Vicari, J.;Sternjakob, A.;Staiger, J.;Kuhn, C.;Podgorska, M.;Weishaar, J.;Doering, M.;Khrystenko, T.;Kim, Y.;Solomayer, E.;Smola, S.

High-grade squamous intraepithelial lesions (HSIL) of the uterine cervix result from transformation by high-risk human papillomavirus (HR-HPV) and are considered precancerous lesions. Depending on the proportion of the epithelium occupied by dysplastic cells, commonly evidenced by a block-like p16INK4a staining pattern, HSIL are subdivided into CIN2 or CIN3. We used immunohistochemistry (IHC) and laser-capture microdissection (LCM) to further characterize these lesions. Broad suprabasal expression of S100A9, a component of the epidermal differentiation complex (EDC), which we had previously found to be up-regulated in genus-beta-HPV-positive skin lesions, was present in the normal exocervix and declined gradually with increasing CIN grade. In CIN2 in particular, the S100A9 staining pattern was nearly complementary to the block-like p16INK4a expression. However, it largely coincided with the expression of CD63, a marker showing strong expression in cervical crypts and in columnar cells within the normal transformation zone, also those overlying reserve cells. We then used LCM to analyze both the p16INK4a- and S100A9/CD63-positive compartments in HSIL separately. Notably, in all samples investigated, we detected the same HR-HPV type (either HPV16, 45 or 51) in both compartments of the same HSIL, while tissue samples dissected from normal exocervix or transformation zone were consistently HR-HPV-negative. Our data suggest that the upper, S100A9-positive maturing compartment of HSILs retaining weak CD63 expression may represent a residual, HR-HPV-infected columnar-cell-derived epithelium, rather than not-yet-transformed squamous epithelium that is progressively being replaced by dysplastic cells. The two-compartment architecture of these HSILs was reminiscent of the staining pattern in the normal transformation zone, where columnar cells overlie reserve cells, and compatible with HR-HPV infection of columnar-derived cells but transformation of reserve cells, the putative origin of cervical squamous cell carcinoma. Our findings have direct clinical implications. Since the upper and lower layers of HSIL appear to form distinct epithelial compartments, our study suggests that the extent and thickness of the HR-HPV-transformed lower compartment itself, rather than the proportion of the epithelium occupied by dysplastic layers, may be the decisive factor in determining the HSIL grade.