Printer Friendly

ppGaINAc-T 13: a new molecular marker of bone marrow involvement in neuroblastoma.

Neuroblastoma (NB) [6] is a common pediatric solid tumor that begins in early childhood and shows extreme clinical, histologic, and genetic heterogeneity. Prognosis is highly variable and is related to tumor staging (localized stage (1-3) vs disseminated stage 4 [stage 4 and 45]) and age at diagnosis for stage 4 disease (<1 year, >1 year). Overall, MYCN [7] amplification is a genetic hallmark of the disease and an independent specific prognostic marker. Favorable NB has been defined as localized stages and stage 4 disease in children <1 year of age (1, 2). High-risk NB, defined as MYCN-amplified tumors and stage 4 tumors in children >1 year of age, is characterized by distant metastases and an aggressive course with a dismal prognosis. So far, numerous studies have been conducted to identify the genes involved in the aggressive forms of this disease but only ~25 genes, including MYCN, have been proven or are likely to be involved in NB tumorigenesis, invasion, and dissemination (2).

Because bone marrow (BM) is the preferential site for NB dissemination, an assessment of BM involvement is required not only for staging NB, but also for evaluating minimal residual disease (MRD) throughout the management of the disease. Although novel treatment strategies such as multimodality treatment with dose-intensive chemotherapy, myeloablative radiotherapy, and 13-cis-retinoic acid therapies have been developed (3), relapse is frequent in the majority of stage 4 NB in children >1 year, giving rise to a systematic investigation of MRD in patients. More than 10 years ago, many qualitative studies showed that tyrosine hydroxylase (TH) mRNA could be used to detect malignant NB both in the blood and BM of children with NB (4). Quantitative analysis seems to have shown that the blood and BM of children with widespread disease at diagnosis (stage 4) express considerably higher TH mRNA values than those of children with locoregional disease (stages 1-3) (5). This is consistent with the fact that prognosis in stages 1-3 is better than in stage 4, and that stage 4 NB children without demonstrable BM involvement have a more favorable prognosis than those with infiltrated marrow (6). The identification of MRD by means of sensitive methods is thus required to pinpoint NB high-risk patients and to find evidence for specific molecular markers of malignancy. In this regard, several new detection assays have been developed and optimized (7), and new markers such as ELAVL4 have recently been proposed (8).

It was with this aim that we studied the human MYCN-amplified NB experimental model derived from a stage 4 disease capable of disseminating to mouse BM, as previously described (9,10). Using oligomicroarray transcriptome analysis, we identified a set of 107 genes that were differentially expressed in the metastatic neuroblasts. The most strongly up-regulated gene of this gene set was GALNTI3, a recently cloned and characterized isoform of the ppGalNAc-T family (11). Our findings showed that GALNTI3 transcript values can be used as an informative marker to monitor MRD in BM in patients with stage 4 NB.

CELL LINES

Briefly, human IGR-N-91 neuroblasts derived from an involved BM collected from a stage 4 disease >1-year-old NB patient were injected subcutaneously into nude mice to give rise to a stage 4 disease. After 2 passages in nude mice, stage 4 disease, BM, and myocardium (Myoc) neuroblasts were collected and cultured in vitro to yield established cell lines, as previously described (9). The SH-SY5Y cell line was purchased from the European Collection of Cell Cultures (ECACC). Cells were maintained as previously described (10).

PATIENT SAMPLES

BM aspirates were obtained from 42 patients with a histologic diagnosis of various stages of neuroblastoma, the majority of which were stage 4 (29 of 42). Written informed consent was obtained from the parents. While the patients were under anesthesia, 1 to 2 mL of BM aspirate samples were collected and pooled in EDTA/ saline buffer from 10 sites, including the sternum and the iliac crest. BM samples were separated by Ficoll centrifugation, washed in RPMI, recovered by centrifugation at 15008 for 30 min, then submitted to a conventional cytologic examination. Thus, samples were classified as involved BM (<30%, 30-60%, or >60% malignant cells) or noninvolved BM (absence of malignant cells). The analysis of the MYCN gene copy number has been performed in patient tumors as previously described (10).

RNA EXTRACTION

Total RNA was extracted from cells and BM with RNAbIe reagent (Eurobio) and purified with the RNeasy System (Qiagen S.A.) in accordance with the manufacturer's recommendations. The quality of total RNA samples was established as having a ratio of 285/185 over 1.7 by an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.).

MICROARRAY EXPERIMENTS

We used 5 [micro]g of total RNA to synthesize cDNA by mouse Maloney murine leukemia virus-reverse transcriptase with a T7-promoter-polyT-primer followed by random hexamers. cRNAs were produced by incubation in a cocktail containing Cy3-dCTP or Cy5-dCTP and T7-RNA polymerase. Labeled samples were hybridized to microarray slides spotted with 60-mer oligonucleotides at 72 [degrees]C for 16 hrs (ref: G4110-17 086 dyes, Agilent). Each cell line from the IGR-N-91 experimental model (stage 4 disease, BM, and Myoc) was analyzed with 8 arrays: 2 replicates and 2 dye-swaps for each of the 2 cell lines, BM and Myoc. Each of these 2 cell lines was compared with stage 4 disease, with dual color Agilent array (one color for BM or Myoc and the other color for stage 4 disease). Slides were scanned by use of an Agilent scanner (resolution, 5 [micro]m). Image analyses were performed with Agilent Feature Extraction Software and then normalized with the Lowess method.

GENE EXPRESSION ANALYSIS

cDNA probes were synthesized with Superscript II (Invitrogen). We verified results of microarray experiments with a real-time quantitative reverse transcription-PC[R.sup.E] (QRT-PCR) with 12.5 ng of cDNAs, 10 pmol of each primer, and 12.5 [micro]L of a master mix containing SYBR[R]-Green in a final volume of 25 [micro]L (Applied Biosystems, Abi Prism 7000 Sequence Detection System). The sample RNA was normalized by the amplification of an endogenous control (185) as previously described (10); the calibrator used was the neuroblastoma SH-SY5Y cell line, and results were quantified by the comparative threshold cycle ([C.sub.T]) method. The oligonucleotide primers (Table 1), were designed with the Oligo 6 Primer Analysis Software (Molecular Biology Insights, Inc.).

ANALYSIS OF MRD IN BM SAMPLES

ppGalNAc-T13 transcripts were quantified by QRT-PCR method using a labeled probe with a minor groove binder (MGB, Applied Biosystems). The cDNAs were prepared as described above and the amplification conditions were as follows: 15 s at 95 [degrees]C, 60 s at 60 [degrees]C, and 60 s at 72 [degrees]C for 40 cycles. These results were quantified with the comparative [C.sub.T] method. An arbitrary threshold was chosen on the basis of the variability of the baseline and was modified manually for each experiment. [C.sub.T] (fractionary cycle at which the PCR curve intercepts the fluorescence threshold value) was assessed as the cycle number at this point. The amount of target gene, normalized to an endogenous house-keeping gene, was then given by [2-.sup.[DELTA][DELTA]CT] where [DELTA][DELTA][C.sub.T] = [DELTA][C.sub.T] (sample)-[DELTA][C.sub.T] (reference), and [DELTA][C.sub.T] was the difference between the [C.sub.T] of the target gene and the [C.sub.T] of the endogenous control gene. The endogenous gene used was 185 and the reference used was the neuroblastoma SH-SY5Y cell line. In the validation of the methods for GALNTI3, samples with no amplification within 40 cycles were considered as zero.

Moreover, we optimized a nested RT-PCR with 1 [micro]g of total RNA for the synthesis of first-strand cDNA in a final volume of 20 [micro]L. The PCR first round was performed with 1 [micro]L of cDNA, 300 nmol/L of each primer, 3 mmol/L Mg[Cl.sub.2], and 1 unit of Taq DNA polymerase (Invitrogen) in a final volume of 25 [micro]L. The amplification conditions were 30 s at 94 [degrees]C, 30 s at 62 [degrees]C, and 60 s at 72 [degrees]C for 30 cycles. The 2nd round was seeded by 1 [micro]L of the first round product in similar conditions except for 1.5 mmol/L Mg[Cl.sub.2] and annealing at 60 [degrees]C for 35 cycles. We analyzed 15 [micro]L of PCR products by electrophoresis on 2% agarose gels by direct visualization after ethidium bromide staining. The sequence of GALNTI3 primers and probe are given in Table 1 for both nested RT-PCR and Q-PCR.

For tyrosine hydroxylase (TH, EC 1.14.16.2), G[D.sub.2] synthase ([beta]1,4-N-acetylgalactosaminyl transferase, EC 2.4.192), and dopa decarboxylase (DDC, EC 4.1.1.28) transcripts, analyses were performed by QRT-PCR with an SDS 7700 instrument, as previously described (5). In short, 1 [micro]L of samples containing 150 ng of RNA extracted from BM samples was diluted with 4 [micro]L Rnase-free water. Then 5 [micro]L of reverse transcriptase was added and, after reaction, the samples were diluted 1:2 to give a total volume of 20 [micro]L for PCR (5). The QRT-PCR step was performed in triplicate with 2.5 [micro]L sample cDNA or 2.5 [micro]L of each calibrator (range [10.sup.0]-[10.sup.5] targets/[micro]L) in each reaction. TH mRNA was analyzed by multiplex QRT-PCR together with G[D.sub.2] mRNA, and DDC was analyzed together with the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT). Predeveloped Assay Reagents (Applied Biosystems) were the sources of primers and probe for HPRT transcript analysis. The sequences of these oligonucleotides were not disclosed by the manufacturer. The analyses of TH, G[D.sub.2] synthase, and DDC were performed with primers (100 nmol/L) and probes (100 nmol/L for TH and DDC and 200 nmol/L for G[D.sub.2] synthase) from Scandinavian Gene Synthesis. The calibrator described earlier (5) was used for TH analysis, and calibrators for G[D.sub.2] synthase and DDC were synthesized similarly. For HRPT, analyzed together with DDC, the response of the DDC calibration curve was used. The primers and probes designed for the analyses as well as for the synthesis of the TH, G[D.sub.2] synthase, and DDC calibrators are given in Table 1. In the PCR step, we added equal volumes (2.5 [micro]L) of sample (cDNA) and calibrator to their respective wells. The calibrator concentration (molecules per microliter) is given for each calibrator point. The calibrator curve was constructed and the concentration of the unknown samples was read from the calibrator curve. Thus, the results were obtained as concentrations (cDNA molecules per microliter) of the solution used for the PCR step. Because the samples were diluted 1:20 before this step, we multiplied the results by 20 to obtain the analyte concentration (number of tran scripts per microliter) in the original sample. Because we had 1 [micro]L from the beginning, the final result equaled the number of transcripts in the 150 ng of starting material. Finally, the results of TH, G[D.sub.2] synthase, and DDC transcripts were normalized to the HPRT results by division.

In the validation of the methods for TH, G[D.sub.2], and DDC, samples with no amplification within 40 cycles were considered as zero. Cord blood samples from 52 newborns and blood samples from 26 children ages 4 months to 16 years were also analyzed for TH mRNA, G[D.sub.2] synthase mRNA, and DDC mRNA, and none of the transcripts were detectable in any sample. We calculated the imprecision as mean (SD) CV of the PCR step with the 3 transcripts measured in blood and BM, according to principles described earlier (5). The results from samples with [C.sub.T]S <40 and with concentrations <100 transcripts/mL were 59 (30) transcripts/mL (51%) for TH, 56 (25) transcripts/mL (44%) for G[D.sub.2] synthase, and 33 (20) transcripts/mL (62%) for DDC respectively. The Q-PCR measurements were applied to the same RNA extract, indicating that the detection limit is similar for the 3 analyses. Therefore, the same cutoff values were used for the 3 transcripts. In the Kaplan-Meier survival studies, we used the data normalized by HPRT.

PRODUCTION OF ANTI-ppGd1NAC-T13 ANTIBODY

A synthetic specific peptide of ppGalNAc-T13 (RSLLPAL RAVISRNQE, accession number BAC54545, Biosynthesis) that had been conjugated by keyhole-limpet hemocyanin was used for rabbit immunization. Antibody titer was determined with the same peptide conjugated to bovine serum albumin. hninunoblots, which have been previously described in detail (10), were probed with ppGalNAc-T13 antibody at a dilution of 1:500 and revealed by Enhanced Chemio Luminescence (Amersham, Pharmacia, Biotech).

STATISTICAL ANALYSIS

Overall survival (defined as the time from diagnosis until the date of death or last follow-up) was used as a follow-up endpoint. Kaplan-Meier survival curves were plotted using Statview Software (SAS Institute Inc). Logrank tests were used to calculate the statistical significance (P-value) of difference between groups.

Results

GENE EXPRESSION PATTERN OF METASTASISASSOCIATED GENES IN AN MYCN-AMPLIFIED NB EXPERIMENTAL MODEL

Our study was designed to pinpoint the genes involved in the metastatic dissemination of MYCN-amplified NB. To this end, we compared the gene expression profiles of metastatic-derived neuroblasts (BM and Myoc) with stage 4 disease profiles from the IGR-N-91 experimental human NB model (9,10) using Agilent long oligonucleotide arrays with 8 arrays (see the section "Microarray Experiments", above). Signals were analyzed with 2 methods. We first used the Resolver[R] system for gene expression analysis (Rosetta Inpharmatics, Inc.), in which the microarray data were processed and combined with a weighted common method (12). By the criteria of a 2-fold change in expression value, we selected 79 genes, with a P-value <0.01 for the combined data. As a second method, we used Significance Analysis of Microarrays (13), in which 82 genes were selected (with 1 sample Significance Analysis of Microarrays test, related to the sample t-test). Moreover, in the second case, we determined the following parameters-[DELTA] = 1.94 and a false discovery rate of 0.79%-to obtain approximately the same number of genes as in the first method. The combination of these 2 sets of genes provided us a list of 107 genes that were found to be considerably differentially expressed in the BM/Myoc metastatic neuroblasts compared with the stage 4 disease neuroblasts (Fig. 1a). Table 2 classifies the 68 up-regulated and 39 down-regulated genes into 9 classes according to their known functions and reports the genes mainly involved in detoxication/ chemoresistance (ABCB1), invasiveness and cell adhesion pathways (ENPP2, EMP2, DCN), and neuronal structure and signaling (PRPH, RELN, SYCP2, MDK). QRT-PCR analyses were performed with 14 gene-specific primers to validate the up- and down-regulation in gene expression for the stage 4 disease and metastatic neuroblasts. Results coincided fully with the up- and down-regulated genes identified by microarray analysis (Fig. 1, B and C).

These data show that GALNTI3 (ppGalNAc-T13) is the most strongly up-regulated gene and confirm that ABCB1, whose higher expression has been previously described (9,10), is the second most strongly up-regulated gene, which highlights the fact that acquired drug resistance is an important cause of neuroblastoma treatment failure. In this model, although established metastatic neuroblasts exhibit a similar MYCN amplification compared with stage 4 disease-derived neuroblasts, a considerably higher MYCN expression is observed that is consistent with their aggressive biological behavior (10). The metastatic neuroblast expression pattern shows a highly aggressive tumorigenic feature defined by a set of genes reflecting alterations in the genes involved in cell adhesion and cell-to-cell interaction. Of particular note among these genes is the increased expression of ENPP, an exoenzyme known to enhance experimental metastasis and angiogenesis, and the down-regulation of many genes (EMP2, DCN, FMOD, LUM, COL1A1) involved in cell-to-cell interaction and the assembly of extracellular matrix. NB is a neuroectodermal tumor derived from primitive cells of the sympathetic nervous system, as assessed by the neuronal feature of metastatic neuroblasts, with the latter being characterized by the up-expression of genes associated with the neural architecture network. The variations in the expression of 17 genes (underlined in Table 2) are involved in specific neuronal pathways, such as neuronal cell structure (GPM6B, PRPH, RELN), neural signaling and development (HOXB2, ELAVLI, EFNB1, NRG2, NELL1, MDK, GFRA1), synaptogenesis and steroid biosynthesis (SYCP2, PCDH17, STX1B2, SCG5, Nstage 4 diseasel, GATA3, NROB1).

The higher differentially up-regulated gene in this set is GALNTI3 (12-fold higher in metastatic neuroblasts compared with primary tumor, P = 9.53 E-44), which encodes for ppGalNAc-T13, a recently identified member of the UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases family, otherwise known as ppGalNAc-Tases, which control the initiation of mucintype O-glycosylation (14). Up to the date of this writing, 15 distinct members have been identified and characterized in humans, all of which share a highly homologous primary sequence, particularly in the predicted catalytic domain. In contrast to ppGalNAc-T1 (84% sequence homology with ppGalNAc-T13), whose expression is distributed throughout the digestive organs, lymphatic organs, and peripheral blood cells, ppGalNAc-T13 is expressed exclusively in the brain, primary cultured neurons, and neuroblastoma cells (11). We found that GALNTI3 gene expression was considerably greater in the BM and Myoc metastatic neuroblasts than in the stage 4 disease cells (P <0.001) as assessed by Q-PCR and nested RT-PCR analyses (Fig. 2, A and B). Western blotting showed a consistent expression of ppGalNAc-T13 in the BM neuroblasts but a lack in the stage 4 disease neuroblasts as well as in metastatic Myoc neuroblasts (Fig. 2C).

DETECTION OF BM DISSEMINATED NEUROBLASTOMA CELLS USING GALNTT3 MRNA

To determine whether GALNTI3 might be a potential marker for NB dissemination, we tested for malignant neuroblasts in BM from patients with various stages of NB by measuring GALNTI3 expression. We compared our data with the conventional cytology data and the data corresponding to other proposed markers for NB MRD, i.e., TH, G[D.sub.2] synthase, and DDC normalized for the housekeeping gene HPRT. We tested BM samples from 42 patients (50 BM samples) whose clinicopathological parameters and overall survival are presented in Table 3. By routine cytological examination, we assessed 23 samples obtained at diagnosis of stage 4 NB as positive (involved BM), and 27 samples (14 samples from stages 1-3 and 45 and 13 samples obtained after chemotherapy) as negative (noninvolved BM). Four BM samples from stage 4 patients obtained only after chemotherapy were determined to be negative (no. 24 to 27).

We calculated a corresponding value for normalized data by dividing the amount of 100 transcript/sample by the mean HPRT transcripts/sample of all the samples. This cutoff was 0.0089. BM samples diagnosed as positive by cytology were confirmed to be involved by mRNA transcripts values measurement in 23 of 23 samples (100% by nested- and QRT-PCR) for GALNTI3, TH, and DDC and in 22 of 23 (96%) patients for GD2 synthase with the 0.0089 cutoff value for the 3 later comparison analyses. In samples with negative cytology, the number of positives was observed in 5 of 27 samples (18%)for GALNTI3 and TH, in 7 of 27 samples (26%) for GD2 synthase, and increased to 6 of 27 (22%)for DDC. Of the 16 patients who died, 14 of 16 (87.5%) showed at least 1 positive marker, and 2 of 16 (12.5%) were negative for all markers (Table 3).

[FIGURE 1 OMITTED]

SURVIVAL ANALYSIS

We compared overall survival of 37 NB patients to their BM involvement as assessed by cytology, or GD2, TH, DDC, and GALNTI3 transcript values. We have analyzed samples collected at diagnosis. According to a KaplanMeier analysis, the best correlation with a poor clinical outcome is observed for GALNTI3 expression (P = 0.043) (Fig. 3).

Discussion

A molecular signature of metastatic potential has been identified recently in many primary tumors, suggesting that malignant primary tumor cells possess a de novo intrinsic metastatic identity (15). An expression profiling method using cDNA microarray was recently defined to predict the prognosis of intermediate-risk NB (16). However, these analyses do not specify the key pathways involved in the metastatic potential of NB. Using an experimental model of BM involvement in NB, we report here, for the first time, the close association between a considerable differential increase in GALNTI3 gene mRNA amounts and the metastatic dissemination of NB disease, and we propose this as a potential new marker for MRD. Indeed, the detection of disseminated tumor cells is essential to clinical oncology: (a) to detect the early occult spread of tumor cells; (b) to assess a relevant risk factor for subsequent metastasis; and (c) to monitor treatment efficacy (17). Genome and transcriptome analyses performed on single micrometastatic cells from different primary tumors have shown that these disseminated tumor cells possess unique gene expression signatures (18, 19).

[FIGURE 2 OMITTED]

Our data show that considerable GALNTI3 transcript values were detected in the involved BM of high-risk patients with stage 4 NB contrasting to the absence of expression of the same gene in the BM of the majority of the study participants with negative cytology. In addition, the GALNTI3 data are in accordance with those from TH, GD2 synthase, and DDC Q-PCR. However, in spite of specific GALNTI3 mRNA detection, the lower limit of the detection of analyses appears less than optimal, and we conclude, therefore, that GALNTI3 expression will be suitable only for detection of malignant neuroblasts at diagnosis or relapse. To be used as a tool for MRD follow-up in clinical practice, this new marker needs to be analyzed in a larger patient cohort.

GALNTI3 is a glycosyltransferase specifically expressed in neuronal tissue (11). Our analysis of GALNTI3 mRNA transcripts in various NB cell lines, as well as in favorable and high-risk neuroblastic tumors, showed that GALNTI3 is highly expressed in malignant neuroblasts but is not correlated with MYCN amplification and/or expression (data not shown). This abnormally high GALNTI3 expression could reflect specific alterations in the O-glycosylation process, as described for ppGalNAcT3 in various adenocarcinomas (20). In fact, recent studies report that aberrant glycosylation patterns are a hallmark of the tumor phenotype and may influence cancer cell behavior (21). Glycans have been shown to regulate tumor progression, including proliferation, invasion, an-Biogenesis, and metastasis processes. ppGalNAc-T13 is a major enzyme responsible for the synthesis of O-glycan, which is specifically able to form a triplet Tn epitope on peptides encoded in syndecan-3 (11), a proteoglycan predominantly expressed in neurons and Schwann cells (22). Syndecans are cell-surface transmembrane heparan sulfate proteoglycans with a variety of functions in the cell. They act, for example, as coreceptors for growth factors and mediators for cell-to-cell and cell-to-matrix adhesion (23). Bearing in mind that GALNTI3 was the most up-regulated gene in the metastatic model as well as the marker best correlated with poor clinical outcome in NB patients, we hypothesize that this enzyme could be an indicator of disseminated neuroblasts in BM. To elucidate the biological role of GALNTI3 in NB, further work is warranted to analyze the molecular mechanisms that regulate this gene expression and to identify the enzyme acceptor substrates potentially involved in metastatic activity of these cells.

[FIGURE 3 OMITTED]

DATA AVAILABILITY

The microarray data related to this paper have been submitted to the Array Express data repository at the European Bioinformatics Institute (http://www.ebi. ac.uk/arrayexpress/) under the accession number E-TABM-44.

The authors thank Dr. Vladimir Lazar and Gwennaelle Le Roux for their skillful assistance in the microarray assay, Dr. Enrique Barrios for statistical analysis, and Dr. Dominique Valteau-Couanet for providing us with clinical data of NB patients. We also thank "Les arms de MarieSophie de St Cloud" for their generous gift. This work was supported by the Ligue contre le Cancer, Comite de Montbeliard, Universite Paris XI (Bonus Quality of Research), the French Ministry of Health (Program Hospitalier de Recherche Clinique 2002 AOM 02 112), and the Swedish Child Cancer Foundation, project number 04/ 027. This study was edited by English Booster.

Received February 1, 2006; accepted June 21, 2006.

Previously published online at DOI: 10.1373/clinchem.2006.067975

References

(1.) Brodeur GM, Maris JM. Neuroblastoma. In : Pizzo PA and Poplack DG, eds. Principles and Practice of Pediatric Oncology. Philadelphia: Lippincott J B Company, 2002:895.

(2.) Maris JM, Matthay K. Molecular biology of neuroblastoma. J Clin Oncol 1999;17:2264-79.

(3.) Matthay KK, Villablanca JG, Seeger RC, Stram D0, Harris RE, Ramsay NK et al. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous BM transplantation, and 13-cis-retinoic acid. N Engl J Med 1999;341:1165-73.

(4.) Burchill SA, Bradbury FM, Smith B, Lewis IJ, Selby P. Neuroblastoma cell detection by reverse transcriptase polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA. Int J Cancer 1994; 57:671-5.

(5.) Treger C, Kogner P, Lindskog M, Ponthan F, Kullman A, Kagedal B. Quantitative analysis of tyrosine hydroxylase mRNA for sensitive detection of neuroblastoma cells in blood and BM. Clin Chem 2003;49:104-12.

(6.) Moss TJ, Reynolds CP, Sather HN, Romansky SG, Hammond GD, Seeger RC. Prognostic value of immunocytologic detection of BM metastases in neuroblastoma. N Engl J Med 1991;324:219-26.

(7.) Swerts K, Ambros PF, Brouzes C, Navarro JM, Gross N, Rampling D et al. Standardization of the immunocytochemical detection of neuroblastoma cells in BM. J Histochem Cytochem 2005;53: 1433-40.

(8.) Swerts K, De Moerloose B, Dhooge C, Vandesompele J, Hoyoux C, Beiske K, et al. Potential application of ELAVL4 real-Time quantitative reverse transcription-PCR for detection of disseminated neuroblastoma cells. Clin Chem 2006;52:438-45.

(9.) Ferrandis E, Da Silva J, Riou G, Benard J. Coactivation of the MDR1 and MYCN in human neuroblastoma during the metastatic process in the nude mouse. Cancer Res 1994;54:2256-61.

(10.) Blanc E, Goldschneider D, Ferrandis E, Barrois M, Le Roux G, Leonce S et al. MYCN enhances P-gp/MDR1 gene expression in the human metastatic neuroblastoma IGR-N-91 model. Am J Pathol 2003;163:321-31.

(11.) Zhang Y, Iwasaki H, Wang H, Kudo T, Kalka TB, Hennet T et al. Characterization of a new human UDP-N-Acetyl-a-D-galactosamine: polypeptide-N-Acetyl galactosaminyl-tranferase, designated pp GaINac-T13, that is specifically expressed in neurons and synthesizes GaINac a-Serine/Threonine antigen. J Biol Chem 2003;278: 573-84.

(12.) Stoughton RS, Dai H. Statistical combining of cell expression profiles, 2002;US Patent No. 6,351,712.

(13.) Tusher VG, Tibshirani R, Chu C. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001;98:5116-21.

(14.) Ten Hagen KG, Fritz TA, Tabak LA. All in the family: the UDPGaINAc:polypeptide N-acetylgalactosaminyltranferases. Glycobiology 2003;13:1-16.

(15.) Ramaswamy S, Ross K, Lander E, Golub T. A molecular signature of metastasis in primary solid tumors. Nature Genetics 2003;33: 49-54.

(16.) Ohira M, Oba S, Nakamura Y, Isogai E, Kaneko S, Nakagawa A et al. Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas. Cancer Cell 2005;7:337-50.

(17.) Braun S, Natme B. Circulating and disseminated tumor cells. J Clin Oncol 2005;23:1623-6.

(18.) Klein CA, Seidl S, Petat-Duffer K, Offner S, Geigl JB, SchmidtKittler 0 et al. Combined transcriptome and genome analysis of single micrometastatic cells. Nat Biotechnol 2002;20:387-92.

(19.) Smirnov DA, Zweitzig DR, Foulk BW, Miller MC, Doyle GV, Pienta KJ et al. Global gene expression profiling of circulating tumor cells. Cancer Res 2005;65:4993-7.

(20.) Nomoto M, Izumi H, Ise T, Kato K, Takano H, Nagatani G et al. Structural basis for the regulation of UDP-N-Acetyl-a-D-galactosamine : polypeptide N-acetylgalactosaminyl transferase-3 gene expression in adenocarcinoma cells. Cancer Res 1999;59:621422.

(21.) Fuster MM, Esko JD. The sweet and sour of cancer: glycans as novel therapeutic targets. Nature Rev Cancer 2005;5:526-42.

(22.) Goutebroze L, Carnaud M, Denisenko N, Boutterin MC, GirauItJA. Syndecan-3 and syndecan-4 are enriched in schwann cell perinodal processes. BMC Neuroscience 2003;4:29-38.

(23.) Woods A, Couchman JR. Syndecans : synergistic activators of cell adhesion. Trends in Cell Biology 1998;8:189-92.

(24.) Cheung IY, Cheung NK. Quantitation of marrow disease in neuroblastoma by real-time reverse transcription-PCR. Clin Cancer Res 2001;7:1698-705.

NORA BEROIS, [2 [dagger]] ETIENNE BLANC, [1 [dagger]] HUGUES RIPOCHE, [3] XENIA MERGUI, [1] FELIPE TRAJTENBERG, [2] SABRINA CANTAIS, [1] MICHEL BARROIS, [4] PHILIPPE DESSEN, [3] BERTIL KAGEDAL [5] JEAN BENARD, [1,4] EDUARDO OSINAGA, [1] and GILDA RAGUENEZ [1 *]

[1] CNRS-UMR 8126, Interactions Moleculaires at Cancer; [3] CNRS-FRE 2939, Groupe de Bioinformafique; and [4] Departement de Biologie et Pathologie Medicales, IFR54 Institut Gustave Roussy, Villejuf, France.

[2] Departamento de Bioquimica, Laboratorio de Oncologia Basica, Facultad de Medicina, Universidad de la Republica, Avda. Montevideo, Uruguay.

[5] University of Linkoping, Faculty of Health Sciences, Division of Clinical Chemistry, Sweden.

[6] Nonstandard abbreviafions: NB, neuroblastoma; MRD, minimal residual disease; QRT-PCR, quanfitafive reverse transcription-PCR; CT, threshold cycle; TH, tyrosine hydroxylase; DDC, dopa decarboxylase; HPRT, hypoxanthine phosphoribosyl transferase; BM, bone marrow; Myoc, myocardium.

[7] Human genes: MYCN, v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian); GALNTI3, UDP-N-acetyl-a-D-galactosamine: polypepfide N-acetylgalactosaminyltransferase 13; ABCB1, ATP-binding cas sette, sub-family B (MDR/TAP), member 1; ENPP2, ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin); EMP2, epithelial membrane protein 2; DCN, decorin; PRPH, peripherin; RELN, reelin; SYCP2, synaptonemal complex protein 2; MDK, midkine (neurite growth-promofing factor 2); FMOD, fibromodulin; LUM, lumican; COL1A1, collagen, type I, a 1; GPM6B, glycoprotein M6B; HOXB2, homeobox B2; ELAVLI, ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R); EFNB1, ephrin-B1; NRG2, neuregulin 2; NELL1, NEL-like 1 (chicken); PCDH17, protocadherin 17; STX1B2, syntaxin 1B2; SCG5, secretogranin V (7B2 protein); NPTX1, neuronal pentraxin I; GAT3, GATA binding protein 3; NROB1, nuclear receptor subfamily 0, group B, member 1.

[dagger] These authors have contributed equally to this work.

* Address correspondence to this author at: CNRS-UMR 8126, Interactions Moleculaires et Cancer, IFR54 Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuf, France. E-mail raguenez@igr.fr.
Table 1. Sequences of primers and probes used in nested and real-time
RT-PCR for gene transcripts analyses in NB.

Gene Forward primer

GALNTi3 - nesled PCR
outer 5'- ACA TCT ATC CGG ACT CCC-3'
Inner 5'-GAG GAA ATC AGT TAT GGG AA-3'
GALNTI3 - QPCR 5'-GCC-ACC-ATA AGA-GAG-GAA-ATC-AGT-TA-A-3'
probe FAM-CAA CGT GAG TCT CTC AGC AT- MGB
MDR1 5'-GTC CCA GGA GCC CAT CCT-3'
C~01 5'-ATG ATT CCA'TTG GCT TAC-3'
ENPP2 5'-CGT GAT AAG TGG ACC AAT-3'
EMP2 5'-GCC TGG TGA GTA GGA-3'
CpL1A1 5'-GAG GAA GGC AAA CCC CAG 3'
SMARCA 2 5'-ATTC ATG AG GCA AAA TCA GTC AAC-3'
DCN 5'-CTG ATG ACC GCG ACT TCG A-3'
PRPH 5'-ACG AGT CCC GAC GCC AGA-3'
RELN 5'-ATT ACC ACC CGT GAC CTA-3'
RGS7 5'-ACC TGG ACG ATA CAC AT-3'
SYCP2 5'-GCT ACA GAA AAC TGA AGA CTA CCT TTG TTA-3'
NRG2 5'-TCG AGG GCA TCA ACC-3'
NROB1 5'-GGC CTG CAG TGC GTG AAG-3'
18S 5'-CGG CTA CCA CAT CCA AGG AA-3'
TH primers 5'-TGG ACC ACC CGG GCT-3'
probe FAM-CCG CCA GCG CAG GAA GCT GCT G-TAMRA
calibrator 5'-TGT CAG AGC TGG ACA AGT GT-3'
DDPA decarboxylase
primers 5'- CCA TCA GGA TTC AGG GCT TAT-3'
probe FAM-CTG ACT ACC GGC ATT GGC AGA TAC CA-TAMRA
calibrator 5'-TTC TGC CAT GTG GGT GAA-3'
GD2 synfhasef *)
primers 5'-GAC AAG CCA GAG CGC GTT A3-'
probe VIC-ACC CAGF CCC TTG CCH AAG GGC-TAMRA
calibrator 5'CCA GGG AGC CCA GTA CAA C-3

Gene Reverse primer

GALNTi3 - nesled PCR
outer 5'-TCA TGT GCC CAA GGT CAT GTT CC-3'
Inner 5'-TAG GCA CCA TTT TGT CTT CTT-3'
GALNTI3 - QPCR 5'-GGT-TCA-TCG-AGA-CAT-TGG-TTA-CTG-TTA-3'
probe
MDR1 5'-TGT ATG TTG GCC TCC TTT GCT-3'
C~01 5'-GCA TGG CAT GTA TCA AA-3'
ENPP2 5'-CCT TCC ACG TAC TGT TT-3'
EMP2 5'-AC-I' C I-I GAA AGC TGT CAT TGA-3'
CpL1A1 5'-TCG GGT TTC CAC ACC TCT-3'
SMARCA 2 5'-TTC CTC GAT TTG GCC TTT TCT-3'
DCN 5'-GGG AAG ATC CTT TGG CAC TTT-3'
PRPH 5'-TCC AGG GCG AAC TGC T-3'
RELN 5'-GCG GAT GAG CTA TCT GT-3'
RGS7 5'-GGC ACT GGA TCT TAT AAA-3'
SYCP2 5'-TAG GGT CAT CAG CTC CAT TY'A A-3'
NRG2 5'-CAT GTA CAA TCG CAA AGG-3'
NROB1 5'-GCC CTT GGT GCG TCA TC-3'
18S 5'-GCT GCA TAT AGG CCT GCA-3'
TH primers 5'-GTC GCC GTG CCT GTA CTG-3'
probe
calibrator 5'-ATA TTG TCT TCC CGG TAG C-3'
DDPA decarboxylase
primers 5'-TTC AAA GAG CGA AAT CTT CTG C'-3'
probe
calibrator 5'-TGC GGA TAT AAG CCT GCA-3'
GD2 synfhasef *)
primers 5'GCC GTG AAG AGG TCG -3'
probe
calibrator

For the quantitative real-time PCR of GALNTI3, the forward and reverse
primers are located in exon 10 (GALNTI3-F) and in exon 11 (GALNTI3-R),
and the MGB probe in overlapped junction exons 10-11. For the nested
PCR, the outer primers are located in exon 8 (GALNTI3-F outer) and in
exon 11 (GALNTI3-R outer), the inner primers are located in exon 10
(GALNTI3-F inner) and in exon 11 (GALNTI3-R inner).
(*) Primers and probes for GD2 synthase were those described by Cheung
and Cheung (24).

Table 2. Differentially expressed genes for Primary Tumor Xenograft
(stage 4 disease) and Bone Marrow (BM) or

Myocardium (Myoc) metastatic neuroblasts.

Sequence Name(s) (*) Sequence description

Chemoresistance/detoxication

1932289 (GALNT 13) Member of the GaINAc-T family
ABCB7(MDRi) P-glycoprotein, Multidrug resistance 1
11100002 Similar to TRAG-3
CDO1 Cysteine dioxygenase 1
TRAG3 Taxol resistance associated gene
MGST1 MicrosomalglutathioneS-transferase 1

Immune response

MAGEE1 MelanomaantgenfamilyEl
RAGE Renal tumor antigen
LOC55901 (THBS1) Containing a type 1 trhombospondin
 domain
TRA7 Tumor rejectron antigen 1

Stress response/Inflammation

FLJ13842 Similar to mouseWigl
HSPA5 Heat shock 70kD protein 5
HERPUDI Homocysteine-inducible ER stress
SERPINA5 Serine proteinase inhibdor member 5

Neuronafceflstmcture

GPM6B Glycoprolein M6B
RELN reelin, role in brain devebpment
PRFH Peripherin, neuronal intermediate
 filament
TMSNB Similar to thymosin beta 4, neuronal
 remoduling
TUB81 Similar to beta-2 tubulin
ACTN2 Alpha actinin 2 P35609 2,3

Motility

ENPP2 Phosphodiesterase-aappha/autotaxin

Cell Adhesion

FLRT2 Simiar to a region of fbromodulin
 (Fmod)
NMA Putative transmembrane protein
EMP2 Eprhelial membrane protein 2
DCN Decorin, a dermatan/chondroitin
 suffate proteoglycan
SMARCA2 SW I-8NF related matrix associated
 actin...
COL1A1 Alpha 1 subund of type I collagen
ANKTMI Ankyrh-like protein
FMOD Fibromodulin
MGC3047 Containing two Immunogbbulin (Ig)
 domains
PCDH17 Containing five cadherin domains
DKFZP564L0862 Similar to ankyrh repeat (Asb1)
LTBP7 Latent transforming growth factor
 beta binding prod
LUM Lumican
CSPG4 Chondrodin suffate proteoglycan 4

Cell signaling

1964086 (IMP-1) IGF-II mRNA-binding protein 1
1_931758 Similar to IGFALS
SYCP2 Synaptonemal complex protein 2
MGC26655 Similar to RaP2 interacting protein
 8, Ras pathway
RGS7 Regulator of G protein sgnaling 7,
 G protein pathway
KCNJ3 Signal Tronsduction/G protein pathway
GFRA7 GDNF family receptor alpha 1
PPARGCI Peroxisome prolif. activ. receptor
 gamma coactivator 1
NELL1 Nel(chicken)-like 1
RGS13 Regulator of G protein sgnaling 1
GDS2 GO-G1 swdch gene 2
MDK Midkine
ARMET Arginine-rich mutated kr early stage
 tumors
STX1B2 Syntaxin 1B
TEX14 Testis expressed sequence 14
KHDRBS3 RNA binding KH domain

Cell signaling

IFRD1 Similar to Rn.3723
MAPK10 Mitogen-activated protein kinase 10
TMSNB Thymosin beta 4
SGNE1 Secretory granule neuroendocrine
 protein 1
SDF2L1 Stromal cell-derived factor 2-like 1
PTGER2 Prostaglandin E receptor 2
TMEFFt Transmembrane protein with EGF and
 follistatin domains
SNK Serum-inducible kinase
LOC84524 Contains 3 CCCH-type zinc finger
 domains
NROBI Nuclear receptor subfamily O group
 B membe rt
NRG2 Neuregulin 2
I_1212867(SEPT10) member of GTP-binding cell division
 family
ELAVL 1 Embryonic lethal abnormal vision-
 like 1
GPR30 G protein-coupled receptor 30
NP7X1 Neuronal pentraxin 1
IGFBP2 Insulin like growth factor binding
 protein 2
OAS3 2-5-oligoadenylate synthetase 3
CAPN6 Calpain 6
EFN81 Ephrin B1
RERG Similar to human RRAS2

Transcription factors

ZNF215 Contains 4CH2 type zync finger domains
1_1152158 Similar to cut (Drosophila)-like 2
 (mouse Cutl2)
XBP 1 X-box binding protein 1
HOXB2 Homeo box 82
ATF3 Activating transcription factor 3
RRN3 RNA polymerase I transcription factor
MEF-2 Myocyte-specific enhancer-binding
 factor
ID4 Inhibitor of DNA-binding 4
GLI2 Gli-Kruppel family member GIi2
TCFL4 Transcription factor-like 4 (/Max-
 like protein X)
GATA3 GATA-binding protein 3
KIAA0711 Contains 6 Ketch motif domarts and
 2 BTB or POZ domains
Metabolism

1960435 Mber of the shortchain dehydrogenase-
 reductase family
ASNS Asparaginasesynthase
EN03 Enolase 3
1929099 Moderate similarity to human HU-K4
1_152190 Srong similariry to beta! subunit of
 Na+/K+-gTPase
FACL6 Fatty acid Coenzyme A ligase
 long-chain 6
ATP1B1 Beta subunit of Na+/K+ATPase
MGLL Monoglyceride lipase
ATP5C1 ATP synthase H+ transporting
 mitochondria) F1 complex
PDHA2 Pyruvate dehydrogenase E1 alpha 2
ASPH Aspartylbeia-hydroxylase
1_965027 Containing a cache domain
PECI Peroxisomal D3, D2-enoyl-CoA isomerase
TXNIP Vitamin D-3 up-regulated protein-1

Unknown

FLJ22662 Unknown
MGC13269 Unknown
AGTPBPt Unknown
TRAP25 Unknown
1_1100083 Unknown
Ctorf21 Unknown
KIAA0125 Unknown
HSPC053 Unknown
DKFZp434G0920 Unknown
1_1151996 Unknown

Myocardium (Myoc) metastatic neuroblasts.

Sequence Name(s) (*) Accession no. FoldChange(c) P-value

Chemoresistance/detoxication

9 (GALNT 13) Q07537 12,1 9,53E-44
ABCB7(MDRi) P08183 7,5 9,53E-44
11100002 Q9YSP2 3,9 5,90E-07
CDO1 Q16878 2,8 2,44E-03
TRAG3 AAC29487.1 2,4 5,87E-03
MGST1 P10620 1,9 1,24E-04

Immune response

MAGEE1 09UBF1 3,7 1,34E-04
RAGE BAA81688.1 2,7 5,06E-18
LOC55901 (THBS1) BAA96553.1 2,3 7,58E-09

TRA7 P14625 2 3,84E-05

Stress response/Inflamm

FLJ13842 BAB14719.1 6 9,53E-04
HSPA5 P11021 3,1 4,32E-18
HERPUDI 015011 2,7 1,58E-08
SERPINA5 P05154 2,5 4,99E-12

Neuronafceflstmcture

GPM6B Q13491 2,6 2,03E-04
RELN P785D9 2,6 1,63E-06
PRFH P41219 2,4 2,72E-07

TMSNB CAC18959.1 1,9 1,18E-09

TUB81 CAC09371.2 2,4 1,96E-03
ACTN2 P35609 2,3 1418

Motility

ENPP2 AAA64785.1 3,3 9,65E-04

Cell Adhesion

FLRT2 Q43155 2,8 2,04E-12

NMA Q13145 1,7 8,38E-07
EMP2 P54851 -7,5 1,89E-26
DCN P07585 -6,1 2,70E-36

SMARCA2 P51531 -3,4 9,76E-14

COL1A1 P02452 -2,7 6,29E-10
ANKTMI CAA71610.1 -2,7 7,20E-05
FMOD Q06828 -2,6 6,66E-14
MGC3047 AAH17312.1 -2,5 9,62E-14

PCDH17 AAB84144.1 -2,3 1,56E-03
DKFZP564L0862 BAA91302.1 -2,2 2,09E-04
LTBP7 P22064 -2,1 5,66E-09

LUM P51884 -1,9 4,19E-15
CSPG4 CAA65529.1 -1,8 2,00E-08

Cell signaling

1964086 (IMP-1) AK022617.1 8,3 9.53E-04
1_931758 BAB31253.1 5,4 2,24E-33
SYCP2 CAA70171.1 5 1,78E-12
MGC26655 BA870882.1 3,9 5,91E-13

RGS7 AAD34290.1 3,8 7,60E-23

KCNJ3 P48549 3,2 1,28E-23
GFRA7 P56159 2,3 3,78E-05
PPARGCI AAF78573.1 2,3 4,63E-09

NELL1 Q92832 2,3 4,60E-09
RGS13 014921 2,2 3,75E-09
GDS2 P27469 2,2 2,43E-12
MDK P21741 2,1 75E-14
ARMET P55145 2,1 1,44E-06

STX1B2 AAK27267.1 2,1 6,10E-03
TEX14 AAK31980.1 2 1,01E-12
KHDRBS3 AAC99294.1 2 2,90E-06

Cell signaling

IFRD1 CAA71366.1 2 6,92E-09
MAPK10 P53779 1,9 1,22E-10
TMSNB CAC18959.1 1,9 1,18E-09
SGNE1 P05408 1,9 2,69E-06

SDF2L1 Q9HCN8 1,8 7,45E-08
PTGER2 P43116 1,8 2,28E-11
TMEFFt AAA64622.1 1,8 1,43E-08

SNK Q9NYY3 1,8 4,33E-06
LOC84524 AAK13496.1 1,7 1,53E-05

NROBI P51843 -5,5 2,37E-09

NRG2 AAF28850.1 -3,3 1,58E-03
I_1212867(SEPT10) BC020502.1 -2,9 9,48E-30

ELAVL 1 Q15717 -2,5 7,94E-17

GPR30 Q99527 -2,5 4,06E-23
NP7X1 Q1581E -2,3 6,31E-06
IGFBP2 P18065 -2,3 9,21E-09

OAS3 Q9Y6K5 -2 1,45E-09
CAPN6 P10070 -2 7,89E-07
EFN81 P98172 -1,7 2,68E-08
RERG P23771 -1,9 9,61E-10

Transcription factors

ZNF215 Q9UL58 2,7 8,30E-08
1_1152158 Q14529 2 7,96E-14

XBP 1 P17861 1,9 2,95E-09
HOXB2 P14652 1,9 4,66E-06
ATF3 P18847 1,9 1,44E-15
RRN3 AAC27823.1 1,8 3,14E-05
MEF-2 AAD43038.1 1,7 1,36E-10

ID4 P47928 -2,4 8,81E-09
GLI2 P10070 -2 7,89E-07
TCFL4 AAG40146.1 -2 5,89E-10

GATA3 P23771 -1,9 9,61E-10
KIAA0711 94819 -2,2 5,50E-05

Metabolism

1960435 CAD19397.1 3,8 5,59E-04

ASNS P08243 2,4 1,96E-06
EN03 P13929 2,2 2,44E-08
1929099 AAC73069.1 2,1 4,33E-04
1_152190 P07340 2,1 1,16E-09

FACL6 Q9UKU0 2 1,25E-15

ATP1B1 P05026 1,9 8,48E-10
MGLL AAH06230.1 -2,4 4,08E-22
ATP5C1 P36542 -2,4 7,54E-27

PDHA2 P29803 -2,3 9,84E-03
ASPH Q12797 -2,2 1,64E-07
1_965027 094819 -2,2 5,50E-05
PECI AAH02668.1 -2,1 2,50E-03
TXNIP BAB18859.1 -1,8 4,56E-11

Unknown

FLJ22662 BAB15442.1 2,6 9,18E-09
MGC13269 AAH06340.1 2 1,06E-04
AGTPBPt BAA91749.1 1,9 1,89E-14
TRAP25 CAC24634.1 1,7 1,91E-10
1_1100083 AAH11405.1 1,7 7,89E-08
Ctorf21 BAB71196.1 1,7 1,25E-06
KIAA0125 014138 -4,5 2,92E-09
HSPC053 AAF29025.1 -2,2 2,69E-08
DKFZp434G0920 CAB66733.1 -2,1 7,72E-14
1_1151996 AAH26269.1 -1,8 9,39E-11

Sequence Name(s) (*) Int.1(b) Int.2(b)

Chemoresistance/detoxication
9 (GALNT 13) 43 570
ABCB7(MDRi) 682 5283
11100002 60 193
CDO1 48 127
TRAG3 74 199
MGST1 362 703

Immune response

MAGEE1 677 3036
RAGE 2079 5643
LOC55901 (THBS1) 542 1270

TRA7 5742 31772

Stress response/Inflammation

FLJ13842 101 610
HSPA5 0944 97030
HERPUDI 4883 13459
SERPINA5 251 618

Neuronafceflstmcture

GPM6B 122 312
RELN 153 411
PRFH 2209 29967

TMSNB 1989 3829

TUB81 25 59
ACTN2

Motility

ENPP2 27 85

Cell Adhesion

FLRT2 53 153

NMA 9060 15174
EMP2 2994 401
DCN 4182 685

SMARCA2 312 91

COL1A1 1797 674
ANKTMI 2550 959
FMOD 1561 590
MGC3047 1517 4693

PCDH17 9506 4501
DKFZP564L0862 337 156
LTBP7 2629 1234

LUM 630 319
CSPG4 4142 2234

Cell signaling

1964086 (IMP-1) 443 3703
1_931758 88 480
SYCP2 32 150
MGC26655 288 1758

RGS7 31 121

KCNJ3 78 253
GFRA7 211 518
PPARGCI 1339 3158

NELL1 2378 5675
RGS13 211 478
GDS2 317 701
MDK 6818 35716
ARMET 0793 66513

STX1B2 51 117
TEX14 296 614
KHDRBS3 2560 5255

Cell signaling

IFRD1 901 1869
MAPK10 383 756
TMSNB 1989 3829
SGNE1 1042 2248

SDF2L1 6447 31150
PTGER2 5524 10348
TMEFFt 1086 1960

SNK 2971 23398
LOC84524 859 1460

NROBI 310 54

NRG2 118 49
I_1212867(SEPT10) 1379 454

ELAVL 1 5527 2187

GPR30 916 366
NP7X1 67 27
IGFBP2 8169 12096

OAS3 253 122
CAPN6 42 213
EFN81 2360 1338
RERG 3402 1793

Transcription factors

ZNF215 285 808
1_1152158 182 379

XBP 1 0692 20608
HOXB2 3078 6133
ATF3 1521 3035
RRN3 237 454
MEF-2 496 878

ID4 169 71
GLI2 429 213
TCFL4 503 251

GATA3 3402 1793
KIAA0711 1629 768

Metabolism

1960435 56 217

ASNS 7245 17952
EN03 5635 12374
1929099 45 100
1_152190 3255 6872

FACL6 102 209

ATP1B1 6383 12772
MGLL 1037 417
ATP5C1 6413 2662

PDHA2 70 42
ASPH 295 137
1_965027 1629 768
PECI 2643 1318
TXNIP 1408 768

Unknown

FLJ22662 733 1986
MGC13269 62 118
AGTPBPt 1842 3672
TRAP25 2457 4346
1_1100083 2165 3841
Ctorf21 943 1633
KIAA0125 3635 851
HSPC053 1212 537
DKFZp434G0920 1433 668
1_1151996 1794 992

A list of 68 up-regulated genes and 39 down-regulated genes
(in italics) for which the absolute value of fold change is >2 and
P-value is <0.01. Functional annotation was performed with the
Online Mendelian Inheritance in Man (OMIM). (a) Genes were classified by
function, as reported in the Agilent database. (b)Int1 and Int2 depict
the average expression level of each gene in PTX and metastases
respectively. (c)The changes for all the genes listed are statistically
significant according to Signifcance
Analysis of Microarrays (SAM) and Resolver.

Table 3. Clinicopathological characteristics of patients and expression
of different markers in BM.

Patient Age Stage Time MYCN Cytology T13 T13
 (months) point copy nbr QPCR nPCR

 1 24 4 Dx 20 30<60 2,092 +
 2 24 4 Dx 1 30<60 0,35 +
 3 24 4 Dx 120 30<60 0,155 +
 4 42 4 Dx 50 30<60 0,167 +
 5 30 4 Dx 1 >60 0,029 +
 6 - 4 Dx - 30<60 0,278 +
 7 36 4 Dx 1 30<60 0,017 NA
 8 42 4 Dx 1 30<60 0,006 +
 9 96 4 Dx 2 <30 0,006 +
10 24 4 Dx 1 30<60 0,05 NA
11 24 4 Dx 27 30<60 0,015 +
12 72 4 Dx 1 30<60 0,009 +
13 48 4 Dx 1 >50 0,002 +
14 180 4 Dx - 30<60 0,002 +
15 16 4 Dx 36 >60 NA +
16 48 4 Dx 1 >60 2,704 +
17 19 4 Dx 19 >60 1,86 NA
18 21 4 Dx 53 30<60 2,639 +
 24 PC - NI 0 -
19 78 4 Dx - 30<60 0,016 +
 78 PC - NI 0 +
 84 PC - NI 0 +
20 36 4 Dx 1 30<60 0,001 +
 42 PC - NI 0,001 +
 48 PC - NI 0 -
21 15 4 Dx 5 30<60 0,018 +
 18 PC - NI 0 -
22 24 4 Dx 1 30<60 0,003 +
 30 PC - NI 0 -
23 42 4 Dx 1 <30 0,006 -
 42 PC - NI 0 -
24 156 4 PC - NI 0 NA
25 24 4 PC - NI 0 -
26 22 4 PC - NI 0 -
27 60 4 PC - NI 0 -
28 1 4S Dx - NI 0 -
29 7 4S Dx - NI 0 -
30 1 1 Dx - NI 0 -
31 9 2 Dx 1 NI 0 -
32 8 2 Dx 1 NI 0 -
33 108 2 Dx 1 NI 0 -
34 24 2 Dx - Nt 0 -
35 60 2 PC - NI 0 -
36 84 2 Dx 1 NI 0 -
37 42 2 Dx 1 NI 0 +
38 30 2 Dx - NI 0 +
39 204 3 Dx - NI 0 -
40 192 3 Dx 1 NI 0 -
41 30 3 Dx 1 NI 0 -
42 60 3 Dx - NI 0 -

Patient Age HPRT TH/HPRT GDsIHPRT DDC/HPRT Clinical
 (months) Evolution

 1 24 38340 1,7202 2,6206 1,4346 A
 2 24 7113 2,4821 4,5771 0,7828 D
 3 24 10560 41,7545 0,1101 2,7720 D
 4 42 37092 7,1062 0,6769 0,8276 A
 5 30 11625 154,3754 0,4652 12,3909 D
 6 - 9752 45,5515 0,4513 2,9023 -
 7 36 594 3097,791 10,0084 89,6347 D
 8 42 42302 0,9253 0,0138 0,1899 D
 9 96 5985 0,1579 0,3761 0,0192 A
10 24 16036 1,6405 0,0162 0,2035 A
11 24 4410 4,7150 1,7079 0,0338 D
12 72 3937 6,6949 0,0589 0,3259 D
13 48 1906 172,6590 1,0273 15,0525 A
14 180 7711 0,6240 0,0252 0,0575 A
15 16 5066 0,2872 3,6630 0,0610 D
16 48 3421 287,9018 0,8603 11,8688 D
17 19 5732 39,5249 1,8128 6,8437 A
18 21 6166 6,3153 0,7331 0,0409
 24 6795 0,0006 0,0044 0,0004 D
19 78 15028 9,1322 0,0230 0,7350
 78 10798 0,0121 0,0051 0,0021
 84 12984 0,1642 0,0031 0,0193 D
20 36 10244 0,2097 0,0134 0,0348
 42 13944 0,0016 0,0014 0,0006
 48 5633 0,0057 0,0023 0,0016 D
21 15 13067 0,1675 0,1296 0,0312
 18 7577 0,0000 0,0026 0,0018 A
22 24 5865 17,6002 0,1168 0,5956
 30 4997 0,0078 0,0226 0,0148 A
23 42 4579 0,3005 0,0024 0,0223
 42 5995 0,0040 0,0157 0,0153 A
24 156 12896 0,0009 0,0015 0,0009 D
25 24 11971 0,0007 0,0029 0,0017 D
26 22 8820 0,0011 0,0037 0,0031 A
27 60 6087 0,0169 0,0238 0,0138 A
28 1 24174 0,0258 0,0057 0,0040 A
29 7 24263 0,0041 0,0011 0,0024 A
30 1 7984 0,0006 0,0016 0,0004 A
31 9 12399 0,0009 0,0005 0,0001 A
32 8 11823 0,0033 0,0035 0,0014 A
33 108 9250 0,0335 0,0627 0,1050 A
34 24 5759 0,0000 0,0030 0,0005 A
35 60 11202 0,0010 0,0087 0,0040 A
36 84 3682 0,0079 0,0071 0,0152 A
37 42 6961 0,0010 0,0007 0,0014 A
38 30 15421 0,0000 0,0128 0,0005 D
39 204 9944 0,0000 0,0103 0,0015 D
40 192 17119 0,0034 0,0027 0,0011 A
41 30 1387 0,0079 0,0224 0,0043 A
42 60 24366 0,0004 0,0044 0,0004 A

Detection of GALNTI3 (T13), TH, GD2 synthase, and DDC transcripts in BM
samples from stage 2, stage 3 and stage 4 NB patients (see Materials
and Methods). The samples were taken at diagnosis, during treatment and
at relapse. (Dx: diagnosis, PC: post-chemotherapy, QRT-PCR:
quantitative-RT-PCR, nRT-PCR: nested RT-PCR, NI: noninvolved, I:
involved, NA: not analyzed, A: alive, D: deceased). Clinical evolution
is an overall survival rate of 2 years after diagnosis. Two of these
(patients 19 and 20) and a 3rd (no. 38), who scored negative with
conventional cytology, scored positive for GALNTI3 expression and died
shortly thereafter. Eleven of 23 of the stage 4 patients are in fact
dead, and each of them was positive for GALNTI3 at diagnosis. Samples
obtained after chemotherapy from 2 stage 4 patients who died
(nos. 24 and 25) were negative for cytology and GALNTI3 expression.
COPYRIGHT 2006 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2006 Gale, Cengage Learning. All rights reserved.

 
Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Cancer Diagnostics
Author:Berois, Nora; Blanc, Etienne; Ripoche, Hugues; Mergui, Xenia; Trajtenberg, Felife; Cantais, Sabrina;
Publication:Clinical Chemistry
Date:Sep 1, 2006
Words:8260
Previous Article:Do the survivin (BIRC5) splice variants modulate or add to the prognostic value of total survivin in breast cancer?
Next Article:Identification of novel brain biomarkers.
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters