Aberrant Methylation Of CpG Islands Research Articles

Identification of 20 genes aberrantly methylated in human breast cancers

Aberrant methylation of CpG islands (CGI) not only plays a role in gene silencing, but is also a potential cancer biomarker. To identify more CGI aberrantly methylated in human breast cancers, we carried out a genome-wide search for aberrant methylation, using methylation-sensitive-representational difference analysis. CGI in 5' upstream regions of 20 genes, TSPAN-2, AK5, LOC284999, HOXD11, FLJ25161, XT3, PCDH10, PCDHGB6, SIM1, LOC346978, COE2, TDH (FLJ25033), LOC346419, FLJ33790, GJB2, AMN, LOC201164, DLX4, DCC and FOXA2, were found to be methylated in at least one of 8 breast cancer cell lines. Fifteen of the 20 genes were methylated in more than one of 21 primary breast cancers in Stages I or II, and especially, those of LOC346978, HOXD11, SIM1, PCDHGB6 and FLJ25161 were methylated in more than 10 cancers. All the breast cancers had some aberrant methylation. Among the 13 genes whose CGI were completely methylated in one or more cell lines, FOXA2 and XT3 were expressed in normal human mammary epithelial cells (HMEC) and were not expressed in cancer cell lines with complete methylation. The other 11 genes examined were barely expressed, or not expressed even in HMEC. Our results showed that breast cancer cells accumulate aberrant methylation of the CGI identified here. This may serve as markers for early-stage breast cancers and suggests that aberrant methylation targets transcriptionally inactive genes in vivo.

Diagnostic and Therapeutic Applications of Epigenetics

Epigenetic abnormalities, such as aberrant methylation of CpG islands, are inherited over cell divisions, and play important roles in carcinogenesis. Aberrant methylation of CpG islands specific to tumor cells can be used as a marker to detect cancer cells or cancer-derived DNA, taking advantage of the high sensitivity of methods to detect aberrant methylation. Methylations of specific genes or methylation patterns of groups of genes were found to be associated with responses to chemotherapeutics and prognosis. Methylation in non-cancerous tissues is now attracting attention as a tumor risk marker, and is emerging as a target for cancer prevention. Epigenetic alterations are potentially reversible. The use of DNA demethylating agents has turned out to be effective for hematological malignancies, and is being tested in solid tumors. Histone deacetylase inhibitors and methods for gene-specific epigenetic modification are being developed. Application of epigenetics to cancer diagnostics and therapeutics, and possibly to cancer prevention, is coming into clinics.

Identification of epigenetic aberrant promoter methylation in pleural fluid DNA is useful for diagnosis of malignant pleural effusion

9567 Lung cancer and malignant mesothelioma are major cause of pleural effusion, and management of lung cancer patients with pleural effusion requires effective treatment based on timely and accurate diagnosis. Thus, differentiating between malignant and nonmalignant pleural effusions is a critical clinical problem, and conventional methods have proven to be inadequate. Aberrant methylation of CpG islands, one of the major mechanisms to inactivate tumor suppressor genes in human cancers, has been found in lung cancer and other malignancies. Recent studies revealed that detection of aberrant methylation of DNA in serum or BAL might be available to diagnose malignancies. Based on these studies, we investigated to identify aberrant promoter methylation of DNA in pleural effusion and evaluate the usefulness of this approach for differential diagnosis of pleural effusion. We examined methylation status of RARβ, p16INK4a, DAPK, RASSF1A, and MGMT in 81 pleural effusion samples using methylation-specific PCR. Forty-seven patients were given a diagnosis of malignancies (median age 69 years; range 29–87; male/female 35/12). 34 patients were non-malignant pleural effusion. In patients with malignancies, DNA methylation of pleural effusion was detected in 6.4% for MGMT, 21.3% for p16INK4a, 27.7% for RASSF1A, 29.8% for DAPK, and 40.4% for RARβ. Of 47 patients with malignancies, 34 (72.3%) had methylation of at least one gene. The total number of methylation in five genes per patient was significantly higher in malignancies (1.26 genes/patient) than in non-malignant pulmonary diseases (0.56 genes/patient, p<0.001). A crude unconditional logistic regression model correlating methylation status and risk of malignancies showed that the patients with at least one methylated gene showed 3.31 times higher probability of having mlignancies compared to patients without any methylated genes (p = 0.01). These results suggested that patients with methylation have significant high risk for malignancies,and identification of aberrant promoter methylation in pleural effusion DNA could be a useful tool for differential diagnosis of pleural effusion. No significant financial relationships to disclose.

Promoter hypermethylation of TMS1 and DAPK were correlated to low chemosensitivity and poor prognosis in patients with recurrent gastric cancer

4280 Background: Promoter hypermethylation plays an important role in the inactivation of tumor suppressor genes during tumorigenesis. Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers. Target of methylation-induced silencing (TMS1) is a novel CpG island-associated gene and induces apoptosis through activation of caspase-9 and inhibited the survival of human breast cancer cells. Methylation of the apoptosis-associated gene death-associated protein kinase (DAPK) has been recently described in stage I lung cancer, in which it portends a poor prognosis. In the present study, promoter hypermethylation of TMS1 and DAPK were investigated and in gastric cancer specimen and compared to chemosensitivity and patient’s prognosis. Methods: Methylation status of forty-one gastric cancer specimens, all of which were formalin-fixed and paraffin embedded, were studied by methylation specific PCR. All the patients were administered 5-FU based chemotherapy for the treatment of recurrent lesion. Results: Hypermethylation of TMS1 and DAPK was observed 36.7% and 37.1% of the specimen, respectively. In TMS1 (DAPK) unmethylated group, 42.3 (45.5) % of the patients responded to the chemotherapy, while 20.0 (23.1)% responded in TMS1 (DAPK) methylated group. Patients with both TMS1 and DAPK methylated cancer showed significant poor survival than the others (p=0.0452, Log-rank test). Conclusions: Promoter hypermethylation of TMS1 and DAPK were supposed to be negative predictive and prognostic factors in patients with recurrent gastric cancer, probably through preventing apoptosis. No significant financial relationships to disclose.

CpG island methylation in aberrant crypt foci and cancers from the same patients

Aberrant methylation of CpG islands is a common alteration in human colon cancer. Methylation of only a limited number of loci has been studied in aberrant crypt foci (ACF), the earliest identified premalignant lesions in the colon, and in only a limited number of lesions from the same patients. Methylation-specific PCR was used to analyze 35 ACF, samples of normal crypts with the same number of crypts as were in each ACF and 22 cancers from the same patients. Aberrant methylation in cellular retinol-binding protein 1 (CRBP1), MINT31 or H-cadherin (CDH13) was identified in 19 of 35 (54%) ACF. Hypermethylation of CRBP1, MINT31 or CDH13 was more frequent (15 of 22, 68%) in cancers. DNA methylation of CRBP1, MINT31 or CDH13 was not correlated between cancers and ACF from the same patients. Aberrant methylation was not correlated with patient age, number of crypts in the ACF or cancer stage. These results suggest that hypermethylation in the promoter region of some genes is an independent event, occurs early in human colon tumorigenesis and may play an important role during the transformation and progression of some lesions to colon cancers.

Modifications épigénétiques et cancer

Epigenetics is defined as "the study of mitotically and/or meiotically heritable changes in gene expression that cannot be explained by changes in the DNA sequence". Setting up the epigenetic program is crucial for correct development and its stable inheritance throughout its lifespan is essential for the maintenance of the tissue- and cell-specific functions of the organism. For many years, the genetic causes of cancer have hold centre stage. However, the recent wealth of information about the molecular mechanisms which, by modulating the chromatin structure, can regulate gene expression has high-lighted the predominant role of epigenetic modifications in the initiation and progression of numerous pathologies, including cancer. The nucleosome is the major target of these epigenetic regulation mechanisms. They include a series of tightly interconnected steps which starting with the setting ("writing") of the epigenetic mark till its "reading" and interpretation will result in long-term gene regulation. The major epigenetic changes associated with tumorigenesis are aberrant DNA methylation of CpG islands located in the promoter region of tumor suppressor gene, global genomic hypomethylation and covalent modifications of histone N-terminal tails which are protruding out from the nucleosome core. In sharp contrast with genetic modifications, epigenetic modifications are highly dynamic and reversible. The characterization of specific inhibitors directed against some key epigenetic players has opened a new and promising therapeutic avenue, the epigenetic therapy, since some inhibitors are already used in clinical trials.

Can Aberrant Promoter Hypermethylation of CpG Islands Predict the Clinical Outcome of Non-Small Cell Lung Cancer After Curative Resection?

Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one cause for the loss of gene function. We examined whether aberrant DNA hypermethylation could be used to predict the clinical outcomes of patients with primary nonsmall cell lung cancer (NSCLC) after curative resection. We tested 61 patients with NSCLC using methylation-specific polymerase chain reaction (MSP) and searched for promoter hypermethylation of the genes p16INK4a, retinoic acid receptor beta-promoter (RARbetaP2), death-associated protein kinase (DAPK), and O6-methylguanine-DNA-methyltransferase (MGMT). The clinical data, the presence of DNA hypermethylation, and clinical outcomes were analyzed. Hypermethylation in the tumor samples was detected in 67% (41 of 61) for p16(INK4a), 49% (30 of 61) for RARbetaP2, 30% (18 of 61) for DAPK, and 62% (38 of 61) for MGMT. Thirty patients (49%) developed recurrence within 33 months; 16 in the remaining lung, 10 in other organs, and 4 in both. We found no correlation between the specific DNA hypermethylation and any of the clinicopathological characteristics of the patients. DNA hypermethylation was not associated with a different survival or recurrence rate. However, the aberrant hypermethylation of RARbetaP2 seemed to be related to the location of cancer recurrence. Although advanced T stage and preoperative chemotherapy were statistically significant in univariate analysis, unmethylation of DAPK (p = 0.030) and hypermethylation of RARbetaP2 (p = 0.014), as well as advanced T stage (p = 0.075) and preoperative chemotherapy (p = 0.025), were significant risk factors in multivariate analysis for early recurrence in the remaining lung. The P2 hypermethylation of the RARbeta gene and unmethylation of DAPK seem to be important factors in predicting early cancer recurrence in the remaining lung and could be used as a prognostic marker in NSCLC. However, the clinical implications of this finding need further investigation.

Comparative epigenomics of leukemia

Proving that aberrant CpG island methylation has a functional role in human tumorigenesis is a chief goal in cancer epigenomics. A study now shows that a predictable mouse model of acute lymphocytic leukemia faithfully recapitulates the pattern, targets and frequency of aberrant methylation observed in its human counterpart and may soon allow the timing, and perhaps even the cause, of aberrant CpG island methylation to be investigated.

The fundamental role of epigenetics in hematopoietic malignancies

The term epigenetics defines a heritable alteration in gene expression without an accompanying change in primary DNA sequence. Two major mechanisms that foster epigenetic changes are DNA methylation at cytosine bases within a CpG dinucleotide and post-translational histone modifications. Disruption of the balanced epigenetic network may have significant impact on chromatin structure and transcriptional activity. DNA methylation patterns are profoundly deranged in human cancer and comprise genome-wide losses as well as regional gains in DNA methylation. Hypermethylation of CpG islands within gene promoter regions in collaboration with deacetylation and other modifications of key histone amino acids is associated with transcriptional inactivation and represents, in addition to genetic aberrations, an important mechanism of gene silencing in the pathogenesis of human cancer. These epigenetic events act as alternatives to mutations and deletions to disrupt tumor suppressor gene function. A large number of genes involving fundamental cellular pathways may be affected by aberrant CpG island methylation in association with transcriptional silencing in virtually all tumor types. Altered DNA methylation patterns may serve as biomarkers for cancer detection, assessment of prognosis, and prediction of response to therapy. Furthermore, clinical trials using epigenetically targeted therapies have yielded promising results in hematopoietic malignancies. The ongoing exploration of basic events involved in altered gene transcription patterns and continued clinical investigative studies are helping to develop novel strategies for the diagnosis, prevention, and treatment of human cancer.

Adjuvant Treatment Strategies for Early Colon Cancer

Colon cancer remains a major cause of death; however, in the last 3 years a number of trials have been published that have led to changes in the treatment of patients with this disease. Initially, the adjuvant treatment of patients following curative resection was based on their Dukes staging; this is now being refined by consideration of other pathological factors, as well as the investigation of newer prognostic markers such as p53, Ki67 and a number of genes on chromosome 18. Tumours generally develop from the progressive accumulation of genetic events, although some develop through mutation or inactivation of DNA mismatch repair proteins leading to microsatellite instability; this is particularly important in Lynch's syndrome. The loss of gene expression can occur by deletion or mutation of genes or by aberrant methylation of CpG islands. In patients with Dukes C colon cancer the standard of care for adjuvant chemotherapy was previously based on bolus fluorouracil (5-fluorouracil) and folinic acid (leucovorin) administered 5 days per month or weekly for 6 months. Recent studies with a combination of infusional fluorouracil, folinic acid and oxaliplatin have been found to be superior. A further study replacing fluorouracil with oral capecitabine has also demonstrated equivalent disease-free survival. Although some debate remains regarding the benefit of adjuvant treatment for patients with Dukes B colon cancer, the emerging consensus is that, for those patients who are younger and have high-risk features, chemotherapy should be discussed. A number of large vaccine trials have also been conducted in the adjuvant setting and, overall, these have been disappointing. This is a rapidly advancing area of therapy and the results of new trials are awaited to determine whether additional benefits can be achieved with biological therapies such as anti-vascular endothelial growth factor and anti-epithelial growth factor receptor monoclonal antibodies, which have already been shown to be effective in setting of metastatic colon cancer.

Genetic and epigenetic alteration of the NF2 gene in sporadic meningiomas

The role of the NF2 gene in the development of meningiomas has recently been documented; inactivating mutations plus allelic loss at 22q, the site of this gene (at 22q12), have been identified in both sporadic and neurofibromatosis type 2-associated tumors. Although epigenetic inactivation through aberrant CpG island methylation of the NF2 5' flanking region has been documented in schwannoma (another NF2-associated neoplasm), data on participation of this epigenetic modification in meningiomas are not yet widely available. Using methylation-specific PCR (MSP) plus sequencing, we assessed the presence of aberrant promoter NF2 methylation in a series of 88 meningiomas (61 grade I, 24 grade II, and 3 grade III), in which the allelic constitution at 22q and the NF2 mutational status also were determined by RFLP/microsatellite and PCR-SSCP analyses. Chromosome 22 allelic loss, NF2 gene mutation, and aberrant NF2 promoter methylation were detected in 49%, 24%, and 26% of cases, respectively. Aberrant NF2 methylation with loss of heterozygosity (LOH) at 22q was found in five cases, and aberrant methylation with NF2 mutation in another; LOH 22q and the mutation were found in 16 samples. The aberrant methylation of the NF2 gene also was the sole alteration in 15 samples, most of which were from grade I tumors. These results indicate that aberrant NF2 hypermethylation may participate in the development of a significant proportion of sporadic meningiomas, primarily those of grade I.

How frequent is SOCS1 methylation in acute myeloid leukaemia?

A recent article by Watanabe et al (2004) described aberrant methylation of the suppressor of cytokine signalling (SOCS)-1 gene in 72% of primary acute myeloid leukaemia samples, and 52% of leukaemia cell lines. In their experiment, they employed methylation specific polymerase chain reaction (MSP) with a pair of primers flanking the CpG islands inside exon 2 of SOCS1. SOCS1 has two exons, an untranslated exon 1 and a translated exon 2. CpG islands exist both in the untranslated region (UTR) 5′ to, as well as inside the coding region of, exon 2 (Fig 1). Aberrant methylation of CpG islands in these two regions and its relationship to SOCS1 suppression have been studied (Yoshikawa et al, 2001; Chen et al, 2003; Fukushima et al, 2003; Galm et al, 2003; Liu et al, 2003; Nagai et al, 2003; Chim et al, 2004; Watanabe et al, 2004) (Fig 1). Initial reports investigated the CpG islands within exon 2. Later reports, however, focussed on CpG islands in the 5′UTR. We have recently shown that MSP with primers located in exon 2, as described by Watanabe et al (2004), detected methylation in five of 12 normal peripheral blood and two of three normal marrow samples (Chim et al, 2004). These results were confirmed by sequencing, suggesting that methylation within SOSC1 exon 2 might not be necessarily involved in regulation of gene transcription. The lack of impact of SOSC1 exon 2 methylation on gene expression was also shown by Watanabe et al (2004). In the two cell lines HL60 and U937, where complete methylation of CpG islands within SOCS1 exon 2 was present, there was still a substantial expression of SOCS1 (Fig 1, Watanabe et al, 2004), thus showing an incomplete correlation between methylation and gene silencing. In fact, methylation in CpG islands within exons are not uncommon and may occur in normal tissues, and is not associated with gene suppression. For instance, in a study involving the HIC1 gene, CpG island methylation inside exon 3 was present in both leukaemia samples and normal controls, and this was not associated with down regulation of HIC1 in normal samples. Schematic diagram of the SOCS1 gene and the positions of primers for methylation specific polymerase chain reaction (MSP). Primers are represented by arrows. Figure not drawn to scale. The frequencies of SOCS1 methylation in different malignancies as detected by different MSP primers are also shown. *Position of primers; MSP, methylation specific polymerase chain reaction; ANT, adjacent non-tumourous tissue; PB, peripheral blood; BM, bone marrow; NS, not specified; 5-AC, 5-azacytadine; UTR, untranslated region; CL, cell lines; PS, primary samples, ND, not done. On the other hand, we showed that with MSP using primers in the 5′UTR, no methylation was demonstrable in normal tissues. Using these primers, we were also unable to show aberrant SOSC1 methylation in any of the myeloma samples that we tested (Chim et al, 2004), nor in any of the leukaemia samples in another study. Therefore, the role of SOCS1 methylation in leukemogenesis must be considered with caution, particularly when results were obtained by investigating exon 2 CpG island methylation. To fully address this problem, SOCS1 exon 2 methylation should be confirmed by concomitant examination of methylation in the 5′UTR. This is because a methylation boundary might exist, which marks methylated and unmethylated regions in DNA of normal cells. If the SOCS1 exon 2 methylation was not restricted to neoplastic cells and occurred in normal cells, a methylation boundary between exon 2 and the 5′UTR might be observed, with the 5′UTR unmethylated. Secondly, methylation-induced silencing of SOCS1 is potentially associated with constitutive activation of Janus kinaseSTAT (signal transducers and activators of transcription) signalling, and thus elevation of phosphorylated STAT (pSTAT) levels. Therefore, SOCS1 down-regulation would be expected to give rise to inappropriate activation of pSTAT, whereas SOCS1 re-expression after gene demethylation would give rise to suppressed pSTAT activation. The investigations of these signalling pathways would address whether SOCS1 exon 2 methylation is biologically important.

Aberrant DNA Methylation of the Src Tyrosine Kinase Hck Is a Frequent Event in Human Leukemia and May Predict for Poor Prognosis in Adult Acute Lymphocytic Leukemia (ALL).

Aberrant DNA methylation of promoter-associated CpG islands is a frequent phenomenon in human leukemias, and in particular in adult ALL. Hck is a member of the Src family of tyrosine kinases, and functionally is located downstream of BCR-ABL signaling in chronic myelogenous leukemia (CML). Hck expression is limitedly to myeloid cells and B cell lymphocytes. Although some evidence indicates that Hck is required for malignant transformation and apoptosis, its role in leukemia is not fully understood. Here we analyze the role of aberrant DNA methylation of Hck in leukemia cell lines and patients. Using BLAT, we first identified the presence of a canonical CpG island in the near proximity of the transcription start site of HcK. To detect and measure DNA methylation, we designed a combined bisulfite restriction PCR assay. Using this assay, we found that Hck was methylated in 13 out of 23 hematopoietic and 8 out of 10 non-hematopoietic cell lines, but not in the bone marrow from 6 healthy individuals. We subsequently studied Hck expression by real-time PCR using GAPDH expression as an internal control. Hck expression was lower (dCT = −14.2± 3.6) in 7 Hck methylated cell lines than in 8 Hck unmethylated ones (dCT= −9.0± 3.5), p=0.017. All the cell lines studied were of myeloid or B cell origin. We then treated the Raji cell line with the hypomethylating agent 5-aza-2-deoxycytidine (DAC). DAC treatment resulted in partial hypomethylation of Hck and in an increment of Hck expression (dCT: −19.37 to −8.47). Subsequently, the effects of DAC treatment on Hck protein expression levels were analyzed using Western blot. These experiments showed a strong correlation between hypomethylation, gene re-expression and protein expression levels. These data therefore indicates that DNA methylation is an important aberrant regulator of Hck expression in leukemia cell lines. Based on the relevance of these findings, we then analyzed the frequency of Hck methylation in patients with leukemia. Using a cut-off of 10%, Hck was found to be methylated in 15 out of 44 (34%) patients with ALL, 9 out 23 pts (39%) with CML, and 3 out 10 pts (30%) with AML. Of importance, the density of Hck methylation was significantly higher in patients with ALL (mean 11.3%; range 0–76) compared to those with CML(5.2%; range 0–12) or AML ( 7.5%, range 0–14), p=0.02. Hck methylation was not associated with a B cell phenotype or the presence of the Philadelphia chromosome in patients with ALL. Nine ALL pts out of 15 with Hck methylation had died compared to 7 out 29 unmethylated (total ALL group n=34). Median survival had not been reached for the group of patients with no Hck methylation (n=29) compared to 116 weeks for those with Hck methylation (n=15) (p=0.08). All pts had been treated with hyperCVAD based chemotherapy. These data indicates that Hck methylation is a frequent phenomenon in human leukemia that maybe associated with a worse prognosis in ALL and suggests that Hck has a tumor suppressor like function in these disorders.

Detection of Aberrant DNA Methylation in Acute Lymphocytic Leukemia (ALL) Using a Real-Time Polymerase Chain Reaction (PCR) Assay.

Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. DNA methylation of specific subsets of genes is associated with poor prognosis and is stable in a majority of patients (pts) at the time of relapse (Clinical Cancer Res 2002; 8:1897), and therefore tracking these epigenetic marks during remission may predict for relapse risk. Most commonly used methods to detect DNA methylation exploit the availability of sodium bisulfite that transforms unmethylated C into A/T leaving methylated C as such. This has allowed the development of several techniques that use conventional PCR or sequencing to detect methylated alleles. Although there are significant differences in the sensitivity and specificity of these assays, with bisulfite sequencing considered the gold standard, most of them are labor intensive and require several days to be performed. To circumvent some of these problems, we have developed a real-time bisulfite PCR assay to detect methylation of p57KIP2, p73, and p15 in samples obtained from bone marrows in pts with ALL at remission. Methylation of these genes has been shown to be common in ALL and to predict for poor prognosis at initial presentation (Blood 2003; 101:4131). This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2μg of DNA for each target gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon γ (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. Methylation density is defined as: Methylation (%) = 2CT of target gene / 2 CT of EFM x 100. CT is the number of cycles threshold, and EFM the estimated 100% fully methylation (EFM) of the target gene in control experiments. Using DNA artificially methylated with SssI in dilution experiments with unmethylated DNA, p57, p15 and p73 methylation density could be detected at dilutions of 0.2%, 1.2% and 0.1%, respectively. We then studied the methylation status of 30 cell lines (22 hematopoietic and 8 no-hematopoietic). Methylation of p57, p15 and p73 gene was detected in 17(57%), 19(63%) and 16(53%) cell lines respectively. Methylation of 3 genes, 2 genes and 1 gene was observed in 9(30%), 8(27%) and 7(23%) cell lines respectively. DNA methylation of p15 and p73 was not detected in the marrow from 6 healthy volunteers but p15 was detected (0.1% methylation) in 1 out of 6 of these specimens. Subsequently, we studied 50 pts with ALL at the time of remission. We found the frequencies of p57, p15 and p73 gene to be 2(4%), 26(52%) and 10(20%), respectively. There was a trend towards a better overall survival for pts with methylation of 0 or 1 gene (209 weeks) compared with those with methylation of 2 or more genes (71 weeks), p=0.1. In conclusion, the real-time bisulfite PCR described here allows for the rapid detection of aberrant DNA methylation in samples obtained at the time of remission in pts with ALL and may have a role in the development of techniques to assess minimal residual disease in this group of pts.

Comparison of Aberrant Methylation of CpG Islands in the p16/CDKN2A and p14/ARF Promoters in Non-Small Cell Lung Cancer and Acute Lymphoblastic Leukemia

Multiplex methylation-sensitive (MSe-PCR) and methylation-specific (MSp-PCR) PCRs were used to detect aberrant methylation of CpG islands in the promoter regions and first exons of p16/CDKN2A and p14/ARF in non-small cell lung cancer (NSCLC, 54 specimens) and B-cell acute lymphoblastic leukemia (B-ALL, 61 specimens). A difference in CpG methylation was observed for individual specimens and for the two malignancies. A high methylation frequency of the first exon of p16/CDKN2A was detected both in NSCLC (68%) and in B-ALL (55%). The CpG island of the p14/ARF first exon proved to be nonmethylated in both malignancies. Particular CpG-rich fragments were examined in the p16/CDKN2A and p14/ARF promoters. It was shown that methylation frequency can differ between the 5′ regions of one promoter. The sensitivity was compared for MSe-PCR and MSp-PCR, which are commonly employed in methylation analysis.

CpG-island methylation and epigenetic control of resistance to chemotherapy

Aberrant methylation of CpG islands (CpG-rich regions of DNA associated with the promoters of many genes) is associated with transcriptional inactivation of genes involved in tumour development. Genes involved in key DNA damage response pathways, such as cell-cycle control, apoptosis signalling and DNA repair can frequently become epigenetically silenced and methylated in tumours. This may lead to differences in intrinsic sensitivity of tumours to chemotherapy, depending on the specific function of the gene inactivated. Furthermore, chemotherapy itself may exert a selective pressure on epigenetically silenced drug sensitivity genes present in subpopulations of cells, leading to acquired chemoresistance. Clinical trials of epigenetic therapies are now in progress, and epigenetic profiling using DNA methylation will provide guidance on optimization of the use of these therapies with conventional chemotherapy, as well as helping to identify patient populations who may particularly benefit from such approaches.

Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma

Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

Use of DNA from Human Stools to Detect Aberrant CpG Island Methylation of Genes Implicated in Colorectal Cancer

Abstract Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16INK4a, APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.

Epigenetic silencing mediated by CpG island methylation: potential as a therapeutic target and as a biomarker

Many genes become transcriptionally silenced during the development of cancer. As well as affecting disease progression, gene silencing has the potential to influence drug resistance and clinical outcome following therapy. In addition to silencing due to gene mutations, covalent epigenetic modifications such as DNA hypermethylation and histone post-translational modifications are associated with transcriptional inactivation of many genes and are an important early event during carcinogenesis and tumour development. Aberrant methylation of CpG islands in promoters is associated with transcriptional inactivation of genes involved in all aspects of tumour development. Genes involved in key DNA damage response pathways, such as cell cycle control, apoptosis signalling and DNA repair, can frequently become methylated and epigenetically silenced in tumours. This may lead to differences in intrinsic sensitivity of tumours to chemotherapy, depending on the specific function of the gene inactivated. Furthermore, it is proposed that chemotherapy itself can exert a selective pressure on epigenetically silenced drug sensitivity genes present in subpopulations of cells, leading to acquired chemoresistance. Since the DNA sequence of epigenetically inactivated genes are not mutated but rather subject to reversible modifications via DNA methyltransferases (DNMTs) or histone modification, it is possible to reverse silencing using small molecule inhibitors. Such compounds show anti-tumour activity and can increase the sensitivity of drug resistant preclinical tumour models. Clinical trials of epigenetic therapies are now underway. Epigenetic profiling, using DNA methylation and histone analysis, will provide guidance on optimisation of these therapies with conventional chemotherapy and will help identify patient populations who may particularly benefit from such approaches.

Methylation of p16 CpG islands associated with malignant transformation of gastric dysplasia in a population-based study.

Inactivation of p16 by aberrant methylation of CpG islands is a frequent event in carcinomas and precancerous lesions of various organs, including the stomach. The aim of this study is to investigate the relationship between p16 methylation and malignant transformation of human gastric dysplasia (DYS) based on follow-up endoscopic screening in a high-risk population. Genomic DNA samples were extracted from paraffin blocks of gastric mucosal biopsies that were histopathologically diagnosed as low-grade DYS from patients who developed gastric carcinomas [GCs (n = 21)] and those that did not do so (n = 21) during 5 years of follow-up. The methylation status of p16 CpG islands of each sample was detected by methylation-specific PCR, denatured high-performance liquid chromatography, and sequencing. Aberrant p16 methylation was observed in 5 of 21 samples of DYS that progressed to GC but in 0 of 21 samples that did not progress to GC (P = 0.048, two-sided). Sequencing results confirmed that all CpG sites were methylated in the analyzed sequence from these five p16-methylated cases. Furthermore, p16 methylation was also observed in the five subsequent GCs. Unmethylated p16 CpG islands were detected in all of the samples without p16 methylation. Our findings suggest p16 methylation is correlated with the malignant transformation of gastric DYS, and p16 methylation might be a useful biomarker for prediction of malignant potential of gastric DYS.