Application of the hybrid capture 2 assay to squamous cell carcinomas of the head and neck: a convenient liquid-phase approach for the reliable determination of human papillomavirus status

Abstract

Background: A growing proportion of head and neck squamous cell carcinoma (HNSCC) is caused by the human papillomavirus (HPV). In light of the unique natural history and prognosis of HPV-related HNSCCs, routine HPV testing is being incorporated into diagnostic protocols. Accordingly, there is an escalating demand for an optimal detection strategy that is sensitive and specific, transferrable to the diagnostic laboratory, standardized across laboratories, cost-effective, and amenable to broad application across specimen types including cytologic preparations.

Methods: Cytologic preparations (fine-needle aspirates [FNAs] and brushes) were obtained from surgically resected HNSCCs and evaluated for the presence of high-risk HPV using the Hybrid Capture 2 assay. HPV analysis was also performed on the corresponding tissue sections using HPV in situ hybridization and p16 immunohistochemistry. In cases in which the immunohistochemical and in situ hybridization results were discordant, HPV status was determined by real-time polymerase chain reaction detection of E7 expression. HPV status in the tissues and corresponding cytologic samples was compared.

Results: Based on benchmark HPV testing of the tissue sections, 14 HNSCCs were classified as HPV positive and 10 as HPV negative. All corresponding cytologic preparations were correctly classified using the Hybrid Capture 2 assay.

Conclusions: The Hybrid Capture 2 strategy, already widely used for the detection of high-risk HPV in cervical brushes, is readily transferrable to HNSCCs. Consistent accuracy in cytologic preparation suggests its potential application in FNAs from patients who present with lymph node metastases, and may eliminate the need to obtain tissue solely for the purpose of HPV testing.

Figure 1

An HPV-related oropharyngeal squamous cell carcinoma. Cytologic specimens were sent for hybrid capture 2 testing after the presence of tumor was confirmed by a Diff Quick stain (A). Tumor in the corresponding tissue sections (B) was determined to be HPV positive on the basis of strong p16 immunohistochemical staining (C) and the presence of in situ hybridization signals within the tumor nuclei (D).

Figure 2

HPV status by sample scores. For the cytologic preparations from head and neck squamous cell carcinomas, the reference scores of >3.0 and <0.85 (dashed lines) was found to have 100% sensitivity and 100% specificity in differentiating HPV positive (red diamonds) from HPV negative (black diamonds). The benign tonsils and lymph nodes (blue diamonds) were also scored as HPV negative. The y-values are shown on log scale.

Figure 3

Representative real-time quantitative PCR curves showed the amplification of E7 of HPV16. Standard curves were generated with series dilution of DNA (ng) from Caski cells. The samples have been normalized for DNA equivalence using actin dilution curves (data not shown). The X axis represents the PCR cycle number as the reaction takes place.