Molecular mechanisms of perineural spread of cutaneous carcinoma: a potential role for receptor tyrosine kinases and other biomarkers

Abstract

Perineural invasion and spread of cutaneous squamous cell carcinoma of the head and neck (cSCCHN) is associated with worse prognosis and high rates of locoregional recurrence. It occurs in less than 5% of cases, but given the high incidence of cSCC in Australia, portends significant morbidity and mortality. Primary tumour biology remains poorly understood and precise molecular mechanisms by which malignant squamous cells invade and progress axially within the perineural space remain unclear. Thus, there is no targeted therapy for perineural spread of cSCCHN or other neurotropic malignancies. Dysregulation of cell membrane receptor trafficking is a hallmark of cancer and such receptors are potential therapeutic targets. Over-expression of the epidermal growth factor receptor (EGFR) is well described in squamous cell carcinoma and the monoclonal antibody cetuximab is approved for advanced mucosal head and neck SCC. Other members of the ErbB family (HER2, ErbB3 and ErbB4) are less well understood in SCC. The erythropoietin-producing hepatocellular carcinoma (Eph) family of RTKs is similarly dysregulated in a wide range of cancers. Given their physiological roles in neural development and skin homeostasis, these receptors represent other potential therapeutic targets in perineural spread. The aim of this thesis was to investigate the expression and cellular distribution of molecular receptors involved in perineural spread, particularly those with targeted therapies in development or clinical use for other indications. Four SCC cell lines and one human keratinocyte line were examined by immunoblotting and immunofluorescence. Human tissue specimens of perineural spread from cSCCHN were collected over an 18-month period and immunostained for EGFR, HER2 and other cancer biomarkers. Ki-67 proliferation index was examined for the first time as a potential prognostic factor and marker of aggressiveness in perineural spread. Cell cycle regulators p16 and p53 were examined as novel biomarkers and to shed light on possible non-RTK dependent mechanisms implicated in the disease process. The work with p53 builds on recent data published by our collaborators suggesting dysregulation of the p53 pathway in clinical perineural invasion. P16 is an established prognostic factor and surrogate marker for carcinogenic viral infection in oropharyngeal SCC not previously investigated in perineural spread cSCCHN. To our knowledge, this is the first study to demonstrate over-expression of the EGFR in perineural spread cSCCHN, in 90% (n=18/20) cases evaluated. This suggests a potential mechanistic role and reveals a valuable therapeutic target. Moreover, half of these cases had strong plasma membrane expression in the absence of cytoplasmic labelling, consistent with dysregulation of receptor trafficking and RTK escape, a new hallmark of cancer. Conversely, perineural spread specimens were uniformly HER2 negative effectively ruling this out as a therapeutic target. These findings will have implications for treatment of advanced cSCCHN with perineural spread of carcinoma. Patients with recalcitrant and/or resistant disease could be offered existing anti-EGFR therapies or enrolled in clinical trials testing novel or combination therapeutics. Ultimately, we aim to improve survival and quality of life for sufferers of this morbid form of tumour spread. Our findings may also have implications for other neurotropic malignancy, including pancreatic, gastric, colorectal and prostate cancers.