Introduction
Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed cancer of the head and neck region, accounting for ~300,000 new cases annually worldwide (1). Despite advances in diagnostic, surgical and chemo-radiation techniques, the 5-year survival rates for patients with OSCC have remained at ~50% for the last five decades (2). We hypothesize that biological programs, including hypoxia and proliferation, within the tumor microenvironment are associated with tumor progression and may help to identify those patients at greatest risk of relapse and mortality. Identifying those mechanisms and using those factors in clinical decision-making could help target adjuvant therapies and improve the clinical management of patients with OSCC.
Hypoxia is common within the oral tumor microenvironment but rare in normal oral epithelial tissue (3). Tumor hypoxia (O2 <5 mmHg) is associated with increased invasiveness, metastasis and an overall poor prognosis (4–6). Several methods can be used to assess hypoxia within tumors. The direct measurement of tissue oxygen tension, using an oxygen electrode, is highly sensitive but intra-tumoral heterogeneity and the accessibility of tumors, due to anatomical restrictions, limit its utility for the clinical assessment of tumor hypoxia (7,8). Exogenous markers, including bioreductive drugs (e.g., pimonidazole), enable targeted imaging but the threshold for activation by hypoxia may not correspond to clinically-relevant tumor hypoxia, and incomplete distribution due to ineffective perfusion may lead to variability in the reliability of staining across the tumor regions (6). Immunohistochemistry (IHC)-based staining for endogenous markers of hypoxia (EMH) is cost effective and routinely available in clinical pathology laboratories; however, no single EMH has been consistently demonstrated to reliably indicate an association with adverse tumor phenotypes and poor prognosis in OSCC (9).
While several hypoxic sensitizers and hypoxia-specific toxins are available as potential therapeutic agents, reliably identifying patients with hypoxic tumors for patient selection in the clinical setting is a consistent barrier to implementing effective hypoxia-targeting/sensitizing therapies (6). A number of studies have reported associations between a poorer prognosis and intratumoral levels of the EMH carbonic anhydrase IX (CAIX) in several cancer sites, including head and neck squamous cell carcinoma (10–14). CAIX expression is regulated by hypoxia inducible factor 1-α (HIF1α) (15), an oxygen-sensitive transcription factor. The stability and activity of HIF1α are predominantly regulated by intracellular pO2 levels; however, elevated transcriptional activation may also occur under normoxic/mildly hypoxic conditions through the stabilization of HIF1α by proliferation-inducing signaling pathways such as phosphatidylinositol 3-kinase (PI3K), nuclear factor-κB (NF-κB), AKT and Her2 (10,16–20). Highly hypoxic tumor cells typically exhibit low proliferation (6,21,22). We previously proposed that the co-localization of proliferation and EMH expression may identify cells responding to proliferative stimuli or EMH regulation by factors other than low pO2 (23), including alternative signaling pathways that control HIF1α stability and activity (10,24). We had also hypothesized that functionally significant hypoxia would be characterized by low proliferation (low Ki-67 index) within areas of high stromal CAIX (sCAIX) expression (23).
Attempts to test these hypotheses led to the observations that sCAIX was more highly associated with patient survival than tumoral CAIX (tCAIX) (13,14), and that there was a strong association between sCAIXhi/Ki-67lo protein levels and reduced disease-specific survival (DSS). However, the literature on prognostic biomarkers is replete with association studies that have failed independent replication (25). Therefore, the present study aimed to replicate our previous findings in an independent cohort of patients with OSCC.
Discussion
Our previous study demonstrated, in two independent cohorts, that sCAIX was more highly associated with patient survival than tCAIX (13,14). In the current study, elevated sCAIX and tCAIX were associated with reduced DSS when analyzed as continuous variables, but only tCAIX was associated with DSS when dichotomized at the 75th percentile. The association between sCAIX and DSS, when analyzed as a continuous variable but not as a categorical variable, suggests that sCAIX may be a biologically important factor in OSCC survival but also that the 75% cut-off point is not appropriate for clinical decision making.
The association between DSS and sCAIX and tCAIX, for each unit increase in expression (AQUA score), was similar (sCAIX: HR, 1.014, 95% CIs, 1.003–1.024 vs. tCAIX: HR, 1.003, 95% CIs, 1.001–1.006). The increased risks associated with each measure of CAIX expression were also similar when dichotomized at their optimized cut points (sCAIX adjusted HR, 2.39, 95% CIs, 1.22–4.67; tCAIX adjusted HR, 2.45, 95% CIs, 1.40–4.31). However, the group defined by the optimized cut-off point for tCAIX contained almost twice as many patients, compared with the group defined by the optimized cut-off point for sCAIX. The strength of the association with DSS was similar for each dichotomized measure of CAIX, suggesting that, tCAIX is a more informative prognostic marker than sCAIX. Determining which of the two, tCAIX or sCAIX, is the more informative measure of tumor CAIX status and defining a biologically meaningful expression threshold remains a necessary step if this marker is to be translated into a clinically useful test.
Our prior study reported the prognostic impact of a combined sCAIX/Ki-67 signature on DSS in OSCC (23). This signature was proposed on the basis of the potential confounding effect of proliferation, and associated signaling pathways, on the association between CAIX expression and prognosis. This was a logical extension of our previous research, reporting significantly reduced survival in patients with high sCAIX and low Ki-67, separately (14,28). The current study did not replicate this proposed ‘functional hypoxia’ signature according to our original definitions of low Ki-67 and high sCAIX. The association between DSS and each component of the functional hypoxia signature was then further examined. The failure of each individual component may explain the lack of association detected in the proposed ‘functional hypoxia’ signature. In contrast to prior reports (14,23), sCAIX was not significantly associated with survival when dichotomized at the 75th percentile in univariate and adjusted analyses (HR, 1.36 and 95% CIs, 0.74–2.51; HR, 1.23 and 95% CIs, 0.68–2.23, respectively). Furthermore, when Ki-67 was analyzed as a continuous variable, there was no association with reduced survival in univariate or adjusted analyses (HR, 1.009 and 95% CIs, 0.995–1.023; HR, 1.009 and 95% CIs, 0.995–1.024, respectively) in the current study.
Hypoxic tumors are typically associated with a poor prognosis; not all tumors are considered to be hypoxic, but a high proportion of tumors have areas of hypoxia and tumors which exhibit increased hypoxia typically have poorer outcomes (22,32). The potential role for hypoxia and proliferation in the prognosis of OSCC, and other types of head and neck cancer, has been investigated by a number of research groups (33–35). Recently, there has been particular interest in the role of CAIX in the stroma as it should not be confounded by somatic gene alterations commonly observed in OSCC. For example, the von Hippel-Lindau (VHL) gene, a negative regulator of HIF1α activity, is commonly deleted in OSCC (36). The loss of VHL leads to an accumulation of HIF1α and drives the hypoxia-independent expression of CAIX (37,38). However, proliferation-associated factors may also enhance sCAIX expression despite the absence of hypoxia. The response of tumor cells to proliferative signaling in the tumor microenvironment is likely to be most relevant to the disease course. Consequently, we assigned proliferation status of the tumor/patient based on the measurement of Ki-67 in the PCK-positive tumor cell compartment. However, paracrine/autocrine growth factor signaling within the tumor microenvironment may contribute to HIF1α stabilization in the tumor stroma, and elevate sCAIX expression independent of hypoxia. Furthermore, local depletion of oxygen and nutrients due to the high tumor cell density may induce peri-cellular hypoxia and consequent elevation in sCAIX expression (10,39).
The cohorts of oral cancer patients from Calgary and FHCRC are broadly similar when comparing common clinical and demographic characteristics (Table II). Therefore, the failure to replicate the previous combined Ki-67-sCAIX association cannot be solely attributed to differences in these populations. However, the limitation of the sub-group sizes and post hoc definition of survival groups in the original report is acknowledged, and emphasizes the requirement for replication in an independent OSCC cohort.
Several mRNA-based multi-parameter prognostic and predictive assays have been proposed for head and neck cancer and for other cancer types, particularly breast cancer (5,40–42). However, due to logistical and cost challenges, few gene expression-based tests are currently in routine clinical use for treatment selection. IHC-based assays, for the purposes of diagnosis and prognostication, are appealing as standard clinical pathology laboratories are equipped to conduct these assays. Recent evidence suggests that the IHC-based tests provide similar assessments of recurrence risk in patients with breast cancer, so the potential to develop an equivalent test in patients with OSCC is present (43).
The current study adhered to the REMARK criteria for the reporting of prognostic studies (27). The present study was limited by its retrospective design and the lack of corroborative measures of intra-tumoral hypoxia, such as directly measured pO2 or pimonidazole distribution. Our results support the role of CAIX in OSCC survival but the relative importance of sCAIX or tCAIX is unclear. The addition of proliferation markers, such as Ki-67, could potentially improve the prognostic utility of sCAIX or tCAIX, but the present study was unable to replicate the prior findings (23). Studies in larger cohorts of patients with OSCC could enable the derivation of prognostic signatures for OSCC or the identification of specific hypoxia-sensitizing/targeting treatments (44).