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

Department of Pediatrics and Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Clinical Cancer Research (Impact Factor: 8.72). 11/2008; 14(21):7020-7. DOI: 10.1158/1078-0432.CCR-08-0541
Source: PubMed


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.
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.
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 PHOX2B in 116 marrows sampled after two treatment cycles was highly prognostic of progression-free and overall survival (P<0.001).
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.

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    • "Novel techniques in marker discovery, such as genome-wide gene expression array analyses, have been successfully applied to NB, leading to identification of useful markers for minimal residual disease diagnosis (124) and prognostic markers (125, 126). Using the same strategy, we studied the transcriptome profiles of malignant neuroblasts established from the human MYCN-amplified IGR-N-91 model (127). "
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    ABSTRACT: Neuroblastoma (NB), accounting for 10% of childhood cancers, exhibits aberrant cell-surface glycosylation patterns. There is evidence that changes in glycolipids and protein glycosylation pathways are associated to NB biological behavior. Polysialic acid (PSA) interferes with cellular adhesion, and correlates with NB progression and poor prognosis, as well as the expression of sialyltransferase STX, the key enzyme responsible for PSA synthesis. Galectin-1 and gangliosides, overexpressed and actively shedded by tumor cells, can modulate normal cells present in the tumor microenvironment, favoring angiogenesis and immunological escape. Different glycosyltransferases are emerging as tumor markers and potential molecular targets. Immunotherapy targeting disialoganglioside GD2 rises as an important treatment option. One anti-GD2 antibody (ch14.18), combined with IL-2 and GM-CSF, significantly improves survival for high-risk NB patients. This review summarizes our current knowledge on NB glycobiology, highlighting the molecular basis by which carbohydrates and protein-carbohydrate interactions impact on biological behavior and patient clinical outcome.
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    • "PHOX2B mutations are often found in association with other neurocristopathies such as Congenital Central Hypoventilation Syndrome (CCHS) and Hirschsprung disease (HSCR), likely modifying susceptibility to NB in the corresponding patients [1], [2], [10], [11], [13]. Moreover, the involvement of PHOX2B and its paralogue PHOX2A in NB pathogenesis seems to be also mediated by a mechanism of gene up-regulation [14], with abundance of PHOX2B transcript shown to be highly prognostic of poorer progression-free and overall survival [15], [16]. Little is known about physiological regulation of the PHOX2 genes transcription, except that PHOX2B expression depends on an auto-regulatory mechanisms in NB cells [17] and regulates transcription of PHOX2A [6]. "
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    ABSTRACT: Neuroblastoma (NB) is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) and the transcription factor Paired-like Homeobox 2B (PHOX2B), in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis. Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target. These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.
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    • "For both assays the evaluation of samples with several antibodies or for multiple mRNA species may increase the specificity of MD and MRD detection in heterogeneous samples. Marker discovery based on differential gene expression profiling, stringent sensitivity and specificity assays, and well-annotated patient samples can rapidly prioritise and identify novel MRD markers of NB (Cheung et al, 2008). Interestingly a combination of techniques or targets might provide not only prognostication about the clinical significance of MD but could also provide additional biologically relevant information. "
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