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ORIGINAL ARTICLE Table of Contents  
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Morphometric analysis of collagen in different grades of oral squamous cell carcinoma using picrosirius red stain and spectrophotometry


1 Department of Oral and Maxillofacial Pathology, GSL Dental College and Hospital, Rajahmundry, Andhra Pradesh, India
2 Department of Periodontics and Implantology, GSL Dental College and Hospital, Rajahmundry, Andhra Pradesh, India

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Date of Submission31-Oct-2020
Date of Decision18-Aug-2021
Date of Acceptance19-Jun-2021
Date of Web Publication05-Jan-2023
 

  Abstract 


Introduction: Oral squamous cell carcinoma (OSCC) is one of the most formidable health problems for mankind. These carcinomas are characterized by invasion of epithelial tumor cells into the stroma, which get embedded in extracellular matrix and collagen producing reactive changes. Such changes in the stroma may alter the biological aggressiveness of the tumor. An attempt was made to evaluate the collagen changes in different grades of OSCC which can contribute to understanding the biologic behavior of oral cancer and predict clinical outcomes. Aims and Objectives: To assess the quantitative changes in collagen in different grades of OSCC using hematoxylin and eosin (H and E) and Picrosirius red (PSR) stain through spectrophotometry and to compare the efficacy of these stains for estimation of collagen. Materials and Methods: The study comprised a total sample size of 60, which were distributed under 4 different groups, each containing 15 samples. Group I to IV consisted of normal buccal mucosa, with well-, moderately-, and poorly-differentiated OSCC, respectively. The tissues of 10 μm thickness were stained with H and E and PSR for spectrophotometric analysis. Results: The quantity of collagen decreased with increasing grades of OSCC. Comparison between two stains showed that PSR can provide a more reliable and accurate result than H and E. Conclusion: Collagen estimation is one of the methods to assess the progression of tumor. The method used in the present study for collagen estimation in different grades of OSCC is reliable and accurate.

Keywords: Collagen, hematoxylin and eosin, oral squamous cell carcinoma, picrosirius red, spectrophotometry


How to cite this URL:
Ashok S, Ashok K P. Morphometric analysis of collagen in different grades of oral squamous cell carcinoma using picrosirius red stain and spectrophotometry. J Microsc Ultrastruct [Epub ahead of print] [cited 2023 Feb 8]. Available from: https://www.jmau.org/preprintarticle.asp?id=362483





  Introduction Top


Cancer is probably as old as the human race. Hippocrates is credited with the first use of the term “cancer,” which means crab in Greek, possibly because the growths reminded him of a moving crab. Although cancer is described in ancient manuscripts, it is only in last 150 years that there has been clear recognition of the nature of the disease.[1]

The most prevalent cancers in developed countries are lung, breast, or colon cancer, whereas in developing countries, it is head and neck cancers. According to the World Health Organization, carcinoma of the oral cavity in males in developing countries is the sixth most common cancer, whereas in females, it is the tenth most common site of cancer after breast, colorectal, lung, stomach, uterus, cervix, ovary, bladder, and liver.[2] Oral cancer represents 5% of all cancers in men and 2% in women. More than 90% of oral cancers are squamous cell carcinoma (SCC) or one of its variants.[3]

The oral cavity is easily accessible to complete examination, due to which the rate of early detection of precancerous and cancerous lesions should be high. However, due to ignorance or inaccessibility of medical care, the disease is often detected in the later stages.[2]

Oral squamous cell carcinomas (OSCCs) are mostly related to tobacco, alcohol, and betel use.[4] In general, risk factors for oral/pharynx cancer can be classified into several categories, including chemical carcinogens, oncogenic viruses, sunlight, oral hygiene, nutritional factors, genetic predisposition, and being immunocompromised.[5]

The biology of OSCC has been recognized, including its progression from dysplasia to carcinoma and the effect of matrix metalloproteinase (MMP) in breaking down of matrix proteins, facilitating invasion. Alterations of the extracellular matrix (ECM) may play a role in the recurrence and invasion of tumor cells. The ECM is composed of ground substance and fibrous components including collagen and elastic fibers.[6] Collagen plays a vital role in maintaining structural integrity and in determining tissue function. Therefore, methods to detect, quantify, and analyze collagen are valuable.[7]

Routine hematoxylin and eosin (H and E) staining and special stains play a critical role in tissue-based diagnosis or research. In spite of advanced staining methods used today, H and E staining still forms a critical part of the diagnosis. In a histology laboratory, all specimens are initially stained with H and E and special or advanced stains are only used if additional information is needed to provide a more detailed analysis. Collagen analysis is one of the methods which require the use of special stains.[8]

Various special stains such as Van Gieson's, Masson's Trichrome, and Picrosirius red (PSR) stain can be used for qualitative collagen analysis. PSR stains thin fibers do not fade, unlike other stains. When examined under a polarizing microscope, collagen exhibits birefringence and differentiates between procollagens, intermediate, and pathologic collagen fibers.[9] Apart from the evaluation of neocollagen genesis, PSR can also be used in quantifying collagen content. Such evaluations can be quantified using morphometry techniques.[10]

A spectrophotometer is one of the morphometric techniques which is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The resultant reading is read in terms of absorbance and transmittance.[11] The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, and molecular biology.[12] Quantitative analysis of collagen can be done using this technique. The absorbency refers to the collagen content in the specimen.[13]

The early diagnosis and prevention of OSCC depend on the understanding of its development and progression. Once the mechanism is known, more effective therapies may be created to reduce the degradation of ECM.[14]

This study was carried out to assess the quantitative changes in collagen fibers in different grades of OSCC and normal buccal mucosa using H and E and PSR stains through spectrophotometry and to compare the efficacy of these stains for the estimation of collagen.


  Materials and Methods Top


The samples for the present retrospective study were retrieved from the archives of the Department of Oral and Maxillofacial Pathology of the author's present institution. The tissues for the control group were obtained from buccal mucosa of patients undergoing minor surgical procedures in the department of oral and maxillofacial surgery. The spectrophotometric analysis for the study was done in the nearest available Defense Laboratory to the institution.

The study comprised a total sample size of 60 cases within the age range of 20–80 years. The samples were distributed in four different groups each involving 15 cases. Group I to Group IV consisted of normal buccal mucosa, well-differentiated SCC, moderately differentiated SCC, and poorly-differentiated SCC, respectively.

Written consent according to Declaration of Helsinki was obtained from 15 patients of Group I. An institutional ethical committee clearance for the study was also taken from Institutional Ethical Board (GSLDC/IEC/2019/010 on 21/03/2019).

Two sections of each tissue from all four groups were cut into two different thicknesses (one of 5 μm thickness and one of 10 μm thickness). All the tissue sections of 5 μm thickness were stained with H and E stain, and histopathological grading was done according to Broder's classification (1927).[15] Similarly, tissues of 10 μm were stained with PSR stain for further spectrophotometric analysis.

The sections prepared for spectrophotometric analysis were scraped off from the slides with the help of a bard parker blade (No. 11), and agglomerated to separate assay tubes containing 1ml of solution of 0.1% of NaOH in absolute methanol[13] [Figure 1]a and [Figure 1]b. The halogen light was passed through the assay tubes placed in the probe of spectrophotometer (Ocean Optics – QU 65000). The detected absorbance was computed in the form of a graph.
Figure 1: (a) Scraping of picrosirius red stained tissue sections with the blade. (b) Assay tubes

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  Results Top


The readings were noted accordingly from the graphs. Statistical analysis of the study consisted of one-way analysis of variance (one-way ANOVA) for multiple group comparisons, followed by Tukey's honestly significant difference (HSD) post hoc test for pairwise comparisons.

The quantitative evaluation of collagen in all the four groups was done in a spectrophotometer using PSR-stained sections. The absorbance for PSR-stained sections was read at 540 nm[13] [Graph 1]a, [Graph 1]b, [Graph 1]c, [Graph 1]d.



The total number of samples in each group was 15 (n = 15). The minimum absorbance values from Group I to Group IV for PSR were 0.10, 0.13, 0.20, and 0.40, respectively. Similarly, the maximum absorbance values in above groups were 0.19, 0.21, 0.41, and 0.64, respectively.

The mean values for each group from Group I to Group IV for PSR stain were calculated as 0.132 ± 0.035, 0.168 ± 0.025, 0.324 ± 0.063, and 0.515 ± 0.063.

The comparison between groups was done using a one way ANOVA. The variation within the groups was statistically highly significant for the used stain (F = 186.98, P ≤ 0.001).

The Tukey's HSD pairwise comparisons among the groups for PSR stain pairwise comparison between Group I and Group II were not significant (P ≤ 0.20), but all other groups showed statistically highly significant results (P ≤ 0.001) [Table 1].
Table 1: Tukey's honestly significant difference post hoc analysis for inter-group comparison for picrosirius red stain

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The results showed that the PSR stain, as a special stain for collagen, can provide a reliable and accurate result for the evaluation of collagen in the tissues of different grades of OSCC.


  Discussion Top


Oral cancer is an important global health concern, accounting for an estimated 275,000 cases and 128,000 deaths annually. Two-thirds of all cases are observed in developing countries. The Indian subcontinent accounts for one-third of the global burden.[16] Oral cancers are the sixth most prevalent cancer in the world.[17]

The term oral cancer encompasses all malignancies that originate in the oral tissues which includes the lip, buccal mucosa, lower and upper alveolar ridges, retromolar gingiva, oropharynx, floor of the mouth, hard palate, and the anterior two thirds of the tongue.[18] Over 80% of these lesions are SCCs.[17]

OSCC consists of a heterogeneous cell population with different biologic characters. For many years a tumor node metastasis staging system has been used clinically for estimating response to therapy and survival. There are many patients who die despite the fact that their neoplasms were considered clinically to be Stages I and II and were treated accordingly. In such patients, a combined assessment of clinical staging and histological grading of neoplasm might serve as more precise measure for predicting the outcome of neoplasm and for determining their treatment.[15]

The prognosis for cancer patients with metastatic disease remains poor. For a tumor to metastasize, the basement membrane and ECM must be degraded to allow the passage of tumor cells from a primary tumor to distinct sites in the body. The primary structural constituent of these physical barriers is collagen.[19] Therefore, the present study was designed for the quantitative evaluation of collagen fibers in different grades of OSCC using PSR for spectrophotometry. To the best of our knowledge, our study is the first of its kind to use PSR stain in a spectrophotometric evaluation of collagen in OSCC.

The spectrophotometric analysis with the PSR stain showed an increase in absorbance peak with increasing grades of OSCC. This indicates that as the severity of OSCC increases, the amount of collagen decreases. This was in accordance with Venigella and Charu.[14] Kawashiri et al.[20] and Venigella and Charu[14] also showed that there was a decrease in collagen content with an increase in grades of OSCC by observing the color changes of fibers using PSR and polarizing microscope. Kawashiri et al.[20] also showed the same fact using an immunohistochemical marker α-smooth muscle actin for myofibroblasts.

Venigella and Charu[14] suspected that the collagen changes observed in different grades of OSCC clearly indicated alterations in the stromal tissue. This could be due to the action of enzymes such as collagenases or the metallo-proteinases secreted by tumor cells, an abnormal disintegration of the matrix by the tumor cells, or an uninhibited proliferation of the dedifferentiated tumor cells with the secretion of their abnormal matrix, disorganized or abortive stroma. This was supported by Stenbäck et al.,[21] who demonstrated the presence of a delicate meshwork (reticular) of Type III collagen at the invading front of the tumor islands in increasing gradations of skin cancer.

Li et al. and Fan et al., in two different studies, proved that there is increased production of MMPs-2, MMP-3, and MMP-9 in OSCC, resulting in degradation of collagen fibers. These collagen fiber changes in human OSCCs are related to unfavorable clinical prognostic factors and decreased survival.[22],[23]

OSCC is not the only malignant tumor that has shown such changes in the collagen of the stromal tissue. Studies on breast cancer by Monsky et al.[24] have also shown that an increase in the collagen content of the ECM increases the mechanical stiffness and transport resistance of the tumors. In a similar manner, Koren et al.[25] observed that follicular thyroid carcinomas had revealed a higher frequency of yellow–green collagen fibers than orange–red fibers at the sites of invasion, whereas orange–red fibers significantly predominated at noninvaded sites.

This ultimately indicates the contribution of the stromal constituents in the progression of the neoplasm; in particular, these stromal changes may enhance the movement of the tumor cells towards the blood vessels or the lymphatic vessels.[14]


  Conclusion Top


OSCC constitutes the most common life-threatening oral cancer. Its prevention can be attempted at a primary and secondary level in clinics and at hospitals. In primary prevention, the risk of cancer is reduced by avoiding the exposure to causative agents. This helps in tackling the problem at the grassroots level. Along with this, secondary prevention is also required. This form of prevention consists of early diagnosis and management of suspected lesions.

The early diagnosis and prevention of OSCC depends on the understanding of its development and progression. Collagen estimation is one of the methods to assess the progression of the tumor. The method used in the present study for collagen estimation is reliable and accurate to study the changes in the ECM in different grades of OSCC. Thus there is a need in further research in this field using a larger sample size.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Correspondence Address:
Sahana Ashok,
Department of Oral and Maxillofacial Pathology, GSL Dental College and Hospital, Rajahmundry, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_111_20



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