Squamous cell carcinoma of the head and neck, focusing on

Epstein-Barr-virus, programmed cell death ligand

1 and serum lipoproteins

Torben Wilms

Department of Medical Biosciences Department of Clinical Sciences

Umeå University, 2021

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7855-485-0 (print) ISBN: 978-91-7855-486-7 (pdf) ISSN: 0346-6612 New Series Number 2118 Cover and figure design: Katharina, Amanda and Julia Electronic version available at: http://umu.diva-portal.org/ Printed by: Cityprint i Norr ABUmeå, Sweden 2021

To my wife and kids who kept me rooted

“Life did not intend to make us perfect. Whoever is perfect belongs in a museum”. Erich Maria Remarque

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Table of Contents

Abstract ............................................................................................. ii Abbreviations ................................................................................... iv Enkel sammanfattning på svenska ................................................... vi List of papers ................................................................................. viii Introduction/Background .............................................................. - 1 -

Head and neck cancer.................................................................................................. - 1 - Squamous cell carcinoma of the oral tongue .............................................................. - 2 - Oncogenic viruses ........................................................................................................ - 4 - EBV ............................................................................................................................... - 6 - PD-L1 and immunotherapy ......................................................................................... - 8 - Lipids, obesity and cancer .......................................................................................... - 11 -

Aims ............................................................................................. - 15 - Materials and Methods ................................................................ - 16 -

Patients....................................................................................................................... - 16 - EBER in situ hybridisation (EBER-ISH) .................................................................. - 17 - Polymerase chain reaction (PCR) of EBV ................................................................. - 17 - Immunohistochemistry ............................................................................................. - 17 - Quick score ................................................................................................................. - 18 - Immune cell analysis using xCell .............................................................................. - 18 - Serum analysis ........................................................................................................... - 18 - Statistical analysis...................................................................................................... - 18 -

Results and Discussion ............................................................... - 20 - EBV (Paper I) ............................................................................................................. - 20 - PD-L1 (Paper II) ........................................................................................................ - 23 - Lipoproteins (Paper III) ............................................................................................ - 27 -

Conclusions .................................................................................. - 31 - Future perspectives ..................................................................... - 32 - Acknowledgements ..................................................................... - 34 - References .................................................................................. - 36 -

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Abstract

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

Materials and methods: Ninety-eight cases of SCCOT and 15 cases of tonsillar squamous cell carcinoma were examined for the presence of EBV-encoded ribonucleic acids (EBERs), EBV deoxyribonucleic acid (DNA) and the protein EBV-encoded nuclear antigen-1 (EBNA-1), using in situ hybridisation, polymerase chain reaction (PCR) and immunohistochemistry respectively. One hundred and one cases of SCCOT were examined for expression of PD-L1 in tumour and surrounding immune cells using Ventana SP263 immunohistochemistry assay and a QuickScore (QS) method. An estimation of tumour-infiltrating immune cells was also performed in 25 of the patients. Circulating levels of PD-L1 were measured using an electrochemiluminescence assay platform in serum from 30 patients. Finally, serum samples from 106 patients and 28 healthy controls were investigated for levels of total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides and lipoprotein(a).

Results: In the first study, using an in situ hybridisation kit no EBER transcripts were detected. No EBV DNA was identified with PCR analysis, and immunohistochemistry for EBNA-1 was also negative. In the second study, higher tumour cell PD-L1 levels were found in females than males (p = 0.019). For patients with low PD-L1 in tumour cells, better survival was shown in males than females (overall survival p = 0.021, disease-free survival p = 0.020). Tumour-infiltrating natural killer (NK) T cells, immature dendritic cells (DCs) and M1 macrophages correlated positively with tumour cell PD-L1 (p < 0.05). In the last study, the only lipoprotein showing significant difference in concentration

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between healthy controls and patients was HDL (p = 0.012). Kaplan-Meier survival curves showed that patients with high levels of total cholesterol or LDL had better survival than patients with normal levels (p = 0.028 and p = 0.007 respectively). Adjusting for the effects of age at diagnosis, TNM stage and weight change, multivariate Cox regression models showed LDL to be an independent prognostic 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 and young patients and highlight the importance of appropriate controls for EBV-encoded RNA in-situ hybridization (EBER-ISH) when investigating EBV in human diseases. Regarding PD-L1, our data supported the significance of gender on tumour cell PD-L1 expression and demonstrated combined effects of gender and PD-L1 levels on clinical outcome in patients with SCCOT. Data also indicated the involvement of specific immune cell types in PD-L1-regulated immune evasion. Looking at serum lipoproteins, we found high LDL levels to be beneficial for survival outcome in patients with SCCHN. Furthermore, the use of cholesterol lowering medicine for prevention or management of SCCHN needs to be carefully evaluated.

iv

Abbreviations

AJCC American Joint Committee on Cancer

Cbl-b Casitas B lineage lymphoma-b

CR1 Complement receptor type 1

CR2 Complement receptor type 2

DCs Dendritic cells

DNA Deoxyribonucleic acid

EBER EBV-encoded RNA

EBER-ISH EBV-encoded RNA in-situ hybridization

EBNA-1 Epstein-Barr virus encoded nuclear antigen-1

EBV Epstein-Barr virus

EGFR Epidermal growth factor receptor

HBB Human β-globin gene

HBV Hepatitis B virus

HDL High-density lipoprotein

HIV Human immunodeficiency virus

HPV Human papillomavirus

IFN-γ Interferon-γ

IgG Immunoglobulin G

IgM Immunoglobulin M

IHC Immunohistochemistry

IM Infectious mononucleosis

IRF-1 Interferon regulatory factor-1

ISH In situ hybridization

Kbp Kilo base pairs

KSHV Kaposi's sarcoma-associated herpesvirus

LDL Low-density lipoprotein

ME/CFS Myalgic encephalomyelitis/chronic fatigue syndrome

miR MicroRNA

v

NK Natural killer

OS Overall survival

OSCC Oral squamous cell carcinoma

PCR Polymerase chain reaction

PD-1 Programmed cell death 1

PD-L1 Programmed cell death ligand 1

PD-L2 Programmed cell death ligand 2

PET-CT Positron emission tomography and computed tomography

QS Quick score

RNA Ribonucleic acid

RSV Rous sarcoma virus

SCC Squamous cell carcinoma

SCCHN Squamous cell carcinoma of head and neck

SCCOT Squamous cell carcinoma of the oral tongue

SV40 Simian virus 40

VCA Viral capsid antigen

WHO World Health Organization

ZAP-70 Zeta-chain-associated protein kinase 70

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Enkel sammanfattning på svenska

Introduktion: Skivepitelcancer i huvud-halsområdet är en stor tumörgrupp som innehåller tumörer i munhålan och svalgområdet och framförallt drabbar äldre män som brukar alkohol och tobak. I munhålan är den vanligaste platsen för den här tumören den rörliga delen av tungan. Skivepitelcancer i tungan blir allt vanligare hos unga individer, varför man misstänker andra än de vanliga riskfaktorerna för sjukdomen. Två vanliga cancerframkallande virus, humant papillomvirus (HPV) och Epstein-Barr virus (EBV) är förknippade med skivepitelcancer i munsvalg (orofarynx) och nässvalg (nasofarynx). PD-1 (programmerad celldöd 1) och det protein den binder till, PD-L1 (programmerad celldöd ligand 1) är särskilt viktiga för att påverka immunsystemet, och är förhöjda i många olika cancerformer. I tidgare studier har man sett ett samband mellan rubbningar i blodfetter och cancer.

Syftet med den här avhandlingen var att undersöka riskfaktorer och förutspå prognos för patienter med skivepitelcancer i huvud-halsområdet, med fokus på EBV, PD-L1 och serum lipoproteiner (blodfetter).

Material och metoder: Nittioåtta fall av oral tungcancer och femton fall av tonsillcancer analyserades för närvaro av EBV. Etthundraett fall av oral tungcancer undersöktes för uttryck av PD-L1 i tumören och omkringliggande immunceller, och med hjälp av ett så kallat quick score (QS) system utvärderades både mängden celler som uttryckte de olika proteinerna, samt intensiteten i uttrycket. En skattning av typ av immunceller utfördes på 25 patienter. Halter av i blodet cirkulerande PD-L1 mättes hos 30 patienter. Dessutom undersöktes serumprover från 106 patienter med huvud- och halscancer och 28 kontrollpersoner för olika blodfetter.

Resultat: I studie ett kunde vi inte påvisa någon infektion med EBV. I studie två såg vi högre halter av PD-L1 hos kvinnor jämfört med män. För patienter med låg halt av PD-L1 i tumörceller påvisades bättre överlevnad för män än för kvinnor. Gällande särskilda immunceller hittades ett positivt samband mellan vissa typer av inflammationsceller och tumörcellernas innehåll av PD-L1. I sista studien påvisades endast för lipoproteinet HDL-kolesterol en signifikant tydlig skillnad mellan patienter och kontrollpersoner. Patienter med höga halter av totalkolesterol och LDL-kolesterol uppvisade en bättre överlevnad jämfört med de med normala halter, och LDL-kolesterol var en oberoende prognosfaktor för överlevnad.

Slutsatser: Vi kunde utesluta EBV som en möjlig påverkande faktor för oral tungcancer bland gamla och unga patienter och kunde påpeka betydelsen av att

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använda lämpliga kontrollmetoder när man undersöker EBV. Gällande PD-L1, kunde vi visa att både kön och halter av PD-L1 påverkar överlevnaden hos patienter med tungcancer. Dessutom visar vår forskning att särskilda immunceller påverkar den till PD-L1 kopplade immunreaktionen. Vid undersökningen av lipoproteiner noterade vi att högt LDL-kolesterol verkar vara gynnsamt för överlevnaden hos patienter med huvud- och halscancer. Slutligen kan behandligen mot blodfettsrubbningar med så kallade statiner vara av betydelse vid huvud- och halscancer.

viii

List of papers

Paper I

Wilms, T., G. Khan, P. J. Coates, N. Sgaramella, R. Fahraeus, A. Hassani, P. S. Philip, L. Norberg Spaak, L. Califano, G. Colella, K. Olofsson, C. Loizou, R. Franco and K. Nylander (2017). "No evidence for the presence of Epstein-Barr virus in squamous cell carcinoma of the mobile tongue." PLoS One 12(9): e0184201.

Paper II

Wilms, T., X. Gu, L. Boldrup, P. J. Coates, R. Fahraeus, L. Wang, N. Sgaramella, N. H. Nielsen, L. Norberg-Spaak and K. Nylander (2020). "PD-L1 in squamous cell carcinoma of the oral tongue shows gender-specific association with prognosis." Oral Dis 26(7): 1414-1423.

Paper III

Wilms, T., L. Boldrup, X. Gu, P. J. Coates, N. Sgaramella, K. Nylander. ”High levels of low-density lipoprotein correlate with improved survival in patients with squamous cell carcinoma of the head and neck.” Submitted 2021

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- 1 -

Introduction/Background

Head and neck cancer

Head and neck cancer is a group of malignant tumours that originate in the head-neck region, including lip, oral cavity, salivary glands, tonsils, oropharynx, nasopharynx, hypopharynx, nasal cavity, middle ear, paranasal sinuses and larynx. Histologically, over 90% of head and neck cancers are squamous cell carcinomas. Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer with 650,000 new cases annually worldwide (Kulasinghe, Perry et al. 2015). In 2016, SCCHN contributed to 5.7% of global cancer-related mortality, which can be compared with breast cancer (6.1%) and pancreatic cancer (4.5%) (Patterson, Fischman et al. 2020).

The TNM staging system of the American Joint Committee on Cancer (AJCC) is used for staging of SCCHN and based on estimation of the extent of disease before treatment (Sood, Wykes et al. 2019). Many patients at diagnosis have involvement of regional lymph nodes, whereas the incidence of distant metastasis in SCCHN remains low when compared to other types of cancers such as breast or lung cancer (Kulasinghe, Perry et al. 2015). Moreover, head and neck cancer patients regularly develop second primary tumours as these share common risk factors (Brands, Smeekens et al. 2019) (Day and Blot 1992). In terms of relapse, the majority develops within the same region as the primary tumour despite aggressive treatment of the primary SCCHN. Local and regional recurrences are the cause of up to 80% of primary treatment failures (Wong, Wei et al. 2003). As the disease progresses, the incidence of distant metastases increases, including lungs, and to some extent also bone and liver. The use of PET-CT (positron emission tomography and computed tomography) is therefore recommended for mapping distant spread of the cancer.

In general, treatment of SCCHN requires a multidisciplinary team. Treatment options are based on tumour site, extent of the primary tumour and lymph node status, and may consist of: Radiation therapy alone, surgery alone or a combination of the above, or even chemotherapy. Recently also immunotherapy has been introduced in treatment of SCCHN. The need for a swift diagnosis and referral of patients to a skilled specialist with expertise in the management of SCCHN is crucial as early diagnosis can lead to a reduction in mortality (Pfister, Ang et al. 2013) (Abrahao, Perdomo et al. 2020). It is recommended to perform a full physical examination on any adult patient with symptoms attributable to the upper aero digestive tract lasting for more than two weeks, or with an asymptomatic cervical tumour. Physical examination is most adequate to detect lesions of the upper aero digestive tract. For example, squamous cell carcinomas

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of the oral tongue (SCCOT) usually generate symptoms linked to the upper aerodigestive tract, including changes in speech, swallowing, breathing and even hearing. Therefore, the examining physician should give emphasis to the following key symptoms: non-healing ulcer on the tongue, tongue pain and changes in the ability to articulate words.

Only 40–50% of SCCHN patients survive 5 years or longer with only little improvement in this number over the past four decades (Kulasinghe, Perry et al. 2015). The prognosis firmly correlates with the stage of the disease at diagnosis. Development of metastases in lymph nodes decreases the survival rate of a patient with a small primary tumour by around 50 % (Abrahao, Perdomo et al. 2020). Other histological predictors for aggressive tumour behavior consist of perineural infiltration and tumour extension beyond the capsule of the lymph nodes (Massano, Regateiro et al. 2006). A recent study found that older patients with advanced disease SCCHN, increased WHO (World Health Organization) score, primary tumour in the hypopharynx, and those given palliative treatment, are more likely than others to die from SCCHN within six months of diagnosis (Talani, Makitie et al. 2019).

Tobacco is regarded to be one of the most important risk factors inducing SCCHN, and it is estimated that around eighty-five percent of head and neck cancers can be related to tobacco consumption (Gandini, Botteri et al. 2008) (Secretan, Straif et al. 2009) (Kelwaip, Fose et al. 2020). Despite the declining consumption of tobacco, the incidence of SCCHN at specific sites, namely the occurrence of SCCOT, increased during the past decades (Harris, Kimple et al. 2010).

Squamous cell carcinoma of the oral tongue

SCCOT is the most common cancer of the oral cavity. Two thirds of SCCOT occur on the lateral surfaces of the middle third of the tongue, whereas one third is located on the ventrolateral or the anterior undersurface of the tongue. Base of tongue cancers, which develop in the posterior one-third of the tongue are located within the oropharynx (Campbell, Netterville et al. 2018).

The tongue is an elongated, mucosa covered muscular organ (consisting of nine individual muscles) in humans and most other vertebrates. It lies at the bottom of the oral cavity and almost completely fills it when jaws are closed (Hiatt and Gartner 2010). The normal adult human tongue weighs on average 70 g (wet weight) and is 9 cm long from the tip to the oropharynx (Sanders, Mu et al. 2013).

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SCCOT in the posterior portion of the tongue metastasises to deep cervical lymph nodes early on, whereas those on the anterior part of the tongue do not metastasise to the deep cervical lymph nodes until advanced disease progression. Since the deep cervical lymph nodes drain into the internal jugular vein, it is extremely important that the disease be identified and treated as early as possible to prevent metastases into deeper structures within the neck.

Large differences in the incidence of oral cavity cancer have been recognised among different geographical regions. The highest incidence of SCCOT has been observed in Asia and has been reported to correspond to the prevalence of certain risk factors, such as the consumption of betel nut (Ho, Ko et al. 2002) (Hu, Zhong et al. 2020) (Kelwaip, Fose et al. 2020) and smokeless tobacco (Niaz, Maqbool et al. 2017). The Indian subcontinent stands for one-third of the oral cancer rates in the world, with oral cancer being the most common cancer among men in India and the second cancer among Pakistani women. In contrast, the lowest rates for oral cancer are found in Western Africa and Eastern Asia, and North African countries also present low incidences (García-Martín J.M. 2019). In some developed countries SCCOT has decreased (the USA or Canada), whereas in others, e.g. certain European countries, it has increased. These differences may be attributed to differing population habits, life expectancies, preventive education and the quality of medical records in various countries (García-Martín J.M. 2019).

Globally, malignant tumours of the tongue are undoubtedly more common in men than in women, which is comparable with the rest of the oral cavity cancers (García-Martín J.M. 2019). Worldwide, the mortality rate of oral cancer is higher in males than females. Death from oral cancer ranks fifteenth place for men and seventeenth for women in the US population. In Europe, it ranked the seventh and tenth, for men and women respectively (García-Martín J.M. 2019).

The risk of SCCOT rises with age, notably after age 50. Most patients are between 50 and 70 years, but SCCOT can also develop in younger patients (Campbell, Netterville et al. 2018). Alcohol by itself contributes to the development of tongue and oral cavity cancer, although it is regarded as a less potent carcinogen than tobacco. For patients who use both tobacco and alcohol, these two risk factors appear to be synergistic and increase the cancer risk significantly (Hashibe, Brennan et al. 2007).

As for the carcinogenic mechanisms, the malignant transformation to SCCOT consists of a multistep progressive process engaging changes related to specific genes, epigenetic events, and signal transduction within the cell (Tanaka and Ishigamori 2011) (Tanaka, Tanaka et al. 2011). Tobacco smoke contains mutagenic agents, and tobacco smoke extracts activate the epidermal growth

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factor receptor (EGFR) (Park and Kim 2018). The activation of EGFR increases as a consequence of the production of prostaglandins, including PGE2 which may enhance EGFR signal transduction (Pai, Soreghan et al. 2002). Furthermore, cyclin-D1 has been shown to be overexpressed in SCCOT, and increased cyclin-D1 activity is triggered by EGFR activation (Sasahira, Kirita et al. 2014) (Angadi and Krishnapillai 2007). As an important step in the epigenetic progression to SCCOT, gene promoter regions can be silenced through hypermethylation (Diez-Perez, Campo-Trapero et al. 2011), which can influence the tumour suppressors p16, DAPkinase and E-cadherin. On the other hand, the viral proteins E6 and E7 can deregulate the cell cycle by p53 inactivation, which may contribute to HPV-mediated carcinogenesis in SCCHN, but this is still unknown for SCCOT (Tornesello, Perri et al. 2014). Furthermore, apart from deletions or mutations of individual genes, aneuploidy, which means numeric chromosomal imbalances, may induce malignant transformation (Shi, Wang et al. 2020).

The concept of field cancerization describes diffuse injury to the epithelium through chronic carcinogenic exposure, which can clinically manifest as mucosal abnormalities like leukoplakia and dysplasia, outside the margins of an SCCOT (Mohan and Jagannathan 2014). This contributes to the lifetime risk of a patient with oral cancer to develop a new cancer, which in the case of leukoplakia is around 5 % (Speight 2007).

Overall, SCCOT occurs as the most frequent of all oral SCC and exhibits a completely different clinical behaviour, with more aggressive local growth and more frequent regional spread than other oral SCC (Shah 2019).

Oncogenic viruses

Oncogenic viruses are significantly pathogenic for humans, pets and farm animals. These pathogens are classified into different virus families such as Flaviviridae, Hepadnaviridae, Papillomaviridae and Retroviridae (Truyen and Lochelt 2006). Oncogenic viruses (tumour viruses) include both DNA and RNA viruses (Klein 2002). Compared to RNA tumour viruses, DNA tumour virus oncogenes encode viral proteins necessary for viral replication. On the other hand, RNA tumour viruses carry changed variants of normal host cell genes, which are not necessary for viral replication (Levine 2009). In DNA viruses, genetic material is directly integrated into the host genome causing neoplasia, whereas RNA viruses have to reverse transcribe RNA to DNA before being transferred into the host genome (Akram, Imran et al. 2017). Oncogenic viruses integrate their genes into the host cell genome, which is indispensable for chromosomal rearrangements, cellular transformations, causing mutations, and

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uncontrolled cell divisions when interfering with cell cycle processes and the mitogenic signaling pathway (Akram, Imran et al. 2017).

In general, oncogenic viruses promote cell transformation, uncontrollable cell generation and lead to development of malignant tumours. Viral oncogenes (v-onc genes) describe the genes in the viral genome that change host cell proliferation control, lead to synthesis of new proteins, and are responsible for transformation characteristics (Gonda and Metcalf 1984). Protooncogenes (c-onc genes) can be described as the cellular counterparts of v-onc genes (Adamson 1987) and their functions are cellular growth and development. The activation of c-onc genes by mutation provokes uncontrolled cell growth (Vats and Emami 1993). Moreover, c-onc genes are transformed into their oncogenic form by point mutation, deletion, amplification or chromosomal translocation. C-onc genes can be classified into different groups related to their protein products, such as growth factors, growth factor receptors, protein kinases and DNA binding proteins (Bell 1988).

Oncoviruses by themselves may not be sufficient for carcinogenesis; other cofactors such as environmental mutagens, chronic inflammation, and immunosuppression are synergistically involved in cancer development, which may explain why the incidence of viral oncogenesis is not much higher (Aoki, Jaffe et al. 1999) (Cao and Li 2018). In this context, inflammation has been recognised as a significant part of tumour progression (Coussens and Werb 2002).

The hepatitis B virus (HBV), with a genome of 3 kilobase pairs (kbp), is the smallest DNA-virus, it can cause chronic hepatitis, which raises the risk for liver carcinoma by around 100 times. Because of this, HBV infection was ranked 15th among all causes of human mortality (Lozano, Naghavi et al. 2012) (MacLachlan and Cowie 2015).

The human papillomavirus (HPV; 8 kbp) with around 60 subtypes infects especially epithelial cells and induces harmless papillomas but also cervical carcinoma, SCCHN and anogenital tumours (Bolt, Foran et al. 2018) (Araldi, Sant'Ana et al. 2018) (Hendawi, Niklander et al. 2020). To the group of herpes virus (100 to 200 kbp) belongs the Epstein-Barr-Virus (EBV), which causes Burkitt-lymphoma, B-cell-lymphomas and nasopharyngeal tumours (Masrour-Roudsari and Ebrahimpour 2017).

The Kaposi's sarcoma-associated herpesvirus (KSHV) can induce multiple tumours at late stage of human immunodeficiency virus (HIV) infection (Zhang and Wang 2017).

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EBV

Epstein-Barr-Virus (EBV, also Human Herpesvirus 4) is a species of human-pathogenic coated double-stranded DNA viruses from the family herpesviridae. It was first described in 1964 by Epstein and Barr and discovered in B-lymphocytes from Burkitt-lymphoma in African patients. EBV is therefore the tumour virus that was first discovered in humans (Esau 2017). EBV belongs, together with HBV, HCV, HPV, HTLV-1 and KSHV to a group of human pathogenic virus that are responsible for 10 to 15 percent of all cancers worldwide (Martin and Gutkind 2008).

The main transmission route of EBV is a droplet infection or a contact infection (especially saliva) or smear infection (Gillet, Frederico et al. 2015) (Alter, Bennett et al. 2015). More rarely are transmissions in the context of transplants or blood transfusions (Smatti, Al-Sadeq et al. 2018). The fact that EBV could also be detected in secretions of the genitals makes the transmission route through sexual contacts conceivable (Thomas, Macsween et al. 2006).

At the molecular level, complement receptor type 1 (CR1) and complement receptor type 2 (CR2) are described as adhesion factors at the cell surface (Tanner, Whang et al. 1988) (Ogembo, Kannan et al. 2013). For entry into cells, EBV uses integrins or HLA-II (HLA class II histocompatibility antigen) (Chesnokova, Nishimura et al. 2009) (Li, Spriggs et al. 1997).

Infection with EBV usually occurs in childhood (Alter, Bennett et al. 2015). While in this case, as a rule, there are no symptoms in childhood, the onset of glandular fever or infectious mononucleosis (IM) occurs in 30-70% of all cases, when previously unexposed adolescents or adults become infected (AbuSalah, Gan et al. 2020). From the age of 35, more than 90% of people are infected with EBV (Balfour, Sifakis et al. 2013). Both after asymptomatic and symptomatic infection, the virus persists in the body for life (Kerr 2019).

EBV can be reactivated, like all herpes viruses. Usually, reactivation is unnoticed by the host and quickly contained by its immune system (Maurmann, Fricke et al. 2003) If there is immunosuppression (e.g., in HIV-infected or organ receptors), the virus can multiply uncontrollably and contribute to complications and to the development of various cancers (Thoden, Rieg et al. 2012).

In the recent past, the suspicion that EBV is associated with a variety of autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and rheumatoid arthritis has been confirmed (Houen, Trier et al. 2020) (Li, Zeng et al. 2019) (Balandraud and Roudier 2018). However, there are also indications

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that an infection with EBV cannot be regarded as the sole cause of later autoimmune diseases.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is also associated with this virus (Shikova, Reshkova et al. 2020).

For about 40 years, there has been a suspicion that EBV plays an important aetiological role in diseases such as Hodgkin’s disease, Burkitt’s lymphoma and other lymphomas and in posttransplanted lymphoproliferative disease (Maeda, Akahane et al. 2009).

In Africa, there is also a locally recurring (endemic) variant of the EBV-associated Burkitt lymphoma. However, EBV alone is not sufficient for the development of cancer, as shown by the low number of cancers compared to the spread of the virus. Other factors play a role here (chromosomal translocation of the c-myc gene) (Farrell 2019). In the case of Burkitt’s lymphoma, malaria and retinoid toxicity are also discussed as potential adjunctive factors (cofactors) (Mawson and Majumdar 2017).

As for diagnostics, in general, an increased proportion of lymphocytes in the total white blood cells (so-called relative lymphocytosis) is almost always found in infection with EBV (Hamad and Mangla 2021). The total number of white blood cells can be reduced, normal or increased.

If a blood smear is examined under the microscope, one sees atypical mononuclear cells with characteristic changes, so-called Downey cells, which sometimes already enable diagnosis (Carter 1966) (Balfour, Schmeling et al. 2020). Serological assessment of EBV status is not always easy (Feder and Rezuke 2020). For the detection of acute infection, so-called rapid tests are used in some places, but, depending on age, up to 20% false positive and 30% false negative results are obtained.

Detection of immunoglobulin G (IgG) antibodies against the nuclear antigen of EBV (EBNA-1-IgG) proves the presence of the virus. If the EBNA-1 IgG test is clearly positive, any further virus detection test for acute disease (mononucleosis) is unnecessary.

In about 95 % of people with previous EBV infection, EBNA-1 IgG antibodies are detectable. If the EBNA-1-IgG is negative, IgG against the viral capsid antigen (VCA)-IgG should be tested. VCA-IgG is a marker for current or previous contact with EBV and usually remains detectable for life. To detect an acute infection, immunoglobulin M (IgM) antibodies against the viral capsid (VCA-IgM) are tested in positive VCA-IgG. If these are positive, this indicates a fresh or recent

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infection (but does not prove this). The VCA-IgM can persist during heavy physical exertion (especially among competitive athletes). It is called prolonged healing if the EBNA-1-IgG test is already positive.

A reliable assessment of EBV reactivations in immunodeficiency is only possible by determining the viral load by polymerase chain reaction (PCR). Direct detection of the virus DNA by PCR is usually not useful in immune competent subjects, since the latent genome can also be detected in the blood of asymptomatic carriers for their lifetime, who also secrete the virus constantly or temporarily in the saliva.

As a rule, the above-mentioned EBV reactivations do not cause any complaints to an immunohealthy person, they merely represent a laboratory diagnostic problem, since the detection of VCA-IgG in high concentrations and possibly recurring VCA-IgM can lead to misinterpretations (Nystad and Myrmel 2007).

PD-L1 and immunotherapy

The immune system has both costimulatory (activating) and inhibitory (inhibitory) signalling pathways (Marin-Acevedo, Dholaria et al. 2018). These control mechanisms influence the strength and intensity of an immune response. Normally, these mechanisms are used to avoid autoimmune reactions. Those signalling pathways with inhibitory effect are called co-inhibitory immunocheckpoints and reduce T-cell activation or T-cell effector function (Sharma and Allison 2015). Immune checkpoints with pro-inflammatory effects are called co-stimulatory immune checkpoints. With regard to tumour disease it is known that tumour cells can use these immunocheckpoints to escape recognition by the immune system (immunoevasion) (Sharma and Allison 2020).

PD-L1, formerly known as B7-H1, is a transmembrane surface protein involved in inhibiting the immune response. PD-L1 is formed in the heart, skeletal muscles, placenta and lungs in higher concentrations, but is also produced at lower concentrations in thymus, spleen, kidney, liver and by activated T- and B- cells, dendritic cells, keratinocytes and monocytes. PD-L1 interacts with the PD-1 receptor on T-cells, thereby inhibiting T-cell activation and promoting tumour-mediated immune evasion (Dong, Strome et al. 2002).

Expression of the PD-L1 gene is regulated by various mechanisms. When interferon-γ (IFN-γ) binds to its receptor, expression of PD-L1 increases in T-cells, NK cells, macrophages, myeloid dendritic cells, B-cells and epithelial cells (Flies and Chen 2007). The transcription factor interferon regulatory factor-1

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(IRF-1) binds to the PD-L1 gene promoter (Lee, Jang et al. 2006) (Yamazaki, Akiba et al. 2002) (Loke and Allison 2003). Dormant cholangiocytes form PD-L1 mRNA, but not protein, due to inhibition by miRNA miR-513 (Gong, Zhou et al. 2009). Other miRNAs, miR-200, miR-197, miRNA-34, also inhibit expression of PD-L1 (Grenda and Krawczyk 2017).

Binding of PD-L1 to PD-1 initiates gene expression of interleukin-10 in monocytes, thus inhibiting the immune response (Said, Dupuy et al. 2010). This also inhibits phosphorylation of the zeta-chain-associated protein kinase 70 (ZAP70) and promotes expression of the ubiquitin ligase casitas B lineage lymphoma-b (CBL-b) (Sheppard, Fitz et al. 2004) (Karwacz, Bricogne et al. 2011). The other known ligand of PD-1 is programmed cell death ligand 2 (PD-L2). Studies having assessed the prevalence and distribution of PD-L2 in human tumours are limited (Pinto, Park et al. 2017).

PD-L1 is strongly formed by some tumours in the course of immunoevasion (Abiko, Mandai et al. 2013) (Alsaab, Sau et al. 2017). The PD-1/PD-L1 axis in the immunosuppressive tumour microenvironment was elucidated in Figure 1. With regard to other diseases, insufficient PD-L1 is formed in patients with lupus erythematosus (Mozaffarian, Wiedeman et al. 2008).

Figure 1. The PD-1/PD-L1 axis in the immunosuppressive tumour microenvironment. Abbreviations: TC, tumour cell; Treg, regulatory T cell; MDSC, myeloid-derived suppressor cell; TAM, tumour-associated macrophage; CD8, CD8+ T cell; CD4, CD4+ T cell; DC, dendritic cell; NKT, natural killer T cell; NK, natural killer cell. This figure was produced together with Dr. Xiaolian Gu.

Whereas inhibition of PD-L1 on the one hand increases pathogenicity of Listeria monocytogenes (Seo, Jeong et al. 2008), it is also investigated for treatment of

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certain chronic infectious diseases such as tuberculosis and HIV (Rao, Valentini et al. 2017) (Velu, Shetty et al. 2015).

Immunotherapy is a collective term for different methods of immune-modulation for cancer treatment (Velcheti and Schalper 2016) (Gupta, Gupta et al. 2020). The classical forms of cancer treatment include surgical removal (resection), radiotherapy and chemotherapy (Yum, Li et al. 2020). Often two or three of these treatment options are combined for one patient. If, despite these therapies, not all cancer cells or metastases are removed, further treatment is significantly more difficult due to the development of tumour resistance (Housman, Byler et al. 2014). Therefore, for years, there has been ongoing research on new treatment methods which have the highest possible selective effect against cancer cells, with the various approaches of cancer immunotherapy having a promising potential.

The immune suppressive milieu of some tumours is generated by production of inhibitory cytokines, recruitment of immune-suppressive immune cells and upregulation of co-inhibitory receptors, the negatively regulating immune checkpoints (Allison, Coomber et al. 2017). This knowledge led to the development of checkpoint inhibitors, which switch off immune-suppressing signals by interrupting a receptor-ligand binding. In this way, the immune system of the organism can recognise and combat the cancer cells (Topalian, Drake et al. 2015).

Treatment with checkpoint inhibitors has revolutionised oncology in recent years, whereby e.g. spread disease such as disseminated melanoma became treatable (Payandeh, Yarahmadi et al. 2019). In other tumours, such as non-small cell lung cancer (NSCLC), treatment with checkpoint inhibitors could replace and chemotherapy (Passiglia, Reale et al. 2021).

Immunocheckpoint inhibitors are currently exclusively monoclonal antibodies (Fritz and Lenardo 2019). After infusion with the antibody, it binds to the proteins acting as immune checkpoints. Thereby, tumour cells that carry one of the immune checkpoint proteins on the cell surface and bind the antibody temporarily (or as long as the antibody circulates in the body) are attacked by immune cells and removed from the body by macrophages (temporary cell depletion) (Pardoll 2012). These processes lead to amplification of the immune response to the tumour, so that its strategy of immune evasion is counteracted (Kok 2020). Immune checkpoint inhibitors are therefore used in cancer immunotherapy, including in combination with cancer vaccines (Mougel, Terme et al. 2019).

Immunocheckpoint inhibitors include antibodies to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Ipilimumab), PD-1 (Nivolumab) and PD-L1

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(Atezolizumab, Durvalumab and Avelumab) (Lee, Lee et al. 2019) (Vaddepally, Kharel et al. 2020). The PD-L1 antibody Avelumab in combination with the vascular endothelial growth factor (VEGF) inhibitor Axitinib was compared with the tyrosine kinase inhibitor Sunitinib in advanced renal cell carcinoma in the JAVELIN renal 101 study, a randomised phase III study. Avelumab and Axitinib achieved a significantly longer recurrence-free survival (Choueiri, Motzer et al. 2020).

However, checkpoint inhibitor therapies can also have significant side effects (Lewis and Miller 2019). The partial elimination of friend-enemy detection of killer cells can lead to life-threatening autoaggression diseases affecting the lungs, liver or kidneys (Marin-Acevedo, Chirila et al. 2019) (Martins, Sofiya et al. 2019).

When cancer immunotherapy is performed, the concentration of PD-L1 is investigated prior to administration of antibodies against PD-1. This assessment might not detect those anti-PD1-sensitive tumours which do not form PD-L1, but still respond partly to a cancer immunotherapy against PD-1 through other pathways (Ribas and Hu-Lieskovan 2016) (Yuasa, Masuda et al. 2017).

Different systems can be used for scoring PD-L1 positivivity. Tumour Proportion Score (TPS) estimates the percentage of PD-L1-positive tumour cells of all vital tumour cells, Immune Cell Score (IC) estimates the percentage of area of PD-L1-positive immune cells out of vital tumour cells and Combined Positivity Score (CPS) which is a combination of TPS and IC, estimates the percentage of PD-L1-positive cells including lymphocytes and macrophages of all vital tumour cells (Witte, Gebauer et al. 2020).

In addition to its utility as a predictive marker for treatment response, PD-L1 status in tumour and tumour-infiltrating immune cells could provide prognostic information. In some tumours like melanomas, non-small cell lung cancer and bladder cancer, high concentration of PD-L1 has shown to be a negative prognostic marker (Wu, Wu et al. 2015). In SCCHN, high PD-L1 levels correlated with tumour size, presence of regional metastases and poor prognosis (Muller, Braun et al. 2017, Yang, Zeng et al. 2018, de Vicente, Rodriguez-Santamarta et al. 2019, Moratin, Metzger et al. 2019).

Lipids, obesity and cancer

Lipoproteins (more precisely plasma-lipoprotein particles) are non-covalent aggregates of lipids and proteins. Lipoproteins are largely distinguished by their physical density, which is recognised in their English names, whereby their

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difference essentially arises from the different protein and lipid components of the respective lipoprotein.

Plasma lipoprotein particles are used in all animal classes to transport water-insoluble lipids (fats) as well as cholesterol and cholesteryl ester in the blood. In principle, all particles contain both triglycerides as well as cholesterol and cholesterol esters, but in very different amounts. Plasma lipoprotein particles are produced in specific cells, released into the blood and have a half-life of only a few days. Both their surface and content are susceptible to oxidation by radicals. There are some indications that the oxidative modification of low density lipoprotein is an important step in the pathogenesis of atherosclerosis (Steinberg, Parthasarathy et al. 1989). For delivery or uptake of the respective transported substance, the plasma lipoprotein particles dock by means of their apoproteins on specific receptor proteins of the target cells.

The role of cholesterol and lipoproteins in cancer development and their role as potential diagnostic biomarkers or therapeutical targets are controversial areas in the cancer community. A positive association between elevated serum cholesterol and risk for certain cancer types has been proposed by some epidemiologic studies (Pelton, Freeman et al. 2012).

Furthermore, the Cancer Genome Atlas (TCGA) project using next-generation sequencing has profiled the mutational status and expression levels of all the genes in different cancer types, including those involved in cholesterol metabolism, and supported a correlation between the cholesterol pathway and cancer development (Fernandez, Gomez de Cedron et al. 2020).

Theoretically, tobacco carcinogens produce free radicals and reactive oxygen, which cause a high rate of oxidation of polyunsaturated fatty acids. This peroxidation reaction releases further peroxide radicals, which alter essential constituents of the cell membrane (Ames 1983). Consequently, this lipid peroxidation requires a greater utilisation of lipids including total cholesterol (TC), lipoproteins and triglycerides (TG) for building new membranes. Tumour cells can fulfill these requirements from the circulation, by synthesis through metabolism, or from degradation of major lipoprotein fractions like low-density lipoproteins (LDL) or high-density lipoproteins (HDL).

The plasma concentrations of lipids are not the single additive function of intake, utilisation and biosynthesis because of continuous cycling of lipids in and out of the bloodstream. The question of whether hypolipidemia at the time of diagnosis is a causative factor or a result of cancer is still unclear.

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Statins are medicinal substances used as cholesterol-lowering agents or lipid-lowering agents. Of all drugs that affect lipid metabolism, they have the highest potency. The free names of their representatives end in -statin. Biochemically, a statin is a drug from the class of 3-hydroxy-3-methylglutaryl coenzyme-A-reductase (HMG-CoA-reductase) inhibitors (also called HMG-CoA reductase inhibitors). The first statin was mevastatin (laboratory name ML-236B, also called compactin), which occurs in the fungus Penicillium citrinum and whose lipid effect was first described by the Japanese researcher Akira Endō in 1976 (Endo 2004).

The effect of statins as lipid sinkers is based on their competitive inhibition of HMG-CoA reductase (Istvan and Deisenhofer 2001). Since HMG-CoA is a substance that the body needs for the biosynthesis of cholesterol, less cholesterol is formed by the body under the influence of statins than without.

Since there is a relative cholesterol deficiency in the cells, they increasingly produce LDL receptors that absorb the low-density lipoprotein from the blood through endocytosis. LDL is primarily responsible for most of the body’s damage caused by excessive cholesterol levels. In this way, LDL is removed from the bloodstream, which reduces the level of LDL in the blood and thus also the effects of LDL such as atherosclerosis.

The administration of statins reduces heart attacks and deaths. The independent research association Cochrane concluded that out of 1000 people who would be treated with statins over a period of five years, one in 18 cardiovascular events could be avoided (Taylor, Huffman et al. 2013).

There are no significant differences in efficacy between pravastatin, simvastatin or atorvastatin in their standard dosage when reducing infarction or death (Zhou, Rahme et al. 2006).

Regarding statins and cancer, a 2006 published meta-analysis of previous studies (N=27) on the effect on cancer with data of approx. 90,000 patients found statin intake ineffective - neutral" - i.e., neither useful nor harmful (Dale, Coleman et al. 2006). There could not be identified cancer types or circumstances in which the incidence, course or prognosis of the disease would be significantly influenced by statins.

A 2012 epidemiological study with data from the Danish Cancer Registry showed that patients over 40 years of age were significantly less likely to die from cancer if a statin was taken in their lifetime. Likewise, overall mortality was reduced to the same extent (Nielsen, Nordestgaard et al. 2012). The study could not show the reasons for this correlation. Other studies associated statin use with lowered

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risk of melanoma, non-Hodgkin lymphoma, endometrial, and breast cancers (Jacobs, Newton et al. 2011) (Murtola, Visvanathan et al. 2014). Furthermore, another report proposed a dose dependent reduction in colorectal cancer mortality with statin use (Cardwell, Hicks et al. 2014). Earlier, a case–control study with 295,925 cancer patients, advocated a link between statin use and a slight reduction in cancer-related mortality for 13 different cancer entities (Nielsen, Nordestgaard et al. 2012).

Obesity is a nutritional and metabolic disease with severe overweight and a positive energy balance, which is characterised by an increase in body fat that exceeds normal levels and often has pathological effects. Body mass index (BMI) is a measurement obtained by dividing a person's weight by the square of the person's height. According to the WHO definition, obesity is present in people with a body mass index (BMI) of 30 kg/m². A distinction is made between three degrees of severity defined by the BMI. Obesity is prevalent in industrialised countries, especially under living conditions characterised by little physical work with a simultaneous abundance of food. In recent years, however, emerging countries have also been increasingly affected, and obesity has obtained global pandemic dimensions.

Obesity has been associated with metabolic dysfunction, including insulin resistance and altered adipokine levels, which promotes tumour cell proliferation. Regarding the association between obesity and worse outcomes for several common cancers, BMI is of significant general interest (Kroenke, Neugebauer et al. 2016, Sun, Zhu et al. 2018, Cha, Yu et al. 2020).

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Aims

Overall aim

To investigate risk factors and prognostic features for SCCHN, and to explore tumour subsite-specific characteristics, by focusing on the oncogenic virus EBV, the immune checkpoint protein PD-L1 and serum lipoproteins.

Specific aims

Paper I

To examine the presence of EBV in squamous cell carcinoma of the oral tongue (SCCOT) and tonsil (Tonsillar SCC).

Paper II

To investigate the clinical relevance of PD-L1 in patients with SCCOT.

Paper III

To evaluate the prognostic importance of serum lipoprotein levels in patients with SCCHN.

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Materials and Methods

Patients

The number of patients analysed in the studies varied between 101-113. In study I, 12 of the patients were retrieved from the Second University of Naples, whereas in study II and III all samples were from the University Hospital in Umeå, either from the archive at Clinical Pathology, or fresh frozen from the ENT-clinic (Table 1). In the EBV study tissue was used, in the PD-L1 study both tissue and serum and in the lipoprotein study only serum samples were analysed. Permission for the studies had been granted by the Regional Ethics Review Board at Umea University (dnr:s 03-201 and 08-003M).

Table 1. Overview of research subjects in different studies

Study

I EBV

II PD-L1

III Lipoprotein

Total number of patients analysed 113 101 106

Nationality Sweden 101 101 106

Italy 12 0 0

Gender Female 56 51 36

Male 57 50 70

Tumour subsite

Oral tongue 98 101 28

Oropharynx 15 0 48

Gingiva 0 0 21

Floor of mouth 0 0 9

TNM stage

I 27 24 35

II 34 37 16

III 15 15 20

IVa, IVb, IVc 37 25 35

Age at diagnosis

≤ 40 years 14 16 8

41 – 65 years 44 40 50

> 65 years 55 45 48

Total number of healthy controls 0 0 28

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EBER in situ hybridisation (EBER-ISH)

For detection of EBER1 and EBER2, RNA-molecules encoded by EBV-infected cells, so called in situ hybridisation (ISH) was used. ISH is a method for detection of nucleic acids (RNA or DNA), where an artificially produced probe via base pairs binds/hybridises to the target.

When using the commercially available EBER-ISH kit (800-2842, Ventana Medical Systems, Roche Diagnostics GmbH, Mannheim, Germany) two slides are needed. The first one to verify the presence of mRNA in the sample, and the second one for tracing EBER1 and EBER2. As results from the EBV study were hard to interpret, our collaborator in this work, professor Gulfaraz Khan, an acknowledged authority within the field of EBV-research who, together with dr Philip Coates developed the EBER-ISH method (Khan, Coates et al. 1992) suggested to try his refined EBER-ISH assay, with additional controls not available commercially. Also, the PCR-analysis for presence of EBV DNA and the staining of the EBNA1 protein were performed in professor Khan’s laboratory.

Polymerase chain reaction (PCR) of EBV

Nine out of 10 SCCOT that were reanalysed with professor Khan’s EBER-ISH kit, were further analysed for the presence of EBV DNA extracted from formalin fixed paraffin embedded (FFPE) sections using EBV specific primers. The analysis was performed in professor Khan’s laboratory.

Immunohistochemistry

Immunohistochemistry (IHC) was further used on some of the samples for detection of the EBV-encoded protein EBNA1. A mouse monoclonal antibody was used (clone D810H, ThermoFisher), and stainings performed in professor Khan’s laboratory.

For detection of the PD-L1 protein, formalin fixed paraffin embedded, FFPE, sections were incubated with the Ventana PD-L1 (SP263) assay (Ventana Medical Systems Inc) and stainings performed at the accredited laboratory at Clinical Pathology, Umeå University Hospital.

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Quick score

The QuickScore (QS) method (Detre et al., 1995) was implemented for evaluation of PD-L1 levels separately in tumour and immune cells. The proportion of stained cells was divided into six categories: 1 = 0%– 4%, 2 = 5%–19%, 3 = 20%–39%, 4 = 40%–59%, 5 = 60%–79% and 6 = 80%–100%. Intensity of staining was divided into four levels: 0 = negative, 1 = weak, 2 = intermediate, and 3 = strong. A QS for each tumour was then calculated by multiplying the two scores, rendering a QS value between 0-18.

Immune cell analysis using xCell

For 25 of the 101 patients included in the PD-L1 analysis, fresh frozen tumour tissue adjacent to the FFPE tissue used in the immunohistochemical analysis previously had been analysed for RNA expression using microarray gene expression profiling (Boldrup, Gu, et al., 2017; Gu et al., 2019). Using the xCell method (https://xcell.ucsf.edu/) (Aran, Hu, & Butte, 2017) the relations between mRNA levels of PD-L1 and infiltration of 34 different kinds of immune cells could be estimated.

Serum analysis

Levels of circulating PD-L1 protein was measured by digital ELISA using R-PLEX Human PD-L1 Antibody Set from Meso Scale Discovery (MSD).

Measurement of levels of total cholesterol, HDL, triglyceride and lipoprotein(a) was performed at the accredited laboratory at Clinical Chemistry, Umeå University Hospital. Levels of LDL were measured at the same laboratory and calculated using the Friedewald equation (Friedewald, Levy et al. 1972).

Statistical analysis

In paper I no statistical analysis was performed.

In papers II and III the Chi-Square test was used to determine associations between categorised clinicopathological variables and categorised levels of PD-L1

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and lipids, respectively. To evaluate correlations between continuous variables, nonparametric Spearman correlation analysis tests were performed.

To study the difference between two or several groups of continuous variables, Mann-Whitney U test or Kruskal-Wallis H tests were applied, respectively.

For survival analysis, the Kaplan-Meier method with log-rank test was used. Univariate and multivariate analyses using the Cox regression model were also conducted to assess the independence of variables.

All statistical tests were conducted in IBM SPSS Statistics 25 (IBM Corp.), and a two-sided p value < 0.05 was considered significant.

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Results and Discussion

The material analysed in the present studies had been consecutively collected, and showed a standard distribution similar to previous studies, regarding sex, age and other clinical factors. Our analysis also showed, in concert with previous studies, that primary tumour size and lymph node metastasis were strongly correlated to prognosis.

EBV (Paper I)

Tobacco and alcohol are well known risk factors in the development of SCCHN. Regarding the aetiology, viruses are becoming increasingly important as potential triggers of SCCHN (Sand and Jalouli 2014) (Metgud, Astekar et al. 2012) (Gondivkar, Parikh et al. 2012) (Prabhu and Wilson 2016). EBV is particularly implicated in the pathogenesis of SCCOT by its prevalence in the oral cavity and its known ability to infect tongue epithelial cells (Hutt-Fletcher 2017). Here, we investigated whether there are indications of a possible role of EBV in the carcinogenesis of SCCOT. A viral aetiology may provide a plausible explanation to account for the increasing incidence of SCCOT in young patients (Llewellyn, Johnson et al. 2001) (Patel, Carpenter et al. 2011) (Tota, Anderson et al. 2017), a patient group which may not have been included in the early studies on EBV

Initially we used the EBER-ISH method in the accredited laboratory at Clinical Pathology at our University hospital, whereby the EBV encoded RNAs EBER 1 and 2 were detected. As the results from this analysis were difficult to interpret, a subgroup of the samples was further analysed with a refined version of the test, developed by professor Khan in UAE, who had developed the EBER-ISH method used in the commercial kit employed in Umeå. In professor Khan’s laboratory, methods were used for detection of the EBV encoded protein EBNA1 and presence of EBV DNA. Taken together, in this study three different methods, ISH, PCR and immunohistochemistry, were applied, giving the same results.

EBER ISH

Using the commercial kit, EBER-ISH was completed on all 113 carcinoma specimens in the Clinical Pathology laboratory in Umea. All investigated samples produced staining with the positive control probe, which underlines that these tissues suit the method. Seven of 15 tonsillar SCCs, (46.5%) were negative, in one

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case (7%) positive nuclei were occasionally detected and seven (46.5%) showed cytoplasmic positivity. In the EBER-positive samples, the staining was not detected in all tumour cells.

Of the 98 SCCOTs, 56 (57%) were negative, 41 (42%) exhibited cytoplasmic positivity and 5 of them also showed isolated cells with nuclear positivity. Only in one case of SCCOT (1%) could a nuclear positivity be determined. However, the staining pattern for EBERs in the SCCOT samples was unlike the previously described pattern associated with EBV-related malignancies, which is strong nuclear staining with nucleoli sparing (Khan, Coates et al. 1992) (Khan, Coates et al. 1992) (Fok, Friend et al. 2006).

To go further, ten of the SCCOT cases with nuclear and/or cytoplasmic EBER reactivity were investigated with in-house prepared probes and reagents for EBER-ISH. Just as with the commercial EBER-ISH kit, staining with similarly weak and diffuse patterns was observed when using the in-house protocol. Surprisingly, the same pattern of staining was achieved after pretreatment with RNase prior to hybridization with anti-sense EBER probes. A second control using non-complementary sense EBER probes produced analogous staining patterns, indicating non-specific hybridisation signals. On the other hand, strong nuclear staining with nucleolar sparing was noticed in the EBV positive controls with anti-sense EBER probes, whereas no signal was seen after RNAse pretreatment or when using sense EBER probes.

Immunohistochemistry

The EBV-encoded nuclear antigen 1 (EBNA1) protein is expressed in all EBV-associated malignancies, where it is required for maintenance, replication and transcription of the EBV genome. The previously described ten SCCOT specimens that had been analysed with the in-house EBER ISH method were further separately tested for EBV with the help of immunohistochemistry for EBNA1.When using a rabbit infected with EBV spleen as a positive control, a strong nuclear staining appeared for EBNA1. However, no EBNA1 staining could be detected in SCCOT samples.

PCR

The previously mentioned ten SCCOT samples were further independently analysed for EBV DNA. Out of the nine cases with sufficient material available for

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PCR analysis, HBB (Human β-globin gene) could be amplified after 30 amplification cycles in seven cases, as an indicator for suitable DNA quality and quantity for this amplification method. None of the SCCOT samples was positive after the same number of cycles for EBV using primers for the BamH1 `W' region. Since the EBV BamHIWregion appears in multiple copies within the viral genome (Cheung and Kieff 1982), PCR amplification of this region is a highly sensitive method for the detection of EBV.

Interpretation paper I

Overall, these results from three analysis methods applied for our cohort indicate that there is no evidence for an association of EBV with SCCOT in either young or old patients. Furthermore, the lack of HPV in our tumour specimens (Sgaramella, Coates et al. 2015) suggests that the increasing incidence of this disease in younger people seen in the west (Llewellyn, Johnson et al. 2001) (Patel, Carpenter et al. 2011) (Tota, Anderson et al. 2017) cannot be attributed to EBV or HPV.

A recent meta-analysis supported that EBV infections are associated with an increased risk of OSCC (She, Nong et al. 2017). And a recent meta-analysis from Brazil of 53 studies that were published from 1990 to 2019, concluded that EBV-infected persons had a 2.5 higher risk for developing OSCC (de Lima, Teodoro et al. 2019). By the use of PCR, significantly more EBV-infected samples were found in industrialised compared to developing countries (Jalouli, Jalouli et al. 2012). Using a different analytical methodology, a recent study from Thailand found overexpression of EBV-encoded latent membrane protein-1 (LMP-1) in OSCC (Rahman, Poomsawat et al. 2019). And a recent study from Saudi Arabia observed remarkably strong EBV EBNA1 expression in an SCCOT cohort (Alsofyani, Dallol et al. 2020). Interestingly an Iranian study showed a statistically significant higher distribution of EBV PCR products in SCCOT at advanced tumour stage, where solely nested PCR was applied as analysis method (Shahrabi-Farahani, Karimi et al. 2018). Moreover, a recent study suggested a possible oncogenic collaboration of microorganisms EBV and bacteria Porphyromonas gingivalis in oral carcinogenesis (Nunez-Acurio, Bravo et al. 2020).

A possible explanation for the results in other studies may be the lack of adequate supporting analytical methods to confirm viral presence (Shamaa, Zyada et al. 2008). Furthermore, in some studies additional analytical methods produced conflicting results (Kobayashi, Shima et al. 1999). This inconsistency may be due to the application of EBER-ISH as the primary method in many studies, which

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we observed could produce misleading staining results in some samples. Furthermore, application of PCR with high numbers of amplification cycles leads to viral detection unrelated to EBV presence in tumour cells, instead representing latent virus in lymphocytes, present in 90% or more of the world population.

These conflicting experimental results could also be associated with the differing anatomical location of sample collection from either the mobile tongue, as in this work, or from the base of tongue. In addition, the contradicting findings from many studies in Asia that link EBV with OSCC (Shimakage, Horii et al. 2002) (Kikuchi, Noguchi et al. 2016) (Kobayashi, Shima et al. 1999) (Higa, Kinjo et al. 2003) (Higa, Kinjo et al. 2002) (Jiang, Gu et al. 2012) might be explained by their different geography with differences in ethnicity, environment and lifestyle. Additionally, genetic heterogeneity of EBV and different epitheliotropism of EBV subtypes may also contribute to differering results when comparing these studies (Tsai, Raykova et al. 2013). A recent study from Pakistan related acantholytic tumour, a rare histological subtype of OSCC, to EBV (Saleem, Baig et al. 2019).

Taken together, the study on EBV used three different analytical techniques to detect EBV and its products EBERs and EBNA-1, and could not demonstrate EBV in SCCOT or tonsillar SCC. Together with our previous data on HPV (Sgaramella, Coates et al. 2015), the present findings can most probably exclude both EBV and HPV from the carcinogenesis of SCCOT. In contrast, a recent Indian pilot study on 108 cases of OSCC recorded co-infection by EBV and HPV in 5.6 % of cases and suggested these oncogenic viruses as independent cofactors of oral cancer development (Vanshika, Preeti et al. 2021). Nevertheless, we could not identify dual infection of EBV and HPV in our OSCC samples, which indicates that this viral co-infection pattern (Sand and Jalouli 2014) (Hermann, Fuzesi et al. 2004) is uncommon in at least these investigated two tumour sites in a European population.

PD-L1 (Paper II)

Since PD-L1 levels in SCCHN may be distinguished related to the anatomic origin of the tumor (Troeltzsch, Woodlock et al. 2017), this work strived to investigate the clinical implications of PD-L1 expression in a cohort of SCCOT patients from Northern Sweden, using an FDA (Food and Drug Administration, USA) approved PD-L1 IHC assay. Regarding PD-L1 expression in SCCOT and tumour infiltrating immune cells, the PD-L1 staining encompassed the membrane and the cytoplasm of both tumour cells and immune cells. The measurement of PD-L1 staining was conducted with the QS method and displayed PD-L1 levels from 0 to 18 in tumour

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cells and 0 to 15 in immune cells. Significant correlation was seen between tumour cell PD-L1 levels and immune cell PD-L1 levels (rho = 0.427, p < .001).

PD-L1 levels in tumour cells

Analysis of the tumour cell compartment revealed that 21 of 101 patients were negative for PD-L1 staining (QS = 0). For further analysis, the patients were divided based on PD-L1 staining QS into three groups, negative (QS = 0), low (QS from 1 to 5), and high (QS from 6 to 18). Using Chi-square test, no significant association was found between categorized PD-L1 levels and clinicopathological characteristics. However, according to Mann-Whitney U test on non-categorised scores PD-L1 levels were significantly higher in females than in males (p = 0.019).

With regard to PD-L1 and patient survival, statistical analysis using Kaplan–Meier with log-rank test was performed. No significant difference in survival among patients with negative-, low- or high-PD-L1 levels was found (overall survival p = 0.789, disease-free survival p = 0.710). Comparing survial between genders, there was also no significant difference (overall survival p = 0.400, disease-free survival p = 0.282). However, for the subgroup of patients with low PD-L1 levels, better surival was seen in males (n = 23) compared to females (n = 22) (overall survival p = 0.021, disease-free survival p = 0.020).

The further investigation of gender differences in clinical variables revealed that female patients were significantly older than males (p = 0.003). However, with regard to the PD-L1-low group, there was no significant difference in age between genders (p = 0.097).

Fresh frozen tumour samples from 25 of the 101 patients included in this study had been previously investigated with microarray gene expression profiling. The gene expression data was uploaded to the xCell webtool to estimate immune cell types in these tumour samples. Then we performed a correlation analysis for the resulting xCell scores for immune cell types with the PD-L1 IHC results. As the main finding, among the 34 immune cell types identified by xCell, levels of natural killer T cells (NKT), immature dendritic cells (iDC), and M1 macrophages positively correlated with tumour cell PD-L1 levels (p < 0.05).

With regard to circulating PD-L1, serum PD-L1 levels were quantified in 30 patients, which showed that the median PD-L1 concentration was 49.97 pg/ml. We compared serum PD-L1 levels among patients with various clinicopathological features, no significant difference was found. Both FFPE sections and blood samples were available from 13 patients. Therefore,

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correlations between tissue PD-L1 and serum PD-L1 levels were investigated, which revealed that tumour cell PD-L1 did not correlate with serum PD-L1 levels (rho = −0.505, p = 0.078).

PD-L1 levels in tumour-infiltrating immune cells

Only 4 patients were PD-L1 negative regarding tumour-infiltrating immune cells, and these 4 patients also had negative PD-L1 tumour cell compartments. There was no gender difference in immune cell PD-L1 levels. In addition, analyses of the impact of immune cell PD-L1 on survival showed no significance even with regard to gender. According to xCell results, immune cell PD-L1 levels did not correlate with any estimated immune cell types. Finally, no correlation between immune cell PD-L1 and circulating PD-L1 was found (rho = −0.123, p = 0.689).

Interpretation paper II

In this work, PD-L1-positive tumour cells were seen in 79% and PD-L1-positive immune cells in 96% of the samples. Previous studies focusing on patients with SCCOT reported PD-L1 positivity in 23% (Yoshida, Nagatsuka et al. 2018), 57.9% (Naruse, Yanamoto et al. 2019), or 79% (Mattox, Lee et al. 2017) of patients. Such differences between studies may be explained by intra-tumour heterogeneity of PD-L1 (Rasmussen, Lelkaitis et al. 2019) and different IHC antibodies/detection protocols and the different scoring criteria used.

With regard to PD-L1 staining in SCCOT cells, two distinct staining patterns of PD-L1 were previously described, either diffuse staining throughout the tumour or peripheral staining around the tumour (Mattox, Lee et al. 2017). In contrast, such patterns were not recorded in the samples examined in this work. It is also worth bearing in mind that PD-L1 staining in nuclei of tumour cells (Naruse, Yanamoto et al. 2019) is likely an artifact resulting from inappropriate sample treatment (Polioudaki, Chantziou et al. 2019).

The above-mentioned studies led to different conclusions regarding the association of PD-L1 with clinicopathological characteristics and clinical outcomes in patients with SCCOT. In accordance with our results, Mattox et al. (Mattox, Lee et al. 2017) revealed no significant associations.

On the other hand, a study investigating 81 patients aged ≤45 years with oral cavity squamous cell carcinoma (86.4% of patients with SCCOT) associated high

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tumour PD-L1 and the presence of tumour-infiltrating lymphocytes with better prognosis among females (Hanna, Woo et al. 2018). In contrast, we did not find any relevance of infiltrating immune cells or PD-L1-positive infiltrating immune cells for clinical variables.

Earlier studies have observed gender differences in immune response (Capone, Marchetti et al. 2018) (Klein and Flanagan 2016). A previous study of oral squamous cell carcinoma revealed a prognostic value of PD-L1 for males (Lin, Sung et al. 2015). There may not be a gender difference regarding the efficacy of immune checkpoint inhibitors (Conforti, Pala et al. 2018) (Wallis, Butaney et al. 2019). Nevertheless, this study further suggests that gender as a critical biological variable is an important issue for future PD-L1 research.

With regard to the tumour-infiltrating immune cell composition in bulk tumour samples the xCell tool was applied for a subset of patients. The xCell approach is described as a transcriptome-based computational method showing high accuracy for well-defined cell type signatures in contrast to the widely used IHC staining approaches for immune cell detection and quantitation (Aran, Hu et al. 2017) (Sturm, Finotello et al. 2019). When xCell-estimated immune cells are correlated with PD-L1 a note of caution is due since information regarding the exact location of the immune cells cannot be obtained through xCell (Galon, Mlecnik et al. 2014).

The immune cell correlation has been exemplified in several reports on oral squamous cell carcinoma where IHC analysis revealed CD8+ T-cells and CD4+ T-cells to be correlated with PD-L1 (Cho, Yoon et al. 2011) (Mattox, Lee et al. 2017) (Satgunaseelan, Gupta et al. 2016), whereas our study did not show any correlation. On the other hand, in our work a positive correlation was seen between NKT, iDC, M1 macrophages and PD-L1 on tumour cells. As mentioned above, the interaction between immune cells and PD-L1-positive tumour cells cannot be clarified using the xCell approach. Further research should be undertaken to investigate how these cells are involved in PD-L1-related immune evasion in SCCOT and how they respond to PD-L1 inhibitors.

Taken together, this is the first work employing QS and xCell methods on PD-L1 in SCCOT according to a standardised FDA approved IHC protocol. Our data support the notion that the impact of PD-L1 on patient survival is gender-related. The use of transcriptome-based computational methods for cellular characterisation could also contribute to the understanding of the tumour microenvironment.

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Lipoproteins (Paper III)

In the last few years, there has been an increase in scientific research on the role that lipoproteins might play in tumour development, but findings from different tumour types are often contradictory (Ganjali, Ricciuti et al. 2019). Nevertheless, several previous studies have shown an involvement of blood lipids in the initiation and development of different types of cancer, including OSCC (Lohe, Degwekar et al. 2010).

In our study, serum samples were obtained before, at, or after surgery, which resulted in a shifting fasting stage. In terms of fasting stage, all patients were required to fast before surgery, while at surgery they received infusion fluid containing glucose, and after surgery the fasting status was unknown. According to these three different collection time points, the blood samples demonstrated significantly different levels of both TGs and lipoprotein(a) (p < 0.05). Therefore, a limitation in this work is that only a subset of serum samples collected at fasting stage were used for the analysis of TGs and lipoprotein(a).

Next, the analysis of the impact of BMI on overall survival revealed that the group of overweight patients (defined as having a BMI of 25-30) showed better survival compared to normal weight (BMI 18.5-24), obese (BMI > 30) and underweight patients (BMI < 18.5) (p < 0.001).

For completeness, overall survival for oropharyngeal cancer in relation to HPV status was also analysed, although no significant difference was seen, only a tendency to worse overall survival in HPV negative tumours (p = 0.177).

Total cholesterol

With regard to total cholesterol in SCCHN patients compared to healthy controls, similar levels were seen (p = 0.208). Comparing cholesterol-high patients to cholesterol-normal patients, no significant differences in clinicopathological features were identified, such as age, gender, alcohol comsumption, smoking status and BMI. However, patients with high cholesterol levels seemed to have better overall survival outcome than patients with normal cholesterol levels (Fisher’s exact test, p = 0.048). Moreover, we explored correlations between total cholesterol and BMI applying the Mann-Whitney U test on non-categorised values. No significant correlation was identified (correlation coefficient = 0.132, p = 0.177).

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The impact of total cholesterol levels on patient survival was further analysed using the Kaplan-Meier method. Longer overall survival was seen in SCCHN patients with high levels of total cholesterol compared to patients with normal levels (p = 0.028). Since 17 patients were on lipid-lowering drugs, a selective analysis of patients without lipid-lowering medication only (n = 89) was carried out, and results still indicated high levels of cholesterol to be beneficial for survival (p = 0.017).

Univariate Cox regression analysis showed that total cholesterol was significantly associated with overall survial (p = 0.007) and disease-free survival (p = 0.023). Multivariate Cox regression with adjustment for the effects of age, TNM stage and weight change indicated that total cholesterol was not an independent prognostic factor for patient survival.

LDL

No significant difference in LDL levels was seen between patients and controls (p = 0.554). Patients with high LDL levels had better survival outcome than patients with normal LDL levels (Fisher’s exact test, overall survival, p = 0.017; disease-free survival, p = 0.030). A positive correlation between LDL and BMI was seen (correlation coefficient = 0.217, p = 0.029).

Levels of LDL correlated to overall survival for SCCHN patients, where high levels of LDL were beneficial (Kaplan-Meier curve, p = 0.007). When we selected only patients without lipid-lowering medication (n = 85), high levels of LDL continued to be significantly correlated to longer survival (p = 0.010).

Univariate Cox regression analysis showed that LDL was significantly associated with overall survial (p = 0.002) and disease-free survival (p = 0.005). Multivariate Cox regression with adjustment for the effects of age, TNM stage and weight change indicated that LDL was an independent prognostic factor for overall survival (p = 0.010) and disease-free survival (p = 0.018).

HDL

There was a significant difference between SCCHN patients and healthy controls with regard to HDL, where levels were lower in patients (p = 0.012). Previous studies have revealed that tumours in different sub-locations of the head and neck vary in biological characteristics (Boldrup, Coates et al. 2011, Boldrup, Coates et

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al. 2012). Therefore, our patient samples were grouped according to sub-location of the tumour. There were significant differences only for HDL in patients with gingival and oropharyngeal SCC (p = 0.003 and 0.013 respectively). Comparing HDL-low patients to HDL-normal patients, no significant differences in any clinicopathological features were identified. Correlating HDL to BMI levels, a negative correlation was found (correlation coefficient = -0.380, p < 0.0001).

TGs and lipoprotein(a)

Since fasting status influences the levels of TGs and lipoprotein(a), we chose to analyse only samples taken at fasting. With regard to overall survival, no significant impact was found for either of these two factors.

Interpretation paper III

To date, only a few studies are available on serum lipid profiles in SCCHN. Consistent with earlier studies (Patel, Shah et al. 2004, Lohe, Degwekar et al. 2010, Ghosh, Jayaram et al. 2011), in this work, a tendency of lower levels of total cholesterol, LDL and HDL was seen in SCCHN patients compared to controls, but HDL was the only lipoprotein that was statistically significantly lower in SCCHN patients in our study. Numerous previous studies described a reduction in HDL, and low HDL is proposed to be an additional cancer predictor and believed to be caused by excess cholesterol utilisation for membrane biogenesis by proliferating tumour cells (Bailwad, Singh et al. 2014).

LDL comprises several proteins and lipids, carrying cholesterol into peripheral tissues and also affecting the metabolism of fatty acids. Previous research has established that LDL can promote breast cancer by affecting cell proliferation and migration to facilitate disease progression (Guan, Liu et al. 2019). In addition, a study of 601 patients with small-cell lung cancer indicated lower LDL to be an independent prognostic factor for longer overall survival (Zhou, Zhan et al. 2017). In ovarian cancer, longer overall survival has been observed for patients with normal compared to elevated levels of LDL (Li, Elmore et al. 2010). However, in our group of SCCHN patients, high levels of LDL were correlated with improved survival. Cholesterol is involved in formation of steroid hormones like estrogen and may have different effects in hormone dependent cancer types (ovary, breast) compared to non-hormone dependent cancers like SCCHN.

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A previous study on SCCHN did not find a correlation between LDL and survival (Li, Da et al. 2016). In this study, Li and co-workers had excluded patients with a family history of lipidemia as well as obese persons, and they also applied different methods for detection of LDL, which might explain the different results.

Cholesterol synthesis is reduced in the liver under statin treatments, which promotes cholesterol uptake from plasma through upregulation of LDL-receptors on the surface of hepatocytes, also increasing LDL degradation in the liver. As the use of statins is a frequent medication, we excluded patients on statin medication (17 patients) in further calculations, but still obtained significantly better survival for patients with high levels of total cholesterol and LDL. Since cholesterol levels were lower in SCCHN patients compared to healthy controls, it could be hypothesised that higher cholesterol levels mimic the normal state.

Another main finding in our work was that the group of overweight, but not obese, patients (pre-treatment BMI of 25-30) showed better survival compared to normal weight, obese and underweight patients. Other studies have proposed that higher BMI is related to better survival and less recurrence and distant metastasis rates in SCCHN (Hollander, Kampman et al. 2015, Karnell, Sperry et al. 2016).

Obesity, on the other hand, which encompasses increased levels of LDL, cholesterol and TGs, as well as decreased HDL (Mika and Sledzinski 2017), has previously been identified as an independent disadvantageous prognostic variable, where obese SCCOT patients had a 5-fold increase in risk of death compared to normal weight patients (Iyengar, Kochhar et al. 2014).

Even if measuring a single pre-treatment LDL level may not adequately reflect the variance in lipoproteins over the clinical course of SCCHN, these data provide one of the first accounts to examine LDL levels as a predictor of outcome in SCCHN. Furthermore, in terms of directions for future research, these results may also suggest a mechanism by which cholesterol altering drugs may be used to influence outcomes.

Overall, based on the present results indicating improved survival for patients with high LDL, it can also be speculated that lipid-lowering drugs should be withdrawn for SCCHN patients during cancer treatment.

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Conclusions

Three different methods were applied for the detection of EBV and its most prevalent products, the EBERs and EBNA-1, without finding evidence for presence of the virus in SCCOT or tonsillar SCC. These contrasting data compared with some previous studies may be related to anatomical, ethnical, lifestyle and methodological differences, and emphasises the importance of appropriate controls for EBER-ISH when investigating EBV in human diseases. Combining the present data with our previous findings showing lack of HPV infection in SCCOT indicates that these two viruses are unlikely to be involved in the pathogenesis of SCCOT in this European population. Regarding PD-L1, our data in a cohort of SCCOT patients from Northern Sweden supported the significance of gender on tumour cell PD-L1 expression and demonstrated significantly higher PD-L1 levels in females. Moreover, better survival outcome was found for females lacking PD-L1-positive tumour cells compared to males lacking PD-L1 on their tumour cells. Our data also exposed the involvement of specific immune cell types in PD-L1-regulated immune evasion. Specifically, NKT, iDC, and M1 macrophages were positively correlated with PD-L1 on tumour cells. In accordance with previous studies, we found HDL to be significantly lower in SCCHN patients than controls. Moreover, high levels of LDL were found to be beneficial for survival outcome in patients with SCCHN. Accordingly, cholesterol-lowering drugs like statins may influence the course of SCCHN.

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Future perspectives

This thesis focuses on EBV, PD-L1 and serum lipoproteins in order to contribute to further understanding of SCCHN and risk factors and prognostic features. To date, a great amount of different clinical and histopathological SCCHN characteristics have been accumulated to possibly contribute to tumour prognosis and outcome, many of which have not been validated as a single decisive prognostic tumour marker.

With regard to EBV and SCCOT, we found no evidence for EBV-infection. This is in contrast to nasopharyngeal cancers which have a clear association with EBV, once again emphasising the sub-site differences we have seen previously when studying the heterogeneous group of SCCHN tumours. Also with regard to the heterogenity in EBV studies seen globally, and the multicultural society we have there are also ethnicity-based features that may further refine individual-based SCCHN tumour diagnosis and therapy.

Regarding PD-L1 and SCCHN, the ongoing research on immune checkpoint inhibitor combination therapies and use of co-factors will possibly lead to a refinement in immune checkpoint treatment efficacy and predictability. The future development in assessing immune cell populations with application of transcriptome-based computational methods for cellular characterisation will provide additional information for understanding the complex tumour microenvironment and its response to tumour cells which show varying degrees of immune checkpoint activity.

As for lipoproteins and SCCHN, in this thesis, measurement of major lipoproteins was performed with classical methods. Concerning lipid metabolism related to SCCHN and other cancers, the alterations in lipid metabolism known so far are most probably barely the tip of the iceberg. In the future, apart from the commonly known lipoproteins, less known lipid species may be analysed for their impact on SCCHN. Refinements of analytical methods are emerging, for example comprising more advanced mass spectrometry lipidomics. Visualising methods such as mass spectrometry imaging can contribute with spatial information about the lipid distrubution in SCCHN cancer samples. Genomic methods (such as data within the Cancer Genome Atlas, TCGA) are expected to deepen our understanding of tumour lipid metabolism pathways.

With regard to lipids as biomarkers for SCCHN, one can expect further development of both lipidomics in biofluids and tumour lipidomics. For example, as a patient-convenient non-invasive method, urine lipidomic profiling may constitute a source of SCCHN markers. Further liquid biopsies such as blood,

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saliva and urine samples may be linked with disease charateristics including favourable or unfavourable prognosis. In the future, refined tumour-characteristic lipid profiles with intraoperative sample analysis may contribute to surgical decision making in the operating theatre.

In spite of a bit of controversy, statins have been associated with a decreased risk of cancer in epidemiological studies. The abundance of statin use for hyperlipidemia in developed countries makes the assessment of statin effects on cancer difficult. Larger epidemiological studies may clarify further the effect of statins. Furthermore, new lipid metabolism modifying agents such as Betulin, Silibin and others are investigated for their impact on cancer.

As for future research, dietary interventions on SCCHN, regarding parametres like lipid consumption and total caloric intake, may be additionally investigated with regard to therapy outcome and other clinical characteristics.

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Acknowledgements

I would like to express my sincere gratitude to all the people who have contributed to the realization of this thesis in different ways.

First of all, I would like to thank all the patients and controls who participated in the studies.

Karin Nylander, my main supervisor, thank you for introducing me to this field of research and all the work and time you have spent making this thesis possible. Thank you for the training I have received, I have learned an incredible amount. Your tireless support even through these difficult times with the clinical work during covid-19 pandemic, your warm-hearted encouragement and up-building enthusiasm are inspiring and unforgettable! Thank you, for always feeling welcome despite all the interruptions during the research studies, and for all the laughter. Without your endless support this work would not have happened, thank you!

Linda Boldrup and Xiaolian Gu, my assistant supervisors, thank you for your support and valuable contributions to the thesis. Thank you for many fine years of hard work, with discussions, both about research and everything else between heaven and earth. You are amazing!

Lena Norberg-Spaak, my assistant supervisor, your positive attitude makes working with you a great pleasure. Thank you for all your help, support and good talks.

Many thanks to all the co-authors and members of the research team for sharing your expertise and constructive advice;

Gulfaraz Khan, Philip Coates, Nicola Sgaramella, Robin Fåhraeus, Lixiao Wang, Sa Chen, Nima Attaran, Amir Salehi and Sivakumar Vadivel Gnanasundram.

I would like to thank you all the members of the department of Medical Biosciences and an extra thank you to Anders Ödin and Clas Wikström for their very helpful IT support and more than that. Special thanks to Richard Palmkvist, Oleg Alexeyev and Maréne Landström for your valuable scientific discussions, your feedback and pep talks.

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At Umeå university, special thanks also to Eva Fhärm, Fredrik Karlsson, Eva Levring Jäghagen, Stefan Söderberg and Per Dahlqvist for their big support in my research training.

A big thank you to Carina Ahlgren, Maud Normark, Helena Harding and Tomas Jacobsson for helping with administration managing and much more.

Gabriella Dekombis and Minna Oscarsson from the printing service, thank you for all your valuable advice.

I would like to thank all my other colleagues and co-workers at all my working places who have helped me in various ways. Former and present staff members at the ENT clinics in Umeå and Örebro, thank you very much for your support of my research. I want to direct special thanks to Magnus Wahlgren (always super working with you), Adrian Ström, Katarina Zborayova, Jan Vokurka, Andrea Donfrancesco, Mats Aadde, Diana Berggren, Sten Hellström, Svante Hugosson, Helena Hidman, Miklós Fülöp, Göran Laurell (an extra thank you for your guidance at the beginning of my research), Thorbjörn Holmlund and Krister Tano, all of whom especially contributed to the completion of this thesis.

Last but not least I thank my family and all friends for their interest and encouragement for this dissertation.

Alf Molin, thank you very much for instilling enthusiasm, aside from the research meetings.

Thanks to Tino, James, Zoltan and Per Erik, who provided stimulating discussions and happy distractions.

To my parents, Peter Wilms and Annegret Wilms, and auntie Edith, thank you for always being supportive and believing in me. My brother Henrik, thank you for always being there for me, in spite of the long distance. A big thank you to Jan Johansson and Marita Johansson, and Carro, Linda and Björn.

My wife, Anna, thank you for your love and patience, and for helping to save this thesis on numerous occasions. You and me! And thank you to our lovely children, Katharina, Amanda and Julia for reminding me of the priorities in life.

The studies were supported by grants from Lion’s Cancer Research Foundation, Umeå University; The Swedish Cancer Society; Region Västerbotten; Acta Oto-Laryngologica and Umeå University.

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