Tumor tissue heterogeneity is a well known feature of several solid tumors. Neuroblastic Tumors (NTs) is a group of paediatric cancers with a great tissue heterogeneity. Most of NTs are composed of undifferentiated, poorly differentiated or differentiating neuroblastic (Nb) cells with very few or absent Schwannian stromal (SS) cells: these tumors are grouped as Neuroblastoma (Schwannian stroma-poor). The remaining NTs are composed of abundant SS cells and classified as Ganglioneuroblastoma (Schwannian stroma-rich) intermixed or nodular and Ganglioneuroma. The importance to understand Nb and SS gene signatures in NTs, is to clarify the complex network mechanism of tumor growth and progression. In order to identify the Nb and SS cells gene signatures, we analyzed the gene expression profiling of 19 cases of neuroblastic tumors: 10 stroma poor (NTs-SP) and 9 stroma rich (NTs-SR), by high density oligonucleotide microarrays. Moreover, the analysis was performed in parallel on both whole and laser microdissected tumor samples: from 4 of 19 cases, was isolated different areas all composed of pure cellular populations.
We performed genome wide expression analysis by using Affymetrix technology and we used two different approaches for data analysis: SAM (Significance Analysis of Microarrays) and a method based on Game Theory (GT), to identify genes differently expressed in SS and Nb cells. Differently from the SAM method, the analysis based on GT (Moretti et al. (2006)) gives the advantage of selecting relevant genes not only according to the expression profile of each single gene, but considering also gene interaction. Keywords: Gene signature of Neuroblastic and Schwannian stromal cells.
Overall design
Tissue samples from 10 primary neuroblastoma stroma-poor (NTs-SP) and 9 ganglioneuroblastoma/ganglioneuroma stroma-rich (NTs-SR) tumors, were obtained at diagnosis from patients at different clinical stage. Frozen samples of NTs-SP had a minimum content of 90% of malignant cells, while NTs-SR had more than 80-90% of schwannian stromal cells. Four out of 19 NTs: 2 neuroblastomas (Schwannian stoma-poor) and 2 ganglioneublastomas (Schwannian stroma-rich), were further analyzed with laser microdissection.
ATP-binding cassette, sub-family A (ABC1), member 8
transport
NM_007168
NT-SR > NT-SP
20
CXCL14
chemokine (C-X-C motif) ligand 14
chemotaxis, inflammatory response, signal transduction, cell-cell signaling, immune response
NM_004887
NT-SR > NT-SP
Total number of rows: 50
Table truncated, full table size 7 Kbytes.
Supplementary Table 3. Validation of microarray data by RT-PCR. Comparison between Affymetrix fold-change and Real-Time PCR fold-change for single gene header descriptions header descriptions
Gene Name
Affymetrix
Affymetrix fold-change. Fold-changes are calculated dividing the mean expression value in NTs-SP by the mean expression value in NTs-SR. In our analysis, fold-change < 0,625 means down-regulated; fold-change > 1.6 means up-regulated genes.
Real-Time PCR
Real-Time PCR fold-change. Fold-change comments same as above.
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