Volume 130, Issue 7, Pages 1950-1961 (June 2006) Association of JC Virus T-Antigen Expression With the Methylator Phenotype in Sporadic Colorectal Cancers  Ajay Goel, Mei–Shu Li, Takeshi Nagasaka, Sung Kwan Shin, Florentine Fuerst, Luigi Ricciardiello, Linda Wasserman, C. Richard Boland  Gastroenterology  Volume 130, Issue 7, Pages 1950-1961 (June 2006) DOI: 10.1053/j.gastro.2006.02.061 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Detection of JCV T-Ag DNA sequences and protein expression in CRCs. (A) PCR amplification of T-Ag DNA sequences using gene specific primers that amplify a 154-bp product from JCV templates. M, size marker; T1–T9, T-Ag sequences detected from 7 different paraffin-embedded CRC samples; B, water blank; JCI, positive control DNA from JCI cells that are persistently infected with JCV. (B) Bar graphs representing the frequency of JCV T-Ag DNA sequences and protein expression in a cohort of 100 sporadic colorectal cancers. DNA sequences were detected in 77% of these cancers, but only 43% of the total (all of which also had JCV DNA detected) also expressed T-Ag protein at IHC. (C) IHC characterization of T-Ag expression in a paraffin-embedded CRC tissue section. The image on the left depicts a low-power (×100) view of the section with normal colonic epithelial glands [N] on the left and an infiltrating cancer [C] on the right. T-Ag-specific (brown) staining is observed only in the nuclei of cancer cells, whereas the normal colonic cells shown do not (blue). The image on the right is a higher magnification view (×400) from the same section (square box), depicting the specific localization of T-Ag staining in the nucleus, whereas the normal colonic cells are unstained in any subcellular location. Gastroenterology 2006 130, 1950-1961DOI: (10.1053/j.gastro.2006.02.061) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 JCV T-Ag DNA in 100 CRCs correlated with MSI status. (A) T-Ag DNA sequences are present in comparable frequencies from CRCs that are MSI-H (78%) or MSS/MSI-L (73%) (P = .95, not significant). (B) By IHC, T-Ag protein expression is detected in 53% of MSI-H cancers and 41% of MSS/MSI-L CRCs (P = .55). Gastroenterology 2006 130, 1950-1961DOI: (10.1053/j.gastro.2006.02.061) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 The promoters of tumor-suppressor genes have a high frequency of hypermethylation in CRCs that express JCV T-Ag. This provides a detailed data profile from 91 CRCs analyzed for methylation status at 9 tumor-suppressor genes. The columns depict the following data: IHC for T-Ag protein expression, presence of JCV T-Ag DNA sequences, MSI analysis using standard markers, LOH status, and MSP data for methylation of tumor-suppressor genes. For IHC, JCV DNA, and LOH, a solid box indicates a positive result. For MSI, a solid box indicates MSI-H, and an open box indicates MSI-L or MSS. For the gene-specific MSP data, the solid boxes represent a methylated gene promoter, and open boxes indicate an unmethylated promoter. The final column indicates the total number of methylated promoters in the DNA from that tumor sample. (A) Methylation profiles in the subset of CRCs (n = 43) that have JCV T-Ag DNA sequences and express T-Ag protein. (Note that the last 12 have neither MSI nor LOH.) (B) Methylation profiles in the subset of CRCs (n = 33) that harbor JCV DNA sequences but do not express T-Ag at IHC. (C) Methylation profiles in the subset of CRCs (n = 15) that do not harbor JCV DNA sequences and do not express the T-Ag protein. The methylation frequencies in subset A (JCV DNA+/T-Ag+) were significantly higher than in the other 2 subsets (P < .01). Gastroenterology 2006 130, 1950-1961DOI: (10.1053/j.gastro.2006.02.061) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 MSI-H CRCs that express JCV T-Ag have higher MIs. The paired bars depict methylation indices for individual tumor-suppressor genes in the CRCs that either express (solid bars on the left) or do not express JCV T-Ag (open bars on the right). The paired bars at the extreme right represent the methylation index (MI), which is the sum of all methylated promoters divided by the total number of CRCs in the subset. (A) The data in the upper series of bars represent the frequency of methylation, by gene, in MSI-H CRCs. A significant increase in methylation frequencies was observed for the hMLH1, PTEN, p16, and RUNX3 genes in CRCs expressing T-Ag compared with the colon cancers, which did not. The MI was significantly greater in T-Ag expressing cancers (P < .0001). (B) The data in the lower series of bars demonstrate that there were no significant differences in methylation frequencies between T-Ag expressing and nonexpressing tumors, with the exception of the PTEN gene (P = .02), in CRCs that were MSI-L/MSS. Gastroenterology 2006 130, 1950-1961DOI: (10.1053/j.gastro.2006.02.061) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions