IGSF3 tissue expression in squamous cell carcinoma of the oropharynx: a novel tool for prognosis assessment in HPV-related and HPV-unrelated disease.

Biomarkers are not broadly used in the management of head and neck cancers (HNCs). Biomarkers have been beneficial in the management of other cancers, however, not in HNCs. Therefore, we observed the immunopositivity of a novel biomarker called immunoglobulin superfamily member 3 (IGSF3) in tumor tissues in HPV-related and HPV-unrelated OPSCC. Two patient cohorts (C1 and C2) from separate time periods were available for this study (total N = 282). Both consisted of OPSCC patients treated at the Helsinki University Hospital (HUS, Helsinki, Finland) during 2000-2016. For HPV determination, HPV mRNA in situ hybridization was used. Immunohistochemistry was used to assess IGSF3 immunopositivity in cancer tissues. Overall survival (OS) was used as endpoint in the statistical analysis. In C1, stronger immunopositivity of IGSF3 in tumor-infiltrating lymphocytes (TILs) correlated with favorable OS (p = 0.005). Stronger IGSF3 immunopositivity in tumor cells (TCs) was associated with HPV negativity (p = 0.017). Stronger IGSF3 immunopositivity in TILs correlated with HPV positivity (p < 0.001). Elevated IGSF3 immunopositivity in TILs associates with HPV-related tumors and may signify favorable prognosis. The immunopositivity of IGSF3 differs between HPV-related and HPV-unrelated OPSCC.

Altered microheterogeneity at several N-glycosylation sites in OPSCC in constant protein expression conditions.

Protein glycosylation responds sensitively to disease states. It is implicated in every hallmark of cancer and has recently started to be considered as a hallmark itself. Changes in N-glycosylation microheterogeneity are more dramatic than those of protein expression due to the non-template nature of protein glycosylation. This enables their potential use in serum-based diagnostics. Here, we perform glycopeptidomics on serum from patients with oropharyngeal squamous cell carcinoma (OPSCC), compared to controls and comparing between cancers based on etiology (human papilloma virus- positive or negative). Using MS2, we then targeted glycoforms, significantly different between the groups, to identify their glycopeptide compositions. Simultaneously we investigate the same serum proteins, comparing whether N-glycosylation changes reflect protein-level changes. Significant glycoforms were identified from proteins such as alpha-1-antitrypsin (SERPINA1), haptoglobin, and different immunoglobulins. SERPINA1 had glycovariance at 2 N-glycosylation sites, that were up to 35 times more abundant in even early-stage OPSCCs, despite minimal differences between SERPINA1 protein levels between groups. Some identified glycoforms' fold changes (FCs) were in line with serum protein level FCs, others were less abundant in early-stage cancers but with great variance in higher-stage cancers, such as on immunoglobulin heavy constant gamma 2, despite no change in protein levels. Such findings indicate that glycovariant analysis might be more beneficial than proteomic analysis, which is yet to be fruitful in the search for biomarkers. Highly sensitive glycopeptide changes could potentially be used in the future for cancer screening. Additionally, characterizing the glycopeptide changes in OPSCC is valuable in the search for potential therapeutic targets.

Occupational variation in incidence of oropharyngeal cancer in the Nordic countries.

PURPOSE: Evaluate the occupational variation in incidence of oropharyngeal cancer (OPC). METHODS: We calculated standardized incidence ratios (SIRs) of OPC in occupational categories in the Nordic countries relative to the entire national populations. The data covered 6155 OPC cases. RESULTS: Among men high risk of OPC was observed, among else, in waiters (SIR 6.28, 95% CI 4.68-8.26), beverage workers (SIR 3.00, 95% CI 1.72-4.88), and artistic workers (SIR 2.97, 95% CI 2.31-3.76). Among women high risk of OPC was observed in waiters (SIR 2.02, 95% CI 1.41-2.81) and packers (SIR 1.73, 95% CI 1.07-2.64). The lowest SIRs were observed in female gardeners (SIR 0.27, 95% CI 0.12-0.51) and male farmers (SIR 0.30, 95% CI 0.25-0.35). CONCLUSION: The 20-fold variation in incidence of OPC between occupations needs further investigation in studies with detailed information on occupational and non-occupational risk factors.

Occupational Etiology of Oropharyngeal Cancer: A Literature Review.

While abundant evidence exists linking alcohol, tobacco, and HPV infection to a carcinogenic impact on the oropharynx, the contribution of inhalational workplace hazards remains ill-defined. We aim to determine whether the literature reveals occupational environments at a higher-than-average risk of developing oropharyngeal cancer (OPC) and summarize the available data. To identify studies assessing the relationship between occupational exposure and risk of OPC, a search of the literature through the PubMed-NCBI database was carried out and, ultimately, 15 original articles meeting eligibility criteria were selected. Only original articles in English focusing on the association between occupational exposure and risk or death of specifically OPC were included. The available data are supportive of a potentially increased risk of OPC in waiters, cooks and stewards, artistic workers, poultry and meat workers, mechanics, and World Trade Center responders exposed to dust. However, the available literature on occupation-related OPC is limited. To identify occupational categories at risk, large cohorts with long follow-ups are needed. Identification of causal associations with occupation-related factors would require dose-response analyses adequately adjusted for confounders.

Cancer incidence among visual artists: 45 years of follow-up in four Nordic countries.

INTRODUCTION: Professional visual artists constitute a heterogeneous vocational group, including, but not limited to painters, photographers, textile artists, and sculptors who may face exposure to workplace hazardous substances and lifestyle factors that may contribute to the development of selected cancers. The objective of this registry-based cohort study was to assess the cancer incidence among Nordic visual artists. MATERIALS AND METHODS: This study is based on data from the Nordic Occupational Cancer (NOCCA) project that combines census data of 15 million people from all Nordic countries and cancer registries from 1961 to 2005. For the present study we selected a cohort of visual artists from Finland, Iceland, Norway, and Sweden. Standardized incidence ratio (SIR) analyses were conducted with the cancer incidence rates for the entire national study populations used as reference rates. RESULTS: In male visual artists, there were statistically significant excesses in cancers of the tongue (SIR 2.91, 95% confidence interval 1.74-4.55), oral cavity (2.09, 1.26-3.27), pharynx (2.18, 1.45--3.15), testis (1.91, 1.11-3.05), renal pelvis (2.48, 1.42-4.03) and bladder (1.33, 1.14-1.55). The risk was significantly decreased for cancers of the lip (0.45, 0.18-0.93) and stomach (0.65, 0.50-0.84). In female visual artists, the only significantly increased risk was observed for breast cancer (1.29, 1.13-1.48) and the only significantly decreased risk for stomach cancer (0.43, 0.17-0.88). The incidence of lung cancer was close to the population average in both sexes. CONCLUSIONS: The non-elevated incidence of lung cancer suggests a similar prevalence of smoking between visual artists and the general population, while the elevated risk of cancers of mouth and pharynx among male visual artists is suggestive of more widespread alcohol drinking. The excess risk of urogenital cancers might be associated with exposure to solvents.

The exposure of visual artists to carcinogens remains unstudied and equivocal. The current study suggests that visual artists carry an overall cancer risk that is slightly above the risk among the general population of the four Nordic countries. We observed in men over two-fold excess risks of cancers of the tongue, oral cavity, pharynx, and renal pelvis, and also a significant risk of testis and bladder cancers.

Liprin-α1 Expression in Tumor-Infiltrating Lymphocytes Associates with Improved Survival in Patients with HPV-Positive Oropharyngeal Squamous Cell Carcinoma.

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.

Occupational variation in the incidence of lip cancer in the Nordic countries.

INTRODUCTION: While certain occupations, such as agriculture and fishery, have been associated with an increased risk of lip cancer, the occupational risk profile of lip cancer and its change over time remain poorly known. This study aims to evaluate the incidence of lip cancer across different occupations in the Nordic countries. METHODS: The Nordic Occupational Cancer Study (NOCCA) covers 14.9 million people and includes 45 years of cancer incidence data, from 1961 to 2005, linked to occupational categories for all the five Nordic populations. Standardized incidence ratios (SIRs) with 95% confidence intervals (CIs) were used to quantify the risk of lip cancer across occupational categories relative to the entire national populations. RESULTS: There were a total of 14,477 male and 3008 female lip cancer patients identified during follow up. The highest SIRs were observed among male fishermen (SIR 2.26, 95% CI: 2.04-2.50), gardeners (SIR 1.60, 95% CI: 1.48-1.72), and farmers (SIR 1.60, 95% CI: 1.55-1.66). A significantly reduced risk of lip cancer (SIR < 0.50) was observed among male physicians, teachers, religious workers, artistic workers, journalists, administrators, printers, waiters, and hairdressers. Among women, no occupations were associated with an increased risk of lip cancer. CONCLUSIONS: The incidence of lip cancer varies widely between outdoor and indoor occupations. Occupations involving outdoor activity and exposure to sunlight show the most elevated SIRs.

Certain outdoor occupations, such as agriculture and fishery, have been associated with an increased risk of lip cancer. However, the occupational risk profile of lip cancer and its change over time remain poorly known. This study highlights the excess risk of lip cancer among men with outdoor occupations and further corroborates previous studies. Efforts to counsel outdoor workers on the risk and prevention of lip cancer are needed to reduce the societal burden of the disease.

Application of artificial intelligence for overall survival risk stratification in oropharyngeal carcinoma: A validation of ProgTOOL.

BACKGROUND: In recent years, there has been a surge in machine learning-based models for diagnosis and prognostication of outcomes in oncology. However, there are concerns relating to the model's reproducibility and generalizability to a separate patient cohort (i.e., external validation). OBJECTIVES: This study primarily provides a validation study for a recently introduced and publicly available machine learning (ML) web-based prognostic tool (ProgTOOL) for overall survival risk stratification of oropharyngeal squamous cell carcinoma (OPSCC). Additionally, we reviewed the published studies that have utilized ML for outcome prognostication in OPSCC to examine how many of these models were externally validated, type of external validation, characteristics of the external dataset, and diagnostic performance characteristics on the internal validation (IV) and external validation (EV) datasets were extracted and compared. METHODS: We used a total of 163 OPSCC patients obtained from the Helsinki University Hospital to externally validate the ProgTOOL for generalizability. In addition, PubMed, OvidMedline, Scopus, and Web of Science databases were systematically searched according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: The ProgTOOL produced a predictive performance of 86.5% balanced accuracy, Mathew's correlation coefficient of 0.78, Net Benefit (0.7) and Brier score (0.06) for overall survival stratification of OPSCC patients as either low-chance or high-chance. In addition, out of a total of 31 studies found to have used ML for the prognostication of outcomes in OPSCC, only seven (22.6%) reported a form of EV. Three studies (42.9%) each used either temporal EV or geographical EV while only one study (14.2%) used expert as a form of EV. Most of the studies reported a reduction in performance when externally validated. CONCLUSION: The performance of the model in this validation study indicates that it may be generalized, therefore, bringing recommendations of the model for clinical evaluation closer to reality. However, the number of externally validated ML-based models for OPSCC is still relatively small. This significantly limits the transfer of these models for clinical evaluation and subsequently reduces the likelihood of the use of these models in daily clinical practice. As a gold standard, we recommend the use of geographical EV and validation studies to reveal biases and overfitting of these models. These recommendations are poised to facilitate the implementation of these models in clinical practice.

Hypopharyngeal carcinoma in Finland from 2005 to 2014: outcome remains poor after major changes in treatment.

PURPOSE: Hypopharyngeal carcinoma (HPC) is typically diagnosed at late stages, the patients tend to have serious co-morbidities, distant relapses are frequent, and the related mortality remains high. The treatment paradigm of HPC has remarkably changed from primary surgical approach toward definitive, platinum-based concomitant chemoradiotherapy (CRT). Our aim was to analyze the HPC treatment approaches and outcome in a nationwide series and to make a comparison with a previously published corresponding nationwide patient cohort from the period 1990-1999. METHODS: We retrospectively reviewed all patients diagnosed with HPC at the five university hospitals in Finland between 2005 and 2014. RESULTS: The cohort comprised 231 patients. Treatment with curative intent was offered for 175 (76%) patients and consisted of definitive radiotherapy (RT) or CRT in 156 (89%) patients, while 20 (11%) patients had primary surgery with or without adjuvant RT or CRT. The 5-year estimates for overall survival (OS) and disease specific survival (DSS) for the whole study group were 22.7% and 36.5%, respectively. For patients treated with curative intent, the 5-year estimates for OS and DSS were 29.4% and 44.3%, respectively. CONCLUSIONS: The treatment approach of HPC in Finland has changed thoroughly, as in the 1990s, 63% of HPC patients with curative treatment intent underwent primary surgery with or without RT, while in the current study, the primary treatment approach was non-surgical in 89% of the patients. However, the survival figures have not changed and remain dismal, but most of the few surviving patients now can retain their larynx.

Occupational risk variation of nasopharyngeal cancer in the Nordic countries.

BACKGROUND: The aim of this study was to estimate occupational risk variation in the incidence of nasopharyngeal cancer (NPC) in a large population-based cohort of the Nordic Occupational Cancer (NOCCA) study. METHODS: This study is based on a cohort of almost 15 million persons from Denmark, Finland, Iceland, Norway and Sweden, with 2898 nasopharyngeal cancer cases diagnosed in 1961-2005. The data on occupations were gathered from population censuses and cancer data from the national cancer registries. Standardized incidence ratios (SIR) with 95% confidence intervals (CI) were estimated using the national NPC incidence rates as the reference. RESULTS: There were 1980 male and 918 female NPC patients. The highest SIRs of NPC were observed among male waiters (SIR 3.69, 95% CI 1.91-6.45) and cooks and stewards (SIR 2.24, 95% CI 1.16-3.91). Among women, launderers had the highest SIR of NPC (2.04, 95% CI 1.02-3.65). Significantly decreased SIRs were found among male farmers (SIR 0.79, 95% CI 0.68-0.92) and male textile workers (SIR 0.49, 95% CI 0.22-0.93). CONCLUSIONS: This study suggests that NPC may be associated with several work-related exposure agents such as smoking, kitchen air pollution and solvents. In future, occupational exposure-risk relations should be studied to understand more about causality and to assess effective prevention strategies.

The Role of Human Chorionic Gonadotropin Beta (hCGß) in HPV-Positive and HPV-Negative Oropharyngeal Squamous Cell Carcinoma.

Background: This study was carried out to observe the upregulation of the free ß-subunit of human chorionic gonadotropin (hCGß) and its prognostic significance in human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma (OPSCC). Materials and methods: A total of 90 patients with OPSCC treated with curative intent at the Helsinki University Hospital (HUS), Helsinki, Finland, during 2012−2016 were included. Serum samples were collected prospectively, and their hCGß concentrations (S-hCGß) were determined by an immunofluorometric assay. The expression of hCGß in tumor tissues was defined by immunohistochemistry (IHC). HPV determination was performed by combining p16-INK4 IHC and HPV DNA PCR genotyping. Overall survival (OS) and disease-specific survival (DSS) were used as survival endpoints. Results: S-hCGß positivity correlated with poor OS in the whole patient cohort (p < 0.001) and in patients with HPV-negative OPSCC (p < 0.001). A significant correlation was seen between S-hCGß and poor DSS in the whole cohort (p < 0.001) and in patients with HPV-negative OPSCC (p = 0.007). In a multivariable analysis, S-hCGß was associated with poor DSS. Of the clinical characteristics, higher cancer stage and grade were associated with S-hCGß positivity. No statistically significant correlation with tissue positivity of hCGß was seen in these analyses. Conclusion: S-hCGß may be a potential independent factor indicating poor prognosis, notably in HPV-negative OPSCC.

Cisplatin overcomes radiotherapy resistance in OCT4-expressing head and neck squamous cell carcinoma.

OBJECTIVES: Cisplatin is combined with radiotherapy for advanced head and neck squamous cell carcinoma (HNSCC). While providing a beneficial effect on survival, it also causes side effects and thus is an important target when considering treatment de-escalation. Currently, there are no biomarkers to predict its patient-selective therapeutic utility. In this study, we examined the role of the stem cell factor OCT4 as a potential biomarker to help clinicians stratify HNSCC patients between radiotherapy and chemoradiotherapy. MATERIALS AND METHODS: OCT4 immunohistochemical staining of a population-validated tissue microarray (PV-TMA) (n = 166) representative of a standard HNSCC patients was carried out, and 5-year survival was analyzed. The results were validated using ex vivo drug sensitivity analysis of HNSCC tumor samples, and further cross-validated in independent oropharyngeal (n = 118), nasopharyngeal (n = 170), and vulvar carcinoma (n = 95) clinical datasets. In vitro, genetically modified, patient-derived HNSCC cells were used. RESULTS: OCT4 expression in HNSCC tumors was associated with radioresistance. However, combination therapy with cisplatin was found to overcome thisradioresistance in OCT4-expressing HNSCC tumors. The results were validated by using several independent patient cohorts. Furthermore, CRISPRa-based OCT4 overexpression in the HNSCC cell line resulted in apoptosis resistance, and cisplatin was found to downregulate OCT4 protein expression in vitro. Ex vivo drug sensitivity analysis of HNSCC tumors confirmed the association between OCT4 expression and cisplatin sensitivity. CONCLUSION: This study introduces OCT4 immunohistochemistry as a simple and cost-effective diagnostic approach for clinical practice to identify HNSCC patients benefitting from radiosensitization by cisplatin using either full or reduced dosing.

Tumor-Associated Trypsin Inhibitor (TATI) as a Biomarker of Poor Prognosis in Oropharyngeal Squamous Cell Carcinoma Irrespective of HPV Status.

BACKGROUND: We studied the role of tumor-associated trypsin inhibitor (TATI) in serum and in tumor tissues among human papillomavirus (HPV)-positive and HPV-negative OPSCC patients. MATERIALS AND METHODS: The study cohort included 90 OPSCC patients treated at the Helsinki University Hospital (HUS), Helsinki, Finland, in 2012-2016. TATI serum concentrations (S-TATIs) were determined by an immunofluorometric assay. Immunostaining was used to assess tissue expression. HPV status was determined with a combination of p16 immunohistochemistry and HPV DNA PCR genotyping. The survival endpoints were overall survival (OS) and disease-specific survival (DSS). RESULTS: A significant correlation was found between S-TATI positivity and poor OS (p < 0.001) and DSS (p = 0.04) in all patients. In HPV-negative cases, S-TATI positivity was linked to poor OS (p = 0.01) and DSS (p = 0.05). In HPV-positive disease, S-TATI positivity correlated with poor DSS (p = 0.01). S-TATI positivity was strongly associated with HPV negativity. TATI serum was negatively linked to a lower cancer stage. TATI expression in peritumoral lymphocytes was associated with favorable OS (p < 0.025) and HPV positivity. TATI expression in tumor and in peritumoral lymphocytes correlated with lower cancer stages. CONCLUSION: Our results suggest that S-TATI positivity may be a biomarker of poor prognosis in both HPV-positive and HPV-negative OPSCC.

Comparing serum protein levels can aid in differentiating HPV-negative and -positive oropharyngeal squamous cell carcinoma patients.

BACKGROUND: The surrogate immunohistochemical marker, p16INK4a, 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 p16INK4a-positive but HPV DNA-negative, or RNA-negative, may be unsuitable for treatment de-escalation aimed at reducing treatment-related side effects. We aimed to identify cost-effective serum markers to improve decision making for patients at risk of misclassification by p16INK4a 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 p16INK4a/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). RESULTS: Of 174 serum proteins identified, complement component C7 (C7), apolipoprotein F (ApoF) and galectin-3-Binding Protein (LGALS3BP) significantly differed between HPV-positive and -negative tumors (AUC ranging from 0.84-0.87). ApoF levels were more than twice as high in the E6/E7 mRNA HPV-positive group than HPV-negative. CONCLUSIONS: Serum C7, ApoF and LGALS3BP levels discriminate between HPV-positive and HPV-negative OPSCC. Further studies are needed to validate these host immunity-related proteins as markers for HPV-associated OPSCC.

Epstein-Barr virus (EBV) and polyomaviruses are detectable in oropharyngeal cancer and EBV may have prognostic impact.

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 HPV-positive 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.

Elevated TLR5 expression in vivo and loss of NF-κΒ activation via TLR5 in vitro detected in HPV-negative oropharyngeal squamous cell carcinoma.

In oropharyngeal squamous cell carcinoma (OPSCC), the expression pattern of toll-like receptors (TLRs), in comparison between human papillomavirus (HPV)-positive and -negative tumors differs. TLRs control innate immune responses by activating, among others, the nuclear factor-κΒ (NF-κΒ) signaling pathway. Elevated NF-κΒ activity is detectable in several cancers and regulates cancer development and progression. We studied TLR5 expression in 143 unselected consecutive OPSCC tumors, and its relation to HPV-DNA and p16 status, clinicopathological parameters, and patient outcome, and studied TLR5 stimulation and consecutive NF-κB cascade activation in vitro in two human OPSCC cell lines and immortalized human keratinocytes (HaCat). Clinicopathological data came from hospital registries, and TLR5 immunoexpression was evaluated by immunohistochemistry. Flagellin served to stimulate TLR5 in cultured cells, followed by analysis of the activity of the NF-κB signaling cascade with In-Cell Western for IκΒ and p-IκΒ. High TLR5 expression was associated with poor disease-specific survival in HPV-positive OPSCC, which typically shows low TLR5 immunoexpression. High TLR5 immunoexpression was more common in HPV-negative OPSCC, known for its less-favorable prognosis. In vitro, we detected NF-κΒ cascade activation in the HPV-positive OPSCC cell line and in HaCat cells, but not in the HPV-negative OPSCC cell line. Our results suggest that elevated TLR5 immunoexpression may be related to reduced NF-κΒ activity in HPV-negative OPSCC. The possible prognosis-worsening mechanisms among these high-risk OPSCC patients however, require further evaluation.

High levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) in the serum are associated with poor prognosis in HPV-negative squamous cell oropharyngeal cancer.

BACKGROUND: An emerging subset of oropharyngeal squamous cell carcinomas (OPSCC) is caused by HPV. HPV-positive OPSCC has a better prognosis than HPV-negative OPSCC, but other prognostic markers for these two different diseases are scarce. Our aim was to evaluate serum levels and tumor expression of matrix metalloproteinase-8 (MMP-8) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and to assess their prognostic role in HPV-positive and HPV-negative OPSCC. MATERIALS AND METHODS: A total of 90 consecutive OPSCC patients diagnosed and treated with curative intent at the Helsinki University Hospital between 2012 and 2016 were included. Serum samples were prospectively collected. An immunofluorometric assay and an enzyme-linked immunosorbent assay were used to determine MMP-8 and TIMP-1 serum concentrations, respectively. HPV status of the tumors was determined using a combination of HPV-DNA genotyping and p16-INK4a immunohistochemistry. The endpoints were overall survival (OS) and disease-free survival (DFS). RESULTS: High TIMP-1 serum levels were strongly and independently associated with poorer OS (adjusted HR 14.7, 95% CI 1.8-117.4, p = 0.011) and DFS (adjusted HR 8.7, 95% CI 1.3-57.1, p = 0.024) among HPV-negative patients; this association was not observed in HPV-positive OPSCC. Although TIMP-1 was immunoexpressed in the majority of the tumor tissue samples, the level of immunoexpression was not associated with prognosis, nor did MMP-8 serum levels. CONCLUSION: Our results indicate that serum TIMP-1 levels may serve as an independent prognostic marker for HPV-negative OPSCC patients.

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

Current human papillomavirus (HPV) detection methods in oropharyngeal squamous cell carcinoma (OPSCC) have varying sensitivity and specificity. We aimed to compare different HPV-detection methods against the test used in clinical practice, ie, p16 immunohistochemistry (IHC) and to evaluate whether another HPV-detection test additional to p16 IHC would be worthwhile in OPSCC specimens. The study cohort comprised 357 consecutive OPSCC patients during two time periods: 2000-2009 and 2012-2016. From tumor tissue slides, HPV mRNA via in situ hybridization (ISH), HPV DNA via ISH and HPV DNA via polymerase chain reaction (PCR) were detected. The results of these methods were compared with p16 IHC results. Additionally, clinicopathological factors were compared with the methods studied. The sensitivity of HPV mRNA ISH, HPV DNA ISH and HPV DNA PCR were 93.4%, 86.3%, and 83.5%, respectively. The corresponding specificity was 92.4%, 95.3%, and 89.1%, respectively. The negative predictive value for p16 IHC was highest (89.0%) when using mRNA ISH, and followed by DNA ISH (83.5%). ISH for high-risk HPV E6/E7 mRNA was found to be a highly specific and sensitive method for detecting HPV in OPSCC. As p16 protein may be overexpressed due to HPV-independent mechanisms, all p16 IHC-positive OPSCCs should be considered for retesting using mRNA ISH in order to verify transcriptionally active HPV. This is especially critical when considering de-escalated treatment approaches for patients with HPV-positive tumors and still maintaining favorable outcomes for this subgroup of patients.

Newly detected DNA viruses in juvenile nasopharyngeal angiofibroma (JNA) and oral and oropharyngeal squamous cell carcinoma (OSCC/OPSCC).

PURPOSE: Approximately 20% of cancers are estimated to have a viral etiology. We aimed to investigate whether DNA of 8 human parvoviruses [bocavirus 1-4 (HBoV1-4), parvovirus B19 (B19V), protoparvoviruses (bufa-, tusa-, and cutavirus)] and 13 human polyomaviruses (HPyV) can be detected in oropharyngeal and oral cavity squamous cell carcinoma (OPSCC/OSCC), and in juvenile nasopharyngeal angiofibroma (JNA) tissue samples. METHODS: Fresh samples of seven JNA tissues and ten paired tissues of OSCC/OPSCC tumor and adjacent healthy tissues were collected. DNA extraction and real-time PCRs were performed to detect HBoV1-4, B19V, bufa- tusa- and cutavirus, and HPyV genomes. RESULTS: JNA specimens were negative for all parvoviruses tested, whereas one JNA sample was Merkel cell polyomavirus (MCPyV) DNA positive. The OSCC/OPSCC samples were negative for the human protoparvoviruses, HBoV1-4, and all human polyomaviruses, except for one patient that was MCPyV DNA positive in both healthy and tumor tissues. Seven OSCC/OPSCC patients were positive for B19V DNA, three of them in both healthy and cancerous tissues and three in only healthy tissues. Three of the B19V DNA-positive patients harbored viral genotype 1, three genotype 2, and one genotype 3B. CONCLUSIONS: These are the first reports of MCPyV and B19V DNA being detected in JNA and OPSCC. The significance of viral DNA positivity is unclear. B19V DNA is known to remain in the tissues lifelong, however, it is of interest that there are some patients with B19 DNA in healthy tissue, but not in the corresponding cancer tissue.

Patients with early-stage oropharyngeal cancer can be identified with label-free serum proteomics.

BACKGROUND: The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) is mainly related to human papillomavirus (HPV) infection. As OPSCCs are often diagnosed at an advanced stage, mortality and morbidity remain high. There are no diagnostic biomarkers for early detection of OPSCC. METHODS: Serum from 25 patients with stage I-II OPSCC, and 12 healthy controls, was studied with quantitative label-free proteomics using ultra-definition MSE. Statistical analyses were performed to identify the proteins most reliably distinguishing early-stage OPSCCs from controls. P16 was used as a surrogate marker for HPV. P16-positive and P16-negative tumours were analysed separately. RESULTS: With two or more unique proteins per identification, 176 proteins were quantified. A clear separation between patients with early-stage tumours and controls was seen in principal component analysis. Latent structures discriminant analysis identified 96 proteins, most reliably differentiating OPSCC patients from controls, with 13 upregulated and 83 downregulated proteins in study cases. The set of proteins was studied further with network, pathway and protein-protein interaction analyses, and found to participate in lipid metabolism, for example. CONCLUSIONS: We found a set of serum proteins distinguishing early-stage OPSCC from healthy individuals, and suggest a protein set for further evaluation as a diagnostic biomarker panel for OPSCC.