Li-Fraumeni syndrome (LFS) is a rare familial dominantly inherited cancer disorder characterized by a wide spectrum of early onset neoplasms. The most prevalent tumors include rhabdomyosarcoma (RMS), osteosarcoma, brain tumor, adrenocortical carcinoma (ADCC), leukemia and early-onset breast cancer. LFS is associated with germline p53 mutations in >75% of reported families. Absence of p53 mutations in the remaining families may result from inadequate current analytical methods to detect functional promoter and intron alterations, or because other novel gene loci may be etiologic candidates. In this study, biologic and genomic approaches were used to examine apoptotic function and gene regulation in a young boy with RMS from a family with familial syndactyly and LFS, in whom no p53 mutation is detected by comprehensive genomic sequencing. Cell cycle analysis of cultured fibroblasts, using propidiumiodite (PI) staining and fluorescence activated cell sorter (FACS) assay, confirmed a p53-independent defect in apoptotic response to radiation-induced DNA damage. The Human U133A Affymetrix microarray system was used to compare gene expression profiles from fibroblasts obtained from this patient to normal control fibroblasts. Expression profiles derived from 22000 genes were analyzed using MAS 5 software (Affymetrix). Over 1000 genes were found to be differentially expressed. Of these, 517 showed very good correlation of expression patterns in replica experiments utilizing a threshold of 1.5-fold, and validated using Q-PCR. PathwayAssist software (Iobion) was employed to create and analyse biological pathways and gene regulation networks of differentially expressed genes. In particular, high expression of IGF2 (8.2-fold) was noted in concordance with previously published reports of elevated expression in RMS, suggesting an important role of this pathway in progression of tumorigenesis in this patient. Aberrant expression of several other genes suggestive of apoptotic pathway failure, implicate these specific pathways in the syndactyly and tumor formation observed in this LFS patient. Evidence for genomic instability was demonstrated by spectral karyotyping (SKY) of early and late passage non-irradiated fibroblasts. In addition to acquisition of numerous non-random numerical and structural chromosomal aberrations with late passage, a balanced translocation (t(1;3)(p21;p25) was revealed. The chromosome 1 breakpoint was refined by chromosome specific M-band anlaysis from p21 to p13/14. Our observations in this informative patient highlight the value of expression microarray analysis in understanding the apoptotic pathway defects associated with Li-Fraumeni syndrome in the absence of germline p53 alterations.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]