The study of elastic fibres in oral precancerous and cancerous lesions using Shikata’s modified orcein stain: a retrospective study

Introduction

Oral cavity cancer is a major global health concern. Squamous cell carcinoma is a malignant epithelial cell neoplasm exhibiting squamous differentiation as characterised by the formation of keratin and/or the presence of intercellular bridges [1–3]. When a potentially malignant disorder progresses to cancer it undergoes series of histological alterations [4, 5]. The stroma especially stromal fibres plays an important role in the invasion and metastasis [6]. The role of mesenchyme in tumours can indicate the ability of the tumour cells to invade and metastasize [7]. Morphologic detection on haematoxylin and eosin and special stains of connective tissue fibres are cost-effective compared to the molecular markers. Invasion and metastasis can be studied effectively by the surgeon with the use of special stains. There are multiple studies on collagen and its association with metastasis but the association of tumour cells and elastic fibres and its effect on the progression of carcinoma is questionable [8]. Tuxhorn et al [9] quoted that the cancer cells can interact specifically with elastin through two elastin-binding proteins and galectin-3. Timar et al [10] Sabnis et al [11] and Kielty et al [12] have suggested that there is a positive correlation between tumour progression and the presence of elastic fibres in the tumour stroma [10–12].

The advantages of the use of orcein for staining elastic fibres in the skin were described in 1891 by Unna [13]. Henwood mentioned that various unpublished studies on elastic tissues failed to demonstrate elastic fibres including the Verhoeffs Van Giesson stain. Therefore, Henwood used Shikata’s orcein technique in his research to demonstrate the hepatitis B surface antigen (HBsAg), copper-associated protein (CAP) and sulfated mucins, which also stained the elastic fibres in the areas of liver fibrosis [14]. In dental literature, Shikata’s modified orcein stain has been used only in lichen sclerosis of the lip [15].

Shikata’s modified orcein stain has been used to study the elastic tissue by Henwood [16, 17].

Verhoeff–van Giesson’s Elastic Stain is used to identify the atrophy of elastic tissue, such as in emphysema, evidence of vascular diseases (arteriosclerosis) and the invasion of tumours into vessels.

In the literature only 2 studies, one by Dineshshankar et al [18] and another by John and Murthy [19] have been done to study the morphology of elastic fibres using special stains. They are as follows: Dineshshankar et al [18] conducted a comprehensive investigation to evaluate the morphological alterations in elastic fibres, a principal component of connective tissue, across varying grades of oral squamous cell carcinoma (OSCC) and epithelial Oral Epithelial Dysplasias. Their study provided valuable insights into the progressive changes in elastic fibre architecture during oral carcinogenesis [18].

In a related study, John and Murthy [19] examined both collagen and elastic fibres at different stages of OSCC. Their research aimed to correlate these stromal changes with two well-established grading systems: Broder’s and Bryne’s. This comparative approach offered a nuanced understanding of how extracellular matrix (ECM) alterations relate to traditional histopathological grading methods in OSCC [19].

Notably, Katsoulas et al [15] employed Shikata’s modified orcein stain in their study of lichen sclerosus of the lip. Their use of this staining technique demonstrates the versatility and potential broader applications of Shikata’s modified orcein stain in visualising elastic fibre changes across various oral lesions [15].

These studies collectively underscore the growing interest and importance of investigating elastic fibre alterations in oral pathologies. They highlight the potential of elastic fibre assessment as a valuable tool in understanding disease progression and potentially aiding in the diagnosis and prognosis of oral lesions. The sparse literature, the lesser number of elastic fibres in lamina propria and the masking effect of overlying inflammatory cells could be the limiting factors in the assessment of elastic fibres in OSCC. This calls for more sensitive labelling systems for assessing elastic fibres (Tables 1 and 2).

This could be is the first study in the literature in which elastic fibres have been evaluated in precancer and cancer using Shikata’s modified orcein stain.

Aims and objectives

To study the elastic fibres in oral potentially malignant disorders and oral cancerous lesions using Shikata’s modified orcein stain and to correlate with the histological grading using hematoxylin and eosin.

Materials and method

Ethical approval was obtained from the Institutional Ethics Committee before the commencement of the study. The IREB Reference Number is IREB/2020/OP/01.

The study was conducted over 1 year, from January to December 2021. Histological sections were obtained from the archives of the Department of Oral Pathology and Microbiology. As controls, ten cases of normal oral mucosa were selected, along with a transverse section of the femoral artery as a positive control.

The study group comprised diagnosed cases of OSCC and potentially malignant disorders, including oral submucous fibrosis (OSMF) and oral leukoplakia (oral epithelial dysplasia). The inclusion criteria were histologically confirmed cases with sufficient epithelial and connective tissue. Cases were divided into two categories OSCC: cancerous and precancerous OSCC. The cancerous group OSCC included 50 cases of OSCC, which were further classified into 30 well-differentiated OSCC, 15 moderately differentiated and 5 poorly differentiated cases according to Broder’s classification [20]. The precancerous group included 21 cases of leukoplakia categorised by the severity of epithelial dysplasia (9 mild, 7 moderate and 5 severe) and 29 cases of OSMF were graded as follows: 18 cases in Grade I, 6 in Grade II, 3 in Grade III and 2 in Grade IV, based on the system proposed by Rajendran et al [21].

Paraffin-embedded tissue sections were retrieved from the archives for further analysis. A single section was prepared from each paraffin block and stained using Shikata’s modified orcein stain, following the technique described by Henwood [14] to assess the elastic fibre component. The elastic fibres were analysed at magnifications of 4×, 10× and 40× for their density, grouping patterns, staining intensity and orientation relative to the overlying epithelium and tumour islands using a Leica research microscope (Model No. DM1000 LED, Leica Microsystems GmbH, Ernst-Leitz-Straße 17–37, 35578 Wetzlar, Germany). Photomicrographs were captured for documentation purposes.

The density of elastic fibres was categorised as scanty, moderate or abundant. Morphological patterns were classified as long, thin, straight fibres present singly or short, thick, wavy fibres present in clusters. The staining intensity, the orientation of elastic fibres to the overlying epithelium and their orientation to tumour islands were carefully evaluated [22].

All histological slides were reviewed independently by two pathologists who were blinded to the patients’ clinical details. In cases of discrepancy, a consensus was reached using a multi-headed microscope. A minimum of four fields of view at 200× magnification were selected for analysis, and digital images were captured for further evaluation. Statistical analysis of elastic fibre parameters across various grades of OSCC, OSMF and oral leukoplakia was performed using Spearman’s test.

Results

Under microscopic examination, the elastic fibres were observed as delicate, undulating structures. These fibres were predominantly located within the connective tissue matrix, forming an intricate network throughout the examined tissue sections. Their distinctive wavy appearance and distribution pattern were characteristic features that aided in their identification and analysis.

Colour of elastic fibres

The elastic fibres stained a deep brown to dark purple colour in the tissue sections, providing a distinct contrast against the background tissue, which stained a lighter shade. This coloration facilitated the clear identification and assessment of the elastic fibres within the oral precancerous and cancerous lesions and positive control sample that is artery wall.

Various morphologic patterns of elastic fibres were observed under the Leica microscope (Tables 3–6).

Control group

The elastic fibres in the control group showed normal long fibres, clumped fibres and fragmented fibres, but long normal-looking fibres were in the majority (Figure 1).

Study group

When the density of the elastic fibres was analysed, abundant to moderate fibres were noted in well differentiated OSCC, moderately differentiated OSCC, OSMF grade 1 and 2 and scanty fibres were noted in poorly differentiated OSCC, OSMF grade 3 and OSMF grade 4; whereas in oral epithelial dysplasias the density was moderate to abundant in mild and moderate oral epithelial dysplasiascases, and scanty in severe oral epithelial dysplasias.

When morphology and pattern of grouping as evaluated in both the subgroups of study groups, the milder histopathological grades showed a combination of short, thick and wavy, whereas the higher grades showed short, thin and straight fibres. The elastic fibres were present in bunches and singly irrespective of the grades in a few cases in OSCC. Elastic fibres were fragmented or thin clusters/bunches in higher grades of the Oral Potentially Malignant disorders and in their initial grades were mostly present in bunches.

The evaluation of morphology noted short, thick, wavy fibres in both well-differentiated OSCC, Moderately differentiated OSCC, OSMF grade 1 and OSMF grade 2 and in mild oral epithelial dysplasias and moderate oral epithelial dysplasias; whereas as the grades progressed, short, thin, straight fibres were seen in poorly differentiated OSCC, OSMF grade 3, OSMF grade 4 and in severe Oral Epithelial Dysplasias. As grade progresses fibres became long/short, thin and straight (poorly differentiated OSCC and severe oral epithelial dysplasias).

The grouping pattern of the elastic fibres were evaluated as present singly, in bunches and fragmented/clustered. The well-differentiated OSCC, OSMF GRADE 1 and mild oral epithelial dysplasia showed elastic fibres singly present and in bunches. Elastic fibres showed fragmentation and were seen as short fibres in thin clusters in most of the higher grades of the cases taken in precancerous lesions/disorders group.

The findings for the staining intensity for precancerous and cancerous lesions with various grades were inconsistent.

The orientation of elastic fibres to epithelium and orientation to tumour islands is parallel in the initial grades of OSCC. Few cases of initial grades showed haphazard/perpendicular arrangement of elastic fibres. The orientation of elastic fibres to epithelium and orientation to tumour islands is haphazard and showed no significant arrangement across various grades of potentially malignant disorders/lesions (Figures 1a–e, 2a–d and 3a–c).

Our study revealed that the elastic fibres were predominantly seen in the initial stages of cancer and potentially malignant disorders/lesions. Scanty or no fibres were seen in the advanced stages of cancer and potentially malignant disorders/lesions. The sparse literature, the lesser number of elastic fibres in lamina propria and the masking effect of overlying inflammatory cells could be the limiting factors in the assessment of elastic fibres in OSCC. This calls for more sensitive labelling systems for assessing elastic fibres (Figures 4–6).

When the density of elastic fibres (abundant, moderate and scanty), were correlated within the OSCC group, the statistical analysis was significant in all earlier grades as compared to advanced grades, with a significant p value < 0.05 (p = 0.002) The statistical analysis showed p value less than 0.05 (p = 0.002) which is significant with Spearman’s ratio.

When the staining intensity (strong, moderate and weak) and the orientation of elastic fibres to overlying epithelium (parallel, perpendicular/haphazard) were correlated within the OSCC group, the statistical analysis was significant in all earlier grades as compared to advanced grades (staining intensity (p = 0.025) and (p = 0.00) for orientation of elastic fibres to overlying epithelium with the Spearman’s ratio).

The statistical analysis for the morphology of elastic fibres in OSCC cases (p > 0.05)] (p = 0.183) (short, thick, wavy, long, thin and straight) and pattern of grouping (present singly, in bunches or clustered/fragmented) p value >0.05 (p = 0.869) was not significant with Spearman’s ratio (Tables 4–6) Figures 4–6.

Most of the cases of the well-differentiated OSCC showed a parallel arrangement of elastic fibres around some tumour islands and to overlying epithelium whereas in some cases of well-differentiated OSCC and higher grades showed haphazard and absence of elastic fibres except for a few cases. The staining intensity was milder in the initial grades of the OSCC and was moderate to strong in higher grades in OSCC except for a few cases.

The statistical analysis for the pattern of grouping (present singly, in bunches or clustered/fragmented) of elastic fibres was significant with the Spearman’s ratio p value <0.05 (p = 0.036) for oral potentially malignant lesions/disorders group. The higher grades of the oral potentially malignant disorders group showed elastic fibres in fragmented/clustered form and in thin bunches. The milder grades of the oral potentially malignant disorder/lesions group showed elastic fibres mostly in thick bunches and presented as singly.

The statistical analysis of elastic fibres for staining intensity (strong, moderate and weak) in Oral potentially malignant lesions/disorders group was not significant with Spearman’s ratio p > 0.05 (p = 0.46) The statistical analysis for density of elastic fibres in oral potentially malignant lesions/disorders group was not significant p > 0.05 (p =0.907).

The statistical analysis for the morphological patterns of elastic fibres in oral potentially malignant lesions/disorders group (OSMF and ORAL LEUKOPLAKIA) was not significant p > 0.05 (p = 0.059) no. 8). The p value was more than 0.05 for all the morphological parameters of elastic fibres (short, thick, wavy, straight, long, thick and thin) and orientation to overlying epithelium (parallel, perpendicular and haphazard). The p value >0.05 for the orientation of elastic fibres to overlying epithelium (parallel, perpendicular or haphazard) (p = 0.734) for the Oral Potentially Malignant lesions group is not significant. Out of all parameters for elastic fibres–density, morphology, staining intensity and orientation of elastic fibres to overlying epithelium, the statistical analysis shows that the p-value was more than 0.05 which is not significant in oral potentially malignant disorders/lesions.

Discussion

In this study, we employed Shikata’s modified orcein stain due to its cost-effectiveness, ease of use for novice researchers and the ready availability of required reagents. This staining method provides an accessible and efficient means of visualising elastic fibres in tissue samples.

The literature has documented therapeutic approaches that incorporate elastic molecules to enhance the healing process. These findings underscore the potential significance of elastic fibres in tissue repair and regeneration [22].

Tosios et al [23] in their investigation of elastofibromatous changes and hyperelastosis in oral mucosa, observed that the connective tissue exhibited chronic and subacute inflammation, accompanied by increased vascularity and fibrosis. They noted a progressive loss of tissue elasticity correlating with the advancing grades of the lesion. Our study of OSMF revealed similar patterns across various grades of the condition [23].

These observations collectively highlight the importance of elastic fibres in maintaining tissue integrity and function, as well as their potential role in the pathogenesis and progression of oral mucosal disorders. The alterations in elastic fibre distribution and morphology may serve as valuable indicators of disease severity and progression in conditions such as OSMF.

Nuclear pleomorphism and perineural invasion have been identified as crucial parameters in assessing cancer aggressiveness, as demonstrated by Jamadar et al [24] These findings underscore the importance of evaluating such areas for elastic fibre distribution and morphology in future studies. The relationship between these histopathological features and elastic fibre alterations could provide valuable insights into the mechanisms of tumour progression and invasion.

Kardam et al [25] conducted a comprehensive analysis of stromal fibrosis in OSCC utilising picrosirius red and Verhoeff’s Van Gieson stains. Their study revealed a notable shift in collagen fibre characteristics as the tumour progressed from well-differentiated to poorly differentiated OSCC. Specifically, they observed a predominance of thin collagen fibres exhibiting a greenish–yellow hue, while thick fibres displayed a diverse colour spectrum as the tumour grade advanced. The collagen fibres were found to be loosely packed and haphazardly arranged, suggesting a significant disruption of the ECM architecture. Our study followed similar guidelines but for elastic fibres using Shikata’s Modified Orcein Stain. The quantitative analysis of collagen and qualitative assessment of elastic fibres in Kardam et al [25]’s study yielded statistically significant results, highlighting the potential of these stromal changes as markers of tumour progression. These findings were corroborated by John and Murthy [19] and align with our observations in both precancerous and cancerous groups.

The intricate relationship between chronic inflammation and cancer development has been the subject of intense research in recent years. Tlsty and Coussens [26] provided compelling evidence that chronic inflammatory disorders significantly enhance the overall risk for cancer development. Our study builds upon this foundation, offering novel insights into the role of elastic fibres in this process.

We observed a striking absence of elastic fibres in areas of inflammation, particularly in advanced grades of the study group. This finding suggests a potential mechanistic link between the inflammatory microenvironment and the degradation of elastic fibres. The correlation between inflammation and elastic fibre alteration that we identified may represent a critical step in the progression from chronic inflammation to neoplastic transformation.

The ECM plays a pivotal role in regulating tumour behaviour and disease progression. As noted in previous studies, inappropriate synthesis or degradation of any ECM component can profoundly alter cell physiology [27]. Our research provides compelling evidence supporting this concept, demonstrating both qualitative and quantitative alterations in elastic fibres across different stages of disease progression.

Specifically, we observed progressive disorganisation and fragmentation of elastic fibres correlating with advancing disease stages. In the early stages, elastic fibres appeared relatively intact, forming an organised network within the ECM. However, as the disease progressed, we noted a marked decrease in fibre density, increased fragmentation and a loss of the normal architectural arrangement.

Quantitative analysis revealed a statistically significant reduction in elastic fibre content in advanced stages of disease compared to early stages and normal tissue. This quantitative change was accompanied by qualitative alterations, including changes in fibre thickness, continuity and spatial distribution. These observations suggest that elastic fibre degradation is not merely a consequence of disease progression but may actively contribute to the creation of a permissive microenvironment for tumour growth and invasion.

The absence of elastic fibres in areas of intense inflammation in advanced disease stages is particularly intriguing. This finding implies that inflammatory processes may directly or indirectly promote elastic fibre degradation, possibly through the action of inflammatory cell-derived proteases or by altering the expression of elastic fibre-associated proteins.

Elastic fibres play a crucial role in shaping the tumour microenvironment (TME). In the initial phases of tumour development, these fibres are abundant, contributing to the structural framework of the tissue [28–30]. However, as cancer progresses, the quantity of elastic fibres significantly decreases. In advanced cancers, the tumour stroma becomes progressively stiffened, a phenomenon that serves to protect the tumour from host defence mechanisms [31–33]. This stiffening is often associated with increased collagen deposition and other ECM modifications, leading to a more rigid microenvironment that promotes tumour survival and invasion. Matrix degradation can additionally promote malignant progression and metastasis as suggested by Erler and Weaver et al [31, 34]. These findings have significant implications for our understanding of disease progression in oral potentially malignant disorders and oral cancer. The alterations in elastic fibres could serve as a valuable biomarker for disease staging and prognosis. Moreover, targeting the processes involved in elastic fibre degradation or promoting elastic fibre preservation could represent a novel therapeutic approach.

The alterations in elastic fibres observed in our study align with several hallmarks of cancer as described by Hanahan and Weinberg [35–38]. Elastic fibre degradation may contribute to ‘sustaining proliferative signalling’ through altered mechanotransduction as suggested by Pickup et al [39], ‘activating invasion and metastasis’ by compromising tissue integrity as mentioned by Lu et al [34], and ‘tumour-promoting inflammation’ via its correlation with inflammatory processes as stated by Tlsty and Coussens and various authors. et al [26, 34, 38, 40]. The disruption of elastic fibre architecture could also play a role in ‘inducing angiogenesis’ [41] and These findings suggest that elastic fibre alterations are not merely a consequence of cancer progression but actively participate in multiple cancer hallmarks, highlighting their significance in tumour biology and potential as therapeutic targets.

They undergo dynamic changes in the accompanying tumour and results in desmoplasia and is associated with the recruitment of inflammatory cells and activation of angiogenic programmes [16].

The TME plays a crucial role in the progression of oral precancerous and cancerous lesions, with cancer-associated fibroblasts (CAFs) emerging as key players in this process. CAFs are a primary source of matrix metalloproteinases (MMPs), enzymes known to degrade ECM components, including elastic fibres according to Monteran and Erez [42]. The degradation of elastic fibres by MMPs may contribute to the altered tissue architecture observed in our study using Shikata’s modified orcein stain.

While CAFs, often identified by markers such as fibroblast activation protein α, have been implicated in promoting tumour growth, metastasis and angiogenesis across various solid tumours [36, 43], our study focuses on the direct visualisation of elastic fibres. The changes in elastic fibre distribution and morphology may reflect the degree of stromal activation and remodelling in oral precancerous and cancerous lesions.

Khalid et al [44] demonstrated an increased expression of myofibroblasts, a CAF subtype, in precancerous and cancerous oral lesions, with higher expression correlating with advanced grades of neoplasia. This finding complements our investigation of elastic fibres, as both may serve as indicators of disease progression and TME alterations.

Neoplasms undergoing malignant transformation exhibit increased architectural disarray at the invasive front of the tumour mass. At this site, there is enhanced production of matrix-remodelling enzymes and synthesis of various ECM components, particularly Type I collagen. MMPs, produced by CAFs and inflammatory cells, degrade key ECM structural components, including both collagens and elastic fibres, facilitating neoplastic progression as stated by Dineshshankar et al [18] Similar findings were observed in our study where advanced stages of potentially malignant and cancerous lesions, where elastic fibres appeared thin, short and fragmented.

Henwood [14] used the modified Shikats’s orcein staining, an old-established method for the demonstration of elastic fibres, thus providing a simple way of distinguishing collapse from fibrosis. It can be used for the demonstration of elastic fibres, HBsAg [14] and CAP in liver biopsies [16].

The density of elastic fibres was abundantly seen in the initial grades of cancerous and potentially malignant disorders group. Short, thick, wavy fibres were mostly observed in the initial grades of OSCC and potentially malignant disorders groups (ORAL LEUKOPLAKIA and OSMF) whereas long, thin, wavy with an insignificant amount of straight elastic fibres were seen in the higher grades of the cases taken for evaluation. Similar findings were seen by Dineshshankar et al [18] and John and Murthy [19] where he used Verhoef’s Van Geisson stain and Picrosirius red stain There was a significantly increased amount of fragmented/short/thin elastic fibres in poorly differentiated/Grade 3 and 4 OSMF/severe Oral Epithelial Dysplasias when compared to normal mucosa. In our study, the findings were inconsistent due to unequal numbers of cases in varying grades. Staining intensity too was inconsistent which might be due to minor changes during each lot of staining. Therefore, immunofluorescence is recommended over histochemical staining because the stain fades over a period of time.

There are various methods to visualise elastic fibres such as special stains, scanning electron microscopy and fluorescent microscopy. Since scanning electron microscopy and fluorescent microscopy are very expensive, the histochemical staining procedure is very effective and reliable [45].

Once prepared by mixing various reagents, the solution has an extended shelf life and can be stored at room temperature. Therefore, Shikata’s modified orcein stain is highly recommended. The OSMF cases in this study were clinically graded as OSMF Grade I, II, III and IV, based on the mouth opening criteria provided by Rajendran and Sivapathasundharam et al [46]. Given that OSMF exhibits extensive connective tissue fibrosis, this study also evaluated stromal elastic fibres in addition to collagen. In terms of the orientation of elastic fibres relative to the overlying epithelium and around tumour islands, the fibres were haphazardly arranged, without any significant parallel or perpendicular pattern.

Some tables showed fewer than 50 cases due to the exclusion of samples where specific parameters could not be accurately assessed. This may be due to factors such as inadequate tissue preservation, staining artefacts or absence of evaluable elastic fibres in some sections. To maintain the integrity of the analysis, only cases with clear, assessable features for each specific parameter were included in the respective tables. This approach ensures that the data presented is of high quality and reliability, even if it results in a reduced sample size for certain analyses. Future studies with larger sample sizes may help to further validate these findings.

Highlight

This would be the first study on the Description of Shikata’s modified orcein stain and its advantages in highlighting elastic fibres in OSCC and oral precancer.

The manuscript provides a valuable contribution to the scientific community by shedding light on the role of elastic fibres in the oral mucosa during the progression of precancerous conditions to cancer. The use of Shikata’s modified orcein stain enhances the precision of the analysis, making the findings particularly relevant for clinicians and researchers working in the field of oral pathology.

Recommendations and future scope

Future studies can be carried out with Shikata’s modified orcein stain to study elastic tissue in the hard tissue sections.

Future studies should aim to elucidate the molecular mechanisms underlying the observed elastic fibre alterations, particularly in the context of inflammation. Investigation of the specific proteases involved in elastic fibre degradation and the signalling pathways regulating their expression could provide valuable targets for intervention. Additionally, longitudinal studies correlating elastic fibre changes with disease outcomes could further establish their prognostic value.

Conclusion

In conclusion, this retrospective study utilising Shikata’s modified orcein stain reveals significant insights into the role of elastic fibres in oral precancerous and cancerous lesions. The density, morphology and pattern of elastic fibres correlated significantly with the grades of OSMF, oral leukoplakia and OSCC, while other parameters showed no significant correlation. In OSCC, elastic fibres exhibited haphazard arrangements and fragmentation, indicating extensive remodelling of the ECM. Moreover, the aggressiveness of tumours was linked to stromal changes, with degradation of elastic fibres at the advancing front. These qualitative and quantitative alterations suggest that elastic fibres could serve as adjunct parameters in histological grading. Ultimately, we conclude that the abundance of elastic fibres decreases as the grade of cancer and potentially malignant disorders increases, emphasising their potential role as biomarkers in tumorigenesis.

Conflicts of interest

The authors declare no conflicts of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

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