Association of an intact <i>E2</i> gene with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome in HPV16 positive head and neck squamous cell carcinoma

<div><p>To assess the relationship of <i>E2</i> gene disruption with viral gene expression and clinical outcome in human papillomavirus (HPV) positive head and neck squamous cell carcinoma, we evaluated 31 oropharyngeal and 17 non-oropharyngeal HPV16 positive carcinomas using two PCR-based methods to test for disruption of <i>E2</i>, followed by Sanger sequencing. Expression of HPV16 <i>E6</i>, <i>E7</i> and <i>E2</i> transcripts, along with cellular <i>ARF</i> and <i>INK4A</i>, were also assessed by RT-qPCR. Associations between <i>E2</i> disruption, E2/E6/E7 expression, and clinical outcome were evaluated by Kaplan-Meier analysis for loco-regional recurrence and disease-specific survival. The majority (n = 21, 68%) of HPV16 positive oropharyngeal carcinomas had an intact <i>E2</i> gene, whereas the majority of HPV16 positive non-oropharyngeal carcinomas (n = 10, 59%) had a disrupted <i>E2</i> gene. Three of the oropharyngeal tumors and two of the non-oropharyngeal tumors had deletions within <i>E2</i>. Detection of an intact <i>E2</i> gene was associated with a higher DNA viral load and increased <i>E2/E6/E7</i>, <i>ARF</i> and <i>INK4A</i> expression in oropharyngeal tumors. Oropharyngeal carcinomas with an intact <i>E2</i> had a lower risk of loco-regional recurrence (log-rank p = 0.04) and improved disease-specific survival (p = 0.03) compared to tumors with disrupted <i>E2</i>. In addition, high E7 expression was associated with lower risk of loco-regional recurrence (p = 0.004) as was high E6 expression (p = 0.006). In summary, an intact <i>E2</i> gene is more common in HPV16 positive oropharyngeal than non-oropharyngeal carcinomas; the presence of an intact <i>E2</i> gene is associated with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome compared to patients with a disrupted <i>E2</i> gene in oropharyngeal cancer.</p></div

Study population characteristics stratified by HPV16 <i>E2</i> gene status and tumor site.

<p>Study population characteristics stratified by HPV16 <i>E2</i> gene status and tumor site.</p

HPV16 E6, E7 and E2 are overexpressed in OPSCCs with an intact <i>E2</i>.

<p>HPV16 E6 (A) and HPV16 E7 (B) expression with HPV16 <i>E2</i> gene disruption. Expression is defined as Y = 2^{maxΔCT- ΔCT} where maxΔCT is the lower limit threshold of detection which equals 20 or 1. ΔCT is the experimental value minus the endogenous control (GAPDH). Statistical significance for difference in HPVE6 and E7 expression by site and between samples with an intact or disrupted HPV16 <i>E2</i> gene was assessed by Mann-Whitney test.</p

Positions of HPV16 E2 primers along the HPV16 E2 gene.

<p>The HPV16 genome encompassing the <i>E2</i> gene region shown in gray. Primer locations of the <i>E2</i> disruption assay and the <i>E2</i> full gene primer set are shown above and numbered according to nucleotides on the whole genome. Overlapping primer design is illustrated below along with product size primer set.</p

Association of local-regional recurrence and disease specific survival with HPV16 <i>E2</i> disruption in the oropharynx.

<p>(A) Samples with an intact <i>E2</i> gene (broken black line) have a lower incidence of local regional recurrence than oropharyngeal cases with a disrupted <i>E2</i> gene (solid black line) (Mantel-Cox test p = 0.04). (B) Samples with an intact <i>E2</i> gene (broken black line) have better disease specific survival than oropharyngeal cases with a disrupted <i>E2</i> gene (solid black line) (Mantel-Cox test p = 0.03). (C and D) Samples were separated into high (broken black) or low (solid black) expression of viral genes based on median cutoff using derived (2maxΔCT- ΔCT) qRT-PCR results compared to GAPDH for: (C) HPV16 E7 (median = 1.69 2maxΔCT- ΔCT; Mantel-Cox test p = 0.004), and (D) HPV16 E6 (median = 1.23 2maxΔCT- ΔCT; Mantel-Cox test p = 0.006).</p