|Year : 2015 | Volume
| Issue : 3 | Page : 104-108
Analysis of reticulin fiber pattern in lymph nodes with metastasis from oral squamous cell carcinoma
Shanmukha Raviteja Yinti, Srikant Natarajan, Karen Boaz, Amitha J Lewis, Jay Pandya, Supriya Nikita Kapila
Department of Oral Pathology and Microbiology, Manipal College of Dental Sciences, Manipal University, Mangalore, Karnataka, India
|Date of Web Publication||28-Aug-2015|
Manipal College of Dental Sciences, Manipal University - 570001, Mangalore, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: The aim of the study was to determine the susceptibility of lymph nodes to metastasis in oral squamous cell carcinoma (OSCC) by analyzing the alterations in reticulin fiber pattern.
Materials and Methods: This retrospective study on 30 cases of OSCC had 15 cases that presented with lymph node metastasis (test group) and 15 cases without metastatic episodes (control group). Four micron thick sections of the tumor proper and the resected lymph nodes of the cases were stained with hematoxylin and eosin, and Gordon and Sweet's stain for reticulin. Statistical analysis of the variations in the staining patterns of reticulin at the invasive tumor front (ITF), involved and uninvolved lymph nodes were done with Statistical Package for Social Sciences (SPSS) 15 version using chi-square test. Results: The assessment of reticulin fiber patterns at the ITF, cortical and medullary areas of lymph nodes in test cases showed thin, long individual fibers, as compared to thick, shorter interlacing fibers that were seen in control cases. The peritumoral and uninvolved areas in metastatic lymph nodes of almost all test cases showed very scant fibers. Conclusion: Reticulin pattern alteration by aggressive tumors may be appreciated as a part of the wide spectrum of "proneoplastic" stromal alterations. The histopathologist can discern these changes and thereby aid the clinician in predicting tumor behavior, the clinical course of the disease, and weighing the prognostic implications.
Keywords: Oral squamous cell carcinoma, metastasis, lymphnode reticulin fiber pattern, premetastatic niche
|How to cite this article:|
Yinti SR, Natarajan S, Boaz K, Lewis AJ, Pandya J, Kapila SN. Analysis of reticulin fiber pattern in lymph nodes with metastasis from oral squamous cell carcinoma. Dent Hypotheses 2015;6:104-8
|How to cite this URL:|
Yinti SR, Natarajan S, Boaz K, Lewis AJ, Pandya J, Kapila SN. Analysis of reticulin fiber pattern in lymph nodes with metastasis from oral squamous cell carcinoma. Dent Hypotheses [serial online] 2015 [cited 2021 Dec 4];6:104-8. Available from: http://www.dentalhypotheses.com/text.asp?2015/6/3/104/163814
| Introduction|| |
Oral squamous cell carcinoma (OSCC), most commonly seen as an ulceroproliferative lesion, is considered a dreaded disease because of its ability to invade and alter the surrounding stroma to best suit its relentless invasive nature, eventually leading to episodes of metastasis. Among the heterogeneous tumor cell population, a small clone of OSCC cells are sufficient to cause metastases to lymph node. Lymph node metastasis is assessed clinically based on the presence of palpable, fixed, or enlarged lymph nodes which may not correlate with the pathological nodal status. This may result in unwarranted radical neck dissections and increasing patient morbidity. The changes within the tumor, at the peritumoral stroma and at the distant site that is predisposed to metastasis are quite obscure. Reticulin fibers, an integral component of the tumor host interaction, show variations in quantity, distribution, and pattern at the invasive tumor front (ITF) as well as in the corresponding draining lymph nodes. , Reticulin fibers are argyrophilic type III collagen fibers, invisible in routine hematoxylin and eosin (H and E) examination, but can be visualized by silver impregnation techniques. Recent investigations revealed the presence of excess collagen fibers along with altered reticulin fiber architecture in the stromal microenvironment. While stromal desmoplasia is known to serve as a prognosticator of OSCC,  it is presumed that assessment of reticular fiber pattern in incisional biopsy specimens at ITF may likewise help predict the occurrence of lymph node metastasis, thereby proving to be beneficial to the patients. The present study aimed to determine the susceptibility of lymph node to metastasis of OSCC by evaluating the architectural pattern of reticulin fibers at the ITF and within the lymph nodes.
| Materials and Methods|| |
On receiving approval from the Institutional Ethics Committee 30 cases of OSCC were retrieved from the archives of the Department of Oral Pathology and Microbiology of our institution. Fifteen pathological node negative (PN0) cases served as controls. Cases (n = 15) having one or more pathological node positivity (PN + ) served as test.
Control and test samples from the tumor proper as well as the lymph nodes were stained with H and E and Gordon and Sweet's stain for reticulin. All lymph nodes (PN0 or PN + ) along with the ITF of all 30 cases were assessed for the reticulin fiber pattern. The pattern of reticulin fibers were assessed and categorized based on the quantity (scant/moderate/abundant), thickness (thin/thick), arrangement (individual/interlacing), and length (short/long) [shown in [Figure 1] at the following sites:
|Figure 1: Grading of reticulin fibers. (a) Scant quantity, (b) Moderate quantity, (c) Abundant quantity, (d) Thin/long/individual fibers, and (e) Thick/ short/interlaced fibers|
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Statistical software SPSS 15 version was employed and chi-square tests were used to compare the reticular pattern between the following:
- ITF of the squamous cell carcinoma tissue in test and control cases.
- The cortex, medulla, and the juxta tumoral region of the involved lymph nodes of test cases.
- The cortex and medulla of the uninvolved lymph nodes of test and control cases.
- ITF of tests and controls.
- PN0 nodes of test and controls at cortex and medulla.
- PN0 and PN + lymph nodes of cases.
| Results|| |
Of the 30 cases included in the study, majority were males, and most of the test and control cases showed the tumor proper to be histologically moderately differentiated [Table 1].
On assessment of the arrangement of reticulin fibers at the ITF, it was seen that test cases showed moderate quantities of thin (64.3%), long (62.5%), and individual fibers (56%) with chi-square value of 2.14, 2.143, and 2.16, respectively (P - value > 0.05) [Figure 2]. Comparison of the uninvolved and involved lymph nodes of cases showed increased presence of thin (87% cortex, 100% medulla), scant, individual, and short fibers (100% cortex and medulla) at the involved lymph nodes. The thickness of the reticulin fibers was found to be significantly lesser in involved lymph nodes (P = 0.008) [Figure 3].
Comparison of the reticular fiber pattern at the cortex between the uninvolved lymph nodes (PN0) of the cases and controls revealed that the test cases had moderate quantities of thin (71.4%), long (66.7%), and individual fibers (57.1%) with chi-square value of 1.677 (P = 0.195), 3.33 (P = 0.06) and 1.429 (P = 0.232), respectively [Figure 4].
|Figure 2: Assessing the arrangement of reticulin fibers at invasive tumor front (ITF) in test and control cases|
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|Figure 3: Assessing the arrangement of reticulin fibers in involved lymph node of test cases|
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|Figure 4: Assessing the arrangement of reticulin fibers in cortex of uninvolved lymph node of test cases and control cases|
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Comparison of the reticular fiber pattern at the medulla between the uninvolved lymph nodes of the cases and controls showed that the test cases had moderate quantities of thin (61.5%), long (60.0%), and individual fibers (52%) with chi-square value of 1.66, 1.007, and 1.934, respectively (P - value > 0.05) [Figure 5].
|Figure 5: Assessing the arrangement of reticulin fibers in medulla of uninvolved lymph node of test cases and control cases|
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| Discussion|| |
Most therapeutic decisions for cancer patients are made on the basis of traditional tumor, node, and metastasis (TNM) staging system. It is commonly observed that this conventional mode of deciding treatment protocol leads to a large group of patients who are over/undertreated. This is owing to the increasingly variable biological behavior of the OSCC. Understanding the tumor cells nature along with its interaction with the stromal component is essential to predict the prognosis of the patient. 
Reticulin is one such component that forms an integral part of the stromal skeleton. It is present beneath the basement membrane, on the surface of adipose cells, muscle cells, and liver and lymphoid tissues. Reticulin fibers (collagen type III) are supporting fibers measuring 20 nm in diameter forming a frame work around lymphoid lobules and medullary sinuses. These fibers provide support for the lymphocytes and the follicular dendritic cells and are essential for the functioning of the lymphoid system. ,,
Reticular fibers form a very scant network within the follicles, whereas those surrounding the follicles are arranged densely as if they are compressed outward and may represent a rapid proliferation of lymphoid cells in the follicle during an immune response.  Fibers in the interfollicular space and deep cortex typically form a thick abundant coarse network. Reticular fibers are perpendicularly oriented and merge with the fibrous capsule resulting in a continuous network throughout the lymph node. Thus, the reticular fiber network undoubtedly acts as a skeletal framework of an organ (Moe, 1963). 
Assessment of reticulin fibers has been done in metastatic carcinomas as well as lymphoid neoplasms. The reticulin fibers are known to enclose the carcinoma tumor islands, whereas they are relatively absent in the lymphomas. , Loss of reticular pattern was seen in aggressive hepatocellular carcinoma.  George et al., 2012, observed significant reduction in intensity of reticulin fiber pattern with increase in collagen fiber intensity in various grades of OSCC. 
In our study, the reticulin fibers at the invasive tumor interface of OSCC, which showed lymph node metastasis, were predominantly thinner and present in individual strands. This pattern offers lesser resistance and act as pathways for the invasive clone of tumor cells. On the other hand, OSCC cases without lymph node metastasis (controls) showed thick intermeshed and condensed reticulin fibers around the ITF. The explanation could be twofold. Either the tumor has learnt to degrade the extracellular matrix by production of metalloproteinases or the host is facilitating the tumor infiltration by degrading the reticulin fibers for the tumor. 
The tumor cells are known to produce a series of interleukins and cytokines which are carried forward by the tissue fluids towards the lymph nodes. These cytokines condition the site of lymph node, making it congenial for the tumor to home into. This concept is known as the "pre-metastatic niche" formation.  In lymph nodes of test cases it can be postulated that the aggressive tumors exert their effects on lymph nodes through direct and indirect mechanisms. While direct involvement results in complete effacement of nodal architecture due to frank tumoral invasion, indirect mechanisms are more subtle and are characterized by alteration of the reticulin meshwork into thin, long, individual fibers; a modulation that paves the way for unrestricted tumor access, thus forming a 'pre-metastatic niche'. Further, it may also be added that certain individuals may be genetically predisposed to episodes of nodal metastasis arising due to inherent defects in reticulin patterning.
The non-involvement of the same level of lymph nodes from the same test case could possibly be explained by the concept of the sentinel lymph node, which is the echelon group of lymph nodes draining the primary tumor. When a tumor from the ITF reaches the sentinel lymph node, the entire basin, that is, the entire level of lymph nodes could be preconditioned to metastasis, thus acting as a pre-metastatic niche. Further involvement of lymph nodes occurs in the sequential order of their drainage. Some of the lymph nodes which were involved in the present study might have actually been the sentinel lymph node that first received the neoplastic cells from primary tumor. In the present study, the uninvolved lymph nodes in the same level of the test cases showed altered reticulin pattern (thin, long and individual fibers indicating a premetastatic niche formation). This could also suggest the need for surgical intervention prior to the involvement of this uninvolved yet pre-metastatically 'altered' lymph node.
| Conclusion|| |
Visualized alterations of the reticulin pattern by tumors such as OSCC can be interpreted as a part of the wide spectrum of "pro-neoplastic stromal" alterations. The present study indicates that the presence of the thin, long and individual reticulin fibers of moderate density at the invasive tumour front and lymphnode can be deciphered as occult yet omnious inroads for aggressive tumour propagation. Even though the grading employed was subjective with few cases, these findings might open new vistas for further research.
Financial support and sponsorship
The authors do not have any financial support.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Aparna V, Charu S. Evaluation of collagen in different grades of oral squamous cell carcinoma by using the picrosirius red stain-a histochemical study. J Clin Diagn Res 2010;4:3444-9.
Kawashiri S, Tanaka A, Noguchi N, Hase T, Nakaya H, Ohara T, et al
. Significance of stromal desmoplasia and myofibroblast appearance at the invasive front in squamous cell carcinoma of the oral cavity. Head Neck 2009;31:1346-53.
George J, Narang RS, Rao NN. Stromal response in different histological grades of oral squamous cell carcinoma: A histochemical study. Indian J Dent Res 2012;23:842-50.
Kaldjian EP, Gretz JE, Anderson AO, Shi Y, Shaw S. Spatial and molecular organization of lymph node T cell cortex: A labyrinthine cavity bounded by an epithelium-like monolayer of fibroblastic reticular cells anchored to basement membrane-like extracellular matrix. Int Immunol 2001;13:1243-53.
Roozendaal R, Mebius RE, Kraal G. The conduit system of the lymph node. Int Immunol 2008;20:1483-7.
Ma B, Jablonska J, Lindenmaier W, Dittmar KE. Immunohistochemical study of the reticular and vascular network of mouse lymph node using vibratome sections. Acta Histochem 2007;109:15-28.
Ushiki T, Ohtani O, Abe K. Scanning electron microscopic studies of reticular framework in the rat mesenteric lymph node. Anat Rec 1995;241:113-22.
Henry K, Symmers W. Thymus, lymph nodes, spleen and lymphatics. Systemic pathology. 3 rd
ed., Vol. 7. Ch 6. London: Churchill Livingstone publication. 1992 p. 908-60.
Yao S, Zhang J, Chen H, Sheng Y, Zhang X, Liu Z, et al
. Diagnostic Value of Immunohistochemical Staining of GP73, GPC3, DCP, CD34, CD31, and reticulin staining in hepatocellular carcinoma. J Histochem Cytochem 2013;61:639-46.
Singhi AD, Jain D, Kakar S, Wu TT, Yeh MM, Torbenson M. Reticulin loss in benign fatty liver: An important diagnostic pitfall when considering a diagnosis of hepatocellular carcinoma. Am J Surg Pathol 2012;36:710-5.
Sleeman JP, Christofori G, Fodde R, Collard JG, Berx G, Decraene C, et al.
Concepts of metastasis in flux: The stromal progression model. Semin Cancer Biol 2012;22:174-86.
Sleeman JP. The metastatic niche and stromal progression. Cancer Metastasis Rev 2012;31:429-40.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
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