Urokinase-type plasminogen activator receptor (uPAR), tissue factor (TF) and epidermal growth factor receptor (EGFR): tumor expression patterns and prognostic value in oral cancer

Oral cavity cancer is the 11th most common cancer worldwide and accounts for substantial mortality and morbidity for individuals affected by this disease [1]. Despite important technological advances in diagnosis and therapy especially in the last decades, the prognosis for OSCC has only moderately improved, and reported overall survival rate has remained at roughly 50% [2]. Surgery is a cornerstone in the treatment of primary OSCC with curative intent, whether the objective is to achieve complete removal of the tumor as well as any regional metastatic disease in the neck. Failure to achieve a clear tumor-resection margin, and to detect residual disease in the surgical bed intraoperatively, has direct major negative impact on the chances for cure not fully compensated for by adjuvant radiotherapy [3, 4]. Intraoperative detection and delineation of cancer is still based on visual inspection and palpation of the tissues obviating reliable assessment of the microscopic extent of the disease. Consequently, non-radical surgery remains a major challenge, and novel imaging technology, that enables accurate planning of surgery and intraoperative tumor detection, is warranted.

The discovery of a large number of tumor-specific biomarkers has stimulated new optimism in the development of targeted imaging and treatment of cancer [5]. Ideally, a biomarker suitable for targeting purposes should have strong expression within the tumor compartment, and absent or insignificant expression in adjacent normal tissue. The expression of a specific biomarker may vary between different types of cancer, and within each specific type of cancer due to tumor heterogeneity, and therefore studies designed to examine the exact histological expression and tumor-specificity of different targets in large patient cohorts, are becoming increasingly important. Furthermore, accumulating evidence has validated biomarker expression and profiling as an important tool for individual risk stratification and planning of patient-tailored treatment [6]. The combined use of a specific biomarker as a prognosticator and a tumor-specific target for theranostic purposes is a novel strategy, which may have potential applications in the development of effective anti-cancer therapy. This study examined specifically the expression of uPAR and TF because our group has developed imaging and treatment agents targeting these to cell membrane receptors [7‐9]. uPAR and TF have consistently been associated with cancer in most types of solid carcinomas [10, 11]. In addition, EGFR expression was investigated because it is an established target for therapy in HNSCC. However, data on the utility of EGFR as target for imaging agents are lacking.

In head and neck squamous cell carcinoma (HNSCC), the role of Endothelial Growth Factor Receptor (EGFR) in cancer progression has been extensively investigated. Several studies found EGFR overexpression to be a negative prognostic factor for local control and survival outcome measures for tumors arising in different sub-sites in the upper aerodigastive tract. However, existing data with regard to the prognostic role of EGFR in OSSC is ambiguous [12]. The recent clinical introduction of anti-EGFR agents (i.e. Cetuximab) for treatment of advanced HNSCC has emphasized the potential of EGFR as a target for anti-cancer therapy [13]. Importantly, a clinical trial on EGFR-targeted intraoperative optical tumor imaging was recently published, and EGFR-directed PET-imaging has been demonstrated in preclinical studies [14, 15].

uPAR signaling stimulates pericellular proteolysis facilitating plasmin-mediated extracellular matrix (ECM) degradation and subsequent tumor cell migration and invasion. Because of abundant implications in the carcinogenesis of numerous types of cancer, uPAR has been regarded as a promising biomarker for targeted molecular imaging and therapy [16‐18]. Also in OSCC the pathophysiological role of the plasminogen activator system has been investigated, and uPAR has been appointed a key role in process of local invasion in the interplay with other cancer-associated proteolytic systems and signaling pathways [19]. However, there is a need to further uncover the prognostic value of uPAR in OSCC and to explore the rational of uPAR-targeted strategies in this cancer entity.

A strong relation between cancer and hemostasis is generally accepted, and aberrant venous thromboembolism is a common manifestation in malignant disease, including HNSCC [20, 21]. TF is a transmembrane protein receptor and the principle initiator of the extrinsic coagulation cascade leading to fibrin formation after activation by its natural ligand factor VII. In addition TF activation has been associated with angiogenesis, tumor growth and invasion through regulation of the proteolytic cascade necessary for ECM degradation and tissue remodeling [22]. To our knowledge, TF expression in HNSCC has not been explored previously.

Accordingly, the aim of this study was to investigate the prognostic value and tumor expression patterns of EGFR, uPAR and TF in OSCC [23].

The retrospective cohort of 191 patients with primary OSCC had a male predominance (66%), the median age at time of surgery was 59 years (range: 23–89 years). The median follow-up was 5.1 years (range: 0.1–15.9 years). Demographics and the clinicopathological variables are listed in Table 1. Overall, the anatomical location of the tumors was distributed almost equally as 98 FOM tumors and 93 tongue tumors. The majority of the patients (86%) presented with early stage disease (S1-S2) and most tumors (90%) were well differentiated (G1-G2). 31% were diagnosed with primary regional nodal disease and 51 patients (27%) had post-treatment relapse. For the entire cohort the 5-year OS and DFS was 51% and 41%, respectively.

This study provides novel data compared to existing literature because it was based on IHC biomarker expression in whole tumor resection specimens of OSCC and not only biopsy specimens. Resection specimens containing both the tumor compartment and the adjacent resection margin of healthy tissue is a prerequisite to accurately examine the utility of a biomarker for tumor-specific molecular targeting. Also, resection specimens allows to assess the heterogeneity of the biomarker expression across the tumor compartment as opposed to studies based on biopsies that only sample a very small fraction of the tumor. To our knowledge, this type of study of uPAR and TF expression in OSCC has not previously been reported.

uPAR and TF both showed an enhanced expression specifically confined to the tumor compartment with very limited expression in the normal tissues surrounding the neoplasm. In contrast, EGFR lacked a tumor-specific expression pattern and therefore the ability to distinguish between malignant and normal tissues. Further, the positive expression rate of uPAR and TF was high, which implies, that a substantial part of OSCC patients should be regarded as candidates for imaging and/or therapy directed against either uPAR or TF. Especially uPAR was found to have a highly tumor-specific pattern, and also with very limited expression in both normal and dysplastic epithelium around the epithelial tumor lesion. In comparison, EGFR exhibited staining of both normal and dysplastic epithelium in most cases, and importantly also a general EGFR expression in salivary gland tissue outside the tumor compartment was seen. A prerequisite to develop highly accurate targeted imaging is that the molecular target exhibits very low expression in normal tissues bordering the tumor to create a high tumor-to-normal tissue ratio. Targeted optical-guided surgery is currently being clinically translated, and this imaging modality will allow intraoperative real-time assessment of resection margins in order to ensure complete removal of tumors. Further, our group and others have recently presented preclinical data derived from animal models of oral cancer, showing that detection of subclinical disease by use of targeted fluorescent probes is possible [28, 29]. However, because an optical imaging signal has a low energy with a limited range in intensity, target binding of an imaging agent outside the tumor compartment in normal tissues would potentially have substantial influence on the ability to detect a reliable tumor-specific signal to guide a tumor resection. Accordingly, in the data from the recently published first phase 1 trial of targeted optical imaging in HNSCC, using the optical agent cetuximab-IRDye800 directed against EGFR, extratumoral signal uptake in normal epithelium and salivary gland tissue in tissue sections was reported [30].

Direct comparison of the positive expression rates of uPAR, TF and EGFR in this study and previous studies is not possible because of difference in scoring systems used and because of different cut-off values to determine positive and negative expression. Three studies investigated expression of uPAR in OSCC and reported a positive expression rate in the range of 39–100% [31‐33]. Positive EGFR expression in OSCC was in the range of 60–100% in previous studies [34‐37]. We found a positive expression rate of 58% for TF, but the expression of TF in OSCC has not previously been investigated. Chen et al. found a TF immunopositivity of 91% in esophageal cancer [38].

To select potential biomarkers for targeted imaging, van Oosten et al. suggested a selection criteria tool identifying seven factors on order of importance: (1) Extracellular receptor location, (2) diffuse enhanced target expression in tumor compartment, (3) high tumor-to-normal tissue target expression, (4) high expression-rate in patients, (5) previous successful targeted imaging in vivo, (6) enzymatic activity of the receptor and (7) target receptor internalization [39]. In relation to the results of the present study, both uPAR, TF and EGFR fulfills the factors 1–5, which underlines the relevance of these receptors for targeted tumor imaging in OSCC. In addition, uPAR is also reported to exhibit enzymatic activity in the tumor microenvironment and internalization to the intracellular space upon ligand-binding, which makes this receptor especially suited for targeting by imaging agents [11].

In the correlation analysis, high TF expression was associated with relapse of disease. However, TF expression did not show significant impact on OS or DFS in the survival analysis, which has been reported in colorectal, breast and esophageal cancer [38, 40, 41]. We found high uPAR expression to be associated with late TNM stage disease (S3-S4). However, no significant association was reached between uPAR and N- or T-stage analyzed separately. In a study of 115 patients with OSCC, Magnusson et al. reported that low expression of uPAR was correlated with reduced disease specific death only in patients with stage 1 (S1) disease [42]. Bacchiochi et al. analyzed the prognostic value of uPAR in 189 patients with OSCC and found enhanced uPAR expression to be associated with increasing tumor cell differentiation, and that low uPAR expression only was associated with increased OS in well differentiated tumors [31]. Our study confirms the findings in the latter study, as high uPAR expression only for the sub-group of well differentiated tumors was associated negatively with OS in the univariate analysis and reached borderline significance in the multivariate analysis. In two studies from the same Japanese group, containing 34 and 54 OSCC patients, respectively, high uPAR expression was associated with an aggressive mode of invasion [43, 44]. Unfortunately, our study did not include the pattern of invasion (cohesive vs. non-cohesive invasive tumor front) as a histopathological variable, but further research in the relation between local tumor aggressiveness and uPAR activity in OSCC is warranted.

In the survival analysis in the present study, enhanced uPAR expression was associated with a significant reduction in OS only, while no significant associations between expression of TF or EGFR and survival outcome could be demonstrated. Accordingly, our data supports findings in previous studies, that uPAR expression seems to be a prognostic factor for survival outcome in OSCC [31, 42, 44]. However, we did not find uPAR to be an independent prognostic factor in multivariate analysis. Also, uPAR was associated with advanced TNM stage. Therefore, uPAR expression could also be a surrogate for advanced stage of disease as well as comorbidity. A larger sample size would be able to clarify the meaning of these findings.

Interestingly, our study is the first to show that uPAR had impact on survival outcome across an entire cohort of patients, and not only in a subgroup analysis of patients defined by a specific clinicopathological variable [31, 42] or a combination of biomarker expressions [44]. We did not find any significant association between EGFR expression and survival outcome, which is consistent with several previous studies, although some studies have associated enhanced EGFR expression with poor clinical outcome [12]. Of note, a limitation of this study was a relatively small sample size with a limited number of outcome events, which may have affected the ability to detect significant correlations between biomarker expression and survival. In addition, assessment of biomarker expression based on immunohistochemistry has an inherent limited accuracy due to intra-observer variability and the design of the scoring systems used.

Consistent with a study of Lindberg et al., we also found that uPAR predominantly was expressed on stromal cells of the tumor compartment [33]. Similar findings have been reported in colorectal and esophageal cancer [45, 46]. Stroma-rich tumors, like OSCC, may be less sensitive to targeted therapy, if the target only is expressed by tumor cells, because of reduced target density in the tumor compartment. Therefore, uPAR expression on both tumor cells and tumor-associated stromal cells may provide uniform target availability in the entire tumor volume. This expression pattern may be a particular advantage in order to achieve high efficacy of uPAR-targeted intervention and therapy.

Existing data indicate, that elevated uPAR expression in OSCC and several other types of cancer within the tumor seem to predict a more aggressive phenotype that carries reduced survival outcome [18]. Therefore uPAR may have a role to play as a reliable prognostic biomarker in future personalized management of OSCC. Based on the favorable properties of uPAR as an imaging target, a clinical phase 2 trial of preoperative uPAR-PET/CT imaging in patients with oral or oropharyngeal SCC is currently being conducted in our institution (NCT02960724). uPAR-PET imaging may provide a non-invasive quantitative assessment of the uPAR expression in the entire volume of individual tumors, thereby surpassing the inherent problems related to surgical biopsies and risk of sampling error due to tumor heterogeneity.

Our results showed that both uPAR and TF had high positive expression rates and tumor-specific expression patterns while EGFR also had regular expression in normal tissues. These findings may suggest that uPAR and TF could potentially be attractive targets for molecular imaging and targeted therapy in OSCC.

High uPAR expression was significantly associated with reduced survival outcome. Accordingly, uPAR seems to be a potential prognostic biomarker in OSCC, which may have applications for risk-stratification and treatment planning.