(489e) Single Molecule, or Near Single Molecule, in-Situ, RNA Imaging of Viral RNA of Human Papillomavirus in Circulating Tumor Cells and Cell Lines in Patients with Head and Neck Cancer

Human papillomavirus (HPV) has become the major causative (etiological) agent for oropharyngeal tumors (Head and Neck Squamous Cell Carcinoma, HNSCC, in the back of the throat/tonsils). Carcinogenicity of HPV is hypothesized to be closely related to cell differentiation; the production of HPV virion occurs in the secondary suprabasal region differentiated from primary HPV-infected basal layer. The expressions of HPV viral sequences, E1-E7, L1 and L2 vary at different differentiation stage, which provide a tool to begin to understand the complex process.

To characterize HPV16 at both tumor tissue level and circulating tumor cell (CTC) level, we developed 6-color integrated RNA in situ Hybridization (ISH) and immunochemical staining. The commercial RNA ISH allows in situ highly amplified labeling of mRNA. The elimination of spectral overlap by spectral deconvolution can reduce the effect of false positivity introduced by background noise and spectral leaking. Meanwhile, the integrated staining incorporates the strengths of RNA ISH and immunochemical assay and provides high throughput screening analysis with single molecular sensitivity on the transcription and expression of recombinant HPV genes. The staining based on HPV16 E2, E6, E7, L1, EGFR, panCK and CD45 can provide a snapshot of the dynamic profile of HPV proliferation in the process of basal cell differentiation in patient specimens.

We have previously established, and demonstrated, our CD45-based negative magnetic separation which can enrich heterogeneous CTCs. Unlike prevailing single tumor marker based positive selection methods, which can only enrich cells positive on certain tumor markers, cells without hematopoietic origin can be mostly retained for subsequent analysis with our quadrupole magnetic separation. The HPV-infected cells at different differentiation stage from oropharyngeal cancer patient can thus be enriched and analyzed.

This presentation will provide an update of our understanding of HPV biology in HNSCC with specific emphasis on our multiparameter staining of patient CTCs and HNSCC cell lines, and model tissues, using not only traditional immunocytochemistry (ICC), but also the use of the new technology, RNA ISH, which allows in situ highly amplified labeling of mRNA. In our case, we use the RNA ISH to target the HPV products, and we are the first to report that recombinant mRNA of HPV can be found in CTCs.