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Supp Figure 3. Association of miRNA expression with water arsenic concentration and creatinine adjusted urine arsenic concentration (Mann-Whitney U test).

Supp Figure 1. Technical validation of Notch Signaling Pathway DNA Methylation PCR Array.

Exposure to toxicants leads to cumulative molecular changes that overtime increase a subject's risk of developing urothelial carcinoma. To assess the impact of arsenic exposure at a time progressive manner, we developed and characterized a cell culture model and tested a panel of miRNAs in urine samples from arsenic-exposed subjects, urothelial carcinoma patients, and controls. To prepare an in vitro model, we chronically exposed an immortalized normal human bladder cell line (HUC1) to arsenic. Growth of the HUC1 cells was increased in a time-dependent manner after arsenic treatment and cellular morphology was changed. In a soft agar assay, colonies were observed only in arsenic-treated cells, and the number of colonies gradually increased with longer periods of treatment. Similarly, invaded cells in an invasion assay were observed only in arsenic-treated cells. Withdrawal of arsenic treatment for 2.5 months did not reverse the tumorigenic properties of arsenic-treated cells. Western blot analysis demonstrated decreased PTEN and increased AKT and mTOR in arsenic-treated HUC1 cells. Levels of miR-200a, miR-200b, and miR-200c were downregulated in arsenic-exposed HUC1 cells by quantitative RT-PCR. Furthermore, in human urine, miR-200c and miR-205 were inversely associated with arsenic exposure (P = 0.005 and 0.009, respectively). Expression of miR-205 discriminated cancer cases from controls with high sensitivity and specificity (AUC = 0.845). Our study suggests that exposure to arsenic rapidly induces a multifaceted dedifferentiation program and miR-205 has potential to be used as a marker of arsenic exposure as well as a maker of early urothelial carcinoma detection. Cancer Prev Res; 8(3); 208–21. ©2015 AACR.

Supp Figure 2A. Promoter methylation is inversely associated with gene expression

Supp Table 1, Supp Table 2. Supp Table 1: A.QMSP probe and primers, B.RT-PCR assay. Supplemental Table 2: The summary of notch methylation array findings

Supp Figure 4. ROC curves for individual micro RNA expression in 177 normal and 32 bladder cancer urine samples were shown.

Supp Figure legends

Current non-invasive approaches for bladder cancer (BC) detection are suboptimal. We report the development of non-invasive molecular test for BC using DNA recovered from cells shed into urine. This “UroSEEK” test incorporates assays for mutations in 11 genes and copy number changes on 39 chromosome arms. We first evaluated 570 urine samples from patients at risk for BC (microscopic hematuria or dysuria). UroSEEK was positive in 83% of patients that developed BC, but in only 7% of patients who did not develop BC. Combined with cytology, 95% of patients that developed BC were positive. We then evaluated 322 urine samples from patients soon after their BCs had been surgically resected. UroSEEK detected abnormalities in 66% of the urine samples from these patients, sometimes up to 4 years prior to clinical evidence of residual neoplasia, while cytology was positive in only 25% of such urine samples. The advantages of UroSEEK over cytology were particularly evident in low-grade tumors, wherein cytology detected none while UroSEEK detected 67% of 49 cases. These results establish the foundation for a new, non-invasive approach to the detection of BC in patients at risk for initial or recurrent disease.

Epithelioid hemangioendothelioma (EHE) is a rare mesenchymal tumor of vascular origin with variable morphological features and unpredictable biological behavior. EHE occasionally involves the pleural fluid. However, the cytomorphology of EHE found in pleural fluid has not been well characterized in the literature. Herein we describe a case of EHE, initially presenting as multiple liver lesions plus several small nodular densities at the bases of the lungs followed by bilateral pleural effusions.