Abstract
The rate of apoptosis varies in malignant tumors, and it can be involved in diminishing tumor size. Different protein-regulators of apoptosis, such as Bcl-2, BclX, and Bax have an influence on the rate of apoptosis in various tumors. In human renal diseases, such as the experimental model of acute renal failure, and many tumors, including Wilms' tumor, the expression of antiapoptotic members of Bcl-2 family is increased, while the expression of proapoptotic members is low. Aim: The aim of our study was to investigate Bcl-XS/L protein expression in Wilms' tumor, to compare it with the expression in normal renal tissue, as well as to see if there is a correlation between Bcl-XS/L expression in Wilms' tumor with tumor stage, histological type, prognostic group, or response to preoperative chemotherapy. Materials and Methods: Twenty-eight cases of Wilms' tumor (two cases with metastasis) and two samples of normal kidney tissue were studied using streptavidin-biotin-complex technique. Bcl-XS/L expression levels were semiquantitatively scored. Results: The expression of Bcl-XS/L was observed in the majority of cases (60.7%), more often in the blastemal than in the epithelial component of Wilms' tumor: 60.7% and 28.6%, respectively (p = 0.02). There was a statistically significant inverse relationship between Bcl-XS/L expression and tumor stage (p = 0.015). Bcl-XS/L was found less frequently in high-risk tumors then in tumors with good prognosis (p = 0.02). Treated Wilms' tumors showed Bcl-XS/L expression more often than nontreated tumors, but the relationship was not statistically significant (p = 0.08). Expression of Bcl-XS/L was detected in various histological types of Wilms' tumor, but there was no statistically significant association (p = 0.82) except in cases with diffuse anaplasia (p = 0.012), which were always negative. No Bcl-XS/L immunostaining was observed in two cases of metastasis or in one case of bilateral Wilms' tumor. Conclusion: Our results suggest that the expression of Bcl-XS/L protein is associated with prognostic group, tumor stage, and presence of anaplasia.
Introduction
Apoptosis is a sequence of successive, morphologically different phases that develops both in pathologic and in physiologic conditions.Citation[1] Lack of cell response to induced apoptosis could be one of the mechanisms included in the progression of malignant tumors as well as their resistance to chemotherapy.Citation[2] It is well known that several proteins are included in regulation of apoptosis. The best described is Bcl-2 protein (B cell lymphoma 2), present in all hematopoietic and lymphoid cells, as well as in numerous epithelial cells and neurons.Citation[3] Members of the same protein family as Bcl-2 are Bcl-XL, Bcl-w, and others. Together with the Bcl-2 protein, they have a negative influence on apoptosis. Another group of similar proteins, containing Bax, Bcl-XS, Bad, and Bak, stimulates apoptosis.Citation[4] Antiapoptotic members of the Bcl-2 family induce an increase in cell number and contribute to tumor development. They are described as oncogens, while proapoptotic members represent genes that prevent tumor development.Citation[3]
Wilms' tumor (nephroblastoma) originates from metanephric blastema. It is one of the most common solid renal tumors in children.Citation[4] Despite a good response to chemotherapy, in 5–10% of cases, either metastases or resistance to chemotherapy occurs.Citation[2] Apoptosis plays an important role in normal development of the kidney. Irregularities of apoptotic regulation could cause tumor occurrence and development.
The aim of our study was to analyze the expression of a proapoptotic member of the Bcl-2 family, proteinBcl-XS/L,Citation[5] in Wilms' tumor, to compare it with the expression in normal renal tissue as well as to see if there is a correlation between Bcl-XS/L expressions in Wilms' tumor with tumor stage, histological type, prognostic group, or tumor response to preoperative chemotherapy.
Materials and Methods
We have investigated 28 specimens of patients with Wilms' tumor that were surgically removed (10 males and 18 females, age 7–132 months). In 19 cases, the patients received chemotherapy before the surgery. We also investigated two cases of Wilms' tumor with metastases, and two samples of normal kidney tissue (control group).
SIOP (International Society of Pediatric Oncology) classification from 2002Citation[6] was used to determine tumor stage, histological type, and prognostic group. According to this classification, 17 out of 28 cases (60.7%) were classified as stage I, four as stage II (14.3%), four (14.3%) as stage III, and two as stage IV (7.1%). One case (3.6%) of bilateral Wilms' tumor (stage V) was also analyzed. Tumor developed in left kidney was classified as stage I, and in right kidney as stage II. Two out of 28 cases (7.1%) were epithelial histological type, 11 blastemal (39.3%), six stromal (21.4%), and four mixed (14.3%). Out of five anaplastic tumors, four (14.3%) showed diffuse anaplasia, and one (3.6%) focal anaplasia. According to the prognostic groups, 18 (64.3%) cases belonged to the group with intermediate risk, and 10 (35.7%) to the group of high risk.
In order to demask the antigen, the tissue was treated in a microwave oven, in citrate buffer (pH = 6.0), three cycles, for 5 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min. To block unspecific staining, normal swine serum was used (dilution 1:10 for 30 min), and after that, primary antibody Bcl-XS/L (S-18, Santa Cruz, USA) was applied (dilution 1:100 for 60 min). Streptavidin-biotin staining method was performed using DAKO LSAB + kit. 3,3 Diaminobenzidine (DAB) was used as a chromogene, and for contrasting Mayer's hematoxylin. Bcl-XS/L expression levels were semiquantitatively scored: absence of staining in all tumor cells −, less than 10% positive cells + (focal expression), 10–50% positive cells ++ (medium expression), and over 50% +++ (diffuse expression). Statistic evaluation was done by using Fisher's test, Mann–Whitney's and student's t-test. P value < 0.05 was considered as statistically significant.
Results
In one case of normal renal tissue, there was no expression of Bcl-XS/L protein, and in the second case, there was a moderate epithelial expression in distal convoluted tubules. From 28 Wilms' tumor investigated cases, in 17 cases (60.7%) Bcl-XS/L expression was found (), both in epithelial and in blastemal tumor components. Statistically, comparison between the cases without or with focal Bcl-XS/L expression in Wilms' tumor (absence of expression) and the cases with moderate and diffuse Bcl-XS/L expression (prominent expression) was done. We have noticed that Bcl-XS/L expression was found more often in blastemal than in epithelial tumor components (60.7%:28.6%). This correlation was statistically significant (p = 0.023). Statistically significant difference was found between Bcl-XS/L expression and the tumor stage (p = 0.015). Tumors with lower tumor stage (1 and 2) showed Bcl-XS/L positivity more often than tumors in stages III, IV, and V (57.1%:5.7%). Analyzing Bcl-XS/L expression separately in the blastemal component (without expression in epithelial component) in comparison to the tumor stage, we did not find a statistically significant correlation (p = 0.136). We observed the same results comparing Bcl-XS/L expression in the epithelial component (without blastemal expression) and tumor stage (p = 0.624). In Wilms' tumor cases from the intermediate risk group, Bcl-XS/L protein expression was more often found in comparison to tumors from the high-risk group (77.8% : 30%), which was statistically significant (p = 0.02). The correlation between Bcl-XS/L expression in the blastemal component (without epithelial expression) and the prognostic group (p = 0.190), as well as between Bcl-XS/L expression only in the epithelial component (without blastemal expression) and the prognostic group was not statistically significant (p = 0.097).
Table 1 Clinical-Morphological Characteristics and Bcl-XS/L Expression in Wilms' Tumor.
Because some of the investigated patients with Wilms' tumor did not receive chemotherapy before surgery, we compared Bcl-XS/L protein expression separately in pretreated patients and those who did not receive chemotherapy before surgery. Pretreated patients with Wilms' tumor showed Bcl-XS/L expression more often than nontreated patients, but this finding was not statistically significant (p = 0.08). Medium tumor diameter in positive Bcl-XS/L cases was 7.8 cm, while in Bcl-XS/L negative cases, it was 9.5 cm. Although this difference was found, it was not statistically significant (p = 0.264). Bcl-XS/L expression was observed in different histological types of Wilms' tumor, but the correlation between certain tumor type and intensity of expression was not statistically significant (p = 0.82). We have investigated four cases of anaplastic Wilms' tumor, and in all of them, we found an absence of Bcl-XS/L expression. This finding is statistically significant in comparison to the intensity of Bcl-XS/L expression in other histological types of Wilms' tumor (p = 0.012). In this study, we also analyzed two cases of metastatic Wilms' tumor (marked as “*” in ) and one case with bilateral Wilms' tumor. These cases showed the absence of Bcl-XS/L expression.
Discussion
Accurate expression of proteins from the Bcl-2 family in normal as well as in damaged or changed kidneys is still poorly investigated. A weak expression of Bcl-2 is found in distal convoluted tubules of normal kidneys, and stronger Bcl-2 positivity was found in the collecting ducts of the inner part of the medulla.Citation[7] Bcl-XS/L protein was detected in normal renal tissue in the proximal and distal convoluted tubules as well as in the parietal layer of Bowman's capsule.Citation[2] In our study, in one out of two normal kidney specimens, we detected Bcl-XS/L expression, but only in distal convoluted tubules, while in the second case, the staining was negative.
In adult kidneys, there is a low degree of apoptosis. In embryonal renal tissue, apoptosis is present in metanephric mesenchyme and in the parietal layer of the Bowman's capsule, and later in embryonal development in tubular epithelium.Citation[8] The expression of apoptosis is not constant in malignant tumors. It can influence tumor growth. Certain proteins, regulators of apoptosis, like Bcl-2,Bcl-L, Bcl-S, Bax, and some others, express their influence to the expression of apoptosis in tumors. In literature, increased expression of several antiapoptotic, and decreased expression of proaortoptotic members is described in different human renal diseases, in experimental models of acute renal failure, as well as in different types of renal tumors, including Wilms' tumor.Citation[2], Citation[7], Citation[9-17] There are only four reports showing the results of immunohistochemical investigations of apoptotic regulators in Wilms' tumor,Citation[2], Citation[15-17] and only one in which Bcl-XS/L expression in Wilms' tumor is described.Citation[2]
We have investigated the expression of Bcl-XS/L, which is like the Bax protein, a proapoptototic member of the Bcl-2 family,Citation[5] in renal tissue, in Wilms' tumor, and in metastases of Wilms' tumor in lungs. Bcl-X protein, as a member of the Bcl-2 family, works as a Bcl-2 independent regulator of programmed cell death. Its two products, Bcl-XL (long) and Bcl-XS (short), express opposite effects. Bcl-XL is similar to the Bcl-2 gene, and its protein product also inhibits apoptosis, but in different cell lines. It looks like Bcl-XS stimulates cell death and inhibits the activity of Bcl-2. The function of Bcl-2 and Bcl-X can be modified by the activity of Bax that accelerates apoptosis by the formation of heterodimers.Citation[3] Literature data about antiapoptotic as well as proaortoptotic protein expression and the influence on Wilms' tumor are controversial. Some investigators think that Bcl-2 is not a regulator of apoptosis. According to them, some other oncogens and tumor suppressors express their influences to apoptosis in Wilms' tumor.Citation[16] Others try to illuminate the role of Bcl-2 in the pathogenesis of Wilms' tumor, pointing out that the blastemal expression of Bcl-2 protein can be the independent prognostic marker.Citation[2]
The results of our investigations showed the expression of Bcl-XS/L in the majority of Wilms' tumor cases (60.7%). Its expression in blastemal components was more prominent compared to epithelial component expression (p = 0.023), opposite to the data in the literature.Citation[2] We presume that this finding can be the result of the type of tumors we investigated, which had greater portions of blastemal components in comparison to epithelial, as well as the fact that in our study, we included the cases that were not pretreated with chemotherapy before surgery (five were predominantly blastemal nephroblastoma). With the increase of tumor stage, Bcl-XS/L expression decreases (p = 0.015). This connection between the weak expression of proaortoptotic proteins and low grade of Wilms' tumor was also detected by other investigators.Citation[10]
In the majority of cases, Bcl-XS/L expression is positive and increased in Wilms' tumor in comparison to the normal renal tissue. On the other hand, in both cases of Wilms' tumor metastases in lungs, Bcl-XS/L expression was negative. Absence of expression in metastatic tissue as well as in the primary Wilms' tumor that is the origin of metastases suggests that diminishing Bcl-XS/L expression could be crucial in the ability of Wilms' tumor to spread in the form of metastases. Such a connection between metastasizing and diminishing of proapoptotic proteins is already well documented in cases of colon carcinomas.Citation[12] Our observations showed that the intensity of Bcl-XS/L expression was not connected with histological type of Wilms' tumor (p = 0.82). The same results we found in the literature apply to Wilms' tumor,Citation[2] as well as to different types of tumors.Citation[10], Citation[18&19]
The anaplastic type of Wilms' tumor is rare, and in comparison to the classical, tricomponent type, it gives metastases more often, it is resistant to therapy, and it has a very bad prognosis. Some investigators suggest that overexpression of antiapoptotic proteins (Bcl-2 and Bcl-XL) can induce metastatic spread of Wilms' tumor, as well as the resistance to therapy. In some cases of anaplastic Wilms' tumor, decreased expression of proapoptotic protein Bax was described.Citation[15] In the literature, authors who investigated Bax and Bcl-XS/L expression did not analyze the correlation of proapoptotic protein expression and anaplastic development of tumor.Citation[2] We have done such analyses and found that the expression of Bcl-XS/L was absent in all diffuse anaplastic Wilms' tumor cases. In one case with focal anaplastic changes in Wilms' tumor, the expression of Bcl-XS/L was present. Anaplastic types of Wilms' tumor more often show absence of Bcl-XS/L expression in comparison to the other histological types (p = 0.012). While all of our classic Wilms' tumor cases in stage III showed a decreased, and in stage IV an absent Bcl-XS/L, expression in comparison to stage I/II, in anaplastic cases, there was a complete absence of Bcl-XS/L expression in stages III/IV. Our conclusion was that the absence of Bcl-XS/L expression is connected with the anaplastic development. However, it is necessary to investigate more cases with focal as well as with diffuse anaplastic changes of Wilms' tumor. As in the anaplastic types of Wilms' tumor, metastases are more often found than in classical types,Citation[15] absence of Bcl-XS/L expression could indicate a possible connection with metastatic development.
Mutation of genes involved in the control of cell death could affect the response of tumor cells to chemotherapy.Citation[3] Data from the literature showed that the intensity of apoptotic markers expression that follows chemotherapy could predict further behavior of the tumor and its prognosis.Citation[2] Numerous clinical studies showed correlation between the intensive antiapoptotic geneexpression and a poor response to therapy in cases with neuroblastoma, pulmonal carcinoma, and chronic myelocyte leukemia,Citation[3], Citation[14] as well as between the absence of proapoptotic gene expression and poor answer to therapy in cases with breast carcinoma, metastatic breast adenocarcinoma, and prostatic carcinoma.Citation[3], Citation[18], Citation[20-22] In our group of Wilms' tumor cases there was no statistically significant difference between the Bcl-XS/L expression in treated and nontreated patients (p = 0.08). Unfortunately, our study could not show if increased proapoptotic protein Bcl-XS/L expression is in correlation with a good tumor response to chemotherapy, because all our cases that received preoperative therapy were treated according to different SIOP protocols. Only in one case that was treated according to SIOP-6 protocol (long preoperative chemotherapy), have we found an absence of Bcl-XS/L expression. However, because it was a case with diffuse anaplastic changes resistant to chemotherapy, such a response was expected. Investigation of the connection between Bcl-XS/L expression and prognostic group of Wilms' tumor showed a statistical significance (p = 0.02). In tumors of intermediate risk, we found a more prominent Bcl-XS/L expression than in the high-risk tumor group.
Conclusion
Our results suggest that decreased expression of Bcl-XS/L protein in Wilms' tumor is in correlation with tumor stage, high-risk prognostic group, and presence of anaplasia.
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