Nasopharyngeal carcinoma in Finland : Epstein-Barr virus, human papillomaviruses, and toll-like receptors as prognostic factors

Nasopharyngeal carcinoma (NPC) is a malignant tumour arising from the surface epithelium of the nasopharynx. NPC is a rare cancer type in Northern Europe, and approximately ten new cases are diagnosed in Finland yearly. NPC carcinogenesis has been linked to Epstein-Barr virus (EBV) infection, but human papillomaviruses (HPVs) have also been found in NPC tumours. Presumably, the host’s innate immunological properties also have an impact on pathological processes. Toll-like receptors (TLRs) are transmembrane proteins, which recognize both microbial and host’s own structures released in tissue damage in order to regulate innate immunity. The expression of TLRs has been observed in many cancer types, but their actual function is still unknown. TLR stimulation may have both pro- or anti-tumoural effects. The aim of this nationwide study was to evaluate NPC incidence, histological subgroups, provided treatments and their adverse effects, and outcome in NPC patients diagnosed and treated in Finland from 1990 to 2009. A total of 207 patients were identified from the Finnish Cancer Registry database during the 20-year study period. In addition, we examined the samples of 150/207 patients for the presence of EBV, high-risk HPVs, and TLR expression (TLR1, TLR2, TLR4, TLR5, TLR7, and TLR9), and compared the results with survival data. EBV was found in 62% and HPVs in 14% of the tumour cases, while 24% of the tumours were EBV/HPV-negative. The patients with EBV-positive tumours had the best 5-year survival rates, and the patients with HPV-positive tumours had a significantly better overall survival than those with EBV/HPV-negative tumours. Moreover, positive TLR7 expression was found to be an independent prognostic factor for favourable outcome

Epstein-Barr virus and human papillomaviruses as favorable prognostic factors in nasopharyngeal carcinoma : A nationwide study in Finland

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.Peer reviewe

Effects of parental exposure to glyphosate-based herbicides on embryonic development and oxidative status: a long-term experiment in a bird model

IntroductionGlyphosate (N-[Phosphonomethyl]glycine)-based herbicides (GBHs) are the most frequently used herbicides globally and also one of the most controversial agrochemicals1. Evidence is accumulating with regard to the potentially negative effects of glyphosate on the development, phenotype, and fitness of most non-target animal taxa from invertebrates to vertebrates, yet, exposure levels (natural exposure load vs. levels used in experimental studies) need to be carefully accounted for2,3,4. Non-target organisms are commonly exposed to GBH residues in the food chain because residues can persist in soil, water, and plants5,6. In particular, the estimated amount of glyphosate introduced into the food chain through genetically modified, i.e. glyphosate tolerant crops (such as soybeans) add up to several thousands of metric tonnes yearly7. Consequently, different regulatory authorities heatedly debate the effects of GBH in our ecosystems.Organisms in early developmental stages are generally more susceptible to external stress compared to adults. In the case of environmental toxins, this may be related to disturbed ontogeny or undeveloped detoxification metabolism in juveniles8. In aquatic animals, embryos can be directly exposed to GBHs via the surrounding water. Glyphosate and commercial products (e.g. RoundUp) made with glyphosate have been repeatedly reported to cause embryo mortality and deformations in fish (zebrafish 10uM to 1 mM pure glyphosate9, 10 mg/L RoundUp or pure glyphosate10), and aquatic amphibians (Xenopus 0.3–1.3 mg/L RoundUp11, 500 pg/egg pure glyphosate12). In contrast, mammal and bird embryos and fetuses are exposed to glyphosate residues only via maternal transfer of the chemicals, which may result in malformations, altered sex ratios, and low sperm quality in rodent models (doses: 500 mg/kg RoundUp13, 5 g/L pure glyphosate14, 50–450 mg/kg RoundUp15). Such effects are referred to as (transmissive) maternal effects sensu16. Furthermore, recent studies suggest that effects of GBHs on the next generation can be mediated via epigenetic paternal effects, for example via alterations of paternal sperm(17, parental generation, a dose of 25 mg/kg BW pure glyphosate in rats18).However, the true maternal and paternal effects of GBH are poorly understood because the majority of the studies are involving direct embryo manipulations with high doses of GBHs. The authors of future studies should take into account that GBHs may influence the quality of the resources allocated to eggs/embryos and thus offspring development, phenotype, and fitness indirectly. Prenatal environmental conditions, and for example hormonal signals from the mother are known to have crucial importance for offspring development and even lasting effects into adulthood19,20,21.In this study we used birds as a model to study the parental and developmental effects of GBHs. Birds are highly underrepresented in studies testing the adverse effects of GBH residues on non-target taxa2, although they have recently been suggested as a key group for biomonitoring with regard to the effects of GBHs22. The importance of poultry in food production also calls for more attention on residues and the effects of GBHs in birds. In the two available studies of poultry and GBH-related maternal effects, a direct injection of a relatively high concentration of RoundUp (10 mg/kg glyphosate) in eggs was found to decrease hatchability, induce oxidative stress and cause damage to lipids in the exposed chicks, as compared to the control group23,24, potentially via the disruption of retinoid acid signaling12.To understand the potential for GBH-induced parental effects, we studied parental exposure of GBHs on embryo development and key physiological biomarkers—embryonic brain oxidative status in a bird model. To our knowledge, this is the first long-term study on parental effects of GBHs in bird taxa. Oxidative stress refers to the imbalance between reactive oxygen species (ROS) and antioxidants: If antioxidants are not able to neutralize ROS, oxidative damage to cell components (proteins, lipids, and DNA) will occur, which then has negative consequences on cell functions25. GBHs have been previously found to induce oxidative stress and damage in a variety of organisms and tissues, including embryos reviewed in2. We quantified glyphosate residues in eggs, but also maternal allocation to eggs (egg, yolk, and shell mass and yolk thyroid hormone concentration) to account for potential indirect GBH effects. Prenatal thyroid hormones (THs) (thyroxine, T4 and triiodothyronine, T3) play a key role in coordinating embryo development20,26, especially brain development27. Embryo THs have been reported to vary with maternal GBH exposure in rats (dose: RoundUp 5 and 50 mg/kg/day28), but generally the effects of GBHs on THs are poorly understood. Japanese quails were selected as the model species because the results can be applied to both wild birds feeding on GBH-contaminated food in the field and to poultry farming. We experimentally exposed parental bird generation to GBHs (ca 200 mg/kg feed) or respective controls from 10 days of age to 12 months. The egg samples were collected at 4 and 12 months to examine the potential cumulative effects of long-term exposure. We measured the potential effects on (1) on egg quality (egg, yolk, and shell mass as well as egg thyroid hormones); (2) on embryo development; and (3) embryo oxidative stress and damage.ResultsWe detected 0.76 mg/kg (S.D. ± 0.16) of glyphosate residue in eggs see also29, which is above the levels reported in the previous literature30. Egg mass from GBH and control parents did not differ after 4 or 12 months of exposure (treatment F1, 17,1 = 0.12, p = 0.73, treatment*exposure duration F1, 270 = 0.02, p = 0.89, Table 1) but was generally larger at 12 months of age (duration: F1, 271 = 8.8, p = 0.003). No differences between GBH exposed and control females in yolk mass, shell mass, or egg T3 and T4 concentrations were detected (Table 1, Suppl Fig. 1).Table 1 Quality of the eggs (egg, yolk, and shell mass; thyroid hormone concentrations: T3 = triiodothyronine, T4 = thyroxine, average ± SD) from GBH (glyphosate based herbicide)-exposed and control females. The egg mass was averaged over all eggs (4 and 12 months of exposure). The other parameters were measured after 4 months of exposure. See text for power analysis.Full size tableEmbryo development was normal in 89% of control eggs, while 76% of GBH eggs had normally developed embryos when pooling data from 3-day and 10-day embryos. The lower percentage of normal development in GBH eggs tended to be statistically significant (treatment F1, 22 = 3.08, p = 0.09) and the trend was similar at both 4 and 12 months of parental exposure (treatment*duration F1, 312 = 0.6, p = 0.43, Fig. 1). The eggs with no or little development were distributed across pairs and for none of the pairs were all eggs classified as undeveloped. Brain mass did not differ between embryos from GBH-exposed and control parents (mean ± SD in mg; GBH: 67.1 ± 12.5, control 68.1 ± 15.5; F1,31 = 0.04, p = 0.84). Brain oxidative status at 12 months of parental exposure was measured from 19 control and 16 GBH embryos. We measured ca 20% higher lipid damage in the GBH embryos than controls. This difference tended to be statistically significant (F1, 16.8 = 3.2, p = 0.088, Table 2, Suppl Fig. 2), yet there were no differences in the activity of antioxidant enzymes (GST, GP or CAT) between the two groups (Table 2, Suppl Fig. 2).Figure 1Embryonic status in relation to glyphosate-based herbicide exposure and duration of the exposure. GBH = glyphosate exposed, CO = controls. The bars are drawn separately for GBH and control eggs and after 4 and 12 months of exposure: we assessed 3-day-old embryos at 4 months and at 10-day-old embryos 12 months. Sample sizes are indicated in parentheses.Full size imageTable 2 Average (±SD) of glutathione-S-transferase (GST), glutathione peroxidase (GP), catalase (CAT) activity, and damage to lipids (MDA) in 10-day-old Japanese quail embryos exposed to maternally-derived glyphosate-based herbicide (GBH) or unexposed embryos (control). Associate statistics from linear mixed models (LMMs) are reported below. See text for power analysis.Full size tableDiscussionOur results indicate that parental exposure to GBHs may lead to weak negative effects on embryo development and physiology. We detected no evidence for changes in egg quality (egg, yolk, shell mass, or egg hormone concentration), suggesting no indirect effects via the altered allocation of resources or hormones to eggs and embryos.The tendency for poorer embryo development in eggs of GBH-exposed parents may be explained by GBH-related effects via either a paternal or maternal route, or both. We did not appear to observe cumulative effects of GBH exposure on embryo viability, as longer exposure (12 vs 4 months) did not lead to less viability. The eggs with no visible development (to the naked eye) could have been completely infertile or showing developmental arrest at an early stage. There are multiple potential mechanisms underlying such effects: For example, early embryos of fish showed developmental anomalies (disproportional head and body size) and increased heart rate (medeka, 100–500 mg/L RoundUp31, neurotoxic effects zebrafish, up to 50 mg/L RoundUp32) and Xenopus showed craniofacial deformities12. Poultry embryos and mice oocytes expressed increased oxidative stress (500 mM pure glyphosate33) and lower hatchability (10 mg/kg egg RoundUp<a title="Fathi, M. A. et al. Effect of in ovo glyphosate injection on embryonic development, serum

Tumor-infiltrating lymphocytes associate with outcome in nonendemic nasopharyngeal carcinoma : a multicenter study

The prognostic significance of tumor-infiltrating lymphocytes (TILs) has been studied recently in many cancers. For the first time in a nonendemic region, we have evaluated the prognostic value of TILs in a whole population based nationwide cohort of nasopharyngeal carcinoma (NPC) in Finland. A total of 115 cases from Finnish hospitals were included. TILs were analyzed using hematoxylin and eosin stained slides according to the criteria of the International Immuno-Oncology Biomarker Working Group. TILs were evaluated separately in stromal and tumor compartments. The log-rank test and univariable and multivariable analyses were used to compare survival in patients with tumors with low and high TILs. A significant positive correlation was observed between the occurrence of intratumoral and stromal TILs (P <.001). In multivariable analysis, NPC cases with low intratumoral TILs had poor overall survival with a hazard ratio (HR) of 2.55 and 95% confidence interval (95% CI) of 1.60 to 4.05 (P <.001). Cases with low intratumoral TILs also had poor disease-specific survival (HR, 2.02; 95% CI, 1.16-3.52; P = .015). Keratinized tumors with low intratumoral TILs were associated with an even poorer overall survival (HR, 3.94; 95% CI, 2.17-7.15; P <.001) and a poor disease-specific survival (HR, 2.97; 95% CI, 1.46-6.05; P = .009). Our study demonstrates that the evaluation of TILs is simple and can be assessed routinely in NPC. (C) 2018 Elsevier Inc. All rights reserved.Peer reviewe

Adaptation of bacteria to glyphosate: a microevolutionary perspective of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase

Glyphosate is the leading herbicide worldwide, but it also affects prokaryotes because it targets the central enzyme (5-enolpyruvylshikimate-3-phosphate, EPSP) of the shikimate pathway in the synthesis of the three essential aromatic amino acids in bacteria, fungi and plants. Our results reveal that bacteria may easily become resistant to glyphosate through changes in the 5-enolpyruvylshikimate-3-phosphate synthase active site. This indicates the importance of examining how glyphosate affects microbe-mediated ecosystem functions and human microbiomes

Epstein-Barr virus and human papillomaviruses as favorable prognostic factors in nasopharyngeal carcinoma: A nationwide study in Finland

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.</p

Glyphosate-based herbicides influence antioxidants, reproductive hormones and gut microbiome but not reproduction: A long-term experiment in an avian model

Controversial glyphosate-based herbicides (GBHs) are the most frequently used herbicides globally. GBH residues in the wild, in animal and human food may expose non-target organisms to health risks, yet the developmental and cumulative effects of GBHs on physiology and reproduction remain poorly understood. We present the first long-term study on the effects of subtoxic GBH exposure (160 mg/kg) on multiple key physiological biomarkers (cellular oxidative status and neurotransmitters), gut microbiome, reproductive hormones, and reproduction in an avian model. We experimentally exposed in Japanese quail females and males (Cotumix japonica) to GBHs and respective controls from the age of 10 days -52 weeks. GBH exposure decreased hepatic activity of an intracellular antioxidant enzyme (catalase), independent of sex, but did not influence other intracellular oxidative stress biomarkers or neurotransmitter enzyme (acetylcholinesterase). GBH exposure altered overall gut microbiome composition, especially at a younger age and in females, and suppressed potentially beneficial microbes at an early age. Many of the microbial groups increased in frequency from 12 to 28 weeks under GBH exposure. GBH exposure decreased male testosterone levels both at sexual maturity and at 52 weeks of exposure, but did not clearly influence reproduction in either sex (maturation, testis size or egg production). Future studies are needed to characterize the effects on reproductive physiology in more detail. Our results suggest that cumulative GBH exposure may influence health and reproduction-related traits, which is important in predicting their effects on wild populations and global poultry industry. (C) 2020 The Authors. Published by Elsevier Ltd

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 patientselective 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 crossvalidated 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 this radioresistance 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.Peer reviewe

Finnish Version of the Eating Assessment Tool (F-EAT-10) : A Valid and Reliable Patient-reported Outcome Measure for Dysphagia Evaluation

Our aim was to validate a Finnish version of the Eating Assessment Tool (F-EAT-10) for clinical use and to test its reliability and validity in a multicenter nationwide study. Normative data were acquired from 180 non-dysphagic participants (median age 57.0 years, 62.2% female). Dysphagia patients (n = 117, median age 69.7 years, 53.0% female) referred to fiberoptic endoscopic evaluation of swallowing (FEES) completed F-EAT-10 before the examination and after 2 weeks. Patients underwent the 100-ml water swallow test (WST) and FEES was evaluated using the following three scales: the Yale Pharyngeal Residue Severity Rating Scale, Penetration-Aspiration Scale, and the Dysphagia Outcome Severity Scale. An operative cohort of 19 patients (median age 75.8 years, 57.9% female) underwent an endoscopic operation on Zenker's diverticulum, tight cricopharyngeal muscle diagnosed in videofluorography, or both. Patients completed the F-EAT-10 preoperatively and 3 months postoperatively. The cut-off score for controls was = 3 is abnormal. Re-questionnaires for test-retest reliability analysis were available from 92 FEES patients and 123 controls. The intraclass correlation coefficient was excellent for the total F-EAT-10 score (0.93, 95% confidence interval 0.91-0.95). Pearson correlation coefficients were strong (p < 0.001) for each of the questions and the total score. Internal consistency as assessed by Cronbach's alpha was excellent (0.95). Some correlations between findings in FEES and 100-ml WST with F-EAT-10 were observed. The change in subjective symptoms of operative patients paralleled the change in F-EAT-10. F-EAT-10 is a reliable, valid, and symptom-specific patient-reported outcome measure for assessing dysphagia among Finnish speakers.Peer reviewe

Finnish Version of the Eating Assessment Tool (F-EAT-10): A Valid and Reliable Patient-reported Outcome Measure for Dysphagia Evaluation

Our aim was to validate a Finnish version of the Eating Assessment Tool (F-EAT-10) for clinical use and to test its reliability and validity in a multicenter nationwide study. Normative data were acquired from 180 non-dysphagic participants (median age 57.0 years, 62.2% female). Dysphagia patients (n = 117, median age 69.7 years, 53.0% female) referred to fiberoptic endoscopic evaluation of swallowing (FEES) completed F-EAT-10 before the examination and after 2 weeks. Patients underwent the 100-ml water swallow test (WST) and FEES was evaluated using the following three scales: the Yale Pharyngeal Residue Severity Rating Scale, Penetration-Aspiration Scale, and the Dysphagia Outcome Severity Scale. An operative cohort of 19 patients (median age 75.8 years, 57.9% female) underwent an endoscopic operation on Zenker's diverticulum, tight cricopharyngeal muscle diagnosed in videofluorography, or both. Patients completed the F-EAT-10 preoperatively and 3 months postoperatively. The cut-off score for controls was = 3 is abnormal. Re-questionnaires for test-retest reliability analysis were available from 92 FEES patients and 123 controls. The intraclass correlation coefficient was excellent for the total F-EAT-10 score (0.93, 95% confidence interval 0.91-0.95). Pearson correlation coefficients were strong (p < 0.001) for each of the questions and the total score. Internal consistency as assessed by Cronbach's alpha was excellent (0.95). Some correlations between findings in FEES and 100-ml WST with F-EAT-10 were observed. The change in subjective symptoms of operative patients paralleled the change in F-EAT-10. F-EAT-10 is a reliable, valid, and symptom-specific patient-reported outcome measure for assessing dysphagia among Finnish speakers