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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 26  |  Issue : 3  |  Page : 370-375
 

Centromere Protein F (CENPF): A novel marker for salivary gland pathology


1 Department of Oral Pathology and Microbiology, Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India
2 Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India

Date of Submission19-Jul-2022
Date of Acceptance26-Jul-2022
Date of Web Publication17-Oct-2022

Correspondence Address:
T A Lakshmi
Department of Oral Pathology and Microbiology Saveetha Dental College and Hospitals, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.jomfp_309_22

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   Abstract 


Background: Salivary gland tumours are relatively uncommon, and there exists considerable diagnostic difficulty. This is due to individual lesions having diverse histopathological features, presence of number of types and variants, and overlapping histological features in different tumour entities.
Aim: The current study aimed at assessing the expression of centromere protein F (CENPF) in benign and malignant salivary gland tumours and to evaluate the efficacy of CENPF as a proliferative marker to aid in the diagnosis of malignancy so that it will help in surgical pathology practice.
Materials and Methods: The study group involved 20 cases of benign salivary gland tumours, 20 cases of malignant salivary gland tumours, and 10 normal salivary gland tissues. All the cases were subjected to immunohistochemical analysis for CENPF expression and were assessed by two independent observers and further taken up for evaluation.
Statistical Analysis: The results were analysed statistically among different groups using analysis of variance (ANOVA) or Kruskal–Wallis test with Chi-squared test using IBM's Statistical Package for the Social Sciences (SPSS) version 17.0.
Results: CENPF expression in normal salivary gland was negative with gradual increase in expression from benign salivary gland tumours to malignant salivary gland tumours. CENPF expression was high in malignant salivary gland tumours.
Conclusion: Findings of the study suggest that CENPF can be regarded as a new cell proliferation marker for malignant salivary gland tumours.


Keywords: Biomarker, CENPF, immunohistochemistry, salivary gland tumours


How to cite this article:
Lakshmi T A, Narasimhan M, Harikrishnan T, Rajan ST. Centromere Protein F (CENPF): A novel marker for salivary gland pathology. J Oral Maxillofac Pathol 2022;26:370-5

How to cite this URL:
Lakshmi T A, Narasimhan M, Harikrishnan T, Rajan ST. Centromere Protein F (CENPF): A novel marker for salivary gland pathology. J Oral Maxillofac Pathol [serial online] 2022 [cited 2022 Dec 3];26:370-5. Available from: https://www.jomfp.in/text.asp?2022/26/3/370/358734





   Introduction Top


Salivary gland tumours make up about 3% of all head and neck neoplasms, and they are relatively uncommon. Most commonly affected sites are parotid gland, submandibular gland, and minor salivary glands, where the sublingual gland is very rarely affected.[1] There exists considerable diagnostic difficulty due to individual lesions having diverse histopathological features, presence of number of types and variants, and overlapping histological features in different tumour entities.

Even though haematoxylin and eosin (H&E) staining is considered as the gold standard method used for the diagnosis, immunohistochemistry can enhance the accuracy and can be a helpful tool in cases that cannot be assessed by histological examination, such as the cell nature and differentiation status, cell proliferation, and tumour protein expression. Immunohistochemistry plays an important role in the diagnosis of salivary gland tumours and is always helpful in supporting the histological assessment. Unfortunately, there is not enough data available regarding immunohistochemical analysis of salivary gland tumours that can be applied to surgical pathology practice.[2]

Various studies have been done in studying the incidence of salivary gland tumours across USA, Brazil, Jordan, Japan, Africa, and Nigeria. But only a few studies have been done so far to see the pattern of distribution of salivary gland lesions in Asian countries especially in India, thus indicating the scarcity of epidemiological data of these lesions.[3]

Centromere protein F (CENPF) is a 350 KD transient kinetochore protein that is also known as mitosin in humans, Lek 1 in mouse, and CMF-1 in chicken. It is located at the outer kinetochore plate and is expressed in a cell-cycle dependent manner. It has a complex and dynamic expression and localization pattern. CENPF level is low in G1 phase but increases sharply in S phase as a nuclear protein. At the G2/M transition, CENPF re-localises to the nuclear envelope where it contributes via NudE/EL to dynein/dynactin recruitment to the nuclear envelope. Following the progression of M phase, mitosin is hyperphosphorylated and exhibits localisation at the kinetochores, spindle poles, and midbody. It is subjected to rapid degradation at the end of mitosis.[4–6]

Various studies show overamplification of CENPF in various malignancies, but to our knowledge literature review shows only one study done on CENPF expression in salivary gland tumours.[7] The current study aimed at assessing the expression of CENPF in benign and malignant salivary gland tumours and to evaluate the efficacy of CENPF as a proliferative marker to aid in the diagnosis of malignancy so that it will help in surgical pathology practice.


   Materials and Methods Top


The current study involved 10% formalin-fixed paraffin-embedded (FFPE) tissues of histopathologically diagnosed cases of 20 benign salivary gland tumours, 20 malignant salivary gland tumours, and 10 normal salivary gland tissues from the archives. The study proposal was approved by the Institutional Ethics Committee, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai. The clinical and histological parameters included in the study were obtained from the records of the patients documented in the department.

The study samples were divided into three groups

  • Group I: Normal Salivary Gland (n = 10)
  • Group II: Benign Salivary Gland Tumours (n = 20)
  • Group III: Malignant Salivary Gland Tumours (n = 20)


Group I comprised of 10 normal salivary gland tissues; group II comprised of 12 pleomorphic adenomas, 3 basal cell adenomas, 1 cystadenoma, 2 myoepitheliomas, and 2 Warthin tumours.; and group III comprised of 6 mucoepidermoid carcinomas, 9 adenoid cystic carcinomas, 1 adenoid cystic carcinoma ex pleomorphic adenoma, 2 acinic cell carcinomas, and 2 carcinoma ex pleomorphic adenomas.

All the study samples were stained with routine H and E stain and were also subjected to immunohistochemical analysis for CENPF expression.

Immunohistochemistry

All the cases were subjected to CENPF immunostaining using CENPF antibody (polyclonal NB 100-88157, Novus Biologicals- Rabbit, dilution 1:200). Ewing's sarcoma was selected as the control group, as given in the protocol of the kit.

The immunostained slides were evaluated semi-quantitatively. The intensity of staining and the percentage of cells were evaluated and scored. The degree of immunostaining was assessed and scored independently by two investigators blinded from the clinical parameters, according to both intensity and extent of staining. Only cells stained in the nucleus were taken into account. The entire tissue sections were observed to assign scores.

Based on the previous studies done,[8],[9] the sum of the intensity scores and extent scores were used as final staining scores (0–7). For the purpose of statistical evaluation, tumours having a final staining score of less than 3 were grouped as low CENPF expression and greater than or equal to 3 were grouped as high CENPF expression.

Statistical analysis

The results were analysed statistically among different groups using analysis of variance (ANOVA) or Kruskal–Wallis test with Chi-squared test using IBM's Statistical Package for the Social Sciences (SPSS) version 17.0. P values less than 0.05 were considered as statistically significant [Table 1] and [Table 2].
Table 1: Significance in expression of CENPF in different study groups

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Table 2: Table showing final scores of CENPF being analysed statistically among different groups using ANOVA and found to be highly significant

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   Results Top


We studied the expression of CENPF in 20 benign salivary gland tumours, 20 malignant salivary gland tumours, and 10 normal salivary gland tissues. Only cells stained in the nucleus were taken into account. The age group of the study sample ranged from 5 years to 83 years. The mean age of the patients in group I, group II, and group III were 31.5 years, 43.8 years, and 48.4 years, respectively. The mean age of patients in the positive group was 49.14 years and the mean age of patients in the negative group was 35.64 years. The distribution of sex in the study was found to be 32 males (64%) and 18 females (36%). Among 32 males, 19 cases (59.4%) expressed CENPF and among 18 females, 9 cases (50%) expressed CENPF.

Site distribution in group II and group III included 6 different sites. Among the 40 cases, 17 (42.5%) were from the parotid gland, 17 (42.5%) were from the palate, 3 (7.5%) were from the submandibular gland, 1 (2.5%) was seen in the sublingual, 1 (2.5%) was seen in the base of the tongue, and 1 case (2.5%) was seen in the retromolar trigone.

Positive expression of CENPF was seen in 14 out of 17 cases (82.4%) of the palate, 11 out of 17 cases (64.7%) of the parotid, 1 out of 3 cases (33.3%) of the submandibular gland, and 1 each (100%) of the sublingual and retromolar trigone. The base of the tongue showed no CENPF expression. Pearson's Chi-squared test was performed, and the result was statistically significant (P < 0.05).

Three out of 20 cases (15%) of benign salivary gland tumours had reactive lymphadenitis. One case (5%) had lymph node involvement in malignant salivary gland tumours. Five out of 20 cases (25%) of malignant salivary gland tumours showed recurrence.

All the cases in group I stained negative for CENPF. Out of 20 cases in benign salivary gland tumours, 40% of cases showed positive CENPF expression. One hundred percent of cases from malignant salivary gland tumours showed positive CENPF expression [Figure 1].
Figure 1: Bar diagram showing expression of CENPF in different study groups

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   Discussion Top


Tumours arising from the salivary glands are relatively uncommon, and they comprise approximately 1% of all neoplasms in the whole body.[2] The aetiology of salivary gland carcinomas remains unclear. Most of the head and neck carcinomas are strongly associated with smoking and alcohol—they do not play a role in the salivary glands.[10] Biomarkers are grouped into several classes like genomic markers, proliferation markers, and differentiation markers.[11] Centromere protein F or CENPF is a 350 kD nuclear phosphoprotein that is involved in cell division.[6] Autoantibodies to CENPF have been observed in various malignant tumours like breast carcinoma and lung carcinoma. The detection of anti-CENPF antibodies indicates increased cell division and dysfunctional cell cycle regulation during carcinogenesis.[7],[12] Therefore, it acts as a suitable marker for evaluating proliferation of cells by immunohistochemistry.[13] CENPF is upregulated in various tumours like lung cancer, non-Hodgkin's lymphoma, mantle cell lymphoma, and oesophageal carcinoma.[14],[15]

Till date, only one study by Shigeishi et al.[7] has been carried out to evaluate the expression of CENPF in salivary gland tumours. In this study, we used polyclonal antibody CENPF for studying its expression in salivary gland tumours using immunohistochemistry. Ewing's sarcoma was used as positive control, and it showed strong nuclear staining with CENPF antibody [Figure 2].
Figure 2: Photomicrograph showing nuclear staining with CENPF (×400) in positive control (Ewing's sarcoma)

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There was negative expression of CENPF in all 10 cases of normal salivary gland [Figure 3]. This is in accordance with the study done by Shigeishi et al.[7] that showed that normal submandibular salivary gland tumours showed no immunoreactivity to CENPF in immunohistochemistry. This may be due to the fact that only intercalated duct reserve cell can undergo proliferation when compared to salivary gland acini.
Figure 3: Photomicrograph showing negative CENPF expression (×100) in normal salivary gland

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Out of 20 cases of benign salivary gland tumours, 8 (40%) showed positive CENPF expression and 12 (60%) showed negative CENPF expression [Figure 4] and [Figure 5]. In a similar study conducted by Shigeishi et al.[7] on CENPF gene expression in human salivary gland tumours, benign salivary gland tumours showed weak CENPF staining.
Figure 4: Photomicrograph showing negative CENPF expression (×100) in pleomorphic adenoma

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Figure 5: Photomicrograph showing low CENPF expression (×400) in Warthin tumour

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Out of 20 cases of malignant salivary gland tumours, all cases showed positive CENPF expression [Figure 6],[Figure 7],[Figure 8]. In a similar study conducted by Shigeishi et al.[7] on CENPF gene expression in human salivary gland tumours, three of eight mucoepidermoid carcinomas, two of four acinic cell carcinomas, two of four adenoid cystic carcinomas, and one of one malignant myoepithelioma were positive for CENPF.
Figure 6: Photomicrograph showing >75% of staining with CENPF (×100) severe intensity in adenoid cystic carcinoma ex pleomorphic adenoma

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Figure 7: Photomicrograph showing neural invasion in adenoid cystic carcinoma (H&E, ×400)

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Figure 8: Photomicrograph of area of neural invasion in adenoid cystic carcinoma showing >75% of staining with CENPF (×400), severe intensity

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In our study, when considering the final score, only group II and group III were taken into account since all cases from group I showed negative (0%) CENPF expression. A final score of less than 3 was considered as low CENPF expression, and a final score of greater than or equal to 3 was considered as high CENPF expression.

Out of 8 cases that showed positive expression of CENPF in benign salivary gland tumours (group II), 4 (50%) showed high CENPF expression and 4 (50%) showed low CENPF expression. Among the 4 cases from benign salivary gland tumours which showed high CENPF expression, 3 cases were pleomorphic adenomas and one case was myoepithelioma. The reason for high CENPF expression in four of the benign salivary gland tumours may be because of the fact that cells may be rapidly proliferating and these lesions have more chances of undergoing malignant transformation.

Among 20 malignant salivary gland tumours, 19 (95%) showed high CENPF expression and 1 (5%) showed low CENPF expression. That one case was diagnosed as mucoepidermoid carcinoma. The reason for low CENPF expression in this case may be because of the fact that mucoepidermoid carcinoma was cystic type and was of low grade.

Malignant salivary gland tumour with lymph node metastasis and recurrence has high CENPF expression.

Thus, in the present study expression of CENPF in various study groups revealed that normal salivary gland tumours did not express CENPF. The gradual increase in the expression of CENPF was noted from benign salivary gland tumours (40%) to malignant salivary gland tumours (100%) with statistical significance [Figure 9].
Figure 9: Image showing summary of study results

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   Conclusion Top


Together with our study results and knowledge about the role of CENPF as a proliferative marker for malignancy, CENPF may play a role in increased tumour aggressiveness. Since the number of cases included in our study was small, further analyses with increased number of cases are needed for further studies. So the present study suggests CENPF may be an additional diagnostic tool for salivary gland carcinomas.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Subhashraj K. Salivary gland tumors: A single institution experience in India. Br J Oral Maxillofac Surg 2008;46:635-8.  Back to cited text no. 1
    
2.
Nagao T, Sato E, Inoue R, Oshiro H, H Takahashi R, Nagai T, et al. Immunohistochemical analysis of salivary gland tumors: Application for surgical pathology practice. Acta Histochem Cytochem 2012;45:269-82.  Back to cited text no. 2
    
3.
Aggarwal P, Saxena S, Reddy V, Sharma P, Agarwal V. Demographics of salivary gland tumors: An institutional study in Western Uttar Pradesh. Int J Oral Med Sci 2014;1375-80.  Back to cited text no. 3
    
4.
Berto A, Yu J, Morchoisne-Bolhy S, Bertipaglia C, Vallee R, Dumont J, et al. Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores. EMBO Rep 2018;19:e44742. doi: 10.15252/embr. 201744742.  Back to cited text no. 4
    
5.
Ma L, Zhao X, Zhu X. Mitosin/CENPF in mitosis, transcriptional control, and differentiation. J Biomed Sci 2006;13:205-13.  Back to cited text no. 5
    
6.
Yang ZY, Guo J, Li N, Qian M, Wang SN, Zhu XL. Mitosin/CENPF is a conserved kinetochore protein subjected to cytoplasmic dyneinmediated poleward transport. Cell Res 2003;13:275-83.  Back to cited text no. 6
    
7.
Shigeishi H, Mizuta K, Higashikawa K, Yoneda S, Ono S, Kamata N. Correlation of CENPF gene expression with tumorproliferating activity in human salivary gland tumors. Oral Oncol 2005;41:716-22.  Back to cited text no. 7
    
8.
Cao JY, Liu L, Chen SP, Zhang X, Mi YJ, Liu ZG, et al. Prognostic significance and therapeutic implications of centromere protein F expression in human nasopharyngeal carcinoma. Mol Cancer 2010;9:237. doi: 10.1186/1476-4598-9-237.  Back to cited text no. 8
    
9.
Dai Y, Liu L, Zeng T, Zhu YH, Li J, Chen L, et al. Characterization of the oncogenic function of centromere protein F in hepatocellular carcinoma. Biochem Biophys Res Commun 2013;436:711-8.  Back to cited text no. 9
    
10.
To VS, Chan JY, Tsang RK, Wei WI. Review of salivary gland neoplasms. ISRN Otolaryngol 2012;2012:872982. doi: 10.5402/2012/872982.  Back to cited text no. 10
    
11.
Liu SC, KleinSzanto AJ. Markers of proliferation in normal and leukoplakic oral epithelia. Oral Oncol 2000;36:145-51.  Back to cited text no. 11
    
12.
Yuen KW, Montpetit B, Hieter P. The kinetochore and cancer: What's the connection? Curr Opin Cell Biol 2005;17:576-82.  Back to cited text no. 12
    
13.
Landberg G, Erlanson M, Roos G, Tan EM, Casiano CA. Nuclear autoantigen p330d/CENP-F: A marker for cell proliferation in human malignancies. Cytometry 1996;25:90-8.  Back to cited text no. 13
    
14.
Pfaltzgraff ER, Roth GM, Miller PM, Gintzig AG, Ohi R, Bader DM. Loss of CENP-F results in distinct microtubule-related defects without chromosomal abnormalities. Mol Biol Cell 2016;27:1990-9.  Back to cited text no. 14
    
15.
Welner S, Trier NH, Houen G, Hansen PR. Identification and mapping of a linear epitope of centromere protein F using monoclonal antibodies. J Pept Sci 2013;19:95-101.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2]



 

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