Exploiting Gene Expression Profiling to Identify Novel Minimal Residual Disease Markers of Neuroblastoma

Purpose: Minimal residual disease (MRD) presents a significant hurdle to curing metastatic neuroblastoma. Biological therapies directed against MRD can improve outcome. Evaluating treatment efficacy requires MRD measurement, which serves as surrogate endpoint. Because of tumor heterogeneity, no single marker will likely be adequate. Genome-wide expression profiling can uncover potential MRD markers differentially expressed in tumors over normal marrow/blood. Experimental Design: Gene expression array was carried out on 48 stage 4 tumors and 9 remission marrows using the Affymetrix U95 gene chip. Thirty-four genes with a tumor-to-marrow expression ratio higher than tyrosine hydroxylase were identified. Quantitative reverse transcription-PCR was done on all 34 genes to study the dynamic range of tumor cell detection and the expression of these genes in normal marrow/blood samples and in stage 4 neuroblastoma tumors. Top ranking markers were then tested for prognostic significance in the marrows of stage 4 patients collected from the same treatment protocol after two cycles of immunotherapy. Results: Based on sensitivity assays, 8 top-ranking markers were identified: CCND1, CRMP1, DDC, GABRB3, ISL1, KIF1A, PHOX2B, and TACC2. They were abundantly expressed in stage IV neuroblastoma tumors (n = 20) and had low to no detection in normal marrow/blood samples (n = 20). Moreover, expression of CCND1, DDC, GABRB3, ISL1, KIF1A, and PHOX213 in 116 marrows sampled after two treatment cycles was highly prognostic of progression-free and overall survival (P < 0.001). Conclusions: Marker discovery based on differential gene expression profiling, stringent sensitivity and specificity assays, and well-annotated patient samples can rapidly prioritize and identify potential MRD markers of neuroblastoma.