DRH1 - a novel blood-based HPV tumour marker

Abstract

Background: To date, no studies have successfully shown that a highly specific, blood-based tumour marker to detect clinically relevant HPV-induced disease could be used for screening, monitoring therapy response or early detection of recurrence. This study aims to assess the clinical performance of a newly developed HPV16-L1 DRH1 epitope-specific serological assay.

Methods: In a multi-centre study sera of 1486 patients (301 Head and Neck Squamous Cell Carcinoma (HNSCC) patients, 12 HIV+ anal cancer patients, 80 HIV-positive patients, 29 Gardasil-9-vaccinees, 1064 healthy controls) were tested for human HPV16-L1 DRH1 antibodies. Analytical specificity was determined using WHO reference-sera for HPV16/18 and 29 pre- and post-immune sera of Gardasil-9-vaccinees. Tumour-tissue was immunochemically stained for HPV-L1-capsidprotein-expression.

Findings: The DRH1-competitive-serological-assay showed a sensitivity of 95% (95% CI, 77^.2-99^.9%) for HPV16-driven HNSCC, and 90% (95% CI, 55^.5-99^.7%) for HPV16-induced anal cancer in HIV-positives. Overall diagnostic specificity was 99^.46% for men and 99^.29% for women ≥ 30 years. After vaccination, antibody level increased from average 364 ng/ml to 37,500 ng/ml. During post-therapy-monitoring, HNSCC patients showing an antibody decrease in the range of 30-100% lived disease free over a period of up to 26 months. The increase of antibodies from 2750 to 12,000 ng/ml mirrored recurrent disease. We can also show that the L1-capsidprotein is expressed in HPV16-DNA positive tumour-tissue.

Interpretation: HPV16-L1 DRH1 epitope-specific antibodies are linked to HPV16-induced malignant disease. As post-treatment biomarker, the assay allows independent post-therapy monitoring as well as early diagnosis of tumour recurrence. An AUC of 0^.96 indicates high sensitivity and specificity for early detection of HPV16-induced disease.

Funding: The manufacturer provided assays free of charge.

Keywords: Antibodies; Blood test; HNSCC; HPV16; Screening; Tumour marker.

Fig. 1

Remark Diagram of study population and patient subsets used for calculation of sensitivity, specificity, positive predictive value, negative predictive value, ROC- and Area under the curve calculation.

Fig. 2

a–d. Characteristic antibody graphs during follow up. Fig. 2a: Classical antibody decrease during follow up, indicating a successful treatment understood as successful removal of tumour cells, which is associated with disease free overall surveillance. The black spotted bar at 1000 ng/ml represents the cut off antibody concentration discriminating positive and negative DRH1 test results. Patient characteristics of patient 10 as described in Table 1: Male, 58 year old, carcinoma of the tonsils, HPV16 DNA positive, p16 positive Therapy: Surgery + adjuvant Radiotherapy Decrease of antibody concentration by 90% within 6 months, and 100% within 18 months.

Fig. 2

a–d. Characteristic antibody graphs during follow up. Fig. 2b: Antibody concentration during follow up of patient No 12. Initial antibody decrease during follow up by 43% after 9 months (in green), indicating a successful treatment, was followed by a sudden increase of antibody concentration (in red) 3 months later– soon after tumour recurrence in the lungs was diagnosed. The black spotted bar at 1000 ng/ml represents the cut off antibody concentration discriminating positive and negative DRH1 test results. Patient characteristics of patient 12 as described in Table 1: Male, 81 year old, base of tongue carcinoma, HPV16 DNA positive, p16 positive Therapy: Radioimmunotherapie with Cetuximab *Time of diagnosis of tumour recurrence

Fig. 2

a–d. Characteristic antibody graphs during follow up. Fig. 2c: Characteristical graph in a patient with an HPV33 associated OPSCC showing HPV16 L1 antibody concentration at a constant low level indicating the type-specificity of the assay. The black spotted bar at 1000 ng/ml represents the cut off antibody concentration discriminating positive and negative DRH1 test results. Patient characteristics of patient 9 as described in Table 1: Male, 72 years old, base of tongue carcinoma, HPV33 DNA positive, p16 positive Therapy: Radiochemotherapy

Fig. 2

a–d. Characteristic antibody graphs during follow up. Fig. 2d: Overview of antibody concentrations of all HNSCC patients from Graz during follow up, baseline characteristics can be seen in Table 1. Green curves: HPV16-L1 immunoassay positive, Orange curves: HPV16-L1 immunoassay negative, Red curves: Increasing antibody titers in three HPV16-L1 immunoassay positive patients.

Fig. 3

Receiver-Operating-Characteristic (ROC)–curve analysis, with an ´area under the curve´ of 0^.96 (95% confidence interval 0.91–1), was calculated for 20 HPV16 driven OPSCCs and 1064 controls.

Fig. 4

: HPV16-L1 capsid protein expression in tumour cells As shown in pictures a - d, about half of the tumour cells show a nuclear staining in red colour for the L1 capsid protein. L1 expression was confirmed by Western Blot analysis. Magnification: 100x (a), 200x (b, c), 400x (d) Pictures e and f show an `inner border like´ staining. This means different clusters of tumour cells next to each other being L1 capsid protein positive (on the right) and L1 negative (on the left). Picture f shows a higher magnification of the area marked by a white circle. Magnification: 400x (e) and 1000x (f) The dot like staining of the nuclei (black arrows) was always associated with a sporadic (g) or an `inner border like´ (h) staining. Like shown here, the ´inner border like´ staining showed a sharp border between the L1 positive and the L1 negative cells. Magnification: 1000x each.