Valeria Santini

Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study

BACKGROUND Drug treatments for patients with high-risk myelodysplastic syndromes provide no survival advantage. In this trial, we aimed to assess the effect of azacitidine on overall survival compared with the three commonest conventional care regimens. METHODS In a phase III, international, multicentre, controlled, parallel-group, open-label trial, patients with higher-risk myelodysplastic syndromes were randomly assigned one-to-one to receive azacitidine (75 mg/m(2) per day for 7 days every 28 days) or conventional care (best supportive care, low-dose cytarabine, or intensive chemotherapy as selected by investigators before randomisation). Patients were stratified by French-American-British and international prognostic scoring system classifications; randomisation was done with a block size of four. The primary endpoint was overall survival. Efficacy analyses were by intention to treat for all patients assigned to receive treatment. This study is registered with ClinicalTrials.gov, number NCT00071799. FINDINGS Between Feb 13, 2004, and Aug 7, 2006, 358 patients were randomly assigned to receive azacitidine (n=179) or conventional care regimens (n=179). Four patients in the azacitidine and 14 in the conventional care groups received no study drugs but were included in the intention-to-treat efficacy analysis. After a median follow-up of 21.1 months (IQR 15.1-26.9), median overall survival was 24.5 months (9.9-not reached) for the azacitidine group versus 15.0 months (5.6-24.1) for the conventional care group (hazard ratio 0.58; 95% CI 0.43-0.77; stratified log-rank p=0.0001). At last follow-up, 82 patients in the azacitidine group had died compared with 113 in the conventional care group. At 2 years, on the basis of Kaplan-Meier estimates, 50.8% (95% CI 42.1-58.8) of patients in the azacitidine group were alive compared with 26.2% (18.7-34.3) in the conventional care group (p<0.0001). Peripheral cytopenias were the most common grade 3-4 adverse events for all treatments. INTERPRETATION Treatment with azacitidine increases overall survival in patients with higher-risk myelodysplastic syndromes relative to conventional care.

Azacitidine Prolongs Overall Survival Compared With Conventional Care Regimens in Elderly Patients With Low Bone Marrow Blast Count Acute Myeloid Leukemia

PURPOSE In a phase III randomized trial, azacitidine significantly prolonged overall survival (OS) compared with conventional care regimens (CCRs) in patients with intermediate-2- and high-risk myelodysplastic syndromes. Approximately one third of these patients were classified as having acute myeloid leukemia (AML) under current WHO criteria. This analysis compared the effects of azacitidine versus CCR on OS in this subgroup. PATIENTS AND METHODS Patients were randomly assigned to receive subcutaneous azacitidine 75 mg/m(2)/d or CCR (best supportive care [BSC] only, low-dose cytarabine (LDAC), or intensive chemotherapy [IC]). RESULTS Of the 113 elderly patients (median age, 70 years) randomly assigned to receive azacitidine (n = 55) or CCR (n = 58; 47% BSC, 34% LDAC, 19% IC), 86% were considered unfit for IC. At a median follow-up of 20.1 months, median OS for azacitidine-treated patients was 24.5 months compared with 16.0 months for CCR-treated patients (hazard ratio = 0.47; 95% CI, 0.28 to 0.79; P = .005), and 2-year OS rates were 50% and 16%, respectively (P = .001). Two-year OS rates were higher with azacitidine versus CCR in patients considered unfit for IC (P = .0003). Azacitidine was associated with fewer total days in hospital (P < .0001) than CCR. CONCLUSION In older adult patients with low marrow blast count (20% to 30%) WHO-defined AML, azacitidine significantly prolongs OS and significantly improves several patient morbidity measures compared with CCR.

Changes in DNA methylation in neoplasia : Pathophysiology and therapeutic implications

Our increasing knowledge of the molecular pathophysiology of cancer is beginning to find applications in the diagnosis and treatment of various neoplastic diseases. In particular, new therapeutic approaches such as targeted agents, differentiation therapy, and immunotherapy promise to yield substantial clinical benefits with relatively few side effects. Recently, aberrant methylation of the cytosine base within the regulatory area of selected genes was shown to be a very common event in neoplasia; it is thought to contribute to the molecular pathogenesis of the disease through inactivation of tumor suppressor genes (1, 2). This finding has increased interest in use of drugs that can inhibit the process of DNA methylation and restore tumor suppressor gene function as a potential strategy to treat various malignant diseases. Hematopoietic neoplasms in particular have a high degree of aberrant methylation (3), and clinical trials have demonstrated significant activity for hypomethylating drugs in this setting. We discuss the importance and prevalence of DNA hypermethylation in cancer and review the potential value of hypomethylating agents in the treatment of human neoplasms. DNA Methylation The presence of 5-methylcytosine in human DNA (4) has genetic and epigenetic effects on cellular development, differentiation, and neoplastic transformation. 5-Methylcytosine differs from cytosine by the presence of a methyl group at the 5 position of the pyrimidine ring (Figure 1). Methylcytosine is formed after replication by addition of a methyl group to a cytosine already present in the DNA strand. Dramatic changes in overall methylation of DNA occur at different periods of embryogenesis, development, and differentiation to adult cells (5). A wave of demethylation initially erases preset methylation patterns in the first days of embryogenesis. This is followed by several waves of de novo methylation that eventually establish adult patterns of gene methylation. In differentiated cells, methylation patterns change relatively little and are perpetuated after DNA replication through the high affinity of DNA methyltransferase for hemimethylated DNA (6) (Figure 2). Unlike cytosine, 5-methylcytosine is a relatively unstable base because its spontaneous deamination leads to uracil. Through evolution, such mutations have resulted in a relative depletion of 5-methylcytosine in human DNA, and they are a major cause of germ-line mutations in inherited disease and of somatic mutations in neoplasia (7). Figure 1. Structure of cytosine, 5-methylcytosine, and hypomethylating 5-methylcytidine analogues. Figure 2. The maintenance methylation process. Top. Middle. Mtase Bottom. left 5-Aza right The functions of DNA methylation in mammalian cells remain poorly defined. Early speculation that attributed a global transcriptional regulation role to cytosine methylation (8) has not yet been confirmed experimentally. In bacteria, methylation plays a role in defense against genomic invasion by foreign DNA sequences (9). In mammalian cells, most normal methylation takes place within highly repeated transposable elements, and it has been proposed that such methylation also plays a role in genome defense by suppressing the potentially harmful effects of expression at these sites (10). This hypothesis was questioned recently (11). Regardless of its global functions, one unequivocal role for DNA methylation is in irreversible gene inactivation in selected cases, such as imprinted genes (12) and genes on the inactivated X chromosome (13). CpG Island Methylation and Gene Silencing In mammalian DNA, normal methylation is restricted to cytosine followed by guanosine (the CpG dinucleotide). These CpG sites are rarer in the human genome than their predicted frequency, presumably because they are eliminated during evolution through C to T mutations of methylcytosine (14). The human genome, however, also contains small regions of DNA called CpG islands, in which the frequency of CpG is normal or higher than expected (14). About half of all human genes (including most housekeeping genes) have CpG islands in their 5-promoter regions. Of note, the promoter regions containing CpG islands are in fact usually unmethylated in normal tissues, regardless of the transcriptional status of the gene. CpG island methylation is associated with changes in chromatin organization and consequent repression of gene transcription (1). In normal tissues, CpG island methylation is limited to exceptional situations, such as imprinted alleles (12) and genes on the inactive X chromosome [13]. These well-studied exceptions to the rule of absent methylation at CpG islands suggest that, once established, gene silencing by CpG island methylation is physiologically irreversible during the lifetime of affected cells. A direct correlation between CpG island methylation and inhibited gene transcription is supported by the facts that 1) cells in which silencing occurs are usually transcriptionally competent for the affected genes [as demonstrated by normal expression of the unmethylated alleles and exogenously inserted unmethylated promoters], 2) demethylation by pharmacologic (15) or genetic [16] manipulation results in reactivation of gene expression, and 3) in vitro methylation substantially reduces gene expression in reporter experiments (1). The mechanism of CpG islandassociated gene silencing appears to involve binding of specific methylated DNA binding proteins, followed by recruitment of a silencing complex that includes histone deacetylases (Figure 3) (17, 18). Figure 3. Effects of methylation and histone deacetylation on gene expression and silencing. top black boxes ovals arrows (left m MBP bottom HDAC right top Aberrant CpG Island Methylation in Cancer Neoplastic cells often have simultaneous global DNA hypomethylation, localized hypermethylation that involves CpG islands, and increased levels of DNA methyltransferase activity (1). Hypomethylation was initially postulated to play a role in carcinogenesis through activation of oncogenes (19), but this hypothesis has not been experimentally confirmed. Hypomethylation has been linked to chromosomal instability in vitro (20), and it may play such a role in neoplasia. Aberrant CpG island hypermethylation in cancer, in contrast, is clearly associated with transcriptional silencing of gene expression, and increasing experimental data suggest that it plays an important role as an alternate mechanism by which tumor suppressor genes are inactivated in cancer (1, 2). Aberrant CpG island methylation in cancer was initially described for the calcitonin (21) and MyoD (22) genes. These two genes are not thought to play a tumor suppressive role in cancer, but these findings prompted additional investigations into the process. The first tumor suppressor gene shown to be inactivated by hypermethylation was the RB1 gene, in which methylation appeared to be a clear alternate to mutations and deletions for eliminating expression of functional protein (23). Several additional tumor suppressor genes have since been shown to be similarly inactivated in some cancers, including VHL (24), P16 (25), E-cadherin (26), and hMLH1 (27). For most of these genes, hypermethylation appears to provide a similar selective advantage as genetic inactivation and is usually associated with absence of coding region or promoter mutations of involved alleles. The list of genes that display hypermethylation-associated inactivation in some sporadic cancers has grown long (Table 1). Multiple cellular systems can be affected by this process, including cell growth and differentiation, cell cycle control, and DNA repair, as well as angiogenesis and invasion. However, hypermethylation in cancer is not invariably associated with repressed transcription. In some cases, the involved CpG island is not in the promoter of the genes (28). In other cases, methylation involves genes that are not normally expressed in the diseased tissues (29). In still others, methylation is relatively sparse, and although it can easily be detected experimentally, it does not lead to substantial decreases in gene expression. Aberrant methylation in cancer therefore functions as a mechanism of generating molecular diversity in neoplasia. In a manner analogous to mismatch repair defects in cancer, methylation defects affect many different loci, only some of which are pathophysiologically relevant to the neoplastic process (30). Table 1. Genes That Are Hypermethylated in Sporadic Cancers The causes of aberrant methylation in cancer remain poorly defined. Both hypomethylation and methyltransferase activation can occur in cells that are induced to proliferate (31), and it is not clear whether the observed changes in malignant cells simply reflect cell cycle deregulation. De novo CpG island methylation, however, is not a feature of proliferating cells, and it appears to represent a true pathologic event in neoplasia. For many genes, hypermethylation begins in normal tissues during the process of aging (32), which may partially explain the dramatic increase in cancer incidence associated with aging. Other genes are methylated exclusively in malignant cells and are presumed to arise from rare chance events that lead to gene inactivation and a selective advantage for affected cells (1, 2). Recent data in multiple neoplasms suggest that some cancers have a high degree of de novo methylation compared with others (33), and specific genetic defects or exposure events may explain these differences. In particular, acute and chronic leukemias have a high degree of aberrant CpG island methylation; genes involved include the cell-cycle regulator p15 (34), the p53 homologue p73 (35), the drug-resistance gene MDR1 (36), ER (37), and HIC1 (38). Therefore, hematologic malignant conditions present unique opportunities for studying the clinical implications of aberrant methylation. Rationale for Use of Methylation Inhibitors in Neop

Multicenter Independent Assessment of Outcomes in Chronic Myeloid Leukemia Patients Treated With Imatinib

BACKGROUND Imatinib slows development of chronic myeloid leukemia (CML). However, available information on morbidity and mortality is largely based on sponsored trials, whereas independent long-term field studies are lacking. PATIENTS AND METHODS Consecutive CML patients who started imatinib treatment before 2005 and who were in complete cytogenetic remission (CCyR) after 2 years (± 3 months) were eligible for enrollment in the independent multicenter Imatinib Long-Term (Side) Effects (ILTE) study. Incidence of the first serious and nonserious adverse events and loss of CCyR were estimated according to the Kaplan-Meier method and compared with the standard log-rank test. Attainment of negative Philadelphia chromosome hematopoiesis was assessed with cytogenetics and quantitative polymerase chain reaction. Cumulative incidence of death related or unrelated to CML progression was estimated, accounting for competing risks, according to the Kalbleisch-Prentice method. Standardized incidence ratios were calculated based on population rates specific for sex and age classes. Confidence intervals were calculated by the exact method based on the χ(2) distribution. All statistical tests were two-sided. RESULTS A total of 832 patients who were treated for a median of 5.8 years were enrolled. There were 139 recorded serious adverse events, of which 19.4% were imatinib-related. A total of 830 nonserious adverse events were observed in 53% of patients; 560 (68%) were imatinib-related. The most frequent were muscle cramps, asthenia, edema, skin fragility, diarrhea, tendon, or ligament lesions. Nineteen patients (2.3%) discontinued imatinib because of drug-related toxic effects. Forty-five patients lost CCyR, at a rate of 1.4 per 100 person-years. Durable (>1 year) negative Philadelphia chromosome hematopoiesis was attained by 179 patients. Twenty deaths were observed, with a 4.8% mortality incidence rate (standardized incidence ratio = 0.7; 95% confidence interval = 0.40 to 1.10, P = .08), with only six (30%) associated with CML progression. CONCLUSIONS In this study, CML-related deaths were uncommon in CML patients who were in CCyR 2 years after starting imatinib, and survival was not statistically significantly different from that of the general population.

Autonomous Proliferation of Leukemic Cells in Vitro as a Determinant of Prognosis in Adult Acute Myeloid Leukemia

BACKGROUND AND METHODS A characteristic of acute myeloid leukemia is the frequent ability of the leukemic cells to sustain their own proliferation in vitro. To determine the clinical importance of this property, we measured the uptake of tritiated thymidine by leukemic cells in serum-free and cytokine-free cultures as a means of determining the rate of spontaneous proliferation in 114 patients with newly diagnosed acute myeloid leukemia. Proliferation was then classified according to three quantitative levels of activity and related to overall survival and to treatment outcome (the response to treatment, the actuarial probability of relapse, and disease-free survival) in 91 patients who were treated with chemotherapy to induce remission. RESULTS Of the 114 patients, 37 had low, 39 had intermediate, and 38 had high levels of proliferation. The probability of survival at three years was 36 percent among patients with low levels of proliferative activity and 3 percent among those with high levels (P < 0.001). Among the patients treated with chemotherapy, those with low rates of proliferative activity had a 68 percent rate of complete remission and a 49 percent probability of remaining free of relapse, whereas those with high rates of proliferative activity had only a 39 percent rate of complete remission (P = 0.04) and an 11 percent probability of remaining in complete remission (P = 0.009). The probability of disease-free survival at three years among the patients in complete remission after chemotherapy was 49 percent among those with low rates of proliferative activity and 9 percent among those with high rates (P = 0.004). Accordingly, patients with low rates of proliferative activity also had a significantly higher rate of overall survival (44 percent vs. 4 percent; P = 0.002). Patients whose cells had intermediate levels of proliferation in vitro had intermediate rates of survival, relapse, and disease-free survival. CONCLUSIONS The capacity of leukemic blasts for autonomous proliferation is associated with highly aggressive acute myeloid leukemia.

Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia.

Variable proportions of mutant alleles are found in patients with JAK2 (V617F)-positive myeloproliferative disorders. This study shows that this variable mutant allele burden influences the clinical phenotype of JAK2 (V617F)-positive essential thrombocythemia. Background Fifty to sixty percent of patients with essential thrombocythemia harbor the JAK2V617F mutation. The impact of this mutation on clinical phenotype is still debated. The aim of this study was to evaluate possible correlations between JAK2V617F mutant allele burden and both clinical presentation and hematologic abnormalities in essential thrombocythemia patients. Design and Methods In this single-center retrospective study, JAK2V617F allele load was measured by sensitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the granulocytes of 260 patients diagnosed as having essential thrombocythemia according to WHO criteria. Results Median V617F allele burden in patients with the mutation (n=165, 63.4%) was 24%, ranging from 1% to 87%; an allele burden greater than 51% was found in 5% of the patients. Older patients presented progressively higher percentages of the V617F allele. Signs of stimulated erythropoiesis and myelopoiesis, as well as higher PRV-1 levels, were found in patients with the mutation, but no linear correlation with load of mutant allele could be ascertained; on the other hand, the frequency of patients with erythropoietin-independent erythroid colonies progressively increased depending on mutant allele load. Splenomegaly and microvessel symptoms were significantly more represented among patients with greater than 50% and 25% JAK2V617F allele burden, respectively. Increasing mutant allele load correlated with higher frequency of arterial thrombosis at diagnosis, as confirmed also in multivariate analysis; the relative risk was 3.0 (95% CI 1.3–6.8; p=0.01) in patients having a greater than 25% mutant allele burden. Conclusions The JAK2V617F mutant allele burden contributes to determining the clinical phenotype in patients with essential thrombocythemia.

Homoharringtonine: History, current research, and future directions

Cephalotoxine esters, including homoharringtonine (HHT), have shown encouraging activity in leukemia in initial studies in China and in later studies in the U.S.BACKGROUND Cephalotoxine esters, including homoharringtonine (HHT), have shown encouraging activity in leukemia in initial studies in China and in later studies in the U.S. METHODS The authors conducted a review of the literature to examine the studies pertinent to HHT in relation to preclinical studies and Phase I-II trials in patients with hematologic malignancies and solid tumors. RESULTS HHT and analogues appear to induce differentiation and apoptosis. Studies from China reported high response rates in patients with leukemia. Trials in the U.S. using short HHT infusions (3-4 mg/m(2) daily for 5 days) resulted in a high incidence of cardiovascular complications that were reduced using continuous infusion schedules of 3-7 mg/m(2) daily for 5-7 days initially, and later lower dose schedules of 2.5 mg/m(2) daily for 7-14 days. Results in solid tumors were negative. However encouraging results were reported in patients with acute myeloid leukemia, myelodysplastic syndrome, acute promyelocytic leukemia, and, most important, chronic myeloid leukemia (CML). In CML patients, HHT has been investigated alone and in combination with interferon-alpha and low-dose cytarabine in late and early chronic phases, with positive results. Additional areas of interest include the potential use of HHT for the treatment of central nervous system leukemia, polycythemia vera, and other nonmalignant conditions such as malaria. New semisynthetic preparations and HHT derivatives that bypass multidrug resistance may improve the efficacy and toxicity profiles, and broaden the range of antitumor efficacy. CONCLUSIONS HHT and its derivatives appear to have promising activity in hematologic malignancies, a finding that needs to be pursued.

Continued Azacitidine Therapy Beyond Time of First Response Improves Quality of Response in Patients With Higher-Risk Myelodysplastic Syndromes

In the AZA‐001 trial, azacitidine (75 mg/m2/d subcutaneously for Days 1‐7 of every 28‐day cycle) demonstrated improved survival compared with conventional care regimens in patients with International Prognostic Scoring System‐defined intermediate‐2‐ or high‐risk myelodysplastic syndrome and World Health Organization‐defined acute myeloid leukemia with 20% to 30% bone marrow blasts.

Management and supportive care measures for adverse events in patients with myelodysplastic syndromes treated with azacitidine

Objective:  Myelodysplastic syndrome (MDS) treatment can initially worsen patients’ clinical condition and they may discontinue therapy before achieving benefit. We present previously unpublished data from two large phase III trials describing common adverse events (AEs) associated with azacitidine and methods to manage them.

Clinical management of myelodysplastic syndromes: update of SIE, SIES, GITMO practice guidelines

Since 2002, date of publication of the previous Italian Society of Haematology (SIE) practice guidelines for management of myelodysplastic syndromes (MDS), novel disease-modifying treatments have been introduced and the SIE commissioned an update. After a comprehensive review of the medical literature published since January 2001, the Expert Panel formulated recommendations for the management of adult and paediatric MDS, graded according to the available evidence. The major updates are: first-line hypomethylating agents in patients with INT2-high-risk disease; controlled use of first-line lenalidomide in low-INT1 risk transfusion-dependent patients with 5q deletion; deferasirox in low-INT1 patients with a relevant transfusional load; first-line high-dose ESA in low-INT1 patients with Hb <10 g/dl and endogenous EPO <500 U/l; allogeneic HSCT first-line therapy for INT2- and high-risk patients <65 years without severe co morbidities.