精准医学杂志

2021 Jan 01

李志鹏, ;范志伟, ;王文龙, ;卜祥斌, ;张凌楠, ;
| DOI: 10.13362/j.pmed.202104024

不同于其他头颈部鳞状细胞癌,人乳头状瘤病毒(HPV)相关口咽鳞状细胞癌(OPSCC)是一类具有不同分子生物学发病机制、危险因素、临床病理特征的特殊疾病。HPV 阳性 OPSCC患者的预后明显优于 HPV 阴性患者,因此 HPV 的检测对 OPSCC的诊断和治疗尤为重要。目前,国内外针对 OPSCC中 HPV 生物分子标志物的检测方法多种多样,但仍然缺乏一个在该领域达成共识的规范化检测标准。本文围绕 OPSCC中 HPV 生物分子标志物的研究现状做一总结,旨在为实验室以及临床上选择适合的生物分子标志物进行 OPSCC诊断提供借鉴。

Detection accuracy of the Cobas HPV assay for high-risk HPV in head and neck FNA biopsy specimens

Cancer cytopathology

2022 Feb 22

Guo, M;Khanna, A;Tinnirello, AA;Hwang, J;Zhang, P;Xu, L;Li, G;Dahlstrom, KR;Sturgis, EM;Stewart, J;
PMID: 35192231 | DOI: 10.1002/cncy.22563

This study evaluated the detection accuracy of the Cobas human papillomavirus (HPV) assay for high-risk human papillomavirus (hrHPV) and HPV-16 in head and neck fine-needle aspiration (FNA) specimens with squamous cell carcinoma.Head and neck FNA biopsy specimens from 2012 to 2020 were retrospectively collected. Cobas HPV testing was performed on 90 FNA specimens with valid Cervista HPV testing results. Results of Cobas HPV and Cervista HPV assays were compared. A Linear Array or SPF10-LiPA25 HPV genotyping assay resolved cases with discrepant results. The κ value and accuracy of Cobas HPV testing were calculated. The accuracy of the Cobas HPV assay was also determined in 42 FNA needle-rinse specimens.Cobas HPV was positive in 82% of the FNA specimens (74 of 90). The concordance between Cobas HPV and Cervista HPV test results was 88.9% (80 of 90) with substantial agreement (κ = 0.669; 95% CI, 0.481-0.856). With HPV genotyping confirmation in cases with discrepant results between the 2 HPV assays, Cobas HPV showed 100% sensitivity and specificity for hrHPV. HPV-16 was detected in 88% of HPV-positive cases (65 of 74). HPV genotyping confirmed 1 false-negative HPV-16 result and 1 false-positive HPV-16 result. Overall, the accuracy of Cobas HPV for HPV-16 was 97.8%. The accuracy of Cobas HPV in FNA needle-rinse specimens was 100%.The Cobas HPV assay is highly accurate for determining the HPV status in head and neck FNA specimens. FNA needle rinse is valid for Cobas HPV testing in patients with squamous cell carcinoma.

Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia

Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology

2016 Jun 22

Bhosale PG, Pandey M, Desai RS, Patil A, Kane S, Prabhash K, Mahimkar MB.
PMID: - | DOI: 10.1016/j.oooo.2016.06.006

Abstract

Objectives

In the present study, we comprehensively analyzed the prevalence of transcriptionally active HPV in tissue samples of Indian patients with leukoplakia - predominantly hyperplastic lesions and HNSCC. In addition, saliva samples from patients with HNSCC were screened for HPV detection.

Study Design

p16 overexpression was analyzed by immunohistochemistry. Leukoplakia (n = 121) and HNSCC (n = 427) tissue samples and the saliva of patients with HNSCC (n = 215) were tested for HPV using nested PCR. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in-situ hybridization.

Results

p16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, either in saliva and/or tissue. E6/E7 mRNA positivity was observed in eight HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx with very little concordance between p16 overexpression and HPV positivity. All patients with HPV positive saliva samples had transcriptionally active HPV present in their tumors.

Conclusion

Presence of HPV-DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV associated tumors.

Human papillomavirus infection and its biomarkers' expressions in laryngeal basaloid squamous cell carcinoma.

J Int J Clin Exp Pathol (2018)

2018 Nov 15

Cui L, Qu C, Liu H.
| DOI: ISSN:1936-2625/IJCEP0085220

Abstract: Aims: To investigate the frequency and transcriptional activity of HPV and its correlation to p16 and p21 expression in basaloid squamous cell carcinoma (BSCC) of the larynx. Methods: We evaluated tissues from 29 patients with BSCC of the larynx for the expressions of p16 and p21 proteins by immunohistochemistry (IHC) and for HPV E6 and E7 mRNA by RNA in situ hybridization (ISH). The presence of genotype-specific HPV DNA was evaluated using PCR-RDB in formalin-fixed paraffin-embedded tissues. P16 and p21 expression and HPV DNA status were correlated with clinicopathological features. Results: HPV DNA was detected in 8 of 29 (27.59%) patients, with HPV-16 being the predominant genotype. P16 and p21-positivity were observed in 7/29 (24.14%) and 8/29 (27.59%) patients, respectively. HPV was not correlated with p16 expression (P > 0.05). However, p21 expression was significantly higher in HPV-positive tumors than in HPV-negative tumors (P < 0.05). No cases exhibited transcriptionally active HPV in our series. Conclusion: Our findings suggest that a small fraction of BSCC of the larynx is HPV DNA-positive in this Chinese population, p21 expression was significantly higher in HPV-positive tumors, and no cases were HPV transcriptionally active in this small cohort. Further research of HPV and its role in BSCC of the larynx are warranted.

Defining the better algorithm for the accurate identification of HPV status among oropharyngeal squamous-cell carcinoma. Results from a pilot study

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.

Detection of Human Papillomavirus in Squamous Lesions of the Conjunctiva Using RNA and DNA In-Situ Hybridization

International journal of molecular sciences

2022 Jun 29

Peterson, C;Parikh, RN;Ahmad, MT;Campbell, AA;Daoud, Y;Mahoney, N;Siadati, S;Eberhart, CG;
PMID: 35806252 | DOI: 10.3390/ijms23137249

In-situ hybridization provides a convenient and reliable method to detect human papillomavirus (HPV) infection in formalin-fixed paraffin-embedded tissue. Cases of conjunctival papillomas, conjunctival intraepithelial neoplasia (CIN), conjunctival carcinoma in situ (cCIS), and invasive squamous cell carcinoma (SCC), in which low-risk (LR) and/or high-risk (HR) HPV types were evaluated by RNA or DNA in-situ hybridization, were retrospectively identified. LR HPV types were frequently detected in conjunctival papillomas (25/30, 83%), including 17/18 (94%) with RNA probes, compared to 8/12 (75%) with DNA probes. None of the CIN/cCIS or SCC cases were positive for LR HPV by either method. HR HPV was detected by RNA in-situ hybridization in 1/16 (6%) of CIN/cCIS cases and 2/4 (50%) of SCC cases, while DNA in-situ hybridization failed to detect HPV infection in any of the CIN/cCIS lesions. Reactive atypia and dysplasia observed in papillomas was generally associated with the detection of LR HPV types. Collectively, our findings indicate RNA in-situ hybridization may provide a high-sensitivity approach for identifying HPV infection in squamous lesions of the conjunctiva and facilitate the distinction between reactive atypia and true dysplasia. There was no clear association between HPV infection and atopy in papillomas or dysplastic lesions.

Am J Otolaryngol. 2014 Jan-Feb;35(1):25-32.

Melkane AE, Mirghani H, Aupérin A, Saulnier P, Lacroix L, Vielh P, Casiraghi O, Griscelli F, Temam S.
PMID: 24112760 | DOI: 10.1016/j.amjoto.2013.08.007.

PURPOSE: HPV-related oropharyngeal squamous cell carcinomas clearly represent a growing entity in the head and neck with distinct carcinogenesis, clinico-pathological presentation and survival profile. We aimed to compare the HPV prevalence rates and clinico-pathological correlations obtained with three distinct commonly used HPV detection methods. MATERIALS AND METHODS: p16-immunohistochemistry (IHC), HPV DNA viral load by real-time PCR (qPCR), and HPV genotyping by a reverse hybridization-based line probe assay (INNO-LiPA) were performed on pretreatment formalin-fixed paraffin-embedded tumor samples from 46 patients treated for single primary oropharyngeal carcinomas. RESULTS: Twenty-eight patients (61%) had a p16 overexpression in IHC. Twenty-nine patients (63%) harbored HPV DNA on qPCR. Thirty-four patients (74%) harbored HPV DNA on INNO-LiPA. The concordance analysis revealed a good agreement between both HPV DNA detection methods (κ=0.65); when both tests were positive, the depicted HPV subtypes were always concordant (HPV16 in 27 cases, HPV18 in 1 case). Agreement was moderate between IHC and qPCR (κ=0.59) and fair between IHC and INNO-LiPA (κ=0.22). CONCLUSIONS: Certain highly sensitive methods are able to detect the mere presence of HPV without any carcinogenetic involvement while other more specific tests provide proof of viral transcriptional activity and thus evidence of clinically relevant infections. The use of a stepwise approach allows reducing false positives; p16-immunostaining seems to be an excellent screening test and in situ hybridization may overcome some of the PCR limitations.

"Transcriptionally Active High-Risk Human Papillomavirus is Not a Common Etiologic Agent in the Malignant Transformation of Inverted Schneiderian Papillomas. "

Head Neck Pathol.

2017 Feb 08

Rooper LM, Bishop JA, Westra WH.
PMID: 28181187 | DOI: 10.1007/s12105-017-0779-0

The role of human papillomavirus (HPV) as an etiologic and transformational agent in inverted Schneiderian papilloma (ISP) is unclear. Indeed, reported detection rates of HPV in ISPs range from 0 to 100%. The true incidence has been confounded by a tendency to conflate high- and low-risk HPV types and by the inability to discern biologically relevant from irrelevant HPV infections. The recent development of RNA in situ hybridization for high-risk HPV E6/E7 mRNA now allows the direct visualization of transcriptionally active high-risk HPV in ISP, providing an opportunity to more definitively assess its role in the development and progression of ISPs. We performed p16 immunohistochemistry and high-risk HPV RNA in situ hybridization on 30 benign ISPs, 7 ISPs with dysplasia, 16 ISPs with carcinomatous transformation, and 7 non-keratinizing squamous cell carcinomas (SCCs) with inverted growth that were unassociated with ISP. Transcriptionally active HPV was not detected in any of the 52 ISPs including those that had undergone carcinomatous transformation, but it was detected in two of seven (29%) non-keratinizing SCCs that showed inverted growth. There was a strong correlation between high-risk HPV RNA in situ hybridization and p16 immunohistochemistry (97%; p < 0.01). These results indicate that transcriptionally active high-risk HPV does not play a common role in either the development of ISP or in its transformation into carcinoma.

Evaluation of Seegene Anyplex II Performance for Detection of Human Papillomavirus Genotypes in Formalin-Fixed, Paraffin-Embedded Cervical Cancer Specimens

Archives of pathology & laboratory medicine

2023 May 23

Haqshenas, G;Molano, M;Phillips, S;Balgovind, P;Garland, SM;Hawkes, D;Brotherton, JM;Machalek, DA;Murray, G;
PMID: 37226838 | DOI: 10.5858/arpa.2022-0317-OA

Detection of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) tissues may identify the cause of lesions and has value for the development of new diagnostic assays and epidemiologic studies. Seegene Anyplex II assays are widely used for HPV screening, but their performance using FFPE samples has not been fully explored.To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples.We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005-2015 and that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples.Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7-98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9-99.3).Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.

Prognostic utility of HPV specific testing in addition to p16 immunohistochemistry in oropharyngeal squamous cell carcinoma.

Ann Oncol.

2018 Aug 08

Sathasivam HP, Santambrogio A, Andoniadou CL, Robinson M, Thavaraj S.
PMID: 30101315 | DOI: 10.1093/annonc/mdy313

Detection of human papillomavirus (HPV) in clinical samples: Evolving methods and strategies for the accurate determination of HPV status of head and neck carcinomas

Oral Oncology, 2014 - Elsevier

Westra WH
PMID: 24932529 | DOI: 10.1016/j.oraloncology.2014.05.004

Much recent attention has highlighted a subset of head and neck squamous cell carcinomas (HNSCCs) related to human papillomavirus (HPV) that has an epidemiologic, demographic, molecular and clinical profile which is distinct from non-HPV-related HNSCC. The clinical significance of detecting HPV in a HNSCC has resulted in a growing expectation for HPV testing of HNSCCs. Although the growing demand for routine testing is understandable and appropriate, it has impelled an undisciplined approach that has been largely unsystematic. The current state of the art has now arrived at a point where a better understanding of HPV-related tumorigenesis and a growing experience with HPV testing can now move wide scale, indiscriminant and non-standardized testing towards a more directed, clinically relevant and standardized approach. This review will address the current state of HPV detection; and will focus on why HPV testing is important, when HPV testing is appropriate, and how to test for the presence of HPV in various clinical samples. As no single test has been universally accepted as a best method, this review will consider the strengths and weaknesses of some of the more commonly used assays, and will emphasize some emerging techniques that may improve the efficiency of HPV testing of clinical samples including cytologic specimens.

In situ hybridization detection methods for HPV16 E6/E7 mRNA in identifying transcriptionally active HPV infection of oropharyngeal carcinoma: an updating

Human Pathology

2017 Oct 06

Volpi CC, Ciniselli CM, Gualeni AV, Plebani M, Alfieri S, Verderio P, Locati L, Perrone F, Quattronea P, Carbone A, Pilotti S, Gloghini A.
PMID: 28993274 | DOI: 10.1016/j.humpath.2017.09.011

The aim of this study is comparing two in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, i.e. the RNAscope™ 2.0 High Definition (HD) and the upgraded RNAscope™ 2.5 HD version. The RNAscope™ 2.5 HD has recently replaced the RNAscope™ 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma (OSCC) with the final goal to apply it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify OSCC that are truly driven by high risk-HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real time reverse transcriptase-polymerase chain reaction (RT-PCR), in a fraction of cases where material for HPV E6/E7 mRNA real time RT-PCR was available. Furthermore, the algorithm that associates p16 immunohistochemistry (IHC) with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 IHC with the identification of HPV DNA by ISH.

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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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