**5. Principles of HPV testing for oropharyngeal carcinoma**

All patients diagnosed with OPSCC should undergo testing for HPV status. Biopsy from the primary lesion or FNAC from an involved node is sufficient for HPV testing. The gold standard is the demonstration of HPV E6/E7 mRNA expression in clinical specimens, which is often impractical. Demonstration of HPV DNA, by polymerase chain reaction (PCR), has high sensitivity, but specificity is low as cross-contamination can occur. In situ hybridization (ISH) technique allows the identification of a single viral copy and is more specific. In the HPV carcinogenesis, E7 mediated Rb inhibition leads to induction of demethylases resulting in overexpression of p16INK4A, which is a cyclin-dependent kinase inhibitor. Hence the immunohistochemistry (IHC) test for P 16 is used as a surrogate marker for HPV status. Various methods for testing the HPV status is summarized in **Table 2**. Infection with non-HPV subtypes or low viral copy numbers cannot be detected by IHC and there can be a 7% disparity between HPV ISH and IHC reports. In the case of an equivocal P16, further testing by ISH can clarify the HPV status. Work up for patients includes thorough history taking, with documentation on pack-years smoked, and clinical examination (inspection, palpation, and endoscopy evaluation to see the extent of the lesion). Imaging using CT or MRI


#### **Table 2.**

*Various methods used for testing HPV status.*

#### *Pharynx - Diagnosis and Treatment*

neck aids in staging detects regional lymphadenopathy including retropharyngeal nodes. MRI neck in treatment position is particularly useful in delineation of the primary lesion for radiotherapy planning. The primary lesions of HPV positive OPSCC often had well-defined borders on imaging with a cystic nodal disease with or without necrosis. A chest X-ray is advisable to assess the baseline pulmonary function. Additionally, they need a dental evaluation for radiotherapy planning. All patients should undergo nutrition, speech, and swallowing evaluation, and smoking cessation counseling should be given if needed. Pre-anesthesia workup is needed if planning for surgery.
