Richard Braunschweig

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation‐sensitive single‐strand conformation analysis (MS‐SSCA) and direct sequencing using bisulfite‐modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT‐expressing telomerase‐positive samples and was absent in telomerase‐negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.

Methylation of APC, TIMP3, and TERT: a new predictive marker to distinguish Barrett's oesophagus patients at risk for malignant transformation.

Barretts associated oesophageal adenocarcinoma (EAC) is one of the most rapidly increasing malignancies in Western countries. Because of its poor prognosis, management of this disease through screening of Barretts oesophagus (BE) patients and identification of those with a high risk of developing an adenocarcinoma seems a promising approach. Early molecular markers of malignant transformation might contribute to such screening approaches. Gene promoter methylation analysis was performed on normal, pre‐neoplastic, and neoplastic lesions from BE patients. All lesions of interest were sampled by microdissection from formalin‐fixed paraffin‐embedded tissue sections. We found that, in 27 adenocarcinomas, APC, TIMP3, TERT, CDKN2A, and SFRP1 promoters were methylated in 93%, 65%, 64%, 48%, and 91%, respectively; in contrast MLH1, RASSF1, RARB, CDH1, and FHIT promoters were methylated in less than 5% of the tumours. In BE mucosa from patients who had progressed to adenocarcinoma (12 samples), APC, TIMP3, and TERT promoters were hypermethylated in 100%, 91%, and 92% of cases, whereas in BE mucosa from patients who had not progressed (16 samples) methylation was found only in 36%, 23%, and 17%, respectively. Furthermore, the epigenetic profile of BE with and without EAC differed significantly with, respectively, 81% and 26% of the PCR samples showing promoter hypermethylation for APC, TIMP3, and TERT (p < 0.0001). Promoter methylation of CDKN2A was infrequently detected in BE samples, while SFRP1 methylation was observed in all samples. Our results suggest that promoter methylation profiling of BE using multiple target genes including APC, TIMP3, and TERT might be used as a predictive marker for increased EAC risk. Copyright

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.

Detection of malignant effusions: Comparison of a telomerase assay and cytologic examination

Telomerase is inactive in most somatic cells, but has been found to be reactivated in a majority of cancers. Our principal goal was to test whether the presence of telomerase activity concurred with positive cytology, and was thus of potential use in detecting cancer cells in effusions. The telomeric repeat amplification protocol (TRAP) assay and cytological examination were performed in a blinded fashion on 91 unselected effusions, for which laboratory processing was done according to standard procedures. In our series, 30% (27/91) of samples were found to be malignant by cytology. Of these, 19 (70%) were also positive in the TRAP assay. Of the 8 telomerase‐negative cytology‐positive samples, RNA integrity was generally poor, indicating suboptimal sample conservation for molecular analysis. Negative cytology in the presence of telomerase activity was observed in 17 effusions. Of these, 11 were from patients with advanced cancer, and thus a diagnosis of malignant effusion should be suspected. The TRAP assay for telomerase activity holds promise in the analysis of effusions, but its routine use as an adjunct to cytology awaits further confirmation of its positive predictive value. Diagn. Cytopathol. 2001;24:174–180.

Imprinting of tumor-suppressor genes in human placenta.

Transcriptional deregulation in cancer has been shown to be associated with epigenetic alterations, in particular to tumor-suppressor-gene (TSG) promoters. In contrast, DNA methylation of TSGs is not considered to be present in normal differentiated cells. Nevertheless, we previously showed that the promoter of the tumor-suppressor gene APC is methylated, for one allele only, in normal gastric cells. Recently, RASSF1A has been shown to be imprinted in normal human placenta. To clarify putative TSG methylation in the placenta, 23 normal placental tissues from the first trimester, both decidua and villi, and four normal non-gestational endometrium were screened for DNA methylation by methylation-sensitive single-strand conformation analysis (MS-SSCA) and sequencing after bisulfite modification, on a panel of 12 genes known to be implicated in carcinogenesis. In all placental villi, 4 TSG promoters - APC, SFRP2, RASSF1A and WIF1 - were hypermethylated, whereas all decidua and normal endometrium did not show any methylation. Allele-specific methylation analysis revealed that this methylation was monoallelic. Furthermore, comparison with maternal DNA indicated that APC and WIF1 were methylated on the maternal allele, whereas SFRP2 was methylated on the paternal allele. Sequence analysis of WIF1 mRNA revealed that only the unmethylated paternal allele was transcribed. The imprinting status of these TSGs is conserved during pregnancy. These results indicate that TSG imprinting is pre-existent in normal human placenta and should not be confused with carcinogenesis or pathology-induced methylation.

Pleomorphic adenoma with predominant plasmocytoid myoepithelial cells: A diagnostic pitfall in aspiration cytology. Case report and review of the literature

Fine‐needle aspiration (FNA) biopsy of the salivary gland is a sensitive and specific diagnostic tool. However, diagnostic problems are sometimes encountered in interpreting some cases, not only in differentiating benign from malignant cases but also in the specific classification of these neoplasms. We report a case of a pleomorphic adenoma with predominant plasmocytoid myoepithelial cells arising in minor salivary glands from the hard palate in a 78‐year‐old patient, which was falsely diagnosed as a carcinoma on liquid‐based cytology (ThinPrep (TP)). The differential diagnosis of salivary gland tumors with predominant myoepithelial cells on FNA biopsy is discussed. Diagn. Cytopathol. 2009.