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Cellular profile of the peritumoral inflammatory infiltrate in squamous cells carcinoma of oral mucosa: Correlation with the expression of Ki67 and histologic grading
© Vieira et al; licensee BioMed Central Ltd. 2008
Received: 29 January 2008
Accepted: 02 September 2008
Published: 02 September 2008
Squamous cells carcinoma is the most important malignant tumor with primary site in the oral cavity and, given the great exposure of mucosa and lips to the etiologic factors of this neoplasm, its incidence is high. Investigation of the prognostic determinants is significant for the expectations of treatment proposal and cure of the patient. The local immune response represented by peritumoral inflammatory infiltrate is a possible prognostic factor.
In this study, oral mucosa samples of squamous cells carcinoma were analyzed, separated according to their histological classification as well as the phenotypical profile of the cells comprising the peritumoral inflammatory infiltrate was investigated by immunohistochemical method, in addiction, the cell proliferation index via protein Ki67 expression was determinated.
The T lymphocytes made up most of this inflammatory infiltrate, and among these cells, there was a predominance of T CD8 lymphocytes relative to the T CD4 lymphocytes. The B lymhocytes were the second most visualized leucocyte cell type followed by macrophages and neutrophils. The immunohistochemical assessment of Ki-67 positive cells revealed a greater expression of this protein in samples of undifferentiated squamous cells carcinoma.
The results suggest that the cellular immune response is the main defense mechanism in squamous cells carcinoma of oral mucosa, expressed by the large number of T lymphocytes and macrophages, and that the greatest intensity of local response may be associated with the best prognosis.
Malignant neoplasms of the mouth comprise a heterogeneous group of lesions that differ among themselves as to risk factors, clinical aspects, and histologic origin. Among the different tumors of this group, squamous cells carcinoma is the most prevalent histologic type and represents most cases of diagnosed oral cancer [1–4]. Despite prevention campaigns of several governmental health care programs and expansion of the network of specialized professionals who prioritize the diagnosis of premalignant lesions of the mouth in the different health care services currently active, 7% of the world's population is affected by oral cancer, and it is, therefore, a worldwide health concern .
Squamous cells carcinoma originates in the epithelium that lines the mouth. This fact is extremely important when we consider the constant exposure of the oral cavity to local intrinsic stimuli associated with the increased risk of this type of cancer. Among the main risk factors, smoking, chronic alcoholic beverage consumption, and chronic mechanical traumas – such as maladapted dental prostheses and fractured teeth – stand out. Furthermore, including primary cancer of the lips, especially of the inferior portion, we should consider continuous exposure to solar radiation as a significant extrinsic factor, especially in leukodermic individuals. Besides local intrinsic factors and extrinsic factors, the patient's genetic predisposition and immune response are determining factors in the risk for cancer [2, 4–10].
In addition to the intensification in prevention activities, several studies have been developed to establish the determining factors of the prognosis for these lesions with the objective of minimizing morbidity and mortality caused by the existing malignant lesion. Many authors have investigated a possible correlation between the patient's immune response, neoplastic behavior, and prognosis [5, 11, 12].
Meneses et al., (1998) demonstrated, in oral squamous cells carcinoma samples, a possible association between tumor size, area of invasion, angiogenesis, and the phenotypical characterization of the peritumoral inflammatory infiltrate predominantly comprised by T lymphocytes and B lymphocytes. Coussens et al., (1999) also found in carcinomas of oral mucosa an association between the predominance of mast cells in the peritumoral infiltrate and a greater development of the stromal angiogenesis, which would provide adequate blood supply for neoplastic nutrition, and consequently, a poorer prognosis.
Along with the evaluation of the peritumoral inflammatory infiltrate and its products, the study of tumoral cellular kinetics, its regulating mechanisms and its inter-relationship with growth factors, oncogenes and anti-oncogenes has also been the target of several studies [8, 15–19].
Among the cellular events that directly determine the tumor's clinical progress, cellular proliferation is significant, i.e., disorders in the number of cells resulting from dysfunctions in the mitotic cycle [20, 21].
The proliferative activity of any tissue or neoplasm can be determined by its growth rate using antibodies directed against specific antigens expressed by proliferating neoplastic cells, allowing the simultaneous analysis of cell proliferation and histology [13, 22, 23]. The Ki67 molecule has been the antigenic marker of choice, since it does not suffer much influence from internal and external factors, and its nuclear expression during a defined period of the cell's cycle represents an advantage in its use as a biological marker of mitotic activity [24–27].
Recently it was demonstrated that Ki-67 gene suffers "over expression" in epithelial cells of pre-malignant and malignant oral lesions.
In this study the cell proliferation index was evaluated by means of the expression of protein Ki67 and the phenotypical profile of cells that comprise the peritumoral inflammatory infiltrate in samples of squamous cells carcinoma of oral mucosa. The results demonstrated a vital participation of the population of T lymphocytes in the composition of the inflammatory infiltrate associated with the neoplasic area. These results also suggest a correlation between the intensity of the peritumoral inflammatory reaction and the proliferation of tumor cells.
Samples of oral mucosa squamous cells carcinoma from treatment naïve patients submitted to diagnostic incisional biopsies. Paraffin-blocked samples (n = 30) were obtained from the archives of the Pathological Anatomy Laboratory of the Medical School of the Dom André Arcoverde Foundation – Valença/RJ.
The blocks were processed histologically to obtain slices 4 μm thick and stained with routine hematoxylin and eosin (HE).
Slides were evaluated by two different examiners, and the tumors were classified histologically according to the International Classification of Diseases for Oncology (ICD-O/2000). Samples were divided into the following groups:
Group 1: well-differentiated squamous cells carcinoma of oral mucosa (n = 10)
Group 2: moderately differentiated squamous cells carcinoma of oral mucosa (n = 10)
Group 3: undifferentiated squamous cells carcinoma of oral mucosa (n = 10).
Histomorphometry of the inflammatory infiltrate
In order to quantify the peritumoral inflammatory infiltrate, Scion Image software and FotoScan software were used for image capture and morphometry of the inflammatory infiltrate. All samples were captured in full extent and fields (original magnification 100×) with inflamed areas around the tumor were measured semi-automatically, with manual selection of the inflamed region and computed analysis of the selected area.
Based on the analysis of each sample, simple arithmetic means the inflamed area per microscopic field were obtained and the results were expressed in percentage of inflamed peritumoral area per group.
Slices with 4 μm thickness arranged on silanized slides (3-aminopropyltriethoxysilane; Sigma Chemical, Co; USA), were deparaffinized in a 60°C chamber, and sequentially hydrated in passages through xylol, absolute alcohol, 70% alcohol, and distilled water.
Antibodies for immunohistochemistry analysis Antibody Marking Brand
Regulatory T Lymphocytes
Helper T Lymphocytes
Negative control of the immunohistochemical reaction was performed by omitting incubation with the primary antibody for some slices. Slices were analyzed and photomicrographs were made with the Nikon Microphot system (Tokyo, Japan).
Reactive cell count for each antibody was made by scanning the entire sample, with 400× magnification, with a count of positive cells in all fields. Results were obtained from the simple arithmetic mean and expressed in percentage of positive cells per microscopic field.
Statistical analysis was performed using Mann-Whitney with a p < 0.05 significance level in order to comparatively evaluate means of the parameters analyzed in the different groups of the experiment.
In all the samples evaluated in this study, the quantitative predominance of the inflammatory infiltrate was directly associated with areas of greatest invasion into tissues subjacent to the tumor parenchyma.
The phenotypical evaluation of the peritumoral infiltrate was carried out by identification of inflammatory cell positivity, characterized by the visualization of light brown intracellular coloring, against a background counter-stained with hematoxylin in each one of the reactions for the antibodies used.
Besides the multifactor etiology involving extrinsic and intrinsic factors, the patient's immune status is also considered to influence the risk of cancer and determine several aspects of its progression [4, 9, 10]. In the 1980s, Scully C. (1983) had already recognized the involvement of the immune response in the development of malignant mouth tumors, and had emphasized that future cancer treatment would be based on immunotherapy through immunomodulation.
Among the events that directly determine tumor clinical progression, those of primary importance include disorders in the number of cells resulting from mitotic cycle dysfunctions and possible failures" in host's immune evasion of the tumors – assessed by the intensity of the peritumoral inflammatory infiltrate, inflammatory cell composition, cytokine production by infiltrate cells, and presence of angiogenesis [11, 12, 14, 20, 21].
According to Costa L. et al., (2005) in a retrospective clinical study with 38 samples of oral squamous cells carcinoma, the TNM classification (tumor staging determined by the size of the primary cancer and the presence of metastases) correlates with the chief histopathologic characteristics of tumoral classification – degree of keratinization, nuclear pleomorphism, and intensity of peritumoral lymphocytic infiltration, and the link among these data is important in determining prognosis and choosing treatment.
The present results showed an inverse correlation between the degree of tumor differentiation and the rate of cell proliferation obtained by the expression of protein Ki67. Results similar to those found in this study are also suggested by Costa et al., (2005) in oral carcinoma, Glen et al., (2006) in malignant pancreatic lesions, Deans et al., (2006) in gastro-esophageal cancers, and Cai et al., (2006) in transitional cell carcinoma of the bladder. The undifferentiated tumors showed an accentuated expression of protein KI-67. Aguiar (1996) demonstrated that the mitotic index increases progressively from normal peritumoral oral mucosa towards tumor areas, as well as in areas of greatest tissue invasion . Pich et al., (2004) in a retrospective study with malignant lesions of the mouth cavity, salivary glands, pharynx, and larynx, observed that the proliferative activity investigated by different methods – such as the AgNORs Index determination and MIB-1 and Ki67 expression by immunohistochemistry – is clinically relevant and valid for proposing treatment and defining prognosis. In this paper, the histologic characteristics exhibited by the malignant tumor were associated with the clinical aggressiveness by the analysis of the malignant grade related to possible determinants of lesion prognosis – local immunologic profile and malignant cell proliferation.
According to the results obtained from morphometry of the peritumoral infiltrate, the average percentage of inflamed area per microscopic field was greater in the undifferentiated tumors (Group 3) when compared to the averages of the moderately differentiated tumor samples (Group 2), followed by the well-differentiated tumor samples (Group 1). In evaluating these data, we noted that there is a correlation between the highest degree of malignity and the greatest inflammatory intensity.
The characterization of the peritumoral infiltrate composition did not reveal a difference between the leukocyte types of the three groups, determined from their grades, but interestingly, the quantities of each cell type were equally proportional in these groups. We note that in all samples, the total number of T lymphocytes and macrophages predominated over the quantification of plasmocytes, characterizing the patient's local reaction as a predominantly cell type immune response.
In addition to the evaluation of the peritumoral inflammatory infiltrate intensity and its cellular components, some of the major determinants of tumor aggressiveness and possible predictors of prognosis are alterations in cell proliferation, i.e., abnormalities in the number of cells resulting from mitotic cycle dysfunctions. This neoplastic proliferative activity can be determined by the growth rate by means of Ki67 expression [18–21, 28–31].
The comparison among the percentages of tumor cells positive for the expression of Ki-67 per microscopic field in the oral mucosa samples of the three groups showed statistically significant differences. The samples from Group 3 (undifferentiated squamous cells carcinoma of the oral mucosa) displayed a greater expression of cells marked positively, followed by the samples of patients from Group 2 (moderately differentiated squamous cells carcinoma of the oral mucosa), and from Group 1 (well-differentiated squamous cells carcinoma of oral mucosa).
As to leukocyte characterization, Sica et al., (2006) reported evidence associated to the predominance of the macrophage population in the peritumoral infiltrate and a greater promotion of tumoral angiogenesis, attributing to these cells a pro-tumoral role, and consequently, a poor prognosis.
In the samples studied of oral carcinoma, the evaluation of peritumoral inflammatory cells demonstrated the importance of cellular immunity in the local antineoplastic response due to the presence of a population predominantly composed of T lymphocytes and macrophages, albeit with no relevant differences as to the infiltrate composition according to the histological grading.
Recently, in samples of invasive breast cancer, we investigated the possible correlation between the intensity of the peritumoral inflammatory infiltrate and the degree of tumor differentiation . We point out that when we analyzed patients with malignant tumors of the same grade, those who progressed satisfactorily showed a more intense peritumoral inflammatory response, while those who experienced tumor relapses and metastatic dissemination developed a less intense peritumoral inflammatory response.
In this study, the parallel between intensity of the inflammation and the patient's prognosis has not yet been fully clarified, and further research is needed. The distribution of the oral mucosa carcinoma samples into groups was made according to histologic grading. The results suggest a positive correlation between inflammatory response intensity and the degree of tumor differentiation. Therefore, undifferentiated tumors present a greater development of the peritumoral inflammatory process when compared to moderately differentiated and well-differentiated tumors. Nevertheless, in order to define this variable of inflammatory response intensity as a prognostic factor in oral cancer, other studies should be carried out so that the intensity of the local response and malignant cell proliferation in tumors of the same histopathologic classification associated to the patients' clinical progress can be evaluated.
Finally, the results presented in this study suggest that the cellular immune response is the main defense mechanism in oral mucosa squamous cells carcinoma, expressed by the large number of T lymphocytes and macrophages.
We further underscore that Ki-67 expression is related to the mitotic index and, consequently, to cellular proliferation and malignant grading of the neoplasm.
Rede mineira de bioterismo. 2824/05 – FAPEMIG; Rede mineira "TOXIFAR". 2827/05 – FAPEMIG; CNPQ
- Costa ALL: Dupla marcação PCNA/AgNOR e Ki-67/AgNOR em carcinoma de células escamosas de boca. PhD Thesis. 1997, São Paulo University (USP), São Paulo, SP, BrazilGoogle Scholar
- Tian Z, Guo W, Zhang WG: The expression of e-cadherin in oral cancer with different biological behavior. Shanghai Kou Qiang Yi Xue. 2002, 11: 350-352.PubMedGoogle Scholar
- do Nascimento-Souza KC, Faria PR, Costa IM, Duriguetto AF, Loyola AM: Oral alveolar soft-part sarcoma: review of literature and case report with immunohistochemistry study for prognostic markers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005, 99: 64-70. 10.1016/j.tripleo.2004.07.002.View ArticlePubMedGoogle Scholar
- Chen PC, Pan CC, Kuo C, Lin CP: Risk of oral nonmalignant lesions associated with human papillomavirus infection, betel quid chewing, and cigarette smoking in Taiwan: an integrated molecular and epidemiologic study. Arch Pathol Lab Med. 2006, 130: 57-61.PubMedGoogle Scholar
- Rizzolo D, Hanifin C, Chiodo TA: Oral cancer: how to find this hidden killer in 2 minutes. JAAPA. 2007, 20: 42-47.PubMedGoogle Scholar
- Lorz M, Meyer-Breiting E, Bettinger R: Proliferating cell nuclear antigen counts as markers of cell proliferation in head and neck cancer. Eur Arch Otorrhinolaryngol. 1994, 251: 91-94. 10.1007/BF02565229.View ArticleGoogle Scholar
- Aguiar MCF: Estudo comparativo das proteínas p53, PCNA, Ki-67 e das regiões organizadoras nucleares (NORs) no epitélio de revestimento da mucosa próximo ao carcinoma de células escamosas da boca. PhD Thesis. 1996, São Paulo University (USP), São Paulo, SP, BrazilGoogle Scholar
- Costa L, Araujo RF, Ramos CC: Correlation between TNM classification and malignancy histological feature of oral squamous cell carcinoma. Rev Bras Otorrinolaringol (Engl Ed). 2005, 71 (2): 181-187.View ArticleGoogle Scholar
- East SC, Stoker JL, Avery BS: Is the two week rule of any benefit to patients with oral cancer?. Br J Oral Maxillofac Surg. 2005, 43: 511-512. 10.1016/j.bjoms.2005.02.013.View ArticlePubMedGoogle Scholar
- Tezal M, Grossi SG, Genco RJ: Is periodontitis associated with oral neoplasms?. J Periodontol. 2005, 76: 406-410. 10.1902/jop.2005.76.3.406.View ArticlePubMedGoogle Scholar
- Meneses A, Verastegui E, Barrera JL, Zinser J, de la Garza J, Hadden JW: Histologic findings in patients with head and neck squamous cell carcinoma receiving perilymphatic natural cytokine mixture (IRX-2) prior to surgery. Arch Pathol Lab Med. 1998, 447-454.Google Scholar
- Sica A, Schioppa T, Mantovani A, Allavena P: Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer. 2006, 42: 717-727. 10.1016/j.ejca.2006.01.003.View ArticlePubMedGoogle Scholar
- Lyons AJ, Jones J: Cell adhesion molecules, the extracellular matrix and oral squamous carcinoma. Int J Oral Maxillofac Surg. 2007, 36: 671-679. 10.1016/j.ijom.2007.04.002.View ArticlePubMedGoogle Scholar
- Coussens LM, Raymond WW, Bergers G, Laig-Webster M, Behrendtsen O, Werb Z, Caughey GH, Hanahan D: Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev. 1999, 13: 1382-1397. 10.1101/gad.13.11.1382.View ArticlePubMedPubMed CentralGoogle Scholar
- Cox SC, Walker DM: Epithelial growth fraction and expression of p53 tumor suppressor gene in oral submucous fibrosis. Aust Dent J. 1996, 41 (2): 91-96.View ArticlePubMedGoogle Scholar
- Bair EL, Massey CP, Tran Nl, Borchers AH, Heimark RL, Cress AE, Bowden GT: Integrin-and caderin-mediated induction of the matrix meteloprotease matrilysin in cultures of malignant oral squamous cell carcinoma cells and dermal fibroblasts. Exp Cell Res. 2001, 207: 259-267. 10.1006/excr.2001.5347.View ArticleGoogle Scholar
- Li W, Wen YM, Wang LH, Yanq Z, Zhanq RL: In vitro and in vivo study on proliferation of adenoid cystic carcinoma cell lines after nm23-h1 – Introduction. Hua Xi Kou Qiang Yi Xue Za Zhi. 2004, 22: 109-111.PubMedGoogle Scholar
- Evans MJ, Gibbons AJ: Advice given in community pharmacies to patients with possible oral carcinoma. Br J Oral Maxillofac Surg. 2005, 43: 253-255. 10.1016/j.bjoms.2004.11.010.View ArticlePubMedGoogle Scholar
- Gordon M, Rishpon S, Gorski M: Delayed diagnosis of carcinoma of the oral cavity. Harefuah. 2005, 144: 243-245.PubMedGoogle Scholar
- Levine AJ: The 1993 Walter Hubert Lecture: The role of p53 tumour suppressor gene in tumorigenesis. Br J Cancer. 1994, 3: 409-416.View ArticleGoogle Scholar
- Komberg IJ, Villaret D, Popp M, Lui L, McLaren R, Brown H, Cohen D, Yun J, McFadden M: Gene expression profiling in squamous cell carcinoma of the oral cavity shows abnormalities in several signaling pathways. Laryngoscope. 2005, 115: 690-698.View ArticleGoogle Scholar
- Hidayatullah G, Munshi , Supurna G, Subhendu M, Wu , Ratna S, Kathleen JM, Sharon S: Proteinase suppression by E-cedherin-mediated cell-cell attachment in pre-malignant oral keratinocytes. J Biol Chem. 2002, 277 (41): 38159-38167. 10.1074/jbc.M202384200.View ArticleGoogle Scholar
- Grabenbauer GG, Suckorada O, Naidobitek G: Imbalance between proliferation and apoptosis may be responsible for treatment failure after postoperative radiotherapy in squamous cell carcinoma of the oropharynx. Oral Oncology. 2003, 39: 459-469. 10.1016/S1368-8375(03)00005-8.View ArticlePubMedGoogle Scholar
- Guillaud P: Quantification and topographical description of Ki-67 antibody labeling during the cell cycle of normal fibroblastic (MRC-5) and mammary tumor cell lines (MCF-7). Anal Cell Pathol. 1989, 1: 25-39.PubMedGoogle Scholar
- Sakaki T, Wato M, Otake S, Shirasu R, Tanuka A: Localization of Ecadherin adhesion molecules in human gingival and gingival carcinoma. Acta Pathol Jpn. 1993, 43: 99-106.PubMedGoogle Scholar
- Ravi DK, Ramadas BS, Mathew KR, Panikkar MK, Nair MR, Pillai : Apoptosis, angiogenesis and proliferation: trifunctional measure of tumor response to radiotherapy for oral cancer. Oral Oncology. 2001, 37: 164-171. 10.1016/S1368-8375(00)00082-8.View ArticlePubMedGoogle Scholar
- Iamaroon A, Khemaleelekul U, Pongsiwit S, Pintong J: Co-expression of p53 and Ki67 and lack of EBV expression in oral squamous cell carcinoma. Journal Oral Patholog Med. 2004, 33: 30-36. 10.1111/j.1600-0714.2004.00192.x.View ArticleGoogle Scholar
- Scully C: Immunology and oral cancer. Br J Oral Surg. 1983, 21: 136-146. 10.1016/0007-117X(83)90059-8.View ArticlePubMedGoogle Scholar
- Lo Muzio L, Santarelli A, Panzarella V, Campisi G, Carella M, Ciavarella D, Di Cosola M, Giannone N, Bascones A: Oral squamous cell carcinoma and biological markers: an update on the molecules. mainly involved in oral carcinogenesis. Minerva Stomatol. 2007, 56: 341-347.PubMedGoogle Scholar
- Fernández MM, García-Rozado A, Parente PL: Is microvascular density an independent prognostic factor in squamous cell carcinoma of the tongue?. Acta Otorrinolaringol Esp. 2007, 58: 341-346.View ArticlePubMedGoogle Scholar
- Lequerica-Fernández P, Astudillo A, de Vicente JC: Expression of vascular endothelial growth factor in salivary gland carcinomas correlates with lymph node metastasis. Anticancer Res. 2007, 27: 3661-3666.PubMedGoogle Scholar
- Estevam FR, Augusto SF, Rodrigues SA, Pinheiro MR, Aarestrup FM: Apoptosis and production of TNF-alpha by tumor-associated inflammatory cells in histological grade III breast cancer. Cancer Immunol Immunother. 2005, 54: 671-676. 10.1007/s00262-004-0639-0.View ArticlePubMedGoogle Scholar
- Glen P, Jamieson NB, McMillan DC, Carter R, Imrie CW, McKay CJ: Evaluation of an inflammation-based prognostic score in patients with inoperable pancreatic cancer. Pancreatology. 2006, 6: 450-453. 10.1159/000094562.View ArticlePubMedGoogle Scholar
- Deans C, Rose-Zerilli M, Wigmore S, Ross J, Howell M, Jackson A, Grimble R, Fearon K: Host Cytokine Genotype is Related to Adverse Prognosis and Systemic Inflammation in Gastro-Oesophageal Cancer. Ann Surg Onco. 2006, 14: 329-339. 10.1245/s10434-006-9122-9.View ArticleGoogle Scholar
- Cai T, Nesi G, Boddi V, Mazzoli S, Dal Canto M, Bartoletti R: Prognostic role of the tumor-associated tissue inflammatory reaction in transitional bladder cell carcinoma. Oncol Rep. 2006, 16: 329-334.PubMedGoogle Scholar
- Pich A, Chiusa L, Navone R: Prognostic relevance of cell proliferation in head and neck tumors. Ann Oncol. 2004, 15: 1319-1329. 10.1093/annonc/mdh299.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/8/25/prepub
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