Background Bladder cancer (BC) is the 5th most common cancer in the USA. Non-muscle invasive bladder cancer represents about 70% of all cases and has generally a favorable outcome. However, recurrence rates as high as 60 to 70% and progression rates of 10 to 20% necessitate intensive surveillance with cystoscopy. The invasiveness and high cost of cystoscopy poses significant burden on BC patients as well as on the healthcare system. In this study we test the feasibility of a simple, sensitive, and non-invasive detection of BC using Bladder CARE test in urine samples. Results Urine from 136 healthy and 77 BC subjects was collected using the at-home Bladder CARE Urine Collection Kit and analyzed with Bladder CARE test. The test measures the methylation level of three BC-specific biomarkers and two internal controls using methylation-sensitive restriction enzymes coupled with qPCR. Bladder CARE showed an overall sensitivity of 93.5%, a specificity of 92.6%, and a PPV and NPV of 87.8% and 96.2%, respectively. Bladder CARE has an LOD as low as 0.046%, which equates to detecting 1 cancer cell for every 2,200 cells analyzed. We also provided evidence that bisulfite-free methods to assess DNA methylation, like Bladder CARE, are advantageous compared to conventional methods that rely on bisulfite conversion of the DNA. Conclusion Highly sensitive detection of BC in urine samples is possible using Bladder CARE. The low LOD of the test and the measurement of epigenetic biomarkers make Bladder CARE a good candidate for the early detection of BC and possibly for the routine screening and surveillance of BC patients. Bladder CARE and the at-home urine sample collection system have the potential to (1) reduce unnecessary invasive testing for BC (2) reduce the burden of surveillance on patients and on the healthcare system, (3) improve the detection of early stage BC, and (4) allow physicians to streamline the monitoring of patients.

Background: DNA methylation clocks have been developed recently to estimate epigenetic age which is associated with chronological age in mouse and human. Changes in epigenetic age were reported in patients with health conditions including cancer. Different from genetic variations, epigenetic properties can be modified by environmental factors. Epigenetic age tests using blood samples have been offered directly to customers recently. We sought to analyze relationships between epigenetic aging and lifestyle factors, medication and aging interventions in the hope of assisting healthier living. Methods: Epigenetic age, called DNAge, is estimated using linear regression of DNA methylation levels at 206 loci containing 1344 CpGs and chronological age. Delta age is defined as the difference of epigenetic age minus chronological age. Correlations between lifestyle factors or medication and delta age of hundreds of whole blood samples were evaluated using biweight midcorrelation. Statistical significance is set at 0.05. Results: Our results showed positive correlations between accelerated epigenetic aging and BMI (bicor=0.148, p=0.009, n=311), current smoker (bicor=0.143, p=0.011, n=315), or stress (bicor=0.088, p=0.128, n=302). Factors with positive correlation but not reaching statistical significance included total cholesterol, alcohol consumption and Keto diet. Factors with negative correlation but not statistically significant included exercise, balanced diet, mostly meat, mostly vegetables, and education. Conclusions: The epigenetic age analysis of real customer data confirmed beneficial and adversarial health effects of certain lifestyle factors. The DNAge test offered proves to be useful as a biomarker for biological age and as a practical tool for monitoring and evaluation of health status which may be used in cancer epidemiology study and help cancer prevention. Citation Format: Wei Guo, Yap Ching Chew, Xiaojing Yang, Michiko Suwoto, Taikun Yamada, Xi Yu Jia. Correlation of lifestyle factors with epigenetic aging unveiled in the practice of a DNA methylation-based age test [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2332.

Introduction: Bladder cancer (BC) is the 5th most common cancer in the USA, with 81,190 new cases and 17,240 deaths in 2018. The high recurrence rate of BC requires a lifelong patient follow-up with cystoscopy. However, the invasiveness of cystoscopy and the high costs associated to this procedure, poses a significant burden on patients as well as on the healthcare system. Several tests for the non-invasive detection of BC have been developed in the past years however, because of relatively low sensitivities and/or specificities (especially for low-grade tumors), these tests have not been adopted for the routine clinical use. Therefore, a non-invasive, simple and sensitive test for detection and routine monitoring of BC is needed. Methods: Bladder CARE is a new non-invasive and quantitative test that measures the methylation levels of three BC specific biomarkers and two internal controls in urine samples. Contrary to other epigenetic tests, Bladder CARE analyzes DNA methylation without converting the DNA with sodium bisulfite, thus minimizing the degradation of the target DNA. In this clinical study, we evaluated the performance of this test using voided urine samples collected from 136 healthy individuals and 77 BC patients with no prior history of bladder cancer. Urine specimens were collected and stabilized using Bladder CARE at-home urine sample collection kit and BC detection was performed by a CLIA-certified and CAP-accredited laboratory (Pangea Laboratory LLC) using Bladder CARE. The results were then compared to the clinical information (cystoscopy). Additionally, the test linearity and limit of detection (LOD) were determined by analyzing artificial samples with Bladder CARE test. Results: Bladder CARE was able to discriminate BC from healthy control samples with a sensitivity of 93.5%, a specificity of 92.6%, and a positive and negative predictive value of 87.8% and 96.2%, respectively. Remarkably, low-grade tumors were detected with a sensitivity of 90.0%. We also found that Bladder CARE test has a LOD of 0.046% (the equivalent of detecting 1 cancer cell in a sample containing 2,200 normal cells). Conclusions: While cystoscopy remains the gold standard to confirm the presence of BC, the superior sensitivity of Bladder CARE test, together with its low LOD, make this test an effective early indicator of low-grade tumors and for routine recurrence monitoring of BC patients. In addition, Bladder CARE urine collection kit allows to collect and stabilize the urine specimen comfortably at-home and mail it to the testing laboratory at room temperature, streamlining the patients' detection and monitoring process. Citation Format: Paolo Piatti, Michiko Suwoto, Taikun Jamada, Yap Ching Chew, Gangning Liang, Saum Ghodossipour, Siamak Daneshmand, Xi-Yu Jia. Bladder care epigenetic test allows the sensitive early detection of bladder cancer from urine samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2295.

disease, and 5 died of disease. Within the same period, other histological variants were found in 48 cases, including 13 micropapillary and 14 plasmacytoid variant cases. Compared to LCV and other variants, LCV had significantly worse recurrence-free survival (HR [ 1.82; 95% CI 1.03e4.73; p [ 0.043) and cancer-specific survival (HR [ 3.40; 95% CI 1.10e10.77; p [ 0.032). On IHCS, adipophilin was positive for cytoplasm of LCV but cytoplasmic vacuoles themselves were negative. In a special case, we firstly demonstrated lipid component in cytoplasmic vacuoles of LCV by oil red O staining using frozen sections. The type of lipid dyed by oil red O staining was triglyceride, and it was proved that cytoplasmic vacuoles in LCV were triglycerides.CONCLUSIONS: Although LCV has been thought to be rare, the incidence of this variant does not seem to be extremely low based on the present study. Patients with this variant had high recurrence rates and poor prognosis. Also, this is the first report in the literature that the component of the cytoplasmic vacuoles of LCV is lipid directly.

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