Probes for INS

Dysregulated expression of oncogenic types of E6 and E7 is necessary for human papillomavirus (HPV)-driven carcinogenesis. An HPV E6/E7 mRNA in situ hybridization (ISH) assay covering 18 common high-risk types ("HR-RISH," aka HR-HPV RNA18 ISH) has not been extensively studied in the anogenital tract or validated on automated technology. We herein compare HR-RISH to DNA polymerase chain reaction (PCR), p16 immunohistochemistry, and a previously available HPV DNA ISH assay in HPV-related anogenital and head and neck (H&N) neoplasia. A total of 102 squamous intraepithelial lesions (16 CIN1, 25 CIN3, 3 AIN1, 12 AIN3, 9 VIN3)/invasive squamous cell carcinomas (17 cervical, 2 anal, 18 H&N) as well as 10 normal and 15 reactive cervix samples were collected. HR-RISH, DNA ISH, and p16 immunohistochemistry were performed on whole formalin-fixed, paraffin-embedded sections. RNA ISH for 6 low-risk HPV types (LR-RISH) was also performed. RNA and DNA ISH assays used automated systems. HR-HPV PCR was performed on morphology-directed formalin-fixed, paraffin-embedded punches. HR-RISH was ≥97% sensitive for PCR+ and p16+ neoplasia, as well as morphologically defined anogenital high grade squamous intraepithelial lesion/invasive squamous cell carcinoma. HR-RISH was also positive in 78% of anogenital low grade squamous intraepithelial lesion, including 81% of CIN1. Furthermore, a subset of PCR-negative/invalid and p16-negative lesions was positive for HR-RISH. Only 1 problematic reactive cervix sample and no normal cervix samples stained. These results demonstrate that HR-RISH is a robust method for the detection of HR-HPV-related neoplasia and provides insight into HPV pathobiology. Performance meets or exceeds that of existing assays in anogenital and H&N lesions and may play a role in resolving diagnostically challenging CIN1 versus reactive cases.

Cervical low-grade squamous intraepithelial lesions (LSIL) (aka cervical intraepithelial neoplasia, grade 1 [CIN1]) can present considerable diagnostic challenges and are associated with poor interobserver reproducibility and overdiagnosis. Furthermore, ancillary studies such as p16 immunohistochemistry have shown little utility in resolving the LSIL versus negative/reactive differential. Human papillomavirus (HPV) RNA in situ hybridization (ISH) has shown promise as a diagnostic aid in this setting, but has not been studied in a large case series. We herein investigate high-risk and low-risk HPV RNA ISH in 126 cervical biopsies originally diagnosed as LSIL/CIN1 and compare HPV RNA ISH results to expert-adjudicated morphologic diagnosis to assess whether this assay can help routine cases attain the existing "gold standard" of morphologic consensus diagnosis. We also assess whether this criterion standard can be further improved by integration of HPV RNA ISH results. A consensus diagnosis of intraepithelial lesion (CIN1) was confirmed in 61% of cases, whereas 57% were HPV RNA. HPV-RNA positivity was 84% sensitive and 86% specific for an expert-adjudicated diagnosis of CIN1. Conversely, consensus diagnosis was 90% sensitive and 78% specific for the presence of HPV RNA. Integrating RNA ISH into morphologic review led to further reclassification of 10% of cases, resulting in 95% sensitivity and 98% specificity of HPV RNA ISH for a CIN1 diagnosis and 98% sensitivity and 92% specificity of CIN1 for the presence of HPV RNA. These findings suggest that judicious use of HPV RNA ISH can improve the accuracy of LSIL/CIN1 diagnosis for morphologically ambiguous cases.

Abstract

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.

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.

AIMS:

Verrucous carcinoma (VC) is a variant of well differentiated squamous cell carcinoma and is in the anal region regarded as synonymous with giant condyloma (Buschke-Löwenstein tumor) (BLT). Etiology, diagnostic criteria and clinical behavior of both lesions are controversial. Recent studies suggest that VC at other sites is not associated with human papillomaviruses (HPV). We hypothesized that anal VC is also not related to HPV, while BLT is a HPV-induced lesion.

METHODS AND RESULTS:

Ten cases of VC and 4 cases of BLT were included. Several techniques were used for HPV detection: in situ hybridization for HPV6, 11, 16 and 18, six different PCR protocols for detection of at least 89 HPV types from Alpha-, Beta-, Gamma- and Mu-PV genera, and in situ hybridization for high risk HPV E6/E7 mRNA. p16 immunohistochemistry and morphometric analysis were also performed. Alpha-, Gamma- and Mu-PVs were not found in any case of VC, while HPV6 was detected in all cases of BLT. p16 overexpression was not present in any of the lesions. Among microscopic features, only the absence of koilocytosis and enlarged spinous cells seem to be useful to distinguish VC from BLT.

CONCLUSIONS:

Our results suggest that anal VC, similarly to VC at other sites, is not associated with HPV infection and must be distinguished from BLT which is associated with low risk HPV. Only with well-set diagnostic criteria will it be possible to ascertain clinical behavior and optimal treatment for both lesions. 

Human Papillomavirus (HPV) is known as causative for squamous cell carcinoma (SCC) of the oropharynx, but is also not infrequently found in carcinomas of the sinonasal tract. Recently, a subset of these carcinomas was recognized to harbour HPV33 and have a significant morphological overlap with adenoid cystic carcinoma (ACC), a rare and aggressive carcinoma originating in the minor salivary glands. Termed HPV-related carcinoma with ACC-like features, only 9 cases have been reported. To clarify the occurrence of these tumours we screened a large material for presence of HPV-related ACC-like carcinoma. The identified tumours were characterized immunohistochemically and with fluorescence in situ hybridization and clinicopathologic information for all cases is presented.

METHODS AND RESULTS:

Forty-seven candidate cases were screened for presence of HPV. Six cases were identified and genotyped as HPV types 33, 35 and 56. All six cases had areas of dysplastic mucosal lining and showed remarkable heterogeneous morphologies. MYB, MYBL1, and NFIB genes were intact and, interestingly, staining for MYB protein was largely negative in contrast to what was found in ACC. One patient experienced a local recurrence 11 years after initial treatment and the remaining five patients were alive without evidence of disease.

CONCLUSION:

We report six new cases of HPV-related ACC-like carcinoma and found that, although in a small material, the prognosis for these patients seems more favourable than for ACC. For the distinction between ACC and HPV-related ACC-like carcinoma, p16, MYB immunohistochemistry, or investigation of MYB, MYBL1, and NFIB gene status are valuable. This article is protected by copyright. All rights reserved.

Abstract

BACKGROUND:

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing among older adults. It is unknown whether these trends can be explained by human papillomavirus (HPV) and whether HPV-related tumors remain associated with an improved prognosis among older patients.

METHODS:

In a retrospective study of OPSCCs diagnosed from 1995 to 2013 at 2 National Comprehensive Cancer Network-designated cancer centers, p16 immunohistochemistry and in situ hybridization (ISH) for HPV-16, high-risk DNA, and/or E6/E7 RNA were performed. The median age at diagnosis was compared by p16 and ISH tumor status. Trends in age were analyzed with nonparametric trends. Survival was analyzed with the Kaplan-Meier method and Cox proportional hazards models.

RESULTS:

Among 239 patients, 144 (60%) were p16-positive. During 1998-2013, the median age increased among p16-positive patients (Ptrend = .01) but not among p16-negative patients (Ptrend = .71). The median age of p16-positive patients increased from 53 years (interquartile range [IQR] in 1995-2000, 45-65 years) to 58 years (IQR for 2001-2013, 53-64 years). Among patients ≥ 65 years old, the proportion of OPSCCs that were p16-positive increased from 41% during 1995-2000 to 75% during 2007-2013 (Ptrend = .04). Among all age groups, including older patients, a p16-positive tumor status conferred improved overall survival in comparison with a p16-negative status.

CONCLUSIONS:

The median age at diagnosis for HPV-related OPSCC is increasing as the proportion of OPSCCs caused by HPV rises among older adults. The favorable survival conferred by an HPV-positive tumor status persists in older adults. Cancer 2018. © 2018 American Cancer Society.

Abstract

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH “low” and RNA CISH “high”. Negative HPV RNA CISH cases were scored as RNA CISH “low”. This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.