However, a considerable lack of independent study and replication of associations for most of the genes studied highlights a need for further investigation in this aspect of methylation in GC. Data from five studies on 4-(Aminomethyl)benzoic acid patients receiving chemotherapy also has suggested that DNA methylation at CHFR, DAPK, TMS1 could be useful predictors of response to chemotherapy. However, from the design of these analyses, it is difficult to determine whether the survival differences were due to inherent prognostic differences or were a function of a treatment interaction, or both. In a study of a different design, Mitsuno et al. reported that patients with p16 methylation gained a survival benefit from chemotherapy, while those without methylation did not. This result suggests that p16 methylation may be a useful marker for predicting response to chemotherapy, and provides (-)-Tetramisole evidence of a treatment interaction. However, this study only examined 56 subjects in a retrospective analysis, and much further work is required to confirm these findings. The findings of this study highlight a promising potential for DNA methylation in GC risk prediction, prognostication and prediction of treatment response. However, many issues relevant to clinical implementation remain unaddressed by the studies. Methodologically, the studies inadequately define optimal approaches for analysis, due to their large variability in assays, PCR primers and probes, PCR conditions, and thresholds for positivity used. Most studies have been based on methylation-specific PCR, for which the non-quantitative nature of analysis presents difficulties to quality control and standardization. With methylation a dynamic event, protocols for sampling are also in need of clarification, both with respect to the region or site of sampling, and time of sampling. The distance of normal tissue from tumour, and time of sampling have all been documented to significantly influence methylation levels. The large variability in the genes and gene panels examined between studies, combined with a lack of validation in independent series and characterization of test performance characteristics, also makes it difficult to define a clinically relevant test. Variation in the interrogation of often functionally different CpG sites between studies of the same gene provides additional complications. Moreover, all the gene methylation events examined in multiple studies and significant associated with GC have been implicated as risk markers of many other cancer types, raising questions about the interpretation of their detection in asymptomatic individuals. Of further consideration are the co-variates of analysis that would be analyzed with methylation. Methylation has been associated with many demographic, clinical and molecular features, including age, gender, smoking, intestinal metaplasia, host genetics, and H. pylori and Epstein Barr virus status.