An Immunohistochemical Study Showing Ki-67 as an Analytical Marker in Oral Malignant and Premalignant Lesions.

INTRODUCTION: Ki-67 is a nuclear protein. It is a proliferation marker that has an essential function in tumorigenesis due to its positive connection with tumor expansion. AIM: The aim of this study was to evaluate the articulation of Ki‑67 as prognostic marker in various grades of oral epithelial dysplasia (OED) and in oral squamous cell carcinoma (OSCC).
MATERIALS AND METHODS: A total of 100 histologically affirmed samples of normal oral mucosa (NOM), OED, and OSCC were divided into three groups-Group I (10 samples of normal oral mucosa), Group II (45 samples of OED), Group III (45 samples of OSCC). Routine hematoxylin and eosin and immunohistochemical staining with Ki-67 monoclonal antibody were carried out in all the samples.
RESULTS: Within Group I, articulation of Ki-67 was constrained to the basal layers. In Group II, cells showing positive expression of Ki-67 were available in the basal, suprabasal, and spinous layers. Cells showing positive expression of Ki-67 among well-differentiated OSCC were presented mainly in the periphery of the tumor nests; in moderately differentiated OSCC, cells were located in both peripheral and part of a center of the tumor nests; and in most cases of poorly differentiated OSCC, cells were diffused. Statistically significant difference in positive expression of Ki-67 was appreciated between three groups.
CONCLUSION: Ki-67 antigen may perhaps be used as a marker for the histological reviewing of OED and OSCC. With the increase in the severity of OED, cells showing positive expression of Ki-67 also increased. Copyright:

INTRODUCTION

The most commonly found malignant neoplasm constitutes oral squamous cell carcinoma (OSCC) that holds up 90% of all types of oral carcinomas.[1] In India, oral cancer is among the top three carcinomas and is hitting the highest point. Among 10 of all carcinomas, four are oral carcinomas, subsequently comprising significant well-being setback in individuals.[2]

The transformation of normal oral epithelium from normal histology to oral epithelial dysplasia (OED) to superficial cancer and invasive squamous cell carcinoma is influenced by chromosomal damage, accumulating mutations, and loss of cellular control functions.[3]

The study of tumors markers can aid in foreseeing the level of dysplasia and its succession to malignancy. Thus facilitating in the diagnosis and prognosis of patients with oral squamous cell carcinoma.[2] Increase in the proliferation of tumor cell is considered as significant natural factor for tumor detection. Ki-67 has appeared to serve a significant part in tumorigenesis because of its positive relationship with tumor multiplication and invasion.[24]

Ki-67 is a monoclonal marker used for proliferation. It is exclusively related to proliferation of cells and aggressiveness of malignant tumors.[5] During cell cycle, the presence of Ki-67 is seen during prophase and metaphase but is absent during resting cells.[6] The aforementioned features make Ki-67 as one of the best markers to calculate cell proliferation, and it can be used as a reagent that helps in determining a patient prognosis for several types of tumor.[7]

This research intended to evaluate the positive appearance of Ki‑67 as prognostic marker in diverse grades of OSCC and OED.

MATERIALS AND METHODS

A total of 100 paraffin-embedded blocks that were formalin fixed were included in the study sample as follows [Figure 1]:

Figure 1

Distribution of samples

Group I (15 samples of normal oral mucosa [NOM])

Group II (45 samples of OED)

Group III (45 samples of OSCC)

All the blocks were collected from archival files of department of oral pathology and microbiology of Kalinga Institute of Dental Sciences, Kalinga Institute of Industrial Technology, Deemed to be University, Bhubaneswar, Odisha, India. OED was further subdivided into 15 mild OED, 15 moderate OED, and 15 severe OED. OSCC was subdivided into 15 well-differentiated OSCC, 15 moderately differentiated OSCC, and 15 poorly differentiated OSCC. Paraffin sections of formalin-fixed tissues were used for both histological and immunohistochemical evaluation. Hematoxylin and eosin stained sections of 4 μ were used for routine histological examination.

Immunohistochemistry

IHC recognition of Ki‑67 was done with Dako antibody detection system. For IHC staining, the segments were sliced at approximately 3-μm thickness using semiautomatic microtome. In case of IHC, segments were positioned on lysine-coated slides. For antigen recovery, the segments were dipped in a 1mM citrate buffer (pH 6), and microwave was used with patterns of high, medium high, low, and very low, each going for 5 min and afterward cooled to room temperature.

The endogenous peroxidase action was blocked with 3% hydrogen peroxide for 10 min, after that washing in 0.05mM Tris‑buffered saline (TBS) at pH 7.4 was done. The segments were incubated with ready to use primary mouse monoclonal antibodies against Ki‑67 1h at 37°C. Subsequent to washing in TBS, the segments were incubated by means of a secondary antibody conjugated amid peroxidase-labeled dextran polymers for half hour at room temperature. Subsequent to washing with TBS, they were treated with 0.5 mg/mL 3, 3’‑diaminobenzidine solution containing 0.001% hydrogen peroxide to envisage reaction products, and for 3 min further stained with Mayer’s hematoxylin.

Statistical Analysis

Labelling Index

Labeling index was calculated in cases of OSCC as the number of positive cells per 100 cells. In cases of OED, nuclear expression of Ki‑67 was confirmed by basal, parabasal, and suprabasal layers [Figure 2].

Figure 2

Mean labeling index for Ki-67 protein in normal oral mucosa, oral epithelial dysplasia, and oral squamous cell carcinoma

RESULTS

Clinicopathologically, age range of 100 participants was between 20 and 75 years (average 40 years), the sex ratio was 1:1.

In Group I, positive Ki-67 cells were constrained in basal layer only. In Group II, positive Ki-67 cells were present in the basal, suprabasal, and spinous layers. Positive Ki-67 cells in well-differentiated OSCC were presented chiefly in the periphery of the tumor nests; in moderately differentiated OSCC, Ki-67 positive cells were located in both peripheral and part of a middle of the tumor nests; and in most cases of poorly differentiated OSCC, positive Ki-67 cells were diffused. The Ki-67 positive cells in various groups were scrutinized for statistical significance with one-way analysis of variance (ANOVA) test. A P value of <0.05 indicated significant difference.

DISCUSSION

Oral mucosa comprises stratified squamous epithelium. Presence of stratifications in the epithelium is due to differentiation and proliferation of cells.[8] Differentiation begins when recently divided cells segregate from underlying extracellular matrix.[9] With time when differentiating cells mature, they move toward the epithelial surface due to force generated in the underlying proliferation compartment.[8] Cell proliferation, imperative natural procedure, is a noteworthy addition to histological tumor classification and has prospective importance such as a marker of treatment success and setback. Numerous investigations have revealed that abnormal cell increase seems to be a precursor and can act as an indicator for tumorigenesis.[10] Different IHC markers are used to identify cell proliferation, of which Ki‑67 is used as an increasingly dependable marker of proliferation in our investigation.

In cases of NOM, the mean labeling index of Ki‑67 protein was 12.56; for OED, it was 28.4; and for OSCC, it was 40.18 [Table 1]. There was a statistical noteworthy distinction in between three groups. Further labeling index of mild OED was found to be 15.42; for moderate OED, it was 29.77; for severe OED, it was 58.5; for well-differentiated OSCC, it was 35.23; for moderately differentiated OSCC, it was 48.22; and for poorly differentiated OSCC, it was 59.13 [Table 2]. No statistical noteworthy distinction was present amid NOE and mild OED, this shows that the pace of carcinomatous change is more in entities that display moderate to severe dysplasia.[11] There was a statistical noteworthy distinction in the expression of Ki-67 among well-differentiated OSCC, moderately differentiated OSCC, poorly differentiated OSCC, and NOM, this shows that the appearance of Ki‑67 amplified progressively with the increase of differentiation of OSCC. Macluskey et al.[12] showed statistical noteworthy relation with mean Ki‑67 in healthy tissue, dysplasia, and in carcinomatous tissue, thus suggestive of increase in epithelial propagation throughout the changeover from dysplasia to cancer [Table 3].

Table 1

Mean labeling index for Ki-67 in normal oral mucosa, oral epithelial dysplasia, and oral squamous cell carcinoma

Group	Ki-67 labeling index (mean ± SD)	P value
Group I	12.56 ± 6.9	0.00
Group II	28.24 ± 13.2
Group III	40.18 ± 15.9

Table 2

Mean labeling index for Ki-67 protein in normal oral mucosa; mild, moderate, and severe oral epithelial dysplasia; and well, moderate, and poor oral squamous cell carcinoma

Group	Grade	Ki-67 (mean ± SD)	P value
Group I	Normal	12.56 ± 6.9
Group II	Mild	15.42 ± 10.13	0.01
	Moderate	29.77 ± 14.02
	Severe	58.5 ± 11.00
Group III	Well	35.23 ± 14.27	0.013
	Moderate	48.22 ± 14.00
	Poor	59.13 ± 16.11

Table 3

Comparison of labeling index for Ki-67 protein in different subgroups

Sample 1	Sample 2 (control)	P value	Significance
Mild dysplasia	Normal oral mucosa	0.71	Nonsignificant
Moderate dysplasia	Normal oral mucosa	0.01	Significant
Severe dysplasia	Normal oral mucosa	0.013	Significant
Well OSCC	Normal oral mucosa	0.00	Significant
Moderate OSCC	Normal oral mucosa	0.00	Significant
Poor OSCC	Normal oral mucosa	0.00	Significant

In Group I, expression of Ki-67 was limited to basal layers. Comparable results were reported by Takeda et al.,[13] Dwivedi et al.,[14] and Birajdar et al.[15]

In Group II, Ki-67 positive cells were present in the basal, suprabasal, and spinous layers. There was an increase in the expression of Ki-67 with the severity of dysplasia. Comparable results have been reported by Gonzalez-Moles et al.[16] and Kumar et al.[17]

In the mild OED, the maximum expression of Ki‑67 was located at the basal and parabasal layers of the epithelium, which showed the least expression. As there was no statistical noteworthy relation among mild OED and NOM, it can be concluded that it is very difficult to predict the prognosis of mild OED lesions, as it is not very violent lesion and its proliferative activity is similar to normal epithelium.

In the moderate OED, the nuclear Ki-67 positivity was found in basal, parabasal, and some of the spinous layers of the epithelium. A statistical noteworthy distinction was observed between the moderate OED and the NOM, and in the severe OED, the nuclear Ki-67 positivity was found in the basal, parabasal, and most of the spinous layers of the epithelium; it can be concluded that the speed of malignant transformation is higher in lesions that have moderate to severe dysplasia. In future, the Ki-67 protein may serve as prognostic tools in finding malignant changes in OED.

This increased proliferation in parabasal layers of premalignant oral epithelium is likely related with the loss of heterozygosity in 3p, 9p, and 17p, which carries on as a marker of precancerous fields and builds the plausibility of developing multiple tumors.[18]

The pace of malignant transformation may rely on the level of dysplasia, and lesions that have high grade of dysplasia may be 4.5-times bound to experience malignant transformation when contrasted with mildly dysplastic lesions.[19]

Ki-67-positive cells in well-differentiated OSCC were displayed fundamentally in the periphery of the tumor nests; in moderately differentiated OSCC, Ki-67-positive cells were situated in both peripheral and part of middle of the tumor nests; and most of the time in poorly differentiated OSCC, Ki-67 positive cells were diffused.

We saw that the cell expansion in OSCC expanded with expanding histological evaluations of OSCC. Comparable outcomes were accounted for by Takkem et al.[11] and Tumuluri et al.[20]

We saw that the cell proliferation in OSCC expanded with increasing histological grades of OSCC. Comparable outcomes were accounted by Takkem et al.[11] and Tumuluri et al.[20]

The high expression of the Ki-67 protein in OSCC tissues may play an important role in the development of OSCC.[21]

As indicated by our examination and past investigations performed by Raju et al.,[22] Dragomir et al.,[23] and Ahmad et al.,[24] Ki‑67 ends up being helpful in surveying tumor aggressiveness. Increased cellular proliferation is associated with more advanced lesions, and the distribution of proliferating cells in tissues may disclose about the regulatory mechanisms that become dysfunctional through carcinogenesis.[25]

Owing to the expression of Ki-67 protein in all proliferating cells and the prognostic significance of the Ki-67 marker in many cancers, Ki-67 protein is a potential beneficial target in cancer, and approaches that inactivate Ki-67 protein region, promising anti-proliferative approach, can have potential applicability in cancer treatment.[26]

CONCLUSION

Ki-67 antigen may possibly be used as a marker for the histological reviewing of OED and OSCC. Expression of Ki-67 expands with the severity of OED.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.