Expression of HIF-1α, c-Met, CA9 and GLUT1 were investigated in the current study. In those markers, HIF-1α and c-Met were associated with LN metastasis and FIGO stage in cervical cancer. HIF-1α and c-Met have been known as contributing factors for cancer invasion and LN metastasis in various cancers [18–20]. Activated HIF-1α under hypoxia reduces tissue integrity through the loss of E-cadherin, a cell adhesion molecule that acts as a suppressor of invasion and metastasis . Conversely, inhibition of HIF-1α with siRNA induces E-cadherin expression, which increases cell to cell adhesion . HIF-1α also facilitates disruption of basement membrane and extracellular matrix which are physical barriers against tumor cell migration . After HIF-1α disrupts cell integrity and membrane, c-Met enhances tumor cell to invade surrounding stroma and migrate into the blood and lymphatic vessels [5, 22].
We investigated correlation of HIF-1α expression with c-Met, GLUT1, or CA9 expression, respectively. Firstly, HIF-1α expression was associated with c-Met expression in cervical cancer, while no correlation between HIF-1α and c-Met was observed in CIN and CIS. Although c-Met is well known to be regulated by HIF-1α in in vitro study, its association has not been confirmed with cervical cancer. In our knowledge, this is the first report of correlation between HIF-1α and c-Met in cervical cancer tissue. For correlation between HIF-1α and c-Met in other cancer tissues, only limited number of pancreatic tissue showed tendency of correlation with c-Met and HIF-1α expression . Furthermore, correlation between HIF-1α and c-Met is not reported in pre-invasive lesions as well. As for pre-invasive cervical lesions, not only was c-Met found to lack correlation with HIF-1α but also expressed much lower. Since CIN primarily comprises of cells that proliferate without the feature of metastasis and invasion, c-Met expression in these cells is expected to remain low. Regulating factors such as TGF-β, proteasomal inhibitor, AP1, PI3K and ERK are involved in the CA9 expression, which are independent of HIF-1α [24–26].
In this study, c-Met was an independent prognostic factor on overall survival in cervical cancer. Similar to our study, c-Met is associated with FIGO stage and LN metastasis, and c-Met expression influences on the adverse survival outcome in cervical cancer [20, 27, 28]. Although the high expression of HIF-1α revealed worse 5-year overall survival on Kaplan-Meier plots, HIF-1α did not show significance on multivariate analysis. In previous studies performed on patients with cervical cancer, prognostic significance of HIF-1α was inconsistent, even though the HIF-1α is involved in tumor cell metabolism, invasion and metastasis [29–31]. Furthermore, even the CA9 and GLUT1, which play crucial roles in tumor cell metabolism associated with tumor survival, did not show survival significance in our research. Survival significance of CA9 expression has been studied in cervical cancer but this remains unclear [12, 32, 33]. In the terms of GLUT1, research on its survival outcome in cervical cancer remains preliminary with very few reported cases in other cancers .
There are numerous potential sources of the conflicting results of hypoxia markers and prognostic significance in cervical cancer. Foremost, sampling site is a factor to be considered. Hypoxia increases as the distance of the sampling site from blood vessel increase, and this contributes to conflicting results. HIF-1α is stabilized in the cytoplasm under hypoxic conditions and translocated to the nucleus as it dimerizes with HIF-1β binding to the hypoxia response element. Degree of cytoplasmic and nuclear expression often varies with different HIF-1α antibodies used which result in mixed and conflicting outcomes. Daponate et al. suggested applying a panel of HIF-1α antibodies to overcome controversy , however this approach introduces even more variables, and makes the application in a clinical setting unappealing. Medical condition of the patient can also affect the expression of hypoxia and metabolic markers. Patients with low level of hemoglobin and hyperglycemia in diabetes mellitus exhibited over-expression of HIF-1α and increased GLUT1 expression, respectively [36, 37]. Manual scoring is time consuming, results in qualitative results of limited dynamic range, and is prone to intra- and inter-pathologist variability. The use of image analysis alleviates the issues of variability, and allows the application of quantitative scoring. The challenge with automated scoring is ensuring the correct cells are being quantitated, and many users rely on manual annotations of regions of interest. Computer-aided image analysis is limited by the same factors that manual interpretation are, mainly the quality of the immunohistochemical staining, and the overall quality of the tissue being stained .
Although HIF-1α expression was significantly higher in precancerous and cancer tissue than in normal tissue which was acquired around cancerous tissue, weak or moderate expression was observed in a large number of normal tissues. This finding was also observed in other study including IHC results of esophageal cancer . Hypoxia may influence tissues around the cancerous lesions through increased interstitial pressure or metabolic product to affect oxygen release and consumption . Similar to our result, Birner et al. reported HIF-1α was highly expressed in high grade CIN and cervical cancer compared to that in normal cervix but no difference was observed between high grade CIN and cancer . Considering such high expression of HIF-1α from high grade CIN, HIF-1α is thought to be involved in early event of tumorigenesis. Since human papillomavirus (HPV) infection is involved in tumorigenesis, much research has been conducted in finding the association between HPV infection and HIF-1α. Lu et al. reported double transgenic mice with HPV 16 and HIF-1α showed highly invasive cervical cancer compared to that in single transgenic mice only with HPV 16 expression . HIF-1α expression with HPV infection facilitates tumor progression from premalignancy to malignancy . Furthermore in other study, HPV E6 oncoprotein, inactivator of p53 tumor suppressor gene, was shown to enhance HIF-1α stability and HIF-1-dependent vascular endothelial growth factor (VEGF) expression in hypoxia . Considering our finding and together with previous studies, stabilization and expression of HIF-1α through high-risk HPV infection is speculated to play an important role in cervical cancer progression. In addition to HIF-1α, c-Met may co-facilitate cancer progression with HPV infection as well. In CIN and anal intraepithelial lesion (AIN), c-Met expression was correlated with oncogenic HPV infection but was not correlated with non-oncogenic HPV infection, condyloma acuminata [42, 43]. However, further research is required to clarify the association between oncogenic HPV and c-Met expression in cervical cancer progression.
In this study, we evaluated protein expressions of HIF-1α and its related markers using automated digital image analysis. Manual interpretation of IHC is highly subjective and may produce conflicting results among studies. In such cases, automated digital image analysis for the interpretation of IHC could serve as an alternative method in presenting reproducible, objective and quantitative measurements. As a result of its ability to predict response to Trastuzumab, which specifically targets human epidermal growth factor receptor 2 (HER2), the assessment of HER2 expression has been incorporated in the diagnostic work-up of breast cancer. Hence, 32.7% of the U.S. laboratories reported to perform image analysis for quantitation of HER2 in a survey conducted in 2008 . At present, fluorescence in situ hybridization (FISH) is commonly used to clarify equivocal expression of HER2 by manual interpretation before administration of Trastuzumab. However, automated image analysis of HER2 has been reported to show excellent concordance to FISH results, while reducing time required for interpretation and being more user-friendly compared to FISH  and therefore, it may substitute FISH in determination of equivocal case. Despite of powerful advantages mentioned above, digital image analysis has not been widely applied to diagnostic work-up outside breast cancer. As previously applied in breast cancer research, when used in gynecologic cancer studies, digital image analysis is expected to improve protein expression analysis from IHC.