in 2011 , about 52,000 men and 17,000 women will be diagnosed with bladder cancer in the united states . before a normal cell transforms into a bladder cancer cell , although we do not fully understand the mechanisms , dna alterations including hypermethylation and somatic mutation are commonly observed events in human cancer . in a recent bladder cancer study fgfr3 mutation in combination with apc , rassf1a , and sfrp2 methylation markers provided a sensitivity of 90% using tissue samples and 62% using paired urine samples to identify the presence of cancer with 100% specificity . in nonsmall cell lung cancer ( nsclc ) and breast cancer , studies showed that rassf1a had different frequencies of methylation depending on histology [ 2 , 3 ] . in nasopharyngeal carcinoma , rassf2a was frequently inactivated by its promoter methylation and the methylation correlated with lymph node metastasis . the ras - association family , also called rassf tumor suppressor genes , currently includes 10 members . all of the rassf proteins contain a ras - association domain on their c - terminus ( rassf 16 ) or n - terminus ( rassf 710 ) . two important issues that are not previously addressed by studies of rassf gene methylation are ( 1 ) whether all of the rassf family members show aberrant methylation in bladder cancer and ( 2 ) whether methylation pattern of rassf genes can be used as a diagnostic biomarker . rassf1a ( ras - association domain family 1 isoform a ) is the first identified rassf family member which is frequently epigenetically inactivated in a wide range of cancer types . as a tumor suppressor gene , the methylation signature of rassf1a is thought to be among the earliest cellular changes in tumorigenesis . as a potential tumor suppressor , rassf2 plays a role in apoptosis and cell cycle arrest and is frequently downregulated in lung tumor cell lines by hypermethylation . although the 5 cpg island of rassf3 has been identified earlier , rassf3 does not show methylation in glioma tumor cell lines . rassf4 is broadly expressed in different human tissues , but its expression is down - regulated by promoter hypermethylation in a majority of tumor cell lines and primary tumors . as a proapoptotic ras effector , rassf5 ( nore1a ) is frequently inactivated by promoter methylation in human tumors like glioma tumor cell lines , colorectal tumors , and lung cancer [ 1215 ] . rassf6 promotes apoptosis by cooperating with activated k - ras to induce cell death and inhibit the tumor cell survival . it appears that all of c - terminal rassf family members have hypermethylation - induced gene inactivation in various types of cancer . while there is extensive literature on rassf1a , other rassf family members have not been studied as widely . high - resolution melting ( hrm ) analysis is a new methodology that monitors the melting behavior of pcr amplicons by using dna intercalating fluorescent dye . originally the lcgreen was used to develop a closed - tube method for genotyping and mutation scanning . new high sensitive dyes such as eva green and syto 9 can be used at saturation concentration to monitor the denaturing process of pcr amplicons . compared to traditional methylation specific pcr ( msp ) method , hrm is a reliable and simple method for dna methylation detection [ 20 , 21 ] . in this study , to examine diagnostic value of rassf gene methylation , we identified the methylation status of cpg islands associated with c - terminal rassf 16 in a group of formalin fixed paraffin embedded bladder cancer samples using a methylation specific hrm assay . universal methylated and unmethylated dna samples ( zymo research corp , orange , ca ) were used as 100% and 0% methylated control . the methylated dna was serially diluted in unmethylated dna to create standard dilutions of 0% , 10% , 50% , and 100% methylated dna . the standard dilutions from 100% to 0% were used to semiquantitatively measure promoter methylation status of c - terminal rassf genes in ffpe samples . ffpe blocks from 48 bladder cancer patients were collected by the department of pathology and the human tissue resource network at the ohio state university . we obtained 16 paired tumor and matched normal adjacent tissues and 32 tumor tissues ( 35 males and 13 females , male - to - female ratio 2.7  :  1 ; median age 67 years , range 2890 years ) . among these patients , 41 cases were diagnosed with transitional cell carcinoma ; 4 cases were small cell carcinoma and 3 cases were squamous cell carcinoma . dna samples were extracted using recover all total nucleic acid isolation kit ( life technologies corporation , carlsbad , ca ) . briefly , 510  mg samples were sliced from paraffin blocks and deparaffinizated by xylene at 50c , followed by 100% ethanol wash . the air - dry tissue samples were digested by proteinase k for 24  hrs in a microtube shaking incubator set at 50c . the digested samples were mixed with appropriate volume of isolation additive and 100% ethanol . after passing the mixture through the filter cartridge , the dna was purified by washing buffer and eluted with 95c nuclease - free water . the ffpe dna bisulfite modification was processed using ez dna methylation kit ( zymo research corp , orange , ca ) . the double - stranded dna was denatured in m - dilution buffer for 15 minutes at 37c , and then ct conversion reagent was added to each sample . the samples were incubated in the dark at 50c for 12 hours followed by 4c for 10 minutes . after mixing with m - binding buffer . finally , the bisulfite - modified dna was eluted by m - elution buffer from column matrix . ms - hrm is based on pcr amplification of bisulfite modified genomic dna with subsequent hrm analysis of pcr amplicons . the primers were designed to amplify both methylated and unmethylated dna . because of dna degradation in ffpe samples , the sizes of amplicons were limited to 80180  bp . the free online tool from methprimer ( http://www.urogene.org/methprimer/index1.html ) was used specifically for primer design in this rassf promoter methylation study . pcr amplification and high - resolution melting analysis were carried out sequentially on a cfx96 real - time pcr system ( biorad , hercules , ca ) . pcr was performed in a 20  l total volume containing : 10  l 2x type - it hrm pcr master mix ( qiagen , hilden , germany ) , 1  l 10 picomol/l ms - hrm primer , 8  l nuclease - free water , and 1  l bisulfite converted dna ( theoretical concentration 10  ng/l ) . the amplification consisted of 10  min at 95c , followed by 40 cycles of 10  s at 95c , 30  s at annealing temperature and 10  s at 72c . high - resolution melting analysis were performed at the temperature ramping from 7095c by 0.2c / s and florescence acquisition was set per manufacturer 's recommendation . the chi - square test was used to examine the significant differences of methylation depending on histology and staging ( p  0.05 ) . to evaluate sensitivity and specificity of methylation as a predictive marker receiver operating characteristic ( roc ) analysis was used . to generate a profile of c - terminal rassf gene epigenetic changes in bladder cancer , 64 bladder ffpe tissue samples were examined by methylation - specific hrm assay . the c - terminal rassf family members ( rassf1a , rassf2a , rassf4 , rassf5 , and rassf6 ) were analyzed in this study . in the ucsc genome browser however , due to the dense cg dinucleotides on the cpg island of the identified rassf3 promoter , no appropriate hrm primers could be designed for this gene . hrm assay uses double - stranded dna binding dyes and requires less pcr optimization than other methods . the principle of hrm depends on recording the melting profile of double - stranded dna samples . as double - stranded dna is denatured , the fluorescence signal from dye bound to double - stranded dna decreases . the high - resolution melting requires smaller temperature increase steps ( < 0.5c / s ) between each fluorescence reading , which can provide detailed information of amplicon melting behavior . ms - hrm is a semi - quantitative method for rapidly assessing the presence of dna methylation . the bisulfite - modified fully methylated dna was diluted in bisulfite - modified fully unmethylated dna to obtain a series of methylation percentage : 0% , 10% , 50% , and 100% methylation . only samples containing more than 10% methylation were counted as methylated samples . promoter methylation was analyzed in tumor and normal adjacent tissues from 16 cases using ms - hrm . 56% ( 9/16 ) of tumor samples were found to have rassf1a promoter methylation , and 25% ( 4/16 ) of tumor samples showed rassf2a promoter methylation , while only 6% ( 1/16 ) of the normal adjacent tissue samples showed rassf1a promoter methylation and none of normal adjacent tissue samples showed rassf2a methylation . rassf 4 , 5 , and 6 were not found to be methylated in either tumor or normal adjacent tissues . figure 2 demonstrates that tumor and normal adjacent tissue showed different rassf1a and rassf2a methylation profiles . among the 48 patients with bladder cancer , rassf1a promoter methylation alone had 71% sensitivity and 94% specificity and an area under the curve ( auc ) of 0.823 to correctly identify bladder cancer tissue whereas rassf1a and rassf2a together had 77% sensitivity and 94% specificity and auc of 0.854 . rassf1a and rassf2a promoter methylation did discriminate bladder cancer tissue from normal adjacent tissue ( p < 0.0001 ) . 41 patients had tumors with transitional cell carcinoma features 4 with small cell carcinoma , and 3 with squamous cell carcinoma . rassf1a was methylated in 68% ( 28/41 ) and rassf2a in 7% ( 3/41 ) of transitional cell carcinoma samples . rassf1a was methylated in 100% ( 4/4 ) and rassf2a in 0% ( 0/4 ) of small cell carcinoma . rassf1a was methylated in 67% ( 2/3 ) and rassf2a in 33% ( 1/3 ) of squamous cell carcinoma ( figure 3 ) . the frequency of rassf1 and rassf2 promoter methylation together showed no significant difference with histology in our study ( p = 0.295 ) . the reasons that lead to aberrant cpg island methylation of rassf1a and rassf2a in transitional cell carcinoma , small cell carcinoma , and squamous cell carcinoma of the bladder are not well understood . investigating nonsmall cell lung cancer showed that rassf1a promoter region cpg islands were methylated in 55% of adenocarcinomas , 25% of large cell carcinomas , and 25% of squamous cell carcinomas . this study indicates that promoter methylation of rassf gene family members might be dependent on histology in nonsmall cell lung cancer . 60 malignant bladder tumor and normal adjacent tissue samples from patients with different t stages ( 16 normal adjacent tissue samples , 9 stage t1 , 11 stage t2 , 18 stage t3 , and 6 stage t4 ) were analyzed to detect the rassf promoter methylation changes of bladder cancer patients . methylated rassf1a promoters were only found in 6% ( 1/16 ) of normal adjacent tissues . the percentage of promoter methylation positive patients increased with t stage , being lower in t1 tumors and higher with higher stage . the percentage of samples with methylated rassf1a genes was 55% ( 5/9 ) in stage t1 , 73% ( 8/11 ) in stage t2 , 78% ( 14/18 ) in stage t3 , and 83% ( 5/6 ) in specimens from t4 tumors ( figure 4 ) . the frequency of rassf1 and rassf2 promoter methylation was not associated with t stage ( p = 0.363 ) . due to lack of samples with rassf2a methylation , no association between rassf2a methylation and t stage was identified in the current data set . the rassf2a promoter methylation was found at low levels ( 018% ) at different t stages . we examined methylation status of rassf1a and rassf2a in 27 bladder tumor samples with stage n0 , 8 samples with stage n1 , and 8 samples with stage n2 . percentage of rassf1a promoter methylation had a range of 5087.5% in tissues from patients with different lymph node metastasis stage . the percentage of samples with methylated rassf2a was 11% ( 3/27 ) for stage n0 , and 6% ( 1/16 ) for stage n1/2 ( figure 5 ) . based on the result of chi - square test , there is no significant difference between the frequency of rassf1 and rassf2 promoter methylation in lymph node positive and negative patients . in nasopharyngeal carcinoma , aberrant methylation of rassf2a promotor was found to be associated with lymph node metastasis . although both rassf1a and rassf2 protein can function as a negative effector of ras protein in tumor formation , rassf2a and rassf1a have apparently different functions in different type of tumors . rassf1 - 6 share a variable n - terminal sequence followed by a ras - association domain . the hrm analysis showed no detectable promoter methylation of rassf4 , rassf5 and rassf6 in bladder cancer and normal adjacent tissue samples . promoter hypermethylation has been proposed as a potential diagnostic or prognostic biomarker in various cancers . recent research showed that urine is potentially useful for bladder cancer screening [ 26 , 27 ] . methylation status of certain genes identified in urine samples showed higher sensitivity than the conventional urine cytology method . these studies indicated that detection of promoter methylation in urine specimen could potentially provide a simple , noninvasive , and sufficiently sensitive method for bladder cancer screening in the future . in our study , a new methodology , methylation - specific - high resolution melting analysis was used to examine the melting behavior of methylated or unmethylated rassf gene amplicons . we studied dna promoter methylation of five rassf family members ( rassf1a , rassf2a , rassf4 , rassf5 , and rassf6 ) in ffpe bladder cancer tissues and normal adjacent tissues . we identified distinctive rassf1a and rassf2a gene promoter methylation profiles that differentiate between bladder cancer and normal adjacent tissue samples . using rassf1a and rassf2a genes together showed an acceptable sensitivity ( 77% ) and high specificity ( 94% ) identifying bladder cancer tissues . previous studies have identified rassf1a promoter methylation as a potentially useful urine biomarker for the presence of invasive bladder cancer [ 26 , 28 , 29 ] . we now show that the addition of rassf2a promoter methylation analysis can improve the sensitivity potentially without compromising specificity . as discussed by serizawa , results also showed that fgfr3 mutation in bladder cancer when combined with methylation markers ( apc , rassf1a and sfrp2 ) provided a sensitivity of 90% to identify bladder tumors . it remains to be shown if the addition of an rassf2a promoter methylation assay to the previously published urine biomarker assay can indeed improve sensitivity when using urine samples . limitations of the study include small sample size or lack of a validation data and sample set . in addition we did not have sufficient data to analyze if rassf gene family promoter methylation could predict the recurrence of bladder cancer . methylation analysis of both rassf1a and rassf2a genes appeared to increase the sensitivity of discriminating cancer from normal adjacent tissue . the addition of rassf2a methylation analysis to recent bladder cancer biomarker signatures has the potential to further increase sensitivity for bladder cancer diagnosis . rassf1a and rassf2a promoter methylation analysis could be useful as a biomarker to detect the presence of bladder cancer recurrence .
promoter hypermethylation , a widely studied epigenetic event known to influence gene expression levels , has been proposed as a potential biomarker in multiple types of cancer . 
 clinical diagnostic biomarkers are needed for reliable prediction of bladder cancer recurrence . in this paper , dna promoter methylation of five c - terminal ras - association family members ( rassf1a , rassf2a , rassf4 , rassf5 , and rassf6 ) 
 was studied in 64 formalin - fixed paraffin - embedded ( ffpe ) bladder cancer and normal adjacent tissues using methylation - specific high - resolution melting ( ms - hrm ) analysis . 
 results showed that 73% ( 30/41 ) of transitional cell carcinoma , 100% ( 3/3 ) of squamous cell carcinoma , and 100% ( 4/4 ) of small cell carcinoma demonstrated promoter methylation of the rassf1a or rassf2a gene , but only 6% ( 1/16 ) of normal tissues had promoter methylation of rassf genes . 
 testing positive for hypermethylation of rassf1a or rassf2a promoter provided 77% sensitivity and 94% specificity for identification of cancer tissues with an area under the curve of 0.854 , suggesting that promoter methylation analysis of rassf1a and rassf2a genes has potential for use as a recurrence biomarker for bladder cancer patients .