Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is significantly represented in the military population with substantial impact on military operations. Two-thirds of new HNSCC cases are first diagnosed at advanced stage III or IV with lymph node metastases. Thus, there is an urgent need of biomarkers for early detection and risk stratification. Archived serum samples from the Department of Defense Serum Repository provide a unique resource for longitudinal studies of HNSCC in the military and the identification of early detection biomarkers. Methods: We identified 175 patients diagnosed with HNSCC with 175 matched healthy controls and retrieved a total of 978 serum samples drawn at the time of diagnosis, 2 and 4 years prior to diagnosis, and 2 years after diagnosis. Following immunoaffinity depletion, serum samples were analyzed by targeted proteomics assays for multiplexed quantification of a panel of 146 candidate protein biomarkers from the curated literature. Results: We derived a 13-protein signature that distinguishes case versus controls based on longitudinal changes in serum protein concentration. The abundances of each of the 13 proteins remain constant over time in control subjects. The area under the curve for the derived random forest classifier was 0.90. Conclusions: This 13-protein classifier is highly promising for detection of HNSCC prior to overt symptoms. Impact: Use of longitudinal samples has significant potential to identify biomarkers for detection and risk stratification.

Candidate targets were selected by searching PubMed for publications containing the keywords ‘biomarkers, HNSCC, oral cancer, oropharyngeal cancer, head and neck cancer’ and curating the results using a genescraper R package10. A total of 277 candidate biomarkers associated with either HNSCC specifically or identified as general cancer-associated proteins were identified in the curated literature. Clinical experts in oral cancer reviewed the list, and down-selected candidates in a stepwise workflow (including MS detectability of the proteins in serum) until a final panel of 146 candidate protein biomarkers was selected for targeted proteomics analysis in the large cohort of DODSR samples using liquid chromatography coupled to selective reaction monitoring (LC-SRM).

DODSR serum specimens All DODSR serum samples used in this study were collected between 2003 and 2013. We identified 175 cases who developed HNSCC while on active duty, and analyzed at least two and up to four serum samples from each case: ‘Post1’, specimen collected closest to and within 365 days after the diagnosis; ‘Pre4’, four years prior to diagnosis; ‘Pre2’, two years prior to diagnosis; and ‘Post2’, two years after diagnosis. Corresponding samples were also obtained from 175 healthy controls matched by age at the time of diagnosis and gender. In total there were 978 serum samples. The average patient age was 45 (ranging from 21 to 64) with 169 males and 6 females. Diagnoses were limited to ICD-O codes corresponding to oropharyngeal primary tumors, irrespective of HPV status.