Histochemical Analysis of Collagen Reorganization at the Tumor-Stromal Interface in Oral Squamous Cell Carcinoma- A Polarizing Microscopic Study

  

    
Category 
    
Parameter 
    
Categories 
    
Well 
    
Mod, Mod-poor 
    
p-value 
  
  

    
Broder’s grading 
    
Thickness
    
Thick
    
10(47%)
    
5(55%)
    
0.782
  
  

    
Thin
    
10(53%)
    
4(45%)
    

  
  

    
Organization
    
Organized
    
8(36%)
    
2(22%)
    
0.351
  
  

    
Disorganized
    
12(64%)
    
7(88%)
    

  
  

    
Colors
      
 
    
Red-orange
    
11(52%)
    
3(33%)
    
0.537
  
  

    
Yellow-orange
    
2(11%)
    
1(11%)
    

  
  

    
Green-yellow
    
7(37%)
    
5(56%)
    

  
  

    
Density
    
Well packed
    
9(42%)
    
3(33%)
    
0.657
  
  

    
Loosely packed
    
11(58%)
    
6(67%
    

  
  

    
Birefringence 
    
Strong
    
11(58%)
    
3(33%)
    
0.225
  
  

    
Weak
    
8(42%)
    
6(67%)
    

  
  

    
Modified
      
Invasive front    grading
    

    
Categories 
    
4-8
    
9-12
    
13-16
    

  
  

    
Thickness
    
Thick
    
1(50%)
    
5(45%)
    
8(50%)
    
0.972
  
  

    
Thin
    
1(50%)
    
6(55%)
    
8(50%)
    

  
  

    
Organization
    
Organized
    
1(50%)
    
4(36%)
    
4(25%)
    
0.686
  
  

    
Disorganized
    
1(50%)
    
7(64%)
    
12(75%)
    

  
  

    
Colors
      
 
    
Red-orange
    
1(50%)
    
6(54%)
    
6(37%)
    
0.224
  
  

    
Yellow-orange
    
1(50%)
    
0
    
2(13%)
    

  
  

    
Green-yellow
    
0
    
5(46%)
    
8(50%)
    

  
  

    
Density
    
Well packed
    
1(50%)
    
4(36%)
    
6(37%)
    
0.934
  
  

    
Loosely packed
    
1(50%)
    
7(64%)
    
10(63%)
    

  
  

    
Birefringence 
    
Strong
    
1(50%)
    
8(72%)
    
6(37%)
    
0.198
  
  

    
Weak
    
1(50%)
    
3(28%)
    
10(63%)
    

  
  

    
Abbreviations: Well= Well Differentiated    Carcinoma; MOD= Moderately Differentiated Carcinoma; Mod-Poor= Moderately to Poorly    Differentiated Carcinoma 
      
 
  

Table 4:  Correlation between the histological grading and nature of collagen
fiber.

Discussion

One of the most prevalent keratinocyte based cancers is squamous cell carcinoma. The highly invasive and metastatic nature of OSCC makes it one of the most frustrating cancers to treat [5]. However, to be invasive, a tumor cell must be able penetrate and move through the stroma [6]. The process of tumor dissemination involves specific interactions with tumor cell-surface adhesion receptors and multiple adhesive components of the extracellular matrix (ECM).

Tumor stroma plays a critical role in carcinogenesis; to grow beyond a minimal size of 1-2 mm, the tumor requires stroma [4]. The stroma is composed of ground substance composed of proteoglycans, glycoproteins and water, and the fibrous component including interstitial collagen and elastic fibers [1]. Which provides the vascular supply, nourishment, oxygen supply, and also limits the influx of inflammatory cells, thereby acting as a barrier for immunological rejection. Fibrous component of stroma has shown to be associated with OSCC and hence stromal changes which indicate the propensity of tumor cells to infiltrate and metastasize are now being studied as one of the prognostic indicators [1].

Van Gieson and trichrome stains may not be ideal for detection of collagen fibers as both these methods fail to reveal thin collagen fibers, stain’s tendency to fade and these disadvantages lead to under estimation of collagen content. This perplexing issue incited Puchtler and colleagues to seek a better method and they found that Sirius red dissolved in a saturated picric acid solution (picrosirius red) consistently stained thin collagen fibers, did not fade, and was appropriate for use with polarized light microscopy [7].

Combination of sirius red and picric acid was first considered to be a special stain for connective tissue in 1964, especially for differentiating the subtype of collagen. It works on the principle that sulphonic group of sirius red reacts with the basic groups in collagen molecules. 126 sirius dye molecules bind to purified collagen types I, II and III. The enhanced birefringence of collagen is due to the attachment of the elongated dye molecules parallel to the long axis of the collagen. The orientation of the fibers to polars and collagen birefringence provides brightness to the collagen. Thickness, density of packing and spatial arrangement determines the polarization birefringence of the collagen. Picrosirius red stain thus helps in better understanding of the collagen function and pathology [8-11]. In addition the picrosirius red stained sections when observed under a polarizing microscope show birefringence due to the anisotropic properties of collagen [12,13].

As proposed in different studies that different patterns of invasion (representing different grades of tumor cell dissociation) are associated with the prognostic outcome in cancer [14]. An attempt has been made in this study to assess the nature of collagen at the invasive front of OSCC using picrosirius red and polarizing microscope.

Previous literature confirmed that Invasive front grading in contrast to Broder’s grading is of high prognostic value, because of the following reasons:

1. Tumors often are more poorly differentiated in invasive than in superficial parts [15].

2. Blood group H antigen is often lost in invasive tumor margins of OSCC and this lost is associated with poor prognosis [16].

3. Increased expression of proliferation associated structure in the invading tumor front [17].

4. Melanoma cells from the deep tumor parts have a higher DNA content than more superficial cells [18].

5. Increased expression of Ki-67, L-myc, c-myc, N,myc, c-erbB-2 oncoprotein in most invasive lung cancer [19,20].

6. Increased labeling of bromodeoxyuridine at the site of invasion [21].

All of these studies show that the key to a better understanding of the invasive and metastatic behavior of cancer cells may reside within the invasive margins of different tumors so in the present study nature of collagen fiber was analyzed with the modified invasive front grading.

As proposed by Bryne, et al. [22], pattern of invasion is divided into four grades,

Grade I: Pushing, well delineated infiltrating borders,

Grade II: Infiltrating, solid, cords, bands and/ or strands,

Grade III: Small groups or cords of infiltrating cells (n >15),

Grade IV: Marked and wide spread cellular dissociation in small groups and/ or in single cells (n< 15).

The cases were divided into 2 groups

a) The majority 19 (63%) of the tumor showed discohesive tumor front represented by grade II, III, IV (66%)

b) Followed by cohesive tumor front 11(37%) represented grade I.

In the present study with respect to the relationship between the collagenous component in the stroma and the invading tumor front, there have been some observable changes with different advancing tumor front i.e. cohesive and discohesive.

In cohesive tumor front densely packed distinct collagen, shows reddish orange color which was mainly concentrated around tumor islands probably to prevent the tumor invasion. This may be due to deposition of type 1 collagen fiber which were in the form of thick bands and composed of densely packed, well organized with strong birefringence (Figure 1A, 1B), while in discohesive tumor front collagen fiber were thin, loosely packed, disorganized, with weak birefringence, probably due to majority of Type III collagen fiber (Figure 2A, 2B, 3 and 4). This feature being consistent with the concept of Jungeria, et al. [23] and Montes, et al. [24]. They stated that the thick fibers were type I collagen fiber and exhibit an intense birefringence of red, orange and yellow color by polarizing microscope and a weak birefringence of green when fiber were thin fibrillar thus consisting type III collagen. Observation is further supported by study by Stenback, et al. [25], the presence of a delicate meshwork (reticular) of type III collagen at the invading front of tumor islands in increasing gradations of skin cancer. Study on “stromal reaction during tumor progression in oral mucosa” by Megumi Yokoyama revealed that type I collagen which was stained red by PSR, were decreased with advanced dysplastic grading [26].

Figure 1A: Photomicrograph of Cohesive tumor front (Picro Sirius red stain without polars, 10X).

    

    

    Figure 1A: Photomicrograph of Cohesive tumor front (Picro Sirius red stain
without polars, 10X).
    
    

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Figure 1B: Photomicrograph of Cohesive tumor front (Picro Sirius red stain with polars, 10X).

    

    

    Figure 1B: Photomicrograph of Cohesive tumor front (Picro Sirius red stain
with polars, 10X).
    
    

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Figure 2A: Photomicrograph of Discohesive tumor front (Picro Sirius red stain without polars, 10X).

    

    

    Figure 2A: Photomicrograph of Discohesive tumor front (Picro Sirius red
stain without polars, 10X).
    
    

Close

Figure 2B: Photomicrograph of Discohesive tumor front (Picro Sirius red stain with polars, 10X).

    

    

    Figure 2B: Photomicrograph of Discohesive tumor front (Picro Sirius red
stain with polars, 10X).
    
    

Close

Figure 3: Photomicrograph of Discohesive tumor front showing thin collagen fibers with yellow orange color birefringence (Picro Sirius red stain 10X).

    

    

    Figure 3: Photomicrograph of Discohesive tumor front showing thin collagen
fibers with yellow orange color birefringence (Picro Sirius red stain 10X).
    
    

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Figure 4: Photomicrograph of Discohesive tumor front showing thin, disorganized, yellow orange color birefringence, loosely packed collagen fibers. (Picro Sirius red stain 4X).

    

    

    Figure 4: Photomicrograph of Discohesive tumor front showing thin,
disorganized, yellow orange color birefringence, loosely packed collagen
fibers. (Picro Sirius red stain 4X).
    
    

Close

Thus the color changes observed in the present study clearly indicate some alteration in the stromal tissue around the tumor island of advancing front, which in turn may be due to carcinogenic agents that are involved in tumorigenesis. The above results are further supported by Brekken, et al. [27] who stated that the tumor progression is influenced by extracellular matrix. This finding is further supported by study on mechanics of capsule formation which reveals that a more robust extracellular matrix and capsule results in slower growth of tumor [28].

Further, nuclear resonance studies on the physical aggregation of the collagen fiber by Sharf, et al. [29] have also revealed a color profile of orange to red while corresponded to the well packed fiber and green to greenish yellow to poorly packed fibers. This collagen may be from the tumor cell in origin, thus benefiting the tumor by reducing the access to the host lymphocytes.

Alternatively, the collagen could be of stromal cell origin, thus benefiting the host by walling off the invading tumor.

Results in the study also revealed that 9 cases (53%), tumor having size less than 4 cm shows densely packed collagen fiber and 10 (83%) tumor having size greater than 4 cm shows loosely packed collagen fiber around the tumor island of advancing front. Hence tumors with excessive collagen in the stroma may respond in this manner as seen in study on breast cancer have also shown that an increase in collagen content of extracellular matrix increase the mechanical stiffness and transport resistance of tumor [30]. As also study on myocardial infarction concluded that the collagen degradation and loss after myocardial infarction is associated with infarct expansion and followed by functional decline [31].

Present study also revealed that majority of cases which were graded as TNM stage I and II 9 (56%) with absence of lymph node metastasis 10 (53%) shows densely packed collagen fiber whereas tumor in stage III and IV, 11 (85%) with evident metastasis 8 (89%), collagen fiber were loosely packed. Alon, et al. [32] in their study on stromal differences in salivary gland tumors found that 50% of collagen fiber in polymorphous low grade adenocarcinoma and adenoid cystic carcinoma were greenish yellow, whereas in pleomorphic adenoma, only 13% of them were greenish yellow. In similar manner study of SCC of skin and lower lip revealed that high grade of tumor cell dissociation, represented by a spray like pattern of invasion (PI), was significantly associated with a high frequency of metastatic as well as recurrent disease [33,34]. It was also reported that non-cohesive (spray like) pattern of invasion was significantly associated with the invasive with lymphovascular space involvement and large tumor size.

Contrary to our result the nature of collagen fiber was not significant with the Broder’s grading system as mentioned in the study done by Venigella [3] and Kalele [4], that collagen fiber in well differentiated carcinoma revealed polarizing colors of reddish orange around the tumor island, which gradually changes into yelloworange in moderately differentiated and greenish yellow with weak birefringence in poorly differentiated carcinoma, but association was observed with the invasive front grading by Bryne, et al. [35]. Previous literatures have also confirmed that Invasive front grading in contrast to the conventional Broder’s grading is of high prognostic value. Irrespective of difference scoring system the common findings of all study was that a discohesive tumor front was associated with metastasis, large tumor size, advanced tumor stage (TNM stage III and IV) increased recurrence and decrease survival.

Conclusion

Based on the above study it is concluded that picrosirius red stain with the use of polarizing microscope is most suitable stain to visualize collagen fibers and application of stain is a relatively simple tool to study the changes in extracellular matrix in particular the structural integrity of collagen fibers at the different invasive front of OSCC.

In the present study an observable change in the collagen with the different pattern of invasion was evinced with the pattern of invasion. Adjacent to the cohesive tumor front represented by pushing borders of invasion is positive correlated with thick bands of collagen which were well organized, and resist the tumor against invasion and metastasis, preventing it to increase in size and thus associated with initial stage of tumor (I&II). In discohesive tumor front the fibers are thin, disorganized which may enhance the movement of tumor cells towards invasion and metastasis.

Determination of collagen fiber nature in different patterns of invasion of oral squamous cell can help for targeting the stroma for various treatment strategies. Further research with larger sample size is with advanced techniques of using immunohistochemistry and collagen gene identification, Second harmonic generation (SHG) microscopy, Confocal laser microscopy are probably more specific and sensitive for collagen detection in this direction.

Drawbacks and Limitations

Though picrosirius red stains very thin collagen fibers in comparison to other collagen stains, Factors like pH, concentration of stain and the duration of staining will attribute to the variation in results.

Sample of the stains can be deteriorated when kept in for more than four years. Under this condition the solution loses its specificity and besides staining collagen, it also stains muscle and epithelium [11].

It is not advisable to use the staining technique on tissue preserved in formalin for many number of days. Hence, researchers must aim at ultra structural features of connective tissue in different stages of OSCC in future.

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Citation: Alrani D, Niranjan KC, Acharya S and Hallikeri K. Histochemical Analysis of Collagen Reorganization at the Tumor-Stromal Interface in Oral Squamous Cell Carcinoma- A Polarizing Microscopic Study. Austin J Dent. 2016; 3(2): 1034. ISSN: 2381-9189

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