Giulia Falconi

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.

Mutations of epigenetic regulators and of the spliceosome machinery in therapy-related myeloid neoplasms and in acute leukemias evolved from chronic myeloproliferative diseases.

Mutations of epigenetic regulators and of the spliceosome machinery in therapy-related myeloid neoplasms and in acute leukemias evolved from chronic myeloproliferative diseases

Impairment of PI3K/AKT and WNT/β-catenin pathways in bone marrow mesenchymal stem cells isolated from patients with myelodysplastic syndromes.

Bone marrow mesenchymal stem cells (BM-MSCs) exhibit multiple abnormalities in myelodysplastic syndromes (MDS), including impaired proliferative and clonogenic capacity, altered morphology, increased senescence, impaired immunoregulatory properties, and reduced hematopoietic support capacity. Common signaling pathways, such as PI3K/AKT and WNT/β-catenin, regulate multiple MSC properties, including proliferation, differentiation, and cell-cell interaction. Here, with polymerase chain reaction arrays, we investigated the expression of 84 genes belonging to the PI3K/AKT signaling pathways in BM-MSCs isolated from patients with MDS, acute myeloid leukemia, and therapy-related myeloid neoplasms, using as a control BM-MSCs isolated from patients with untreated early-stage lymphomas without BM involvement. Statistically significant downregulation of GSK3β, SOS1, RASA1, and MTCP1 gene expression was observed in BM-MSCs isolated from patients with de novo MDS, as compared with controls. Moreover, expression of the GSK3β protein was reduced in MDS-derived MSCs, and was associated with concomitant reduction of phosphorylation at Ser-9. The role of GSK3β in the downstream WNT/β-catenin signaling pathway was assessed. We investigated β-catenin protein levels and expression of 84 genes belonging to the WNT target gene pathway using PCR arrays in MDS BM-MSCs, as compared with control BM-MSCs. GSK3β impairment translated into decreased β-catenin protein levels and downregulation of several WNT/β-catenin target genes (SOX9, EGR1, WISP1). These findings suggest that deregulation of genes involved in the PI3K/AKT and WNT signaling pathways may contribute to the phenotypical abnormalities of MDS BM-MSCs.

The BCL2L10 Leu21Arg variant and risk of therapy-related myeloid neoplasms and de novo myelodysplastic syndromes

Abstract Therapy-related myeloid neoplasms (t-MNs) are an increasingly recognized complication in patients previously treated with radiotherapy and/or chemotherapy for cancer or autoimmune disease. Single nucleotide variants (SNVs) in genes involved in the cellular pathways of detoxification, DNA repair and apoptosis may modify the individual risk of developing a t-MN. We studied the frequency of the SNVs of six genes involved in xenobiotic detoxification (CYP3A4, NQO1, GSTA1, GSTM1, GSTP1 and GSTT1), two DNA repair genes (RAD51 and XRCC3) and one key regulator of apoptosis (BCL2L10) in a case–control study including 111 cases of t-MN and 259 controls. This is the first report on the prevalence of BCL2L10 Leu21Arg polymorphism in myeloid malignancies. In this line, we also tested 146 cases of de novo myelodysplastic syndrome (MDS) and 109 cases of de novo acute myeloid leukemia (AML). Our results showed a significantly lower frequency of the BCL2L10-21Arg allele in patients with t-MN and de novo MDS compared to controls (Leu/Arg + Arg/Arg: 50.6% vs. 65.9%, p = 0.017 and 45.8% vs. 65.9%, p = 0.0003, respectively). Carriers of the BCL2L10-21Arg variant have a reduced risk of developing t-MN and de novo MDS.

Fanconi anemia gene variants in therapy-related myeloid neoplasms

Therapy-related myeloid neoplasms (t-MN) include myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) occurring as a late effect of chemotherapy and/or radiotherapy for a primary malignancy or for autoimmune diseases.1, 2 The incidence of this complication has been raising in the past years because of the prolonged survival and the higher number of treated patients. Still, <5% of patients exposed to cytotoxic drugs and radiotherapy develop a t-MN, suggesting an underlying individual susceptibility. Primary malignancies most frequently associated to t-MN are breast cancer and lymphoproliferative diseases. Other recurrent clinical characteristics are presence of multiple primary neoplasms in the same individual and cancer familiarity.2 So far, higher frequency of single-nucleotide variants of detoxification and DNA-repair enzymes, alone or in association, have been reported in t-MN, but none has been validated as significant risk factor in large patient groups.3, 4, 5, 6

SETBP1 mutations in 106 patients with therapy-related myeloid neoplasms

Therapy-related myeloid neoplasms (t-MN) are myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) developing in patients treated with radiotherapy and/or chemotherapy for cancer or autoimmune diseases. Cytotoxic therapy may induce chromosomal alteration and

Clonal evolution in therapy-related neoplasms

Therapy-related myeloid neoplasms (t-MN) may occur as a late effect of cytotoxic therapy for a primary malignancy or autoimmune diseases in susceptible individuals. We studied the development of somatic mutations in t-MN, using a collection of follow-up samples from 14 patients with a primary hematologic malignancy, who developed a secondary leukemia (13 t-MN and 1 t-acute lymphoblastic leukemia), at a median latency of 73 months (range 18-108) from primary cancer diagnosis. Using Sanger and next generation sequencing (NGS) approaches we identified 8 mutations (IDH1 R132H, ASXL1 Y591*, ASXL1 S689*, ASXL1 R693*, SRSF2 P95H, SF3B1 K700E, SETBP1 G870R and TP53 Y220C) in seven of thirteen t-MN patients (54%), whereas the t-ALL patient had a t(4,11) translocation, resulting in the KMT2A/AFF1 fusion gene. These mutations were then tracked backwards in marrow samples preceding secondary leukemia occurrence, using pyrosequencing and a NGS protocol that allows the detection of low variant allele frequencies (≥0.1%). Somatic mutations were detectable in the BM harvested at the primary diagnosis, prior to any cytotoxic treatment in three patients, while they were not detectable and apparently acquired by the t-MN clone in five patients. These data show that clonal evolution in t-MN is heterogeneous, with some somatic mutations preceding cytotoxic treatment and possibly favoring leukemic development.

Methylenetetrahydrofolate reductase polymorphisms in myelodysplastic syndromes and therapy-related myeloid neoplasms.

Myelodysplastic syndromes (MDS) are characterized by altered methylation patterns, with frequent methylation of CpG islands of tumor suppressor gene promoter regions, and hyper- and hypomethylation in CpG islands not included in promoter regions and in intercoding sequences [1,2]. In recent years, hypomethylating agents have produced encouraging results in terms of complete and partial responses (CR and PR), delayed leukemia progression and prolonged overall survival in the setting of higher-risk MDS [3,4]. Interestingly, there is not a clear correlation between hypermethylation of CpG islands at the promoter level and clinical response, nor between baseline methylation and hypomethylating drug response, suggesting a wider mechanism of action of these treatments and a complex interaction among drugs, host polymorphisms and methylation patterns [5]. Aberrant hypermethylation is also a frequent feature of therapy-related myeloid neoplasms (t-MN), including t-MDS and t-acute myeloid leukemia (t-AML), a late complication of cancer treatment, characterized by a poor prognosis with standard chemotherapy and improved response rates to hypomethylating treatment [6]. Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in folate cell metabolism: it catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the prevalent form of serum folate. Th is product acts as a co-substrate of methionine synthetase in remethylation of homocysteine to methionine, leading to the production of S-adenosylmethionine, a methyl group donor. Ultimately these enzymes participate in recycling of methyl groups, which are involved in purine and pyrimidine synthesis and methylation, contributing to the regulation of gene expression, DNA integrity and stability, chromosomal modifi cation and development of mutations [7]. Some single nucleotide polymorphisms (SNPs) involving the MTHFR gene have been reported. Th e C677T variant results in an alanine-to-valine substitution at the binding site of the cofactor fl avine adenine dinucleotide, which generates a more labile enzyme with decreased activity. Th e A1298C variant results in an alanine-to-glutamate substitution in the S-adenosyl-methionine regulatory domain, so that binding of S-adenosyl-methionine inhibits enzyme activity [7]. Th e aim of this study was to investigate the role of MTHFR genetic variants as susceptibility and prognostic factors for hypomethylating treatment in the setting of de novo MDS and t-MN. MTHFR C677T and A1298C polymorphisms were analyzed by restriction fragment length polymorphismpolymerase chain reaction (RFLP-PCR) on genomic DNA, as previously described [7]. DNA had been extracted from the

Somatic mutations as markers of outcome after azacitidine and allogeneic stem cell transplantation in higher-risk myelodysplastic syndromes

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Longitudinal detection of DNMT3A R882H transcripts in patients with acute myeloid leukemia

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