Mapping of Human Polyomavirus in Renal Cell Carcinoma Tissues

Abstract

Worldwide, the incidence of renal cell carcinoma (RCC) is rising, accounting for approximately 2% of all cancer diagnoses and deaths. The etiology of RCC is still obscure. Here, we assessed the presence of HPyVs in paraffin-embedded tissue (FFPE) resected tissue from patients with RCC by using different molecular techniques. Fifty-five FFPE tissues from 11 RCC patients were included in this study. Consensus and HPyV-specific primers were used to screen for HPyVs. Both PCR approaches revealed that HPyV is frequently detected in the tissues of RCC kidney resections. A total of 78% (43/55) of the tissues tested were positive for at least one HPyV (i.e., MCPyV, HPyV6, HPyV7, BKPyV, JCPyV, or WUyV). Additionally, 25 tissues (45%) were positive for only one HPyV, 14 (25%) for two HPyVs, 3 (5%) for three HPyVs, and 1 one (1%) tissue specimen was positive for four HPyVs. Eleven (20%) RCC specimens were completely devoid of HPyV sequences. MCPyV was found in 24/55 RCC tissues, HPyV7 in 19, and HPyV6 in 8. The presence of MCPyV and HPyV6 was confirmed by specific FISH or RNA-ISH. In addition, we aimed to confirm HPyV gene expression by IHC. Our results strongly indicate that these HPyVs infect RCC and nontumor tissues, possibly indicating that kidney tissues serve as a reservoir for HPyV latency. Whether HPyVs possibly contribute to the etiopathogenesis of RCC remains to be elucidated.

Keywords: BKPyV; HPyV6; HPyV7; JCPyV; Merkel cell polyomavirus; RCC; WUPyV; adjacent tissues; small DNA viruses; tumorigenesis.

Figure 1

Schematic representation of tissue sampling of RCC and non-tumoral kidney tissues. tc: tumor core; tt: tumor transition; t1: 1 cm, t2: 2 cm, and t3: 3 cm distance to tumor. (Created by Bio—render.com).

Figure 2

HPyV consensus DNA PCR. Multiple PCR products after amplifying DNA of RCC tissues with HPyV-consensus primers. On the far right, the MKL-1-positive control reveals a specific PCR product at the expected size of 186 bp. All PCR products in the range of 150 to 220 bp were sequenced and analyzed.

Figure 3

(A) Summary of the total HPyV PCR results for both consensus and HPyV-specific DNA PCR in 55 tissues. (B) Venn diagram showing cases with simultaneous different HPyVs as tested by consensus and specific PCR.

Figure 4

(A) Detection of MCPyV on DNA in FFPE of RCC and adjacent tissues. Merged green (FITC) and blue (nuclei were counterstained with DAPI) show specific renal cells. Merged green (FITC) and blue (nuclei were counterstained with DAPI) show specific green signals. Example of results of FISH specific nuclear MCPyV in the nuclei of epithelial RCC and the adjacent tissue. WaGa cell lines served as a positive control for the MCPyV probe. The images were taken at 630× magnification, and a red square area was magnified 6× in the top right corner of each figure. (B) Detection of HPyV6 on the DNA in FFPE of RCC and adjacent tissues. Merged green (FITC) and blue (nuclei were counterstained with DAPI) show specific renal cells. Merged green (FITC) and blue (nuclei were counterstained with DAPI) show specific green signals. Example of results of FISH specific nuclear HPyV6 in the nuclei of epithelial RCC and the adjacent tissue. HEK-HPyV6 cell lines served as a positive control for the HPyV6 probe. The images were taken at 630× magnification, and a red square area was magnified 6× in the top right corner of each figure. The representative cases shown above correspond to the case numbers listed in Table 2.

Figure 5

(A) Detection of MCPyV on the transcriptional level in FFPE RCC tissue by RNA-ISH; the RCC patient tissue section was hybridized with 20 set labeled probes to detect MCPyV LTAg mRNA using an RNAscope RNA in situ hybridization assay. WaGa cell lines served as a positive control for the MCPyV probe. Positive red signals were detected using fast red chromogen. MCPyV LTA transcript seen as red signals were detected using fast red chromogen. MCPyV LTA transcript seen as red signals in RCC and adjacent tissue. The images were taken at 200× magnification, and a red square area was magnified 6× in the top right corner of each figure. (B) Detection of HPyV6 on the transcriptional level in FFPE RCC tissue by RNA-ISH; the RCC patient tissue section was hybridized with 20 set labeled probes to detect HPyV6 LTAg mRNA using an RNAscope RNA in situ hybridization assay. HEK-HPyV6 cell lines served as a positive control for the HPyV6 probe. Positive red signals were detected using fast red chromogen. HPyV6 LTA transcript seen as red signals were detected using fast red chromogen. HPyV6 LTA transcript seen as red signals in RCC and adjacent tissue. The images were taken at 400× magnification, and a red square area was magnified 6× in the top right corner of each figure. The representative cases shown above correspond to the case numbers listed in Table 2.

Figure 6

(A) Detection of MCPyV on the translational level in FFPE of RCC and adjacent tissues. Representative examples of IHC using CM2B4 antibodies show the specific immunoreactivity in the nucleus (brown) of RCC tissues. WaGa cell line served as a positive for MCPyV antibodies. The images were taken at 200× magnification, and a red square area was magnified 6× in the top right corner of each figure. (B) Detection of HPyVs on the translational level in FFPE of RCC and adjacent tissues. Representative examples of IHC using PAb416 antibodies show the specific nuclear immunoreactivity (brown) of RCC tissues. HEK-HPyV6 cell lines were used as positive control, while WaGa cell line served as a negative for PAb416 antibodies. The images were taken at 200× magnification, and a red square area was magnified 6× in the top right corner of each figure. The representative cases shown above correspond to the case numbers listed in Table 2.

Figure 7

Flow chart depicting the details of the clinicopathologic parameters of this RCC cohort. yrs, years; CCRCC, clear cell renal cell carcinoma; PRCC, papillary renal cell carcinoma.