Research Article

A Retrospective Correlative Study between Histologic Tumor Thickness and Nodal Dendritic Cells in Oral Squamous Cell Carcinoma.

Shailja Chatterjee, Sumita Mahajan

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Abstract

Aims and Objectives: Histological tumor thickness is an important parameter in prediction of nodal metastasis over T of TNM staging system. This study analyzed tumor thickness in proven cases of squamous cell carcinoma of buccal mucosa (n=60) to derive a significant depth at which the nodes are histologically positive for metastasis. Dendritic cells are potent antigen presenting cells that play a role in antitumor immune response. Counting of paracortical dendritic cells‟ population in regional lymph nodes was done for estimation of immune response in metastatic and non-metastatic nodes in oral squamous cell carcinoma. Material and Methods: Tumor thickness was calculated by means of an ocular graticule standardized at 4X magnification. Nodal biopsies (n=60) from these cases were selected and immunohistochemistry performed using Streptavidin Biotin method using anti-S100 antibody in both metastatic (n=30) and non-metastatic (n=30) lymph nodes. Results: Study results showed that a tumor thickness exceeded more than 2.25 mm, lymph node metastasis was present. A cut-off value of 15 dendritic cells per unit area was derived below which the nodes show evidence of metastasis. Two-tailed T test was done for statistical analysis. A significant correlation (P=0.028) between nodal status and dendritic cell population was found. Conclusion: The cut-off values obtained in tumor thickness and nodal dendritic cell counting can aid in predicting the immunocompetence of an individual suffering from oral squamous cell carcinoma.

Key words: Dendritic Cells;Paracortex;Lymph Nodes;Squamous Cell Carcinoma;Metastasis.

Shailja Chatterjee, Sumita Mahajan A Retrospective Correlative Study between Histologic Tumor Thickness and Nodal Dendritic Cells in Oral Squamous Cell Carcinoma. International Journal of Oral & Maxillofacial Pathology; 2011:2(1):4-8. ©International Journal of Oral and Maxillofacial Pathology. Published by Publishing Division, Celesta Software Private Limited. All Rights Reserved.

Received on: 25/01/2011 Accepted on: 21/03/2011

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Introduction

Squamous cell carcinoma (SCC) is the most common intra-oral epithelial malignancy. Numerous prognostic determinants have been studied in the past and play an important role in determining the disease prognosis. Nodal status in oral SCC is an important prognostic determinant. It has been suggested that the risk of lymph node metastasis increases with tumor thickness. In the present study, tumor thickness has been evaluated to derive a significant depth at which tumor metastasis to lymph nodes can be suspected in absence of a clinically palpable lymph node1.

Interdigitating dendritic cells are antigen presenting cells confined to the T zone or paracortical areas of lymph nodes. The increase or decrease in these cell populations determines the prognosis and is an indicator of host immune response. Attempts have been made to enumerate and characterize dendritic cells in the microenvironment of squamous cell carcinoma of head and neck, and the number of dendritic cells present has been described as a highly significant prognostic as well as survival biomarker.

In the present study, a morphometric analysis of tumor thickness and mean dendritic cell population per unit area was done to evaluate the local immunosurveillance in lymph nodes with or without metastasis positivity. “Mean Dendritic cell population per unit area” in this study is defined as the total number of dendritic cells divided by 0.6125 mm2 i.e., area calculated in „20 population dense‟ areas. This paper analyzes the host immune response in OSCC patients by means of morphometric counting of dendritic cells in both metastasis positive and negative lymph nodes and derivation of a correlation between nodal status and dendritic cell population. The aim of this study was to do a morphometric analysis of tumor thickness and dendritic cell population in regional lymph nodes for evaluating significant depth of invasion and local host immune response in oral squamous cell carcinoma.

Materials and Methods

Retrospective analysis of 60 histopathologically proven cases of squamous cell carcinoma of buccal mucosa was undertaken for measurement of tumor

thickness at which nodes were positive for metastasis. Nodal biopsies from the study group selected were subjected to immunohistochemical analysis using S100 antibody for cell counting.

For evaluation of tumor thickness, 5 µm thick tissue sections were cut from wax blocks, mounted on Mayer‟s albumin coated slides and stained with routine Harris‟ hematoxylin and eosin method. Breslow‟s grading criteria was used for evaluating tumor thickness and a „200 point plus grid‟ was used for the purpose under 4X magnification1. Tumor thickness was measured from stratum granulosum to the deepest invading epithelial front. Hyperkeratotic layers were avoided and in ulcerated areas adjacent normal epithelium was taken as reference.

In 4X magnification, 90 units of „plus grid‟ constituted 1.1mm of graduated scale of stage micrometer. Therefore, 1 unit constituted = 0.012 mm. The units were standardized as per Breslow‟s criteria and standardization was achieved1. (Table 1)

Grading

Tumor Thickness (in mm) (According to Breslow)

No. of units (4X) according to standardization

I

< .76 mm

< 62 units

II

.76 1.5mm

62 – 123 units

III

1.5 3.9 mm

124 327 units

IV

> 4 mm

> 327 units

Tabel 1: Breslow‟s criteria

To ensure representativeness of tumor thickness in each case, sections were cut from all available blocks, evaluated for histopathologic grading using Bryne‟s invasive front grading and the thickness measured2. The tumor thickness in each section was compared and the maximum thickness recorded was taken as the representative thickness in that particular case.

Retrospective analysis of dendritic cell population in lymph node biopsies obtained from 60 histopathologically proven cases of oral squamous cell carcinoma was done by means of anti-S-100 (anti-mouse, DAKO Cytomation, Denmark) immunostaining. For dendritic cell counting, 30 nodes with frank carcinomatous metastasis and 30 with no histopathologically evident metastasis were chosen. Eight lymph nodes were chosen as controls based upon following inclusion criteria: Age-match; No known malignancy and no loss of nodal architecture on histopathological examination.

An institutional ethical committee clearance was obtained and permission was obtained either from subjects or from their relatives (if deceased) and none of the personal patient details was disclosed. For dendritic cell counting, m thick paraffin embedded nodal tissue sections were mounted on poly-L-Lysine coated slides. Sections were deparaffinized and hydrated through graded alcohol series. Endogenous peroxidase was blocked using 1% hydrogen peroxide. Antigen retrieval was performed using pressure cooker method, bench cooled, washed (x 2 times) with phosphate buffered saline (PBS, pH=5.8) and incubated with primary antibody, S-100 for 45 minutes. Washed with PBS (two times), slides were wiped and treated with biotinylated secondary (link) antibody for 30 minutes. Washed as before and treated with Streptavidin-Horseradish peroxidase (1:200) for 30 minutes. Washed with PBS as before and treated with DAB chromogen for 15 minutes. Slides were washed with PBS, wiped and counterstained with Mayer‟s hematoxylin, placed in xylene for one minute. They were mounted with DPX.

Dendritic cells were counted using an ocular grid at 400X magnification with a Leitz HM-Lux3 bright field microscope (Fig 1). The ocular grid had 49 equal squares, with a total area of 0.030625 mm2. Dendritic cells were counted at 20 „population-dense‟ areas in paracortices. Hence, the total surface area examined in one lymph node was calculated to be 0.6125 mm2. Therefore, the formula derived is as under:

Mean number of dendritic cells in 20 population-dense‟ areas = Number of dendritic cells/ mm2 divided by 0.6125 mm2.

Fig 1: 400X magnification with a Leitz HM-Lux3 brightfield microscope with ocular grid.

Results and Observations

According to statistical analysis, tumor thickness showed a significant association (P=0.003) with nodal metastasis and the cutoff value for tumor thickness beyond which nodal metastasis takes place was found to be 2.5 mm (Table 3).

When the tumor thickness exceeded more than 2.25 mm, lymph node metastasis was seen in 46.9% of cases as compared to 3.12% of cases with metastasis when thickness was lesser than 2.25mm (Table 2).

The Negative Predictive Value (NPV) was calculated to be 90%, which meant that if tumor thickness is less than 2.25mm, the absence of metastasis can be predicted with 90% accuracy. 83.3% of cases with no histopathological evidence of metastasis were found to have a mean dendritic cell count >15 cells/mm2 whereas, 93.5% node positive cases were found to have a dendritic cell count of <15 cells/mm2. T test for equality of means was done to derive a correlation between mean dendritic cell population per unit area and presence/absence of metastasis in regional lymph nodes. T-test showed highly significant correlation ((P=0.028) between mean dendritic cell population per unit area and presence/absence of nodal metastasis (Table 3).

NEGATIVE

Total (n=30) Percentage

POSITIVE

Total (n=30) Percentage

<15cells

5

16.7%

28

93.3%

>15 cells

25

83.3%

2

6.7%

Table 2: Correlation between nodal status and mean dendritic cell population per unit area

Parameters

Lymph Node Metastasis

N

Mean

Standard Deviation

Standard Error Mean

T- Test

Significance (2 Tailed)

Mean dendritic cell population

NEGATIVE

POSITIVE

15

15

12.2125

5.7094

6.61056

2.04672

1.65264

.51168

3.759

.001

Mean dendritic cell population per unit area

NEGATIVE

POSITIVE

15

15

19.9713

11.8144

10.83002

9.14450

2.70750

2.28613

2.302

.028

Table 3: T test for group statistics

Discussion

Lymph node metastasis is an important prognostic determinant in oral squamous cell carcinoma. Askage et al identified tumor thickness as having a powerful predictive value for postoperative cervical metastasis irrespective of site3. It has been suggested that the risk of lymph node metastasis increases with tumor thickness. In the present study, tumor thickness showed a significant association (P=0.003) with nodal metastasis and the cutoff value for tumor thickness beyond which nodal metastasis takes place was found to be 2.5 mm (Table 2).

Shingaki et al suggested that the depth of stromal invasion is a valid parameter in predicting lymph node metastasis because the incidence of lymph node metastasis was only 8.3% in patients with tumors of less than 4mm in depth whereas, it increased to 35.3% for lesions 4 to 8 mm in depth and to 83.3% for lesions greater than 8 mm in depth. Therefore, the author considered tumor thickness of 8 mm to be a valuable cutoff point in the assessment of lymph node metastasis4. Spiro et al reported that the prognosis was excellent in patients with 2 mm or less of thickness5. Mirseyed et al‟s study showed a strong correlation of tumor thickness with the appearance of later metastasis in the neck. It appeared that the patients with lesions <1.5 mm should be followed with observation as the only therapy, since cervical metastasis developed in less than 2% of such patients. On the other hand, this incidence increased to 33% when the lesion was 1.6 to 3.5 mm in thickness, and to 60% for those lesions thicker than 3.6 mm6. The concept that tumor thickness is an important prognostic feature for predicting regional spread of mucosal cancer and possibly other cancers of the aero digestive mucosa has received support from a number of investigators. The applicability of Breslow‟s tumor thickness in head and neck squamous cell carcinoma was first assessed by Spiro et al and they found it to be a highly reproducible parameter5. Askage et al in their study evaluated tumor thickness for prediction of cervical metastasis in patients with Stage I/II of carcinoma of tongue. Multivariate analysis demonstrated that only tumor thickness > 4 mm had a predictive value for cervical metastasis.3

In a study by Stein and Tahan, the workers found that the depth of tumor thickness of carcinoma lip exceeding 2 mm was associated with metastasis (P= 0.028) and all carcinomas deeper than 6mm metastasized7. Woolgar estimated the critical thickness in carcinoma of tongue to be 8 mm8.

Present study results showed that if the histological tumor thickness exceeded more than 2.25 mm, lymph node metastasis was seen. 46.9% of cases were found to have

metastasis if tumor thickness exceeded 2.25mm compared to 3.12% of cases with metastasis when thickness was lesser than 2.25mm (Tables 2 & 3). The Negative Predictive Value (NPV) was calculated to be 90%, which meant that if tumor thickness is less than 2.25mm, the absence of metastasis can be predicted with 90% accuracy.

This variance in critical thickness may be due to differences in laboratory techniques such as the extent of sampling, tissue processing, the direction of sectioning, and the accuracy of identifying satellite islands and individual tumor cells ahead of the main invasive front.

Dendritic cells are potent antigen presenting cells with the ability to initiate primary immune response. They populate both lymphoid and nonlymphoid tissues like the epidermis and mucosal epithelium. They exist in immature form in nonlymphoid areas and in both mature as well as immature states in lymphoid tissues. Tumor immunity is critically dependent on dendritic cells. The CD8+ cytotoxic T cells recognize and kill the tumor cells presented to them by the tumor antigen-laden dendritic cells. They act by targeting the tumor cells‟ MHC Class I molecules loaded with peptide derived from tumor-specific antigens. The failure to mount effective antitumor response may have multiple causes like, low density of tumor-infiltrating cells, or by inhibition through immunosuppressive cytokines, IL-109. Numerous study results have shown that high numbers of dendritic cells are associated with certain tumors like malignant melanoma, tongue carcinoma, gastric carcinoma, thyroid carcinoma and uterine cervix10,11,12,13,14.

Studies on recombinant human CXCL14 and CXCL14-positive head and neck squamous cell carcinoma cell lines induced dendritic cells in vitro and increased tumor infiltration by dendritic cells in vivo in chimeric animal models. The activation of dendritic cells with recombinant human CXCL14 was accompanied by up-regulation of NF-κβ activity15. Thus, the prognostic chances of an individual increase. However, studies like this are at their initial stages and their application in human subjects is still undergoing trials, for example, dendritic cell vaccine. Moreover, there are limited numbers of set-ups worldwide that are actually involved in these trials. Statistical analysis gave a mean cut-off value of fifteen dendritic cells per unit area. Metastasis was evident in 83.3% cases with a dendritic cell count of less than fifteen whereas it was 93.5% in nonmetastatic lymph nodes. A significant correlation (P=0.028) was derived between histopathological nodal status and mean dendritic cell population per unit area. These results show that a lower density of dendritic cell population relates to metastasis to lymph nodes and is an indicator of depressed immunological capacity in an individual diagnosed with oral squamous cell carcinoma. Hence, it can be concluded that dendritic cells population is compromised in metastatic nodes thereby, indicating an important immunological function in transportation and presentation of tumor antigens (cells) to the CD8+ lymphocyte population in regional lymph nodes.

Affiliations of Authors: 1. Shailja Chatterjee, Assistant Professor, M.M. College of Dental Sciences and Research, Mullana, Haryana. 2. Late.Sumita Mahajan, former Prof & Head, Manipal College of Dental Sciences and Research, Mangalore -575001, Karnataka.

Acknowledgement: All Staff in the Department of Oral Pathology, Manipal College of Dental Sciences and Research Mangalore - 575001, Karnataka.

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Corresponding Author

Shailja Chatterjee,

Assistant Professor,

Department of Oral & Maxillofacial Pathology,

M.M. College of Dental Sciences & Research,

Mullana (Ambala) 133203, Haryana, India.

Email id: shailjachatterjee@gmail.com

Source of Support: Nil, Conflict of Interest: None Declared.

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