Aberrant CpG island methylation in early-onset sporadic gastric carcinoma

Purpose: Gastric carcinoma more commonly affects older patients, and it is thought that cases of early-onset gastric carcinoma may develop with a different molecular profile different from that of carcinoma occurring at a later age. We assayed the methylation status and genetic changes in genes associated with the APC-β-catenin axis and the mismatch repair system in relatively early-onset gastric carcinoma samples to determine their association with gastric carcinogenesis. Methods: Tumor and normal tissue DNA samples were obtained from 40 patients with early-onset (< 50 y) gastric carcinomas and assayed for APC and CTNNB1 mutations, microsatellite instability, and methylation of the promoters of the hMLH1, TIMP3, THBS1, DAP- K, GSTP1 , APC, and MINT2. Results: Promoter methylation at these seven loci ranged from 12.5 to 62%, with 38/40 tumors (95%) showing promoter methylation at more than one locus. The CpG island methylation phenotype (CIMP) was classified as high in 16 tumors (40%), low in 22 tumors (55%), and negative in 2 tumors (5%). Two concurrent missense mutations (E1685G, R1763L) in the APC mutation cluster region were detected in two tumors, nine tumors showed loss of APC heterozygosity (LOH), and two showed both LOH and promoter methylation. Conclusions: Our results indicate that, unlike in colorectal carcinoma, APC and CTNNB1 mutations do not appear to be highly implicated in early-onset gastric carcinogenesis. In contrast, our data show that promoter methylation is a prevalent phenomenon in early-onset gastric carcinoma and may be related to gastric carcinogenesis.     

Aberrant methylation of multiple tumor suppressor genes in acute myeloid leukemia

Hypermethylator phenotype, a propensity of tumors to incur nonrandom concurrent methylation, has been described in several tumors, including acute myeloid leukemia (AML). More recent studies identified methylation of other tumor suppressor genes, DAP-kinase and SOCS1, singly in AML. We therefore assessed the methylation status of these genes concurrently with other known targets of methylation. We used methylation-specific PCR or COBRA to determine the extent of methylation of 10 genes in 28 AML samples from Turkey. In addition to DAP-kinase and SOCS1, we included ER, p15, and E-cadherin (reported to be frequently methylated) as well as p16, GSTP1, and HIC1 (reported as rarely methylated). We also included RARbeta and p73 for which only minimal data in AML is available. All samples were methylated at least in one locus and all except one demonstrated methylation of DAP-kinase, SOCS1, p15, and/or ER. DAP-kinase is the most frequently methylated gene in both pediatric (70%) and adult AML (55%). RARbeta is methylated in 18% and p73 in 10% of AMLs. Methylation of E-cadherin and RARbeta occurs preferentially in AMLs with high methylation index (MI), while epigenetic lesions in SOCS1, DAP-kinase, and p15 appear to be independent. MI may be age-dependent, with a peak in young adults. FAB M3 demonstrated a higher extent of methylation than M2/M4. This study provides an impetus for larger studies to define if the extent and pattern of methylation in subgroups of AML are clinically relevant.     

Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes

Death-associated protein kinase (DAP-kinase), a proapoptotic serine/threonine kinase, is a candidate tumor suppressor gene. We studied the methylation status of DAP-kinase of 194 bone marrow samples from 160 patients with acute myeloid leukemia (AML) and 34 with a myelodysplastic syndrome (MDS) at the time of initial diagnosis by polymerase chain reaction (PCR). Hypermethylation of DAP-kinase was present in 27.5% (44 of 160) of AML and in 47% (16 of 34) of MDS specimens and significantly correlated to loss of DAP-kinase expression (P =.008). It was significantly more frequent in AML secondary to therapy for other malignancies (s-AML; 14 of 29, 48.3%), as compared to de novo AML (30 of 131, 22.9%, P =.01). DAP-kinase hypermethylation in AML was associated with myelodysplastic changes in the bone marrow at the time of the initial diagnosis (P =.002) and with the presence of cytogenetic abnormalities (P =.02). Alteration in the apoptotic response due to the loss of DAP-kinase function may be an early event in the transformation pathway to secondary leukemia via myelodysplasia.     

Prognostic factors at relapse for adults with acute myeloid leukemia

To identify prognostic factors for patients with acute myeloid leukemia in first relapse we have analyzed the courses of 57 patients undergoing intensive reinduction therapy. The median duration of first remission was 9 months. A second complete remission was achieved by 31 patients (54%). The median duration of second remission was 5 months (range 1-142 + months). Two patients are still disease-free with 42 months and 142 months follow-up. Clinical, laboratory, and treatment variables at the time of original diagnosis and at relapse were examined for possible associations with the outcome of reinduction chemotherapy. Only duration of first remission consistently correlated with ability to achieve a second remission, second remission duration, and overall survival from reinduction. The second complete remission rate was 69% for those with a first remission longer than 9 months but only 39% for those with a first remission less than 9 months (P less than 0.05). The patients with longer first remissions also had longer second remissions (8 months median vs. 4 months, P = 0.02) and a longer median survival following reinduction chemotherapy (8 months vs. 5 months, P = 0.02). We conclude that the duration of a patient's first remission should be considered a useful prognostic factor both in treatment planning and in the interpretation of clinical trials of patients in relapse.     

Predicting aberrant CpG island methylation

Epigenetic silencing associated with aberrant methylation of promoter region CpG islands is one mechanism leading to loss of tumor suppressor function in human cancer. Profiling of CpG island methylation indicates that some genes are more frequently methylated than others, and that each tumor type is associated with a unique set of methylated genes. However, little is known about why certain genes succumb to this aberrant event. To address this question, we used Restriction Landmark Genome Scanning to analyze the susceptibility of 1,749 unselected CpG islands to de novo methylation driven by overexpression of DNA cytosine-5-methyltransferase 1 (DNMT1). We found that although the overall incidence of CpG island methylation was increased in cells overexpressing DNMT1, not all loci were equally affected. The majority of CpG islands (69.9%) were resistant to de novo methylation, regardless of DNMT1 overexpression. In contrast, we identified a subset of methylation-prone CpG islands (3.8%) that were consistently hypermethylated in multiple DNMT1 overexpressing clones. Methylation-prone and methylation-resistant CpG islands were not significantly different with respect to size, C+G content, CpG frequency, chromosomal location, or promoter association. We used DNA pattern recognition and supervised learning techniques to derive a classification function based on the frequency of seven novel sequence patterns that was capable of discriminating methylation-prone from methylation-resistant CpG islands with 82% accuracy. The data indicate that CpG islands differ in their intrinsic susceptibility to de novo methylation, and suggest that the propensity for a CpG island to become aberrantly methylated can be predicted based on its sequence context.     

CpG island methylation of the hTERT promoter is associated with lower telomerase activity in B-cell lymphocytic leukemia

OBJECTIVE: Expression of the catalytic subunit of the telomerase enzyme hTERT is essential for prolonging the replicative lifespan and is the rate-limiting step in cellular immortalization and carcinogenesis. Because hTERT expression is positively correlated with telomerase activity, its regulation is suggested as the major determinant of enzymatic activity. The hTERT promoter region contains two CpG islands, which are known to be target sites for de novo DNA methylation. To elucidate the impact of this epigenetic mechanism on telomerase activity, we analyzed the degree of hTERT promoter methylation in 30 patients with B-cell chronic lymphocytic leukemia. MATERIALS AND METHODS: hTERT promoter methylation was assessed using a methylation-specific competitive polymerase chain reaction assay. The assay is based on digestion of genomic DNA with a methylation-sensitive restriction enzyme before amplification with an internal standard. RESULTS: Patients exhibiting high telomerase activity showed significantly less methylation of the hTERT promoter core domain than patients with low enzyme activity. In addition, telomerase activity was significantly associated with telomere length and overall survival. CONCLUSIONS: Our data show that the degree of CpG island methylation of the hTERT promoter exhibits an impact on telomerase activity in a subgroup of patients with B-cell chronic lymphocytic leukemia and therefore is assumed to play a role in regulating hTERT gene expression in these patients.     

Aberrant methylation of the major breakpoint cluster region in chronic myeloid leukemia

Isolated hypomethylated sites exist in the major breakpoint cluster region (M-bcr) where most Philadelphia chromosome (Ph) breakpoints are located. Twenty of 50 (40%) chronic myeloid leukemia (CML) patients were found to have aberrant hypermethylation of these sites on the rearranged M-bcr when compared with control marrows. The aberrancy correlated strongly with M-bcr breakpoint location; 19 of 20 cases had breakpoints located 5' of the M-bcr Sca I site, and 28 of 30 cases with normal M-bcr methylation had breakpoints located 3' of the M-bcr Sca I site. Sequence analysis of the Ph M-bcr breakpoints failed to find an M- bcr nucleotide position that delineated the transition between abnormally and normally methylated cases, indicating that the translocation of a critical M-bcr sequence was not responsible for the methylation abnormality. In 3 of 8 CML patients, cells without the t(9;22) were found to have abnormally methylated, unrearranged M-bcrs. The data indicate that abnormally methylated rearranged M-bcrs are present in CML cases with Ph breakpoints 5' of the M-bcr Sca I site and that the M-bcr in Ph- cells of patients with CML may also be abnormally methylated.     

Immunotherapeutic strategies for relapse control in acute myeloid leukemia

Despite that the initial phases of chemotherapy induce disappearance of leukemic cells in many patients with acute myeloid leukemia (AML), the prevention of life-threatening relapses in the post-remission phase remains a significant clinical challenge. Allogeneic bone marrow transplantation, which is available for a minority of patients, efficiently prevents recurrences of leukemia by inducing immune-mediated elimination of leukemic cells, and over the past decades, numerous immunotherapeutic protocols have been developed aiming to mimic the graft-versus-leukemia reaction for the prevention of relapse. Here we review past and present strategies for relapse control with focus on overcoming leukemia-related immunosuppression in AML. We envisage future treatment protocols, in which systemic immune activators, such as vaccines, dendritic cell-based therapies, engineered variants of IL-2, or IL-15, are combined with agents that counter immunosuppression mediated by, e.g., the PD/PDL interaction, CTLA-4, CD200, reactive oxygen species, IDO expression, CXCR4, or the KIR/class I interaction, based on characteristics of the prevailing malignant clone. This combinatorial approach may pave the way for individualized immunotherapy in AML.     

Central nervous system involvement at first relapse in patients with acute myeloid leukemia

The risk factors for and incidence of central nervous system involvement at first relapse in adult patients with acute myeloid leukemia have not been established. This single-center study analyzed the prognostic factors for and cumulative incidence of meningeal relapse in 458 adult patients achieving complete remission. Before 1990, patients received old chemotherapy approaches without stem cell transplantation that often included prophylactic intrathecal chemotherapy. Since 1990, modern protocols included stem cell transplantation without intrathecal prophylaxis. Meningeal relapse occurred in 6 patients (overall 5-year cumulative incidence 1.3%). The 5-year cumulative incidence of meningeal relapse in patients treated with old and modern protocols were 3.9% and 0.3%, respectively. Univariate and multivariate analyses showed that the chemotherapy approach was the main prognostic factor for central nervous system relapse (P=0.02). This study shows an extremely low incidence of meningeal relapse in adult patients with acute myeloid leukemia treated with modern protocols including stem cell transplantation without intrathecal prophylaxis.     

Updates on DNA methylation modifiers in acute myeloid leukemia

Annals of Hematology - Acute myeloid leukemia (AML)&nbsp;is the most common acute leukemia in adults. Chemotherapy with cytotoxic agents is the standard of care, but is associated with a high...     

Busulfan pharmacokinetics do not predict relapse in acute myeloid leukemia

The purpose of this study was to evaluate the influence of busulfan (BU) pharmacokinetics on survival, grades II-IV acute graft-versus-host disease (GVHD), non-relapse mortality (NRM) and relapse in a group composed of 45 children (<18 years) and seven adults with acute myeloid leukemia (AML) in first complete remission and undergoing hematopoietic stem cell transplantation (SCT). Fifty-two patients underwent autologous (n = 25) or allogeneic (n = 27) SCT. The median age was 8.9 years (range 0.6-53 years). Conditioning therapy consisted of BU and cyclophosphamide. Improved disease-free survival was found in those patients with a steady-state concentration of BU (CssBU) below the median (<578 mg/ml, P = 0.05), and the same trend was noted for overall survival (P = 0.07). This was secondary to a higher incidence of NRM in the group of patients with CssBU above the median (P = 0.06). There was no significant correlation with CssBU and relapse (P = 0.31). No association between CssBU and GVHD was found in allogeneic patients (P = 0.30). Relapse was evaluated among the subgroups of age (< or >10 years) and transplant type (allogeneic or autologous) with no statistically significant association observed among these factors. Multiple regression analysis for relapse revealed no significant correlation with CssBU above or below the median, age, or transplant type. In this study, CssBU below the median did not correlate with an inferior outcome for patients with AML. Pharmacokinetic dosing of BU may be important for prevention of NRM but does not appear to influence the risk of relapse in this largely pediatric population with AML.     

Genome-wide methylation profiling in decitabine-treated patients with acute myeloid leukemia

The outcome of older (≥ 60 years) acute myeloid leukemia (AML) patients is poor, and novel treatments are needed. In a phase 2 trial for older AML patients, low-dose (20 mg/m(2) per day for 10 days) decitabine, a DNA hypomethylating azanucleoside, produced 47% complete response rate with an excellent toxicity profile. To assess the genome-wide activity of decitabine, we profiled pretreatment and post treatment (day 25/course 1) methylomes of marrow samples from patients (n = 16) participating in the trial using deep-sequencing analysis of methylated DNA captured by methyl-binding protein (MBD2). Decitabine significantly reduced global methylation compared with pretreatment baseline (P = .001). Percent marrow blasts did not correlate with global methylation levels, suggesting that hypomethylation was related to the activity of decitabine rather than to a mere decrease in leukemia burden. Hypomethylation occurred predominantly in CpG islands and CpG island-associated regions (P ranged from .03 to .04) A significant concentration (P < .001) of the hypomehtylated CpG islands was found in chromosome subtelomeric regions, suggesting a differential activity of decitabine in distinct chromosome regions. Hypermethylation occurred much less frequently than hypomethylation and was associated with low CpG content regions. Decitabine-related methylation changes were concordant with those previously reported in distinct genes. In summary, our study supports the feasibility of methylome analyses as a pharmacodynamic endpoint for hypomethylating therapies.     

Outcome of older patients with acute myeloid leukemia in first relapse

To provide data for future drug evaluation, we analyzed the outcome of 393 patients aged 50 years or older (median, 64 years) with AML in first relapse after treatment in recent ALFA trials. Salvage options were retrospectively classified as follows: best supportive care (BSC), low‐dose cytarabine (LDAC), gemtuzumab ozogamicin (GO), intensive chemotherapy (ICT), or ICT combined with GO. Second complete remission (CR2) rate was 31% and median post‐relapse survival was 6.8 months (0, 17, 42.5, 53, and 80% and 3.2, 5.6, 8.9, 9, and 19.8 months in BSC, LDAC, GO, ICT, and ICT + GO subsets, respectively). Age, performance status, WBC, CR1 duration, and favorable AML karyotype, but not other cytogenetic or molecular features, influenced post‐relapse outcome. Multivariate adjustment and propensity score matching showed that intensive salvage (ICT/ICT+GO/GO versus LDAC/BSC) was associated with longer post‐relapse survival, at least in patients with CR1 duration ≥12 months (P = 0.001 and 0.0005, respectively). Of interest, GO appeared to be as effective as standard ICT, and ICT + GO combination more effective than standard ICT. In conclusion, older patients with CR1 duration ≥12 months appeared to benefit from intensive salvage and results observed with GO‐containing salvage suggest that GO combination studies should be actively pursued in this setting. Am. J. Hematol. 88:758–764, 2013. © 2013 Wiley Periodicals, Inc.     

Characterization of childhood acute leukemia with multiple myeloid and lymphoid markers at diagnosis and at relapse

To define the clinical and biologic significance of childhood acute mixed-lineage leukemia diagnosed by stringent criteria, we studied 25 cases of acute lymphoblastic leukemia expressing greater than or equal to 2 myeloid-associated antigens (My+ ALL), and 16 cases of acute myeloid leukemia expressing greater than or equal to 2 lymphoid associated antigens (Ly+ AML). These cases represented 6.1% of 410 newly diagnosed ALLs (two treatment protocols) and 16.8% of 95 AMLs (two protocols). T-lineage--associated antigens were identified in 9 of the My+ ALL cases and in 14 of those classified as Ly+ AML; all but 1 of the 19 cases that could be subclassified had an early thymocyte stage of differentiation. The My+ ALL cases had an increased frequency of French-American-British (FAB) L2 morphology (36%); the Ly+ AML cases were characterized by FAB M1 or M2 morphology, low levels of myeloperoxidase reactivity and combined populations of myeloperoxidase-positive large blasts and small blasts generally of hand-mirror morphology. Karyotypic abnormalities included t(9;22)(q34;q11) in three cases of My+ ALL, 11q23 translocations in two cases of My+ ALL, and 14q32 translocations in three My+ ALL and five Ly+ AML cases. Mixed-lineage expression lacked prognostic significance in either ALL or AML; however, the findings indicate that some patients with Ly+ AML may respond to prednisone, vincristine, and L-asparaginase after failing on protocols for myeloid leukemia. At relapse, two My+ ALLs had converted to AML and two Ly+ AMLs to ALL; one case in each group showed complete replacement of the original karyotype. Acute mixed-lineage leukemia does not adequately describe the heterogeneity of the cases identified in this study and should be replaced by a set of more restrictive terms that indicate the unique biologic features of these leukemias.