Viral Integration Plays a Minor Role in the Development and Prognostication of Oral Squamous Cell Carcinoma

Abstract

Viruses are well known drivers of several human malignancies. A causative factor for oral cavity squamous cell carcinoma (OSCC) in patients with limited exposure to traditional risk factors, including tobacco use, is yet to be identified. Our study aimed to comprehensively evaluate the role of viral drivers in OSCC patients with low cumulative exposure to traditional risk factors. Patients under 50 years of age with OSCC, defined using strict anatomic criteria were selected for WGS. The WGS data was interrogated using viral detection tools (Kraken 2 and BLASTN), together examining >700,000 viruses. The findings were further verified using tissue microarrays of OSCC samples using both immunohistochemistry and RNA in situ hybridisation (ISH). 28 patients underwent WGS and comprehensive viral profiling. One 49-year-old male patient with OSCC of the hard palate demonstrated HPV35 integration. 657 cases of OSCC were then evaluated for the presence of HPV integration through immunohistochemistry for p16 and HPV RNA ISH. HPV integration was seen in 8 (1.2%) patients, all middle-aged men with predominant floor of mouth involvement. In summary, a wide-ranging interrogation of >700,000 viruses using OSCC WGS data showed HPV integration in a minority of male OSCC patients and did not carry any prognostic significance.

Keywords: human papillomavirus (HPV); oral squamous cell carcinoma; viruses.

Figure 1

Schematic diagram demonstrating oral cavity anatomy. Oral cavity is depicted in red and oropharynx in blue.

Figure 2

Integrated Genome Viewer image of HPV integration in OSCC tumour genomic sample. A black arrow demonstrates a genomic breakend of an HPV viral insertion.

Figure 3

Mutational signatures (COSMIC3) of patient cohort (n = 28). The HPV-positive patient (patient 28 with HPV35) demonstrates high proportions of SBS2 and SBS13.

Figure 4

Frequent single nucleotide variants (SNVs) in patient cohort.

Figure 5

(A) Haematoxylin and eosin (H&E) stained sections of OSCC with HPV integration. There are expanded nests of keratinocytes with scanty cytoplasm and angulated hyperchromatic nuclei. Comedonecrosis is seen. There is scanty keratinization at the periphery of the nests (magnification ×400); (B) p16 immunohistochemical staining, showing strong, cytoplasmic and nuclear block-like positive staining in >75% of the cells (magnification ×400); (C) HPV RNA ISH demonstrating punctate tumour cell staining (magnification ×400); (D) H&E stained section of OSCC with weak/patchy p16 immunohistochemical staining (magnification ×400); (E) p16 immunohistochemical staining with patchy weak p16 staining considered negative for p16 (magnification ×400); (F) HPV RNA ISH demonstrating lack of HPV integration (magnification ×400).

Figure 6

(A) Overall survival in OSCC by p16 status; (B) disease free survival by p16 status; (C) Overall survival in OSCC by HPV status; (D) disease free survival by HPV status; (E) Kaplan–Meier survival curves by radiotherapy and p16 status in OSCC patients.