The Disease-specific Survival From Plasma Il-1 and Il-6 Families Cytokines With Associated Soluble Receptors at Diagnosis in Patients With Head and Neck Squamous Cell Carcinoma Importance of Tnm Stage and Smoking History

doi:10.21203/rs.3.rs-3639927/v1

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The Disease-specific Survival From Plasma Il-1 and Il-6 Families Cytokines With Associated Soluble Receptors at Diagnosis in Patients With Head and Neck Squamous Cell Carcinoma Importance of Tnm Stage and Smoking History

https://doi.org/10.21203/rs.3.rs-3639927/v1

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The acute-phase cascade (APC) has been correlated with outcome in many different cancers, including head and neck squamous cell carcinoma (HNSCC). A main driver of the APC are the cytokines in the IL-6, IL-1 and TNF cytokine families.

We have studied the systemic acute phase soluble cytokine/cytokine receptor profiles in serum (s) for patients with HNSCC. Systemic levels the of Interleukin-6 family cytokines/s-receptors (IL-6, IL-27, IL-31, OSM, CNTF, gp130,IL-6Rα), IL-1 family members (IL-1RA, IL-33Rα/ST2) and TNF-α at diagnosis from 144 HNSCC patients. Five-year disease-specific survival (DSS) was then studied.

Increased levels of CRP (p < .001), IL-6 (p < .001), IL-31 (p = .044), IL-1RA (p = .004), IL-6Rα (p = .022) and gp130 (p = .007) were predictors among human papilloma virus (HPV) infection negative (−) patients. Only the IL-6 levels predicted DSS among HPV positive (+) patients. High TNF-α levels showed a trend toward predicting DSS (p = .078) when considering all patients and adjusted for HPV. Adjusting for TNM stage and smoking history, IL-6Rα, gp130, and IL-33Rα/ST2 retained DSS survival prediction. Factor analysis suggests two underlying factors for the present results. The first factor contains the cytokines and the second contains the soluble cytokine receptors.

To conclude: High serum levels of IL-6, IL-31 and IL-1RA as well as the soluble receptors IL-6Rα and gp130, and to some extent TNF-α at the time of diagnosis predicted lowered DSS. Clinically, IL-6 levels above 2.5 pg/ml yielded more than 70% specificity and sensitivity for DSS. The cytokine predictions were associated with TNM stage and smoking history, whereas IL-6Rα, gp130 and IL-33Rα/ST2 levels predicted DSS more uniquely.

Head and neck squamous cell carcinoma

HPV

IL-6

IL-31

gp130

In 2020, over 900,000 new cases of head and neck cancer (HNC) (lip, oral cavity, pharynx, larynx or salivary glands) were reported worldwide. Approximately 50% of the newly diagnosed HNC patients are alive at 5 years [1]. The pathogenesis and prognosis of HN squamous cell carcinoma (HNSCC) are related to mucosal inflammation caused by local irritants such as tobacco, alcohol and betel nuts [2], or chronic viral infections, predominately human papilloma virus (HPV) and to a lesser degree EBV. [3]. Type and localization of inflammation clearly influence clinical and biological characteristic of HNSCC. This is most evident in the case of HPV positive (HPV(+)) tumors, which primarily manifest in the oropharynx (tonsils and base of tongue), exhibit a characteristic mutational signature [4, 5], and demonstrate superior survival rates compared to HPV negative (HPV(−)) tumors [6]. Several previous studies have shown that the host inflammatory response to the tumor has significant positive prognostic value and might guide the selection of patients benefiting from immunotherapy.

The acute-phase cascade (APC) includes proteins with a significant change in response to infection or tissue injury [7, 8]. The APC response is seen at the cellular, intercellular and systemic level. Prominent members of the APC are C-reactive protein (CRP) [9], the cytokines in the interleukin (IL)-6 family, those in the IL-1 family [7, 10] and TNF families [11], in the given order. Unlike other cytokines, which primarily exert local effects, these APC role players differ in that they also exhibit hormone-like effects on distant organ systems [12, 13]. This is most prominent for IL-6, where systemic inflammatory markers such as CRP, leukocytosis and thrombocytosis are closely correlated with its activity. Systemic inflammatory response, including the systemic levels of acute-phase cytokines, has an independent prognostic impact in several cancers, including HNSCC [14–29].

IL-6 is secreted in response to IL-1 family and TNF family cytokines. IL-6 is a pleiotropic hormone that plays an important in tissue homeostasis in the bone marrow, liver cells and mucosa [30–32], but it is also a driver of acute and chronic inflammation [33]. The pleiotropic effects of IL-6 is explained through the various mode of IL6 signaling; the classical IL-6 signaling, IL-6 trans-signaling and IL-6 cluster signaling [13]. In classic signaling IL6 binds directly to the membrane-bound IL6Rα/gp130 receptor complex and does only occur on cells expressing these receptor, i.e. hepatocytes, neutrophils, monocytes, macrophages and some lymphocytes. [12]. In IL-6 trans-signaling IL-6 binds solubleIL6Rα and then binds to membrane-bound gp130 [34] [34, 35]. IL-6 trans-signaling is blocked by a soluble dimeric version of gp130 that specifically inhibits the pro-inflammatory effects of IL-6 [36]. Furthermore, the IL6 cytokine family [12, 37] includes IL-11, IL-27, IL-31, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), and cardiotrophin-like cytokine factor 1 (CLC) [38]. These cytokines share gp130 as their signal-transductor, but do also mediate cytokine-specific effects through a ligand-specific receptor [38]. Mostly, the association of these cytokines with HNSCC is not known.

We have found it pertinent to study the interaction between cytokine prediction and underlying clinically used predictors of HNSCC survival. Although previous studies have demonstrated a significant correlation with IL-6 and long-term survival in HNSCC, a common challenge with simultaneously analyzing effects of different cytokines and clinical outcome is statistical interaction due to covariation of the different cytokines. To overcome this challenge in the current study, we analyzed the association of the IL-6 family cytokines, IL-1 family cytokines and TNF-α cytokines in relation to clinical characteristics and clinical outcome using factor analysis.

Patients included in the study

The study included 144 consecutive patients newly diagnosed with HNSCC admitted to Department of Otolaryngology and Head and Neck Surgery, Haukeland University Hospital (Bergen, Norway) from 2013 until 2018. Only patients treated with curative intent were included. Patients with autoimmune disease or on systemic corticosteroid therapy were excluded from the study. Patient characteristics are given in Table 1. The survival by the end of July 2023 was determined from the Norwegian Population Registry; at this time point 105 patients were alive, 29 were dead due to the index HNSCC as determined from hospital records.

Laboratory analyses

The biopsy analyses for human papilloma virus (HPV) infection have been described in detail previously [39, 40].

CRP serum levels was analyzed using an immuoturbidimetic assay provided by Roche (Basel, Switzerland) collected from standard laboratory analyses.

Peripheral venous blood samples were collected during initial hospital admission for any HNSCC treatment. Samples were stored at room temperature for less than two hours before they underwent centrifugation and collection of plasma prepared by gradient centrifugation with Lymphoprep®, aliquoted and stored at -80^o C until analysis.

The samples were thawed and centrifuged at 16,000 x g for 4 minutes before analysis. IL-33Rα and IL-1RA levels were analyzed by using Luminex analyses (R&D Systems Europe Ltd, Abingdon, UK). IL-6 levels were determined by a high-sensitivity IL-6 Luminex kit (Invitrogen, Biosource, Carlsbad, CA, USA) and IL-1β by a High-Sensitivity ELISA kit (R&D Systems). IL-1β, sgp130, IL-6Rα, IL-27, IL-31, OSM, CNTF and TNF-α were determined using the Human Premixed Multi-Analyte Kit for Luminex technology (R&D Systems), whereas CNTF was analyzed with a kit from EMD Millipore Corporation, Billerica, MA, USA. All analyses were performed strictly according to the manufacturer’s instructions using a Luminex® 100™ (Luminex Corporation, Austin, TX, USA). All results are presented as the mean level of duplicate determinations.

One of the patient samples was included in all assays to evaluate inter-platelet variation. No substantial differences were detected between the different assays. The variation between duplicates was generally < 10% of the mean concentration. Cytokine levels showed no significant correlations with sample storage time.

Tobacco history scores

From the hospital records information about smoking history were obtained and scored as follows as pack-years (defined as 20 cigarettes daily for one year) cumulative: 0 = Never smoked. 1 = Less than 10 pack-years. 2 = Probably less than 10 pack-years. 3 = Probably more than 10 pack-years. 4 = More than 10 pack-years.

Statistical analyses

The IBM® SPSS® Statistics software, version 29.0 (IBM Corp., Armonk, NY, USA) was employed. Comparison of categorized descriptive data was performed by cross-tables and exact Chi-square test. Mann-Whitney U test was used for comparison of different groups and Pearson correlations was used for correlation analyses. Receiver operating characteristic (ROC) analysis was employed to estimate sensitivity and specificity by cut-off values in predicting IL-6 levels regarding disease-specific survival (DSS). Kaplan-Meier analyses were used to estimate survival curves and the Log-Rank test to compare survival between groups. Factor analysis was used to determine internal relationships between relevant variables, using Varimax rotation and a criterion that Eigenvalues should exceed one. Factor scores were generated by the Regression analysis. Multivariate Cox proportional hazard models were used to analyze the effect of introducing covariates to cytokine/soluble cytokine receptor survival predictions. The results are reported as relative risk (RR) with 95% confidence intervals. The cytokines/receptors were either dichotomized by the median (CRP, IL-6) or quartiles (CRP, IL-6, IL-1RA, IL-1, TNF-α, s-IL-6Rα, s-gp130, ST2/IL33Rα). The age of the patients at diagnosis was incorporated into the analyses in the form of quartiles. TNM stage and smoking history were introduced as originally scored. All analysis were performed as two-tailed analyses. A p-value < 0.05 was regarded as statistically significant.

Patients included in the study

Consecutive patients (N=144) were included in the study (Table 1). The median age at diagnosis was 63 years, ranging from 37 to 82 years. 73% (N = 105) of patients were male. 57 patients (40 %) of all tumors were HPV(+) tumors with 48 patients (84%) being positive for HPV-16 followed by HPV-18 (3 patients), HPV-33 (3 patients), HPV-35, HPV-58 and HPV-73 (one patient each). Forty-five patients were HPV(─). The remaining 43 patients were not subjected to testing, but were labeled HPV negative as they had primary tumors outside the oropharynx area [41].

Clinical parameters

Differences in clinical parameters for HPV(─) and HPV(+) were as expected; patients in the HPV(+) group were predominantly younger (62.9±10.3 years vs. 57.7±9.5 years; p <.01) with a higher proportion of males (HPV(+) 46/11 (male/female) vs. HPV(-) 59/28 ; p <.05). While no difference in T stage was observed, HPV(+) patients had more extensive lymph node pathology than the HPV(─) patients (p <.001) (Table 1). The HPV(─) had a more frequent smoking history than the HPV(+) patients (p <.001). Advanced T stage was associated with inferior prognosis in both HPV(+) (p =.02) and HPV(─) (p <.001) patients. N stage influenced survival negatively in HPV(─)patients only (p <.001). The level of smoking carried a strong trend in relation to the 5-year DSS among HPV(─) patients (p =.064); all as reported in Table 1.

Correlation between cytokines/ soluble receptors, age, TNM stage, smoking history and CRP by HPV status

Regarding the HPV(−) patients the cytokine values are shown correlated to clinical variables.

Significant levels of correlations were only observed in HPV(─) patients and is presented in Supplementary table 1. A significant but weak correlation was observed between IL-6 and age (r = 0.32; p < .01), T stage (r = 0.45; p < .01), N stage (r = 0.29; p < .01) and smoking history (r = 0.32; p < .01), while IL-31 levels correlated with N stage (r = 0.27; p < .05). IL-1RA levels correlated with N stage (r = 0.30; p < .01) and tobacco history (r = 0.24; p < .05). Lastly, IL-33Ra/ST2 correlated with tobacco history (r=.24; p < .05). CRP levels correlated to T-stage (r=0.31, (p<.001), L-6 levels (r= 0.43; p<.001) and s-IL-33-Ra/ST2 (r=0.31; p<.01).

CRP is associated with prognosis in HNSCC

By analyzing survival by Kaplan-Meier estimates by stratifying patients by CRP levels (quartiles), inferior survival was observed for all patients (p < .001) (data not shown) and for the HPV(-) subgroup (p < .001; Fig. 1a+b). A significant difference in survival was also observed when using the median value of IL-6 to dichotomize the patients subsets (p < .001) (data not shown), with all patients included (p < .001), as well as stratified by HPV, the HPV(─) (p < .001) showed survival prediction with a trend towards survival prediction regarding the HPV(+) patients (p = .08; Fig.1c+d).

Individual acute phase cytokines are associated with prognosis in HNSCC

By analyzing survival by Kaplan-Meier estimates by stratifying patients by IL-6 levels (quartiles), inferior survival was observed for all patients(p < .001) (data not shown) and for the HPV(+) and HPV(─) subgroups (p < .001; Fig. 2a+b). A significant difference in survival was also observed when using the median value of IL-6 to dichotomize the patients subsets (p < .001) (data not shown), as well as irrespective of HPV status (HPV(─) p < .001 and HPV(+) (p = .038; Fig. 2c+d). IL-31 level did also correlate with 5-year DSS in HPV(─) patients (p = .044; Fig. 3a). When studied by HPV status, we also found that the level of IL-6Ra predicted survival among HPV(─) patients (p = .022; Fig. 3c). Furthermore, when examining gp130, high levels were associated with worse 5-year DSS among HPV(─) patients (p = 0.007; Fig. 3d). IL-1RA also predicted 5- year DSS among HPV(─) patients (p = .004; Fig.3b). When TNF-a levels were studied a combined analysis of results sorted according to HPV status showed a trend towards DSS prediction (p=.079; Fig. 4). All predicting results showed worse prognosis with increasing values.

Factor analysis of cytokine/soluble receptor levels and subsequent survival analysis of the calculated two factors generated from these analyses.

To better describe the correlation structure amongst the cytokines and receptors that were associated with survival (IL-6, IL-31, IL-1RA, gp130, IL-6Ra), a factor analysis was performed. Two factors with Eigenvalue above one were generated (Supplementary table 2). Kaplan-Meier analyses were subsequently performed analyzing the generated factors binomially divided by median value. Factor I showed significant 5-year DSS prediction for HPV(─) patients (p = .019; Fig. 5a+b) and factor II predicted 5- year DSS among the HPV(─) patients (p = .021; Fig 5c+d).

Prognostic impact of individual cytokine, cytokine receptors and factors from factor analysis by Cox regression analysis

Multivariate Cox regression survival analyses was performed including age, gender, T and N stage, smoking history and one of the indicated cytokine/soluble receptors or the factor analysis-generated factor values. Analyses were performed for HPV(─) and HPV(+) patients separately.

Concerning IL-6, IL-31 and IL-1RA, the above mentioned covariates accounted for the survival predictions (Table 2). If the soluble cytokine receptors were introduced to this analysis the IL-6Ra (RR = 1.44; CI = 1.0-2.33; p=.041), gp130 (RR = 1.73; CI = 1.2-2.5; p=.004) and IL-33Ra (RR = 1.58; CI = 1.1-2.3; p=.019) all still predicted significant prognostic value (Table 2). When the factors derived from factor analysis were included in this analysis, Factor I (RR = 2.91; 1.03=8.28; p=.045) showed statistical survival prediction, whereas Factor II did not (Table 3). Survival curves were also calculated employing Cox regression analysis with factor I and II studied separately with age at diagnosis, TN stage and smoking history included to these analyses (Fig. 5b+d). Only factor I generated significant prediction (p=.009) from these analyses.

ROC analysis

Receiver operating characteristic (ROC) analysis was employed to estimate a cut-off level of IL-6 which can be used clinically to estimate DSS. The ROC curves by HPV are shown in Figure 6. If HPV(─), the area under the curve was 0.77with 95% confidence interval (CI) 0.661-0.89; (p<.001). HPV(+) patients area under the curve being 0.821 (CI 0.69 – 0.951) (p=.063). With both HPV(−) and HPV(+) patients a cut off at IL-6 = 2.5 pg/ml provided more than 70% sensitivity and 70% specificity.

Receiver operating characteristic (ROC) analysis was employed to estimate a cut-off level of IL-6 which can be used clinically to estimate DSS. The ROC curves by HPV are shown in Fig. 6. With HPV(−) patients, the area under the curve (AUC) was 0.77(95% CI 0.661-0.89; p<.001). Employing HPV(+) the patients, AUC was 0.821 (95% CI 0.69 – 0.951, p=.063. With both HPV(−) and HPV(+) HNSCC, a cut-off value of IL-6 = 2.5 pg/ml provided more than 70% sensitivity and specificity.

In the present study, we investigated CRP with the underlying cascade of acute-phase cytokine levels of IL-1, IL-6 family and TNF-α cytokines with associated soluble receptors at time of diagnosis in relation to 5-year DSS in a cohort of 144 HNSCC patients. High levels of CRP predicted impaired prognosis. Regarding the IL-6 family, high values of IL-6 and IL-31, along with the associated receptors s-IL-6Rα and s-gp130, predicted worse DSS among HPV(─) patients. From the IL-1 family molecules, IL-1RA, but not IL-33Rα/ST2 also predicted DSS with adjustment by tumor HPV status. The IL-6/IL-31/IL-1RA predictions were primarily associated to tumor extent and smoking history. With tumor extent and smoking history adjustment the soluble receptors IL-6Rα, gp130 and IL-33Rα/ST2 still predicted survival.

High CRP and IL-6 levels [15, 22–29] are associated with an adverse prognosis in cancer, HNSCC included. We have currently confirmed this together with a dose-response relationship. IL-6 is a member of the IL-6 cytokine family; i.e. several cytokines with common receptor characteristics [7, 35]. IL-31, a member of the IL-6 family, also predicted 5-year DSS. The other investigated IL-6 family cytokines i.e. IL-27, OSM and CNTF, did not generally predict 5- year DSS. As IL-6Rα predicted survival among the HPV(─) tumor patients activation of both the classical and trans intracellular pathway seem to be associated with an increased HNSCC-caused mortality. On the other hand, gp130 is regarded as an inhibitory system [42], but also showed that increased concentrations were associated with decreased 5-year DSS.

Other cytokines beside IL-6 family members, are also viewed as secondary drivers of the APC [7], and for this reason we also included the IL-1 family mediators IL-1β, IL-1RA, IL-33Rα and TNF-α. IL-33 was not included because it is expressed by the cancer cells and we therefore regard local release as more important than distant release [43, 44]. Unfortunately, IL-1β was not measurable from plasma. We found that high IL-1RA predicted decreased 5-year DSS. We have also determined a trend towards survival prediction to the worse by increased TNF-α. Thus, we have shown that the pro-inflammatory cytokine cascade from CRP to TNF-α was associated with HNSCC prognosis.

Generally, we have demonstrated that examples of both stimulating and inhibiting factors of the inflammatory system, if elevated at diagnosis, predict worse prognosis. This aligns with the idea that the inflammatory system exhibits multifaceted interactions with HNSCC tumors, and it is probably the net effect that matters. To provide a clearer overview, we performed factor analyses, resulting in two factors, both of which predicted survival by primary Kaplan-Meier analyses. This suggest that two fundamentally different factors contribute to these predictions.

Levels of these inflammatory markers can also be influenced by inflamm-aging, i.e. a smoldering pro-inflammatory phenotype that can be a part of the aging process [45]. Therefore, we adjusted for the age of the patients. This adjustment did not alter the survival prediction. Disease extent, as measured by the TN stage, predict HNSCC survival [46]. Smoking is a part of HNSCC biology, as well as an inflammatory driver [47] and tied to worse survival [48]. We have therefore studied smoking level and TN stage as covariates in Cox regression analyses of the cytokine survival predictions. The 5-year DSS cytokine predictions were mostly statistically secondary to a combination of TNM stage and smoking history. On the other hand, higher levels of the studied soluble IL receptors also predicted survival even after adjusting for age, gender, TN stage and smoking history. These present findings furthermore underline the importance of trans IL-6R caused activation as an important part of HNSCC tumor biology.

The present study has included cancers with fundamentally different causes; i.e. tobacco and smoking versus HPV [6]. These diseases have many common properties, but differ fundamentally as to molecular biology [5] and prognosis [49]. The association between inflammation and HNSCC is closer regarding HPV(─) than HPV(+) tumor patients. Due to the excellent prognosis of HPV(+) patients, however, this study has a low sensitivity of investigations based on survival among such patients. This study supports that these phenotypic inflammation characteristics reflect different underlying drivers, as similarly observed in other comparable phenomena [7].

The present observations show that the HNSCC cytokine/cytokine receptor levels likely reflect separate aspects of the cancer-associated systemic acute-phase cytokine response with one dimension associated with IL-6, IL-31 and IL-1RA, whereas another dimension is associated to IL-6Rα, gp130 levels and IL-33Rα/ST2 levels. A potential explanation may be that malignant tumors have a higher overall cellular turnover compared to their more benign counterparts [50] with consequently more receptor shedding. Increased number of immune cells in to HNSCC blood have also been shown to signal worse DSS [51], and increased number of soluble cytokine receptors could be related to these findings.

IL-1RA is an acute-phase marker and high levels have previously been associated with aggressive disease and/or unfavorable prognosis in various malignancies, including T-cell lymphoma [52], sarcoma [53], colorectal cancer [54, 55] and thyroid cancer [56]. Our studies are the first, to our knowledge, suggesting that this is also true in HNSCC.

We have included only patients primarily treated with curative intent. Including newly diagnosed palliative patients would have moved especially the HPV(─) patient group to have included additional patients with substantial inflammation present at diagnosis. Our results thus apply more to the HNSCC disease before end-stage of the disease.

The DSS risk is tied to the IL-6 values dose-dependent. We have therefore studied the actual IL-6 values generated by ROC analysis. A cut-off at 2.5 pg/ml provide both and sensitivity and specificity of more than 70% of risk to succumb to disease-specific death. Such information could be used to personalize the treatment, particularly to maximize treatment for clinically limited disease cases. In addition, an individualized analysis, excluding patients with other explanatory causes for high IL-6 values, could improve this prognostic factor.

Another question is that only one blood sample has been drawn from each patient. The half-life of the studied cytokines in blood is unknown, and the blood cytokine concentration may vary substantially over time. However, we have previously conducted studies, with renal carcinoma as experimental system, that have demonstrated the cytokines levels in blood to be surprisingly stable [57] making this methodical problem less.

The fact that the immune system seems to be important for patient prognosis encourages further explorations of immune-modulating treatments for HNSCC patients [58]. The present results suggest targeting the general inflammatory dimension of the HNSCC disease. Additional research into the interactions between carcinomas and the immune system is needed.

To conclude, increased IL-6, IL-31, IL-1RA, IL-6Rα and gp130 blood levels predicted worse HNSCC 5-year DSS. We suggest that the cytokines reflect one HNSCC inflammatory dimension, whereas soluble IL-33Rα/ST2, IL-1RA, gp130 and IL-6Rα represent separate HNSCC inflammatory dimension. The soluble cytokine receptors we have studied in particular warrant further investigation in HNSCC patients. The strong association between IL-6 values and DSS invites clinical use of this relation.

ACKNOWLEDGEMENT

The technical assistance of Kristin Paulsen Rye and Gry Hilde Nilsen is gratefully acknowledged.

Funding

The study received financial support from the Norwegian Cancer Society (grant 182609). This work is supported by the Norwegian Central Health West authorities with grant number F-12125.

Competing interests

All authors declare that they have no financial or non-financial interests to disclose.

Author contribution:

Dr. H.H. Aarstad constructed, analyzed, quality-assured the first version of the data base, co-analyzed the data, and wrote first version of the manuscript.

Dr. S.E. Moe updated the data base and performed data analysis.

Dr. S. Lybak quality-assured the data base.

Dr. Ø. Bruserud had the basic responsibility for laboratory quality including correctness of the results, and participated in conceptualization of the work.

Dr. T.H.A. Tvedt was responsible for writing and conceptualization of the work.

Dr. H.J. Aarstad had the original idea to the work and responsibility for the data base, statistics, and writing of the paper.

All authors took part in paper writing and have given their consent to publication.

Ethics approval:

The study was approved by the Norwegian regional ethics committee (REK VEST 2011-125) and performed in accordance with the Declaration of Helsinki. All samples were collected after written informed consent.

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STATEMENTS & DECLARATONS

Table I. Clinical patient characteristics at diagnosis by tumor HPV status

Variable		Distribution		HPV(-) vs. HPV(+) distribution	Prediction of 5-Year DSS
		HPV(-)	HPV(+)		HPV(-)	HPV(+)
Age at diagn.	Years Mean ± SD	62.9±10.3	57.7±9.5	p<.01	n.s.	p=.019
Gender				p<.05	n.s	n.s.
	Males	59	46
	Females	28	11
T stage				n.s.	p<.001	p=.02
	0	3	4
	1	29	13
	2	34	29
	3	8	5
	4	13	6
N stage				p<.001	p<.001	n.s.
	0	57	11
	1	9	13
	2	20	31
	3	1	2
Smoking				p<.001	p=.064	n.s.
	0	21	24
	1	1	7
	2	8	6
	3	5	4
	4	50	16
Site (ICD-10)				n.a.
	Oropharynx	17	54
	Oral cavity	61	1
	Others	9	2

Total patients included		87	57
DSS : Disease specific survival analysis by Kaplan-Meier analysis with log rank comparison between groups
n.s.: not significant
n.a.: not applicable
TNM stage according to 7th TNM edition of classification of malignant tumors by the International Union Against Cancer
All patients were M0 at diagnosis
One pack-year defined as 20 cigarettes per day for 365 days

Table 2 Cox multivariate regression analyses studying 5 year DSS with gender, age, TN stage, tobacco-smoking history as adjusting variables and each indicated cytokine/soluble cytokine receptor in separate analyses for both HPV(-) and HPV(+) status. With the exception of sgp130, only results from cytokine/receptors included to analyses are shown.

	HPV-status		p-value	RR (95% CI)
Block + gp130	HPV(─)	Gender	.205	0.46 (0.1-1.5)
		Age	.273	0.97 (0.9-1.0)
		T stage	<.001	2.34 (1.5-3.7)
		N stage	<.001	4.13 (2.2-7.8)
		Tobacco	.097	1.39 (0.9-2.1)
		gp130	.004	1.73 (1.2-2.5)

Block + gp130	HPV(+)	Gender	.900	7.41
		Age	.536	1.84
		T stage	.451	334
		N stage	.774	89
		Tobacco	.405	94
		gp130	.926	0.43

Block results not shown + indicated cytokine/cytokine receptor	Block + IL-6Ra
	HPV(─)	IL-6Ra	.041	1.44 (1.0-2.1)
	HPV(+)	IL-6Ra	.936	n.s.

	Block + IL-33Ra/ST2
	HPV(─)	IL-33Ra	.019	1.58 (1.1-2.3)
	HPV(+)	IL-33Ra	.696	n.s.

	Block + IL-6
	HPV(─)	IL-6	.476	n.s.
	HPV(+)	IL-6	.835	n.s.
All cytokine/cytokine receptor values were analyzed binomially based on quartiles

Table 3 Cox multivariate regression 5-year DSS analysis including gender, age, TN stage, tobacco smoking history of patients as well as the two estimated factor analysis generated factors reported in suppl. table 2.

Covariates	Sig.	HR (95% CI)
T stage	<.001	2.63 (1.6-4.2)
N stage	<.001	4.06 (2.1-7.8)
HPV	.020	0.12 (0.05-0.8)
Tobacco	.048	1.48 (1.0-8.28
Gender	.359	0.60 (0.2-1.83)
Age	.791	.943 (0.96-1.08)
Factor 1 (bin)	.045	2.91 (1.03-8.28)
Factor 2 (bin)	.363	1.78 (0.52-6.1)

No competing interests reported.

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The Disease-specific Survival From Plasma Il-1 and Il-6 Families Cytokines With Associated Soluble Receptors at Diagnosis in Patients With Head and Neck Squamous Cell Carcinoma Importance of Tnm Stage and Smoking History

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Tobacco history scores

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