Involvement of the mutated ALK receptor in neuroblastoma oncogenesis and development


Sympathetic nervous system

neural crest





Neuroblastoma (NB) is a pediatric cancer which arise from the sympathetic nervous system. It is the most common extracranial cancer of early childhood. The estimated incidence is of 1/8,000 to 1/10,000 births (Maris 2007). NB arises from the sympathetic nervous system. Sixty-five percent of primary tumors occur within the abdomen, mostly in the adrenal medulla or sympathetic paraspinal ganglia. Other common locations are the neck when NB arise from the superior cervical ganglion (SCG), the thorax and the pelvis. Around 40% of patients present with a localized disease, others will present a metastatic disease. The most frequently observed metastatic sites are bone marrow, bone, lymph nodes, liver of subcutaneaous tissue. Among the metastatic patients, 10% have metastases in liver, skin or bone marrow that spontaneously regress. They compose the stage 4S (S=special) of the International Neuroblastoma Staging System (INSS) based on the anatomical presence of NB at diagnosis.

The hallmark of neuroblastoma is its clinical heterogeneity whereas half of all cases classified as high-risk have an overall survival rate less than 40%, a subset of tumors will undergo spontaneous regressions. These tumors that regress spontaneously are more frequent than we expected. Autopsies of infants whose diagnosed cause of death was not cancer have shown an incidence of neuroblast precancer 40-fold higher than the incidence of the clinical disease (Beckwith 1963). In addition, mass infant-screaning programs for the neuroblastoma tumor marker of catecholamines in urine samples found a two-fold higher incidence (Woods 1996).

In 1984, Shimada and colleagues (Shimada 1984) classified tumors according to histopathological features. They were divided into 2 groups: stroma-poor and stroma-rich according to their organizational pattern (stromal development). These two groups were divided in subgroups associated with favorable or unfavorable cases.


\label{predispo} Although the vast majority of NB cases are sporadic, rare familial and syndromic presentations are observed suggesting a genetic predisposition. Familial cases represent about 1% of all cases (Shojaei-Brosseau 2004).

The first predisposition mutation identified in neuroblastoma was in the paired-like homeobox 2b (PHOX2B) gene (Trochet 2004). This transcription factor promotes cell cycle exit and neuronal differentiation. Two types of mutations were identified in the PHOX2B gene. PHOX2B protein harbors a homeodomain and 2 polyalanine stretches. Expansions of the second polyalanine stretch is often associated with congeital central hypoventilation syndrome (CCHS also refered as Ondine’s curse) alone whereas missense mutations or small insertions/deletions, non-polyalanine repeat expansion mutations (NPARMs), are associated with CCHS, NB, and Hirshprung’s disease. Hirshprung’s disease is due to a defect of migration of neural crest cells so it is characterized by the absence of the enteric ganglia in the distal part of the gastrointestinal tract. The patients are affected by chronic occlusions.

About half of familial cases are explained by an activating mutation in the Anaplastic Lymphoma Kinase (ALK) gene. This will be described in details in the section \ref{sectionALK}.

The presence of constitutional PHOX2B and ALK mutations in familial neuroblastoma shows that these genes are involved in an early phase of tumor development that establishes susceptibility to NB.

The RASopathies are developmental syndromes caused by germline mutations in genes that alter the Ras subfamily and Mitogen-activated protein kinases (MAPK) that control signal transduction (see figure \ref{fig:FIGURE_RAS}). Few cases of NB were described in the familial tumor syndrome neurofibromatosis type 1 (NF1) well as in Noonan and Costello syndromes.

\label{fig:FIGURE_RAS} The RAS/RAF/MEK/ERK signal transduction pathway and the RASopathies. From A-M Nyström et al. J Med Genet 2008;45:500-506 (Nyström 2008)

More recently, a novel syndromic presentation associating congenital NB with severe encephalopathy and abnormal shape of the brainstem on brain MRI was reported in two unrelated sporadic cases harboring de novo, germline, heterozygous ALK gene mutations (de Pontual 2011). These mutations were the F1174V and the F1245V. The patients were hypomotile, had no or poor sucking and swallowing reflexes. They both needed respiratory support due to severe episodes of apneas or desaturation. They died within one year of age.

As it was done in other diseases, genome-wide association study (GWAS) identified several single-nucleotide polymorphisms (SNPs) present more frequently in the neuroblastoma patients than in the global population. Among them, 6 SNPs found in the BARD1 gene (Capasso 2009), one within the LIN28B gene (Diskin 2012) and another in the LMO1 gene (Wang 2011) can be highlighted. These three genes has been shown to be oncogenic in NB.

Somatic genetic of neuroblastoma

In 20-25% of primary tumors, an amplification (more than 10 copies) of the MYCN gene was detected (Brodeur 1984). This amplification is strongly correlated with poor outcome. MYCN for Neuroblastoma-Myc oncogene is part of the MYC family. It binds to DNA at conserved elements and seems to interact as direct amplifiers of transcriptionally active genes. Its expression in normal developpement is high in the early post-migratory neural crest but reduces in differentiating sympathetic neurons.

Analysis of DNA copy number profiles of NB tumors showed that they could be classified in two different groups: numerical cases or segmental cases. The numerical cases present with gain or loss of whole chromosomes whereas the segmental cases present breaks within at least one chromosome associated with a gain or loss of a part of a chromosome including MYCN amplification. These categories are associated with prognosis: no disease-associated death was observed in the numerical cases whereas the segmental cases were associated with bad prognosis even without MYCN amplification (Janoueix-Lerosey 2009). The number of breakpoints increases with age in NB patients (Schleiermacher 2010).

Genome sequencing studies of NB tumors has revealed a low mutation rate (about 0.6 mutation per Mb in exons). Activating mutations of ALK are present in around 8% of cases (see section \ref{sectionALK} for more details). Heterozygous missense PTPN11 and NRAS mutations were identified in around 2.9% and 0.8% of cases respectively. PTPN11 encodes the SHP2 protein which is part of the RAS/MAPK pathway and its germline mutation cause Leopard or Noonan syndrome (figure \ref{fig:FIGURE_RAS}). Moreover, mutations of MYCN in non-amplified cases were recurrently observed. Furthermore, mutations or structural variations targeting genes i