The American journal of surgical pathology, 37(9):1349–1356.
Mehrad M, Carpenter DH, Chernock RD, Wang H, Ma XJ, Luo Y, Luo J, Lewis JS Jr, El-Mofty SK. (2013).
PMID: 23797720 | DOI: 10.1097/PAS.0b013e318290427d.
A relationship between human papillomavirus (HPV) infection and papillary squamous cell carcinoma (PSCC) has been suggested. However, to date, no studies have thoroughly and directly evaluated for transcriptional activity of the virus or the clinicopathologic significance of HPV-positive PSCC. Forty-eight cases of PSCC were retrieved from our surgical pathology database and were reviewed by 4 study pathologists, with tumors defined as SCC with a significant component of papillary growth in the tumor. Immunohistochemical analysis for p16 and p53 was performed. Overexpression of p16 was used as a surrogate marker of transcriptionally active HPV. Transcriptional activity was also directly evaluated using RNA in situ hybridization to detect high-risk HPV E6/E7 mRNA. Clinical follow-up data were obtained by chart review. Seven cases were located in the oral cavity, 19 in the oropharynx, and 22 in the larynx. Two morphologic types of PSCC were identified: keratinizing type, in which the epithelial cells showed a maturation trend with minimal surface parakeratin, and nonkeratinizing type, in which the papillae were completely covered by immature basaloid cells. Transcriptionally active HPV was present in 23 of 43 (53.4%) tumors. The majority of tumors harboring transcriptionally active HPV arose in the oropharynx, showed nonkeratinizing morphology, were p16 positive, and p53 negative. Transcriptionally active HPV was also present in many laryngeal and oral cavity PSCCs. Overall survival, disease-specific survival, and disease-free survival were favorable and did not significantly differ by anatomic subsite. However, HPV-related tumors showed a trend toward better survival.
Eur Arch Otorhinolaryngol. 2014 Dec 27
Strojan P, Zadnik V, Šifrer R, Lanišnik B, Didanović V, Jereb S, Poljak M, Kocjan BJ, Gale N.
PMID: 25542247
Incidence trends in head and neck squamous cell carcinoma in Slovenia, 1983-2009: role of human papillomavirus infection.
Wang H, Wang MX, Su N, Wang LC, Wu X, Bui 1, Nielsen A, Vo HT, Nguyen N, Luo Y, Ma XJ (2014).
PMID: 24637627doi
The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.
Histopathology. May; 60(6):982-91.
Lewis JS Jr1, Ukpo OC, Ma XJ, Flanagan JJ, Luo Y, Thorstad WL, Chernock RD (2012)
PMID: 22360821 | DOI: 10.1111/j.1365-2559.2011.04169.x.
AIMS:
Human papillomavirus is well established in oropharyngeal squamous cell carcinoma as both causative and prognostic, but its significance in non-oropharyngeal tumours is unclear. In particular, the significance of finding viral DNA is not known. We sought to evaluate nonoropharyngeal squamous cell carcinomas for transcriptionally-active human papillomavirus and to compare this with the presence of viral DNA.
METHODS:
We evaluated an 87 patient tissue microarray cohort of oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas for high risk human papillomavirus DNA and E6 and E7 mRNA transcripts by in situ hybridization, and for p16 expression by immunohistochemistry.
RESULTS:
We found only two of the 73 (2.7%) evaluable cases to harbour transcriptionally-active human papillomavirus. Both of these tumours were from the larynx, one was positive for human papillomavirus DNA by in situ hybridization, and both were extensively positive for p16. All oral cavity and hypopharyngeal tumours were negative for human papillomavirus.
CONCLUSIONS:
Transcriptionally-active human papillomavirus appears to be rare in laryngeal, hypopharyngeal, and oral cavity squamous cell carcinomas. As such, it appears unlikely to be a 'driver' or to be clinically significant in most established tumours.
Ruuskanen M, Irjala H, Minn H, Vahlberg T, Randen-Brady R, Hagström J, Syrjänen S, Leivo I.
PMID: 30549170 | DOI: 10.1002/hed.25450
Abstract BACKGROUND: Nasopharyngeal carcinoma (NPC) is related to Epstein-Barr virus (EBV) in endemic areas; however, the role of viruses in nonendemic countries is unclear. Our nationwide study investigated the prevalence and prognostic significance of EBV and human papillomaviruses (HPVs) in Finnish NPC tumors. METHODS: We analyzed samples from 150 patients diagnosed between 1990 and 2009. Viral status was determined using EBV and HPV RNA in situ hybridizations, and p16 immunohistochemistry. Patient and treatment characteristics were obtained from patient records. RESULTS: In our white patient cohort, 93 of 150 (62%) patients were EBV-positive and 21/150 (14%) patients were HPV-positive with no coinfections. Thirty-six (24%) tumors were negative for both viruses. The 5-year disease-specific survival for patients with EBV-positive, HPV-positive, and EBV/HPV-negative tumors was 69%, 63%, and 39%, respectively. In multivariable-adjusted analysis, overall survival was better among patients with EBV-positive (P = .005) and HPV-positive (P = .03) tumors compared to patients with EBV/HPV-negative tumors. CONCLUSIONS: In our low-incidence population, EBV and HPV are important prognostic factors for NPC.
Cancer Cytopathol. 2015 Aug 4.
Jalaly JB, Lewis JS Jr, Collins BT, Wu X, Ma XJ, Luo Y, Bernadt CT.
PMID: 26242494 | DOI: 10.1002/cncy.21600.
Abstract BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is a unique form of carcinoma that is important to identify for prognosis and treatment. Immunohistochemistry (IHC) for p16 (also known as cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1) is used as a surrogate marker for transcriptionally active, high-risk HPV. The primary objective of this study was to correlate p16 IHC of cell blocks from fine-needle aspirations (FNAs) with surgical pathology specimens of HPV-related oropharyngeal SCC. METHODS: In total, 48 patients who had a diagnosis of oropharyngeal or nonoropharyngeal SCC and also had an FNA that demonstrated metastatic SCC with available cell block material were identified. IHC for p16 was evaluated on both FNA cell blocks and surgical pathology specimens. In situ hybridization for high-risk HPV messenger RNA was performed on 31 of the FNA cell blocks. RESULTS: Although partial p16 staining was observed in the majority of cell blocks, there was concordance in 47 of 48 FNAs (98%) with surgical pathology specimens when strong positive p16 staining of at least 15% of tumor cells in FNA cell block material was present. In addition, high-risk HPV RNA in situ hybridization demonstrated a high correlation with p16 staining in surgical pathology specimens (96%) and FNAs (93%). CONCLUSIONS: There was excellent correlation between p16 IHC of FNA cell blocks and surgical pathology specimens using a cutoff of at least 15% positive staining in cell blocks. The recommended threshold (70% positive staining) for surgical pathology specimens may yield a high rate of false-negative results if applied to FNA cell blocks.
Head and neck pathology, 7(2):113–21.
Ukpo OC, Thorstad WL, Lewis JS Jr (2012).
PMID: 23179191 | DOI: 10.1007/s12105-012-0406-z.
Human papillomavirus (HPV) is associated with oropharyngeal squamous cell carcinomas. Persistent viral infection is postulated to lead to carcinogenesis, although infection of benign adjacent epithelium is not typically observed. It is known that immune evasive tumor cells can provide an ideal niche for a virus. The B7-H1/PD-1 cosignaling pathway plays an important role in viral immune evasion by rendering CD8+ cytotoxic T cells anergic. We hypothesized that HPV-related oropharyngeal squamous cell carcinomas express B7-H1 as a mechanism for immune evasion. A tissue microarray was utilized, for which HPV E6/E7 mRNA by in situ hybridization was previously performed. Immunohistochemistry was performed to detect B7-H1 and staining was characterized by pattern, distribution, and intensity. B7-H1 was expressed by 84 of the 181 (46.4%) cases. Both tumor cell membranous and cytoplasmic expression were present and cytoplasmic expression was identified in some peritumoral lymphocytes. Expression was analyzed in several different ways and then considered binarily as positive versus negative. Tumors expressing B7-H1 were more likely to be HPV positive (49.2 vs. 34.1 %, p = 0.08). B7-H1 expression showed no correlation with disease recurrence in the entire cohort (OR = 1.09, p = 0.66), HPV positive cohort (OR = 0.80, p = 0.69) or HPV negative cohort (OR = 2.02, p = 0.22). However, B7-H1 expression intensity did correlate with the development of distant metastasis (p = 0.03), and B7-H1 intensity of 3+ (versus all other staining) showed a strong trend towards distant metastasis in the HPV positive (OR = 6.67, p = 0.13) and HPV negative (OR = 9.0, p = 0.13) cohorts. There was no correlation between B7-H1 expression and patient survival for any of the different ways in which staining was characterized, whether binarily, by distribution, intensity, or combined scores. B7-H1 is expressed in the majority of oropharyngeal squamous cell carcinomas with transcriptionally-active HPV. This suggests that B7-H1 expression by tumor cells may play a role in harboring persistent HPV infection.
McDowell LJ, Young RJ, Johnston ML, Tan TJ, Kleid S, Liu CS, Bressel M, Estall V, Rischin D, Solomon B, Corry J.
PMID: 26881928 | DOI: 10.1002/cncr.29901.Abstract BACKGROUND: The incidence of p16 overexpression and the role of human papillomavirus (HPV) in cutaneous head and neck squamous cell carcinoma (cHNSCC) are unclear. METHODS: One hundred forty-three patients with cHNSCC lymph nod
Abstract
BACKGROUND:
The incidence of p16 overexpression and the role of human papillomavirus (HPV) in cutaneous head and neck squamous cell carcinoma (cHNSCC) are unclear.
METHODS:
One hundred forty-three patients with cHNSCC lymph node metastases involving the parotid gland were evaluated for p16 expression by immunohistochemistry. The detection of 18 high-risk HPV subtypes was performed with HPV RNA in situ hybridization for a subset of 59 patients. The results were correlated with clinicopathological features and outcomes.
RESULTS:
The median follow-up time was 5.3 years. No differences were observed in clinicopathological factors with respect to the p16 status. p16 was positive, weak, and negative in 45 (31%), 21 (15%), and 77 cases (54%), respectively. No high-risk HPV subtypes were identified, regardless of the p16 status. The p16 status was not prognostic for overall (hazard ratio, 1.08; 95% confidence interval [CI], 0.85-1.36; P = .528), cancer-specific (hazard ratio, 1.12; 95% CI, 0.77-1.64; P = .542), or progression-free survival (hazard ratio, 1.03; 95% CI, 0.83-1.29; P = .783). Distant metastasis-free survival, freedom from locoregional failure, and freedom from local failure were also not significantly associated with the p16 status.
CONCLUSIONS:
p16 positivity is common but not prognostic in cHNSCC lymph node metastases. High-risk HPV subtypes are not associated with p16 positivity and do not appear to play a role in this disease. HPV testing, in addition to the p16 status in the unknown primary setting, may provide additional information for determining a putative primary site.
Jiang RT, Wang JW, Peng S, Huang TC, Wang C, Cannella F, Chang YN, Viscidi RP, Best SRA, Hung CF, Roden RBS.
PMID: 28515303 | DOI: 10.1128/JVI.00699-17
Mus musculus Papillomavirus1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not common laboratory strains. To facilitate study of immune control, we sought an outbred and immune competent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) by scarification on their tail with MmuPV1 resulted in three clinical outcomes: 1) persistent (>2 months) papillomas (∼20%), 2) transient papillomas that spontaneously regress typically within 2 months (∼15%), 3) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated using a candidate preventive/therapeutic naked DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and E7 (mE7) early proteins and residues 11-200 of late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and failure of disease to recur after CD3 T cell depletion. Vaccination induced a strong mE6 and mE7 CD8+ T cell response in all mice, although significantly lower in mice that initially presented with persistent warts as compared with those that spontaneously cleared their infection. An HPV16-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with HPV16 pseudovirus. Thus MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearance of HPV-related disease.IMPORTANCE High risk type human papillomaviruses (hrHPV) cause 5% of all cancer cases worldwide, notably cervical, anogenital and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries, and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas, and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Further an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16 suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.
Augustin J, Outh-Gauer S, Mandavit M, Gasne C, Grard O, Denize T, Nervo M, Mirghani H, Laccourreye O, Bonfils P, Bruneval P, Veyer D, Péré H, Tartour E, Badoual C.
PMID: 29684499 | DOI: 10.1016/j.humpath.2018.04.006
It is now established that HPV plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), notably oropharyngeal squamous cell carcinomas (SCCs). However, it is not clear which test one should use to detect HPV in oropharyngeal (OP) and non-OP SCCs. In this study, using 348 HNSCCs (126 OP SCCs and 222 non-OP SCCs), we evaluated diagnostic performances of different HPV tests in OP and non-OP SCCs: PCR, p16 immunostaining, in situ hybridization targeting DNA (DNA-CISH) and RNA (RNA-CISH), combined p16 + DNA-CISH, and combined p16 + RNA-CISH. HPV DNA (PCR) was detected in 26% of all tumors (44% of OP SCCs and 17% of non-OP SCCs). For OP SCCs, RNA-CISH was the most sensitive standalone test (88%), but p16 + RNA-CISH was even more sensitive (95%). Specificities were the same for RNA-CISH and DNA-CISH (97%) but it was better for p16 + RNA-CISH (100%). For non-OP SCCs, all tests had sensitivities below 50%, and RNA-CISH, DNA-CISH and p16 + DNA-CISH had respectively 100%, 97% and 99% specificities. As a standalone test, RNA-CISH is the most performant assay to detect HPV in OP SCCs, and combined p16 + RNA-CISH test slightly improves its performances. However, RNA-CISH has the advantage of being one single test. Like p16 and DNA-CISH, RNA-CISH performances are poor in non-OP SCCs to detect HPV, and combining tests does not improve performances.
Br J Cancer. 2015 Feb 17.
Young RJ, Urban D, Angel C, Corry J, Lyons B, Vallance N, Kleid S, Iseli TA, Solomon B, Rischin D.
PMID: 25688737 | DOI: 10.1038/bjc.2015.59.
Background:Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed.Methods:We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.Results:Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36-1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26-1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23-2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19-2.03, P=0.43).Conclusions:p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.
Roopera LM, Gandhib M, Bishop JA, Westraa WH
PMID: - | DOI: 10.1016/j.oraloncology.2016.02.008
Objectives
Evaluation of human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC) has become increasingly important for prognostication and clinical trial enrollment. This assessment is confounded in OPSCCs that are p16 positive by immunohistochemistry (IHC) but HPV negative by DNA in situ hybridization (DISH). This study evaluates whether E6/E7 mRNA in situ hybridization (RISH) can detect transcriptionally active HPV in these problematic cases.
Materials and methods
Eighty-two head and neck squamous cell carcinoma cases that had previously undergone p16 IHC and HPV DISH were evaluated with two RISH platforms and a second-generation DISH probe. The study included 21 p16+/DISH+ concordant cases, 19 p16−/DISH− concordant cases, and 42 p16+/DISH− discordant cases.
Results
RISH identified E6/E7 mRNA in 37 (88%) p16+/DISH− cases, 21 (100%) p16+/DISH+ cases, and 0 (0%) p16−/DISH− cases. RISH signals were clearly visible at low to medium magnification in 97% of positive cases, facilitating almost-perfect inter-observer reproducibility. The performance of the manual and automated RISH platforms were equivalent (kappa = 0.915). Only 29% of carcinomas that demonstrated E6/E7 mRNA transcriptional activity were positive using the 2nd generation DISH probe.
Conclusions
HPV RISH is a highly sensitive and specific platform that can clarify the HPV status of those perplexing OPSCCs that are p16 positive by IHC but HPV negative by DISH. Moreover, it is easy to interpret, readily adaptable to the clinical laboratory, and provides direct evidence of HPV transcriptional activity. E6/E7 RISH should be considered as a first-line platform for determination of HPV status in OPSCCs.
