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Year : 2011  |  Volume : 22  |  Issue : 1  |  Page : 16-21
Morphological and ultrastructural characteristics of extracellular matrix changes in oral squamous cell carcinoma

Institute of Pathology, Indian Council of Medical Research, New Delhi, India

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Date of Submission04-May-2009
Date of Decision30-Oct-2010
Date of Acceptance11-Nov-2010
Date of Web Publication25-Apr-2011


Background: The biology of oral squamous cell carcinoma (OSCC), including its progression from dysplasia to carcinoma, "field effects", genetic changes in tumor associated mucosa (TAM) and effect of matrix metalloproteinases in breaking down of matrix proteins to facilitate invasion, has been well documented. However, what remains to be done is to extrapolate this knowledge to improve patient care.
Aim: The aim of this study was to observe the extracellular matrix (ECM) changes with the routine histochemical stains available to most histopathologists.
Materials and Methods: The study includes 72 cases of OSCC in which the tumor and adjacent normal appearing areas were sampled to study the ECM changes with hematoxylin and eosin (H and E) and Verhoeff's-Van Gieson elastic stain (VVG).
Results: Basophilic fragmentation of collagen (H and E) and clumped short elastic fibers (VVG) were seen in 12 (16.7%) cases. Of the remaining cases, 18 (25%) had a dense lymphocytic infiltrate and had no demonstrable elastic fibers. Those cases with H and E changes were further studied and compared with normal mucosa for ultrastructural changes. The ultrastructural study demonstrated an increase in oxytalan, elaunin and elastic fibers and decrease in collagen fibers with some transformation changes associated with OSCCs and lymph node metastasis.
Changes in transformation of collagen to elastic fibers and also the loss of both the fibers in areas of lymphocytic infiltration possibly indicate degradation of ECM fibers by factors released from the lymphocytes or tumor cells and the limiting effect on the tumor by ECM remodeling.

Keywords: Oral squamous cell carcinoma, extracellular matrix, elastic fibers

How to cite this article:
Agrawal U, Rai H, Jain AK. Morphological and ultrastructural characteristics of extracellular matrix changes in oral squamous cell carcinoma. Indian J Dent Res 2011;22:16-21

How to cite this URL:
Agrawal U, Rai H, Jain AK. Morphological and ultrastructural characteristics of extracellular matrix changes in oral squamous cell carcinoma. Indian J Dent Res [serial online] 2011 [cited 2019 Nov 13];22:16-21. Available from:
The biology of oral squamous cell carcinoma (OSCC), including its progression from dysplasia to carcinoma, "field effects", genetic changes in tumor associated mucosa (TAM) and effect of matrix metalloproteinases in breaking down of matrix proteins to facilitate invasion, has been well documented. However, what remains to be done is to extrapolate this knowledge to improve patient care. The incidence of oral carcinomas in India is high, contributed in part by the habit of tobacco and betel quid chewing. [1],[2] The morbidity of this disease is further increased due to a tendency for invasion and metastasis as well as the propensity to develop either second primary tumors or second field tumors. [3] The recent concept of epithelial-mesenchymal transition (EMT) has added further to our knowledge about the mechanisms involved in metastasis. [4] Factors in the microenvironment, such as extracellular matrix (ECM) proteins, growth factors and host immune response, play a role in extension, invasion and metastasis of the tumor. Alterations of ECM may play a role in the recurrence and in facilitating the invasion of tumor cells. [5]

The ECM is composed of ground substance composed of proteoglycans, glycoproteins and water, and the fibrous component including collagen and elastic fibers. In the oral cavity, the ECM subjacent to squamous epithelial lining is in the form of a delicate layer of connective tissue - the lamina propria composed of a few elastic and collagenous fibers and the submucosa of loosely arranged connective tissue. [6] The elastic fibers which are present in the ECM of oral cavity are oxytalan fibers which do not stain with the routinely used stains for elastic fibers as they do not contain the protein elastin. Changes in ECM and ECM matrix proteinshave been studied in the metastatic progression of colon, lung, prostate and cervical cancer. [7],[8],[9],[10] It has also been reported that the ECM produced by transformed cells differs from normal cells. [11] Characterization of these changes has mostly been based on immunohistochemical and genetic studies. In this study, the fibrous component of ECM has been studied for changes associated with squamous cell carcinoma of the oral cavity. The changes were observed under both light and electron microscopy and compared with normal mucosa. The ultrastructural changes in malignant transformation of oral mucosa have been reported, [12] but to the best of our knowledge, this is the first report on the ultrastructural demonstration of collagen changes associated with OSCC.

   Materials and Methods Top

Patients with a biopsy-proven malignant oral ulcer/mass were treated with wide excision of tumor and radical neck dissection and constituted the study group (n = 72). The patients' age ranged from 22 to 71 years (median = 51 years). Among these, patient's with dysplasia (n=12) were not subjected to further IHC or ultrastructural examination. History of tobacco use in the form of smoking and betel (areca) quid and nut chewing was also elicited. Specimens were received in 10% neutral buffered formalin for light microscopy and in phosphate-buffered 3% glutaraldehyde for ultrastructural studies. Sections were obtained from tumor areas, adjacent normal appearing areas, resection margins and lymph nodes. They were fixed with 10% neutral buffered formalin, dehydrated through an increasing ethanolseries and xylene and embedded in paraffin. A representative series of sections, 3 μm thick, were cut fromeach specimen and stained with hematoxylin and eosin (H and E). Histopathological diagnosis of OSCC was confirmed, graded by Broder's grading system, [13] and ECM changes, if any, were noted. Sections were studied with Verhoeff's-Van Gieson (VVG) method for demonstration of elastic fibers, Masson's trichrome stain to identify collagen and Von Kossa stain to demonstrate calcification. Lymph nodes were studied in all cases for evidence of metastasis. The presence of ECM changes was compared with the grade of tumor and lymph node metastasis.

For ultrastructural studies, tissue was postfixed in phosphate-buffered 1% osmium tetroxide and embedded in epoxy resin. Ultrathin sections were stained with aqueous saturated solution of uranyl acetate and lead citrate and these contrasted sections on the grid were examined under HITACHI 7500 TEM. Electron microscopy was carried out at 40-60 kV accelerating voltage as it is necessary for clear visualization of the detailed ultrastructure. Sections from resection margins and adjacent normal areas in the wide excision specimens were taken as controls for comparison for both histochemistry and ultrastructure examination.

   Results Top

Light microscopy

Of the 72 cases examined, 28 (38.8%), 34 (47.3%) and 10 (14.9%) were in OSCC Grades I, II and III, respectively. Tobacco usage was elicited in 68 (80.9%) patients (males: 54 and females: 14). The cases were classified into three groups: one with no morphologic change in stroma around tumor; the second group showing dense lymphocytic infiltrate and the last group with ECM changes evidenced by the presence of elastic fibers [Table 1]. In 42 cases, the ECM around the tumor cell nests showed collagen bundles seen as uniform eosinophilic fibrous bundles arranged parallel to the epithelium. Of the rest of the patients in the study group, 18 (25%) were seen to have a dense lymphocytic infiltrate and these patients showed minimal or no demonstrable elastic fibers around tumor cells, possibly masked by the dense lymphocytic infiltrate. HandE stained sections of the tumor showed ECM changes around the infiltrating tumor cell nests in 12 (16.7%) cases. The ECM around the malignant cells was seen as basophilic, fragmented bundles of randomly arranged fibers, some in thick bundles [Figure 1]a. While the ECM changes were seen to be increased with the grade of tumor, it was not statistically significant (P > 0.05). The ECM changes were not seen to be associated in the adjacent normal-appearing tissue and resection margins. While there was a significant association between tobacco use and grade of tumor (P = 0.002), no association was found with ECM changes (P > 0.05). However, lymphocytic infiltration of ECM was significantly associated with tobacco use (P = 0.030) possibly reflecting a local immune response to absorbed toxins or friction due to areca nut chewing.
Figure 1: (a) Basophilic fragmented degeneration of collagen on H and E; (b) elastic fibers demonstrated with VVG and stained black in adjacent ECM; (c) fragmentation and clumping of elastic fibers; (d) squamous cell carcinoma with adjacent stroma showing a dense infiltration of lymphocytes and paucity of elastic fibers; (e) a few normal-appearing elastic fibers adjacent to tumor and (f) fragmented elastic fibers in VVG stained section adjacent to the tumor

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Table 1: Distribution of ECM changes among various grades of OSCC

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Histochemical stains

The 12 cases showed basophilic fibers in bundles stained blue-black with VVG, confirming them to be elastic fibers [Figure 1]b, c, e. However, these changes were not uniform, and variation in size, thickness and density of the bundles was observed. The cases with a dense lymphocytic infiltrate around the tumor cell nests revealed minimal or no elastic fibers on VVG [Figure 1]d, f and a few collagen bundles were identified with Masson's trichrome. Von Kossa stain did not reveal any evidence of calcification in any of these cases. The 42 cases which were not showing morphologic changes on H&E did not show elastic fibers with VVG. Collagen bundles were identified in stroma around the tumor by Masson's trichrome stain.

Ultrastructural morphology

Electron microscopic examination of the control grids showed predominantly collagen bundles with oxytalan fibers and an occasional elastic fiber interspersed in between. Ultrastructural examination of the cases with changes on light microscopy showed the presence of numerous oxytalan, elaunin and elastic fibers interspersed with collagen bundles, which were seen both in cross section and longitudinal section [Figure 2]a. The collagen fibrils were identified by their banding pattern and periodicity. Electron dense oxytalan fibers were seen around tumor cells. Elaunin fibers were also identified but the predominant fibers were elastic fibers which were seen to be increased in ECM around tumor cells. The elastic fibers were of varying sizes and on longitudinal section appeared to have a jagged outline and variable electron density [Figure 2]a, b, e, f. Cross-sectional view of certain fibers showed a central electron-lucent area with surrounding electron dense fibrils or electron dense deposit. Further, amorphous deposition was seen within the collagen bundles and elastic fibers were seen merging with the collagen fibers [Figure 2]c, d. Compared to the normal mucosa, there was a marked increase of these elastic fibers in the ECM around tumor cells. The cases with dense lymphocytic infiltrate showed only occasional collagen bundles in between. Oxytalan fibers were also seen in areas subjacent to normal epithelium.
Figure 2: Ultrastructure shows (a) collagen bundles both in cross section and longitudinal section; (b) electron dense oxytalan fibers; (c) elastic fibers merging with the collagen fiber bundle; (d) amorphous deposition within the collagen bundles; (e) elastic fibers of varying sizes and (f) longitudinal section of elastic fiber appeared to have a jagged outline and variable electron density

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Lymph node metastasis

Lymph node metastasis was seen in 23/72 (31.9%) cases. These constituted 11 (61.1%) cases with dense lymphocytic infiltration and 3 (25%) cases with elastic fibers in fragmented bundles. Lymph node involvement was seen in 9 (21.4%) cases which had no ECM changes. There was significant association found between ECM changes and metastasis (P = 0.009) specifically associated with lymphoid infiltrate.

   Discussion Top

Oral cancer constitute 5.5% of the tumor burden, especially among the lower socioeconomic groups in a country like India. [1] In addition to the habit of tobacco and betel quid chewing, the etiology of this group of cancers has been attributed to bad oral hygiene, ill-fitting dentures and delay in seeking medical attention. The treatment is surgery followed by radiotherapy. The histopathology report guides the clinician to plan further treatment (radiotherapy or chemotherapy) and to assess prognosis according to the grade of tumor and adequacy of resection margins. Hence, the importance of detecting changes in tumor adjacent tissue comprising both mucosa and ECM cannot be denied.

The present study addresses the changes in ECM. While 42 (58.3%) cases had no change either on morphology or ultrastructure, 18 (25%) cases showed a host immune response in the form of lymphocytic infiltration and loss of collagen on ultrastructure. The remaining 12 (16.7%) cases showed evidence of ECM remodeling and presence of elastic fibers in excess of normal. Lymph node metastasis was seen to be significantly increasing with grade (P = 0.017) and with lymphocytic infiltration in ECM (P = 0.009). Metastasis to regional lymph nodes was 21.4% in those cases with normal ECM, and it was seen to be increasing with grade (P = 0.023) and hence can be attributed to poorer differentiation in this subgroup of patients.

Among the 18 cases with lymphocytic infiltration around the tumor cells, 11 (61.1%) metastasized to the regional lymph nodes (P = 0.019). These cases did not show the presence of elastic fibers on VVG staining or on ultrastructural examination, and collagen bundles were also less in the ECM. So, it appears that there is collagen degradation or lysis in these areas. Tumor cells secrete collagenase which helps in lysis of the basement membrane. The damage may possibly result in the recruitment of lymphocytes to this area as part of the host immune response. Lymphocytes release cytokines such as interleukin (IL)-1 which induces enzymes like matrix metalloproteinases (MMPs), especially MMP-1. [14] MMPs cause collagen degradation allowing the tumor cells to infiltrate, progress and metastasize. The proteolytic degradation of ECM leads to changes in cell-cell and cell-matrix interactions with an increase in invasive potential. [5]

The lamina propria of the oral cavity is normally dense and thick collagen fibers are present in bundles. [6] Elastic fibers in oral cavity are commonly the oxytalan fibers and these are seen scattered among the collagen bundles. Elastic fibers are of three types (oxytalan, elaunin and elastic) and these vary in their histochemical properties because of the varying amount of elastin in them. [15] Oxytalan is composed of a bundle of microfibrils without elastin; elaunin fibers have dispersed elastin intermingled among the microfibrils and elastic fibers have a central solid cylinder of elastin surrounded by tubular microfibrils, giving it a frayed appearance. [16] These fibers are generally demonstrated by Weigert's resorcein-fuchsin or Verhoeff's elastic stain which stain the elastin component of the fibers. [15] Hence, histochemistry demonstrates elastic fibers on VVG, oxytalan fibers after pre-oxidation with peracetic acid [17] and elaunin not at all. So, while routine histochemical stains for elastic fibers are expected to be negative because of the known paucity of these fibers in the oral mucosa, the present study demonstrated varying amounts, sizes and thickness of these fibers in 12 cases.

Ultrastructure demonstrated not only the presence of all the three types of fibers but also an increase in the number of elastic fibers. There was also evidence of remodeling demonstrated as deposition of amorphous electron-dense elastin within collagen fibers and this has not previously been reported. Lymph node metastasis was present in only 3 (25%) of these cases (P = 0.087), indicating the presence of elastic fibers and tissue remodeling as a limiting factor in spread and metastasis of tumor. The response of the ECM to this tumor cell and lymphocyte-induced damage may be similar to the fibrosis of healing [18] and results in increased production of elastic fibers over time, explaining the varying amount of fibers found in these 12 cases. Elastic fibers provide tensile strength but the present study suggests that in addition, the remodeling of ECM limits tumor cell invasion. In the three cases which had lymph node metastasis, it is possible that this remodeling is in the early stages or has occurred too late to prevent the spread.

Whatever the cause, the association of these cases with lymph node metastasis appears to indicate the presence of lymphocytes in addition to lysis of collagen as an indicator of poor prognosis and modification of collagen to elastin as a good prognostic indicator. The importance of matrix changes has to be understood in the overall perspective of tumor biology, which implies modification of both the epithelium and mesenchyme. Most studies in oncology focus on the tumor grade and stage, lymph node metastases and therapeutic response. ECM changes which indicate the propensity of tumor cells to infiltrate and metastasize are only now being studied as one of the prognostic indicators.

It has been propounded that field effects could possibly be due to exposure to a carcinogen such as tobacco smoke which could result in polyclonal abnormalities. [19] The same carcinogens possibly induce tumor cell damage, transforming them to produce ECM different from that produced by normal cells. The study of the continuum of morphologic changes in the ECM related to the degradation, modification and reformation is of as much importance as the study of genetic changes in the tumor cells itself. The present study indicates the importance of the host immune response, the effects of this cell-mediated immunity on the ECM and the modification of ECM leading to elastic fiber proliferation resulting in limiting the tumor cell invasion and metastasis.

Genetic and immunohistochemical (IHC) studies help us in understanding the biology of invasion, recurrence and metastasis. However, routine reporting, treatment and follow-up are hampered by the inaccessibility and the delay inherent with these techniques. While many markers are now available to study the tumor-stroma interface and the ECM, morphologic detection on H&E and VVG stain for elastic fibers is easier to perform, cost-effective and available at most hospitals even in the outer reaches of our country. Moreover, the facility of studying ECM changes by ultrastructure may not be available at all centers. Hence, it is proposed that routine histochemical techniques, which are easily accessible, can be used to identify these changes in oral biopsies so as to advise the surgeon regarding the propensity for invasion and metastasis, thus allowing him to plan the surgery accordingly. However, the study is limited because of the small numbers, and larger cohort studies devoted to relating these factors with outcome would help to identify a predictive factor. Further studies should be aimed to understand not only the spectrum of ECM changes which cause tissue remodeling and facilitate metastasis of tumors but also the relation of various etiological factors such as tobacco in causation of these changes.

   References Top

1.National Cancer Registry Project. Indian Council of Medical Research Population based cancer registries under North-East regional cancer registry first report 2003-2004, incidence and distribution of cancer. 2004.  Back to cited text no. 1
2.Sankaranarayanan R. Oral cancer in India: An epidemiologic and clinical review. Oral Surg Oral Med Oral Path 1990;69:325-30.  Back to cited text no. 2
3.Braakhuis BJ, Brakenhoff RH, Leemans CR. Second Field Tumors: A New Opportunity for Cancer Prevention? Oncologist 2005;10:493-500.  Back to cited text no. 3
4.Savagner P. Leaving the neighbourhood: molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays 2001;23:912-23.  Back to cited text no. 4
5.DeClerck YA, Mercurio AM, Stack SM, Chapman HA, Zutter MM, Muschel RJ,et al. Proteases, Extracellular Matrix, and Cancer. A Workshop of the Path B Study Section. Am J of Path 2004;164:1131-9.  Back to cited text no. 5
6.Balogh K, Pantanowitz L. Mouth, Nose, and Paranasal sinuses. In: Mills SE, editor. Histology for Pathologists. 3 rd ed. Philadelphia: Lippincott Williams and Wilkins; 2007. p. 403-30.  Back to cited text no. 6
7.Ohtaka K, Watanabe S, Iwazaki R, Hirose M, Sato N. Role of extracellular matrix on colonic cancer cell migration and proliferation. Biochem Biophys Res Commun 1996;220:346-52.  Back to cited text no. 7
8.Rintoul RC, Sethi T. The role of extracellular matrix in small-cell lung cancer. Lancet Oncol 2001;2:437-42.   Back to cited text no. 8
9.Nagle RB. Role of the extracellular matrix in prostate carcinogenesis. J Cell Biochem 2004;91:36-40.  Back to cited text no. 9
10.oldberg I, Davidson B, Lerner-Geva L, Gotlieb WH, Ben-Baruch G, Novikov I, et al. Expression of extracellular matrix proteins in cervical squamous cell carcinoma--a clinicopathological study. J Clin Pathol1998;51:781-5.  Back to cited text no. 10
11.Lelièvre SA, Weaver VM, Nickerson JA, Larabell CA, Bhaumik A, Petersen OW, et al. Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus. Proc Natl Acad Sci U S A 1998;95:14711-6.  Back to cited text no. 11
12.Cheng LH, Hudson J. Ultrastructural changes in malignant transformation of oral mucosa. Br J Oral Maxillofac Surg 2002;40:207-12.  Back to cited text no. 12
13.Broders AC. The microscopic significance of histologic grading of cancer. Surg Clin North Am 1941;21:947-62.  Back to cited text no. 13
14.Saito S, Katoh M, Masumoto M, Matsumoto S, Masuho Y. Collagen degradation induced by the combination of IL-1a and plasminogen in rabbit articular cartilage explant culture. J Biochem 1997;122:49-54.  Back to cited text no. 14
15.Fullmer HM. A comparative study of elastic, pre-elastic and oxytalan connective tissue fibres. J Histochem Cytochem 1960;8:290-5.  Back to cited text no. 15
16.Cotta-Pereira G, Guerra RF, Bittencourt-Sampaio S. Oxytalan, elaunin, and elastic fibers in the human skin. J Invest Dermatol 1976;66:143-8.  Back to cited text no. 16
17.Fullmer HM, Lillie RD. The oxytalan fiber: a previously undescribed connective tissue fibre. J Histochem Cytochem 1958;6:425-30.  Back to cited text no. 17
18.Shuttleworth L, Black RA, Ferguson MW, Herrick SE. Deposition of elastic fibres in a murine cutaneous wound healing model. Eur Cells and Materials 2005;10:18.  Back to cited text no. 18
19.Waridel F, Estreicher A, Bron L, Flaman JM, Fontolliet C, Monnier P, et al. Field cancerisation and polyclonal p53 mutation in the upper aero-digestive tract. Oncogene 1997;14:163-9.  Back to cited text no. 19

Correspondence Address:
Usha Agrawal
Institute of Pathology, Indian Council of Medical Research, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.79968

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