, 39.5 (2-409)×10 9/L for platelets and 0.8 (0.1-8.6)×10 9/L for neutrophils. By the data cut of June 11th, 2023, the median duration of follow-up was 5.6 months (95%CI:3.6-7.9). Among the 22 efficacy evaluable patients who received initial treatment of ≥4 months, overall response rate (ORR) was 81.8% (18/22), including 36.4%(8/22) complete response (CR) rate, 22.7%(5/22) marrow CR (mCR) with hematologic improvement (HI), 9.1% (2/22) HI and 13.6% (3/22) mCR alone. Among the 17 efficacy evaluable patients who received initial treatment of ≥6 months, ORR was 88.2%(15/17), including 41.2% (7/17) CR, 29.4% (5/17) mCR with HI, 5.9% (1/17) HI and 11.8% (2/17) mCR alone. The median duration of response (DoR) was not reached. Biomarker data showed mutation burdens of several markers, including TP53, DNMT3A, ASXL1, U2AF1, were dramatically reduced on study treatment. The most frequent treatment related adverse events (TRAEs) (≥20%) were leukopenia (85.2%), thrombocytopenia (72.2%), neutropenia (66.7%), lymphopenia (57.4%), anemia (44.4%), vomiting (44.4%), pyrexia (33.3%), infusion related reaction (33.3%), constipation (29.6%), nausea (25.9%), hypoalbuminemia (22.2%) and infection (20.4%). The most common ≥G3 TRAEs (≥10%) included leukopenia (81.5%), thrombocytopenia (68.5%), neutropenia (66.7%), lymphopenia (57.4%), anemia (44.4%) and infection(16.7%). These AEs were consistent with the AE profile of the AZA monotherapy in treatment-naive MDS as commonly reported in China. Without using of a low priming dose, no Grade ≥3 hemolysis occurred. The study is ongoing. Conclusions: Preliminary data from IMM01 (without low-dose priming) combined with AZA were well tolerated and showed exciting efficacy results in patients with treatment-naive higher-risk MDS.

Intensive Chemotherapy (IC) Followed By Oral Azacitidine (AZA) Maintenance Versus Venetoclax (VEN) Plus AZA for Patients (pts) with Acute Myeloid Leukemia (AML): Retrospective Analysis of an Electronic Medical Record (EMR) Database in the United States

Introduction:Frontline therapy with IC is usually recommended for eligible pts with newly diagnosed (ND) AML. Pts who achieve remission with induction and are transplant-ineligible can receive oral AZA (Oral-AZA) maintenance. For pts ineligible for IC, VEN with injectable AZA (VEN-AZA) is the recommended treatment. Selection of frontline therapy can be challenging for pts considered reasonable candidates for either IC or VEN-AZA. A prior analysis suggested that survival outcomes may be comparable in matched pts with ND AML treated with either IC or VEN-AZA (Zeidan AM, et al, Annal Hematol 2023;102:749-754). However, the impact of Oral-AZA maintenance was not accounted for. In this analysis, we compare the survival outcomes of pts who received IC followed by Oral-AZA maintenance (IC-Oral-AZA) versus those who received frontline VEN-AZA and achieved remission, before and after matching for baseline and clinical characteristics Methods:In this retrospective, observational, study using Flatiron Health's database, de-identified data from adult pts with ND AML who received either frontline IC-Oral-AZA or frontline VEN-AZA and achieved remission were analyzed. Exclusion criteria included diagnosis of acute promyelocytic leukemia, treatment with arsenic trioxide/tretinoin, clinical trial participation, or prior hematopoietic stem cell transplant (HSCT). Treatment with Oral-AZA for IC-Oral-AZA pts had to commence prior to a 2nd line of therapy or disease relapse, and after Sept 1, 2020. Cohorts were matched 1:1 on baseline demographic and clinical characteristics using propensity scores to minimize differences. Matched characteristics included sex, age at diagnosis (18-64, 65-74, ≥ 75 years), race/ethnicity, region, ECOG performance status (PS), BMI, previous diagnosis of myelodysplastic syndromes or myeloproliferative neoplasm, cytogenetic risk category, whether AML is treatment-related or was diagnosed as mixed phenotype. Baseline molecular data were insufficient. Relapse-free survival (RFS) and overall survival (OS) were estimated for unmatched and matched cohorts using Kaplan-Meier analysis from date of remission, censoring pts at the time of HSCT/end of follow-up. Cox regression analyses were conducted for RFS and OS, both on an unadjusted basis and after adjusting for any remaining differences in age between the matched cohorts. Confirmatory landmark analysis is being conducted. Results:In total, 50 IC-Oral-AZA and 403 VEN-AZA pts met the selection criteria prior to matching. The matching process yielded 33 pts in each cohort, with median follow-up from start of therapy of 13.1 and 11.2 months for IC-Oral-AZA and VEN-AZA cohorts, respectively. The cohorts matched well on median age at diagnosis (63 years for both), sex (54.5%, 51.5% males), ECOG PS 2+ (18.2%, 21.2%), cytogenetic risk (46.7% poor, 26.7% intermediate, 26.7% favorable risk for both groups, among those with known risk status), whether AML was treatment-related (12.1%, 21.2%), race/ethnicity, region, and BMI. Three pts (9.1%) in the IC-Oral-AZA cohort and 8 (24.2%) in the VEN-AZA cohort received HSCT during follow-up. Median time from remission to start of Oral-AZA for the IC-Oral-AZA cohort was 128 days. For the unmatched cohorts, median RFS was significantly longer for IC-Oral-AZA (20.0 months [95% CI, 13.4-not reached (NR)]) than for VEN-AZA (9.3 months [8.3-10.7]; P<0.001) as was median OS (IC-Oral-AZA, NR [15.5-NR]) versus VEN-AZA (13.4 months [10.7-15.5]; P < 0.001). For the matched cohorts, median RFS was significantly longer for IC-Oral-AZA (20.0 months [95% CI, 9.9-NR]) than for VEN-AZA (8.7 months [4.1-15.5]; P = 0.002) as was median OS (IC-Oral-AZA, NR [14.6-NR] versus VEN-AZA, 15.2 months [7.4-19.7]; P = 0.002) (Figures A and B). The Cox analyses confirmed the superior outcomes of the IC-Oral-AZA cohort for both RFS (unadjusted HR, 0.33 [95% CI, 0.17-0.65]; P < 0.01, adjusted HR, 0.34 [0.17-0.70]; P < 0.01) and OS (unadjusted HR, 0.28 [0.11-0.68]; P < 0.01, adjusted HR, 0.29 [0.11-0.74]; P < 0.01). Conclusion:Pts treated with frontline IC followed by Oral-AZA as maintenance had significantly better survival outcomes than pts who were treated and achieved remission with frontline VEN-AZA. These results suggest that pts with ND AML eligible for frontline IC should receive this regimen followed by Oral-AZA maintenance, if they are not candidates for transplant at remission.

Phase 3b Study Design for a Comparison of Treatment Preference between Oral Decitabine/Cedazuridine and Azacitidine in Adult Patients with IPSS-R Intermediate Myelodysplastic Syndrome, IPSS Intermediate-2 or High-Risk Myelodysplastic Syndrome, Low-Blast Acute Myeloid Leukaemia, or Chronic Myelomonocytic Leukaemia

Myelodysplastic syndromes (MDS) are heterogeneous clonal bone marrow neoplasms characterised by marrow dysplasia, low blood counts and risk of progression to acute myeloid leukaemia (AML). Hypomethylating agents (HMAs) decitabine and azacitidine (AZA) are effective treatments for hematologic cancers including MDS. AZA is indicated to treat International Prognostic Scoring System (IPPS) defined Intermediate-2 (Int-2) and high-risk (HR) MDS, low blast AML (LB-AML; 20-30% blasts) with multi-lineage dysplasia, and chronic myelomonocytic leukaemia (CMML) with 10-29% blasts without myeloproliferative disorder. Oral decitabine/cedazuridine is currently the only oral hypomethylating agent indicated to treat IPSS Int-1, Int-2 or HR MDS and CMML, in the United States, Canada and Australia. AZA administration requires daily visits to a hospital or clinic for 7 days every 28 days, for either intravenous infusions, or large-volume subcutaneous (SC) injections for a minimum of 6 cycles.This places a burden on this patient population, resulting in low treatment adherence, early discontinuation, and subsequently low treatment uptake. Oral decitabine/cedazuridine is administered as one tablet daily for the first 5 days of a 28-day cycle for a minimum of 4 cycles. This allows patients to take their medication at a location of their choice. Alongside previously reported preference for oral therapy, patients may favour oral decitabine/cedazuridine over SC AZA due to improved treatment convenience and reduced discomfort. However, this is untested in a controlled setting. This study aims to evaluate preference between oral decitabine/cedazuridine and SC AZA treatment for adult patients with Revised International Prognostic Scoring System (IPSS-R) intermediate, IPSS Int-2 or HR MDS, LB-AML or CMML. This phase 3b, open-label, multi-centre study (NCT05883956) has sites planned in Australia (4 sites) and New Zealand (5 sites), to compare preference for oral decitabine/cedazuridine (Treatment A) and SC AZA (Treatment B). The study design includes 28 days of screening, four continuous 28-day cycles of study treatment, and a follow-up period with two 28-day cycles of continued therapy. Patients will be randomised to two balanced treatment sequences: ABBA or BAAB (Figure 1). Patients will express a preference twice in the study; after completing Cycle 1 and 2, and after completing Cycle 3 and 4. This cross-over design is twice the length of a typical 2-period AB vs BA design and allows patients to act as their own controls. The study design allows estimation of the treatment effect while mitigating factors that may influence preference. These include the order of treatments, and fluctuations in the patient's physical and disease state over the course of the trial. All available preferences expressed throughout the trial will be used for primary analysis. A total of 42 patients are planned for this study. The primary objective of this study is to compare patients' treatment preference for oral decitabine/cedazuridine and SC AZA for the treatment of MDS, LB-AML or CMML. Secondary objectives include evaluation of carers' and clinicians' treatment preference between oral decitabine/cedazuridine and SC AZA for the treatment and continued treatment of adult patients with MDS, LB-AML or CMML. The primary objective will be assessed by a treatment preference in myelodysplasia questionnaire (pTPMQ), which instructs patients to indicate a preference for injection, oral/tablets or no preference. The secondary objectives will be evaluated by a carer treatment preference in myelodysplasia questionnaire (cTPMQ) and a medical clinician treatment preference in myelodysplasia questionnaire (mTPMQ). Treatment discontinuation rates, quality of life and safety of SC AZA and oral decitabine/cedazuridine will also be compared. The primary analysis will be conducted with a multilevel logistic regression model analysing all available preferences. Descriptive statistics including frequencies, proportions, mean, standard deviation, median, and interquartile range will be used to summarize the study data. When complete, this study will address an evidence gap in the comparison of patient preference for oral decitabine/cedazuridine and SC AZA, and subsequently inform the value of oral decitabine/cedazuridine for the treatment of MDS, LB-AML and CMML.

Low-Dose Decitabine Plus Venetoclax As Post-Transplant Maintenance for High-Risk Myeloid Malignancies

Background: Allogeneic stem cell transplantation (allo-SCT) is potentially curative for patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, post-SCT relapse remains a major cause of treatment failure for these patients and strategies to prevent post-transplant relapse are urgently needed. The combination of venetoclax (VEN) with either azacitidine (AZA) or decitabine (DEC) has shown promising results in older or unfit patients with AML and is now being investigated in younger patients as well. Herein, we report our experience of administering low-dose DEC and VEN as post-transplant maintenance in patients with high-risk myeloid malignancies. Methods: A retrospective chart review was performed to evaluate outcomes of patients with AML and MDS that received post-SCT maintenance with DEC and VEN at the University of Alabama at Birmingham. DEC was administered at 10mg/m 2 on days 1-3 per cycle and VEN was given at a target dose of 200mg on days 1-28 for AML and days 1-14 for MDS. The dose of VEN was reduced to 20mg when administered with voriconazole or posaconazole or 100mg when administered with fluconazole or isavuconazium. The cycle length was 4 weeks. Results: We identified 19 patients between March 2021 and May 2023 that received post-SCT DEC/VEN for AML and MDS (Table 1). The median follow-up was 10m (range 3-26m). The median age was 60y (range 34-69y). There were 10 (53%) females and 13 (68%) non-Hispanic whites (NHWs). Fourteen patients had AML and 5 had MDS. For patients with AML, 10 had poor-risk and 4 had intermediate-risk disease per ELN 2017. At the time of transplant, 11 patients with AML were in CR1 (CR1=6, CRi1=5), 2 were in CR2 (CR2=1, CRi2=1) and 1 was in CR3. Three patients with AML had MRD+ at the time of transplant (CRi1=2, CR3=1). For patients with MDS, 2 had very-high and 2 had high-risk disease per R-IPSS. One patient had therapy-related MDS. Two patients with MDS had CR, 2 had stable disease and 1 had hematological-improvement at the time of transplant. Overall, 6 patients (32%) had a TP53 mutation. Donor type was matched-related in 5 patients, matched-unrelated in 12 patients and haploidentical in 2 patients. Seventeen patients received reduced-intensity conditioning (RIC). GVHD prevention consisted of tacrolimus and methotrexate in 14 patients and post-transplant cyclophosphamide-based in 5 patients. The median time to initiating maintenance therapy was 69d (range 54-200d) post-SCT. The median number of cycles administered was 6 (range 2-24). Grade III-IV neutropenia, anemia and thrombocytopenia occurred in 5, 1 and 2 patients, respectively. Two patients had febrile neutropenia and one patient had candida esophagitis. Five patients required a reduction or interruption in the dose due to adverse events. In all cases, the VEN duration was shortened by 1 week. The cumulative incidence (CI) of day 100 grade II-IV and III-IV aGVHD was 13% and 5%, respectively. There were no grade IV cases. The 1y CI of moderate-severe cGVHD was 26%. To date, one patient who received a RIC haplo-SCT for poor-risk AML in CR2 relapsed 14m after SCT. The 1y non-relapse mortality was 11%. The 1y OS for the entire cohort was 84% (Figure 1). Conclusion: Our preliminary data suggests that the combination of low-dose DEC plus VEN is a safe and potentially effective strategy to reduce the risk of post-SCT relapse. Adverse effects mainly included manageable cytopenias with no increase in risk of GVHD. The efficacy of the combination appears to be promising but needs longer follow-up as well as confirmation in large, randomized studies.

Azacitidine Treatment in MDS: A Systematic Literature Review and Meta-Analysis Comparing the Efficacy of Real World Data with Randomized Controlled Trials

Background: Myelodysplastic Syndromes (MDS) are a group of myeloid malignancies affecting the hematopoietic stem and progenitor cells and occurring mainly in elderly individuals. Two hypomethylating agents (HMA), Azacitidine (AZA) and Decitabine (DAC), have shown efficacy in treatment of MDS patients and received approval based on randomized controlled trials (RCTs) more than a decade ago. Since then, many publications have reported real world experience on HMA treatment in MDS patients, which showed a reduced efficacy of HMA than the RCT. The reasons for this difference have been extensively discussed but a systematic review never performed. Aim: We performed a systematic literature review and meta-analysis to compare the efficacy of real world data with randomized controlled trials and to identify potential cofactors for different outcomes. Material and Methods: We performed a systematic literature search from observational studies (OBSTs) describing real world experience of AZA treatment in MDS patients using Embase, Ovid Medline, and Web of Science databases. Patient characteristics, IPSS/IPSS-R, and outcome (response rates, overall survival) were collected from OBSTs that were performed between 2003 and 2023. Kaplan-Meier curves with log-rank tests were used to assess differences in survival outcomes. An inverse variance weighted meta-analysis was performed by using restricted mean survival times or proportions (response rate outcome). The restricted mean survival time and the standard error at 12, 24 and 36 months was calculated by using the R package survival. The pooled effect and prediction intervals from random effect models were reported. Results: In our systematic literature search, data from OBSTs describing real world experience of AZA treatment in MDS patients could be extracted and compared to the only RCT published with AZA (Fenaux P et al., Lancet Oncology 2009). Patient and disease characteristics were similar for AZA-RCT and OBST (respect of gender ratio, median age, IPPS/IPSS-R). Restricted mean survival time and standard error of the OBSTs and AZA-RCT showed no significant difference for the time points at 12 months (10.0 [95%-CI: 9.6; 10.3] months (OBSTs) vs 10.0 [95%-CI: 9.5; 10.5] months (AZA-RCT); figure part A) and 24 months (15.5 [95%-CI: 14.6; 16.5] months (OBSTs) vs. 16.9 [95%-CI: 15.7; 18.2] months (AZA-RCT); figure part B). At 36 months, the restricted mean survival time of OBSTs was 18.7 [95%-CI: 17.3; 20.1] months compared to 22.3 [95%-CI: 20.2; 24.5] months of the AZA-RCT (figure part C), which was different in favor of the RCT. Using random effect models, the AZA treatment response rate was 0.51 [95%-CI: 0.47; 0.56] for the OBSTs and 0.49 [95%-CI: 0.42; 0.57] for the AZA-RCT (figure part D). Conclusion: Compared to AZA-RCT data, restricted mean survival time (for time points 12 months and 24 months) and response rate of OBSTs describing real world data of AZA treatment in MDS patients are comparable. Regarding the observation time point at 36 months, the lower restricted mean survival time of the OBSTs compared to AZA-RCT may be most likely explained by study patient selection bias.

A Phase Ib/II Study Evaluating Navitoclax after Failure of Hypomethylating Agent and Venetoclax for Treatment of Relapsed or Refractory High-Risk Myelodysplastic Syndrome

BACKGROUND AND SIGNIFICANCE Myelodysplastic syndrome (MDS) is a heterogeneous group of blood cancers of myeloid progenitor cells. They vary widely in genetic landscape, risk of progression to acute myeloid leukemia, and risk of death, as predicted by the revised international prognostic score (IPSS-R). The hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DEC) are the standard of care for treatment of high risk (HR)-MDS, however complete response rates are <15% and most patients (pts) eventually progress. Outcomes after HMA failure are dismal. Defective apoptosis is a major feature of MDS. Increased expression of anti-apoptotic members of the BCL-2 family (BCL-2, BCL-X L and BCL-W) correlates with disease progression and poor outcomes. The addition of venetoclax (VEN), a selective BCL-2 inhibitor, has shown benefit when added to AZA in pts with relapsed or refractory (R/R) MDS (Zeidan, et al., AJH, 2023). However, patterns of BCL-2 family proteins are heterogeneous and response to inhibition of BCL-2 family proteins depends on the balance of pro- and anti-apoptotic proteins. Navitoclax (NAV) is a broad oral inhibitor of BCL-2, BCL-X L, and BCL-W. Increased expression of BCL-X L has been shown to make MDS cells insensitive to VEN. Further, VEN-resistant myeloid cell lines derived by prolonged VEN exposure showed up-regulation of BCL-X L, driving acquired drug resistance in these cell lines. Thus, the addition of NAV to the combination of VEN and HMA is a logical strategy to induce response in pts with R/R after HMA with or without prior VEN exposure. STUDY DESIGN AND METHODS We will perform a phase Ib/II, dose escalation and expansion study to evaluate escalating doses of NAV in combination with standard dose VEN and DEC in pts with R/R HR-MDS after failure of HMA (NCT05564650). The primary objective of the phase Ib portion of the study will be to evaluate the safety profile and determine the recommended phase II dose (RP2D) of NAV in the combination. The primary objective of the phase II portion of the study will be to evaluate the efficacy of combination therapy NAV/VEN/DEC in pts with R/R HR-MDS after failure of HMA/VEN. During cycle 1, to limit the risk of tumor lysis, pts will be treated with a short dose ramp-up with VEN 200 mg daily on day 1 and 400 mg daily on days 2-16, DEC 20 mg/m2 on days 3-7 and NAV 25 mg, 50 mg or 100 mg daily on days 3-16 during cycle 1. During cycles 2 and beyond, dosing will be similar to cycle 1 without dose ramp-up ( Figure 1). We will employ the Bayesian optimal interval (BOIN) design to identify the maximum tolerated dose and RP2D of NAV. Subsequently, the study will proceed to phase II to further evaluate the safety and efficacy of the combination. The study will enroll pts with IPSS-R ≥3 who have been previously treated with HMA/VEN. Pts in the phase I portion of the trial may be enrolled if they have not received VEN. Pts must have adequate performance status and organ function. Pts with a WBC >25,000 cells/µL and those taking prohibited medications will be excluded. Correlative studies will be performed to determine the impact of treatment on molecular targets and will include profiling of BCL-2 family members and single cell gene expression patterns pre- and post-treatment and at relapse. Pts who achieve a complete response (CR) or modified CR (mCR) will continue on the study until disease progression, dose limiting toxicity, availability of an alternative therapy or withdrawal of consent. Pts who have hematologic improvement but who do not attain CR or mCR can continue the study at the discretion of their treating physician in conjunction with the primary investigator.