Comparative Evaluation of Immunohistochemical Expression of p16 with p16 Microsatellite Marker by PCR in Surgical Margins of Oral Squamous Cell Carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) is most common oral cancer with multifactorial etiology. Surgical therapy is treatment of choice but known to have recurrence. The main reason for recurrence is associated with surgical margins which need to be tumor free. Changes at genetic level cannot be ascertained only through routine light microscopy in surgical margins, even though they are tumor free. Detection of early marker like p16 can help in predicting the risk of recurrence. Hence study aimed to detect p16 microsatellite marker (D9s1747) in surgical margins of oral squamous cell carcinoma and compare the same with p16 marker through immunohistochemistry. Total of 40 paraffin embedded tissue samples diagnosed and surgically treated cases of OSCC were included. From each sample one tumor proper and one surgical margin was obtained. From paraffin embedded tissue sample 2 sections of 4 µm thick was obtained from tumor proper and tumor margin. One section was stained with hematoxylin and eosin and other section was stained immunohistochemically using p16 antibody. DNA extraction was done for tumor proper and surgical margin tissue and PCR analysis was carried for p16 microsatellite marker (D91747). Out of 40 cases 37 cases showed positivity in tumor proper for p16 with IHC. Out of 37 cases 23 cases showed positivity for both tumor proper and surgical margin. There were 3 cases negative for tumor proper. Out of these 3 cases, 1 (33.3%) case was positive for surgical margin. Out of 40 cases 27 cases showed positivity for tumor proper with p16 microsatellite marker. Out of 27 cases 16 cases showed positivity for both tumor proper and surgical margin. There were 13 cases negative for tumor proper. However there were 8 (61.5%) cases negative which were in tumor proper but showed positivity for surgical margin. Other 5 cases were negative in both tumor proper and surgical margin. Our study reveals that surgical margins of OSCC exhibit alteration in p16 markers both by IHC and PCR techniques. p16 and p16 microsatellite marker detection in margins indicates field change. Further studies with larger sample size comparing expression with clinical and histological parameter and follow up has to be done to substantiate our findings.

Introduction

Traditionally the surgical margin is classified by surgeons and pathologists as surgical margins involved (margin ≤ 1 mm), close margins (margin = 1–5 mm) and clear margins (margin > 5 mm). Molecular transmutations in the surrounding normal tumor margins and in the overall oral mucous membrane is one of the prime reason for high recurrence rate (25–45%) and metastasis of oral squamous cell carcinoma(OSCC) [1]. Therefore it is important to identify risk factors which lead to the relapse in cases with clear margins. As histopathological diagnosis alone is insufficient to predict the local recurrence, molecular analysis of these margins may help clinicians to establish prognosis and treatment planning.

Several genetic and epigenetic studies on surgical margins to evaluate local recurrence in OSCC patients have been carried out over the last decade. The margins were analysed by immunohistochemistry for p53, p16, Chk2, Laminin-5 and glycosylated oncofetal fibronectin [2, 3]. Among them p53 has been studied in surgical margins traditionally. Immunohistochemical analysis with the molecular marker eIF4E was studied in tumors and surgical margins [4]. Gene amplification (c-Myc and HER2) and promoter methylation (p14 and p16) were studied in the tumours, tumour margins, and unaffected oral mucosa [5]. Epigenetic markers such as p16 (INK4A), cytoglobin, E-cadherin, and TMEFF2 were studied in surgical margins [6]. Amongst the plethora of genetic aberrations useful for the early detection and progression of OSCC, microsatellite markers are one such aberration.

Microsatellites are extensions of DNA in which a short motif is repeated 5–100 times and is susceptible to inaccurate repetition during DNA replication. These microsatellite alterations are of two types: microsatellite instability (MSI) and loss of heterozygosity (LOH). The LOH markers 9p21 (D9s1747, RPS6, D9s162) and 17p13 (TP53) and the immunostaining results of the corresponding mutant P53, P14, P15, and P16 proteins were analyzed in surgical margins of OSCC for local recurrence and disease free survival. It was observed that loss of p16 occurs in stages of oral carcinogenesis [1].

The p16 INK4a (p16) is a tumor suppressor gene (TSG) located on the chromosome 9p, locus 21, involved in cell cycle progression blocking process. It is inactive in wide range of human malignancies. A loss of p16 INK4a immunosuppression has been observed in the early stages of oral carcinogenesis and has been considered as first TSGs to be inactivated in OSCC [7]. Genomic changes leading to OSCC remains unclear. Early changes include loss of tumor suppressor genes 3p, 9p and 17p. In head and neck squamous cell carcinoma allelic loss on chromosome 9p occurs most frequently at 9p21-22, the locus for p16 and p14ARF [8]. Since there are very small numbers of studies carried on surgical margins with p16 microsatellite marker we aimed to detect p16 microsatellite marker (D9s1747) in surgical margins of oral squamous cell carcinoma and compare the same with p16 marker through immunohistochemistry.

Materials and Methods

After obtaining the Institutional Review Board and Ethical Committee approval, a retrospective study was conducted from January 2015 to July 2017. The study group included 40 patients diagnosed with OSCC and who underwent tumor resection with radical neck dissection. From paraffin embedded tissue sample 2 sections of 4 µm thick was obtained from tumor proper and tumor margin. One section was taken on albumin coated slide for hematoxylin and eosin and other section was obtained on glass slide coated with 3-aminopropyltriethylsilane (APES Sigma-Aldrich, St Louis, MO) for immunohistochemistry using p16 antibody. Demographic data and other related clinical history and habit history was obtained from the archives. All the samples were reevaluated for histopathological grading according to WHO (Broder criteria) [9] Fig. 1a, b c. Clinical staging was determined for every case according to American Joint Committee for Cancer (8th Edition, AJCC) TNM staging [10].

Fig. 1.

Photomicrograph showing, a well differentiated squamous cell carcinoma (H&E 10 X), b p16 staining in well differentiated squamous cell carcinoma (IHC 10 X), c moderately differentiated squamous cell carcinoma (H&E 10 X), d p16 staining in moderately differentiated squamous cell carcinoma (IHC 10 X), e poorly differentiated squamous cell carcinoma (H&E 10 X), f p16 staining in poorly differentiated squamous cell carcinoma (IHC 10 X), g surgical margin (H&E 10 X), h p16 staining surgical margin (IHC 10 X)

For immunohistochemistry, the section was collected on glass slides coated with 3-aminopropyltriethylsilane. The section was deparaffinized on a slide warmer at 60 °C for 40–45 min, immersed in two changes of xylene I and II for 5 min, then rehydrated with distilled water for 5 min. The slide was then washed with distilled water. Antigen retrieval was performed by using a Thermo Ultravision Quanto detection system (TL-015- QHD; Pathnsitu Biotechnologies Pvt. Ltd., Hyderabad, India) in tris EDTA buffer.

Section was then allowed to cool at room temperature, washed with distilled water, and then with tris-buffered saline (TBS) for 5 min. Blocking of endogenous peroxidase activity was done by incubation with 3% hydrogen peroxide for 10 min. The sections were washed with distilled water and TBS for 5 min. After draining the excess buffer, sections were incubated for 2 h with primary antibody against p16 (Pathnsitu clone: G175-405, source: mouse Monoclonal, place: Hydrabad, India), washed with distilled water, excess buffer was drained off and section were incubated with target binder for 10 min. Section were washed in TBS for 5 min, then covered with polymer horseradish peroxidase for 10 min. Further sections were rinsed in TBS three times for 5 min each. To visualize the reaction, sections were incubated with diaminobenzidine for 5 min, counterstained with Harris’ hematoxylin for 30 s and bluing was done by keeping the slides under running tap water. Stained slide were dehydrated through a graded alcohol series, cleared in xylene and coverslips were mounted using disterene dibutylphthalate xylene (DPX Lot. no. 4356791014; Fisher Scientific, Mumbai, India). The sections were not allowed to dry at any time during the staining procedure. Finally sections were stored in slide incubator at room temperature till further analysis.

Evaluation of Immunostaining

IHC staining was assessed based on both intensity and number of cells stained. PBS slides were taken as negative control. Intensity of staining degree was scored as follows: score 0: no staining; score 1: light yellow; score 2: yellow; score 3: brown. Number of cells stained was scored as follows: score 1:0–25%, score 2: 26–50%, score 3: 51–75%, score 4: 76–100% of the cells. Both scores were then multiplied and a final score was obtained. After calculating a median score, the cases with scores equal to or more than median were defined as high expression and those with scores less than median were defined as low expression. Statistical significance was calculated for the association between OSCC and surgical margin for the presence or absence of p16 marker using Fisher exact test. Also measure of agreement between the technique (IHC and PCR) was done using Kappa measure of agreement.

DNA Extraction

DNA was extracted from tissue sections after deparaffinization using xylene and alcohol. Modified proteinase-k method was used to extract the DNA. Lysis buffer I containing 1 m Tris, 0.5 m EDTA and Lysis buffer II containing 50 mm Tris–HCL, 2.5 mm MgCl₂, 0.45% tween 20 and 0.45% Nodient P-40 was added by 10 mg 1 ml proteinase k. The tubes were kept at 60 °C for 2 h and then in the boiling water bath for 10 min to deactivate the enzyme. The tubes were centrifuged and supernatant containing DNA was collected in a fresh tube and stored at − 20 °c until PCR was performed.

Polymerase Chain Reaction

For PCR analysis, p16 microsatellite marker (D91747) was selected with sequence of forward 5′-ATTCAACG AGTGG GATGAAG-3′ and reverse 5′-TCCAGGTTGCTGCAAATGCC-3′. An expected PCR product size of 130-150 bp was used.

Reaction mixture was prepared by using Ampli Taq red master mix containing 2 mM MgCl2, 0.2 of dNTPs, 1U of Taq DNA polymerase. Primer was added at a concentration of 0.4 µM and 3 µl of DNA template was added to the mixture. The amplification was carried out in a veriti thermal cycler. (Applied Biosystems, California, USA). The thermal cycling conditions were as follows. 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 54 °C for 1 min, and 72 °C for 1 min. final extension was done at 72 °C for 5 min.

15 µl of the sample was loaded on 2% agarose gel and subjected to electrophoresis at 80 V for 1 h. The gel was stained with 0.5 µg/ml ethidium bromide and observed under UV gel documentation system (Major science, Saratoga, CA, USA).

The molecular weight of the band was confirmed by comparing the location with standard molecular weight marker ladder (100–1500 bp)

Results

Clinical and Histopathological Evaluation

Among the 40 cases considered, 67.5% (n = 27) were males and 32.5% (n = 13) were females. The age of the patients ranged from 16 to 75 years, with mean age range of 49.92 years. Buccal mucosa was most common site for OSCC 47.5% (n = 19) followed by tongue 32.5% (n = 13). Majority were in stage II. On the basis of histologic grading, 70% (n = 26) cases were of well-differentiated squamous cell carcinoma, 20% (n = 7) cases were moderately differentiated squamous cell carcinoma, and 10% (n = 4) cases were poorly differentiated squamous cell carcinoma. All the patients gave history of tobacco habits (in either smoked or smokeless forms or both).

Out of total 40 cases 37 cases showed positivity in tumor proper for p16 with IHC. Out of 37 cases 23 cases showed positivity for both tumor proper and surgical margin (Fig. 1b, d, f). Other 14 cases showed negativity for surgical margin. There were 3 cases negative for tumor proper. Out of these 3 cases, 1 (33.3%) case was positive for surgical margin (Fig. 1h and Table 1).

Table 1.

p16 Immunoexpression in tumor proper and surgical margin

IHC result	Surgical margin		Total	Test of significance
	Negative	Positive
Tumor
Negative	2	1	3	Fisher’s exact test p value 0.553, Not significant
	66.7%	33.3%	100.0%
Positive	14	23	37
	37.8%	62.2%	100.0%
Total	16	24	40
	40.0%	60.0%	100.0%

Out of 40 cases 27 cases showed positivity for tumor proper with p16 microsatellite marker. Out of 27 cases 16 cases showed positivity for both tumor proper and surgical margin. Other 11 cases showed negativity for surgical margin. There were 13 cases negative for tumor proper. However there were 8 (61.5%) cases negative which were in tumor proper but showed positivity for surgical margin. Other 5 cases were negative in both tumor proper and surgical margin (Fig 2, Table 2).

Fig. 2.

Photograph showing 2% Agarose gel electrophoresis for p16 microsatellite marker. *NC: Negative control (water), 1: Tumor proper, 2: Surgical margin, M: Standard molecular lab marker ladder (100–1000 bp)

Table 2.

p16 Microsatellite marker in tumor proper and surgical margin

PCR results	Surgical margin		Total	Test of significance
	Negative	Positive
Negative	5	8	13	Fisher’s exact test p value > 0.05, Not significant
	38.5%	61.5%	100.0%
Positive	11	16	27
	40.7%	59.3%	100.0%
Total	16	24	40
	40.0%	60.0%	100.0%

Amongst the tumors analyzed, 37 cases were positive when analyzed for p16 by IHC. However, only 24 cases (64.9%) were positive for p16 microsatellite marker when analyzed by PCR. Three cases which were negative for IHC turned out to be positive when analyzed by PCR (Table 3). In surgical margin all the cases negative for p16 microsatellite marker with PCR remained negative for p16 with IHC. Also all the cases positive for PCR were also positive for IHC (Table 4). p16 marker with IHC showed high positivity in tumor proper compared to p16 microsatellite marker (Tablse 3, 4).

Table 3.

Comparison of p16 immunoexpression with p16 microsatellite marker in tumor proper

IHC positivity in tumour	PCR positivity in Tumour		Total	Statistical test	Measurement of agreement
	Negative	Positive
Negative	0	3	3	Fisher’s exact test p value 0.538, Not significant	Kappa value- 0.139 Degree of agreement poor p value 0.211, Not significant
	0.0%	100.0%	100.0%
Positive	13	24	37
	35.1%	64.9%	100.0%
Total	13	27	40
	32.5%	67.5%	100.0%

Table 4.

Comparison of p16 immunoexpression with p16 microsatellite marker in surgical margin

IHC positivity in surgical margin	PCR positivity in surgical margin		Total	Test of significance	Measurement of agreement
	Negative	Positive
Negative	16	0	16	Fisher’s exact test p value < 0.001, significant	Kappa value-1 Degree of agreement excellent p value < 0.001, Significant
	100.0%	0.0%	100.0%
Positive	0	24	24
	0.0%	100.0%	100.0%
Total	16	24	40
	40.0%	60.0%	100.0%

For both tumor proper and surgical margin

Kappa value: 0.533, Degree of agreement: Average, p value < 0.001, Significant

The degree of agreement between IHC and PCR methods were poor for tumor proper. However degree of agreement was excellent for surgical margin (Tables 3 and 4).

Discussion

OSCC accounts for 95% of all oral cavity and oropharyngeal cancers and consistently ranks as sixth most common cancers with approximately 300,000 new cases annually [11]. The etiologies remain multifactorial, consumption of alcohol or tobacco or a combination of both is the main risk factor for OSCC. Surgical therapy remains the main treatment approach, while adjuvant therapy such as radiation or chemotherapy may be used in combination with either treatment in advanced cases [12].

Despite the attainment achieved concerning prognosis and therapy of oral squamous cell carcinoma, millions of deaths occur each year throughout the world. The overall survival rate of less than 60% remains generally poor, with prognosis heavily relying on the TNM staging system. The disease staging, extracapsular spread, tumor thickness and resection free margins of the tumor has enormous acceptance which has influence on disease outcome [13] The status of surgical free margins is one of the important factors for local recurrence [14].

Brennan et al. in 1955 was a first person to introduce molecular assessment of surgical margins in head and neck patients by using marker p53 mutation. Mutations of genes have been found to increase the possibility of loco regional recurrence in OSCC patients [15]. One of the genes which is associated with loco regional recurrence p16 gene is one of the negative regulators of cell cycle. The p16 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 by disrupting the regulation of G1 to S phase [16, 17]. More than 80% of head and neck tumors show early inactivation of p16 where as deregulation of p53 and cyclin D1 occur later in sequence of molecular progression to frank carcinoma. In head and neck cancer p16 protein often inactivated by 3 different methods: homozygous deletion, promoter methylation or point mutations [18]. Majority of studies demonstrated loss of p16 in head and neck SCC. Even though there are numerous studies on OSCC, not many genetic changes has been identified in surgical margins to accurately predict which OSCC patients are at high risk for local recurrence.

In the present study we observed higher expression of p16 and p16 Microsatellite marker in tumor proper. The observed increase in pl6 marker levels in tumors can be the consequence of retinoblastoma (pRb) pathway disruption, an event occurring in most, if not all tumor cells [19]. Inactivated pRb by phosphorylation will promote cell cycle progression. Studies have shown variable p16 expression in OSCC revealing both under expression and over expression [20]. The expression of p16 was also seen associated with HPV infection. The observation of our finding (over expression) could be associated with HPV infection. Literature supports that patients positive for HPV are at the high risk of developing HPV related head and neck squamous cell carcinoma (HNSCC) [21–23]. However our aim was not to correlate HPV status with p16 expression. Hence we did not test samples for HPV.

In our study out of 40 OSCC cases 23 (62.2%) cases showed positivity in both tumor proper and surgical margin for p16 with IHC. Out of 27 cases positivity in both tumor proper and surgical margin, 16 (59.3%) cases showed positivity for p16 microsatellite marker with PCR. Deletions and mutations of p16 in OSCC are described in various articles. Our findings are in accordance with previous findings which show surgical margin being tumor free but showed positivity for p16 marker which suggests that the margins are not clear, they show field changes. Shaw et al. in their study observed p16 methylation changes in surgical margins of OSCC. Three out of 4 recurrences from tumors demonstrating p16 methylation, also showed p16 promoter methylation in the deep margins. p16 down regulation has a possible role in tumor recurrence [6]. Wang et al. observed 23 cases with genetic alterations in histological normal mucosa developed local recurrence. TP53 LOH and 9p21 LOH appear to be associated with local recurrence in OSCC¹. Supic G and co authors found cancer related genes hypermethylation detected in normal-appearing surgical margins or the adjacent normal mucosa of OSCC and HNSCC [24].

We found 3 cases with p16 with IHC negativity in tumor proper, 1 (33.3%) case turned out to be positive in surgical margin. Molecular tests applied to paraffin-embedded tissues are less accurate than those performed in fresh material. This molecular test is only considered when the diagnosis of OSCC is indicated by a simple hematoxylin and eosin stain. At this time, PCR can be performed on paraffin embedded material in association with IHC to increase the diagnostic accuracy. The status of the surgical margins is one of the important factors because tumor cells or dysplastic epithelia that remain in the margins may lead to the local recurrence in OSCC patients. Local recurrence occurs in up to half of patients with even microscopically negative surgical margins. This is because of sampling errors inherent in the assessment of thin sections of large piece of tissue and interpretive errors by the pathologist. Handling of surgical specimens by the surgeons and pathologist and collection of damaged tissue at the margin by electrocautery procedure that is difficult to interpret (non diagnostic) is another source of error. Residual tumor cells in negative surgical margins with molecular analysis are a major strength. [25].

There were 13 cases with PCR negativity in tumor proper when compared with matched surgical margin there were 8 (61.5%) cases with PCR negativity in tumor but showed positivity in surgical margin. p16 expression results in OSCC reveal variable findings in different studies [25]. Studies also have shown that in advanced stages of the OSCC there is reduction in p16 expression [25] The reason could be the neoplastic cells that loose cell cycle control mechanism favor uncontrolled cell replication but p16 blocks the cell cycle progression from G1- to S phase [23]. The reduction in p16 expression is related to oral cancer and precancer is believed that its inactivation is an early and progressive event as advances in tumor stage and degree of dysplasia of premalignant lesion and disappears in neoplastic cells [26]. Hence the 13 cases which were negative for p16 expression might have favored uncontrolled cell replication.

When the results were compared for both IHC and PCR techniques for p16 and p16 microsatellite marker the p16 marker with IHC sowed high positivity in tumor proper compared to p16 microsatellite marker. The possible reasons could be p16 staining is affected by preanalytical, analytical, and postanalytical variables. Preanalytical variables include mostly tissue fixation and processing, whereas analytical variables associated to protocol, variability in reagent, and technician experience. Assessment of controls, result interpretation and reporting, and experience of the pathologist are factors accounting for postanalytical variables [27].

The degree of agreement (kappa value 0.139) was poor in tumor proper and non significant but was excellent for surgical margin (kappa value 1) and was significant (p value < 0.0001).The degree of agreement was average when it is compared in both tumor proper and surgical margins. This variation is because of 13 cases showed negativity in the tumor proper which favored uncontrolled replication. The surgical margins both the techniques (IHC, PCR) showed excellent degree of agreement.

Considering the corresponding protein p16 and p16 microsatellite marker will have limited value in predicting local recurrence. However predictive value will be more sensitive when p16 and p16 microsatellite marker are combined. To help surgeons infer which patients with surgical margins have a high risk of local recurrence p16 immunohistochemical staining should be performed followed by p16 microsatellite marker. Because detection of markers in surgical margins can easily be performed within 2 weeks of surgery and before histological alteration detected. This procedure may be of help in making postoperative decision [1].

We must point out the limitations of our study 1.The extent of validity of findings is limited as it was conducted at single institute, 2. Comparison between histopathological grading, TNM staging and also correlation with habit was not done due to small sample size, 3. Utilization of various antibodies result in variation of p16 staining, 4. Follow up to check out recurrence.

Conclusion

Our study reveals that surgical margins of OSCC exhibit alteration in p16 markers both by IHC and PCR techniques. p16 and p16 microsatellite marker detection in margins indicates field change. Early changes in OSCC can be detected in margins using p16 microsatellite markers and it may be worthwhile method for predicting local recurrence in OSCC patients. Surgical excision of entire affected mucosa is not feasible, but the inclusion of more rigorous treatment and more intensive surveillance during follow-up in patients with early detection may provide an enhanced overall survival. Above all the study observation suggest to consider both IHC for p16 and determine p16 microsatellite analysis for cases of OSCC and surgical margin, so that early preventive measures can be taken by conducting regular following up of OSCC cases to avoid recurrence. Further studies with larger sample size comparing expression with clinical and histological parameter and follow up has to be done to substantiate our findings.