Background: Myelodysplastic syndrome (MDS) is a clinically heterogenous disease of hematopoietic ... more 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. The paradigm of MDS therapy is now shifting to combinatorial drug treatment to overcome single-agent HMA resistance in higher-risk MDS patients. Rigosertib (RIGO), a Ras mimetic which had been shown to interfere with the Ras-Raf binding domain, has limited single-agent activity (ORR 15%) and failed to provide a survival benefit compared to standard of care in MDS patients failing an HMA. RIGO combined with AZA produced an ORR of 90% in HMA naïve patients and 54% in patients who failed HMA (Navada SC, et al. EHA Library 2019). This represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The mechanism is still unclear. Method: We therefore investigated the pathways that are perturbed by AZA and RIGO monotherapy and in combination (RIGO-AZA). We used the MDS-L cell line as a model to limit the heterogeneity observed in MDS patients. The cells were treated with RIGO, AZA, and RIGO-AZA for 48 hrs and further analyzed by qPCR and western blot. Result: We found an increase in H3K9ac protein expression with RIGO and RIGO-AZA; AZA and RIGO alone each had similar effects on H3K4me3, however, its expression was markedly upregulated with RIGO-AZA (Figure A). Effects on H3K36me3 were comparable in all treated cells. We observed marked effects on the repression marks H3K9me3 and H3K27me3 by RIGO and RIGO-AZA combination (Figure A). Furthermore, we studied the expression of bacterial sensing TLRs (1, 2 and 6), viral sensing endosomal TLRs (3 and 9), and cytosolic viral particle sensing receptors like Retinoic acid inducible gene (RIG)-I, Melanoma differentiation-associated protein 5 (MDA5) and Stimulator of interferon genes (STING); their intermediate adaptor molecules Myeloid differentiation factor 88 (MYD88) (for all TLRs except TLR3), mitochondrial antiviral signaling (MAVS) gene (for RIG-I and MDA5); and interferon regulatory factor (IRF)-3 and -7 by qPCR. We observed that AZA, RIGO, and RIGO-AZA significantly inhibit the expression of TLR1, 2 and 6 (Figure B). However, TLR-3, 9, RIG-I, MDA5, STING, MAVS, MYD88, and IRF-3, 7 were significantly inhibited by RIGO and RIGO-AZA (Figure C-E). Conclusion: RIGO has effects on innate immune signaling and histone modification of both activator and repressor marks. Further studies are underway to determine the correlation of the histone modification and innate immune signaling changes, and if these mechanisms contribute to the improvement in hematopoiesis in MDS patients. Figure 1 Figure 1. Disclosures Navada: Janssen Pharmaceuticals, Inc.: Current Employment. Reddy: Onconova Therapeutics, Inc.: Current equity holder in publicly-traded company.
Myelodysplastic syndromes (MDS) represent a collection of heterogeneous malignant bone marrow ste... more Myelodysplastic syndromes (MDS) represent a collection of heterogeneous malignant bone marrow stem cell disorders that result in the production of dysplastic and ineffective blood cells. The disease is marked by gradually worsening cytopenias and a variable risk for the eventual transformation to acute myelogenous leukemia (AML). The risk of developing MDS increases with age, and disease onset before 50 years is unusual. Several morphologic subtypes of MDS have been identified. Each of these subtypes has specific prognostic and morphologic and/or cytogenetic features which make it unique. The International Prognostic Scoring System (IPSS) was developed to aid in determining the prognosis of patients with MDS; this system categorizes patients into four risk groups for both overall survival and transformation to AML: low, intermediate-1, intermediate-2, and high. The management of MDS is based on the goal of controlling cytopenia-related symptoms, improving survival, improving quality of life, and decreasing risk of progression to AML. Treatment strategies include supportive care, iron chelation, treatment with hematopoietic growth factors,immunosuppressive therapies including lenalidomide, antithymocyte globulin, chemotherapy (eg, azacitidine, decitabine, low-dose Ara-C, 7+3 chemotherapy), and stem cell transplantation. However, selecting the appropriate therapy for each individual patient is critical to optimize clinical benefit. This monograph discusses treatment selection for the MDS patient,including a discussion of the overall survival and maintenance of MDS patients, how an appropriate therapy should be chosen in the community setting, and how MDS classification and risk stratification impacts treatment decisions.
Overview The existence of a hematopoietic disorder characterized by anemia and dyspoiesis precedi... more Overview The existence of a hematopoietic disorder characterized by anemia and dyspoiesis preceding the onset of acute myelocytic leukemia (AML) has been recognized since the early part of the twentieth century. Initially designated as preleukemia, the syndrome was ill defined and could only be established with certainty retrospectively. Moreover, the terminology itself conveyed an unwarranted confidence in predicting the outcome that often belied the facts. The more accurately descriptive and appropriate designation as a myelodysplastic syndrome (MDS) was adopted in 1976 by the French–American–British (FAB) study group. The FAB classification permitted the prospective identification of patients within this heterogeneous clonal disorder. MDS, derived from a multipotent hematopoietic stem cell, is characterized clinically by a hyperproliferative bone marrow, reflective of ineffective hematopoiesis, and is accompanied by one or more peripheral blood cytopenias. Bone marrow failure results, leading to death from bleeding and infection in the majority, while transformation to acute leukemia occurs in up to 40% of patients. The evolution of the disease proceeds in accordance with the multistep pathogenesis theory of carcinogenesis and can thus serve as an important model in furthering our understanding of the processes involved in neoplastic transformation. This constellation of findings raises the question of whether MDS represents a frank neoplastic state or is merely a preneoplastic condition in transition. The syndrome appears to represent a spectrum where the initial lesion in the genome, though clinically undetectable, subsequently evolves, with the acquisition of additional genetic and epigenetic lesions, to a state of frank neoplasia. The designation of this disorder as an MDS, rather than preleukemia, permits its distinction from other abnormalities that are known to be associated with the development of acute leukemia. These latter include the classic myeloproliferative syndromes (polycythemia vera, chronic myelocytic leukemia, agnogenic myeloid metaplasia, essential thrombocythemia), aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH), as well as Fanconi, Bloom, and Down syndromes. These particular “preleukemic states” are beyond the scope of this chapter. MDS can be further divided into primary and secondary syndromes. The former arise de novo and are of indeterminate etiology, while the latter are induced by identifiable environmental, occupational, or iatrogenic causes. Estimates of the incidence of MDS range from a frequency equal to that of AML or approximately 14,000 new cases per year in the United States to almost twice that of AML. The SEER database now tracks the disease; thus more accurate data will be available in the future. An estimate based on medical insurance claims indicated a total of 107,000 cases in the observation period from 2009 through 2011. The consensus is that the incidence is increasing owing to a number of factors, including greater awareness, greater diagnostic precision, and the aging of the population. Keywords: myelodysplastic syndrome; epigenetics; treatment; hypomethylating agents; azacitidine; decitabine; lenalidomide
The two most frequent cytogenetic abnormalities in pts with MDS involve rearrangements of chromos... more 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.
Valproic acid (VPA), acetylates histones, induces apoptosis and synergizes with All Trans Retinoi... more Valproic acid (VPA), acetylates histones, induces apoptosis and synergizes with All Trans Retinoic Acid (ATRA), in promoting G1 arrest and leukemic cell differentiation in vitro. Based on these observations we designed a Phase I/II pilot study of VPA alone and in combination with ATRA in patients with relapsed, refractory or poor risk acute myeloid leukemia (AML). VPA was initiated at 15mg/kg/day (day 1 IV followed by daily po) and escalated weekly by 10mg/kg (max 60 mg/kg/day) targeted to reach a serum concentration of 80–120mg/ml. For the combination, ATRA was to be added initially at 25mg/M2/day (first 3 patients) and titrated, in the absence of toxicity, to 45mg/M2/day, in two divided doses, following a minimum of 7 days of VPA therapy alone. Treatment was planned to continue for 45 days following achievement of a VPA level of >80mg/ml on two separate, consecutive measurements. To date we have treated 8 patients (7 tAML/MDS, 1 de novo AML) with VPA alone. Two of 8 additional patients to be treated with VPA + ATRA (1 tAML/ET; 1 tAML/MDS), are too early in their treatment upon which to report. Of the VPA alone treated patients, Median age was 63 (range 52–78). No Prior therapy: 3/8 pts; 5F:3M; ECOG PS 0 (1), 1 (5), 2 (2);Cytogenetic abnormalities: normal (1); t(3;18) (1); complex karyotype (6), including trisomy 8 (3), abnormalities of 11 (2) or monosomy 7 (2). Baseline bone marrow blast percentage > 50%; (8); median bone marrow cellularity 84% (range 35–90). Treatment: Median duration of treatment 45 days (range 19–99). Therapy discontinued before 45 days; withdrawl (2); death at home on study (day 33 of treatment), (1); reversible grade 3 toxicity(2). VPA levels: Serum concentrations of VPA of > 80mg/ml was achieved in all patients; Hematologic effects: Changes in CBC: increase in WBC, ANC and blast count (BLC)(3), followed by decrease in WBC and BLC (2); increase in WBC/ANC &decrease in BLC (1); decrease in WBC, ANC and BLC (3); &initial decrease in BLC &increase in ANC (1). Marrow: decrease in marrow cellularity (2)( 90% to 30%); loss of detectable trisomy 8 population (1); &decrease in bone marrow blasts (1) (64% to 5%) was observed. One patient reverted back to MDS, lasting 5 months. Side Effects: somnolence (grade 2–4) was observed in 4/8 patients, and was the major cause of withholding treatment. We also observed a constellation of symptoms (weight gain, fever, elevated WBC, shortness of breath, elevated LDH, bone pain) and chest x-ray findings consistent with &reminiscent of APL syndrome, within seven days of achieving the targeted serum VPA levels (> 80mg/ml), in 2 pts. Decadron was started two days (1) and immediately (1) following the onset of symptoms: signs and symptoms abated quickly &resolved subsequently within three days. In retrospect, one additional patient had comparable symptoms, for which VPA was stopped, following admission to an outside hospital for fever, pulmonary infiltrates and altered mental status. Correlative studies: serial assessment of bulk histone acetylation (H3, H4) was performed in all 8 patients. 7/8 patients showed evidence of histone hyperacetylation in mononuclear cells from peripheral blood, during treatment with VPA. Additional patients are being treated with VPA + ATRA. Analysis of these studies will be forthcoming.
Epigenetic modifications tightly regulate the gene expression and cellular function of haematopoi... more Epigenetic modifications tightly regulate the gene expression and cellular function of haematopoietic stem cells. Histone deacetylase inhibitors (HDACIs) alter the gene expression profile of cord blood (CB) CD34(+) cells by controlling the genes involved in chromatin modification, thereby influencing the self-renewal, maintenance and expansion of haematopoietic stem and progenitor cells (HSPCs). The class I and II HDACIs, valproic acid and scriptaid, were utilized to expand CB-CD34(+) cells ex vivo. The gene profiling was performed on HSPC using Illumina microarray, GeneGO MetaCore(™) and Ingenuity pathway analyses. The molecular analyses were performed using Q-PCR and Western blotting. Each HDACI treatment of CB-CD34(+) cells created unique epigenetic and molecular signatures that governed chromatin modification required for cellular and functional behaviour of stem cells. GeneGO MetaCore(™) and Ingenuity pathway analyses established the molecular understanding of epigenetically regulated HSPCs in the presence of scriptaid and VPA that revealed different network(s) of potential regulators during erythropoiesis. VPA induced transcriptional activation of the glucocorticoid receptor (GCR) and an increase in the intracellular signalling of signal transducers and activators of transcription (STAT) required during stress erythropoiesis. Canonical Wnt signalling and many epigenetically regulated chromatin remodellers were significantly influenced so as to establish maintenance and regulation of HSPC. Treatment with Individual HDACIs has demonstrated significantly unique epigenetic and molecular signatures of CB-HSPC. This study identifies potential key regulators of HSPC and gives insights into the clinically important processes of HSPC expansion and haematopoietic lineage development for transplantation purposes.
Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic... more Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.
The implication of DNA hypermethylation in the pathogenesis of myelodysplastic syndromes (MDS) pr... more The implication of DNA hypermethylation in the pathogenesis of myelodysplastic syndromes (MDS) provides a rationale for using hypomethylating agents such as azacitidine. Growing evidence suggests that azacitidine may reverse epigenetic gene silencing at specific genomic targets. AZA-001 established azacitidine as the first agent to provide a significant overall survival benefit in MDS patients. These data confirmed that azacitidine has a progressively cumulative effect on the MDS clone and support the value of maintenance therapy. Prolonged survival was independent of achieving complete response. Azacitidine in combination with histone deacetylase inhibitors might offer better efficacy by modulating the methylation and acetylation states of silenced genes.
The International Prognostic Scoring System (IPSS) provides relatively homogeneous prognoses for ... more The International Prognostic Scoring System (IPSS) provides relatively homogeneous prognoses for untreated patients with MDS in terms of survival and transformation to AML. In many cases, cytogenetic results are not available and prevent use of the IPSS. Our objective was to create and validate a dynamic prognostic model that would allow identification of high-risk MDS patients when cytogenetics are unavailable. We followed the methodological approach used by Greenberg et al (Blood1997;89:2079) and used our new model to re-analyze the FAB-based CALGB data (JCO2002;20:2429). The variables used in the prognostic model were identified as statistically significant independent variables by Greenberg and included % marrow blasts, number of cytopenias, the multivariate risk factors (age and gender), the univariate risk factor (FAB classification), and time since diagnosis because CALGB data were collected at randomization and IPSS at diagnosis. Both blasts and FAB were included in our model since there is considerable outcome heterogeneity within FAB subtypes. Predicted survival for each of the 191 CALGB patients was determined from a stratified proportional hazards regression model fitted to patients’ survival times. The 191 patients were stratified using their randomized treatment assignment (azacitidine or supportive care). All patients with predicted survival ≤ 1.2 years (median survival of IPSS INT-2 risk group) comprised a high-risk subgroup; those with predicted survival > 1.2 years formed the low-risk subgroup. To assess its predictive accuracy, our model was validated by demonstrating significant segregation of risk groups in an independent dataset of 2,318 untreated primary MDS patients from the MDS Registry, Düsseldorf. In all, 964 (42%) registry cases and 72 (38%) CALGB cases fit into the high-risk subgroup. Log-rank analyses of overall survival and time to AML transformation demonstrated clear separation of the low- and high-risk subgroups (both p-values < 0.0001). Figure 1: Validation of Pregnostic Model with MDS Registry, Düsselderf, Overall Survival Figure 1:. Validation of Pregnostic Model with MDS Registry, Düsselderf, Overall Survival There was fair agreement between the low and high subgroups from our model with the low (low+INT-1) and high (INT-2+high) subgroups from the IPSS, Cohen’s Kappa = 0.30 (95% CI: 0.21 to 0.39) in 547 patients from the MDS Registry, Düsseldorf. A sensitivity analysis of the CALGB efficacy results was conducted by varying the ≤ 1.2 years survival criterion from our model. High-risk subgroups, defined by using predicted survival of 1.0 to 2.8 years in increments of 0.1 years, were investigated. In general, the sensitivity analyses revealed that clinically meaningful benefit was statistically demonstrated in the smaller subgroups but lost significance as the subgroups approached the full data set. The smaller subgroups were more homogeneous in predicted survival. In conclusion, our model is a valid prognostic model that bridges reasonably well to the IPSS high-risk group and identified a high-risk homogeneous subgroup of CALGB patients to further investigate the effects of azacitidine.
CXCL12 (stromal cell-derived factor, SDF-1), is an 8 kDa peptide chemokine. The interaction betwe... more CXCL12 (stromal cell-derived factor, SDF-1), is an 8 kDa peptide chemokine. The interaction between CXCL12 and its receptor, CXCR4, plays a pivotal role in the trafficking of hematopoietic stem cells between bone marrow and peripheral blood. The CXCL12/CXCR4 axis may play a role in the pathogenesis of myeloid neoplasms. We developed a technique for the determination of intact (full length) CXCL12 and its protease(s)-induced truncation products in plasma from patients with myeloproliferative neoplasms (Cancer Res., 70, 3402, 2010). In the present work, we are extending our observations to the myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). CXCL12 concentrations in normal subjects were 16.6 ± 9.4 ng/mL (n=10). In primary myelofibrosis (PMF), the concentrations were 5.8 ± 4.0 ng/mL (n=15), and in polycythemia vera (PV) 7.9 ± 3.6 ng/mL (n=21). Now we report that in MDS/AML, the concentrations are 2.2 ± 2.1 ng/mL (n=8). In normal subjects, truncation products, due to proteolysis, were not detectable (≥ 1.0 ng/mL). The loss of 2, 3, 4, and 5 amino acids (aa) from CXCL12 were confirmed by molecular masses measured using electrospray ionization mass spectrometry. Quantification was completed with synthetic standards. In MDS/AML patients the concentration of the truncation product corresponding to −2 aa (KP removed),-3 aa (KPV removed), −4 aa (KPVS removed) and −5 aa (KPVSL removed) were 2.7 ± 3.2 ng/mL, 2.4 ± 2.1 ng/mL, 3.8 ± 3.0 ng/mL, and 2.5 ± 2.4 ng/mL, respectively. The total concentration of all truncation products was 11.3 ± 5.8 ng/mL. For comparison, the concentration of total truncation products was 28.7 ± 19.9 for PMF, and 31.1 ± 7.8 for PV. Patients with these myeloid malignancies have lower CXCL12 concentrations compared to normal controls and high concentrations of proteolytic truncation products which are absent in normal plasma. Of the 8 patients with MDS (n=3) and AML (n=5), treated with continuous infusion of ON 01910.Na (rigosertib), 650 - 1,700 mg/m2, in a Phase I dose escalation study, 4 patients attained a partial or complete bone marrow remission according to International Working Group criteria. The concentration of intact CXCL12 rose at 72 h in responding patients, whereas those who failed to respond had a decrease. These data suggest that monitoring intact CXCL12 and its truncation products may provide a marker of response to treatment with ON 01910.Na, as well as insight into the role of the CXCL12/CXCR4 axis in the pathobiology of these bone marrow diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2675. doi:1538-7445.AM2012-2675
A patient with myelodysplastic syndrome (MDS) and a 47,XY,+21 karyotype at diagnosis, was documen... more A patient with myelodysplastic syndrome (MDS) and a 47,XY,+21 karyotype at diagnosis, was documented to have a clonal chromosome 21 rearrangement, i(21q), four months before transformation to acute biphenotypic leukemia. For 4 months after transformation, isochromosome 21 persisted while the patient was receiving treatment with zidovudine. Vitamin D3 was added to zidovudine for an additional month, during which time the trisomy 21 clone reappeared as the predominant cell population. The unique aspects of this patient are the atypical evolution of chromosome 21, the transformation to biphenotypic leukemia, and the occurrence of i(21q) associated with biphenotypic leukemia evolving from an MDS.
Abstract 3998 We analyzed bone marrow (BM) response and overall survival (OS) in 48 patients (pts... more Abstract 3998 We analyzed bone marrow (BM) response and overall survival (OS) in 48 patients (pts) with a myelodysplastic syndrome (MDS) and WHO/FAB subtypes of refractory anemia with excess blasts (RAEB) -1,- 2 or -t and acute myeloid leukemia (AML), enrolled in 4 independent ongoing clinical trials of the novel small molecule ON 01910.Na. Pts received ON 01910.Na administered as a continuous intravenous infusion (CIV) from 2 to 6 days weekly or every other week with BM response initially assessed per protocol by week 4 or 8. A BM complete response (CR) (>50% decrease from baseline BM blast and decrease below 5% for at least 4 weeks, per MDS IWG 2006 criteria) or an initial 50% decrease of BM blasts by week 4 to 8 was documented in 19/48 (40%) treated pts and was associated with a significant increase in overall survival (OS) (p = 0.0001) by the method of Kaplan-Meier (Figure 1 and Table 1). This relationship was still significant when excluding AML patients (p = 0.008). Six pts (3 RAEB-1, 3 RAEB-2) had complete BM response. Five of these six pts previously failed to respond or relapsed after azacitidine/decitabine and five out of six are alive (17 to 113 weeks follow-up; one death at 68 weeks). Eleven pts (5 AML, 3 RAEB-t, 2 RAEB-2 and 1 RAEB-1) could not be assessed at 4–8 weeks with follow-up BM evaluation; their median OS was 7.5 weeks; 9 of these 11 pts previously failed to respond or relapsed after azacitidine/decitabine. Among 10 pts with trisomy 8 cytogenetics (4 had an initial BM response), median survival was 25 weeks. FAB/WHO classification of all 48 pts was also significantly correlated with survival (Table 2, p=0.003). Overall, ON 01910.Na infusions were well tolerated. Eight pts had hematological improvements at various time points after starting therapy. Median OS was 33 weeks in the subset of 29 RAEB-1,-2,-t pts refractory or relapsing after azacitidine/decitabine, and a significant association (p = 0.056) between BM response and OS was also found in these pts (Table 1). The median survival of MDS pts who had failed to respond to prior treatment with decitabine has been reported to be approximately 17 weeks (Jabbour et al, Cancer 2010, in press). These results suggest a strong correlation between BM blast response and OS and the predictive value of BM response to ON 01910.Na for estimating overall survival of higher risk MDS or AML pts. Table 1. Overall Survival of Pts Treated with ON 01910.Na by BM CR and Initial (4-8 Weeks) Blast Response 4–8 Week BM Blast Reduction (%) BM CR > 50% Initial Response < 50% Initial Response Not Assessed P value Logrank test Figure Legend Red Blue Black Green RAEB-1, RAEB-2, RAEB-t, & AML pts, N = 48 6 13 18 11 Median Survival (weeks) Not reached 36 28 7.5 P = 0.0001 RAEB-1, RAEB-2, RAEB-t pts relapsed/refractory to azacitidine/decitabine, N = 29 5 6 12 6 Median Survival (weeks) 68 Not reached 33 3.5 P = 0.056 Table 2. Overall Survival by FAB/WHO Classification of 48 RAEB and AML Pts Treated with ON 01910.Na FAB/WHO Classification RAEB-1 RAEB-2 RAEB-t AML (>30% BM blasts) P value Logrank test N pts 11 18 10 9 Median Survival (weeks) Not reached 28 20 11 P = 0.003 Disclosures: Silverman: Onconova Therapeutics Inc: Research Funding, Research support of Clinical Trial. Raza:Onconova Therapeutics Inc: Research Funding, Research support for clinical trial. Greenberg:Onconova funding of clinical trial: Research Funding, Research support from Onconova for performance of this trial. Wilhelm:Onconova Therapeutics Inc.: Employment, Equity Ownership.
Background: The hypomethylating agent azacitidine (azaC) which can reverse epigenetic silencing, ... more 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
13137 Background: The benzyl styryl sulfone analog ON 01910.Na is a novel anticancer agent that i... more 13137 Background: The benzyl styryl sulfone analog ON 01910.Na is a novel anticancer agent that inhibits mitotic progression and induces apoptosis; it has activity against most human cancer cells in vitro and against a broad spectrum of human xenografts in mice. Cell kill effects are exposure time-dependent in vitro. After 3d exposure 200 nM ON 01910.Na killed > 90% of Daudi lymphoma cells, whereas 40-fold higher drug concentration killed only 50% of cells after 24h exposure. Dogs that received CI of 325 mg/kg/d ×3 failed to reach MTD. Methods: Starting dose 50 mg/m2/d as a 72 hr CI was 1/10th MTD of rats’ daily dose given × 28 d. Treatment cycles were repeated every 2 wks until progressive disease, intolerable toxicity, or withdrawal of consent. Dose was escalated by Fibonacci progression in single pts until grade 2 toxicity when cohorts of 3 are to be studied. Volunteers may be retreated at a higher dose if tolerated by a preceding naïve subject. Results: One man and four women (61–77 yrs) have been studied in 21 cycles in 5 mos as of 1/10/06. Doses of 50, 100, 150 and 250 mg/m2/d × 3 have been given for 1 to 10 cycles. Grade 1 granulocytopenia (2/4 pts with carcinoma) indicates biologic activity. Grade 1 fatigue (3/5) was less than in prior chemotherapy regimens. No grade 2 toxicity has yet occurred. Cumulative toxicity has not been seen. Two pts have had stable disease 6+ and 22+ wks. After deproteinization with acetonitrile, plasma or serum samples were measured by mass spectrometry. At 100 mg/m2/d steady state levels were 730 nM after the 1st cycle and 1190 nM after the 4th cycle. Drug levels were maximal at 3 to 6 h despite CI suggesting induction of a metabolizing pathway. Levels fell precipitously at the end of infusion on the 1st cycle but a low level persisted in one pt for 48h after the major drop in the 4th cycle, suggesting drug accumulation. Conclusion: Levels effective in vitro have been obtained in vivo by CI without limiting toxicity. Hints of activity already seen suggest that this compound has clinical promise. This phase I study continues. [Table: see text]
Background: Myelodysplastic syndrome (MDS) is a clinically heterogenous disease of hematopoietic ... more 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. The paradigm of MDS therapy is now shifting to combinatorial drug treatment to overcome single-agent HMA resistance in higher-risk MDS patients. Rigosertib (RIGO), a Ras mimetic which had been shown to interfere with the Ras-Raf binding domain, has limited single-agent activity (ORR 15%) and failed to provide a survival benefit compared to standard of care in MDS patients failing an HMA. RIGO combined with AZA produced an ORR of 90% in HMA naïve patients and 54% in patients who failed HMA (Navada SC, et al. EHA Library 2019). This represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The mechanism is still unclear. Method: We therefore investigated the pathways that are perturbed by AZA and RIGO monotherapy and in combination (RIGO-AZA). We used the MDS-L cell line as a model to limit the heterogeneity observed in MDS patients. The cells were treated with RIGO, AZA, and RIGO-AZA for 48 hrs and further analyzed by qPCR and western blot. Result: We found an increase in H3K9ac protein expression with RIGO and RIGO-AZA; AZA and RIGO alone each had similar effects on H3K4me3, however, its expression was markedly upregulated with RIGO-AZA (Figure A). Effects on H3K36me3 were comparable in all treated cells. We observed marked effects on the repression marks H3K9me3 and H3K27me3 by RIGO and RIGO-AZA combination (Figure A). Furthermore, we studied the expression of bacterial sensing TLRs (1, 2 and 6), viral sensing endosomal TLRs (3 and 9), and cytosolic viral particle sensing receptors like Retinoic acid inducible gene (RIG)-I, Melanoma differentiation-associated protein 5 (MDA5) and Stimulator of interferon genes (STING); their intermediate adaptor molecules Myeloid differentiation factor 88 (MYD88) (for all TLRs except TLR3), mitochondrial antiviral signaling (MAVS) gene (for RIG-I and MDA5); and interferon regulatory factor (IRF)-3 and -7 by qPCR. We observed that AZA, RIGO, and RIGO-AZA significantly inhibit the expression of TLR1, 2 and 6 (Figure B). However, TLR-3, 9, RIG-I, MDA5, STING, MAVS, MYD88, and IRF-3, 7 were significantly inhibited by RIGO and RIGO-AZA (Figure C-E). Conclusion: RIGO has effects on innate immune signaling and histone modification of both activator and repressor marks. Further studies are underway to determine the correlation of the histone modification and innate immune signaling changes, and if these mechanisms contribute to the improvement in hematopoiesis in MDS patients. Figure 1 Figure 1. Disclosures Navada: Janssen Pharmaceuticals, Inc.: Current Employment. Reddy: Onconova Therapeutics, Inc.: Current equity holder in publicly-traded company.
Myelodysplastic syndromes (MDS) represent a collection of heterogeneous malignant bone marrow ste... more Myelodysplastic syndromes (MDS) represent a collection of heterogeneous malignant bone marrow stem cell disorders that result in the production of dysplastic and ineffective blood cells. The disease is marked by gradually worsening cytopenias and a variable risk for the eventual transformation to acute myelogenous leukemia (AML). The risk of developing MDS increases with age, and disease onset before 50 years is unusual. Several morphologic subtypes of MDS have been identified. Each of these subtypes has specific prognostic and morphologic and/or cytogenetic features which make it unique. The International Prognostic Scoring System (IPSS) was developed to aid in determining the prognosis of patients with MDS; this system categorizes patients into four risk groups for both overall survival and transformation to AML: low, intermediate-1, intermediate-2, and high. The management of MDS is based on the goal of controlling cytopenia-related symptoms, improving survival, improving quality of life, and decreasing risk of progression to AML. Treatment strategies include supportive care, iron chelation, treatment with hematopoietic growth factors,immunosuppressive therapies including lenalidomide, antithymocyte globulin, chemotherapy (eg, azacitidine, decitabine, low-dose Ara-C, 7+3 chemotherapy), and stem cell transplantation. However, selecting the appropriate therapy for each individual patient is critical to optimize clinical benefit. This monograph discusses treatment selection for the MDS patient,including a discussion of the overall survival and maintenance of MDS patients, how an appropriate therapy should be chosen in the community setting, and how MDS classification and risk stratification impacts treatment decisions.
Overview The existence of a hematopoietic disorder characterized by anemia and dyspoiesis precedi... more Overview The existence of a hematopoietic disorder characterized by anemia and dyspoiesis preceding the onset of acute myelocytic leukemia (AML) has been recognized since the early part of the twentieth century. Initially designated as preleukemia, the syndrome was ill defined and could only be established with certainty retrospectively. Moreover, the terminology itself conveyed an unwarranted confidence in predicting the outcome that often belied the facts. The more accurately descriptive and appropriate designation as a myelodysplastic syndrome (MDS) was adopted in 1976 by the French–American–British (FAB) study group. The FAB classification permitted the prospective identification of patients within this heterogeneous clonal disorder. MDS, derived from a multipotent hematopoietic stem cell, is characterized clinically by a hyperproliferative bone marrow, reflective of ineffective hematopoiesis, and is accompanied by one or more peripheral blood cytopenias. Bone marrow failure results, leading to death from bleeding and infection in the majority, while transformation to acute leukemia occurs in up to 40% of patients. The evolution of the disease proceeds in accordance with the multistep pathogenesis theory of carcinogenesis and can thus serve as an important model in furthering our understanding of the processes involved in neoplastic transformation. This constellation of findings raises the question of whether MDS represents a frank neoplastic state or is merely a preneoplastic condition in transition. The syndrome appears to represent a spectrum where the initial lesion in the genome, though clinically undetectable, subsequently evolves, with the acquisition of additional genetic and epigenetic lesions, to a state of frank neoplasia. The designation of this disorder as an MDS, rather than preleukemia, permits its distinction from other abnormalities that are known to be associated with the development of acute leukemia. These latter include the classic myeloproliferative syndromes (polycythemia vera, chronic myelocytic leukemia, agnogenic myeloid metaplasia, essential thrombocythemia), aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH), as well as Fanconi, Bloom, and Down syndromes. These particular “preleukemic states” are beyond the scope of this chapter. MDS can be further divided into primary and secondary syndromes. The former arise de novo and are of indeterminate etiology, while the latter are induced by identifiable environmental, occupational, or iatrogenic causes. Estimates of the incidence of MDS range from a frequency equal to that of AML or approximately 14,000 new cases per year in the United States to almost twice that of AML. The SEER database now tracks the disease; thus more accurate data will be available in the future. An estimate based on medical insurance claims indicated a total of 107,000 cases in the observation period from 2009 through 2011. The consensus is that the incidence is increasing owing to a number of factors, including greater awareness, greater diagnostic precision, and the aging of the population. Keywords: myelodysplastic syndrome; epigenetics; treatment; hypomethylating agents; azacitidine; decitabine; lenalidomide
The two most frequent cytogenetic abnormalities in pts with MDS involve rearrangements of chromos... more 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.
Valproic acid (VPA), acetylates histones, induces apoptosis and synergizes with All Trans Retinoi... more Valproic acid (VPA), acetylates histones, induces apoptosis and synergizes with All Trans Retinoic Acid (ATRA), in promoting G1 arrest and leukemic cell differentiation in vitro. Based on these observations we designed a Phase I/II pilot study of VPA alone and in combination with ATRA in patients with relapsed, refractory or poor risk acute myeloid leukemia (AML). VPA was initiated at 15mg/kg/day (day 1 IV followed by daily po) and escalated weekly by 10mg/kg (max 60 mg/kg/day) targeted to reach a serum concentration of 80–120mg/ml. For the combination, ATRA was to be added initially at 25mg/M2/day (first 3 patients) and titrated, in the absence of toxicity, to 45mg/M2/day, in two divided doses, following a minimum of 7 days of VPA therapy alone. Treatment was planned to continue for 45 days following achievement of a VPA level of >80mg/ml on two separate, consecutive measurements. To date we have treated 8 patients (7 tAML/MDS, 1 de novo AML) with VPA alone. Two of 8 additional patients to be treated with VPA + ATRA (1 tAML/ET; 1 tAML/MDS), are too early in their treatment upon which to report. Of the VPA alone treated patients, Median age was 63 (range 52–78). No Prior therapy: 3/8 pts; 5F:3M; ECOG PS 0 (1), 1 (5), 2 (2);Cytogenetic abnormalities: normal (1); t(3;18) (1); complex karyotype (6), including trisomy 8 (3), abnormalities of 11 (2) or monosomy 7 (2). Baseline bone marrow blast percentage > 50%; (8); median bone marrow cellularity 84% (range 35–90). Treatment: Median duration of treatment 45 days (range 19–99). Therapy discontinued before 45 days; withdrawl (2); death at home on study (day 33 of treatment), (1); reversible grade 3 toxicity(2). VPA levels: Serum concentrations of VPA of > 80mg/ml was achieved in all patients; Hematologic effects: Changes in CBC: increase in WBC, ANC and blast count (BLC)(3), followed by decrease in WBC and BLC (2); increase in WBC/ANC &decrease in BLC (1); decrease in WBC, ANC and BLC (3); &initial decrease in BLC &increase in ANC (1). Marrow: decrease in marrow cellularity (2)( 90% to 30%); loss of detectable trisomy 8 population (1); &decrease in bone marrow blasts (1) (64% to 5%) was observed. One patient reverted back to MDS, lasting 5 months. Side Effects: somnolence (grade 2–4) was observed in 4/8 patients, and was the major cause of withholding treatment. We also observed a constellation of symptoms (weight gain, fever, elevated WBC, shortness of breath, elevated LDH, bone pain) and chest x-ray findings consistent with &reminiscent of APL syndrome, within seven days of achieving the targeted serum VPA levels (> 80mg/ml), in 2 pts. Decadron was started two days (1) and immediately (1) following the onset of symptoms: signs and symptoms abated quickly &resolved subsequently within three days. In retrospect, one additional patient had comparable symptoms, for which VPA was stopped, following admission to an outside hospital for fever, pulmonary infiltrates and altered mental status. Correlative studies: serial assessment of bulk histone acetylation (H3, H4) was performed in all 8 patients. 7/8 patients showed evidence of histone hyperacetylation in mononuclear cells from peripheral blood, during treatment with VPA. Additional patients are being treated with VPA + ATRA. Analysis of these studies will be forthcoming.
Epigenetic modifications tightly regulate the gene expression and cellular function of haematopoi... more Epigenetic modifications tightly regulate the gene expression and cellular function of haematopoietic stem cells. Histone deacetylase inhibitors (HDACIs) alter the gene expression profile of cord blood (CB) CD34(+) cells by controlling the genes involved in chromatin modification, thereby influencing the self-renewal, maintenance and expansion of haematopoietic stem and progenitor cells (HSPCs). The class I and II HDACIs, valproic acid and scriptaid, were utilized to expand CB-CD34(+) cells ex vivo. The gene profiling was performed on HSPC using Illumina microarray, GeneGO MetaCore(™) and Ingenuity pathway analyses. The molecular analyses were performed using Q-PCR and Western blotting. Each HDACI treatment of CB-CD34(+) cells created unique epigenetic and molecular signatures that governed chromatin modification required for cellular and functional behaviour of stem cells. GeneGO MetaCore(™) and Ingenuity pathway analyses established the molecular understanding of epigenetically regulated HSPCs in the presence of scriptaid and VPA that revealed different network(s) of potential regulators during erythropoiesis. VPA induced transcriptional activation of the glucocorticoid receptor (GCR) and an increase in the intracellular signalling of signal transducers and activators of transcription (STAT) required during stress erythropoiesis. Canonical Wnt signalling and many epigenetically regulated chromatin remodellers were significantly influenced so as to establish maintenance and regulation of HSPC. Treatment with Individual HDACIs has demonstrated significantly unique epigenetic and molecular signatures of CB-HSPC. This study identifies potential key regulators of HSPC and gives insights into the clinically important processes of HSPC expansion and haematopoietic lineage development for transplantation purposes.
Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic... more Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.
The implication of DNA hypermethylation in the pathogenesis of myelodysplastic syndromes (MDS) pr... more The implication of DNA hypermethylation in the pathogenesis of myelodysplastic syndromes (MDS) provides a rationale for using hypomethylating agents such as azacitidine. Growing evidence suggests that azacitidine may reverse epigenetic gene silencing at specific genomic targets. AZA-001 established azacitidine as the first agent to provide a significant overall survival benefit in MDS patients. These data confirmed that azacitidine has a progressively cumulative effect on the MDS clone and support the value of maintenance therapy. Prolonged survival was independent of achieving complete response. Azacitidine in combination with histone deacetylase inhibitors might offer better efficacy by modulating the methylation and acetylation states of silenced genes.
The International Prognostic Scoring System (IPSS) provides relatively homogeneous prognoses for ... more The International Prognostic Scoring System (IPSS) provides relatively homogeneous prognoses for untreated patients with MDS in terms of survival and transformation to AML. In many cases, cytogenetic results are not available and prevent use of the IPSS. Our objective was to create and validate a dynamic prognostic model that would allow identification of high-risk MDS patients when cytogenetics are unavailable. We followed the methodological approach used by Greenberg et al (Blood1997;89:2079) and used our new model to re-analyze the FAB-based CALGB data (JCO2002;20:2429). The variables used in the prognostic model were identified as statistically significant independent variables by Greenberg and included % marrow blasts, number of cytopenias, the multivariate risk factors (age and gender), the univariate risk factor (FAB classification), and time since diagnosis because CALGB data were collected at randomization and IPSS at diagnosis. Both blasts and FAB were included in our model since there is considerable outcome heterogeneity within FAB subtypes. Predicted survival for each of the 191 CALGB patients was determined from a stratified proportional hazards regression model fitted to patients’ survival times. The 191 patients were stratified using their randomized treatment assignment (azacitidine or supportive care). All patients with predicted survival ≤ 1.2 years (median survival of IPSS INT-2 risk group) comprised a high-risk subgroup; those with predicted survival > 1.2 years formed the low-risk subgroup. To assess its predictive accuracy, our model was validated by demonstrating significant segregation of risk groups in an independent dataset of 2,318 untreated primary MDS patients from the MDS Registry, Düsseldorf. In all, 964 (42%) registry cases and 72 (38%) CALGB cases fit into the high-risk subgroup. Log-rank analyses of overall survival and time to AML transformation demonstrated clear separation of the low- and high-risk subgroups (both p-values < 0.0001). Figure 1: Validation of Pregnostic Model with MDS Registry, Düsselderf, Overall Survival Figure 1:. Validation of Pregnostic Model with MDS Registry, Düsselderf, Overall Survival There was fair agreement between the low and high subgroups from our model with the low (low+INT-1) and high (INT-2+high) subgroups from the IPSS, Cohen’s Kappa = 0.30 (95% CI: 0.21 to 0.39) in 547 patients from the MDS Registry, Düsseldorf. A sensitivity analysis of the CALGB efficacy results was conducted by varying the ≤ 1.2 years survival criterion from our model. High-risk subgroups, defined by using predicted survival of 1.0 to 2.8 years in increments of 0.1 years, were investigated. In general, the sensitivity analyses revealed that clinically meaningful benefit was statistically demonstrated in the smaller subgroups but lost significance as the subgroups approached the full data set. The smaller subgroups were more homogeneous in predicted survival. In conclusion, our model is a valid prognostic model that bridges reasonably well to the IPSS high-risk group and identified a high-risk homogeneous subgroup of CALGB patients to further investigate the effects of azacitidine.
CXCL12 (stromal cell-derived factor, SDF-1), is an 8 kDa peptide chemokine. The interaction betwe... more CXCL12 (stromal cell-derived factor, SDF-1), is an 8 kDa peptide chemokine. The interaction between CXCL12 and its receptor, CXCR4, plays a pivotal role in the trafficking of hematopoietic stem cells between bone marrow and peripheral blood. The CXCL12/CXCR4 axis may play a role in the pathogenesis of myeloid neoplasms. We developed a technique for the determination of intact (full length) CXCL12 and its protease(s)-induced truncation products in plasma from patients with myeloproliferative neoplasms (Cancer Res., 70, 3402, 2010). In the present work, we are extending our observations to the myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). CXCL12 concentrations in normal subjects were 16.6 ± 9.4 ng/mL (n=10). In primary myelofibrosis (PMF), the concentrations were 5.8 ± 4.0 ng/mL (n=15), and in polycythemia vera (PV) 7.9 ± 3.6 ng/mL (n=21). Now we report that in MDS/AML, the concentrations are 2.2 ± 2.1 ng/mL (n=8). In normal subjects, truncation products, due to proteolysis, were not detectable (≥ 1.0 ng/mL). The loss of 2, 3, 4, and 5 amino acids (aa) from CXCL12 were confirmed by molecular masses measured using electrospray ionization mass spectrometry. Quantification was completed with synthetic standards. In MDS/AML patients the concentration of the truncation product corresponding to −2 aa (KP removed),-3 aa (KPV removed), −4 aa (KPVS removed) and −5 aa (KPVSL removed) were 2.7 ± 3.2 ng/mL, 2.4 ± 2.1 ng/mL, 3.8 ± 3.0 ng/mL, and 2.5 ± 2.4 ng/mL, respectively. The total concentration of all truncation products was 11.3 ± 5.8 ng/mL. For comparison, the concentration of total truncation products was 28.7 ± 19.9 for PMF, and 31.1 ± 7.8 for PV. Patients with these myeloid malignancies have lower CXCL12 concentrations compared to normal controls and high concentrations of proteolytic truncation products which are absent in normal plasma. Of the 8 patients with MDS (n=3) and AML (n=5), treated with continuous infusion of ON 01910.Na (rigosertib), 650 - 1,700 mg/m2, in a Phase I dose escalation study, 4 patients attained a partial or complete bone marrow remission according to International Working Group criteria. The concentration of intact CXCL12 rose at 72 h in responding patients, whereas those who failed to respond had a decrease. These data suggest that monitoring intact CXCL12 and its truncation products may provide a marker of response to treatment with ON 01910.Na, as well as insight into the role of the CXCL12/CXCR4 axis in the pathobiology of these bone marrow diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2675. doi:1538-7445.AM2012-2675
A patient with myelodysplastic syndrome (MDS) and a 47,XY,+21 karyotype at diagnosis, was documen... more A patient with myelodysplastic syndrome (MDS) and a 47,XY,+21 karyotype at diagnosis, was documented to have a clonal chromosome 21 rearrangement, i(21q), four months before transformation to acute biphenotypic leukemia. For 4 months after transformation, isochromosome 21 persisted while the patient was receiving treatment with zidovudine. Vitamin D3 was added to zidovudine for an additional month, during which time the trisomy 21 clone reappeared as the predominant cell population. The unique aspects of this patient are the atypical evolution of chromosome 21, the transformation to biphenotypic leukemia, and the occurrence of i(21q) associated with biphenotypic leukemia evolving from an MDS.
Abstract 3998 We analyzed bone marrow (BM) response and overall survival (OS) in 48 patients (pts... more Abstract 3998 We analyzed bone marrow (BM) response and overall survival (OS) in 48 patients (pts) with a myelodysplastic syndrome (MDS) and WHO/FAB subtypes of refractory anemia with excess blasts (RAEB) -1,- 2 or -t and acute myeloid leukemia (AML), enrolled in 4 independent ongoing clinical trials of the novel small molecule ON 01910.Na. Pts received ON 01910.Na administered as a continuous intravenous infusion (CIV) from 2 to 6 days weekly or every other week with BM response initially assessed per protocol by week 4 or 8. A BM complete response (CR) (>50% decrease from baseline BM blast and decrease below 5% for at least 4 weeks, per MDS IWG 2006 criteria) or an initial 50% decrease of BM blasts by week 4 to 8 was documented in 19/48 (40%) treated pts and was associated with a significant increase in overall survival (OS) (p = 0.0001) by the method of Kaplan-Meier (Figure 1 and Table 1). This relationship was still significant when excluding AML patients (p = 0.008). Six pts (3 RAEB-1, 3 RAEB-2) had complete BM response. Five of these six pts previously failed to respond or relapsed after azacitidine/decitabine and five out of six are alive (17 to 113 weeks follow-up; one death at 68 weeks). Eleven pts (5 AML, 3 RAEB-t, 2 RAEB-2 and 1 RAEB-1) could not be assessed at 4–8 weeks with follow-up BM evaluation; their median OS was 7.5 weeks; 9 of these 11 pts previously failed to respond or relapsed after azacitidine/decitabine. Among 10 pts with trisomy 8 cytogenetics (4 had an initial BM response), median survival was 25 weeks. FAB/WHO classification of all 48 pts was also significantly correlated with survival (Table 2, p=0.003). Overall, ON 01910.Na infusions were well tolerated. Eight pts had hematological improvements at various time points after starting therapy. Median OS was 33 weeks in the subset of 29 RAEB-1,-2,-t pts refractory or relapsing after azacitidine/decitabine, and a significant association (p = 0.056) between BM response and OS was also found in these pts (Table 1). The median survival of MDS pts who had failed to respond to prior treatment with decitabine has been reported to be approximately 17 weeks (Jabbour et al, Cancer 2010, in press). These results suggest a strong correlation between BM blast response and OS and the predictive value of BM response to ON 01910.Na for estimating overall survival of higher risk MDS or AML pts. Table 1. Overall Survival of Pts Treated with ON 01910.Na by BM CR and Initial (4-8 Weeks) Blast Response 4–8 Week BM Blast Reduction (%) BM CR > 50% Initial Response < 50% Initial Response Not Assessed P value Logrank test Figure Legend Red Blue Black Green RAEB-1, RAEB-2, RAEB-t, & AML pts, N = 48 6 13 18 11 Median Survival (weeks) Not reached 36 28 7.5 P = 0.0001 RAEB-1, RAEB-2, RAEB-t pts relapsed/refractory to azacitidine/decitabine, N = 29 5 6 12 6 Median Survival (weeks) 68 Not reached 33 3.5 P = 0.056 Table 2. Overall Survival by FAB/WHO Classification of 48 RAEB and AML Pts Treated with ON 01910.Na FAB/WHO Classification RAEB-1 RAEB-2 RAEB-t AML (>30% BM blasts) P value Logrank test N pts 11 18 10 9 Median Survival (weeks) Not reached 28 20 11 P = 0.003 Disclosures: Silverman: Onconova Therapeutics Inc: Research Funding, Research support of Clinical Trial. Raza:Onconova Therapeutics Inc: Research Funding, Research support for clinical trial. Greenberg:Onconova funding of clinical trial: Research Funding, Research support from Onconova for performance of this trial. Wilhelm:Onconova Therapeutics Inc.: Employment, Equity Ownership.
Background: The hypomethylating agent azacitidine (azaC) which can reverse epigenetic silencing, ... more 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
13137 Background: The benzyl styryl sulfone analog ON 01910.Na is a novel anticancer agent that i... more 13137 Background: The benzyl styryl sulfone analog ON 01910.Na is a novel anticancer agent that inhibits mitotic progression and induces apoptosis; it has activity against most human cancer cells in vitro and against a broad spectrum of human xenografts in mice. Cell kill effects are exposure time-dependent in vitro. After 3d exposure 200 nM ON 01910.Na killed > 90% of Daudi lymphoma cells, whereas 40-fold higher drug concentration killed only 50% of cells after 24h exposure. Dogs that received CI of 325 mg/kg/d ×3 failed to reach MTD. Methods: Starting dose 50 mg/m2/d as a 72 hr CI was 1/10th MTD of rats’ daily dose given × 28 d. Treatment cycles were repeated every 2 wks until progressive disease, intolerable toxicity, or withdrawal of consent. Dose was escalated by Fibonacci progression in single pts until grade 2 toxicity when cohorts of 3 are to be studied. Volunteers may be retreated at a higher dose if tolerated by a preceding naïve subject. Results: One man and four women (61–77 yrs) have been studied in 21 cycles in 5 mos as of 1/10/06. Doses of 50, 100, 150 and 250 mg/m2/d × 3 have been given for 1 to 10 cycles. Grade 1 granulocytopenia (2/4 pts with carcinoma) indicates biologic activity. Grade 1 fatigue (3/5) was less than in prior chemotherapy regimens. No grade 2 toxicity has yet occurred. Cumulative toxicity has not been seen. Two pts have had stable disease 6+ and 22+ wks. After deproteinization with acetonitrile, plasma or serum samples were measured by mass spectrometry. At 100 mg/m2/d steady state levels were 730 nM after the 1st cycle and 1190 nM after the 4th cycle. Drug levels were maximal at 3 to 6 h despite CI suggesting induction of a metabolizing pathway. Levels fell precipitously at the end of infusion on the 1st cycle but a low level persisted in one pt for 48h after the major drop in the 4th cycle, suggesting drug accumulation. Conclusion: Levels effective in vitro have been obtained in vivo by CI without limiting toxicity. Hints of activity already seen suggest that this compound has clinical promise. This phase I study continues. [Table: see text]
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