Clinical Chemistry 52: 1701-1712, 2006.
First published July 27, 2006; 10.1373/clinchem.2006.067975
(Clinical Chemistry. 2006;52:1701-1712.)
© 2006 American Association for Clinical Chemistry, Inc.
ppGalNAc-T13: A New Molecular Marker of Bone Marrow Involvement in Neuroblastoma
Nora Berois4,3,
Etienne Blanc1,3,
Hugues Ripoche2,
Xénia Mergui1,
Felipe Trajtenberg4,
Sabrina Cantais1,
Michel Barrois3,
Philippe Dessen2,
Bertil Kågedal5,
Jean Bénard1,3,
Eduardo Osinaga4 and
Gilda Raguénez1,a
1 CNRS-UMR 8126, Interactions Moléculaires at Cancer; 2
CNRS–FRE 2939, Groupe de Bioinformatique; and 3
Département de Biologie et Pathologie Médicales, IFR54 Institut Gustave Roussy, Villejuif, France.
4 Departamento de Bioquímica, Laboratorio de Oncologia Basica, Facultad de Medicina, Universidad de la República, Avda. Montevideo, Uruguay.
5 University of Linköping, Faculty of Health Sciences, Division of Clinical Chemistry, Sweden.
aAddress correspondence to this author at: CNRS-UMR 8126, Interactions Moléculaires et Cancer, IFR54 Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif, France. E-mail raguenez{at}igr.fr.
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Abstract
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Background: To identify new molecular markers of bone marrow dissemination in human neuroblastoma (NB), we studied the transcriptome profiles of malignant neuroblasts established from the human MYCN-amplified IGR-N-91 model.
Methods: This experimental model includes human neuroblastoma cells derived from a subcutaneous stage 4 disease, myocardium (Myoc) and bone marrow (BM) metastatic cells.
Results: Gene expression profiles obtained with Agilent oligo microarrays revealed a set of 107 differentially expressed genes in the metastatic neuroblasts. This set included up-regulated genes involved in chemoresistance, cell motility, neuronal structure/signaling, and the recently characterized GALNT13 gene encoding a glycosyltransferase that initiates mucin-type O-glycosylation. Because the glycosylation process is involved in the progression of primary tumor to metastatic disease, we investigated whether the most strongly up-regulated gene, GALNT13, might be a marker of bone marrow involvement in stage 4 NB patients. Importantly, in the BM of healthy adults no GALNT13 transcript was detected with analysis by quantitative (n = 3) and nested reverse transcription-PCR (n = 4) assays. In contrast, GALNT13 transcripts were detected in 23/23 cytologically involved BM samples obtained at diagnosis of stage 4 NB patients and in 5/27 cytologically noninvolved BM samples obtained from patients with stage 1–4 and 4S and treated stage 4 NB. The quantitative measurements of tyrosine hydroxylase (TH), ganglioside D2 synthase, dopa decarboxylase, and GALNT13 transcript values were compared in the same NB patients, and the results showed that GALNT13 expression was most highly correlated to poor clinical outcome at diagnosis.
Conclusion: We propose ppGalNAc-T13 as a new informative marker for the molecular diagnosis of BM involvement and the follow-up of minimal residual disease in NB patients.
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Introduction
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Neuroblastoma (NB)1
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)(2)(3) vs disseminated stage 4 [stage 4 and 4S]) and age at diagnosis for stage 4 disease (<1 year, >1 year). Overall, MYCN2
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 GALNT13, a recently cloned and characterized isoform of the ppGalNAc-T family(11). Our findings showed that GALNT13 transcript values can be used as an informative marker to monitor MRD in BM in patients with stage 4 NB.
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cell lines
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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).
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patient samples
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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 1500g 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).
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rna extraction
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Total RNA was extracted from cells and BM with RNAble 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 28S/18S over 1.7 by an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.).
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microarray experiments
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We used 5 µg 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 °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 µm). Image analyses were performed with Agilent Feature Extraction Software and then normalized with the Lowess method.
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gene expression analysis
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cDNA probes were synthesized with SuperScript II (Invitrogen). We verified results of microarray experiments with a real-time quantitative reverse transcription-PCRE (QRT-PCR) with 12.5 ng of cDNAs, 10 pmol of each primer, and 12.5 µL of a master mix containing SYBR®-Green in a final volume of 25 µL (Applied Biosystems, Abi Prism 7000 Sequence Detection System). The sample RNA was normalized by the amplification of an endogenous control (18S) as previously described(10); the calibrator used was the neuroblastoma SH-SY5Y cell line, and results were quantified by the comparative threshold cycle (CT) method. The oligonucleotide primers (Table 1
), were designed with the Oligo 6 Primer Analysis Software (Molecular Biology Insights, Inc.).
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Table 1. Sequences of primers and probes used in nested and real-time RT-PCR for gene transcripts analyses in NB.
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analysis of mrd in bm samples
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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 °C, 60 s at 60 °C, and 60 s at 72 °C for 40 cycles. These results were quantified with the comparative CT method. An arbitrary threshold was chosen on the basis of the variability of the baseline and was modified manually for each experiment. CT (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-
CT where CT = CT (sample)-CT (reference), and CT was the difference between the CT of the target gene and the CT of the endogenous control gene. The endogenous gene used was 18S and the reference used was the neuroblastoma SH-SY5Y cell line. In the validation of the methods for GALNT13, samples with no amplification within 40 cycles were considered as zero.
Moreover, we optimized a nested RT-PCR with 1 µg of total RNA for the synthesis of first-strand cDNA in a final volume of 20 µL. The PCR first round was performed with 1 µL of cDNA, 300 nmol/L of each primer, 3 mmol/L MgCl2, and 1 unit of Taq DNA polymerase (Invitrogen) in a final volume of 25 µL. The amplification conditions were 30 s at 94 °C, 30 s at 62 °C, and 60 s at 72 °C for 30 cycles. The 2nd round was seeded by 1 µL of the first round product in similar conditions except for 1.5 mmol/L MgCl2 and annealing at 60 °C for 35 cycles. We analyzed 15 µL of PCR products by electrophoresis on 2% agarose gels by direct visualization after ethidium bromide staining. The sequence of GALNT13 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), GD2 synthase (ß1,4-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 µL of samples containing 150 ng of RNA extracted from BM samples was diluted with 4 µL Rnase-free water. Then 5 µL of reverse transcriptase was added and, after reaction, the samples were diluted 1:2 to give a total volume of 20 µL for PCR(5). The QRT-PCR step was performed in triplicate with 2.5 µL sample cDNA or 2.5 µL of each calibrator (range 100–105 targets/µL) in each reaction. TH mRNA was analyzed by multiplex QRT-PCR together with GD2 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, GD2 synthase, and DDC were performed with primers (100 nmol/L) and probes (100 nmol/L for TH and DDC and 200 nmol/L for GD2 synthase) from Scandinavian Gene Synthesis. The calibrator described earlier(5) was used for TH analysis, and calibrators for GD2 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, GD2 synthase, and DDC calibrators are given in Table 1
. In the PCR step, we added equal volumes (2.5 µL) 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 transcripts per microliter) in the original sample. Because we had 1 µL from the beginning, the final result equaled the number of transcripts in the 150 ng of starting material. Finally, the results of TH, GD2 synthase, and DDC transcripts were normalized to the HPRT results by division.
In the validation of the methods for TH, GD2, 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, GD2 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 CTs <40 and with concentrations <100 transcripts/mL were 59 (30) transcripts/mL (51%) for TH, 56 (25) transcripts/mL (44%) for GD2 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 analytes. Therefore, the same cutoff values were used for the 3 transcripts. In the Kaplan-Meier survival studies, we used the data normalized by HPRT.
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production of anti-PPGALNAC-T13 antibody
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A synthetic specific peptide of ppGalNAc-T13 (RSLLPALRAVISRNQE, 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. Immunoblots, 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).
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statistical analysis
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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). Log-rank tests were used to calculate the statistical significance (P-value) of difference between groups.
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Results
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gene expression pattern of metastasis-associated 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® 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— = 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).
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Figure 1. (A) Gene expression pattern in BM/Myoc metastatic neuroblasts in the IGR-N-91 model.
Green and red respectively designate the down- and up-regulation of a gene in metastatic neuroblasts compared with stage 4 disease neuroblasts. Grey or black spots indicate no considerable change in gene expression. Experiments were performed in duplicate with 2 dye-swap experiments; BM vs stage 4 disease: lanes 1–2 duplicate (Cy3/Cy5); lanes 3 and 4, duplicate (Cy5/Cy3). Myoc vs stage 4 disease: lanes 5 and 6 duplicate (Cy3/Cy5); lanes 7 and 8, duplicate (Cy5/Cy3). The colors of lanes 3, 4, 7, 8 (dye-swap) were reversed to improve legibility. The expression level of selected genes was measured using QRT-PCR. Results are expressed as the mean(SD) for 3 independent experiments. The expression level of each gene was considerably altered in both BM or Myoc neuroblasts compared with stage 4 disease, as shown in (B) and (C) for the up- and down-regulated genes respectively (*** P <0.001, unpaired Student t-test).
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Table 2. Differentially expressed genes for Primary Tumor Xenograft (stage 4 disease) and Bone Marrow (BM) or Myocardium (Myoc) metastatic neuroblasts.
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These data show that GALNT13 (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, ELAVL1, EFNB1, NRG2, NELL1, MDK, GFRA1), synaptogenesis and steroid biosynthesis (SYCP2, PCDH17, STX1B2, SCG5, Nstage 4 disease1, GATA3, NR0B1).
The higher differentially up-regulated gene in this set is GALNT13 (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 mucin-type 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 GALNT13 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
).
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Figure 2. Analyses of GALNT13 expression in stage 4 disease and metastatic neuroblasts performed by quantitative (A) and nested (B) RT-PCR show a higher level of mRNA in BM neuroblasts (*** P <0.001, unpaired Student t-test).
ppGalNAc-T13 protein expression was also increased in BM neuroblasts, as shown by western blotting analysis (C).
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detection of BM disseminated neuroblastoma cells using galnt13 mrna
To determine whether GALNT13 might be a potential marker for NB dissemination, we tested for malignant neuroblasts in BM from patients with various stages of NB by measuring GALNT13 expression. We compared our data with the conventional cytology data and the data corresponding to other proposed markers for NB MRD, i.e., TH, GD2 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 4S 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 GALNT13, 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 GALNT13 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
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survival analysis
We compared overall survival of 37 NB patients to their BM involvement as assessed by cytology, or GD2, TH, DDC, and GALNT13 transcript values. We have analyzed samples collected at diagnosis. According to a Kaplan-Meier analysis, the best correlation with a poor clinical outcome is observed for GALNT13 expression (P = 0.043) (Fig. 3
).
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Figure 3. Kaplan-Meier analysis for overall survival of 37 patients according to the expression of (A) GALNT13/T13, (B) GD2 synthase, (C) TH, (D) DDC, and (E) cytological analysis in BM samples at diagnosis.
The transcripts were normalized to appropriate housekeeping genes.
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Discussion
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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 GALNT13 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).
Our data show that considerable GALNT13 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 GALNT13 data are in accordance with those from TH, GD2 synthase, and DDC Q-PCR. However, in spite of specific GALNT13 mRNA detection, the lower limit of the detection of analyses appears less than optimal, and we conclude, therefore, that GALNT13 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.
GALNT13 is a glycosyltransferase specifically expressed in neuronal tissue(11). Our analysis of GALNT13 mRNA transcripts in various NB cell lines, as well as in favorable and high-risk neuroblastic tumors, showed that GALNT13 is highly expressed in malignant neuroblasts but is not correlated with MYCN amplification and/or expression (data not shown). This abnormally high GALNT13 expression could reflect specific alterations in the O-glycosylation process, as described for ppGalNAc-T3 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, angiogenesis, 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 GALNT13 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 GALNT13 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.
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.
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Acknowledgments
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The authors thank Dr. Vladimir Lazar and Gwennaëlle 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 amis de Marie-Sophie de St Cloud" for their generous gift. This work was supported by the Ligue contre le Cancer, Comité de Montbéliard, Université 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.
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Footnotes
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3 These authors have contributed equally to this work. 
1 Nonstandard abbreviations: NB, neuroblastoma; MRD, minimal residual disease; QRT-PCR, quantitative reverse transcription-PCR; CT, threshold cycle; TH, tyrosine hydroxylase; DDC, dopa decarboxylase; HPRT, hypoxanthine phosphoribosyl transferase; BM, bone marrow; Myoc, myocardium.
2 Human genes: MYCN, v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian); GALNT13, UDP-N-acetyl-
-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 13; ABCB1, ATP-binding cassette, 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-promoting factor 2); FMOD, fibromodulin; LUM, lumican; COL1A1, collagen, type I, 1; GPM6B, glycoprotein M6B; HOXB2, homeobox B2; ELAVL1, 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; GATA3, GATA binding protein 3; NR0B1, nuclear receptor subfamily 0, group B, member 1.
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Abstract
Introduction
