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. 2020 Jul 14:15:46.
doi: 10.1186/s13027-020-00310-x. eCollection 2020.

Multiplex HPV RNA in situ hybridization/p16 immunohistochemistry: a novel approach to detect papillomavirus in HPV-related cancers. A novel multiplex ISH/IHC assay to detect HPV

Federica Zito Marino  1 , Andrea Ronchi  1 , Marianna Stilo  1 , Immacolata Cozzolino  1 , Elvira La Mantia  1 , Nicola Colacurci  2 , Giuseppe Colella  3 , Renato Franco  1
Affiliations

Multiplex HPV RNA in situ hybridization/p16 immunohistochemistry: a novel approach to detect papillomavirus in HPV-related cancers. A novel multiplex ISH/IHC assay to detect HPV

Federica Zito Marino et al. Infect Agent Cancer. .

Abstract

Background: High-risk human papillomavirus (HR-HPV) is notoriously associated with tumor progression in a broad spectrum of malignancies. Detection of HR-HPV is clinically important in the management of HPV-related carcinomas, particularly in cervical cancer and oropharyngeal squamous cell carcinoma (OPSCC). Several methods for HPV detection are currently available including Polymerase chain reaction (PCR)-based techniques, DNA in situ hybridization (ISH), RNA ISH, and p16 immunohistochemistry (IHC). Currently, the guidelines for HPV detection in cervical carcinoma are available, while no clear consensus has not yet been reached on the gold standard for HPV testing in OPSCC. Multimodality testing could help to reliably identify patients with transcriptionally active high-risk HPV-positive.

Methods: We propose a multiplex approach carrying out HPV RNA ISH and p16 IHC on the same slide to detect simultaneously HPV E6/E7 transcripts and p16INK4a overexpression. We tested this assay in two different series one of the cervical cancers with p16-positive, as control, and the other of oropharyngeal squamous cell carcinomas with blind p16 status.

Results: The multiplex HPV RNA ISH /p16 IHC results in the series both of the cervical cancers and the oral-oropharyngeal cancers were fully concordant with the previous results achieved through the classic p16 IHC and HPV RNA scope carried out on two different slides.

Conclusions: Our results suggesting several advantages of this technical approach, namely an easy interpretation fully in the light field, the feasibility in formalin-fixed paraffin-embedded tissue sections, complete automation and a potential wide spreadable for routine testing in several clinical laboratories.

Keywords: Cervical Cancer and oral cancer; HPV; ISH; p16.

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Conflict of interest statement

Competing interestsNo competing interest for each author.

Figures

Fig. 1
Fig. 1
Schematic workflow of multiplex HPV RNA ISH/p16 IHC assay
Fig. 2
Fig. 2
Representative results of a cervical cancer with high p16 and HPV RNA expression. a positive p16 IHC, DAB staining (100x); b positive HPV RNA in situ hybridization, DAB staining (100x); c-d multiplex HPV RNA ISH/p16 IHC: positive p16 IHC Fast Red staining and positive HPV RNA ISH DAB staining (50x)
Fig. 3
Fig. 3
Representative results of an oropharyngeal squamous cell carcinoma with high p16 and HPV RNA expression. a positive p16 IHC, DAB staining (100x); b positive HPV RNA in situ hybridization, DAB staining (50x); c multiplex HPV RNA ISH/p16 IHC: positive p16 IHC Fast Red staining and positive HPV RNA ISH DAB staining (100x); d multiplex HPV RNA ISH/p16 IHC: positive p16 IHC Fast Red staining and positive HPV RNA ISH DAB staining (50x)
Fig. 4
Fig. 4
Representative results of an oropharyngeal squamous cell carcinoma with low p16 and HPV RNA expression. a positive p16 IHC, DAB staining (50x); b positive HPV RNA in situ hybridization, DAB staining (50x); c-d multiplex HPV RNA ISH/p16 IHC: positive p16 IHC Fast Red staining and positive HPV RNA ISH DAB staining (50x)
Fig. 5
Fig. 5
HPV diagnostic alghoritm for oropharyngeal squamous cell carcinoma OPSCC) including the multiplex HPV RNA ISH/p16 IHC assay
See this image and copyright information in PMC

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