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Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15INK4B, CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34+ population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage–associated variant histone γ-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination “epigenetic” therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179.

Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15INK4B, CDH-1, DAPK-1, and SOCS-1)were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34+population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage–associated variant histone γ-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination “epigenetic” therapy. This trial is registered with http://www.clinicaltrials.govunder NCT00101179.

BackgroundMyelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis with peripheral blood cytopenias, leading to death from infection or bleeding. Azacitidine (AZA), a hypomethylating agent (HMA) is the standard of care for treatment of MDS patients (pts) with higher-risk MDS [Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017]. Responses to AZA occur in 50% of pts with significant effects on hematopoiesis ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence [Silverman LR, et al. Leukemia, 1993]. AZA treatment is associated with global DNA hypomethylation, including human endogenous retroviruses (HEV) which further activates innate immune signaling [Chiappinelli KB, et al.Cell, 2015]. The exact mechanism by which AZA improves hematopoiesis is unknown. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment with worsening cytopenias or transformation to leukemia [Silverman LR, et al. B. J Clin Oncol, 2002; Cancer, 2011]. Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical. Our clinical data demonstrate that AZA combined with Rigosertib (RIGO), a novel Ras mimetic that inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al.Cell, 2016], yields a response rate of 54% of pts who were HMA failures [Navada SC, et al. EHA 2019]. The response was associated with significant improvement in hematopoiesis and represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The precise mechanism that leads to reversal of the resistance phenotype is poorly understood.

Navada: Onconova Therapeutics Inc: Research Funding. Reddy:Onconova Therapeutics Inc: Research Funding. Silverman:Celgene: Research Funding; Medimmune: Research Funding; Onconova Therapeutics Inc: Patents & Royalties, Research Funding.

Background:

Background:Myelodysplastic syndrome (MDS) is a clinically heterogenous disease of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis, uni/multi-lineage dysplasia and a high tendency to transform into acute myeloid leukemia. Aberrant chromosomal and genetic lesions contribute to MDS pathogenesis which has been associated with chronic activation of the innate immune response and a hyperinflammatory microenvironment (Barryero L, et al.Blood, 2018). Dysfunction of Toll like receptors (TLR) and downstream effectors has been associated with the loss of progenitor function and differentiation of bone marrow (BM) cells in MDS patients. Azacitidine (AZA), a hypomethylating agent (HMA), is the mainstay of therapy for patients with higher-risk MDS (Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017) and carries an overall response rate (ORR) of 50% in patients with significant effects on hematopoiesis, ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence with survival benefits (Silverman LR, et al., Leukemia, 1993). The response to AZA is not durable and all patients relapse with worsening bone marrow failure.

Navada: Janssen Pharmaceuticals, Inc.: Current Employment. Reddy: Onconova Therapeutics, Inc.: Current equity holder in publicly-traded company.

The action of thrombin on blood platelets has been studied under the electron microscope. In control experiments with preparations treated with normal plasma or Tyrode fluid, the normal structure of platelets in their different forms of extension and flattening on the film has been observed. Thrombin (Parke-Davis Co.) in concentrations of 25 and 50 units per ml. has a lytic effect on platelets. The number of platelets affected by this treatment increases in the period of 5 to 30 minutes. Disintegration proceeds in the hyalomere from the periphery to the center with the formation of long fibrillar processes constituted by microvesicles. These processes resemble myelin figures. The granulomere turns into an amorphous dense body which also undergoes lysis. The final residue of disintegration is constituted by microvesicular material and dense amorphous bodies. The purified bovine thrombin of Seegers has exactly the same action. The lytic effect is observed with concentrations of thrombin as low as 2.5 units per ml. The number of platelets undergoing disintegration increases proportionally to the logarithm of the thrombin concentration. The probable role of thrombin and the lysis of platelets in normal blood clotting is discussed.

Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

Navada: Onconova Therapeutics Inc: Research Funding. Reddy:Onconova Therapeutics Inc: Equity Ownership, Research Funding. Silverman:Medimmune: Research Funding; Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding.

Navada: Onconova Therapeutics Inc: Research Funding. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Atallah:Helsinn: Consultancy; Jazz: Consultancy; Helsinn: Consultancy; Jazz: Consultancy; Novartis: Consultancy; Takeda: Consultancy, Research Funding; Pfizer: Consultancy. Shammo:Otsuka: Consultancy, Honoraria; CTI Pharma: Research Funding; Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Alexion: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onconova: Research Funding; Apellis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Speakers Bureau; Astex Pharma: Research Funding. Griffiths:Abbvie, Inc.: Consultancy, PI on a clinical trial; Celgene, Inc: Consultancy, Research Funding; Genentech, Inc.: Research Funding; Novartis Inc.: Consultancy; Novartis Inc.: Consultancy; Genentech, Inc.: Research Funding; Boston Scientific: Consultancy; Boston Scientific: Consultancy; Persimmune: Consultancy; Persimmune: Consultancy; New Link Genetics: Consultancy; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Abbvie, Inc.: Consultancy; Partner Therapeutics: Consultancy; Celgene, Inc: Consultancy, Research Funding. Khaled:Alexion: Consultancy, Speakers Bureau; Omeros: Consultancy; Daiichi Sankyo: Other: Travel support. Adesanya:Onconova Therapeutics, Inc.: Employment. Zbyszewski:Onconova Therapeutics, Inc.: Employment. Woodman:Onconova Therapeutics, Inc.: Employment. Fenaux:Jazz: Honoraria, Research Funding; Aprea: Research Funding; Celgene Corporation: Honoraria, Research Funding; Astex: Honoraria, Research Funding. Silverman:Medimmune: Research Funding; Celgene: Research Funding; Onconova Therapeutics Inc: Patents & Royalties, Research Funding.

Background: MDS is characterized by ineffective hematopoiesis and multiple cytopenias. Azacitidine (AZA), a hypomethylating agent (HMA), the standard therapy for higher-risk MDS patients (pts), improves hematopoiesis in 50% of MDS pts, with a median response of 14-24 months. Those pts who initially respond to AZA either relapse or progress with bone marrow failure and have a median survival of 4 to 6 months. Both primary and secondary resistance is a significant challenge and results in poor survival. Rigosertib (RIGO), a small molecule Ras mimetic, as a single agent improved hematopoiesis in 15% of MDS pts who had failed a prior HMA. In vitrodata of synergy of RIGO combined with AZA that was sequence dependent (Skiddan et al. AACR 2006), led to a Phase I/II study of the combination of RIGO/AZA and demonstrated an overall response rate of 90% in HMA naïve and 54% in HMA failures pts (Navada et al. ASH 2018). Restoration of functional hematopoiesis in response to treatment with AZA when combined with RIGO in pts, who had failed an HMA, is a unique observation in overcoming the HMA clinical resistance phenotype. Elucidating mechanisms leading to restoration of HMA effects on hematopoiesis could have profound clinical benefit.

Background: Abnormalities of the immune system and innate immune signaling have been described in patients (pts) with MDS, however the interaction between the MDS tumor cells and the immune compartment is poorly defined. Immune modulatory therapies are being explored in MDS and a better understanding of the interaction and potential cross-talk with immune components and MDS tumor cells is critical in developing new therapeutic strategies.

Background:

Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

Navada: Onconova: Research Funding. Atallah:Pfizer: Consultancy; Abbvie: Consultancy; Jazz: Consultancy; BMS: Consultancy; Novartis: Consultancy. Shammo:Incyte: Consultancy, Honoraria, Research Funding; Onconova: Other: research support; Alexion: Honoraria, Other: research support; Novartis: Consultancy, Honoraria; Celgene: Other: research support. Griffiths:Novartis, Inc.: Research Funding; Celgene, Inc: Honoraria, Research Funding; Astex/Otsuka Pharmaceuticals: Honoraria, Research Funding; Pfizer, Inc.: Research Funding; Alexion Inc.: Honoraria, Research Funding. Khaled:Juno: Other: Travel Funding; Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy. Pemmaraju:abbvie: Research Funding; stemline: Consultancy, Honoraria, Research Funding; celgene: Consultancy, Honoraria; Affymetrix: Research Funding; samus: Research Funding; SagerStrong Foundation: Research Funding; cellectis: Research Funding; daiichi sankyo: Research Funding; novartis: Research Funding; plexxikon: Research Funding. Zbyszewski:Onconova Therapeutics, Inc: Employment, Equity Ownership. Maniar:Onconova Therapeutics, Inc: Employment, Equity Ownership. Petrone:Onconova Terapeutics Inc.: Employment, Equity Ownership. Fruchtman:Onconova Therapeutic Inc: Employment, Equity Ownership. Silverman:Johnson and Johnson: Research Funding; Onconova Therapeutics Inc.: Patents & Royalties, Research Funding; Bayer: Research Funding; Celgene: Research Funding; Medimmune: Research Funding; Mount Sinai School of Medicine: Employment.

Navada: Onconova: Research Funding. Silverman:Mount Sinai School of Medicine: Employment; Onconova Therapeutics Inc.: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding; Johnson and Johnson: Research Funding; Bayer: Research Funding.

Background: MDS is a clonal stem cell disorder characterized by abnormal maturation and differentiation of hematopoietic cells. Azacitidine (AZA), a hypomethylating agent (HMA), was approved by the FDA for patients (pts) with MDS and is the standard of care as 1stline therapy for higher- risk disease. About 50% of MDS pts respond to AZA, with a median duration of response of 14 - 24 month. For those pts responding to an HMA, most either relapse or progress with worsening bone marrow failure (BMF) and have a median survival of 4 to 6 months after treatment failure. Both primary and secondary resistance remains significant clinical problems and result in poor survival. A recent study reveals that AZA activates several immunomodulatory pathways leading to activation of the antiviral defense pathway and upregulation of interferon signaling (IFN) (Chiappinelli et al, 2017). INF has multiple effects on hematopoiesis and appears highly regulated. Interferon can stimulate hematopoietic stem cells (HSC) which may be countered through activation of p38 MAPK; inhibition of the latter may improve hematopoiesis. Short-term effects of IFN stimulate myeloid and erythroid hematopoiesis.

Background: AZA is first line therapy for pts with HR-MDS with an overall response rate (ORR) between 35 and 60% in various publications; with CR/PR rates ranging between 23 and 29% in 3 randomized phase II/III studies (Silverman et al., JCO 2002; Fenaux et al., Lancet Oncol 2009; Sekeres, JCO 2017). AZA also has demonstrated efficacy in older pts with AML (Dombret et al., Blood 2015; Fenaux et al., JCO 2010). Rigosertib interferes with the RAS-binding domains of RAF kinases and inhibits the RAS-RAF-MEK and the PI3Ks pathways (Athuluri-Divakar, Cell 2016). RAS and other genes in this pathway are frequently mutated in HR MDS and putatively drive the malignant clone (Sperling et al., Nat Rev CA 2017). In vitro, the combination of rigosertib with AZA synergistically inhibits growth and induces apoptosis of leukemic cells in a sequence-dependent fashion (Skidan et al; AACR 2006 Abstract 1310).

Rigosertib (RIG) which functions as a RAS-mimetic, binds to the RAS-binding domain (RBD) impacting many pathways that involve RBD-mediated interactions, including the RAS-Raf and AKT/PI3-kinase pathways (Divakar et al., Cell 2016). Recently, we demonstrated that RIG also functions as an epigenetic modifier in MDS. RIG alone or in combination with Azacitidine (AZA) exerted epigenetic effects on the global histone post-translational modifications (PTMs), chromatin remodelers, HDACs, and DNMTs and caused enhanced apoptosis in MDS (MDS-L) and AML (BW-90) cell lines as well as in bone marrow cells (BM) derived from pts with MDS (Chaurasia et al ASCO 2016). In clinical trials, the combination of RIG and AZA demonstrated an overall response rate of 76% in all MDS patients; 62% response in pts who had failed prior hypomethyating agent (HMA) (Navada et al ASH 2016) and 85% in HMA naive pts. The growing evidence suggests that combined therapy may be more effective than either agent alone. To further study these effects, we explored activity of RIG in combination with Vorinostat (VOR), a histone deacetylase inhibitor, on the cell lines. Combined treatment in sequential order VOR/RIG and RIG/VOR potentiates far greater effects on the global PTMs, HDACs, DNMTs, chromatin remodelers, cell cycle checkpoint proteins and PI3/AKT pathway than RIG/AZA or AZA/RIG.

Navada: Onconova Therapeutics, Inc.: Research Funding. Daver:Karyopharm: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Ariad: Research Funding; Otsuka: Consultancy, Honoraria; Kiromic: Research Funding; BMS: Research Funding. DiNardo:Agios: Other: advisory board, Research Funding; Novartis: Other: advisory board, Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Fenaux:Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding. Petrone:Onconova Therapeutics, Inc.: Employment. Zbyszewski:Onconova Therapeutics, Inc.: Employment. Fruchtman:Onconova: Employment. Silverman:Onconova Therapeutics, Inc.: Patents & Royalties: Co-Patent holder for the combination of azacitidine and rigosertib, Research Funding.

Off Label Use: IVIG.

Silverman: Onconova Therapeutics Inc: Honoraria, Patents & Royalties: co-patent holder on combination of rigosertib and azacitdine, Research Funding. Daver:ImmunoGen: Other: clinical trial, Research Funding. DiNardo:Novartis: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Fenaux:CELGENE: Honoraria, Research Funding; JANSSEN: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; NOVARTIS: Honoraria, Research Funding. Fruchtman:Onconova Therapeutics Inc: Employment. Azarnia:Onconova Therapeutics Inc: Employment.

Background:Myelodysplastic Syndrome (MDS) and Aplastic Anemia (AA) are often associated with clinical immune manifestations. An abnormal profile of the T-cell repertoire can be detected in these patients (pts) and is thought to play a role in bone marrow (BM) insufficiency. The presence of a co-existent large granular lymphocytic (LGL) clone may exacerbate cytopenias independent of the primary disease mechanism and offers another target for therapeutic intervention. Treatment for LGL proliferation is usually immunosuppressive therapy but there is no accepted standard of care.

Wilhelm: Onconova Therapeutics, Inc: Employment, Equity Ownership. Demakos:Onconova: Consultancy. Azarnia:Onconova Therapeutics, Inc: Employment. Silverman:Onconova: with Icahn School of Medicine at Mount Sinai Patents & Royalties.

Background:Rigosertib is a small molecule anti-cancer agent targeting PI3/polo-like kinase pathways that promotes G2/M arrest and has effects on the B-Raf and Ras pathways. It is currently being tested as a single agent with the intravenous (IV) formulation in patients (pts) who have relapsed or are refractory to hypomethylating agents (HMAs) as well as with the oral formulation in lower-risk, red-cell transfusion-dependent MDS patients. Azacitidine (AZA) is first-line therapy for pts with higher-risk MDS. In vitro, the combination of rigosertib with AZA acts synergistically to inhibit growth and induce apoptosis of leukemic cells (Skidan et al 2006). This effect appears to be sequence dependent, requiring exposure to rigosertib first, followed by AZA. These nonclinical results provided the rationale to combine the 2 agents in a phase I/II study in pts with MDS and AML.

Rigosertib is a small molecule anti-cancer agent which inhibits the PI-3K and PLK pathways, promotes G2/M arrest, and selectively induces apoptosis in cancer cells. We have previously reported results of a phase I/II study of rigosertib in patients (pts) with MDS and AML who had relapsed or were refractory to hypomethylating agents (HMA), a population for which there are currently no approved second line therapies. Rigosertib appeared to be well tolerated in this pt population and to have biologic activity with reduction or stabilization of bone marrow (BM) blasts and improvement in the peripheral blood (PB) counts in few treated pts. Reduction in BM blasts by rigosertib was associated with increased survival. In the current analysis, we evaluate pt characteristics that may predict for response.We analyzed the results of a phase I/II study of Rigosertib that was conducted in pts with MDS and AML. In the phase I component, pts were entered in cohorts of escalating doses in a classic 3+3 design ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) administered for 72 to 144 hours. A MTD of 1375 mg/m2 was identified for the phase II component, and subsequent pts were treated with this dose as a CIV for 72 hours. BMs were performed at baseline, week 4, 8, and then q3 months.Twenty-two pts with MDS or AML refractory or relapsing to a HMA have been treated with rigosertib. The study cohort comprised pts with a diagnosis of int-2 MDS (2), high risk MDS (6), CMMOL (1), and AML (13 pts all with antecedent MDS). Responses according to IWG 2006 criteria were observed in the BM and PB: marrow CR (4), marrow PR (2). Two pts also had hematologic improvement of the erythroid (1) and platelet (1) lineages. Four pts had stable disease (SD) after treatment but their courses were complicated by infections requiring hospitalization and removal from study. Three pts were deemed to be inevaluable because they received < 2 cycles of treatment or did not have a follow-up BM evaluation. Thus, 10/19 evaluable pts (53%) demonstrated either a BM/PB response (6) or SD (4).The median overall survival (OS) of pts with marrow CR+PR (n=6) was 12 months versus 1.8 months for those without a BM response (n=9) (p=0.0159, log-rank test). Age was not a predictor of response. 1 out of 6 responders had a major elimination in the size of the clonal population. Non-responders did not have a change in the magnitude of the clone. Prior response to HMA was not a predictor for response to rigosertib. Those who did have a marrow CR or PR responded early with median time to response of 2-4 cycles. Those with higher blast counts were less likely to respond. At study entry, the median blast percentage of responders was 16% versus 44% for non-responders. Less than 20% blasts at study entry was a positive predictor of response (p=0.047). Of those pts who did not respond or were inevaluable, the majority (75%) had AML, many with a proliferative course. Nine of 19 pts developed cystitis manifested by dysuria and/or hematuria as a side-effect of therapy. Among responding patients, 5 of 6 had cystitis [grade (GR) 1 (2); GR 2 (1); GR 3 (2)] compared with 3 of 9 non-responders [GR 1(1); GR 2 (1); GR 3(1)] (p=0.08). In one responding pt with grade 3 cystitis, a cystoscopy was performed which revealed polypoid inflammatory changes of the mucosa with hemorrhage. Biopsy showed neutrophilic inflammation without malignant cells. Upon resolution of symptoms, treatment was restarted at 50% of the original dose without complications. Pts who developed symptomatic cystitis were treated with sodium bicarbonate with improvement. The relationship between cystitis and response is being investigated.Rigosertib appears to have biologic activity with reduction in BM blasts associated with increased survival and improvement in the PB counts in a subset of treated pts. Pts with <20% blasts at study entry had a greater likelihood of response. Pts with proliferative disease with rapidly rising or high wbc did not respond. Age, cytogenetic profiles, and response to prior therapy were not predictors of response. Cystitis may be a response related biomarker and requires further analysis. A phase III multicenter randomized trial is underway to compare rigosertib to best supportive care with a primary endpoint of OS in pts with higher risk MDS who have failed, progressed, or relapsed after treatment with HMA, and can be used to validate the observations reported here in a larger study.Reddy: Onconova: Equity Ownership, Research Funding. Holland:Onconova: Research Funding. Wilhelm:Onconova Therapeutics: Employment, Equity Ownership. Silverman:Onconova: Research Funding.

The hypomethylating agent (HMA) azaC reverses epigenetic silencing and is the first agent demonstrated to improve survival in patients with higher-risk MDS (Silverman et al JCO 2002, Fenaux et al Lancet Oncology 2009). Time to initial response with single agent azaC, is 3 to 4 cycles, the CR rate ranges from 7 to 17% and overall response is 45-50%. Vorinostat, a histone deacetylase inhibitor (HDACI) which inhibits class I and II HDAC, has demonstrated single agent activity in patients with MDS with responses of 20% (Garcia-Manero Blood 2006). In vitro the 2 agents are synergistic in reactivating epigenetically silenced genes. The effect is sequence-dependent requiring exposure to the HMA first followed by the HDAC. We conducted a phase I pilot study of escalating and de-escalating doses of the 2 drugs in combination in 8 cohorts and demonstrated broad activity with responses ranging up to 80% across all of the cohorts tested (Blood 2008).To determine the response rate of patients treated with the combination of vorinostat and azacitidine at the doses established as safe and effective in Phase I in an expanded cohort of patients with MDS.In the phase II component 3 schedules of the combination were chosen based on response and adverse event profile for further evaluation. Eligible patients were entered into one of 3 cohorts with the combination (see table 1): cohort 1: azaC 55 mg/m2/d 1-7 SC, vorinostat 200mg po Bid d3-16; cohort 2: azaC 75 mg/m2/d 1-7 SC, vorinostat 300mg po Bid d3-9; cohort 3: azaC 55 mg/m2/d 1-7 SC, vorinostat 300mg po Bid d3- 9. Patients with IPSS int-1, -2 and high-risk disease were eligible. Patients with secondary MDS were eligible, those with AML were excluded. Using a simon 2 stage design 13 patients were entered in each cohort and assessed for response, scored according to IWG 2006 criteria and toxicity.40 patients have been entered and 39 (21 male, 18 female) are evaluable for toxicity and 33 for response, 1 pt was registered but never treated. IPSS classification among the 39 patients: int-1 8; int-2 12; high-risk 12; unclassified 7, with median age 67 (23-79). Responses among evaluable patients have occurred in 23 of 33 (70%); 10 CR, 4 CRi, (CR+CRi=42%) 9 HI, 5 SD, 5 NR. Median time to response is 2 cycles (8 weeks). Responses by cohort (table 1) are 70%, 73% and 67% for cohort 1, 2 and 3, respectively. Analysis for the MDS clone at the time of best response demonstrated the persistence of the clone in 45% of patients as marked by cytogenetic or FISH abnormalities, suggesting a modulating rather than cytotoxic effect of the combination on the clone. Cycles administered, range from 1 to 26+ with a median of 6 cycles. Median duration of response is 16 months overall and 9.5, 23 and 27 months, respectively for cohorts 1, 2 and 3. Eighteen patients have come off study for: death or due to disease complications (6); co-morbidities (2); consent withdrawal (5); and withdrawal for stem cell transplant (3). Median OS is 21.1 months and is 10.1, 37.4 and 19 months for cohorts 1, 2, and 3 respectively (NS). Grade 3 fatigue occurred during cycle 1, 2 or 3 in cohort 1 (8%), cohort 2 (16%) and cohort 3 (8%). GI toxicity grade 3 (vomiting, diarrhea, dehydration) occurred in 8% of patients in each of the cohorts. There was no suggestion of cumulative toxicity for either fatigue or GI adverse events. Correlative biologic study analysis is underway.The combination of azaC and vorinostat can be safely administered, and is well tolerated in repetitive cycles. The dose of azaC in cohort 2 adheres to the FDA approved dose and schedule. OR and CR rates and time to initial response are comparable among the cohorts and these data suggest that the combination is superior to published results of azaC alone. Cohorts 2 and 3, with vorinostat administered for 7 days, appears to be associated with longer response duration and OS. An intergroup study (SWOG-S1117) comparing azaC and vorinostat to either azaC alone or combined with lenalidomide is underway utilizing the doses and schedule in cohort 2 from this study. Correlative studies that may shed light on mechanism of action or guide patient selection are being conducted. Supported in part by NYCC- N01 CM-62204 and the Henry and Mickey Taub Foundation.Silverman: Merck: Research Funding. Off Label Use: vorinostat for treatment of patients with a myelodysplastic syndrome investigational use.

Rigosertib is a small molecule anti-cancer agent which inhibits the PI-3K and PLK pathways, promotes G2/M arrest, and selectively induces apoptosis in cancer cells. We have previously reported results of a phase I/II study of rigosertib in patients (pts) with MDS and AML who had relapsed or were refractory to hypomethylating agents (HMA), a population for which there are currently no approved second line therapies. Rigosertib appeared to be well tolerated in this pt population and to have biologic activity with reduction or stabilization of bone marrow (BM) blasts and improvement in the peripheral blood (PB) counts in few treated pts. Reduction in BM blasts by rigosertib was associated with increased survival. In the current analysis, we evaluate pt characteristics that may predict for response.

The hypomethylating agent azacitidine (azaC) which can reverse epigenetic silencing, is the first agent demonstrated to alter the natural history of MDS and improve survival in higher-risk patients (Silverman JCO 2002, Fenaux Lancet Oncology 2009). AzaC also produces comparable rates of response in patients with non-proliferative AML and appears to affect survival (Silverman JCO 2006). The response rate to single agent azaC is about 50% with median duration of response about 14 to 22 months. Patients who are refractory to or relapse following first line therapy with azaC have a median survival of 4 to 6 months (Jabbour 2010, Prebet 2011). Options for these patients failing first-line therapy with azaC are limited and there is no standard of care. Investigational therapies are being explored but not always widely available for these patients. Prior studies with azaC explored higher doses but were inconclusive to a dose response effect secondary to the slow time to response with a median of 2 to 3 cycles. In vitro, azaC has a wide range of concentrations that can induce differentiation up to 4 μm. In patients the standard clinical dose achieves a plasma level of 1. 25 μm thus suggesting that higher doses might have a clinical benefit (Marcucci 2005).Patients who were refractory to or relapsing following treatment with an azaC based regimen for MDS where alternative investigational options were not available and who were not rapidly progressing (i. e rapidly rising WBC or myeloblast %), were treated with higher doses of single agent azaC. The dose was increased by 33% from the baseline prior failing regimen (eg 55 to 75 or 75 to 100 mg/m2). The azaC was administered SC × 7 days q 28 days; response status was assessed after 2 cycles. Patients stable or responding were continued on 28 day cycles. Myeloid cytokine support was utilized for patients with ANC < 200 and ESA support for patients who were RBC dependent.As of the data cut for this submission 13 patients are evaluable for toxicity and response. Among the 13 patients the median age was 68 and 11 were male. All had been treated with azaC based regimens before and 10 had responded: 4 CR; 1 PR; 5 HI; 2 NR; 1 unknown. The immediate therapy prior to increased dosing included: azaC + histone deacetylase inhibitor (HDACi) (6); single agent azaC (5); investigational treatment (2). 6 had MDS (int-1 (2); int-2 (3); high (1) and 7 AML (all transformed following MDS all smoldering). Responses among evaluable patients have occurred in 11 of 13 (85%); 1 PR, 7 HI (4 CRm), 1 CRm, (CR+CRi=53%) 2 PRm, 1 SD, 1 NR. Although responses occurred, the abnormal MDS/AML clone persisted, suggesting that the higher dose did not have a cytotoxic effect on the malignant clone. A total of 117 cycles have been administered, range 2 to 17 with a mean 8 cycles. Median time to treatment failure was 11. 6 months and median survival is 17. 5 months. Eight patients have come off treatment for progression (5); relapse (2); co-morbidities (1). No grade 3 or 4 non-hematologic toxicities were observed. Hematologic toxicity was similar to that seen with standard dose azaC.Modification of the dose of azaC in select patients, who lack alternative options including investigational agents or stem cell transplant, may improve blood counts and reduce the bone marrow blasts. The quality of the response to the increased dose after primary failure does not attain the level of the original response in most, however, this approach may increase therapeutic options in a poor risk population. Investigations into the potential mechanisms of action are being explored.Silverman: celgene: Speakers Bureau. Demakos:celgene: Speakers Bureau.

Rigosertib is a small molecule anti-cancer agent with a multi-targeted mechanism of action. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to rigosertib compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Chen Proc AACR 2008). Azacitidine is first-line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating agents have a short life expectancy of approximately 4 to 6 months (Jabbour 2010, Prebet 2011). There are no approved second line therapies for this patient population.A phase I/II study of Rigosertib is being conducted in pts with MDS and AML. Pts with higher-risk disease had to have failed a hypomethylating agent. In the phase I component, pts were entered in cohorts of escalating doses in a classic 3+3 design ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments were administered every 3 to 4 weeks. A maximum tolerated dose of 1375 mg/m2 was identified for the phase II component, and subsequent pts were treated with this dose as a CIV for 72 hours. A CBC was performed weekly and a bone marrow (BM) was performed at baseline and week 4, 8, and then q3 months afterwards.Twenty-one patients with MDS or AML refractory/relapsed to a hypomethylating agent have been treated with rigosertib. The study cohort comprised pts with a diagnosis of intermediate-2 MDS (2 pts), high risk MDS (5 pts), chronic myelomonocytic leukemia (1 pt), and AML (13 pts) (all AML had an antecedent MDS). The median age was 79 years. 86% of pts were male. Patients received between 1–19 cycles of treatment. Their cytogenetic profiles were diverse with the most recurrent abnormalities including a complex karyotype (5 pts), normal (5 pts), monosomy 7 (4 pts), and trisomy 8 (2 pts). Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: marrow CR (4), hematologic improvement (HI) (2); erythroid (1) platelet (1). Time to response was 2–4 cycles. An additional 2 pts had a >50% BM blast decrease from baseline but not to < 5%. Three pts had stable disease after treatment but their courses were complicated by infections requiring hospitalization and removal from the study. Three pts were deemed to be inevaluable because they received less than 2 cycles of treatment or did not have a follow-up bone marrow evaluation. Thus, 9/18 evaluable pts (50%) demonstrated either a bone marrow/peripheral blood response (6) or stable disease (3). The median overall survival of those with marrow CR+PR was 10.1 months versus 2 months for those without a bone marrow response (p=0.0011, log-rank test). Of those pts who did not respond or were inevaluable, the majority (83%) had AML, many with a proliferative course. The most frequent grade 1–2 side-effects included dysuria, hematuria, fatigue, anorexia, nausea, and diarrhea. Possibly related grade 3 side-effects included fatigue, hematuria, and dyspnea, each in one pt. Six of 21 pts developed cystitis manifested by dysuria and/or hematuria. Among responding or stable patients, 5 of 9 had cystitis compared with 1 of 12 non-responders. Patients who developed symptomatic cystitis were treated with sodium bicarbonate with improvement. The relationship between dysuria and/or cystitis and response is being investigated.Rigosertib appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. It has biologic activity with reduction in BM blasts and improvement in the peripheral blood counts in a subset of treated pts, and these effects are associated with increased survival. Dysuria/cystitis may be a response related biomarker and requires further analysis. Data regarding pharmacokinetics and pharmacodynamics will be presented and correlated to response. Given promising initial results, a phase III multicenter randomized trial is underway to compare rigosertib to best supportive care with a primary endpoint of overall survival in patients with higher risk MDS who have failed, progressed, or relapsed after treatment with hypomethylating agents.Reddy: Onconova: Research Funding. Holland:Onconova: Research Funding. Wilhelm:Onconova: Employment, Equity Ownership. Silverman:Onconova: Research Funding.

Abstract 4932

Abstract 3794

ON 01910.Na a novel benzyl styryl sulfone derivative is under clinical development in hematologic malignancies. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to ON 01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). Azacitidine (AzaC), is first line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating based therapies have a poor prognosis and there are no accepted effective second line treatments, thus a need for new agents.A phase I/II study of ON 01910.Na is being conducted in pts with hematological malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks. A CBC is performed weekly and a bone marrow (BM) is performed at baseline and week 4, 8, and then q3 months thereafter. Pts with higher-risk disease had to have failed a hypomethylating agent.Ten pts with MDS or AML relapsed/refractory to a hypomethylating agent have been treated with ON 01910.Na thus far (table 1). The study cohort comprised pts with a diagnosis (Dx) RAEB-2 (4 pts), RAEB-T (1 pt), and AML (5 pts) (median age of 75 years). Their cytogenetic profile included 1 pt with normal, 2 with intermediate (+8), and 7 pts with poor risk cytogenetics (monosomy 7 and/or complex). Patients were treated between 5 and 70 weeks. Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: Marrow CR (3), hematologic improvement (HI-P) (2); erythroid (1) platelet (1). An additional 2 pts had a >50% BM blast decrease from baseline but not to <5%. Thus, 5/10 (50%) demonstrate a bone marrow response. Survival of these pts was 7.3, 15.7, and 16.4 months; one patient remains on study 5+ months. Four of the five responders had MDS at the initiation of treatment: RAEB-2 (3), CMMoL (1), AML (1). Responders had monosomy 7 (2), trisomy 8 (1) and complex cytogenetics (2). One pt had an elimination of the MDS clone and the others had persistence of the abnormal karyotype throughout their treatment course. Five pts had SD without HI at 4 weeks, 2 pts progressed to AML. All 5 non-responders had AML; 4 with a proliferative course. These latter received only 2 (2) or 3 (3) cycles before succumbing to disease related infectious complications. Survival for these patients ranged from 1.3 – 2 months with a median duration on study of 42 days. The most frequent side effects grade2 2 for all pts included fatigue, anorexia, nausea, and dysuria in patients receiving extended duration infusions. One pt had a grade 3 urinary frequency. No hematologic toxicities occurred and no bone marrow toxicity or hypoplasia was noted. Pharmacokinetic studies are ongoing, data to date demonstrate no evidence of drug accumulation in patients who are treated repeatedly.ON 01910.Na appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. ON 01910.Na has biologic activity with reduction in BM blasts, eradication of the MDS clone and improvement in the peripheral blood counts in some pts. These effects are associated with increased survival albeit in limited numbers of pts treated thus far. Further study of ON 01910.Na is warranted to better define biologic activity, appropriate target populations and to define mechanism of action.Silverman: Onconova Therapeutics Inc: Research Funding, Research support of Clinical Trial. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership.

Abstract 2944

ON 01910.Na, a novel benzyl styryl sulfone derivative is currently under clinical development as an anticancer agent in solid tumors and hematologic malignancies. ON 01910.Na selectively targets tumor cells at the mitotic phase of the cell cycle, arrests leukemia cells at the G2/M stage and induces apoptosis in a time and dose dependent manner. ON 01910.Na affects multiple cellular pathways, has multikinase activity and has been shown to target multiple genetic pathways, including those that are regulated by cyclin dependent kinases. Our studies indicate that leukemic cells exhibit significantly higher levels of sensitivity of ON01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). AzaC alters the natural history of higher risk MDS with effects on marrow function, reduction in the risk of leukemic transformation and increased overall survival (Silverman JCO 2002, 2006; Fenaux Lancet 2009). However, 50% of patients (pts) do not respond to AzaC and upon progression after treatment their prognosis is poor, thus, a need for alternative agents.A phase I/II study of ON 01910.Na is being conducted in pts with heme malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CI) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks.This report details two pts with progressive disease following treatment for MDS with AzaC at a dose of 75mg/m2/d x 7 days SC every 4 weeks. Upon progression they were treated with ON 01910.Na at 1050 mg/m2/d CI x 72h to 144 hours. Patient 002 presented with MDS int-2 with myelofibrosis and mild splenomegaly, 8% marrow blasts, normal karyotype, and RBC transfusion dependence. AzaC therapy was initiated in 9/2004. A hematologic improvement (HI) was achieved according to modified IWG criteria with marrow myeloblasts < 5%, RBC transfusion independence and a persistence of mild splenomegaly. A stable HI was maintained for 4 years on monthly AzaC therapy until relapse. At progression (10/2008), marrow myeloblasts increased to 11%, RBC transfusion requirement resumed, platelets fell to 20 × 109/L, the spleen enlarged and a new cytogenetically abnormal clone emerged, 46,XY,del(20)(q11q13),i(21)(q10),der(21)t(1;21)(q12;p11) resulting in trisomy for 1q. Administration of ON 01910.Na 1050 mg/m2/d x 72 hours CI was initiated 3 months after the last dose of AzaC. After 2 cycles of treatment evaluation demonstrated a reduction in: marrow myeloblast to 1%, spleen size by 50% and the MDS clone by 74% by standard cytogenetic analysis. Following the 4th and 7th cycles of treatment with ON 01910.Na, marrow myeloblasts ranged from 1 to 3%, palpable splenomegaly resolved, RBC transfusions decreased by 33% and platelets rose to 80 × 109/L. Although the conventional karyotype normalized after the 4th cycle, all 3 abnormalites were detected by FISH in 30% of cells following the 4th and 7th cycles suggesting a loss of proliferative capacity of the MDS clone. Patient 004 presented with high risk MDS, 19% marrow blasts, pancytopenia and a complex karyotype. AzaC treatment was initiated in 9/2002 leading to a stable HI, with marrow myeloblasts < 5%, RBC transfusion independence, and persistence of the MDS clone with no change in the complex karyotype, including deletion of the MLL locus. The HI was maintained for 6.5 years with monthly AzaC therapy until progression. At relapse marrow myeloblasts increased to 19%, and the complex karyotype persisted unchanged from initial diagnosis. ON 01910.Na treatment started (3/2009) and evaluations at the 2nd, 4th and 7th cycles revealed: a) reduction in % marrow myeloblasts to 12%, 8% and 7% respectively; b) persistence of abnormal karyotype in over 90% of metaphase and interphase cells, irrespective of the decreased % blasts; c) increased platelets from 20 to 40 × 109/L. Patient 002 and 004 remain on treatment with ON 01910.Na at 8+ and 5+ months, respectively.Preliminary data suggest that ON 01910.Na has an anti-proliferative modulatory effect on the MDS clone in pts progressing after AzaC therapy and should be explored in a broader MDS population.Silverman: Onconova Therapeutics: Research Funding. Off Label Use: ON01910Na is investigational and not FDA approved. Wilhelm:Onconova: Employment. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Holland:Onconova Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 4839

Background: The hypomethylating agent azacitidine (azaC) which can reverse epigenetic silencing, is the first agent demonstrated to alter the natural history of MDS and improve survival in higher-risk patients (Silverman JCO 2002, Fenaux Blood 2007). AzaC also produces comparable rates of response in patients with non-proliferative AML and appears to affect survival (Silverman JCO 2006). Time to response is slow with single agent azaC, requiring a median of 3 to 4 cycles to initial response and the CR + PR rates ranges from 7 to 27%. Vorinostat, a histone deacetylase inhibitor (HDACI) which inhibits class I and II HDAC, has demonstrated single agent activity in patients with MDS and AML with responses of 25% (Garcia-Manero Blood 2006). In vitro the 2 agents are synergistic in reactivating epigenetically silenced genes. The effect is sequence dependent requiring exposure to the hypomethylating agent first followed by the HDACI. This study was designed to test the safety of the combination and to determine the response rate and effects on the MDS/AML clone. Methods: In the phase I component eligible patients were entered into one of eight cohorts with the combination of vorinostat and azaC in a 3+3 dose escalating de-escalating design (see table). Results: As of the data cut for this submission 21 patients are evaluable for toxicity and response. Accrual to all 8 cohorts has been completed, full data will be available for the meeting. Among the 28 patients entered: 20 have MDS and 8 AML with median age 68. Responses among evaluable patients have occurred in 18 of 21 (86%); 9 CR, 2 CRi, (CR+CRi=53%) 7 HI, 2 SD. Median time to response is 2 cycles. Among patients with high risk MDS and AML 10/12 (83%) responded (5CR, 1CRi, 4 HI). Responses including CR occurred, but in 57% of patients the abnormal MDS/AML clone persisted, suggesting a modulating effect of the combination on the clone. A total of 171 cycles have been administered, range 1 to 17 with a mean 5 cycles. Eight patients have come off study for progression (1); relapse (2); co-morbidities (2); or consent withdrawal (3). No grade 3 or 4 non-hematologic toxicities in cycle 1 were observed. Grade 2 fatigue occurred during cycle 1 in 89% of patients in cohorts 2, 3 and 4. Grade 2 anorexia and fatigue occurred in cycle 1 in 31.6% and 57.9% of patients, respectively. The fatigue correlates with the scheduled duration of vorinostat administration with 14 days vorinostat (cohorts 1–4) associated with grade 2 and 7 days (cohorts 5–7) producing grade 1. Some suggestion of cumlative fatigue (grade 3) occurred in cycles 2 and beyond in cohorts 1, 3 and 4 with 14 days of vorinostat. However it did not result in discontinuation of therapy. Correlative biologic studies are underway. Conclusion: The combination of azaC and vorinostat can be safely combined, is well tolerated in repetitive cycles and is active in both lower and higher risk/AML patients with an OR and CR rate superior to azaC alone. AzaC at a dose of 55 mg/m2 appears to be optimal for combination. The phase II study will utilize an optimization design to further identify the optimal biologic/epigenetic effects among the three vorinostat schedules of 3, 7 or 14 days when combined. Cohort No of Pts AzaC dose mg/m2/d 1–7SQ Vorinostat dose Mg/d Total dose AzaC/Vorinostat Adverse Events anorexia/fatigue Response 2 patients withdrew consent before treatmen and were replaced in cohorts. 1 3 55 200 bid × 14d 385/5600 1;0;0/2;1;2 CR;CRi;CR 2 3 55 200 tid × 14d 385/8400 2;2;2/2;2;2 CR;HI;CR 3 3 75 200 tid × 14d 525/8400 1;0;1/2;0;2 NR;CR;CRi 4 3 75 200 bid × 14d 525/5600 2;1;2/2;2;2 IE;CR;HI 5 4 75 300 bid × 7d 525/4200 1;2;1/2;1;1 IE;SD;HI;SD 6 3 55 300 bid × 7d 385/4200 0;0;0/0;0;0 CR;HI;CR 7 5 55 200 bid ×7d 385/2800 0;2;2/0;1;1 IE;IE;CR;HI;HI 8 4 55 300 bid × 3d 385/1800 NA IE;HI;TE;TE

Background:The hypomethylating agent azacitidine (azaC) which can reverse epigenetic silencing, is the first agent demonstrated to alter the natural history of MDS and improve survival in higher-risk patients (Silverman JCO 2002, Fenaux Blood 2007). AzaC also produces comparable rates of response in patients with non-proliferative AML and appears to affect survival (Silverman JCO 2006). Time to response is slow with single agent azaC, requiring a median of 3 to 4 cycles to initial response and the CR + PR rates ranges from 7 to 27%. Vorinostat, a histone deacetylase inhibitor (HDACI) which inhibits class I and II HDAC, has demonstrated single agent activity in patients with MDS and AML with responses of 25% (Garcia-Manero Blood 2006). In vitro the 2 agents are synergistic in reactivating epigenetically silenced genes. The effect is sequence dependent requiring exposure to the hypomethylating agent first followed by the HDACI. This study was designed to test the safety of the combination and to determine the response rate and effects on the MDS/AML clone.

The genetic hallmark of MDS is a gain or loss of chromosomal loci identified in bone marrow (BM) cells in ∼50% of pts at diagnosis. We previously demonstrated in longitudinal chromosome study a modulating effect of chronic AZA C therapy on the MDS clone which subdivided patients into five distinct cytogenetic groups with statistically significant differences in survival (P=0.0003) (Najfeld et al, ASH 2004). Our goal was, therefore, to investigate whether PB cells can be used in substitute of BM cells for detection of genomic defects to monitor the clone during AzaC-based therapy. Chromosomal and interphase (I-) FISH studies were performed using BM and PB cells at baseline and following AzaC-based therapy. I-FISH studies were evaluated with a panel of six probes [EGR1 (5q31), D7S522 (7q31), D8Z2 (8p11.1-q11.1), MLL (11q23), Rb1 (13q14), D20S108 (20q12)]. Of the 47 pts studied, 25 (53%) had a normal karyotype and disomy for the MDS panel of six probes. The other 22 pts (47%) were cytogenetically abnormal, showing concordant results in 16 of 22 pts (73%) for cytogenetic and I-FISH genomic defect in BM cells (showing ≤20% frequency difference). The remaining 6 pts had a mean of 77% (range 37.5–100%) of abnormal metaphase cells and a mean of 42% (range 1.8–73.3%) of abnormal BM interphase nuclei. The 35% difference in frequency seen between metaphase and interphase BM cells is attributed to the proliferative advantage of metaphase cells with a complex karyotype. The frequency of the abnormal MDS-marked clone in BM and PB cells was concordant in 13 of 20 pts (65%). Hematological response of these pts was PR=1, hematological improvement=3, stable disease=2, too early for evaluation=6, and 1 patient had no hematological response after 10 months of AZA C treatment. The remaining 7 pts had a mean of 59% (range 48–68%) of abnormal BM metaphase cells and a mean of 21% (range 7–41%) of abnormal PB interphase nuclei. The MDS abnormal clone was detectable in more than two-fold higher frequency in BM compared to PB prior to treatment in 35% of pts. Preliminary sequential studies in discordant pts revealed a transition between the BM and PB cells to concordant frequencies within 4–5 months after AzaC-based therapy. These early observations suggest that monitoring AzaC-based therapy can be achieved using peripheral blood cells in 80% of pts. Remarkably, one pt with monosomy 7, a notoriously poor IPSS indicator, demonstrated a hematological improvement, full cytogenetic and 97% FISH remission, both in the BM and PB after 4 months of AzaC-based therapy. To examine AzaC’s response on cell lineage involvement in MDS, purified BM and PB cells were subjected to FISH analysis before and during treatment from patients who were cytogenetically abnormal at baseline. Similar frequencies of genomic imbalances were seen in purified BM and PB derived CD34+ (97% vs. 96%) and CD15+ cells (94% vs. 98%), indicating that these cell populations had a similar response to AZAC therapy, as monitored in PB or BM, during the initial treatment period (4–5 months). Purified BM and PB-derived T-lymphocytes (CD3+/4+/8+) had normal disomic patterns before and during AzaC-based therapy, indicating that T-cells were not involved in the MDS clone in these pts. In contrast, BM and PB derived B-cells (CD19+) had slightly discordant results, showing a mean of 83% vs. 56% respectively of MDS-marked clone, indicating a trend towards greater response in PB- derived B-cells when compared to BM-derived CD19+ cells. In summary, our results demonstrates that although the MDS abnormal clone may be detected in both the BM and PB at the start of therapy, due to the proliferative advantage of the abnormal clone the optimal tissue should be bone marrow. This is the first study demonstrating that tracking the MDS-marked clone during the AZA-C therapy is feasible in peripheral blood cells.

The two most frequent cytogenetic abnormalities in pts with MDS involve rearrangements of chromosomes 5 and 7. Monosomy 7 or deletion 7q, alone or in a complex karyotypes are poor-risk abnormalities and associated with a low response rate to conventional therapies. As a part of our comprehensive longitudinal study of 220 patients treated with AZA C, we asked the question whether pts with −7/del(7q) alone or as a part of the complex clone, may achieve hematological and/or cytogenetic response. A minimum of three follow-up cytogenetic and FISH analyses were required as an inclusion criterion. A normal karyotype was observed in 129 of 229 pts (56%) and 100 pts (44%) had an abnormal karyotype at baseline. Among the 100 pts with an abnormal karyotype 29 pts (29%) had chromosome 7 abnormalities prior to the AZA C treatment. In an additional 12 pts chromosome 7 abnormalities developed during the course of disease and AZA C therapy (range 4 months to 5 years). Response of the abnormal chromosome 7 clone to AZA C therapy was observed in 3 patterns:

The two most frequent cytogenetic abnormalities in pts with MDS involve rearrangements of chromosomes 5 and 7. Monosomy 7 or deletion 7q, alone or in a complex karyotypes are poor-risk abnormalities and associated with a low response rate to conventional therapies. As a part of our comprehensive longitudinal study of 220 patients treated with AZA C, we asked the question whether pts with −7/del(7q) alone or as a part of the complex clone, may achieve hematological and/or cytogenetic response. A minimum of three follow-up cytogenetic and FISH analyses were required as an inclusion criterion. A normal karyotype was observed in 129 of 229 pts (56%) and 100 pts (44%) had an abnormal karyotype at baseline. Among the 100 pts with an abnormal karyotype 29 pts (29%) had chromosome 7 abnormalities prior to the AZA C treatment. In an additional 12 pts chromosome 7 abnormalities developed during the course of disease and AZA C therapy (range 4 months to 5 years). Response of the abnormal chromosome 7 clone to AZA C therapy was observed in 3 patterns: pts who had −7/del(7q) present at baseline without any further cytogenetic change during the AZA C treatment (20 pts; 69%). Eight of these pts had hematological improvement (HI); Pts who had either reduction or elimination (complete cytogenetic response=CCR) of the abnormal chromosome 7 clone as a results of treatment (9 pts, 31%). Four of 29 pts (14%) had a reduction in the size of the abnormal clone from 100% to a mean of 23% (range 8%–50%) as judged by conventional cytogenetics. The median time to achieve reduction was 6 months (range 2 to 9 months) with median duration of 10.5 months (range 6 to 24 months) during maintenance therapy with AZA C. CCR was observed in 5 of 29 pts (17%) and occurred within a mean of 4.2 months (range 3–6 months). Repeated cytogenetic studies showed a normal karyotype and the CCR lasted a mean of 5.2 months (range 3–9 months) during therapy. FISH studies showed 2–5% cells with −7/del(7q) during the CCR. Four of the 5 pts had HI and one pt had CR. These patients relapsed with the diagnostic −7/del(7q) clone without additional cytogenetic abnormalities. 3) Of the 12 pts who developed −7/del(7q) while on Aza C therapy, 4 pts had a normal karyotype at baseline and developed −7/del(7q) after a mean of 16 months (range 6–39 months) therapy. One had PR and 3 pts had a stable disease. The other 8 pts were cytogenetically abnormal at baseline and developed −7/del(7q) as a subclonal evolution during therapy in 4 pts (range 4 months to 5 years) and following disease progression in 4 pts. Six pts had HI and two had a stable disease. In conclusion, our longitudinal study allowed us to delineate 3 categories of AZA C response to abnormal chromosome 7 clone: 31% had cytogenetic response: 17% complete and 14% major and 69% had no change in the abnormal −7/del(7q) clone. In additional 5% (12/229) pts, −7/del(7q) emerged either as a new abnormal clone or as a subclonal evolution during therapy or at progression. AZA C-based therapy can either stabilize or reduce/eradicate the abnormal chromosome 7 clone as determined by both, cytogenetics and FISH.

Optimal re-expression of genes silenced through promoter methylation requires treatment with a DNA methyltransferase inhibitor (DNMTi) followed by the addition of a histone deacetylase inhibitor (HDACi). MS-275 is an orally bioavailable HDACi with a 50 hour half-life. A phase I study combining 5-azacitidine (5AC; 30, 40, or 50 mg/m2/dose) and MS-275 (2, 4, 6, 8 mg/m2/dose) was conducted in patients with MDS, CMMoL, and AML. Subcutaneous 5AC was self-administered daily for 10 days as per the most successful dose schedule of a prior study with 5AC plus sodium phenylbutyrate. MS-275 was administered on days 3 and 10 of each 28 day treatment cycle, following the in vitro paradigm requiring initial exposure to the DNMTi alone for optimal reversal of epigenetic transcriptional silencing. 31 patients were treated: 13 MDS, 4 CMMoL (8 IPSS Int-1, 7 Int-2, 2 proliferative CMMoL), 14 AML (7 AML with trilineage dysplasia [AML-TLD], 4 relapsed, 3 primary-refractory). Median age was 63 years (35 – 84). 5 patients received < 4 cycles and are not considered evaluable for response (declining performance status- 3, sepsis and death - 1, recurrent dose limiting toxicity [DLT]-1). DLT occurred in four patients treated with MS-275 8 mg/m2/dose (5AC 40 and 50 mg/m2/dose): laryngeal edema (2), delayed neutrophil recovery > 21 days (1), asthenia (1). No DLT occurred at any combination with lower doses of MS-275; however, grade 2 fatigue was common at MS-275 6 mg/m2/dose and was considered excessive for chronic administration. 12/27 evaluable patients responded, including 2 CR, 4 PR, 6 bilineage hematologic improvement (2000 IWG criteria). Responses occurred at all dose combinations, in patients with MDS (7), CMMoL (1), AML-TLD (3), and relapsed AML (1). Combining MDS and AML-TLD, responses developed in 10/20. The median number of cycles administered to date to responding patients is 11 (6 – 22). Median time to first objective hematologic response was 2 cycles (1 – 5); median time to best hematologic response was 4 cycles (2 – 9). 20 patients had clonal cytogenetic abnormalities including 6 clinical responders: all 6 had a minimum decrease in abnormal metaphases of 50%, with 4 cytogenetic CR. Median time to best cytogenetic response was four cycles (4 – 6). Following 3 days of 5AC alone, H3 or H4 acetylation (PBMC) was increased in 10/30 patients. Overall, H3 acetylation increased in 25/29 patients (median 2.5 fold, range 1.5 - >1000-fold); H4 acetylation increased in 28/29 patients (median 4-fold, range 1.5 −154-fold). The DNA damage-associated H2AXγ was induced in 7/29 patients after 3 days of 5AC (PBMC) and 20/29 patients after the administration of MS-275. Median fold-increase in H2AXγ-expression was 5.3 (range 1.5 - >1000). The 5AC/MS-275 combination is clinically tolerable and leads to substantive cytogenetic remissions. The relative contribution of the HDACi is under examination in a current Intergroup study randomizing patients to the 5AC/MS-275 combination versus the comparable dose schedule of 5AC alone.

Optimal re-expression of genes silenced through promoter methylation requires treatment with a DNA methyltransferase inhibitor (DNMTi) followed by the addition of a histone deacetylase inhibitor (HDACi). MS-275 is an orally bioavailable HDACi with a 50 hour half-life. A phase I study combining 5-azacitidine (5AC; 30, 40, or 50 mg/m2/dose) and MS-275 (2, 4, 6, 8 mg/m2/dose) was conducted in patients with MDS, CMMoL, and AML. Subcutaneous 5AC was self-administered daily for 10 days as per the most successful dose schedule of a prior study with 5AC plus sodium phenylbutyrate. MS-275 was administered on days 3 and 10 of each 28 day treatment cycle, following the in vitro paradigm requiring initial exposure to the DNMTi alone for optimal reversal of epigenetic transcriptional silencing. 31 patients were treated: 13 MDS, 4 CMMoL (8 IPSS Int-1, 7 Int-2, 2 proliferative CMMoL), 14 AML (7 AML with trilineage dysplasia [AML-TLD], 4 relapsed, 3 primary-refractory). Median age was 63 years (35 – 84). 5 patients received < 4 cycles and are not considered evaluable for response (declining performance status- 3, sepsis and death - 1, recurrent dose limiting toxicity [DLT]-1). DLT occurred in four patients treated with MS-275 8 mg/m2/dose (5AC 40 and 50 mg/m2/dose): laryngeal edema (2), delayed neutrophil recovery > 21 days (1), asthenia (1). No DLT occurred at any combination with lower doses of MS-275; however, grade 2 fatigue was common at MS-275 6 mg/m2/dose and was considered excessive for chronic administration. 12/27 evaluable patients responded, including 2 CR, 4 PR, 6 bilineage hematologic improvement (2000 IWG criteria). Responses occurred at all dose combinations, in patients with MDS (7), CMMoL (1), AML-TLD (3), and relapsed AML (1). Combining MDS and AML-TLD, responses developed in 10/20. The median number of cycles administered to date to responding patients is 11 (6 – 22). Median time to first objective hematologic response was 2 cycles (1 – 5); median time to best hematologic response was 4 cycles (2 – 9). 20 patients had clonal cytogenetic abnormalities including 6 clinical responders: all 6 had a minimum decrease in abnormal metaphases of 50%, with 4 cytogenetic CR. Median time to best cytogenetic response was four cycles (4 – 6). Following 3 days of 5AC alone, H3 or H4 acetylation (PBMC) was increased in 10/30 patients. Overall, H3 acetylation increased in 25/29 patients (median 2.5 fold, range 1.5 - >1000-fold); H4 acetylation increased in 28/29 patients (median 4-fold, range 1.5 −154-fold). The DNA damage-associated H2AXγ was induced in 7/29 patients after 3 days of 5AC (PBMC) and 20/29 patients after the administration of MS-275. Median fold-increase in H2AXγ-expression was 5.3 (range 1.5 - >1000). The 5AC/MS-275 combination is clinically tolerable and leads to substantive cytogenetic remissions. The relative contribution of the HDACi is under examination in a current Intergroup study randomizing patients to the 5AC/MS-275 combination versus the comparable dose schedule of 5AC alone.

MDS are heterogeneous hematopoietic disorders characterized by dyspoiesis and cytopenias. Most patients with high-risk MDS die within 1 year from infection or hemorrhage usually attributable to bone marrow failure or progression to AML. In a phase 3, open-label, multicenter, randomized study comparing azacitidine (Vidaza®) plus supportive care (N=99) versus supportive care alone (N=91) in patients with MDS (JCO2002;20:2429), treatment with azacitidine was associated with worsening of pre-existing cytopenias in up to 78% (115/147) of patients with pre-existing cytopenias. The median time to first occurrence of any worsening cytopenia was 8 days (interquartile range: 6 to 15 days). All patients were treated with the same initial dose of azacitidine (75 mg/m2/day); no dose adjustments were made for pre-existing cytopenias despite the severity. We examined rates of infection and bleeding associated with azacitidine therapy in comparison with the rates seen with the underlying disease (supportive care). Our results are based on re-collected data from patients described in the published report; our analyses were conducted separately and independently. Total exposure for azacitidine was the cumulative time from the first dose to the end of study (30 days after last dose), and for supportive care was the cumulative time from randomization to withdrawal from study or day prior to crossover. The azacitidine group included all patients exposed to azacitidine (N=150), which included patients randomized to azacitidine (N=99) and patients after crossing over from supportive care (N=51). Similar to the rate of infections previously reported in patients with MDS (AJM1991; 90:338), infections occurred in patients in the supportive care group at nearly one per patient-year. However, infections occurred at an overall lower rate (patient-years) in the azacitidine group (0.64) compared with the supportive care group (0.95). (Table). Similarly, the overall rate (patient-years) of bleeding events was comparable between azacitidine (0.56) and supportive care (0.60). In particular, bleeding events in the gastrointestinal system occurred at a similar rate between the azacitidine group (0.26) and the supportive care group (0.25). This was also true for bleeding events in the central nervous system (azacitidine: 0.01, supportive care: 0.02). The rates of infection and bleeding were 3 to 4 times higher during cycles 1 and 2 in both treatment groups, reflecting the severity of the underlying disease at study entry. In summary, despite the potential to exacerbate pre-existing cytopenias early in therapy, treatment with azacitidine did not increase the rate of infection and bleeding above the rate due to the underlying disease. Infection Rates (Patient-Years of Exposure) in Protocol 9221

MDS are heterogeneous hematopoietic disorders characterized by dyspoiesis and cytopenias. Most patients with high-risk MDS die within 1 year from infection or hemorrhage usually attributable to bone marrow failure or progression to AML. In a phase 3, open-label, multicenter, randomized study comparing azacitidine (Vidaza®) plus supportive care (N=99) versus supportive care alone (N=91) in patients with MDS (JCO 2002; 20:2429), treatment with azacitidine was associated with worsening of pre-existing cytopenias in up to 78% (115/147) of patients with pre-existing cytopenias. The median time to first occurrence of any worsening cytopenia was 8 days (interquartile range: 6 to 15 days). All patients were treated with the same initial dose of azacitidine (75 mg/m2/day); no dose adjustments were made for pre-existing cytopenias despite the severity. We examined rates of infection and bleeding associated with azacitidine therapy in comparison with the rates seen with the underlying disease (supportive care). Our results are based on re-collected data from patients described in the published report; our analyses were conducted separately and independently. Total exposure for azacitidine was the cumulative time from the first dose to the end of study (30 days after last dose), and for supportive care was the cumulative time from randomization to withdrawal from study or day prior to crossover. The azacitidine group included all patients exposed to azacitidine (N=150), which included patients randomized to azacitidine (N=99) and patients after crossing over from supportive care (N=51). Similar to the rate of infections previously reported in patients with MDS (AJM 1991; 90:338), infections occurred in patients in the supportive care group at nearly one per patient-year. However, infections occurred at an overall lower rate (patient-years) in the azacitidine group (0.64) compared with the supportive care group (0.95). (Table). Similarly, the overall rate (patient-years) of bleeding events was comparable between azacitidine (0.56) and supportive care (0.60). In particular, bleeding events in the gastrointestinal system occurred at a similar rate between the azacitidine group (0.26) and the supportive care group (0.25). This was also true for bleeding events in the central nervous system (azacitidine: 0.01, supportive care: 0.02). The rates of infection and bleeding were 3 to 4 times higher during cycles 1 and 2 in both treatment groups, reflecting the severity of the underlying disease at study entry. In summary, despite the potential to exacerbate pre-existing cytopenias early in therapy, treatment with azacitidine did not increase the rate of infection and bleeding above the rate due to the underlying disease.