ALK (anaplastic lymphoma kinase) is oncogenic in several tumours and has recently been identified as a predisposition gene for familial NB (neuroblastoma) harbouring mutations in the TKD (tyrosine kinase domain). We have analysed a large set of sporadic human NB primary tumours of all clinical stages for chromosomal re-arrangements using a CGH (comparative genomic hybridization) array (n=108) and mutations of the ALK gene (n=90), and expression of ALK and related genes (n=19). ALK amplification or in-gene re-arrangements were found in 5% of NB tumours and mutations were found in 11%, including two novel not previously published mutations in the TKD, c.3733T>A and c.3735C>A. DNA mutations in the TKD and gene amplifications were only found in advanced large primary tumours or metastatic tumours, and correlated with the expression levels of ALK and downstream genes as well as other unfavourable features, and poor outcome. The results of the present study support that the ALK protein contributes to NB oncogenesis providing a highly interesting putative therapeutic target in a subset of unfavourable NB tumours.

INTRODUCTION

NB (neuroblastoma), the most common solid tumour of childhood, is an embryonal tumour of the postganglionic sympathetic nervous system. The most common genetic features of NB are amplification of the proto-oncogene MYCN (v-myc myelocytomatosis viral-related oncogene, NB-derived), deletions of parts of chromosome arms 1p and 11q, gain of parts of chromosome 17q and triploidy. MYCN amplification, 1p loss and 17q gain are strongly associated with aggressive tumours and a poor outcome for the patient, whereas triploidy is associated with low-stage NBs with a good outcome [13].

ALK (anaplastic lymphoma kinase; OMIM 105590) is dominantly expressed in the neural system and the gene encoding it is located on the short arm of chromosome 2 (2p23.2) [4,5]. It is a tyrosine kinase that was first identified as part of a fusion gene between NPM (nucleophosmin) and ALK, which is the result of the translocation t(2;5)(p23;q35) in anaplastic large cell lymphoma [6,7]. The ALK kinase is constitutively activated by gene amplification of ALK in three NB cell lines and it has been shown that the suppression of activated ALK induces apoptosis through reduced phosphorylation of ShcC, MAPKs (mitogen-activated protein kinases) and Akt [8]. The ALK locus has previously been reported to be amplified in single cases of primary NB tumours with MYCN amplification [812]. In a recent study by Mosse et al. [13], ALK was identified as a major familial NB predisposition gene with activating germline mutations mapping to the TKD (tyrosine kinase domain). They also detected ALK amplifications in sporadic tumours mainly of metastatic stage and with poor outcome, and ALK mutations in a subset of high-risk NBs [13]. We recently performed a CGH (comparative genomic hybridization) array study of 92 NB tumours [14] and detected a few cases of amplification of the ALK gene in chromosome region 2p that was distinct from the amplification of MYCN (also in 2p, but distal of the ALK gene). The results of our previous study [14], together with the recent finding by Mosse et al. [13], prompted us to perform the present detailed study of ALK gene copy number, amplification, rearrangement and mutation, as well as expression patterns of ALK and related genes in sporadic childhood NB tumours of all clinical stages.

EXPERIMENTAL

Patient and control material

A panel of 90 primary NB tumours of all clinical stages according to the INSS (International Neuroblastoma Staging System), 11 stage 1, 12 stage 2, 13 stage 3, 46 stage 4, four stage 4S and four tumours of unknown stage, from children without known familial history of NB, was used for mutation analysis. In addition, normal tissue (blood samples) from some of the patients with identified mutations was used. Genomic DNA was extracted with a DNeasy blood and tissue kit (Qiagen) according to the manufacturer's protocol. RNA from 19 of the tumours used for mutation analysis was extracted from collected tumour material after homogenization by TissueLyser (Qiagen), using the Totally RNA kit (Ambion). Genomic DNA was removed with the DNA-free kit (Ambion) and the purity and integrity of the RNA samples were assayed with the ND-1000 spectrophotometer (NanoDrop) and RNA 6000 Nano Bioanalyzer (Agilent) respectively. Seventy of the tumours used for mutation screening have also been analysed with SNP (single nucleotide polymorphism) arrays, together with 38 additional tumours; this material has been described previously [14]. Informed consent was obtained from the parents of the patients and the study was approved by the relevant ethics committees.

SNP microarray analysis

The DNA microarray experiments have been described previously [14]. Briefly, Affymetrix 250K arrays were used and primary data analysis was performed using GDAS (GeneChip® DNA Analysis software; Affymetrix), whereas further statistical studies were performed using CNAG (Copy Number Analyzer for Affymetrix GeneChip Mapping arrays software, version 3.0; Genome Laboratory, Tokyo University, http://www.genome.umin.jp) [15,16].

DNA amplification and sequencing

Primers were designed for exons and flanking intronic sequences of the TKD and surrounding exons of the ALK transcript [uc002rmy.1/NM_004304 from UCSC (University of California Santa Cruz) Genome Browser March 2006 (http://genome.ucsc.edu/); exons 20–26]. Primers were designed using the Exonprimer feature of the UCSC Genome Browser and primers were ordered from Invitrogen. Touch-down PCR was performed in 10 μl reactions containing 1×Coral Load PCR Buffer (Qiagen), 0.2 mM dNTP mix, 0.25 units of Hot Star TaqPlus DNA polymerase (Qiagen), 10 μM forward and reverse primer respectively and 25 ng of tumour DNA. The PCR programme was performed as follows: 95 °C for 5 min, before 20 cycles of 95 °C for 30 s, 65 °C for 30 s (decreasing by 0.5 °C in every cycle) and 72 °C for 1 min, followed by 20 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and ending with an extension step at 72 °C for 7 min. The specificity of products was inspected by agarose gel electrophoresis before they were purified using Agencourt AMPure magnetic beads (Beckman Coulter) using the Biomek NX pipetting robot (Beckman Coulter) and eluted in distilled H2O.

Sequence PCR was performed using the BDT (BigDye Terminator) v3.1 Cycle Sequence Kit (Applied Biosystems) in 10 μl reactions containing 6 μl of 1:3 diluted PCR/template DNA, 1 μl of BDT, 1×BDT buffer and 1.6 μM forward or reverse PCR primer. Sequence PCR was run under the following conditions: 94 °C for 3 min, followed by 50 cycles of 96 °C for 30 s, 50 °C for 10 s and 60 °C for 3 min each. Sequencing products were purified using CleanSeq magnetic beads (Agencourt) using the Biomek NX and re-suspended in 10 μl of High Dye formamide (Applied Biosystems). The sequencing products were separated with gel electrophoresis on a 3730 DNA analyser (Applied Biosystems) and the output data were viewed and analysed using SeqScape v2.5 (Applied Biosystems). All of the fragments were analysed with both forward and reverse primers and all of the findings were confirmed by sequencing of a new PCR product.

Expression microarray analysis

Microarray expression analysis from 19 tumours including samples without ALK gene aberrations and samples with ALK gene rearrangement, amplification and mutation of the TKD was performed using the HU133A and HU133plus2 expression arrays from Affymetrix. Expression analysis of the five tumour cases, NB56, NB18, NB41, NB42 and NB30, was performed on HU133A chips (A. Wilzén, S. Nilsson, R. M. Sjöberg, P. Kogner, T. Martinsson and F. Abel, unpublished work), whereas the remaining 14 tumour cases were analysed by the HU133plus2 platform at Aros Applied Biotechnology (http://www.arosab.com). The total RNA samples used for the HU133A chips were labelled according to standard procedures from Affymetrix, whereas the total RNA samples used for the HU133plus2 analysis were labelled using the NuGEN whole transcriptome amplification WT-Ovation™ FFPE system.

The downstream genes assumed to be positively regulated by ALK were selected from Bohling et al. [17]. Gene profiles from two fusion gene experiments [TPM3 (tropomyosin 3)–ALK and NPMALK] were compared, and ALK-specific downstream targets were identified. In the present study, we chose the eight most up-regulated genes from these expression profiles; BCL10 (B-cell CLL/lymphoma 10), CEBPB (CCAAT/enhancer binding protein β), INHBA (inhibin βA), IL2RB (interleukin 2 receptor β), COL6A3 (collagen, type VI, α3), TNC (tenascin C), IL1R1 (interleukin 1 receptor, type I), and FCGR3A (Fc fragment of IgG, low affinity IIIa, receptor)/FCGR3B (Fc fragment of IgG, low affinity IIIb, receptor), to represent the ALK pathway activation. A Pearson correlation calculation was performed on our expression data from these eight genes, and they were found to correlate well. To add up the total activation of the ALK downstream pathway, the mean of the log2 expression value from the eight genes was calculated and then raised to the power of 2.

RESULTS

SNP array analysis

Four tumours harboured amplification of the ALK gene and one tumour showed a re-arrangement inside ALK (Figure 1). In addition, ALK was amplified in the IMR-32 NB cell line. All cases with amplification also showed amplification of MYCN. A gain of chromosome 2p was detected in 20 cases; 18 of these included the ALK gene and five showed MYCN amplification (Figure 2). Moreover, five NB cell lines showed a gain of chromosome 2p; two of them also showed amplification of MYCN. It should be noted that cell line SH-SY-5Y is derived from cell line SK-N-SH, and as expected they showed identical gain of chromosome 2p.

Amplification of the ALK gene in NB tumours and cell lines

Figure 1
Amplification of the ALK gene in NB tumours and cell lines

Chr, chromosome; ampl., amplification.

Figure 1
Amplification of the ALK gene in NB tumours and cell lines

Chr, chromosome; ampl., amplification.

Gain of chromosome 2p in 20 NB tumours and five NB cell lines

Figure 2
Gain of chromosome 2p in 20 NB tumours and five NB cell lines

Note that cell line SH-SY-5Y is derived from cell line SK-N-SH. MNA, MYCN amplification.

Figure 2
Gain of chromosome 2p in 20 NB tumours and five NB cell lines

Note that cell line SH-SY-5Y is derived from cell line SK-N-SH. MNA, MYCN amplification.

DNA sequencing

Five different missense mutations in a total of ten tumours were detected in the TKD (Table 1). In exon 20 (outside the TKD), at base 3182, a G>A substitution was detected in one NB tumour. This resulted in an amino acid change from an arginine to a glutamine residue. At positions 3520 and 3522, the mutations T>A (one tumour) and C>A (three tumours) respectively, were detected, both changing the amino acid residue phenylalanine into isoleucine and leucine respectively (Figure 3). Two novel mutations, 3733T>A and 3735C>A (not previously published), were detected in exon 24, giving rise to an amino acid change from a phenylalanine residue to isoleucine and leucine, in one tumour each. In four NB tumours, a mutation was detected at position 3824 (G>A), resulting in the transition from the amino acid residue arginine to glutamine. In addition, one silent change (3633C>A) was found in three tumours and one non-coding mutation (IVS22+18 C>T) was detected in five tumours.

Table 1
DNA mutations and chromosomal abberations in the TKD of the ALK gene

AWD, alive with disease; DOD, dead of disease; NED, no evidence of disease; Chr2, chromosome 2; neg, negative; MNA, MYCN amplification; WCG, whole chromosome gain; n.a., not available.

Mutation/abberationGene positionBase changePredicted protein changePatientNB stageOutcomeSurvival after diagnosisAdditional Chr 2 featuresNormal tissue from the patient
Mutation Exon 23 3520T>A F1174I NB92 AWD 4+ neg n.a. 
  3522C>A F1174L NB27 DOD MNA n.a. 
  3522C>A F1174L NB28 DOD 10 MNA n.a. 
  3522C>A F1174L NB36 DOD 29 MNA C/C 
 Exon 24 3733T>A F1245I NB61 AWD 66+ neg n.a. 
  3735C>A F1245L NB29 DOD 21 WCG C/C 
 Exon 25 3824G>A R1275Q NB64 AWD 2+ MNA n.a. 
  3824G>A R1275Q NB70 NED 30+ 2p gain G/G 
  3824G>A R1275Q NB90 AWD 4+ MNA n.a. 
  3824G>A R1275Q NB91 NED 4+ MNA n.a. 
Amplification    NB30 DOD 12 MNA  
    NB42 NED 127+ MNA  
    NB19 DOD MNA  
    NB104 NED 14+ MNA  
Structural abberation    NB10 NED 190+ 2p gain  
Mutation/abberationGene positionBase changePredicted protein changePatientNB stageOutcomeSurvival after diagnosisAdditional Chr 2 featuresNormal tissue from the patient
Mutation Exon 23 3520T>A F1174I NB92 AWD 4+ neg n.a. 
  3522C>A F1174L NB27 DOD MNA n.a. 
  3522C>A F1174L NB28 DOD 10 MNA n.a. 
  3522C>A F1174L NB36 DOD 29 MNA C/C 
 Exon 24 3733T>A F1245I NB61 AWD 66+ neg n.a. 
  3735C>A F1245L NB29 DOD 21 WCG C/C 
 Exon 25 3824G>A R1275Q NB64 AWD 2+ MNA n.a. 
  3824G>A R1275Q NB70 NED 30+ 2p gain G/G 
  3824G>A R1275Q NB90 AWD 4+ MNA n.a. 
  3824G>A R1275Q NB91 NED 4+ MNA n.a. 
Amplification    NB30 DOD 12 MNA  
    NB42 NED 127+ MNA  
    NB19 DOD MNA  
    NB104 NED 14+ MNA  
Structural abberation    NB10 NED 190+ 2p gain  

Missense mutations in the TKD of the ALK gene in NB primary tumours

Figure 3
Missense mutations in the TKD of the ALK gene in NB primary tumours

For each mutation, the top panel shows the chromatogram of normal tissue from a patient with the mutation (N* indicates when available), the middle panel shows the mutation in primary tumours (T) and the bottom panel shows a reference sequence (Ctrl). The tumour case represented in the bottom panel for each mutation is underlined. aa, amino acid.

Figure 3
Missense mutations in the TKD of the ALK gene in NB primary tumours

For each mutation, the top panel shows the chromatogram of normal tissue from a patient with the mutation (N* indicates when available), the middle panel shows the mutation in primary tumours (T) and the bottom panel shows a reference sequence (Ctrl). The tumour case represented in the bottom panel for each mutation is underlined. aa, amino acid.

Expression analysis

Gene expression of ALK was analysed in 19 NB tumours by DNA microarrays (Figure 4). The highest expression was detected in the three unfavourable tumours, NB29, that carried an ALK mutation, and NB42 and NB30, both carrying ALK amplification. In addition, eight ALK downstream positively regulated transcripts were analysed (Figure 4) and their expression levels correlated significantly (P<0.01) with one another according to Pearson's correlation test (results not shown). Results from six tumours, NB21, NB56, NB18, NB41, NB42 and NB30 indicate activation of both ALK and the ALK downstream pathway.

Relative ALK expression from 19 NB tumours

Figure 4
Relative ALK expression from 19 NB tumours

The expression values are generated from Affymetrix HU133A or HU133plus2 expression arrays (A. Wilzén, S. Nilsson, R. M. Sjöberg, P. Kogner, T. Martinsson and F. Abel, unpublished work). Bars represent the relative ALK expression. Lines represent the mean expression from eight ALK-downstream positively regulated transcripts (see the text for details). White bars represent favourable tumour cases, whereas grey bars represent unfavourable tumours. Cases showing genomic amplification (NB30, NB42) or a re-arrangement (NB10) of ALK, and the tumour case showing a mutation in the TKD of ALK (NB29) are marked. rearr, rearrangement; mut, mutation; ampl, amplification.

Figure 4
Relative ALK expression from 19 NB tumours

The expression values are generated from Affymetrix HU133A or HU133plus2 expression arrays (A. Wilzén, S. Nilsson, R. M. Sjöberg, P. Kogner, T. Martinsson and F. Abel, unpublished work). Bars represent the relative ALK expression. Lines represent the mean expression from eight ALK-downstream positively regulated transcripts (see the text for details). White bars represent favourable tumour cases, whereas grey bars represent unfavourable tumours. Cases showing genomic amplification (NB30, NB42) or a re-arrangement (NB10) of ALK, and the tumour case showing a mutation in the TKD of ALK (NB29) are marked. rearr, rearrangement; mut, mutation; ampl, amplification.

DISCUSSION

The ALK gene has been shown to be involved in several chromosomal translocations or inversions contributing to oncogenesis and providing a putative therapeutic target in several different tumour types, as reviewed by Chiarle et al. [18]. The most common is the translocation t(2;5)(p23;q35) in anaplastic large cell lymphomas that gives rise to the oncogenic NPM–ALK fusion protein. Fusion proteins involving ALK and other partner proteins have also been identified in diffuse large B-cell lymphomas and inflammatory myofibroblastic tumours [19,20].

From the present study and recent data on NB [13] it is suggested that ALK is activated and may contribute to tumour development also through gene amplification and specific mutations targeting the TKD as indicated by the elevated expression of ALK and downstream genes, and constitutive kinase phosphorylation [13]. In a recent study screening various molecular targeted inhibitors in a large panel of cell lines [21], it was found that a selective inhibitor of ALK, TAE684, potently suppresses the growth of a subset of NB cell lines and the authors suggest that NB tumours with ALK amplification or re-arrangements may be responsive to treatment by ALK kinase inhibition [21]. ALK is therefore an attractive target for novel therapeutic strategies in NB and it is noteworthy that several ALK kinase inhibitors are in development for specific targeted cancer therapy, including early clinical testing [22].

In a recent study using a CGH array of 92 NB primary tumours, we identified rare cases with ALK amplification [14]. In the present study we more thoroughly investigated the frequency of ALK abnormalities in NB using data from the CGH array, expression arrays and DNA sequencing. In our present study, the first to investigate both mutations and copy number alterations in a large set of sporadic NB tumours of all clinical stages and different biological subsets, we detected ALK aberrations in a subset of advanced tumours from children with an unfavourable outcome. ALK amplification and intragenic re-arrangements were detected in five tumours (Figure 1), giving a frequency of chromosomal abnormalities of 4.6% (5/108), with 3.7% being amplifications, close to the 3.3% amplification rate in sporadic cases published recently [13]. In addition, a previously unreported amplification of ALK was also detected in the IMR-32 NB cell line. Also, 18 additional tumours harboured a gain of chromosome 2p which included ALK (16.7% compared with 22.8% as reported previously [13]) (Figure 2). Five of them also had an amplification of the MYCN gene. The gene target of the 2p gain in NB tumours is not known. Both MYCN and/or ALK are potent targets that could contribute to transformation when involving extra copies. In this context, it is interesting to note that 18 out of 20 2p gain cases contain ALK in the gain region.

Mosse et al. [13] sequenced the coding exons of ALK in NB and reported alterations in the TKD of ALK in familial NB and in high-risk sporadic NB tumours. In the present study, we also focused on the TKD and surrounding exons and sequenced them in 90 NB tumours. Mutations resulting in amino acid changes were found in 11 cases (ten located in the TKD; Table 1). Three of the mutations in the TKD have been reported previously and they have a predicted high probability of resulting in oncogenic activation [13], whereas two have not been described previously. Interestingly, they target the same amino acid as one of the mutations reported previously, phenylalanine at amino acid position 1245. The frequency of amino acid substitutions in the TKD was 11.1% in the tumours used in the present study from all clinical stages, compared with 12.4% in high-risk tumours as investigated by Mosse et al. [13]. Counting DNA mutations inside the TKD and chromosomal abnormalities gives a frequency of 16% of tumours where ALK is affected in the present study (2p gain cases are not included). Interestingly, although investigating the full spectrum of NB tumours, ALK-activating gene aberrations were only detected in advanced tumours either metastatic or with big primaries (large or unresectable stage 2 or 3 according to the INSS) including several with extensive tumour growth into the spinal canal (results not shown). Children with small localized tumours available for radical surgery never showed mutations in the TKD, ALK amplifications or unbalanced 2p gain (P=0.002, Fisher's exact test). Furthermore, ALK aberrations correlated significantly with unfavourable outcome with an inferior survival probability of 33%, 50% and 52% for children with TKD mutations, amplifications and 2p gain respectively. However, all children dying from disease had additional unfavourable clinical and biological prognostic features, apart from ALK alterations. One patient, (harbouring the F1245I mutation; Table 1) with extensive regional residual tumour without known adverse prognostic factors, is a long-term survivor despite the absence of therapy for remaining viable tumour tissue. The clinical role of ALK mutations still seems unclear since, both among familial cases [13] and in our material of sporadic tumours, there are several long-term survivors, indicating that ALK mutations themselves are not an adverse factor.

The expression analysis agrees well with the data on gene amplification and DNA mutations. The three tumours with the highest expression of ALK had either ALK amplification (two cases) or a mutation (F1245L, one case; Figure 4). Moreover, high ALK expression appears to up-regulate ALK downstream targets, although the re-arrangement in tumour NB10 does not appear to affect the expression of ALK, and it only slightly affects downstream genes. Moreover, the F1245L mutation found in case NB29 does not appear to affect the ALK downstream targets selected in the present study. This may be explained by the mutation-specific activation of selected downstream pathways. Also, the eight selected targets (see the Experimental section) probably do not cover all downstream effects of ALK. We should also bear in mind that we did not include analysis of ALK protein expression or phosphorylation status. Interestingly, there are four cases (NB21, NB56, NB18 and NB41) that both show high ALK expression and indicate strong downstream activation, but with no detectable ALK amplification or mutation. These cases will be the subject of further detailed mutation and re-arrangement studies of ALK.

In conclusion, we have analysed chromosomal re-arrangements of the ALK gene, performed a mutation screening of the ALK TKD and investigated the expression of ALK and related genes in a large number of sporadic NB primary tumours from all clinical and biological subsets. We present two novel mutations in the TKD of ALK. ALK mutations were found in 11% of NB tumours, and gene amplification or re-arrangements were found in 5%. DNA mutations and gene amplifications generally showed a correlation with the expression level of ALK and downstream genes of ALK and unfavourable clinical features, such as extensive primary tumours or metastatic stage and poor prognosis. The results shown in the present study and those previously published by Mosse et al. [13] provide clear evidence that ALK plays an important role in a significant fraction of advanced sporadic NB tumours as well as in the small number of cases with familial tumours. These studies also open new options for specific targeted therapy in children with NB.

We would like to thank the Genomics Core Facility resource unit at the University of Gothenburg for access to the ABI 3730 Sequencer.

Abbreviations

     
  • ALK

    anaplastic lymphoma kinase

  •  
  • BDT

    BigDye Terminator

  •  
  • CGH

    comparative genomic hybridization

  •  
  • INSS

    International Neuroblastoma Staging System

  •  
  • MYCN

    v-myc myelocytomatosis viral-related oncogene, neuroblastoma-derived

  •  
  • NB

    neuroblastoma

  •  
  • NPM

    nucleophosmin

  •  
  • SNP

    single nucleotide polymorphism

  •  
  • TKD

    tyrosine kinase domain

FUNDING

This work was supported by the Swedish Cancer Society [grant number 070544]; the Children's Cancer Foundation [grant number 07-22103]; the Assar Gabrielsson Foundation [grant number FB07-27]; the Wilhelm and Martina Lundgren Research Foundation [grant number 2008-305]; the Sahlgrenska University Hospital Foundation [grant number ALFGBG-11537]; the Stockholm Cancer Society [grant number 061172]; and the Stockholm County Council [grant number 20060643]. H. C. is the recipient of a fellowship from the Swedish Knowledge Foundation through the Industrial Ph.D. Programme in Medical Bioinformatics at the SDO (Strategy and Development Office) at the Karolinska Institutet. F. A. is the recipient of a post-doctoral position from the Swedish Medical Council. T. M. is the recipient of a senior cancer researcher position from the Swedish Cancer Society.

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