Urine Sediment DNA Research Articles

Identification and validation of methylated PENK gene for early detection of bladder cancer using urine DNA

BackgroundEarly detection of bladder cancer (BCa) offers patients a favorable outcome and avoids the need for cystectomy. Development of an accurate and sensitive noninvasive BCa diagnostic test is imperative. DNA methylation is an early epigenetic event in the development of BCa. Certain specific aberrant methylations could serve as useful biomarkers. The aim of this study was to identify methylation biomarkers for early detection of BCa.MethodsCpG methylation microarray analysis was conducted on primary tumors with varying stages (T1—T4) and paired nontumor tissues from nine BCa patients. Bisulfite-pyrosequencing was performed to confirm the methylation status of candidate genes in tissues and urine sediments (n = 51). Among them, PENK was selected as a potential candidate and validated using an independent set of 169 urine sediments (55 BCa, 25 benign urologic diseases, 8 other urologic cancers, and 81 healthy controls) with a quantitative methylation-specific real time PCR (mePENK-qMSP). All statistical analyses were performed using MedCalc software version 9.3.2.0.ResultsCpG methylation microarray analysis and stepwise validation by bisulfite-pyrosequencing for tissues and urine sediments supported aberrant methylation sites of the PENK gene as potential biomarkers for early detection of BCa. Clinical validation of the mePENK-qMSP test using urine sediment-DNA showed a sensitivity of 86.5% (95% CI: 71.2 – 95.5%), a specificity of 92.5% (95% CI: 85.7 – 96.7%), and an area under ROC of 0.920 (95% CI: 0.863 – 0.959) in detecting Ta high-grade and advanced tumor stages (T1-T4) of BCa patients. Sensitivities for Ta low-grade, Ta high-grade, T1 and T2-T4 were 55.6, 83.3, 88.5, and 100%, respectively. Methylation status of PENK was not correlated with sex, age or stage, while it was associated with the tumor grade of BCa.ConclusionsIn this study, we analyzed the comprehensive patterns of DNA methylation identified that PENK methylation possesses a high potential as a biomarker for urine-based early detection of BCa. Validation of PENK methylation confirms that it could significantly improve the noninvasive detection of BCa.

Detecting and monitoring bladder cancer with exfoliated cells in urine.

Current methods for the diagnosis and monitoring of bladder cancer are invasive and have suboptimal sensitivity. Liquid biopsy as a non-invasive approach has been capturing attentions recently. To explore the ability of urine-based liquid biopsy in detecting and monitoring genitourinary tumors, we developed a method based on promoter-targeted DNA methylation of urine sediment DNA. We used samples from a primary bladder cancer cohort (n=40) and a healthy cohort (n=40) to train a model and obtained an integrated area under the curve (AUC) > 0.96 in the 10-fold cross-validation, which demonstrated the ability of our method for detecting bladder cancer from the healthy. We next validated the model with samples from a recurrent cohort (n=21) and a non-recurrent cohort (n=19) and obtained an AUC > 0.91, which demonstrated the ability of our model in monitoring the progress of bladder cancer. Moreover, 80% (4/5) of samples from patients with benign urothelial diseases had been considered to be healthy sample rather than cancer sample, preliminarily demonstrating the potential of distinguishing benign urothelial diseases from cancer. Further analysis basing on multiple-time point sampling revealed that the cancer signal in 80% (4/5) patients had decreased as expected when they achieved the recurrent-free state. All the results suggested that our method is a promising approach for noninvasive detection and prognostic monitoring of bladder cancer.

Abstract 5101: Urine based dynamically monitoring reflect pathologic response in MIBC patients with neoadjuvant immunotherapy

Abstract Background: The recommended standard of care treatment for nonmetastatic muscle-invasive bladder cancer (MIBC) is radical cystectomy (RC). Even with urinary diversion, radical cystectomy has a major impact on patients' quality of life. Although high rates of overall and disease-specific survival were observed in patients managed with bladder preservation, who achieved a clinically complete response to neoadjuvant therapy. Frequent evaluation of tumor response based cystoscopy implementing personalized bladder-sparing treatment paradigms. Case Presentation: Here, we report on a case of urine-based dynamically monitoring during neoadjuvant immunotherapy in a MIBC patient treated with RC. A 57-year-old male presented macrohematuria and was diagnosed with bladder urothelial carcinoma by transurethral resection of bladder tumor (TURBT) with clinical stage IIIA (high-grade, multiple, cT2N0M0). He received 3 cycles of Tislelizumab monotherapy and sequential radical cystectomy. The RC specimen showed a complete pathological response (pCR). Four urine samples were collected prior to TURBT, on a day after TURBT, at beginning of neoadjuvant immunotherapy, and on the day of CR. Mutations status in 17 genes and methylation level of ONECUT2 were analyzed by a high-throughput sequencing-based urine test panel (genetron-uro-V1). In urine sediment DNA, which was collected before TURBT, the TERT promoter mutations C228T, TP53 D281H, and ERBB2 D277Y were detected in urine sediment DNA at variant allele frequencies (VAF) of 31.8%, 10.5%, and 0.5%, respectively. After removing in most parts of tumor, the VAF of TERT C228T decreased to 19.6% and mutation in TP53 and ERBB2 could not be detected. Without neoadjuvant immunotherapy, the VAF of TERT C228T still stayed above 12%. After 3 cycles of Tislelizumab monotherapy on the day of CR, all mutation was below the detection limit in urine sediment DNA, which was consistent with pCR in the pathological report of RC specimen. Conclusions: This case suggested that urine-based dynamically monitoring reflected pathologic response, which could support personalized care and help select patients for bladder-sparing treatment. Citation Format: Xiao Yang, Lingkai Cai, Qiang Cao, Min Shi, Yiming Liang, Yurong Qu, Qiang Lu. Urine based dynamically monitoring reflect pathologic response in MIBC patients with neoadjuvant immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5101.

Accuracy of an urine-based liquid biopsy genetic test in detecting renal pelvic cancer.

e16500 Background: Renal pelvis cancer is accounting for about two-thirds of upper tract urinary carcinoma. Lesions in renal pelvis often lead to a differential diagnosis that requires ureteroscopic biopsy. Besides causing discomfort and pain, ureteroscopy prior to surgery increases risk of tumor implantation and recurrence. Although urine cytology and FISH were applied, more effective non-invasive diagnostic method is imperative for diagnosis of renal pelvic cancer. Methods: A total of 141 patients with one or more lesions in renal pelvis detected by ultrasound were enrolled. 50-ml first-void urine sample was collection before ureteroscopic biopsy and was analysis by Genetron uro-18 genes panel, by which mutations in 17 genes and methylation status of ONECUT2 were analysis using high-throughput sequencing. If mutations and/or ONECUT2 methylation were detected in urine sediment DNA, the patients were considered as high risk of renal pelvis cancer. The results of the urine-based liquid biopsy genetic test were compared with pathology report and urine FISH results of urinary cells. Results: According to pathological analysis with ureteroscopic biopsy, 42 out of 141 patients (29.7%) were diagnosis with renal pelvic cancer: 13 low-grade, 24 high-grade, 5 equivocal. For 33 renal pelvic cancer patients with FISH testing result of urinary cells, only 54.5% (18/33) samples were positive. By Genetron uro-18 genes test, 95 patients (67.4%) were classified as high risk and 46 (32.6%) patients were low risk. The overall specificity was 88.0% (88/95) and overall sensitivity was 92.9% (39/42), which is significant higher than FISH test of urinary cells. Conclusions: With high specificity and sensitivity, Genetron uro-18 genes test could be incorporated in renal pelvic cancer diagnosis, especially for patients with a ultrasound detectable lesions. Ureteroscopy prior to surgery could be reduced, which may lead to decrease risk of tumor implantation and recurrence.

EP425 - A robust and sensitive method for the detection of mitochondrial DNA mutations from urine sediment and other low-quality DNA sources

eP425 - A robust and sensitive method for the detection of mitochondrial DNA mutations from urine sediment and other low-quality DNA sources

Low-Coverage Sequencing of Urine Sediment DNA for Detection of Copy Number Aberrations in Bladder Cancer.

PurposeChromosomal copy number aberrations (CNAs) are a hallmark of bladder cancer and a useful target for diagnostic explorations. Here we constructed a low-coverage whole-genome sequencing method for the detection of CNAs in urine sediment DNA from patients with bladder cancer.Patients and MethodsWe conducted a prospective study using urine sediment samples from 65 patients with bladder tumors, including 54 patients with bladder cancer and 11 patients with benign bladder tumors. Forty-three healthy individuals were included as normal controls. DNA was extracted from urine sediments and analyzed by low-coverage whole-genome sequencing to compare differences in CNAs among these three groups. CNAs are defined by arbitrary R values (normal range ± 2). When these values exceed ± 0.2 of normal range, gain/duplication or loss/deletion are suspected.ResultsWith this method, CNAs were detected in 39 of 51 patients with bladder cancer, 2 of 10 patients with benign bladder tumors, and 8 of 39 normal controls. The lengths of DNA deletion and duplication were significantly larger in patients with bladder cancer than in patients with benign tumors or normal controls (P < 0.05). Bladder cancer duplicate CNAs mainly occurred on chromosomes 1q, 5p, 6p, 7p, 8q, and 13q, while deletions mainly occurred on 2q, 8p, 9q, 9p, and 11p. Those regions contained bladder cancer tumor-related genes, such as STK3, COX6C, SPAG1, CDKAL1, C9orf53, CDKN2A, CDKN2B, MIR31, and IFNA1. The number of CNAs detected in urine sediment DNA during the follow-up period was significantly reduced.ConclusionOur sequencing method is highly sensitive and can detect a minimal chromosome repeat/microdeletion change of 0.15 Mb. The use of 0.1~0.3× low-coverage whole-genome sequencing can be used to detect bladder cancer CNAs in urine sediment DNA. This method provides a promising method for noninvasive diagnosis of bladder cancer, but still needs further verification in a larger sample size.

Evaluation of TERT promoter mutations in urinary cell-free DNA and sediment DNA for detection of bladder cancer

BackgroundCell-free DNA (cfDNA) is proposed to be a valuable source of biomarkers in liquid biopsies for various diseases as it is supposed to partially originate from tumor cells. However, data about the diagnostic implications of cfDNA in urine for the detection of bladder cancer (BCa) is sparse. MethodsWe evaluated the usability of urinary cfDNA for diagnostic purposes compared to urine sediment DNA (sDNA) in 53 BCa patients and 36 control subjects by analyzing two abundant point-mutations (C228T/C250T) in the TERT promoter using Next-Generation Sequencing. ResultsMutations were detected in 77% of the urinary sDNA compared to 63% of the cfDNA samples. Moreover, the TERT mutation allele frequencies (MAF) were highly correlated in cfDNA and sDNA. In comparison, the accuracy of the TERT assay was higher in sDNA (84%) compared to cfDNA or voided urine cytology (both 77%). Interestingly, MAFs from leukocyte-rich urines were higher in cfDNA than in sDNA, indicating a diagnostic advantage of cfDNA in such urines. ConclusionsUrine-based mutation detection has the ability to augment and surpass voided urine cytology as the current gold-standard for the non-invasive detection and surveillance of BCa. The analysis of cell-free DNA provides no general diagnostic advantage compared to urine sediment DNA.

AB242. An epigenetic biomarker combination of eomes, pcdh17, and pou4f2 detects bladder cancer accurately from methylation analyses of urine sediment DNA in Han Chinese

BackgroundTo recognize a novel and optimized combination of epigenetic biomarkers capable of returning higher sensitivity and specificity synergistically in pathologically varied sample groups for urine sediment examination of bladder cancer (BlCa).MethodsMethylation levels of seven candidate biomarkers (EOMES, GDF15, NID2, PCDH17, POU4F2, TCF21, and ZNF154) in 58 urothelial cell carcinoma (UCC) patients, 20 infected urinary calculi (IUC) patients, 20 kidney cancer (KC) patients, and 30 healthy volunteers (HV) were quantified by qMSP using the sediment DNA of urine samples. Standard curves and receiver operating characteristic (ROC) curves were generated for each biomarker. The combining predictors of all the possible biomarker combinations were calculated through logistic regression model. Subsequently, the ROC curves of the three combinations that have yielded the best performing combining predictors were constructed.ResultsFrom the samples provided, the top three biomarker combinations (C) returned high sensitivity and specificity of 91.38% and 94.29% from PCDH17 + POU4F2 (C1), 91.38% and 90.00% from PCDH17 + POU4F2 + NID2 (C2), and 93.10% and 92.86% from PCDH17 + POU4F2 + EOMES (C3) consequently. These combinations provide greater sensitivities and specificities than any examined single biomarkers, except for C2, which has a lower specificity compare to POU4F2 (92.86%).ConclusionsCompound biomarker panels were shown to be capable of detecting BlCa effectively by utilizing urine sediment DNA. Due to the performance of higher sensitivity and specificity when compared to single biomarker examinations in BlCa patients and the diverse control groups, these panels are particularly suitable for mass-scale routine BlCa screening.

An epigenetic biomarker combination of PCDH17 and POU4F2 detects bladder cancer accurately by methylation analyses of urine sediment DNA in Han Chinese.

To develop a routine and effectual procedure of detecting bladder cancer (BlCa), an optimized combination of epigenetic biomarkers that work synergistically with high sensitivity and specificity is necessary. In this study, methylation levels of seven biomarkers (EOMES, GDF15, NID2, PCDH17, POU4F2, TCF21, and ZNF154) in 148 individuals—which including 58 urothelial cell carcinoma (UCC) patients, 20 infected urinary calculi (IUC) patients, 20 kidney cancer (KC) patients,20 prostate cancer (PC) patients, and 30 healthy volunteers (HV)—were quantified by qMSP using the urine sediment DNA. Receiver operating characteristic (ROC) curves were generated for each biomarker. The combining predictors of possible combinations were calculated through logistic regression model. Subsequently, ROC curves of the three best performing combinations were constructed. Then, we validated the three best performing combinations and POU4F2 in another 72 UCC, 21 IUC, 26 KC and 22 PC, and 23 HV urine samples. The combination of POU4F2/PCDH17 has yielded the highest sensitivity and specificity of 90.00% and 93.96% in all the 312 individuals, showing the capability of detecting BlCa effectively among pathologically varied sample groups.

AB164. An epigenetic biomarker combination of EOMES, pcdh17, and pou4f2 detects bladder cancer accurately from methylation analyses of urine sediment DNA in Han Chinese

ObjectiveTo recognize a novel and optimized combination of epigenetic biomarkers capable of returning higher sensitivity and specificity synergistically in pathologically varied sample groups for urine sediment examination of bladder cancer (BlCa).MethodsMethylation levels of seven candidate biomarkers (EOMES, GDF15, NID2, PCDH17, POU4F2, TCF21, and ZNF154) in 58 urothelial cell carcinoma (UCC) patients, 20 infected urinary calculi (IUC) patients, 20 kidney cancer (KC) patients, and 30 healthy volunteers (HV) were quantified by qMSP using the sediment DNA of urine samples. Standard curves and receiver operating characteristic (ROC) curves were generated for each biomarker. The combining predictors of all the possible biomarker combinations were calculated through logistic regression model. Subsequently, the ROC curves of the three combinations that have yielded the best performing combining predictors were constructed.ResultsFrom the samples provided, the top three biomarker combinations (C) returned high sensitivity and specificity of 91.38% and 94.29% from PCDH17 + POU4F2 (C1), 91.38% and 90.00% from PCDH17 + POU4F2 + NID2 (C2), and 93.10% and 92.86% from PCDH17 + POU4F2 + EOMES (C3) consequently. These combinations provide greater sensitivities and specificities than any examined single biomarkers, except for C2, which has a lower specificity compare to POU4F2 (92.86%).ConclusionsCompound biomarker panels were shown to be capable of detecting BlCa effectively by utilizing urine sediment DNA. Due to the performance of higher sensitivity and specificity when compared to single biomarker examinations in BlCa patients and the diverse control groups, these panels are particularly suitable for mass-scale routine BlCa screening.

1056 Detection of FGFR3 mutations from urine sediment DNA to predict the risk of intravesical recurrence after radical nephroureterectomy for upper tract urothelial carcinoma

1056 Detection of FGFR3 mutations from urine sediment DNA to predict the risk of intravesical recurrence after radical nephroureterectomy for upper tract urothelial carcinoma

C110 Genetic diversity of urinary bladder cancer and the risk of recurrence based on mutation assay using tumor and urine sediment DNA

C110 Genetic diversity of urinary bladder cancer and the risk of recurrence based on mutation assay using tumor and urine sediment DNA

Abstract 4908: Detection of bladder tumor by quantitative methylation specific real time PCR from urine sediment DNA

Abstract Bladder cancer is the fifth most frequent human cancer and causes approximately 3% of all cancer-related deaths. DNA hypermethylation is a common epigenetic abnormality in cancer and may serve as a useful marker to clone new cancer-related genes as well as a marker of the disease activity. Recently, we developed a high throughput genome wide methylation assay, Methylated CpG Island Amplification Microarray (MCAM). Using MCAM, we investigated methylation changes in about 8,000 genes in bladder tumors. Through this screening method and validation by bisulfite pyrosequencing, we have developed novel biomarkers for diagnosis and prognosis of bladder tumor. We characterized a group of genes that was found to be methylated in 80-90% of the tested bladder tumor samples. These candidate genes (DBC1, MYO3A, SOX11, SLC16A12, NPTX2, NKX6-2, A2BP1, PENK and CA10) were used to screen for bladder cancer in urine samples by quantitative methylation specific real time PCR, thus representing a non-invasive method for early detection. Based on these data, we established a panel of 6 biomarkers (MYO3A, SOX11, NKX6-2, PENK, CA10 and quantity of DNA in test urine) that can be used for bladder tumor screening in urine samples. The method showed 89.1 % sensitivity and 91.4 % specificity for detection of bladder tumor urines (n=128) compared to normal control urine (n=105) by 3 or more than 3 positive out of 6 biomarkers. Analyzing by stage of bladder tumors, 5 out of 5 (100 %) in Tis, 21 out of 29 (72 %) in Ta, 18 out of 19 (95 %) in T1, 57 out of 60 (95 %) in T2, 6 out of 6 (100 %) in T3 and 2 out of 2 (100 %) in T4 showed 3 or more than 3 positive out of 6 biomarkers. Thus, 83% of Tis-Ta-T1 stages and 95.6 % of T2-T3-T4 stages were detected in analyzed bladder tumor urines. This DNA methylation biomarkers panel will help the early detection and monitoring of bladder tumor patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4908.

Increase sensitivity in detecting superficial, low grade bladder cancer by combination analysis of hypermethylation of E-cadherin, p16, p14, RASSF1A genes in urine

Objectives To identify a better set of DNA methylation markers to detect superficial, low grade cancer cell in urine sediment for improving cancer treatment, morbidity, and mortality. Materials and Methods Methylation-specific PCR (MSP) assay was used to detect promoter hypermethylation in 4 genes (E-cadherin, p16, p14, and RASSF1A) to identify reliable biomarkers for bladder cancer diagnosis in primary tumor DNA and urine sediment DNA from 57 bladder cancer patients. Urine DNA was compared with 20 healthy controls. Results Fifty-one (90%) tumor DNA and 47 urine DNA (83%) samples from bladder cancer patients revealed hypermethylation in at least 1 of the 4 analyzed genes, whereas all urine samples from normal controls were negative. The sensitivity of MSP assay for detecting E-cadherin, p16, p14 and RASSF1A in tumor cells in voided urine was 35%, 35%, 33%, and 65%, respectively. Diagnostic sensitivity was 75% for combining RASSF1A and p14, and 83% for RASSF1A, p14 and E-cadherin. Urine cytology, however, detect only 13 (28%) cases of cancer or suspicious cancer. For detecting superficial and invasive bladder tumor, urine cytology revealed a sensitivity of 23% (6/26) and 35% (7/20), respectively. In contrast, MSP detected hypermethylation in the urine of 80% (37/46) bladder cancer patients. Moreover, hypermethylation analysis of E-cadherin, p14 or RASSF1A genes in urine sediment DNA detected in 85% (22/26) of superficial, 85% (11/13) of low grade, 75% (15/20) of invasive and 79% (26/33) of high grade bladder cancers. Importantly, hypermethylation was detected in the urine DNA of 90% (18/20) superficial tumors with negative or atypia cytology. Conclusions Hypermethylation of E-cadherin, p14 or RASSF1A in urine sediment DNA is a potential biomarker for detecting superficial, low grade cancer. Besides, hypermethylation of these 3 genes is a valuable adjunct diagnostic marker to urine cytology, which can enhance the diagnostic accuracy and follow-up treatment of bladder cancer patients.

Molecular Methods for Simultaneously and Noninvasively Ascertaining Ovarian Status, Insemination Status, and Urogenital Microbial Status from Urine Sediment DNA: Progress.

Molecular Methods for Simultaneously and Noninvasively Ascertaining Ovarian Status, Insemination Status, and Urogenital Microbial Status from Urine Sediment DNA: Progress.

Tissue Inhibitor of Metalloproteinases-3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer

TIMP-3 (tissue inhibitor of metalloproteinases-3) is 1 of 4 members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases. We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from patients with bladder cancer with well characterized clinicopathological parameters, including patient outcome. We examined urine sediment DNA for aberrant methylation of 9 genes, including TIMP-3, by quantitative fluorogenic real-time polymerase chain reaction. Using an optimal cutoff value by TaqMan(R) quantitation we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (HR 1.99, 95% CI 1.12 to 3.27, p = 0.01 and HR 1.66, 95% CI 1.00 to 2.76, p = 0.05, respectively) than in patients without/lower TIMP3 and ARF methylation in urine. A significant correlation was also seen between the risk of death and stage 3 tumor (HR 2.73, 95% CI 1.58 to 4.72, p = 0.003) and metastasis (HR 3.32, 95% CI 1.98 to 5.57, p = 0.0001). Multivariate analysis subsequently revealed that TIMP-3 methylation was an independent prognostic factor for bladder cancer survival with stage and metastasis (p = 0.001 and 0.02, respectively). These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.

High Concordance of Gene Methylation in Post-Digital Rectal Examination and Post-Biopsy Urine Samples for Prostate Cancer Detection

We evaluated the concordance between post-digital rectal examination and post-prostate biopsy urine samples using conventional methylation specific polymerase chain reaction analysis of 3 gene promoters in patients with suspected or confirmed prostate cancer. Voided urine specimens were collected from 17 men after 15-second digital rectal examination and again after transrectal ultrasound guided biopsy of the prostate for suspected malignancy or for followup biopsy as part of an expectant management protocol. Urine sediment DNA was isolated and subjected to bisulfite modification. Methylation of GSTP1, EDNRB and APC promoters was determined by conventional methylation specific polymerase chain reaction analysis in post-digital rectal examination and post-biopsy samples, and correlated with clinical information. Prostate cancer was detected on prostate biopsy in 12 of 17 patients (71%). Promoter methylation was detected in post-digital rectal examination urine specimens for GSTP1 (24%), APC (12%) and EDNRB (66%). Promoter methylation was detected in post-biopsy urine specimens for GSTP1 (18%), APC (18%) and EDNRB (77%). The concordance between post-digital rectal examination and post-biopsy urine samples was 94% for GSTP1 and APC, and 82% for EDNRB. Overall 100% of patients with biopsy proven prostate cancer had at least 1 gene methylated in urine vs 60% of those without evidence of prostate cancer on biopsy. Gene analysis using conventional methylation specific polymerase chain reaction is a reliable method for detecting abnormal DNA methylation in voided urine samples obtained following digital rectal examination or prostate needle biopsy. The concordance between post-digital rectal examination and post-biopsy urinary samples for promoter methylation is high (82% to 94%), suggesting that urine collected after digital rectal examination may be used for genetic analysis with results similar to those in post-biopsy urine samples.

Quantitation of Promoter Methylation of Multiple Genes in Urine DNA and Bladder Cancer Detection

The noninvasive identification of bladder tumors may improve disease control and prevent disease progression. Aberrant promoter methylation (i.e., hypermethylation) is a major mechanism for silencing tumor suppressor genes and other cancer-associated genes in many human cancers, including bladder cancer. A quantitative fluorogenic real-time polymerase chain reaction (PCR) assay was used to examine primary tumor DNA and urine sediment DNA from 15 patients with bladder cancer and 25 control subjects for promoter hypermethylation of nine genes (APC, ARF, CDH1, GSTP1, MGMT, CDKN2A, RARbeta2, RASSF1A, and TIMP3) to identify potential biomarkers for bladder cancer. We then used these markers to examine urine sediment DNA samples from an additional 160 patients with bladder cancers of various stages and grades and from an additional 69 age-matched control subjects. Data were analyzed on the basis of a prediction model and were internally validated using a jacknife procedure. All statistical tests were two-sided. For all 15 patients with paired DNA samples, the promoter methylation pattern in urine matched that in the primary tumors. Four genes displayed 100% specificity. Of the 175 bladder cancer patients, 121 (69%, 95% confidence interval [CI] = 62% to 76%) displayed promoter methylation in at least one of these genes (CDKN2A, ARF, MGMT, and GSTP1), whereas all control subjects were negative for such methylation (100% specificity, 95% CI = 96% to 100%). A logistic prediction model using the methylation levels of all remaining five genes was developed and internally validated for subjects who were negative on the four-gene panel. This combined, two-stage predictor produced an internally validated ROC curve with an overall sensitivity of 82% (95% CI = 75 % to 87%) and specificity of 96% (95% CI = 90% to 99%). Testing a small panel of genes with the quantitative methylation-specific PCR assay in urine sediment DNA is a powerful noninvasive approach for the detection of bladder cancer. Larger independent confirmatory cohorts with longitudinal follow-up will be required in future studies to define the impact of this technology on early detection, prognosis, and disease monitoring before clinical application.

Specific and sensitive detection of prostate cancer using urine sediment DNA

Specific and sensitive detection of prostate cancer using urine sediment DNA

Specific and sensitive detection of prostate cancer using urine sediment DNA

Specific and sensitive detection of prostate cancer using urine sediment DNA