LAUSR

Tangible therapeutic advances for aggressive and potentially lethal forms of cutaneous squamous cell carcinoma (cSCC), one of the most common forms of cancer, are currently lacking. As our aging population continues to suffer from its relentless increase in incidence, defining the molecular events that drive cSCC, and importantly the context in which those events occur, clearly represent an urgent research need. Our recent data provides compelling support for the proposed tumor suppressor role of TGF-b signaling in skin tumourigenesis, as well as contextual insight into how loss of TGF-b signaling, when targeted to specific skin stem cell compartments, permits rapid tumor formation. In essence, evidence of a key tumor suppressor role for TGFb signaling in human squamo-proliferative disease and invasive cSCC already exists. Convincing reports include the genotypephenotype correlation between inactivating TGFBR1 mutation and familial multiple self-healing squamous epithelioma (MSSE), as well as reports of spontaneous cSCC arising secondary to systemic treatment with the pan-TGF-b ligand antibody (GC1008). In addition, TGF-b receptor mutations have also been detected in RAF-kinase induced skin tumors; which is where our study began. We initially performed targeted deep sequencing of 39 squamo-proliferative lesions from patients treated with vemurafenib (a BRAF inhibitor used to treat advanced melanoma) and identified mutations of both TGF-b receptors (TGFBR1 and TGFBR2) in 28% of lesions. Next, using the same targeted sequencing profile, we interrogated 91 sporadic human cSCC and 21 human primary cSCC cell lines and detected mutations of both TGF-b receptors in 43% of samples. Crucially, normal blood samples as well as matched normal distant and perilesional skin controls harboured no TGF-b receptor mutations, indicating that these mutations appeared to be lesion-specific non-germline events. Consolidating these findings, we detected a similar high frequency of TGF-b receptor mutations in a separate cohort of 30 cSCC analyzed by whole exome sequencing (WES) – with alterations in a total of 53% of samples analyzed using this platform with no mutations in matched normal samples. Factors such as varying efficiencies in deep sequencing techniques, an exceptionally high mutational burden of cSCC tumors (producing significant background passenger mutation rates) and tumor heterogeneity all contribute to the challenge of confirming driver gene status in cSCC. A robust analytical approach combining computational and statistical models, a functional in-vitro analysis of detected mutations and goldstandard in-vivo mouse models, not only provided substantial evidence to suggest these mutations were indeed driver mutations, but also the novel finding that Tgfbr1 deletion appears to drive tumourigenesis when specifically targeted to the bulge stem cell compartment of the hair follicle. A stringent combination of analytical software programs was used to predict the functional consequence of mutation. This approach predicted that 50% of TGFBR1 and 70% of detected TGFBR2 mutations were likely to be functionally damaging. The functional consequence of mutations detected by WES was further assessed by MutsigCV and IntOgen algorithms. Although these algorithms failed to detect TGFBR1 or TGFBR2 mutations as significant on an individual basis, it was apparent that this approach failed to account for the potential bias of detecting mutually exclusive mutations that may disrupt signaling pathways. IntOgen analysis also predicts the significance of mutation in signaling pathways and confirmed that the TGF-b signaling pathway was significantly mutated. In addition, analysis of variant allelic frequencies (VAF) demonstrated a significant proportion of tumors containing TGF-b receptor mutations exhibited the highest VAF’s in those mutations compare with VAF’s of other common drivers of disease, suggesting they were more likely to be initiating events. In strong supporting this hypothesis, clonal analysis of the WES data using ABSOLUTE predicted that 7 of the 8 TGF-b receptor mutations were indeed clonal and therefore likely to represent driver events. Functional evidence out-strips prediction programmes and algorithms. In-vitro TGF-b reporter gene analysis of a panel of TGF-b receptor mutants demonstrated a significant proportion of TGFBR1 mutants and all TGFBR2 mutants tested failed to restore active TGF-b signaling. In addition, restoring wild-type TGFBR2 receptor to TGFBR2null cSCC cells restored growth arrest. Taken together, these findings provide convincing evidence that loss of TGF-b tumor suppressor function is a common event in cSCC.