Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

Baldwin, Don A.; Feldman, Michael; Alwine, James C.; Robertson, Erle S.

The ability of the PathoChIP to combine saturation probe sets and RNA and DNA detection enhances the screening for known oncogenic pathogens. If sufficient target copies are present in a sample, inferences regarding genomic structural variation and RNA expression levels may be possible. For HPV16, probes mapping to early gene transcripts produced more overall signal than those for late genes in OSCC samples; this is consistent with studies of actively infected cells and the oncogenic effects of E6/E7 expression (32, 33) . Moreover, the transcription of HPV at the level of detail that we are probing here is greater than previously investigated and suggests a potential role for other transcripts, including the E4 ORF and related antigen, in maintenance of the transformed state in the tumors. Signals from probes to late gene sequences were similar to or somewhat higher than those of probes to intergenic HPV16 sequences. Therefore, the boost in early gene signals is likely due to the RNA portion of the sample target preparation. Among the early genes, strong signals were observed for E6/E7 sequences, and for the E4 region of E2, which is known to be a highly abundant RNA splicing product from the primary transcript (34) . Detailed interpretation of these data is complicated by the ability of HPV16 to exist in episomal and multiple host-genome integrated forms (35) , but probe signal differences within and between tumors will provide the identification of potential agents having oncogenic activities and so lead to new lines of investigation as a follow-up to the initial screening experiment.