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allele is expressed as to the expression outcome, e.g., this can occur in ovarian cancer [14,15] . Gene expression
by the non-methylated allele is likely for some genes so leading to continued expression despite the other
allele being methylated. In their study, Losi et al. investigated 41 ovarian cancer associated promotor
[16]
genes, and observed an intermediate level of hypermethylation (~50%) for most hypermethylated genes.
Since there were > 70% of tumor cells present in each tumor sample employed, they would have expected
either a high or a low methylation level. Combining their data with those described in the literature, where
most studies dealt with very few genes, they proposed that this might be a general event of intra-tumoral
heterogeneity existing for epigenetic changes.

Hypermethylation also has its effects with that of promoter CpG islands causing the silencing of tumor
suppressor genes whilst methylation of CpG islands results in the inhibition of transcription factor
suppressors.

Gene selection is also important for the study of ovarian cancer. Whilst Losi et al. and Choi et al. listed
[17]
[16]
genes involved in cancer in general those epigenetically modifiable promoter genes relevant to ovarian cancer
are given in Table 1. Genes involved in cisplatin/carboplatin resistance/sensitization of ovarian cancer are
presented in Table 2.

Finally, it appears to be necessary to consider the effect of the degree of methylation of each gene with
respect to the histological type of ovarian cancer being investigated, e.g., epithelial, serous, endometrioid
and mucinous since each may offer a different response depending upon the degree of methylation of a
particular gene .
[16]
Methods for determining DNA methylation
When determining methylation levels, there are a number of approaches. The first consideration concerns
tissue preparation before selection of that which is to be analysed. Thus, the tissue may be either histologically
processed, often wax embedded or, alternatively, the tissue may be either as fixed or unfixed, frozen sections.
The selected material to be analysed is isolated from the sections after microscopical analysis. Hence, there
are three different factors involved relating to the tissue sample examined, namely, chemical fixation of the
tissue, dewaxing of embedded material and the use of unfixed, frozen tissue.

Once the selected material has been removed from the sections, a relevant method for analysis of methylation
levels needs to be selected. Some methods utilized for the detection of DNA methylation levels [42-45] :
Digestion-based assay (PCR, qPCR, RT-PCR, cold PCR); High resolution melting luminometric methylation
assay; HPLC-UV; Mass spectrometry; ELISA-based methods; Amplified fragment length polymorphism;
Restriction fragment length polymorphism; Luminometric methylation assay; Pyrosequencing; Bisulfite
sequencing; ELISA; Methylation ligation-dependent macro-array; High-throughput measurement
technologies.

An in-depth analysis of the majority of the available methods was given by Olkhov- Olkhov-Mitsel &
Bapat to detect methylated and hydroxyl-methylated DNA biomarkers. The methods are grouped under
[42]
bisulphite-based strategies, restriction enzyme based methods and affinity-based strategies. Subsequently,
Kurdyukov and Bullock developed a simple algorithm for selecting the most appropriate method for the
[43]
material to be analyzed and for the identification of the form of methylation to be determined through either
whole genome methylation profiling or identification of differentially-methylated regions or the methylation
status of specific genes or digestion based assays or differentially-methylated loci or hydroxymethyl cytosine
determination.

Clearly, given the range of methods available for the determination of different aspects of DNA methylation
used by different authors, it becomes somewhat difficult to routinely compare results in the case of applied