Gender effects on cytidine analogue metabolism and myelodysplastic syndrome treatment outcomes

In vivo, half-lives of cytidine analogues such as 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), are determined largely by cytidine deaminase (CDA), an enzyme that rapidly metabolizes these drugs into inactive uridine counterparts. Genetic factors influence CDA activity, and hence, could impact 5-azacytidine/decitabine levels and efficacy, a possibility requiring evaluation. Using an HPLC assay, plasma CDA activity was confirmed to be decreased in individuals with the CDA SNP A79C. More interestingly, there was an even larger decrease in females. Explaining the decrease in enzyme activity, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass-spectrometry, were significantly higher in females. In mathematical modeling, the detrimental effect of shortening half-life of S-phase specific therapy was amplified in low S-phase fraction disease (e.g., MDS). Accordingly, in multivariate analysis of MDS patients treated with 5-azacytidine/decitabine, overall survival was significantly worse in males

Analysis of Immunogenetic Factors in Myelodysplastic Syndromes (MDS) Reveals Potential Pathogenic Role Cytokine Genotypes Such as TGF-β

Abstract Pathophysiologic and clinical features of MDS are related to the phenotype of the dysplastic hematopoietic clone. The immunogenetic background resulting from complex genetic predisposition traits may influence the quality of the immune response and shape the clinical features of MDS, including the severity of cytopenias and speed of malignant evolution. To test this hypothesis we selected the following immunogenetic factors: KIR and KIR-ligand (KIR-L) genotype, as well as cytokine/receptor single nucleotide polymorphisms (SNP). We genotyped a cohort of 90 patients with MDS/sAML (30 RA/RCMD, 30 RARS/ RCMD-RS, 30 RAEB/sAML); 60 healthy donors matched for ethnicity were analyzed as a control. A group of 66 patients with aplastic anemia (AA) was used as a reference. HLA type, KIR, KIR-L haplotypes, and the following SNPs were analyzed: IL-1α (−889 T/C),IL2 (−330 T/G +166 G/T), IL4 (−1098 T/G -590 T/C -33 T/C, IL-1R (−1970 C/T), IL-1RA (mspa 111100 T/C), IL-4RA (+ 190 G/A), IL-1 β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, -819 C/T), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), INF- γ (+874 A/T), TNF- α (−308 G/A, -238 G/A), CTLA-4 (exon 1, +49 A/G), FcgIIIR (+559 G/T) as well as SNPs in the CD45 gene (exon 4 +77 C/G, +138 A/G). In the MDS cohort, no difference in the frequency of KIR genotype constellations was identified. However, a higher frequency of 2DS5 (66% vs. 26%, p=.01) and a decreased frequency of 2DL3 (62% vs. 87% p=.02) was found when patients with hypocellular MDS (N=10) were analyzed separately. No significant difference in KIR-L C1/C2 genotype frequency between the group was found. However, an increased incidence of C2/C2 was found in high grade MDS/sAML (RAEB/sAML 44% vs. 13%, p=.02). In MDS, there was a decreased frequency of stimulatory 2DS1/C2 mismatch consistent with potentially enhanced cytotoxicity (17% vs. 44%, p=.01). No significant difference in the MDS cohort compared to control and when MDS subgroup were compared to each other, was found for the SNPs in IL-4RA, IL-12, IL-1α, IFN-γ, IL-2, IL-1 α, IL-1R, and IL4. However, when we examined the frequency of TGF- β genotypes, the MDS population showed a higher rate of TT codon 10 variant (59% vs. 32% in controls, p=.002) and of GG codon 25 variant (71% vs. 35% in controls, p=.0001), consistent with a “high secretor phenotype”. Of note is that, when AA was examined and compared to controls, a higher frequency of TGF-β high secretor genotype was found (GG codon 25 variant; 61% vs 35% in controls, p=.03). We also found a higher incidence of A/A genotype for CTLA-4 in MDS (47 vs 27, p=.001). This relationship was even more pronounced in hypocellular MDS. Moreover, hypocellular MDS was characterized by a higher prevalence of IL10 -819 T/T and -592 A/A phenotypes (40% vs 12% p=.03), which are functionally associated with a lower secretion. In sum, our findings demonstrate that various immunogenetic factors may be demonstrated in MDS patients, which may likely influence the quality of immune response and shape clinical features of MDS. Certain genotypic constellations (e.g., TGF gene variants) resemble, in particular in hypocellular MDS, a constellation seen in AA

A phase 2 trial of combination therapy with thalidomide, arsenic trioxide, dexamethasone, and ascorbic acid (TADA) in patients with overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN) or primary myelofibrosis (PMF)

BACKGROUND. Primary myelofibrosis (PMF) and overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal hematopoietic disorders that share similar clinical features and molecular abnormalities, such as the Janus kinase 2 (JAK2) valine to phenylalanine mutation at codon 617 (V617F) and the tet methylcytosine dioxygenase 2 (TET2) mutation. There are limited therapeutic options available for these diseases, and single agents have only modest efficacy. In this phase 2 study, the authors combined multiple active agents (thalidomide, arsenic trioxide, dexamethasone, and ascorbic acid [TADA]) to treat patients with these disorders. METHODS. This multicenter trial was conducted from January 2005 to July 2007. The primary endpoint was to evaluate the efficacy of TADA therapy. Patients received the combination for one 12-week cycle followed by maintenance thalidomide for an additional 3 months. Response was assessed using International Working Group criteria. RESULTS. Among 28 enrolled patients, the median age was 66.5 years; 15 patients had MDS/MPN-unclassifiable, 8 patients had chronic myelomonocytic leukemia type 1, and 5 patients had PMF. Approximately 60% of the patients had normal cytogenetics. The JAK2V617F mutation was detected in 5 of 14 tested patients, and TET2 mutations were detected in 2 of 8 tested patients. Almost half of the patients had splenomegaly. With a median on-study follow-up of 5.7 months, 21 patients (75%) completed the entire 12-week course of therapy, and 6 patients (29%) responded to TADA. With a median extended follow-up of 24.1 months for 15 evaluable patients, the median progression-free survival was 14.4 months, and the median overall survival was 21.4 months. CONCLUSIONS. The TADA regimen yielded clinical responses in patients with PMF and MDS/MPN. To the authors' knowledge, this study represents the first trial targeting this patient population. The results indicated that it is reasonable to incorporate multiple novel agents in the treatment of these rare diseases. Cancer 2012. (c) 2011 American Cancer Society

Gender Is A Major Determinant of Cytidine Analogue Metabolism and May Contribute to Differences in Treatment Outcomes

Abstract Abstract 1434 Cytidine analogues are mainstays in hematologic malignancy therapy. One documented limitation of therapy is interindividual variability in cytidine analogue pharmacokinetics that compromises predictability, safety and efficacy. Causes of interindividual variability include single nucleotide polymorphisms (SNPs) in cytidine metabolizing enzymes, particularly cytidine deaminase (CDA): the CDA SNP A79C (rs2072671) is present in >70% of Caucasians and decreases CDA enzyme activity. Gender is another known genetic determinant of cytidine analogue clinical activity, although the pathway is unknown (Dimopoulou et al, Ann Oncol 2004). With high dose cytarabine therapy, CDA may be saturated, dampening the clinical impact of CDA SNPs. However, the recently approved cytidine analogues 5-azacytidine and decitabine are used as single agents at relatively low doses, and interindividual variability could have greater clinical significance, since drug levels may be close to minimum thresholds required for efficacy. To evaluate this possibility, the impact of A79C and gender on treatment outcomes was examined in a cohort of patients with myelodysplastic syndrome (MDS, n=127) and acute myeloid leukemia (AML, n=74) classified by WHO criteria, initiated on treatment between 1/2002 and 12/2007, with IRB approved tissue-banked bone marrow samples available for analysis for the A79C SNP by sequencing and SNP array, and with verifiable follow-up and survival annotation. Other variables analyzed were those known to have major prognostic importance in patients with MDS and AML (bone marrow myeloblast percentage, karyotype, age). Patients were analyzed in three treatment groups: (i) did not receive cytidine analogues (n=35), (ii) treated with cytarabine (n=76), (iii) treated with 5-azacytidine or decitabine (n=90). In all three treatment groups, heterozygosity or homozygosity for A79C was not associated with significant differences in overall survival (OS). Similarly, there was no significant difference in overall survival in females versus males who were treated with cytarabine or who did not receive cytidine analogues. However, in the 5-azacytidine/decitabine treatment group, females had significantly longer overall survival than males (median 1033 v 563 days, Log Rank p=0.014). In multivariate analysis incorporating age, karyotype, bone marrow blast percentage and A79C, only age, gender and karyotype were significantly (Cox Proportional Hazards p<0.01) associated with survival, with the hazard ratio for female gender 0.356 (95%CI 0.165–0.766). An important caveat is that MDS in females has a less aggressive natural history (Nosslinger et al, Ann Oncol 2010), therefore, it is possible that gender differences in cytidine analogue metabolism do not underlie the difference in overall survival. Nonetheless, in AML myeloblasts (GSE15434) and normal liver tissue (measured by microarray), CDA mRNA expression in females was significantly lower than in males (Wilcoxon test p=0.005 and p=0.025 respectively). To measure functional CDA activity, uridine conversion from cytidine by plasma was measured by uHPLC (Dionex). As expected, there was a significant 2-fold decrease in mean enzyme activity in individuals homozygous for the A79C variant (CC, n=32) compared to individuals homozygous for the ancestral allele (AA, n=32, t-test p=0.02). However, the decrease in mean enzyme activity in females (n=48) compared to males (n=48) was even greater (3-fold, t-test p<0.001). In an analysis of decitabine pharmacokinetics after administration of oral decitabine 1 mg/kg to female (n=18) and male (n=18) adult mice, plasma levels of decitabine measured by LCMSMS were consistently higher in females at all time-points (1.169-4.375 fold at the different time points, paired t-test p=0.001). In conclusion, decreased cytidine analogue conversion to uridine counterparts in females, most likely from differences in CDA expression, could be relevant to gender differences in treatment outcomes with 5-azacytidine or decitabine. Disclosures: No relevant conflicts of interest to declare

Increased CDA Expression/Activity in Males Contributes to Decreased Cytidine Analog Half-Life and Likely Contributes to Worse Outcomes with 5-Azacytidine or Decitabine Therapy

Purpose: The cytidine analogs 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase 1 (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. Experimental design: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity, and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated patients with MDS (n = 90) and cytarabine-treated patients with acute myeloid leukemia (AML) (n = 76). Results: By high-performance liquid chromatography (HPLC), plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females than in males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared with high S-phase fraction disease (e.g., MDS vs. AML), because in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female patients with MDS treated with 5-azacytidine/decitabine. Conclusions: Increased CDA expression/activity in males contributes to decreased cytidine analog half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy. Clin Cancer Res; 19(4); 938-48. (c) 2012 AACR

Identification of Oncogenic EZH2 Mutations In Myelodysplastic Syndromes and Related Myeloid Malignancies

Abstract Abstract 607 Using single nucleotide polymorphism arrays (SNP-A) as a karyotyping tool led to the recognition that segmental somatic uniparental disomy (UPD) is a common and important defect in myelodysplastic syndromes (MDS) and related conditions. Homozygous mutations associated with UPD have been found. Additionally, SNP-A-detected submicroscopic deletions have proven helpful in narrowing the search for mutant genes to shared regions of loss of heterozygosity (LOH). Mapping of such microdeletions was instrumental in the identification of TET2 and CBL mutations, frequently found in a homozygous constellation within UPD of the corresponding chromosomal regions. UPD7q is one of the most frequent areas of UPD present in MDS, MDS/MPN and sAML. We hypothesized that this region may be associated with the presence of pathogenic mutations in a homozygous configuration. We utilized two strategies: i) because of the large size of the area commonly affected by UPD7, we applied next generation sequencing (NGS) for the identification of affected genes, and ii) we systematically sequenced genes contained in the overlapping deleted regions. In total, 15 patients with UPD7q were identified. Exome libraries were generated from 2 cases with UPD7q and subjected to sequencing. In one, a missense C to T mutation was found at nt148137376 of EZH2 (100% of sequences), resulting in R690H. The somatic origin of this homozygous mutation was confirmed by sequencing of DNA from a skin biopsy. Simultaneously, we identified 2 patients with overlapping microdeletion involving 7q36.1. The minimally affected area involved 2 genes: CUL1 and EZH2. Sequenced all exons of both genes revealed no mutations in CUL1. However, a mutation in EZH2 exon 19 was identified in proximity to the mutation detected through NGS. The mutation involved position Ile715 producing a frame shift. We detected EZH2 mutations in 10/351 patients (7% of MDS/MPN, 2% of MDS and 0.9% of AML). We found 8 different EZH2 mutations present in 4/15 (27%) of cases with UPD7q, 2/30 (7%) patients with deletion 7q and 4/306 (1%) without LOH. Of note is that both hemizygous mutations were found in patients with microdeletions and none of the patients with large del(7q) or monosomy 7 harbored EZH2 mutation. There were 8 missense and 2 frame shift mutations, located in the SET domain of the EZH2 gene. Diagnoses included 3 cases of refractory cytopenia with multilineage dysplasia, while 5 had myelomonocytoid malignancies, and one each with MDS/MPN unclassifiable and atypical chronic myeloid leukemia (aCML). Overall, EZH2 mutations were detected in 4/53 (8%) cases of CMML, consistent with the high prevalence of somatic UPD7 in this disease. Interestingly, no EZH2 mutant case had a chromosome 7 abnormality by metaphase cytogenetics, but SNP-A karyotyping detected cryptic LOH7q in 6/10 patients with EZH2 mutations. EZH2 is a polycomb associated gene encoding a methyltransferase targeting H3K27, thereby producing a repressive mark. Loss of function or hypomorphic mutations of EZH2 would thus be predicted to result in abrogation of inhibitory chromatin marks and chromatin decompaction, conducive to expression of oncogenes. Immunofluorescence and western blot showed that EZH2 mutations in myeloid malignancies lead to decreased methylation of H3K27 and thereby are functionally relevant. Expression analysis of EZH2 did not show significantly decreased expression in MDS or haploinsuffciency in patients with del7/7q. In a patient with aCML and UPD7q, homozygous R690H EZH2 mutation was detected and trimethylated H3K27 was abrogated while H3K9 methylation appeared normal, consistent with functional impairment of the SET domain. Interestingly, an EZH2 mutation was associated with heterozygous TET2 S733fsX21 and heterozygous ASXL1 W538X mutations, suggesting multiple genes associated with epigenetic regulation may be mutated and act synergistically in malignant evolution. These triple mutant cells ultimately formed lethal tumors when injected to NOD SCID gamma mice. In summary, our investigations demonstrate EZH2 mutations in patients with myeloid malignancies frequently associated with UPD7q, or 7q36.1 microdeletion, but not monosomy 7 and del(7q). These findings suggest that an increasing possibility that mutations in the polycomb gene family represent a new class of molecular lesions conveying a clonal epigenetic instability phenotype and can constitute leukemogenic events. Disclosures: No relevant conflicts of interest to declare

New TET2, ASXL1 and CBL Mutations Have Poor Prognostic Impact In Systemic Mastocytosis and Related Disorders

Abstract Abstract 3076 Mastocytosis is a heterogeneous hematopoietic neoplasm characterized by proliferation and organ infiltration by clonal mast cells (MC). The disease spectrum encompasses chronic indolent forms such as cutaneous mastocytosis (CM)/indolent systemic mastocytosis (ISM) to more aggressive types such as SM with associated clonal hematologic non-mast-cell disease (SM-AHNMD), the latter most closely related to myeloproliferative neoplasms (MPN) or MDS/MPN overlap syndromes. Molecular pathogenesis of mastocytosis involves acquisition of c-KIT mutations, particularly D816V, which is present in many cases and confers resistance to imatinib. TET2 mutations are often found in MPN and MDS/MPN and also in ∼20% of SM patients without noticeable impact on survival. We have hypothesized that analysis of molecular defects in mastocytosis may shed light on disease pathogenesis and possibly convey prognostic information that may help in diagnosis and selection of rational therapies. To investigate these molecular events, we have applied single nucleotide polymorphism array-based karyotyping (SNP-A) (Affymetrix 6.0) to identify recurrent areas of loss of heterozygosity and performed a broad screen for mutations which could be present in mastocytosis including c-KIT, TET2, CBL gene family (CBL, CBLB, CBLC), ASXL1, IDH1/IDH2, which have been found in hematologic disorders related to or associated with SM. Overall survival (OS) was analyzed using the Kaplan-Meier method (Log-Rank). We studied a total of 35 mastocytosis patients classified using WHO criteria (CM, N=9; ISM, N=14; SM-AHNMD, N=9; [CMML, N=6; AML, N=1; NHL, N=2], aggressive SM (ASM) N=2; MC sarcoma, N=1). Median age of the cohort was 51 yrs (13-71). SNP-A showed a total of 20 new lesions (13 gains, 3 losses and 4 uniparental disomy [UPD]) in 10 patients (CM=1, ISM=4, SM-AHNMD=4, ASM=1). The most frequently affected chromosomes were 2, 7, 12, 13, 14 and X. UPD was only found in SM-AHNMD and ASM and it involved chromosomes 2p, 4q, 7p and 13q. No OS difference were observed between patients with new SNP lesions compared to those without (47 mo vs. 38 mo; p=.84). c-KIT sequencing showed D816V in 29% of patients (ISM=29%; SM-AHNMD=44%, ASM=100%). A total of 15 additional mutations were found in 9/35 patients. TET2 mutations were found in 8/35 (23%), including 2 patients with biallelic mutations (3 frameshift, 2 nonsense and 5 missense). TET2 mutational frequencies for CM, ISM and SM-AHNMD (only CMML) were 22% (2/9), 7% (1/14) and 56% (5/9). Majority of TET2 mutations were heterozygous, except one that was homozygous. These mutations have not been previously described in mastocytosis. We have also detected ASXL1 mutations in 3/35 (9%) patients, with biallelic mutation seen in one patient (1 frameshift, 1 nonsense and 2 missense). ASXL1 mutations were seen in 1/14 ISM and 2/9 SM-AHNMD (with CMML). To our knowledge, ASXL1 mutations have not been described in mastocytosis. A heterozygous CBL mutation was found in 1/35 patients with SM-AHNMD (CMML). No mutations were found in CBLB, CBLC and IDH1/IDH2. Interestingly, 5 patients were found to have >1 mutation, c-KIT and TET2 in 2, c-KIT/TET2/ASXL1 in 2 and TET2/CBL in 1 patient. The median OS of the cohort was 18 mo (1-85). As expected, for patients with only SM (excluding CM cases), c-KIT mutants had a worse OS than wild type (WT) c-KIT patients (17 mo vs. 52 mo; p=.02). SM patients with TET2, ASXL1 or CBL mutations, independently of c-KIT, had a worse OS than those with WT genes (17 mo vs. 52; p=.01). SM patients with c-KIT mutation who carry additional mutations had a worse OS, c-KIT + any mutation [11 mo] vs. TET2/ASXL1/CBL mutant [32 mo] vs. c-KIT mutant alone [NR] vs. WT [NR]; p<.0001. Similarly, when TET2 and c-KIT mutations were analyzed independent of CBL and ASXL1, patients with mutant c-KIT and TET2 had the poorest OS in the group (c-KIT plus TET2 [10 mo] vs. TET2 alone [32 mo] vs. c-KIT alone [NR] vs. WT [NR]; p<.0001). All patients with CM were still alive at the time of analysis. In conclusion, SNP-A lesions including UPD are karyotypic changes also seen in mastocytosis. TET2 mutations are frequently found in mastocytosis, particularly in SM-AHNMD (CMML). Novel molecular mutations frequently found in MDS and MPN, as ASXL1 and CBL, are also found in mastocytosis but at lower frequencies. More importantly, these new mutations may affect prognosis, as demonstrated by poor OS in patients who carry these mutations independently of c-KIT. Disclosures: No relevant conflicts of interest to declare

Increased CDA Expression/Activity in Males Contributes to Decreased Cytidine Analog Half-Life and Likely Contributes to Worse Outcomes with 5-Azacytidine or Decitabine Therapy

PURPOSE: The cytidine analogues 5-azacytidine and decitabine, used to treat myelodysplastic syndromes (MDS), produce a molecular epigenetic effect, depletion of DNA-methyltransferase (DNMT1). This action is S-phase dependent. Hence, genetic factors that decrease the half-lives of these drugs could impact efficacy. Documentation of such impact, and elucidation of underlying mechanisms, could lead to improved clinical application. DESIGN: Cytidine deaminase (CDA) rapidly inactivates 5-azacytidine/decitabine. The effect of CDA SNP A79C and gender on CDA expression, enzyme activity and drug pharmacokinetics/pharmacodynamics was examined in mice and humans, and the impact on overall survival (OS) was evaluated in 5-azacytidine/decitabine-treated MDS patients (n=90) and cytarabine-treated acute myeloid leukemia (AML) patients (n=76). RESULTS: By HPLC, plasma CDA activity was decreased as expected in individuals with the SNP A79C. Interestingly and significantly, there was an even larger decrease in females compared to males. Explaining this decrease, liver CDA expression was significantly lower in female versus male mice. As expected, decitabine plasma levels, measured by mass-spectrometry, were significantly higher in females. In mathematical modeling, the detrimental impact of shorter drug half-life (e.g., in males) was greater in low compared to high S-phase fraction disease (e.g., MDS versus AML), since in high S-phase fraction disease, even a short exposure treats a major portion of cells. Accordingly, in multivariate analysis, OS was significantly worse in male versus female MDS patients treated with 5-azacytidine/decitabine. CONCLUSIONS: Increased CDA expression/activity in males contributes to decreased cytidine analogue half-life and likely contributes to worse outcomes with 5-azacytidine or decitabine therapy