Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 10  |  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

Correspondence Address:
Seema Gupta
Department of Radiotherapy, C.S.M. Medical University, Chowk City, Lucknow - 226 003, Uttar Pradesh
India

Abstract

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.



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;10:258-264


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 2021 Jan 27 ];10:258-264
Available from: https://www.cancerjournal.net/text.asp?2014/10/2/258/136549


Full Text

 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. [1] 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. [2],[3]

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. [4],[5],[6]

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. [7],[8]

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.

Statistical analysis

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



Patient characteristics

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}{Table 1}

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}

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}

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}

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}

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}{Table 6}

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}

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. [2] 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. [3],[6],[9] 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. [9]

In a recent study in oral cancer in 2009 Anak Iamaroon and Suttichai Krisanaprakornkit [3] 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. [10]

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. [11]

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. [3] 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.

References

1Al-Sarraf M. Treatment of locally advanced head and neck cancer: Historical and critical review. Cancer Control 2002;9:387-99.
2Gold KA, Kim ES. Role of molecular markers and gene profiling in head and neck cancers. Curr Opin Oncol 2009;21:206-11.
3Bussink J, van der Kogel AJ, Kaanders JH. Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer. Lancet Oncol 2008;9:288-96.
4Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv Cancer Res 1996;68:67-108.
5Motokura T, Arnold A. Cyclin D and oncogenesis. Curr Opin Genet Dev 1993;3:5-10.
6Musgrove EA. Cyclins: Roles in mitogenic signaling and oncogenic transformation. Growth Factors 2006;24:13-9.
7Shiraki M, Odajima T, Ikeda T, Sasaki A, Satoh M, Yamaguchi A, et al. Combined expression of p53, cyclin D1 and epidermal growth factor receptor improves estimation of prognosis in curatively resected oral cancer. Mod Pathol 2005;18:1482-9.
8Pesutiæ-Pisac V, Punda A, Glunciæ I, Bedekoviæ V, Praniæ-Kragiæ A, Kunac N. Cyclin D1 and p27 expression as prognostic factor in papillary carcinoma of thyroid: Association with clinicopathological parameters. Croat Med J 2008;49:643-9.
9Liang J, Slingerland JM. Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression. Cell Cycle 2003;2:339-45.
10Iamaroon A, Krisanaprakornkit S. Overexpression and activation of Akt2 protein in oral squamous cell carcinoma. Oral Oncol 2009;45:E175-9.
11Schuurbiers OC, Kaanders JH, van der Heijden HF, Dekhuijzen RP, Oyen WJ, Bussink J. The PI3-K/AKT-pathway and radiation resistance mechanisms in non-small cell lung cancer. J Thorac Oncol 2009;4:761-7.