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.
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.
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-risk genotypes such as genotypes 16 and also 18 are responsible for most cervical cancers and anal cancers and play a role in HNSCC oncogenesis in variable proportions as accounted for by regional epidemiological data2.
Several tools are available for the detection of HPV infection. As a high-risk HPV infection leads to the expression of viral oncogenic proteins E6 and E73, the detection of E6 and E7 transcripts is widely viewed as the gold standard for active HPV infection identification4. HPV RNA CISH can be performed on FFPE samples that are quite easily obtained from patients suffering from various HPV-related diseases. Its performance has been evaluated in squamous intraepithelial neoplasia in the cervix, the anus, and the vagina, and in invasive squamous cell carcinoma in the cervix, the anus, and the upper aerodigestive tract5: it achieves a sensitivity of over 98% among HPV DNA polymerase chain reaction (PCR)-positive cases. This is slightly better than p16 immunostaining (93%) and HPV DNA in situ hybridization (DNA ISH: 97%), which are more commonly used. In another cohort of 57 patients with squamous cell carcinoma (SCC) arising from the head and neck region, the genital region, the skin, and the urinary tract, compared to HPV DNA ISH, HPV RNA CISH achieved better sensitivity (100% versus 88%) and specificity (87% versus 74%)6.
P16 immunostaining is an indirect marker reflecting cell cycle disruption that may be caused (but not exclusively) by HPV infection4,7. This cost-effective test possesses good sensitivity and a negative predictive value and is recommended as a surrogate marker of high-risk HPV infection in oropharynx cancer (OPC) by the College of American Pathologists (CAP) and by the Union for International Cancer Control (UICC)8.
Though this paper solely focuses on the detection of HPV in HNSCC, HPV RNA CISH is clinically relevant in various other conditions that involve HPV infection. For instance, this technique may improve the accuracy of the diagnosis of low-grade squamous intraepithelial lesions of the cervix (LSIL, formerly known as cervical intraepithelial neoplasia, grade 1 [CIN1]) for morphologically ambiguous cases9. Regarding oropharyngeal SCC, HPV RNA CISH allows the identification of HPV-related SCC, labeled as distinct from HPV-unrelated oropharyngeal SCC in the recent eighth edition of the TNM Classification of Head and Neck Cancer (of the Union for International Cancer Control [UICC])10. Since HPV-related SCC exhibits a better prognosis with longer survival and enhanced radiotherapy and chemotherapy sensitivity than HPV-unrelated SCC11,12,13, the detection of HPV infection may impact patient management14,15. Besides, HPV RNA CISH can be used for the diagnosis of HPV-related multiphenotypic sinonasal carcinoma with a higher signal than HPV DNA CISH16. Several multivariate analyses suggest that the detection of E6 and E7 transcripts is correlated with a better prognosis in oropharyngeal SCC overall7,15,17,18 and in the subgroup of p16-positive oropharyngeal SCC19,20.
Here we present the protocol for manual HPV RNA CISH performed on FFPE slides with a kit obtained from the manufacturer.
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
2. Adhesion enhancing and deparaffinization in the fume hood
NOTE: Start the protocol with 3–5 µm-thick histological samples mounted on unstained slides.
3. Tissue pretreatment
NOTE: These steps follow the “standard” pretreatment recommendation according to the manufacturer’s instructions for head and neck samples. The timing of sections 3.1 and 3.2 may need to be adjusted depending on the manipulated tissue.
4. Running the assay
NOTE: Do not let sections dry out between the incubation steps.
5. Signal detection with 3,3'-diaminobenzidine
CAUTION: Diaminobenzidine (DAB) is toxic. Follow appropriate precautions and safety guidelines when disposing of and handling this chemical.
6. Counterstaining
7. Dehydration
8. Slide mounting
9. Sample evaluation
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+ with 10 dots or more per tumor cell6; in post hoc analyses, only 2+ and 3+ signals, which were easier to interpret, were taken into account. In another study, the results were divided into two scores: RNA CISH “high” and RNA CISH “low”. RNA CISH “high” score was defined by more than 50% of stained cancer cells, or by staining covering more than 80% of the cell surface (nucleus and cytoplasm) in at least 30% of the cancer cells, as observed with a 20x objective (Figure 1)21.
Regarding positive controls, the PPIB signal should be visible as punctate dots within cell nuclei at 20x–40x magnification. As for negative control slides, one dot to every 10 cells displaying background DAB staining per 20x microscope field is acceptable.
Figure 1: Examples of “low” and “high” RNA CISH staining. (A and B) RNA CISH “low” score staining in oropharyngeal squamous cell carcinomas. Staining is observed in under 50% of the tumor cells and covers less than 80% of the cell surface. (C and D) RNA CISH “high” score staining in oropharyngeal squamous cell carcinomas. Staining is observed in more than 50% of the tumor cells and, in this case, the staining surface exceeds 80% in more than 30% of the tumor cells. This figure has been modified from Augustin et al.21 Please click here to view a larger version of this figure.
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 on the manipulated tissue.
HPV E6-E7 mRNA expression signal is detected with a precise spatial resolution, thereby ruling out any contamination from HPV-infected nonneoplastic cells adjacent to the tumor. Detection of HPV E6 and E7 RNA is functionally relevant since these transcripts are needed for HPV-induced cell transformation through their interaction with the cellular p53 and pRb proteins4. Therefore, in precancerous lesions of the uterine cervix, HPV RNA CISH may help discriminate low-grade intraepithelial lesions (LSIL) from high-grade intraepithelial lesions (HSIL) according to the localization of the signal: in most cases of LSIL, abundant diffusely stained nuclei are labeled throughout the epithelial thickness, indicating a productive phase. HSIL exhibit either abundant diffusely stained cell nuclei in the superficial layer, coexisting with strong nuclear and cytoplasmic punctuate signals in the lower layer (in lesions formerly known as CIN2), or strong nuclear staining with cytoplasmic dots throughout the thickness of the epithelium, indicating the transformative phase of HPV infection (in lesions formerly known as CIN3)22.
Although there is as yet no standard recommendation for the semiquantitative evaluation of the signal, some authors report a clinical relevance since the semiquantitative evaluation of HPV E6 and E7 transcripts has allowed the identification of two prognostic groups among HPV-related HNSCC patients21. It has been postulated that the detection of HPV DNA without E6 and E7 transcripts or with only low levels of E6 and E7 transcripts would be functionally irrelevant and that such patients should be pooled with HPV-negative cancer patients21,23.
Regarding limitations of this procedure, it is postulated that a few nonspecific cross hybridizations might happen in some cases, as hypothesized by Dreyer et al.23 RNA CISH may not be suitable for discrimination between E6/E7 RNA transcripts and viral DNA, as the protocol includes a 100 °C heat treatment step, which is suspected to allow for viral DNA denaturation23. This is supported by the observation of two types of signals in positive cases, namely strong staining mainly localized in tumor cell nuclei, as well as a fine granular signal in the cytoplasm. As the probe used in this protocol specifically binds HPV genotypes 16 and 18, which are involved in a vast majority of HPV-related HNSCC4, occasional HPV-associated cases may be missed by RNA CISH, either because of certain HPV genotypes that are not included in the probe, or because of mutations or deletions of primer binding sites. Finally, this method requires costly reagents and devices and is not easily accessible in routine practice.
For every sample, a total of three sections are needed: one to perform the HPV assay, one for the positive control, and one for the negative control. As RNA is a fragile molecule and might deteriorate over time in FFPE samples, the quality of the transcripts has to be checked for every sample, using positive control probes such as PPIB, DNA-directed RNA polymerase II subunit RPB1 (Polr2a), or polyubiquitin-C (UBC), which are human housekeeping genes. Moreover, when there is a semiquantitative approach, the signal must be normalized to the housekeeping gene control probe21. The recommended positive control for each tissue may be found in the manufacturer's instructions. However, a recent study confirms that mRNA expression can be robustly studied both in prospective and retrospective samples, showing comparable mRNA levels between samples from 2004 and from 2008. This is in favor of the relative integrity of mRNA over time24. The recommended negative control probe used to avoid unspecific staining is the bacterial gene coding for DAPB.
Here the chromogenic in situ hybridization of HPV RNA is described as performed manually. This technique may also be performed with fluorescent labeling and/or combined with conventional immunostaining.
The authors have nothing to disclose.
The authors thank the department of Pathology of Hopital Européen Georges Pompidou and Necker (Laurianne Chambolle, Elodie Michel, and Gisèle Legall); the Histology platform of PARCC, Hopital Européen Georges Pompidou (Corinne Lesaffre); Virginia Clark for language editing; Alexandra Elbakyan for her contribution.
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 |