Emiliano Fabiani

Valproic Acid at Therapeutic Plasma Levels May Increase 5-Azacytidine Efficacy in Higher Risk Myelodysplastic Syndromes

Purpose: Epigenetic changes play a role and cooperate with genetic alterations in the pathogenesis of myelodysplastic syndromes (MDS). We conducted a phase II multicenter study on the combination of the DNA-methyltransferase inhibitor 5-azacytidine (5-AZA) and the histone deacetylase inhibitor valproic acid (VPA) in patients with higher risk MDS. Experimental Design: We enrolled 62 patients with MDS (refractory anemia with excess blasts, 39 patients; refractory anemia with excess blasts in transformation, 19 patients; and chronic myelomanocytic leukemia (CMML), 4 patients) and an International Prognostic Scoring System (IPSS) rating of Intermediate-2 (42 patients) or high (20 patients). VPA was given to reach a plasma concentration of >50 μg/mL, then 5-AZA was added s.c. at 75 mg/m2 for 7 days in eight monthly cycles. Results: The median overall survival was 14.4 months. At a median follow-up of 12 months (range, 0.7-21.0), the disease progressed in 20 patients, with 21% cumulative incidence of progression. Of 26 patients who completed eight cycles, 30.7% obtained complete or partial remission, 15.4% had a major hematologic improvement, whereas 38.5% showed stable disease. Drug-related toxicity was mild. Favorable prognostic factors for survival were IPSS Intermediate-2 and plasma VPA of ≥50 μg/mL (log rank = 0.013 and 0.007, respectively). Analysis of polymorphisms important for the metabolism of the drugs used in the trial showed that carriers of the CYP2C19*2 variant of cytochrome P450 required higher VPA doses to achieve the target VPA plasma concentration of 50 μg/mL on day 1 of 5-AZA treatment (P = 0.0021). Conclusion: Our data show that the 5-AZA/VPA combination is active and safe in patients with MDS with a poor prognosis. Achievement of VPA therapeutic levels may indeed increase 5-AZA efficacy.

Reduced BRCA1 expression due to promoter hypermethylation in therapy-related acute myeloid leukaemia

BRCA1 plays a pivotal role in the repair of DNA damage, especially following chemotherapy and ionising radiation. We were interested in the regulation of BRCA1 expression in acute myeloid leukaemia (AML), in particular in therapy-related forms (t-AML). Using real-time PCR and Western blot, we found that BRCA1 mRNA was expressed at barely detectable levels by normal peripheral blood granulocytes, monocytes and lymphocytes, whereas control BM-mononuclear cells and selected CD34+ progenitor cells displayed significantly higher BRCA1 expression (P=0.0003). Acute myeloid leukaemia samples showed heterogeneous BRCA1 mRNA levels, which were lower than those of normal bone marrows (P=0.0001). We found a high frequency of hypermethylation of the BRCA1 promoter region in AML (51/133 samples, 38%), in particular in patients with karyotypic aberrations (P=0.026), and in t-AML, as compared to de novo AML (76 vs 31%, P=0.0002). Examining eight primary tumour samples from hypermethylated t-AML patients, BRCA1 was hypermethylated in three of four breast cancer samples, whereas it was unmethylated in the other four tumours. BRCA1 hypermethylation correlated to reduced BRCA1 mRNA (P=0.0004), and to increased DNA methyltransferase DNMT3A (P=0.003) expression. Our data show that reduced BRCA1 expression owing to promoter hypermethylation is frequent in t-AML and that this could contribute to secondary leukaemogenesis.

Epigenetic changes in therapy-related MDS/AML

Therapy-related Myelodysplastic Syndromes/Acute Myeloid Leukemias (t-MDS/AML) are one of the most compelling long term adverse events occurring in cancer survivors treated with chemo-radiotherapy regimes. Beside several well-described genetic lesions, a growing amount of data suggests that abnormalities in DNA methylation profile contribute to multistep secondary leukemogenesis. Two distinct alterations of normal DNA methylation patterns may occur in cancer: a global hypomethylation resulting in chromosomal instability and loss of genetic integrity, and promoter specific DNA hypermethylation which leads to silencing of tumor suppressor genes. Cytotoxic drugs and radiation have been shown to affect tissue DNA methylation profile. Radiation is able to induce a stable DNA hypomethylation in both target and bystander tissues. Gene promoter methylation is a common finding in t-MDS/AML and has been associated to a shorter latency period from the treatment of the primary tumor. Among the studied genes, p15 methylation correlated to monosomy/deletion of chromosome 7q, suggesting that it could be a relevant event in alkylating agent-induced leukemogenesis. We found frequent methylation of DAPK in the t-MDS/AML group, especially in patients with a previous lymphoproliferative disease. In patients studied for concurrent methylation of several promoters, t-MDS/AML were significantly more frequently hypermethylated in 2 or more promoter regions than de novo MDS or AML suggesting that promoter hypermethylation of genes involved in cell cycle control, apoptosis and DNA repair pathways is a frequent finding in t-MDS/AML and may contribute to secondary leukemogenesis. However, how the epigenetic machinery is disrupted after chemo/radiotherapy and during secondary carcinogenesis is still unknown, warranting further studies.

Prognostic role of glutathione S-transferase polymorphisms in acute myeloid leukemia.

Glutathione S-transferases (GSTs) are phase II detoxification enzymes involved in the metabolism of carcinogens and anticancer drugs, known also to interact with kinase complexes during oxidative or chemical stress-induced apoptosis. We were interested whether their polymorphic variants may account for differences in outcome of patients with acute myeloid leukemia (AML) following chemotherapy. We studied the prognostic role of polymorphisms in three GST genes (GSTP1/M1/T1) in a large patient cohort of the German Austrian Acute Myeloid Leukemia Study Group, treated according to prospective multicenter clinical trials (AML HD98A: 254 patients; AML HD98-B: 100 patients), with a median follow-up of 46 months. Looking at short-term adverse drug reactions, homozygous carriers of the GSTP1*105 Val allele had a faster neutrophil and platelet recovery (P=0.002 and 0.02, respectively) and a reduced need of red cell and platelet transfusions (P=0.01 and 0.03, respectively). Response to induction chemotherapy did not vary according to GST polymorphisms. Multivariable Cox regression models revealed a significant better relapse-free (RFS) and overall survival for the GSTP1*105 Val (P=0.003 and 0.03, respectively), whereas GSTT1 and GSTM1 genotypes had no significant impact. The favorable impact of GSTP1*105 Val on RFS seems to be restricted to the subgroup of patients exhibiting a normal karyotype.

Role of BCL2L10 methylation and TET2 mutations in higher risk myelodysplastic syndromes treated with 5-azacytidine

Role of BCL2L10 methylation and TET2 mutations in higher risk myelodysplastic syndromes treated with 5-Azacytidine

Analysis of genome-wide methylation and gene expression induced by 5-aza-2'-deoxycytidine identifies BCL2L10 as a frequent methylation target in acute myeloid leukemia

Epigenetic changes play a role in the pathogenesis of myeloid malignancies, and hypomethylating agents have shown efficacy in these diseases. We studied the apoptotic effect, genome-wide methylation, and gene expression profiles in HL60 cells following 5-aza-2′-deoxycytidine (decitabine; DAC) treatment, using microarray technologies. Decitabine treatment resulted in a decrease in global DNA methylation, corresponding to 4876 probeset IDs with significantly reduced methylation levels, while the expression of 2583 IDs was modified. The integrated analysis identified 160 genes demethylated and up-regulated by decitabine, mainly including development and differentiation pathway genes. Gene targets of Polycomb group protein regulation were overrepresented in this group. Apoptosis was induced by decitabine, and apoptosis-specific PCR arrays more precisely indicated decitabine-induced up-regulation of 13 apoptosis-related genes, in particular DAP-kinase 1 and BCL2L10. Correspondingly, in primary patient samples, BCL2L10 was hypermethylated in 45% of AML, 43% of therapy-related myeloid neoplasms, 12% of MDS, and in none of the controls. In conclusion, decitabine induces global demethylation and gene expression, in particular of Polycomb target genes involved in development and differentiation pathways. The apoptotic gene BCL2L10 is a frequent target for aberrant promoter methylation in patients with acute leukemia, de novo and therapy-related.

Why methylation is not a marker predictive of response to hypomethylating agents.

The azanucleotides azacitidine and decitabine have been shown to induce hematologic response and prolong survival in higher-risk myelodysplastic syndromes. They are inhibitors of DNA methyltransferase-1 and induce DNA-hypomethylation. Induction of apoptosis is also clinically relevant, in particular during the first treatment cycles, when cytopenia is a frequent side-effect. Since the hypomethylating effect is reversible, and the malignant clone has been shown to persist in most responding patients, several cycles are necessary to achieve and maintain responses, while treatment interruption is associated with rapid relapse. Methylation studies have shown global and gene-specific hypermethylation in myelodysplastic syndromes, but there seems to be little relation between the degree of demethylation following hypomethylating treatment and hematologic response. The presence of concurrent genomic hypermethylation and hypomethylation may impair the predictive power of current detection techniques. This scenario has been complicated by the identification of epigenetic enzyme mutations, including TET2, IDH1/2, DNMT3A and EZH2, which are important for response to hypomethylating treatment. Changes in azanucleotide metabolism genes may also play a role. In the future, methylation analysis concentrating not only on promoters, but also on gene bodies and intergenic regions, may identify key genes in patients with the highest probability of response to azanucleotides and allow a patient-tailored approach.

Promoter methylation of DAPK1, E-cadherin and thrombospondin-1 in de novo and therapy-related myeloid neoplasms

DNA methylation is one of the major epigenetic changes in human cancers, leading to silencing of tumor suppressor genes, with a pathogenetic role in tumor development and progression in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methylation of key promoter regions, induced by cytotoxic therapy together with complex genetic changes, is important in the biology of therapy-related myeloid neoplasms (t-MN). We were interested in the characterization of the methylation pattern of AML and MDS de novo and therapy-related. We studied 385 patients (179 females, 206 males), of a median age of 66 years (range 16-98 years). There were 105 MDS, 208 de novo AML and 72 t-MN (45 MDS and 27 AML). Using a methylation-specific PCR, we studied the promoter methylation status of E-cadherin (CDH1), TSP1 and DAP-Kinase 1. These genes have been shown to be involved in the malignant transformation, interfering with angiogenesis, interaction with micro-environment, apoptosis and xenobiotic detoxification. We found no associations between promoter hypermethylation and gender or age at the time of initial diagnosis. In patients with MDS, there were no associations between hypermethylation and clinical characteristics, including IPSS score, WHO classification and cytogenetics. DAPK1 was more frequently methylated in t-MDS/AML when compared to de novo MDS and AML (39% vs 15.3% and 24.4%, p=0.0001), while methylation of CDH1 was similar in t-MDS/AML and AML (51% and 53.4%), but less frequent in de novo MDS (29%) (p=0.003). In the t-MDS/AML group, we found that the methylation pattern appeared to be related to the primary tumor, with DAPK1 more frequently methylated in patients with a previous lymphoproliferative disease (75% vs 32%, p=0.006). On the other hand, methylation of CDH1 was associated to radiotherapy for the primary malignancy (84.5% vs 38%, p=0.003). TSP1 hypermethylation was rare and not characteristic of t-MDS/AML. In 177 patients studied for concurrent methylation of several promoters, t-MN and AML de novo were significantly more frequently hypermethylated in 2 or more promoter regions than de novo MDS (20% vs 12.4%, p<0.001). Chemotherapy and individual genetic predisposition have a role in t-MDS/AML development, the identification of specific epigenetic modifications may explain complexity and genomic instability of these diseases and give the basis for targeted-therapy. The significant association with previous malignancy subtypes may underlie a likely susceptibility to methylation of specific targets and a role for constitutional epimutations as predisposing factors for the development of therapy-related myeloid neoplasm.

Polymorphisms of detoxification and DNA repair enzymes in myelodyplastic syndromes.

The genetic background may modify the individuals risk of developing cancer. We performed a case-control study to test the impact of genomic polymorphisms of 9 genes involved in xenobiotics detoxification and DNA repair in myelodysplastic syndromes (MDS). Frequencies of polymorphic variants of RAD51, XRCC3, NQO1, GSTA1, GSTM1, GSTT1, CYP3A4 and XPD enzymes were similar in patients and controls. On the other hand, the GSTP1-105Val allele was associated to an increased risk of MDS (O.R. 1.66; p=0.04) and to higher probability of overall survival in the low/intermediate-1 IPSS risk group (p=0.008). The GSTP1-Ile105Val polymorphism is likely to influence MDS risk and prognosis.

Characteristics and outcome of therapy-related myeloid neoplasms: Report from the Italian network on secondary leukemias

Therapy‐related myeloid neoplasms (t‐MN) are a complication of cytotoxic treatment for primary tumors and autoimmune diseases. We report data on 277 t‐MN patients, recruited between 1999 and 2013 by the Italian Network on Secondary Leukemias (104 retrospectively and 173 prospectively registered). Median age at t‐MN diagnosis was 64 years (range, 21–87). Most frequent primary malignancies (PMs) were lymphoproliferative diseases and breast cancer. One hundred and thirty‐three patients had received chemotherapy (CHT), 43 patients radiotherapy (RT), and 101 patients combined CHT/RT for PM. Median time between cytotoxic treatment and t‐MN was 5.7 years, with t‐MN following RT alone associated with significantly longer latency, compared to CHT or combined CHT/RT (mean, 11.2 vs. 7.1 years, P = 0.0005). The addition of topoisomerase‐II inhibitors to alkylating agents was associated with shorter latency compared to alkylating agents alone (median, 6 vs. 8.4 years, P = 0.02). Median survival was 14.6 months from t‐MN diagnosis, and was significantly longer in patients treated with allogeneic stem cell transplantation. Significant factors for survival at the multivariable analysis included age, adverse karyotype, and degree of anemia. Our data underline the prognostic importance of karyotype and age in t‐MN, similar to de novo acute myeloid leukemia. Treatment approaches should not preclude the use of conventional treatments for younger t‐MN patients, including allogeneic stem cell transplantation as potentially curative approach. Am. J. Hematol. 90:E80–E85, 2015.