Probes for P16

Abstract

Objectives

In the present study, we comprehensively analyzed the prevalence of transcriptionally active HPV in tissue samples of Indian patients with leukoplakia - predominantly hyperplastic lesions and HNSCC. In addition, saliva samples from patients with HNSCC were screened for HPV detection.

Study Design

p16 overexpression was analyzed by immunohistochemistry. Leukoplakia (n = 121) and HNSCC (n = 427) tissue samples and the saliva of patients with HNSCC (n = 215) were tested for HPV using nested PCR. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in-situ hybridization.

Results

p16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, either in saliva and/or tissue. E6/E7 mRNA positivity was observed in eight HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx with very little concordance between p16 overexpression and HPV positivity. All patients with HPV positive saliva samples had transcriptionally active HPV present in their tumors.

Conclusion

Presence of HPV-DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV associated tumors.

HPV-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk human papillomavirus (HR-HPV) infection. Using HR-HPV mRNA in situ hybridization (ISH), we reported six new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma (ACC). Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified six HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium and one also had invasive squamous cell carcinoma (SCC). All HMSC were SOX10-positive whereas the overlying atypical squamous epithelium and the SCC were SOX10-negative. One atypical HMSC-like case was also identified which was positive for HR-HPV mRNA ISH, HR-HPV DNA ISH, SOX10 IHC, but negative for p16 IHC. This study showed that HR-HPV mRNA ISH was a useful tool to diagnose HMSC and had stronger signals than HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium as well as the invasive SCC. A combination of p16 and SOX10 IHC will be a useful screening panel for HMSC followed by confirmatory HR-HPV mRNA ISH test.

Our understanding of adenoid basal tumors of the cervix has evolved over time. Most of the proliferations referred to as adenoid basal carcinoma have a clinically benign course-leading some to suggest the term "adenoid basal epithelioma." However, rarely, these may be associated with invasive carcinomas. These tumors have been etiologically linked with high-risk human papillomavirus (HR-HPV) infection. Here, we investigate the use of p16 immunohistochemistry and HR-HPV RNA in situ hybridization (ISH) in the classification of adenoid basal tumors of the cervix. Seventeen cases of adenoid basal tumors of the cervix were included. The patients' age ranged from 19 to 79 yr (average, 59 yr). p16 immunostain was performed on all cases and RNA ISH was performed in 4 cases with available formalin-fixed paraffin-embedded tissue. There were 11 low-grade tumors, 5 frankly invasive carcinomas, and 1 with histologic features that were intermediate between the former 2 categories. p16 immunostain was negative or showed patchy cytoplasmic staining in the low-grade tumors and was strongly and diffusely positive in the invasive carcinomas. HR-HPV RNA ISH was negative in the 3 low-grade tumors and was positive in 1 case of invasive carcinoma including the adenoid basal component. Distinct p16 immunostaining and HR-HPV RNA ISH patterns exist between low-grade adenoid basal tumors and invasive adenoid basal carcinomas. Our study indicates that p16 immunostaining and HR-HPV RNA ISH can be employed as useful ancillary tools in differentiating between noninvasive and invasive adenoid basal tumors along with careful histopathologic evaluation.

Abstract

BACKGROUND:

The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC.

METHODS:

Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope^® (a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus.

RESULTS:

HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH.

CONCLUSION:

We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.

High-risk human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas have a more favorable prognosis than HPV-negative ones. p16 immunohistochemistry has been recommended as a prognostic test in clinical practice. Several p16 antibodies are available, and their performance has not been directly compared. We evaluated three commercially available p16 antibody clones (E6H4, JC8 and G175-405) utilizing 199 cases of oropharyngeal squamous cell carcinoma from a tissue microarray, read by three pathologists with three different cutoffs for positivity: any staining, >50% and >75%. Positive predictive values for high-risk HPV status by RNA in situ hybridization for the E6H4, JC8 and G175-405 clones were 98%, 100% and 99% at the 75% cutoff, but negative predictive values were much more variable at 86%, 69% and 56%, respectively. These improved using the 50% cutoff, becoming similar for all three antibodies. Intensity varied substantially, with 85% of E6H4, 72% of JC8 and 67% of G175-405 showing strong (3+) intensity. With Kaplan-Meier survival plots at the 75% cutoff, the E6H4 clone showed the largest differential in disease specific and overall survival between p16-positive and -negative results. Decreasing the cutoff to 50% increased correlation with HPV in situ hybridization and improved the survival differential for the JC8 and G175-405 clones without worsening of performance for the E6H4 clone. Interobserver agreement was also assessed by kappa scores and was highest for the E6H4 clone. Overall, these study results show modest but important performance differences between the three different p16 antibody clones, suggesting that the E6H4 clone performs best because of strongest staining intensity, greatest differential in outcomes between positive and negative results, lowest interobserver variability, and lowest background, nonspecific staining. The results also suggest that a 75% cutoff is very functional but that, in this patient population with high HPV incidence, 50% and any staining cutoffs may be more effective, particularly for the non-E6H4 clones.