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DNA Methylation Architecture Provides Insight into the Pathogenesis of Upper Tract Urothelial Carcinoma: A Systematic Review and Meta-Analysis

  • Author Footnotes
    # These authors contributed equally to the work.
    Yifei Lin
    Footnotes
    # These authors contributed equally to the work.
    Affiliations
    West China Hospital, Sichuan University, Chengdu, PR China
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  • Author Footnotes
    # These authors contributed equally to the work.
    Ling Lin
    Footnotes
    # These authors contributed equally to the work.
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China
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  • Author Footnotes
    # These authors contributed equally to the work.
    Yong Yang
    Footnotes
    # These authors contributed equally to the work.
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China
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  • Mei Li
    Affiliations
    Medical Equipment Innovation Research Center, West China School of Medicine, Med+X Center for Manufacturing, West China Hospital, Sichuan University, Chengdu, Sichuan, P.R.China
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  • Xin Jiang
    Affiliations
    Medical Equipment Innovation Research Center, West China School of Medicine, Med+X Center for Manufacturing, West China Hospital, Sichuan University, Chengdu, Sichuan, P.R.China
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  • Tingting Fu
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China

    Medical Device Regulatory Research and Evaluation Center, Frontiers Science Center for Disease-related Molecular Network, West China Hospital/West China School of Medicine, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,College of Life Sciences, Sichuan University, Chengdu, Sichuan, P.R.China.
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  • Youlin Long
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China

    Chinese Evidence-Based Medicine Centre, West China Hospital, Sichuan University, Chengdu, PR China
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  • Qiong Guo
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China
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  • He He
    Affiliations
    Department of laboratory medicine, West China Hospital, Sichuan University, Chengdu, PR China
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  • Zhenglong Chen
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China
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  • Liang Du
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China
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  • Ga Liao
    Affiliations
    State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, PR China
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  • Banghua Liao
    Correspondence
    Address for correspondence: Banghua Liao, MD, Department of Urology, Institute of Urology (Laboratory of Reconstructive Urology), West China Hospital, Sichuan University, #37 Guoxue Alley, Chengdu 610041, PR China.
    Affiliations
    Department of Urology, Institute of Urology (Laboratory of Reconstructive Urology), West China Hospital, Sichuan University, Chengdu, PR China
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  • Jin Huang
    Correspondence
    Address for correspondence: Jin Huang, MD, Medical Device Regulatory Research and Evaluation Centre, West China Hospital, #37 Guoxue Alley, Sichuan University, Chengdu 610041 PR China.
    Affiliations
    Medical Device Regulatory Research and Evaluation Centre, West China Hospital, Sichuan University, Chengdu, PR China

    Medical Equipment Innovation Research Center, West China School of Medicine, Med+X Center for Manufacturing, West China Hospital, Sichuan University, Chengdu, Sichuan, P.R.China
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  • Author Footnotes
    # These authors contributed equally to the work.
Open AccessPublished:October 20, 2022DOI:https://doi.org/10.1016/j.clgc.2022.10.008

      Abstract

      Purpose

      Numerous studies suggested methylation modifications play an important role in upper tract urothelial carcinoma (UTUC), but few have depicted DNA methylation architecture on the pathological process of UTUC. We aimed to better understand the pathogenesis of UTUC and provide precision medicine references when managing UTUC patients.

      Methods

      PubMed, Cochrane Library, EMBASE, and Scopus were searched for UTUC until December 31, 2020. Methodological quality assessment was conducted according to NIH recommendations. Meta-analysis was conducted to assess the prognostic effect of methylated genes. Kaplan-Meier survival analyses were performed to validate methylated genes and cytosine-phosphate-guanine (CpG) sites.

      Results

      Eleven studies (3619 patients) were eligible to investigate 12 methylated genes and 10 CpGs. The quality of all the studies was fair to good. Meta-analysis found the pooled effect of eligible methylated genes had a low risk of tumor recurrence (HR = 0·67; 95% CI: 0·51-0·87; P = ·003), but a high risk of tumor progression (HR = 1·60; 95% CI: 1·17-2·18; P = ·003) and cancer-specific mortality (HR = 1·35; 95% CI: 1·06-1·72; P = ·01). For individual methylation status of GDF15, HSPA2, RASSF1A, TMEFF2, and VIM, the pooled effect of each gene was found pleiotropic on both diagnosis and prognosis. Survival analysis suggested higher methylation of SPARCL1 had a better disease-specific survival (P = ·048).

      Conclusion

      We combined meta-analysis and Kaplan-Meier survival analysis using the most updated evidence on the methylation of UTUC. Candidate biomarkers with essential diagnosis and prognosis function might provide precision medicine references for personalized therapies.

      Keywords

      Abbreviations:

      CpG (cytosine-phosphate-guanine), CI (confidence interval), FFPE (formalin-fixed paraffin-embedded tissue), HRs (hazard ratios), OS (overall survival), UCs (urothelial carcinomas), UCB (urothelial carcinoma of the bladder), UTUC (upper tract urothelial carcinoma)

      Introduction

      Upper tract urothelial carcinoma (UTUC) is a rare malignant tumor that accounts for 5% to 10% of urothelial carcinomas (UCs),
      • Siegel RL
      • Miller KD
      • Jemal A.
      Cancer statistics, 2020.
      with an incidence of approximately 2 per 100,000 inhabitants.
      • Rouprêt M
      • Babjuk M
      • Burger M
      • et al.
      European association of urology guidelines on upper urinary tract urothelial carcinoma: 2020 update.
      However, the symptoms of UTUC are so varied and non-specific; thus, it was hard to distinguish UTUC early and precisely or improve the prognosis satisfactorily.
      Due to the involvement of both the environment and genetics, the risk factors and prognostic factors of UTUC remain unclear. Previous studies have concluded that environmental factors, including cigarette smoking
      • Rink M
      • Xylinas E
      • Margulis V
      • et al.
      Impact of smoking on oncologic outcomes of upper tract urothelial carcinoma after radical nephroureterectomy.
      and aristolochic acid,
      • Chen IH
      • Luo HL
      • Su YL
      • et al.
      Aristolochic acid affects upper tract urothelial cancer behavior through the mapk pathway.
      are carcinogens for UTUC. In addition, genetic predisposition also plays an important role in the pathogenesis of UTUC. A previous study found novel genomic and proteomic technologies could help identify numerous molecular alterations for UTUC.
      • Hassler MR
      • Bray F
      • Catto JWF
      • et al.
      Molecular characterization of upper tract urothelial carcinoma in the era of next-generation sequencing: a systematic review of the current literature.
      However, DNA methylation modifications were rarely discussed using quantitative methods.
      As increasing interest in epigenetics has been accompanied by technological breakthroughs,
      • Portela A
      • Esteller M.
      Epigenetic modifications and human disease.
      the new evidence of methylation modifications emerged recently to explore the susceptibility of UTUC. Although various sequencing and experimental studies found that DNA methylation in urine
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      or tissue,
      • Wang J
      • Zhao Y
      • Xu H
      • et al.
      Silencing NID2 by DNA hypermethylation promotes lung cancer.
      can be considered as specific biomarkers for diagnosis or prognosis of UTUC, very few could gain a comprehensive picture of the DNA methylation mechanism of UTUC.
      To better understand the pathogenesis of UTUC, we systematically evaluated the available literature on DNA methylation modifications of UTUC. Through meta-analysis and Kaplan-Meier survival analysis, we aimed to detect the methylation architecture of UTUC and hoped to find novel methylation molecular biomarkers to help to improve the prediction and treatment of UTUC.
      We present the following article following the PRISMA reporting checklist.

      Methods

      Search Strategy

      This study was conducted following the preferred reporting items for systematic reviews and meta-analyses statement (PRISMA) guidelines
      • Liberati A
      • Altman DG
      • Tetzlaff J
      • et al.
      The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration.
      and with the reporting recommendations for tumor marker prognostic studies (REMARK) guidelines.
      • McShane LM
      • Altman DG
      • Sauerbrei W
      • Taube SE
      • Gion M
      • Clark GM.
      Reporting recommendations for tumor marker prognostic studies.
      To identify all relevant literature, we searched PubMed, The Cochrane Library, EMBASE, and Scopus for studies of UTUC between January 1, 2004 and December 31, 2020. The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) under number CRD42021271633. Details of the search terms and the search strategies are available in the Supplementary Note.

      Inclusion and Exclusion Criteria

      All studies had to meet the following inclusion criteria:1) samples including tumor tissue, serum, and urine are from patients with UTUC regardless of age, sex, area, or country; 2) samples that were analyzed for DNA methylation used next gene sequencing, methylation-sensitive PCR, microarray assays and other molecular biotechnology; and 3) molecular alterations regarding DNA methylation were the primary outcomes of the studies. The following exclusion criteria were also applied in this study:1) animal and other tumor studies; 2) case reports/series, systematic reviews, reviews, editorials, abstracts, conference and overlapped studies; and 3) studies published in languages other than English.

      Study Selection and Data Extraction

      Through bibliographic citation management software (EndNote X9, Thomson Reuters, USA), 2 researchers (L.L. and Y.L.) independently screened the topics and abstracts and further screened the studies that met the eligibility criteria by reading the full text. When researchers faced conflicts in the screening phase, disagreements should be resolved by reaching a consensus after discussion or consulting with a third reviewer (B.L.). The following data were extracted independently from each included study by 2 reviewers: first author name, region, year of publication, study design, gender, sample size, source of the sample, analysis methods/platforms, significant biomarkers studied, follow-up, and clinical application. The extracted data were subsequently cross-checked by a third reviewer (B.L.).

      Methodological Quality Assessment

      Two researchers independently assessed the risk of bias in the included studies using study quality assessment tools (https://www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools), and the inconsistent results were resolved through discussion or a third reviewer. The detailed content of study quality assessment tools differed depending on various studies including observational cohorts, cross-sectional studies, and case-control studies.

      Meta-analysis

      For those UTUC prognostic studies, significant biomarkers with hazard ratios (HRs) and associated 95% confidence interval (CI) of survival, from multivariate analyses, were pooled to assess the overall effect size. Heterogeneity was tested using the χ2 test, and its magnitude was further assessed using I2 statistics which was based on Cochran's Q statistic. If considerable heterogeneity (P < .05, I2 > 50%) was detected, sources of heterogeneity were then analyzed. After excluding the influence of obvious clinical heterogeneity, the random effect model will be used for meta-analysis. Otherwise, the fixed-effect model was applied.
      Subgroup analysis was additionally performed based on different prognosis outcomes. Specifically, for tumor recurrence, patients were categorized as the first bladder/intravesical recurrence, second bladder recurrence, contralateral recurrence, and systemic recurrence group. For tumor progression, patients were classified into higher tumor grade (G3), higher tumor stage (T3 & T4), and pN+ groups. Furthermore, subgroup analyses for different methylated genes were performed based on different prognosis outcomes, including tumor recurrence, tumor progression, and cancer-specific mortality.
      Publication bias was evaluated using Egger's test and funnel plots. All statistical analyses were performed using Review Manager (Version 5·3; Cochrane Collaboration, Oxford, UK). The threshold for the statistical significance of all outcomes was set to a 2-sided α of 0·05.

      Survival Analysis

      Kaplan-Meier survival analysis was performed for all the included biomarkers based on Cancer Genome Atlas (TCGA) using UCSC Xena browser(https://xenabrowser.net/). We extracted the methylation levels of gene methylation sites from different clinic samples and set them as high or low methylation according to the upper and lower quartiles. The log-rank test was applied to examine if risk differed by levels of methylation. As UTUC was uncommon and not included in TCGA, we studied the effect of DNA methylation modification on urothelial carcinoma of the bladder (UCB) instead. There is a similar histologic appearance between UTUC and UCB. Due to the relative preponderance of UCB, much of the clinical decision-making regarding UTUC was extrapolated from the evidence that was based on UCB cohorts.
      • Yang K
      • Yu W
      • Liu H
      • et al.
      Comparison of genomic characterization in upper tract urothelial carcinoma and urothelial carcinoma of the bladder.
      Thus, we believe the exploration of UCB dataset (434 patients) can still be valuable to reflect the effect of DNA methylation on UTUC.
      • Robinson BD
      • Vlachostergios PJ
      • Bhinder B
      • et al.
      Upper tract urothelial carcinoma has a luminal-papillary T-cell depleted contexture and activated FGFR3 signaling.
      P < ·05 is considered to be statistically significant.

      Results

      Study Selection Process and Characteristics of Eligible Studies

      After an initial search of selected electronic databases, we identified a total of 6380 articles regarding the sequencing platform results of UTUC and methylation molecular markers of UTUC. Finally, 11 studies
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      ,
      • Monteiro-Reis S
      • Leça L
      • Almeida M
      • et al.
      Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      • Zhang L
      • Xiong G
      • Fang D
      • et al.
      Contralateral upper tract urothelial carcinoma after nephroureterectomy: the predictive role of DNA methylation.
      • Xing Y
      • Xiong G
      • Fang D
      • Yang X
      • Li X
      • Zhou L.
      Prognostic value of gene methylation and clinical factors in non-muscle-invasive upper tract urothelial carcinoma after radical nephroureterectomy.
      • Fang D
      • He S
      • Xiong G
      • et al.
      Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      • Xiong G
      • Yao L
      • Hong P
      • et al.
      Aristolochic acid containing herbs induce gender-related oncological differences in upper tract urothelial carcinoma patients.
      • Luo HL
      • Chiang PH
      • Huang CC
      • et al.
      Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma.
      • Fujimoto M
      • Arai E
      • Tsumura K
      • et al.
      Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.
      • Xu Y
      • Ma X
      • Ai X
      • et al.
      A urine-based liquid biopsy method for detection of upper tract urinary carcinoma.
      were included in the final analysis. A flow diagram of the detailed selection process was presented in Figure 1. The risk of bias in 11 eligible studies was evaluated based on different domains, that was, clear objective or research question, rationalized design, and appropriate follow-up treatment. In general, the quality of the studies on DNA methylation alterations was fair to good (Supplemental Table S2).
      Figure 1
      Figure 1Flow diagram of the detailed selection process.
      The baseline characteristics of all eligible studies were summarized in Table 1 (Detailed characteristics can be found in Supplementary Table S1). Among the total 11 studies,
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      ,
      • Monteiro-Reis S
      • Leça L
      • Almeida M
      • et al.
      Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      • Zhang L
      • Xiong G
      • Fang D
      • et al.
      Contralateral upper tract urothelial carcinoma after nephroureterectomy: the predictive role of DNA methylation.
      • Xing Y
      • Xiong G
      • Fang D
      • Yang X
      • Li X
      • Zhou L.
      Prognostic value of gene methylation and clinical factors in non-muscle-invasive upper tract urothelial carcinoma after radical nephroureterectomy.
      • Fang D
      • He S
      • Xiong G
      • et al.
      Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      • Xiong G
      • Yao L
      • Hong P
      • et al.
      Aristolochic acid containing herbs induce gender-related oncological differences in upper tract urothelial carcinoma patients.
      • Luo HL
      • Chiang PH
      • Huang CC
      • et al.
      Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma.
      • Fujimoto M
      • Arai E
      • Tsumura K
      • et al.
      Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.
      • Xu Y
      • Ma X
      • Ai X
      • et al.
      A urine-based liquid biopsy method for detection of upper tract urinary carcinoma.
      samples were extracted from urine, blood, tissue or formalin-fixed paraffin-embedded tissue (FFPE) from 3619 patients. Overall, 12 genes of DNA methylation and 10 CpGs of UTUC were reported, some of which were investigated repeatedly aiming for multiple functions. Specifically, 8 studies
      • Monteiro-Reis S
      • Leça L
      • Almeida M
      • et al.
      Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      • Zhang L
      • Xiong G
      • Fang D
      • et al.
      Contralateral upper tract urothelial carcinoma after nephroureterectomy: the predictive role of DNA methylation.
      • Xing Y
      • Xiong G
      • Fang D
      • Yang X
      • Li X
      • Zhou L.
      Prognostic value of gene methylation and clinical factors in non-muscle-invasive upper tract urothelial carcinoma after radical nephroureterectomy.
      • Fang D
      • He S
      • Xiong G
      • et al.
      Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      • Xiong G
      • Yao L
      • Hong P
      • et al.
      Aristolochic acid containing herbs induce gender-related oncological differences in upper tract urothelial carcinoma patients.
      • Luo HL
      • Chiang PH
      • Huang CC
      • et al.
      Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma.
      reported 11 genes of methylation modification on UTUC prognosis. Meta-analysis was performed based on different prognostic outcomes and different methylated genes. The rest 4 studies
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      ,
      • Monteiro-Reis S
      • Leça L
      • Almeida M
      • et al.
      Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
      ,
      • Fujimoto M
      • Arai E
      • Tsumura K
      • et al.
      Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.
      ,
      • Xu Y
      • Ma X
      • Ai X
      • et al.
      A urine-based liquid biopsy method for detection of upper tract urinary carcinoma.
      were descriptively reviewed on diagnosis based on 7 genes and 10 CpGs.

      DNA Methylation Modification on Tumor Recurrence

      A total of 5 methylated genes from 7 studies
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      • Zhang L
      • Xiong G
      • Fang D
      • et al.
      Contralateral upper tract urothelial carcinoma after nephroureterectomy: the predictive role of DNA methylation.
      • Xing Y
      • Xiong G
      • Fang D
      • Yang X
      • Li X
      • Zhou L.
      Prognostic value of gene methylation and clinical factors in non-muscle-invasive upper tract urothelial carcinoma after radical nephroureterectomy.
      • Fang D
      • He S
      • Xiong G
      • et al.
      Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      • Xiong G
      • Yao L
      • Hong P
      • et al.
      Aristolochic acid containing herbs induce gender-related oncological differences in upper tract urothelial carcinoma patients.
      • Luo HL
      • Chiang PH
      • Huang CC
      • et al.
      Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma.
      (3011patients) were identified to be significantly associated with the recurrence of UTUC after surgery (Figure 2A, Table 2, Supplemental Figure S1). Pooled results showed significant low risk for tumor recurrence for methylation of all genes (HR = 0·67; 95% CI: 0·51-0·87; P = ·003). Subgroup analyses also found a significantly low risk for first bladder/intravesical recurrence for methylation of GDF15, RASSF1A, TMEFF2, and VIM (HR = 0·62; 95% CI: 0·52-0·74; P < ·00001). However, no significant high risk was found for the second bladder recurrence after pooling effect sizes of methylation of both GDF15 and VIM (HR = 1·02; 95% CI: 0·13-7·92; P = ·98). Methylation of SPARCL1 was associated with a high risk of systemic recurrence (HR = 2·89; 95% CI: 1·28-6·53; P = ·01).
      Figure 2
      Figure 2Forest plots of pooled outcomes based on different prognosis outcomes: A) A forest plot of pooled tumor recurrence; B) A forest plot of pooled tumor progression. C) A forest plot of pooled cancer-specific mortality. *Asterisk means the same study but compared separately in different gender; - Hyphen means the same study but compared separately in different tumor recurrence situation. SBR: second bladder recurrence; BR: first bladder recurrence; IR: intravesical recurrence (same as BR); CR: contralateral recurrence; SR: systemic recurrence.

      DNA Methylation Modification on Tumor Progression

      A total of 7 methylated genes from 2 studies
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      ,
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      (772 patients) were reported to have an effect on UTUC tumor progression, defined as the presence of a pathologically confirmed, muscle-invasive tumor during follow-up,
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      including higher tumor grade, higher tumor stage, and pN+(Figure 2B, Table 2, Supplemental Figure S2). The pooled results, consistent with the results of most of the single genes, showed an overall positive association between included methylated genes and the risk of UTUC. (HR = 1·60; 95% CI: 1·17-2·18; P = ·003). However, subgroup analysis revealed that the pooled results of methylation of BRCA1, GDF15, HSPA2, RASSF1A, and THBS1 were not significant for G3 tumor grade (HR = 1·27; 95% CI: 0·77-2·09; P = ·36).

      DNA Methylation Modification on Cancer-specific Mortality

      A total of 8 methylated genes from 5 studies
      • Monteiro-Reis S
      • Leça L
      • Almeida M
      • et al.
      Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
      ,
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      ,
      • Xing Y
      • Xiong G
      • Fang D
      • Yang X
      • Li X
      • Zhou L.
      Prognostic value of gene methylation and clinical factors in non-muscle-invasive upper tract urothelial carcinoma after radical nephroureterectomy.
      ,
      • Fang D
      • He S
      • Xiong G
      • et al.
      Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
      ,
      • Xiong G
      • Yao L
      • Hong P
      • et al.
      Aristolochic acid containing herbs induce gender-related oncological differences in upper tract urothelial carcinoma patients.
      (2313 patients) were identified to be related to UTUC-specific mortality (Figure 2C, Table 2). The pooled results showed a significantly high risk across all the methylation genes, including VIM, TMEFF2, SALL3, RASSF1A, HSPA2, GDF15, BRCA1, and ABCC6 (HR = 1·35; 95% CI: 1·06-1·72; P = ·01). No subgroup was reported about mortality among the included studies.

      Pleiotropy of Methylated Genes in Different Prognosis Outcomes

      Although only methylated HSPA2 and TMEFF2 were associated with a higher risk of mortality of UTUC (all HR > 1, all P < ·0001) in terms of the pooled results, we further found the other methylated genes, namely GDF15, RASSF1A, and VIM, pleiotropic across diverse prognosis outcomes in subgroup analysis (Table 3, Supplemental Figure S3-S7). Similarly, pooled results showed both methylation of GDF15 and RASSF1A had a significantly low risk of tumor recurrence (GDF15: HR = 0·63; 95% CI: 0·46-0·87; P = ·005; RASSF1A: HR = 0·51; 95% CI: 0·41-0·64; P < ·00001). However, they showed separately significantly high risks on tumor progression (methylated RASSF1A: HR = 1·60; 95% CI: 1·26-2·03; P = ·0001) and cancer-specific mortality (methylated GDF15: HR = 1·66; 95% CI: 1·03-2·67; P = ·04). Moreover, no significant prognostic finding was detected from the overall risk of methylated VIM (P = ·98), while only 1 study
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      showed it could increase the risk of tumor progression (HR = 4·91, 95% CI:1·11-21·72, P = ·04).
      Table 1Basic Characteristics of all Included Epigenetic Studies on UTUC
      NoAuthorYearCountry/RegionBiomarker TypeSample SizeSource of SampleAnalysis Methods/PlatformSignificant Biomarkers StudiedFollow-up (months)Clinical Application
      CaseControl
      1Monteiro Reis2014PortugalMethylationUTUC: 57 (G1/>G1 = 15/42)Normal upper tract urothelium: 36FFPE, UrineMethylation-sensitive PCR (MSP)GDF15, TMEFF2, VIMMedian: 31 (1-146)Diagnosis, Cancer-specific mortality
      UTUC: 22 (G1/>G1 = 3/19)Non-UTUC: 20
      2Zhang2015ChinaMethylationContralateral recurrence UTUC: 30Non-Contralateral recurrence UTUC: 634FFPEMethylation-sensitive PCR (MSP)RASSF1AMedian: 48 (3-144)Contralateral UTUC Recurrence
      3Xiong2015ChinaMethylationUTUC with methylated genes: 98-434UTUC without methylated genes: 253-589FFPEMethylation-sensitive PCR (MSP)GDF15, BRCA1, RASSF1A, TMEFF2, THBS1median: 65 (3-144)Bladder Recurrence,Cancer-specific mortality, Tumor progression
      4Xing2016ChinaMethylationnone-muscle-invasive UTUC with methylated genes: 19-109none-muscle-invasive UTUC without methylated genes: 83-173FFPEMethylation-sensitive PCR (MSP)GDF15, ABCC6, RASSF1AMedian: 65 (3-144)Cancer-specific mortality, Intravesical Recurrence
      5Fang2018ChinaMethylationRenal pelvic tumors with methylated genes: 51-228Renal pelvic tumors without methylated genes:113-290FFPEMethylation-sensitive PCR (MSP)TMEFF2, GDF15, RASSF1A, SALL3, ABCC6, HSPA2, VIMMedian: 64Bladder Recurrence,Cancer-specific mortality
      Ureteral tumors with methylated genes: 30-165Ureteral tumors without methylated genes:106-241
      6Guan2018ChinaMethylationFirst bladder recurrence UTUC with methylated genes: 16-56First bladder recurrence UTUC without methylated genes: 29-69FFPEMethylation-sensitive PCR (MSP)GDF15, VIM, CDH1Median: 51 (5-161)Second Bladder Recurrence, Tumor progression
      7Guo2018ChinaMethylationUTUC: 98 (G1/>G1 = 26/63)Non-UTUC: 113UrineMethylation-sensitive PCR (MSP) /FISHCDH1, HSPA2, VIM, RASSF1A,TMEFF2,GDF15NRDiagnosis
      8Xiong2018ChinaMethylationUTUC with methylated genes: 173-434UTUC without methylated genes: 253-514BloodMethylation-sensitive PCR (MSP)RASSF1A, ABCC6 (Female), TMEFF2 (Male)median: 52 (2-139)Bladder Recurrence,Cancer-specific mortality
      9Luo2019TaiwanMethylationUTUC: 3 (high-grade/stage)Normal urothelium adjacent to low-grade/stage UTUC: 3FFPE, TissueMethylation microarray assays/pyrosequencing/real-time PCR/tissue microarraysSPARCL139.1 ± 30.2/37.8 ± 31.9Systemic UTUC Recurrence
      UTUC: 25Paired adjacent urothelium: 25
      UTUC: 55Matched adjacent normal tissues: 55
      UTUC with methylated SPARCL1: 22UTUC with unmethylated SPARCL1: 56
      10Xu2020ChinaMethylationMalignant UTUC: 64 (low/high = 17/47)Non-malignant UTUC: 86Urine, TissueNext-Generation Sequencing /methylation-sensitive PCR (MSP)ONECUT2Total: 24Diagnosis
      11Fujimoto2020JapanMethylationUTUC: 72 (low/high = 15/57)Non-cancerous urothelium from UTUC: 72TissueGenome-wide DNA methylation screening/pyrosequencing10 CpGNRDiagnosis
      Normal control urothelial tissue: 26
      Urinary bladder urothelial carcinoma: 14 (low/high = 1/13)
      Abbreviations: UTUC = upper tract urothelial carcinoma; FFPE = formalin fixed paraffin-embedded; PCR = polymerase chain reaction; FISH = Fluorescence in situ hybridization; NR = not reported.
      10CpGs: cg01921432, cg07197785, cg07418387, cg08364561, cg10256242, cg10874111, cg14302471, cg14851578, cg15822765, and cg24035245.
      Detailed characteristics of included studies can be found in Supplementary Table S1.

      DNA Methylation Modification on the Diagnosis of UTUC

      A total of 4 studies
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      ,
      • Monteiro-Reis S
      • Leça L
      • Almeida M
      • et al.
      Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
      ,
      • Fujimoto M
      • Arai E
      • Tsumura K
      • et al.
      Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.
      ,
      • Xu Y
      • Ma X
      • Ai X
      • et al.
      A urine-based liquid biopsy method for detection of upper tract urinary carcinoma.
      reported the results of the diagnosis of UTUC (Table 1, Supplemental Table S1). To be specific, the 5 aforementioned pleiotropic methylated genes in urine, along with ONECUT2 and CDH1, were also regarded as diagnostic biomarkers for UTUC.
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      ,
      • Monteiro-Reis S
      • Leça L
      • Almeida M
      • et al.
      Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
      ,
      • Xu Y
      • Ma X
      • Ai X
      • et al.
      A urine-based liquid biopsy method for detection of upper tract urinary carcinoma.
      Other than that, Fugimoto recognized 10 CpGs in urine samples as a robust, noninvasive diagnostic method, using genome-wide DNA methylation analysis.
      • Fujimoto M
      • Arai E
      • Tsumura K
      • et al.
      Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.
      Table 2Pooled Outcomes of all the Subgroups Based on Different Prognosis Outcomes
      OutcomesSubgroupsNo. of StudiesHR95% CITest for Overall EffectHeterogeneity
      Tumor recurrenceFirst bladder/intravesical recurrence40.62[0.52, 0.74]Z = 5.38 (P< .00001)P = .22; I2 = 25%
      Second bladder recurrence1
      Results of GDF15 and VIM methylation for second bladder recurrence were from (Guan, 2018) study
      1.02[0.13, 7.92]Z = 0.02 (P = .98)P = .002 ; I2 = 89%
      Contralateral recurrence10.22[0.05, 0.93]Z = 2.06 (P = .04)NA
      Systemic recurrence12.89[1.28, 6.53]Z = 2.55 (P = .01)NA
      Tumor progressionHigher tumor grade (G3)1
      The results of RASSF1A, GDF15, HSPA2, BRCA1 and THBS1 methylation for higher tumor grade (G3) were from (Xiong, 2015) study; the result of RASSF1A for pN+ was from the same study NA = not applicable
      1.27[0.77, 2.09]Z = 0.92 (P = .36)P< .00001; I2= 89%
      Higher tumor stage (T3 & T4)21.86[1.44, 2.39]Z = 4.79 (P< .00001)P= .29; I2= 20%
      pN+1
      The results of RASSF1A, GDF15, HSPA2, BRCA1 and THBS1 methylation for higher tumor grade (G3) were from (Xiong, 2015) study; the result of RASSF1A for pN+ was from the same study NA = not applicable
      2.31[1.29, 4.14]Z = 2.82 (P = .005)NA
      Cancer-specific mortalityCancer-specific mortality51.35[1.06, 1.72]Z = 2.47 (P = .01)P< .00001; I2 = 75%
      a Results of GDF15 and VIM methylation for second bladder recurrence were from (Guan, 2018) study
      b The results of RASSF1A, GDF15, HSPA2, BRCA1 and THBS1 methylation for higher tumor grade (G3) were from (Xiong, 2015) study; the result of RASSF1A for pN+ was from the same studyNA = not applicable

      Survival Analysis of Included Genes

      Based on all the included studies,
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      ,
      • Zhang L
      • Xiong G
      • Fang D
      • et al.
      Contralateral upper tract urothelial carcinoma after nephroureterectomy: the predictive role of DNA methylation.
      • Xing Y
      • Xiong G
      • Fang D
      • Yang X
      • Li X
      • Zhou L.
      Prognostic value of gene methylation and clinical factors in non-muscle-invasive upper tract urothelial carcinoma after radical nephroureterectomy.
      • Fang D
      • He S
      • Xiong G
      • et al.
      Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
      • Guan B
      • Xing Y
      • Xiong G
      • et al.
      Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
      • Xiong G
      • Yao L
      • Hong P
      • et al.
      Aristolochic acid containing herbs induce gender-related oncological differences in upper tract urothelial carcinoma patients.
      • Luo HL
      • Chiang PH
      • Huang CC
      • et al.
      Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma.
      • Fujimoto M
      • Arai E
      • Tsumura K
      • et al.
      Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.
      • Xu Y
      • Ma X
      • Ai X
      • et al.
      A urine-based liquid biopsy method for detection of upper tract urinary carcinoma.
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      a total of 12 methylated genes and 10 CpGs were finally selected to conduct Kaplan-Meier survival analysis, based on the TCGA bladder cohort (Figure 3A-3L), Supplemental Figure S8-S17, Supplemental Table S3-S24). Only methylation of SPARCL1 exhibited a better disease-specific survival (Figure 3I, P = .04765), while no significant difference was discovered among the rest methylated genes and CpGs.
      Figure 3
      Figure 3A Kaplan-Meier curves for disease-specific survival based on different methylation expression of 12 included genes. A) A Kaplan-Meier curves of ABCC6. B) A Kaplan-Meier curves of BRCA1. C) A Kaplan-Meier curves of CDH1. D) A Kaplan-Meier curves of GDF15. E) A Kaplan-Meier curves of HSPA2. F) A Kaplan-Meier curves of ONECUT2. G) A Kaplan-Meier curves of RASSF1. H) A Kaplan-Meier curves of SALL3. I) A Kaplan-Meier curves of SPARCL1. J) A Kaplan-Meier curves of THBS1. K) A Kaplan-Meier curves of TMEFF2. L) A Kaplan-Meier curves of VIM.

      Publication Bias

      Publication bias was evaluated using the Begg's funnel plots. The shape of the funnel plots appeared symmetric in the UTUC case and control groups, suggesting no evidence of publication bias (Supplemental Figure S18-S27).

      Discussion

      Based on a substantial amount of available literature from 4 databases on methylation modifications of UTUC, we performed a meta-analysis and a descriptive systematic review to explore the overall effect of all methylation of UTUC-related genes. Our pooled results indicated that the generally pooled effect of all the methylation alteration had a lower risk for tumor recurrence, especially first bladder/intravesical recurrence, but a high risk of progression and mortality. The correlation between the gene methylation and progression indicated that once UTUC developed to progression, patients would suffer from higher stages of tumor or cancer-related death. Thus, the diagnosis or treatment of the progression of UTUC should target gene methylation modification.
      In addition, we would highlight the pleiotropy effects on both diagnosis and prognosis from the 5 methylations of genes, ie: RASSF1A, GDF15, TMEFF2, HSPA2, and VIM. To be specific, our results indicated that methylation of RASSF1A and GDF15 might play key roles in the development of UTUC, because they could not only be regarded as a diagnosis marker
      • Guo RQ
      • Xiong GY
      • Yang KW
      • et al.
      Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
      but also had diverse effects on tumor prognosis including recurrence, progression, and mortality (Table 3). RASSF1A, a tumor-suppressor gene, was involved in various molecular signaling mechanisms including Ras/Rho GTPases, microtubules, and the Hippo pathway of cancer,
      • Dubois F
      • Bergot E
      • Zalcman G
      • Levallet G.
      RASSF1A, puppeteer of cellular homeostasis, fights tumorigenesis, and metastasis-an updated review.
      while GDF15, which encoded a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins, could also contribute to the development of UTUC. Liu and Xiong agreed with our results by presenting that the methylation status of RASSF1A, was capable of assessing the aggressiveness of UTUC.
      Table 3Pooled Outcomes of all the Subgroups Based on Different Methylated Genes
      GeneOutcomesNo. of StudiesHR95% CITest for Overall EffectHeterogeneity
      RASSF1ATumor recurrence50.51[0.41, 0.64]Z = 5.92  (P < .00001)P= .43; I2= 0%
      Tumor progression1
      Results of RASSF1A and HSPA2 methylation for all subgroups of tumor progression were from (Xiong, 2015) study.
      1.60[1.26, 2.03]Z = 3.88  (P = .0001)P = .40; I2= 0%
      Cancer-specific mortality11.15[0.78, 1.70]Z = .71  (P =.48)NA
      Total50.79[0.53,1.16]Z = 1.20 (P =.23)P< .00001; I2 = 84%
      GDF15Tumor recurrence30.63[0.46, 0.87]Z = 2.82  (P = .005)P= .26; I2 = 26%
      Tumor progression10.48[0.34, 0.68]Z = 4.17  (P < .0001)NA
      Cancer-specific mortality21.66[1.03, 2.67]Z = 2.10  (P = .04)P = .12; I2 = 59%
      Total40.78[0.46, 1.32]Z = 0.92  (P = .36)P< .00001; I2= 89%
      VIMTumor recurrence20.90[0.57, 1.43]Z = 0.44 (P = .66)P = .007; I2 = 86%
      Tumor progression14.91[1.11, 21.72]Z = 2.10 (P = .04)NA
      Cancer-specific mortality20.84[0.59, 1.21]Z = 0.93 (P = .35)P= .005; I2 = 87%
      Total31.01[0.48,2.15]Z = 0.03 (P = .98)P= .0005; I2 = 80%
      HSPA2Tumor progression1
      Results of RASSF1A and HSPA2 methylation for all subgroups of tumor progression were from (Xiong, 2015) study.
      1.77[1.39, 2.25]Z = 4.66  (P < .00001)P = .11; I2= 60%
      Cancer-specific mortality1
      Results of HSPA2 methylation for cancer-specific mortality, and TMEFF2 methylation for bladder recurrence were from the same included study (Fang, 2018). NA = not applicable.
      1.52[1.03, 2.24]Z = 2.11  (P = .04)NA
      Total21.70[1.38, 2.08]Z = 5.07  (P < .00001)P = .23; I2= 32%
      TMEFF2Tumor recurrence1
      Results of HSPA2 methylation for cancer-specific mortality, and TMEFF2 methylation for bladder recurrence were from the same included study (Fang, 2018). NA = not applicable.
      0.91[0.60, 1.38]Z = 0.44  (P = .66) NA
      Cancer-specific mortality31.58[1.31, 1.91]Z = 4.71 (P < .00001)P= .94; I2 = 0%
      Total31.44[1.21, 1.71]Z = 4.10 (P < .0001)P = .13; I2= 47%
      a Results of RASSF1A and HSPA2 methylation for all subgroups of tumor progression were from (Xiong, 2015) study.
      b Results of HSPA2 methylation for cancer-specific mortality, and TMEFF2 methylation for bladder recurrence were from the same included study (Fang, 2018).NA = not applicable.
      Moreover, we also discovered both methylations of HSPA2 and TMEFF2 had an overall significantly high risk on prognosis (Table 3). As HSPA2 encoded a 70-kDa heat shock chaperone protein while TMEFF2 encoded a transmembrane protein of the tomoregulin family,
      • Horie M
      • Mitsumoto Y
      • Kyushiki H
      • et al.
      Identification and characterization of TMEFF2, a novel survival factor for hippocampal and mesencephalic neurons.
      their role in the pathogenesis of UTUC was also supported by previous researchers.
      • Xiong G
      • Liu J
      • Tang Q
      • et al.
      Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
      ,
      • Fang D
      • He S
      • Xiong G
      • et al.
      Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
      Furthermore, Luo claimed that SPARCL1 encoding an extracellular matrix glycoprotein was also considered a prognostic marker for clinical practice. Notably, our survival analysis also approved the significance of this included study. Nevertheless, our survival analysis did not find a significant effect of other methylation of genes in the general bladder cancer cohort. Although UTUC has traditionally been considered an uncommon type of UC, an increasing number of evidence recently defined UTUC and UC as “disparate twins” for the discrepancies in pathological and biological basis.
      • Kawashima A
      • Kanazawa T
      • Jingushi K
      • et al.
      Phenotypic analysis of tumor tissue-infiltrating lymphocytes in tumor microenvironment of bladder cancer and upper urinary tract carcinoma.
      • Sanguedolce F
      • Cormio L.
      The complex relationship between upper urinary tract and bladder cancer: clinical and predictive issues.
      • Green DA
      • Rink M
      • Xylinas E
      • et al.
      Urothelial carcinoma of the bladder and the upper tract: disparate twins.
      Thus, the results of UC may not truly reflect the pathogenesis of UTUC. Besides, included gene methylation played an essential role in various tumors. For example, Hu found that hypermethylated RASSF1A can be a potential prognostic biomarker in colorectal cancer based on a meta-analysis and TCGA analysis.
      • Hu F
      • Chen L
      • Bi MY
      • et al.
      Potential of RASSF1A promoter methylation as a biomarker for colorectal cancer: Meta-analysis and TCGA analysis.
      More importantly, our discovery of the genes might provide insights into the future investigation of the prediction, diagnosis, and treatment of UTUC.
      However, we are aware of the limitations of this study. First, most included studies were from single-center institutions and lacked long follow-ups, so the selection bias of patients cannot be ignored. Second, we did not perform a meta-analysis for diagnostic studies due to a lack of enough data, but a descriptive systematic review was conducted. Third, although we performed Kaplan-Meier survival analysis for whole genes in UCB patients, validations using a large-scale sample size in a prospective cohort and experimental analysis on patients suffering from UTUC are still needed. In addition, the Kaplan-Meier curve cannot account for the competing risk of cancer-specific mortality. At last, we did not perform subgroup analysis based on the experimental technique or source of biospecimen, because either germline or somatic mutation identified could provide practical value for the clinical management of UTUC.
      This study has several strengths. First, as numerous evidence of methylation pathogenesis sprung up, it was the first systematic review focusing exclusively on methylation modifications of UTUC with the most updated evidence. Second, we combined both meta-analysis and Kaplan-Meier survival analysis to comprehensively depict the architecture of methylation of UTUC, from the perspectives of diagnosis and prognosis. Third, the methylation of genes found in our study could help decision-making in clinical practice.
      In conclusion, this is currently the first systematic review with the most updated evidence on the methylation architecture of UTUC. Our results could contribute to a comprehensive understanding of the methylation of UTUC. Novel biomarkers with potential diagnosis and prognosis function, could not only provide promising and innovative insights for future investigations but also offer precision medicine references when managing individuals with UTUC.

      Clinical Practice Points

      • Few sequencing and experimental studies could gain a comprehensive picture of the DNA methylation mechanism of UTUC.
      • 11 studies (3619 patients) were eligible to investigate 12 methylated genes and 10 cytosine-phosphate-guanine (CpG) sites.
      • Meta-analysis found the pooled effect of eligible methylated genes had a low risk of tumor recurrence, but a high risk of tumor progression and cancer-specific mortality.
      • For individual methylation status of GDF15, HSPA2, RASSF1A, TMEFF2 and VIM, the pooled effect of each gene was found pleiotropic on both diagnosis and prognosis.
      • Survival analysis suggested higher methylation of SPARCL1 had better disease-specific survival.
      • The methylation of genes with essential diagnosis and prognosis function found in this study might provide precision medicine references for personalized therapies.

      Authors’ contributions

      BL, JH: Conception and design; YL, LL, YY, TF, BL: Collection and assembly of data; YL, LL, YY, ML, XJ, TF, YL, QG, HH, ZC: Data analysis and interpretation; All authors: Manuscript writing.
      Final approval of manuscript: All authors.

      Data Availability

      All the data analyzed in this study are publicly available and can be found in the supplemental tables.

      Ethics approval

      The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

      Disclosure

      This work was supported by the National Key R&D Program of China [ 2020YFC2003405 to J.H.]; Key Program of Science and Technology Department of Sichuan Province [ 2020YFS0047 to J.H.] and National Natural Science Foundation of China [ 32171285 to J.H., 32101206 to YF.L.].
      The authors have no relevant financial or non-financial interests to disclose.

      Acknowledgments

      None

      Reference

        • Siegel RL
        • Miller KD
        • Jemal A.
        Cancer statistics, 2020.
        CA Cancer J Clin. 2020; 70: 7-30https://doi.org/10.3322/caac.21590
        • Rouprêt M
        • Babjuk M
        • Burger M
        • et al.
        European association of urology guidelines on upper urinary tract urothelial carcinoma: 2020 update.
        Eur Urol. 2021; 79: 62-79https://doi.org/10.1016/j.eururo.2020.05.042
        • Rink M
        • Xylinas E
        • Margulis V
        • et al.
        Impact of smoking on oncologic outcomes of upper tract urothelial carcinoma after radical nephroureterectomy.
        Eur Urol. 2013; 63: 1082-1090https://doi.org/10.1016/j.eururo.2012.06.029
        • Chen IH
        • Luo HL
        • Su YL
        • et al.
        Aristolochic acid affects upper tract urothelial cancer behavior through the mapk pathway.
        Molecules (Basel, Switzerland). 2019; 24https://doi.org/10.3390/molecules24203707
        • Hassler MR
        • Bray F
        • Catto JWF
        • et al.
        Molecular characterization of upper tract urothelial carcinoma in the era of next-generation sequencing: a systematic review of the current literature.
        Eur Urol. 2020; 78: 209-220https://doi.org/10.1016/j.eururo.2020.05.039
        • Portela A
        • Esteller M.
        Epigenetic modifications and human disease.
        Nat Biotechnol. 2010; 28: 1057-1068https://doi.org/10.1038/nbt.1685
        • Guo RQ
        • Xiong GY
        • Yang KW
        • et al.
        Detection of urothelial carcinoma, upper tract urothelial carcinoma, bladder carcinoma, and urothelial carcinoma with gross hematuria using selected urine-DNA methylation biomarkers: A prospective, single-center study.
        Urol Oncol. 2018; 36 (e15-342.e23): 342https://doi.org/10.1016/j.urolonc.2018.04.001
        • Wang J
        • Zhao Y
        • Xu H
        • et al.
        Silencing NID2 by DNA hypermethylation promotes lung cancer.
        Pathol Oncol Res: POR. 2020; 26: 801-811https://doi.org/10.1007/s12253-019-00609-0
        • Liberati A
        • Altman DG
        • Tetzlaff J
        • et al.
        The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration.
        BMJ (Clinical research ed). 2009; 339: b2700https://doi.org/10.1136/bmj.b2700
        • McShane LM
        • Altman DG
        • Sauerbrei W
        • Taube SE
        • Gion M
        • Clark GM.
        Reporting recommendations for tumor marker prognostic studies.
        J Clin Oncol: Official J American Society Clin Oncol. 2005; 23: 9067-9072https://doi.org/10.1200/jco.2004.01.0454
        • Yang K
        • Yu W
        • Liu H
        • et al.
        Comparison of genomic characterization in upper tract urothelial carcinoma and urothelial carcinoma of the bladder.
        Oncologist. 2021; 26: e1395-e1405https://doi.org/10.1002/onco.13839
        • Robinson BD
        • Vlachostergios PJ
        • Bhinder B
        • et al.
        Upper tract urothelial carcinoma has a luminal-papillary T-cell depleted contexture and activated FGFR3 signaling.
        Nat Commun. 2019; 10: 2977https://doi.org/10.1038/s41467-019-10873-y
        • Monteiro-Reis S
        • Leça L
        • Almeida M
        • et al.
        Accurate detection of upper tract urothelial carcinoma in tissue and urine by means of quantitative GDF15, TMEFF2 and VIM promoter methylation.
        Eur J Cancer (Oxford, England: 1990). 2014; 50: 226-233https://doi.org/10.1016/j.ejca.2013.08.025
        • Xiong G
        • Liu J
        • Tang Q
        • et al.
        Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
        Epigenomics. 2015; 7 (Aug): 733-744https://doi.org/10.2217/epi.15.34
        • Zhang L
        • Xiong G
        • Fang D
        • et al.
        Contralateral upper tract urothelial carcinoma after nephroureterectomy: the predictive role of DNA methylation.
        J Experimental & Clin Cancer Res: CR. 2015; 34: 5https://doi.org/10.1186/s13046-015-0120-2
        • Xing Y
        • Xiong G
        • Fang D
        • Yang X
        • Li X
        • Zhou L.
        Prognostic value of gene methylation and clinical factors in non-muscle-invasive upper tract urothelial carcinoma after radical nephroureterectomy.
        Clin Genitourin Cancer. 2016; 14: e371-e378https://doi.org/10.1016/j.clgc.2016.02.009
        • Fang D
        • He S
        • Xiong G
        • et al.
        Comparison of clinicopathologic characteristics, epigenetic biomarkers and prognosis between renal pelvic and ureteral tumors in upper tract urothelial carcinoma.
        BMC Urol. 2018; 18: 22https://doi.org/10.1186/s12894-018-0334-7
        • Guan B
        • Xing Y
        • Xiong G
        • et al.
        Predictive value of gene methylation for second recurrence following surgical treatment of first bladder recurrence of a primary upper-tract urothelial carcinoma.
        Oncol Lett. 2018; 15: 9397-9405https://doi.org/10.3892/ol.2018.8498
        • Xiong G
        • Yao L
        • Hong P
        • et al.
        Aristolochic acid containing herbs induce gender-related oncological differences in upper tract urothelial carcinoma patients.
        Cancer Manag Res. 2018; 10: 6627-6639https://doi.org/10.2147/cmar.S178554
        • Luo HL
        • Chiang PH
        • Huang CC
        • et al.
        Methylation of SPARCL1 Is Associated with Oncologic Outcome of Advanced Upper Urinary Tract Urothelial Carcinoma.
        Int J Mol Sci. 2019; 20https://doi.org/10.3390/ijms20071653
        • Fujimoto M
        • Arai E
        • Tsumura K
        • et al.
        Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.
        Epigenetics. 2020; 15: 1289-1301https://doi.org/10.1080/15592294.2020.1767374
        • Xu Y
        • Ma X
        • Ai X
        • et al.
        A urine-based liquid biopsy method for detection of upper tract urinary carcinoma.
        Front Oncol. 2020; 10597486https://doi.org/10.3389/fonc.2020.597486
        • Xiong G
        • Liu J
        • Tang Q
        • et al.
        Prognostic and predictive value of epigenetic biomarkers and clinical factors in upper tract urothelial carcinoma.
        Epigenomics. 2015 Aug; 7: 733-744https://doi.org/10.2217/epi.15.34
        • Dubois F
        • Bergot E
        • Zalcman G
        • Levallet G.
        RASSF1A, puppeteer of cellular homeostasis, fights tumorigenesis, and metastasis-an updated review.
        Cell Death & Disease. 2019; 10: 928https://doi.org/10.1038/s41419-019-2169-x
        • Horie M
        • Mitsumoto Y
        • Kyushiki H
        • et al.
        Identification and characterization of TMEFF2, a novel survival factor for hippocampal and mesencephalic neurons.
        Genomics. 2000; 67: 146-152https://doi.org/10.1006/geno.2000.6228
        • Kawashima A
        • Kanazawa T
        • Jingushi K
        • et al.
        Phenotypic analysis of tumor tissue-infiltrating lymphocytes in tumor microenvironment of bladder cancer and upper urinary tract carcinoma.
        Clin Genitourin Cancer. 2019; 17: 114-124https://doi.org/10.1016/j.clgc.2018.11.004
        • Sanguedolce F
        • Cormio L.
        The complex relationship between upper urinary tract and bladder cancer: clinical and predictive issues.
        Transl Androl Urol. 2018; 7 (Suppl): S248-S251https://doi.org/10.21037/tau.2018.05.07
        • Green DA
        • Rink M
        • Xylinas E
        • et al.
        Urothelial carcinoma of the bladder and the upper tract: disparate twins.
        J Urol. 2013; 189: 1214-1221https://doi.org/10.1016/j.juro.2012.05.079
        • Hu F
        • Chen L
        • Bi MY
        • et al.
        Potential of RASSF1A promoter methylation as a biomarker for colorectal cancer: Meta-analysis and TCGA analysis.
        Pathol Res Pract. 2020; 216153009https://doi.org/10.1016/j.prp.2020.153009