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

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.

Detection of human papillomavirus in oropharyngeal squamous cell carcinoma

“…None of the developed assays seems to have both very high specificity and sensitivity. Therefore, multimodal testing that integrates two HPV detection methods could help to reliably identify patients with transcriptionally active high-risk HPV-positive OPSCC and to avoid the possibility of false-positive and false-negative cases (Marino et al, 2020). The use of highly sensitive, specific, and accurate methods is critical, especially when considering de-escalation treatment approaches for HPVpositive OPSCC patients.…”

“…The combined testing of p16 INK4a IHC/HPV DNA PCR showed a sensitivity similar to either p16 INK4a IHC or HPV DNA PCR alone, but was significantly more specific than either separate test (Prigge et al, 2017). Marino et al (2020) recently established a novel multiplex HPV RNA ISH/p16 IHC assay to detect both HPV E6/E7 transcripts and p16 INK4a overexpression simultaneously. There are advantages from combining various detection assays to achieve an accurate and reliable HPV status; however, it is technically inconvenient, may produce discordant results, and requires more cost and time.…”

Understanding the Pattern of Oropharyngeal Cancers from North-East Romanian Patients

“…The results obtained by this double testing was similar with the results previously obtained by classical p16 and HPV RNA testing. The authors are underlying the ease of applicability of this assay on FFPE samples, with complete automation, and possibility for implementation in clinical laboratories [41]. Researchers from Finland, with recognized experience in the HNSCC field, analyzed OPSCC samples by different methods, including HPV mRNA ISH.…”

The Expression of HPV E6/E7 mRNA in Situ Hybridization in HPV Typing-negative Cervical Cancer

“…Images of the RISH slides were taken at 20× magni cation using a Motic light microscope (Motic BA600 Mot, Germany). A positive RISH test result was de ned as positive if any of the malignant cells showed brown punctuate dot-like nuclear and/or cytoplasmic positivity [13,14].…”