Abstract: SA-PO487

Targeted Sequencing of BKPyV in Urothelial Carcinomas in Transplant Recipients

Session Information

Category: Transplantation

  • 1702 Transplantation: Clinical and Translational

Authors

  • Farkash, Evan A., University of Michigan, Ann Arbor, Michigan, United States
  • Harake, Edward Samir, University of Michigan, Ann Arbor, Michigan, United States
Background

Some urothelial carcinomas arising in transplant recipients show strong, universal expression of BKPyV Large T antigen (LTA). BKPyV is closely related to known oncoviruses SV40 and MCPyV; we hypothesize that BKPyV acts as oncovirus in immunosuppressed transplant reciepients. Determining the genomic structure of BKPyV in urothelial tumors may provide clues to the mechanism of oncogenesis.

Methods

We designed 26 sets of fully nested 5’ and 3’ primers using the BKPyV DIK strain and validated on a DIK isolate. DNA was extracted from paraffin blocks of 2 urothelial carcinomas with diffuse LTA expression (QIAMP FFPE). The BKPyV genome was amplified by 2x overlapping nested PCRs and Sanger sequenced. Sequences were aligned and compared to 312 full length BKPyV sequences from the NCBI database, as well as 1 tumor previously sequenced by a different method (Megalign, Figtree).

Results

Tumor 1 (fatal) arising 9.7 years after transplant in 59 year old male harbors a clade Ib1 virus (84.8% sequenced). Tumor 2 (nonfatal) arising 8.9 years after transplant in a 68 year old female harbors a clade IV virus (31.2% sequenced). Tumor 3 (previous work) contains a clade 1a virus. Clade I has a worldwide distribution, and clade IV is enriched in Asian and Japanese regions.

Conclusion

A targeted amplification strategy was partially successful at sequencing BKPyV from urothelial carcinoma tumors from FFPE tissue. Identification of viruses from clades Ia, Ib1 and IV provides evidence that potential oncogenecity from BKPyV in transplant recipients is not restricted to a single clade. No viral integration sites or flanking DNA were identified, and a linker DNA technique is likely needed to map potential integration sites.

Phylogenetic analysis of BKPyV from 3 urothelial carcinomas and 312 full length BKPyV sequences. Roman numerals indicate viral clade.

Funding

  • Other NIH Support