|Year : 2018 | Volume
| Issue : 1 | Page : 19-25
Quantitative analysis of eosinophils and mast cells in potentially malignant disorder and squamous cell carcinoma using special stains
Treville Pereira, Neha Tiwari, Avinash Tamgadge, Sandhya Tamgadge, Subraj J Shetty, Swati Gotmare
Department of Oral and Maxillofacial Pathology and Microbiology, Dr D. Y. Patil University, School of Dentistry, Navi Mumbai, Maharashtra, India
|Date of Web Publication||20-Dec-2018|
Dr. Treville Pereira
Department of Oral and Maxillofacial Pathology and Microbiology, Dr D. Y. Patil University, School of Dentistry, Sector 7, Nerul, Navi Mumbai, Maharashtra - 400 706
Source of Support: None, Conflict of Interest: None
Background: Oral squamous cell carcinoma (OSCC) develops from potentially malignant lesions. Cells of the immune system are comprised of lymphoid series and myeloid progenitor series cells. Mast cells (MCs) and tissue eosinophils are both granulocytes which come under the myeloid progenitor series of the immune cells system. Inflammatory cells are found to play a crucial role and therefore should be studied in detail using various simple histochemical stains. Aims and Objectives: To evaluate the presence and compare infiltration of MCs and eosinophils in potentially malignant disorders and OSCC using special stains. Materials and Methods: Twenty-five cases of previously diagnosed cases of OSCC, 13 cases of oral submucous fibrosis, and 12 cases of oral leukoplakia (50 cases) were retrieved from the archives. Congo red and toluidine blue staining were performed for eosinophils and MCs, respectively. Analysis was done using analysis of variance and independent t-test, and P ≤ 0.05 was considered for statistical significance. Results: The mean number of MC and eosinophils was more in OSCC when compared to potentially malignant disorders. There was a significant correlation between eosinophils and MCs in potentially malignant disorder and OSCC. Intact MCs were more in number in potentially malignant disorder than in OSCC, and the mean number of degranulated MC was more in OSCC than in potentially malignant disorder. Conclusion: Eosinophil chemo-attractant factor released by MC invites more number of eosinophils during tumor progression. MCs and eosinophils may serve as a novel therapeutic target for cancer treatment, and inhibiting their function may inhibit tumor progression. Eosinophils and MCs can be visualized using Congo red and toluidine blue stain, respectively, which is accurate and easy to perform.
Keywords: Coloring agents, Congo red, eosinophils, mast cells, special stain, toluidine blue
|How to cite this article:|
Pereira T, Tiwari N, Tamgadge A, Tamgadge S, Shetty SJ, Gotmare S. Quantitative analysis of eosinophils and mast cells in potentially malignant disorder and squamous cell carcinoma using special stains. Niger J Exp Clin Biosci 2018;6:19-25
|How to cite this URL:|
Pereira T, Tiwari N, Tamgadge A, Tamgadge S, Shetty SJ, Gotmare S. Quantitative analysis of eosinophils and mast cells in potentially malignant disorder and squamous cell carcinoma using special stains. Niger J Exp Clin Biosci [serial online] 2018 [cited 2019 Mar 23];6:19-25. Available from: http://www.njecbonline.org/text.asp?2018/6/1/19/248007
| Introduction|| |
Cancer is the second most common cause of death after heart diseases in developed countries and third leading cause of mortality following heart and diarrheal diseases in developing countries. Cancer of oral cavity accounts for approximately 3% of all malignancies and found in 270,000 patients annually worldwide. Oral squamous cell carcinoma (OSCC) comprises 92%–95% of all oral cancers. Although 95% of oral cancers occur in individuals older than 40, it is noteworthy that many OSCCs develop from potentially malignant disorders. Correct diagnosis and timely treatment of potentially malignant disorders may help prevent malignant transformation. Leukoplakia is a precancerous lesion defined as a predominantly white lesion of the oral mucosa that cannot be characterized as any other definable lesion. Oral submucous fibrosis (OSF) is a chronic, progressive, scarring, precancerous condition which is characterized by mucosal rigidity. Solid tumors do not consist of neoplastic cells in isolation, rather they include a range of inflammatory cell infiltrate. The chief inflammatory cells mounting the host response to tumorigenic cells include lymphocytes, macrophages, neutrophils, plasma cell, mast cells (MCs), and eosinophils. It is becoming accepted that multiple cell types in stromal microenvironment are involved in tumorigenesis. In this setting, MC displays a diversity of roles that may contribute to the defense against tumors or tumor progression. Eosinophils are thought to become active following the action of MC as these cells secrete histamine and eosinophil chemo-attractant factor (ECF) which attract eosinophils in tissues.,
Eosinophils have been hypothesized to affect the tumor cells directly by releasing cytotoxic proteins and also indirectly by enhancing the permeability into tumor cells by facilitating penetration of cytokine-induced killer cells (T-lymphocytes). Recent studies have shown that MCs are significantly increased in human lip carcinoma and OSCC, being associated with tumor-favoring effects. Moreover, in an experimental model of carcinogenesis, MC density has been associated with carcinoma development through the upregulation of angiogenesis during premalignant and malignant stages of squamous epithelial carcinogenesis. On the other hand, some studies suggest a tumor antagonistic effect of MC, given that MC-deficient mice had an increased tumor incidence after treatment with a carcinogenic agent. Furthermore, MC mediators have an in vitro inhibitory effect on keratinocyte proliferation.
Intact eosinophils and MCs can be identified in tissue sections stained with hematoxylin and eosin staining. However, these granulocytes assume an uncommon morphology making their recognition difficult in routinely stained sections. MCs are not readily identified in hematoxylin and eosin stains because their metachromatic granules are refractile and do not take up the stain. This metachromasia is due to the high concentrations of the sulfated mucopolysaccharide heparin. Hence, this study aimed at evaluating the infiltration of these inflammatory cells (MCs and eosinophils) in OSCC and potentially malignant disorders using toluidine blue and Congo red stain, respectively, and its possible use as a prognosticator in OSCC.
| Materials and Methods|| |
The study comprised a total of 50 cases, of which 25 were of OSCC and 25 of potentially malignant disorders (13 cases of OSF and 12 cases of oral leukoplakia). Formalin-fixed paraffin-embedded tissue blocks of these cases were retrieved from the archives of the Department of Oral and Maxillofacial Pathology. The control group consisted of 10 samples of normal oral mucosa. All the cases were diagnosed on the basis of clinical and histopathological correlation. Serial sections of 4–5 μm thickness were used for staining with hematoxylin and eosin and special stains. The study was carried out for 6 months.
Two special stains; toluidine blue was used for MC staining and Congo red stain was used for tissue eosinophils staining. High-density areas of infiltration of these cells were selected randomly in section and the cells were counted in high-power fields (HPFs). For evaluation of the inter/intra-examiner consistency, the slides were observed by two or more examiners for counting of these cells.
Congo red staining procedure
First, sections were deparaffinized, hydrated through graded alcohols to water, and then placed in 1% Congo red solution for 8 min followed by washing in water. Then, differentiation was done in 2.5% potassium hydroxide solution by dipping once. Sections were counterstained with hematoxylin for 8 min and then washed under running tap water. Differentiation was done in 1% acid alcohol by dipping once. Finally, the sections were dehydrated through alcohol and cleared in xylene. Sections were mounted with distyrene plasticizer xylene (DPX). Results showed eosinophil granules red in color while the nuclei stained blue [Figure 1].
|Figure 1: Photomicrograph of potentially malignant disorder and oral squamous cell carcinoma stained with Congo red showing eosinophils (×40)|
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Toluidine blue staining procedure
Add 100 ml distilled water in 0.5 g toluidine blue. Mix it properly and then filter the final solution to remove undissolved particles or dust from solution. The day of preparation of this solution was noted as this prepared staining solution has shelf life of about 6 months. Final solution can be stored at room temperature. After preparation of 0.5% toluidine blue solution, sections were stained in this solution for 30 s. Results showed MC granules took up a red/purple color while the tissue showed varying shades of blue [Figure 2].
|Figure 2: Photomicrograph of potentially malignant disorder and oral squamous cell carcinoma stained with toluidine blue showing mast cells (×40)|
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Common steps after staining
Sections were washed in tap water, then dehydrated through ascending grades (70%, 80%, 90%, and 100%) of alcohol, and cleared and mounted in DPX. Slides were examined under microscope.
MCs and eosinophils were analyzed quantitatively by counting the total number of MCs and eosinophils in toluidine blue and Congo red stained sections, respectively. Further, qualitative analysis was done by counting the number of intact and degranulated MCs. Cells were counted using software grid under a magnification of ×40.
The toluidine blue-stained sections and Congo red-stained sections were first screened at low power (×10). Cell counting was then performed under ×40. Five high-density areas were selected and software grid (10 × 10) was created with an area of 0.04 mm2 which was calibrated. The cells were counted throughout each of the tissue sections in five representative and consecutive grid fields (×40). The mean of five values was calculated and expressed as mean ± standard deviation (SD) per mm2. The fields were studied in a step ladder fashion and care was taken to prevent the overlapping of fields. The cells extending over other squares were counted in first square.
Based on intensity of metachromasia, MCs were categorized into two groups.
- Intact MCs exhibiting intense metachromasia and dense granules obscuring the nucleus
- Degranulated MCs with less intense metachromasia and a clear outline of the nucleus.
Statistical analysis used
The results were then analyzed with IBM SPSS 20 (Chicago, IL, USA.) software using the descriptive data including mean, SD, and percentages and were calculated for each group. The association was done using analysis of variance and independent ttest. For all the tests, a P ≤ 0.05 was considered for statistical significance.
| Results|| |
In the present study, of 13 cases of OSF, it was observed that the age range for the patients was 20–45 years, occurring in 12 males and one female. Buccal mucosa was the single most common site of involvement. In 25 cases of OSSC, the age range was found to be 29–75 years, with a mean age of 51.96 years. Of 25 cases, 19 were males while six were females [Graph 1] and [Graph 2]. Among all cases of OSCC, buccal mucosa was found to be the most common site of involvement (9 cases) followed by alveolus (8 cases), tongue (5 cases), and lip, floor of the mouth, and labial mucosa (1 case each).
In the present study, we observed that the mean of MC/sq. mm in normal oral mucosa, potentially malignant disorder, and OSCC which were 0.4 ± 0.6, 4.8 ± 1.5, and 13.9 ± 1.1 cells/mm2, respectively [[Table 1] and [Graph 3]]. On statistical analysis, we found that MC density has significantly increased from normal oral mucosa to potentially malignant disorder (P < 0.0001) and from normal oral mucosa to OSSC (P < 0.0001). The increase in the MC density from potentially malignant disorder to OSSC was also found to be statistically significant (P < 0.0001).
|Table 1: Correlation between mast cells and eosinophils in potentially malignant disorders and oral squamous cell carcinoma|
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The qualitative assessment of MC was also done as the intact and degranulated MC/mm2. In patients of potentially malignant disorder, the mean of intact MCs was found to be 4.4 ± 0.5/sq. mm while the mean of degranulated MC was found to be 0.48 ± 0.3/sq. mm. In the patients of OSCC, mean of intact MC was found to be 2.2 ± 0.4/sq. mm while mean of degranulated MC was found to be 5.2 ± 1.4/sq. mm [[Table 2] and [Graph 4]]. On comparing the mean of degranulated MC density in various groups, we found that mean of degranulated MC density has significantly increased from potentially malignant disorder to OSSC (P < 0.0001).
|Table 2: Distribution of intact mast cells and degranulated mast cells in potentially malignant disorder and oral squamous cell carcinoma|
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| Discussion|| |
Tissue eosinophils and MCs both are granulocytes which come under myeloid progenitor series of immune cells system. Eosinophils were first described by Wharton Jones in 1846 as “coarse granule cells.” The eosinophils are 8 μm in diameter and characterized by its bright red granules. Their nuclei are usually bilobed although three or more lobes are often observed. An eosinophil is a granular leukocyte which is normally found in the bloodstream and the gut lining. They contain proteins that help the body to fight infection from parasitic organisms, such as worms. However, in certain diseases, these proteins can damage the body. The term eosinophilia refers to conditions in which abnormally high amounts of eosinophils are found in either the blood or in body tissues.
MCs were first described by Paul Ehrlich in 1879. Some years later, Elie Metchnikoff in 1892 suggested that MCs on account of their phagocytic capacity might contribute to host defense. MCs are round or elongated in shape with characteristic metachromatically staining cytoplasmic granules. They can be appreciated as large cells with a diameter varying from 5 to 25 μm. These cells are derived from multipotent stem cells in the bone marrow. The undifferentiated precursors are carried by the bloodstream to their final tissues of deposition. The main functions of MC are probably regulation of vascular functions at the initiation of an inflammatory response and activation of other cells. MCs also initiate immediate hypersensitivity reactions, modulate allergic inflammation, and participate in the immune response to parasitic infections. MCs, when stimulated, can exocytose a fraction of their secretory granules in the surrounding tissue, a process called degranulation. These cell infiltrations can be seen in mucocutaneous diseases, periodontal diseases, OSF, and OSCC. Eosinophils are thought to become active following the action of MC as MCs secrete histamine and ECF which attract eosinophils in the tissue.
In the present study, of 13 cases of OSF, it was observed that the age range for the patients was 20–45 years and occurred in 12 males and one female. Buccal mucosa was the single most common site of involvement.
Similar results have been reported by Mortazavi et al., who showed that OSF occurred more frequently in men from 20 to 40 years of age and the most common site of involvement was buccal mucosa. In 12 cases of hyperkeratotic lesion, the age range was found to be 20–56 with male predominance. In the present study, the buccal mucosa was the single most common site of involvement followed by labial mucosa.
Bokor-Bratic et al. in their study reported that hyperkeratotic lesions occurred more frequently in men over 40 years of age, with buccal mucosa being the most common site of involvement. Markopoulos in his study reported male preponderance with mean age of 53.5 years. He also reported that buccal mucosa was the most commonly affected site because of frequent exposure to carcinogens.
In the present study, MCs were stained with metachromatic dye, 1% toluidine blue, and eosinophils were stained with 0.5% Congo red. Average number of cells was counted at ×40 using software grid (0.04 mm2) in five grid fields.
It was also observed that toluidine blue staining revealed MCs as large, purple, oval, and highly granulated cells and Congo red staining revealed eosinophils as large, bright red, round, and highly granulated cells. In normal oral mucosa, MCs and eosinophils were very few and looked small without signs of activation around blood vessels. In case of potentially malignant disorder, MCs looked larger and intact; the histologic location was in the underlying connective tissue and predominantly around the blood vessels and eosinophils looked larger and round; the histologic location was in superficial lamina propria just below the epithelium. In case of OSCC, MC looked enlarged and mostly degranulated in the underlying connective tissue, predominantly around the blood vessels growing into the epithelial lesion, and eosinophils were numerous and in clusters predominantly around blood vessels.
Ankle et al. found an increase in the number of MC in tissues sections of oral leukoplakia, OSF, lichen planus, and OSCC as compared to normal mucosa. They observed that MC hyperplasia was present in all the conditions compared to normal oral mucosa.
Jandinski et al. showed an increase in number of MC from normal to benign hyperkeratotic and dyskeratotic tissues, but this increase was not significant while the MC number increased significantly from normal tissue to low-grade carcinoma. They suggested that this increase in number is because of antigenic stimulation.
Iamaroon et al. compared the MC density and mean vasculature density in cases of OSCC, premalignant dysplasia, oral hyperkeratosis, and normal oral mucosa and reported that MC and microvascular counts were significantly higher in OSCC than in premalignant dysplasia and normal oral mucosa. They suggested that MCs play a significant role in tumor angiogenesis in OSCC probably by releasing potent angiogenic factors such as tryptase.
Researches carried out by certain workers also led to the results that were contrary to our findings. A decrease in the number of MC in OSCC and leukoplakia when compared with control was observed by Oliveira-Neto et al. This decrease in the number of MC could be attributed to the migration failure of these cells, possibly reflecting an important modification in the microenvironment during tumor initiation and progression. These differences could be related to differences in the stage of the pathological process.
In the present study, we observed that the increase in the MC density from potentially malignant disorder to OSCC was also found to be statistically significant.
In the present study, it was observed that upon comparison of MC with age or sex, the results were not significant in either of the groups. This was in accordance to the study conducted by Oliveira-Neto et al., who also did not find any correlation of MC density with age or sex.
In the present study, the quantitative assessment of MC was also done as the intact and degranulated MC/mm2. In normal oral mucosa and potentially malignant disorder groups, the number of mean of intact MC/mm2 was observed to be more than that of mean of degranulated MC/mm2. In OSCC group, more number of MC were observed to undergo degranulation, and therefore, the number of mean of degranulated MC/mm2 was observed to be more than that of intact MC/mm2.
Walsh et al. studied the relationship between MC degranulation and inflammation within the oral cavity. They observed that in normal and perilesional tissue the mean proportion of degranulated MC was 4%, while in lesional tissues, the mean proportion of degranulated MC was 93%. Their study elicited that the MC degranulation is a common feature of inflammatory lesion.
Flynn et al. in experimental carcinogenesis demonstrated sequential MC migration and degranulation toward progressive mucosal dysplasia and subsequent development of squamous cell carcinoma.
Yano et al. found that many tumor-associated MCs underwent degranulation and released granular components such as heparin and histamine, which have been shown to potentiate endothelial cell migration and proliferation and to induce adhesion molecule expression on epithelial cells, potentially leading to increased tumor angiogenesis and metastasis.
Further, in the present study, we observed that eosinophil count also had significantly increased from normal mucosa to potentially malignant (P < 0.0001) and from normal oral mucosa to OSCC (P < 0.0001). The increase in eosinophils from potentially malignant disorder to OSCC was also found to be statistically significant (P < 0.0001).
Alrawi et al. demonstrated elevated eosinophilic counts in invasive squamous cell carcinoma compared to noninvasive tumors of head and neck region. Eosinophilic counts were elevated focally and/or diffusely more frequently in invasive squamous cell carcinoma than in noninvasive tumors. The increased eosinophilic counts, specifically >10/HPF and >20/10 HPF, were both significantly associated with stromal invasion. They concluded that the elevated eosinophilic counts in biopsies and eosinophilic indices in specimens of squamous cell carcinoma of the aerodigestive tract is a histopathologic marker associated with tumor invasion and a clinical predictor for aggressive tumor biology. Similarly, the presence of eosinophils meeting these thresholds in an excisional specimen should indicate the need for additional therapeutic measures and close surveillance to detect earlier locoregional recurrence and possible distant metastasis.
Jain et al. reported that mean eosinophilic count in OSCC group was found to be significantly higher than dysplasia group, suggesting that they might have a role in stromal invasion. Nonmetastatic cases showed higher counts than metastatic carcinomas. Hence, it is concluded that eosinophilia is a favorable histopathological prognostic factor in oral cancer. Moreover, higher eosinophil counts in carcinoma group compared to dysplasia group proved that they might have a role in stromal invasion, thus suggesting that quantitative assessment of tissue eosinophilia should become a part of the routine histopathological diagnosis for oral precancer and OSCC. Further, Dorta et al. found that intense tissue eosinophilia was associated with 72% of 5-year disease-free cumulative survival, whereas only 32% and 44% were associated with absent/mild and moderate tissue eosinophilia, respectively.
Debta et al. also found that increase infiltration of eosinophils and MC in OSCC was associated with favorable prognosis.
Kargahi et al. reported that the number of eosinophils progressively increased from normal mucosa to dysplastic and squamous cell carcinoma. In addition, it increased from mild to severe at different levels of dysplastic mucosa and from well differentiation to poor differentiation in squamous cell carcinoma. Accordingly, eosinophils can be regarded as an indicator of a developing malignancy along with other indicators, and they possibly can be used to develop the prognosis for the disease.
In the present study, we also used special stain such as toluidine blue and Congo red for detection of MCs and eosinophils, respectively. The MCs stained purple with light blue background and eosinophils stained bright red with light red blue background, which is easy and quick to identify.
Kargahi et al. in their study suggested that for better diagnosis of eosinophils, a Congo red staining technique could be used where eosinophils with shiny red cytoplasm were analyzed more effectively than they could have been done using the hematoxylin and eosin staining method. In addition, Joseph et al. suggested the total number of cells was greater with toluidine blue than with thionin.
The results of the present study, therefore, elicited that the number of MCs and eosinophils increased with the progression of lesions from normal oral mucosa to potentially malignant disorder to OSCC. In case of OSSC, there is progressive degranulation of MC and the mean degranulated MC density is more than that of mean intact MC density. These degranulated MCs probably play a role in facilitating tumor progression by release of factors contained within the MC granules. In addition, Congo red and toluidine blue staining for eosinophils and MC, respectively, are economical, efficient, practical, and reliable.
| Conclusion|| |
OSCC is becoming one of the most common cancers within India; there is an imminent need to counter the disease at its early stage and decrease disease morbidity. This is however only possible if the disease is understood in its entirety and all possible mechanisms of cancer growth and progression are identified. As one of the hallmarks of cancer is tumor-promoting inflammation, the present study focused on finding the correlation of inflammatory cells with degree of malignancy.
Based on the present study, we conclude that the role of MC and eosinophils in tumors may have direct clinical relevance following important clinical implications. Correlation between eosinophils and MC may suggest that ECF released by MC invite more number of eosinophils during tumor progression. Therefore, inflammatory cells (MCs and eosinophils) may serve as a novel therapeutic target for cancer treatment and inhibiting their function may inhibit tumor progression. The present study has not shown that the MC and eosinophils may potentiate invasive malignancy because these cells may be a failed response to invasion rather than the cause. Congo red stain for eosinophils and toluidine blue stain for MCs are economical, accurate, and easy to perform for routine purpose.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]