Markers expression of cell proliferation and apoptosis in basal cell carcinoma

Abstracts

FUNDAMENTOS: O carcinoma basocelular é o câncer mais comum em humanos. Estudos que utilizam recursos da biologia molecular e genética, associados à histomorfologia, permitem a identificação de fatores de risco no desenvolvimento de lesões mais recorrentes e agressivas. OBJETIVO: Correlacionar a expressão dos marcadores de apoptose (p53 e Bcl-2) e proliferação celular (Ki-67 e PCNA) com os indicadores histológicos de gravidade do tumor. MÉTODOS: Estudaram-se cinco amostras das formas nodular, morfeiforme e superficial, respectivamente, e um grupo-controle com três pacientes livres de lesão. Empregou-se o teste de Mann-Whitney na comparação da expressão desses marcadores com a forma de apresentação do carcinoma basocelular. RESULTADOS: Verificou-se que a marcação do Bcl-2 foi expressiva nos CBCs ditos agressivos (variantes morfeiforme e nodular). Dos tumores estudados, 66,7% (n = 10) indicaram fortemente o p53. Nossos resultados mostram maior expressão do Ki-67 no carcinoma basocelular nodular e superficial, sem expressão nos controles. O PCNA mostrou forte marcação em todos os tipos de tumores e nos controles. CONCLUSÃO: Os achados nos permitem concluir que o Bcl-2 e o p53 apresentam tendência para diagnosticar gravidade do carcinoma basocelular e o Ki-67, por seu comportamento variável, não pode ser considerado como marcador de gravidade, assim como o PCNA, que não foi um bom marcador de proliferação celular.

carcinoma basocelular; imunoistoquímica; marcadores biológicos de tumor; neoplasias cutâneas; proteínas de ciclo celular

INVESTIGATION

Markers expression of cell proliferation and apoptosis in basal cell carcinoma^*

Marília de Pádua Dornelas Corrêa^I; Ana Paula Ferreira^II; Ângela Maria Gollner^III; Michele Fernandes Rodrigues^IV; Magno Cunha de Souza Guerra^V

^IJoint Professor, Department of Surgery, Faculdade de Medicina da Universidade Federal de Juiz de Fora (UFJF) – Juiz de Fora (MG), Brazil

^IIPh.D. in Immunology, Universidade de São Paulo (USP), Associate Professor, Department of Parasitology, Microbiology and Immunology, Universidade Federal de Juiz de Fora (UFJF) – Juiz de Fora (MG), Brazil

^IIIPh.D. in Pathology, UFF, Joint Professor, Department of Pathology, Universidade Federal de Juiz de Fora (UFJF) – Juiz de Fora (MG), Brazil

^IVMaster studies in Healthcare Sciences under course, Major in Immunology, Universidade Federal de Juiz de Fora (UFJF) – Juiz de Fora (MG), Brazil

^V6th year Undergraduate, Medical School, Universidade Federal de Juiz de Fora (UFJF) – Juiz de Fora (MG), Brazil

Mailing Address

ABSTRACT

BACKGROUND: - Basal cell carcinoma is the most common form of human cancer. Studies employing molecular and genetic biology techniques, associated with histomorphology, lead to the identification of risk factors in the development of more recurring and aggressive lesions.

OBJECTIVE - To correlate markers expression of apoptosis (p53 and bcl-2) and cell proliferation (Ki-67 and PCNA) with histological indicators of tumor severity.

METHODS - Five samples of the nodular, morpheaform and superficial types of carcinoma were studied, espectively.One control group with three lesion-free patients was also included in the study. The Mann-Whitney test was used to compare these markers expression with the manifestation form of basal cell carcinoma.

RESULTS - Bcl-2 expression was significant in basal cell carcinomas said to be aggressive (morpheaform and nodular types). Of the studied tumors, 66.7% (n =10) strongly expressed p53.Our results show a greater expression of Ki-67 in nodular and superficial basal cell carcinoma, with no

expression in the controls. PCNA showed a strong expression in all types of tumors and in the controls.

CONCLUSION - The findings allow us to conclude that Bcl-2 and p53 show a tendency to indicate the severity of basal cell carcinoma. In contrast, Ki-67, due to its variable behavior, cannot be considered a marker of severity. Also, PCNA was not a good marker of cell proliferation.

Keywords: carcinoma, basal cell; immunohistochemistry; tumor markers, biological; skin neoplasms; cell cycle proteins

INTRODUCTION

Basal cell carcinoma (BCC) is the most common type of cancer in humans, affecting primarily men over the age of 50 years, with skin type I. It is preferably located on the head (facial upper third) and neck ¹.

It is manifested as an infiltrative papule or nodule, skin-colored, erythematous or brown, with telangectasias and pearled aspect. The different clinical forms range in aspect and malignant potential, and the most frequent ones are nodular or nodular-ulcerative, superficial or pagetoid, sclerosing or morpheiform, cystic and pigmented ².

It normally has slow growth and low aggressiveness and rarely generates metastases, being effectively treated by different therapeutic options. However, a minority of these cases has aggressive biological behavior, causing extensive tissue destruction and even metastases. In such patients, treatment may cause functional and cosmetic deformities, generating high costs to the healthcare system ³.

As a result of increased incidence of BCC, studies of the histogenesis of this neoplasm with molecular and genetic biology resources, associated with histomorphologic assessment and epidemiological inquiries, have enabled the determination of parameters with prognostic implications ⁴. Consequently, it is an attempt to identify risk factors for the development of more aggressive lesions, with greater potential for recurrence and metastases ⁵.

The complementation of histological and morphological study of BCC with biological markers of cell proliferation and apoptosis, considering the immunohistochemical techniques (IHC), enables the understanding of morphofunctional abnormalities in the process of neoplastic transformation and early identification of malignant transformation ⁶. Information about biological behavior of the neoplasms may help prevention and definition of the best treatment to be followed, leading to development of new drugs directed specifically to cell molecules that facilitate tumor propagation ⁷.

Cell proliferation and programmed cell death (apoptosis) are considered important events in carcinogenesis. It is known that ultraviolet light causes damage to epidermal DNA that can be repaired or eliminated by apoptosis, preventing the development of cancer ⁸. Abnormalities to the controlling genes of apoptosis mechanisms (p53 and Bcl-2) and cell proliferation markers (Ki-67 and PCNA) are associated with predisposition to cancer development ⁹.

Gene p53 transcription results in nuclear phosphoprotein that induces cell apoptosis, when there is a defect in the mechanism that repairs DNA damage, preventing proliferation of mutant cells. P53 mutations may indicate malignant transformation of a specific lesion ¹⁰. Another gene considered as "apoptosis regulating key" is Bcl-2, codifying a protein that inhibits apoptosis. Its abnormal expression may lead to cell immortalization, predisposing to development of neoplasm ¹¹.

There are two important markers of cell proliferation in the assessment of BCC: proliferative cell nuclear antigen (PCNA) and Ki-67. PCNA was considered by previous studies as a good alternative to identify cell proliferation compartments in blocked tumor samples ¹². PCNA expression is enhanced in cutaneous carcinogenesis related with ultraviolet light ¹³. Immunohistochemical analysis of Ki-67, product of a gene sequence, was considered by a recent study as an effective method to assess the fraction of growth of human neoplasm, providing valuable prognostic information about the disease ¹⁴.

Considering the possibility of determining the potential of aggressiveness of the increasing number of BCC cases based on the referred immunohistochemical markers, the present study intended to assess the expression of apoptosis markers (p53 and Bcl-2) and cell proliferation markers (Ki-67 and PCNA) in histology patterns nodular, sclerosing and superficial in subjects undergoing routine surgical procedures. It is known that this investigation may contribute to prevention, management and reduction of recurrence of basal cell carcinoma.

MATERIAL AND METHOD

We studied five patients with nodular BCC, five with sclerosing BCC and five with superficial BCC, already submitted to surgery and with confirmed clinical pathology diagnosis. The control group contained three normal subjects (without lesions) that had undergone cosmetic blepharoplasty. All surgical procedures were performed at Plastic Center - Clínica de Cirurgia Plástica e Medicina Estética (private practice of Plastic Surgery and Cosmetic Medicine), in Juiz de Fora (MG), after signature of the Free Informed Consent Term.

Skin samples obtained from surgery went through histological and immunohistochemical analyses. The material was fixed with formol 10% and later it was submitted to routine histology processing, fixed in block with paraffin and taken to microtomy. Sections measuring 3-5? were made and the stained with hematoxylin-eosin (HE) and assessed under optical microscopy (Olympus BX40 microscope) for clinical pathology diagnosis.

For immunohistochemical analysis, we investigated the involvement of apoptotic proteins and cell proliferation in basal cell carcinoma, using immunohistochemical reaction to detect proteins Bcl-2, p53, PCNA and Ki-67, as shown in Table 1.

In general lines, immunohistochemical study comprised the following stages: (1) deparaffinization and rehydration of skin sections; (2) blockage of endogenous peroxidase using H2O2 at 3%; (3) antigenic recovery at high temperature with citrate buffer 0.01M, pH 6.0; (4) incubation with monoclonal primary antibodies; (5) incubation with secondary biotinylated antibody (Universal Detection Pathway EP-USA - Easy Path®); (6) incubation with complex Streptoavidin-Biotin Peroxidase; (7) development with chromogene substrate diaminobenzidine (DAB); (8) contra-staining with Harris hematoxylin, and (9) dehydration and set up of slides to be analyzed by optical microscopy. Slides containing palatine tonsil sections were used as positive controls for markers Bcl-2, PCNA and Ki-67; for p-53, breast carcinoma sections were used for the same purpose.

In immunohistochemical tests, we considered to be positive strictly nuclear reactions for markers p53, PCNA and Ki-67; for Bcl-2, cytoplasmatic marker was defined as positive.

In view of the low positivity with some markers, we decided to perform descriptive analysis of the expression of all markers, using a system of crosses. The classification, based on the observation of a microscopic field with 200x magnification, ranged from zero to four crosses (4+), defined for p53, Ki-67 and PCNA: 4+ to all stained nuclei; 3+ in case some few nuclei did not show staining; 2+ in case half of them had no staining; 1+ for few stained nuclei and total absence of reaction. For Bcl-2, marking is cytoplasmatic and linear around the external cell membrane; we used the same observation and the same criteria for nuclear marking.

In the statistical analysis, we used non-parametric Mann-Whitney test to assess the significance of expression of apoptosis and cell proliferation markers, according to severity of basal cell carcinoma.

The Project was approved by the Research Ethics Committee, Universidade Federal de Juiz de Fora (UFJF), under number 277/2005. The study was performed between February 2006 and October 2008.

RESULTS

BCC samples were studied by optical microscopy and classified according to the predominant histology pattern. Nodular variant is characterized by multiple rounded nodules of varied sizes, with well defined contour, amidst the stroma, in addition to retraction of tumor blocks, creating an opening around it. Superficial BCC shows proliferation of atypical basaloid cells, forming a parallel axis to epidermal surface. The samples of sclerosing form evidence thick columns of basaloid cells, involved in densely collagen stroma and contain many fibroblasts.

Neoplastic cells that comprised basal cell carcinoma were delimited by a retraction in the interface with the surrounding stroma. Such characteristic is more marked in nodular and superficial variants, and less common in sclerosing BCC (Figures 1A, 1B and 1C).

Palpebral tissue considered in the control group (Figure 1D), free from pathology, presented histology abnormalities that resulted from aging and sun exposure, such as atrophy and solar elastosis, without epithelial atypias.

Through immunohistochemical analysis we could observe the expression of apoptosis markers (p53 and Bcl-2) and cell proliferation markers (Ki-67 and PCNA) in clinical forms of BCC and the control tissue (healthy eyelid tissue), as shown in Graph 1.

The expression of protein p53 (represented by brownish staining restricted to the nucleus) was very similar in all variants of BCC, but the highest in nodular type, in which 80% (n=4) of the patients expressed 4+. In sclerosing and superficial forms, 60% (m=3) of the cases had maximum score (4+). None of the patients in the control group presented expression of this marker (Table 2).

The expression of PCNA (brownish staining, granular and delicate, limited by the cell nucleus) was the maximum in all patients who had nodular and sclerosing variation. In superficial BCC, in 80% of the cases (n=4), there was expression of 4+ and in 20% (n=1), it was +2. In palpebral samples of the control group, more than 60% (n=2) showed markings of 4+ and the remaining subjects had no expression of PCNA (Table 3).

Marking standard for Ki-67 (brownish granulation, more intense in cells with atypia or mitosis) was maximum for 60% of the cases (n=3) of nodular BCC and in 40% (n=2) of patients with superficial variation. In the other samples of these two forms there was no other expression. In sclerosing BCC, there was reduced intensity in all samples: 80% (n = 4) in 3+ and 20% (n = 1) in 1+. In palpebral controls, there was Ki-67 staining, as shown by Table 4.

Cytoplasmatic immunestaining of Bcl-2 (characterized by granular aspect, brownish color, in addition to linear marking at the external cell membrane level) was very expressive in the sclerosing form, presenting as 4+ in 80% of the cases (n=4) and as 2+ in 20% of them (n=1). In the nodular type, staining was 4+ in 60% of the samples (n=3) and 3+ in the others. In superficial BCC, staining was smaller: in 60% (n=3), it was absent; in 20% (n=1), it showed 3+ expression, and in the others, it showed 2+. In the control group, there was expression 3+ in only one case (n=1), which occurred on the basal cell layer of the epidermis (Table 5).

According to the non-parametric Mann-Whitney test, we observed strong tendency to indicate severity of the tumor according to markers Bcl-2 (p = 0.04) and Ki-67 (p = 0.04). As to p53, we detected p=0.02 in BCC of superficial type, indicating high significance in low malignancy tumor, as shown in Table 6.

DISCUSSION

In the present study, we compared the expression of markers Bcl-2, p53, Ki-67 and PCNA in subjects with BCC of sclerosing, nodular and superficial types, with negative controls represented by tumor-free upper lid skin.

Bcl-2 staining was highly expressive in BCC said to be aggressive (Figure 2). Sclerosing form was the one that expressed the most this marker, followed by nodular form, which confirms the bibliographic data. Previous studies have indicated that BCC variants, without exception, expressed moderate to high level of protein Bcl-2, and the most aggressive clinical forms, such as sclerosing form, showed immunoreactivity greater than that of nodular basal cell carcinoma ¹⁵.

Negative significant correlation between expression of p53 and/or Bcl-2 with development of BCC has been already described. P53 mutations or overexpression of Bcl-2 are enough to enhance the formation of BCC by suspension of apoptosis. Even though in most studies expression of Bcl-2 is related with tumor severity, a review study concluded that the "apoptosis survival gene" (Bcl-2) is increased in nodular and superficial BCC ¹⁶.

p53 is frequently abnormal in human tumors, including skin cancer, in which these mutations were shown as a result of sun exposure. P53 abnormalities are found in 50% of the cases, but it is difficult to differentiate whether this defect of "the most known gene suppressor" (p53) is the cause of development of BCC or whether it is merely a marker of damage caused by ultraviolet light ¹⁷. However, a previous study concluded that synthesis or stabilization of p53 is essential to induce apoptosis of basal cell carcinoma.

Out of all studied tumors, 66.7% (n = 10) strongly expressed p53 and in the control group, only one subject showed low expression of this marker, only in basal layer cells (Figure 3), confirming the statements made by previous studies ¹⁸.

Mutations of protein p53 are common in some malignant neoplasms, and immunohistochemical detection reflects gene abnormalities, given that normal p53 is not easily detected because it has short half-life and low levels ¹³. However, not all positive tumors for p53 show gene mutations, and absence of staining does not mean absence of gene involvement. Immunohistochemical assessment of protein p53 needs careful interpretation, because false-positive and false-negative results may happen ¹⁹. Such inconsistent findings may be attributed primarily to the complex biology of this protein.

Based on the results of the present study, in agreement with the relevant literature, we can suggest that superficial BCC may seem to have high percentage of p53 mutations ²⁰. However, many tumors have to be assessed to define the clinical correlation between this protein and basal cell carcinoma.

Ki-67 expression makes us infer about the life cycle of a specific cell, informing whether the cell is active in the cycle. As a result, it is possible to have a neoplasm that has high proliferation rate and low percentage of positive cells for a specific marker ⁸.

Some studies have shown that Ki-67 is not subject to the influences of internal and external factors, such as PCNA, reason why it is superior as a marker of cell proliferation ¹⁵. The results of the present study showed greater expression of Ki-67 in nodular and superficial BCC, and in the latter, 4+ marking occurred in only 40% of the cases (n=2). In the controls, there had been no staining. Therefore, we cannot state, as the literature advocates, that Ki-67 is a marker that supports diagnosis and severity of BCC owing to the great variation of its cell expression ⁸.

The high rate of positivity for PCNA found in the studied tumors had also been described by other authors and it was considered the cause of low sensitivity for investigation of proliferation index, when compared to other markers ¹⁵. Without knowing the exact moment of the cell cycle, it is not possible to determine whether the high number of positive-PCNA cells in a neoplasm is due to high turnover or whether it indicates large proportion of cells divided into a prolonged cycle ^8,15.

There is a great variation in intensity of staining for positive-PCNA cells, which may be attributed to long half-life. Owing to the inability to learn whether the less-stained cell is in cycle or not, it is suggested that all cells should be considered marked, regardless of the staining intensity, exactly as considered in the present paper to assess the staining with PCNA ^8,15.

The use and reliability of these biomarkers are directly related with better characterization, and more studies that correlate detection and clinical data and other assessment methods of proliferation, already standardized, such as analysis using morphometrics.

To present, it seems that there is no absolutely reliable and conclusive test. However, many markers assessed by the present study seem to support the severity analysis of these three subtypes of BCC studied here.

CONCLUSION

According to the immunohistochemical results of expression of p53, PCNA, Ki-67 and Bcl-2 in variants sclerosing, nodular and superficial of BCC and on eyelid skin of patients free from the pathology, we could conclude that Bcl-2 and protein p53 present a tendency to diagnose severity of basal cell carcinoma.

PCNA, owing to its long life and cell marking outside the cell cycle, cannot be considered a good marker for the diagnosis of severity of BCC, which also applies to Ki-67, which has unstable behavior.

REFERENCES

• 1.  Lage IR, Ramírez ELA, Ayalas JAR, Lage MR. Epidemiología Del Cáncer de Piel no Melanoma (Hospital Provincial de Villa Clara). Rev Cuba Oncol. 2001;17:43-7.
• 2.  Murphy GF, Sellheyer K, Mihm MC. A pele. In: Kumar V, Abbas AK, Fausto N, editores. Robbins e Cotran - Patologia: Bases patológicas das Doenças. 7 ed. Rio de Janeiro: Elsevier; 2005. p. 1283-1330.
• 3.  Ishi LA, Pereira IC, Schellini AS, Marques MEA, Padovani CR. Carcinoma basocelular de pálpebras - fatores relacionados com a recidiva tumoral. An Bras Dermatol. 2004;79:423-30.
• 4.  Silva FB, Souza SMG. Aplicação da Biologia Molecular na Odontologia: Conceitos e Técnicas. Rev Fac Odont de Lins. 2002;14:7-14.
• 5.  Crowson, AN. Basal cell carcinoma: biology, morphology and clinical implications. Mod Pathol. 2006;19:127-47.
• 6.  Stratigos AJ. Immunophenotipic analysis of the p53 gene in non-melanoma skin cancer and correlation with apoptosis and cell proliferation. J Eur Acad Dermatol Venereol. 2005;19:180-6.
• 7.  Kopke LFF, Schmidt M. Carcinoma Basocelular. An Bras Dermatol. 2002;77:249-85.
• 8.  Arisawa EAL, Moraes E, Rocha RF, Almeida JD. Marcadores biológicos: PCNA e Ki-67. Breve revisão. Rev Fac Odont. 1999;2:54-60.
• 9.  Hwa Jee S, Chuan Shen S, Ren Tseng C, Ching Chiu H, Liang Kuo M. Curcumin Induces a p53-dependent apoptosis in Human Basal cell carcinoma cells. J Invest Dermatol. 1998;111:656-61.
• 10.  Cavalcanti GB. Citometria de Fluxo, Imunocitoquímica e Western Blot na Detecção da Expressão da Proteína p53 em Células Tumorais: uma Análise Comparativa. Rev Bras Anal Clin. 2003;35:135-42.
• 11.  Isoherranen K. UV irradiation induces downregulation of Bcl-2 expression in vitro and in vivo. Arch Dermatol. 1999;291:212-6.
• 12.  Murphy M, Mabruk MJEMF, Lenane P, Liew A, Buckley A, Flatharta CO et al. Comparison of the expression of p53, p21, bax and the induction of apoptosis between patients with basal cell carcinoma and normal controls in response to ultraviolet irradiation. J Clin Pathol. 2002;55:829-33.
• 13.  Tweddle DA, Malcolm AJ, Cole M, Pearson ADJ, Lunec J. p53 Cellular Localization and Function in Neuroblastoma: Evidence for Defective G1 Arrest Despite WAF1 Induction in MYCN-Amplified Cells. Am J Pathol. 2001;158:2067-77.
• 14.  Sousa FAC, Brandão AAH, Almeida JD, Rosa LEB. Alterações gënicas e cäncer bucal - uma breve revisáo. Rev Bras Patol Oral. 2004;3:20-25.
• 15.  Rubin AI, Chen EH, Ratner D. Basal-cell carcinoma. N Engl J Med. 2005;353:2262-69.
• 16.  Lear W, Dahlke E, Murray CA. Basal Cell Carcinoma: Review of Epidemiology, Pathogenesis, and Associated Risk Factors. J Cut Med Surg. 2007;11:19-30.
• 17.  Tilli CMLJ, Van Steensel MAM, Krekels GAM, Neumann HAM, Ramaekers FCS. Molecular aetiology and pathogenesis of basal cell carcinoma. Br J Dermatol. 2005;152:1108-24.
• 18.  Kim KH, Park EJ, Seo YJ, Cho HS, Kim CW, Kim KJ, Park HR. Immunohistochemical study of cyclooxygenase-2 and p53 expression in skin tumors. J Dermatol. 2006;33:319-25.
• 19.  Ansarin H, Daliri M, Soltani-Arabshahi R. Expression of p53 in aggressive and non-agressive histologic variants of basal cell carcinoma. Eur J Dermatol. 2006;543-47.
• 20.  Park HR, Min SK, Cho DC, Kim KH, Shin HS, Park YE. Expression profiles of p63, p53, survivin an htert in skin tumors. J Cut Pathol. 2004;31:544-9.

Publication Dates

• Publication in this collection
  26 Feb 2010
• Date of issue
  Dec 2009

History

• Received
  10 June 2009
• Accepted
  21 July 2009