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Oral Oncology
2016 Oct 15
Mirghani H, Casiraghi O, Guerlain J, Amen F, He MX, Ma XJ, Luo Y, Mourareau C, Drusch F, Lakdhar AB, Melkane A, St Guily L, Badoual C, Scoazec JY, Borget I, Aupérin A, Dalstein V, Vielh P.
PMID: - | DOI: http://dx.doi.org/10.1016/j.oraloncology.2016.10.009
Abstract
Background
Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status.
Methods
To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2).
Results
105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%.
The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07–0.51, p = 0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08–0.66, p = 0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1–0.65, p = 0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13–0.81, p = 0.02).
Conclusions
Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.
Am J Surg Pathol.
2017 May 01
Mills AM, Dirks DC, Poulter MD, Mills SE, Stoler MH.
PMID: 28403015 | DOI: 10.1097/PAS.0000000000000800
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.
Virchows Arch. 2015 Jul 31.
Laco J, Sieglová K, Vošmiková H, Dundr P, Němejcová K, Michálek J, Čelakovský P, Chrobok V, Mottl R, Mottlová A, Tuček L, Slezák R, Chmelařová M, Sirák I, Vošmik M, Ryška A.
PMID: 26229021
The aim of the study was to investigate prevalence of high-risk human papillomavirus (HR-HPV) infection in sinonasal carcinomas by immunohistochemistry, in situ hybridization, and polymerase chain reaction, detecting p16INK4a protein (p16) expression and presence of both HPV DNA and HPV E6/E7 messenger RNA (mRNA). The study comprised 47 males and 26 females, aged 23-83 years (median 62 years), mostly (67 %) with a squamous cell carcinoma (SCC). Of the tumors, 53 % arose in the nasal cavity, 42 % in the maxillary sinus, and 5 % in the ethmoid complex. The follow-up period ranged 1-241 months (median 19 months). HPV16, HPV18, or HPV35 were detected in 18/73 (25 %) tumors, 17 SCCs, and 1 small cell neuroendocrine carcinoma. There was a strong correlation between results of HPV detection methods and p16 expression (p < 0.005). HPV-positive SCCs occurred more frequently in smokers (p = 0.04) and were more frequently p16-positive (p < 0.0001) and nonkeratinizing (p = 0.02), the latter occurring more commonly in nasal cavity (p = 0.025). Median survival for HPV-positive SCC patients was 30 months, while for HPV-negative SCC patients was 14 months (p = 0.23). In summary, we confirm that HR-HPV is actively involved in the etiopathogenesis of a significant subset of sinonasal SCCs. p16 may be used as a reliable surrogate marker for determination of HPV status also in sinonasal SCCs. Although we observed a trend toward better overall survival in HPV-positive SCCs, the prognostic impact of HPV status in sinonasal carcinomas needs to be elucidated by further studies.
Head and neck pathology, ;7(3):250–257.
Watson RF, Chernock RD, Wang X, Liu W, Ma XJ, Luo Y, Wang H, El-Mofty SK, Lewis JS Jr (2013).
PMID: 23536041 | DOI: 10.1007/s12105-013-0438-z.
Spindle cell carcinoma is an uncommon variant of squamous cell carcinoma characterized by spindled or pleomorphic cells which appear to be a true sarcoma but are actually epithelial. Some head and neck squamous cell carcinoma variants can be human papillomavirus (HPV)-related and have improved outcomes. We sought to determine if spindle cell carcinomas are associated with transcriptionally-active HPV. Cases of spindle cell carcinoma were retrieved from department files. Transcriptionally-active HPV was determined by mRNA in situ hybridization for high risk HPV E6 and E7 transcripts and by a surrogate marker, p16 immunohistochemistry, with a 50% staining cutoff. RT-PCR for high risk HPV mRNA was performed on the cases that were technical failures by in situ hybridization. Medical records and follow up information were retrieved for all patients. Of 31 cases, 5 were from the oropharynx, 12 from the oral cavity, and 14 from the larynx or hypopharynx. One purely spindled oral cavity spindle cell carcinoma was HPV positive. It was also diffusely positive for p16. Another laryngeal spindle cell carcinoma was HPV positive in both the squamous and spindle cell components, but was negative for p16. None of the five oropharyngeal spindle cell carcinomas were positive for p16 or HPV RNA. The HPV positive patients both presented at high stage (IV) and died with disease within 2 years of diagnosis. The majority of spindle cell carcinomas of the head and neck, including those arising in the oropharynx, are not related to transcriptionally active HPV. Although the number of cases is too small for any definitive conclusions, for the rare HPV positive spindle cell carcinoma cases, positive viral status does not appear to confer any prognostic benefit.
Head & Neck Pathology, 5(2):108–116.
Masand RP, El-Mofty SK, Ma XJ, Luo Y, Flanagan JJ, Lewis JS Jr (2011).
PMID: 21305368 | DOI: 10.1007/s12105-011-0245-3.
Adenosquamous carcinoma (ADSC) of the head and neck is an aggressive variant of squamous cell carcinoma (SCC). Certain variants of head and neck SCC are human papillomavirus (HPV)-related and have better prognosis. The relationship of HPV to head and neck ADSC has not been investigated. We searched our files for the term "adenosquamous" and head and neck subsites and found cases from 1998 to 2009. The requisite histologic criteria were the presence of SCC combined with distinct gland formation and/or intracellular mucin. DNA in situ hybridization for high-risk HPV, RNA in situ hybridization for high risk HPV E6 and E7 transcripts, and immunohistochemistry for p16 and p53 were performed. The existing literature on ADSC was also reviewed. Of the 18 cases, eight were from the larynx and hypopharynx, four from the oral cavity, three from the oropharynx, and three from the nasal cavity. Three cases (16%) showed both high risk HPV E6 and E7 and p16 expression, one from the nasal cavity and two from the oropharynx. Both oropharyngeal carcinoma patients were alive and disease free at 34 and 103 months, respectively. ADSCs of the head and neck are a heterogeneous group of tumors. A small minority of cases harbor HPV and most of these, particularly those occurring at sites with known high prevalence of HPV, show active viral transcription with detectable E6 and E7 and overexpression of p16. The HPV-related oropharyngeal cases, though rare, appear to do very well clinically, while the remaining cohort of ADSC patients do quite poorly. Head and neck ADSC appears to be a mixed variant that can be further classified according to its HPV status.
Mod Pathol.
2015 Sep 25
Mirghani H, Casiraghi O, Amen F, He M, Ma XJ, Saulnier P, Lacroix L, Drusch F, Ben Lakdhar A, Saint Guily JL, Badoual C, Scoazec JY, Vielh P.
PMID: 26403782 | DOI: 10.1038/modpathol.2015.113
Abstract
Accurate screening of HPV-driven head and neck squamous cell carcinoma is a critical issue. Although there are commercial direct and indirect assays for HPV-related head and neck squamous cell carcinoma, none are ideal. Recently, a novel RNA in situ hybridization test (the RNAscope HPV-test) has been developed for the detection of high-risk HPV E6/E7 mRNA in formalin-fixed paraffin-embedded tissue. However, validation of this assay against the 'gold standard' (identification of high-risk HPV E6/E7 mRNA in fresh-frozen tissue by quantitative real-time (qRT)-PCR) has only been reported by one team. Formalin-fixed paraffin-embedded samples from 50 patients with tonsil or tongue base carcinoma were tested using the RNAscope HPV-test, p16 immunohistochemistry, and chromogenic in situ hybridization for high-risk HPV-DNA. The results were compared with those of qRT-PCR on matched fresh-frozen samples. Compared with the reference test, the sensitivity, specificity, positive, and negative predictive values of the RNAscope HPV-test and of p16 immunohistochemistry were 93%, 94%, 96%, 88% and 96%, 93%, 96%, and 93%, respectively. Five cases were discrepant between the RNAscope HPV-test and p16-immunohistochemisrty. The RNAscope HPV-test demonstrated excellent analytical performance against the 'gold standard' and is easier to interpret than chromogenic in situ hybridization. p16-immunohistochemistry also performed very well, however its main weakness is that it is an indirect marker of the presence of HPV. These data suggest that the RNAscope HPV-test is a promising test that could be developed as a clinical standard for the precise identification of HPV-driven oropharyngeal squamous cell carcinoma.
Pathology - Research and Practice
2016 Sep 22
Wanga D, Fu L, Shah W, Zhang J, Yan Y, Ge X, He J, Wang Y, Xu Li.
PMID: - | DOI: dx.doi.org/10.1016/j.prp.2016.09.009
Background and aims
The causative role of high risk human papillomavirus (HR-HPV) in breast cancer development is controversial, though a number of reports have identified HR-HPV DNA in breast cancer specimens. Nevertheless, most studies to date have focused primarily on viral DNA rather than the viral transcription. The aim of this study was to investigate the presence of HR-HPV in breast cancer tissues at HPV DNA level and HPV oncogenes mRNA level by in situ hybridization (ISH).
Methods
One hundred and forty six (146) cases of breast invasive ductal carcinoma(IDC) and 83 cases of benign breast lesions were included in the study. Type specific oligonucleotide probes were used for the DNA detection of HPV 16,18 and 58 by ISH. HR-HPV oncogenes mRNA was assayed by novel RNAscope HR-HPV HR7 assay ISH. p16 protein expression was evaluated by immunohistochemistry (IHC).
Results
HR-HPV 16,18 and 58 DNA were detected in 52 out of 146 (35.6%) IDC and in 3 out of 83 (3.6%) benign breast lesions by ISH. The HR-HPV mRNAs was detected only in a few specimens with strong HPV DNA positivity(4/25) in a few scattered cancer cells with very weak punctate nuclear and/or cytoplasmic staining. p16 over-expression did not correlate with the HPV DNA positive breast cancer samples(17/52 HPVDNA+ vs 28/94 HPV DNA-, p = 0.731).
Conclusions
HR-HPVs certainly exist in breast cancer tissue with less active transcription, which implies that the causal role of HPV in breast cancer development need further study.
Histopathology.
2016 Dec 24
Zidar N, Langner C, Odar K, Hošnjak L, Kamarádová K, Daum O, Pollheimer MJ, Košorok P, Poljak M.
PMID: 28012208 | DOI: 10.1111/his.13158
AIMS:
METHODS AND RESULTS:
CONCLUSIONS:
Cancer.
2018 Apr 30
Windon MJ, D'Souza G, Rettig EM, Westra WH, van Zante A, Wang SJ, Ryan WR, Mydlarz WK, Ha PK, Miles BA, Koch W, Gourin C, Eisele DW, Fakhry C.
PMID: 29710393 | DOI: 10.1002/cncr.31385
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.
Head and neck pathology
2022 Jul 08
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.
WCRJ 2015; 2 (1): e497
Gloghini A, Volpi CC, Gualeni AV, Cortellazzi B, Perrone F, Pilotti S.
PMID: //www.wcrj.net/wp-content/uploads/2015/04/WCRJ-2015-2-1-e497-Gloghini-WCRJ.pdf
Abstract: Background: The recognition of tumor infection by human papilloma virus (HPV) in oropharyngeal squamous-cell carcinoma (OSCC) is emerging as a valid biomarker to more accurate selection of patients for specific treatment, surveillance and tumor staging. To this aim, the HPV detection strategy in OSCC must dissect between HPV that is acting as a driver of malignant transformation, and transcriptionally silent virus involved in productive infection. The aim of this study is to define the better method for the accurate identification of HPV status among OSCC. Patients and Methods: Thirty-six patients were selected for HPV status assessment combining different methods, such as immunohistochemistry (IHC) for p16, in-situ hybridization (ISH) for high risk (HR)-HPV DNA and HR-HPV E6/E7 mRNA along with real-time PCR of HPV16 E6/E7 mRNA. All these cases were originally classified as HPV negative by DNA-based ISH but p16 positive by the IHC. Results: Twenty-six cases showed concordance between methods; whereas, nine cases resulted negative for HPV E6/E7 mRNA RT-PCR but positive for HPV E6/E7 mRNA ISH. Conclusion: By considering that the bright field HPV E6/E7 mRNA ISH could be more sensitive than mRNA-based real-time RT-PCR, and that it provides the precise identification of transcriptionally active HPV infected cells, a randomized analysis to validate the robustness of this preliminary assay will be undertaken.
Diagnostic Histopathology
Moutasim KA, Robinson M, Thavaraj S.
PMID: 10.1016/j.mpdhp.2015.02.002
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.
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| Description | ||
|---|---|---|
| sense Example: Hs-LAG3-sense | Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe. | |
| Intron# Example: Mm-Htt-intron2 | Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection | |
| Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G) | A mixture of multiple probe sets targeting multiple genes or transcripts | |
| No-XSp Example: Hs-PDGFB-No-XMm | Does not cross detect with the species (Sp) | |
| XSp Example: Rn-Pde9a-XMm | designed to cross detect with the species (Sp) | |
| O# Example: Mm-Islr-O1 | Alternative design targeting different regions of the same transcript or isoforms | |
| CDS Example: Hs-SLC31A-CDS | Probe targets the protein-coding sequence only | |
| EnEm | Probe targets exons n and m | |
| En-Em | Probe targets region from exon n to exon m | |
| Retired Nomenclature | ||
| tvn Example: Hs-LEPR-tv1 | Designed to target transcript variant n | |
| ORF Example: Hs-ACVRL1-ORF | Probe targets open reading frame | |
| UTR Example: Hs-HTT-UTR-C3 | Probe targets the untranslated region (non-protein-coding region) only | |
| 5UTR Example: Hs-GNRHR-5UTR | Probe targets the 5' untranslated region only | |
| 3UTR Example: Rn-Npy1r-3UTR | Probe targets the 3' untranslated region only | |
| Pan Example: Pool | A mixture of multiple probe sets targeting multiple genes or transcripts | |
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