Archives of Medical Science - Civilization Diseases
Nascimento, M;Silva Galbiatti-Dias, A;Oliveira-Cucolo, J;Pavarino, É;Goloni-Bertollo, E;
| DOI: 10.5114/amscd.2022.119197
The presence of human papillomavirus (HPV) in patients with head and neck squamous cell carcinoma (HNSCC) can guide medical management. The aim of this study was to highlight the importance of HPV diagnosis, verifying which diagnostic techniques are most used in identifying HPV and the differences between these techniques, in the research aimed at establishing a consensus on the gold standard method. We verify that HPV infection is associated with the development of HNSCC. The techniques most commonly used for diagnosis of HPV are immunohistochemistry (IHC), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Our study concludes that detection of E6/E7 DNA by PCR is the most accepted method of diagnosis. The standardization of an accurate HPV diagnostic method can reduce morbidity and mortality in HNSCC, especially in emerging countries, where few screenings are performed, in addition to improving the social and economic impact of the disease.
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.
Human pathology, 44(8):1672–1680.
Scantlebury JB, Luo J, Thorstad WL, El-Mofty SK, Lewis JS Jr (2013).
PMID: 23566410 | DOI: 10.1016/j.humpath.2013.01.021.
Human papillomavirus-related oropharyngeal squamous cell carcinoma has a unique biology and improved prognosis. A new focus is to identify prognostic biomarkers specifically in this human papillomavirus-positive cohort. We analyzed cyclin D1 immunostaining on a tissue microarray of patients with known clinical follow-up and p16 and human papillomavirus status (by E6/E7 RNA in situ hybridization). Cyclin D1 staining was read visually and digitally. Cutoffs of 5%, 10%, and 30% were separately analyzed as was linear intensity data derived from the image analysis. For the 202 tumors, cyclin D1 expression was > 10% in 25.7% (visual) and 35.5% (digital) of the cases. It was > 30% in 15.8% (visual) and 16.5% (digital) of the cases. High cyclin D1 by both methods, cutoffs, and expression intensity was associated with poorer overall, disease-free, and disease-specific survival in univariate analysis. However, low cyclin D1 expression was also tightly associated with human papillomavirus RNA (P < 1.0 × 10(-18) for all cutoffs) and p16 positivity (P < 1.0 × 10(-14) for all cutoffs). In multivariate analysis using the digital 30% cutoff (the strongest cyclin D1 assessment method), only T stage, p16 status, smoking, and treatment approach associated with survival. Intensity of cyclin D1 expression did, however, significantly substratify the human papillomavirus RNA-positive patients into prognostic subgroups independent of other variables. In summary, cyclin D1 overexpression correlates strongly with patient survival in oropharyngeal squamous cell carcinoma, but its relationship with human papillomavirus status is very tight, and the complex nature of this correlation likely limits any clinical application for cyclin D1 assessment.
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.
Diagnostics (Basel, Switzerland)
Bumrungthai, S;Ekalaksananan, T;Kleebkaow, P;Pongsawatkul, K;Phatnithikul, P;Jaikan, J;Raumsuk, P;Duangjit, S;Chuenchai, D;Pientong, C;
PMID: 36980391 | DOI: 10.3390/diagnostics13061084
The current practice of determining histologic grade with a single molecular biomarker can facilitate differential diagnosis but cannot predict the risk of lesion progression. Cancer is caused by complex mechanisms, and no single biomarker can both make accurate diagnoses and predict progression risk. Modelling using multiple biomarkers can be used to derive scores for risk prediction. Mathematical models (MMs) may be capable of making predictions from biomarker data. Therefore, this study aimed to develop MM-based scores for predicting the risk of precancerous cervical lesion progression and identifying precancerous lesions in patients in northern Thailand by evaluating the expression of multiple biomarkers. The MMs (Models 1-5) were developed in the test sample set based on patient age range (five categories) and biomarker levels (cortactin, p16INK4A, and Ki-67 by immunohistochemistry [IHC], and HPV E6/E7 ribonucleic acid (RNA) by in situ hybridization [ISH]). The risk scores for the prediction of cervical lesion progression ("risk biomolecules") ranged from 2.56-2.60 in the normal and low-grade squamous intraepithelial lesion (LSIL) cases and from 3.54-3.62 in cases where precancerous lesions were predicted to progress. In Model 4, 23/86 (26.7%) normal and LSIL cases had biomolecule levels that suggested a risk of progression, while 5/86 (5.8%) cases were identified as precancerous lesions. Additionally, histologic grading with a single molecular biomarker did not identify 23 cases with risk, preventing close patient monitoring. These results suggest that biomarker level-based risk scores are useful for predicting the risk of cervical lesion progression and identifying precancerous lesion development. This multiple biomarker-based strategy may ultimately have utility for predicting cancer progression in other contexts.
Lewis, JS;Smith, MH;Wang, X;Tong, F;Mehrad, M;Lang-Kuhs, KA;
PMID: 35802245 | DOI: 10.1007/s12105-022-01467-0
HPV-associated oral cavity squamous cell carcinoma (SCC) is not well-characterized in the literature, and also has a clinical significance that is poorly understood.We gathered a cohort of oral cavity (OC) SCC with nonkeratinizing morphology, either in the invasive or in situ carcinoma (or both), tested for p16 by immunohistochemistry and high risk HPV E6/E7 mRNA by RTPCR (reference standard for transcriptionally-active high risk HPV) and gathered detailed morphologic and clinicopathologic data.Thirteen patients from two institutions were proven to be HPV-associated by combined p16 and high risk HPV mRNA positivity. All 13 patients (100%) were males, all were heavy smokers (average 57 pack/year), and most were active drinkers (9/11 or 81.8%). All 13 (100%) involved the tongue and/or floor of mouth. All had nonkeratinizing features, but maturing squamous differentiation varied widely (0-90%; mean 37.3%). Nonkeratinizing areas had high N:C ratios and larger nests, frequently with pushing borders, and minimal (or no) stromal desmoplasia. The carcinoma in situ, when present, was Bowenoid/nonkeratinizing with cells with high N:C ratios, full thickness loss of maturation, and abundant apoptosis and mitosis. HPV was type 16 in 11 patients (84.6%) and type 33 in two (15.4%). Nine patients had treatment data available. These underwent primary surgical resection with tumors ranging from 1.6 to 5.2 cm. Most had bone invasion (6/9-66.7% were T4a tumors), and most (6/9-66.7%) had extensive SCC in situ with all 6 of these patients having final margins positive for in situ carcinoma.HPV-associated OCSCC is an uncommon entity that shows certain distinct clinical and pathologic features. Recognition of these features may help pathologic diagnosis and could potentially help guide clinical management.
Diagnostic Histopathology
Assessment of human papillomavirus (HPV) status is a requirement for the diagnosis of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) and metastatic squamous cell carcinoma in cervical lymph nodes where the location of the primary neoplasm is unknown. Within the diagnostic histopathology laboratory, there should be a validated and reproducible HPV testing strategy that can provide HPV status within a reasonable timeframe to inform patient care. Although these requirements are recognized by the head and neck oncology community, there is no internationally accepted standard for HPV testing. A two-tiered approach incorporating p16 immunohistochemistry with specific HPV testing by DNA in situ hybridization is a pragmatic way of providing HPV testing in clinical practice. A novel RNA in situ hybridization methodology targeting E6 and E7 mRNA has been validated and is likely to be available as an in vitro diagnostic device soon. This review will outline the current concepts around the diagnosis of HPV-associated head and neck SCC and suggest a diagnostic algorithm that can be instituted in most diagnostic cellular pathology laboratories.
