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Clinical Chemistry 52: 438-445, 2006. First published December 29, 2005; 10.1373/clinchem.2005.059485
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(Clinical Chemistry. 2006;52:438-445.)
© 2006 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Potential Application of ELAVL4 Real-Time Quantitative Reverse Transcription-PCR for Detection of Disseminated Neuroblastoma Cells

Katrien Swerts1,a, Barbara De Moerloose1, Catharina Dhooge1, Jo Vandesompele2, Claire Hoyoux3, Klaus Beiske4, Yves Benoit1, Geneviève Laureys1 and Jan Philippé5

1 Department of Pediatric Hematology and Oncology, 2 Center for Medical Genetics, and 5 Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium.
3 Department of Pediatric Hematology and Oncology, Hôpital de la Citadelle, Liège, Belgium.
4 Department of Pathology, Rikshospitalet, Oslo, Norway.

aAddress correspondence to this author at: Department of Pediatric Hematology and Oncology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. Fax 32-9-240-4985; e-mail Katrien.Swerts{at}UGent.be.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Reliable detection of neuroblastoma cells in bone marrow (BM) is critical because BM involvement influences staging, risk assessment, and evaluation of therapeutic response in neuroblastoma patients. Standard cytomorphologic examination of BM aspirates is sensitive enough to detect single tumor cells. Consequently, more sensitive and specific detection methods are indispensable.

Methods: We used real-time quantitative reverse transcription-PCR (QPCR) of the tyrosine hydroxylase (TH), GD2 synthetase (GALGT), and embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) genes to detect disseminated neuroblastoma cells. We assessed assay sensitivity by addition experiments and then analyzed 97 neuroblastic tumor, BM, peripheral blood (PB), or peripheral blood stem cell (PBSC) samples from 30 patients. The QPCR results were compared with those of a standardized immunocytochemical assay.

Results: The molecular markers were highly expressed in all evaluated tumor samples. In addition, 32%, 11%, and 38% of all BM, PB, and PBSC samples scored positive for TH, GALGT, or ELAVL4, respectively. The TH and ELAVL4 assays could detect 1 neuroblastoma cell in 106 mononuclear cells. By contrast, the GALGT QPCR assay could detect 1 neuroblastoma cell in 104 mononuclear cells. We assessed the potential prognostic value of TH, GALGT, and ELAVL4 QPCR by analyzing subsequent samples from 3 patients with stage 4 disease. Preliminary results indicated that persistence of high ELAVL4 expression has prognostic value.

Conclusions: ELAVL4 QPCR can be used to detect residual neuroblastoma cells in clinical samples. However, combination of several molecular markers and screening techniques should be considered to ensure reliable detection of rare neuroblastoma cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neuroblastoma (NB), 1 the most common extracranial malignant solid tumor in children, originates from sympathetic nervous tissue in the neural crest. The clinical behavior of NB ranges from a localized tumor with a good prognosis to disseminated disease with unfavorable outcome. Many clinical and biological factors have been shown to affect the prognosis of NB patients (1).

Evaluation of bone marrow (BM) metastasis is crucial for correct clinical staging and risk assessment at diagnosis. Detection of residual NB cells in BM is also important for monitoring therapeutic response during treatment. In addition, screening of autologous stem cell preparations is crucial because reinfusion of contaminated stem cell products could lead to systemic recurrence (2)(3)(4).

According to the International Neuroblastoma Staging System, conventional cytology of BM smears is still the only accepted technique for the detection of residual NB cells (5). The sensitivity of this method is limited, however, because tumor cells present at <0.1% (1 cell in a background of 1000 nontumor cells) cannot be detected by conventional cytomorphology (6)(7). The development of more sensitive and specific detection methods is therefore necessary.

During the last few decades, sensitive assays based on immunocytology (8)(9)(10)(11), automatic immunofluorescence plus fluorescence in situ hybridization (12), and flow cytometry (13)(14)(15)(16)(17) have been evaluated. In addition, several molecular tests based on reverse transcription-PCR have been developed, and the usefulness of tumor-specific gene transcripts such as tyrosine hydroxylase (TH) (18)(19)(20), GAGE (21)(22), MAGE-1 to -4 (23), neuroendocrine protein PGP 9.5 (24)(25), GD2 synthetase (GALGT; ß1,4-N-acetylgalactosaminyltransferase) (26)(27), dopamine decarboxylase(28), chromogranin A (29), and doublecortin(30) has been assessed.

Most NBs (90%–95%) are characterized by high catecholamine production. The first and rate-limiting step in the biosynthesis of catecholamines is catalyzed by TH; this enzyme is therefore often used as a molecular marker for the detection of disseminated NB cells in peripheral blood (PB), BM, and peripheral blood stem cell (PBSC) preparations.

GALGT catalyzes the transfer of 1,4-N-acetylgalactosamine to GD3 ganglioside and plays a key role in GD2 disialoganglioside biosynthesis (31). Because GALGT is strongly expressed in neural crest-derived tumors such as melanoma and NB (32), the enzyme is frequently used as a molecular marker for detection of minimal residual disease (MRD) in NB.

Embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4), or Hu antigen D (HuD), which belongs to the elav gene family of Drosophila melanogaster, has been reported to be highly specific for neuroectodermally derived tumors (33), but to date, the use of ELAVL4 mRNA for MRD detection in NB has not been reported.

In this study, we developed and evaluated real-time quantitative reverse transcription-PCR (QPCR) tests for TH, GALGT, and ELAVL4 mRNA. We assessed the sensitivity of the assays by use of both addition experiments and clinical samples. Thirty PB or BM samples from patients without malignant disease were included as negative controls. Expression of the TH, GALGT, and ELAVL4 genes was analyzed in 18 tumor, 70 BM, 5 PB, and 4 PBSC samples collected at diagnosis or during treatment. Finally, the molecular results were compared with those of a standardized anti-GD2 immunocytochemical (IC) reference assay.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and sample preparation
Between April 2001 and October 2004, we examined 18 tumor, 70 BM, 5 PB, and 4 PBSC samples, taken at diagnosis or during treatment, from 1 patient with ganglioneuroblastoma (GNB), 1 patient with ganglioneuroma (GN), and 28 patients with NB. Bilateral BM samples were considered as 2 different samples because they were analyzed separately. Patient characteristics are summarized in Table 1 . MYCN and 1p status was evaluated by fluorescence in situ hybridization according to the guidelines of the European Neuroblastoma Quality Assessment group (34).


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  		Table 1. Patient characteristics.

The NB patients were diagnosed and staged according to the International Neuroblastoma Staging System (5). Treatment depended on the patient’s age, tumor stage, and biological risk factors. The Ethics Committee approved the study, and informed consent was obtained from the patients and/or the patients’ parents.

From fresh tumor samples, we prepared single-cell suspensions, using collagenase (1000 kU/L in RPMI for 1 h at 37 °C). Cells were subsequently washed, and cell clumps were removed by use of a 70 µm nylon membrane filter.

BM, PB, and PBSC samples were collected into EDTA-containing tubes. Immediately after collection, mononuclear cells were isolated by density gradient centrifugation on Fycoll-Hypaque (Nycomed). All cells were resuspended in 750 mL/L ethanol and stored at –80 °C until analysis.

control samples
The NB cell lines SJ-NB-10, SJ-NB-8, NMB, STA-NB-3, STA-NB-8, STA-NB-10, SK-N-BE, SK-N-SH, and CLB-GA were included in the experiments as positive controls. BM and PB samples from 30 adults without malignant disease were used as negative controls.

sensitivity
We performed addition experiments to evaluate the sensitivity of the TH, GALGT, and ELAVL4 QPCR assays. RNA from the NB cell line IMR32 was added to 1 µg of RNA isolated from healthy PB mononuclear cells. Five 10-fold dilutions containing 10 000 to 10 pg of IMR32 RNA were analyzed.

We also assessed the sensitivity of the molecular assays by use of patient samples. QPCR results were compared with those of an IC reference assay (detection limit, 1 NB cell in a background of 106 cells), which is the generally accepted method (11). All samples were handled as described below.

rna isolation and reverse transcription
Total RNA was extracted from 5 x 106 to 1 x 107 cells by use of TRIzol Reagent (Invitrogen). The cells were pelleted by centrifugation and lysed by addition of 800 µL of TRIzol Reagent. In addition, 10 µL of tRNA from baker’s yeast (1 mmol/L) and 160 µL of chloroform (Sigma-Aldrich) were added. The homogenized samples were incubated for 15 min at room temperature, and after centrifugation at 12 000g for 15 min at 4 °C, the aqueous phase was transferred to a fresh tube and 400 µL of isopropyl alcohol (Sigma) was added. The samples were incubated for 10 min at room temperature and centrifuged at 12 000g for 10 min at 4 °C. The supernatant was removed, and the RNA pellet was washed with 800 µL of 750 mL/L ethanol (Merck). After centrifugation at 7500g for 6 min at 4 °C and removal of the supernatant, the RNA pellet was air-dried and resuspended in RNase-free water. The concentration and purity of the recovered RNA were determined by the absorbance at 260 and 280 nm. The integrity of the isolated RNA was verified by QPCR for human ß2-microglobulin (B2M) and ubiquitin C (UBC).

Total RNA (1 µg in 5 µL) was denatured at 95 °C for 5 min in an OmniGene thermal cycler (Thermo Electron) and subsequently placed on ice. An equal volume of the reverse transcription reaction mixture (5 µL) was added. Each 10-µL reaction contained (final concentration) 1 mM each deoxynucleotide triphosphate (Amersham Biosciences), 8 mM MgCl2 (Sigma-Aldrich), 0.3 µg of Random Hexamer Primers (Invitrogen), 8 units of RNA Guard (Amersham Biosciences), and 10 U of Moloney murine leukemia virus reverse transcriptase (Amersham Biosciences) in 1x TaqMan Buffer A (Applied Biosystems). The RNA was reverse-transcribed at 37 °C for 1 h, after which the enzyme was inactivated by incubation at 95 °C for 5 min.

qpcr
Detection chemistry.
TH, GALGT, and ELAVL4 transcripts were quantified by use of the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). A fluorogenic probe, labeled with a reporter (6-carboxyfluorescein) at the 3' end and a quencher (6-carboxytetramethylrhodamine) at the 5' end, was used. All primers and probes were designed by use of Primer Express software (Applied Biosystems; see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue3/). To avoid the amplification of genomic DNA, primers and probes were designed to be complementary to successive exons, which are not influenced by alternative splicing.

Quantification.
To construct relative calibration curves for the quantification of TH, GALGT, and ELAVL4 transcripts, we used serial dilutions of cDNA from the NB cell line CLB-GA. For each assay, we analyzed 10-fold serial dilutions, starting from a relative copy number of 100 000 to a relative copy number of 10, in duplicate (see Fig. 1A in the online Data Supplement). Calibration curves were constructed by plotting the threshold cycle vs the logarithm of the relative copy number (see Fig. 1B in the online Data Supplement).

Normalization.
Accurate normalization of gene expression is a prerequisite to obtain reliable results. Because the expression of a single housekeeping gene can vary considerably, several reference genes should be combined to calculate a normalization factor (35). In this study, we normalized TH, GALGT, and ELAVL4 transcript numbers to 2 reference genes, B2M (36) and UBC, by dividing the relative transcript numbers of the target genes by the geometric means of the transcript numbers of the 2 reference genes.

PCR conditions.
All PCR reactions were performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems). A 3 x 3 primer matrix (combination of 300, 600, and 900 nM each of the forward and reverse primers) was analyzed to determine the optimal primer concentrations. In addition, 3 different MgCl2 concentrations (3, 5, and 7 mM) were evaluated. The concentrations giving the lowest threshold cycle and the highest fluorescent signal were chosen.

PCR samples were prepared as follows: 5 µL of cDNA was added to the reaction mixture, and every sample was analyzed in duplicate. The amplification mixture for TH (25 µL) contained 1x TaqMan Buffer A, 5 mM MgCl2, 1.2 mM each deoxynucleoside triphosphate, 150 nM probe, 600 nM each of the forward and reverse primers, and 0.6 U of AmpliTaq Gold (Applied Biosystems). The amplification mixtures for GALGT and ELAVL4 (25 µL) contained 1x TaqMan Buffer A, 7 mM MgCl2, 1.2 mM each deoxynucleoside triphosphate, 150 nM probe, 600 nM each of the primers, and 0.6 U of AmpliTaq Gold.

Because of their high expression, B2M and UBC were more efficiently amplified in 50 µL than in 25 µL. The reaction mixture (50 µL) contained 1x TaqMan Buffer A, 5 mM MgCl2, 1.2 mM each of the deoxynucleoside triphosphates, 150 nM probe, 600 nM each of the primers, and 1.25 U of AmpliTaq Gold.

The PCR conditions were 10 min at 95 °C, 50 cycles at 95 °C for 30 s, and 60 °C for 1 min.

immunocytochemistry
Cytospins were immunocytochemically stained as described previously (11). Briefly, we prepared large-diameter (17 mm) cytospins containing 5 x 105 mononuclear cells by centrifuging no more than 7 x 105 cells down on precoated (e.g., poly-L-lysine) glass slides in a Hettich centrifuge (Hettich Zentrifugen). The slides were air-dried overnight and stored in air-tight boxes at –24 °C until IC was performed. Before staining, the slides were thawed in closed boxes to avoid the formation of condensation. After fixation with 40 g/L p-formaldehyde for 10 min, the slides were incubated for 30 min with 30 µL of an unlabeled monoclonal mouse anti-human GD2 disialoganglioside antibody (clone 14.G2a; BD Biosystems) diluted 1:100 in 10 g/L bovine serum albumin (BSA; Gibco) in 1x phosphate-buffered saline (Gibco). Controls in which the primary antibody was replaced by an isotypic IgG2a control antibody (Dako Corporation) were included to evaluate nonspecific staining. During a second and third incubation step, cells were incubated for 30 min with 30 µL of an unlabeled rabbit anti-mouse antibody (Dako Corporation) diluted 1:20 in 10 g/L BSA in 1x phosphate-buffered saline and 30 µL of the APAAP complex (Dako Corporation) diluted 1:20 in 10 g/L BSA in 1x phosphate-buffered saline, respectively. The bound alkaline phosphatase complexes were stained using the Fuchsin+TM Substrate Chromogene System (Dako Corporation), prepared as indicated by the manufacturer. The cells were counterstained with hematoxylin (Sigma) until an appropriate blue nuclear stain was obtained. Finally, slides were examined under a light microscope by 2 independent observers. Preferably 6 slides, each containing 5 x 105 cells, were analyzed. Cells with a round nucleus, a granular chromatin structure, a scarce amount of cytoplasm, and strong staining around the entire cell were considered positive. Cells with an aberrant morphology or pink staining restricted to a subcellular compartment were called negative. The numbers of positive single cells and positive cells that were part of a Homer Wright rosette or cell clump were counted. The number of evaluated mononuclear cells was also reported. When the 2 local observers disagreed or when fewer than 10 positive cells were found, samples were reviewed by a panel of external experts. We were able to detect 1 NB cell in 106 mononuclear cells with this IC staining method (11).

statistics
After classification of the results in a frequency table, we performed a {chi}2 test with Yates correction for continuity to study the relationship between the different categories. The Fisher exact test was computed when a cell in the 2 x 2 table had an expected frequency <5, and a {kappa} test was used to measure the agreement among the different assays. In addition, Pearson correlation coefficients were calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
expression of molecular markers in negative control samples
TH, GALGT, and ELAVL4 expression was evaluated in 15 PB and 15 BM samples from patients without malignant disease (see Table 2 in the online Data Supplement).

Because TH, GALGT, and ELAVL4 transcripts were detected in healthy PB and BM samples, a threshold for positivity, based on the mean normalized transcript number and the standard deviation, was defined. All clinical samples with a normalized relative transcript number higher than the mean + 4 SD were considered positive.

sensitivity of the qpcr assays
We assessed the sensitivity of the TH, GALGT, and ELAVL4 QPCR assays by means of addition experiments and use of clinical samples.

Different amounts of IMR32 RNA were added to 1 µg of RNA isolated from healthy PB mononuclear cells. Five 10-fold dilutions (10 000 to 10 pg of IMR32 RNA) were evaluated. The TH and ELAVL4 QPCR assays could detect 10 pg of IMR32 RNA in 1 µg of normal RNA. The detection limit of the GALGT assay was higher; only 100 pg of IMR32 RNA was reliably detected.

Both QPCR and IC were used to screen for residual NB cells in patient samples. TH and ELAVL4 transcripts were detected in clinical samples containing NB cells at the concentration of 1 in 106 mononuclear cells. The detection limit of the GALGT QPCR assay was higher; no GALGT transcripts were found in clinical samples containing <100 NB cells per 106 mononuclear cells.

reproducibility
To evaluate the within- and between-run reproducibility of the ELAVL4 QPCR assay, we added RNA from the NB cell line CLB-GA to RNA isolated from healthy PB mononuclear cells and analyzed three 100-fold dilutions containing 1 µg to 100 pg of CLB-GA RNA 5 times. The within-run reproducibility [mean (SD) relative transcript number (CV)] for the 3 dilutions was as follows: 3.73 (1.40) x 10–1 (37%); 1.96 (0.22) x 10–3 (11%); and 4.16 (1.02) x 10–5 (25%). The between-run reproducibility was 3.23 (0.46) x 10–1 (14%), 1.42 (0.28) x 10–3 (20%), and 4.33 (1.32) x 10–5 (31%).

ELAVL4 expression in cell lines
We evaluated ELAVL4 expression in 8 NB cell lines (SJ-NB-10, SJ-NB-8, NMB, STA-NB-3, SK-N-SH, STA-NB-10, SK-N-BE, and STA-NB-8), a Ewing sarcoma cell line (SK-N-MC), a breast cancer carcinoma cell line (MCF-7/AZ), a colon carcinoma cell line (HCT-8/S11), a chronic myelogenous leukemia cell line (K562), a lymphoma cell line (Daudi), and an adrenal gland cortex carcinoma (AGCC) sample. ELAVL4 was highly expressed in all evaluated NB cell lines. The expression in the SK-N-MC, Daudi, HCT-8/S11, and MCF-7/AZ cell lines was lower (SK-N-MC, Daudi, HCT-8/S11, and MCF-7/AZ) or undetectable (AGCC and K562). The mean normalized transcript number in the NB cell lines was 1.36, whereas the mean normalized transcript number in the other cell lines and tumor sample was 7.59 x 10–4.

TH, GALGT, and ELAVL4 expression in clinical samples
We evaluated TH, GALGT, and ELAVL4 expression in 18 tumor, 70 BM, 5 PB, and 4 PBSC samples from 1 GNB patient, 1 GN patient, and 28 NB patients. The 3 markers were highly expressed in all primary tumors (1 stage 1, 1 stage 2, 6 stage 3, 7 stage 4, 1 stage 4S, and 1 GNB). Two tumor samples from NB patients without increased catecholamine also scored positive for TH. In addition, 32% (25 of 79), 11% (9 of 79), and 38% (30 of 79) of all BM, PB, and PBSC samples scored positive for TH, GALGT, or ELAVL4, respectively (see Table 3 in the online Data Supplement). On the basis of these results, we found a clear association between the different QPCR assays: TH and GALGT, Fisher exact test, P <0.0001; {kappa} = 0.44; TH and ELAVL4, {chi}2 = 20.15 (P <0.0001); {kappa} = 0.53; GALGT and ELAVL4, Fisher exact test, P <0.0001; {kappa} = 0.35. We also calculated the correlation between the different molecular markers: TH and GALGT, r = 0.14; P = 0.22; TH and ELAVL4, r = 0.94; P <0.0001; GALGT and ELAVL4, r = 0.46; P <0.0001.

comparison of qpcr with ic
We compared the QPCR results for 68 BM, PB, and PBSC samples with those obtained with an anti-GD2 IC assay. The latter was considered to be the generally accepted method. When we compared the TH and ELAVL4 QPCR results with the IC results, we found only a few discordant results. The association between the QPCR and IC results was good: TH QPCR and IC, {chi}2 = 33.74; P <0.0001; {kappa} = 0.74; ELAVL4 QPCR and IC, {chi}2 = 37.27; P <0.0001; {kappa} = 0.77; Table 2 ).


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  		Table 2. Comparison of QPCR and IC results.

When we compared GALGT QPCR and IC, we found more discordant results. Fourteen samples were QPCR–/IC+, whereas none were QPCR+/IC–. This reflects the difference in sensitivity between the assays. The association between GALGT QPCR and IC was moderate (Fisher exact test, P <0.0001; {kappa} = 0.46).

We also analyzed 14 diagnostic BM samples from 9 patients with stage 4 disease (see Table 4 in the online Data Supplement). Seven samples scored positive for all 3 markers. The NB cells were also detected by IC and conventional cytomorphologic analysis. In 1 BM sample, only TH and ELAVL4 were highly expressed, and another sample scored positive only for TH QPCR. In these samples, no disseminated NB cells were found by IC or cytomorphologic analysis. Five BM samples were negative for QPCR, IC, and cytomorphology.

We also evaluated TH, GALGT, and ELAVL4 expression in 12 diagnostic BM samples from 7 patients with stage 3 disease. None of them scored positive for all 3 markers; however, 5 BM samples from 3 patients with a stage 3 NB without MYCN amplification scored positive for TH or ELAVL4 at diagnosis. All samples were negative for IC and cytomorphology. In 1 of these patients, a distal pathologic lymph node was detected after diagnosis, suggesting that this patient was in stage 4 instead of stage 3. Fourteen months after diagnosis, an additional BM sample from this patient was analyzed, but no residual NB cells were found. All 3 patients were still alive and in complete remission at the conclusion of this study.

potential prognostic value of qpcr detection of disseminated nb cells
To investigate whether the detection of TH, GALGT, and ELAVL4 transcripts could be used to study MRD in NB, we studied 30 subsequent BM, PB, or PBSC samples from 3 patients with stage 4 disease. The samples were taken at diagnosis, during treatment, at relapse, or during follow-up. We compared the QPCR results with IC and cytomorphologic data (see Table 4 in the online Data Supplement). In 14 of 30 samples (47%), the results were concordant. Four samples scored positive in each assay. In 10 samples, no residual NB cells were found.

Patient 19, a 6-year-old boy diagnosed with a stage 4 NB with a 1p deletion and no MYCN amplification, repeatedly tested negative at different time points during therapy. This boy was still alive and in complete remission at the end of the study.

In all PB and BM samples (n = 6) from patient 26, a 6-month-old girl with a stage 4 NB without MYCN amplification or 1p deletion, residual NB cells were detected by IC, TH QPCR, and/or ELAVL4 QPCR. This patient was in complete remission at the conclusion of this study; however, the follow-up time was short (3 months) at that point.

Patient 27, a 7.5-year-old girl suffering from a stage 4 NB with MYCN amplification and 1p deletion, repeatedly tested positive by IC, TH QPCR, and ELAVL4 QPCR. In fact, ELAVL4 was highly expressed in every PB or BM sample taken during therapy. This child relapsed 14 months after diagnosis and died of malignant disease shortly thereafter.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The detection of residual NB cells in BM has important therapeutic and prognostic implications because BM disease is usually associated with unfavorable outcome (37)(38). Moreover, the detection of disseminated tumor cells in autologous stem cell preparations may signify the need for ex vivo manipulations such as tumor cell purging and/or CD34+ cell selection (39). In the 4 hematopoietic stem cell preparations analyzed in this study, no disseminated NB cells were detected by QPCR or IC.

Cytomorphologic screening of BM smears is still the only accepted technique for the detection of disseminated NB cells. The sensitivity of this method is limited, however. Therefore, alternative approaches, based on immunology or molecular biology, have been developed to improve the detection of NB cells present in very low numbers.

In this study, we used QPCR for TH, GALGT, and ELAVL4 to detect NB cells. TH and GALGT are frequently used molecular markers for MRD detection in NB, whereas the use of ELAVL4 QPCR has not been described previously in the literature. ELAVL4 plays an essential role in the development of the nervous system and is highly expressed in neuroectodermally derived tumors (33). By contrast, ELAVL4 expression in hematopoietic cells is very low. Consequently, ELAVL4 is a potential marker for tumor cell detection in PB, BM, and PBSC samples from patients with NB.

QPCR offers several advantages over other methods. In contrast to conventional PCR, no labor-intensive post-PCR processing such as gel electrophoresis and hazardous radioactive hybridization is needed to detect and quantify PCR products. In addition, carryover contamination is minimized because both amplification and detection are performed within a closed system.

We assessed TH, GALGT, and ELAVL4 expression in 17 primary NB tumors. Each tumor sample scored positive for all 3 markers. Moreover, ELAVL4 was strongly expressed in 8 NB cell lines. These findings suggest the potential application of ELAVL4 QPCR in the study of NB.

We also evaluated expression of these 3 molecular markers in 79 BM, PB, or PBSC samples; 32%, 11%, and 38% of the samples scored positive for TH, GALGT, or ELAVL4, respectively. Results were concordant for 52 of 79 samples, whereas results were discordant for 34% of the samples. TH transcripts were found in 6 GALGT–/ELAVL4– samples. Eleven samples scored positive only for ELAVL4 QPCR. No GALGT transcripts were detected in 10 TH+/ELAVL4+ samples. Some of these samples might be false positive, but the discrepancies could also be attributable to sample variability or differences in sensitivity.

Using addition experiments, we demonstrated that 10 pg of IMR32 RNA in 1 µg of healthy PB mononuclear cell RNA could be detected by TH or ELAVL4 QPCR. The detection limit of the GALGT assay was ~10-fold higher. We also assessed the sensitivity of the molecular assays by use of patient samples, comparing QPCR results with those obtained by the standardized IC assay considered the generally accepted method. QPCR detected TH and ELAVL4 transcripts in clinical samples containing NB cells at a concentration of 1 in 106 mononuclear cells. This is in accordance with previously published data (40). The detection limit of GALGT QPCR was higher; no GALGT transcripts were found in clinical samples containing up to 100 NB cells per 106 mononuclear cells. These results are in contrast with those published by Cheung and coworkers (41)(42). They used GALGT QPCR to evaluate the efficacy of adjuvant immunotherapy in NB patients; the assay was able to detect 1 NB cell in 106 healthy mononuclear cells. From our data, we conclude that ELAVL4 and TH QPCR can be used to study BM involvement in clinical samples collected during therapy or follow-up. By contrast, GALGT QPCR may be not sensitive enough to study MRD and can be used only at diagnosis or relapse.

When we compared TH or ELAVL4 QPCR with IC, we found only a few discordant results. The discrepancies were probably attributable to sample variability (related to Poisson law) because only a few TH or ELAVL4 transcripts were detected in the QPCR+/IC– samples. In addition, we found <10 NB cells per 106 mononuclear cells in QPCR–/IC+ samples.

Discordant results were more numerous when we compared GALGT QPCR and IC. Fourteen samples scored positive in the IC assays, but no GALGT transcripts were detected. This reflects the difference in detection limits between the assays (1 NB cell in 106 mononuclear cells for IC and 1 NB cell in 104 mononuclear cells for GALGT QPCR).

We assessed the potential prognostic value of the detection of TH, GALGT, and ELAVL4 transcripts by analyzing 30 subsequent BM, PB, and PBSC samples from 3 patients with stage 4 NB. One patient repeatedly tested negative at different time points during therapy and was still alive and disease free at the end of his treatment. In 6 PB and BM samples from the second patient, residual NB cells were detected by IC, TH QPCR, and/or ELAVL4 QPCR. This child was still in complete remission at the conclusion of this study, but the follow-up time was short (3 months). The third patient repeatedly tested positive by IC, TH QPCR, and ELAVL4 QPCR. In fact, ELAVL4 was highly expressed in every PB or BM sample taken during therapy. This child relapsed 14 months after diagnosis and died of malignant disease shortly thereafter. These preliminary data suggest that the persistence of high ELAVL4 expression has prognostic value. However, additional multicenter studies with large groups (n >100) of homogeneously treated patients are needed to confirm these findings.

In conclusion, QPCR for ELAVL4 mRNA may be applicable to the study of MRD in NB, but the combination of multiple molecular markers (e.g., ELAVL4 and TH) with IC should be considered to avoid false-positive or -negative results. Preliminary results indicate that persistence of high ELAVL4 expression has a prognostic value. However, additional studies with larger patient groups are required to confirm these results.


   Acknowledgments
 
We thank Prof. Dr. F. Speleman for providing the NB cell lines CLB-GA and IMR32. In addition, we gratefully acknowledge Prof. Dr. B. Verhasselt for technical assistance and use of the ABI PRISM 7700 Sequence Detection System. We are grateful to D. Claeys for skillful assistance in the IC assays. We also thank Drs. B. Brichard, C. De Valck, N. Francotte, E. Michiels, A. Uyttebroeck, and J. van der Werff for providing us with samples from NB patients. Katrien Swerts was supported by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). Jo Vandesompele is supported by a postdoctoral research grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). This work was funded by the Children’s Cancer Fund "Kinderkankerfonds".


   Footnotes
 
1 Nonstandard abbreviations: NB, neuroblastoma; BM, bone marrow; TH, tyrosine hydroxylase; PB, peripheral blood; PBSC, peripheral blood stem cell; GALGT, GD2 synthetase; MRD, minimal residual disease; ELAVL4, embryonic lethal, abnormal vision, Drosophila-like 4; QPCR, real-time quantitative reverse transcription-PCR; IC, immunocytochemistry; GNB, ganglioneuroblastoma; GN, ganglioneuroma; B2M, ß2-microglobulin, UBC, ubiquitin C; and BSA, bovine serum albumin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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