|Year : 2014 | Volume
| Issue : 1 | Page : 24-30
|The correlation between p16 expression and INK4a locus mutation with grades and stages in oral squamous cell carcinoma
Soudabeh Sargolzaei1, Sareh Farhadi2, Bahram Kazemi3, Mojgan Bandehpour3, Mohammad Javad Kharazifard4
1 Departments of Oral and Maxillofacial Pathology, Faculty of Dentistry, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Oral and Maxillofacial Pathology, Shahed University, Tehran, Iran
3 Department of Cellular and Molecular Biology Research, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Dental Research, Faculty of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
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|Date of Web Publication||17-Apr-2014|
| Abstract|| |
Objective: p16INK4a is a tumor suppressor gene playing a critical role. Researches have indicated the gene to be altered in oral squamous cell carcinoma. Present studies have tried to assess the correlation between p16INK4a expression and INK4a locus mutation in relation to grades and stages of this tumor. Materials and Methods: Expression of p16INK4a was studied immunohistochemically in 58 oral squamous sell carcinoma samples and INK4a locus mutation was determined by polymerase chain reaction (PCR) and conformation sensitive gel electrophoresis (CSGE). Results: Expression of p16INK4a was higher in stage1 compared to stage 2, 3, and 4 (P = 0.234). The difference was not significant in grade 1, 2, and 3 (P = 0.671). The average values of total score (TS) were significantly higher in stage1 compared to stage 2, 3, and 4 (P = 0.035). The average values of complete score (CS) were higher in stage 1 compared to stage 2, 3, and 4 (P = 0.061). The research did not show a significant correlation between lymph node involvement and p16INK4a expression (P = 0.491). It seems that 5.1% (3/58) of samples have mutation in INK4a locus. Conclusion: Loss of p16INK4a expression occurred in initial stages of oral squamous cell carcinoma. Evaluation of TS and CS for p16INK4a might be a useful clinical indicator concerning the tumor. However, gene mutation is believed to have minor rate of genetic alteration in carcinogenesis.
Keywords: Oral squamous cell carcinoma, p16INK4a , stage, grade
|How to cite this article:|
Sargolzaei S, Farhadi S, Kazemi B, Bandehpour M, Kharazifard MJ. The correlation between p16 expression and INK4a locus mutation with grades and stages in oral squamous cell carcinoma. Indian J Pathol Microbiol 2014;57:24-30
|How to cite this URL:|
Sargolzaei S, Farhadi S, Kazemi B, Bandehpour M, Kharazifard MJ. The correlation between p16 expression and INK4a locus mutation with grades and stages in oral squamous cell carcinoma. Indian J Pathol Microbiol [serial online] 2014 [cited 2020 Feb 19];57:24-30. Available from: http://www.ijpmonline.org/text.asp?2014/57/1/24/130877
| Introduction|| |
Oral cancer represents 3-5% of all human malignant neoplasms worldwide.  Oral squamous cell carcinoma (OSCC)is the most common malignancy of the oral cavity, which accounts for more than 90% of malignant neoplasms in this area.  squamous cell carcinoma of Head and Neck is the sixth most prevalent cancer worldwide.  In spite of diagnostic and therapeutic advancements made as well as increased understanding of the molecular mechanism of tumor progression, the overall survival rate of OSCC has not improved , , as remained constant at approximately 50% over past 30 years. 
Multiple genetic alterations consisting of inactivation of tumor suppressor genes and activation of proto-oncogenes are thought to be responsible for malignant transformation.  Alterations in cell cycle regulators were reported in several types of human cancers including oral cancer.  Therefore, alterations of their marker expression and gene mutations may be a biomarker for screening more invasive behaviors in cancer progression.
The p16 INK4a gene is one of the negative regulators of cell cycle. The p16 INK4a protein binds to the cyclin-dependent kinases (CDKs) CDK4 and CDK6 and then inhibits their activities, which results in hypophosphorylation of retinoblastoma protein (pRb) as well as inhibition of cell cycle progression. Many human tumor cell lines and some primary tumoral tissues show low expression of p16 INK4a protein.  Further, they reveal alterations of its gene during the processes of cancer progression and metastasis. Surprisingly, a recent study demonstrates increased expression level of p16 INK4a protein in some cases of OSCC.  In addition to this, a number of other studies have also pointed to its involvement in the correlation of its expression and the clinical behaviors concerning the stage and the grade of disease.
We have extensively analyzed and examined the expression level of p16 protein and the probable mutation in INK4a locus of p16 gene in primary tumors of OSCC, and their correlation with the stage and the grade of the disease. In the following sections is given a summary of our research.
| Materials and Methods|| |
A total of 58 archival primary OSCC specimens were obtained from general pathology department of Taleghani hospital and oral and maxillofacial department of Shahid Beheshti University's dental school. All specimens were fixed in formalin 10% and embedded in paraffin. At the grading and staging phases, the specimens were carefully evaluated and analyzed in accordance with the standards of the World Health Organization (WHO) criteria. 
The majority of samples were from buccal mucosa (18 cases), followed by tongue (16 cases), palate and lip (each 7 cases), floor of the mouth (4 cases), and gingiva and retromolar (each 3 cases). There were 14 cases in stage 1, 16 in stage 2, 15 in stage 3, and 13 cases in stage 4. Furthermore, 36 cases of well-differentiated OSCC, 19 moderately differentiated cases, and 3 poorly differentiated cases were analyzed in this study.
Three-micrometer-thick tissue sections were deparaffinized and rehydrated. The staining procedure was performed according to the instructions given in the CINtec™ p16 INK4a histology kit (MTMLabs, Heidelberg, Germany). Antigen retrieval procedure was briefly conducted by immersing the sections in Antigen Retrieval Epitope (following the kit details) and boiled in a microwave oven in three stages for 5 min (180 W), 10 min (600 W), and 5 min (450 W), respectively. Next, the slides were washed thoroughly in wash buffer (MTMLabs; cat No. 8550) and allowed to cool at room temperature. In order to inhibit endogenous peroxidase activities, the samples were carefully treated with peroxidase-blocking reagent for 30 min at 37°C. The slides were subsequently incubated with ready-to-use mouse anti-human p16 INK4a antibody clone E6H4 CINtec™ histology kit (MTMLabs) or negative control reagent for 30 min at 37°C.At the next step,, the slides were treated with visualization reagent for 30 min at 37°C for elimination of cross-reactivity with human immunoglobulins. By then, 3,3΄-diaminobenzidine (DAB) chromogen, containing DAB substrate liquid, was applied for the visibility of reaction products. The sections were washed in running tap water, stained with Hematoxylin, dehydrated, and mounted. A section of uterus cervical squamous cell carcinoma was used as a positive control in each run.
Interpretation of p16 expression
Each p16-stained slide was evaluated by two oral pathologists. For quantification, the number of positive keratinocytes was counted under a light microscope (400× magnification). We considered the staining positive when nuclear and/or cytoplasmic staining was compatible with that of positive control tissue. Positive cases were defined as having at least five stained cells [Figure 1].
|Figure 1: Severe p16INK 4a expression in moderately differentiated SCC (200× magnification)|
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In specimens with a large number of positive cells, a total of 1000 cells were randomly assessed by counting 10 fields including 100 cells and the average number of positive cells was presented as the percentage. Briefly, the quantification of positivity was categorized into three expression scores (ES): "0" for specimens with less than five positive cells, "1" for those having 20% positive cells or less, and finally, "2" for those having more than 20% of positive cells.
The staining intensity score was recorded as intensity score (IS) of "1" or "weak," "2"or "moderate," and "3" or "severe," when compared with positive control [Figure 1].
Based on the correlation between p16 expression and clinicopathologic factors, we evaluated the total score (TS) for all specimens using the formula: TS = ES × IS. To our knowledge, this was the first evaluation of TS for p16 in OSCC. Finally, in order to evaluate the proportion, intensity, and location of p16 expression consequently, we introduced a new score (complete score or CS) using the formula: CS = TS × LS (location score). The location score was "1" for positivity of nuclear or cytoplasmic expression and "2' for the presence of both.
Preparation of DNA
For each OSCC sample, a 5-μm-thick section was prepared for DNA extraction. Xylene and ethanol were used in order to remove paraffin. The deparaffinized tissues were incubated with lysis buffer (Tris 10 mM, SDS (sodium dodecyl sulphate) 10%) at 37°C overnight. DNA extraction was carried out by phenol-chloroform method, followed by ethanol precipitation.
Polymerase chain reaction
A fragment of 500 bp DNA was designed for p16INK4a gene amplification using the primers (CinnaGen, Tehran, Iran) shown in [Table 1]. The reaction mixture consisted of polymerase chain reaction (PCR) 10X buffer [100 mM Tris-HCl, pH = 8.3, 50 mM KCl, 1.5 mM MgCl 2 , and 0.1% w/v gelatin], 0.2 mM dNTP, 10 pmol of each primer, 1.5 units of Taq polymerase, and 0.5 μg genomic DNA. Blood sample was used as positive control in each run. PCR amplification consisted of 30 cycles of denaturation at 94°C for 40 sec, annealing at 63°C for 60 sec, and extension at 72°C for 60 sec. The procedure was conducted at 94°C and 72°C for 5 min before and after PCR cycling, respectively. PCR products were analyzed by electrophoresis on a 2% agarose gel containing cyber green [Figure 2].
|Figure 2: 1.5% agarose gel electrophoresis, 500 bp p16 PCR product (left), 100 bp DNA ladder (right)|
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For screening of mutation in the samples, we used conformation sensitive gel electrophoresis (CSGE) method. A mixture of 10-15 μl PCR product of each sample and control PCR product, together with 1 μl of loading buffer was loaded in Universal Detection System. Acrylamide-BAP (bis-acrolylpiperazine) (40% w/v) gel and 20X TE buffer, pH = 9 [Tris 1.78 mM, taurine 570 mM, and ethylene diamine tetra acetic acid (EDTA) 4 mM] were used for electrophoresis which lasted approximately 6 h. The specimens with mutation had a different rate during electrophoresis compared to those without it. The difference was shown as double bands [Figure 3].
In order to derive significant staining differences in the four stages and find the correlation between staining scores and clinical features, the results were statistically analyzed by binary logistic regression test, backward method, and also Fisher's exact test, using SSCP software version 16. P < 0.05 was considered significant.
| Results|| |
The expression of p16INK4a and its representative scores were examined in 58 OSCC samples including those of 29 men and 29 women with an average age of 59.2 years.
p16 INK4a expression in four stages
The p16INK4a expression was studied in each stage and found to be in 50% (7/14), 25% (4/16), 26.6% (4/15), and 30.7% (4/13) of samples in stage 1, 2, 3, and 4, respectively [Table 2]. The p16 immunopositivity was obviously lower in stages 2-4 compared to stage 1, although the difference was not statistically significant (P = 0.234).
TS and CS in four stages
The average TS values were 1.28, 0.71, 0.75, and 0.8 in stage 1, 2, 3, and 4, respectively [Figure 4]. They were significantly lower in stages 2-4 compared to stage 1 (P = 0.035). Also, the average CS values were 2.07, 1.18, 1.25, and 1.60 in stage 1, 2, 3, and 4, respectively. They were obviously lower in stages 2-4, although the difference was not significant (P = 0.061) [Figure 5].
|Figure 4: The average of TS values of p16INK 4a expression in four stages|
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|Figure 5: The average of CS values of P16INK 4a expression in four stages|
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p16 INK4a expression in three histological grades
33.3%, 31.6%, and 33.3% of well-differentiated, moderately differentiated, and poorly differentiated OSCCs, respectively, were positive for p16INK4a expression [Table 3], although the differences were not statistically significant (P = 0.671).
p16 INK4a expression in different sites
The p16INK4a expression level was found to be 71.4%, 42.9%, 37.5%, 25%, 22.2%, 0%, and 0% in the specimens of lip, palate, tongue, floor of the mouth, buccal mucosa, gingival, and retromolar areas, respectively [Table 4]. This expression was obviously higher in lip squamous cell carcinomas compared to OSCCs (71.4% vs. 27.4%) [Figure 6], but because of lesser number of lip tumors, this finding was not analyzed statistically.
p16 INK4a expression in tumoral islands and beyond dysplastic epithelium
The difference between p16 expression in tumoral islands and beyond dysplastic epithelium was analyzed statistically in each stage and was not found to be significant (P = 0.074) [Table 5].
|Table 5: p16INK 4a expression in tumoral islands of OSCC and dysplastic|
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Correlation between p16 INK4a expression and involvement of lymph nodes
The correlation between p16INK4a expression and involvement of lymph nodes is shown in [Table 6] with no significant correlation. (P = 0.491) [Table 6].
|Table 6: p16INK 4a expression in positive and negative lymph node|
specimens of OSCC
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Analysis of gene mutation
PCR and CSGE
0INK4a locus in chromosome 9 (9p21) was analyzed by CSGE mutation detection system in 58 OSCC samples. [Figure 2] reveals amplification of the 500 bp DNA fragment that contains the mentioned locus, and [Figure 3] shows mutation in p16INK4a gene.
It seems that 3 of total 58 cases of OSCC (5.1%) have mutation in INK4a locus, which showed double bands in CSGE screening method [Figure 3].
Correlation of p16 INK4a expression and INK4a locus mutation
Study of p16INK4a expression and INK4a locus mutation revealed that all the three suspected cases containing point mutation in INK4a locus showed loss of p16 expression also.
| Discussion|| |
Regulation of cell cycle is explained by a series of events leading to cell duplication using two types of molecules which are cylins and CDKs. In this process, two gene families, CIP/KIP and INK4a/ARF, act as tumor suppressors and prevent cell cycle progression. , p21, p27, and p57 genes are believed to be members of the cip/kip family. The rest includes p16INK4a and p14ARF genes. The former binds to CDK4 and the latter prevents p53 degradation. 
The chromosome locus 9p21 harbors three genes: p14ARF , p15INK4b , and p16INK4a . Inactivation of these genes by homozygous deletion or gene methylation is probably one of the most common molecular events in cancers.  Previous studies have found the loss of heterozygosity at 9p21 locus to occur with a high frequency in various cancers including OSCC. , Furthermore, alterations of this locus have been reported in cancer cell lines and primary tumors including melanoma, leukemia, lymphoma, esophageal and lung carcinomas,  as well as squamous cell carcinoma of head and neck. ,,,, In order to delve further into this research, we went ahead and assessed the importance of the mutational pathway in the inactivation of INK4a locus in OSCC.
To our knowledge, as far as grade and stage are concerned, a few studies have been conducted in order to understand the correlations between the alterations and clinicopathologic characteristics of OSCC and have reported differing results. ,,,
Here, we have reported our findings of p16INK4a expression level and its representative scores in four stages and three histological grades of OSCC, as well as the rate of INK4a locus mutation (we also studied the correlation between p16INK4a expression and INK4a locus mutation and the clinicopathologic characteristics of this tumor).
Briefly, p16INK4a expression was found in 50%, 25%, 26%, and 30.7% of samples in stage 1, 2, 3, and 4, respectively. Also, the average CS values were 2.07, 1.18, 1.25, and 1.60 in stage 1, 2, 3, and 4, respectively. The average TS values were 1.28, 0.71, 0.75, and 0.8 in stage 1, 2, 3, and 4, respectively. 33.3%, 31.6%, and 33.3% of well-differentiated, moderately differentiated, and poorly differentiated OSCCs, respectively, were positive for p16 expression. Three of 58 cases of this tumor (5.1%) were suspected to have mutation in INK4a locus.
Pande et al,  concluded that the p16INK4a expression has a significant correlation with the staging and progression of OSCC; however, they considered negative p16 expression for lower than 1%. However, some researchers believe that the loss of this expression is correlated with oral tumorigenesis. ,, Accordingly, in the present study as well as in the study of Buajeeb et al.,  negative expression was defined for those specimens having less than five stained cells. Differing samples and scoring methods may be a way to explain and determine such situations. Moreover, the utilization of various monoclonal antibodies which results in variation in p16INK4a staining may be another cause.  In our study, there was higher level of p16INK4a expression in stage 1 compared to stages of 2, 3, and 4. Although the difference was not statistically significant, it was in line with the studies that pointed to the loss of p16INK4a expression as an early event in oral tumorigenesis. ,,,, The findings also support the hypothesis of Buajeeb et al.  that p16INK4a has a main role in the initial invasion of tumor cells into the underlying connective tissue.
Tsai et al.  and Suzuki et al.  reported that the alteration of p16INK4a gene may be attributed to lymph node metastasis; however, in our endeavor, we were unable to find any correlations between p16INK4a expression and lymph node involvement. Tsai et al.  indicated a slight elevation of p16INK4a expression according to histological grade, while the elevation in our study was 33.3%, 31.6%, and 33.3% in the three grades, respectively. Notwithstanding the differences which may be related to the diversity in distribution of different grades in our cases, the elevated trend in the two studies shows some similarities. Due to the fact that few cases were examined with p16 gene mutation in our study, the findings were not statistically analyzed to evaluate its correlation with grade and stage.
Few researchers, i.e. Buajeeb et al.,  have taken into account the nuclear and/or cytoplasmic p16 expression in the absence of any basis for statistical analysis. Though this is worth mentioning, only Chen et al.  reported a significantly higher cytoplasmic staining than nuclear one.
To the best of our knowledge, our study is the first one to evaluate TS for p16 in OSCC, and fortunately, a statistically significant correlation between TS decease and increased stage level was found. In addition, we have introduced a new score (CS) to evaluate the proportion, intensity, and location of expression consequently. Although it did not show statistical significance, in our research, we have investigated the correlations between decrease in CS and increase in stage levels.
Our findings are in line with those of Tokman et al.'s  study and have revealed that p16 expression is more common in lip SCCs compared with intraoral ones. As it seems, other factors like p53 alteration play a greater role in tumor genesis of OSCC compared with lip tumors.  We could not find any significant correlations between p16 expression in neoplastic tissue and dysplastic epithelium. However, our findings are in concordance with the findings of Buajeeb et al.,  which showed no significant differences in p16INK4a expression between OSCC and oral leukoplakia with and without dysplasia.
A number of investigations have attempted to analyze the alteration of p16INK4a gene in head and neck squamous cell carcinomas and suggested that homozygous gene deletion and hypermethylation of p16 promoter are the predominant inactivating events in this alteration and, as a result, p16INK4a gene mutation is believed to have a minor role in the development of head and neck squamous cell carcinomas. ,,, The mutation rate of p16INK4a gene (INK4a locus) in this tumor was 3-6%. ,,, Uzawa et al.  also reported it in 6% of OSCCs and Kannan et al.  and Ohta et al.  reported it in 9% of cases.
The present mutation rate of 5.1% is in line with all these studies; however, Poi et al.  reported 22% point mutation in p16 gene of head and neck squamous cell carcinoma cases. The difference probably refers to sample location, sensitivity of evaluation procedure, and a larger number of samples used. Although, we recommend DNA sequencing to confirm the result.
| Conclusion|| |
Loss of p16INK4a expression seems to occur in early stages of OSCC progression. Evaluation of TS and probably CS for p16INK4a could prove to be a useful marker to determine the progression of this tumor. Moreover, p16INK4a gene (INK4a locus) mutation has minor rate of genetic alteration in this tumorigenesis, compared with other alterations.
| Acknowledgments|| |
The present manuscript is extracted from postgraduate thesis by Dr. Farhadi, which was successfully completed under the supervision of Dr. Sargolzaei and with the close co-operation of Molecular and Cellular Research department, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Shahed University, No. 39, Italia st, Vesal st, Keshavarz Blvd, Tehran
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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| ||Asian Pacific Journal of Cancer Prevention. 2014; 15(10): 4281 |
|[Pubmed] | [DOI]|
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