Quantitative assessment of p16 expression in FNA specimens from head and neck squamous cell carcinoma and correlation with HPV status

Abstract

Background This study investigated p16 by immunohistochemistry (IHC) on cellblocks (CBs) and human papillomavirus (HPV) by polymerase chain reaction (PCR) in fine‐needle aspiration (FNA) of head and neck squamous cell carcinoma (HNSCC). Methods Receiver operating characteristic (ROC) curve analysis was used to assess test performance in CBs compared with p16 IHC in 42 surgical specimens from patients with HNSCC and in correlation with HPV by PCR in cytology specimens. The study assessed HPV by PCR in FNA specimens as a substitute for p16 IHC in surgical specimens. Results Of 42 cases, 38 CBs showed malignant cells as cohesive clusters of viable cells with or without single tumor cells, whereas 4 specimens were composed exclusively of single tumor cells and degenerated cells. All p16‐negative surgical specimens showed an absence of p16 staining in the corresponding CBs (n = 16). In the p16‐positive surgical cases (n = 26), corresponding CBs with tumor clusters (n = 23) showed heterogeneous p16 expression ranging from 40% to 100%; however, scoring single cells was challenging and unreliable because of cellular degradation. ROC curve inspection showed the optimal threshold to be at least 40% p16 staining in tumor clusters with 100% sensitivity and specificity. In cases with inadequate CBs, HPV by PCR on needle rinse showed 88% sensitivity and 100% specificity for p16 expression in surgical specimens. Conclusions A cutoff of at least 40% p16 expression in tumor clusters may be appropriate for p16 positivity in cytology CB specimens. A positive HPV finding by PCR on needle rinse can be used as a substitute for p16 expression in surgical specimens.

Detection of Human Papillomavirus in Squamous Lesions of the Conjunctiva Using RNA and DNA In-Situ Hybridization

“…Immunohistochemical approaches, including direct detection of HPV-encoded proteins or alterations in p16 expression, have been investigated, but more recent studies suggest that these methods are prone to false-positive results [13,22,41,46,49,51,53,55,56,58,59,82]. In particular, it has been found that many lesions negative for HPV express p16 [49,58].…”

“…Considerable variability in the correlation of HPV infection with squamous lesions has been reported, and this may be due to the variety of methods for both sampling and viral detection [31,35,46,47]. Immunohistochemical approaches, including direct detection of HPV-encoded proteins or alterations in p16 expression, have been investigated, but more recent studies suggest that these methods are prone to false-positive results in conjunctival lesions [13,22,34,41,46,[48][49][50][51][52][53][54][55][56][57][58][59]. Historically, HPV DNA was detected with Southern blotting or in-situ hybridization assays, which can have poor sensitivity, and polymerase chain reactions (PCR), with high false-positive rates [31,47].…”

Detection accuracy of the Cobas HPV assay for high‐risk HPV in head and neck FNA biopsy specimens

“…Second, the interpretation of p16 IHC in FNA cell blocks is challenging because of staining variation and controversial cutoffs for a positive result. 12,[15][16][17][18][19] The use of RNAScope can confirm p16 IHC results, but it does not provide the HPV-16 status, which is a requirement for clinical trials at our institution. For these reasons, we previously validated and used 2 commercial HPV testing assays, the Cervista HPV-16/18 and HPV HR assays (Hologic, Inc, Bedford, Massachusetts), for determining the HPV status in head and neck FNA specimens.…”

“…First, cell block material is not always available, but when it is, low cellularity and extensive necrosis often preclude p16 IHC. Second, the interpretation of p16 IHC in FNA cell blocks is challenging because of staining variation and controversial cutoffs for a positive result 12,15‐19 . The use of RNAScope can confirm p16 IHC results, but it does not provide the HPV‐16 status, which is a requirement for clinical trials at our institution.…”

Immunocytochemistry for diagnostic cytopathology—A practical guide

“…While a positive p16 IHC result in surgical pathology specimens is defined as “greater than or equal to 70% nuclear and cytoplasmic staining of at least moderate intensity”, 71 the criteria for detecting HPV using p16 in cytological material are not well defined, and the threshold for a positive p16 result needs to be individually validated in each laboratory 72–75 . A cut‐off of greater than or equal to 40% expression in tumour cell clusters may be appropriate for p16 positivity in CB specimens 76 . Negative HPV testing results in cytological material should be repeated if a subsequent surgical pathology specimen becomes available 71 .…”