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Journal of Clinical Oncology, Vol 22, No 20 (October 15), 2004: pp. 4127-4134 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.02.032 GABAergic System Gene Expression Predicts Clinical Outcome in Patients With NeuroblastomaFrom the Center for Biomarker Discovery, Division of Pediatric Hematology/Oncology, Department of Pediatrics, Division of Biostatistics, Department of Public Health and Preventive Medicine, Oregon Health & Science University, Portland, OR; Children's Center for Cancer and Blood Diseases, Children's Hospital, Los Angeles; and Children's Cancer Group, Arcadia, CA Address reprint requests to Srinivasa R. Nagalla, MD, Department of Pediatrics, NRC-5, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239; e-mail: nagallas{at}ohsu.edu
PURPOSE: Neuroblastoma (NB) is a common childhood malignancy characterized by heterogeneous clinical behavior. The purpose of this study was to identify potential NB biomarkers that may improve outcome prediction. PATIENTS AND METHODS: The suppression subtractive hybridization (SSH) technique was used to identify the genes differentially expressed between NB and control tissue. RNA isolated from 235 primary NB tumor samples obtained from the Children's Cancer Group was evaluated for expression of the candidate markers using quantitative reverse transcriptase polymerase chain reaction (Taqman assays). The association between the mRNA expression levels in the identified candidate genes and clinical outcome was evaluated.
RESULTS: SSH analysis identified differential expression of members of the GABAergic gene family in NB. Lower levels of gamma-aminobutyric acid (GABA) receptor–associated protein (GABARAP) gene expression predict decreased survival among all patients. GABAA CONCLUSION: Dysregulation of the GABAergic system may constitute a fundamental event in the development of NB, and assessment of GABAergic system gene expression could provide improved patient stratification and potential new therapies.
Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for nearly 10% of all childhood cancers.1 It has highly variable biologic properties, ranging from spontaneous regression without treatment to aggressive, therapy-resistant disease. Although highly curable when diagnosed at an early stage, the majority of patients will present with stage IV, metastatic disease. The survival for these patients remains less than 40%, despite aggressive therapy including stem-cell transplantation.2,3 At present, patients are classified into risk categories on the basis of a combination of clinical and biologic markers. These include age at diagnosis, stage of disease, histopathology findings, and amplification of the proto-oncogene N-myc (MYCN).4-7 Additional markers, including loss of heterozygosity at chromosome 1p36,8 neurotrophic factor receptors TrkA and TrkB expression levels,9,10 and telomerase expression,11 have also been reported. To date, MYCN amplification remains the single most important prognostic factor in NB. Patients with MYCN amplification (> 10 gene copies) have aggressive disease and a poor clinical outcome. However, a subset of patients with MYCN amplification will be cured with aggressive therapy, whereas some without amplification will ultimately prove to have aggressive, incurable disease.2 The underlying biologic mechanisms for this diversity of behavior remain obscure. Therefore, the identification of new, biologically based prognostic markers that predict outcome independently of the current risk stratification markers is a priority. Various genomic approaches have been used to identify differentially expressed genes among different tissue and tumor types, including differential hybridization screening,12 representational difference analysis,13 cDNA and oligonucleotide microarrays,14 serial analysis of gene expression,15 and suppression subtractive hybridization (SSH).16 SSH uses the technique of suppression polymerase chain reaction (PCR) and allows one to identify both high- and low-abundance transcripts in a single step.16 We took advantage of the ability of SSH to provide unbiased whole genome screening to identify potential new prognostic biomarkers in NB. In this study we report the identification of the GABA gene family as a potential new biomarker in NB, and confirmation of its prognostic significance in a large clinical sample set.
SSH Total RNA was isolated from 10 NB samples, two from each of the five Evans stages (I to IV-S) using a CsCl gradient.17 This RNA was pooled to create the reference NB RNA sample for the SSH experiments. Normal adrenal gland total RNA was purchased from Clontech (Palo Alto, CA). Two SSH experiments were performed in forward (NB RNA as tester and adrenal gland as driver) and reverse (adrenal gland as tester and NB as driver) directions to identify mRNA species that were upregulated or downregulated in NB. The Clontech PCR-Select cDNA Subtraction Kit was used according to the manufacturer's protocol: the tester and driver cDNAs were digested with the restriction enzyme Rsa1. The tester cDNA was then divided into two portions and each ligated with a different cDNA adaptor, which resulted in two populations of tester-adaptor cDNA. An excess of driver cDNA was added to each sample of tester cDNA for the first hybridization. The mixed tester-driver samples were heat-denatured and allowed to reanneal. The two primary hybridization samples were then mixed together with new driver cDNA, allowed to hybridize, and then subjected to PCR amplification. The resulting PCR products were subcloned into the pGEM-T vector per the manufacturer's protocol (Promega, Madison, WI) and transformed into Escherichia coli. Approximately 1,000 colonies (500 from each direction of subtraction) were randomly picked and amplified by PCR using universal primers. The resulting PCR products (100 ng/spot) were arrayed onto nylon membranes (Strategene, La Jolla, CA) and ultraviolet cross-linked for additional evaluation by dot blot analysis.
Dot Blot Analysis
Sequence Analysis
Clinical Samples
Quantitative Real-Time Reverse Transcriptase PCR We measured mRNA expression of 21 different GABA system genes, including the gamma-aminobutyric acid (GABA) receptors and their subunits (GABAA  1 to 6, ß1-3,  2 and 3,  ,  and  , GABAB, and GABAC), the GABA metabolizing enzymes GAD1 and GAD2, GABA receptor–associated protein (GABARAP), an expressed sequence tag (EST) N1 (now CGI-100), and MYCN, in each of the 235 NB samples. Two micrograms of total RNA was reverse transcribed for 1 hour at 37°C and 30 minutes at 45°C in a 20-µL reaction. The resulting cDNA was diluted 1:10 and 5 µL was used for quantitative real-time reverse transcriptase (RT) -PCR. Matching primers and probes sets were designed using Primer Express software (PE Applied Biosystems). Full sequences for all primers and probes used are available on the authors' Web site (http://medir.ohsu.edu/ geneview/). Quantitative PCR was run on the ABI Prism 7700 Sequence Detector at 95°C for 15 seconds and 60°C for 1 minute per cycle for a total of 40 cycles according to the manufacturer's protocol (PE Applied Biosystems). 18S ribosomal RNA was used as an internal control housekeeping gene. Relative copy numbers for each of the measured mRNAs were derived using serial-dilution standard curves of the 18s rRNA internal control and compared with the target genes.20 Measured MYCN expression levels were compared with MYCN copy number status previously determined by the CCG reference laboratory. Elevated MYCN expression levels correlated with MYCN amplification status in more than 90% of all samples (data not shown).
Statistical Analysis Univariate analysis. The clinical characteristics evaluated were age at diagnosis, Evans stage, and MYCN amplification status. The international NB pathology classification system (the Shimada system)21 was not included because 58 tumor samples, of which 39 were from stage 4 patients, had missing information. DNA ploidy information was unavailable because the CCG did not collect these data. Evaluation of the association between GABAergic system gene expression and clinical outcomes, both among all patients and within each Evans stage, was performed. Outcomes were measured by both overall and event-free survival (EFS). Given that no substantial differences were seen among overall EFS, EFS was used throughout. Events were defined as death as a result of any cause, disease progression, or relapse. Association with clinical characteristics was assessed by the Kruskal-Wallis test.22 Association with EFS was evaluated using the Kaplan-Meier method and log-rank test.23 Multivariate analysis. Multivariate analysis using a Cox regression model was performed to evaluate any additional contribution of GABAergic system genes, and the GABAA profile (GAP) score, after controlling for Children's Oncology Group (COG) risk classification status.2,24 Principal components analysis. Principal components analysis was used to determine if any GABAA receptor subunits were preferentially coexpressed. Weighted sums of GABAA subunits' expression levels were calculated and two principle components were extracted. Two standardized component scores (mean, 0; standard deviation, 1) were calculated for each sample. Differences in standardized component scores between patients with each of the clinical characteristics were assessed using one-way analysis of variance.
Identification of Differential Expression of the GABAergic System in NB We screened approximately 1,000 clones obtained by SSH using dot blot analysis. Sequence analysis performed on a subset of 200 clones from this initial screening established both the redundancy of this library ( 15%) and sequence identification of genes differentially expressed between NB and normal adrenal gland tissue (Fig 1). Sequence homology search revealed that approximately 40% of the clones identified had known functions, whereas the remaining were novel ESTs. Several of the most abundant transcripts identified were LINE-1 transposons. LINE-1 transposable elements are common transposed sequences that have been associated with other tumors.25-27 In addition, multiple transcripts from the GABAergic gene family, including various GABAA receptor subunits and GABARAP, were detected with high frequency. Several well-known genes associated with neoplasia were also identified and confirmed by the secondary analysis. Among the ESTs with unknown function, CGI-100 (accession number NM_016040; named neurolin 1 by our group) was the most abundant transcript and was overexpressed in NB. Table 2 shows a partial list of the transcripts identified by SSH and sequence analysis.
The consistent finding of GABAergic system transcripts, which had not been associated previously with NB, combined with the knowledge that the GABAergic system plays an important role in nervous system maturation, prompted us to evaluate further the potential of GABAergic gene expression as a new prognostic marker for this disease.
Association Between GABAergic System Gene Expression and Current Clinical Markers
GABAergic System Gene Expression and Overall Outcome
GABAergic Gene Expression and Clinical Outcome by Evans Stage Clinical stage at diagnosis is one of the most important predictors of outcome. We evaluated the ability of GABAergic system gene expression levels to predict outcome after dividing patients according to their Evans stage at diagnosis. There were no statistically significant associations seen for stages I to IV. Within the subset of the stage IV-S patients, GABAA  receptor subunit expression was predictive of outcome. Stage IV-S NB is defined as a primary tumor with metastatic disease limited to the liver, skin, and less than 10% of the bone marrow in a child younger than 1 year of age. Although the majority of these tumors will undergo spontaneous regression, often without any treatment, a subset will behave like typical aggressive, metastatic disease. The underlying biology that accounts for this variety of behavior remains unknown. Because of the rarity of MYCN amplification in this subset of patients, it has not been a useful prognostic marker.28 Only two of the 23 patients with stage IV-S disease in this study had MYCN amplification. Figure 3 shows a Kaplan-Meier plot of survival among the 23 patients with stage IV-S disease and GABAA receptor subunit expression. Patients with detectable GABAA receptor subunit expression had an EFS of 100%, whereas those without expression had a significantly worse prognosis (P = .016). Infants with stage IV-S NB who are younger than 2 months of age at the time of diagnosis have a worse prognosis than older infants.28 In our study, no significant difference in outcome was observed on the basis of GABAA receptor subunit expression levels after these patients were stratified further by age.
Principal Components Analysis and GABAA Profile Score Physiologically, functional GABAA receptors exist as pentamers; therefore, we evaluated whether any of the GABAA receptor subunits were preferentially coexpressed in these tumors. Principal components analysis was performed and identified a cluster of five GABAA receptor subunits ( 2, ß1, ß2, and ß3, and 2) that were preferentially coexpressed, and a weighted score was created. We termed this the GAP score. Expression of more of the individual receptor subunits in this component gives a higher overall GAP score, and a higher GAP score independently predicted a favorable outcome. Elevated expression levels of any particular subunit within this component did not further improve survival once the combined expression had been accounted for, suggesting that expression of all of these specific receptor subunits, rather than the actual expression level of any individual subunit, is the important factor in conferring a survival advantage. A second component identified, consisting of seven different GABAA receptor subunits (1, 3, 6, , , , and ), was not predictive of outcome (data not shown). Figure 4 presents a graph of a Cox regression model showing a poor clinical outcome in those patients with a GAP score less than –1 compared with those with scores greater than –1 after adjusting for MYCN status, stage of disease, and age at diagnosis (P = .006).
Multivariate Analysis The current COG risk classification system assigns NB patients to risk-based therapy regimens on the basis of the combination of the prognostic factors of age, stage, MYCN amplification status, Shimada histopathology classification, and DNA ploidy. We performed Cox proportional hazards regression analyses to assess whether the GABAergic system genes retained their prognostic significance after taking the COG risk classification status into account. In addition to the risk classification status, lack of GABAA 2 receptor subunit expression, a GAP score of less than –1, and decreased GABARAP expression each remained significant predictors of outcome. Table 3 shows the hazard ratios, 95% CIs, and P values of the models selected by the stepwise procedure.
In this study, SSH provided an unbiased isolation of cDNAs preferentially expressed in NB. Through this process, we identified a number of interesting genes, many of which have not been associated previously with this disease. The discovery of Line-1 transposable elements as one of the most abundant and overexpressed transcripts in the NB cDNA pool was surprising. Line-1 elements have been proposed to contribute to oncogenesis through their insertion into important regulatory oncogenes, leading to disruption of the gene and subsequent loss of function.25-27 The high abundance of these transposons in the sequenced clones suggests this may be an important event in NB pathogenesis. In addition, several important cell cycle regulatory genes, including the RB1 and p53 genes, were also identified. Additional studies to determine their roles in NB are needed.
The diverse behavior of NB, which includes spontaneous regression, tumorigenesis along the migration pathway of neuroblasts, and heterogenous clinical outcome, suggests that dysregulation of neuroblast maturation contributes to the pathogenesis of this disease. The specific pathways that are dysregulated are unknown. However, it is likely that interactions between neurotrophic factors, including nerve growth factor and brain-derived neurotrophic factor, and their receptors (TrkA, TrkB, and TrkC)9,10 are at least partially responsible for the clinical diversity seen. The inhibitory neurotransmitter and neurotrophic factor GABA also play an important role in the regulation of normal neural differentiation.29,30 The GABAA receptor is the most abundant of the GABA receptor subtypes (GABAA, GABAB, and GABAC) and is a ligand-gated ion channel formed from combinations of five subunits derived from eight subclasses ( During early nervous system development, GABA exerts a stimulatory effect on neuroblasts through upregulation of nerve growth factor, brain-derived neurotrophic factor, and basic fibroblast growth factor.38-40 As development progresses, however, GABA inhibits additional proliferation.41,42 This functional change appears to be mediated by changes in GABAA receptor subunit configurations.33,43 GABA also stimulates spinal neuroblast migration in vitro.44 This promigratory effect of GABA, although necessary for normal neural development, has been implicated recently in the processes of invasion and metastasis in prostate cancer cell lines through upregulation of matrix metalloproteinases.45
In our study, low expression levels of GABARAP, the GABAA
Stage IV-S disease remains an enigma. Although the majority of these patients have tumors that will undergo spontaneous regression, a significant subset will ultimately prove to have aggressive disease. Currently, MYCN amplification is used to stratify these patients; however, because of the rarity of amplification in this patient subset, it is inadequate. We have shown that low or absent GABAA
Loss of heterozygosity at 1p36.3 is found in Treatment is a critical variable in any prognostic study, and our results may have been effected by the different therapies these patients received. The results involving the stage IV and IV-S patients are particularly at risk. Some stage IV-S patients received supportive care only, whereas others received radiation or chemotherapy. During the course of this study, the treatment for high-risk patients changed significantly with addition of autologous stem-cell transplantation after intensive multimodality induction therapy. This change has been shown to have improved survival in this subset of patients.3 Although treatment changes have occurred, we believe that the important role of GABA as a neurotrophic factor in normal nervous system development argues strongly that the different outcomes seen represent true differences in the underlying tumor biology. Given the strong independent prognostic ability of GABAergic system gene expression seen in this study, a confirmatory prospective trial is warranted. Our findings represent the first association between the GABAergic system and clinical outcome in a human malignancy. The regulatory role that GABA exhibits over both neuroblast migration and other neurotrophic factors during normal neural development suggests that loss of GABA-directed growth inhibition and differentiation in primitive neuroblasts may represent a fundamental event in the pathogenesis of NB. The discovery that a specific set of five GABAA receptor subunits is both coexpressed and predictive of overall outcome supports this concept. Functional studies to define the specific role GABA plays are needed. Given the ubiquitous presence of GABA receptors throughout the central and peripheral nervous systems, exploration of potential associations between the GABAergic system and other nervous system malignancies is also warranted. Finally, many clinically available, well-tolerated medications act via the GABA system. Modulation of GABAergic signaling through the use of these medications represents a potential new therapeutic option in the treatment of NB.
The authors indicated no potential conflicts of interest.
We thank the Children's Cancer Group (CCG) and the Children's Oncology Group (COG) for providing the neuroblastoma (NB) patient samples and outcome data, and the many health professionals who cared for the children entered onto the CCG/COG NB trials.
Supported in part by grants from the National Cancer Institute, The Lematta Foundation, and The Dickinson Foundation to S.R.N. Additional support was received from the Biostatistics & Bioinformatics Shared Resource of the Oregon Health & Science University Cancer Institute (NCI CCSG: 5P30 CA69533-04). Presented in part at the Children's Oncology Group Neuroblastoma Biology Committee meeting, October 2002, St Louis, MO, and the American Association for Cancer Research Annual Meeting, July 12, 2003, Washington, DC. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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45. Azuma H, Inamoto T, Sakamoto T, et al: Gamma-aminobutyric acid as a promoting factor of cancer metastasis: Induction of matrix metalloproteinase production is potentially its underlying mechanism. Cancer Res 63: 8090-8096, 2003 46. Maris JM, Guo C, Blake D, et al: Comprehensive analysis of chromosome 1p deletions in neuroblastoma. Med Pediatr Oncol 36: 32-36, 2001[CrossRef][Medline] 47. Emberger W, Windpassinger C, Petek E, et al: Assignment of the human GABAA receptor delta-subunit gene (GABRD) to chromosome band 1p36.3 distal to marker NIB1364 by radiation hybrid mapping. Cytogenet Cell Genet 89: 281-282, 2000[CrossRef][Medline] Submitted February 4, 2004; accepted August 5, 2004. This article has been cited by other articles:
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
–1 had no detectable expression of at least three of the five subunits identified as part of the GAP.
1 to 
