Human papillomavirus and other molecular markers in cancer of different oropharyngeal sub-sites

Background: Human papillomavirus (HPV) is a known risk factor for oropharyngeal cancer (OPSCC), and over the past few decades OPSCC has increased drastically due to an HPV epidemic. The oropharynx contains different sub-sites, where sub-sites rich in lymphoid tissue, such as the tonsils and the tongue base, are suggested to be more prone to harbor an HPV infection, and cancer of these sub-sites is more often HPV-positive (HPV+). Interestingly, patients with an HPV+ tonsillar or base of tongue cancer (TSCC or BOTSCC) generally have a better survival compared to patients with corresponding HPV-negative (HPV-) tumors. However, HPV does not have the same prognostic value in the other OPSCCs, and has been suggested to differ even depending on the histology of normal tissue surrounding TSCC. Moreover, we have previously shown that low levels of HLA class I (which presents antigens to the immune system) in HPV+ TSCC and BOTSCC was associated with a good prognosis, whereas the opposite was shown in HPV- tumors. Since current treatment often leads to severe side effects, de-escalation trials for patients with a predicted excellent prognosis would be an attractive alternative. Therefore, there is a need to understand the differences between OPSCC sub-sites, and find biomarkers that together with HPV status would identify patients that could benefit of de-escalated or targeted therapy. Aims: To identify new prognostic markers in OPSCC and to study the importance of subdividing OPSCC. To study expression of proteins involved in antigen processing and presentation, in HPV+ and HPV- OPSCC, and how expression is affected by irradiation. Results: In paper I and II, the expression of the antigen processing machinery (APM) components were evaluated both in the nucleus and in the cytoplasm. We showed that LMP10 and LMP7 had prognostic value in both HPV+ and HPV- TSCC and BOTSCC. We also found that APM components TAP2, LMP2, LMP7, and LMP10 were commonly suppressed in both HPV+ and HPV- TSCC and BOTSCC, and that LMP2 and LMP7 expression was correlated to HLA class I expression. In paper III, we found that radiotherapy had the ability to increase cell surface HLA class I expression in some HPV+ head and neck cancer cell lines, without an observed change at the transcriptional level. Our results from paper IV, show that HPV was significantly more prevalent in TSCC and BOTSCC as compared to the other oropharyngeal sub-sites. As described in paper V, the histology adjacent to TSCC varies and can be divided into TSCC, where normal tonsil-like adjacent tissue is present (specified TSCC (STSCC)), and absent (non-specified TSCC (NSTSCC)). We show that HPV is significantly more common in STSCC compared to NSTSCC. HPV+ STSCC patients have a better clinical outcome compared to HPV+ NSTSCC patients. However, no differences in clinical outcome was observed in patients with HPV- STSCC and NSTSCC. Conclusions: This thesis provides increased understanding of differences between HPV+ and HPV- status in the context of OPSCC sub-sites. In addition, we have identified several prognostic biomarkers that together with HPV status and OPSCC sub-site, can contribute to improved personalized medicine for patients with OPSCC

TLR5 and TLR7 are differentially expressed in human papillomavirus-positive and negative base of tongue squamous cell carcinoma, and TLR7 may have an independent prognostic influence

Background: Human papillomavirus-positive (HPV+) base of tongue squamous cell carcinoma (BOTSCC) has a better outcome than corresponding HPV- cancer. TLR5 and TLR7 expression was previously shown to differ depending on HPV - status and correlate with outcome in oropharyngeal squamous cell carcinoma. Aims/objectives: For validation, TLR5 and TLR7 were analyzed in a BOTSCC-cohort for correlation with HPV, survival, CD4(+) and CD8(+) tumor-infiltrating lymphocyte (TIL) counts, the latter being a well-documented prognostic marker. Materials and methods: BOTSCC biopsies, (49HPV(+)/28HPV(-)) were analyzed by immunohistochemistry for TLR5 and TLR7, and correlated with the above parameters. Results: TLR5 expression was more frequently absent/weak than medium/strong in HPV+ compared to HPV- BOTSCC (p <.001). The opposite was observed for TLR7 (p <.007). TLR5 and TLR7 expression did not correlate to survival in either the HPV- or HPV+ cases, or to CD4(+) TILs. TLR5, (but not TLR7) expression was correlated to CD8(+) TIL counts (p = .023).Peer reviewe

Changes in Cervical Human Papillomavirus (HPV) Prevalence at a Youth Clinic in Stockholm, Sweden, a Decade After the Introduction of the HPV Vaccine

Aim: This study aimed to follow the impact of human papillomavirus (HPV) catch-up and vaccination on the very high cervical HPV-prevalence in women at a youth clinic in central Stockholm during the period 2008–2018.Background: 2008–2010, cervical HPV-prevalence (69.5%) and HPV16 prevalence (34.7%) were high in non-vaccinated women at a youth clinic in Stockholm. 2013–2015, after the introduction of the quadrivalent-Gardasil® HPV-vaccine, HPV16 and HPV6 prevalence had decreased. Here, cervical HPV-prevalence was investigated 10 years after primary sampling.Material and Methods: 2017–2018, 178 cervical swabs, from women aged 15–23 years old, were tested for 27 HPV types by a bead-based multiplex method. HPV-prevalence data were then related to vaccination status and age and compared to HPV-prevalence in 615 samples from 2008 to 2010 and 338 samples from 2013 to 2015 from the same clinic, and to HPV types in 143 cervical cancer cases during 2003–2008 in Stockholm.Results: The proportion of vaccinated women increased from 10.7% (2008–2010) to 82.1% (2017–2018). The prevalence of all 27 HPVs, all high-risk HPVs (HR-HPVs) and the combined presence of the quadrivalent-Gardasil® types HPV16, 18, 6, and 11, was lower in vaccinated compared to unvaccinated women (67.4 vs. 93.3%, p = 0.0031, 60.1 vs. 86.7%, p = 0.0057 and 5.8 vs. 26.7%, p = 0.002, respectively). Furthermore, HPV16 prevalence in non-vaccinated women 2017–2018 was lower than that in 2008–2010 (16.7 and 34.7%, respectively, p = 0.0471) and similar trends were observed for HPV18 and 11. In both vaccinated and non-vaccinated women, the most common non-quadrivalent-Gardasil® vaccine HR-HPV types were HPV39, 51, 52, 56, and 59. Together they accounted for around 9.8% of cervical cancer cases in Stockholm during 2003–2008, and their prevalence tended to have increased during 2017–2018 compared to 2008–2010.Conclusion: Quadrivalent-Gardasil® vaccination has decreased HPV-vaccine type prevalence significantly. However, non-vaccine HR-HPV types remain high in potentially high-risk women at a youth clinic in Stockholm

Time to change perspectives on HPV in oropharyngeal cancer. A systematic review of HPV prevalence per oropharyngeal sub-site the last 3 years

Objectives: Human papillomavirus (HPV) as a risk factor in oropharyngeal squamous cell carcinoma (OPSCC) is well established. However, accumulating data imply that the OPSCC concept is too unspecific with regard to HPV prevalence and clinical importance. To further study the role of HPV in OPSCC by sub-site, a systematic review and meta-analysis was performed. Material and method: PubMed was searched and all studies reporting HPV data (p16/HPV DNA/RNA) in both âlymphoepithelial associatedâ (i.e. tonsillar and base of tongue cancer; TSCC and BOTSCC respectively) and ânon-lymphoepithelialâ (âotherâ OPSCC) OPSCC were included. Pooled odds ratios by HPV detection method were analysed using a random effects model. Results: In total, 58 unique patient cohorts were identified. Total HPV prevalence in TSCC/BOTSCC was 56%, 95%CI: 55â57% (59%, 95%CI: 58â60% for TSCC only) as compared to 19%, 95%CI: 17â20%, in âotherâ OPSCC. Significant association of HPV to TSCC/BOTSCC vs. âotherâ OPSCC was observed no matter HPV detection method used, but statistical homogeneity was only observed when studies using algorithm based HPV detection were pooled. Conclusion: HPV prevalence differs markedly between OPSCC sub-sites and while the role of HPV in TSCC/BOTSCC is strong, the role in âotherâ OPSCC is more uncertain and needs further evaluation. Keywords: HPV, Tonsillar cancer, Base of tongue cancer, Oropharyngeal cancer, Meta-analysis, Prevalenc

Reduced Expression of the Antigen Processing Machinery Components TAP2, LMP2, and LMP7 in Tonsillar and Base of Tongue Cancer and Implications for Clinical Outcome

OBJECTIVES: Patients with human papillomavirus (HPV)–positive tonsillar squamous cell carcinoma (TSCC) and base of tongue squamous cell carcinoma (BOTSCC) have a better clinical outcome than those with corresponding HPV-negative tumors. Moreover, there is a strong positive correlation between absent/low as opposed to strong HLA class I expression and favorable clinical outcome for HPV-positive tumors, while the reverse applies to HPV-negative tumors. The expression of the antigen processing machinery (APM) components TAP1, TAP2, LMP2, and LMP7 in these tumors in relation to HPV status, HLA class I expression, each other, and clinical outcome was therefore investigated. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded TSCC and BOTSCC, derived from 151 patients and previously analyzed for HPV DNA, HLA class I, and LMP10 expression were stained by immunohistochemistry for TAP1, TAP2, LMP2, and LMP7. RESULTS: Absent/low TAP2, LMP2, and LMP7 expression, similar to HLA class I and LMP10, was common in TSCC and BOTSCC, irrespective of HPV status. Expression of TAP1 and TAP2 was correlated, as was LMP2 to LMP7. LMP2 and LMP7 expression was also associated to HLA class I expression. Moreover, absence of LMP7 was linked to increased disease-free survival in both HPV-positive and HPV-negative cases. CONCLUSION: Reduced expression of TAP2, LMP2, and LMP7 was frequent in TSCC and BOTSCC and their expression as well as that of TAP1 was often interrelated. Furthermore, low LMP7 expression correlated to better clinical outcome and may, together with HPV status, potentially be used for prediction of treatment response

Correlation of LMP10 expression and clinical outcome in Human Papillomavirus (HPV) positive and HPV-Negative tonsillar and base of tongue cancer.

To examine LMP10 expression and its possible impact on clinical outcome in human papillomavirus (HPV) positive and HPV-negative tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC).Outcome is better in HPV-positive TSCC and BOTSCC compared to matching HPV-negative tumours, with roughly 80% vs. 40% 5-year disease free survival (DFS) with less aggressive treatment than today's chemoradiotherapy. Since current treatment often results in harmful side effects, less intensive therapy, with sustained patient survival would be an attractive alternative. However, other markers together with HPV status are necessary to select patients and for this purpose LMP10 expression is investigated here in parallel to HPV status and clinical outcome.From 385 patients diagnosed between 2000 and 2007 at the Karolinska University Hospital, 278 formalin fixed paraffin embedded TSCC and BOTSCC biopsies, with known HPV DNA status, were tested for LMP10 nuclear and cytoplasmic expression (fraction of positive cells and staining intensity). The data was then correlated to clinical outcome.An absent/low compared to a moderate/high LMP10 nuclear fraction of positive cells was correlated to a better 3-year DFS in the HPV-positive group of patients (log-rank p = 0.005), but not in the HPV-negative group. In the HPV-negative group of patients, in contrast to the HPV-positive group, moderate/high LMP10 cytoplasmic fraction and weak/moderate/high LMP10 cytoplasmic intensity correlated to a better 3-year DFS (p = 0.003 and p = 0.001) and 3-year overall survival (p = 0.001 and 0.009).LMP10 nuclear expression in the HPV-positive group and LMP10 cytoplasmic expression in the HPV-negative group of patients correlated to better clinical outcome

No evidence for human papillomavirus having a causal role in salivary gland tumors

Abstract Background Salivary gland malignancies are a very heterogeneous group of cancers, with histologically > 20 different subtypes, and prognosis varies greatly. Their etiology is unknown, however, a few small studies show presence of human papillomavirus (HPV) in some subtypes, although the evidence for HPV having a causal role is weak. The aim of this study was to investigate if HPV plays a causal role in the development of different parotid salivary gland tumor subtypes. Methods DNA was extracted from 107 parotid salivary gland formalin fixed paraffin embedded tumors and 10 corresponding metastases, and tested for 27 different HPV types using a multiplex bead based assay. HPV DNA positive tumors were stained for p16INK4a overexpression by immunohistochemistry. Results One of the 107 malignant parotid salivary gland tumors (0.93%) and its corresponding metastasis on the neck were positive for HPV16 DNA, and both also overexpressed p16INK4a. The HPV positive primary tumor was a squamous cell carcinoma; neither mucoepidermoid nor adenoid cystic tumors were found HPV positive. Conclusions In conclusion, HPV DNA analysis in a large number of malignant parotid salivary gland tumors, including 12 different subtypes, did not show any strong indications that tested HPV types have a causal role in the studied salivary gland tumor types

Protein Expression in Tonsillar and Base of Tongue Cancer and in Relation to Human Papillomavirus (HPV) and Clinical Outcome

Human papillomavirus (HPV) is a major etiological factor for tonsillar and the base of tongue cancer (TSCC/BOTSCC). HPV-positive and HPV-negative TSCC/BOTSCC present major differences in mutations, mRNA expression and clinical outcome. Earlier protein studies on TSCC/BOTSCC have mainly analyzed individual proteins. Here, the aim was to compare a larger set of cancer and immune related proteins in HPV-positive and HPV-negative TSCC/BOTSCC in relation to normal tissue, presence of HPV, and clinical outcome. Fresh frozen tissue from 42 HPV-positive and 17 HPV-negative TSCC/BOTSCC, and corresponding normal samples, were analyzed for expression of 167 proteins using two Olink multiplex immunoassays. Major differences in protein expression between TSCC/BOTSCC and normal tissue were identified, especially in chemo- and cytokines. Moreover, 34 proteins, mainly immunoregulatory proteins and chemokines, were differently expressed in HPV-positive vs HPV-negative TSCC/BOTSCC. Several proteins were potentially related to clinical outcome for HPV-positive or HPV-negative tumors. For HPV-positive tumors, these were mostly related to angiogenesis and hypoxia. Correlation with clinical outcome of one of these, VEGFA, was validated by immunohistochemistry. Differences in immune related proteins between HPV-positive and HPV-negative TSCC/BOTSCC reflect the stronger activity of the immune defense in the former. Angiogenesis related proteins might serve as potential targets for therapy in HPV-positive TSCC/BOTSCC

High Levels of FGF11 Correlate with Poor Survival in Patients with Human Papillomavirus (HPV)-Positive Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favourable prognosis. It has therefore been suggested that treatment should be individualized and separated by HPV status. However, additional prognostic markers are still needed before treatment can be individualized for this patient group. For this purpose, all patients diagnosed with HPV and p16-positive OPSCC in Stockholm 2000–2009, identified as having a partial/nonresponse to treatment and having viable tumour cells in their neck specimen with material available were categorized as cases. These were matched to controls (complete responders), and the differences in the gene expression were analysed. Two separate verification cohorts were identified including patients with HPV- and p16-positive OPSCC, and the data from the case-control study were verified by qPCR and immunohistochemistry (IHC) in the respective cohorts. A separation of gene expression in correlation with survival was observed in the case-control study, and FGF11 expression was identified as significantly differently expressed between the two groups. The prognostic role of FGF11 was validated in the two cohorts on the RNA and protein levels, respectively. Taken together, our findings suggest that FGF11 may indicate a poor prognosis in HPV-positive OPSCC and may serve as a prognostic biomarker