Compared to gene-expression based markers, DNA methylation patterns are more stable and can be detected using various approaches requiring relatively small amounts of patient materials. In addition, these markers can be objectively identified and quantified, providing potential improvement to reliability in histological subjective diagnoses such as tumor grades or compared to immunohistochemical staining patterns which rely on individual interpretation of intensity and location of staining. For example, the markers identified and independently validated in this study hold promise as biomarkers useful in bladder cancer screening and follow-up for recurrence and progression and could be applied and tested for clinical utility in easy to collect urine sediments 
. In addition, discovery of genes and pathways epigenetically altered in bladder tumors suggest novel targets for therapeutic intervention, especially when considering the poor prognosis associated with invasive bladder cancers. Identification of important and useful DNA methylation based markers is not without challenge, particularly as there is often a high degree of correlation between these alterations 
. Therefore appropriate statistical approaches must be utilized to examine these alterations and to identify those alterations with the greatest clinical utility. We believe that our 2 stage approach provides for the most robust and generalizable identification of DNA methylation alterations which can be further examined in prospective studies. Using several different statistical approaches, our selection strategy for informative markers allowed us to identify a manageable number of potentially useful and robust biomarkers for laboratory validation as well as replication in independent samples. Most importantly, our validation of these markers in an independent group of patients provides compelling evidence of their potential utility and biological importance.
Previous studies examining panels of candidate genes have identified specific genes and panels of loci which are predictive of both the presence of tumor when examined in urine sediment 
and the progression from non-invasive to invasive disease 
when examined within the tumor tissue. Specific genes such as RASSF1A
and the SFRP
family have been associated with higher grade and higher stage bladder cancer 
. Our strategy did not identify these specific genes, and this discrepancy may be due to a number of differences between our approach and those previously utilized. We have made use of a population-based case series sample, while many previous studies have utilized hospital-based convenience series, and thus may have had some bias introduced in the samples examined. The array platform used in this study is limited in that it examines only a subset of all genes and only 1 or 2 specific CpG sites for those genes, and thus may miss the specific genes of loci previously described. Finally, our analytical approach, as it is based on the identification of panels of genes may identify different loci than those used in candidate analyses, but hypermethylation of those previously described candidates, may, in fact, be highly correlated to the methylation patterns identified herein. Further study combining previously identified loci with our own new targets in a prospective fashion is needed to more definitively determine those loci with the greatest clinical utility.
The biologic underpinnings of non-invasive yet recurrent disease versus invasive disease remain to be fully elucidated 
. Our findings indicate that there are large differences in methylation between invasive and non-invasive tumors, with invasive tumors exhibiting a general increase in methylation compared to non-invasive tumors. As DNA methylation at any one CpG is a binary measure (either the cytosine is methylated or not) the difference in the degree of methylation may reflect potentially greater homogeneity in the selection of epigenetically altered cells in the development of invasive disease, a result consistent with our previous work examining only a limited panel of gene promoters in a single series of bladder tumors 
. This greater degree of methylation may be driving the features of the invasive phenotype, or alternatively may be a consequence of selective pressures related to this phenotype. Although our approach in examining incident disease cannot fully distinguish these possibilities, it provides data illustrative of the urgent need for further examination of this epigenetic phenomenon.
Identification of specific genes whose DNA methylation pattern is associated with tumor phenotypes, including stage and grade also illuminates the particularly important biological processes and pathways required for genesis of these histologically defined tissue states. The FRZB
gene (also known as SFRP3
) is a member of the secreted frizzled receptor family of soluble proteins which binds to and antagonizes the WNT receptor. We and others have previously shown that hypermethylation of SFRP
genes is strongly and significantly associated with invasive bladder cancer, confirming the importance of this pathway in this phenotype 
. Of the SFRP
genes, only SFRP1
, (in addition to FRZB
) is profiled on the array, and at a different CpG location than the primers used in our and previous reports of its methylation 
. This may explain why this loci was not identified in our screening approach. Additional, more genomically dense investigation of the WNT pathway is warranted, as this may suggest a novel route for therapeutic intervention for this disease.
is a member of the homeobox family of transcription factors, and is encoded on chromosome 17 as part of a gene cluster with other HOX family members. Interestingly, we observe that non-diseased bladder epithelium exhibits a similar extent of methylation at HOXB2
as the non-invasive disease, while at FRZB
the non-diseased tissue has a significantly lower extent of methylation than either of the 2 tumor types. We also observe, in the human bladder carcinoma cell line HTB-9, which demonstrates an extent of methylation similar to that observed in invasive tumors, that treatment with the methylation inhibitor 5-aza-2′-deoxycytidine leads to increased expression of HOXB2
, suggesting that methylation of this gene may be functionally leading to the inactivation of this gene. The results in HOXB2
are consistent with the literature which has described a bivalent domain structure of the chromatin of homeobox genes, wherein both pluripotent and terminally differentiated tissue exhibit marks of both active and repressive chromatin within the same region 
, and thus an intermediate extent of DNA methylation. Invasive bladder cancers appear targeted for hypermethylation of this region, a phenomenon previously reported across polycomb group controlled genes in colorectal cancer cells 
. Such a differential alteration of a key developmental gene may be critical in defining the phenotypes of these tumors and explain the behavior and outcome from these disparate forms of this disease.
encodes cytokeratin-13 which has been shown to have specific expression in cervical squamous tumors and in mucinous cervical type adenocarcinomas 
; to our knowledge, epigenetic alterations to this gene have not been reported. Epigenetic alteration of this gene may affect its expression pattern, and such changes may be signs of a more de-differentiated phenotype, considered characteristic of aggressive, high grade tumors.
We demonstrated in an independent confirmation series of tumors that the extent of methylation of HOXB2
is associated with tumor grade, independent of each other and of TP53 protein immunohistochemical staining. TP53 staining has previously been examined and has been touted as a useful prognostic marker in this disease 
, although there is conflicting data on its utility 
. A recent meta-analysis examining its utility suggests that there is not appropriate evidence to suggest utilizing this marker clinically 
, as it may provide no addition prognostic information other than a strong association with the invasive phenotype of the disease 
. We cannot yet suggest that the markers discovered using our approach hold any more clinical utility than pathological grading and staging of tumors, but we believe that our data suggest specific cellular pathways that are disrupted in this disease. Thus, these pathways could be explored intensively as novel targets for therapeutic strategies. In addition, we suggest that future work, examining the utility of these markers in a prospective fashion is needed, as they may be useful in determining which tumors may progress. Indeed, these markers might also contribute to clinical efforts to follow patients with less invasive methods, to determine if their tumor has recurred or progressed, thereby requiring more aggressive treatment approaches.