Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Summary

Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

Abstract

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since "low" or "high" HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Introduction

HPV RNA CISH is a powerful tool for the detection of active HPV infection, which may prove crucial in benign or malignant lesions in various locations such as the oropharynx or the uterine cervix. The detection of an active HPV infection may support the diagnosis of an HPV-induced lesion and, thereby, influence its treatment and prognosis.

HPV is the most frequent sexually transmitted infection, and over 100 viral genotypes have been described¹. Schematically, low-risk genotypes such as genotypes 6 and 11 are known to induce genital warts, recurrent respiratory papillomatosis, and other benign lesions, whereas high-r....

Protocol

The protocol follows ethical guidelines and was approved by the Ethical Committee (Comité-de-Protection-des-Personnes Ile-de-France-II, #2015-09-04).

1. Preparation of the materials

1. Preparation of 1x wash buffer
   1. Prepare 3 L of 1x wash buffer by adding 2.94 L of distilled water and one bottle (60 mL) of wash buffer (50x) (see the Table of Materials) to a large carboy. Mix well.
      NOTE: The 1x wash buffer may be prepared ahead of time and stored at room temperature for up to 1 month.
2. Preparation of counterstaining reagents

Results

As described here, in head and neck squamous cell cancer, a case may be considered positive in the presence of brown punctiform staining in the cytoplasm or in the nuclei of tumor cells. In most studies, the signal is considered as either “positive” or “not detected”¹⁴. Methods of semiquantification of the signal have been reported but lack standardization between teams. For example, in some studies, the signals were scored as 1+ with 1–3 dots per tumor cell, 2+ with 4–9 dots per tumor cell, or 3+.......

Discussion

HPV RNA CISH performed with a purchased kit is a powerful tool for the detection of viral transcripts and it indicates active HPV infection. Performed manually, the steps of the protocol are overall easy to follow, and the purchased kit is convenient. This technique allows the staining of 19 histological samples plus one control slide at once, and the assay lasts around 8 h. It is critical not to let the samples dry out between steps unless otherwise mentioned. The pretreatment condition may need to be adjusted depending.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hematoxylin solution, Gill No. 1	Merck	GHS132
HybEZ Oven (110v)	Advanced Cell Diagnostics Inc.	321710
HybEZ slide rack	Advanced Cell Diagnostics Inc.	300104
ImmEdge Hydrophobic Barrier Pen	Advanced Cell Diagnostics Inc.	310018
RNAscope 2.5 HD Detection Reagents-BROWN	Advanced Cell Diagnostics Inc.	322310	This kit includes amplification reagents AMP1, AMP2, AMP3, AMP4, AMP5 and AMP6, and detection reagents DAB-A and DAB-B
RNAscope 3-Plex Negative Control Probe	Advanced Cell Diagnostics Inc.	320871	DAPB
RNAscope 3-Plex Positive Control Probe	Advanced Cell Diagnostics Inc.	320861	PPIB
RNAscope H202 & Protease Plus Reagent	Advanced Cell Diagnostics Inc.	322330	Hydrogen Peroxyde x2 and Protease Plus x 1
RNAscope Probe- HPV16/18	Advanced Cell Diagnostics Inc.	311121
RNAscope Target Retrieval Reagents	Advanced Cell Diagnostics Inc.	322000
RNAscope Wash Buffer Reagents	Advanced Cell Diagnostics Inc.	310091	Wash Buffer 50X x4