AKT1 Loss Correlates with Episomal HPV16 in Vulval Intraepithelial Neoplasia

Anogenital malignancy has a significant association with high-risk mucosal alpha-human papillomaviruses (alpha-PV), particularly HPV 16 and 18 whereas extragenital SCC has been linked to the presence of cutaneous beta and gamma–HPV types. Vulval skin may be colonised by both mucosal and cutaneous (beta-, mu-, nu- and gamma-) PV types, but there are few systematic studies investigating their presence and their relative contributions to vulval malignancy. Dysregulation of AKT, a serine/threonine kinase, plays a significant role in several cancers. Mucosal HPV types can increase AKT phosphorylation and activity whereas cutaneous HPV types down-regulate AKT1 expression, probably to weaken the cornified envelope to promote viral release. We assessed the presence of mucosal and cutaneous HPV in vulval malignancy and its relationship to AKT1 expression in order to establish the corresponding HPV and AKT1 profile of normal vulval skin, vulval intraepithelial neoplasia (VIN) and vulval squamous cell carcinoma (vSCC). We show that HPV16 is the principle HPV type present in VIN, there were few detectable beta types present and AKT1 loss was not associated with the presence of these cutaneous HPV. We show that HPV16 early gene expression reduced AKT1 expression in transgenic mouse epidermis. AKT1 loss in our VIN cohort correlated with presence of high copy number, episomal HPV16. Maintained AKT1 expression correlated with low copy number, an increased frequency of integration and increased HPV16E7 expression, a finding we replicated in another untyped cohort of vSCC. Since expression of E7 reflects tumour progression, these findings suggest that AKT1 loss associated with episomal HPV16 may have positive prognostic implications in vulval malignancy

Keratinocyte differentiation marker expression in epidermis, vulva and cervix.

<p>Immunohistochemical analysis of AKT1, loricrin and keratin 1 expression in extragenital cutaneous epidermis, vulval external epithelium and cervix. Bar 50 µm.</p

AKT1 expression is lost in a subset of VIN and the epidermis of the K14-HPV16 mouse.

<p>A. Histology and AKT1 expression in two representative VIN. Inset shows specific AKT1 expression associated with nucleated upper epidermal cells. Secondary alone control of upper epidermis is also shown. B. Immunohistochemical analysis of AKT1 in the dorsal epidermis of the K14-HPV8 complete early region (CER) and ear epidermis of the K14-HPV16 complete early region mouse and corresponding wildtype controls (wildtype). Arrowheads indicate AKT1 positivity in the granular layer of controls. Sec alone is a no primary antibody control for staining specificity. Bar (A–B) 50 µm.</p

Early gene expression in VIN.

<p>Expression of the early genes 16E1∧E4 and 16E7 in a representative AKT1 negative (AKT1 −ve) and AKT1 positive (AKT1 +ve) VIN. Note the increased 16E7 expression in AKT1 positive VIN, and the change of 16E1∧E4 expression from perinuclear low expression in AKT1 negative VIN to high nuclear expression in AKT1 positive VIN (see insets). Bar 50µm.</p

AKT1 expression and HPV16E7 expression in vSCC cohort.

<p>Representative data of immunohistochemistry of the archive vSCC cohort. AKT1 expression and HPV16E7 expression were analysed by immunohistochemistry. Loss of AKT1 associated with low HPV16E7 levels, while maintained AKT1 correlated with high HPV16E7 expression. Bar 50 µm.</p

Summary of nested PCR analysis and sequencing for HPV16/18, and Beta-PV types.

<p>-, sample is negative for this PV-type. AKT1 indicates the presence (pos) or absence (neg) of AKT1 expression in the epidermis of the VIN/vSCC. Copy no. indicates the HPV16 copy number per cell in each sample. Integrated? denotes the integration status of HPV16 based on the HPV16 genomic PCR. N.B. Several samples were PCR-positive for cutaneous alpha-PV species 4, however sequencing showed this was due to non-specific amplification of HPV16 in all cases; gamma and mu/nu types were not detected. E1-E4 and E7, summary of the immunohistochemistry for HPV16 EE4 and E7. n.d – not determined.</p