Originally published as JCO Early Release 10.1200/JCO.2007.13.6531 on October 6 2008

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PHOX2B Is a Novel and Specific Marker for Minimal Residual Disease Testing in Neuroblastoma

Janine Stutterheim, Annemieke Gerritsen, Lily Zappeij-Kannegieter, Ilona Kleijn, Rob Dee, Lotty Hooft, Max M. van Noesel, Marc Bierings, Frank Berthold, Rogier Versteeg, Huib N. Caron, C. Ellen van der Schoot, Godelieve A.M. Tytgat

From the Department of Pediatric Oncology, Emma Children's Hospital; Dutch Cochrane Centre; Department of Human Genetics, Academic Medical Center; Sanquin-AMC (Academic Medical Center) Landsteiner Laboratory, Amsterdam; Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Erasmus Medical Center, Rotterdam; Department of Pediatric Hematology, Wilhelmina Children's Hospital, Utrecht Medical Center, Utrecht, the Netherlands; and Department of Pediatric Oncology and Hematology, Children's Hospital, University of Cologne, Germany

Corresponding author: C. Ellen van der Schoot, MD, PhD, Plesmanlaan 125,1066 CX Amsterdam, the Netherlands; e-mail: e.vanderschoot{at}sanquin.nl

	ABSTRACT

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Purpose Polymerase chain reaction (PCR)–based detection of minimal residual disease (MRD) in neuroblastoma can be used to monitor therapy response and to evaluate stem cell harvests. Commonly used PCR markers, tyrosine hydroxylase (TH) and GD2 synthase, have expression in normal tissues, thus limiting MRD detection. To identify a more specific MRD marker, we tested PHOX2B.

Patients and Methods To determine PHOX2B, TH, and GD2 synthase expression in normal tissues, it was measured by real-time quantitative PCR in samples of normal bone marrow (BM; n = 51), peripheral blood (PB; n = 37), and peripheral-blood stem cells (PBSCs; n = 24). Then, 289 samples of 101 Dutch patients and 47 samples of 43 German patients were tested for PHOX2B and TH; these samples included 52 tumor, 214 BM, 32 BM, and 38 PBSC harvests. Of the 214 BM samples, 167 were compared with cytology, and 47 BM samples were compared with immunocytology (IC).

Results In contrast to TH and GD2 synthase, PHOX2B was not expressed in any of the normal samples. In patient samples, PHOX2B was detected in 32% cytology-negative and in 14% IC-negative samples and in 94% of cytology-positive and in 90% of IC-positive BM samples. Overall, PHOX2B was positive in 43% compared with 31% for TH. In 24% of all samples, TH expression was inconclusive, which is similar to expression found in normal tissues. In 42% of these samples, PHOX2B expression was positive.

Conclusion PHOX2B is superior to TH and GD2 synthase in specificity and sensitivity for MRD detection of neuroblastoma by using real-time quantitative PCR. We propose to include PHOX2B in additional prospective MRD studies in neuroblastoma alongside TH and other MRD markers.

	INTRODUCTION

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Neuroblastoma (NB) accounts for 10% of all childhood cancers. Approximately 40% of patients with NB suffer from high-risk disease that has dissemination in bone marrow (BM), bone, distant lymph nodes, liver, and/or other organs. In patients older than 1 year, the presence of marrow disease is a strong indicator of high-risk NB; despite intensive treatment schedules, this has a poor prognosis.^1,2 The current golden standard to measure BM infiltration (ie, cytology) is mostly unable to detect tumor cell infiltration less than the level of 0.1%.^3,4 Therefore, more sensitive techniques have been developed for monitoring of minimal residual disease (MRD), such as real-time quantitative polymerase chain reaction (RQ-PCR)⁵ and immunocytology (IC).⁶ Anti-GD2 IC is used in clinical practice and can detect one single GD2-positive cell among 10⁶ or more mononuclear cells. Molecular approaches that use RQ-PCR have the same sensitivity as IC.^7,8

The ideal MRD marker is tumor specific and has no expression in the normal compartments, such as BM, peripheral blood (PB), and peripheral-blood stem cells (PBSCs). No specific NB marker has been found yet. Anti-GD2 IC analysis is highly sensitive, but GD2 is also expressed on normal cells.⁹ Cytogenetic analysis of immunologically positive cells increases specificity significantly.⁶ With RQ-PCR, the most applied PCR targets, tyrosine hydroxylase (TH)^5,7,10,11 and GD2 synthase,^12,13 also show low expression in a sizable fraction of normal tissues.^5,7,8,12-16

For other targets, like GAGE,^17,18 MAGE,¹⁹ PGP9.5,^20,21 DDC,²² CyclinD1,²³ ELAVL4,⁸ and ST8Siall,²⁴ background expression has also been described. The presence of marker-transcripts in normal BM limits the sensitivity of MRD detection.¹⁴ Therefore, identification of better molecular markers without expression in normal tissues is important. Here, we describe a new, specific marker for MRD detection of NB mRNA, PHOX2B. This marker was validated in normal BM, PB, and PBSC samples and in a clinical series of BM aspirates of patients with NB and was found to be superior to the commonly used markers.

	PATIENTS AND METHODS

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Patients and Samples
Retrospectively, all available Dutch samples between 1986 and 2007 (52 tumor samples, 60 BM samples at diagnosis, 107 BM samples during treatment, 38 PBSC harvests, and 32 BM harvests) were collected from 101 patients (six with ganglioneuroblastoma, 95 with NB). Patients were treated at Emma Children's Hospital/Academic Medical Center, Amsterdam, or Sophia Children's Hospital/Erasmus Medial Center, Rotterdam, the Netherlands, according to disease stage (staged according to the International Neuroblastoma Staging System²⁵). Patients' characteristics, clinical data, and genetic data are listed in Table 1, and individual clinical data, genetic data, and origin of the samples are listed in Appendix Table A1 (online only). BM punctures were performed at diagnosis (staging), at designated time points during treatment (morphology to determine disease status), and before high-dose chemotherapy, according to the treatment protocol. After informed consent was given, stored remains were used for this study (research purposes). The study was approved by the Medical Research Ethics Committee of the Academic Medical Center.

In total, 336 clinical samples of 144 patients with NB (289 samples of 101 Dutch patients and 47 samples 43 of GPOH patients) were analyzed with RQ-PCR.

Control Samples
To determine background expression levels of TH, GD2 synthase, and PHOX2B, normal BM samples (n = 51) were collected from 51 children during or after treatment for acute leukemia in molecular remission (determined by antigen-receptor RQ-PCR).²⁶ PB (n = 37) and PBSC (n = 24) samples were obtained from 61 healthy volunteers and patients (adults and children) in complete remission (CR) of malignancies other than NB.

IC
Cytospins were immunocytologically stained and were evaluated by the BM laboratory of the GPOH NB group (Cologne, Germany) according to the standardized European BM method.²⁷

RNA Extraction and Reverse Transcription
Total cellular RNA from tumor, BM, PB, and PBSC samples (5 to 10 million cells) was extracted with the RNAbee (Campro Scientific, Veenendaal, the Netherlands) according to the manufacturer's instructions. cDNA was synthesized (45 minutes, 42°C) by using 1 µg of total RNA, random hexamers (25 µmol/L; Gibco-BRL, Life Technologies, Breda, the Netherlands), dNTPs (1 mmol/L; Promega, Leiden, the Netherlands), and M-MLV reverse transcriptase (100 U; Gibco-BRL, Life Technologies, Breda, the Netherlands), to provide a total reaction volume of 20 µL. Finally, the reverse transcriptase was inactivated by heating (3 minutes, 99°C), and the volume was diluted to 50 µL.

Primer/Probes
Primers and probes for GD2 synthase and PHOX2B were designed by using Primer Express 1.5 (Applied Biosystems, Foster City, CA) and Oligo 6 (Molecular Biology Insights Inc, Cascade, CO) on the basis of published gene sequences (ie, National Center of Biotechnology Information and Celera databases). All amplicons spanned an intron of at least 500 base pairs (bps), and no amplification of genomic DNA was observed. Primers and probes were synthesized by Eurogentec (Liege, Belgium). Primer-probe combinations for PHOX2B were as follows: forward primer, 5'-GGC-ACC-CTC-AGG-GAC-CA-3'; reverse primer, 5'-CTG-CGC-GCT-CCT-GCT-T-3'; and probe, 5'FAM-CCA-GAA-CCG-CCG-CGC-CAA-3'TAMRA. Primer-probe combinations for GD2 synthase were as follows: forward primer, 5'-CTGGACCAA-CTC-AAC-AGG-CAA-3'; reverse primer, 5'-CAT-GTC-CCT-CGG-TGG-AGA-A-3'; and probe, 5'FAM-TAC-AAC-TGG-TCA-CTT-ACA-GCA-GCC-GAA-GC-3'TAMRA. The primer/probe combination for TH and β-glucuronidase (GUS) have been described before.^8,28

RQ-PCR
RQ-PCR was performed in an ABI PRISM 7900 (PE Biosystems, Darmstadt, Germany). Reactions were carried out in 25 µL (12.5 µL TaqMan Universal PCR Mastermix; (Applied Biosystems, Nieuwekerk a/d IJssel, the Netherlands), 4.5 µL H₂O, 300 nmol/L forward and reverse primer, 200 nmol/L TaqMan probe, 5 µL cDNA (input 100 ng) and started with 10 minutes at 95°C followed by 50 cycles (15 seconds on 95°C followed by 1 minute on 60°C).

The housekeeping gene GUS was used as the gene for normalization, because it was stably expressed in all NB tumors tested and because its expression was equal to that in hematologic cells.²⁸ To correct for differences in the amount of total RNA input and for reverse transcription efficiency, the quantity of the marker transcript was normalized to the amount of GUS gene transcripts (normalized Ct [Ct] = Ct GUS – Ct marker). All RQ-PCR experiments were carried out at least in duplicate, and the mean values were used. The number of GUS copies was determined by absolute quantification with GUS-plasmid DNA (Ipsogen, Marseille, France) dilutions to generate a standard curve. Samples with a low input of GUS (Ct > 25, which corresponds to < 5,000 copies) were excluded. The mean Ct of GUS for all samples tested was 22.6 ± 1.4.

Assay Sensitivity by In Vitro Serial Dilutions
Sensitivity of the RQ-PCR assays were assessed by seeding N206 and NGP NB cells into PB cells (one tumor cell in 10² to 10⁷ nucleated normal cells) to obtain a dilution range from 10^–2 to 10^–7. Sensitivity was defined as the lowest dilution in which the mean Ct value was 3.0 Ct lower than the mean Ct of normal PB (equal to background). The quantitative range was defined as the lowest dilution to give a reproducible amplification (Ct of all replicates 1.5), with all Ct values 3.0 lower than mean Ct of background. These criteria have been adapted from those defined by the European Study Group on MRD detection in acute lymphatic leukemia (ALL).²⁹

Data and Statistical Analysis
When a marker showed amplification in normal tissue, a threshold for positivity was determined. This threshold was defined by using the rules adapted from the European Study Group on MRD detection in ALL.²⁹ Clinical samples were scored as positive if the Ct value was less than 40 and mean Ct was 3.0 Ct values lower than the mean Ct of the normal tissue. Samples with Ct values of 40 or of one Ct above the background were scored as negative. Samples with a Ct value between these were scored as inconclusive and were in the background range. The mean Ct in normal tissues was determined by averaging the Ct values of samples that showed amplification. The Ct values were expressed as means ± standard deviations. Expression levels were compared by using a paired t test.

	RESULTS

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Selection of PHOX2B As a Candidate MRD Marker
To identify a new MRD marker for NB, we compared mRNA expression profiles of 11 NB tumors and cell lines with normal tissues obtained by the serial analysis of gene expression technology, as described before.^30,31 A series of candidate MRD markers, including GD2 synthase, PGP9.5, Neurofilamin-M, and GAGE, were identified, and their expressions were tested by RQ-PCR on NB tumor and normal BM samples. These results and the initial testing of these markers will be published elsewhere (Gerritsen et al, manuscript in preparation). The most promising candidate MRD marker, PHOX2B, was selected for additional testing in parallel to the current most widely used MRD markers, TH and GD2 synthase.

Expression of TH and PHOX2B in NB tumors
Fifty-two NB tumor samples, mostly from patients with stage 3 or 4 disease (Appendix Table A1), were tested by RQ-PCR for TH, GD2 synthase, and PHOX2B expression. All three markers were readily detected in all samples (Fig 1). The mean level of TH expression (Table 2) was higher than those of PHOX2B and GD2 synthase (P < .001).

View larger version (14K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Normalized PHOX2B, tyrosine hydroxylase, and GD2 synthase expression in neuroblastoma tumors (n = 52), normal bone marrow (BM; n = 51), peripheral blood (PB; n = 37), and peripheral-blood stem cells (PBSCs; n = 24), as determined by real-time quantitative polymerase chain reaction. Blue boxes, tumor samples; yellow circles, BM; gray triangles, PBSCs; red x, PB.

Sensitivity of the RQ-PCR Assays
The sensitivity of the markers was tested by assessing in vitro dilutions of NB cells from two different cell lines (NGP and N206) in normal PB cells. In NGP cells, PHOX2B had a sensitivity equivalent to one tumor cell in 10⁷ PB cells (10^–7) and a quantative range (QR) of 10^–6 (Table 3). The sensitivity for TH was one tumor cell in 10⁶ PB cells (10^–6), and the QR was 10^–5. In N206 cells, the sensitivity was equal for TH and PHOX2B (10^–6), whereas the QR was higher for TH (10^–6 v 10^–5 for PHOX2B). In both cell lines, GD2 synthase was the least sensitive; it had a sensitivity and QR of 10^–4. Because of the high amount of positive control samples and the low sensitivity, GD2 synthase was excluded from additional analysis.

PHOX2B and TH Detection in Diagnostic BM Samples and in Harvests of BM and PBSC
To compare the applicability of PHOX2B and TH for MRD testing, both markers were tested by RQ-PCR on a large panel of clinical samples (N = 237) from 101 patients with NB. Of these 237, 60 were BM samples at initial diagnosis, 107 were BM samples during treatment, 32 were BM harvests for transplantation, and 38 were PBSC harvests for transplantation (Tables 5 to 9).

The analysis of clinical samples showed that PHOX2B is superior to TH in MRD detection. The higher sensitivity of PHOX2B is most evident in samples that had low tumor infiltration, like BM samples during treatment (Table 7). The higher specificity of PHOX2B allows conclusive results in virtually all samples, including in the 24% of TH-inconclusive samples.

	DISCUSSION

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Many investigators have used immunologic and molecular approaches to detect MRD in patients who have NB.^32,33 Until now, a NB tumor–specific target had not yet been found. In this article, we describe PHOX2B as a new, NB-specific RQ-PCR molecular marker. Recently, PHOX2B has been of much interest, because it was identified as the first gene for which germ-line mutations predispose to NB.^34,35 Analysis of PHOX2B revealed that PHOX2B mutations were found in 2.3% to 6.4% of the cases, which implicated a role in oncogenesis of some sporadic NBs.^36,37

An ideal RQ-PCR target should have a high expression in tumors and no expression in normal tissues, BM, PB, and PBSC. We have previously generated high-throughput expression profiles of NB,³¹ and we now identified PHOX2B as a candidate MRD marker. During the course of this study, a database search of Son et al³⁸ also identified PHOX2B as the most highly expressed NB-specific gene. Here, we have experimentally confirmed that PHOX2B is highly expressed in NB tumors. Validation of PHOX2B in normal BM, PB, and PBSC showed that this marker has no detectable expression in these tissues at all, which thus excludes false-positive results. This represents a major advantage of PHOX2B compared with TH, GD2 synthase, and other NB markers studied. Comparison of cytologic and IC evaluation of BM with PHOX2B RQ-PCR showed better and comparable sensitivities, respectively.

TH and GD2 synthase, the most commonly used RQ-PCR markers for MRD detection of NB, share the problem that, in a subset of individuals, these markers display expression in normal BM, PB, and PBSC samples.^5,7,8,14-16 This low TH and GD2 synthase expression in part of the BM, PB, and PBSC samples of control individuals has posed MRD methodological problems for researchers and has strongly limited the interpretation of the data.

The major question was how to define a cutoff level above which gene expression was considered an indication for tumor infiltration. Several different approaches have been used.^5,7,8,16 In the ongoing, prospective, MRD study by the Society of Paediatric Oncology European Neuroblastoma Group (SIOPEN) that is based on TH RQ-PCR, it was decided to let the statistical analyses of the clinical results define what constitutes a positive and a negative result.⁵ In accordance with the guidelines for RQ-PCR results for trials aimed at therapy intensification, and as agreed upon by the European Study Group on MRD testing in ALL,²⁹ we chose to avoid false-positive results. First, we defined the levels of expression in normal tissues. We are the first to report on the expression of TH, GD2 synthase, and PHOX2B in large numbers of normal tissues, which include normal BM, PB, and PBSC. Then, a cutoff level clearly apart from the background was chosen; we used a cutoff level of 3Cts above the average Ct of the normal samples that had a positive TH expression. A sizable fraction of samples of patients with NB displayed TH levels that did not permit a clear-cut interpretation. The use of PHOX2B overcomes this problem, as a positive PHOX2B result clearly implies the presence of NB mRNA. However, validation of the results has to be done in prospective studies.

In addition, the seeding experiments also showed that the sensitivity of PHOX2B is high. Analysis of BM and PBSC samples of patients with NB also showed that nine samples were negative for TH but were positive for PHOX2B, and PHOX2B as a marker identified 23 samples as positive that had inconclusive TH expression. TH identified three samples that were negative for PHOX2B. All together, TH detected 31% positive samples, and PHOX2B identified 43% of the samples as infiltrated.

RQ-PCR results showed tumor-specific transcripts, not only in BM samples from patient who had stage 4 disease, but also from patients with lower-stage NBs. Stage 4s NBs usually go in spontaneous regression, but they can have circulating tumor cells.²⁵ Both PHOX2B and TH detected NB mRNA in six and four BM examinations, respectively, of patients with localized disease. One of these patients experienced relapse. In the literature, positive BM in low stages is a matter of controversy. These positive BM identifications have been defined both as real tumor cells^39,40 and as false-positive results.⁶ However, even if they are true positive results, it is still not clear whether this indicates clinically relevant tumor cell infiltration.^6,39,40 Recently, Corrias et al⁴⁰ showed that detection of tumor cells in patients with low-stage disease is correlated with relapse.⁴⁰ Prospective studies are needed to determine the clinical relevance of MRD positivity in patients with low- and intermediate-risk disease.

In this study, we present a new, sensitive, and specific RQ-PCR marker for MRD detection in NB—PHOX2B. The data presented suggest that inclusion of PHOX2B as a marker in prospective MRD studies of NB, in addition to TH and other markers, will be valuable.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

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Conception and design: Janine Stutterheim, Annemieke Gerritsen, Ilona Kleijn, Marc Bierings, Huib N. Caron, C. Ellen van der Schoot, Godelieve A.M. Tytgat

Provision of study materials or patients: Max M. van Noesel, Marc Bierings, Frank Berthold, Rogier Versteeg, Huib N. Caron, Godelieve A.M. Tytgat

Collection and assembly of data: Janine Stutterheim, Annemieke Gerritsen, Lily Zappeij-Kannegieter, Ilona Kleijn, Rob Dee, Max M. van Noesel, Marc Bierings, Frank Berthold, Godelieve A.M. Tytgat

Data analysis and interpretation: Janine Stutterheim, Annemieke Gerritsen, Lily Zappeij-Kannegieter, Rob Dee, Lotty Hooft, Frank Berthold, Huib N. Caron, C. Ellen van der Schoot, Godelieve A.M. Tytgat

Manuscript writing: Janine Stutterheim, Rob Dee, Rogier Versteeg, Huib N. Caron, C. Ellen van der Schoot, Godelieve A.M. Tytgat

Final approval of manuscript: Janine Stutterheim, Annemieke Gerritsen, Lilly Zappeij-Kannegieter, Ilona Kleijn, Rob Dee, Lotty Hooft, Max M. van Noesel, Marc Bierings, Frank Berthold, Rogier Versteeg, Huib N. Caron, C. Ellen van der Schoot, Godelieve A.M. Tytgat

	Appendix

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	NOTES

 
published online ahead of print at www.jco.org on October 6, 2008.

Supported by Grant No. SKK02-01 of Stichting Kindergeneeskundig Kankeronderzoek and Grant No. UVA 2006-3546 of KWF Kankerbestrijding (Dutch Cancer Society).

Presented in part at the 35th International Society of Paediatric Oncology SIOP, Cairo, Egypt, October 8-11, 2003, and at the 36th International Society of Paediatric Oncology, Oslo, Norway, September 16-19, 2004.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted July 28, 2007; accepted July 2, 2008.

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