Distinct CpG methylation profiles characterize different clinical groups of neuroblastic tumors.

The hypermethylation of CpG islands within gene promoter regions is an epigenetic phenomenon that is often, but not always, associated with the transcriptional silencing of downstream genes and contributes to carcinogenesis. We have determined the pattern of methylation of several genes involved in distinct biological pathways, including cell proliferation and apoptosis, in neuroblastoma and in the nonmalignant ganglioneuroma. The purpose of this work was to search for epigenetic signatures that could be associated with defined clinical and biological parameters and that, in prospective, could identify specific risk categories among the patients. We have analysed 31 malignant neuroblastoma with or without MYCN amplification and 13 benign ganglioneuroma and we have observed dramatic differences in the methylation pattern of five genes (CASP8, 14.3.3sigma, DeltaN-p73, RASSF1A and DCR2) between these tumors indicating that this phenomenon is not tissue-specific and can be considered as cancer-dependent. Furthermore, the methylation pattern of 14.3.3sigma, RASSF1A and of an intragenic segment of CASP8 was significantly different between MYCN amplified and single copy neuroblastoma suggesting a specific role of epigenetic alterations in aggressive neuroblastoma.

DNA methylation in neuroblastic tumors.

Epigenetic modifications and particularly the methylation of cytosines 5' of guanine residues (CpGs) in gene promoter regions is an essential regulatory mechanism for normal cell development. DNA methylation can inactivate tumor suppressor genes by inducing C > T transitions in somatic and germline cells and by altering gene transcription. On the other hand, hypomethylation of specific sequences may reactivate the expression of potential oncogenes. Thus, aberrant hyper- and hypomethylation are considered crucial steps leading to cancer development. Until recently, differently from most adult tumors, only limited information was available on the methylation aberrations in neuroblastoma. In the last 2 years, however, this situation has drastically changed and many information has been gained on the relevance of methylation in this tumor. In this review, we summarize the latest findings on the role of methylation in neuroblastoma and in particular to its clinical significance.

Caspase-8 gene expression in neuroblastoma.

Neuroblastoma (NB) is a solid tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic/differentiation program. Indeed, the level of expression of molecules involved in the regulation of apoptosis, such as p73 or survivin, is a prognostic factor in NB patients. The caspase-8 gene (CASP8) encodes a key enzyme at the top of the apoptotic cascade. Although methylation of a putative regulatory region of the CASP8 gene reportedly inhibits its transcription in some MYCN-amplified NB, our results indicate that the transcriptional inactivation of caspase-8 occurs in a subset of primary NB independently of MYCN amplification or CpG methylation. In addition, the apoptotic agent fenretinide (4HPR) and interferon-gamma (IFN-gamma) induce caspase-8 expression without modifying the methylation status of this gene. Nevertheless, the methylation level of CASP8 intragenic and promoter regions is higher in MYCN-amplified tumors as compared to nonamplified samples. This phenomenon might reflect the existence of distinct DNA methylation errors in MYCN-amplified and MYCN-single copy tumors. To gain information on the mechanisms that regulate the expression of this crucial apoptotic gene, we searched for potential CASP8 regulatory regions and cloned a DNA element at the 5' terminus of this gene that functionally acts as a promoter only in NB cell lines that express caspase-8. The retinoic acid analogue 4HPR, IFN-gamma, and the demethylating agent 5-aza-cytidine activate this promoter in NB cells that lack endogenous caspase-8, indicating that this element may regulate both constitutive and inducible CASP8 expression. These results indicate also that demethylation of the cellular genome may upregulate CASP8 through the action of trans-acting factors. Our results provide new insights to the regulation of CASP8, a gene with an essential role in a variety of physiologic and pathologic conditions.

Meth-DOP-PCR: an assay for the methylation profiling of trace amounts of DNA extracted from bodily fluids.

Cancer cells release their DNA into the patient's bodily fluids and cancer-specific signatures can be recognized in the circulating DNA. The aberrant methylation of CpG-rich regions in gene promoter sequences is an early marker of cell transformation whose specificity and optimal sensitivity can be achieved by assessing the methylation status of multiple genes ('methylation profiling'). Most of the current technologies for methylation analysis rely upon the combination of chemical conversion of the DNA and PCR analysis for the detection of methylated and unmethylated alleles. However, the small amount of circulating DNA, and its fragmentation, dramatically reduces the template DNA molecules making difficult the methylation profiling. To overcome this limitation, we have developed the Meth-DOP-PCR assay, a combination between a modified degenerate oligonucleotide primed PCR (DOP-PCR) and methylation-specific PCR (MSP), for the high-throughput methylation analysis of trace-amount of circulating DNA. We have demonstrated the concordance between Meth-DOP-PCR and MSP and shown the application of this technique for the methylation analysis of DNA extracted from the serum of lung cancer patients. We have estimated that through this procedure it is possible to obtain at least a 25-fold increase of the number of determinations allowing the methylation profiling from less than 1 ml of serum. Thus, Meth-DOP-PCR appears as a simple, cost-effective and efficient technique, for the development of novel methylation-based diagnostic assays.

p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma.

The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma.