Originally published as JCO Early Release 10.1200/JCO.2005.03.163 on March 7 2005

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Promoter Hypermethylation Is Associated With Tumor Location, Stage, and Subsequent Progression in Transitional Cell Carcinoma

From the Academic Urology Unit, Institute for Cancer Studies, and Academic Pathology Unit, University of Sheffield; Department of Pathology, Royal Hallamshire Hospital, Sheffield, United Kingdom; Départements d'Urologie, Universités Paris, Paris, France

Address reprint requests to James Catto, MD, Academic Urology Unit, K Floor, Royal Hallamshire Hospital, Glossop Rd, Sheffield, S10 2JF United Kingdom; e-mail: J.Catto{at}sheffield.ac.uk

	ABSTRACT

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PURPOSE: Transitional cell carcinoma (TCC) is a pan-urothelial disease characterized by multiplicity. Although little is known about the molecular events in upper-tract TCC, similar carcinogenic mechanisms are thought to occur throughout the urinary tract. However, we have previously shown that distinct patterns of microsatellite instability occur in upper and lower urinary tract TCC, suggesting biologic differences between these tumors. Here we investigate the extent of promoter hypermethylation in TCC throughout the urinary tract.

PATIENTS AND METHODS: Tissue was obtained from 280 patients (median follow-up, 56 months) whose tumors comprised 116 bladder and 164 upper-tract tumors (UTT). Analysis for hypermethylation at 11 CpG islands, using methylation-sensitive polymerase chain reaction and bisulfite sequencing, was performed for each sample and compared with the tumor's clinicopathologic details, microsatellite instability status, and subsequent behavior.

RESULTS: Promoter methylation was present in 86% of TCC and occurred both more frequently and more extensively in UTT (94%) than in bladder tumors (76%; P < .0001). Methylation was associated with advanced tumor stage (P = .0001) and higher tumor progression (P = .03) and mortality rates (P = .04), when compared with tumors without methylation. Multivariate analysis revealed that methylation at the RASSF1A and DAPK loci, in addition to tumor stage and grade, were associated with disease progression (P < .04).

CONCLUSION: Despite morphologic similarities, there are genetic and epigenetic differences between TCC in the upper and lower urinary tracts. Methylation occurs commonly in urinary tract tumors, may affect carcinogenic mechanisms, and is a prognostic marker and a potential therapeutic target.

	INTRODUCTION

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Transitional cell carcinoma (TCC) is a disease of the entire urothelium characterized by multiplicity. The majority of TCC occurs in the urinary bladder, with upper-tract tumors (UTTs) accounting for approximately 5% of occurrences. The distribution and frequency of TCC recurrence depends on the anatomic location of the primary tumor. Although recurrence in the bladder occurs commonly (25% to 75%) after removal of a UTT,¹ the converse is not true, and only 1% to 4% of bladder cancer patients develop subsequent upper-tract disease.² Little is known about the molecular events of UTTs, outside investigations for tumor clonality.³ The few studies of UTTs have shown both similar and lower levels of chromosomal instability in UTTs than in bladder cancer^4,5 and similar alterations in immunohistochemical expression of several tumor-related genes in UTTs and bladder cancer.⁶ Thus, with little information, it is thought that the mechanisms of carcinogenesis are similar throughout the urinary tract.^1,4,7 However, epidemiologic data suggest otherwise. Although all TCCs, regardless of location, share common carcinogens such as cigarette smoking, an increased risk of UTTs is present in several syndromes, including hereditary nonpolyposis colon cancer (HNPCC), Balkans nephropathy, analgesic abuse, and Chinese herb nephropathy.¹ Recent molecular work has now supported these clinical observations, showing that mono- and dinucleotide microsatellite instability (MSI), a feature of tumors with deficient mismatch repair (MMR) typical of HNPCC, is more common in upper than lower urinary tract cancers.^8,9 In contrast, bladder cancers have more frequent nucleotide instability at selected tetranucleotide loci than do UTTs.⁹ Although the mechanism for instability at selected tetra-nucleotides is unknown, it appears unrelated to deficient MMR and may be mediated by a p53 repair pathway.^10,11

Advances in our understanding of the molecular events in cancer reveal that histologically identical tumors may arise through different molecular mechanisms. Genomic instability, for example, may occur at either the chromosomal or nucleotide level, with the latter being characterized by widespread instability of the DNA microsatellite regions (MSI) resulting from deficient DNA MMR.¹² Loss of MMR may occur from either an inherited (in the HNPCC syndrome) or acquired gene mutation, chromosomal deletion, or promoter hypermethylation.^13,14 The methylation of the cytosine residue of a cytosine-guanine dinucleotide (CpG islands), which are concentrated within gene promoter regions, prevents gene transcription and thus is a method of tumor suppressor gene inactivation. Sporadic tumors with MSI are commonly found to inactivate MMR by promoter methylation (of the hMLH1 locus), and may possess a hypermethylator phenotype.¹⁴ Although the presence of the hypermethylator phenotype is debated,¹⁵ authors have found evidence that tumors with frequent methylation have distinctive genotypes,¹⁶ reflecting the individual susceptibility of genes to inactivation by methylation.

Although CpG methylation has been reported to occur frequently in bladder cancer and to be associated with increased tumor stage and grade,^17,18 to our knowledge it has not been studied in UTTs. Investigation of the methylator and mutator phenotypes in colorectal cancer has revealed that the extent of each differs according to tumor location.¹⁹ In this study we investigate whether the same is true for tumors of the urinary tract.

	PATIENTS AND METHODS

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Patients and Specimens
We analyzed 280 patients with primary TCC (Table 1). The patients were treated according to tumor stage at either the Royal Hallamshire Hospital Sheffield, United Kingdom (all bladder and 62 UTT), or at the Institut Mutualiste de Montsouris (n = 23), Hôpital Tenon (n = 56), and Hôpital St Louis (n = 23), Paris, France. Although all superficial bladder tumors were removed by transurethral resection, with adjuvant intravesical chemotherapy or immunotherapy administered as necessary,¹ radical cystectomy was the treatment for muscle-invasive disease. Nephroureterectomy was performed for all of the UTTs in this series, regardless of tumor stage. Postoperative surveillance for tumor relapse was performed in all patients, for a median of 56 months (range, 1 to 216 months) or until death. The patients were representative of a Western population with 67% males (n = 188), 58% smokers (n = 165), and a median age of 70 years (range, 34 to 90 years). Although clinicopathologic data were missing in eight UTT patients, in no cases were there multiple tumors from the same patient. We have previously^20,21 assessed these TCCs for the presence of MSI. For analysis, recurrence was defined as the appearance of an additional TCC with a stage and grade similar to or lower than the original; progression was defined as when an additional TCC occurred with more advanced stage or grade; and relapse was defined as when either recurrence or progression occurred for both superficial and invasive TCC. Local ethics committee approval was obtained before the commencement of this study.

Methylation Analyses
Eleven genomic regions with CpG dinucleotide concentrations (CpG islands) were investigated by methylation-specific polymerase chain reaction (msPCR). The regions were chosen to include the promoters of two MMR genes responsible for MSI (hMLH1 and hMSH2), genes important for urothelial carcinogenesis (p14, p16, and E-cadherin), and other genes found to be methylated frequently in human cancer (RARB, DAPK, MGMT, RASSF1A, and GSTP1). The final CpG island, MINT31, was chosen because it is believed to detect the presence of the hypermethylator phenotype.¹⁴ As previously used,¹⁸ the degree of methylation for each tumor was calculated as a methylation index (MI; number of methylated islands/total number of successfully analyzed islands [as a percentage]). msPCR was performed with Accuprime Taq polymerase (Invitrogen, Paisley, United Kingdom) using primers and conditions described elsewhere.^18,22,23 PCR products were analyzed in adjacent lanes of a 3% agarose gel and visualized using ethidium bromide and UV light.

To confirm the msPCR results, the bisulfite-modified CpG islands of representative tumors were sequenced using the EXCEL II Sequencing Kit-LC (Epicenter, Cambridge, United Kingdom). Sequencing primers were fluorescence labeled and the products were read on an automated sequencer (LI-COR Biosciences, Cambridge, United Kingdom).

Statistical Methods
Two-tailed statistical analyses were performed using SPSS version 12 (SPSS Inc, Chicago, IL). Categoric variables were compared using the ² test and continuous variables were analyzed with a Student's t test. Recurrence, progression, relapse, and survival probabilities after tumor resection are defined elsewhere,²⁰ and were analyzed using the Kaplan-Meier method and log-rank test. Multivariate analysis for predictors of tumor progression was performed using Cox regression analysis. A P value of less than .05 was interpreted as statistically significant.

	RESULTS

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CpG Island Methylation
The frequency of aberrant methylation varied from 0% (GSTP1, hMSH2) to 72% (E-cadherin; Table 2; Fig 1). For all loci, except DAPK, more frequent methylation was present in UTTs than in the bladder cancers and reached statistical significance for hMLH1, RARB, E-Cadherin, p16, and MINT31. hMLH1 and MINT31 were rarely methylated in bladder tumors, in contrast to UTTs, suggesting they play a role in upper-tract carcinogenesis but not in bladder cancer. Individual CpG hypermethylation was associated with increased tumor stage for RARB (² P < .0001), DAPK (² P = .05), E-cadherin (² P = .013), MINT31 (² P = .001), and RASSF1A (² P < .0001), when compared with tumors lacking methylation at these islands. Only RASSF1A methylation was associated with tumor grade, with more methylation in poorly differentiated than well-differentiated tumors (² P = .017). Higher DAPK methylation rates were present in nonsmokers than smokers (² P = .035) and in females than males (² P = .005). The female preponderance of DAPK methylation may be explained by the smoking relationship, given that most nonsmokers in this series are women.

View larger version (60K): [in this window] [in a new window]   Fig 1. Transitional cell carcinoma (TCC) methylation analyses. (A) Methylation-specific polymerase chain reaction analysis reveals representative methylated and unmethylated tumors for each locus. For hMSH2 and GSTP1, positive controls are shown. (B) Reverse strand of hMLH1 promoter bisulfite sequencing reveals () methylated tumors and () unmethylated tumors without guanine bases (methylated cytosines are protected from bisulfite modification).

View larger version (19K): [in this window] [in a new window]   Fig 2. Tumor behavior according to methylation status. Tumors with (A) RASSF1a, (B) MINT31, and (C) any locus methylation have higher progression rates than those without methylation. (D) Superficial noninvasive transitional cell carcinomas (TCCs) with higher methylation indices (MIs) have higher progression rates. (E) Muscle-invasive TCC with higher MI (> 20% shown) have lower progression rates (log-rank shown).

MIs
Methylation was present in the majority of TCC (241 of 280; 86%) and was more frequent in UTTs (93%) than bladder cancer (76%; ² P < .0001). Furthermore, when methylation was present, it was more extensive in UTTs than in bladder tumors (t test P = .0001; Fig 3A), regardless of stage inequalities between the tumor populations (Table 3). In addition to tumor location, the MI varied according to tumor stage, with more frequent and extensive methylation in muscle-invasive tumors compared with superficial cancers (t test P = .00001; Fig 3B; Table 3). Interestingly, superficially invasive (pT1) tumors had a similar degree of methylation to that in deeply invasive (pT2-4) tumors. Although there was a trend toward an association between MI and tumor grade, the small number of grade 1 tumors may have prevented this reaching significance.

View larger version (24K): [in this window] [in a new window]   Fig 3. Methylation indices according to tumor location (A) and stage (B). (A) Upper-tract tumors (B) and invasive transitional cell carcinomas (TCCs) have more extensive methylation than bladder cancers and noninvasive (Ta) tumors (t test values shown). Note the similarity in methylation between superficial tumors with early invasive (pT1) and muscle-invasive (pT2-4) tumors.

As with individual CpG islands, when tumors were stratified for stage there were additional associations between progression and MI. For pTa disease, tumors with methylation more frequently progressed to invasion than those without methylation (log-rank P = .03). Progression was also effected by the degree of methylation, with higher progression rates seen in tumors with higher MI (for example, pTa tumors with MI < 10%; log-rank P = .0028; Fig 2D). In pT2-4 tumors only high methylation levels were associated with progression; tumors with a higher MI (eg, MI > 20%) had lower mortality rates (log-rank P = .023; Fig 2E) than those with a lower MI.

Methylation and MSI
Although the hMSH2 promoter was never methylated, hMLH1 promoter hypermethylation occurred in 20 (7%) of TCC, 19 of which were UTTs. hMLH1 methylation was significantly associated with the presence of mono- and dinucleotide MSI-High (MSI-H 16 of 20; ² P < .0000001), but not instability at selected tetranucleotide loci. Only one bladder tumor (which was MSI-H) had hMLH1 methylation, in contrast to 12% of UTTs (² P = .001). There were no significant associations between hMLH1 methylation status and clinicopathologic factors or tumor behavior. Tumors with hMLH1 methylation were found to have a higher MI than those without hMLH1 methylation (mean, 31.4 v 18.4; t test P = .00001), which is suggestive of a hypermethylator phenotype. Interestingly, none of the suspected HNPCC patients were found to have hMLH1 methylation.

	DISCUSSION

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It was believed previously that bladder cancers and UTTs have similar carcinogenic mechanisms,^1,4,7 reflecting common pathogens. Our results, combined with previous studies,^8,9 now provide evidence that the extent of the mutator and methylator phenotypes in TCC differs with tumor location, and perhaps suggest that carcinogens affect the urinary tract in different ways. Our finding is not surprising, given that epidemiologic data reveal several etiologic risks for UTTs but not bladder cancer, of which HNPCC is the best understood. In the majority of HNPCC families, an inherited mutation predisposes to MMR deficiency, which occurs when the remaining normal MMR allele is lost.²⁴ However, although each cell in the body carries this predisposition to MMR loss, the majority of tumors within the HNPCC syndrome occur in specific anatomic locations (eg, proximal colon, endometrium, stomach, and upper urinary tract).²⁵ That sporadic cancers in these anatomic locations have significantly more frequent MSI than tumors elsewhere suggests that factors unrelated to HNPCC may be responsible for this anatomic specificity.

Our results suggest that in sporadic UTTs, as in colon cancer,²⁴ promoter methylation of hMLH1 is a common method of MMR loss leading to MSI (accounting for 10% of UTTs and 1% of bladder cancers in our series). Although the molecular mechanisms of aberrant promoter methylation in cancer are poorly understood,²⁶ its anatomic specificity suggests that either environmental or embryologic factors are responsible. For both the colon and urinary tract, the embryologic origins of those regions with differential methylation differ. Environmental factors have also been suggested to explain the pattern of methylation in human cancer and it is possible that the upper urinary tract receives a higher dose of a methylating factor than the bladder; for example, a urinary-based mutagen that is subsequently inactivated in the bladder, such as proposed for the development of UTTs in Balkans nephropathy.²⁷ Dietary factors have been suggested to account for the anatomic specificity of promoter methylation in colon cancer.²⁸

Our results confirm the importance of methylation in the molecular pathogenesis of TCC, with the majority of tumors having one or more CpG islands methylated.¹⁸ Previous work has suggested that superficial and invasive bladder tumors arise by different molecular pathways.²⁹ Our data are consistent with this hypothesis and suggest that frequent methylation is more typical of the invasive than superficial tumor pathways. In addition, the similarities of methylation between early invasive (pT1) and muscle-invasive (pT2-4) tumors, suggests that epigenetic changes occur at an early stage of the invasive pathway. Certainly the association between hMLH1 methylation and tumor MSI-H supports this model of early promoter hypermethylation. Previous workers have shown that promoter methylation leads to loss of hMLH1 expression, resulting in MMR deficiency and MSI-produced carcinogenesis.²²

The association between the presence and extent of methylation and subsequent tumor progression suggests a potential role as a prognostic biomarker. In particular, the presence of methylation was a good indicator of superficial tumor progression (Fig 2D), as reflected by the epigenetic similarities between pT1 and pT2-4 tumors (Fig 3B). The detection of methylation has benefits over previously suggested genetic biomarkers, as methylation always occurs in the same DNA location and is therefore easier to detect than gene mutation. In addition, methylation appears to be a general process affecting the whole genome; thus, only a small number of CpG islands need to be analyzed. Finally, with msPCR the significant test result is a positive signal and therefore is easily seen, in contrast to loss of heterozygosity detection in which absence of a PCR product is the important result and can be lost with contamination from normal tissue.

Perhaps the most exciting area of current epigenetic investigation is the development of novel therapies. Although to correct a gene deletion or mutation the normal gene must be reinserted into the genome, a task that has proved difficult, genes inactivated by methylation are wild type. Chemical methods that remove the methyl group result in gene re-expression and in cell culture models can suppress the growth of malignant cells.³⁰ An orally active demethylating agent has recently been described that reduces the growth of bladder tumors in mice.³¹

In conclusion, we have shown that there are genetic and epigenetic differences between upper and lower urinary tract TCC and approximately 10% of UTTs arise from MSI, which results following hMLH1 inactivation by methylation. Methylation is an important molecular mechanism in TCC and could be used as a prognostic and diagnostic marker. In addition, the high frequency of methylation in TCC suggests that this tumor would be a good target for the development of novel therapies with demethylating agents.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	NOTES

 
Supported by grants from the Medical Research Council, the 2000 British Urological Foundation/Merck Sharpe, and Dohme Scholarship and Yorkshire Cancer Research.

Presented in part at the 95th Annual Meeting of the American Association for Cancer Research, March 27-31, 2004, Orlando, FL, and the Annual Meeting of the British Association of Urological Surgeons, June 21-25, 2004.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
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Submitted March 27, 2004; accepted September 24, 2004.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Catto, J. W.F. Articles by Hamdy, F. C. Search for Related Content PubMed PubMed Citation Articles by Catto, J. W.F. Articles by Hamdy, F. C. Related Articles Related Editorial