Next-Generation Sequencing-Based Molecular Profiling of Conjunctival Squamous Cell Carcinoma and Its Potential Application for Therapy

Abstract

Objective: Targeted next-generation sequencing-based genomic and transcriptomic analyses of conjunctival squamous cell carcinoma (cSCC) samples using a panel of >1700 cancer-related genes.

Design: Prospective case series.

Participants: Twenty patients with invasive cSCC consecutively managed at an academic ocular oncology setting.

Methods: Integrative exome and transcriptome analysis of fresh-frozen tumor and matching normal samples.

Main outcome measures: Molecular characterization of invasive cSCCs (for somatic mutations, tumor mutation burden (TMB) and signatures, structural variations, viral transcripts, outlier gene expression) and its potential clinical applications.

Results: Of the 20 invasive cSCCs, only 2 were positive for human papillomavirus (HPV). All 18 HPV-negative tumors had genetic alterations in TP53 and 16 also had alterations in CDKN2A. The 2 HPV-positive tumors showed no alterations in these cell cycle regulators but harbored mutations in PIK3CA. Other frequently altered genes included KMT2C /D (70%), FAT1/3 (65%), and NOTCH1/2/3 (60%), which were implicated in both the HPV-negative and positive tumors. The average TMB was 58.41 mutations per megabase (Mut/Mb). The TMB was >20 Mut/Mb in 13 cases (65%, range: 49.3-160.8 Mut/Mb), of which 11 had ultraviolet (UV) mutational signature, 3 had APOBEC signature, and 2 had microsatellite instability. Most UV-driven tumors (8 of 11) also harbored TERT promoter mutations. The most recurrent large-scale copy number alterations were the deletions affecting chromosomes 3p, 9p, and 14q. Uncommon but potentially drug-targetable copy number gains were also detected affecting several oncogenes. The most frequent gene expression outlier was the aberrantly expressed TP63 found in 19 tumors.

Conclusions: In our invasive cSCC cohort, HPV infection was not a major contributor to tumor etiopathogenesis. Our results confirmed the central role of TP53 genetic alterations and the common presence of UV signature in the HPV-negative cSCCs. Irrespective of HPV status, other commonly observed genetic alterations included those affecting chromatin modifiers followed by Hippo or Notch pathway-related genes. Ultraviolet-driven TERT promoter mutations co-occurred with other driver gene alterations. The commonly observed high TMB makes immunotherapy a good treatment choice for invasive cSCC. Moreover, several cSCC-associated molecular alterations represent potentially actionable targets, while further studies are necessary to understand their roles in cSCC development and invasion.

Financial disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Keywords: Conjunctival squamous cell carcinoma; Copy number alterations; Mutations; Tumor mutation burden; UV signature.

Figure 1

Invasive conjunctival squamous cell carcinoma. A, Slit lamp examination of the left eye showed a nodular amelanotic lesion on the temporal bulbar conjunctiva, which was histopathologically confirmed to be invasive squamous cell carcinoma. B, Ultrasound biomicroscopy showed the conjunctival squamous cell carcinoma had invaded the underlying sclera along the limbus.

Figure 2

The locations of invasive squamous cell carcinomas over the conjunctiva. Red stars: HPV-negative tumors with UV signature; blue stars: HPV-negative tumors without UV signature; green stars: HPV-positive tumors without UV signature (created with BioRender.com). HPV = human papillomavirus; UV = ultraviolet.

Figure 3

Oncoprint for somatic genetic alterations in our cohort of invasive conjunctival squamous cell carcinomas, grouped by HPV status. When there were several mutations in a gene, for the sake of clarity, only 1 type of mutation was selected to depict, prioritized in the following order: stop-gain, frameshift, splice, and missense. A hotspot mutation was defined as one detected in >10 patients in the Catalogue of Somatic Mutations in Cancer (COSMIC). HPV = human papillomavirus; MSI = microsatellite instability; Mut/Mb = mutations per megabase; UV = ultraviolet.

Figure 4

Structural genetic variations in our cohort of invasive conjunctival squamous cell carcinomas. Upper panel: copy number alteration heatmap for gain, loss, and copy neutral loss of heterozygosity (CN-LOH). Lower panel: examples of focal amplification that lead to overexpression.