|Year : 2014 | Volume
| Issue : 2 | Page : 258-264
Prognostics of Cyclin-D1 expression with chemoradiation response in patients of locally advanced oral squamous cell carcinoma
Huma Khan1, Seema Gupta1, Nuzhat Husain2, Sanjeev Misra3, Navin Singh1, MPS Negi4
1 Department of Radiotherapy, C.S.M. Medical University, Lucknow, Uttar Pradesh, India
2 Department of Pathology, C.S.M. Medical University, Lucknow, Uttar Pradesh, India
3 Department of Surgical Oncology, C.S.M. Medical University, Lucknow, Uttar Pradesh, India
4 Biometry and Statistics Division, Institute for Data Computing and Training, Lucknow, Uttar Pradesh, India
|Date of Web Publication||14-Jul-2014|
Department of Radiotherapy, C.S.M. Medical University, Chowk City, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Objective: Cyclin-D1 has been strongly implicated in cell cycle proliferation particularly in the G1/S checkpoint in the cell cycle, and prognosis in many human malignancies. The present study evaluates its prognostic significance with chemoradiation response in patients of locally advanced oral squamous cell carcinoma (OSCC).
Materials and Methods: A total of 97 OSCC patients (females = 19 and males = 78), aged 20-67 years and stage III/IV were recruited. Treatment response was assessed according to World Health Organization criteria. Cyclin-D1 expression in tumor tissue was estimated by immunohistochemical method and quantified as percentage positive nuclei.
Results: The Cyclin-D1 expression showed significant (P < 0.01 or P < 0.001) association with tumor size, lymph node status, and clinical stage. After chemoradiation, there were 53.6% complete response (CR) and 34.0% partial response (PR) in primary tumor, and 49.5% CR and 39.2% PR in lymph node; giving an overall response rate of 85.6%. Further, the mean Cyclin-D1 expression showed significant (P < 0.05 or P < 0.001) and inverse association with chemoradiation responses (tumor size, lymph node status and overall treatment response). The 2-year progression-free and overall survival (OS) was 95.89% and 83.31% respectively. Multivariate Cox regression analysis found site of primary tumor, clinical stage, and Cyclin-D1 expression the significant (P < 0.05 or P < 0.01) and independent prognostic markers of OS and among these Cyclin-D1 expression showed the worst prognosis. The high Cyclin-D1 expression (>50%) also showed significantly lower survival in OSCC patients when compared with those had low (<10%) and moderate expressions (10-50%) (Logrank test: χ2 = 44.42, P < 0.001).
Conclusion: The high Cyclin-D1 expression may serve as a poor prognostic marker in OSCC.
目的：细胞周期蛋白D1主要在细胞周期的G1 / S检查点发挥作用，与细胞增殖高度相关，特别是，并与多种恶性肿瘤的预后相关。本研究评估其在局部晚期口腔鳞状细胞癌患者放化疗反应（OSCC）的预后意义。
材料和方法：共97例口腔癌患者（女性19，男性为78），20岁到67岁， III / IV期患者。治疗反应是根据世界卫生组织的标准进行评估。在肿瘤组织中Cyclin D1的表达采用免疫组化方法估计并按阳性核百分比定量。
结果：细胞周期蛋白D1的表达与肿瘤大小，淋巴结状态，临床分期显著相关（P＜0.01或P＜0.001）。放化疗后，有53.6%的病人完全缓解（CR），34%部分缓解（PR），在淋巴结有49.5% CR和39.2% PR；整体反应率为85.6%。此外，平均细胞周期蛋白 D1的表达与放化疗反应（肿瘤大小，淋巴结状态和整体治疗的反应）呈显著负相关（P＜0.05或P＜0.001）。2年的无进展生存和总体生存率（OS）分别为95.89%和83.31%。多因素Cox回归分析发现原发肿瘤的位置、临床分期，和细胞周期蛋白D1的表达显著相关（P＜0.05或P＜0.01），是独立的预后标志，在细胞周期蛋白中，D1高表达的病例预后最差。在口腔鳞状细胞癌患者中，细胞周期蛋白D1的高表达组（＞50%）与低表达组（＜10%）和中表达组（10%~50%）相比生存率较低（对数秩检验：χ2＝44.42，P＜0.001）。
Keywords: Chemoradiation, cyclin-D1, immunohistochemistry, squamous cell carcinoma
|How to cite this article:|
Khan H, Gupta S, Husain N, Misra S, Singh N, Negi M. Prognostics of Cyclin-D1 expression with chemoradiation response in patients of locally advanced oral squamous cell carcinoma. J Can Res Ther 2014;10:258-64
|How to cite this URL:|
Khan H, Gupta S, Husain N, Misra S, Singh N, Negi M. Prognostics of Cyclin-D1 expression with chemoradiation response in patients of locally advanced oral squamous cell carcinoma. J Can Res Ther [serial online] 2014 [cited 2019 Nov 22];10:258-64. Available from: http://www.cancerjournal.net/text.asp?2014/10/2/258/136549
| > Introduction|| |
It is the commonest cancer in India with a large fraction of cases occurring in males in their productive years of life. Mostly cases (80%) present in late stages and survival is poor with all modalities of treatment.  Response to treatment varies with patients of same stage disease, therefore there is need to develop new treatment strategies to improve survival in these patients. Variability in the clinical course of the disease possibly due to poor monitoring of oral squamous cell carcinoma (OSCC) in absence of a reliable biomarkers increases morbidity and mortality. ,
In radiation response expression and activation of cyclins, cyclin-dependent kinases (Cdks) and Cdk inhibitors which are known to be inhibitors of apoptosis play vital role through activation of survival pathways. ,,
Therefore, identification of suitable marker that could provide prognostic assessment of the disease and would help in designing most appropriate and effective treatment strategies for OSCC is warranted, so that limited resources available to patients can be conserved and undue treatment can be avoided.
The current study is hence proposed to assess the levels of Cyclin-D1 and its prognostic significance with treatment response in oral cancer patients undergoing chemoradiation.
| > Materials and Methods|| |
Totally 97 histologically proven cases of locally advanced stage (III, IV) oral cancer with World Health Organization (WHO) performance status of grade 0/1 attending radiotherapy out patient department at C.S.M. Medical University, Lucknow (UP), India, in the year 2008-2011 were enrolled in the study. These cases were assessed thoroughly (history, clinical examination, and investigations). The study was approved by the ethics committee of the C.S.M. Medical University and written informed consent was obtained from all patients before enrolment.
All the patients were given two cycles of induction taxol (175 mg/m 2 day 1) and cisplatin (50 mg/m 2 day 2) chemotherapy and were subjected for radiation along with concurrent cisplatin (35 mg/m 2 ) 4-weeks from completion of induction chemotherapy. Radiotherapy was given by External beam Conventional Method (200 CGy/fraction to a total dose of 70 Gy in 35 fractions in 7 weeks by cobalt 60 to primary tumor site and neck.
The protocol plan was continued despite mucositis or dermatitis. However, the dose of cisplatin was reduced to 50% if the calculated creatinine clearance level was 30-50 ml/min. No cisplatin was given if the creatinine clearance level was less than 30 ml/min. In presence of myelosuppression white blood cell (count <4000/mm 3 or platelets count less than 100,000/mm 3 ), persistent fever that exceeded 38°C or other clinically apparent infections, chemoradiation was postponed for 1 week or interrupted.
For histopathologic and immunohistochemical studies, tumor samples from the lesion site was fixed in 10% buffered formalin and then embedded in paraffin. Paraffin-embedded formalin-fixed tissues were processed, routine H and E, stained sections evaluated to confirm the diagnosis of squamous cell carcinoma and to grade the lesion. Further sections were processed for Cyclin-D1 biomarker by immunohistochemistry using primary monoclonal antibody to Cyclin-D1 from Dakopatts Denmark and a polymer-based secondary antibody detection kit from Dakopatts, Denmark. Standard immunohistochemistry protocol was used. In short deparaffinized rehydrated sections were blocked for endogenous peroxidases in 0.3% hydrogen peroxide in methanol, followed by a rinse in distill water. Antigen retrieval was achieved at 121°C in 10 mm citrate buffer (pH 6.0) for 10 min using Pascal retrieval system from Dakopatts, Denmark. Slides cooled to room temperature were washed thrice with Tris-buffered saline (TBS) and incubated overnight with primary antibody Cyclin-D1 at 4°C. After washing with TBS, the sections were incubated for 30 min with secondary antibody. Cyclin-D1 was visualized with Dako Denmark liquid diaminobenzidine substrate chromogen and counterstained with diluted Mayer's hematoxylin. Sections mounted with dibutyl phthalate xylene mounting media were inspected under a Zeiss Z2 imager and photographed at ×40 magnification.
The immunohistochemical evaluation was carried out in tumor hotspots including the invasion front, which was regarded as most indicative of the biologic activity of the tumor, in 10 high power fields. About 1500-2000 tumor cells were observed in all tumors at a magnification of ×40 in 10 selected fields. Cyclin-D1 in tumors was labeled as negative if <10%, moderately positive between 10% and 50%, and strongly positive if >50%, tumor cells expressed the antigen. ,
Assessment of tumor response was done by clinical examination, radiologic investigations (computed tomography [CT]-scan) 4-6 weeks after completion of treatment. Biopsy or fine needle aspiration cytology to determine pathologic response was not performed routinely; it was done only in case of partial response (PR)/suspected lesion to confirm the presence of disease. After chemoradiation, patients were followed-up to 2 years.
The definitions of treatment response viz. complete response (CR), PR, and no response (NR) (stable disease [SD] + progressive disease [PD]) were based on the standard definitions established by WHO (1979).
End point was to evaluate clinical benefits of chemoradiation on response rate, 2 year overall survival (OS) and prognostic significance of Cyclin-D1 expression with OS in locally advanced squamous cell carcinoma of oral cavity.
Cyclin-D1 expression (%) data were summarized as Mean ± SD The associations of Cyclin-D1 expression with basic characteristics (demographic and clinicopathologic) and chemoradiation response in OSCC patients were compared by non-parametric Mann-Whitney U test and Kruskal-Wallis one-way analysis of variance. Survival between groups was carried out by Kaplan-Meier method and the difference between the groups was carried out by Logrank (χ2 ) test. The univariate and multivariate Cox proportional hazard analysis was used to assess the prognostic significance of different variables on OS. A two-sided (α =2) P < 0.05 was considered statistically significant.
| > Results|| |
The frequency distributions of basic characteristics (demographic, clinicopathologic, and Cyclin-D1 expression) of 97 OSCC patients were summarized in [Table 1]. The age of all patients ranged from 20 to 67 years with mean (± SD) 50.09 ± 12.15 years and median 52 years. Most of the patients were >50 years (50.5%), mostly males (80.4%) and mostly having good Eastern cooperative oncology group performance status (56.7%). The buccal mucosa was the most prevalent site of primary tumor (24.7%), mostly with well-differentiated histologic grading (67.0%), high tumor size T4 (75.3%), negative lymph node status N0 (35.1%), and high clinical stage IV (72.2%). Of total patients, 12 patients (12.4%) had low Cyclin-D1 expression (<10%), 63 (64.9%) moderate (10-50%), and 22 (22.7%) high (>50%) [Figure 1].
|Figure 1: Microphotograph showing immunohistochemical expression of Cyclin-D1 in oral squamous cell carcinoma (a) Showing negative nuclei. (b) Showing moderately positive stained nuclei. (c) Showing strongly positive stained nuclei 3,3'-diaminobenzidine (×125 × digital magnification)|
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|Table 1: Frequency distribution of demographic, clinicopathological and Cyclin D1 expression of oral squamous cell carcinoma patients (n=97)|
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Cyclin D1 expression
The associations of Cyclin-D1 expression with basic and clinicopathologic characteristics of OSCC patients were summarized in [Table 2]. [Table 2] showed that the mean expression of Cyclin-D1 had a significant and positive (direct) correlation with tumor size (T3 < T4, P = 0.008), lymph node status (N0 < N1 < N2, P < 0.001), and clinical stage (III < IV, P < 0.001). However, the mean expression of Cyclin-D1 did not (P > 0.05) showed any association with age, sex, ECOG performance status, site of primary tumor. and histologic grading [Figure 1].
|Table 2: Association of Cyclin D1 expression with demographic and clinicopathological characteristics in oral squamous cell carcinoma patients (n=97)|
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Treatment response and survival
The chemoradiation response of OSCC patients were summarized in [Table 3]. After chemoradiation, there were 53.6% CR and 34.0% PR in tumor, and 49.5% CR and 39.2% PR in lymph node status; giving response rate of 87.6% and 88.7%, respectively. The overall response rate of chemoradiation was 85.6% whereas NR rate was 14.4%.
|Table 3: Treatment response in oral squamous cell carcinoma patients (n=97)|
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The relationship between Cyclin-D1 expression and chemoradiation responses (tumor size, lymph node status, and overall treatment response) in OSCC patients were summarized in [Table 4]. The mean Cyclin-D1 expression showed significant (P < 0.001) inverse associations with all chemoradiation responses, i.e. as response decreases, Cyclin-D1 expression increases. Further, for each chemoradiation response, comparing the mean Cyclin-D1 expression among responses (CR, PR and NR), the expression of it in PR and NR of all chemoradiation responses was found to be significantly (P < 0.001) different and higher when compared with CR except tumor size, between CR and PR. Furthermore, in all chemoradiation responses, the expression of Cyclin-D1 was also found to be significantly (P < 0.05 or P < 0.001) different and higher in NR when compared with PR [Figure 1].
|Table 4: Association between Cyclin D1 expression and chemoradiation response in oral squamous cell carcinoma patients (n=97)|
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After chemoradiation, to see the survival, patients were followed up to 2 years. Of total, 15 (15.5%) patients died due to progression of disease. The 2-year progression-free survival (CR + PR), OS (CR + PR + NR), and survival according to treatment response (CR, PR and NR) were summarized in [Figure 2]. The 2 year mean (± SE) progression free survival and OS rate was 95.89 ± 2.33% and 83.31 ± 3.95%, respectively [Figure 2]. Further, the 2 year OS in patients with CR and PR were significantly different and higher when compared with patients with NR (Logrank test: χ2 = 109.80, P < 0.001) [Figure 2], indicating that treatment significantly improved the OS in OSCC patients.
|Figure 2: The 2-year progression-free survival. (a) Overall survival. (b) And survival according to treatment response (c) In oral squamous cell carcinoma patients|
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Prognostic of Cyclin-D1 expression
The prognostic significance of basic characteristics, clinicopatholigic features, and Cyclin-D1 expression with 2 year OS were assessed by univariate and multivariate Cox regression hazard analysis and summarized in [Table 5] and [Table 6], respectively. The univariate analysis [Table 5] showed that the site of primary tumor (Lip vs. Alveolus: Hazard ratio [HR] =2.47, 95% confidence interval = 1.06-5.76, P = 0.037), histologic grading well differentiated ( vs. PD: HR = 0.32, 95% CI = 0.13-0.80, P = 0.014), lymph node status (N0 vs. N1 + N2: HR = 0.65, 95% CI = 0.43-1.00, P = 0.047), clinical stage (III vs. IV: HR = 0.63, 95% CI = 0.40-0.99, P = 0.046), and Cyclin-D1 expression (Low vs. Moderate: HR = 0.39, 95% CI = 0.19-0.80, P = 0.010; Low vs. High: HR = 0.40, 95% CI = 0.24-0.67, P < 0.001) was significantly associated with OS [Figure 1].
|Table 5: Univariate Cox proportional hazards analysis of overall survival according to demographic, clinicopatholical and Cyclin D1 expression in oral squamous cell carcinoma patients (n=97)|
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|Table 6: Multivariate Cox proportional hazards analysis of overall survival according to significant clinicopathological and Cyclin D1 expression in oral squamous cell carcinoma patients (n=97)|
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The multivariate analysis [Table 6] of significant variables identified in univariate analysis (site, histologic grading, lymph node status, clinical stage, and Cyclin-D1 expression) showed that the site (Lip vs. Alveolus: HR = 3.08, 95% CI = 1.29-7.34, P = 0.011), clinical stage (III vs. IV: HR = 0.60, 95% CI = 0.37-0.97, P = 0.038), and Cyclin-D1 expression (Low vs. Moderate: HR = 0.35, 95% CI = 0.16-0.77, P = 0.009; Low vs. High: HR = 0.37, 95% CI = 0.21-0.67, P = 0.001) were significant and independent prognostic (poor) markers of OS in OSCC patients [Figure 1].
The 2-year OS according to site of primary tumor, histologic grading, tumor size, lymph node status, clinical stage, and Cyclin-D1 expression were further analyzed by Kaplan-Meier survival analysis and summarized graphically in [Figure 3]. The Kaplan-Meier survival analysis also revealed that the lymph node status and Cyclin-D1 expression associated significantly (P < 0.05 or P < 0.001) with the OS whereas site of primary tumor, histologic grading, tumor size, and clinical stage did not (P > 0.05) show any association with the OS. However, patients with positive lymph node status (N1 + N2) and high Cyclin-D1 expression showed significantly lower survival when compared with patients with negative lymph node status (N0) (Logrank test: χ2 = 5.86, P = 0.016) and both low and moderate Cyclin-D1 expression (Logrank test: χ2 = 44.42, P < 0.001). The multivariate Cox regression hazard analysis and Kaplan-Meier survival analysis both concluded Cyclin-D1 expression the better prognostic marker among the studied markers (variables).
|Figure 3: The 2 year survival according to site of primary tumor. (a) Histologic grading. (b) Tumor size. (c) Lymph node status. (d) Clinical stage (e) And Cyclin D1 expression (f) Of oral squamous cell carcinoma patients|
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Immunohistochemical expression of Cyclin-D1
Microphotograph of immunohistochemical expression of Cyclin-D1 in OSCC patients also correlated well with findings of higher nuclear positivity for cyclin-D1 with tumor stage and treatment outcome. Patients with high Cyclin-D1 expression showed significantly lower survival when compared with patients with low and moderate Cyclin-D1 expression [Figure 1].
| > Discussion|| |
Chemoradiation is the mainstay of treatment in oral cancer worldwide, but the response rate overall is about 30% only, which may vary among individuals.  Most of these patients eventually develop resistance to treatment through a mechanism that remains obscure, which could be explained probably by difference in the molecular pathology of the disease.
Cyclins, Cdks, serine/threonine glycogen synthetase kinase 3, Cdk inhibitors and serine/threonine protein kinase 1, AKT2, AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase which are also associated with regulation of cell cycle progression and prevention of apoptosis which is associated with tumor genesis and resistance to apoptosis, chemotherapy, and ionizing radiation. ,, These could be the potential targets for overcoming the treatment resistance.
Uncontrolled cell division leading to tumorigenesis coordinated by regulatory proteins including cyclins, Cdks, and Cdk inhibitors can be modulated by activation of PI3K/AKT signaling. 
In a recent study in oral cancer in 2009 Anak Iamaroon and Suttichai Krisanaprakornkit  have demonstrated that Akt2 and p-Akt were overexpressed in OSCC and may be involved in carcinogenesis and suggests that post-transcriptional modification of Akt2 in Oral squamous cell cancer may occur. 
Activation of this survival pathway comprising of AKT/PIK3, Cdks, Cyclin-D1 can be the result of stimulation of receptor tyrosine kinases such as epidermal growth factor receptor or vascular endothelial growth factor receptor or from mutations or amplification of PI3-K or AKT itself, these have been documented in non-small cell lung cancer. 
Furthermore, several treatment modalities such as radiotherapy, chemotherapy can also stimulate this survival pathway. Monitoring and manipulation of this signal transduction pathway which forms the basis of treatment resistance to chemoradiation may have important implications for the management of cancer.  Direct targeting and inhibition of this pathway may increase radiosensitivity by antagonizing the radiation-induced cellular defence mechanisms especially in tumors that have activated the PI3-K/AKT cascade. More importantly, specific targeting of this pathway in combination with radiotherapy or chemotherapy may enhance tumor control by antagonizing cellular defence in response to treatment.
Although many markers have been studied and have given new understanding of cancer pathogenesis and progression, they are not yet ready to be used in routine clinical investigative and therapeutic procedures in patients with these tumors. Tumor stage, patient's age, and performance status still remains the basis for therapeutic decisions. In view of this further studies are needed urgently to understand more specific and sensitive markers aiding in tumor diagnosis, selection of treatment modality, monitoring of response to therapeutic interventions, early detection of tumor recurrence, prediction of the results from treatment modality and identification of subsets of patients with unfavorable outcome during the therapeutic interventions and follow-up which would aid in prognosticating, predicting, and deciding therapeutic interventions to achieve beneficial outcome in response and survival of patients with these tumors.
| > Conclusion|| |
It is important to identify the patients likely to respond favorably to treatment so that the limited resources can be used judiciously. The patients with unfavorable expression of Cyclin-D1 invariably do not respond to standard chemoradiation treatment regimen in oral cancer, this helps in the selection and prediction of treatment outcome. Study found patients with positive lymph node status and high Cyclin-D1 expression had significantly lower survival than patients with negative lymph node status, and low and moderate Cyclin-D1 expression. Thus, positive lymph node status and high Cyclin-D1 expression may be the poor prognostics markers of chemoradiation response with patients of locally advanced OSCC.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]