The Study Of The Correlation Between The Aberrant Methylation Of RASSF1A And The Pathological Classification Of Neuroblastic Tumors

ObjectiveNeurblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN) are the tumors of the sympathetic nervous system that arise from primitive sympathogonia and are referred to collectively as neuroblastic tumors (NTs). GNB are classified into two categories:intermixed ganglioneuroblastoma (GNBi) and nodular ganglioneuroblastoma (GNBn). According to the International Classification, Tumors in the categories of intermixed ganglioneuroblastoma and ganglioneuroma all are classified into an favorable histology (FH) group, whereas all nodular ganglioneuroblastoma (GNBn) tumors and neuroblastoma are classified into an unfavorable histology (UH) group. Favorable type can regress spontaneously, or it can have a better outcome after a series of treatments. But whatever the therapy is used for the unfavorable type, the mortality rate still has no change. Therefore, how to change the prognosis related factors plays an important role in the treatment for the neuroblastic tumors.Gene RASSF1A(Ras association domain protein family 1 A) is located on 3p21.3. It is an anti-oncogene about 120kb, which is cloned from the high frequency homologous missing zone. Since the gene P16, the gene is one of the genes which can be methylated in the highest degree and in the most widespread of the tumor. A great number of studies show that the both expressing deletion and the high methylation of the promoter in RASSF1A can be found in a wide variety of tumor (such as breast, lung, non-small cell cancer, gastric adenocarcinoma and so on). So the gene is expected to play a major role in the early diagnosis and the prognosis of tumors.This experiment is designed to detect the abnormal methylation of gene RASSF1A in NTs by methylation-specific polymerase chain reaction (MSP). According to the International Neuroblastoma Pathology Classification (INPC), all of the specimens were divided into favorable histology (FH) group and unfavorable histology (UH) group. In this study, we analyzed the positive rate of methylation of two groups and estimated whether there is a relationship between this gene methylation and pathological classification.Materials and methodsIt was used methylation-specific polymerase chain reaction (MSP) to test the abnormal methylation of gene RASSF1A of neuroblastic tumor. Extract DNA of tissues according to the manufacture of the kit. For the genomic DNA as the positive methylation group, we made it SssI(CpG) methyltransferase processing. For the non-processing placental tissue DNA as the negative methylation group, we had it base hydrosulfite modification and PCR amplification. Then we had the equal deionized water as blank contrast and adrenal medulla DNA as test contrast. Then we had PCR amplification and made the products of PCR have gel electrophoresis by 2% of the agarose. At last, we used the gel imaging system analyzer to take photo and analyze the data.According to the International Neuroblastoma Pathology Classification, all of the specimens were divided into favorable histology (FH) group and unfavorable histology (UH) group by two specialists from pathological department.Results1.There is no expression of gene RASSF1A methylation in normal adrenal medullary tissue.2. No correlation between RASSF1A methylation and known prognostic factors including stage and age was detected.3. The positive rate of gene RASSF1A methylation:unfavorable histology (UH) is 77%, which is greater than favorable histology (FH). The association does not reach statistically significant(P=0.08).ConclusionThe positive rate of gene RASSF1A methylation:unfavorable histology (UH) is higher than favorable histology (FH). Although there is a trend between RASSF1A methylation and the pathological classification of neuroblastic tumors, it does not reach statistically significant.Thus RASSF1A cannot be used as an independent prognostic marker of NTs.