Differential Expression of Neuronal Genes Defines Subtypes Of

Imaging, Diagnosis, Prognosis

Differential Expression of Neuronal Genes Defines Subtypes of Disseminated Neuroblastoma with Favorable and Unfavorable Outcome Matthias Fischer,1Andre¤ Oberthuer,1Benedikt Brors,3 Yvonne Kahlert,1Matthias Skowron,1 Harald Voth,1PatrickWarnat,3 Karen Ernestus,1, 2 Barbara Hero,1and Frank Berthold1

Abstract Purpose: Identification of molecular characteristics of spontaneously regressing stage IVS and progressing stage IV neuroblastoma to improve discrimination of patients with metastatic disease following favorable and unfavorable clinical courses. Experimental Design: Serial analysis of gene expression profiles were generated from five stage IVS and three stage IV neuroblastoma. Differential expression of candidate genes was evaluated by real-time quantitative reverse transcription-PCR in 76 pretreatment tumor samples (stage IVS n = 27 and stage IV n = 49). Gene expression-based outcome prediction was determined by Prediction Analysis for Microarrays using 38 tumors as a training set and 38 tumors as a test set. Results: Comparison of serial analysis of gene expression profiles from stage IV and IVS neuroblastoma revealed f500 differentially expressed transcripts. Genes related to neuronal differentiation were observed more frequently in stage IVS tumors as determined by associating transcripts to Gene Ontology annotations. Forty-one candidate genes were evaluated by quantitative reverse transcription-PCR and18 were confirmed to be differentially expressed (P V 0.001). Classification of patients according to expression patterns of these 18 genes using Prediction Analysis for Microarrays discriminated two subgroups with significantly differing event-free survival (96 F 6% versus 40 F 8% at 3 years; P < 0.0001) and overall survival (100% versus 72 F 7% at 3 years; P = 0.0003). This classifier was the only independent covariate marker in a multivariate analysis considering the variables stage, age, MYCN amplification, and gene signature. Conclusions: Spontaneously regressing and progressing metastatic neuroblastoma differ by specific gene expression patterns, indicating distinct levels of neuronal differentiation and allow- ing for an improved risk estimation of children with disseminated disease.

Neuroblastoma is a malignant embryonal tumor of the regression to fatal tumor progression. These contrasting courses sympathetic nervous system accounting for 7% to 8% of of disease may even occur in patients with disseminated childhood cancers. The biological and clinical behavior of the neuroblastoma: Whereas most metastatic tumors of children tumor is remarkably variable ranging from spontaneous at stage IVare characterized by aggressive growth and many patients succumb to their disease despite intensive treatment, those defined as stage IVS (age V12 months and dissemination restricted to liver, bone marrow, and/or skin) regularly show 1 Authors’ Affiliations: Department of Pediatric Oncology and Hematology and spontaneous regression resulting in an excellent patient’s Center of Molecular Medicine Cologne, University Children’s Hospital; 2Department of Pathology, University Hospital, Cologne, Germany; and outcome (1, 2). Although survival rates of stage IVand IVS 3Department of Theoretical Bioinformatics, German Cancer Research Center, patients differ markedly, the precise delineation of a regressive Heidelberg, Germany phenotype remains challenging, and markers, such as age V12 Received 4/21/06; revised 6/13/06; accepted 6/23/06. months and metastatic pattern, are still a matter of debate (3, 4). Grant support: Deutsche Krebshilfe grant 50-2719, Bundesministerium fu« r Bildung und Forschung through the National Genome Research Network 2 grants However, because treatment strategies of these patients may vary 01GS0456 and 01GR0450, Competence Network Pediatric Oncology and from a ‘‘watch and wait’’ approach to myeloablative mega- Hematology, and Fo« rdergesellschaft Kinderkrebs-Neuroblastom-Forschung e.V. therapy with autologous stem-cell rescue, precise risk group The costs of publication of this article were defrayed in part by the payment of page assignment is critical for therapeutic decisions. charges. This article must therefore be hereby marked advertisement in accordance The molecular mechanisms underlying the process of with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research spontaneous regression are still unknown. Whereas some Online (http://clincancerres.aacrjournals.org/). authors postulated that natural immunologic tumor defense Requests for reprints: Matthias Fischer, Department of Pediatric Oncology and mechanisms could account for this phenomenon (5, 6), others Hematology, University Children’s Hospital, Kerpener Str. 62, 50924 Cologne, suggested that spontaneous involution of the tumor might be Germany. Phone: 49-221-478-6816; Fax: 49-221-478-4689; E-mail: matthias. [email protected]. a result of developmentally regulated programmed cell death F 2006 American Association for Cancer Research. of neuroblasts (1, 7–10). Current models of neuroblastoma doi:10.1158/1078-0432.CCR-06-0985 tumorigenesis propose that there are at least two distinct

Clin Cancer Res 2006;12(17) September 1, 2006 5118 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Gene Expression Patterns of Metastatic Neuroblastoma subtypes of neuroblastoma differing in their tumor cell biology (15). In brief, 5 Ag total RNA was converted into first-strand cDNA in a (9), one with the ability to regress or differentiate spontane- volume of 52.5 AL. PCR amplification was done using standard A A ously and the other showing an aggressive phenotype. These conditions in a volume of 30 L containing 0.4 L of 1:10 diluted first- A Â two subtypes have been shown to differ from one another by strand cDNA, 26.8 Lof1 SYBR Green PCR Master Mix (Applied Biosystems), and 1.4 ALof2.5Amol/L forward and reverse primer each several cytogenetic aberrations, such as MYCN amplification (Eurogentec, Seraing, Belgium). Primer sequences are available from the and deletions of the chromosomal regions 1p, 3p, and 11q authors on request. Serial cDNA dilutions of the neuroblastoma cell line (del1p, del3p, and del11q), all of which are associated with IMR-32 were used for standard curve calculation. Target gene expression poor outcome (1, 2), and more recently by distinctive gene levels were normalized to the geometric mean of transcript levels of the expression signatures with strong prognostic effect (11, 12). control genes SDHA and HPRT1, which have been shown to be However, tumors of stage IVS disease were either absent or consistently expressed in stage IVand IVSneuroblastoma (15), and represented in minor numbers in the latter studies, and efforts calibrated to the minimal expression value within the total set of tumors. of other studies to identify characteristic gene expression Data analysis and statistics. SAGE profiles of stage IVand IVS t patterns of stage IVS tumors in comparison with stage IV neuroblastoma were compared using test statistics, ANOVA, and neuroblastoma failed thus far (13, 14). Fisher’s exact test with Bonferroni correction. Calculations were carried out in R (version 2.1.0, The R Foundation for Statistical Computing; In this study, gene expression patterns of stage IVand IVS http://www.r-project.org). To identify GO categories (http://www. tumors were investigated using serial analysis of gene expres- geneontology.org) overrepresented in the tumor subtypes, the UniGene sion (SAGE) and quantitative reverse transcription-PCR IDs were mapped to the Locus Link/Entrez entries. Associated GO (QPCR) to uncover transcripts related to spontaneous regres- categories were obtained from the Bioconductor (16) library human- sion and progression of disseminated neuroblastoma. Differ- LLmappings (version 1.10.0). Only GO categories in the ‘‘biological entially expressed transcripts were associated to Gene Ontology process’’ section of GO were used. Overrepresentation of GO categories (GO) annotations to identify potential biological processes was tested by using the hypergeometric test implemented in the occurring within the transcriptome of spontaneously regressing Bioconductor package GOstats (version 1.4.0). Distributions of metastatic neuroblastoma. Finally, a supervised class prediction normalized gene expression levels determined by QPCR in stage IV U analysis was done to evaluate the prognostic effect of the and IVS tumors were compared using the Mann-Whitney test. For supervised class prediction analysis, the nearest shrunken centroids identified candidate genes. method [Prediction Analysis for Microarrays (PAM)] was applied (17) after expression data had been transformed by calculating the log ratio Materials and Methods of expression values to the median expression value of each gene. The implementation in the Bioconductor package pamr (version 1.25) was Characteristics of patients and tumor samples. A total of 76 used. To consider all the information from the QPCR data, the PAM 10- pretreatment tumor samples from patients with disseminated neuro- fold cross-validation of the training set was done with a fixed threshold blastoma were obtained from the German tumor tissue bank (stage IV of zero for no shrinkage, thereby estimating the classification accuracy n = 49 and stage IVS n = 27), 8 of which were used for gene expression of the gene signature in the training set. The threshold of zero was also profiling by SAGE (see Table 1). Tumor samples were collected used for class prediction of patients in the test set. Unsupervised cluster from patients registered in the German multicenter neuroblastoma analysis was done using the Genesis Microarray Software Suite version study between 1992 and 2003 and treated according to the German 1.4.0 (http://genome.tugraz.at/Software/GenesisCenter.html). Kaplan- NB90-NB97 trial protocols. Informed consent was obtained before Meier estimates for event-free survival (EFS) and overall survival (OS) analyses of tumor samples. Median age at diagnosis was 1,029 and 118 were calculated and compared by log-rank test. Recurrence, progression, days for patients of stage IVand IVSdisease, respectively, and median and death of disease were considered as events. Cox’s proportional follow-up of patients was 3.3 years. hazards regression model built on EFS was used for multivariate Sample preparation and RNA extraction. Snap-frozen tissue (20-50 analysis. mg) was cryosliced into sections and homogenized by the FastPrep FP120 cell disrupter (Qbiogene, Inc., Carlsbad, CA). Tumor content of Results each sample was assessed by a pathologist (K.E.) and only samples with z60% tumor cells were included. Total RNA was extracted from Identification of genes differentially expressed in stage IVand homogenized samples using the TRIzol reagent (Invitrogen, Karlsruhe, IVS neuroblastoma. Gene expression profiles were generated Germany) according to the supplier’s instructions. Polyadenylate-RNA from five samples of spontaneously regressing stage IVS disease was purified using the MessageMaker messenger isolation system and from three samples of fatal stage IVdisease using SAGE (Invitrogen). Integrity of total RNA samples was examined by RNA Nano Chip assays on the 2100 Bioanalyzer (Agilent Technologies, (18). A total of 213,235 SAGE tags (211,889 tags after Waldbronn, Germany). exclusion of linker tags) were extracted from 7,684 sequence Serial analysis of gene expression. SAGE libraries were constructed files with single profiles varying between 19,601 and 29,407 from 100 to 200 ng polyadenylate-RNA or 20 Ag total RNA using the tags. SAGE data of this study have been deposited in National I-SAGE kit (Invitrogen) following the manufacturer’s protocol. Con- Center for Biotechnology Information Gene Expression Om- catemers were cloned into a plasmid vector, and clones containing nibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible inserts were automatically sequenced (MWG-Biotech, Ebersberg, through Gene Expression Omnibus Series accession no. Germany). Extraction of SAGE tags from sequence files and exclusion GSE4991. SAGE tags that were detected only once in the of duplicate ditags and linker tags was done using the SAGE2000 profiles were excluded from the study, leaving a total of software version 4.12 (http://www.sagenet.org/sage_protocol.htm). 177,992 tags (19,547 unique tags) for statistical analyses. t test Genes corresponding to the tags were identified by comparison with the human UniGene reference database, Build 166 (http:// statistics revealed 429 tags differentially represented in stage IV P www.ncbi.nlm.nih.gov/SAGE). and IVS tumors ( < 0.05), 277 of which were up-regulated Quantitative reverse transcription-PCR. QPCR was done using the and 152 were down-regulated in the latter subtype (Supple- SYBR Green I reagent on the ABI PRISM 7700 Sequence Detection System mentary Table S1). Similarly, 517 and 508 tags with a distinct (Applied Biosystems, Weiterstadt, Germany) as described elsewhere representation were detected by ANOVA and Fisher’s exact test

www.aacrjournals.org 5119 Clin Cancer Res 2006;12(17) September 1, 2006 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Imaging, Diagnosis, Prognosis with Bonferroni correction after pooling profiles of each were related to neuronal differentiation or neuronal functions subgroup, respectively (P < 0.05; data not shown). The largest (e.g., ‘‘positive regulation of neuronal differentiation,’’ ‘‘den- overlap identified by two tests was observed between t test and drite morphogenesis,’’ ‘‘neuron migration,’’ ‘‘neurotransmitter ANOVA (276 tags), whereas it was 143 tags between ANOVA receptor biosynthesis,’’ and ‘‘synaptic vesicle transport’’; Sup- and Fisher’s exact test and 96 tags between t test and Fisher’s plementary Table S2A). In contrast, only two such categories exact test. A total of 65 tags were identified by all three tests. were found among the GO classes of stage IVneuroblastoma Because Fisher’s exact test disregards information about the (‘‘vesicle organization’’ and ‘‘biogenesis and regulation of variance of tag counts within each subgroup but calculates neurotransmitter secretion’’; Supplementary Table S2B), which differences based on averaged counts, and because t test and suggests that these tumors differ from stage IVS by gene ANOVA results largely overlapped, we considered t test results expression patterns, reflecting distinct levels of neuronal for further analysis. differentiation. A less pronounced difference was observed Genes corresponding to tags identified by the t test were regarding GO categories related to programmed cell death or associated to GO annotations to examine whether these growth control, with three of them being overrepresented in transcripts can be categorized into functional classes that may stage IVS tumors (‘‘induction of apoptosis by intracellular provide information about the molecular basis of the distinct signals,’’ ‘‘cell cycle arrest,’’ and ‘‘positive regulation of tumor phenotypes. Fifty-two and 27 GO categories were overrepre- necrosis factor-a biosynthesis,’’ Supplementary Table S2A). The sented in stage IVS and IV tumors, respectively (Supplementary GO class ‘‘apoptosis’’ was overrepresented in stage IVtumors; Table S2). Nine of the 52 GO categories of stage IVS tumors however, subcategories of this class comprise genes with

Table 1. Characteristics of patients and tumors analyzed in this study (n = 76)

Patient Stage PAM MYCN 1p 3p 11q Age Histology Therapy Status EFS OS

NB42 IV F 1het het del 11sp,ud/pd HR No event 67 67 NB51IV F 1ND ND del 12ND HR No event 43 43 NB26 IV F 1 het ND het 12 sp, ud/pd HR No event 14 14 NB59 IV F 1 del ND del 20 sp, ud/pd HR No event 13 13 NB27 IV F 1het het del 48 GNB HR No event 37 37 NB58 IV F 1ND ND ND 66 sp, diff HR No event 79 79 NB60 IV UF 1im het im 12sp, ud/pd HR No event 39 39 NB34 IV UF 1het het im 13sp, ud/pd HR No event 43 43 NB33 IV UF 1ND ND ND 13sp, ud/pd HR No event 9191 NB52 IV UF 1het ND het 14sp, ud/pd HR No event 56 56 NB39 IV UF 1het ND del 16sp, ud/pd 8 CT No event 8181 NB54 IV UF 1het ND del 16sp, diff HR No event 67 67 NB44 IV UF 1het het del 31GNB HR Died of disease 28 51 NB7* IV UF 1het del del 31sp,diff HR Died of disease 1648 NB37 IV UF 1het del del 32 sp, ud/pd HR Relapse/progression 20 37 NB25 IV UF 1del ND del 33 GNB HR No event 37 37 NB61IV UF 1ND ND ND 36 sp, diff HR No event 45 45 NB23 IV UF 1im het het 40 sp, ud/pd HR Died of disease 32 46 NB30 IV UF 1im ND het 41GNB 1CT Died of disease 03 05 NB36 IV UF 1het ND het 47 sp, ud/pd HR Relapse/progression 23 46 NB31IV UF 1ND ND ND 47 GNB HR Died of disease 30 41 NB50 IV UF 1het het het 54 sp, ud/pd HR No event 09 09 NB48 IV UF 1het het del 57 sp, ud/pd HR No event 30 30 NB45 IV UF 1im del im 59 sp, ud/pd HR No event 53 53 NB43 IV UF 1del ND het 61sp,ud/pd HR Died of disease 1821 NB56 IV UF 1 im ND im 61 sp, ud/pd HR Relapse/progression 14 15 NB62 IV UF 1 het ND del 64 sp, ud/pd HR No event 15 15 NB55 IV UF 1het het del 80 sp, ud/pd HR No event 36 36 NB49 IV UF 1het het del 84 sp, ud/pd HR Died of disease 27 48 NB12* IV UF 1 het het het 112 sp, ud/pd HR Died of disease 12 16 NB64 IV UF 1 het del del 145 sp, ud/pd HR Died of disease 16 43 NB53 IV UF 1 het het het 157 GNB HR Relapse/progression 18 44 NB9* IV UF 1 het del het 180 sp, ud/pd HR Died of disease 18 25 NB57 IV UF 1 het ND het 295 sp, ud/pd HR No event 13 13 NB40 IV UF 2 del del del 31sp, diff HR Relapse/progression 18 24 NB66 IV UF 2 del het del 48 sp, diff HR No event 66 66 NB21IV UF 4 ND ND del 32 sp, ud/pd HR Died of disease 1720 NB38 IV UF 15 het ND ND 53 ND HR Died of disease 01 05 NB63 IV UF 20 het ND het 06 sp, ud/pd HR No event 77 77 NB35 IV UF 20 ND ND ND 23 sp, diff HR Died of disease 2128 NB29 IV UF 30 del het het 34 sp, ud/pd HR No event 28 28

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proapoptotic and antiapoptotic functions, which hampers 0.14, respectively). In the combined set of 76 samples, interpretation of this finding. expression levels of all 18 genes were significantly different in Although SAGE results strongly suggested that neuroblasto- stage IVand IVStumors (P V 0.001 for each transcript; Fig. 1). ma of stage IVand IVSdisease harbor distinct gene expression Functional characteristics of these 18 genes are summarized in patterns, the small sample numbers of this analysis might have Table 2. Expression levels of 17 genes were increased in stage generated an overestimation of differentially expressed tran- IVS tumors, whereas one gene (PRAME) was down-regulated in scripts. We therefore evaluated 41 genes with differential this subset as reported previously (19). Taken together, SAGE expression according to SAGE that are related to functional and QPCR analyses showed different gene expression patterns categories of neuronal differentiation and/or apoptosis or in stage IVS and IV neuroblastoma that indicate distinct levels growth control using QPCR in a cohort of 38 neuroblastoma of neuronal differentiation of the tumor cells. (stage IV n = 19 and stage IVS n = 19; Supplementary Table S3). Definition of neuroblastoma subtypes according to gene In this set, 18 (44%) genes showed significantly distinct expression signatures. Given the fact that each single gene transcript abundances between the two subtypes (P < 0.05). delineated in Fig. 1 was differentially expressed with strong Expression levels of these 18 genes were then evaluated in an statistical significance between stage IVS and IV neuroblastoma independent series of 38 tumors (stage IV n = 30 and stage IVS that usually correspond to regressing and progressing pheno- n = 8). Differential expression was confirmed for 16 of 18 genes types, respectively, we asked whether the combined analysis of in this second set (P < 0.05), and a tendency was observed for these transcripts might be able to accurately predict the the two remaining genes CADPS and PRAME (P = 0.10 and biological behavior of the tumors and consequently the clinical

Table 1. Characteristics of patients and tumors analyzed in this study (n = 76) (Cont’d)

Patient Stage PAM MYCN 1p 3p 11q Age Histology Therapy Status EFS OS

NB32 IV UF 32 del del del 63 sp, ud/pd HR Died of disease 07 07 NB65 IV UF 40 del del het 22 sp, ud/pd HR No event 123 123 NB46 IV UF 64 del ND del 19 sp, ud/pd HR Relapse/progression 31 31 NB28 IV UF 64 del ND ND 32 sp, ud/pd HR Relapse/progression 39 40 NB41 IV UF 100 del het het 04 sp, ud/pd HR Died of disease 11 13 NB47 IV UF 100 ND ND ND 41 sp, ud/pd HR Died of disease 01 01 NB22 IV UF 120 im het het 19 sp, ud/pd HR Died of disease 10 13 NB24 IV UF 150 del het het 21 sp, ud/pd HR Died of disease 28 39 NB11* IVS F 1 het het im 00 sp, ud/pd Ob No event 37 37 NB5* IVS F 1ND ND het 00 sp, ud/pd Ob No event 56 56 NB81IVS F 1het het het 00 sp, ud/pd Ob No event 34 34 NB71IVS F 1het het het 00 ND Ob No event 45 45 NB76 IVS F 1het het het 01sp,ud/pd Ob No event 24 24 NB10* IVS F 1 het het het 02 sp, ud/pd Ob No event 44 44 NB2* IVS F 1het het het 02 sp, ud/pd Ob No event 4141 NB73 IVS F 1het het het 02 sp, ud/pd Ob No event 35 35 NB88 IVS F 1het het het 03 sp, ud/pd 1CT No event 59 59 NB77 IVS F 1het ND ND 03 sp, ud/pd Ob No event 75 75 NB83 IVS F 1het het het 04 sp, ud/pd Ob No event 37 37 NB86 IVS F 1 het het het 04 sp, ud/pd Ob No event 19 19 NB89 IVS F 1ND ND ND 04 ND Ob No event 48 48 NB75 IVS F 1 het ND het 04 sp, ud/pd Ob No event 13 13 NB85 IVS F 1het het het 05 sp, ud/pd Ob No event 28 28 NB84 IVS F 1het het het 05 sp, ud/pd Ob No event 47 47 NB74 IVS F 1het het het 06 sp, ud/pd 4 CT No event 05 05 NB78 IVS F 1 het het het 06 sp, ud/pd Ob No event 102 102 NB79 IVS F 1 ND ND ND 07 sp, ud/pd Ob No event 16 16 NB82 IVS F 1het het het 07 sp, ud/pd Ob Relapse/progression 1056 NB1* IVS F 1 het het het 08 sp, ud/pd Ob No event 61 61 NB92 IVS F 1 het ND het 10 sp, ud/pd Ob No event 19 19 NB90 IVS F 5 ND ND het 03 sp, ud/pd Ob No event 43 43 NB87 IVS UF 1 het het het 00 sp, ud/pd 1 Ob, 2 HR Relapse/progression 10 14 NB80 IVS UF 1het ND ND 04 sp, ud/pd Ob No event 08 08 NB91 IVS UF 1 het het del 05 sp, ud/pd 1. Ob, 2. HR Died of disease 05 14 NB72 IVS UF 25 del het het 07 sp, ud/pd HR No event 57 57

NOTE: Patient’s ID; tumor stage; PAM voting (F, favorable; UF, unfavorable); MYCN copy number; status of chromosomal regions 1p, 3p, and 11q (het, heterogeneous; del, deletion; im, imbalance; ND, not determined); age at diagnosis (mo); histology of the tumor (sp; stroma poor; ud/pd, undifferentiated or poorly differentiated; diff, differentiating; GNB, nodular ganglioneuroblastoma; ND, not determined); therapy received by the patients (HR and Ob, treatment according to the German high-risk and observation protocol, respectively; CT, number of chemotherapy cycles in cases of patients that were not treated according to the respective protocol); current status of disease of the patients; and EFS and OS (mo). *Tumors used for SAGE analysis.

www.aacrjournals.org 5121 Clin Cancer Res 2006;12(17) September 1, 2006 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Imaging, Diagnosis, Prognosis courses of the patients. A supervised class prediction analysis signature (n = 43; 3-year EFS 100% versus 40 F 8%; P = 0.04; was therefore done using the PAM algorithm (17) to test the Fig. 3E; 3-year OS 100% versus 73 F 7%; P = 0.1). Examination prognostic value of these 18 genes. Classification accuracy was of cytogenetic aberrations of these six favorable tumors revealed evaluated in the first set of 38 patients by cross-validation. that four of five patients harbored a del11q, whereas only one Whereas PAM voting for most children was analogous to patient had a del1p and none of them a MYCN amplification. staging of disease, three patients with stage IVand two patients Three children of this subgroup were f1 year old at diagnosis with stage IVS tumors were classified into the opposite category (321, 359, and 371 days), but the remaining three patients were (Fig. 2A). PAM prediction was then used to classify tumors of substantially older (619, 1,468, and 2,003 days). Similarly, the second series of patients (n = 38) based on expression levels infants with stage IVS disease and favorable PAM voting of the selected 18 genes. Again, three stage IVand two stage IVS (n = 23) showed a superior EFS (96 F 4% at 3 years) compared tumors were categorized into the opposite subgroup (Fig. 2B). with those with an unfavorable gene signature (n = 4; 3-year Hierarchical clustering of the patients using these 18 genes EFS 38 F 29%; P = 0.002; Fig. 3F). The latter subgroup visualized comparable gene expression patterns of tumors comprised two stage IVS patients that experienced progression showing a similar clinical behavior (Fig. 2C). Kaplan-Meier into stage IVneuroblastoma, one of them with fatal outcome. estimates for EFS and OS of patients with a favorable (n = 29) One of these two patients (NB87) lacked any of the prognostic and an unfavorable PAM voting (n = 47) differed with a higher cytogenetic aberrations MYCN amplification, del1p, del3p, and level of significance (3-year EFS 96 F 6% versus 40 F 8%; del11q, whereas the other one had a del11q (NB91; Table 1). P < 0.0001; 3-year OS 100% versus 72 F 7%; P = 0.0003; Fig. 3) Of the remaining two patients, one (NB72) harbored a MYCN than those of subgroups defined by stage (3-year EFS stage IVS 88 amplification and a del1p and is currently in complete remis- F 6% versus stage IV46 F 8%; P = 0.0027; 3-year OS 96 F 4% sion after intensive treatment according to the German high- versus 76 F 7%, respectively; P = 0.0044; Fig. 3A and B). risk protocol, whereas the other patient (NB80) lacked Outcome prediction by PAM was then evaluated in clinical cytogenetic aberrations and is currently in complete remission subgroups defined by age at diagnosis and stage of disease. without cytotoxic treatment. Within the group of stage IVS Infants <1 year old with an unfavorable signature (n =6) patients with a favorable signature, one child had relapsed from displayed a significantly poorer outcome than those with a favor- disease but reached complete remission without chemotherapy able signature (n = 25; 3-year EFS 42 F 22% versus 96 F 4%; thereafter (3-year OS of this group is 100%). P = 0.0008; 3-year OS 60 F 22% versus 100%; P = 0.001; Finally, the prognostic variables gene signature, stage, age, Fig. 3C and D). Patients with stage IVdisease and a favorable and MYCN status were evaluated in a multivariate Cox gene signature (n = 6) had a significantly better EFS and tended regression model built on EFS for the whole set of 76 patients. to have a better OS than stage IVpatients with an unfavorable Outcome prediction by the gene signature turned out to be a

Fig. 1. Relative expression levels of 18 transcripts in stage IV (4)andIVS(4S) neuroblastoma as determined by QPCR. Transcript abundances of each gene were calibrated to the minimal expression value of the total tumor set. Boxes, median expression values and 25% and 75% quartiles; bars, SD; circles and asterisks, outlying and extreme values, respectively.

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Table 2. Genes differentially expressed in stage IVS and IV neuroblastoma as determined by QPCR

Symbol Gene name Chromosome Category Function Ref. A2BP1 Ataxin-2-binding protein 1 16p13.3 GC/ND Expression in mature neural tissues (31) Binding partner ataxin-2 sensitizes (32) neuroblastoma cells for apoptosis CHRNA7 Cholinergic receptor, nicotinic, 15q14 GC/ND Expression in differentiating neuronal (33, 34) a polypeptide 7 cells and mature neural tissues Involved in synaptic functions or transmitter (35) release Induction of apoptotic cell death (36) CNR1Cannabinoid receptor 1(brain) 6q14-q15 GC/ND Expression in differentiating neuronal (37, 38) cells and mature neural tissues Induced or up-regulated on retinoid acid (39) triggered cellular differentiation Induction of apoptotic cell death (40, 41) Inhibition of proliferation and induction of cell (41) cycle arrest DST Dystonin 6p12-p11 GC/ND Expression in differentiating neuronal cells (42, 43) Loss of expression in malignant cells (44, 45) IGSF4 Immunoglobulin superfamily, 11q23.2 GC/ND Expression in mature neural tissues; contribution (46) member 4 to specific processes of neuronal differentiation; involved in synaptic functions Induction of apoptotic cells death; inhibition (47) of proliferation Decrease or loss of expression in malignant cells; (48) tumor growth suppression in nude mice MEIS1Myeloid ecotropic viral integration 2p14-p13 GC/ND Expression in differentiating neuronal cells and (49) site 1homologue (mouse) mature neural tissues; contribution to specific processes of neuronal differentiation Induced or up-regulated on retinoid acid (39, 50) triggered cellular differentiation Induction of apoptotic cell death (51) ROBO1Roundabout, axon guidance receptor, 3p12 GC/ND Expression in differentiating neuronal cells and (52, 53) homologue 1(Drosophila) mature neural tissues Essential for axon path-finding and navigation of (54) migrating neurons in neural development Heterozygous mice with a targeted disruption of (55) ROBO1are prone to develop neoplasias with loss of ROBO1expression and methylation of its promoter region

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superior, independent prognostic marker in this series of because MYCN amplification is observed in only a minor patients (P < 0.001; hazard ratio, 21.7; 95% confidence fraction (f30-35%; refs. 1, 9) of unfavorable neuroblastoma interval, 3-160). and results in distinctive effects on the tumor’s transcriptome on its own (20, 21), which may mask general mRNA alterations Discussion of aggressive neuroblastoma. Comparison of the SAGE profiles of stage IVand IVStumors using various statistical tests revealed Disseminated neuroblastoma exhibits a remarkable hetero- f500 differentially expressed genes. Because tumor numbers geneity in its clinical and biological behavior. The contrasting analyzed by SAGE were small and thus might have resulted in phenotypes have been suggested to be due to distinct genetic an overestimation of transcriptomic differences, 41 genes that programs of the tumor cells, which are supposed to be were supposed to be differentially expressed were selected and mirrored by characteristic differences on the transcriptome expression levels were determined by QPCR in a series of 76 level (1, 7–10). Thus, identification of genes differentially tumors of stage IVand IVSdisease. Eighteen genes showed expressed in stage IVand IVStumors may contribute to the significantly diverging transcript abundances, suggesting that elucidation of the molecular mechanisms of spontaneous f50% of the f500 transcripts identified by various statistical regression and provide novel prognostic markers that are able tests are differentially expressed in these two subtypes. This to accurately predict outcome of patients suffering from finding is in contrast to results from previous microarray metastatic neuroblastoma. analyses (13, 14) that failed to reliably discover discriminating In the present study, gene expression profiles of untreated gene expression patterns of stage IVS and IV neuroblastoma, neuroblastoma of stage IVand IVSdisease that followed which may in part result from intrinsic methodologic differ- characteristic clinical courses were generated by SAGE. MYCN- ences of SAGE and microarrays (22). However, it seems more amplified tumors were excluded from the SAGE analysis, likely that the discrepant findings are mainly due to the fact that

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Table 2. Genes differentially expressed in stage IVS and IV neuroblastoma as determined by QPCR (Cont’d)

Symbol Gene name Chromosome Category Function Ref. TFAP2B Transcription factor AP-2h 6p21-p12 GC/ND Expression in differentiating neuronal cells (56) (activating enhancer-binding protein 2h) Up-regulated in a reverse transformation gene (57) signature accompanying inhibition of cancer cell growth and tumor regression in animal models DMC1Down-regulated in multiple cancer 117q25.1 GC Loss of expression in malignant cells (58) PCBP4 Poly(rC)-binding protein 4 3p21GC Induction of apoptotic cell death; inhibition of (59) proliferation and induction of cell cycle arrest Loss of expression in malignant cells (60) RBM5 RNA-binding motif protein 5 3p21.3 GC Induction of apoptotic cell death; inhibition of (61) proliferation and induction of cell cycle arrest Decreased expression in malignant cells; tumor (62) growth suppression in nude mice Scotin Scotin 3p21.31 GC Induction of apoptotic cell death (63) CADPS Ca2+ -dependent secretion activator 3p14.2 ND Expression in mature neural tissues (64) Involved in transmitter release (65) CLSTN3 Calsyntenin 3 12p13.31 ND Expression in mature neural tissues; involved (66) in synaptic functions MAP7 Microtubule-associated protein 7 6q23.3 ND Expression in differentiating neuronal cells (67) Induced or up-regulated on retinoid acid (68) triggered cellular differentiation SNAP91Synaptosomal-associated protein, 6q14.2 ND Expression in differentiating neuronal cells and (69, 70) 91-kDa homologue (mouse) mature neural tissues Involved in synaptic functions (71, 72) SYN3 Synapsin III 22q12.3 ND Expression in differentiating neuronal cells (73, 74) Contribution to specific processes of neuronal (73, 75) differentiation Involved in synaptic functions and transmitter (75) release Induced or up-regulated on retinoid (76) acid – triggered cellular differentiation PRAME Preferentially expressed antigen in 22q11.22 — Cancer-testis-antigen (77) melanoma High expression associated with poor outcome (19) of neuroblastoma patients

NOTE: Gene symbols and names, chromosomal localization of each gene, functional category (GC, growth control; ND, neuronal differentiation), a short summary of each gene’s function described thus far, and corresponding references.

previous studies either analyzed small sample numbers of stage that dedifferentiation occurs in neoplastic cells of the latter IVS and IV tumors (n = 9 each; ref. 14) or used a microarray subtype going along with their malignant transformation. that covers only a minor fraction (4,608 cDNAs) of the human Alternatively, these two subtypes may originate from precursor transcriptome (13), whereas in the present study a combined cells of distinct developmental stages. Similarly, a less approach of genome-wide expression profiling followed by an differentiated molecular phenotype of stage IVneuroblastoma independent evaluation of single candidate transcripts in a has been observed in comparison with favorable tumors of large set of tumors was applied. stage I disease recently (10, 23). However, this result might Association of transcripts identified by t test statistics to GO have been expected, because localized neuroblastoma of stage I annotations revealed a predominance of genes related to or II often present a more differentiated histologic phenotype neuronal differentiation and neuronal functions in stage IVS (24, 25), and development into mature ganglioneuroma has compared with stage IVtumors, and differential expression been described to be one of the potential clinical courses in levels of candidate genes were confirmed by QPCR analysis. these stages of disease (26). In contrast, the majority of both Thirteen of the 17 transcripts found to be up-regulated in stage tumor cells of stage IVS and IV disease exhibit a histologically IVS neuroblastoma by QPCR have been shown to contribute to poorly differentiated stage of development (24, 25), which specific processes of neuronal differentiation, to be expressed at applied for the set of neuroblastoma of the present study as well specific stages of neuronal development, to represent markers (Table 1). Moreover, neuroblastoma of stage IVS disease rarely of mature neural tissues, or to be involved in synaptic functions differentiate into benign ganglioneuroma but regress complete- or transmitter release (Table 2). These findings may either ly in the majority of cases (27, 28). High expression levels of indicate that processes of neuronal differentiation are ongoing genes that are induced by the differentiating agent retinoic acid in cells of stage IVS tumors leading to a more differentiated in stage IVS tumors (SYN3, MAP7, CNR1, and MEIS1; Table 2) molecular phenotype compared with cells of stage IVtumors or may further support the notion that molecular development

Clin Cancer Res 2006;12(17) September 1, 2006 5124 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Gene Expression Patterns of Metastatic Neuroblastoma has been proceeded in cells of this subtype compared with The classification accuracy of clinical outcome by this gene those of unfavorable metastatic neuroblastoma. In addition, 12 signature was examined using the PAM algorithm in a training of the genes up-regulated in stage IVS tumors have been and test set of 38 tumors each. Analysis of EFS and OS of suggested to be involved in cell growth control or to represent patients with a favorable and an unfavorable PAM prediction tumor suppressors, and 5 of these are located at chromosomal showed that the gene signature reliably distinguished patients regions 3p and 11q that are frequently deleted in unfavorable with beneficial and adverse outcome (Fig. 3). As expected, neuroblastoma (PCBP4, RBM5, ROBO1, IGSF4, and scotin; classifications by PAM and by stage (stage IVversus IVS)were Table 2). This observation may indicate that several mecha- highly concordant but with some remarkable exceptions. Four nisms of growth control are disrupted in stage IVtumors and stage IVS tumors were classified into the unfavorable subset may add evidence to the hypothesis that spontaneous (Figs. 2 and 3F), two of which were characterized by prog- regression in neuroblastoma is due to a delayed activation of ression into stage IVdisease and another by a MYCN developmentally regulated programmed cell death (1, 9, 10). It amplification. A second stage IVS tumor with a MYCN copy remains to be determined, however, whether down-regulation number of five revealed a favorable gene expression pattern. of the genes described here is causally involved in the However, the prognostic influence of a few additional MYCN malignant phenotype of aggressive neuroblastoma. copies has been reported to be questionable (29, 30); indeed,

Fig. 2. Cross-validation probability plot of the first set of tumors (stage IV n = 19 and stage IVS n =19;A)and prediction probability plot of the second set of tumors (stage IV n = 30 and stage IVS n =8;B)usingPAM. P < 0.5 was counted as voting for the opposite tumor class. As a fixed threshold of zero for the PAM algorithm was set, the cross-validation results can be compared with a supervised tumor class prediction. Blue dots, stage IV patients; orange dots, stage IVS patients. Top, patients’outcome (red, event; green, no event) and PAM voting (red, unfavorable; green, favorable). C, hierarchical cluster analysis of the total set of tumors according to the 18 signature genes (variables: Manhattan distance and average linkage). Red, high expression; blue, low expression. Top, patients’ stage (red, stage IV; green, stage IVS), outcome (red, event; green, no event), and PAM voting (red, unfavorable; green, favorable).

www.aacrjournals.org 5125 Clin Cancer Res 2006;12(17) September 1, 2006 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Imaging, Diagnosis, Prognosis this patient reached complete remission and is free of events occur in some stage IVtumors and probably even in patients since 3.5 years without any cytotoxic treatment. On the other >2 years old. Although this hypothesis has to be verified hand, six children with stage IVneuroblastoma were catego- carefully in prospective studies, confirmation of this finding rized into the favorable subgroup. Although three of them were could result in a substantial reduction of treatment intensity for substantially older than 1 year at diagnosis and most tumors these patients. One has to consider, however, that all stage IV harbored cytogenetic aberrations of chromosome 11q, this patients classified into the favorable subgroup by PAM voting subgroup was characterized by exceptionally benign clinical had received cytotoxic treatment, which might have influenced courses (Fig. 3E). The strong association of favorable outcome the outcome of these children. Finally, the prognostic value and gene expression patterns resembling that of regressing stage of the gene signature was evaluated in a multivariate analysis, IVS tumors indicates that spontaneous involution may also including age, stage, and MYCN status. In this set of tumors,

Fig. 3. Kaplan-Meier curves for EFS and OS of neuroblastoma patients with favorable (F) and unfavorable (UF)gene expression signatures. EFS (A)andOS(B) of patients classified according to favorable or unfavorable PAM voting (gray)in comparison with patients classified according to stage IV or IVS (black). EFS (C)andOS(D)ofinfants(V12 months) with favorable and unfavorable gene expression signatures. EFS of stage IV (E) and IVS (F) patients with favorable and unfavorable gene expression signatures.

Clin Cancer Res 2006;12(17) September 1, 2006 5126 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Gene Expression Patterns of Metastatic Neuroblastoma the PAM classifier turned out to be a superior, independent these two subtypes. In addition, we provide strong evidence predictor of patient’s outcome, thereby supporting findings of that gene expression signatures are suitable to distinguish recent reports, suggesting that gene expression signatures may spontaneously regressing and progressing tumors at the time of be highly accurate prognostic markers for neuroblastoma diagnosis and thus may contribute to an improved risk patients (11, 12). estimation of patients with disseminated neuroblastoma. In conclusion, this is the first study that clearly shows that stage IVS and IV neuroblastoma differ by specific gene Acknowledgments expression patterns, and classification of differentially expressed transcripts indicate distinct levels of neuronal differentiation of We thank Julia Ollenschla« ger and Caroline Kallwass for technical support.

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Matthias Fischer, André Oberthuer, Benedikt Brors, et al.

Clin Cancer Res 2006;12:5118-5128.

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