Background Nasopharyngeal carcinoma (NPC) is related to Epstein‐Barr virus (EBV) in endemic areas; however, the role of viruses in nonendemic countries is unclear. Our nationwide study investigated the prevalence and prognostic significance of EBV and human papillomaviruses (HPVs) in Finnish NPC tumors. Methods We analyzed samples from 150 patients diagnosed between 1990 and 2009. Viral status was determined using EBV and HPV RNA in situ hybridizations, and p16 immunohistochemistry. Patient and treatment characteristics were obtained from patient records. Results In our white patient cohort, 93 of 150 (62%) patients were EBV‐positive and 21/150 (14%) patients were HPV‐positive with no coinfections. Thirty‐six (24%) tumors were negative for both viruses. The 5‐year disease‐specific survival for patients with EBV‐positive, HPV‐positive, and EBV/HPV‐negative tumors was 69%, 63%, and 39%, respectively. In multivariable‐adjusted analysis, overall survival was better among patients with EBV‐positive ( P = .005) and HPV‐positive ( P = .03) tumors compared to patients with EBV/HPV‐negative tumors. Conclusions In our low‐incidence population, EBV and HPV are important prognostic factors for NPC.

In situ hybridization for high-risk HPV E6/E7 mRNA is a superior method for detecting transcriptionally active HPV in oropharyngeal cancer

Background The etiological role of human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) is confirmed. However, the role of other oncoviruses in OPSCC is unknown. Materials and methods A total of 158 consecutive OPSCC patients treated with curative intent were included. DNA extracted from tumor sections was used to detect Epstein-Barr virus (EBV), HPV, and the following polyomaviruses: John Cunningham virus (JCV), Simian virus 40 (SV40), and BK virus (BKV) with PCR. In addition, p16 expression was studied by immunohistochemistry, and EBV-encoded small RNA (EBER) transcripts were localized by in situ hybridization. The effect of viral status on overall survival (OS) and disease-free survival (DFS) was analyzed. Results A total of 94/158 samples (59.5%) were HPV-positive, 29.1% contained BKV DNA, 20.3% EBV DNA, 13.9% JCV DNA, and 0.6% SV40 DNA. EBER was expressed only in stromal lymphocytes adjacent to the tumor and correlated with HPV positivity (p = 0.026). p16 expression associated only with HPV. None of the three polyomaviruses had an impact on survival. Patients with EBER-positive but HPV-negative OPSCC had significantly poorer OS and DFS than those with HPVpositive OPSCC and slightly worse prognosis compared with the patients with EBER-negative and HPV-negative OPSCC. Conclusion Polyomaviruses are detectable in OPSCC but seem to have no impact on survival, whereas HPV was the strongest viral prognostic factor. EBER expression, as a sign of latent EBV infection, may have prognostic impact among patients with HPV-negative OPSCC. EBER analysis may identify a new subgroup of OPSCCs unrelated to HPV. Keywords Epstein-Barr virus • Human papillomavirus • Oropharyngeal cancer • Polyomavirus • Prognosis Abbreviations BKV BK virus DFS Disease-free survival EBER Epstein-Barr virus (EBV)-encoded small RNA * Timo Carpén

Background The surrogate immunohistochemical marker, p16 INK4a , is used in clinical practice to determine the high-risk human papillomavirus (HPV) status of oropharyngeal squamous cell carcinomas (OPSCC). With a specificity of 83%, this will misclassify some patients compared with direct HPV testing. Patients who are p16 INK4a-positive but HPV DNA-negative, or RNAnegative, may be unsuitable for treatment de-escalation aimed at reducing treatmentrelated side effects. We aimed to identify cost-effective serum markers to improve decision making for patients at risk of misclassification by p16 INK4a alone. Methods Serum proteins from pre-treatment samples of 36 patients with OPSCC were identified and quantified using label-free mass spectrometry-based proteomics. HPV-status was determined using p16 INK4a /HPV DNA and E6/E7 mRNA. Serum protein expressions were compared between groups of patients according to HPV status, using the unpaired t-test with a Benjamini-Hochberg correction. ROC curves (AUC) were calculated with SPSS (v25).

Objectives: In oropharyngeal squamous cell carcinoma (OPSCC), toll-like receptors (TLR) 5 and 7 associate with the tumor's human papilloma virus (HPV) status [1]. TLR 2, on the other hand, has been linked to head and neck squamous cell carcinoma (HNSCC), and to oral carcinogenesis [2,3]. Here we investigated the presence of TLR 2 and 4 in HPV-positive and HPV-negative OPSCC, and their relationship to opportunistic oral pathogen Treponema denticola chymotrypsin-like protease (Td-CTLP) immunoexpression, clinical parameters, and patient outcome. Materials and methods:Clinicopathological data of 198 unselected consecutive OPSCC patients came from hospital registries. Immunoexpression of TLRs 2 and 4 we evaluated by immunohistochemistry, and earlier in this patient series we studied immunoexpression of Td-CTLP and HPV DNA, HPV mRNA, and p16 status.Results: Immunoexpression of both TLRs 2 and 4 showed a significant association with HPV-status.Strong expression was associated with HPV-positivity and mild expression with HPV-negativity.Patients with strong TLR 2 immunoexpression in the HPV negative subgroup had significantly poorer 5-year DSS (58%) than did patients with mild TLR 2 expression (77%), and strong TLR 2 immunoexpression remained as an independent factor linked to increased disease mortality in the multivariable setting (P=0.019). No association existed between TLR 2 or 4 and Td-CTLP expression. Conclusion:Our results support the role of TLR 2 receptor as a possible target for development of therapeutics as earlier proposed [2]. The involvement of Td and other oral pathogens in carcinogenesis of OPSCC, remains open and calls for further study. KeywordsOropharyngeal squamous cell carcinoma, human papillomavirus, toll-like receptor, Treponema denticola, chymotrypsin-like protease, dentilisin Recently, Td has appeared also in oral-and gastrointestinal-tumor samples [28,29]. Furthermore, Fusobacterium nucleatum has been associated with colorectal cancer and shown to promote colorectal carcinogenesis [30]. Oral carcinogenesis, on the other hand, was in in vitro and in vivo studies promoted by F. nucleatum and P. gingivalis via their interaction with oral epithelial cells through TLR 2 [3]. In an in vitro and in vivo model of head and neck squamous cell carcinoma (HNSCC), the activation of TLR 2 promoted organoid growth, supporting its pro-tumorigenic role [2].Earlier, we showed an association between Td key virulence factor, a chymotrypsin-like protease (Td-CTLP), and HPV-negative OPSCC [29]. We additionally observed that strong Td-CTLP expression associated with poor disease-specific survival. Here we aimed to investigate the presence of TLRs 2 and 4 in HPV-positive and HPV-negative OPSCC, and the relation of these to Td-CTLP expression, clinical parameters, and patient outcome in vivo in a series of 198 unselected consecutive OPSCC patients. Furthermore, we earlier proposed the use of HPV E6/E7 mRNA ISH detection method as a verifying test for p16-positive tumors [31], the concept which we here evaluate as a HPV...

Background/Aim: The oral bacteria involved in the development of periodontitis alter the tissue conditions and modify immune responses in a way that may also influence tumor development. We investigated the prevalence of R gingipain (Rgp), a key virulence factor of the oral pathobiont Porphyromonas gingivalis, and the tissue-destructive enzymes matrix metalloproteinase 8 (MMP-8) and 9 (MMP-9) in 202 unselected consecutive oropharyngeal squamous cell carcinoma (OPSCC) samples. We further investigated the relationships between these factors and human papillomavirus (HPV) status, Treponema denticola chymotrypsin-like proteinase (Td-CTLP) immunoexpression, clinical parameters, and patient outcome. Patients and Methods: Clinicopathological data were derived from university hospital records. Rgp, MMP-8, and MMP-9 immunoexpression was evaluated by immunohistochemistry; the immunohistochemistry of Td-CTLP and HPV has been described earlier for this patient series. HPVnegative OPSCC (SHR=3.5; p=0.001). Positive immunoexpression of Rgp in inflammatory cells was associated with favorable outcome among all patients (SHR=0.5, CI=0.2-0.9, p=0.021) and among those with HPV-negative disease (SHR=0.4, CI=0.2-0.9, p=0.022). Conclusion: Our results suggest that tumoral MMP-9 may be related to poor outcome in OPSCC, especially in HPV-negative disease, while Rgp immunoexpression in inflammatory cells is associated here with better disease-specific survival (DSS). Cox regression analysis including death by causes other than OPSCC as a competing risk served to assess sub distribution hazard ratios. Results: In multivariable survival analysis, positive tumoral MMP-9 immunoexpression predicted poor prognosis among all patients [sub distribution hazard ratio (SHR)=2.4; confidence interval (CI)=1.2-4.4, p=0.008], and especially among those with

Background Liprin-α1 is a scaffold protein involved in cell adhesion, motility, and invasion in malignancies. Liprin-α1 inhibits the expression of metastatic suppressor CD82 in cancers such as oral carcinoma, and the expression of these proteins has been known to correlate negatively. The role of these proteins has not been previously studied in human papillomavirus (HPV)-related head and neck cancers. Our aim was to assess the clinical and prognostic role of liprin-α1 and CD82 in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) in comparison to HPV-negative OPSCC. Methods The data included 139 OPSCC patients treated at the Helsinki University Hospital (HUS) during 2012–2016. Immunohistochemistry was utilized in HPV determination and in biomarker assays. Overall survival (OS) was used in the survival analysis. Results Stronger expression of liprin-α1 in tumor-infiltrating lymphocytes (TILs) was linked to lower cancer stage (p < 0.001) and HPV positivity (p < 0.001). Additionally, we found an association between elevated expression of liprin-α1 and weak expression of CD82 in tumor cells (p = 0.029). In survival analysis, we found significant correlation between favorable OS and stronger expression of liprin-α1 in TILs among the whole patient cohort (p < 0.001) and among HPV-positive patients (p = 0.042). Conclusions Increased liprin-α1 expression in the TILs is associated with favorable prognosis in OPSCC, especially among HPV-positive patients.