Low potential of circulating interleukin 1 receptor antagonist as a prediction marker for squamous cell carcinoma of the head and neck

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J Oral Pathol Med. 2021;00:1–10. wileyonlinelibrary.com/journal/jop | 1

1 | INTRODUCTION

Squamous cell carcinoma of the head and neck (SCCHN), a hetero- geneous group of tumours arising from squamous epithelium of the oral cavity, oropharynx, larynx, hypopharynx and nasopharynx, was the seventh most common cancer worldwide in 2018.^1,2 Despite in- tense research and advances in treatment, the 5 years survival rate is

still poor, around 60%.³ Plausible reasons are late detection and the aggressive nature of this tumour including a tendency for local invasion and a high incidence of cervical lymph node metastasis already at diagnosis.^2,4 Patients are treated equally, irrespective of histolog- ical and genetic features of the individual tumours.⁵

The circulatory system reflects physiological and patholog- ical states and circulating markers such as micro RNA (miRNA), DOI: 10.1111/jop.13187

O R I G I N A L A R T I C L E

Low potential of circulating interleukin 1 receptor antagonist as a prediction marker for squamous cell carcinoma of the head and neck

This is an open access article under the terms of the Creative Commons Attribution- NonCommercial- NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made.

1Department of Medical Biosciences, Umeå University, Umeå, Sweden

2Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic

3Institute of Molecular Genetics, University of Paris St. Louis Hospital, Paris, France

4Department of Clinical Sciences, Umeå University, Umeå, Sweden

Correspondence

Karin Nylander, Department of Medical Biosciences, Umeå University, Building 6M Analysvägen 9, Umeå, Västerbotten 901 87, Sweden.

Email: karin.nylander@umu.se Funding information

This work was supported by grants from the Cancer Research Foundation in Northern Sweden, Lion´s Cancer Research Foundation, Umeå University, the Swedish Cancer Society Contract number 20 0754 PjF 01H and Region Västerbotten in Sweden and grants by Ministry of Health, Czech Republic— conceptual development of research organization (MMCI, 00209805). Biobank Sweden is supported by the Swedish Research Council (VR 2017- 00650). The role of funding is to cover salary and costs for running the projects.

Abstract

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

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

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

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

K E Y W O R D S

IL- 1Ra, plasma, squamous cell carcinoma

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circulating tumour cells (CTCs), cell- free tumour DNA (ctDNA), mRNA, metabolites, exosomes and proteins are attractive for the development of screening tests that can offer additional information in terms of early detection and diagnosis.^6,7 The body fluids most commonly used in these analyses are plasma and serum; however, saliva and urine are also quite often used.

Previous studies of serum and plasma from patients with SCCHN have revealed differences in the levels of angiogenic factors,^8,9 cytokines, chemokines and growth factors ^{10– 13} between healthy controls, premalignant cases and SCCHN. In a previous study, we screened for 146 cancer- related circulating proteins in patients with squamous cell carcinoma of the oral tongue (SCCOT) and found 23 with dysregulated expression, including cytokines, growth factors and cell adhesion proteins. The only up- regulated protein in our study was interleukin 1 receptor antagonist (IL- 1Ra).¹⁴

IL- 1Ra is a member of the interleukin 1 (IL- 1) family, which is import- ant in regulating innate immunity and inflammation but also has a role in tumour development and progression.^15,16 IL- 1Ra is the major en- dogenous antagonist of IL- 1 and exerts anti- inflammatory functions by inhibiting binding of IL- 1 to its receptor. There is one secreted soluble form and two intracellular isoforms which are considered a reservoir of IL- 1Ra.¹⁶ Immune cells,¹⁷ epithelial cells ¹⁸ and adipocytes ¹⁹ have been reported to produce IL- 1Ra. In this study, in order to confirm and expand our previous findings, we measured plasma levels of IL- 1Ra in a larger cohort of patients with SCCHN, using another sensitive detection platform from Mesoscale diagnostics. More importantly, pre- diagnostic plasma samples from 44 SCCOT patients were also studied to evaluate the potential role of IL- 1Ra as a cancer prediction marker.

2 | MATERIALS AND METHODS 2.1 | Patients and controls

A cohort of 87 patients with primary SCCHN: 27 SCCOT, 11 base of tongue, 11 floor of mouth, 22 gingiva and 16 tonsil were included, along with 28 healthy controls. Patients were enrolled between 2008 and 2017. Mean follow- up time is 45 months, ranging from 1 to 139 months. Human papillomavirus (HPV) status was determined for tumours at the base of tongue and tonsil, respectively, and HPV positivity reported in 9 patients with base of tongue cancer and in 10 with tonsillar cancer. Fasting status differed within the tumour population, with 63 being fasting, 12 non- fasting and 12 patients with unknown fasting status. Samples from 26 of the 28 controls were obtained under non- fasting conditions.

After informed consent, 3 ml of peripheral blood was collected before treatment in connection with diagnostic examination/surgical procedure using standardised venipuncture into vacutainers (BD^® vacutainers EDTA, # 367864), at Norrland's University Hospital, Sweden. Tubes were left standing for at least 30 min at room temperature and centrifuged at 1300 g for 10 min at room temperature, and the plasma layer was aliquoted and stored at −80°C until fur- ther use. The study was approved by the local Ethical Committee

(Dnr 08– 003 M) at Umeå University, Sweden. Clinical information, including age, gender, TNM and BMI, for patients and controls, obtained by Swedish medical journal system, is shown in Table 1. TNM was classified according to the 7^th edition classification system, and most cases were diagnosed by one of the authors (KN) who is ac- tively working within head and neck pathology.

2.2 | Pre- diagnostic SCCOT cases and matched controls

Samples from the Västerbotten Intervention Programme (VIP) and the Northern Sweden Monica Project (MONICA), large ongoing population- based cohorts established in the late 1980s,²⁰ all collected in Biobank Norr were used. Forty- four patients who had developed SCCOT three months or longer after the sample was collected were included, each pre- diagnostic SCCOT sample was age and sex matched with two controls who had not developed any kind of cancer, in total 88 controls. A set of 32 fasting controls, age and sex matched to the diagnostic SCCOT samples, were also selected from Biobank Norr. All samples had been collected in EDTA tubes in the morning, after at least 8 h of fasting according to a standardised protocol. After centrifugation, samples were separated into buffy coat, plasma and erythrocyte fractions, aliquoted and frozen within 1 h of collection, either directly at −80°C or first at −20°C for up to one week before transferal to a central storage facility. In the present study, the plasma fraction was used.

2.3 | Mesoscale diagnostics biomarker assays

Plasma from all 87 SCCHN patients and 28 healthy controls was analysed with the U- PLEX biomarker group 1, a multiplex plate containing IL- 1Ra, IL- 6, IL- 12p70 and TNF- β (MSD, Mesoscale Diagnostics), according to the manufacturer's protocol. In brief, individually U- PLEX- coupled antibody solutions were made by mixing 200 µl of each biotinylated antibody with 300 µl of the assigned linker. The antibody solutions were then combined in a single tube and the U- PLEX plate coated with 50 µl of the solution in each well, incubated at room temperature for 1 h and washed 3 times with PBS- T (PBS + 0.05% Tween- 20). Plasma samples were diluted twofold with diluent 43 from MSD before adding to the wells together with standard solutions and incubated for 1 h at room temperature. After washing, 50 µl of detection antibody solution was added and incubated for 1 h at room temperature under shaking. Plates were washed once more before adding 150 µl MSD GOLD read buffer to each well and analysed with QuickPlex SQ 120 (Mesoscale Diagnostics). Concentrations (pg/ml) were calculated from the calibrator standard curve.

Twenty- three of the SCCOT patients, the 28 healthy controls, the 44 pre- diagnostic samples with 88 matched controls and 32 fasting controls matched to the diagnostic SCCOT from Biobank Norr were analysed using the V- PLEX human IL- 1Ra kit (MSD, Mesoscale Diagnostics) comprising human IL- 1Ra only. The protocol from the manufacturer was

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TA B L E 1 Clinical characteristics

Sample ID Gender Age Fasting IL−1Ra MSD

Controls

C1^a F 82 NF 192

C2^a M 70 NF 275.4

C3 F 64 NF 184.6

C4 F 26 NF 93.8

C5^a M 38 NF 136.6

C6 M 23 NF 216.2

C7^a M 51 NF 118.2

C8 F 21 NF 208.4

C9 F 24 NF 119.8

C10 F 22 NF 129

C11 F 22 NF 71.6

C12 F 20 NF 87.2

C13 F 51 NF 181.8

C14^a M 47 NF 166.4

C15^a M 59 NF 286.2

C16^a F 59 NF 83.2

C17^a M 68 NF 112.2

C18^a M 50 NF 117.6

C19^a M 83 NF 138.8

C20^a F 54 NF 168.8

C21^a F 62 NF 124

C22^a M 58 NF 146.8

C23 F 41 NF 115.2

C24^a M 62 NF 161.6

C25^a F 56 NF 118.2

C26^a F 40 F 94.8

C27^a F 39 NF 125.8

C28^a F 39 NF 194.6

Sample

ID Gender Age Fasting

IL−1Ra

MSD BMI

TNM (clinical,

7th edition) LNS Status Cause Rec Smoking Other diseases Tongue

61^a M 69 NA 156.4 23.7 T4aN0 M0 Neg Dead DDF No S

65^a F 81 NA 163.2 27.7 T2N0 M0 Neg Alive ADF No ?

68^a M 62 NA 250.6 26.1 T2N0 M0 Neg Dead DOD Yes PS

82^a F 19 NA 284.4 27.3 T4N0 M0 Neg Dead DOD Yes PS

83^a F 64 NA 209.8 21.8 T1N0 M0 Neg Alive ADF No ?

98^a M 31 NA 115.4 26.0 T2N0 M0 Neg Alive ADF No NS/P

99^a M 64 NA 280.4 20.7 T4aN2 cM0 Pos Alive ADF No S

105^a M 63 F 237 29.7 T1N0 M0 Neg Alive ADF No S

111^a F 31 F 900.2 28.4 T1N0 M0 Neg Alive ADF No S

119^a M 66 F 128.8 24.2 T2N0 M0 Neg Alive ADF No NS/P

124^a M 54 F 356.8 22.7 T4aN2bM0 Pos Dead DOD ? S Multi diseases

127^a M 27 F 114.2 25.6 T1pN1 M0 Pos Alive ADF No S

131^a F 74 F 250.8 27.6 T2N0 M0 Neg Alive ADF No NS/P

137^a F 71 F 210.4 21.6 T2N0 M0 Neg Alive ADF No NS/P Diabetes

138^a M 50 F 264.2 19.9 T2N1 M0 Pos Alive ADF No S

(Continues)

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Sample

IL−1Ra

MSD BMI

TNM (clinical,

7th edition) LNS Status Cause Rec Smoking Other diseases

149^a F 69 F 287.6 33.6 T1N0 M0 Neg Dead DAD No PS

154^a F 42 F 148.2 20.2 T1N1 M0 Pos Alive ADF No NS/P

155 F 84 F 329 23.4 T2N0 M0 Neg Dead ? No PS

156 M 65 F 87 20.8 T1N0 M0 Neg Alive ADF No S Diabetes

157 M 68 NF 176 37.0 T1N0 M0 Neg Alive ADF No S

187 F 73 F 722 37.8 T1N0 M0 Neg Alive ADF No PS

197 M 58 F 103 28.0 T1N0 M0 Neg Alive ADF No NS/P

204 F 73 F 152.4 27.8 T1N0 M0 Neg Alive ADF No NS/P Candidiasis

206 F 71 F 323 25.2 T1N0 M0 Neg Alive ADF No NS/P

212 M 52 NA 420.2 36.7 T4aN2bM0 Pos Dead DOD Yes NS/P

213 F 72 F 249.4 24.4 T2N0 M0 Neg Alive ADF No S

Gingiva

89^a F 80 461.6 40.4 T4aN2bM0 Pos Dead DOD ? ?

91^a M 54 NF 660 35.9 T4aN0 M0 Neg Alive ADF ? S

100^a M 52 NF 440 42.1 T4aN0 M0 Neg Dead DOD ? S

101 M 69 NF 119 22.8 T4aN1 M0 Pos Alive ADF ? NS/P

104^a F 69 F 219.6 20.7 T4aN0 M0 Neg Alive ADF ? NS/P

109^a M 70 F 127.6 26.9 T4aN0 M0 Neg Dead DOD Yes NS/P

132^a M 67 F 709 35.3 T4apN2bM Pos Alive ADF No NS/P

133^a M 33 F 125.2 28.1 T4bN2bM0 Pos Dead DOD Yes NS/P

134^a F 84 F 200 22.0 T4bN0 M0 Neg Dead ? No PS

143^a F 37 NF 147.6 20.1 T4aN0 M0 Neg Dead DOD ? NS/P

146^a F 81 F 560.6 24.7 T4bN0 M0 Neg Alive ADF Yes NS/P

162 F 78 F 242.2 28.0 T1N0 M0 Neg Dead DOD Yes PS

170 M 67 F 439.6 21.6 T4aN0 M0 Neg Dead ADF No NS/P Diabetes,

immune suppressed

184 F 80 F 121 20.5 T4aN0 M0 Neg Alive ADF No NS/P

188 M 55 F 169.6 25.7 T4aN0 M0 Neg Alive ADF No PS

195 M 53 F 181 22.7 T4aN2bM0 Pos Alive ADF No S

199 M 56 F 310.2 22.6 pT4apN0 M Pos Alive ADF No S

200 F 65 NF 335.2 24.5 T1N0 M0 Neg Alive ADF No S

210 M 73 F 305.8 24.9 T4aN0 M0 Neg Alive ADF No PS

211 F 83 F 189.8 23.2 T2N0 M0 Neg Alive ADF No NS/P

215 M 69 F 191 19.8 T4aN3 M0 Pos Dead DOD ? NS/P

Floor of mouth

62^a M 75 NA 153.8 23.9 T4aN2bM0 Pos Dead ? ?

63^a M 62 NA 121 20.5 T1N0 M0 Neg Dead DDF No ?

69^a M 39 NA 132.2 25.7 T2N0 M0 Neg Alive ADF No ?

90^a F 49 NF 356.8 26.8 T2N0 M0 Neg Alive ADF ? PS

96^a F 51 NF 118.4 22.0 T1N0 M0 Neg Dead DOD Yes S

97^a M 68 NF 753.4 23.9 T4aN2 cM0 Pos Dead DOD Progr S

172 M 61 F 250.2 28.1 T4aN0 M0 Neg Alive ADF No S

174 M 36 NF 160 23.1 T2pN2bM0 Pos Alive ADF No S

TA B L E 1 (Continued)

(Continues)

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followed; in brief, pre- coated plates were washed, and samples (4X diluted) were added and incubated for 1 h at room temperature. Thereafter, plates were washed, and 25 µl detection antibody solution was added and incubated 1 h at room temperature. After washing, 150 µl 2X read buffer T was added before analysis in the MSD instrument.

2.4 | Statistical methods

Mann– Whitney U test was used for the comparisons of differences in IL- 1Ra levels between: controls and SCCHN with U- PLEX assay, controls and SCCOT with V- PLEX assay, tumour size, lymph node state

and fasting. For comparison of plasma IL- 1Ra levels in different sub- groups of SCCHN, BMI and smoking status Kruskal– Wallis H test with Dunn's post hoc test was used. Wilcoxon signed- rank test was used for comparing pre- diagnostic cases with paired controls, a mean of the two controls was used. For correlation analysis between IL- 1Ra levels from Olink panel and MSD U- PLEX assay, BMI and time to diagnosis Spearman correlation analysis was performed. Cox's regression model was used to estimate the impact of IL- 1Ra (treated as continuous vari- ables) on patient survival. SPSS version 26 was used for Kaplan– Meier curves and all statistical calculations; p- values < 0.05 were considered statistically significant. Graphs for circulating levels of IL- 1Ra were prepared in GraphPad Prism 8 (GraphPad Prism software).

Sample

IL−1Ra

MSD BMI

TNM (clinical,

7th edition) LNS Status Cause Rec Smoking Other diseases

189 M 73 F 343.8 23.5 pT4apN1 M Pos Dead DOD Yes PS

190 F 68 F 423.4 18.1 T4aN2 cM0 Pos Dead DOD ? PS

202 F 78 F 75.8 17.4 T2N2bM0 Pos Dead DOD Yes S

Tonsil

60^a M 56 NA 412.4 28.7 T2N2 cM0 Pos Alive ADF No ?

113^a M 56 F 102.2 24.8 T2N2bM0 Pos Dead DOD Yes S

117^a F 57 F 159.4 22.2 T2N0 M0 Neg Alive ADF No PS

118^a M 52 F 1737.4 29.8 T2N0 M0 Neg Alive ADF No S

130^a F 49 F 211.4 20.6 T3N2bM0 Pos Alive ADF No ?

142^a M 66 F 340.6 21.4 T4aN2bM0 Neg Dead DOD Yes S

158 M 64 F 118.4 29.0 T2N0 M0 Neg Alive ADF No NS/P

160 M 71 F 77.2 18.0 T2N2bM0 Pos Dead DDF No S

163 F 77 NF 242 24.0 T2N2bM0 Pos Dead DOD Yes NS/P

166 M 56 F 182.2 25.6 T2N2bM0 Pos Alive ADF No NS/P

168 M 58 F 263.2 27.2 T4aN2bM0 Pos Alive ADF No PS

180 M 61 F 188.8 23.8 T4bN2bM0 Pos Alive ADF No S

192 M 64 F 199.4 20.8 T4aN2bM0 Pos Dead DOD ? S

194 M 48 F 113 24.0 T4aN0 M0 Neg Alive ADF No S

214 M 44 F 187.8 25.9 T2N2aM0 Pos Alive ADF No NS/P

217 M 60 F 192.2 23.5 T4aN2bM0 Pos Alive ADF No PS

Base of tongue

106 F 79 F 289.6 20.6 T4N2 cM0 Neg Alive ADF ? S

165 F 51 F 143.4 22.0 T2N2bM0 Pos Alive ADF No NS/P

175 M 59 F 263 31.2 T1N3 M0 Pos Dead DOD Yes PS

176 M 63 F 323.8 27.7 T4aN2 cM0 Pos Alive ADF No PS

181 M 49 F 316 28.3 T2N2aM0 Pos Alive ADF No NS/P

185 M 61 F 168 30.4 T3N0 M0 Neg Alive ADF No S

196 F 63 F 1828 27.0 T4aN0 M0 Neg Alive ADF No NS/P

198 M 68 F 378.2 21.3 T4aN2 cM0 Pos Alive ADF No PS

203 M 66 F 272.8 35.9 T4aN2 cM0 Pos Alive AWD ? PS

205 M 59 F 521.8 31.8 T4aN0 M0 Neg Alive ADF No NS/P

209 F 74 F 273 27.2 T3N0 M1 Neg Alive ADF No S

Abbreviations: ?, Not known; ADF, Alive disease free; AWD, Alive with disease; DAD, Dead of other disease; DDF, Dead disease free; DOD, Dead of disease; F, Fasting; LNS, Lymph node status; NA, No information; NF, Non- fasting; NS/P, non smoker or party smoker; PS, previous smoker; Rec, Recurrence; S, smoker.

aAnalysed previous with Olink (Boldrup et al., 2017).

TA B L E 1 (Continued)

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3 | RESULTS

3.1 | Significant increase in plasma levels of IL- 1Ra in several sub- sites of SCCHN

Forty- seven per cent of the SCCHN cases and 64% of the controls had previously been analysed using Olink's Inflammation I panel.¹⁴ In this study, we used the U- PLEX system to detect IL- 1Ra in more SCCHN and control plasma samples. Comparing the results from the U- PLEX system with the previous Olink panel showed an excellent correlation between the two methods, ρ = 0.952, p < 0.01 (Figure 1).

The levels of IL- 6, IL- 12p70 and TNF- β were below detection range and thus excluded from the study. Compared to controls, levels of circulating IL- 1Ra were increased in patients with SCCOT (p < 0.01), gingival SCC (p < 0.01) and SCC in base of tongue (p < 0.01). A trend towards higher levels was also seen in patients with SCC in floor of mouth and SCC in tonsil, although this was not statistically significant (p = 0.2376 respective 0.2012) (Figure 2).

3.2 | Tumour size and BMI correlate to plasma levels of IL- 1Ra

When analysing IL- 1Ra levels against clinical factors for all SCCHN samples, patients with larger tumours (T3 and T4) showed higher levels of IL- 1Ra than patients with smaller tumours (T1 and T2) (p < 0.01; Table 2). However, the presence of lymph node me- tastasis did not affect IL- 1Ra levels (p = 0.942; Table 2). Patients with higher body mass index (BMI) also had higher levels of IL- 1Ra (p = 0.013; Table 2) (ρ = 0.309; Figure 3A). When dividing patients into different WHO groups according to BMI (<18.5 = underweight,

18.5– 25 = normal weight, 25– 30 = fat and >30 = obese), fat and/

or obese patients showed highest IL- 1Ra levels in all sub- locations except floor of the mouth tumours (Figure 3B).

3.3 | Age, smoking status and fasting do not correlate with levels of IL- 1Ra

No correlation was found between circulating IL- 1Ra and age of patients (p = 0.432) or smoking status (p = 0.101). The effect of fasting on circu- lating IL- 1Ra levels was analysed both in fasting patients (n = 63) com- pared to non- fasting patients (n = 12) and fasting controls from Biobank Norr (n = 32) compared to non- fasting controls (n = 26). No significant difference was found, neither in tumours nor in controls (Table 2).

3.4 | Levels of circulating IL- 1Ra do not influence prognosis in patients with SCCHN

Using univariate Cox regression model, no impact of IL- 1Ra on overall survival or disease- free survival was seen (p = 0.666 and 0.742, respectively). Due to the small sample size and number of events, tumour subsite- specific survival analysis was not performed.

3.5 | Levels of plasma IL- 1Ra are not significantly increased before diagnosis in SCCOT patients

We also evaluated the potential predictive diagnostic value of IL- 1Ra in samples taken before diagnosis of SCCOT compared with age- and sex- matched healthy controls. Plasma levels of IL- 1Ra were similar in people who subsequently developed SCCOT and in matched

F I G U R E 1 Plasma levels of IL- 1Ra measured with two different methods. In 41 SCCHN patients and 18 controls, the levels of IL- 1Ra were measured with both Olink's Inflammation I panel and MSD U- PLEX IL- 1Ra assay, showing a perfect correlation between the two methods, ρ = 0.952, p < 0.01

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controls, p = 0.05 (Figure 4A), without any correlation between lev- els and time to diagnosis, ρ = 0.042 (Figure 4B).

4 | DISCUSSION

Development of an SCCHN tumour is a long process shaped by genetic/epigenetic alterations and interactions between tumour cells and the microenvironment, reviewed in Ref.5. Inflammation affects most aspects of tumour development and progression.²¹ Inflammatory cells and mediators including cytokines, chemokines and enzymes together create an inflammatory microenvironment that can drive cancer development, accounting for the association of cancer with chronic inflammatory environments.²² On the other hand, cancers are subject to immunoediting, where inflammatory immune cells recognise and eliminate tumour cells.^23,24 Tumours therefore evolve mechanisms to avoid immune elimination, including induction of immune checkpoint molecules such as PD- 1/PDL- 1, or production of an anti- inflammatory environment to reduce immune cell activities.²⁵

One of the most abundant cytokines in the tumour microenvironment is IL- 1, which is involved in tumourigenesis, invasion and progression as well as activation of anti- tumour immunity.^15,16 IL- 1 is upstream in a hierarchy of proinflammatory and immunomodu- latory cytokines,²⁶ and previous studies using ELISA have docu- mented secretion of IL- 1 directly by SCCHN cells in culture and in clinical samples.²⁷ Although in our previous study using OLINK

assay; Inflammation I,¹⁴ IL- 1 was only detected at low levels in only one out of 18 controls and four out of 18 tongue cancer patients.

IL- 1Ra inhibits IL- 1 by binding to its receptor and thus acts as an anti- inflammatory cytokine. We found that patients with T3 and T4 tumours had higher levels of IL- 1Ra than patients with smaller tumours (T1 and T2). The increase in IL- 1Ra was seen in all sub- locations of SCCHN and could be a response to an increased inflammatory process in the tumour microenvironment, thereby inhibiting anti- tumour immune responses and allowing continued growth.

However, not only size of the tumour but also size of the patient, measured as BMI, was accompanied by an increase in circulating IL- 1Ra. The easiest explanation for this is the fact that obese patients, defined as having a body mass index (BMI) ≥30 kg/m², also have an increase in adipose tissue (including in the tongue), resulting in an increased inflammation.²⁸ Even if the number of patients is quite limited, it is interesting to note that there were normal weight and overweight (fat) patients in all sub- groups of SCCHN, whereas obese patients were seen only among patients with SCCOT (n = 4), gingival (n = 1) and base of tongue SCC (n = 4), the three sub- groups showing significantly higher levels of IL- 1Ra compared to controls.

According to our data, the role of circulating levels of IL- 1Ra as an early marker of tumour was not evident, even though a trend of increasing levels of circulating IL- 1Ra was seen in pre- diagnostic samples from patients that subsequently developed clinical SCCOT.

F I G U R E 2 Significant increase of plasma level of IL- 1Ra in several sub- sites of SCCHN. Plasma levels of IL- 1Ra were significantly up- regulated in SCCOT (p < 0.01), gingival SCC (p < 0.01) and SCC in base of tongue (p < 0.01). Levels were not significantly increased in patients with SCC in floor of mouth (p = 0.2376) and tonsil (p = 0.2012)

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A major concern analysing markers in plasma and serum is the variation between studies, emphasising the importance of validating and confirming markers published in only one or a few studies.²⁹ A strength with the present study is the excellent concordance between different antibody- based methods, here the ECL kits from MSD and previously the OLINK proximity extension assays.¹⁴ The different MSD assay also had a good concordance (results not shown) although the single V- PLEX assay was slightly more sensitive detecting IL- 1Ra than the multi- U- PLEX assay.

Another aspect to have in mind when analysing plasma for clinical use is the fasting status of the patients. The effect of fasting differs among proteins in the circulation, and in the present study,

IL- 1Ra was measured in fasting and non- fasting states for both tumour patients and controls, showing that fasting state did not affect the levels of circulating IL- 1Ra.

In summary, our results show increased levels of IL- 1Ra in sub- groups of SCCHN analysed. That levels of this cytokine follow size of both the tumour itself and its host seems logic, as both tumour growth and increased body weight are known to cause increased inflammation.³⁰ Importantly, we once again showed that tumours arising in different sub- locations of the head and neck area are diverse in their clinicopathological characteristics, emphasising the value of studies that investigate individual subtypes and the potential for personalised prognostic and predictive therapeutic approaches.

TA B L E 2 Statistics for IL- 1Ra levels against clinical factors

n Mean ± SD Median p

Different MSD assays

U- PLEX Controls 28 148.9± 53.8 132.8 <0.001^a

SCCHN 87 302.0± 280.7 237

V- PLEX Controls 28 236.5± 111.3 219.2 0.001^a

SCCOT 23 434.8± 236.8 408.9

SCCHN sub- sites

U- PLEX Tongue 27 262.5 ± 180.6 237 0.009^b

Floor of mouth 11 262.6 ± 199.4 160 0.2376^b

Gingiva 22 308.6 ± 183.5 230.9 <0.001^b

Tonsils 16 295.5 ± 394.1 190.5 0.2012^b

Base of tongue 11 434.3 ± 473.0 289.6 <0.001^b

Smoking status

U- PLEX Smoker 30 319.8 ± 331.3 243.2 0.101^c

Previous smoker 19 303.6 ± 123.3 284.4

Non- smoker or party smoker 30 301.4 ± 329.6 188.8

Tumour size

U- PLEX T1 and T2 43 261.9± 277.9 187.8 0.005^a

T3 and T4 44 341.1 ± 280.9 276.7

Lymph node state

U- PLEX Lymph node positive 36 265.2 ± 154.8 226.7 0.942^a

Lymph node negative 51 327.9 ± 342.1 237

BMI

U- PLEX Under (<18.5) 3 192.1 ± 200.3 77.2 0.013^C

Normal (18.5– 25) 43 235.3 ± 128.8 199.4

Fat (25– 30) 29 361.2 ± 424.9 250.2

Obese (> 30) 12 425.2 ± 198.0 430.1

Fasting status

V- PLEX Fasting controls 32 301.1 ± 189.4 250.3 0.285^a

Non- fasting controls 26 229.9 ± 98.5 219.2

U- PLEX Fasting tumours 63 311.1± 297.4 237 0.644^a

Non- fasting tumours 12 315.7± 209.8 261.2

aMann– Whitney U test.

bKruskal– Wallis test with Dunn's correction.

cKruskal– Wallis test.

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ACKNOWLEDGEMENTS

We thank the Biobank Research Unit at Umeå University, Västerbotten Intervention Programme, the Northern Sweden MONICA study and the County Council of Västerbotten for pro- viding data and samples and acknowledge the contribution from Biobank Sweden.

CONFLIC T OF INTEREST

The authors declare no conflict of interest.

AUTHOR CONTRIBUTION

Linda Boldrup: Conceptualization; Data curation; Formal analysis;

Investigation; Methodology; Project administration; Writing- original F I G U R E 3 Correlation of plasma levels of IL- 1Ra with BMI. (A) Levels of IL- 1Ra correlated moderately with BMI, ρ = 0.309; p < 0.01. (B) Mean plasma levels of IL- 1Ra for underweight, normal, fat and obese patients for the different sub- locations; tongue (black bars), floor of mouth (grey bars), gingiva (blue bars), tonsil (green bars) and base of tongue (red bars)

F I G U R E 4 Levels of plasma IL- 1Ra in pre- diagnostic SCCOT patients. (A) Samples taken before SCCOT diagnosis had similar detectable levels of plasma IL- 1Ra compared to age- and sex- matched healthy controls. (B) No correlation between levels and time to diagnosis was seen

(10)

draft; Writing- review & editing. Philip Coates: Conceptualization;

Methodology; Supervision; Writing- original draft; Writing- review &

editing. Xiaolian Gu: Conceptualization; Formal analysis; Methodology;

Writing- review & editing. Lixiao Wang: Formal analysis; Software;

Writing- review & editing. Robin Fahraeus: Conceptualization;

Supervision; Writing- review & editing. Torben Wilms: Data curation;

Writing- review & editing. Nicola Sgaramella: Data curation; Writing- review & editing. Karin Nylander: Conceptualization; Funding acqui- sition; Investigation; Writing- original draft; Writing- review & editing.

ETHIC S STATEMENT

The study was reviewed and approved by the Regional Ethics Review Board, Umeå, Sweden (Dnr 08– 003 M), and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.

PEER RE VIEW

The peer review history for this article is available at https://publo ns.com/publo n/10.1111/jop.13187.

DATA AVAIL ABILIT Y STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ORCID

Linda Boldrup https://orcid.org/0000-0002-1399-592X Philip Coates https://orcid.org/0000-0003-1518-6306 Xiaolian Gu https://orcid.org/0000-0002-6574-3628 Karin Nylander https://orcid.org/0000-0002-4831-4100

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How to cite this article: Boldrup L, Coates P, Gu X, et al. Low potential of circulating interleukin 1 receptor antagonist as a prediction marker for squamous cell carcinoma of the head and neck. J Oral Pathol Med. 2021;00:1–10. https://doi.

org/10.1111/jop.13187