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Circulating lipoproteins as risk factors or prognostic indicators for various cancers have been investigated previously; however, no clear consensus has been reached. In this study, we aimed at evaluating the impact of serum lipoproteins on the prognosis of patients with squamous cell carcinoma of the head and neck (SCCHN). Levels of total cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), triglycerides and lipoprotein(a) were measured in serum samples from 106 patients and 28 healthy controls. We found that HDL was the only lipoprotein exhibiting a significant difference in concentration between healthy controls and patients (p = 0.012). Kaplan–Meier survival curves indicated that patients with high levels of total cholesterol or LDL had better overall survival than patients with normal levels (p = 0.028 and p = 0.007, respectively). Looking at patients without lipid medication (n = 89) and adjusting for the effects of TNM stage and weight change, multivariate Cox regression models indicated that LDL was an independent prognostic factor for both overall (p = 0.005) and disease-free survival (p = 0.013). In summary, our study revealed that high LDL level is beneficial for survival outcome in patients with SCCHN. Use of cholesterol-lowering medicines for prevention or management of SCCHN needs to be evaluated carefully.

Background: Circulating markers are attractive molecules for prognosis and management of cancer that allow sequential monitoring of patients during and after treatment. Based on previous protein profiling data, circulating interleukin 1 receptor antagonist (IL-1Ra) was evaluated as a potential diagnostic and prognostic marker for squamous cell carcinomas of the head and neck (SCCHN). In this study, we aimed at confirming the clinical relevance of plasma IL-1Ra in SCCHN and exploring its potential as a prediction marker for SCCHN.

Methods: Plasma from 87 patients with SCCHN, control plasma from 28 healthy individuals and pre-diagnostic plasma from 44 patients with squamous cell carcinoma of the oral tongue (SCCOT) and 88 matched controls were analysed with IL-1Ra electrochemiluminescence immunoassays from mesoscale diagnostics.

Results: Plasma IL-1Ra was found to be up-regulated in patients with oral tongue, gingiva and base of tongue tumours compared to healthy individuals (p < 0.01). IL-1Ra levels positively correlated with tumour size (p < 0.01) and body mass index (p = 0.013). Comparing pre-diagnostic plasma to the matched controls, similar IL1-Ra levels were seen (p = 0.05).

Conclusion: The anti-inflammatory cytokine IL-1Ra could be a diagnostic marker for SCCHN, whereas its potential as a cancer prediction marker was not supported by our data.

Background: Squamous cell carcinoma of the head and neck (SCCHN)comprises a large group of tumours including the oral cavity and nasopharyngealarea, and typically affects older males in association with alcohol/tobacco usage.Within the oral cavity, the mobile tongue is the most common site for tumourdevelopment. The incidence of squamous cell carcinoma of the oral tongue(SCCOT) is increasing in younger people, which has been suggested to associatewith other than the traditional risk factors for this disease. Two common humanoncogenic viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV)are connected to certain types of SCCHN, in oropharynx and nasopharynxrespectively. The receptor programmed cell death 1 (PD)-1 and its ligandprogrammed cell death ligand 1 (PD-L1) are particularly relevant in immunecheckpoint control, and elevated levels have been seen in various cancer types. Alink between hyperlipidemia and cancer risk has previously been suggested. Theaim of this thesis was to investigate risk factors and prognostic features forSCCHN, by focusing on EBV, PD-L1 and serum lipoproteins.

Materials and methods: Ninety-eight cases of SCCOT and 15 cases of tonsillarsquamous cell carcinoma were examined for the presence of EBV-encodedribonucleic acids (EBERs), EBV deoxyribonucleic acid (DNA) and the proteinEBV-encoded nuclear antigen-1 (EBNA-1), using in situ hybridisation,polymerase chain reaction (PCR) and immunohistochemistry respectively. Onehundred and one cases of SCCOT were examined for expression of PD-L1 intumour and surrounding immune cells using Ventana SP263immunohistochemistry assay and a QuickScore (QS) method. An estimation oftumour-infiltrating immune cells was also performed in 25 of the patients.Circulating levels of PD-L1 were measured using an electrochemiluminescenceassay platform in serum from 30 patients. Finally, serum samples from 106patients and 28 healthy controls were investigated for levels of total cholesterol,low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides andlipoprotein(a).

Results: In the first study, using an in situ hybridisation kit no EBER transcriptswere detected. No EBV DNA was identified with PCR analysis, andimmunohistochemistry for EBNA-1 was also negative. In the second study, highertumour cell PD-L1 levels were found in females than males (p = 0.019). Forpatients with low PD-L1 in tumour cells, better survival was shown in males thanfemales (overall survival p = 0.021, disease-free survival p = 0.020). Tumourinfiltrating natural killer (NK) T cells, immature dendritic cells (DCs) and M1macrophages correlated positively with tumour cell PD-L1 (p < 0.05). In the laststudy, the only lipoprotein showing significant difference in concentration iiibetween healthy controls and patients was HDL (p = 0.012). Kaplan-Meiersurvival curves showed that patients with high levels of total cholesterol or LDLhad better survival than patients with normal levels (p = 0.028 and p = 0.007respectively). Adjusting for the effects of age at diagnosis, TNM stage and weightchange, multivariate Cox regression models showed LDL to be an independentprognostic factor for both overall (p = 0.010) and disease-free survival (p =0.018).

Conclusion: We excluded EBV as a potential player in SCCOT in both old andyoung patients and highlight the importance of appropriate controls for EBVencoded RNA in-situ hybridization (EBER-ISH) when investigating EBV inhuman diseases. Regarding PD-L1, our data supported the significance of genderon tumour cell PD-L1 expression and demonstrated combined effects of genderand PD-L1 levels on clinical outcome in patients with SCCOT. Data also indicatedthe involvement of specific immune cell types in PD-L1-regulated immuneevasion. Looking at serum lipoproteins, we found high LDL levels to be beneficialfor survival outcome in patients with SCCHN. Furthermore, the use of cholesterollowering medicine for prevention or management of SCCHN needs to be carefullyevaluated.

Studies have shown lower treatment-related morbidity when using transoral robotic surgery (TORS) compared to conventional surgery. Patients investigated for oro- and hypopharyngeal cancer (T1, T2) were compared concerning quality of life (QoL) after tonsillectomy and TORS using validated QoL questionnaires: QLQ-C30 and QLQ-H&N35. The patients treated with TORS showed a higher pain score and thus also a higher need for painkillers, whereas they had lower values on self-assessment of anxiety/depression using the Hospital Anxiety and Depression Scale score. The pre- and postoperative information given did not meet the expectations of the patients treated with conventional surgery. The present data show advantages of the TORS technique from the patients' perspective. Even if patients treated with TORS are in need of more painkilling treatment, they cope better with the long-term effects of treatment, as judged by self-assessment of anxiety and depression.

Oral cancers are surrounded by epithelium that histologically might seem normal, but genetically has aberrations. In patients with squamous cell carcinoma of the oral tongue (SCCOT), it is therefore important to study not only the tumor but also the clinically tumor-free contralateral tongue tissue that remains in the patient after treatment to map changes of prognostic and/or diagnostic value. The transporter associated with antigen processing (TAP) dimer is a key factor in the process of activating cytotoxic T cells. By downregulating the expression of TAP, tumor cells can escape cytotoxic T cell recognition. Biopsies from tumor and clinically tumor-free contralateral tongue tissue in 21 patients with SCCOT were analyzed together with tongue biopsies from 14 healthy individuals, which served as the control group. Dividing patients into TAP1-high and TAP1-low groups according to the median TAP1 level in tumor-free samples showed that patients with lower TAP1 mRNA levels in tumor-free samples had better overall (p = 0.003) and disease-free survival (p = 0.002). The results showing that TAP1 levels in tumor-free tongue tissue contralateral to the SCCOT correlate with survival is an important contribution to early diagnosis and follow up of SCCOT.

Objective: To use alternative quantitation approaches to clarify the clinical implication of programmed cell death ligand 1 (PD‐L1) in squamous cell carcinoma of the oral tongue (SCCOT).

Materials and Methods: Ventana SP263 immunohistochemistry assay and a multiplicative QuickScore method were applied to quantify PD‐L1 in tumor and surrounding immune cells from 101 patients with SCCOT. Tumor‐infiltrating immune cells were estimated from bulk tissue transcriptional profiles of 25 patients. Circulating PD‐L1 levels were measured in serum from 30 patients using an electrochemiluminescence assay platform.

Results: We found higher tumor cell PD‐L1 levels in females than males (p  = .019). For patients with low PD‐L1 in tumor cells, better survival was seen in males than females (overall survival p  = .021, disease‐free survival p  = .020). Tumor‐infiltrating natural killer T cells, immature dendritic cells, and M1 macrophages were positively associated with tumor cell PD‐L1 (p  < .05).

Conclusions: Our data confirmed the significance of gender on tumor cell PD‐L1 expression and demonstrated combined effects of gender and PD‐L1 levels on clinical outcome in patients with SCCOT. The data also indicated the involvement of specific immune cell types in PD‐L1‐regulated immune evasion.

Transfer-RNA-derived fragments (tRFs) are a class of small non-coding RNAs that are functionally different from their parental transfer RNAs (tRNAs). tRFs can regulate gene expression by several mechanisms, and are involved in a variety of pathological processes. Here, we aimed at understanding the composition and abundance of tRFs in squamous cell carcinoma of the head and neck (SCCHN), and evaluated the potential of tRFs as prognostic markers in this cancer type. We obtained tRF expression data from The Cancer Genome Atlas (TCGA) HNSC cohort (523 patients) using MINTbase v2.0, and correlated to available TCGA clinical data. RNA-binding proteins were predicted according to the calculated Position Weight Matrix (PWM) score from the RNA-Binding Protein DataBase (RBPDB). A total of 10,158 tRFs were retrieved and a high diversity in expression levels was seen. Fifteen tRFs were found to be significantly associated with overall survival (Kaplan-Meier survival analysis, log rank test p-value < 0.01). The top prognostic marker, tRF-20-S998LO9D (p < 0.001), was further measured in tumor and tumor-free samples from 16 patients with squamous cell carcinoma of the oral tongue and 12 healthy controls, and was significantly upregulated in tumor compared to matched tumor-free tongue (p < 0.001). Results suggest that tRFs are useful prognostic markers in SCCHN

A growing number of long non-coding RNAs (lncRNAs) have been linked to squamous cell carcinoma of the head and neck (SCCHN). A subclass of lncRNAs, termed enhancer RNAs (eRNAs), are derived from enhancer regions and could contribute to enhancer function. In this study, we developed an integrated data analysis approach to identify key eRNAs in SCCHN. Tissue-specific enhancer-derived RNAs and their regulated genes previously predicted using the computational pipeline PreSTIGE, were considered as putative eRNA-target pairs. The interactive web servers, TANRIC (the Atlas of Noncoding RNAs in Cancer) and cBioPortal, were used to explore the RNA levels and clinical data from the Cancer Genome Atlas (TCGA) project. Requiring that key eRNAs should show significant associations with overall survival (Kaplan-Meier log-rank test, p < 0.05) and the predicted target (correlation coefficient r > 0.4, p < 0.001), we identified five key eRNA candidates. The most significant survival-associated eRNA was AP001056.1 with ICOSLG encoding an immune checkpoint protein as its regulated target. Another 1640 genes also showed significant correlation with AP001056.1 (r > 0.4, p < 0.001), with the "immune system process" being the most significantly enriched biological process (adjusted p < 0.001). Our results suggest that AP001056.1 is a key immune-related eRNA in SCCHN with a positive impact on clinical outcome.

Background The incidence of squamous cell carcinoma of the oral tongue (SCCOT) is increasing in people under age 40. There is an urgent need to identify prognostic markers that help identify young SCCOT patients with poor prognosis in order to select these for individualized treatment. Materials and methods To identify genetic markers that can serve as prognostic markers for young SCCOT patients, we first investigated four young (<= 40 years) and five elderly patients (>= 50 years) using global RNA sequencing and whole-exome sequencing. Next, we combined our data with data on SCCOT from the cancer genome atlas (TCGA), giving a total of 16 young and 104 elderly, to explore the correlations between genomic variations and clinical outcomes. Results In agreement with previous studies, we found that SCCOT from young and elderly patients was transcriptomically and also genomically similar with no significant differences regarding cancer driver genes, germline predisposition genes, or the burden of somatic single nucleotide variations (SNVs). However, a disparate copy number variation (CNV) was found in young patients with distinct clinical outcome. Combined with data from TCGA, we found that the overall survival was significantly better in young patients with low-CNV (n = 5) compared to high-CNV (n = 11) burden (P = 0.044). Conclusions Copy number variation burden is a useful single prognostic marker for SCCOT from young, but not elderly, patients. CNV burden thus holds promise to form an important contribution when selecting suitable treatment protocols for young patients with SCCOT.

Immune cells and cytolytic activity within the tumor microenvironment are being intensively studied. Through transcriptome profiling, immune cell enumeration using the xCell tool and cytolytic activity quantification according to granzyme A (GZMA) and perforin (PRF1) mRNA levels, we investigated immunoreactivity in tumor and/or tumor‐free tongue tissue samples from 31 patients with squamous cell carcinoma of the oral tongue and 14 healthy individuals (control tongue tissues). We found significantly altered immune cell compositions (p < 0.001) and elevated cytolytic activity (p < 0.001) in tumor compared to tumor‐free samples, and altered infiltration of a subset of immune cells (e.g. CD8⁺ T cells, p < 0.01) as well as increased cytolytic activity (p < 0.001) in tumor‐free compared to control samples. Controlling for patient age at diagnosis and tumor stage, Cox regression analysis showed that high cytolytic activity in tumor‐free samples associated with improved disease‐free survival (hazard ratio= 4.20, 95% CI = 1.09–16.20, p = 0.037). However, the degree of cytolytic activity in tumor samples did not provide prognostic information. Taken together, our results show the presence of cancer‐related immune responses in clinically tumor‐free tongue in patients with squamous cell carcinoma of the oral tongue. Measuring cytolytic activity in tumor‐free tongue samples contralateral to tumor might thus be an effective approach to predict clinical outcome.