Distinct DNA Methylation Profiles in Ovarian Tumors: Opportunities for Novel Biomarkers.

Aberrant methylation of multiple promoter CpG islands could be related to the biology of ovarian tumors and its determination could help to improve treatment strategies. DNA methylation profiling was performed using the Methylation Ligation-dependent Macroarray (MLM), an array-based analysis. Promoter regions of 41 genes were analyzed in 102 ovarian tumors and 17 normal ovarian samples. An average of 29% of hypermethylated promoter genes was observed in normal ovarian tissues. This percentage increased slightly in serous, endometrioid, and mucinous carcinomas (32%, 34%, and 45%, respectively), but decreased in germ cell tumors (20%). Ovarian tumors had methylation profiles that were more heterogeneous than other epithelial cancers. Unsupervised hierarchical clustering identified four groups that are very close to the histological subtypes of ovarian tumors. Aberrant methylation of three genes (BRCA1, MGMT, and MLH1), playing important roles in the different DNA repair mechanisms, were dependent on the tumor subtype and represent powerful biomarkers for precision therapy. Furthermore, a promising relationship between hypermethylation of MGMT, OSMR, ESR1, and FOXL2 and overall survival was observed. Our study of DNA methylation profiling indicates that the different histotypes of ovarian cancer should be treated as separate diseases both clinically and in research for the development of targeted therapies.

DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM).

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples.

BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells.

Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis.

An unusual uterine tumor with signet ring cell features.

In 2004, a 56-year-old woman was diagnosed with Stage IA follicular lymphoma in a cervical lymph node biopsy. The patient experienced total remission after local radiation therapy. In 2009, a control computed tomography scan evidenced a pelvic mass, prompting total hysterectomy. The latter harbored a 4.8-cm intramural uterine tumor corresponding to a mostly diffuse and focally nodular proliferation of medium to large cells, with extensive, periodic acid-Schiff negative, signet ring cell changes, and a pan-keratin negative, CD20+, CD10+, Bcl2+, Bcl6+ immunophenotype. Molecular genetic studies showed the same clonal IGH gene rearrangement in the lymph node and the uterus, establishing the uterine tumor as a relapse of the preceding follicular lymphoma, although no signet ring cells were evidenced at presentation. Uterine localization of lymphomas is rare, and lymphomas with signet ring cell features are uncommon. This exceptional case exemplifies a diagnostically challenging situation and expands the differential diagnosis of uterine neoplasms displaying signet ring cell morphology.

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells.

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.

TERT promoter methylation and protein expression as predictive biomarkers for recurrence risk in patients with serous borderline ovarian tumours.

Epithelial ovarian neoplasms can be divided into three distinct clinicopathological groups: benign, malignant and borderline tumours. Borderline tumours are less aggressive than epithelial carcinomas, with an indolent clinical course and delayed recurrence. However, a subset of these cases can progress to malignancy and relapse, and death from recurrent disease can occasionally occur. Telomerase activation is a critical element in cellular immortalisation and cancer. The enzyme telomerase comprises a catalytic subunit (TERT) expressed in various types of cancers and regulated by promoter methylation mainly in epithelial tumours. The aim of this study was to investigate the promoter methylation status and the expression of TERT in 50 serous borderline tumours (SBTs) and their correlation with clinicopathological features and outcome. TERT methylation was analysed by bisulfite pyrosequencing and TERT expression by immunohistochemistry. Methylation of TERT promoter was only observed in four SBTs. A good correlation with immunostochemistry was found: nuclear positivity for TERT expression was observed in the methylated cases, whereas no expression was detected in unmethylated tumours. One of these patients had a recurrence after 7 years and another patient died from the disease. SBTs with hypomethylated tumours and absence of TERT expression showed a good clinical behaviour. Our study highlights the low presence of TERT methylation in SBTs, confirming that these tumours have a different biology than serous carcinomas. Furthermore, the concordance between TERT promoter methylation and TERT expression and their association with clinical outcomes leads to consider TERT alteration as a potential predictive biomarker for recurrence risk identifying patients who should undergo a careful and prolonged follow-up.

Specific association between the methyl-CpG-binding domain protein 2 and the hypermethylated region of the human telomerase reverse transcriptase promoter in cancer cells.

Human telomerase reverse transcriptase (hTERT) is expressed in most cancer cells. Paradoxically, its promoter is embedded in a hypermethylated CpG island. A short region escapes to this alteration, allowing a basal level of transcription. However, the methylation of adjacent regions may play a role in the maintenance of low hTERT expression. It is now well established that methyl-CpG binding domain proteins mediate the transcriptional silencing of hypermethylated genes. The potential involvement of these proteins in the control of hTERT expression was firstly investigated in HeLa cells. Chromatin immunoprecipitation assays showed that only methyl-CpG-binding domain protein 2 (MBD2) associated the hypermethylated hTERT promoter. In MBD2 knockdown HeLa cells, constitutively depleted in MBD2, neither methyl CpG binding protein 2 (MeCP2) nor MBD1 acted as substitutes for MBD2. MBD2 depletion by transient or constitutive RNA interference led to an upregulation of hTERT transcription that can be downregulated by expressing mouse Mbd2 protein. Our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT in HeLa cells. This specific transcriptional repression was also observed in breast, liver and neuroblastoma cancer cell lines. Thus, MBD2 seems to be a general repressor of hTERT in hTERT-methylated telomerase-positive cells.

Superficial primitive Ewing's sarcoma: a clinicopathologic and molecular cytogenetic analysis of 14 cases.

Superficial primitive Ewing's sarcomas are rare and have been reported to be of favorable prognosis compared to conventional deep-seated tumors. In the skin and subcutis, the diagnosis is often difficult, and performing molecular cytogenetic techniques may be helpful. We performed a retrospective analysis of 14 cases of superficial Ewing's sarcomas, all confirmed by molecular cytogenetics. Clinical, histological, immunohistochemical, molecular cytogenetic, therapeutic, and follow-up data are reported. There were 11 female and 3 male patients aged from 12 to 77 years (median: 17 years). Seven tumors occurred in the extremities, five in the trunk wall, and two in the head. Tumor size ranged from 1 to 5 cm (median, 3 cm). They were all small round-cell proliferations with a strong membranous positivity for CD99. Ewing's sarcoma translocations/fusion gene transcripts were detected in eight cases, both by FISH and reverse transcriptase (RT)-PCR. Four tumors were positive by RT-PCR alone (FISH not done in three cases and not interpretable in one case), and two cases were positive by FISH alone (RT-PCR not done). Surgical resection was performed in all patients. Chemotherapy was given in ten patients and radiotherapy in six. At last medical examination (median follow-up, 47 months), two patients who underwent surgical resection alone had died from the tumor. Our results confirm that superficial Ewing's sarcomas are of good prognosis. Given the difficulty of the diagnosis and the importance of an adapted treatment, a confirmation of the diagnosis by molecular or cytogenetic techniques is recommended when dealing with a superficial tumor.

Serrated polyps of the large intestine: a molecular study comparing sessile serrated adenomas and hyperplastic polyps.

AIMS: To compare the molecular profile of a series of sessile serrated adenomas (SSAs) and hyperplastic polyps (HPs), in order to distinguish these lesions, SSAs having a potential role in the genesis of serrated adenocarcinomas through a serrated pathway in which methylation plays a key role. METHODS AND RESULTS: Twelve HPs and sixteen SSAs of the right and left colon were investigated for microsatellite instability, DNA mismatch repair genes, p53, p16, and beta-catenin expression, MLH1 and p16 (CDKN2A) gene methylation, and KRAS and BRAF mutations. Both SSAs and HPs were microsatellite stable. MLH1 and MSH2 protein silencing, aberrant cytoplasmic expression and methylation of p16 were found to be exclusive to right-sided SSAs. The MLH1 promoter gene was frequently methylated in right-sided SSAs in contrast with HPs. Abnormal p53 and beta-catenin expression was present in both SSAs and HPs. BRAF and KRAS mutation were mutually exclusive, but KRAS mutation was present only in left-sided SSAs and HPs. CONCLUSIONS: HPs and SSAs may be related lesions. However, at least right-sided SSAs differ from left-sided SSAs and HPs in the occurrence of MLH1 and p16 methylation, supporting the hypothesis that SSAs could be precursors of serrated adenocarcinomas.

Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors.

BORIS, like other members of the 'cancer/testis antigen' family, is normally expressed in testicular germ cells and repressed in somatic cells, but is aberrantly activated in cancers. To understand regulatory mechanisms governing human BORIS expression, we characterized its 5'-flanking region. Using 5' RACE, we identified three promoters, designated A, B and C, corresponding to transcription start sites at -1447, -899 and -658 bp upstream of the first ATG. Alternative promoter usage generated at least five alternatively spliced BORIS mRNAs with different half-lives determined by varying 5'-UTRs. In normal testis, BORIS is transcribed from all three promoters, but 84% of the 30 cancer cell lines tested used only promoter(s) A and/or C while the others utilized primarily promoters B and C. The differences in promoter usage between normal and cancer cells suggested that they were subject to differential regulation. We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter. Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters. These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

Involvement of epigenetic modification of TERT promoter in response to all-trans retinoic acid in ovarian cancer cell lines.

BACKGROUND: All-trans retinoic acid (ATRA) is currently being used to treat hematological malignancies, given the ability to inhibit cell proliferation. This effect seems to be related to epigenetic changes of the TERT (Telomerase Reverse Transcriptase) promoter. When hypomethylated, ATRA-inducible TERT repressors can bind the promoter, repressing transcription of TERT, the rate-limiting component of telomerase. Ovarian carcinomas are heterogeneous tumors characterized by several aberrantly methylated genes among which is TERT. We recently found a hypomethylation of TERT promoter in about one third of serous carcinoma, the most lethal histotype. Our aim was to investigate the potential role of ATRA as an anticancer drug in a sub-group of ovarian carcinoma where the TERT promoter was hypomethylated. METHODS: The potential antiproliferative and cytotoxic effect of ATRA was investigated in seven serous ovarian carcinoma and one teratocarcinoma cell lines and the results were compared to the methylation status of their TERT promoter. RESULTS: The serous ovarian carcinoma cell line OVCAR3, harboring a hypomethylated TERT promoter, was the best and fastest responder. PA1 and SKOV3, two cell lines with an intermediate methylated promoter, revealed a weaker and delayed response. On the contrary, the other 5 cell lines with a highly methylated promoter did not respond to ATRA, indicative of ATRA-resistant cells. CONCLUSIONS: Our results demonstrate an inverse correlation between the methylation level of TERT promoter and ATRA efficacy in ovarian carcinoma cell lines. Although these results are preliminary, ATRA treatment could become a new powerful, personalized therapy in serous ovarian carcinoma patients, but only in those with tumors harboring a hypomethylated TERT promoter.

Epigenetic alteration of the Wnt inhibitory factor-1 promoter occurs early in the carcinogenesis of Barrett's esophagus.

The role of Wnt antagonists in the carcinogenesis of esophageal adenocarcinoma (EAC) remains unclear. We hypothesized that downregulation of the Wnt inhibitory factor-1 (WIF-1) might be involved in the neoplastic progression of Barrett's esophagus (BE). We analyzed the DNA methylation status of the WIF-1 promoter in normal, preneoplastic, and neoplastic samples from BE patients and in EAC cell lines. We investigated the role of WIF-1 on EAC cell growth and the chemosensitization of the cells to cisplatin. We found that silencing of WIF-1 correlated with promoter hypermethylation. EAC tissue samples showed higher levels of WIF-1 methylation compared to the matched normal epithelium. In addition, we found that WIF-1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF-1 in cell lines where WIF-1 was methylation-silenced resulted in growth suppression. Restoration of WIF-1 could sensitize the EAC cells to the chemotherapy drug cisplatin. Our results suggest that silencing of WIF-1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF-1 might be used as a new targeted therapy for the treatment of this malignancy.

Heterogeneity of human glioblastoma: glutathione-S-transferase and methylguanine-methyltransferase.

The DNA repair and detoxifying enzymes, O(6)-methylguanine-DNA-methyltransferase (MGMT) and glutathione-S-transferase (GST), may be responsible fpr poor response to alkylating agents in glioblastoma treatment. The methylation of MGMT promoter and the expression of MGMT and GST were highly heterogeneous in surgical specimens of human glioblastoma and in established human glioblastoma cells under 2-D and 3-D culture conditions, suggesting an intrinsic property of these cells. MGMT and GST expression did not predict the sensitivity of glioblastoma cells to alkylating agents. Combination of alkylating agents with inhibitors of GST disclosed additive effects, suggesting that inhibition of GST should be considered in glioblastoma therapy.

Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group.

Low-grade fibromyxoid sarcomas (LGFMS) bear either the t(7,16) (q32-34;p11) or t(11,16) (p11;p11) translocations, resulting in FUS-CREB3L2 or FUS-CREB3L1 fusions, respectively. Heretofore, fusion transcripts were mainly detected in frozen tissues, using reverse transcription-polymerase chain reaction. In this study, we aimed to develop a reliable method to detect these in paraffin-embedded tissues, and to examine the clinicopathologic characteristics of a series of translocation-positive LGFMS. Sixty-three neoplasms with typical morphologic features of LGFMS and 66 non-LGFMS tumors selected for their resemblance to LGFMS (LGFMS-like tumors) were examined. RNA of sufficient quality could be extracted from 111/129 (86%) cases (59 LGFMS, 52 non-LGFMS). Of all, 48/59 (sensitivity, 81%) LGFMS contained detectable transcripts (45 FUS-CREB3L2, 3 FUS-CREB3L1). Most relevant clinicopathologic features of fusion-positive LGFMS included predominance in lower extremities (22/48; thigh: 13/48), deep situation (46/48), and occasional presence of unusual histologic features, for example, hypercellular areas (16/48), foci of epithelioid cells (13/48), and giant rosettes (6/48). Most tumors expressed EMA (41/45), at least focally, CD99 (38/41) and bcl-2 (36/41) while being essentially negative for CD34 (2/45), mdm2 (1/41), smooth muscle actin (1/45), S100 protein (0/46), desmin (0/44), h-caldesmon (0/42), keratins (0/44), and CD117 (0/40). Eleven presumed LGFMS were fusion negative. Of all, 7/52 non-LGMFS neoplasms contained FUS-CREB3L2 transcripts, of which 4 had been diagnosed as sclerosing epithelioid fibrosarcoma. In conclusion, FUS-CREB3L1/L2 fusion transcripts can be detected in paraffin-embedded LGFMS in a sensitive manner, using reverse transcription-polymerase chain reaction. Most fusion-positive LGFMS are EMA-positive and CD34/S100/smooth muscle actin negative. The presence of epithelioid cells and fusion transcripts in both LGFMS and a subset of sclerosing epithelioid fibrosarcoma suggest that these neoplasms might be related.

Loss of Smad4 expression predicts liver metastasis in human colorectal cancer.

Distant metastases represent the major cause of death after curative surgery of colorectal cancer. The aim of this study was to evaluate the role of Smad4 and KRAS genetic alterations in colorectal metastases taking into account both the site (hepatic versus extrahepatic) and the time (synchronous versus metachronous) of recurrence. We examined the immunohistochemical expression of Smad4 and frequency of KRAS mutation in primary colorectal tumors and in their corresponding metastatic tissues. Loss of Smad4 expression was noted in 37% (26/71) of the primary tumors and the corresponding metastases. Absence of Smad4 protein was more frequently observed in hepatic metastases, whether they were metachronous or synchronous, than in extrahepatic metastases (p<0.005). The frequency of KRAS mutations was high in the synchronous and extrahepatic metachronous metastases (68-80%), but was significantly lower in the hepatic metachronous metastases (11%). Our results indicate that absence of Smad4 expression correlated significantly with liver metastases regardless of the time of their occurrence and represents a promising new biomarker to predict liver metastasis in colorectal cancer patients. Therefore, this group of patients could benefit from a specific and appropriate pre- and/or post-operative therapy.

CTCF binds the proximal exonic region of hTERT and inhibits its transcription.

The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. Previously, we detected a transcriptional repressor effect of the proximal exonic region (first two exons) of the hTERT gene. To better understand the mechanism involved and to identify a potential repressor, we further characterized this region. The addition of the hTERT proximal exonic region downstream of the hTERT minimal promoter strongly reduced promoter transcriptional activity in all cells tested (tumor, normal and immortalized). This exonic region also significantly inhibited the transcriptional activity of the CMV and CDKN2A promoters, regardless of the cell type. Therefore, the repressor effect of hTERT exonic region is neither cell nor promoter-dependent. However, the distance between the promoter and the exonic region can modulate this repressor effect, suggesting that nucleosome positioning plays a role in transcriptional repression. We showed by electrophoretic mobility shift assay that CCCTC-binding factor (CTCF) binds to the proximal exonic region of hTERT. Chromatin immunoprecipitaion assays confirmed the binding of CTCF to this region. CTCF is bound to hTERT in cells in which hTERT is not expressed, but not in telomerase-positive ones. Moreover, the transcriptional downregulation of CTCF by RNA interference derepressed hTERT gene expression in normal telomerase-negative cells. Our results suggest that CTCF participates in key cellular mechanisms underlying immortality by regulating hTERT gene expression.

De novo concurrent papillary renal cell carcinoma and angiomyolipoma in a kidney allograft: evidence of donor origin.

In the general population, renal cell carcinoma (RCC) is a relatively common neoplasm; however, the papillary RCC subtype is infrequent and represents only 10 to 15% of all RCC. Angiomyolipoma is a well-known common benign tumor. The occurrence of RCC in association with angiomyolipoma is a rare event, with only approximately 50 cases reported in the nontransplantation setting. In transplant recipients, RCC can develop in native kidneys, but its occurrence "de novo" in the renal allograft is very rare with an estimated incidence of less than 0.5%. We report here the case of a 39-year-old woman who underwent cadaveric renal transplantation in 1990. No lesion was observed in the allograft during the pre- and perioperative period or on early postoperative ultrasounds. No graft rejection occurred under a standard triple immunosuppressive therapy. Thirteen years later, during a routine ultrasonography, 2 solid masses were discovered in the allograft, both of them richly vascularized. She underwent allograft nephrectomy and the histologic findings revealed that one of the tumors was a chromophilic (type 1) papillary RCC (2.5 cm in diameter) and the other, an angiomyolipoma (1.5 cm). Microsatellite analysis of the allograft, as compared with the recipient peripheral blood leukocytes, demonstrated that the 2 tumors (1 malignant and 1 benign) were of donor origin. To our knowledge, this is the first report of de novo concurrent papillary RCC and angiomyolipoma in a renal allograft.

A cost-effective algorithm for hereditary nonpolyposis colorectal cancer detection.

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.

Monoallelic methylation of the APC promoter is altered in normal gastric mucosa associated with neoplastic lesions.

Adenomatous polyposis coli (APC) promoter hypermethylation has been reported frequently in normal gastric mucosa, but it remained to be clarified whether this occurs in every individual. In this study, methylation of the APC promoter was analyzed in histologically normal-appearing gastric mucosa samples by methylation-sensitive single-strand conformation analysis and by a methylation-sensitive dot blot assay. Epithelial cell samples were collected by microdissection from tissue sections. Equal amounts of methylated and unmethylated APC alleles were found in all gastric mucosa samples from patients without any gastric lesions (20 samples). Allele-specific methylation analysis showed that the methylation of the APC promoter was monoallelic; however, which allele was methylated depended on the cell type. Increased or decreased methylation was found in 10 of 36 (28%) normal gastric mucosa samples adjacent to a gastric or esophageal adenocarcinoma. No allelic loss was found at the APC locus. Modification of the methylation status was also found in 3 of 21 (14%) normal-appearing gastric mucosa samples adjacent to intestinal metaplasia. In contrast, all normal mucosa samples in cases with chronic gastritis but without metaplasia or dysplasia showed a monoallelic methylation pattern. Our results indicate the following: (a) In normal gastric mucosa, the APC promoter shows monoallelic methylation, which is not due to imprinting but most likely due to allelic exclusion; (b) the excluded allele differs between foveolar and glandular epithelial cells; (c) the APC methylation pattern is frequently altered in normal-appearing gastric mucosa of gastric or esophageal adenocarcinoma patients; and (d) such alterations also occur in normal gastric mucosa adjacent to intestinal metaplasia.

Promoter methylation and downregulated expression of the TBX15 gene in ovarian carcinoma.

TBX15 is a gene involved in the development of mesodermal derivatives. As the ovaries and the female reproductive system are of mesodermal origin, the aim of the present study was to determine the methylation status of the TBX15 gene promoter and the expression levels of TBX15 in ovarian carcinoma, which is the most lethal and aggressive type of gynecological tumor, in order to determine the role of TBX15 in the pathogenesis of ovarian carcinoma. This alteration could be used to predict tumor development, progression, recurrence and therapeutic effects. The study was conducted on 80 epithelial ovarian carcinoma and 17 control cases (normal ovarian and tubal tissues). TBX15 promoter methylation was first determined by pyrosequencing following bisulfite modification, then by cloning and sequencing, in order to obtain information about the epigenetic haplotype. Immunohistochemical analysis was performed to evaluate the correlation between the methylation and protein expression levels. Data revealed a statistically significant increase of the TBX15 promoter region methylation in 82% of the tumor samples and in various histological subtypes. Immunohistochemistry showed an inverse correlation between methylation levels and the expression of the TBX15 protein. Furthermore, numerous tumor samples displayed varying degrees of intratumor heterogeneity. Thus, the present study determined that ovarian carcinoma typically expresses low levels of TBX15 protein, predominantly due to an epigenetic mechanism. This may have a role in the pathogenesis of ovarian carcinoma independent of the histological subtype.