Tongue cancer remains a difficult disease to overcome. Despite the availability of a number of therapeutic modalities and marked advances in techniques to diagnose head and neck cancer, the 5-year survival rate of patients with tongue cancer is approximately 50% [
]. Multimodality therapy combining surgery, radiotherapy, and chemotherapy is generally indicated for advanced tongue cancer. However, in the past few decades, little improvement has been noted in its prognosis.
In general, tongue cancer is more common in older people, but even in the young, the incidence is higher than that of other types of head and neck squamous cell carcinomas (HNSCCs). In addition to chronic stimulation by contact with the teeth and certain environmental factors, alcohol intake and smoking are risk factors for tongue cancer [
], while genetic background also appears to be a strong determinant of risk, particularly in the young [
]. A previous study of genetic abnormalities in HNSCC revealed more frequent loss of heterozygosity (LOH) at loci on chromosomes 3p, 9p, and 17p [
]. Tumor suppressor genes p16 and p53 are located at loci on chromosomes 9p and 17p, respectively, and both are reported to show genetic alterations, such as mutations and methylation, in approximately 50% of tumor specimens from HNSCC patients [
]. Recently, whole exome sequencing of HNSCC revealed that dysregulation of
, which regulate squamous differentiation, is a driver of HNSCC carcinogenesis, similar to mutations of
]. Gross et el. found that a
mutation is frequently accompanied by loss of chromosome 3p, and that the combination of both events is associated with poor outcomes [
]. Although 3p loss was determined by evaluating 12 genes located in 3p14.2, it remains unclear which factor encoded on 3p is responsible for the interaction with TP53. Asakawa et al. previously demonstrated that LOH of
(3p25.3), a tumor suppressor gene, occurs at a high frequency in tongue cancer, similar to that of 3p14.2 [
]. However, the biological effect of
loss on tongue cancer remains unclear.
gene, which is responsible for VHL disease, was identified at loci on chromosome 3p as a tumor suppressor gene in clear cell renal cell carcinoma (RCC) [
]. pVHL forms a multimeric complex with Elongin B and C, Culine2, and Rbx1 proteins, which then binds to the α-subunit of hypoxia-inducible factor-1 (HIF-1α) in cytoplasm to induce the ubiquitination and further degradation of HIF-1 [
]. HIF-1 induces vascular endothelial growth factor and other angiogenic factors, thereby promoting angiogenesis. Therefore, pVHL serves to negatively regulate angiogenesis. In addition, pVHL is reported to play a role in control of the cell cycle [
Here, to clarify the relationship between pVHL expression and the pathology of tongue cancer, we conducted immunohistochemical staining to detect the expression of pVHL in cancer tissues and other lesions from patients with tongue cancer.
The present study involved 19 patients (eight men and 11 women) with primary tongue cancer, who were treated at Nihon University Itabashi Hospital [
]. Clinicopathological classification of carcinoma and histopathological grading of tumor tissues were based on the Cancer Staging Classification (6
edition) of the International Union Against Cancer [
]. Histological findings of dysplasia included intraepithelial neoplasia lesions lacking infiltration, which were categorized as mild, moderate, and severe dysplasia based on current World Health Organization classifications [
was not included in the present analysis. For normal tongue epithelium, areas of normal epithelium contained in tissue specimens from patients with invasive tongue cancer were used for investigation. This study was approved by the Ethics Committee of Nihon University School of Medicine (Approval number 118–1). Informed consent was obtained from each patient prior to the start of the study.
IHC staining was performed using anti-pVHL (556347; BD Biosciences, San Jose, CA, USA), anti-CK13 (NCL-CK13; Leica Biosystems, Nussloch GmbH, Germany), and anti-CK17 (clone E3 IR620; DAKO, Glostrup, Denmark) monoclonal antibodies. Formalin-fixed, paraffin-embedded (FFPE) sections (4-μm thick) of the tissue specimens were deparaffinized in xylene and incubated for 15 min in 5% hydrogen peroxide to inactivate endogenous peroxidases. The treated sections were immersed in 0.01 M citrate buffer (pH 6.0; Muto Pure Chemicals, Tokyo, Japan) and heated for 5 min in an autoclave for antigen retrieval. Each tissue section was then immersed in blocking solution (5% dry skim milk) at 37 °C for 30 min. After removal from the blocking solution, the tissue section was reacted with the primary antibody (anti-pVHL antibody) at a 100-fold dilution in phosphate-buffered saline (PBS) at 37 °C for 60 min. After removal from the primary antibody solution, the tissue section was washed three times (5 min per wash) with PBS.
Chromogenic detection of pVHL was achieved using Histofine Simple Stain MAX-PO (Nichirei Bioscience, Tokyo, Japan) in accordance with the manufacturer’s protocol. For CK13 and CK17, the primary antibody reaction was performed as described above. The subsequent chromogenic detection was performed in two steps: each tissue section was first treated with the Envision Plus kit (EnVision™ FLEX Mini Kit, DAKO) and then with the chromogenic substrate diaminobenzidine (Histofine DAB, Nichirei Bioscience) for 5 min. Each tissue section was counterstained using hematoxylin.
In this study, we characterized pVHL staining in tongue tissues and cancer as follows. (1) In normal stratified squamous epithelium, pVHL staining was localized to the cytoplasm of cells in the basal layer and parts of the cytoplasm in the spinous layer adjacent to the basal layer. (2) In dysplasia, a precancerous condition, expansion of the range of positivity was observed mainly in dysplastic cells. (3) In invasive cancer, pVHL staining was observed in all specimens, regardless of the differentiation stage. These findings suggest that pVHL will be useful as an adjunctive marker in the histopathological diagnosis of dysplasia.
Here, using our method for pVHL staining, we demonstrated that FFPE sections can be stained with mouse monoclonal antibodies (Ig32) using antigen retrieval procedures. Staining patterns of pVHL in normal renal tissues and clear cell RCC corresponded well with the results of staining using frozen tissue specimens reported by Corless et al. [
]. Claudio et al. [
] stained FFPE sections of clear cell RCC using the same antibody (Ig32) but a different method to ours. A similar staining pattern has been reported using microwaving for antigen retrieval. The present results may be considered as highly reliable. To date, only one other study has reported staining of tongue cancer specimens for pVHL. In that study, 10 of the 27 (37%) tongue cancers were positive for pVHL. Details of this previous study were not well described, but the reason for the substantial difference in the positive rate in our present study remains unclear [
]. However, it is likely a result of the different antibodies used and staining conditions.
To our knowledge, this is the first report of pVHL staining in the basal layer and the spinous layer adjacent to the basal layer of normal squamous epithelium. These results suggest that both stem cells and undifferentiated cells may be positive for pVHL because these cells exist in the same region. pVHL staining has been observed in the cytoplasm of normal epithelial cells in other tissues [
]. In particular, intense staining was noted in renal proximal tubular cells that are considered to be the origin of clear cell RCC [
]. Similarly, tongue cancer appears to develop from abnormal proliferation of stem cells in basal or parabasal cell layers of normal epithelium, which were positive for pVHL.
The clinical condition leukoplakia includes a wide range of lesions, from “reactive” to “precancerous”. Differentiation between reactive and neoplastic tissues is often difficult, particularly in biopsy diagnosis where the observation target is limited to small tissue sections. Our present comparison of IHC staining for CK13/CK17 and pVHL suggests that pVHL staining may be a useful procedure in the evaluation and diagnosis of dysplasia. CK13 and CK17 are useful to evaluate dysplastic grades of certain specimens, such as those with pattern A staining. In pattern B, however, the staining patterns of CK13 and CK17 were typically observed in malignant lesions such as invasive cancer. In contrast, pVHL was stained positively in dysplasia following hematoxylin and eosin (HE) staining. In pattern C, CK13 staining was reduced greatly, while CK17 staining remained slightly positive, a pattern that hampers determination of the dysplastic grade. Nevertheless, pVHL staining was observed in the same dysplastic regions as those stained with HE, and may have superior sensitivity to stain CK13 and CK17 as adjunctive markers to detect dysplastic regions. The present report is the first to investigate the utility of pVHL staining for the diagnosis of tongue dysplasia. However, the small number of specimens examined is a limitation in this study, particularly with regard to dysplasia patterns B and C. An increased number of specimens and another large cohort study are necessary to validate the utility of pVHL in the conclusive diagnosis of preneoplastic lesions and tongue cancer. It would also be helpful to confirm IHC staining of other proliferative markers such as Ki-67 in pVHL-positive dysplasia. Because Ki-67 staining is well correlated with CK17 staining in tongue dysplasia [
], pVHL- and CK17-positive dysplastic regions, at least, may be positive for Ki-67.
Our unexpected finding is that all tongue cancers were positive for pVHL. Because no tongue cancers had nonsynonymous mutations in the present study [
], wild-type pVHL tended to be produced in more differentiated and well-defined cancers. In a HIF-1α-independent pathway, pVHL interacts directly with fibronectin and collagen IV, resulting in their assembly into the extracellular matrix (ECM) and suppression of tumorigenesis, angiogenesis, and cell invasion [
]. Therefore, even in invasive tongue cancer, it is possible that pVHL plays a role in regulation of the ECM and decrease of the invasive ability. Roland et al. demonstrated that poorly differentiated tongue cancers have a poor prognosis [
], and poorly differentiated tongue cancers were weakly stained for pVHL in the present study. In clear cell RCC, pVHL expression is also associated with a low histological grade and better prognosis [
]. Thus, pVHL in cancer possibly functions in the suppression of tumor progression. In the present study, we noted no clear relationship between LOH of the
gene and the staining pattern of pVHL in tongue cancer. Schraml et al. similarly reported the lack of a relationship between these variables in clear cell RCC [
]. These findings suggest that LOH of the
gene does not affect expression of pVHL, regardless of the cancer type. Considering the small number of specimens, these results should be considered as preliminary, particularly with regard to dysplasia and poorly differentiated tongue cancers. Therefore, further studies are needed on the topic.
We thank the late Dr. Sohei Endo for his initial conception of the study. We also thank Dr. Tomohiro Igarashi for providing clinical samples and data of clear cell RCC.
The authors have nothing to declare.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
HH carried out the IHC staining, interpreted the data, and drafted the manuscript. YK participated in IHC experiments and diagnosed histopathological features. TA collected the clinical samples and performed the LOH analysis. MM determined the optimum condition for IHC and carried out the staining. TOi diagnosed histopathological features. TF and TOs participated in the clinical diagnosis and study coordination. ME designed the study and helped draft the manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Nihon University School of Medicine (Approval number 118–1). Informed consent was obtained from each patient prior to the start of the study.
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