Comparing serum protein levels can aid in differentiating HPV-negative and -positive oropharyngeal squamous cell carcinoma patients

Abstract

Background: The surrogate immunohistochemical marker, p16INK4a, is used in clinical practice to determine the high-risk human papillomavirus (HPV) status of oropharyngeal squamous cell carcinomas (OPSCC). With a specificity of 83%, this will misclassify some patients compared with direct HPV testing. Patients who are p16INK4a-positive but HPV DNA-negative, or RNA-negative, may be unsuitable for treatment de-escalation aimed at reducing treatment-related side effects. We aimed to identify cost-effective serum markers to improve decision making for patients at risk of misclassification by p16INK4a alone.

Methods: Serum proteins from pre-treatment samples of 36 patients with OPSCC were identified and quantified using label-free mass spectrometry-based proteomics. HPV-status was determined using p16INK4a/HPV DNA and E6/E7 mRNA. Serum protein expressions were compared between groups of patients according to HPV status, using the unpaired t-test with a Benjamini-Hochberg correction. ROC curves (AUC) were calculated with SPSS (v25).

Results: Of 174 serum proteins identified, complement component C7 (C7), apolipoprotein F (ApoF) and galectin-3-Binding Protein (LGALS3BP) significantly differed between HPV-positive and -negative tumors (AUC ranging from 0.84-0.87). ApoF levels were more than twice as high in the E6/E7 mRNA HPV-positive group than HPV-negative.

Conclusions: Serum C7, ApoF and LGALS3BP levels discriminate between HPV-positive and HPV-negative OPSCC. Further studies are needed to validate these host immunity-related proteins as markers for HPV-associated OPSCC.

Fig 1. Workflow.

Serum samples and FFPE samples were analysed from patients with OPSCC. 1. Serum proteins were identified and quantified using nano ultra-performance liquid chromatography—ultra definition mass spectrometry. 2. From formalin-fixed paraffin-embedded samples, HPV-status was determined using the following methods: E6/E7 mRNA was detected using RNAScope, to detect transcriptionally active HPV; p16 was detected using p16INK immunohistochemistry; HPV DNA was detected using PCR. 2A shows a representative TMA spot positive for E6/E7 mRNA; 2B shows a representative TMA spot negative for E6/E7 mRNA; 2C shows the positive PPIB probe for RNA analysis; 2D shows DAPB negative probe for the RNA analysis; 2E shows a representative p16 positive FFPE slide; 2F shows a representative p16 negative FFPE slide. Magnification is 100x and 200x. 3. Combining the serum proteomic data with the determined HPV-status, we compared the protein abundance between the groups.