All-trans Retinoic Acid In Patients Research Articles

Abstract CT223: Dormancy reprograming therapy of 5-azacitidine (AZA) and all-trans retinoic acid (ATRA) in biochemically recurrent prostate cancer

Abstract Microenvironmental signals and epigenetic changes can instruct proliferative disseminated tumor cells to enter dormancy. The combination of Azacitidine (AZA) and All-trans retinoid acid (ATRA) can reprogram active prostate cancer (PCa) cells into a dormant state. Herein, we evaluated the safety and clinical activity of AZA + ATRA in patients (pts) with biochemically recurrent PCa. Eligible pts were those with diagnosis of PCa post-local therapy with rising PSA (PSADT < 10 mo). Pts were excluded if they had radiographic evidence of disease or if they were a candidate for salvage radiation therapy (RT). Eligible pts were randomized 1:1 to arm A vs. B. All pts received Lupron 7.5 mg IM at the start of study. Four weeks later, pts entered a 24-week treatment (tx) phase. Pts in arm A received AZA 40 mg/m2 SQ x 5 days and ATRA 45 mg/m2 PO x 5 days Q4W for 12 weeks (w), followed by 12w of observation. Pts in arm B underwent 12w of observation, followed by 12w of AZA and ATRA at same dosing as arm A. All pts underwent follow up until initiation of next tx. The primary endpoint was safety and tolerability as assessed by CTCAE v5.0. Key secondary endpoints included % of pts with prolongation of PSADT pre- and post-tx and time to next treatment (TTNT). Levels of dormancy biomarkers, BMP4 and BMP7, were quantified using ELISA. Circulating tumor cells (CTCs) were analyzed for positive expression of NR2F1 using immunofluorescence. Wilcoxon signed rank test was used for comparing the PSADT. All statistical analysis was performed in R. Fourteen pts were enrolled (A: N=6, B: N=8) with a median age of 66 (range 54-77). At baseline, mean PSA was 4.75 ng/ml (A: 7.11, B: 2.99) and mean PSADT was 2.89 months (A: 3.51, B: 2.43). Eight (57.1%) pts had a Gleason score > 8 at initial diagnosis (A: 83.3%, B: 37.5%). There were no reported Grade > 3 treatment-related adverse events (TRAEs) and no treatment discontinuations. The most common TRAEs included hot flashes, white blood cell decreased and lymphocyte count decreased (all 8.3% each), and anemia (5.6%). There was prolongation of PSADT pre- and post-tx in 6 (46%) pts, however, overall difference in PSADT was non-significant [p>0.1]. 13 out of 14 (93%) pts had TTNT of > 6 months. One pt developed radiographic progression prior to completion of tx phase and a total of 6 pts (42.8%) within 3 months of tx phase. Out of 8 pts, 37.5% and 75% of pts showed increased levels of BMP4 and BMP7 respectively. Upon tx, CTC count decreased in 3 out of 5 pts analyzed, while CTCs were undetectable in two pts. Upon tx, NR2F1 expression could not be detected due to non-detection of CTCs, except for one pt who showed 100% NR2F1 positive CTCs in follow up. AZA + ATRA appears safe and well tolerated, and there was clinical activity in a small subset of pts potentially correlated with dormancy. Additional prospective clinical studies are needed to elucidate dormancy mechanisms further and optimize dosing strategy. Citation Format: Vaibhav G. Patel, Deepak Singh, Himanshu Joshi, Bobby Liaw, Che-Kai Tsao, Matthew Galsky, Lindsay Diamond, Julio Agguire-Ghiso, William Oh. Dormancy reprograming therapy of 5-azacitidine (AZA) and all-trans retinoic acid (ATRA) in biochemically recurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT223.

Levels of retinol and retinoic acid in pancreatic cancer, type-2 diabetes and chronic pancreatitis.

Retinoids participate in multiple key processes in the human body e.g., vision, cell differentiation and embryonic development. There is growing evidence of the relationship between retinol, its active metabolite- all-trans retinoic acid (ATRA) - and several pancreatic disorders. Although low levels of ATRA in pancreatic ductal adenocarcinoma (PDAC) tissue have been reported, data on serum levels of ATRA in PDAC is still limited. The aim of our work was to determine serum concentrations of retinol and ATRA in patients with PDAC, type-2 diabetes mellitus (T2DM), chronic pancreatitis (CHP) and healthy controls. High performance liquid chromatography with UV detection (HPLC) was used to measure serum levels of retinol and ATRA in 246 patients with different stages of PDAC, T2DM, CHP and healthy controls. We found a significant decrease in the retinol concentration in PDAC (0.44+/-0.18 mg/L) compared to T2DM (0.65+/-0.19 mg/L, P<0.001), CHP (0.60+/-0.18 mg/L, P< 0.001) and healthy controls (0.61+/-0.15 mg/L, P<0.001), significant decrease of ATRA levels in PDAC (1.14+/-0.49 ug/L) compared to T2DM (1.37+/-0.56 ug/L, P<0.001) and healthy controls(1.43+/-0.55 ug/L, P<0.001). Differences between early stages (I+II) of PDAC and non-carcinoma groups were not significant. We describe correlations between retinol, prealbumin and transferrin, and correlation of ATRA and IGFBP-2. Significant decrease in retinol and ATRA levels in PDAC compared to T2DM, healthy individuals and/or CHP supports existing evidence of the role of retinoids in PDAC. However, neither ATRA nor retinol are suitable for detection of early PDAC. Correlation of ATRA levels and IGFBP-2 provides new information about a possible IGF and retinol relationship.

All Trans Retinoic Acid: A Cerebral Friend or Foe? (P3-5.002)

<h3>Objective:</h3> NA <h3>Background:</h3> All-trans retinoic acid (ATRA) is an initial treatment for acute promyelocytic leukemia (APL). Studies suggest that ATRA has neuroprotective effects since it reduces atherosclerosis and neural inflammation. We present a case series of patients that developed acute recurrent ischemic strokes and demonstrated rapid progression of intracranial and extracranial stenosis in the setting of ATRA therapy. <h3>Design/Methods:</h3> NA <h3>Results:</h3> <h3>Case 1:</h3> A 47-year-old female with a history of hypertension presented with symptoms concerning for myocardial infarction, deep venous thrombosis, and thrombocytopenia which led to a diagnosis of APL. She was treated with ATRA and two days later developed left gaze deviation, facial droop, and left hemiparesis. Vessel imaging revealed a right middle cerebral artery (MCA) occlusion. Despite successful thrombectomy, the patient continued to decline. Repeat imaging showed re-occlusion of the right MCA as well as a new left MCA infarct. She later developed diffuse cerebral edema and fatal herniation. <h3>Case 2:</h3> A 41-year-old female with a history of hypertension and APL treated with ATRA had multiple recent strokes involving bilateral frontal, left occipital and watershed territories. Vessel imaging during the first admission did not show any stenosis. Over the course of the next seven months, she had multiple admissions for recurrent strokes with rapid progression and worsening of her intracranial stenosis despite dual antiplatelet and statin therapy. Cerebrospinal fluid analysis including cell count, protein, cytology and flow cytometry was unremarkable. She subsequently developed extensor posturing with paraparesis due to malignant cerebral edema and died. <h3>Conclusions:</h3> The underlying mechanism of accelerated vessel stenosis in the above cases is unknown. Further research should explore whether ATRA use in APL is indeed associated with the progression and/or poor clinical outcomes in the setting of acute ischemic stroke. Clinicians should remain vigilant about the concomitant use of ATRA in patients with a history of vascular risk factors. <b>Disclosure:</b> Mannyam, MD (Jackson, MS); Maryam

Arsenic Trioxide Reprograms the Bone-Marrow Microenvironment to Sensitize Minimal Residual Disease in Acute Myeloid Leukemia

Eliminating minimal residual disease (MRD) in order to prevent relapse is an unmet need in acute myeloid leukemia (AML). One of the success stories is acute promyelocytic leukemia (APL), a unique subtype of AML. The use of arsenic trioxide (ATO) in combination with all-trans retinoic acid (ATRA) has essentially eliminated disease relapse (<1% ATRA+ATO vs. ~18% ATRA+ chemotherapy - NEJM (2013), 369(2), 111-121). This is even more impressive as neither agent alone results in significant cure of patients with APL. In fact, single agent ATRA induces remission without cure due to bone marrow stroma expression of CYP26 enzymes (PloS one (2015), 10(6), e0127790), which are induced by ATRA, inactivate retinoids and protect APL MRD (Leukemia (2020), 34(11), 3077-3081). While ATO may synergize with ATRA by restoring PML nuclear bodies (Nature medicine (2014), 20(2), 167-174) and rescuing RAR-α dependent transcription (Nature reviews. Cancer (2018), 18(2), 117-127) in the bulk of the APL tumor cells, it is unclear how these mechanisms contribute to elimination of MRD, particularly given the hypothesized lack of ATRA in the bone marrow niche. It was previously reported that combination of ATRA with ATO may be associated with high incidence of transaminitis and headaches (NEJM (2013), 369(2), 111-121). Since these may be clinical signs of ATRA toxicity, we investigated if addition of ATO changes the pharmacokinetics (PKs) of ATRA in patients with APL. We determined the area under the plasma concentration versus time curve (AUC) for ATRA in patients suspected of APL and treated with single agent ATRA (“preATO”) and in the same patients after the diagnoses of APL was confirmed and the patients received ATO (“postATO”). Even though ATRA should have an expected drop in AUC during the first 3-4 days of therapy (JPET (2017), 361(2), 246-258), addition of ATO resulted in improved ATRA PKs (Figure A). Since ATRA levels are mainly controlled by clearance via hepatic CYP26s, we tested if treatment with ATO changes the levels of CYP26 or its induction by ATRA. We found that exposure to ATO decreases both baseline and ATRA-induced upregulation of CYP26B1 in HepG2 cells (Figure B). Similarly, ATO decreases CYP26B1 levels in human bone marrow derived mesenchymal stroma cells (MSCs) at baseline (21%±7% of NO ATO control, n=3, p<0.01) and after ATRA-induced upregulation (66%±14% of ATRA only control, n=3, p<0.01). These changes resulted in improved ATRA PKs in vitro in the presence of bone marrow MSCs (Figure C). Next, we used NB4-EvAsR cells (NB4R), ATO-resistant APL cells to test if ATO-induced improvement of MSC-dependent ATRA PKs may contribute to elimination of APL MRD. We found that ATO sensitizes NB4R to ATRA in the presence of BM MSCs but not in stroma free conditions (Figure D). ATO had no effect on ATRA-induced differentiation of NB4R in the presence of CYP26B1 knockout stroma (this stroma offers no protection from ATRA) or if stroma expresses CYP26B1 under the control of a viral promoter and thus, resistant to ATO downregulation. This data suggest that ATO-induced changes of BM MSC expression of CYP26 may contribute to elimination of MRD in APL. Stromal CYP26 protects not only APL but also non-APL AML cells from retinoids (Leukemia (2020), 34(11), 3077-3081). Retinoids are synergistic with FLT3 inhibitors to eliminate mutant FLT3 AML cells (Blood (2016), 127(23), 2867-2878) and stromal CYP26 breaks this synergism (Blood (2015) 126 (23): 790). We tested if ATO sensitizes mutant FLT3 AML cells, MV4;11 to gilteritinib in the presence of BM MSCs. For this, we derived MV4.11 cells resistant to ATO (MV4.11R) via long term exposure to increasing concentration of ATO. As seen with NB4R, ATO treatment synergizes with gilteritinib to decrease the IC50 of gilteritinib against the clonogenic activity of MV4.11R in the presence of stroma but not in stroma free conditions (Figure E). In an effort to fully explore the translational potential of these findings, we are currently investigating the mechanism by which ATO changes CYP26 levels.Taken together these findings support a model in which ATO changes the bone marrow niche and it's ability to metabolize retinoids and thus, sensitizes MRD in AML to targeted therapy. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Ghiaur: Syros Pharmaceuticals: Consultancy; Menarini Richerche: Research Funding.

Efficacy and safety of daratumumab combined with all-trans retinoic acid in relapsed/refractory multiple myeloma

AbstractThe efficacy of daratumumab depends partially on CD38 expression on multiple myeloma (MM) cells. We have previously shown that all-trans retinoic acid (ATRA) upregulates CD38 expression and reverts daratumumab-resistance ex vivo. We therefore evaluated the optimal dose, efficacy, and safety of daratumumab combined with ATRA in patients with daratumumab-refractory MM in a phase 1/2 study (NCT02751255). In part A of the study, 63 patients were treated with daratumumab monotherapy. Fifty patients with daratumumab-refractory MM were subsequently enrolled in part B and treated with daratumumab (reintensified schedule) combined with ATRA until disease progression. The recommended phase 2 dose of ATRA in combination with daratumumab was defined as 45 mg/m2. At this dose, the overall response rate (ORR) was 5%, indicating that the primary endpoint (ORR ≥15%) was not met. However, most patients (66%) achieved at least stable disease. After a median follow-up of 43 months, the median progression-free survival (PFS) for all patients was 2.8 months. Patients who previously achieved at least a partial response or minimal response/stable disease with prior daratumumab monotherapy had a significantly longer PFS compared with patients who immediately progressed during daratumumab as single agent (median PFS 3.4 and 2.8 vs 1.3 months). The median overall survival was 19.1 months. The addition of ATRA did not increase the incidence of adverse events. Flow cytometric analysis revealed that ATRA temporarily increased CD38 expression on immune cell subsets. In conclusion, the addition of ATRA and reintensification of daratumumab had limited activity in patients with daratumumab-refractory MM, which may be explained by the transient upregulation of CD38 expression. This trial was registered at www.clinicaltrials.gov as #NCT02751255.

Efficacy and Tolerability of First Line Arsenic Trioxide in Combination With All-Trans Retinoic Acid in Patients With Acute Promyelocytic Leukemia: Real Life Experience.

Acute promyelocytic leukemia is a variant of acute myeloid leukemia characterized by t(15;17) and PML/RAR alfa fusion gene. The discovery of the molecular pathogenesis has led to entitle all-trans retinoic acid (ATRA) as the first targeted therapy for acute leukemia. It is usually associated to anthracycline-based chemotherapy with high response rates, but potential long-term sequelae including therapy-related malignancies have been observed. Arsenic trioxide (ATO) was added to obviate these complications and investigational trials aimed to a new strategy with the incorporation of arsenic trioxide (ATO) into initial therapy instead of chemotherapy in selected patients. ATRA plus ATO without chemotherapy was the first attempt to treat low and intermediate-risk patients with APL. Our study aims to describe a monocentric cohort of patients with newly diagnosed APL effectively treated with ATO plus ATRA underlying its efficacy together with the high grade of tolerability of this association. From January 2009 to December 2019 23 APL patients were diagnosed and treated with ATO plus ATRA regimen: 14 males and 9 females patients with a median age of 45 years (range 18-72), for the majority intermediate risk (15 patients, 65%). The treatment was well tolerated and all patients achieved molecular remission after a median time of 3 months (range 1-6 months). All patients proceeded to consolidation phase as outpatients, they maintained complete molecular response at a median time of 44 months (range 15-127) except for 1 patient. All but one patient are alive and in response at a median follow-up of 48 months (range 9-141) without late effects. ATO plus ATRA regimen shows advantages in comparison to chemotherapy; in fact it allowed to treat patients in which chemotherapy could even not be applicable and it did not show secondary hematological diseases. The association of ATO to ATRA as chemo-free regimen enabled to treat APL even without chemotherapy.

Predicting Resistance to the Combination of ATO and ATRA in APL Patients with PML-Rara Fusions, Using a Computational Biology Modeling Approach: Mycare-021-01

Background: Acute promyelocytic leukemia (APL) is a biologically and clinically distinct subtype of acute myeloid leukemia (AML) with unique molecular pathogenesis, clinical manifestations, and treatment. APL is cytogenetically characterized by a balanced translocation t(15;17) (q24;q21), which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the promyelocytic leukemia (PML) gene on chromosome 15 that results in a PML-RARA fusion gene (PMID: 30575821). The PML-RARA fusion gene is the most critical event involved in the pathogenesis of APL, reported in 99% of APL patients (PMID: 32182684). The fusion confers a selective sensitivity to the targeted drugs, arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA), with response rates over 90% (PMID: 31635329). However, the mechanism of resistance in the minority of non-responders is not well understood. This study used the Cellworks Omics Biology Model (CBM) to predict response to the combination of ATO-ATRA in patients harboring the PML-RARA fusion and identify mechanisms of resistance. Methods: Outcomes of 30 APL patients treated with ATRA or ATRA plus ATO were compared with outcomes predicted by CBM (Table 1). Genomic data from 6 publications (Table 2) derived from whole exome sequencing (WES), targeted next-generation sequencing (NGS), copy number variation (CNV) and/or karyotype data were used. All data was anonymized, de-identified and exempt from IRB review. The available genomic data for each profile was entered into the CBM which generates a patient-specific disease protein network model using PubMed and other online resources. The CBM predicts the patient-specific biomarker and phenotype response of a personalized diseased cell to drug agents, radiation and cell signaling. Disease biomarkers that are unique to each patient were identified within the protein network models. ATO and ATRA were simulated on all 30 patient cases. The treatment impact was assessed by quantitatively measuring the drug's effect on a cell growth score which is a composite of the quantified values for cell proliferation, survival, and apoptosis, along with the simulated impact on each patient-specific disease biomarker score. Each patient-specific model was also digitally screened to identify response to ATO and ATRA. Results: The CBM correctly predicted the response to ATO and ATRA in 28 of 30 cases. The overall prediction accuracy was 93% with a PPV of 100%, NPV of 60%, sensitivity of 93%, and specificity of 100%. In 2 of 30 patients who did not respond to ATO and ATRA, the CBM identified clinically relevant deletions to EZH2, KMT2E, and HIPK2 genes. All three genes are located on chromosome 7 and these non-responders had monosomy 7. Conclusions: The Cellworks Omics Biology Model predicted response to ATO and ATRA in APL patients harboring PML-RARA fusions. Predicting non-response to ATO and ATRA in patients with PML-RARA fusion up-front could prevent ineffective treatment, avoid unnecessary adverse events and reduce treatment costs. Additionally, computational modeling can identify new mechanisms of resistance and suggest alternative regimens for non-responding patients by targeting the patient-specific disease biomarkers unique to each. Disclosures Howard: Servier: Consultancy, Other: Speaker; Boston Scientific: Consultancy; Sanofi: Consultancy, Other: Speaker; EUSA Pharma: Consultancy; Cellworks: Consultancy. Nair:Cellworks Research India Private Limited: Current Employment. Grover:Cellworks Research India Private Limited: Current Employment. Tyagi:Cellworks Research India Private Limited: Current Employment. Kumari:Cellworks Research India Private Limited: Current Employment. Prasad:Cellworks Research India Private Limited: Current Employment. Mitra:Cellworks Research India Private Limited: Current Employment. Lala:Cellworks Research India Private Limited: Current Employment. Azam:Cellworks Research India Private Limited: Current Employment. Gupta:Cellworks Research India Private Limited: Current Employment. Mohapatra:Cellworks Research India Private Limited: Current Employment. G:Cellworks Research India Private Limited: Current Employment. Mundkur:Cellworks Group Inc.: Current Employment. Macpherson:Cellworks Group Inc.: Current Employment. Kapoor:Cellworks Research India Private Limited: Current Employment.

To PARP or not to PARP?-Toward sensitizing acute myeloid leukemia stem cells to immunotherapy.

A new study reveals that leukemia stem cells (LSCs) in acute myeloid leukemia downregulate natural killer cell-activating receptor ligands to evade immune surveillance via the transcriptional co-factor PARP1. The inhibition of PARP1 sensitizes LSCs to immunotherapy, highlighting its potential as a therapeutic target.

Pharmacologic LSD1 Inhibition Preserves Human Granulocytic While Expanding Monocytic Progenitors

Introduction: Epigenetic regulatory mechanisms such as the modification of histone residues are perturbed in myeloid neoplasms. Amongst others, the methylation status of Lysine residue 4 on Histone 3 (H3K4) represents one of the central dysregulated histone marks in acute myeloid leukemia (AML). Demethylation of H3K4 is mediated by the Lysine (K)-specific demethylase 1A (LSD1/ KDM1A). Remarkably, LSD1 expression is de-regulated in AML and therefore represents an attractive therapeutic target. In fact, pharmacologic LSD1 inhibition was shown to induce AML cell differentiation and apoptosis in combination with all-trans-retinoic acid (ATRA) exposure. Recently, a phase 1 trial with the irreversible LSD1 inhibitor IMG-7289 (IMG) ± ATRA in patients with advanced myeloid malignancies commenced (ClinicalTrials.gov Identifier: NCT02842827). However, the effect of a LSD1-inhibitory therapy on normal human progenitors has not been studied in detail.Methods: Experiments were performed with mobilized CD34+ cells of deceased multiple myeloma and lymphoma patients. The usage of these cells was approved by the local ethics committee. The following experimental approaches were employed: (1) studying in vitro colony forming unit (CFU) potential of human CD34+ progenitors in the presence of increasing IMG concentrations, (2) investigating the CFU potential in re-plating experiments of primary IMG-7289 exposed CD34+ cell CFU cultures and (3) studying the impact of IMG on in vitro cytokine (SCF, IL-3, IL-6, Flt3L and TPO for 7 days)-driven CD34+ progenitor expansion.Results: Performing CFU assays in the presence of increasing IMG concentrations revealed a shift from diverse colony types in DMSO control treated CFU assays to increasing phenotypic monocytic (CFU-M) and decreasing granulocytic (CFU-G) and erythroid (BFU-E) colony types (Fig. 1A). Accordingly, increased CFU-M and decreased CFU-G and BFU-E colony numbers were observed (Fig. 1B). In contrast, sole ATRA treatment abrogated CFU-M colony formation, increased CFU-G and slightly decreased BFU-E colony numbers (Fig. 1B). To study the impact of LSD1 inhibition in synergy with ATRA-driven differentiation therapy we treated CFU assays with increasing IMG concentrations in combination with a constant ATRA concentration (10 nM). Strikingly, IMG and ATRA co-exposure severely depleted CFU-M and BFU-E colonies while preserving CFU-G colony formation (Fig.1B). In order to study the impact of IMG on progenitor self-renewal we re-plated IMG-exposed CFU assays in the absence of IMG. In the re-plated dishes we observed a 2.9- and 3.7-fold increase of CFU-G colony numbers compared to the primary CFU-G IMG exposed (100 nM and 1µM, respectively) colony numbers. Finally, we investigated the impact of IMG exposure on in vitro cytokine-driven CD34+ progenitor expansion. In DMSO treated cultures 7 day cytokine incubation led to a 37-fold expansion of CD34+ cell numbers while increasing the total CFU numbers 1.8-fold. Compared to the DMSO control cultures, IMG exposure (100nM and 1µM) boosted CFU-M colony numbers 1.7- and 2.3-fold, respectively. Strikingly, also CFU-G colony numbers were slightly increased by IMG exposure (1.3- and 1.6-fold at 100nM and 1µM, respectively). To investigate how IMG exposure during cytokine-driven CD34+ progenitor expansion alters the transcriptional program of human CD34+ progenitors we analyzed the expression of the hematopoietic regulatory genes GFI1, GFI1B, IRF8 and RARA by real-time PCR. IMG exposure (100 nM) increased the expression of these genes 2.2-, 1.7-, 1.9- and 1.6-fold, respectively. De-repression of GFI1 and GFI1B expression by LSD1 inhibition was expected and the observed upregulation of the monocytic regulator IRF8 is in accordance with the observed increased monocytic colony numbers under LSD1 inhibition. Lastly, RARA upregulation suggested that LSD1 inhibition promotes retinoic acid receptor signaling.Conclusions: Our findings reveal that pharmacologic LSD1 inhibition increases human monocytic progenitor numbers. Furthermore, in vitro CFU-re-plating and also progenitor expansion experiments point to LSD1 inhibition not depleting granulocytic progenitors. Collectively, our data suggest that expected side effects of a LSD1-targeted therapy such as neutropenia will be reversible. [Display omitted] DisclosuresRienhoff:Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Dührsen:Janssen: Honoraria; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Research Funding; Gilead: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Göthert:AOP Orphan Pharmaceuticals: Other: travel support; Proteros Biostructures: Consultancy; Novartis: Honoraria; Pfizer: Consultancy; Incyte: Consultancy, Honoraria, Other: travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: travel support.

Effects of all-trans retinoic acid (ATRA) in addition to chemotherapy for adults with acute myeloid leukaemia (AML) (non-acute promyelocytic leukaemia (non-APL)).

Acute myeloid leukaemia (AML) is the most common acute leukaemia affecting adults. Most patients diagnosed with AML are at advanced age and present with co-morbidities, so that intensive therapy such as stem cell transplantation (SCT) is impossible to provide or is accompanied by high risks for serious adverse events and treatment-related mortality. Especially for these patients, it is necessary to find out whether all-trans retinoic acid (ATRA), an intermediate of vitamin A inducing terminal differentiation of leukaemic cell lines, added to chemotherapy confers increased benefit or harm when compared with the same chemotherapy alone. This review aims to determine benefits and harms of ATRA in addition to chemotherapy compared to chemotherapy alone for adults with AML (not those with acute promyelocytic leukaemia (non-APL)). We searched the Central Register of Controlled Trials (CENTRAL), MEDLINE, study registries and relevant conference proceedings up to July 2018 for randomised controlled trials (RCTs). We also contacted experts for unpublished data. We included RCTs comparing chemotherapy alone with chemotherapy plus ATRA in patients with all stages of AML. We excluded trials if less than 80% of participants were adults or participants with AML, and if no subgroup data were available. Patients with myelodysplastic syndrome (MDS) were included, if they had a refractory anaemia and more than 20% of blasts. Two review authors independently extracted data and assessed the quality of trials. We contacted study authors to obtain missing information. We used hazard ratios (HR) for overall survival (OS) and disease-free survival (DFS; instead of the pre-planned event-free survival, as this outcome was not reported), and we calculated risk ratios (RR) for the other outcomes quality of life, on-study mortality and adverse events. We presented all measures with 95% confidence intervals (CIs). We assessed the certainty of evidence using GRADE methods. Our search resulted in 2192 potentially relevant references, of which we included eight trials with 28 publications assessing 3998 patients. Overall, we judged the potential risk of bias of the eight included trials as moderate. Two of eight trials were published as abstracts only. All the included trials used different chemotherapy schedules and one trial only evaluated the effect of the hypomethylating agent decitabine, a drug know to affect epigenetics, in combination with ATRA.The addition of ATRA to chemotherapy resulted in probably little or no difference in OS compared to chemotherapy only (2985 participants; HR 0.94 (95% confidence interval (CI) 0.87 to 1.02); moderate-certainty evidence). Based on a mortality rate at 24 months of 70% with chemotherapy alone, the mortality rate with chemotherapy plus ATRA was 68% (95% CI 65% to 71%).For DFS, complete response rate (CRR) and on-study mortality there was probably little or no difference between treatment groups (DFS: 1258 participants, HR 0.99, 95% CI 0.87 to 1.12; CRR: 3081 participants, RR 1.02, 95% CI 0.96 to 1.09; on-study mortality: 2839 participants, RR 1.02, 95% CI 0.81 to 1.30, all moderate-certainty evidence).Three trials with 1428 participants reported the adverse events 'infection' and 'cardiac toxicity': There was probably no, or little difference in terms of infection rate between participants receiving ATRA or not (RR 1.05, 95% CI 0.96 to 1.15; moderate-certainty evidence). We are uncertain whether ATRA decreases cardiac toxicity (RR 0.46, 95% CI 0.24 to 0.90; P = 0.02, very low certainty-evidence, however, cardiac toxicity was low).Rates and severity of diarrhoea and nausea/vomiting were assessed in two trials with 337 patients and we are uncertain whether there is a difference between treatment arms (diarrhoea: RR 2.19, 95% CI 1.07 to 4.47; nausea/vomiting: RR 1.46, 95% CI 0.75 to 2.85; both very low-certainty evidence).Quality of life was not reported by any of the included trials. We found no evidence for a difference between participants receiving ATRA in addition to chemotherapy or chemotherapy only for the outcome OS. Regarding DFS, CRR and on-study mortality, there is probably no evidence for a difference between treatment groups. Currently, it seems the risk of adverse events are comparable to chemotherapy only.As quality of life has not been evaluated in any of the included trials, further research is needed to clarify the effect of ATRA on quality of life.

Oral Arsenic Plus Retinoic Acid Versus Intravenous Arsenic Plus Retinoic Acid for Non-High Risk Acute Promyelocytic Leukemia: A Multicenter Randomized Controlled Trials

ObjectiveIntravenous arsenic trioxide (ATO) plus all-trans retinoic acid (ATRA) without chemotherapy is the standard of care for non high-risk acute promyelocytic leukemia (APL), resulting in cure rates exceeding 95%. Pilot study of treatment with oral arsenic named the Realgar-Indigo naturalis formula (RIF) plus ATRA without chemotherapy has shown high efficacy, convenient and economical. This randomized, multicenter, phase 3 noninferiority trial was designed to test the efficacy and safety of an oral RIF and ATRA compared with intravenous ATO for newly diagnosed non high-risk APL patients.MethodsWe conducted a phase 3, multicenter trial comparing RIF plus ATRA with ATO plus ATRA in patients with non high-risk APL (white-cell count, ≤10×109 per liter) between 2014 and 2017. In all, 109 patients were randomly assigned (2:1) to oral RIF (60 mg/kg) plus ATRA (25 mg/m2) or ATO (0.16mg/kg) plus ATRA (25 mg/m2) as induction therapy until complete hematologic remission. Postremission therapy included RIF or ATO on a schedule of 4 weeks on and 4 weeks off and ATRA on a schedule of 2 weeks on and 2 weeks off for 7 months.The study was designed as a noninferiority trial to show that the difference between the rates of event-free survival (EFS) at 2 years in the two groups was not greater than 10%.ResultsComplete remission was achieved in all 69 patients in the RIF-ATRA group who could be evaluated (3 withdraw during the induction) and in 34 of 36 patients in the ATO-ATRA group (2 died and another one withdraw during the induction) (100% vs. 94.4%, p= 0.12).The median follow-up was 32 months. Two-year EFS rates were 97.1% in the RIF-ATRA group (n=69) and 94.4% in the ATO-ATRA group (n=36). The EFS rate difference was 2.7% (95% CI,-5.8% to11.1%). The lower limit of the 95%CI for EFS rate difference was greater than-10% noninferiority margin, confirming noninferiority (noninferiority P=0.0017). There is no difference about relapse rate and overall survival between two groups (all p >0.05).ConclusionsRIF plus ATRA is not inferior to ATO plus ATRA in the treatment of patients with non high-risk APL (Chinese Clinical Trial Registry number, ChiCTR-TRC-13004054). DisclosuresNo relevant conflicts of interest to declare.

Low-dose decitabine plus all-trans retinoic acid in patients with myeloid neoplasms ineligible for intensive chemotherapy

In our previous in vitro trials, decitabine and all-trans retinoic acid (ATRA) demonstrated synergistic effects on growth inhibition, differentiation, and apoptosis in SHI-1 cells; in K562 cells, ATRA enhanced the effect of decitabine on p16 demethylation, and the combination of the two drugs was found to activate RAR-β expression (p16 and RAR-β are two tumor suppressor genes). On the rationale of our in vitro trials, we used low-dose decitabine and ATRA to treat 31 myeloid neoplasms deemed ineligible for intensive chemotherapy. The regimen consisted of decitabine at the dose of 15mg/m(2) intravenously over 1h daily for consecutive 5days and ATRA at the dose of 20mg/m(2) orally from day 1 to 28 except day 4 to 28 in the first cycle, and the regimen was repeated every 28days. After 6 cycles, decitabine treatment was stopped, and ATRA treatment was continued for maintenance treatment. Treated with a median of 2 cycles (range 1-6), 7 patients (22.6%) achieved complete remission (CR), 7 (22.6%) marrow CR (mCR), and 4 (12.9%) partial remission (PR). The overall remission (CR, mCR, and PR) rate was 58.1%, and the best response (CR and mCR) rate was 45.2%. The median overall survival (OS) was 11.0months, the 1-year OS rate was 41.9%, and the 2-year OS rate was 26.6%. In univariate analyses, age, performance status, comorbidities, white blood cell counts and platelets at diagnosis, percentage of bone marrow blasts, karyotype, and treatment efficacy demonstrated no impacts on OS (P > 0.05, each). Main side effects were tolerable hematologic toxicities. In conclusion, low-dose decitabine plus ATRA is a promising treatment for patients with myeloid neoplasms judged ineligible for intensive chemotherapy.

In Acute Myeloid Leukemia (AML), Targeting the Histone Methyltransferase EZH2 Promotes Differentiation, Impairs Clonogenic Survival and Augments the Anti-Leukemic Effects of the Retinoid, All-Trans-Retinoic Acid (ATRA)

The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes.In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks.Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effects of all-trans retinoic acid (ATRA) in addition to chemotherapy for adults with acute myeloid leukaemia (AML) (non-acute promyelocytic leukaemia (APL))

BACKGROUND Acute myeloid leukaemia (AML) is the most common acute leukaemia affecting adults. Most patients diagnosed with AML are at advanced age and present with co-morbidities, so that intensive therapy such as stem cell transplantation (SCT) is impossible to provide or is accompanied by high risks for serious adverse events and treatment-related mortality. Especially for these patients, it is necessary to find out whether all-trans retinoic acid (ATRA), an intermediate of vitamin A inducing terminal differentiation of leukaemic cell lines, added to chemotherapy confers increased benefit or harm when compared with the same chemotherapy alone. OBJECTIVES This review aims to determine benefits and harms of ATRA in addition to chemotherapy compared to chemotherapy alone for adults with AML (not those with acute promyelocytic leukaemia (non-APL)). SEARCH METHODS We searched the Central Register of Controlled Trials (CENTRAL), MEDLINE, study registries and relevant conference proceedings up to July 2018 for randomised controlled trials (RCTs). We also contacted experts for unpublished data. SELECTION CRITERIA We included RCTs comparing chemotherapy alone with chemotherapy plus ATRA in patients with all stages of AML. We excluded trials if less than 80% of participants were adults or participants with AML, and if no subgroup data were available. Patients with myelodysplastic syndrome (MDS) were included, if they had a refractory anaemia and more than 20% of blasts. DATA COLLECTION AND ANALYSIS Two review authors independently extracted data and assessed the quality of trials. We contacted study authors to obtain missing information. We used hazard ratios (HR) for overall survival (OS) and disease-free survival (DFS; instead of the pre-planned event-free survival, as this outcome was not reported), and we calculated risk ratios (RR) for the other outcomes quality of life, on-study mortality and adverse events. We presented all measures with 95% confidence intervals (CIs). We assessed the certainty of evidence using GRADE methods. MAIN RESULTS Our search resulted in 2192 potentially relevant references, of which we included eight trials with 28 publications assessing 3998 patients. Overall, we judged the potential risk of bias of the eight included trials as moderate. Two of eight trials were published as abstracts only. All the included trials used different chemotherapy schedules and one trial only evaluated the effect of the hypomethylating agent decitabine, a drug know to affect epigenetics, in combination with ATRA.The addition of ATRA to chemotherapy resulted in probably little or no difference in OS compared to chemotherapy only (2985 participants; HR 0.94 (95% confidence interval (CI) 0.87 to 1.02); moderate-certainty evidence). Based on a mortality rate at 24 months of 70% with chemotherapy alone, the mortality rate with chemotherapy plus ATRA was 68% (95% CI 65% to 71%).For DFS, complete response rate (CRR) and on-study mortality there was probably little or no difference between treatment groups (DFS: 1258 participants, HR 0.99, 95% CI 0.87 to 1.12; CRR: 3081 participants, RR 1.02, 95% CI 0.96 to 1.09; on-study mortality: 2839 participants, RR 1.02, 95% CI 0.81 to 1.30, all moderate-certainty evidence).Three trials with 1428 participants reported the adverse events 'infection' and 'cardiac toxicity': There was probably no, or little difference in terms of infection rate between participants receiving ATRA or not (RR 1.05, 95% CI 0.96 to 1.15; moderate-certainty evidence). We are uncertain whether ATRA decreases cardiac toxicity (RR 0.46, 95% CI 0.24 to 0.90; P = 0.02, very low certainty-evidence, however, cardiac toxicity was low).Rates and severity of diarrhoea and nausea/vomiting were assessed in two trials with 337 patients and we are uncertain whether there is a difference between treatment arms (diarrhoea: RR 2.19, 95% CI 1.07 to 4.47; nausea/vomiting: RR 1.46, 95% CI 0.75 to 2.85; both very low-certainty evidence).Quality of life was not reported by any of the included trials. AUTHORS' CONCLUSIONS We found no evidence for a difference between participants receiving ATRA in addition to chemotherapy or chemotherapy only for the outcome OS. Regarding DFS, CRR and on-study mortality, there is probably no evidence for a difference between treatment groups. Currently, it seems the risk of adverse events are comparable to chemotherapy only.As quality of life has not been evaluated in any of the included trials, further research is needed to clarify the effect of ATRA on quality of life.

Effect of All-transretinoic Acid on Th17 and T Regulatory Cell Subsets in Patients with Ankylosing Spondylitis

We compared Th17 and T regulatory cells in patients with ankylosing spondylitis (AS) and in healthy controls. The effect of all-transretinoic acid (ATRA) was studied on cultured CD4+ T cells of patients with AS compared to controls. Eighteen patients with AS and 18 age- and sex-matched healthy controls were included. CD4+ T cells were separated and cultured in conditions of anti-CD3 and anti-CD28 stimulation with and without ATRA. Intracellular and secreted cytokines, transcription factors, and gene expression were evaluated after 72 h. The frequency of CD4+IL-17+ T cells was significantly higher in patients with AS compared to controls, and ATRA could significantly decrease it. The frequency of forkhead box protein 3 (FOXP3)+ retinoic acid-related orphan receptor γt (RORγt) negative T-bet negative CD4+ cells was significantly lower in cases compared to controls. Intracellular and secreted interferon-γ (IFN-γ) was not significantly different between cases and controls. ATRA significantly increased intracellular IFN-γ in cases but not in controls. Tumor necrosis factor-α (TNF-α) secretion was significantly higher and interleukin 10 secretion was significantly lower in culture supernatant of cases compared to controls. ATRA could significantly decrease TNF-α secretion in cases. Our findings favor a pathogenic role for Th17 cells in AS. Th1 cells did not seem to contribute in the pathogenesis of this disease. The effect of ATRA as an immunomodulator on deviated immune cells was associated with decreased inflammatory markers. This association could be a reason for a clinical trial of ATRA in patients with AS.

All-Trans Retinoic Acid Enhances the Cytotoxic Effect of T Cells with Anti-CD38 Chimeric Receptor in Acute Myeloid Leukemia

Abstract 1901Majority of AML blasts express CD38, while human hematopoietic stem cells express CD34 but not CD38. Accordingly, we focused on CD38 for a therapeutic target and developed a cellular immunotherapy by using T cells bearing anti-CD38-chimeric antigen receptor (CAR). We recently reported T cells with anti-CD38-CAR efficiently eliminated B-cell lymphoma cells expressing CD38 in vitro and in vivo. However, intensity of CD38 in lymphoma cells is much higher than that in AML cells. Additionally, leukemic stem cells(LSCs) phenotypically express CD34+CD38−. Thus, to fully employ anti-CD38-CAR against AML blasts including LSCs, intensity of CD38 expression need to be raised for the clinical application. All-trans retinoic acid (ATRA) is widely used for treatment of patients with acute promyelocytic leukemia (APL). Interestingly, it has been reported that ATRA enhances CD38 expression on the surface of AML cells. In this study, we investigated whether human peripheral T cells retrovirally transduced with anti-CD38-CAR kills AML cells and furthermore, whether the cytotoxicity is enhanced in the presence of ATRA. First of all, we confirmed that T cells bearing anti-CD38-CAR expressed GFP as well as the anti-goat mouse IgG-PerCP, whose expression is consistent with the expression of anti-CD38-CAR on the T cells. Next, we evaluated the cytotoxic effect of CD38-specific T cells against two AML cell lines (THP-1 and CMK), which highly express CD38 (>99%). Lactate dehydrogenase (LDH) releasing assay and flow cytometry were performed for cytotoxicity. We co-incubated THP-1 or CMK cells with T cells transduced at a variety of effector (E): target (T) ratios for 3 consecutive days in vitro. Interestingly, these assays showed that T cells bearing anti-CD38-CAR were cytotoxic against THP-1 and CMK cells in dose- and time-dependent manners. However, in the cases of KG1a, U937 and HL60 cells which partially express CD38, killing effect was restricted to CD38+ cells by T cells bearing anti-CD38-CAR, and CD38− AML cells remained alive. These results suggested that augmentation of CD38 expression is essential for the sufficient cytotoxicity against AML cells not expressing CD38 by T cells bearing anti-CD38-CAR. We, then, investigated whether ATRA augmented CD38 expression and resultantly enhanced the cytotoxicity against AML cell lines. Interestingly, ATRA augmented CD38 expression in HL60 cells as well as even in KG1a and U937. However, ATRA by itself exerted no effect on cytotoxicity or proliferating activity in KG1, U937 and even in HL60 cells. These results showed that ATRA contributed to enhancement of CD38 expression, but not to cytotoxicity and proliferation on AML cell lines. Next, we attempted to examine cytotoxic effect of T cells with anti-CD38-CAR on KG1, U937 or HL60 cells following ATRA treatment. AML cell lines were co-cultured with the effector T cells transduced for 3 days in the presence of ATRA. The killing effect of T cells bearing anti-CD38-CAR against AML cell lines was limited in the absence of ATRA. Intriguingly, T cells with anti-CD38-CAR exerted enhanced cytotoxic effect on AML cells in the presence of ATRA. Next, we applied our settings above to AML cells freshly isolated from AML patients. Firstly, we confirmed that CD38 expression was enhanced by ATRA in AML cells from patients as observed in AML cell lines. Furthermore, 3-day incubation of patients’ AML cells with T cells bearing anti-CD38-CAR abrogated AML cells in the presence of ATRA. These results indicated that T cells expressing anti-CD38-CAR efficiently eliminated AML cells from patients as well as AML cell line cells through the enhancement of CD38 expression by ATRA. Here, we propose that pre-treatment of ATRA in patient with AML enhances CD38 expression on the leukemic cell surface, which augments the cytotoxic effect of T cells bearing CAR to eradicate leukemic cells including LSCs. Disclosures:No relevant conflicts of interest to declare.

Nutlin-1 strengthened anti-proliferation and differentiation-inducing activity of ATRA in ATRA-treated p-glycoprotein deregulated human myelocytic leukemia cells

Unlike its cytotoxicity in p53-functional cell lines, Nutlin-1, the small-molecule inhibitor of murine double minute (MDM2), significantly enhanced the differentiation-inducing activity of all-trans retinoic acid (ATRA) in HL60 and NB4 cells (p53-nonfunctional) but not in U937 cells (p53 wild-type). Moreover, we demonstrated that the synergistic differentiation-inducing activity of Nutlin-1 combined with ATRA appeared in a p53-independent manner. In the present study, we found that ATRA could selectively induce expression of p-glycoprotein (p-gp) in HL60 and NB4 cells but not in U937 cells. Investigation of p-gp-ATPase activity showed that Nutlin-1 and ATRA were likely to act as p-gp transport substrates. Furthermore, Nutlin-1 enhanced the ability of ATRA to induce expression of the myeloid differentiation-related transcription factor C/EBPβ and to reduce expression of c-myc. Additionally, the expression of retinoic acid receptor α (RARα) was further reduced in cells treated with ATRA in combination with Nutlin-1. Taken together, the mechanisms of synergistic differentiation-inducing activity of Nutlin-1 combined with ATRA could be attributed to Nutlin-1 competitive binding to p-gp, leading to ATRA efflux inhibition, and then the differentiation pathways involved were therefore further activated. Nutlin-1 might be a useful adjuvant with ATRA for patients with retinoid-resistant leukemia induced by overexpression of p-gp.

Phase 2 clinical trial of 5-azacitidine, valproic acid, and all-trans retinoic acid in patients with high-risk acute myeloid leukemia or myelodysplastic syndrome.

In this Phase 2 study, we evaluated the efficacy of combination of 5-azacitidine (AZA), valproic acid (VPA), and all-trans retinoic acid (ATRA) in patients with high-risk acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Treatment consisted of six cycles of AZA and VPA for 7 days, followed by ATRA for 21 days. Sixty-five patients were enrolled (median age, 72 years; 55 AML including 13 relapsed/refractory patients, 10 MDS; 30 unfavorable karyotypes). Best responses included 14 CR and 3 PR (26%), 75% of the responders and 36% of the non-responders achieving an erythroid response. Median overall survival (OS) was 12.4 months. Untreated patients had a longer OS than relapsed/refractory patients. In patients who fulfilled the 6 planned cycles, OS did not appear to depend on CR/PR achievement, suggesting that stable disease while on-treatment would be a surrogate for survival with this approach. During therapy, early platelet response and demethylation of the FZD9, ALOX12, HPN, and CALCA genes were associated with clinical response. Finally, there was no evidence for the restoration of an ATRA-induced differentiation during therapy. Epigenetic modulation deserves prospective comparisons to conventional care in patients with high-risk AML, at least in those presenting previously untreated disease and low blast count.

Epigenetic Therapy with 5-Azacitidine, Valproic Acid, and ATRA in Patients with High-Risk AML or MDS: Results of the French VIVEDEP Phase II Study

Introduction: Promising results have been reported last year with a combination of 5-azacitidine (AZA), valproic acid (VPA), and all-trans retinoic acid (ATRA) in patients with AML/MDS treated at MDACC in a Phase I/II study (Soriano et al. Blood 2007). We report here on a similar study conducted in 9 centers between 7/2006 and 8/2007.Methods: Patients with high-risk AML (AML in patients aged 70y+ unsuitable for intensive chemotherapy or early relapsing/refractory AML) or MDS (int-2/high IPSS without possibility of allogeneic SCT) were eligible. Treatment consisted of 6 cycles with AZA 75 mg/m2/d SC (d1-7), VPA 35 to 50 mg/kg/d PO (d1-7), and ATRA 45 mg/m2/d PO (d8-28). Cycle 1 was initiated at the hospital but cycles 2–6 were planned monthly in out-patients. Response was assessed after cycle 1, 3, and 6 (IWG AML criteria). VPA was started at 35 mg/kg/d and then increased at 50 mg/kg/d if well tolerated. Sixty-three patients were enrolled and 51 are evaluable.Results: Patients characteristics were: M/F, 27/24; median age, 73y (50–87); median WBC, 2.3 × 109/L; PS 0-1/2, 45/6 patients; median follow-up, 13 months. Forty-two patients had AML (31 de novo, 9 therapy-related, 2 post-MDS/MPD) and 9 had MDS. Only 6 patients had received prior intensive therapy. Cytogenetics was available in 46 patients with high-risk features in 26 of them (complex, -7). Twenty-nine patients stopped the treatment after 1–5 cycles: 13 due to disease progression, 11 due to toxic events (mostly infection), and 5 due to physician decision despite stable disease. VPA was associated with notable CNS toxicity at 50 mg/kg, but not at the 35 mg/kg dose level. ATRA-related symptoms (headaches, mucosal dryness) were noted. In the 22 patients who received the 6 cycles, re-hospitalization rate was 27, 41, 23, 18, 20, and 14% after cycle 1 to 6, respectively. Among these patients, 11 reached CR (6 after cycle 3) and 5 reached PR (4 after cycle 3). The CR/PR rate was thus 31%, reaching 35% in the 46 patients who did not interrupt the treatment in the absence of progression or toxic event. Table 1 gives CR/PR rate according to various patient subsets. In patients with de novo AML, CR/PR rate was 45%. Advanced age and high-risk cytogenetics did not influence the response rate. In multivariate analysis, cytogenetics but not age remained, however, a poor risk factor for OS (PS, WBC, and MDS/secondary AML being other significant factors). Ten of the 16 responders relapsed after a median response duration of 10.6 months. Median OS was 12 months (not reached in the responders). Results of sequential DNA methylation and gene expression profiles monitoring (#17 patients) will be presented.Conclusion: This study confirms that epigenetic therapy with AZA, VPA, and ATRA yields a 35% response rate in patients with high-risk AML/MDS. Although randomized studies are needed (AZA ± HDAC inhibitors), this combined approach appears to be a good option to treat older patients with low WBC and favorable PS, whatever their cytogenetics. Maintenance options should be investigated in responding patients.Table 1CR/PRNo CR/PRP valuesAge < 75y7 (27%)19Age ≥ 75y9 (45%)110.23Standard-risk cytogenetics8 (44%)10High-risk cytogenetics7 (29%)170.35De novo AML13 (45%)16MDS/secondary AML3 (18%)140.11PS 0-116 (40%)24PS 20 (0%)60.08WBC < 5.109/L15 (43%)20WBC ≥ 5.109/L1 (9%)100.07

Safety and clinical activity of the combination of 5-azacytidine, valproic acid, and all-trans retinoic acid in acute myeloid leukemia and myelodysplastic syndrome

The combination of a DNA hypomethylating agent with a histone deacetylase inhibitor has synergistic antileukemia activity and may restore sensitivity to all-trans retinoic acid (ATRA). We conducted a phase 1/2 study of the combination of 5-azacitidine (5-AZA), valproic acid (VPA), and ATRA in patients with acute myeloid leukemia or high-risk myelodysplastic syndrome. 5-AZA was administered subcutaneously at a fixed dose of 75 mg/m(2) daily for 7 days. VPA was dose-escalated and given orally daily for 7 days concomitantly with 5-AZA. ATRA was given at 45 mg/m(2) orally daily for 5 days, starting on day 3. A total of 53 patients were treated. Their median age was 69 years (range, 5-84 years). The maximum tolerated dose of VPA in this combination was 50 mg/kg daily for 7 days. Dose-limiting toxicity was reversible neurotoxicity. The overall response rate was 42%. In previously untreated older patients, the response rate was 52%. Median number of courses to response was 1 (range, 1-3 courses). Median remission duration was 26 weeks, and median survival has not been reached. A significant decrease in global DNA methylation and induction of histone acetylation were achieved. VPA blood levels were higher in responders (P < .005). In conclusion, the combination studied is safe and has significant clinical activity. This clinical trial was registered at www.clinicaltrials.gov as no. NCT00326170.