LRG1 and SDR16C5 protein expressions differ according to HPV status in oropharyngeal squamous cell carcinoma.

The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) is primarily due to human papillomavirus, and understanding the tumor biology caused by the virus is crucial. Our goal was to investigate the proteins present in the serum of patients with OPSCC, which were not previously studied in OPSCC tissue. We examined the difference in expression of these proteins between HPV-positive and -negative tumors and their correlation with clinicopathological parameters and patient survival. The study included 157 formalin-fixed, paraffin-embedded tissue samples and clinicopathological data. Based on the protein levels in the sera of OPSCC patients, we selected 12 proteins and studied their expression in HPV-negative and HPV-positive OPSCC cell lines. LRG1, SDR16C5, PIP4K2C and MVD proteins were selected for immunohistochemical analysis in HPV-positive and -negative OPSCC tissue samples. These protein´s expression levels were compared with clinicopathological parameters and patient survival to investigate their clinical relevance. LRG1 expression was strong in HPV-negative whereas SDR16C5 expression was strong in HPV-positive tumors. Correlation was observed between LRG1, SDR16C5, and PIP4K2C expression and patient survival. High expression of PIP4K2C was found to be an independent prognostic factor for overall survival and expression correlated with HPV-positive tumor status. The data suggest the possible role of LRG1, SDR16C5 and PIP4K2C in OPSCC biology.

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.

Inflammation parameters predict fatal outcome in male COVID-19 patients in a low case-fatality area - a population-based registry study.

BACKGROUND: Male sex predicts case-fatality in SARS-CoV-2 (COVID-19) - a phenomenon linked to systemic inflammation. We compared sex-related associations of inflammation parameters and outcome in a population-based setting with low case-fatality prior to wide use of immunosuppressives. METHODS: A population-based quality registry with laboratory-confirmed COVID-19 cases of specialized hospitals of the Capital Province of Finland were analysed to compare inflammatory parameters by sex during the first COVID-19 wave February-June 2020. RESULTS: Altogether, 585 hospitalized patients (54% males) were included. Males required more often intensive care unit (ICU) treatment (26.9 vs. 17.5%) and had higher 90-d case-fatality (14.9 vs. 7.8%) compared with females. Highest association with case-fatality in males was seen for high neutrophil counts (median; interquartile range) (8.70; 7.10-9.10 vs. 5.60; 3.90-7.80) (E9/l), low monocyte (0.50; 0.20-1.50 vs. 0.70; 0.50-0.90) (E9/l) and lymphocyte (0.90; 0.70-1.40 vs. 1.50; 1.10-2.00) (E9/l) counts, and high levels of d-dimer (3.80; 1.80-5.30 vs. 1.10; 0.60-2.75) (mg/l) and C-reactive protein (CRP) (190; 85.5-290 vs. 77.0; 49.0-94.0) (mg/l). In females, low lymphocyte (0.95; interquartile range 0.60-1.28 vs. 1.50; 1.10-2.00) (E9/l) and thrombocyte counts (196; 132-285 vs. 325; 244-464) (E9/l) and high CRP values (95.0; 62.0-256 vs. 66.0; 42.5-89.0) (mg/l) were associated with case-fatality. In multivariable analysis for males, lymphocyte cut-off 0.85 (E9/l) (OR 0.02; 95% CI 0.002-0.260), d-dimer cut-off 1.15 (mg/l) (OR 7.29; 1.01-52.6) and CRP cut-off 110 (mg/l) (OR 15.4; 1.87-127) were independently associated with case-fatality. In female multivariable analysis, CRP cut-off 81 (mg/l) (OR 7.32; 1.44-37.2) was the only inflammatory parameter associated with case-fatality. CONCLUSIONS: COVID-19 results in higher inflammation parameter levels in male vs. female patients irrespective of outcome. This study suggests that low lymphocyte, high d-dimer and high CRP cut-off values may serve as potential markers for risk stratification in male patients.

Association between first language and SARS-CoV-2 infection rates, hospitalization, intensive care admissions and death in Finland: a population-based observational cohort study.

OBJECTIVES: Motivated by reports of increased risk of coronavirus disease 2019 (COVID-19) in ethnic minorities of high-income countries, we explored whether patients with a foreign first language are at an increased risk of COVID-19 infections, more serious presentations, or worse outcomes. METHODS: In a retrospective observational population-based quality registry study covering a population of 1.7 million, we studied the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), admissions to specialist healthcare and the intensive care unit (ICU), and all-cause case fatality in different language groups between 27th February and 3rd August 2020 in Southern Finland. A first language other than Finnish, Swedish or Sámi served as a surrogate marker for a foreign ethnic background. RESULTS: In total, 124 240 individuals were tested, and among the 118 300 (95%) whose first language could be determined, 4005 (3.4%) were COVID-19-positive, 623 (0.5%) were admitted to specialized hospitals, and 147 (0.1%) were admitted to the ICU; 254 (0.2%) died. Those with a foreign first language had lower testing rates (348, 95%CI 340-355 versus 758, 95%CI 753-762 per 10 000, p < 0.0001), higher incidence (36, 95%CI 33-38 versus 22, 95%CI 21-23 per 10 000, p < 0.0001), and higher positivity rates (103, 95%CI 96-109 versus 29, 95%CI 28-30 per 1000, p < 0.0001). There was no significant difference in ICU admissions, disease severity at ICU admission, or ICU outcomes. Case fatality by 90 days was 7.7% in domestic cases and 1.2% in those with a foreign first language, explained by demographics (age- and sex-adjusted HR 0.49, 95%CI 0.21-1.15). CONCLUSIONS: The population with a foreign first language was at an increased risk for testing positive for SARS-CoV-2, but when hospitalized they had outcomes similar to those in the native, domestic language population. This suggests that special attention should be paid to the prevention and control of infectious diseases among language minorities.

Male predominance in disease severity and mortality in a low Covid-19 epidemic and low case-fatality area - a population-based registry study.

BACKGROUND: Men reportedly suffer from a more severe disease and higher mortality during the global SARS-CoV-2 (Covid-19) pandemic. We analysed sex differences in a low epidemic area with low overall mortality in Covid-19 in a population based setting with patients treated in specialized healthcare. METHODS: We entered all hospitalized laboratory-confirmed Covid-19 cases of all specialized healthcare hospitals of the Capital Province of Finland, into a population-based quality registry and described demographics, severity and case-fatality by sex of the first Covid-19 wave February-June 2020. RESULTS: Altogether 5471 patients (49% male) were identified. Patients hospitalized in the specialist healthcare (N = 585, 54% male, OR 1.25; 95% CI 1.05-1.48) were of the same age. Men had less asthma and thyroid insufficiency and more coronary artery disease compared to women. Mean time from symptom onset to diagnosis was at least one day longer for men (p=.005). Men required intensive care unit (ICU) more often (27% vs. 17%) with longer lengths-of-stays at ICU. Male sex associated with significantly higher case-fatality at 90-days (15% vs. 8%) and all excess male deaths occurring after three weeks from onset. Men with fatal outcomes had delays in both Covid-19 testing and hospital admission after a positive test. The delays in patients with the most severe and fatal outcomes differed markedly by sex. In multivariable analysis, male sex associated independently with case-fatality (OR 2.37; 95% CI 1.22-4.59). CONCLUSIONS: Male sex associated with higher disease severity and case-fatality. Late presentation of male fatal cases could represent different treatment-seeking behaviour or disease progression by sex.

Mass spectrometry-based lipidomics of oral squamous cell carcinoma tissue reveals aberrant cholesterol and glycerophospholipid metabolism - A Pilot study.

Lipid metabolic reprogramming is one hallmark of cancer. Lipid metabolism is regulated by numerous enzymes, many of which are targeted by several drugs on the market. We aimed to characterize the lipid alterations in oral squamous cell carcinoma (OSCC) as a basis for understanding its lipid metabolism, thus identifying potential therapeutic targets. We compared lipid species, classes, and glycerophospholipid (GPL) fatty acid species between paired tumor tissue and healthy oral tongue mucosa samples from 10 OSCC patients using a QExactive mass spectrometer. After filtering the 1370 lipid species identified, we analyzed 349 species: 71 were significantly increased in OSCC. The GPL metabolism pathway was most represented by the lipids differing in OSCC (P = .005). Cholesterol and the GPLs phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols were most significantly increased in OSCC tissue (FC 1.8, 2.0, 2.1, and 2.3 and, P = .003, P = .005, P = .002, P = .007). In conclusion, we have demonstrated a shift in the lipid metabolism in these OSCC samples by characterizing the detailed landscape. Predominantly, cholesterol and GPL metabolism were altered, suggesting that interactions with sterol regulatory binding proteins may be involved. The FA composition changes of the GPLs suggest increased de novo lipogenesis.

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.

Tonsillar surface swab bacterial culture results differ from those of the tonsillar core in recurrent tonsillitis.

OBJECTIVES/HYPOTHESIS: We aimed to determine whether there was a difference between core and surface bacteriology of Finnish adults with recurrent or chronic tonsillitis to understand whether a surface swab is worthwhile and which bacteria are involved. STUDY DESIGN: Case series. METHODS: Uninflamed tonsillar surface swabs and core biopsies were taken prior to and during surgery, respectively, in 103 patients aged 16 to 66 years undergoing tonsillectomy for recurrent or chronic tonsillitis. The McNemar test was used to determine differences between the surface and core in the most prevalent bacterial species. RESULTS: Twenty-seven bacterial species were isolated in addition to normal flora and were more commonly found in the core (1.11 surface and 4.75 core bacteria isolated per patient). Viridans group streptococci were the most commonly detected bacteria, found in 88% of the patients, mainly in the core. The bacteria in general were mainly isolated from the core. Of the 10 most prevalent bacteria, only group C ß-hemolytic streptococci showed no difference between detection from core and surface swabs. Other bacteria found mainly in the core include Prevotella melaninogenica, Staphylococcus aureus, and fusobacteria. CONCLUSIONS: There is discord between the surface and core bacteria. A different population of bacteria exists in the core, especially anaerobic bacteria, suggesting that a core sample may be useful in evaluating recurrent and chronic tonsillitis. LEVEL OF EVIDENCE: 4 Laryngoscope, 2019.

Exhaled breath analysis in the diagnosis of head and neck cancer.

Head and neck cancer (HNC) comprises a heterogeneous group of upper aerodigestive tract malignant neoplasms, the most frequent of which is squamous cell carcinoma. HNC forms the eighth most common cancer type and the incidence is increasing. However, survival has improved only moderately during the past decades. Currently, early diagnosis remains the mainstay for improving treatment outcomes in this patient population. Unfortunately, screening methods to allow early detection of HNC are not yet established. Therefore, many cases are still diagnosed at advanced stage, compromising outcomes. Exhaled breath analysis (EBA) is a diagnostic tool that has been recently introduced for many cancers. Breath analysis is non-invasive, cost-effective, time-saving, and can potentially be applied for cancer screening. Here, we provide a summary of the accumulated evidence on the feasibility of EBA in the diagnosis of HNC.

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.

Presentation of second primary cancers in young laryngeal carcinoma patients.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is rare in the young. OBJECTIVES: We characterized the clinical behavior of LSCC and assessed the presentation of second primary tumors (SPCs) in this patient population. MATERIALS AND METHODS: Data from the Finnish Cancer Registry (FCR) were used to identify an epidemiological series of LSCC patients diagnosed at the age of 40 years or under, during 1953-2012 in Finland. Data regarding primary treatment, survival, and SPCs were available. To further characterize the comorbidity and lifestyle factors of young patients with LSCC, institutional data were collected of patients treated at the Helsinki University Hospital during 1967-2012. RESULTS: We identified 151 patients, with a mean follow-up of 252 months. The 10-year overall survival (OS) was 75% and the disease-specific survival was 84%. SPCs were diagnosed in 26% (n = 39), with a median delay of 28 years. Of the 35 patients in the institutional series from Helsinki, 22 (63%) were current or former smokers. LSCC recurred in 28% of patients. CONCLUSIONS AND SIGNIFICANCE: The delay to SPCs in young patients was significantly longer compared with the general LSCC population. As factors underlying this phenomenon cannot be identified by this retrospective study, further studies are warranted.

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.

Label-free tissue proteomics can classify oral squamous cell carcinoma from healthy tissue in a stage-specific manner.

OBJECTIVES: No prognostic or predictive biomarkers for oral squamous cell carcinoma (OSCC) exist. We aimed to discover novel proteins, altered in OSCC, to be further investigated as potential biomarkers, and to improve understanding about pathways involved in OSCC. MATERIALS AND METHODS: Proteomic signatures of seven paired healthy and OSCC tissue samples were identified using ultra-definition quantitative mass spectrometry, then analysed and compared using Anova, principal component analysis, hierarchical clustering and OPLS-DA modelling. A selection of significant proteins that were also altered in the serum from a previous study (PMID: 28632724) were validated immunohistochemically on an independent cohort (n = 66) to confirm immunopositivity and location within tumour tissue. Ingenuity Pathways Analysis was employed to identify altered pathways. RESULTS: Of 829 proteins quantified, 257 were significant and 72 were able to classify healthy vs OSCC using OPLS-DA modelling. We identified 19 proteins not previously known to be upregulated in OSCC, including prosaposin and alpha-taxilin. KIAA1217 and NDRG1 were upregulated in stage IVa compared with stage I tumours. Altered pathways included calcium signalling, cellular movement, haematological system development and function, and immune cell trafficking, and involved NF-kB and MAPK networks. CONCLUSIONS: We found a set of proteins reliably separating OSCC tumour from healthy tissue, and multiple proteins differing between stage I and stage IVa OSCC. These potential biomarkers can be studied and validated in larger cohorts.