Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma

Abstract

High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those that persisted and recurred. Viral oncogene alternate transcripts were assessed, and cellular sites of viral integration were mapped and sequenced. Effects of integration on gene expression were assessed by transcript analysis at the integration sites. All of the tumors demonstrated active viral oncogenesis, indicated by expression of HPV E6 and E7 oncogenes and alternate E6 splicing. In the responsive tumors, HPV integration occurred exclusively in intergenic chromosome regions, except for one tumor with viral integration into TP63. Each recurrent tumor exhibited complex HPV integration patterns into cancer-associated genes, including TNFRSF13B, SCN2A, SH2B1, UBE2V2, SMOC1, NFIA, and SEMA6D Disrupted cellular transcripts were identified in the region of integration in four of the seven affected genes.

Implications: Integration of transcriptionally active hrHPV into cellular intergenic regions associates with tumor behavior by altering gene expression. Mol Cancer Res; 14(10); 941-52. ©2016 AACR.

Figure 1

HPV Oncogene Transcript-Specific Quantitative RT-PCR and E6–E7 RT-PCR in HPV16-Positive Responsive Tumors. Bar graphs represent TaqMan quantitative PCR relative expression, and electrophoretic gel images represent E6–E7 RT-PCR. Panel A. RT-PCR Strategy for Transcript-Specific E6–E7 Oncogene Evaluation. Panel B. TaqMan Quantitative RT-PCR Strategy for Transcript-Specific E6 Oncogene Evaluation. Primers within the spice region or across splice junctions allow for exclusive amplification of full length E6 or alternate E6 transcripts. Panel C. 1733, Panel D. 1769, Panel E. 1804, Panel F. 1971, Panel G. 2148. Arrows indicate sizes of expected amplicon bands: HPVE6 FullLength_E7=499bp, HPVE6*I_E7= 454bp, and HPVE6*II_E7= 338bp. NO RT=no reverse transcriptase negative control. M= 100bp ladder.

Figure 2

HPV Oncogene Transcript-Specific Quantitative RT-PCR and E6–E7 RT-PCR in HPV16-Positive Recurrent Tumors. Bar graphs represent TaqMan quantitative PCR relative expression, and electrophoretic gel images represent E6–E7 RT-PCR. Panel A. 0732, Panel B. 0843, Panel C. 1040, Panel D. 2049, Panel E. 2238. Arrows indicate sizes of expected amplicon bands: HPVE6 FullLength_E7=499bp, HPVE6*I_E7= 454bp, and HPVE6*II_E7= 338bp. NO RT=no reverse transcriptase negative control. M= 100bp ladder.

Figure 3

Schematic Representation of Integration Events in HPV16-Positive Responsive and Recurrent Tumors. Panel A. Linear organization of the HPV genome, Panel B. 1733, Panel C. 1769, Panel D. 1804, Panel E. 1971, Panel F. 2148, Panel G. 2049, Panel H. 0843, Panel I. 2238, Panel J. 0732, Panel K. 1040. Closed arrow direction indicates orientation of genes. Solid black lines represent HPV, Solid gray lines represent cellular regions, Dashed black arrows represent viral-specific primers, Dashed gray arrows represent the adapter-specific primer.

Figure 4

Schematic Representation of Transcript Analysis of Integration Events in HPV16-Positive Responsive and Recurrent Tumors. Panel A. Tumor 1971, Panel B. Tumor 2049, Panel C. 0843, Panel D. 2238, Panel E. 0732, Panel F. 1040. Solid black lines represent HPV, Solid gray lines represent cellular regions, Double black lines represent nonsense (NS) sequence, Dashed black arrows represent viral-specific primers, Dashed gray arrows represent the cellular exon-specific primers, and White circles represent exon-exon boundaries. X= no transcript was produced across the indicated sequences.