Molecular discrimination of cutaneous squamous cell carcinoma from actinic keratosis and normal skin

Abstract

Actinic keratosis is widely believed to be a neoplastic lesion and a precursor to invasive squamous cell carcinoma. However, there has been some debate as to whether actinic keratosis is in fact actually squamous cell carcinoma and should be treated as such. As the clinical management and prognosis of patients is widely held to be different for each of these lesions, our goal was to identify unique gene signatures using DNA microarrays to discriminate among normal skin, actinic keratosis, and squamous cell carcinoma, and examine the molecular pathways of carcinogenesis involved in the progression from normal skin to squamous cell carcinoma. Formalin-fixed and paraffin-embedded blocks of skin: five normal skins (pooled), six actinic keratoses, and six squamous cell carcinomas were retrieved. The RNA was extracted and amplified. The labeled targets were hybridized to the Affymetrix human U133plus2.0 array and the acquisition and initial quantification of array images were performed using the GCOS (Affymetrix). The subsequent data analyses were performed using DNA-Chip Analyzer and Partek Genomic Suite 6.4. Significant differential gene expression (42 fold change, Po0.05) was seen with 382 differentially expressed genes between squamous cell carcinoma and normal skin, 423 differentially expressed genes between actinic keratosis and normal skin, and 9 differentially expressed genes between actinic keratosis and squamous cell carcinoma. The differentially expressed genes offer the possibility of using DNA microarrays as a molecular diagnostic tool to distinguish between normal skin, actinic keratosis, and squamous cell carcinoma. In addition, the differentially expressed genes and their molecular pathways could be potentially used as prognostic markers or targets for future therapeutic innovations.

Keratoacanthoma and squamous cell carcinoma are distinct from a molecular perspective

“…The ten cases of squamous cell carcinoma that were used for the comparison were previously reported. 8,9 They consisted of well-to-moderately differentiated squamous cell carcinoma arising from skin showing actinic damage with solar elastosis and/or actinic keratosis. The ten cases of keratoacanthoma included sporadic keratoacanthomas and BRAF inhibitor-induced keratoacanthomas.…”

“…10 Methods of RNA isolation, quality control, target preparation and microarray hybridization, and data analysis are described below and in our previous studies. 8,9 RNA Isolation and Quality Control Total RNA was isolated using the Ambion RecoverAll (Applied Bio systems/Ambion, Austin, TX, USA) kit according to the manufacturer's instructions. Briefly, formalin-fixed and paraffin-embedded samples were deparaffinized by using a series of xylene and ethanol washes and then subjected to a proteinase K digestion at 50 1C for 16 h to release RNA from covalently linked proteins.…”

WNT Signaling in Cutaneous Squamous Cell Carcinoma: A Future Treatment Strategy?

“…Direct evidence of this has subsequently been found by a number of gene expression array analyses in cSCC samples, which have identified increases in the mRNA levels of WNT ligands and their receptors, and in some samples also the down-regulation of endogenous secreted WNT inhibitors such as secreted FZD-related proteins (SFRPs) that antagonise WNT-FZD interactions (Haider, et al 2006;Ra, et al 2011;Watt, et al 2011). For example, Haider and colleagues found that WNT5A and FZD6 were both upregulated in cSCC (Haider, et al 2006), whilst a study by Ra et al identified the WNT signaling network as the most significantly enriched set of molecular pathways in gene expression comparisons between cSCC and normal skin (Ra, et al 2011). Evidence for enhanced β-catenin signaling in cSCC comes from a number of immunohistochemical staining studies on cSCC tumors, looking at β-catenin levels in tumours.…”

“…One of the earliest studies using comparative genomic hybridization found frequent amplification of chromosomes 7q, 8q, 11q and 17q in cSCC lines, which all contain WNT and/or FZD genes, suggesting that expression of these genes may 7 be increased in cSCC (Popp, et al 2002). Direct evidence of this has subsequently been found by a number of gene expression array analyses in cSCC samples, which have identified increases in the mRNA levels of WNT ligands and their receptors, and in some samples also the down-regulation of endogenous secreted WNT inhibitors such as secreted FZD-related proteins (SFRPs) that antagonise WNT-FZD interactions (Haider, et al 2006;Ra, et al 2011;Watt, et al 2011). For example, Haider and colleagues found that WNT5A and FZD6 were both upregulated in cSCC (Haider, et al 2006), whilst a study by Ra et al identified the WNT signaling network as the most significantly enriched set of molecular pathways in gene expression comparisons between cSCC and normal skin (Ra, et al 2011).…”

“…To progress this aim, expression array profiling of cSCC tumours by our group and others has been used to identify the most dysregulated molecular pathways. Such studies have identified WNT signaling as significantly altered in cSCC (Haider, et al 2006;Ra, et al 2011;Watt, et al 2011). Functional evidence also exists for a role of WNT signaling in cSCC and here we discuss these findings.…”