Equivocal p16 immunostaining in squamous cell carcinoma of the head and neck: staining patterns are suggestive of HPV status

Abstract

p16 immunohistochemistry (IHC) is commonly used as a surrogate marker for human papillomavirus (HPV) detection in squamous cell carcinomas of the head and neck (SCCHN). However, the HPV status of tumors not staining strongly for p16 is difficult to interpret and may require the use of PCR, not available in all laboratories, as a final arbiter. We aim to determine if staining pattern in equivocal p16 staining and correlation to the percentage of positively stained tumor cells is predictive of HPV status. A retrospective review was performed on all SCCHN that underwent p16 IHC and PCR in our institution from 2007 to 2010. Descriptors of staining pattern in the original IHC report were retrieved. All available IHC slides were reviewed and reclassified using consensus staining pattern descriptors. Original and reclassified descriptors were compared to the final PCR HPV status for statistical significance using the χ(2) test. An estimate of the percentage of tumor cells that showed any form of staining was performed. Thirty-two SCCHN cases that underwent PCR HPV testing had equivocal p16 IHC results. Twenty-six cases available for review were reclassified into four staining patterns. Comparing age, sex, tumor site and diagnosis to HPV PCR status showed no statistically significant findings. However, comparing original descriptors to HPV status was statistically significant with isolated staining associated with negative HPV status (p = 0.0002). Analysis using reclassified descriptors showed strong association of membranous/cytoplasmic staining of isolated cells with negative HPV status and faint, diffuse nuclear and cytoplasmic staining with positive HPV status (p = 0.00006). HPV-negative cases with the former pattern had no more than 30 % positively-stained tumor cells and HPV-positive cases with the latter pattern had 50-90 % positively-stained cells. Our results suggest that pattern of staining in p16 IHC is associated with HPV status. For instance, a diffuse nuclear and cytoplasmic staining pattern, regardless of intensity, is associated with HPV positivity. The HPV-positive cases determined by staining pattern were also associated with a higher percentage of stained tumor cells.

Fig. 1

Strong, diffuse nuclear and cytoplasmic p16 immunostaining of HPV + SCCHN (tonsil) with a basaloid morphology (left p16 IHC; right H&E)

Fig. 2

Example of category A “isolated cells with membranous/cytoplasmic staining at periphery of nests” IHC pattern in an HPV-negative conventional keratinizing squamous cell carcinoma of the palate (left p16 IHC; right H&E)

Fig. 3

Example of category B “faint, diffuse nuclear and cytoplasmic staining” IHC pattern in an HPV type 35-positive, mixed keratinizing/non-keratinizing squamous cell carcinoma metastatic to neck lymph node (left p16 IHC; right H&E)

Fig. 4

Category C “isolated, faint nuclear and cytoplasmic staining” IHC pattern in a single case of conventional keratinizing squamous cell carcinoma of the oral cavity positive for HPV types 16 and 33 (left p16 IHC; right H&E)

Fig. 5

Category D “faint, diffuse staining with patches of strong staining” IHC pattern in a single case of non-keratinizing squamous cell carcinoma of the tongue base positive for HPV type 16 (left p16 IHC; right H&E)