Viral Integration Analysis Reveals Likely Common Clonal Origin of Bilateral HPV16-Positive, p16-Positive Tonsil Tumors

Abstract

Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number of oropharyngeal squamous cell carcinoma (OPSCC), with infection rates currently rising at epidemic rates in the western world. Synchronous bilateral HPV+ tumors of both tonsils are a very rare event whose understanding, however, could provide important insights into virus-driven tumor development and progression and whether such integration events are of clonal origin. In this study we analyzed a single case of a bilateral tonsillar p16+ HPV+OPSCC. The viral integration status of the various tumor samples was determined by integration-specific PCR methods and sequencing, which identified viral insertion sites and affected host genes. Integration events were further confirmed by transcript analysis. Analysis of the tumors revealed common viral integration events involving the CD36 gene, as well as a unique event in the LAMA3 gene which resulted in loss of LAMA3 exon one in both tissues that had lost the complex viral LAMA3 integration event. In addition, there were several integration events into intergenic regions. This suggests a common origin but individual evolution of the tumors, supporting the single-clone hypothesis of bilateral tumor development. This hypothesis is further supported by the fact that the two cellular genes LAMA3 and CD36 as targets of viral integration are involved in cell migration and ECM-receptor interactions, which provides a possible mechanism for clonal migration from one tonsil to another.

Association of human papillomavirus integration with better patient outcomes in oropharyngeal squamous cell carcinoma

“…Viral‐host fusion transcripts have been reported by other groups to increase E6/E7 expression 18‐20 . Previous work from our group has shown that viral‐cellular fusion transcripts may or may not form depending on the location of the integration site within the gene (within an intron vs exon) 20,30,43 . It is possible that some integrations within introns are spliced out and therefore do not produce a fusion transcript, while others may alter splice acceptor/donor sites such that they are retained at the transcript level.…”

“…[18][19][20] Previous work from our group has shown that viral-cellular fusion transcripts may or may not form depending on the location of the integration site within the gene (within an intron vs exon). 20,30,43 It is possible that some integrations within introns are spliced out and therefore do not produce a fusion transcript, while others may alter splice acceptor/donor sites such that they are retained at the transcript level. We attempted to amplify the predicted fusion transcripts based on the DNA-level information we obtained from DIPS-PCR in six samples but were only successful in amplifying the fusion in one sample (BMT-1159).…”

Causes and Consequences of HPV Integration in Head and Neck Squamous Cell Carcinomas: State of the Art

“…An interesting finding using HPV integration detection for studying the clonal relationship between bilaterally developing TSCCs was reported by Pinatti et al [68]. In a case study, six integration events were detected by DIPS-PCR, including two intragenic events in the genes CD36, involved in fatty acid import and LAMA3, involved in cell adhesion, migration and differentiation of keratinocytes.…”

“…Both TRAF3 and CYLD play a role in both negatively regulating NF-κB canonical and noncanonical pathways while simultaneously stimulating a potent and first-line antiviral response through type I IFN signaling. Mutations in these genes will therefore lead to constitutive activation of NF-κB, which promotes cell survival and an impaired innate immunity against viral infections [68,95,96]. Moreover, it is suggested that maintenance of episomal HPV even pressures cells to mutate TRAF3/CYLD.…”

Synchronous tonsillar tumors with differing histopathology: A case report and review of the literature

“…In contrast to the two distinct karyotypes seen in our patient, Pinatti et al suggested a common origin of a tumor in their case report on an HPV+ p16+ bilateral TSCC. 7 Through analysis of HPV integration events, the authors ultimately supported single‐clone hypothesis of bilateral tumor development, although, again, no differing histopathological specimens were noted between tonsils. More generally, all three hypotheses of bilateral tonsillar carcinoma origin have validity and the weight of their respective contribution varies on a case‐by‐case basis.…”