UvA-DARE (Digital Academic Repository) Minimal residual ... · Chapter 2 PHOX2B; a novel speciﬁc neuroblastoma MRD marker Chapter 2 PHOX2B is a novel and speciﬁc marker for minimal

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Minimal residual disease detection and monitoring in children with neuroblastoma

Stutterheim, J.

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Citation for published version (APA):Stutterheim, J. (2011). Minimal residual disease detection and monitoring in children with neuroblastoma.

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PHOX2B is a novel and specific marker for minimal residual disease testing in neuroblastoma. (J Clin Oncol. 200 Nov 20;26(33): 5443-9)

Janine Stutterheim1,2,7, Annemieke Gerritsen1,2, Lily Zappeij-Kannegieter2, Ilona Kleijn2, Rob Dee2, Lotty Hooft3, Max M. van Noesel4, Marc Bierings5, Frank Berthold6, Rogier Versteeg7, Huib N. Caron1, C. Ellen van der Schoot1,2, Godelieve A.M.Tytgat1.

1 Department of Pediatric Oncology, Emma Children’s Hospital, Academic Medical Center, Amsterdam, the Netherlands 2 Sanquin Research at CLB and Landsteiner Laboratory of the AMC, Amsterdam, the Netherlands 3 Dutch Cochrane Centre, Academic Medical Center, Amsterdam, the Netherlands 4 Department of Pediatric Oncology/Hematology, Sophia Children’s Hospital, Erasmus Medical Center, Rotterdam, The Netherlands 5 Department of Pediatric Hematology, Wilhelmina Children’s Hospital, Utrecht Medical Center, Utrecht, the Netherlands 6 Department of Pediatric Oncology and Hematology, Childrens Hospital, University of Cologne, Germany 7 Department of Human Genetics, Academic Medical Center, Amsterdam, the Netherlands

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Abstract

PurposePCR-based detection of minimal residual disease (MRD) in neuroblastoma can be used for monitoring therapy response and for evaluation of stem cell harvests. Commonly used PCR markers, tyrosine hydroxylase (TH) and GD2 synthase have expression in normal tissues limiting MRD detection. To identify a more specific MRD marker, we tested PHOX2B.

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

ResultsIn 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 14% IC-negative samples and in 94% of cytology-positive and 90% IC-positive BM samples. Overall, PHOX2B was positive in 43% compared to 31% positive samples for TH. In 24% of all samples TH expression was inconclusive, being similar to expression found in normal tissues. In 42% of these samples, PHOX2B expression was positive.

ConclusionPHOX2B is superior to TH and GD2 synthase in specificity and sensitivity for MRD detection of neuroblastoma using RQ-PCR. We propose to include PHOX2B in further prospective MRD studies in neuroblastoma alongside TH and other MRD markers.

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Introduction

Neuroblastoma (NB) accounts for 10 % of all childhood cancers. Approximately 40% of neuroblastoma-patients suffer from high-risk disease, with 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 neuroblastoma and despite intensive treatment schedules, this has a poor prognosis.1,2 The current golden standard to measure BM infiltration, i.e. cytology, is mostly unable to detect tumor cell infiltration below 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 PCR (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 using RQ-PCR have the same sensitivity as IC.7,8

The ideal MRD marker is tumor specific with no expression in the normal compartments such as BM, peripheral blood (PB) and peripheral blood stem cells (PBSC). No specific neuroblastoma marker has yet been found. For IC, anti-GD2 analysis is very sensitive, but GD2 is also expressed on normal cells.9 Cytogenetic analysis of immunologically-positive cells increases specificity significantly.6 For RQ-PCR, the most applied PCR targets, tyrosine hydroxylase (TH)5,7,10,11 and GD2 synthase12,13 also show low expression in a sizable fraction of normal tissues.5,7,8,12-16

For other targets, like GAGE17,18, MAGE19, PGP9.520,21, DDC22, CyclinD123, ELAVL48 and ST8Siall24, background-expression has also been described. The presence of marker-transcripts in normal BM limits the sensitivity of MRD detection.14 Identification of better molecular markers without expression in normal tissues is therefore important. Here we describe a new specific marker for MRD detection of neuroblastoma 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 found to be superior to the commonly used markers.

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Materials and Methods

Patients and samplesRetrospectively, all available Dutch samples between 1986 and 2007, (52 tumor, 60 BM samples at diagnosis, 107 BM samples during treatment, 38 PBSC -and 32 BM harvests) were collected from 101 patients (6 ganglioneuroblastoma, 95 neuroblastoma). Patients were treated at the Emma Children’s Hospital/ AMC, Amsterdam or Sophia Children’s Hospital/ EMC, Rotterdam in the Netherlands, according to their disease stage (staged according to the International Neuroblastoma Staging System25). Patients´ characteristics, clinical and genetic data are shown in table 1 and individual clinical data and genetic data and origin of the samples in supplementary table 1. BM punctures were performed at diagnosis (staging), at designated time points during treatment (morphology to determine disease status) and prior to high dose chemotherapy, according to the treatment-protocol. Stored remains were, after informed consent was given, used for this study (research purposes). The study was approved by the Medical Research Ethics Committee of the Academic Medical Centre.

Furthermore for comparing RQ-PCR with IC, cDNA of 47 BM samples and clinical data of 43 patients with NB (table 1, supplementary table 2) were kindly provided by prof. F. Berthold, German Pediatric Oncology and Hematology Group (GPOH).

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 samplesTo 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 neuroblastoma.

Immunocytology Cytospins were immunocytologically stained and evaluated by the bone-marrow-laboratory of the GPOH neuroblastoma group (Cologne) according to the standardized European BM method.27

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table 1. Patient characteristics

patient characteristics (n=144)

Dutch (n=101) GPOH (n=43)

age (months)

median 23 34

range 1-186 1-150

gender

male 53 28

female 48 15

INSS stage

stage 1 3 2

stage 2 6 4

stage 3 12 3

stage 4 72 32

stage 4s 8 2

MYCN

amplification 26 12

single copy/ MYCN gain 71 31

not analyzed 4 0

pathology

neuroblastoma 95 43

ganglioneuroblastoma 6 0

RNA extraction and reverse transcriptionTotal cellular RNA from tumor, BM, PB and PBSC samples (5-10 million cells) was extracted with the RNAbee according to the manufacturer’s instructions (Campro Scientific, Veenendaal, the Netherlands). cDNA was synthesized (45 min 42ºC) using 1μg of total RNA, random hexamers (25μM), dNTP’s (1mM) and M-MLV reverse transcriptase (100U), total reaction volume of 20μl (all reagents from Gibco-BRL, Life Technologies, Breda, The Netherlands except for the dNTP’s: Promega, Leiden, The Netherlands). Finally, the reverse transcriptase was inactivated by heating (3 min, 99ºC) and volume diluted to 50μl.

Primer/ProbesPrimers and probes for GD2 synthase and PHOX2B were designed using Primer Express 1.5 (Applied Biosystems, Foster City, CA, U.S.A) and Oligo 6 (Molecular Biology Insights Inc., Cascade, CO, USA),

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amplicons spanned an intron of at least 500 bp, and no amplification of genomic DNA was observed. Primers and probes were synthesized by Eurogentec (Liege, Belgium).

Primer-probe combinations for PHOX2B were: 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 and for GD2 synthase: forward primer 5´-CTG-GAC-CAA-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. Primer/probe combination for TH, �-glucuronidase (GUS) have been described before.5,8,28

Real-time quantitative PCRRQ-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 nM forward and reverse primer, 200 nM Taqman probe, 5 μl cDNA (input 100 ng) starting with 10 min 95ºC followed by 50 cycles (15 sec 95ºC, 1 min 60ºC). The housekeeping gene GUS was used as gene for normalization because it was stably expressed in all neuroblastoma tumors tested and its expression was equal to that in hematological cells.28 To correct for differences in amount of total RNA input and for RT-efficiency, the quantity of the marker transcript was normalized to the amount of GUS gene transcripts (normalized Ct (ΔCt ) = 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 using GUS-plasmid DNA (Ipsogen, Marseille, France) dilutions to generate a standard curve. Samples with a low input of GUS (Ct value >25, corresponding to less than 5000 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 neuroblastoma 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 ΔCt value was ≥ 3.0 Ct

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of the mean ΔCt of normal PB (=background). The quantitative range was defined as the lowest dilution giving a reproducible amplification (ΔCt of all replicates ≤ 1.5), with all Ct values ≥ 3.0 of mean ΔCt of background. These criteria have been adapted from those defined by the European Study Group (ESG) on MRD detection in acute lymphatic leukemia (ALL).29

Data and statistical analysisWhen a marker showed amplification in normal tissue a threshold for positivity was determined. This threshold was defined using the rules adapted from the ESG on MRD detection in ALL.29 Clinical samples were scored positive if the Ct value was < 40 and mean ΔCt ≥ 3.0 Ct of the mean ΔCt of the normal tissue. Samples with Ct value of ≥ 40 or 1 Ct of the background were scored negative. Samples with a ΔCt value in between were scored inconclusive, being in the background range. The mean ΔCt in normal tissues was determined by averaging the ΔCt values of samples that showed amplification. ΔCt values are expressed as mean ± standard deviation. Expression levels were compared using a paired t-test.

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Results

Selection of PHOX2B as candidate MRD markerTo identify new MRD marker for neuroblastoma, we compared mRNA expression profiles of 11 neuroblastoma tumors and cell lines and normal tissues obtained by the SAGE technology as described before.30,31 A series of candidate MRD markers, among which GD2 synthase, PGP9.5, Neurofilamin-M and GAGE, were identified and their expression was tested by RQ-PCR on neuroblastoma tumor and normal BM samples. These results and the initial testing of these markers will be published elsewhere (Stutterheim et al., manuscript in preparation). The most promising candidate MRD-marker PHOX2B was selected for further testing in parallel to the currently most widely used MRD markers, TH and GD2 synthase.

Expression of TH and PHOX2B in neuroblastoma tumors.Fifty-two neuroblastoma tumor-samples, mostly from patients with stage 3 or 4 disease (supplementary table 1), were tested by RQ-PCR for TH, GD2 synthase and PHOX2B expression. All three markers were readily detected in all samples (figure 1). The mean level of TH expression (table 2) was higher than PHOX2B and GD2 synthase (p<0.001).

Expression of the markers in normal tissue.To define threshold for positivity, we determined the expression of the markers in normal BM (n=51), PB (n=37) and PBSC (n=24) (figure 1 and table 2). PHOX2B was not expressed in any of the normal samples. Any expression of PHOX2B can be interpreted as a positive result; no threshold could be established. TH showed amplifications in 14/51 (27%) normal BM samples, in 10/37 (27%) PB samples and in 7/24 (29%) PBSC tested (figure 1, table 2). GD2 synthase was expressed in almost all normal samples tested: in 49/51 (96%) BM samples, 30/37 (81%) PB samples and 19/24 (79%) PBSC samples.

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figure 1. Normalized PHOX2B, TH and GD2 synthase expression in neuroblastoma tumors (n=52), normal bone marrow (n=51), peripheral blood (n= 37) and peripheral blood stem cells (n=24), determined by real time quantitative PCR

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Abbreviations: BM: bone marrow; PB: peripheral blood; PBSC: peripheral blood stem cells

table 2. Mean normalized expression levels in neuroblastoma tumors (n= 52), normal BM (n=51), normal PBSC (n=24) and normal PB (n=37) measured by PHOX2B, TH and GD2 synthase real time quantitative PCR

Tumor BM PBSC PB

Marker positive tumor samples†

Expres-sion *

positive BM samples†

Expres-sion*

Treshold‡ positive PB samples†

Expres-sion*

Treshold‡ positive PB samples†

Expres-sion*

Treshold‡

PHOX2B 52/52 -1,6 (±2.1)

0/51 No ampli-fication

no treshold


no treshold


no treshold

TH 52/52 2.2 § (±2.1)

15/51 -15.3 (±1.1)

-12.3 7/24 -17.4 (±1.1)

-14.4 10/37 -15.2 (±0.8)

-12.2

GD2synthase 52/52 -2.3 (±1.7)

49/51 -13.8 (±1.5)

-10.8 19/24 -16.5 (±1.5)

-13.5 30/37 -14.4 (±1.1)

-11.4

Note:* mean (± sd) normalized Ct values (ΔCt = Ct GUS – Ct Marker) † number of positive BM, PBSC or PB samples out of total numbers tested ‡ threshold for positivity (ΔCt values) was defined as the ΔCt ≥ 3.0 Ct of the mean ΔCt of the normal tissue § TH expression is significantly higher than PHOX2B and GD synthase (p < 0.001) Abbreviations: BM: bone marrow; B: peripheral blood; PBSC: peripheral blood stem cells

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Table 3. Detection of neuroblastoma cells (NGP and N206) diluted in PB cells measured by PHOX2B, TH and GD2 synthase real time quantitative PCR

sensitivity1 quantative range2

NGP

PHOX2B 10-7* 10-6

TH 10-6 10-5

GD2 synthase 10-4 10-4

N206

PHOX2B 10-6 10-5

TH 10-6 10-6

GD2 synthase 10-4 10-4

Note:1 sensitivity was defined as the lowest dilution in which the mean �Ct value was � 3.0 Ct of the mean �Ct of normal PB (=background);2 quantative range was defined as the lowest dilution giving a reproducible amplification (�Ct of all replicates ��1.5), with all Ct values ��3.0 of mean��Ct of background; * detection of 1 tumor cell in 10 million normal nucleated cells

Sensitivity of the RQ-PCR assaysThe sensitivity of the markers was tested by assessing in vitro dilutions of neuroblastoma cells from 2 different cell lines (NGP and N206) in normal PB cells. With NGP cells PHOX2B was most sensitive with a sensitivity of an equivalent of 1 tumor cell in 107 PB cells (10-7) and a quantative-range (QR) of 10-6 (table 3). The sensitivity for TH was 1 tumor cell in 106 PB cells (10-6) and the QR was 10-5. With N206 cells the sensitivity was equal for TH and PHOX2B (10-6), while the QR was higher for TH, namely 10-6 versus 10-5 for PHOX2B. In both cell lines, GD2 synthase was the least sensitive, with a sensitivity and QR of 10-4. Because of the high amount of positive control- samples and the low sensitivity, GD synthase was excluded for further analysis.

Comparison of RQ-PCR results with bone marrow cytology and immunocytology RQ-PCR results of all available Dutch BM samples (60 diagnosis and 107 during treatment) of 79 patients were compared to BM cytology. For stage 4 and 4s, PHOX2B was positive in 50/53 (94%) positive BM-samples and in 31/99 (31%) negative BM-samples (table 4). TH was positive in 48/53 (90%) positive BM-examinations, remained inconclusive in 3/53 -and negative in 2/53 of the positive BM-examinations. In the 99 negative BM-examinations TH was positive in 11/99 and inconclusive in 26/99 of the samples.

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Table 4. Comparison of real time quantitative PCR and cytology in bone marrow samples of stage 4 patients and patients with stage 1, 2, 3 or 4s

bone marrow cytology

stage 4* stage 4s* stage 1,2* stage 3*

+ - + - + - + -

TH

+ 45 11** 3 0 0 3 0 1

inconclusive 3 24 0 2 0 3 0 4

- 2 61 0 1 0 0 0 4

PHOX2B

+ 47 30† 3 1 0 4 0 2

- 3 66 0 2 0 2 0 7

*n=146 samples of 58 patients

*n= 6 patients

*n= 6 patients

*n= 9 patients

** n = 11 samples of 11 patients † n = 30 samples of 27 patientsAbbreviations: PCR, polymerase chain reaction; TH, tyrosine hydroxylase.

This shows that PHOX2B and TH detect more positive samples than cytological examination and that PHOX2B is somewhat more sensitive and much more specific than TH. Interestingly, PHOX2B also detected 4 positive BM-samples in stage 1,2 patients and 2 in stage 3 patients, while TH was positive in respectively 3 and 1 of these PHOX2B positive samples (table 4). One of these patients, N166, relapsed to stage 4 (supplementary table 1).We also compared RQ-PCR of TH and PHOX2B with anti-GD2 immunocytology in 47 BM samples of 43 patients (supplementary table 2). PHOX2B was negative in 2/19 IC positive-samples while TH was negative in 1/19 IC positive-samples. PHOX2B was positive in 4 and negative in 24 out of 28 IC negative-samples. TH was positive in 3, negative in 14 and inconclusive in 11 out of 28 IC negative-samples. When RQ-PCR results of TH and PHOX2B were combined, the sensitivity for RQ-PCR was the same as for IC. However, RQ-PCR detected neuroblastoma mRNA in 5/28 IC negative samples. (supplementary table 3)

PHOX2B and TH detection in diagnostic BM samples and in harvests of BM and PBSCTo 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

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Table 5. Comparison of PHOX2B and TH real time quantitative PCR assays

a. all samples*

TH

PHOX2B + - inconclusive total

+ 70 9 23** 102

- 3 99 33 135

total 73 108 56 237

* n= 237 samples of 101 patients ** n= 32 samples of 26 patients

c. BM examinations during treatment*

TH


+ 20 5 15** 40

- 3 53 11 67

total 23 58 26 107

* n= 107 samples of 50 patients ** n= 20 samples of 17 patients

e. PBSC harvests*

TH


+ 0 1 1 2

- 0 23 13 36

total 0 24 14 38

* n= 38 samples of 36 patients

Abbreviations: BM: bone marrow; PBSC: peripheral blood stem cells ; + : expression of marker; - : no expression of marker; inconclusive: result in range of background of TH expression total: total samples analyzed

initial diagnosis, 107 BM samples during treatment, 32 BM harvests and 38 PBSC harvests for transplantation (table 5).

At diagnosis, PHOX2B detected 7 positive-samples (of 7 patients) of which 2 were TH negative and 5 TH-inconclusive (table 5b). Looking at BM samples collected during treatment, when the tumor load is largely reduced, PHOX2B detected NB mRNA in 20 samples of 17 stage 4 patients while for these samples TH gave inconclusive or negative results (table 5c). Furthermore PCR-based tumor cell detection of stem cells harvests showed that PHOX2B detected more positive samples than TH (table 5d and 5e).

b. BM examinations at initial diagnosis*

TH


+ 40 2 5 47

- 0 9 4 13

total 40 11 9 60


d. BM harvests*

TH


+ 10 1 2 13

- 0 14 5 19

total 10 15 7 32


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In total, 9 (9%) out of 102 PHOX2B-positive samples were negative for TH and 22% (23/102) inconclusive for TH (table 5a). Since PHOX2B has no background expression these results indicate the presence of NB mRNA in these BM samples. Of the 135 PHOX2B-negative samples, 24% (33/135) were PHOX2B-neg/TH-inconclusive and only 2% (3/135) were PHOX2B-neg/TH-positive. The latter three samples probably contained tumor cells with a relatively low PHOX2B and high TH expression. Indeed, analysis of a tumor biopsy corresponding to one of these samples showed that PHOX2B expression was 6.5 Ct lower than TH expression.

The analysis of clinical samples shows that PHOX2B is superior to TH in MRD detection. The higher sensitivity of PHOX2B is most evident in samples with low tumor infiltration, like BM samples during treatment (table 5c). The higher specificity of PHOX2B allows conclusive results in virtually all samples, including in the 24% of TH inconclusive samples.

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Discussion

Many investigators have used immunological and molecular approaches to detect minimal residual disease in patients with NB.32,33 Up to now, a neuroblastoma tumor-specific target has not yet been found. In this paper we describe PHOX2B as a new neuroblastoma-specific RQ-PCR molecular marker. Recently PHOX2B has taken much interest because it was identified as the first gene for which germ-line mutations predispose to neuroblastoma.34,35 Analysis of PHOX2B revealed that PHOX2B mutations were found in 2.3% to 6.4% of the cases, implicating a role in oncogenesis of some sporadic neuroblastomas.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 neuroblastoma31 and now identified PHOX2B as a candidate MRD marker. During the course of this study, a database search of Son and coworkers also identified PHOX2B as the most highly expressed neuroblastoma specific gene.38 Here we have experimentally confirmed that PHOX2B is highly expressed in neuroblastoma tumors. Validation of PHOX2B in normal BM, PB and PBSC showed that this marker has no detectable expression in these tissues at all, thus excluding false positive results. This represents a major advantage of PHOX2B over TH, GD2 synthase and other neuroblastoma markers studied. Comparison of cytological and immunocytological 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 neuroblastoma, 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 researchers for methodological problems and strongly limited the interpretation of the data.

The major question was how to define a cut-off level above which gene expression was considered as an indication for tumor infiltration. Several different approaches have been used.5,7,8,16 In the ongoing prospective MRD study based on TH RQ-PCR of the SIOP Europe Neuroblastoma group (SIOPEN) it was decided to let the statistical analyses of the clinical results define what constitutes a positive and negative result.5 In accordance with the guidelines for RQ-PCR results for trials aimed at therapy intensification as agreed by the European Study Group on MRD testing in ALL29, 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

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synthase and PHOX2B in large numbers of normal tissues, being normal BM, PB and PBSC. Then a cut-off level clearly apart from the background was chosen; we used a cut-off level of 3Ct’s above the average ΔCt of the normal samples with a positive TH expression. A sizable fraction of samples of patients with NB displayed TH levels that do not permit a clear-cut interpretation. The use of PHOX2B overcomes this problem, as a positive PHOX2B result clearly implies the presence of neuroblastoma mRNA. However, validation of the results has to be done in prospective studies.

Furthermore, not only the seeding experiments showed that the sensitivity of PHOX2B is high. Analysis of BM and PBSC samples of patients with NB also showed that 9 samples were negative for TH, but positive for PHOX2B, and PHOX2B identified 23 samples, with an inconclusive TH expression, as positive. TH identified 3 samples which were negative for PHOX2B. All together, TH detected 31% positive samples, while PHOX2B identified 43% of the samples as being infiltrated.

RQ-PCR results showed tumor specific transcripts not only in BM samples from stage 4 patients, but also from patients with lower stage neuroblastomas. Stage 4s neuroblastomas usually go in spontaneous regression, but it is known that they can have circulating tumor cells.25.Both PHOX2B and TH detected neuroblastoma mRNA in respectively, 6 and 4 BM examinations of patients with localized disease. One of these patients relapsed. In literature, positive BM in low stages is a matter of controversy. These positive BM have been defined both as real tumor cells39,40 and as false positives6. But even if they are true positives, it is still not clear whether this indicates clinically relevant tumor cell infiltration.6,39,40 Although, recently Corrias et al. showed that detection of tumor cells in low stage patients is correlated with relapse.39 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 absolute specific RQ-PCR marker for MRD detection in neuroblastoma; PHOX2B. The data presented, suggest that including PHOX2B as marker in prospective MRD studies of neuroblastoma, alongside TH and other markers, will be very valuable.

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2 Hartmann O, Valteau-Couanet D, Vassal G et al. Prognostic factors in metastatic neuroblastoma in patients over 1 year of age treated with high-dose chemotherapy and stem cell transplantation: a multivariate analysis in 218 patients treated in a single institution. Bone Marrow Transplant 1999;23:789-795.

3 Cheung NK, Heller G, Kushner BH et al. Detection of metastatic neuroblastoma in bone marrow: when is routine marrow histology insensitive? J Clin Oncol 1997;15:2807-2817.

4 Mehes G, Luegmayr A, Kornmuller R et al. Detection of disseminated tumor cells in neuroblastoma: 3 log improvement in sensitivity by automatic immunofluorescence plus FISH (AIPF) analysis compared with classical bone marrow cytology. Am J Pathol 2003;163:393-399.

5 Viprey VF, Corrias MV, Kagedal B et al. Standardisation of operating procedures for the detection of minimal disease by QRT-PCR in children with neuroblastoma: quality assurance on behalf of SIOPEN-R-NET. Eur J Cancer 2007;43:341-350.

6 Mehes G, Luegmayr A, Ambros IM et al. Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow. Clin Cancer Res 2001;7:1969-1975.

7 Lambooy LH, Gidding CE, van den Heuvel LP et al. Real-time analysis of tyrosine hydroxylase gene expression: a sensitive and semiquantitative marker for minimal residual disease detection of neuroblastoma. Clin Cancer Res 2003;9:812-819.

8 Swerts K, De Moerloose B, Dhooge C et al. Potential application of ELAVL4 real-time quantitative reverse transcription-PCR for detection of disseminated neuroblastoma cells. Clin Chem 2006;52:438-445.

9 Martinez C, Hofmann TJ, Marino R et al. Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: a novel surface marker for the identification of MSCs. Blood 2007;109:4245-4248.

10 Horibe K, Fukuda M, Miyajima Y et al. Outcome prediction by molecular detection of minimal residual disease in bone marrow for advanced neuroblastoma. Med Pediatr Oncol 2001;36:203-204.

11 Parareda A, Gallego S, Roma J et al. Prognostic impact of the detection of microcirculating tumor cells by a real-time RT-PCR assay of tyrosine hydroxylase in patients with advanced neuroblastoma. Oncol Rep 2005;14:1021-1027.

12 Cheung IY , Cheung NK. Quantitation of marrow disease in neuroblastoma by real-time reverse transcription-PCR. Clin Cancer Res 2001;7:1698-1705.

13 Hoon DS, Kuo CT, Wen S et al. Ganglioside GM2/GD2 synthetase mRNA is a marker for detection of infrequent neuroblastoma cells in bone marrow. Am J Pathol 2001;159:493-500.

14 Ifversen MR, Kagedal B, Christensen LD et al. Comparison of immunocytochemistry, real-time quantitative RT-PCR and flow cytometry for detection of minimal residual disease in neuroblastoma. Int J Oncol 2005;27:121-129.

15 Kuci Z, Seitz G, Kuci S et al. Pitfalls in detection of contaminating neuroblastoma cells by tyrosine hydroxylase RT-PCR due to catecholamine-producing hematopoietic cells. Anticancer Res 2006;26:2075-2080.

16 Trager C, Kogner P, Lindskog M et al. Quantitative analysis of tyrosine hydroxylase mRNA for sensitive detection of neuroblastoma cells in blood and bone marrow. Clin Chem 2003;49:104-112.

17 Cheung IY , Cheung NK. Molecular detection of GAGE expression in peripheral blood and bone marrow: utility as a tumor marker for neuroblastoma. Clin Cancer Res 1997;3:821-826.

18 Oltra S, Martinez F, Orellana C et al. Minimal residual disease in neuroblastoma: to GAGE or not to GAGE. Oncol Res 2004;14:291-295.

19 Cheung IY, Barber D, Cheung NK. Detection of microscopic neuroblastoma in marrow by histology, immunocytology, and reverse transcription-PCR of multiple molecular markers. Clin Cancer Res 1998;4:2801-2805.

20 Gilbert J, Norris MD, Marshall GM et al. Low specificity of PGP9.5 expression for detection of micrometastatic neuroblastoma. Br J Cancer 1997;75:1779-1781.

21 Mattano LA, Jr., Moss TJ, Emerson SG. Sensitive detection of rare circulating neuroblastoma cells by the reverse transcriptase-polymerase chain reaction. Cancer Res 1992;52:4701-4705.

22 Bozzi F, Luksch R, Collini P et al. Molecular detection of dopamine decarboxylase expression by means of reverse transcriptase and polymerase chain reaction in bone marrow and peripheral blood: utility as a tumor marker for neuroblastoma. Diagn Mol Pathol 2004;13:135-143.

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23 Cheung IY, Feng Y, Vickers A et al. Cyclin D1, a novel molecular marker of minimal residual disease, in metastatic neuroblastoma. J Mol Diagn 2007;9:237-241.

24 Cheung IY, Vickers A, Cheung NK. Sialyltransferase STX (ST8SiaII): a novel molecular marker of metastatic neuroblastoma. Int J Cancer 2006;119:152-156.

25 Brodeur GM, Pritchard J, Berthold F et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993;11:1466-1477.

26 Verhagen OJ, Willemse MJ, Breunis WB et al. Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia. Leukemia 2000;14:1426-1435.

27 Swerts K, Ambros PF, Brouzes C et al. Standardization of the immunocytochemical detection of neuroblastoma cells in bone marrow. J Histochem Cytochem 2005;53:1433-1440.

28 Beillard E, Pallisgaard N, van dV, V et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program. Leukemia 2003;17:2474-2486.

29 van dV, V, Cazzaniga G, Schrauder A et al. Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia 2007;21:604-611.

30 Boon K, Caron HN, van Asperen R et al. N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO J 2001;20:1383-1393.

31 Caron H, van Schaik B, van der MM et al. The human transcriptome map: clustering of highly expressed genes in chromosomal domains. Science 2001;291:1289-1292.

32 Beiske K, Ambros PF, Burchill SA et al. Detecting minimal residual disease in neuroblastoma patients-the present state of the art. Cancer Lett 2005;228:229-240.

33 Reynolds CP Detection and treatment of minimal residual disease in high-risk neuroblastoma. Pediatr Transplant 2004;8 Suppl 5:56-66.

34 Mosse YP, Laudenslager M, Khazi D et al. Germline PHOX2B mutation in hereditary neuroblastoma. Am J Hum Genet 2004;75:727-730.

35 Trochet D, Bourdeaut F, Janoueix-Lerosey I et al. Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma. Am J Hum Genet 2004;74:761-764.

36 van L, V, Schramm A, van Lakeman A et al. The Phox2B homeobox gene is mutated in sporadic neuroblastomas. Oncogene 2004;23:9280-9288.

37 Raabe EH, Laudenslager M, Winter C et al. Prevalence and functional consequence of PHOX2B mutations in neuroblastoma. Oncogene 2007;

38 Son CG, Bilke S, Davis S et al. Database of mRNA gene expression profiles of multiple human organs. Genome Res 2005;15:443-450.

39 Corrias MV, Parodi S, Haupt R et al. Detection of GD2-positive cells in bone marrow samples and survival of patients with localised neuroblastoma. Br J Cancer 2008;98:263-269.

40 Shono K, Tajiri T, Fujii Y et al. Clinical implications of minimal disease in the bone marrow and peripheral blood in neuroblastoma. J Pediatr Surg 2000;35:1415-1420.

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Appendix Chapter 2

Supplementary data

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supplementary table 1. Individual clinical, genetic data of 101 Dutch patients and origin of the 289 Dutch samples

patient nr

MYCN 1p deletion

pathology age at Dx (ms) INSS stage material tested

28 0 0 NB 13 4 BM harvest


40 1 1 NB 29 3 BM harvest + tumor

41 0 0 NB 46 1 PBSC*


45 0 0 NB 186 4 d/a therapy + BM harvest

60 0 0 NB 30 4 d/a therapy + BM harvest +tumor

62 0 99 NB 1 3 ini


74 0 0 GNB 28 4 BM harvest

92 0 1 NB 12 2 BM harvest*

93 1 1 NB 14 4 ini + tumor


127 0 0 NB 3 4s ini

130 1 1 NB 20 4 ini + d/a therapy + BM harvest +tumor



165 0 0 NB 33 4 d/a therapy + PBSC + tumor

166 0 0 NB 8 2 ini + d/a/ therapy + BM harvest+ tumor*

167 0 0 NB 1 4s BM harvest



205 0 0 NB 15 2 ini + tumor

247 0 0 NB 1 4s ini + tumor

248 0 0 NB 45 4 d/a therapy + tumor


341 0 99 NB 50 4 d/a therapy + BM harvest




410 1 1 NB 26 4 ini + d/a therapy + PBSC +tumor

415 0 0 NB 57 4 ini + d/a therapy +tumor




442 1 0 NB 14 4 d/a therapy + BM harvest +tumor

458 1 1 NB 26 4 ini + d/a therapy +tumor

462 0 0 NB 26 4 ini + d/a therapy + PBSC +tumor

479 0 99 NB 44 4 PBSC

482 0 0 NB 1 2 ini + tumor

483 0 0 GNB 51 4 PBSC



507 1 1 NB 20 3 d/a therapy+BM harvest+PBSC+tumor

511 0 0 GNB 10 1 d/a therapy + PBSC*



527 0 99 NB 32 4 PBSC

538 1 1 NB 22 4 ini+ d/a therapy+ BM harvest+ PBSC+ tumor

539 0 0 NB 18 4 d/a therapy + BM harvest + PBSC(2x)

540 1 1 NB 20 4 ini, d/a therapy + PBSC + tumor

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542 0 1 NB 1 2 ini

545 0 0 NB 1 3 ini + tumor

546 0 0 NB 8 3 ini + tumor


557 0 0 NB 18 4 ini + PBSC+ tumor

559 1 0 NB 20 4 ini, d/a therapy + PBSC + tumor

576 0 0 NB 55 4 ini+ d/a therapy+ BM harvest+ PBSC+ tumor

579 1 1 NB 15 4 ini + tumor

580 1 1 NB 42 4 ini + tumor

583 1 1 NB 22 4 ini + d/a therapy + PBSC + tumor

584 0 0 NB 3 3 ini

585 0 0 NB 137 4 ini + tumor

589 0 0 NB 8 4 ini

591 0 1 NB 31 4 ini + tumor

592 1 1 NB 35 4 ini

LegendsMYCN amplification: ‘1’ MYCN amplification (>3 MYCN copies); ‘0’ no MYCN amplification (<3 copies); 1p deletion: ‘1’ 1p deletion; ‘0’ no 1p deletion; ‘99’ not analyzed;BM/bone metastasis: BM or bone metastasis at diagnosis as measured by cytology smears and/or bone trephines histology; remark: BM metastasis was measured by bone marrow cytology, bone trephines histology or MIBG scanning; material tested: material tested in this study per patient: ini = initial diagnosis; d/a therapy = during or after therapy; BM harvest = bone marrow harvest; PBSC = peripheral stem cell harvest* relapsed in BM; upstaged to stage 4

patient nr

MYCN 1p deletion

pathology age at Dx (ms) INSS stage material tested

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supplementary table 2. Individual clinical, genetic data of 43 GPOH patients and origin of the 47 GPOH sample

patient nr

MYCN INSS stage treatment protocol

material tested

1 1 4 NB04 ini

2 0 4 NB04 d/a therapy

3 0 4 NB04 ini

4 0 4 NB90 relapse

5 0 4 NB04 ini + d/a therapy

6 1 3 NB04 relapse

7 0 2 NB97 relapse

8 0 4 NB97 relapse

9 0 4 NB97 relapse




13 0 4 NB97 relapse

14 0 4 NB04 ini



17 0 2 NB04 ini


19 1 4 NB04 ini

20 1 4 NB04 ini


22 1 1 NB04 relapse



25 0 1 NB04 ini


27 1 4s NB04 ini


29 0 4s NB04 d/a therapy

30 0 4 NB04 relapse



33 0 3 NB04 ini


35 0 2 NB04 ini


37 1 4 NB04 relapse

38 0 4 NB04 ini

39 0 3 NB97 relapse

40 0 4 NB97 relapse

41 0 2 NB04 ini

42 0 4 NB97 relapse

43 0 4 NB97 relapse

LegendsMYCN amplification: ‘1’ MYCN amplification (according to NB2004 protocol: >4 copies);’0’ MYCN single copy/ MYCN gain (0-4 copies); treatment protocol: treated according to NB97 or NB04; material tested: material tested in this study per patient: ini = initial diagnosis; d/a therapy = during or after therapy; relapse = at relapse

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supplementary table 3. Comparison of real time quantitative PCR and immunocytology in BM samples of stage 4 patients

IC*

+ - total

TH

+ 17 3 20

inconclusive 1 11 12

- 1 14 15

Total 19 28 47

PHOX2B

+ 17 4 21

- 2 24 26

Total 19 28 47

PHOX2B/TH

+ 19 5 24

- 0 23 23

total 19 28 47


Abbreviations: RQ-PCR, real time quantitative -PCR; +, presence of neuroblastoma cells or mRNA; -, absence of neuroblastoma cells or mRNA; total, total bone marrow examinations analyzed; inconclusive, result in range of background of TH expression

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UvA-DARE (Digital Academic Repository) Minimal residual ... · Chapter 2 PHOX2B; a novel speciﬁc neuroblastoma MRD marker Chapter 2 PHOX2B is a novel and speciﬁc marker for minimal