Analysis of allelic imbalances and local DNA copy number changes in neuroblastic tumors

Neuroblastoma (NB) is a neoplasm of sympathetic nervous system and the most frequent extra cranial solid tumor of early childhood. The tumors have variable clinical presentation, ranging from a benign tumor which is regress spontaneously or mature into ganglioneuroma to an aggressive and metastatic malignant progression. NB has very complex and heterogeneous genetic alterations. The most common alterations are ploidy changes, amplification of the MYCN, deletions of chromosomes arms 1p, 3p and 11q and gains of 17q arm. In this study, we analyzed genomic scale of the genetic alterations and clinical parameters of the primary neuroblastic tumors in 249 cases with MLPA. Also, we investigated a significance of association between clinical (age, tumor stage and histology) and genetic (MYCN amplification, 1p, 3p, 11q deletions and 17q gain) parameters with multivariate logistic regression analyses. In addition, array CGH analyses were observed in tumors with 11q and 3p deletions. At the same time, we thought that amplicons observed in cell lines may be replicated at any stage of the cell cycle. MLPA analyses of multiple loci at chromosomes 1, 2, 3, 4, 7, 9, 12, 14, and 17 were performed using more than one hundred probes. Amplified PCR products were separated with a capillary electrophoresis and fragments were analyzed with it. Major anomalies in NB were MYCN amplification (23.3%), 1p (31.7%), 3p (20.8%), 11q (26.2%) deletions and 17q (77.2%) gain. There was a highly significant amplification of MYCN gene and NAG, DDX1 and ALK amplifications. Replication of double minutes on SiMa cell line was shown dependent on the cell cycle (S phase). Genetic analysis of multiple loci by MLPA revealed four different tumor groups: (Grup1) Tumors with MYCN amplification and 1p deletion or at least one of them, (Grup2) Tumors with 11q and 3p deletions or at least one of them, (Grup3) MYCN amplification and/or 1p deletion and 11q and/or 3p deletions at least one of them, (Grup4) tumor with anomalies other than 1p, 3p, 11q deletions or MYCN amplification. The majority of the patients in the Group1, 2 and 3 were detected over 12 months of age, high stage and poor histology. In conclusion, our results show that MLPA can confidently and effectively be utilized to detect multiple genomic imbalances at a time, and these changes can be classified into genetic subtypes of NB. Also, MLPA applications could be effectively supported by treatment protocols.