High‐risk human papilloma virus was not detected in a Norwegian cohort of oral squamous cell carcinoma of the mobile tongue

Abstract Objectives The presence of and the causative role of high‐risk human papilloma virus (HPV) is a subject of controversy in oral squamous cell carcinoma (OSCC). The disagreement can be related to the misclassification of OSCC as oropharyngeal squamous cell carcinoma and/or lack of standard detection methods. This study aimed to examine the presence of transcriptionally active high‐risk HPV in a homogenous Norwegian cohort of primary and second primary OSCC of the mobile tongue (oral tongue squamous cell carcinoma—OTSCC). Methods Tissue microarrays containing formalin‐fixed and paraffin‐embedded cores of 146 OTSCC from the anterior 2/3 of the tongue (n = 128 primary and n = 18 second primary) from a multicentric Norwegian cohort were examined for the presence of high‐risk HPV by DNA‐ and RNA‐in situ hybridization (ISH) assays and p16 immunohistochemistry. Results Transcriptionally active HPV (E6/E7 mRNA) was not identified in any of the OTSCC specimens. In parallel, no tumors were positive for HPV by DNA ISH. Although, 61 (42%) OTSCC demonstrated p16 positivity with varying staining intensity and subcellular localization, only two cases demonstrated strong and uniform p16‐staining (both cytoplasmic and nuclear) in >70% of cancer cells. The absence of transcriptionally active high‐risk HPV in this cohort of OTSCC indicates that high‐risk HPV is an unlikely causative factor in the present material.


| INTRODUCTION
The oral cavity is considered to be a separate anatomical location from the oropharynx. Here, squamous cell carcinoma (SCC) accounts for the majority of malignant tumors (Markopoulos, 2012). The 5-year overall survival for oral squamous cell carcinoma (OSCC) is about 64% and is closely related to the tumor stage (Zanoni et al., 2019). In a recent systematic review, the mobile tongue is shown to be the most common site of oral cancer among the patients below 45 years of age (Hussein et al., 2017). This is of great concern since patients with oral tongue squamous cell carcinoma (OTSCC) have a significantly more unfavorable prognosis than those at other oral cavity sites (Rusthoven, Ballonoff, Raben, & Chen, 2008).
The HPV positive OPSCC is considered to be a biologically distinct entity and is associated with a higher survival rate as compared to the conventional HPV negative (tobacco-induced) OPSCC (Ang et al., 2010). In 1983, Sÿrjanen proposed that HPV could be a possible etiological factor for a subgroup of OSCC (Syrjänen, Syrjänen, Lamberg, Pyrhönen, & Nuutinen, 1983). Since then, several studies have focused on HPV detection in OSCC, however, with conflicting results (Lewis Jr et al., 2012;Yete, D'Souza, & Saranath, 2018).
Secondly, lack of standard methodological approach for HPV testing can significantly lead to over-or underestimation of HPV positivity (Braakhuis, Brakenhoff, Meijer, Snijders, & Leemans, 2009;Lewis Jr et al., 2012;Westra, 2014). In a systematic literature review of approximately 4,000 oral cavity cancer specimens, the weighted prevalence of polymerase chain reaction (PCR)-based HPV DNA detection was found to be 20.2% (Isayeva, Li, D, & Brandwein-Gensler, 2012). However, the high sensitivity of DNA PCR analysis increases the risk of false positive results. Moreover, HPV DNA detection does not distinguish an active (driver) HPV infection from passenger/bystander infection (Kim, Lewis Jr., & Chen, 2018;Madrigal, Bishop, & Faquin, 2018). In recent years, the mRNA E6/E7 in situ hybridization (ISH) technique has become increasing popular and it allows direct visualization of viral transcripts in routinely processed tissues, thereby reflecting the active HPV infection (Wang et al., 2014). To our knowledge, only a few studies using relatively limited numbers of OSCC have evaluated the presence of high-risk HPV in OSCC by this technique (Bishop et al., 2012;Lewis Jr et al., 2012;Poling et al., 2014). Their results indicate that high-risk HPV prevalence is very low in OSCC and challenge the view that HPV is a possible etiological factor in OSCC. This underscores the importance of studies aimed at identifying active HPV infection in a large and homogenous OSCC cohort.
The current work represents a sub-study of a joint initiative (Norwegian Oral Cancer [NOROC] multicenter study) between the four University hospitals in Norway treating OSCC (Bjerkli et al., 2020

| Tissue microarray generation
Tissue microarrays were constructed from formalin-fixed, paraffinembedded (FFPE) tissue blocks in a fully automated tissue microarray machine (TMA Master II, 3DHISTECH). Two to four tissue cores (both invasive front and more superficial parts of the tumors) with a diameter of 2 mm were arrayed on the recipient paraffin blocks. The stained TMA-sections were scanned (Pannoramic® MIDI scanner, Thermo Fisher Scientific) and evaluated using the CaseViewer™ software F I G U R E 1 Flowchart illustrating the selection procedure for OTSCC used in this study. OSCC, oral squamous cell carcinoma; OTSCC, oral tongue squamous cell carcinoma; RT, radiation therapy; TMA, tissue microarray (3dhistech.com). For scanned images with inadequate focus, the original glass slides were examined by a Leitz Aristoplan microscope.     Table 2.

| HPV DNA ISH
Based on the evaluation criteria described above, all tumors tested negative for HPV DNA (Figure 3a), including the two cases with p16

| DISCUSSION
The pathogenic role of HPV in OPSCC has been well established however, its role in OSCC carcinogenesis is a subject of a controversy.  Careful selection of the detection technique and viral target is extremely important to obtain reliable and clinically meaningful data.
However, the commonly used assays such as p16 IHC and PCR have limitations. For example, p16 overexpression may be caused by molecular mechanisms independent of the presence of high-risk HPV.
DNA or RNA extraction procedures in PCR techniques destroy the tumor tissue context of importance for morphological correlation (Wang et al., 2014). Furthermore, the detection of HPV DNA (either PCR-based or by ISH) cannot discern an incidental virus from a persistent viral oncogene expression (Westra, 2014). In contrast, RNA ISH OSCC out of 45 in their study (Lewis Jr et al., 2012). In contrast, a recent systematic review and meta-analysis reported a higher prevalence of HPV DNA positivity in South and Central America and Asia, as compared to that in North America and Europe (Ndiaye et al., 2014).
In line with the DNA/RNA ISH results, only two OTSCC were p16 positive (Score 3). However, both of the p16 positive OTSCC were negative for HPV DNA and RNA ISH. This suggests that the p16 expression in those cases might be related to non-HPV mechanisms and supports the view that p16 is not a reliable surrogate marker for HPV in OTSCC (Bishop et al., 2012;Muller, 2017). A recent metaanalysis showed that 6.7% of p16 positive head and neck squamous cell carcinomas were not related to transcriptionally active HPV infection and that p16 IHC as a single test does not fulfill the criterion to distinguish between bystander HPV and truly HPV-driven cancers (Albers, Qian, Kaufmann, & Coordes, 2017).
Although we aimed to investigate conventional OSCC, two nonkeratinizating carcinomas were also included in the current material.
One of the two OTSCC with p16 Score 3 staining was a basaloid carcinoma (non-keratinizating). This is an interesting observation since p16 positive OPSCC usually are non-keratinizing. However, another non-keratinizing carcinoma included in this study was p16 negative.
A general increase in the incidence of OTSCC has been reported globally with a shifting trend toward female and/or younger patients with OTSCC (Ng, Iyer, Tan, & Edgren, 2017). In this study, such a trend was not obvious in Norway in the period 2005-2010. Here, the majority of the patients were males and 66% of the patients were 60 years or older. The current study benefitted from the use of a homogenous cohort of OSCC only including carcinomas from the anterior 2/3rd of the tongue. As the Norwegian population is homogeneous regarding ethnic origin, lifestyle, and OSCC-related risk habits, the carcinomas can be considered similar with respect to etiology and biology, thereby minimizing the potential biases.
Additionally, to minimize the possible bias caused by tumor heterogeneity, tissue cores representing both the invasive front and the more superficial parts of each tumor were included in the TMA block.
From the majority of the OTSCC, four tissue cores were prepared.
From the rest of the tumors, two tissue cores were made. Four cores should achieve a high degree of concordance when comparing results from whole sections with those of TMA cores (Goethals et al., 2006).

| CONCLUSION
None of the 146 OTSCC (128 primary and 18 second primary) diagnosed from 2005 until 2010 were found to be positive for high-risk HPV. In parallel, only two OTSCC were p16 (Score 3) positive. Our results suggest that high-risk HPV is an unlikely causative factor in the present material and will not influence future biomarker studies utilizing the current material.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of this article.