JICDRO is a UGC approved journal (Journal no. 63927)

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ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 1  |  Page : 48-51

Tissue eosinophils in different grades of oral squamous cell carcinoma – A special stain (carbol chromotrope) study


Department of Oral Pathology and Oral Microbiology, Vinayaka Mission's Sankarachariyar Dental College, Vinayaka Mission's Research Foundation (Deemed to be University), Salem, Tamil Nadu, India

Date of Submission19-Nov-2019
Date of Decision17-May-2020
Date of Acceptance29-Jul-2020
Date of Web Publication26-Jun-2021

Correspondence Address:
Dr. Diana Prem
Department of Oral Pathology and Oral Microbiology, Vinayaka Mission's Sankarachariyar Dental College, Vinayaka Mission's Research Foundation (Deemed to be University), Ariyanoor, Salem - 636 308, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jicdro.jicdro_59_19

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   Abstract 


Background: In recent days, tissue eosinophils are considered to have more impetus role in the biological behavior of various tumors. Compared to routine hematoxylin and eosin stain, the special stains such as carbol chromotrope for eosinophils are considered to be more specific and definitive for its evaluation in routine histopathology. Aim: This study aims to evaluate and compare the infiltration of tissue eosinophils in different grades of oral squamous cell carcinoma (OSCC) using a special stain – carbol chromotrope and to investigate the prognostic role of tissue eosinophils in OSCC. Methodology: The study was carried out with the sample size of fifty histopathologically diagnosed cases of OSCC, of which twenty cases were well differentiated, twenty cases were moderately differentiated, and ten cases were poorly differentiated, and comparison of infiltration of tissue eosinophils in the different grades of OSCC was done using special stain. The tissue sections were stained with carbol chromotrope special stain. Results: In the present study, more number of tissue eosinophils were seen in well-differentiated OSCC cases than the moderately and poorly differentiated OSCC cases, and the analysis of variance test showed P < 0.001. Tukey B's post hoc range test showed that mean scores fell in three different subsets, and the least mean score was observed in the poorly differentiated group (2.20) and the highest mean score was observed in the well-differentiated group (34.75). Conclusion: The results proved that the presence of more number of tissue eosinophils in well-differentiated OSCC is associated with favorable prognosis and that quantitative assessment of tissue eosinophilia should become a part of the routine histopathological diagnosis for the different grades of OSCC.

Keywords: Carbol chromotrope, eosinophils, oral squamous cell carcinoma, special stain, tissue eosinophilia


How to cite this article:
Prem D, Murugesan A, Sekar B, Ramesh M. Tissue eosinophils in different grades of oral squamous cell carcinoma – A special stain (carbol chromotrope) study. J Int Clin Dent Res Organ 2021;13:48-51

How to cite this URL:
Prem D, Murugesan A, Sekar B, Ramesh M. Tissue eosinophils in different grades of oral squamous cell carcinoma – A special stain (carbol chromotrope) study. J Int Clin Dent Res Organ [serial online] 2021 [cited 2021 Sep 24];13:48-51. Available from: https://www.jicdro.org/text.asp?2021/13/1/48/319529




   Introduction Top


In India, oral cancer ranks third among the top three types of cancer in the country.[1] The incidence and pattern of the disease varies according to the combined effect of aging of the population and the regional differences in the prevalence of disease-specific risk factors.[2] The number of people living with the disease is estimated to be around 2.5 million, with over 0.8 million new cases and 0.55 million deaths occurring each year.[3] Recently, the outlook of cancer has changed from bulk of malignant mass to a complex tumor microenvironment which has cancer cells corrupted subpopulation to form a self-sufficient biological structure. The stromal components composed of multiple different cell types such as cancer-associated fibroblasts, neutrophils, macrophages, regulatory T cells, myeloid-derived suppressor cells, natural killer cells, platelets, and mast cells. These cells interact with each other and promote increase in growth factors and cytokines and help in the development of cancer.[4]

Wharton Jones first described eosinophils as “coarse granular cells,” and later, Paul Ehrlich described it as “eosinophils.”[5] The presence of abundant cytoplasm with coarse reflective granules and is notable by their tinctorial properties showing bright red staining with acid aniline dyes.[6],[7] They are involved in initiation and propagation of diverse inflammatory responses as well as modulators of innate and adaptive immunity. Extensive tissue eosinophilia has been studied in many cancers including oral squamous cell carcinoma (OSCC).[6] Tumor-associated tissue eosinophilia (TATE) is defined as stromal infiltration of eosinophils in a tumor but not associated with tumor necrosis or ulceration. It was first described in cervical carcinoma by Przewoski, in 1896.[8] TATE seen in association with different malignancies and the correlation of tissue eosinophilia serves as a better prognostic indicator whose presence has been related to a varied inference ranging from favorable to unfavorable or even having no influence on prognosis.[9],[10] In this study, the infiltration of tissue eosinophils in different grades of OSCC is evaluated and compared its expression using hematoxylin and eosin (H and E) stain and carbol chromotrope.


   Methodology Top


Fifty cases of histologically proven cases of OSCC were selected from the Department of Oral Pathology and Oral Microbiology, Vinayaka Missions Sankarachariyar Dental College. From the selected cases, paraffin-embedded tissue blocks of twenty well-differentiated, twenty moderately differentiated, and ten poorly differentiated cases were taken. Participants treated with chemotherapy or radiotherapy before surgery and/or tumors with extensive ulceration and necrosis are not included in this study. Two sets of slides were prepared from each paraffin-embedded tissue blocks and used for staining with routine H and E and Carbol chromotrope.

Method of preparation of carbol chromotrope stain and staining method

Procedure - 1 g of phenol was melted in a flask by immersing flask in hot water bath. A quantity of 0.5 g of carbol chromotrope was added and dissolved; to the resultant sludge 100 ml of distilled water was added and stored in a Coplin jar.

All slides were stained with Harries hematoxylin for 5 min then differentiate with 1% acid alcohol. Then, counterstaining with carbol chromotrope was done for 15 min for one set of slides, and the other set was stained with eosin for a minute and then washed with tap water, dehydrated, cleared, and mounted.

The eosinophil granules stained light pink in colour with H&E stain [Figure 2], whereas the same stained bright red color when stained by the carbol chromotrope stain [Figure 1] which stood out from the light pink eosin stained sections [Figure 2].
Figure 1: tissue eosinophils in oral squamous cell carcinoma – carbol chromotype stain

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Figure 2: tissue eosinophils in oral squamous cell carcinoma – routine H and E stain

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The eosinophils were counted in 10 consecutive (×40) high power fields (hpf) using an optical binocular microscope and were recorded as a number of eosinophils/10 hpf.

Statistical analysis

The collected data were transferred to the excel sheet and subjected to statistical analysis using analysis of variance (ANOVA) test and Tukey B's post hoc tests. P < 0.001 was considered statistically significant.

Ethical Clearance

Ethical approval for this study (Ethical Committee VMSDC/IEC/Approval No. 184) was provided by the Ethical Committee of Vinayaka Mission's Sankarachariyar Dental College, Salem, Tamilnadu on 24 April 2021.


   Results Top


In the present study, quantitative assessment of eosinophil counts among different grades of OSCC was analyzed using the inferential statistics of ANOVA test, and the results of the analysis are given in [Table 1] and mean score in [Graph 1].
Table 1: Mean score regarding tissue counts

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[Table 1] shows the descriptive statistics of the sample size, mean, and standard deviation. The table also shows that the F-statistics calculated as the ratio of the variances. The column P value shows the probability value from the F-distribution, respectively and the P < 0.001. This shows that there is a highly significant difference in the mean tissue eosinophil counts with respect to the different grades of OSCC.

Once we determined that differences exist among the means, Tukey B's post hoc range tests were done to determine which means differ. This test identified the homogeneous subsets of means that are not different from each other, and the results are given in [Table 2].
Table 2: Tukey B's post hoc tests

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Since the mean scores fall in three different subsets, it showed that the average tissue eosinophil counts differed among all the three groups significantly. Hence, the least mean score was observed in the poorly differentiated group and the highest mean score was observed in the well-differentiated group. When compared to H and E stain, carbol chromotype showed better and specific staining for eosinophils.


   Discussion Top


Our immune system responds to foreign substance either by acting against tumor-specific antibodies and/or against tumor-associated antibodies and tries to eliminate them. Immunotherapy against cancer can boost the immune system of the patient and help in controlling cancer.[11] The role of tumor-associated eosinophils in OSCC is unclear. Still, apart from the common prognostic factors (location and size of tumor, recurrence and metastasis of tumor., etc.), eosinophils role in tumor defense after immunotherapy showed a favorable prognosis. In earlier studies by Fernandez-Acenero et al., in 2000,[12] Dorta et al., in 2002,[13] Debta et al., in 2016,[14] Yellapurkar et al., in 2016,[15] and Vaibhav et al., in 2018,[6] tumor-associated eosinophils had a favorable prognosis. Still, in the studies by Horiuchi et al., in 1993[16] and Joshi et al., in 2013,[17] no correlation of eosinophils with prognosis was noted.

In the present study, in 76% of cases (38 cases out of 50), eosinophil expression was seen. The mean expression in well, moderate, and poorly differentiated OSCC is 34.75, 16.0, and 2.2, respectively. The high eosinophil expression in well-differentiated OSCC denotes the immune activity is more in early stage of cancer which is similar to the study conducted by Jain et al., in 2014[9] and Sahni et al., in 2015[18] who found that mean eosinophil count was more in well-differentiated OSCC when compared to moderately and poorly differentiated OSCC. Low expression of eosinophil in poorly differentiated OSCC noted in our study was similar to the study conducted by Rahrotaban et al., in 2011.[19] In our study, when compared to H and E, carbol chromotrope prominently showed tissue eosinophils similar to the study conducted by Vaibhav et al., in 2018[6] and Debta et al., in 2012[20] eosinophil. To understand the promising role of tissue eosinophils, its expression must be compared in both metastatic and nonmetastatic OSCC, which was the only limitation in our study.

Extensive eosinophil seen in cancer and its role remains unclear, still it has been hypothesized by Lowe et al., in 1981[5] that the eosinophilactic substance released by the tumor cells is responsible for their presence in tumor. Furthermore, Jain et al., in 2014[9] hypothesized that the tumoricidal activity of TATE enhances the cytotoxic protein release and allows permeability and penetration of tumor-killing cytokines. Hence, the presence of immune cells in the tumor stroma determines the host immune response to cancer.


   Conclusion Top


It has been shown that the well-differentiated OSCC cases presented a higher overall survival rate when compared to the moderately and poorly differentiated cases. This concept, along with the presence of an increased number of tissue eosinophils in well-differentiated OSCC when compared to moderately and poorly differentiated OSCC in the present study, determines a good prognostic role in OSCC. Hence, the presence of TATE can be an indicator of prognosis where the eosinophils presence indicates an active role against tumor invasion and its spread. To confirm this, further studies with larger sample size have to be conducted to prove TATE as a better prognosticator.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Coelho KR. Challenges of the Oral Cancer Burden in India. J Cancer Epidemiol 2012;2012:1-17.  Back to cited text no. 1
    
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Nandakumar A, Gupta PC, Gangadharan P, Visweswara RN, Parkin DM. Geographic pathology revisited: Development of an atlas of cancer in India. Int J Cancer 2005;116:740-54.  Back to cited text no. 3
    
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Peltanova B, Raudenska M, Masarik M. Effect of tumor microenvironment on pathogenesis of the head and neck squamous cell carcinoma: A systematic review. Mol Cancer 2019;18:63.  Back to cited text no. 4
    
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Lowe D, Jorizzo J, Hutt MS. Tumour-associated eosinophilia: A review. J Clin Pathol 1981;34:1343-8.  Back to cited text no. 5
    
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Vaibhav SL, Priya PL, Sonam CK, Supriya K, Garima Y, Sabeer S, et al. Evaluation of tumor-associated tissue eosinophilia in different stages of oral squamous cell carcinoma using special stains: An in vitro histopathological study. J Contemp Dent Pract 2018;19:579-86.  Back to cited text no. 6
    
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Hogan SP, Rosenberg HF, Moqbel R, Phipps S, Foster PS, Lacy P, et al. Eosinophils: Biological properties and role in health and disease. Clin Exp Allergy 2008;38:709-50.  Back to cited text no. 7
    
8.
Przewoski E. On the local eosinophilia in cancer, together with remarks on the importance of eosinophil cells in general. Centralbl f General Path u Path Anat 1896;5:177-91.  Back to cited text no. 8
    
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Jain M, Kasetty S, Sudheendra US, Tijare M, Khan S, Desai A. Assessment of tissue eosinophilia as a prognosticator in oral epithelial dysplasia and oral squamous cell carcinoma – An image analysis study. Pathol Res Int 2014;2014:1-6.  Back to cited text no. 9
    
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Nagpal B, Hegde U, Deshpande P, Dey S. Tumor associated tissue eosinophilia: A case report with review. Int J Otorhinolaryngol Head Neck Surg 2018;4:285-8.  Back to cited text no. 10
    
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Finn OJ. Cancer immunology. J Med 2008;358:2704-15.  Back to cited text no. 11
    
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Fernandez-Acenero MJ, Galindo-Gallego M, Sanz J, Aljama A. Prognostic influence of tumor-associated eosinophilic infiltrate in colorectal carcinoma. Cancer 2000;88:1544-9.  Back to cited text no. 12
    
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Dorta RG, Landman G, Kowalski LP, Lauris JR, Latorre MR, Oliveira DT. Tumour-associated tissue eosinophilia as a prognostic factor in oral squamous cell carcinomas. Histopathology 2002;41:152-7.  Back to cited text no. 13
    
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Debta P, Debta FM, Chaudhary M, Bussari S. Evaluation of myeloid cells (tumorassociated tissue eosinophils and mast cells) infiltration in different grades of oral squamous cell carcinoma. Indian J Med Paediatr Oncol 2016;37:158-67.  Back to cited text no. 14
[PUBMED]  [Full text]  
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Yellapurkar S, Natarajan S, Boaz K, Baliga M, Shetty P, Manaktala N, et al. Tumour-associated tissue eosinophilia in oral squamous cell carcinoma – A boon or a bane? J Clin Diagn Res 2016;10:ZC65-8.  Back to cited text no. 15
    
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Horiuchi K, Mishima K, Ohsawa M, Sugimura M, Aozasa K. Prognostic factors for well-differentiated squamous cell carcinoma in the oral cavity with emphasis on immunohistochemical evaluation. J Surg Oncol 1993;53:92-6.  Back to cited text no. 16
    
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Joshi PS, Kaijkar MS. A histochemical study of tissue eosinophilia in oral squamous cell carcinoma using Congo red staining. Dent Res J (Isfahan) 2013;10:784-9.  Back to cited text no. 17
    
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Sahni P, Patel A, Shylaja M, J. hallur, Gujjar PK. Tumor associated tissue eosinophilia in oral squamous cell carcinoma: A histo chemical analysis. Malays J Med Sci 2015;22:21-5.  Back to cited text no. 18
    
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Rahrotaban S, Khatibib A, Allam A. Assessment of tissue eosinophilia in head and neck squamous cell carcinoma by Luna staining. Oral Oncol 2011;47:S74-156.  Back to cited text no. 19
    
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Debta P, Debta FM, Chaudhary M, Dani A. Evaluation of infiltration of immunological cells (tumour associated tissue eosinophils and mast cells) in oral squamous cell carcinoma by using special stains. Br J Med Med Res 2012;2:75-85.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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