Oral squamous cell carcinoma (OSCC) constitutes a major proportion of head and neck squamous cell carcinoma in the Taiwan and South-East Asia [
Osteopontin (OPN) is an arginine-glycine-aspartate-containing adhesive glycoprotein expressed in the kidney, macrophages, vascular smooth muscle cells, and many cells of the epithelial linings [
Therefore, the purpose of this study was to evaluate whether OPN expression can affect the treatment response and survival in patients with OSCC treated with cisplatin-based IC followed by CCRT. The role OPN might play in cisplatin’s effect on one oral cancer cell line was also investigated.
A total of 121 patients with pathologically proven locally advanced stage IVA/B OSCC were treated with IC followed by CCRT between January 1, 2006, and January 1, 2012, at Kaohsiung Chang Gung Medical Center (Taiwan). To be included, all the patients had to have a biopsy-proven nonmetastatic IV (M0) oral squamous cell carcinoma, have no synchronous primary tumors, and be ≥18 years old. In addition, the patients had to have a performance status (PS) of ≤2 on the Eastern Cooperative Oncology Group (ECOG) scale, adequate bone marrow, hepatic and renal function (creatinine clearance >60 mL/min), and a computed tomography or magnetic resonance image scan of the head and neck region within three weeks prior to the initiation of treatment. Clinicopathological information including age, gender, tumor (T) stage, nodal (N) status, TNM stage, and survival was obtained from the patients’ clinical records. The histories of betel nut chewing, alcohol drinking and tobacco use were obtained by oral interview and detailed questioning during the patients’ first visit to the otolaryngology clinic of the hospital. The IC consisted of cisplatin 75 mg/m2 and fluorouracil (5-FU) (1000 mg/m2) given as a continuous 24 h infusion for four days or docetaxel 60 mg/m2, cisplatin 75 mg/m2, and fluorouracil 600 mg/m2/day continuous 24 h infusion for four days. After IC, all patients received CCRT. During the CCRT, cisplatin was administered weekly at a dose of 40 mg/m2. RT was delivered 6-7 weeks after the completion of the IC with a linear accelerator. The response to IC followed by CCRT was assessed according to the World Health Organization (WHO) criteria. Surgery was performed six to twelve weeks after completion of IC followed by CCRT regimen for patients who had residual disease. Surgery was also allowed for patients who did not complete chemoradiation and had resectable residual disease at the primary site or in the neck. Patients were evaluated by CT scan or MRI of the head and neck every three months. The protocol for this study was approved by the Institutional Review Board of Chang Gung Medical Center (Taiwan).
Representative formalin-fixed, paraffin-embedded tissue blocks were retrieved and sectioned for the IHC study. The sections were deparaffinized, treated with 3% hydrogen peroxide for 10 min, and then microwaved in 10 mM citrate buffer (pH 6.0) to unmask the epitopes. The sections were then incubated with diluted OPN antibody (AKm2A1, 1 : 100) for 1 hr. After washing, horseradish peroxidase/Fab polymer conjugate (PicTure-Plus kit; Zymed, South San Francisco, CA) was applied to the sections for 30 min. Finally, the sections were incubated with diaminobenzidine for 5 min to develop the signals. A negative control study was simultaneously performed by omitting the primary antibody. All sections were evaluated by a pathologist, unaware of the clinical data. In the assessment of OPN expression, the sections were examined under a microscope at 200x magnification. Positive OPN immunostaining was defined as detectable immunoreactivity in the perinuclear and/or other cytoplasmic regions in at least 10% of the cancer cells. To evaluate expression of OPN, 10 fields (within the tumor and at the invasive front) were selected, and expression in 1000 tumor cells (100 cells/field) was evaluated using high-power (×400) microscopy [
Human SAS cells were obtained from American Type Culture Collection, cultured in DMEM with 10% FBS, 1% (v/v) penicillin-streptomycin solution, and maintained at 37°C in 5% CO2 humidified air. SAS cells (5 × 104) were seeded into 6-well dishes and cultured at 37°C in 5% CO2 humidified air. After 24 hours, shOPN and shControl plasmids were transfected into the cells with lipofectamine reagent according to manufacturer’s instructions, followed by further incubation for 24 hours at 37°C in 5% CO2. SAS cells stably expressing target genes were selected with 450
Cell pellets were lysed in RIPA lysis buffers (1 mM Na3VO4, 25 mM NaF, and 1x protease inhibitor cocktail protease inhibitor cocktail). Protein concentrations were determined by spectrophotometry. Sample was electrophoresed by 10% SDS-PAGE gel and transferred to PVDF membranes. The membranes were then blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with primary antibodies. Monoclonal anti-antibodies, OPN and
Cells were plated onto 6 wells at 1 × 105 cells/well and transfected with shControl or shOPN plasmids before incubation overnight. The next day, the cells were treated with 0 or 20
Cells were treated for the indicated time with chemotherapeutic drug (cisplatin) at the concentration for assay of cell survival.
Statistical analyses of 2 × 2 tables of categorical variables were performed using Pearson’s
We enrolled 121 eligible patients (117 males; 96.7%) from January 2006 to January 2012. The median age was 50.4 years (range 32–72 years). Thirty-five patients had IVa disease and 86 had stage IVb. All patients completed IC and received follow-up CCRT. Eighty-one (66.9%) patients received IC with PF and 40 received IC with TPF.
Before treatment, tumor OPN expression levels were assessed in all patients. To investigate whether there was a positive expression of OPN associated with various prognostic factors, such as age, gender, and TNM pathologic classification, we classified the patients into two groups based on their immunohistochemical results (negative
Correlation between expression of OPN and clinicopathological factors of stage IVa/b OSCC.
Number of patients | OPN |
|
Multivariates analysis |
| ||
---|---|---|---|---|---|---|
Negative expression | Positive expression | OR (95% CI) | ||||
Age | ||||||
|
102 (84.3%) | 22 (21.6%) | 80 (78.4%) | 0.764 | ||
|
19 (15.7%) | 5 (26.3%) | 14 (73.7%) | |||
Gender | ||||||
Male | 117 (96.7%) | 24 (20.5%) | 93 (79.5%) | 0.034 |
1 | |
Female | 4 (3.3%) | 3 (75%) | 1 (25%) | 0.067 (0.06–0.791) | 0.032 | |
Grade | ||||||
Well | 77 (63.6%) | 17 (22.1%) | 60 (77.9%) | |||
Moderate | 40 (33.1%) | 10 (25%) | 30 (75%) | 0.517 | ||
Poor | 4 (3.3%) | 0 (0%) | 4 (100%) | |||
Stage | ||||||
Iva | 35 (28.9%) | 12 (34.3%) | 23 (65.7%) |
|
1 | |
IVb | 86 (71.1%) | 15 (17.4%) | 71 (82.6%) | 2.755 (1.042–7.279) | 0.041 | |
T stage | ||||||
1/2 | 28 (10.5%) | 5 (17.9%) | 23 (82.1%) | 0.612 | ||
3/4 | 93 (89.5%) | 22 (23.7%) | 71 (76.3%) | |||
N stage | ||||||
Negative | 40 (21.1%) | 14 (35.0%) | 26 (65.0%) |
|
1 | |
Positive | 81 (78.9%) | 13 (16.0%) | 68 (84.0%) | 3.534 (1.363–9.164) | 0.009 | |
Alcohol drinking | ||||||
Never | 30 (24.8%) | 7 (23.3%) | 23 (76.3%) | 0.877 | ||
Yes | 91 (75.2%) | 20 (22.0%) | 71 (78.0%) | |||
Smoking | ||||||
Never | 29 (24.0%) | 11 (37.9%) | 18 (62.1%) |
|
||
Yes | 92 (76.0%) | 16 (17.4%) | 76 (82.6%) | |||
Betel nuts | ||||||
Never | 31 (25.6%) | 10 (32.3%) | 21 (67.7%) | 0.123 | ||
Yes | 90 (74.4%) | 17 (18.9%) | 73 (81.1%) |
OR, odds ratio; CI, confidence interval.
Immunostaining for OPN expression in OSCC patient samples. (a) The representative case of OSCC shows absence of OPN immunoreactivity (400x magnification). (b) The representative case of OSCC shows positive of OPN immunoreactivity in the cytoplasm of the neoplastic squamous epithelial cells (200x magnification). (c) Adjacent noncancerous tissue showed no OPN expression. (d) Statistical analysis showed that OPN expression levels in OSCC sample significantly correlated with gender, stage, N stage, and smoking.
The overall response rate after CCRT for all patients was 43.0%, 52/121 with 24 complete responses and 28/121 with partial responses. Thirty-four had stable disease and 35 had progressive disease. In our univariate analyses, patients with negative expression of OPN had a higher treatment response (19/27, 70.4%) than the positive expression group (33/94, 50%) (
Relationship between treatment response and clinicopathological factors.
Treatment response | Multivariates | ||||
---|---|---|---|---|---|
SD/PD | CR/PR |
|
OR (95% CI) |
| |
Age | |||||
|
58 (56.9%) | 44 (43.1%) | 1.000 | ||
|
11 (57.9%) | 8 (22.1%) | |||
Gender | |||||
Male | 67 (57.3%) | 50 (42.7%) | 1.000 | ||
Female | 2 (50%) | 2 (50%) | |||
Grade | |||||
Well | 41 (53.2%) | 36 (46.8%) | |||
Moderate | 24 (60%) | 16 (40%) | 0.165 | ||
Poor | 4 (100%) | 0 (0%) | |||
Stage | |||||
IVa | 13 (37.1%) | 22 (62.9%) |
|
1 | |
IVb | 56 (65.1%) | 30 (34.9%) | 0.310 (0.128–0.752) |
|
|
T stage | |||||
1/2 | 15 (53.6%) | 13 (46.4%) | 0.674 | ||
3/4 | 54 (58.1%) | 39 (41.9%) | |||
N stage | |||||
Negative | 16 (40.0%) | 24 (60.0%) |
|
1 | |
Positive | 53 (65.4%) | 28 (34.6%) | 0.354 (0.151–0.834) |
|
|
Chemotherapy | |||||
PF | 51 (63%) | 30 (37%) | 0.147 | ||
TPF | 18 (45%) | 22 (55%) | |||
Alcohol drinking | |||||
Never | 17 (56.7%) | 13 (43.3%) | 0.964 | ||
Yes | 52 (57.1%) | 39 (42.9%) | |||
Smoking | |||||
Never | 11 (37.9%) | 18 (62.1%) |
|
||
Yes | 58 (63.0%) | 34 (37.0%) | |||
Betel nuts | |||||
Never | 16 (51.6%) | 15 (48.4%) | 0.480 | ||
Yes | 53 (58.9%) | 37 (41.1%) | |||
OPN | |||||
Positive | 8 (29.6%) | 19 (70.4%) |
|
1 | |
Negative | 61 (64.9%) | 33 (35.1%) | 0.320 (0.120–0.854) |
|
CR, complete response; PR, partial response; SD, stable disease; PD, disease progression; PF, cisplatin/fluorouracil; TPF, docetaxel/cisplatin/fluorouracil; OR, odds ratio; CI, confidence interval.
The median follow-up was 27.3 months (3–75 months). The 5-year PFS rate was 24.2%, and the 5-year OS rate was 30.2%. The 5-year PFS for patients with negative expression of OPN was 61.8%, compared with 13.2% for patients with positive expression of OPN (
Correlation between the clinicopathological features and 5-year progression-free survival in oral squamous cell carcinoma.
Variables | Number of patients | Cumulative 5-year PFS rate |
|
5-year PFS |
|
---|---|---|---|---|---|
HR (95% CI) | |||||
Age | |||||
|
102 | 23.2% | 0.992 | ||
|
19 | 26.3% | |||
Gender | |||||
Male | 117 | 23.2% | 0.372 | ||
Female | 4 | 50.0% | |||
Stage | |||||
IVa | 35 | 38.7% |
|
1 | |
IVb | 86 | 20.0% | 2.097 (1.255–3.505) |
|
|
T stage | |||||
1-2 | 28 | 26.2% | 0.735 | ||
3-4 | 93 | 23.4% | |||
N stage | |||||
Negative | 40 | 47.3% |
|
1 | |
Positive | 81 | 12.0% | 1.940 (1.148–3.279) | 0.013 | |
Chemotherapy | |||||
PF | 83 | 23.0% | 0.465 | ||
TPF | 38 | 26.4% | |||
Alcohol | |||||
Never | 30 | 33.1% | 0.206 | ||
Yes | 91 | 20.8% | |||
Smoking | |||||
Never | 29 | 39.8% |
|
||
Yes | 92 | 19.0% | |||
Betel nuts | |||||
Never | 31 | 22.7% | 0.337 | ||
Yes | 90 | 23.5% | |||
OPN | |||||
Negative | 27 | 61.8% | <0.001 |
1 | |
Positive | 94 | 13.2% | 2.509 (1.256–5.009) |
|
CI, confidence interval; HR, hazard ratio.
Correlation between the clinicopathological features and 5-year overall survival in oral squamous cell carcinoma.
Variables | Number of patients | Cumulative 5-year overall survival rate |
|
HR (95% CI) |
|
---|---|---|---|---|---|
Age | |||||
|
102 | 31.0% | 0.628 | ||
|
19 | 26.3% | |||
Gender | |||||
Male | 117 | 29.4% | 0.415 | ||
Female | 4 | 50.0% | |||
Stage | |||||
IVa | 35 | 64.5% |
|
1 | |
IVb | 86 | 15.3% | 3.936 (2.129–7.277) |
|
|
T stage | |||||
1-2 | 28 | 38.7% | 0.488 | ||
3-4 | 93 | 27.4% | |||
N stage | |||||
Negative | 40 | 51.5% |
|
1 | |
Positive | 81 | 20.5% | 2.436 (1.422–4.175) |
|
|
Chemotherapy | |||||
PF | 83 | 24.7% | 0.123 | ||
TPF | 38 | 41.0% | |||
Alcohol | |||||
Never | 30 | 33.3% | 0.538 | ||
Yes | 91 | 29.1% | |||
Smoking | |||||
Never | 29 | 48.0% |
|
1 | |
Yes | 92 | 24.4% | 1.869 (1.014–3.355) | 0.036 | |
Betel nuts | |||||
Never | 31 | 33.0% | 0.096 | ||
Yes | 90 | 29.2% | |||
OPN | |||||
Negative | 27 | 53.0% |
|
1 | |
Positive | 94 | 23.7% | 2.036 (1.080–3.839) |
|
CI, confidence interval; HR, hazard ratio.
Kaplan-Meier estimates of the probability of survival. (a) Progression-free survival (PFS) and OPN expression. (b) Overall survival (OS) and OPN expression.
To elucidate the role of OPN in cell proliferation, recombinant human OPN was executed to SAS cells (human tongue carcinoma cell line) to determine if increased OPN protein could confer a proliferative advantage to SAS cells. The proliferation rate was significantly increased in matricellular-OPN in a dose-dependent manner in SAS cells (Figure
OPN promoted cell proliferation and drove cisplatin resistance in an oral cancer cell line. (a) SAS cells stimulated with OPN protein promoted cell growth. SAS cells were treated with indicated concentrations of OPN, and cell growth was analyzed on days 1–4 by MTT assay. Data were normalized against the OD570 value on day 1 of each treatment. The results represent the mean ± SD of three independent experiments. (b) OPN affected the chemosensitivity of OSCC cells to cisplatin. SAS cells were cultured in the presence or absence cisplatin and/or OPN in a dose-dependent manner, and their viability was measured. (c) OPN stimulated SAS cells were incubated with increasing concentrations of cisplatin for 72 hours, and their viability was measured and was expressed in percentage.
To gain further insight into the biological effect that OPN might have on chemoresistance to cisplatin, we performed MTT assay to assess cell viability in cells incubated with OPN and/or cisplatin treatment. As shown in Figure
The expression level of OPN was determined by western blotting and Q-RT-PCR in SAS cell transiently transfected with shControl or shOPN (Figures
Endogenous OPN depletion by OPN shRNA sensitized the cytotoxicity to cisplatin in head and neck cancer cells. (a and b) The endogenous expression level of OPN was determined by western blotting and quantitative RT-PCR in SAS cell transfected with shControl or shOPN. Data are representative of three independent experiments. (c) SAS cell transfected with shControl or shOPN were incubated with 20
In this study, we wanted to find out if there was a correlation between OPN expression and treatment response and clinical outcomes in OSCC patients and we performed experiments to determine whether OPN might play an important role in cisplatin resistance in an oral cancer cell line. OPN was positively expressed in 78 percent of our patients with stage IVA/B unresectable OSCC. Patients with this positive expression had a lower treatment response rate to IC followed by CCRT than those with negative expression of OPN (
OPN (Osteopontin), also known as SPP1, is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family and is secreted in mammalian cells. Secreted OPN has been discovered in tissue matrix, including bone, fibroblast, osteocyte, dendritic cells, macrophages, and activated T cells [
Only few studies, however, have addressed the role of OPN in chemotherapy resistance, though one previous report has shown pretreatment low plasma OPN expression led to better treatment response and longer survival for locally advanced HNSCC patients treated by CCRT [
Betel nut chewing, cigarette smoking, and alcohol consumption are important risk factors for the development of OSCC in South-East Asia and Taiwan, where they represent major threats to public health. It is of special interest that in our study specimens obtained from our patients who with habitually smoked cigarettes predominantly had a positive expression of OPN. Cigarette smoking has a long-standing association with cardiovascular disease and there is a wealth of evidence concerning the effects of smoking on key pathological processes such as vascular injury, vascular dysfunction, inflammation, and thrombosis [
Prognosis for nonmetastatic stage IV OSCC patients is dismal, though it has been found that IC followed by CCRT can prolong survival. Resistance to chemotherapy is always a major problem in progressive cancer, when tumor cells begin to amplify their proliferation, metastasis, and invasion to distant organs, leading to poor outcome and low survival [
It is of special interest in the current study that we did not find IC with TPF followed by CCRT to have a better response than PF followed by CCRT in OSCC. Moreover, we did not find stage IVa/b OSCC patients receiving TFP to have a better PFS and OS than those receiving PF. In locally advanced disease groups of patients, IC is known to improve response rates, quickly reduce symptoms, possibly predict subsequent radioresponsiveness, and lead to a reduced rate of distant metastases [
Our study is limited in that only a small number of our patients received TPF. Furthermore, studies of a more homogeneous, larger number of patients may be needed to validate our findings.
In conclusion, this study found that the positive expression of OPN predicts a poor response and survival to cisplatin-based IC followed by CCRT in patients with locally advanced stage IVa/b OSCC. Thus, we conclude that OPN mediated cisplatin resistance contributes to a poorer clinical outcome in patients with locally advanced inoperable OSCC treated with cisplatin-based IC and CCRT. In this context, it is strongly recommended that tissue be collected to assess OPN expression before cisplatin-based IC and CCRT are started. If patients with lymph node metastases and advanced tumors may have higher change of positive OPN expression, then other treatment modalities may be considered.
The authors report no declarations of interest.
Yi-Ju Chen and Sheng-Dean Luo conceived the study design, carried out and coordinated immunohistochemical examinations of tumor specimens and data analysis, and drafted the paper. Chang-Han Chen and Hsin-Ting Tsai participated in the interpretation of data and conducted immunohistochemistry analysis. Chien-Ting Liu, Yi-Ching Chen, and Kun-Ming Rau collected the clinical data of patients and performed statistical data analysis. Tai-Jan Chiu and Chang-Han Chen coordinated the study and were involved in drafting the paper and revised it critically. All authors read and approved the final paper. Chang-Han Chen and Tai-Jan Chiu contributed equally to this study.
This study was funded by grants obtained by Dr. Chang-Han Chen from Kaohsiung Chang Gung Memorial Hospital Taiwan (Grant nos. CMRPG8A0391-2, CMRPG8B1251-3, and CMRPG8C0591-2) and from the Ministry of Science and Technology (MOST-102-2320-B-010, MOST-103-2320-B-182A-105, and MOST-104-2320-B-182A-010). This study was also funded by grants obtained by Dr. Tai-Jan Chiu from Kaohsiung Chang Gung Memorial Hospital Taiwan (Grant nos. CMRPG8C0531 and CMRPG8D0801). The authors thank the Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, for providing them with the instruments for this study (CLRPG871343).