Mutational landscape of aggressive cutaneous squamous cell carcinoma

Abstract

Purpose: Aggressive cutaneous squamous cell carcinoma (cSCC) is often a disfiguring and lethal disease. Very little is currently known about the mutations that drive aggressive cSCC.

Experimental design: Whole-exome sequencing was performed on 39 cases of aggressive cSCC to identify driver genes and novel therapeutic targets. Significantly, mutated genes were identified with MutSig or complementary methods developed to specifically identify candidate tumor suppressors based upon their inactivating mutation bias.

Results: Despite the very high-mutational background caused by UV exposure, 23 candidate drivers were identified, including the well-known cancer-associated genes TP53, CDKN2A, NOTCH1, AJUBA, HRAS, CASP8, FAT1, and KMT2C (MLL3). Three novel candidate tumor suppressors with putative links to cancer or differentiation, NOTCH2, PARD3, and RASA1, were also identified as possible drivers in cSCC. KMT2C mutations were associated with poor outcome and increased bone invasion.

Conclusions: The mutational spectrum of cSCC is similar to that of head and neck squamous cell carcinoma and dominated by tumor-suppressor genes. These results improve the foundation for understanding this disease and should aid in identifying and treating aggressive cSCC.

Figure 1

Aggressive features of cSCC. Aggressive features used to define this cohort are shown in red. Features that result in up-staging by AJCC criteria are shown in blue.

Figure 2

Mutation frequency and types in cSCC. A) Mutation frequency in cSCC compared to other tumor types. Median value is shown and indicated by the horizontal line. Non-cSCC samples are TCGA data from Lawrence et.al. (24). B) Mutation types in cSCC compared to other tumor types. Mutation type frequencies were calculated for each sample and then averaged across the cohort to eliminate bias from highly mutated samples. HNSCC and LUSC are TCGA data from Kandoth et.al. (20) Melanoma data are from Hodis et.al. (14) Abbreviations: HNSCC – head and neck squamous cell carcinoma, LUSC – lung squamous cell carcinoma, SKCM – skin cutaneous melanoma, cSCC – cutaneous squamous cell carcinoma, DNP – dinucleotide polymorphism.

Figure 3

Key mutations in cSCC. Total number of mutations per patient is shown on the top. Inactivating mutations include nonsense, frame-shift, and splice site events. Mutation frequency for each gene is shown on the right.

Figure 4

Mutations in NOTCH1 and NOTCH2 appear to be inactivating in cSCC. A) The spectrum and location of NOTCH1 mutations observed in cSCC, HNSCC (TCGA data), are compared to T-cell acute lymphoblastic leukemia (COSMIC data) where NOTCH1 alterations are instead activating. In both cSCC and HNSCC, most missense mutations cluster in the N-terminal EGF-like repeats where ligand binding occurs, and truncating mutations are distributed throughout the gene; whereas, in leukemia truncating mutations are confined to the C-terminal PEST domain responsible for degradation of activated intracellular NOTCH1 and missense mutations cluster in the heterodimerization domain where they cause ligand-indpendent activation. B) NOTCH2 alterations in cSCC and HNSCC have a similar pattern because missense mutations are clustered in the EGF-like domains and truncating mutations are scattered throughout the gene, suggesting the alterations inactivate function. Red dots indicate truncating mutations (splice, frameshift, or nonsense), green dots indicate missense mutations, black dots indicate inframe insertions or deletions, and purple dots indicate residues affected by different mutation types. NOTCH domains are indicated with different colors. Light green = EGF-like repeat, dark green = calcium binding EGF domain, yellow = LNR (negative regulatory) repeat, blue = heterodimerization domain (HD), orange = transmembrane (TM) region, pink = RAM domain, purple = ankyrin repeats, blue = transactivation domain (TAD), and red = Proline-Glutamic acid-Serine-Threonine rich (PEST) domain.

Figure 5

Mutation status and clinical parameters. A) Association between mutation status and various clinical parameters. B) KMT2C mutation is associated with shorter time to recurrence. C) KMT2C mutation is associated with worse recurrence-free survival. Censored events are indicated by a vertical bar.