Keywords: Melanoma, H-Tert, Caspases, Apoptosis, Breslow, Clark
Introduction
Malignant degeneration of the melanocyte gives rise to the tumor known as melanoma. The locally invasive characteristic of this tumor involves vertical penetration through the skin and into the dermis and subcutaneous tissues of the malignant melanocytes. Melanoma may develop anywhere on the body. Melanoma is one of the fastest growing cancers world-wide. The risk of melanoma over humans is estimated up to 1.33% and is rising every year. Only in 2010 over 60.000 developed melanoma [1]. It is the sixth most common cancer type in men and the seventh in woman and has a poor prognosis with a median survival of 6-10 months and a 5 year survival rate of about 10% [2]. Advanced melanoma has a poor prognosis since chemotherapy is mostly ineffective due to the resistance to anti-neoplastic agent's melanoma cells have. The reasons for this chemoresistance are under study. Through induction of apoptosis many anti-cancer drugs kill susceptible cells. Some tumor cells can resist drug therapy and it appears to have altered apoptotic pathways [3]. Human melanomas are more likely to have altered apoptotic pathways. The complexity of the molecular variants involved in signal transduction along apoptotic pathways suggests that cell may have a variety of possibilities for regulating apoptosis and generating apoptotic deficiency. Deregulation of the apoptotic pathway and clinical-histopathological parameters of melanomas are firmly related to chemo-endurance and poor prognosis [4].
Furthermore, expression of telomerase leads to cell growth and can be considered an important subject for research regarding several types of malignancy [5,6]. Telomerase is a key enzyme ribonucleo-protein involved in the stabilization of telomeres in transformed cells as well as in tumor cells. Evidence suggests that activation of the telomerase enzyme is a pivotal step in the development of all forms of skin cancer. In fact, telomerase activity has been observed in 75-85% of all forms of skin cancer and has also been observed in normal skin exposed to solar ultraviolet radiation. Thus, it is evident that stabilization and maintenance of telomeres is a central event in cellular immortalization and carcinogenesis [7,8].
The objective of this research is to examine the relationship between crucial clinical and histopathological parameters with the deregulation of the apoptotic pathway and the expression of telomerase. Telomerase as well as the members of the apoptotic pathway Bcl 2, caspase 3 and caspase 8 have been correlated in order to prove a relationship between protein expression disruption in the cutaneous melanoma and crucial clinical and histopathological parameters. In regards to our knowledge, this research consists of the first research that combines the method of Tissue Microarray (TMA) and computerized image analysis (CIA) for the combined estimation of the malignant melanoma indicators.
Materials and Methods
Study group
For the purpose of the study, we obtained 25 paraffinembedded histologically confirmed cutaneous melanoma tissue samples derived from patients who had undergone wide local excision after the first diagnostic biopsy. Tissue sections from benign-appearing epithelia adjacent to malignant tissue were used as normal control group. The local Ethical Committee consented to the use of these tissues delivered from the Department of Pathology (417 VA Hospital-NIMITS, Athens, Greece) for research purposes. The archival samples were fixed in 10% neutral-buffed formalin. Hematoxylin and eosin (H&E)- stained slides of the corresponding samples were reviewed for confirmation of histopathological diagnosis. All melanomas were staged according to WHO Pathological Classification of melanoma.
Tissue microarrays construction
Tissue Microarrays (TMAs) are produced by taking small punches from a series of paraffin-embedded (donor) tissue blocks and transferring these tissue cores into a positionally encoded array in a recipient paraffin block. Though TMAs are not used for clinical diagnosis, they have several advantages over using conventional whole histological sections for research. Tissue from multiple patients or blocks can be examined on the same slide, and only a very small amount of reagent is required to stain or label an entire array. Areas of interest were identified using tiles stained with hematoxylin and eosin stain, in combination with a conventional Olympus BX50 microscope. Using the TMArrayer-100 (Chemi-Con International USA), all of the source blocks were cored and 2mm diameter tissue cylindrical cores were implanted from the donor to the recipient block. The final constructed TMA block contained 25 cylindrical tissue specimens. After a micro incision of 3mm and the use of the H&E stain, the final TMA density was 96%, as was observed microscopically, due to the failure of one of the 25 samples (Figure 1).
Antibodies and Immunohistochemistry
Immunohistochemistry (IHC) refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Monoclonal antibodies were selected and applied. The IHC staining was done with the use of the anti-bcl 2 (124-Dako, Denmark) at dilution 1:60, anti-caspase 3 (3CSP03-Neomarkers, USA) 1:50, anti-caspase 8 (11B6, Novocastra, USA) 1:20 and anti-h Tert antibodies (44F12, Novocastra, USA) 1:40. Initially 2 TMA sections were deparaffinized in xylene and rehydrated through graded ethanol. The process was carried out with the use of an automated IHC BIOGENEX 16000 staining system. After peroxidase blocking, the sections were incubated with the primary antibody at room temperature. The time needed was different for each antibody. Afterwards the tissue was incubated with HRP polymer for 30 minutes. With the use of DAB as a chromogen substrate the antigen-antibody reaction was revealed. Following the procedure, TMA sections were dehydrated and mounted. The primary antibodies were omitted for negative control (Figure 2).
Computerized Image Analysis (CIA)
The contribution of CIA for performing morphometric studies is considered significant. So, in this study, three categorizes were used for the evaluation of the IHC staining, which offered increased sensitivity in measurements, thereby providing a more accurate assessment of the end result. The evaluation of the IHC protein expression was done using semiautomatic CIA of digitized images. The stain density measurements of bcl 2, caspase 3 and caspase 8 occurred in five fields per case and using a magnification of 400X per incident studied, while the expression of telomerase, was calculated by measuring the IHC stained cell nuclei per field of view (Nuclear Labeling Index -NLI). Normal epithelial cells were used for the control group and the staining density was characterized as Low (L), Moderate (M), and High (H) between 256 distinct values and the stained cell NLI.
Statistical Analysis
The statistical analysis was done using the SPSS v 11.0 software, the Chi square tests and Spearman value were carried out and the value of P<0.05 was evaluated as statistically important. Correlations were carried out between the levels of the protein expression of the molecules in study and the clinic and histopathological parameters. These were gender, age, Clark level, Breslow's Thickness, the presence of ulceration, the stage of the disease and more.
Results
According to CIA process and group definition, high level of protein overexpression was observed in 1/24 (4, 1%) for h-Tert and caspase 3, 2/24 (8, 2%) for caspase 8 and 4/24 (16, 4%) for bcl-2, respectively. Moderate level was observed in 29, 1%-32, 2%- 20,2% and 32,2% of cases. Whereas reduced or absent expression was demonstrated in 65%-60, 2%-68, 5% and 50% respectively. Statistical significance was noted correlating age to caspace 3 (p=0.05), Breslow's thickness to telomerase (p=0.013) as well as Clark's level to telomerase (p=0.008) and to bcl-2 (p=0.022). Kappa analysis regarding caspase 3 and 8 showed a medium value (kappa=0.442). At the same time bcl-2 in correlation with caspase 3 demonstrated low value (kappa=0.250). Total IHC results and p-values are mentioned in Tables 1-6.
Discussion
Malignant melanoma is an aggressive neoplasm, extremely chemo-resistant, with an ever increasing number of incidents reported worldwide, especially in Caucasians. The malignant transformation of melanocytes is induced by the deregulation of molecular pathways. Change in the apoptotic dynamic of the cells in combination with the overexpression of the molecules that promote cell proliferation such as telomerase, results in uncontrolled mitotic divisions that ultimately leads to this phenotype of cancer. Some studies have already examined the potential effectiveness of targeted inhibition of the telomerase molecular activity using specific suppressor factors to induce its expression.
The evaluation of the indicator "immortalized" cell (h TERT) was made with the index factors of the apoptotic pathway BCL 2, Caspase 3 and Caspase 8 in order to detect possible instabilities in their expression, correlating them with the clinical pathological parameters of the tumor and its general biological behavior [9,10].
Telomeres are small nucleotide size repetitive DNA sequences, coating the ends of the chromosomes, contributing to the protection and the stability of the genetic material [11]. At the end of every replicative process, the telomeres are progressively
shortened and thus this mechanism acts as a tumor suppressor preventing the ceaseless copy of the chromosomes [12]. During
the embryogenic process the protein levels increase, while in mature cells they are found in limited numbers. The increased protein expression is associated with the stabilization of the length of the telomere sequence, partly reflecting the neoplastic diversion of the cells in over 90% of human malignancies. This is the main reason that the telomerase is characterized as an independent indicator of cellular "immortalization". In vitro immortalized cells and stem cell lines of actively proliferating tissues show a high level of telomerase expression [13-15].
The already differentiated melanocytes, when exposed in vitro to high doses of ultraviolet radiation, exhibit elevated levels of p53. In these cell populations, inhibiting apoptosis factors, such as the mitochondrial protein BCL 2, are underexpressed unlike more assisting factors such as the bax protein, which is expressed in high levels. The antiapoptotic factor BCL 2 as a unique melanoma oncogene is located on chromosome 15q. It is essential for melanomas survival in which it is amplified and its overexpression is interesting to note that promote tumor genesis [16]. Therefore as proliferating melanocytes and melanoma cells accumulate high levels of BCL 2 consequently escape the programmed cell death program. In reality, a negative regulator of the cell cycle, the protein of the p16 tumor suppressor gene, plays a key role in promoting the apoptotic process in human melanocytes after exposure to ultraviolet radiation, as it suppresses the exact levels of the BCL 2 protein [17].
A combination of factors can occur tumor growth such as mutation in cell cycle gene which removes the restrains of cell growth, combined with mutations in caspases. The caspase family, which is composed of proteolytic proteins', promotes with the sequential activation of its own members the apoptotic process due to their specific cysteine protease activity. A cascade of biochemical reactions involving caspases 3, 7, 8 and 9 leads the cell to its apoptotic death through the process of segmenting its genetic material and the creation of apoptotic particles. Caspase 3 plays a crucial role in the initiation of the signals in order to stimulate melanoma tumor growth [18,19].
Studies of cell cultures of malignant melanocytes have shown that the latter is relatively resistant to most apoptotic stimuli. These cells also express a variety of anti-apoptotic factors of which the surviving protein appears to be the most frequently expressed agent with inhibitory anti-apoptotic activity which at the same time is not expressed in normal melanocytes. Many studies have demonstrated the central role that telomerase plays in the development of the neoplasm and the mechanisms of the enzyme action are still under investigation.
The reduction of the enzymes activity promote the appearance of a dendritic morphology combined with an increase in melanin
and the tyrosinase enzyme production. At the molecular level, the inhibition of the telomerase activity had an effect on the expression of glycolytic pathways with a corresponding reduction in the consumption of glucose and the production of lactose. Thus, the expression of telomerase appears to significantly affect the glycolytic pathway, first it changes the energy state of the cancer cells and as a result reduces the activity of tyrosinase and melanin production [20].
The telomerase in our study correlated with, other than the Breslow thickness and Clark level, the state of ulceration. Many relevant studies have shown a strong correlation between the activity of telomerase and major histopathological factors associated with poor prognosis such as ulceration, vascular invasion, microsatellites, as well as a high number of mitoses. Overexpression of the telomerase was observed in dysplastic nevi, Spitz nevi and primary melanoma, in which case possibly the expression of the telomerase did not appear to be a reliable factor in the histologic diagnosis of the primary melanocytic lesions. In our study, we found moderate and high levels of telomerase in a number of superficial spreading melanomas which was also shown in the results of another study [21], while being absent in primary nodular melanoma, so we may assume that the h Tert is associated with deregulatory mechanisms that are related more to superficial spreading melanoma [22].
Deregulation of apoptotic (Caspase 3 and 8) and anti-apoptotic molecules (bcl 2) were observed in our study. Strong correlations emerged between the bcl 2, the Breslow thickness as well as the Clark level, factors that are associated with poor prognosis as demonstrated by studies which show that the overexpression of bcl 2 is associated with advanced stages of disease and poor prognosis. Even so, there is conflicting data on the expression of bcl 2 and its role in the development and prognosis of melanoma. Protein analysis depends on several parameters, but the results in our study were based on the CIA that is characterized by high precision in relation to its conventional microscopic analysis.
The deregulation of Caspase 3 and 8 are responsible for the low apoptosis in melanoma. The Caspase 3 in our study was associated with the age of the patients. Moreover, co-expression of the two proteins showed a significant rate in kappa analysis and was low in value when associated with bcl 2, reflecting an imbalance between modulators that promote and suppress apoptosis. This fact led some studies to suggest new treatment regimens with the use of H1 histamine receptor antagonists such as terfenandine, which causes DNA damage and the activation of caspases 2, 3, 6, 8 and 9. According to the results of a study, surviving partially blocks the activation of caspases, mitochondrial release of apoptotic -inducing factor (AIF), cytochrome c and Smac induced by ultraviolet B radiation.
Conclusions
Current research study helped clarify the role of the deregulation of the apoptotic mechanism through the sub expression of the caspase 3 and caspase 8 in conjunction with the relative overexpression of the anti-apoptotic factor bcl 2, in the aggressiveness of the neoplasm. It also proved that both the geographical spread of the tumor and poor prognosis are directly linked with the strong correlations between the h Tert and the anti-apoptotic factor bcl 2 with the Breslow thickness and the Clark levels and also between the h Tert with the ulcerations. Furthermore, it appears that the strong anti-apoptotic reaction of bcl 2 is an important parameter for the aggressiveness of the melanoma since it's associated clinically with chemo-endurance. Additionally, it is with great significance the fact that this aggressive biological behavior of the neoplasm is also related to the suppression of the apoptosis in combination with the moderate overexpression of the h Tert.
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