Molecular Pathogenesis of Peripheral Neuroblastic Tumors

I Janoueix-Lerosey1, G Schleiermacher1,2 and O Delattre1,3

1INSERM U830, Laboratoire de Ge´ne´tique et Biologie des Cancers, Institut Curie, Paris Cedex 05, France; 2De´partement de Pe´diatrie, Institut Curie, Paris Cedex 05, France and 3Unite´ de Ge´ne´tique Somatique, Institut Curie, Paris Cedex 05, France

Neuroblastoma (NB) is an embryonal cancer of the thetic nervous system (Brodeur, 2003; Maris et al., sympathetic nervous system observed in early childhood, 2007). Although most primary tumors occur in the characterized by a broad spectrum of clinical behaviors, abdomen (65%), often in the adrenal medulla, other ranging from spontaneous regression to fatal outcome develop in the paraspinal sympathic ganglia, at various despite aggressive therapies. NB accounts for 8–10% of sites including neck (5%), chest (20%) and pelvis (5%). pediatric cancers and 15% of the deaths attributable to The clinical presentation of NB can be very hetero- malignant conditions in children. Interestingly, NB may geneous as reported more than 30 years ago (Evans occur in various contexts, being mostly sporadic but also et al., 1971). Until recently, the most widely used staging familial or syndromic. This review focuses on recent system was the international NB staging system (INSS) advances in the identification of the genes and mechanisms that classified NB tumors into five stages according to implicated in NB pathogenesis. Although the extensive the clinical presentation and age. This INSS classifica- characterization of the genomic aberrations recurrently tion distinguished localized (stages 1 and 2), advanced observed in sporadic NBs provides important insights locoregional disease (stage 3) or metastatic (stage 4 or into the understanding of the clinical heterogeneity of this 4s) tumors (Brodeur et al., 1993) (Figure 1). A new neoplasm, analysis of familial and syndromic cases also system called International Neuroblastoma Risk Group unravels essential clues on the genetic bases of NB. Staging System (INRGSS), based on clinical criteria and Recently, the ALK gene emerged as an important NB image-defined risk factors, now classifies NB into L1 gene, being implicated both in sporadic and familial cases. (localized disease without image-defined risk factors The identification of gene expression signatures associated precluding surgical resection), L2 (localized disease with with patient’s outcome points out the potential of using image-defined risk factors), M (metastatic tumors) and gene expression profiling to improve clinical management Ms (metastatic tumors with metastases confined to the of patients suffering from NB. Finally, based on recent skin, liver and/or bone marrow in children younger than observations integrating genomic analyses, biological data 18 months of age) (Cohn et al., 2009; Monclair et al., and clinical information, we discuss possible evolution/ 2009). The signs and symptoms of NB are extremely progression schemes in NB. variable, depending on age, primary tumor site, presence Oncogene (2010) 29, 1566–1579; doi:10.1038/onc.2009.518; of metastasis and occasionally on associated paraneo- published online 25 January 2010 plastic syndromes. Heterogeneity also characterizes the clinical course and outcome of NB patients: whereas a Keywords: neuroblastoma; genetic alterations; suscept- subset of tumors will undergo spontaneous regression, ibility; ALK mutations; expression profiling; progression others show relentless progression in spite of multi- modal treatment. The outcome of children with NB is extremely different according to the INSS stage, with long-term survival rates still lower than 40% for patients Neuroblastoma clinical features with a high-risk clinical phenotype (Figure 1).

Clinical presentation Histology Neuroblastoma (NB) was first described more than one An International Pathology Classification System century ago, by three physicians including Robert (INPC), based on the Shimada classification, distin- Hutchison, William Pepper and James Homer Wright guishes favorable and unfavorable tumors depending on who recognized the neural nature of the tumor the degree of neuroblastic differentiation, the amount of (Rothenberg et al., 2009). NB is indeed a disease of Schwannian stromal content, the mitosis-karyorrhexis the sympaticoadrenal lineage of the neural crest and index (MKI) and the age at diagnosis (Shimada et al., tumors may develop at various sites along the sympa- 1999). Schematically, the unfavorable group includes undifferentiated tumors or high MKI cases at any age, Correspondence: Dr I Janoueix-Lerosey, INSERM U830, Laboratoire poorly differentiated tumors or intermediate MKI in de Geńe´tique et Biologie des Cancers, Institut Curie, 26 rue d’Ulm, children older than 18 months, differentiating tumor or Paris Cedex 05 75248, France. E-mail: [email protected] low MKI over 60 months. The favorable group includes Received 14 September 2009; revised 10 December 2009; accepted 18 all the other cases as well as ganglioneuroma (GN) or December 2009; published online 25 January 2010 ganglioneuroblastoma intermixed. The last category is Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1567

Stages 1, 2 or 4s (n=248) 100 ≤ 18 months (n=340) 100

80 80 Stage 3 (n=87)

60 60

40 40 > 18 months (n=153) Stage 4 (n=151) Overall survival (%) Overall survival (%) 20 20

0 0 0 40 80 120 160 200 240 0 40 80 120 160 200 240 Time (months) Time (months) Figure 1 NB is a heterogeneous disease with respect to clinical presentation and outcome. (a) Metastatic NB revealed by an MIBG scintigraphy; (b) localized thoracic NB; (c) example of NB unfavorable histology; (d, e) overall survival of NB patients according to the INSS stage or age at diagnosis, respectively. The Kaplan–Meier survival curves correspond to the series of 493 NB patients reported in our recent paper (Janoueix-Lerosey et al., 2009). nodular ganglioneuroblastoma, with well-defined areas program in Japan, in which more than 15 million children of ganglioneuromatous and neuroblastic components, were screened at 6 months between 1984 and 2003, may be the prognosis of which is determined by evaluating more encouraging (Hiyama et al., 2008). Indeed, though differentiation and MKI of the neuroblastic component. this report confirms the higher incidence of favorable NB in the screened cohort as compared with the unscreened, it also reports a decreased mortality in the screened group Mass screening associated with a decreased incidence of stage 4 tumors. Most of NBs secrete catecholamine whose metabolites can This therefore suggests that screening at 6 months may be found in the urine including dopamine, vanillylmandelic detect some cases that would otherwise evolve toward acid and homovanillic acid. This property has an more aggressive tumors at a later age. Given these results important diagnostic value and is also extremely useful and with the goal of decreasing overdiagnosis and for patient’s follow-up. In the 1980–1990s it provided a increasing effectiveness, pilot screening programs at 18 rationale to propose mass screening programs for NB months have recently been launched in Japan. Although detection at birth or at 6–12 months of age, mainly in encouraging it has to be kept in mind that the Hiyama Japan, Quebec and Germany (Schilling et al., 2002; study is retrospective and should hence be confirmed by a Yamamoto et al., 2002; Barrette et al., 2006). Quite prospective, case–control study to fully document the disappointingly, the main early observation of these benefit of screening (Maris and Woods, 2008). programs was an overall increased incidence of NB without significant decrease in mortality. This therefore indicated that screening mainly detected NB that would Epidemiology otherwise have regressed spontaneously but was relatively NB prevalence is about one case in 7000–10 000 live inefficient for an early detection of clinically relevant high- births. There is no strong evidence for difference in risk cases. However, the recent results of a nationwide disease occurrence according to ethnical origin. No

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1568 major risk factor was identified with respect to the (Figure 2) (Mosse et al., 2004; McConville et al., 2006; potential role of parental occupation and of environ- Raabe et al., 2007). A recent survey of the genotype– mental factors in the pre- and perinatal periods phenotype correletions performed on 174 patients affec- (reviewed in (Heck et al., 2009)). More convincingly, a ted with Congenital Central Hypoventilation Syndrome, recent study investigating the role of birth character- either isolated or associated with Hirschsprung’s disease istics, congenital malformations and maternal reproduc- and/or neuroblastic tumors confirmed that NB predis- tive history reported a very significant association of NB position was mainly linked to missense and frame- with congenital malformation in children less than 1 shift mutations (Trochet et al., 2005b). Although the year (Munzer et al., 2008). involvement of PHOX2B in familial cases of NB is compelling, its contribution to the development of sporadic NB is much less obvious as somatic mutations are extremely rare (van Limpt et al., 2004; McConville Hereditary predisposition to NB et al., 2006; Raabe et al., 2007). Several studies have shown that the various PHOX2B A familial history of NB is observed in about 1% of mutations lead to stable mutant proteins (Trochet et al., patients. A survey of 426 successive NB cases at the 2005a; Raabe et al., 2007), however, the molecular Institut Gustave Roussy in France identified a positive mechanisms underlying the role of these mutations in family history of NB or GN in 5 of the 426 cases (1.2%) NB predisposition remain unclear. The observations (Shojaei-Brosseau et al., 2004). In this cohort, the risk that most frameshift and missense mutations decrease of NB among sibs was estimated at 0.2% (95% or abolish the in vitro transactivation activity of CI 0.004–1.0%) and penetrance in hereditary cases ¼ PHOX2B on the dopamine-b-hydroxylase promoter was estimated at 11.4%. NB pedigrees are usually and can interfere with cellular differentiation suggest consistent with a dominant autosomal pattern of that defects in normal developmental and differentiation inheritance with incomplete penetrance. A remarkable pathways may contribute to NB predisposition (Trochet heterogeneity of the clinical presentation is observed et al., 2005a, 2009; Raabe et al., 2007). within pedigrees in terms of age at diagnosis, histology and aggressiveness. Two main neural-crest-derived conditions are asso- Germline ALK mutations in NB families ciated with an increased risk to develop NB: Hirsch- More recently, the ALK gene was identified as a second sprung’s disease, a developmental disorder characterized NB predisposition gene (see section below for the by an absence of ganglion cells in the distal colon description of ALK gene). The group of J Maris used resulting in functional obstruction and Ondine’s curse, a linkage analysis approach to delineate a putative also named Congenital Central Hypoventilation Syn- NB gene-encoding chromosomal region at 2p23–p24. drome, a disorder characterized by a failure of the Within this large region, ALK was considered as a autonomic control of ventilation during sleep. The two strong candidate and further analyses identified 8 last diseases are frequently associated and most cases are germline mutations out of the 20 studied families linked to mutation of the PHOX2B gene. Interestingly, (Mosse et al., 2008). Our group also documented ALK associations between neuroblastic tumors and various germline mutations in NB pedigrees (Janoueix-Lerosey ‘neuro-cardio-facial-cutaneous’ syndromes, including et al., 2008). Three types of germline mutations have Noonan and Costello syndromes, as well as neurofi- been described to date in NB families, the R1275Q bromatosis type 1, defined by the constitutive activation mutation being the most frequent (Figure 2). Although of the RAS–MAPK pathway (Bentires-Alj et al., 2006), detailed clinical information is still lacking for several have been also reported (Denayer et al., 2008). families, it is apparent that the penetrance of these mutations may be incomplete and that neuroblastic tumors of variable aggressiveness can be observed in PHOX2B and NB predisposition ALK mutation carriers. The paired-like homeobox gene PHOX2B, localized at 4p12, encodes a transcription factor composed of 314 AA (Figure 2), which is a main regulator of the Other clues to NB susceptibility development of the autonomous system. This is under- Germline mutations of NF1, ALK and PHOX2B, three lined by the death in utero of homozygous null mice with genes implicated in nervous system development, do not absence of autonomic, enteric, sympathetic and para- account for all familial NB cases. Interestingly, various sympathetic ganglia (Pattyn et al., 1999). Polyalanine types of abnormal constitutional karyotypes have been expansions and frameshift mutations of PHOX2B gene observed in patients with NB, including constitutional were initially identified in Congenital Central Hypoven- copy number anomalies, balanced and unbalanced tilation Syndrome (Amiel et al., 2003). Subsequently, translocations as well as specific chromosome deletions missense mutations, affecting residues localized in (Mosse et al., 2003; Satge et al., 2003). Although most the homeodomain of PHOX2B, were described in genes targeted by such constitutional anomalies remain two NB families (Trochet et al., 2004). The association to be identified, the NBPF1 (Neuroblastoma Breakpoint of PHOX2B mutation with NB predisposition was Family) gene is worth being pointed out (Vandepoele confirmed by various groups that also reported missense et al., 2005, 2008). It encodes a member of a novel and or frameshift mutations outside the homeodomain intricate human gene family of unknown function and it

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1569 G234fs

R100L G197D

198 158 241 260 314

PHOX2B HD 9 20 4p12

161 170

R141G A226fs

113761122 1620 ALK TK 2p23

1 2 6 R1192P G1128A R1275Q Figure 2 Constitutional PHOX2B and ALK mutations in NB families. PHOX2B missense and frameshift mutations have been identified to date in five NB families and ALK activating mutations have been reported in nine distinct NB families. Mutations are indicated by arrows, with the number of mutations presently identified at each position being indicated beneath the arrow.

was shown to be disrupted by a constitutional Spitz et al., 2006; Stallings et al., 2006; Carr et al., 2007; t(1;17)(p36.2;q11.2) translocation in an NB patient. George et al., 2007; Michels et al., 2007; Janoueix- Very interestingly, a 1q21.1 copy number variant Lerosey et al., 2009). encoding NBPF23, another member of this gene family, was recently shown by a genome-wide association study Numerical chromosome alterations to be associated with NB susceptibility, with an odd Numerical chromosome alterations, that is whole ratio of 2.49 (Diskin et al., 2009). This recurrent chromosome gains or losses, are frequently observed involvement of NBPF family genes in NB susceptibility leading to ploidy variations, with DNA indexes in the may indicate they have critical roles in NB development. near-triploid range. Although one may hypothesize that In addition to the NBPF23 copy number variant, the underlying mechanism leading to such numerical genome-wide association study also identified single chromosome variations is probably linked to a failure of nucleotide polymorphisms (SNPs) at chromosome band chromosomes to properly segregate during mitosis, the 6p22 within the putative FLJ22536 and FLJ44180 genes precise molecular nature of this anomaly remains and at 2q35 within the BARD1 genes that were unknown. In contrast to diploidy or tetraploidy, near- associated with a moderate risk to develop aggressive triploidy is usually associated with a good outcome NB (Maris et al., 2008; Capasso et al., 2009). Indeed, the (Look et al., 1984, 1991; Ladenstein et al., 2001; allelic odds ratios were 1.39–1.40 for the three con- Schneiderman et al., 2008). We recently confirmed the secutive SNPs at 6p22 and odds ratios were of 1.68 each excellent survival of patients with tumors harboring for the two most significant SNPs at 2q35. exclusively whole chromosome copy number variations (type A tumors) (Janoueix-Lerosey et al., 2009).

Somatic genetic changes in sporadic NB Segmental copy number alterations Segmental copy number alterations, mainly including Although NBs may occur in familial and syndromic deletions of chromosome 1p, 3p, 11q and gain of 1q, 2p contexts, most cases occur sporadically. With the aim to and 17q, have been reported to occur recurrently in NB identify the genes and mechanisms implicated in NB and to be usually associated with a poor outcome development and/or progression, many groups includ- (Schleiermacher et al., 1994, 2004; Bown et al., 1999; ing ours have carried out in-depth characterization of Caron et al., 2001; Plantaz et al., 2001; Attiyeh et al., the genomic aberrations recurrently observed in spora- 2005). Although strongly associated with MYCN dic NBs. The genetic alterations of NB tumors and amplification, 1p deletion is also observed in cases cell lines have been largely explored through a panel without such amplification. In contrast, 11q and 3p of techniques, including array-CGH using various types deletions are mainly observed in tumors without MYCN of platforms (Chen et al., 2004; Mosse et al., 2005; amplification (Plantaz et al., 2001). Partial 17q gain is

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1570 frequently observed in primary tumors exhibiting staining regions (hsrs) are predominatly observed in NB segmental alterations, whatever MYCN status. These cell lines. The size of the MYCN amplicon varies from recurrent segmental alterations are thought to lead to o30 kb to several megabases. The DDX1 and NAG the loss of putative tumor suppressor genes and/or to genes have been shown to be co-amplified with MYCN the gain of oncogenes, the exact nature of which remains in 50 and 20% of NB cases, respectively (George et al., to be determined. Interestingly, the use of cytogenetic 1997; Wimmer et al., 1999). The MYCN protein belongs methods revealed that most of these acquired, focal to the MYC oncoproteins family that are transcription copy number alterations were associated with unba- factors of the basic-helix-loop-helix-zipper class (Ad- lanced translocations (Van Roy et al., 2001; Schleier- hikary and Eilers, 2005; Eilers and Eisenman, 2008; macher et al., 2003). In 1994, Caron et al. suggested that Meyer and Penn, 2008). Interestingly, recent data show nonhomologous mitotic recombination in the S/G2 that high MYCN/c-MYC target gene expression is a phase of the cell cycle between 1p and 17q could be a hallmark of malignant NB progression and that c-MYC mechanism leading to unbalanced t(1;17) translocation, probably has a role in agressive tumors without MYCN associated with 1p deletion and 17q gain (Caron et al., amplification (Fredlund et al., 2008; Westermann et al., 1994). In such a model, after segregation of the 2008). Several lines of evidence indicate that over- derivatives in daughter cells and considering a selective expression of MYC proteins has a major role in advantage provided by the translocation, tumor forma- oncogenesis through the deregulation of growth and tion will arise from the cells exhibiting the der(1)t(1;17). proliferation (Vita and Henriksson, 2006). In particular, Another hypothesis relies on a defective intra-S check- transgenic mice overexpressing MYCN in neuroecto- point, or other defects in DNA maintenance or repair dermal cells under the tyrosine hydroxylase promoter pathways that may serve as a starting point for break- develop tumors in peripheral neural-crest-derived struc- induced replication, a mechanism well described in tures and show typical histopathologic features of the yeast Saccharomyces cerevisiae, that could lead human NB, therefore demonstrating that MYCN can to the accumulation of unbalanced translocations contribute to the transformation of neuroblasts in vivo (Malkova et al., 1996). Interestingly, we unambiguously (Weiss et al., 1997; Chesler et al., 2007). Unlike c-MYC, established that unbalanced translocations in NB cell which is ubiquitous, MYCN has a very restricted lines occur preferentially in early replicating regions expression pattern and is critical in the development of (Schleiermacher et al., 2003; Janoueix-Lerosey et al., the peripheral and central nervous system. Mice homo- 2005). Moreover, interstitial telomere sequences lying at zygous for a targeted disruption of the MYCN gene die chromosome breakpoints have been observed in NB between 10.5 and 12.5 days of gestation with under- cells, suggesting that a telomere maintenance pathway developed hearts and reduction of the cranial and spinal may also be altered in these cells (Schleiermacher et al., ganglia (Charron et al., 1992). MYCN conditional 2005). A thorough analysis of the S-phase checkpoint disruption in neuronal progenitor cells further shows may help to understand the molecular basis of these that this gene is essential during neurogenesis, affecting closely linked replication and telomere abnormalities proliferation and differentiation of progenitor cells in NB cells. (Knoepfler et al., 2002). Interestingly, in humans, germline Importantly, we recently demonstrated that the mutations in or deletions of the MYCN gene have been presence of segmental alterations, whatever their type, shown to be responsible for the Feingold syndrome, a was the strongest predictor of relapse in NB patients and disease characterized by variable combinations of eso- that the analysis of the overall genomic pattern, rather phageal and duodenal atresias, microcephaly, learning than the analysis of individual markers, should be disability, syndactyly and cardiac defect (van Bokhoven included in future treatment stratification (Janoueix- et al., 2005). The observed mutations result in haploin- Lerosey et al., 2009). Importantly, this study also clearly sufficiency of both the canonical MYCN protein and of a indicated that tumors with both numerical and seg- shortened MYCN isoform (delta MYCN) (Marcelis et al., mental abnormalities (type D) shared the higher risk of 2008), suggesting that multiple aspects of early embry- relapse of segmental-only (type B) tumors. Tumors with ogenesis and postnatal brain growth in humans are tightly MYCN amplification can either harbor segmental-only regulated by MYCN dosage. alterations (type C) or mixed, segmental and numerical, Other regional amplifications targeting various sites, profiles (type E). nonsyntenic with the MYCN locus and including 1p34, 1p36, 2p14, 5q33, 8q24, 11q13, 12q13–14 and 16q22 have been described in NB (Jinbo et al., 1989; Molenaar Genomic amplifications et al., 2003; Fix et al., 2004, 2008; Su et al., 2004; Mosse Amplification of the MYCN oncogene at chromosome et al., 2005, 2007; Van Roy et al., 2006; Carr et al., 2p24 is observed in about one fourth of NB cases and is 2007). None of these amplicons exhibits a recurrence as associated with advanced-stage disease and rapid tumor high as that of the MYCN amplicon and most often, progression (Seeger et al., 1985). MYCN status is they occur concomitantly with MYCN amplification routinely used in clinical practice to assign therapeutic (Fix et al., 2008). Their contribution to the oncogenic intensity. Although MYCN amplification is mainly transformation therefore remains uncertain at this time. observed cytogenetically as acentric autonomously Of interest, high-level amplifications of the ALK locus replicating double minute chromosomes (dmins) in NB were reported by various groups in a subset of NB primary tumors, intrachromosomally homogeneously samples long before ALK gene emerged as a major actor

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1571 of NB oncogenesis (Lamant et al., 2000; Miyake et al., mutations affecting the F1174 residue have not been 2002; Osajima-Hakomori et al., 2005; George et al., detected in the germline, possibly related to an 2007; Fix et al., 2008). embryonal lethality, whereas mutations of the R1192 residue have been observed in familial but not in sporadic forms of NB. This suggests differences in ALK signaling Somatic ALK activating mutations in sporadic NB through specific mutations, which remain to be further Despite the extensive description of copy number characterized by functional analyses. alterations in NB, only a few mutations in bona fide The existence of a potential link between ALK cancer genes have been reported in NB primary tumors. mutations and the behavior of the tumor remains to The identification of somatic and activating mutations be fully determined. In our screening of sporadic of the ALK gene last year undeniably represents a tumors, ALK mutations did not correlate with aggres- breakthrough in the understanding of NB pathogenesis sive forms of NB (stage 4). However, the other three (Chen et al., 2008; George et al., 2008; Janoueix-Lerosey groups (Caren et al., 2008; Chen et al., 2008; Mosse et al., 2008; Mosse et al., 2008). et al., 2008) reported a higher frequency of ALK The ALK gene encodes a transmembrane receptor mutations in advanced stages of the disease. Interest- tyrosine kinase (RTK) comprising 1620 AA, preferen- ingly, a recent meta-analysis for ALK mutation status in tially expressed in the central and peripheral nervous relation to genomic and clinical parameters performed systems (Chiarle et al., 2008). The extracellular region of on 709 cases indicated that mutations were found in ALK comprises a 26-AA N-terminal signal peptide both low-risk and high-risk NB tumors and that the sequence, two MAM (for meprin, A5 protein and F1174 mutations were significantly overrepresentated in receptor protein tyrosine phosphatase mu) domains tumors presenting with MYCN amplification (Frank (264–427 and 480–626) that are thought to have Speleman, personal communication). Combined occur- adhesive function, one LDLa domain that is expected rence of these two alterations was associated with a to form a binding site for LDL and calcium (453–471) particular poor outcome. and a glycin-rich region (816–940) (Palmer et al., 2009). The group of J Maris has previously reported that The binding site for the two putative ALK ligands, ALK is differentially expressed in human primary NB pleiotrophin and midkine, has been mapped between with higher expression generally seen in the most residues 391 and 401 (Stoica et al., 2001, 2002; Chiarle aggressive subset of tumors (Wang et al., 2006). In et al., 2008). A transmembrane-spanning segment con- agreement with this observation, ALK protein expres- nects the extracellular region with the intracellular sion has recently been shown to be significantly region that includes a juxtamembrane segment (1058– upregulated in adavanced/metastatic tumors compared 1122), the tyrosine kinase domain (1122–1376) and the with localized NB (Passoni et al., 2009). Moreover, two C-terminal region. The function of the full-length ALK reports suggest that higher levels of ALK mRNA are receptor is poorly understood (Chiarle et al., 2008). expressed in NB samples harboring ALK mutations as Mice homozygous for a deletion of the ALK tyrosine compared with ALK wild type (Caren et al., 2008; kinase domain have a normal appearance and no Mosse et al., 2008). The analysis of larger series of obvious tissue abnormality, but preliminary observa- NBs will undoubtedly provide further information tions detected an increase in the number of the with respect to the precise relationship between ALK progenitor cells of the hippocampus and modifications mutations, ALK expression, other genomic alterations in adult brain function (Bilsland et al., 2008). The ALK and tumor phenotype including clinical presentation protein has been described as a new member of the and outcome. growing family of dependence receptors, which may Several lines of evidence indicate that the observed exert antagonist functions, that is apoptotic or anti- mutations are kinase activating and oncogenic: (1) high apoptotic, depending on the absence or presence of the levels of the Y1586- or Y1604-phosphorylated form of ligand (Mourali et al., 2006). This property of the ALK ALK, (that have been associated earlier with activation receptor needs to be confirmed in vivo and requires of the ALK protein (Rikova et al., 2007)) are detected in a true identification and characterization of the NB cell lines harboring mutated ALK (Chen et al., 2008; ALK ligand(s). George et al., 2008; Janoueix-Lerosey et al., 2008; To date, five papers reported a total of 54 ALK Mosse et al., 2008); (2) the mutant versions of ALK mutations, affecting 12 different residues with two exhibit a strong in vitro kinase activity (Janoueix- hotspots of mutations at positions 1174 and 1275, in Lerosey et al., 2008); (3) the F1174L and R1275Q 680 sporadic NB cases that have been analyzed (8% of mutated versions of ALK promote cytokine-indepen- cases) (Figure 3) (Caren et al., 2008; Chen et al., 2008; dent growth of the BaF3 cell line, an immortalized George et al., 2008; Janoueix-Lerosey et al., 2008; murine bone marrow-derived pro-B-cell line the growth Mosse et al., 2008). Although the three dimensional and proliferation of which depends on the presence of structure of the ALK protein has not been resolved to IL-3 (George et al., 2008); (4) overexpression of the date, homology models with other RTK indicate that F1174L and K1062M mutants in NIH3T3 cells induce the F1174 residue is located at the end of the C helix and the formation of foci in soft agar assays and of the R1275 residue is part of the activation loop. subcutaneous tumors in nude mice (Chen et al., 2008). Interestingly, the spectrum of somatic and germline Importantly, the knockdown of ALK expression in ALK mutations are different. Indeed, up to now, ALK-mutated cells, but also in several cell lines over-

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1572

1 1030 1122 1620

1058 1376

TM TK

1 11 1 1 1 1 1 2 6 I1250 T1087 T1151 T1166 D1091 R1061 20 A1234 18 Y1278 I1171 F1245 F1174 R1275

Figure 3 ALK aberrations in NB. (a) Amplification of the ALK locus detected by SNP-array analysis. Only chromosome 2 is displayed. The x axis shows the physical distance (in megabases) from the telomere of the short arm. The y axis shows the ratios of copy number analysis (tumor/normal); (b) amplification of ALK visualized by fluorescent in situ hybridization analysis using the LSI ALK Dual Color, Break Apart Rearrangement Probe (Abbott-Vysis, Abbott Park, IL, USA) in the NB cell line CLB-BAR; (c) immunoperoxidase labeling of paraffin-embedded section of an NB tumor presenting with a R1275Q mutation detected with a polyclonal antibody raised against a portion of the ALK intracellular domain; (d)54ALK mutations have been reported to date in 680 sporadic NB cases. Mutations are indicated by arrows, with the number of mutations presently identified at each position being indicated beneath the arrow. The mutations are mainly located in the kinase domain, with two hotspots at positions 1174 and 1275.

expressing an apparently wild-type ALK, leads to a cell lung cancers (Soda et al., 2007). Most fusion dramatic decrease in cell proliferation (Janoueix-Ler- proteins studied to date are characterized by constitutive osey et al., 2008; Mosse et al., 2008; Passoni et al., 2009). activation of the ALK tyrosine kinase activity through In agreement with these observations, NB cell lines spontaneous dimerization and induce cell transforma- harboring activating ALK mutations respond to the tion (Chiarle et al., 2008; Palmer et al., 2009). The ALK inhibitors NVP-TAE684 and PF-02341066 (vide potent oncogenic activity of the NPM(nucleophosmin)– infra) (George et al., 2008; Janoueix-Lerosey et al., ALK and EML4–ALK fusions have been demonstrated 2008). Finally, although the exact link between MYCN in mouse models by the occurrence of lymphoma and and ALK remains to be defined, the effect of siRNA or lung adenocarcinoma in transgenic mice expressing shRNA targeting ALK was observed in a number of cell NPM–ALK and EML4–ALK, respectively (Chiarle lines also presenting with MYCN amplification includ- et al., 2003; Turner et al., 2003; Soda et al., 2008). ing IMR32, CLB-MA, KELLY and IMR5 (Janoueix- Oncogenic signaling through the NPM–ALK protein Lerosey et al., 2008; Mosse et al., 2008; Passoni et al., has been extensively studied and recently reviewed 2009). Altogether, these data provide a strong molecular (Chiarle et al., 2008; Palmer et al., 2009). The pathways rationale for ALK-targeted therapy in NB. engaged by NPM–ALK include the PLCg, PI3K/AKT, The ALK gene has been previously implicated in other RAS/MAPK and JAK/STAT pathways. The other human malignancies: indeed, the ALK gene is recur- ALK-fusion proteins are assumed to function mainly rently involved in gene fusions induced by chromosome in a similar manner. translocations in different types of cancer including As mentioned earlier, several NB cell lines presen- anaplastic large cell lymphomas (Morris et al., 1994), ting with ALK amplification exhibited constitutively diffuse large B-cell lymphomas (Adam et al., 2003; activated ALK. In these samples, it has been shown Onciu et al., 2003), inflammatory myofibroblastic that activated ALK forms a stable complex with tumors (Griffin et al., 1999) and a subset of non-small hyperphosphorylated ShcC, an adaptator protein, and

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1573 deregulates the responsiveness of the MAPK pathway to include high MYC or b-catenin pathway activity, high growth factors (Osajima-Hakomori et al., 2005). expression of cell cycle and/or chromosome segregation The success of various tyrosine kinase inhibitors in genes and low expression of late neural differentiation different cancers, as well as the expanding number of genes (Krasnoselsky et al., 2005; Fredlund et al., 2008; human malignancies, both in children and adults, Liu et al., 2008; Westermann et al., 2008; Bourdeaut involving aberrant ALK activity encourages the search et al., 2009). In an interesting meta-analysis, Vermeulen for ALK-selective small-molecules inhibitors. Different et al. (2009) recently re-analyzed the seven published categories of putative ALK inhibitors have been micro-array gene expression studies that totalize 690 explored and recently reviewed (Webb et al., 2009). patients with a uniform method, that is prediction The two inhibitors, NVP-TAE684 and PF-2341066, analysis of micro-arrays, and consequently generated targeting the ATP-binding pocket of ALK thereby seven new prognostic gene sets. Fifty-nine genes were blocking ATP binding, have been identified through then selected from these seven sets and their expression small-molecule library screenings (Galkin et al., 2007; level was investigated by quantitative RT–PCR in two Zou et al., 2007; McDermott et al., 2008). Unlike NVP- large cohorts of NB patients from Europe and the US. TAE684 that is not currently being tested as a clinical In both the cohorts this multi-gene expression signature agent, the PF-2341066 compound, which is a dual distinguished high- from low-risk patients. Importantly, inhibitor targeting both ALK and MET kinases, has multivariate logistic regression analyses showed that the entered phase I clinical trials (www.ClinicalTrials.gov) signature was still an independent and statistically and is administered orally to patients with advanced significant parameter in models adjusted for MYCN cancer. At the 2009 ASCO Annual Meeting, Wood and status, age, INSS stage and ploidy. Combined with colleagues reported that PF-02341066 caused complete DNA profiles, it can be anticipated that the analysis of regression of ALK R1275Q NB xenografts, but caused such gene expression signatures that can be easily only minimal growth delay in ALK F1174L and WT assessed in a clinical setting by quantitative RT–PCR, xenografts. Therefore, the development of more than may rapidly constitute an essential parameter for the one ALK-selective inhibitor will likely be required to clinical management of patients. optimally treat NB patients. Further important ques- A strong interest in the role of microRNAs in NB tions remain to be elucidated with respect to the development has recently emerged. Two recent studies implication of ALK in NB pathogenesis. Is the invole- have reported differential microRNA expression in NB ment of ALK in NB limited to the cases with bona fide (Welch et al., 2007; Schulte et al., 2008). Although the mutations or amplification? The issues of ALK ligand(s) size of these series was too limited to address the and signaling pathways are especially crucial in the predictive value of microRNA expression profiles it can understanding of ALK function because abnormalities be foreseen that further studies will identify microRNAs of ALK ligand(s) or downstream targets may constitute linked to aggressiveness. Some micro-RNAs that may be alternative mechanisms to ALK activation. The devel- regulated by MYCN or by retinoic acid have been opment of mouse models will be critical to document the described (Fukuda et al., 2005; Laneve et al., 2007; precise role of ALK mutations in NB oncogenesis. Such Welch et al., 2007). Mir-34-A, a direct target of p53 that models should be extremely useful and powerful for regulates cell cycle arrest and apoptosis, is down- testing anti-ALK therapeutics. regulated in NB through the loss of 1p36 and is expected to have a role in tumorigenesis (Welch et al., 2007; Cole et al., 2008; Wei et al., 2008).

Expression analysis

Numerous groups have used gene expression profiling to Progression schemes in NB seek for gene signatures that may be helpful to classify NB depending on their favorable or unfavorable As mentioned above, the genetic analysis of NB samples outcomes (Berwanger et al., 2002; Ohira et al., 2005; identified several genomic profiles characterized by Schramm et al., 2005; Asgharzadeh et al., 2006; specific abnormalities and associated with distinct Oberthuer et al., 2006; Wang et al., 2006). Importantly, prognosis (Lastowska et al., 2001; Vandesompele these different publications clearly establish that gene et al., 2005; Michels et al., 2007; Mosse et al., 2007; expression profiles strongly differ between favorable and Tomioka et al., 2008; Janoueix-Lerosey et al., 2009). aggressive cases therefore pointing out the potential of Whether these profiles distinguish different, unrelated gene profiling in clinical setting. entities, or whether they constitute snapshots at different The genes involved in the prognostic signature differ times of one entity that may evolve from one profile to from one report to the other, a difference that may be the other, is important. Both hypotheses may be actually attributed to the relatively small size of the series, to the valid. On the one hand, it seems very unlikely that good different array platforms and to the distinct types of prognosis pseudo-triploid tumors may evolve into bioinformatic analyses that were used in these studies. highly aggressive diploid tumors. These two categories However, datamining of these prognostic gene expres- rather constitute clearly distinct entities. On the other sion signatures revealed some important pathways that hand, some recent data argue that, in some occasions, may have critical roles in tumor aggressiveness. These triploid tumors with only numerical chromosome

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1574 Good prognosis neuroblastoma

de novo Type A

Numerical alterations

ganglioneuroma Clonal expansion Segmental Alterations secondary (+/- MYCN) Glial-neural Glial and neuronal progenitor differentiation Type D, E Segmental Alterations (+/- MYCN) Type B, C

de novo

Aggressive neuroblastoma

Schwann cell Neuroblastic cell

Figure 4 A putative model for NB progression. Most NB arise from de novo transformation of neuroblastic precursor cells (bold arrows). Nevertheless, in some instances, glial–neural progenitor cells may give rise to a clonal proliferation of cells that retain the ability to differentiate into Schwann and ganglionic cells (GN) (dashed arrows). No obvious genetic alteration can be detected at this stage (Bourdeaut et al., 2008). Neural cells may then acquire two types of genetic alterations. Occurrence of numerical chromosome abnormalities gives rise to slow-growing pseudo-triploid tumors without abnormality of chromosome structure (good prognosis neuroblastoma type A). At the opposite, unbalanced translocations, which can be associated or not with MYCN ampliﬁcation, lead to aggressive di/tetraploid tumor with no or very few whole chromosome numerical changes (types B and C). Secondarily, type A tumors may acquire additional segmental alterations that lead to aggressive type D or E tumors. Aggressive NB can then occur directly from the neural precursor (de novo occurrence) or after evolution of a good prognosis type A tumor (secondary occurrence).

changes (type A) may secondarily acquire segmental tioned report (Hiyama et al., 2008). In that situation, alterations and progress into more aggressive triploid one would expect that screening does not decrease the tumors with mixed profiles (triploid type D) (Figure 4). number of segmental-only cases but rather decreases the First, the genetic background of these triploid type D number of cases with mixed genetic profiles. cases is highly related to that of purely numerical cases, Although most NB are presumed to arise de novo, with numerical changes concerning the same chromo- without a GN premalignant step, the evolution from somes (G Schleiermacher et al., unpublished data). GN into NB may nevertheless be possible. Indeed, some Secondly, the study of paired samples at diagnosis and lines of arguments suggest that such a progression may at relapse clearly shows that segmental alterations may occur. First, X-methylation analysis indicates that appear at relapse in cases that were purely numerical at though most GNs are polyclonal, a subset of cases diagnosis (G Schleiermacher et al., unpublished data). It harbors a skewed X-inactivation pattern highly sugges- is therefore likely that some tumors with a mixed tive of monoclonality (Bourdeaut et al., 2008). A second numerical and segmental profile arose through evolution argument comes from the analysis of nodular gang- of tumors with a purely numerical profile at onset. In lioneuroblastoma that presents NB nodules within a this scheme, it is of interest to underline that the age at stroma-rich background. In those cases, the Schwannian diagnosis of mixed profile tumors is significantly higher component does not harbor the genetic abnormalities than the age at diagnosis of numerical-only cases observed in the neuroblastic area, indicating that it does therefore suggesting that this progression may require not derive from this last component (Bourdeaut et al., a relatively long time. Importantly, such a progression 2008). However, in most cases that we studied, the scenario may account for the results of recent screening Schwannian and neuroblastic components were mono- programs in Japan that were described above. Indeed, clonal with identical X-methylation profiles therefore though it is clear that de novo, rapidly developing strongly suggesting that both components were derived aggressive tumors can hardly be detected by screening from a common progenitor. A likely hypothesis is programs at 6 months, slowly growing type A tumors therefore that monoclonal expansion of a glial–neural that have a risk to ultimately progress into aggressive precursor first gave rise to a GN with both Schwannian type D may be detected at an early stage (Figure 4). and ganglionic components. Secondarily, through the Consequently, this may result in the decreased frequency occurrence of genetic alterations, neural cells became of NB cases later in life that is noted in the aforemen- fully transformed giving rise to NB. Although it remains

Oncogene Molecular pathogenesis of peripheral neuroblastic tumors I Janoueix-Lerosey et al 1575 to be clearly demonstrated that GN can evolve into NB, neoplasm. Furthermore, genome-wide association study this seems a likely hypothesis at least in a subset of cases. studies still broaden the spectrum of NB susceptibility The observation that PHOX2B mutation carriers can with the discovery of various SNPs associated with an have tumors with very different aggressiveness, includ- increased risk to develop tumors. ing GN, also supports the hypothesis of benign tumors Even more importantly, the ALK receptor emerged as secondarily evolving into more aggressive NB in some a potential therapeutic target through the characteriza- cases. It remains to be documented whether ALK tion of somatic and activating mutations in sporadic mutation also predisposes to GN but it was already NB. Although the link between ALK mutations and stated above that a large spectrum of aggressiveness can the other genomic aberrations present in NB cells still also be noted in ALK mutation carriers. remains to be elucidated, in vitro experiments demon- Altogether, these different observations lead to strated that downregulation of the ALK receptor propose the model indicated in Figure 4. The real leads to a marked decrease in cell proliferation. These frequency of these progression scenarios remains to be observations are consistent with the phenomenon of defined. GN may in some occasions be monoclonal and ‘oncogene addiction,’ stipulating that growth and account for the clonal expansion of a common survival of cancer cells may be impaired by the progenitor that still have the ability to differentiate into inactivation of a single oncogene, despite the occurrence neural and glial lineages (this may constitute a first hit of multiple genetic abnormalities (Weinstein, 2002; in neuroblastic tumor development). Secondarily, the Weinstein and Joe, 2008). Such an addiction provides occurrence of new genetic alterations may lead to a molecular rationale for targeted therapies based on full transformation into NB with a complete block of ALK-selective small-molecules inhibitors, which are differentiation and hence constitute a second hit in NB currently under development. Studies on the use of development. Finally, purely numerical cases may imatinib to inactivate BCR-ABL or c-KIT in CML or evolve, with a frequency that remains to be established, gastrointestinal stromal tumor patients illustrate the into more aggressive tumors with mixed numerical and fantastic power of such approaches to treat patients. segmental profiles. Up to now, there was little evidence, if any, in the Such a model is reminiscent of some progression literature indicating that genetically or biologically schemes in hematopoietic malignancies and particularly favorable neuroblastic tumors may evolve into unfavor- in chronic myeloid leukemia (CML) (Melo and Barnes, able cases. Some of our recent data indicate that such 2007). Indeed, at the chronic phase of the disease to which progression schemes are actually possible. Although the we may tentatively compare the monoclonal GN stage, exact frequency of such scenarios remains to be CML monoclonal cells retain the ability to differentiate determined, they have important clinical consequences into the different hematopoietic lineages. Subsequently, and should therefore be considered. CML cells acquire additional genetic lesions and progress In a review published in 2003 (Brodeur, 2003), Garrett into acute leukemia with lost of the ability to differentiate Brodeur noted that ‘Few tumors have engendered as terminally. In our model, this chronic to acute phase much fascination and frustration for clinical and labora- progression may correspond to the GN to NB evolution tory investigators as NB, the most common and deadly for which the nodular ganglioneuroblastoma may repre- solid tumor of childhood.’ Our feeling is that fascination sent a transition stage. However, as most acute leukemias will still remain because of the huge heterogeneity of this do not rise from a previous CML, the scenario that is tumor, with respect to biology, genetics and outcome, hypothesized here for NB may only account for whereas we hope that frustration will continue to decrease a minority of tumors. In most cases NB would arise with the improvement of the understanding of NB de novo, without prior GN phase. pathogenesis and targeted therapies emerging.

Conclusion Conflict of interest Biological knowledge is essential to understand NB heterogeneity with the aim to improve patients’ manage- The authors declare no conflict of interest. ment, with respect to diagnosis, prognosis and therapeutics. The identification of homogeneous groups of patients is critical to provide optimal therapeutic Acknowledgements strategies and, probably in a near future, new tumor parameters, defined through large genomic and expres- We thank V Marty, P Vielh and G Vassal from the Institut sion profiling studies, will be taken into account to Gustave Roussy for providing the image of ALK immunohis- assign therapeutic strategies. tochemistry. We are grateful to F Bourdeaut for critical Understanding of familial and syndromic NB reading of the paper. Our work was supported by grants from the Agence Nationale pour la Recherche, the Institut National recently has made considerable progress with the du Cancer, the Ligue Nationale contre le Cancer (Equipe identification of PHOX2B and ALK as NB susceptibility labelliseé), the APAESIC (Association des Parents et des Amis genes. Interestingly, both genes are implicated in des Enfants Soigne´sa` l’Institut Curie), the Association Hubert nervous system development, underlying the link Gouin, Les Bagouz a` Manon, les amis de Claire and Enfance between development and cancer in this pediatric et Sante´.

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