Serum antibodies to the HPV16 proteome as biomarkers for head and neck cancer

Abstract

Background: Human papillomavirus (HPV) type 16 is associated with oropharyngeal carcinomas (OPC). Antibodies (Abs) to HPV16 E6 and E7 oncoproteins have been detected in patient sera; however, Abs to other early HPV-derived proteins have not been well explored.

Methods: Antibodies to the HPV16 proteome were quantified using a novel multiplexed bead assay, using C-terminal GST-fusion proteins captured onto Luminex beads. Sera were obtained from untreated patients with OPC (N=40), partners of patients with HPV16+ OPC (N=11), and healthy controls (N=50).

Results: Oropharyngeal carcinomas patients with known virus-like capsid particle+ Abs had elevated serum Abs to HPV16 E1, E2, E4, E6, and E7, and L1 antibody levels, but not E5. The ratios of specific median fluorescence intensity to p21-GST compared with controls were E1: 50.7 vs 2.1; E4: 14.6 vs 1.3; E6: 11.3 vs 2.4; E7: 43.1 vs 2.6; and L1: 10.3 vs 2.6 (each P≤0.01). In a validation cohort, HPV16 E1, E2, and E7 antibody levels were significantly elevated compared with healthy control samples (P≤0.02) and partners of OPC patients (P≤0.01).

Conclusion: Patients with HPV16+ OPC have detectable Abs to E1, E2, and E7 proteins, which are potential biomarkers for HPV-associated OPC.

Figures

Figure 1

HPV bead array ELISA. (A) Schematic of bead array ELISA for the detection of HPV Abs. Individual HPV cDNA's encoding full-length HPV antigens are expressed as C-terminal GST-tagged proteins using reticulocyte lysate. The expressed protein is then captured onto Luminex microspheres via anti-GST antibody that is coupled onto the beads. For IgG detection from sera, beads tagged with HPV antigens are mixed and added to human sera. Bound human IgG is detected with biotin-anti-IgG and streptavidin-PE. (B) GST expression of HPV16 gene products and HPV18 E7. GST expression of N- and C-terminal HPV16 E2 fragments is shown in the right panel. GST protein on the beads was measured by addition of anti-GST-PE antibody as mean fluorescence index (MFI). Background protein expression (vector mean plus 10%) is indicated by the dotted line. (C) Antibodies to HPV16 early gene proteins are detected at >1 : 10 000 dilution in two OPC patient sera. Patient sera were diluted two-fold from 1 : 40 to 1 : 40 960, and IgG Abs to HPV-derived proteins and p21-GST control protein are shown in order of peak MFI signal intensity. Inserts: zoom-in views of (i) and (ii), respectively.

Figure 2

Specific detection of multiple early gene Abs in 10 patients with HPV16 VLP Abs compared with 20 controls. HPV16 proteins, as well as HPV18 E7 and p21 proteins, were expressed and captured on Luminex beads, and the MFI ratio (MFI (HPV)/MFI (p21-GST)) of IgG detected in sera is shown. Training set serum IgG responses were measured in age- and sex-matched healthy controls (‘Controls’), patients with HPV16 VLP Abs (‘HPV16 VLP+’), and patients with HPV18 VLP Abs (‘HPV18 VLP+’). HPV16-specific Abs to E1, E4, E6, E7, and L1 proteins (but not E5 and L2) are detected in HPV16 VLP Ab+ patients compared with controls or patients with HPV18 VLP Abs. Top middle and top right: HPV16 NE2 and CE2 Abs are also detected in cases with HPV18 VLP Abs. Bottom right middle and far right: HPV16 E7 Abs do not cross-react with HPV18 E7.

Figure 3

Specific detection of multiple early gene Abs in untreated OPC patients, but not partners. The MFI ratio of HPV16 proteins to p21 protein detected in sera is shown. The validation set serum IgG responses were measured in an independent set of healthy controls (‘Controls’, N=30), OPC patients (‘Cases’, N=30), and partners of OPC patients (N=11). HPV16-specific Abs to E1, E2, E4, E6, and E7, and L1 proteins are specifically detected in patients compared with controls.

Figure 4

Unsupervised hierarchical clustering of HPV16-specific Abs of validation set patient, partner, and control sera. There is a subset of patients (group I) with multiple HPV-specific Abs, including E1, E2, E6, and E7. Another subset (group II) has E1 and E2, but low or absent E7- and/or E6-specific Abs. There is a subset (group III) of healthy controls with increased (but weak) levels of E6 and L1 Abs.