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.
Oral oncology, 50(1):1–9.
Mirghani H1, Amen F2, Moreau F3, Guigay J4, Ferchiou M5, Melkane AE6, Hartl DM7, Lacau St Guily J (2014).
PMID: 24169585 | DOI: 10.1016/j.oraloncology.2013.10.008.
High risk Human Papilloma virus (HR-HPV) associated oropharyngeal cancers are on the increase. Although, the scientific community is aware of the importance of Human Papilloma Virus (HPV) testing, there is no consensus on the assays that are required to reliably identify HR-HPV related tumors. A wide range of methods have been developed. The most widely used techniques include viral DNA detection, with polymerase chain reaction (PCR) or In Situ Hybridization, and p16 detected by immunohistochemistry. However, these tests provide different information and have their own specific limitations. In this review, we summarize these different techniques, in light of the recent literature. p16 Overexpression, which is an indirect marker of HPV infection, is considered by many head and neck oncologists to be the most important marker for patient stratification. We describe the frequent lack of concordance of this marker with other assays and the possible reasons for this. The latest developments in HPV testing are also reported, such as the RNAscope™ HPV test, and how they fit into the existing framework of techniques. HPV testing must not be considered in isolation, as there are important interactions with other parameters, such as tobacco exposure. This is an important and rapidly evolving field and is likely to become pivotal to staging and choice of treatment of oropharyngeal carcinoma in the future.
Head and neck pathology, 1–7.
Chernock RD, Nussenbaum B, Thorstad WL, Luo Y, Ma XJ, El-Mofty SK, Lewis JS Jr (2013).
PMID: 24151062.
Over the past several decades, it has become clear that human papillomavirus (HPV) is important for the development and progression of many head and neck squamous cell carcinomas, particularly those arising in the oropharyngeal tonsillar crypts. Yet, our understanding of HPV's role in premalignant squamous lesions remains relatively poor. This is in part because premalignant lesions of the oropharyngeal tonsillar crypt tissue, where most HPV-related carcinomas arise, are difficult if not impossible to identify. Recent evidence does suggest a role for HPV in a subset of premalignant lesions of the surface epithelium, especially the oral cavity, despite the rarity of HPV-related invasive squamous cell carcinomas at this site. Furthermore, these HPV-related oral cavity dysplasias appear to have unique, bowenoid histologic features described as 'basaloid' with full-thickness loss of squamous maturation, mitotic figures and apoptosis throughout. Here, we present a unique case of an HPV-related premalignant lesion (squamous cell carcinoma in situ) extensively involving the surface epithelium of the oral cavity, oropharynx and larynx that had 'nonkeratinizing' histologic features typical of HPV-related invasive squamous cell carcinoma. This case was strongly p16 positive by immunohistochemistry and harbored transcriptionally active HPV as demonstrated by E6/E7 RNA in situ hybridization. Furthermore, the patient had an excellent response to radiation treatment.
The Journal of Molecular Diagnostics, 14(1), 22–29.
Wang, F, Flanagan, J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y. (2012).
PMID: 22166544 | DOI: 10.1016/j.jmoldx.2011.08.002.
In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.
Case Reports in Otolaryngology
Brobst T, García J, Rowe Price KA, Gao G, Smith DI, Price D.
PMID: - | DOI: -
Abstract
Background:
Although alcohol and tobacco use are known risk factors for development of squamous cell carcinoma in the head and neck, human papillomavirus (HPV) has been increasingly associated with this group of cancers. We describe the case of a married couple who presented with HPV-positive oropharynx squamous cell carcinoma within two months of each other.
Methods:
Tumor biopsies were positive for p16 and high-risk HPV in both patients. Sanger sequencing showed a nearly identical HPV16 strain in both patients. Both patients received chemoradiation, and one patient also underwent transoral robotic tongue base resection with bilateral neck dissection.
Results:
Both patients showed no evidence of recurrent disease on follow-up PET imaging.
Conclusions:
New head and neck symptoms should be promptly evaluated in the partner of a patient with known HPV-positive oropharynx cancer. This case expands the limited current literature on concurrent presentation of HPV-positive oropharynx squamous cell carcinoma in couples.
Tendron, A;Classe, M;Casiraghi, O;Pere, H;Even, C;Gorphe, P;Moya-Plana, A;
PMID: 35454782 | DOI: 10.3390/cancers14081874
Sinonasal squamous cell carcinoma (SNSCC) is a rare and aggressive malignancy with poor prognosis. Human papilloma virus (HPV) can induce SNSCC although its incidence and impact on patients' outcomes remains unclear. We performed a retrospective cohort study of patients with SNSCC treated consecutively in a comprehensive cancer center. HPV status was determined with p16 immunohistochemistry followed by RNA in situ hybridization (RNAscope). The incidence, clinical characteristics, and oncologic outcomes of HPV+SNSCC were assessed. P16 prognostic value was evaluated. Fifty-nine patients were included. Eleven (18.6%) SNSCC were p16+ with five (8.4%) doubtful cases. RNAscope was positive in nine cases (15.2%). Patients with HPV+SNSCC were younger (p = 0.0298) with a primary tumor originating mainly in nasal fossa (p < 10-4). Pathologic findings were not different according to HPV status. Among patients who were curatively treated, overall survival was better for HPV+SNSCC (p = 0.022). No prognostic value of p16 expression was reported. Patients with HPV+SNSCC have better oncologic outcomes, probably due to earlier tumor stage with primary location predominantly in the nasal fossa, a more suitable epicenter to perform a surgical resection with clear margins. P16 expression seems not to be a good surrogate of HPV status in SNSCC.
Bishop JA, Westra WH.
PMID: 26457358 | DOI: 10.1097/PAS.0000000000000521.
Although human papillomavirus (HPV)-related oropharyngeal carcinomas (HPV-OPCs) are generally regarded as "poorly differentiated," they actually maintain a close resemblance to the lymphoepithelium of the tonsillar crypts from which they arise: they are basaloid, exhibit minimal keratinization, and are often permeated by lymphocytes. In rare cases, the presence of cilia in a primary HPV-OPC and their persistence in lymph node metastasis can confound the distinction between a benign and malignant process. Three cases of ciliated HPV-OPCs were identified from the archives of The Johns Hopkins Head and Neck Pathology consultation service. HPV status was determined using p16 immunohistochemistry and high-risk HPV in situ hybridization. All 3 patients presented with a cystic lymph node metastasis without a known primary carcinoma. One metastasis was originally diagnosed as a branchial cleft cyst only to regionally recur 7 years later. In 2 cases, a primary HPV-OPC was found in the tonsil. The carcinomas exhibited both nonkeratinizing squamous epithelium and cystic/microcystic spaces lined by ciliated columnar cells. Both the squamous and ciliated cells were HPV positive. This report draws attention to a novel variant of HPV-related head and neck cancer that exhibits ciliated columnar cells. This variant challenges prevailing notions that: (1) HPV-OPCs are uniformly poorly differentiated cancers; (2) cilia are an infallible feature of benignancy; and (3) presence of cilia is a reliable criterion for establishing branchial cleft origin when dealing with cystic lesions of the lateral neck.
Montella, M;Sabetta, R;Ronchi, A;De Sio, M;Arcaniolo, D;De Vita, F;Tirino, G;Caputo, A;D'Antonio, A;Fiorentino, F;Facchini, G;Lauro, GD;Perdonà, S;Ventriglia, J;Aquino, G;Feroce, F;Borges Dos Reis, R;Neder, L;Brunelli, M;Franco, R;Zito Marino, F;
PMID: 35592855 | DOI: 10.3389/fmed.2022.874213
Penile cancer (PC) is an extremely rare malignancy, and the patients at advanced stages have currently limited treatment options with disappointing results. Immune checkpoint inhibitors anti-programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) are currently changing the treatment of several tumors. Furthermore, the microsatellite instability (MSI) and the deficient mismatch repair system (dMMR) proteins represent predictive biomarkers for response to immune checkpoint therapy. Until present, few data have been reported related to PD-L1 expression and MSI in PC. The main aim of our study was the evaluation of PD-L1 expression in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) in immune cells and the analysis of dMMR/MSI status in a large series of PCs.A series of 72 PC, including 65 usual squamous cell carcinoma (USCC), 1 verrucous, 4 basaloid, 1 warty, and 1 mixed (warty-basaloid), was collected. Immunohistochemistry (IHC) was performed to assess PD-L1 expression using two different anti-PD-L1 antibodies (clone SP263 and SP142 Ventana) and MMR proteins expression using anti-MLH1, anti-PMS2, anti-MSH2, and anti-MSH6 antibodies. PCR analysis was performed for the detection of MSI status.Of the 72 PC cases analyzed by IHC, 45 (62.5%) cases were TC positive and 57 (79%) cases were combined positive score (CPS) using PDL1 SP263. In our cohort, TILs were present in 62 out of 72 cases (86.1%), 47 (75.8%) out of 62 cases showed positivity to PDL1 clone SP142. In our series, 59 cases (82%) had pMMR, 12 cases (16.7%) had lo-paMMR, and only 1 case (1.3%) had MMR. PCR results showed that only one case lo-paMMR was MSI-H, and the case dMMR by IHC not confirmed MSI status.Our findings showed that PD-L1 expression and MSI status represent frequent biological events in this tumor suggesting a rationale for a new frontier in the treatment of patients with PC based on the immune checkpoint inhibitors.
Journal of Medical Virology
Phusingha P, Ekalaksananan T, Vatanasapt P, Loyha K, Promthet S, Kongyingyoes B, Patarapadungkit N, Chuerduangphui J, Pientong C.
PMID: 27935063 | DOI: 10.1002/jmv.24744
Human papillomavirus (HPV) is an independent risk factor for development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of HPV infection and the trend in percentage of HPV-associated OSCC over a five-year period in northeastern Thailand. In this case-control study, 91 exfoliated oral cell samples and 80 lesion cell samples from OSCC cases and exfoliated oral cells from 100 age/gender-matched controls were collected. HPV infection was investigated by PCR using GP5+/GP6+ primers followed by HPV genotyping using reverse line blot hybridization. Quantitative RT-PCR was used to evaluate HPV oncogene transcription. Temporal trends of HPVinfection were evaluated in archived formalin-fixed paraffin-embedded (FFPE) OSCC tissues using in situ hybridization. HPV DNA was found in 17.5% (14/80) of lesion samples from OSCC cases and 29.7% (27/91) of exfoliated oral cell samples from the same cases. These values were significantly higher than in exfoliated oral cell samples from controls (13%, 13/100). HPV-16 was the genotype most frequently found in OSCC cases (92.8%, 13/14 infected cases). Interestingly, HPV oncogene mRNA expression was detected and correlated with OSCC cases (P < 0.005). Of 146 archived FFPE OSCC samples, 82 (56.2%) were positive for high-risk HPV DNA and 64 (43.8%) cases were positive for HPV E6/E7 mRNA expression. There was a trend of increasing percentage of HPV-associated OSCC from 2005 to 2010. This was especially so for females with well-differentiated tumors in specific tongue sub-sites. We suggest that HPV infection plays an important role in oral carcinogenesis in northeastern Thailand.
Human papillomavirus (HPV) oncogenic activity is the result of viral oncogene E6 and E7 expression in infected cells. Oncogene expression analysis is however not part of the routine diagnostic evaluation of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) since it requires fresh tumor tissue. We compared the diagnostic accuracy of several methods commonly employed for HPV characterization in OPSCC with the results of the newly available HPV E6/E7 mRNA in situ hybridization (ISH) on formalin-fixed, paraffin-embedded biopsy samples, in order to establish if the latter should be introduced in the diagnostic routine to increase accuracy when fresh tissue is not available. p16 immunostain, DNA ISH for high risk (HR) HPV genotypes, SPF LiPA amplification and genotyping, and HPV16 E6 amplification were performed on 41 consecutive OPSCC samples. Twenty (48,7%) cases were positive by mRNA ISH; sensitivity and specificity were 100% and 90% for p16, 90% and 100% for DNA ISH, 70% and 76% for SPF10 LiPA, 90% and 76% for E6 amplification. A diagnostic algorithm considering p16 immunostain as first step followed by either HR HPV DNA ISH or HPV16 E6 amplification in p16-positive cases correctly characterized 90% of mRNA-positive and all mRNA-negative cases; combining the 3 tests correctly identified all cases. While no stand-alone test was sufficiently accurate for classifying HPV-associated OPSCC, the high sensitivity and specificity of the established combination of p16 immunostain, DNA ISH and HPV16 DNA amplification suggests that the introduction of labour- and cost-intensive mRNA ISH, is not necessary in the diagnostic routine of oropharyngeal tumors.
PLoS One. 2014 Mar 13;9(3):e91142
Evans MF, Peng Z, Clark KM, Adamson CSC, Ma XJ, Wu X, Wang H, Luo Y, Cooper K
PMID: 24625757 | DOI: 10.1371/journal.pone.0091142.eCollection2014.
Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay’s high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when ‘LSIL vs. HSIL’ assignment is equivocal.
