The role of salivary cytokine biomarkers in tongue cancer invasion and mortality

Summary

Squamous cell carcinoma of the tongue (TSCC) has one of the poorest prognoses of head and neck cancers. This study aims to improve early detection of the disease by identifying salivary biomarkers that can identify a spectrum of patients progressing from high-risk to TSCC. We also examine the mortality of exophytic and endophytic TSCC, expecting the elevated cytokine levels in endophytic patients to be associated with a shorter survival. Saliva was collected from patients with TSCC and controls and cytokine protein levels were measured. Specimens were collected from the Los Angeles County (LAC) + University of Southern California (USC) and USC University Hospital clinics. A convenience sample of patients with TSCC was divided into endophytic (n = 10) and exophytic (n = 8) cancer by physician diagnosis. Controls were divided into 4 groups of 14 based on their high-risk smoking and drinking behaviors. Main outcome measures: The levels of IL-1α, IL-6, IL-8, VEGF-a and TNF-α in saliva were measured using quantitative ELISA and compared using two-way ANOVA. All five cytokines were elevated in the endophytic TSCC group compared to other groups, which correlated with the decreased survival rate (10.4 months) in this group compared to exophytic TSCC (24 months). IL-1α, IL-6, TNF-α and VEGF were also elevated in the exophytic TSCC group compared to smoking–drinking controls. Salivary levels of IL-1α, IL-6, IL-8, VEGF-a and TNF-α can identify the progression of TSCC from high-risk to neoplasm, serving as potential biomarkers for cancer screening and early detection. The correlation with survival implies a prognostic benefit and could serve as a tool for management decisions and future treatment targets.

Introduction

Despite advances in cancer detection and treatment, the prognosis of squamous cell carcinoma of the tongue (TSCC) has not improved in the last few decades and remains one of the most common and fatal head and neck cancers worldwide.¹ Studies have found that the tongue is the most common site for squamous cell carcinoma of the oral cavity and oropharynx (OSCC), comprising around 41%.³ Of particular concern is the rising incidence in the population and the significant increase of TSCC in patients under 40 years of age.2, 4

TSCC has been found to have a poorer prognosis when compared to similar stage lesions in other locations.¹ Studies have shown that a T2 TSCC lesion has a worse prognosis than T3/T4 oropharyngeal tumors.¹ These results are attributed to the higher recurrence rate, poorer locoregional control, and early nodal metastasis.5, 6, 7, 8 Metastasis has been found to be the single most important predictor of prognosis and is unfortunately present on diagnosis 40% of the time.⁹ This is partially attributable to the lack of screening mechanisms for TSCC and therefore the increased frequency of cases that present at advanced stage.¹⁰

These unfortunate aspects of TSCC point to early detection as the key for improving outcomes, especially for high risk groups that smoke tobacco and drink alcohol.¹¹ One method for early detection is biomarker technology, based on the research indicating that mutations present in primary tumors can also be found in the fluids that drain the affected organ, often before the cancer phenotype is expressed.¹² Previous research has identified both nucleic acid and protein biomarkers that are elevated in the serum of patients with OSCC including proinflammatory cytokines interleukins 1, 6 and 8 (IL-1, IL-6 and IL-8).13, 14, 15 In a comparison of up-regulated genes in OSCC, Ye et al. found a roughly 5.8-fold increase in IL-8 genomic expression and 2.5-fold increase in TNF-α expression.¹⁴ On a proteomic level, IL-6 has been shown to have up to an 80-fold increase in serum of OSCC patients.¹³ Chen et al. also found significantly elevated protein levels of IL-6, IL-8 and vascular endothelial growth factor (VEGF) to be secreted by OSCC tumor cell cultures, indicating the elevated cytokine levels are not merely a stromal response to cancer presence. They supported the potential for these cytokines as OSCC biomarkers by finding these levels to be elevated above that of patients with benign squamous papilloma.¹⁵

These cytokines have also been found to be elevated in the saliva of OSCC patients.13, 16, 17 In our collaboration with St John et al., we identified significantly elevated levels of IL-8 protein and mRNA in the saliva of OSCC patients as well as increased IL-6 in the serum of the same group.¹³ Based on our previous evidence, we chose the same cytokine set to further examine their role as biomarkers in screening and diagnosis. Saliva is non-invasive, easily and efficiently accessed, and in direct contact with the oral mucosa and cancerous lesions. It therefore has the potential for widespread screening and detection of early lesions. If levels of cytokines increase with disease progression, salivary biomarkers also have the potential for prognostic determination or recurrence detection. It is possible that a more invasive cancer will have higher detectable cytokine levels. An ideal example to investigate is endophytic TSCC, which has an ulcerative or invasive growth pattern compared to fungating exophytic TSCC. Several groups have described patterns of growth in TSCC based on clinical exam as well as histopathological features. Grossly, exophytic growth pattern has been described as a papillary mass with a broad base or narrow stalk whereas the endophytic growth pattern is seen in tumors that are deeply infiltrative or ulcerative.19, 30 Furthermore, during analysis of growth patterns and predictive outcomes, some authors have used an exophytic/endophytic ratio; the exophytic component is measured from the surface of the tumor to the level of tongue mucosa, and the endophytic component is determined from the level of the mucosa to the deepest extent of the tumor.³¹ Endophytic TSCC growth patterns are associated with higher recurrence rate, increased rate of lymph node metastasis, poorer response to chemotherapy and shorter survival.18, 19, 20, 32 Because endophytic versus exophytic growth patterns are not reported in all studies examining TSCC, the prevalence of each type of growth pattern is unknown.

While biomarkers have the potential to improve survival in all forms of OSCC, this study will focus specifically on TSCC. Aside from the rising incidence of the disease and the dismal prognosis, TSCC was also observed as having the highest levels of IL-6 and IL-8 among OSCC patients.¹³ This supports the findings that levels of IL-6 and IL-8 are correlated with increased tumor burden, poorer prognosis and increased occurrence of metastases.15, 21, 22, 23 A possible link between these elevated cytokines and poor prognosis is angiogenesis, which is also associated with increased risk of metastases and recurrence in OSCC.24, 25, 26 All of the cytokines selected play a role in the angiogenesis cascade and potentially serve as important angiogenic factors in the development of TSCC.²⁷

This study investigates IL-1, IL-6, IL-8, VEGF and TNF-α as potential TSCC salivary biomarkers. To evaluate their potential for early detection in high-risk patients, we include control subjects who smoke and drink. Similarly, we have divided the TSCC cases into endophytic and exophytic TSCC and will compare the mortality between the two groups. We hypothesize that the salivary biomarkers will increase along a spectrum of disease progression and will be highest in the endophytic TSCC group with a significantly increased mortality.