Immunohistochemical assessment of the MIB, CK-14, p63, p16, Cal A, and Cys A markers in spindle cell squamous cell carcinomas of the lip

Avaliação imuno-histoquímica dos marcadores MIB, CK-14, p63, p16, Cal A e Cys em carcinomas espinocelulares de lábio

Marília Gerhardt de Oliveira^I ; Patrícia W. Fregapani Worm^II; Juliana Dreyer da Silva de Menezes^III; Luciana Maria Pereira Ramalho^IV; João Feliz Duarte de Moraes^V; Ferdinando De Conto^VI

^I Full Professor, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil.
^II Oral and Maxillofacial Surgeon, PUCRS - Porto Alegre - Brazil
^III Dentist, PUCRS, Porto Alegre, Brazil ]]> ^IV Professor, School of Dentistry, UFBA, Salvador, Brazil
^V Professor, School of Mathemathics, UFRGS, Porto Alegre, Brazil
^VI Professor, School of Dentistry, UPF, Passo Fundo, Brazil

Endereço para correspondência

ABSTRACT

Objective: with the objective of testing the expression of the protein markers MIB, CK14, p63, p16, Cal A, and Cys A in the pathogenesis of oral spindle cell carcinoma, we conducted an immunohistochemical study of the expression of the protein markers MIB, CK14, p63, p16, Cal A, and Cys A in human biopsy specimens of these lesions. Methods: fifteen histological specimens of spindle cell squamous cell carcinoma of the lower lip were obtained from the Department of Oral Pathology, Bahia Federal University. Immunohistochemical analyses were performed at the Molecular Biology Laboratory of the Department of Otorhinolaryngology, Heidelberg University, Germany. Results: statistical analysis revealed no association between markers. There was strong positive staining for Ck14, MIB, and Cal A in 93.3% of cases, thus establishing a strong association. Conclusion: p63, p16, MIB, Cal A, Cys A are markedly expressed and p16 is strongly suppressed in oral cavity tumors, which suggests that the latter protein may play a role in negative regulation of cell cycle progression.

Keywords: Diagnostic. Oral cancer.

RESUMO

Objetivo: O objetivo deste estudo foi avaliar, através de iomunohistoquímica, a expressão dos marcadores protéicos MIB, CK14, p63, p16, Cal A e Cys A em lesões bucais com diagnóstico histopatológico de carcinoma espinocelular. Métodos: 15 amostras histológicas com diagnóstico confirmado localizado em lábio inferior obtidas do Serviço de Patologia Bucal da Universidade Federal da Bahia foram examinadas no Laboratório de Biologia Molecular do Departamento de Otorrinola Otorrinolaringologia da Universidade de Heidelberg, Alemanha. Resultados: O estudo estatístico não revelou associação entre os marcadores. Os genes Ck14, MIB e Cal A apresentaram intensa marcação nas células do tecido, em 93,3% dos casos, estabelecendo assim, forte relação. Conclusão: Os resultados suportam a investigação revelando que os genes MIG, CK14, p63, Cal A e Cys A se apresentam fortemente evidentes nos tumores de cavidade oral e o p16 suprimido, sugerindo que esta proteína pode exercer um papel de regulador negativo do ciclo celular.

Palavras-chave: Cãncer bucal. Diagnóstico. Imuno-histoquímica.

Introduction

Immunohistochemical identification of molecular genetic events in the progression of preneoplastic lesions to spindle cell squamous-cell carcinoma enables early detection of lesions with the potential for malignant progression, thus permitting timely intervention^1,2.

The various markers that enable assessment of the progression of preneoplastic lesions to spindle cell carcinoma include the p16 protein, which halts the cell cycle and induces apoptosis by pRb-mediated phosphorylation of cyclin-dependent kinase 4 (CDK4). Functional loss of p16 may lead to uncontrolled cell proliferation^3,4. Another protein, calgranulin A (Cal A), is involved in the regulation of several cell processes, including the cell cycle and cell differentiation. Its expression has recently been associated with the development of severe dysplasias^5-8. Cystatin A (Cys A), a cysteine protease inhibitor, is a precursor of proteins involves in keratinocyte keratinization, and is expressed during the late phase of differentiation of these cells. Studies suggest that expression of cystatin A is inversely associated with malignant progression of cancer⁹.

Other markers, such as retinoblastoma and p53, may be related with early steps of carcinogenesis in oral cavity squamous cell carcinoma. Furthermore, higher Rb expression has also been observed in malignant lesions¹⁰. The p63 protein, a homologue of p53, may be associated with tumor formation in the epithelial tissue, acting as an oncogene^11,12. Expression of p63 is almost exclusively restricted to epithelial cells, mutations in this gene are infrequent, and its expression is increased in a variety of solid tumors, particularly those of the head and neck area^12,13. MIB (Ki-67) is one of the cell cycle regulator proteins that is found during duplication, being higher in carcinomas than in hyperplasias, indicating poor prognosis^14,15. CK14 is positive in basaloid squamous cell carcinoma, and is mostly distributed diffusely in the basaloid cells; it is a parameter of proliferative activity and metastatic potential of squamous cell carcinoma of the lung^16,17.

Methods

Sample selection

Fifteen histological specimens of spindle cell squamous cell carcinoma of the lower lip were obtained from the Department of Oral Pathology, Bahia Federal University. Preliminary histological analysis with H&E staining was performed to grade lesions according to degree of keratinization, nuclear polymorphism, pattern of invasion, and lymphoplasmacytic infiltrate, using the system developed by Byrne et al. (1992)² (Table 1). Changes were scored on a scale of 1 to 4 points according to intensity, and scores were added to yield a mean lesion grade for each slide.

Morphological parameters

Immunohistochemistry

After biopsy selection, immunohistochemical analyses were performed at the Molecular Biology Laboratory of the Department of Otorhinolaryngology, Heidelberg University, Germany.

Specimens were sliced into 4 μm sections, yielding one slide for each tested antibody. After removal of paraffin by xylol immersion and rehydration in a graded alcohol series and deionized water, antigen recovery was performed for the MIB, CK14, p63, p16, Cal A, and Cys A proteins. Sections were placed in citrate buffer (10 mmol/L), microwaved for 3 minutes at 700W and 10 minutes at 200W, left to cool at room temperature, and rinsed with deionized water and PBS. After inactivation of endogenous peroxidase and soaking in horse serum for 1 hour, sections were incubated in primary anti-MIB, anti-CK14, anti-p63, anti-p16, anti-Cal A, and anti-Cys A antibodies.

Criteria for positive immunohistochemical staining.

Immunohistochemistry findings were analyzed semiquantitatively. Expression was considered absent (-) in slides with ≤ 10% positive cells; moderate (+) in those with 10-50% positive cells; and marked (++) in those with ≥ 50% positive cells. Preliminary findings showed differences in expression of the MIB, CK-14, p63, p16, Cal A, and Cys A markers.

Results

Descriptive statistics were used to assess the immunohistochemical expression of CK-14, MIB, p63, Cys A, Cal A, and p16 in lower lip spindle cell carcinoma specimens. Fisher's exact test was used to tabulate data.

All patients were male. Mean age was 55 (range, 43-71 years) (Table 2).

Statistical analysis revealed no association between markers. There was strong positive staining for Ck14, MIB, and Cal A in 93.3% of cases, thus establishing a strong association Table 3.

There was strong, positive tissue staining for the p63 and Cys A genes in 86.7% of cases, as shown in Table 4.

There was no positive association with the p16 gene, as staining for this marker was absent in 93.3% of cases (Table 5):

To overcome this challenge and gain a better understanding of cell survival and apoptosis, cell proliferation and tumor suppression markers have been studied in the search for more reliable indicators for detection and prediction of the progression of preneoplastic or malignant lesions^18-21. A better understanding of the molecular mechanisms that involve control of neoplasm cell growth may enable identification of the factors involved in tumor regulation and progression. From the simple count of mitoses, which provides generic evidence^2,22, to the regulatory mechanisms of the cell cycle, the relationship between growth factors, oncogenes, tumor suppressor genes, and their protein products plays an important role in the detection and quantification of proliferating cells1. Therefore, biomarkers are relevant to the clinical management of patients with malignant neoplasms, as they assist in diagnosis, staging, assessment of treatment response, detection of recurrence, and prognostication. Despite their major clinical application, there is little scientific evidence in this area^23,24, which justifies further research, such as this study.

The possibility of analyzing mRNA from the archives of pathology laboratories is exciting, as it allows for large retrospective studies. Formalin is the most common fixative used in the surgical pathology routine, and its promotion of nucleic acid degradation is well known. In addition, the proteins extracted from formalin-fixed and paraffin-embedded tissue seem to be the same compared with those extracted from fresh frozen tissue. Additionally, easier- to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed.^25-27

Among the various markers reported to be positive in premalignant and malignant lesions, p16 stands out²¹. This study evaluated the p16 protein due to its important role in the pathogenesis of head and neck cancer²⁸, as cell cycle progression depends on the action of cyclin-CDK complexes. This process is inhibited by a group of proteins that modulate the action of these complexes, known as cyclin-CDK complex inhibitors (CKIs)²⁹, which includes p16. Functional loss of this gene may be associated with development and progression of a variety of malignant neoplasms³⁰. Under normal conditions of controlled cell division, p16 acts as a negative regulator of the cell cycle, which may predispose to occurrence of the initial steps required for malignization²⁸. In this study, there was no expression of p16 in 93% of cases, which confirms the hypothesis that p16 is a tumor suppressor gene.

The p63 protein, a p53 homologue, may be associated with tumor formation in epithelial tissues, thus acting as an oncogene^11,12. There has been substantial recent interest in the role of p63 as a regulator of cell proliferation and differentiation in potentially and frankly malignant lesions. As expression of p63 is almost exclusively restricted to epithelial cells, mutations are infrequent, and its expression is increased in a variety of solid tumors, particularly those of the head and neck area^12,13. In our sample, approximately 86% of specimens were strongly positive on immunohistochemical staining, which is consistent with the literature. Even more markedly, MIB (Ki-67) and CK-14 were equally expressed (93%) in our specimens, corroborating previous studies that reported a worse prognosis in tumors that exhibit affinity for this marker^14,15,31.

Cys A expression has been reported in polymorphonuclear granulocytes, keratinocytes, and liver and spleen tissue³². This protein plays an important role in inhibition of apoptosis caused by caspase-3 inhibition, giving the cell more time to promote DNA repair³³. In this study, Cys A was detected very often (86.7%) in specimens of lower lip cancer, which suggests an association with tumor pathogenesis.

The Cal A protein is closely related to regulation of intracellular activities such as abnormal growth, cell cycle progression, transcription, and cellular differentiation6. The Cal A gene has been the object of particular research attention due to its involvement in a variety of human diseases, such as rheumatoid arthritis, acute inflammation, cardiomyopathies, Alzheimer's disease, and cancer¹³. Expression of this protein in normal epithelial tissue, including the oral mucosa, is well established. The role of Cal A in neoplasms is still unclear, but appears to be closely tied to the differentiation potential of epithelial tissue, as it is strongly expressed in keratinocyte hyperproliferation. Dysregulation of this gene has been described in epithelial cell carcinoma of the head and neck, as in a study that revealed strong expression in nasopharyngeal tumors⁵.

Dysregulation of S100 expression may be associated with development of tumors and metastases⁶. Furthermore, protein expression studies have detected increased levels of S100-like proteins, such as Cal A, in a variety of tumor tissues as compared to their corresponding abundance in normal tissue¹³. This is consistent with the data reported in this study, in which approximately 94% of samples exhibited strong immunohistochemical staining for this protein, which suggests it is closely related to spindle cell carcinoma.

Conclusion

Acknowledgements

This work was supported by the National Council for Scientific and Technological Development (CNPq), Brazil; and Heidelberg University, Germany.

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Address for Correspondence:
Ferdinando De Conto
Rua Teixeira Soares, 1075 sala 1002 Edifício Tamandaré
99010-080 - Passo Fundo-RS
e-mail: ferdi@upf.br

Recebido: 26/04/2012
Aceito: 01/08/2012