| Abstract |
The Wilms Tumor or nefroblastoma represents one of the most common neoplasms in childhood, as well as the most frequent intra-abdominal neoplasm in children under the age of ten years. The neoplasm shows about the same frequency in both genders, and the average age at diagnosis is three years old. An important feature of Wilms tumor is the association with birth defects, the most common of which is the benign abnormalities of urogenital system, the hemihypertrophy and the sporadic aniridia. It is a tumor that can be located anywhere in the kidneys. It usually occurs as an asymptomatic mass and discovered mostly by the parents or by the pediatric routine examination. 50% of affected children exhibit abdominal pains, vomiting, weight loss and anorexia. Hypertension, referred to in 60% of patients occurs because renal ischemia, usually due to pressure effects on the renal artery by the tumor. Occasionally the tumor might bleed causing nuisance in kidney area and hematuria. The mass, in general, is smooth and compact and rarely passes the middle line. Macroscopically, the tumor has clear boundaries and is surrounded by a capsule. Microscopically, it seems to be derived from mesodermal stem cells and consists of a mixture of three distinct cell types: stromal, epithelial and blastemal cells, with variety in the degree of differentiation for each subtype. Full dedifferentiation, and anaplasia, observed in 10% of cases classified as blastemal element and has the worst prognosis, since corresponds to 60% of deaths. This histological type, with unfavorable prognosis, characterized by cells that have three times the normal size, with hyperchromatic nuclei and abnormal mitosis, is advancing rapidly and gives blood-borne metastasis to lungs. One other factor associated with the prognosis is and the existence of nephrogenic rests either around either within the renal parenchyma. In the effort to investigate the molecular mechanism of tumor growth, it has been found that two gene loci are involved and found on chromosome 11: the 11p13 (WT1) and the 11p15 (WT2). The classical deletions of one of these gene loci, 11p13, associated also with two rare syndromes which include in their manifestation Wilms tumors: a) the WAGR syndrome (tumor, aniridia, genitourinary malformations and mental retardation) and b) Denys-Drash syndrome (tumor, nephropathy and abnormal genital tract). The existence of the second genital 11p15 loci, can interpret the correlation of Wilms tumor with Beckwith-Wiedemann syndrome, a congenital syndrome characterized by several types of embryonic tumors, hemihypertrophy, makroglossia and splanchnomegaly. The development of Wilms tumor is a complex process and probably other genetic loci are involved. In adult kidney cancer, an effort is being made in recent years to control the expression of the mutated Ras and Raf genes, which belong to the MAP kinases cascade.
The Ras gene was initially identified as the oncogene "Rat Sarcoma Virus". Then found that the Ras protein kinase pathways implicated in six categories: a) growth regulation of fibroblasts, b) transformation of fibroblasts, c) differentiation and maturation of hematopoietic cells, d) activation of T-lymphocytes, e) neuronal differentiation of cells in pheochromocytoma and f) inhibition of maturation of epithelial cells. It seems therefore that the Ras gene and the corresponding membrane protein that is coding are involved in signal transduction pathway of growth factor (GF) and therefore in mitosis of the cell. Specifically, when the GF attached to the membrane of the receptor activates the inactive form of the protein GDP-Ras and is converted to the active form of GTP-Ras. With immediate effect of GTP-Ras to Raf kinase, activates the kinase catalytic activity and through phosphorylation activates the cascade of kinases Raf/MEK/ERK, catalyzing the induction mitogenic signals to the nucleus of the cell resulting in cell cycle control. With the action of GTPase, the active form of Ras hydrolyzed to GDP-Ras and the cell cycle is checked. In mutations of these genes, the mitogenic signal is not interrupted because of which, promotes cell cycle with no extracellular control. Recent studies have shown that in addition to the induction of mitogenic signals, the Ras protein indirectly affects the levels of cyclins influencing the passage of the G1 phase to S phase of the cell cycle. Three members of the family of Ras genes are known, genes H-K-and N-Ras. The H-Ras gene is located in chromosome position 11p15, the K-Ras in 12p12 and N-Ras in position 1p13, while mutations usually involve codons 12, 59 and 61 respectively. Such mutations present in various cancers and precancerous conditions, such as in malignant melanoma, in tumors of the thyroid gland, cancers of the colon and bladder.
As it was already mentioned, the Raf protein forms part of the trail Ras/Raf/MEK/ERK. This is encoded by the Raf gene. The gene group includes B-Raf, A-Raf 1 and Raf 1 genes. The A-Raf 1 found in location Xp11.3-11.23 and the protein that it encodes has serine-threonine kinase action with signal transduction role. The Raf 1 gene detected in 3p25 position, the corresponding protein is part of ras proteins transduction and is involved in ERK kinase signal cascade, with the process of phosphorylation of MEK proteins. The role of the gene is associated with the cell cycle, the proliferation, differentiation and apoptosis. The B-Raf gene is located in chromosome position 7q34. It is a functional gene and the protein it encodes is serine-threonine protein kinase and contributes to induction of mitogenic signals from the cytoplasmic membrane to the nucleus of the cell. It is involved in the cell cycle, while it seems to have and anti-apoptotic action. Point mutations in the regulatory section of the kinase result in constant induction, implicating so in the process of cell cycle. The lack of significant knowledge of the role of these genes in childhood kidney tumors, as well as the participation of WT1 gene and WT2 on paths of MAP kinases, lead to the thought to investigate genes Ras and Raf in Wilms tumors.
The experimental procedure was the following. 32 paraffin embedded tissues were collected diagnosed with Wilms tumor from the Pediatric Hospital "Agia Sofia" of Athens and by the G.N. «G. Gennimatas» of Thessaloniki in a period of 3 years. Also 10 paraffin embedded tissues were collected from normal renal parenchyma, from kidneys removed for non-neoplastic pathology. DNA extraction was followed and polymerase chain reaction with restriction enzymes (endonucleases), especially for genetic sites of more frequent mutations of genes under consideration. This procedure revealed any possible relevance of these genes and possible mutations in the pathophysiology of Wilms tumors. The result was negative and none of the 32 samples analyzed was a carrier of a mutation. Then, RNA extraction followed from the same tissues as well as from the normal ones. Using the RNA, rt-PCR was performed and cDNA was synthesized. Real time PCR was then applied, investigating the expression of these specific genes in tumors compared to normal tissues, using as reference the gene b-actin gene. Statistical analysis of the results of the experiments was performed and recording of significant results. The expression of mRNA levels of these genes, revealed some interesting results. First of all, a total overexpression of genes was observed in tumor samples, compared with normal controls. In particular, a mild increase in the H-Ras and K-Ras while a greater increase in B-Raf and N-Ras. Also, the overexpression especially for the K-Ras and the B-Raf was greater for larger tumors (> 10cm) as compared with smaller ones. Regarding expression of H-Ras, there has been a correlation with tumor necrosis rate. Indeed, greater extent of necrosis entailed with decreased expression and vice versa. Greater extent of tumor necrosis also resulted in a deregulation in the expression of K-Ras. But the more impressive statistical result had to do with the different expressive profiles of these specific genes in relation to the histological features of the tumor. More specifically, depending on the percentage of individual histological characters, epithelial, stromal or blastemal, there was a different involvement in gene expression. So, the N-Ras seemed to be expressed when outweighs the blastemal or stromal element, unlike to B-Raf expression, which overexpressed in tumors that primary histological feature was the epithelial component. The other two genes (H-and K-Ras) experienced no corresponding fluctuations. Another interesting result was related to the co-expression profile of these genes. While in normal samples, there was generally an independent expression of the genes studied, in cases of tumors a much more frequent association was detected between expressions of mRNA.
The etiology of Wilms tumor occurrence is not yet fully interpreted, despite the successful progress in treatment and survival rates. Perinatal and environmental factors seem to participate in pathophysiology as well as histological markers such as nephrogenic rests. Gene loci of genes WT 1 and WT 2 also have been implicated and identified as part of the mechanism of occurrence of the neoplasm. Genes Ras and B-Raf belong to the most frequent genes involved either with mutations or with participation in molecular paths in which their proteins are part of, in tumors of various organs. The idea that they may play a role in this pediatric neoplasm has been researched only partially and so our study, is trying to add some new findings in this field. Although mutations of these genes were not detected in the samples studied, there was a disturbance in their expression. The genes K-Ras and B-Raf have been found to have positive correlation regarding their expression with the size of various tumors, including pancreatic cancer, mesothelioma, hepatocellular carcinoma, melanoma, thyroid carcinoma, etc. We noticed a matching correlation with the size of Wilms tumors as well. Also, the correlation with the proportion of necrosis and decreased expression of H-Ras, it can be associated with the hypothesis based on other investigations, that wild-type H-Ras acts as a safeguard and induces apoptosis in cases of deregulation of the cell cycle. Finally, the fact of different expression profiles of N-Ras and B-Raf genes according to the histological characteristics of tumors was impressive. Stromal and blastemal histology that is presumptively worst-case prognosis from the epithelial, showed a higher expression of ratio with N-Ras, while the epithelial component, which is usually a positive predictor, had more connection with overexpression of B-Raf. This study apart from some interesting results, presents some weaknesses and limitations, such as the relatively small number of incidents. Of course, the country's population and the prevalence of this neoplasm do not allow easy collection of more samples. The same is true for the normal samples, since it does not occur in everyday surgical practice a non-neoplastic kidney from a child. In conclusion, this study managed to produce some interesting conclusions about the pathophysiology of Wilms tumor and the involvement of Ras and B–Rafgenes. Results, originally presented as a suspended international communication conference and eventually published in the journal of Medical Oncology, under the title "K-Ras, H-Ras, N-Ras and B-Raf mutation and expression analysis in Wilms tumors: Association with tumor growth»
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Ανίχνευση και διερεύνηση του ρόλου σημειακών μεταλλάξεων των ογκογονιδίων H-, K- και N-ras, καθώς και του B-raf σε όγκους Wilms
