Phosphorylated (activated) STAT3 (pSTAT3) is a regulator of numerous genes that play an essential part in the onset, development and progression of cancer; it is involved in cell proliferation and preventing apoptosis, and in invasion, angiogenesis, and the evasion of immune surveillance. This study aimed mainly to investigate the potential prognostic role of pSTAT3 expression in oral tongue squamous cell carcinoma (SCC).
Phospho-ser727 STAT3 immunolabeling was correlated with prognostic parameters in 34 consecutive cases of pT1-T2 tongue SCCs undergoing primary surgery. Computer-based image analysis was used for the immunohistochemical reactions analysis.
Statistical analysis showed a difference in disease-free survival (DFS) when patients were stratified by pN status (
In spite of these results, it is worth further investigating the role of pSTAT3 (serine- and tyrosine-pSTAT3) in oral tongue SCC in larger series because preclinical models are increasingly showing that several anticancer strategies would benefit from STAT3 phosphorylation inhibition.
In 2008, with an estimated 263,000 new cases, oral (including lip) squamous cell carcinoma (SCC) ranked as the 15th most common cancer worldwide when the global incidence and related mortality of 27 malignancies was estimated for 182 countries by the International Agency for Research on Cancer as part of the GLOBOCAN scheme [
Despite advances in surgery (including reconstructive techniques) and radiotherapy and chemotherapy, the five-year survival rate for oral cancer has not improved significantly over the past several decades and it is still around 50% to 55%. There is an undeniable need for novel, more effective diagnostic and therapeutic strategies to improve the overall survival of patients with oral SCC, particularly in advanced cases. Very recent research has focused on targeted molecular therapy, immunotherapy, and latent/residual tumor cell ablation. Although many promising therapies have been tested, only anti-epidermal growth factor receptor (anti-EGFR) therapy has been approved by the U.S. Food and Drug Administration in recent years for use in SCCs of the head and neck.
STAT3 is a member of the STAT family that takes part in the normal cellular responses to cytokines and growth factors, such as transcription factors. STAT3 occurs in the cytoplasm under basal conditions and is activated as a consequence of the ligand-mediated receptor activation induced by Janus tyrosine kinases or growth factors. It seems that, while STAT2, STAT4, and STAT6 are only activated under normal conditions, STAT1, STAT3, and STAT5 would appear to have an important part to play in cancer (STAT1 as a tumor suppressor, and STAT3, and STAT5 as oncogenes) [
The aim of this study was to conduct a preliminary (because of the retrospective setting and the limited number of considered cases) investigation into the potentially prognostic role of pSTAT3 expression in a series of consecutive cases of pT1-T2 of primary oral tongue SCC. This is the first time anti-phospho-ser727 STAT3 antibody has been used in oral tongue SCC.
The study was approved by the Padova University Otolaryngology Section's Ethical Committee. It involved 34 consecutive cases (25 male patients, 9 female ones; mean age, 66.2±12.2 years) of primary pT1-2 tongue SCC. Twenty-eight out of 34 cases had been previously evaluated for angiogenin, endoglin, and Mib-1 expressions [
After primary partial glossectomy with unilateral or bilateral cervical lymph node dissection (at the Otolaryngology Section of Padova University), the final histopathology report confirmed that all patients had negative surgical margins. According to the TNM classification of the International Union Against Cancer (Union Internationale Contre le Cancer) and the American Joint Committee on Cancer (7th edition) [
The clinical follow-up schedule (adjustable to patients' individual characteristics) was as follows: (1) once a month for the 1st year after treatment; (2) every 2 months in the 2nd year; (3) every 3 months in the 3rd year; (4) every 4 months in the 4th year; (5) every 6 months in the 5th year; and (6) every 12 months thereafter. Neck ultrasonography and chest X-rays were also performed at least yearly. Contrast-enhanced CT of the neck, total body positron emission tomography, chest CT, and liver ultrasonography were repeated as necessary.
Immunohistochemical reactions were obtained on sections 4- to 5-µm-thick obtained from the 34 formalin-fixed and paraffin-embedded tumor samples. No cases were excluded because of the lack of representative carcinoma tissue. All immunohistochemical stains were handled automatically (Bond maX, Menarini, Florence, Italy), as described elsewhere [
An image analysis (IA) workstation was used to assess nuclear pSTAT3 expression in all samples. The system comprised a conventional Zeiss Axioskop light microscope (Zeiss, Jena, Germany) with a color digital, Peltier-cooled video camera (MicroPublisher 5.0 RTV, QImaging, Surrey, BC, Canada) that was connected to a PC with Image-Pro Plus software, Version 7 for Windows (Media Cybernetics Inc., Bethesda, MD, USA). Five areas of tumor tissue or morphologically normal mucosa 1,378 µ × 954 µ in size were always examined blindly with the aid of a 495-point sampling grid by that the program superimposed on images acquired with a 50× field of view; the analysis relied on a tailored subroutine (Winrec, Immagini & Computer, Milano, Italy) that enabled the points intercepting the positive and negative areas to be identified and counted interactively. In compliance with stereological best practice, the proportion of the area found positive was calculated as a percentage (%).
Fisher exact test and the Mann-Whitney
The average follow-up was 61.7±42.3 months (median, 56.0 months). Twelve patients developed SCC recurrences (9 local and 3 loco-regional) after a mean 34.6±33.6 months (median, 19 months). The Mann-Whitney
The tongue SCCs covered the whole range from well to poorly differentiated neoplasms (
pSTAT3 immunostaining in normal tongue mucosa was considered first with a view to determine its usual pattern of expression. pSTAT3 showed a basal to surface gradient in this stratified squamous epithelium: there was ample, strong nuclear labeling in the basal and spinous layers, while staining in the granular layer was weak and scarce, and there was no immunoreaction in the keratin layer. In normal mucosa, IA measured a mean of 30.4%±1.1% pSTAT3 immunohistochemically positive nuclei. Most of tumors extensively showed an intense nuclear immunoreaction to pSTAT3 (
All 34 oral tongue SCCs revealed some degree of pSTAT3 immunoreactivity: the intensity of this pSTAT3 nuclear reaction was classed as 1+ in six cases, 2+ in 12, and 3+ in 16. IA indicated a mean nuclear pSTAT3 expression of 80.7%±23.8%, in tongue carcinoma that was remarkably higher compared with of normal mucosa's one (30.4%); the median expression value in carcinoma (90.0%) was used as threshold for the following analyses.
Statistical analysis excluded any significant difference in pSTAT3 expression between pT1 SCCs and pT2 cases (Mann-Whitney
The Mann-Whitney
Given that this was a preliminary investigation, the main strength of the study lies in the homogeneity of the series of patients considered, i.e., (1) they all had primary pT1-T2 oral tongue SCCs; (2) they underwent surgery performed consecutively by the same team; (3) pathological T and N staging was available for all cases; (4) only surgical specimens (not biopsies) of oral tongue SCC were assessed. In addition, the present study focused on the expression of serine-pSTAT3, which has been considerably less thoroughly investigated than tyrosine-pSTAT3 in head and neck SCC, and a computer-based IA system was used to ensure a highly accurate, precise, and reproducible analysis of the immunostained slides. The main weaknesses of our investigation, on the other hand, concern the retrospective setting and the limited number of cases considered.
From a conventional pathological viewpoint, pN stage correlated significantly with prognosis (DFS) in the present series, as already reported extensively for oral tongue SCC. Local invasion and spread to regional lymph nodes in the neck are common in oral SCC, facilitated by the carcinoma's highly-invasive behavior and by the lymphatic drainage in the cervical region [
STAT3 is a transcription factor with important roles in cancer formation and progression; it is involved in several cellular mechanisms, such as proliferation, inhibition of apoptosis, immune escape, epithelial-mesenchymal transition, invasion, and angiogenesis [
Investigating the human oral SCC cell line MIKS81-5, Naher et al. [
Although our results suggest that pSTAT3 has no prognostic value, its strong and diffuse expression in tongue SCC remains intriguing. Different pathways (transforming growth factor-α/EGFR, IL-6/gp130/Jak, Src, and nicotine) converge on pSTAT3, which might ultimately have a pivotal role in cell proliferation [
This study was supported in part by grant No. 60A07-1341/12 (G. Marioni) from the University of Padova. The authors thank Frances Coburn for correcting the English version of the paper.
Variable | No. of cases | No loco-regional recurrence | With loco-regional recurrence | DFS (month) | pSTAT3 nuclear expression (%) in tongue carcinoma cells | Hazard ratio for recurrence |
---|---|---|---|---|---|---|
Primary tongue lesions | pT1 vs. pT2 | |||||
pT1 | 16 | 12 | 4 | 66.5±51.3 | 88.4±12.0 | 4.5 times higher for pN+ |
pT2 | 18 | 10 | 8 | 39.6±28.6 | 73.9±29.1 | (95% CI, 0.64–35.0; |
Regional lymph nodes | pN+ vs. pN0 | |||||
pN0 | 26 | 19 | 7 | 57.9±45.4 | 84.4±18.8 | 2.4 times higher for pT2 |
pN+ | 8 | 3 | 5 | 33.8±27.1 | 68.6±32.9 | (95% CI, 0.45–14.0; |
Grade | ||||||
G1 | 12 | 9 | 3 | 54.8±37.5 | 87.5±16.4 | |
G2 | 13 | 7 | 6 | 56.2±52.4 | 80.6±21.3 | |
G3 | 9 | 6 | 3 | 43.2±32.3 | 71.8±31.4 |
Values are presented as mean±standard deviation.