The implications of ALK and the RAS-MAPK pathway in primary NB tumors
Many different studies have reported
ALK mutations in approximately 6–10% of sporadic primary NB tumors and around 12–14% of the high-risk category [
9‐
11,
15]. To understand the relative importance of
ALK in the development of NB we compared the frequency of
ALK mutations in NB with the frequencies of
ALK mutations in other tumor types.
ALK is found to be mutated more frequently in NB than in brain, colorectal, lung, and blood cancers. Only breast and skin cancers have higher
ALK mutation rates than NB, suggesting that
ALK mutations play a more significant role in NB development than in other common cancer types (Table
1).
ALK mutations in NB are mainly single amino acid substitutions; in 90% of cases they are located in the kinase domain, with hot spots at amino acids F1174, R1275, and F1245 [
9‐
11,
15]. Interestingly, in approximately 50% of NB cases,
ALK mutation is found together with
MYCN amplification and correlated to poor prognosis [
11].
Interestingly, in an additional 2–3% of NB samples, ALK was found to be activated by gene amplification, increasing both protein expression and activity [
19‐
21].
ALK amplification, which is mutually exclusive of point mutation, is also almost always associated with
MYCN amplification, with both genes located in the same locus (2p23–24, Table
2) and associated with poor prognosis [
11,
13,
15,
22].
Table 2
Chromosomal locations of the RAS-MAPK pathway genes and most common chromosomal anomalies in NB
Although
ALK mutations have been observed in all clinical risk groups, a recent study analyzing a large cohort of more than 1500 NB patients demonstrated that the presence of an
ALK mutation was independently correlated with survival: patients with an
ALK mutation showed a 1.4-fold greater risk of an event within 5 years than patients without one. Statistically significant correlations were independently observed for all the groups of
ALK mutations:
ALK aberration,
ALK copy number gain, and
ALK amplification [
15]. For a more general review of
ALK and NB, we recommend the recent review by Trigg et al. [
23].
Different studies have also reported somatic RAS-MAPK pathway gene alterations (mutation, amplification, or deletion) leading to the activation of this pathway in large cohorts of sporadic NB patients, with a frequency of around 4–10% [
21,
24,
25]; the most frequently mutated genes were
PTPN11 (2.9%),
NRAS (0.8%), and
NF1 [
14,
24]. Typically, the first mutations observed in
NRAS were the known activators Q61K and C181A, which were initially discovered in SK-N-SH and SK-N-AS cell lines, respectively [
16,
26‐
28]. Also,
HRAS and
KRAS can be mutated, notably by G12D and Q61K substitution, for instance [
16,
29].
Most recently, Ackerman et al. sequenced the genomes of 418 pre-treated NB. In line with a previous study, they found that 9% had
ALK mutations, but also that 6% had alterations in the RAS-MAPK pathway genes (
NF1: 1.2%;
HRAS: 1%;
PTPN11: 1%:
NRAS: 0.5%;
BRAF: 0.5%
KRAS: 0.2%; and
FGFR1: 0.2%). ALK-RAS pathway alterations were detected in all NB risk categories and were found to be strongly correlated to poor outcomes, even in the high-risk category [
25]. Interestingly, patients harboring RAS pathway mutations were found to have a worse prognosis than those with
ALK mutations. Furthermore, Ackermann et al. also investigated the relationship between ALK-RAS alterations and telomere maintenance mechanisms (defined by
MYCN,
TERT, and APB alterations). Although the presence of that telomere maintenance mechanism was associated with worse outcomes compared to those without those mechanisms, an additional mutation in the ALK-RAS pathway increased NB aggressiveness, resulting in a higher mortality rate. In contrast, in NB patients without a telomere maintenance mechanism, the presence of an ALK-RAS alteration did not affect patient outcomes [
25]. Eleveld et al. were recently able to define an mRNA signature, consisting of six genes (
ETV4,
ETV5,
DUSP6,
MAFF,
ETV1, and
DUSP4) that was correlated with an increase in RAS-MAPK pathway activity. A high expression of these signature genes was associated with poor survival in primary NB but also with MEK1/2 and ERK1/2 phosphorylation and sensitivity to different MEK1/2 inhibitors in cell lines [
30].
Mutations in the genes coding for the RAF family of proteins are very rare in NB.
BRAF aberrations have been observed in 1% of NB cases, whereas the study by Shukla et al. reported no alterations in
ARAF and
CRAF [
26]. However, there was a greater expression of all the RAF proteins in the high-risk NB category [
31]. The main mutation found in
BRAF is the V600E substitution [
26]. This mutation is located in BRAF’s kinase domain and leads to a constitutively active form of BRAF [
32]. Interestingly, a tandem duplication in the
BRAF gene was also detected in a relapse NB. This duplication resulted in the expression of a
BRAF transcript that encodes a protein with two kinase domains. Accordingly, the protein was twice as active, which made the RAS-MAPK signaling pathway more active [
16].
A
MEK1/2 mutation is a very rare event in NB, with a frequency of 0.5% [
26]. In one NB sample, a
MEK1 somatic activating mutation (K57N) was observed, located between the protein’s nuclear export signal and its kinase domain. This genetic lesion leads to constitutive activation of the RAS-MAPK pathway in vitro [
33]. On the contrary, no reports of
ERK mutations have been found in NB.
Comparisons of NB with six other major cancer types showed significant differences in the frequency of mutations in the major RAS-MAPK pathway genes. NB, brain and breast tumors do not demonstrate high mutation frequencies along this pathway (Table
1), while colorectal, blood, lung, and skin cancers have high frequencies in at least one of the RAS-MAPK pathway genes. Interestingly, two genes,
RAF1 (3p25.2) which encodes for the CRAF protein, and GRB2 (2p23-2p23.1), were found to be located in regions that sustain frequent chromosomal abnormalities in NB (Table
2).
Finally, different studies have also reported alterations in genes associated with the RAS-MAPK pathway, such as
CDK4,
LIN28B,
CCND1,
SMO,
SOS1,
CIC,
DMD, and
DUSP5 [
24,
25,
30,
34,
35].
ALK and RAS-MAPK mutations at relapse
Different studies have reported that
ALK mutations were more frequently detected in NB relapse samples, with a frequency of 15–40% [
16,
24,
36]. In 2015, Eleveld et al. analyzed 23 paired diagnostic and relapsed NB samples. They were able to show not only a clonal evolution from diagnosis to relapse sample but also that 78% (18/23) of the relapsed NB samples harbored a genetic aberration predicted to activate the RAS-MAPK pathway (
ALK (10/23);
NF1 (2/23);
PTPN11 (1/23);
FGFR1 (1/23);
NRAS (1/23);
KRAS (1/23);
HRAS (1/23); and
BRAF (1/23)) [
16]. In line with these results, Padovan-Merhar et al. reported that suspected
ALK driver mutations were present in 7% (3/43) of samples at diagnosis, 17% (7/41) of the post-treatment samples, and 20% (11/54) of the samples at relapse. Furthermore, they observed more suspected oncogenic
ALK mutations in relapsed disease than at diagnosis, as well as enrichment of RAS-MAPK pathway mutations at relapse [
24]. Also, single-nucleotide variants (SNVs) in RAS proteins are more frequent in relapsed NB, notably, for instance, in
HRAS with the somatic Q61K mutation [
16,
24,
26], resulting in the constitutively active form of RAS due to inactivation of the GTPase domain [
37].
Familial NB, sporadic NB germline susceptibility genes, and RASopathies
Familial cases of NB are very rare, representing 1–2% of all NB cases, and they are inherited in an autosomal-dominant manner. In recent years, many advances have been made in our genetic comprehension of these cases.
PHOX2B (4p12), a gene known
to be an essential regulator of the development of a normal autonomic nervous system and frequently associated with neurocristopathies, was the first to be identified in this setting in 2004 [
38,
39]. However, it was only detected in a small subset of familial NB (approximately 10%). In 2008, two different, independent studies reported
ALK (2p23) as a gene involved in more than half of familial NB cases. Additional studies finally reported
ALK germline mutations in almost 80% of these families [
15,
17,
40]. The majority of these mutations are located in the
ALK tyrosine kinase domain, resulting in constitutive activation of the kinase [
9,
10]. The penetrance is variable, highly related to the activating effect of the mutation [
15], but estimated at around 50% overall [
41]. As yet, no other genes have been identified to explain the 10% of familial cases without the
PHOX2B or
ALK germline mutations.
Interestingly, NB susceptibility genes were recently identified in the germline DNA of a patient with sporadic NB, suggesting that common germline polymorphisms with low penetrance (i.e.,
NBAT-1,
CASC15,
BARD1,
LMO1,
HSD17B12,
DUSP12,
LIN28B,
HACE1,
SPAG16,
NEFL,
MLF1/RSRC1,
CPZ,
CDKN1B,
SLC16A1,
MSX1, MMP20, KIF15) or more rare variants with higher penetrance (i.e.,
ALK,
BARD1,
CHEK2,
AXIN2,
TP53, APC, BRCA2, SDHB, SMARCA4, LZTR1, BRCA1) may also have a relevant role in NB carcinogenesis [
17,
18]. Remarkably, when investigating the associations between these genes and the RAS-MAPK pathway, many of them were reported to interact with this pathway either in NB or other cancers (Table
3). This further supports the view that the RAS-MAPK pathway is one of the central targets in the process of tumorigenesis.
Table 3
Interactions of the RAS-MAPK pathway with NB associated genes
Low penetrance genes | CASC14/NBAT-1 | Loss of function | 6p22 | MAPK1/3 inhibition | [1, 2] |
CASC15 | | 6p22 | | |
BARD1 | Gain of function | 2q35 | MAPK1a | [3–6] |
LMO1 | Gain of function | 11p15.4 | MAPK1/3 activation | [7, 8] |
HSD17B12 | | 11p11.2 | | |
DUSP12 | NA | 1q23.3 | MAPK1, HRAS inhibition | [9–12] |
LIN28B | Gain of function | 6q16 | MAPK1/3 activation | [13, 14] |
HACE1 | Loss of function | 6q16 | MAPK1/3 inhibition | [13, 15, 16] |
SPAG16 | | 2q34 | | |
NEFL | | 8p21 | | |
MLF1/RSRC1 | NA | 3q25 | ARAFa | [17] |
CPZ | | 4p16 | | |
CDKN1B | Inhibited by GRB2 through p27Kip1 | 12p13.1 | MAPK1 | [16, 18–22] |
GRB2 |
SLC16A1 | NA | 1p13.2 | KRASa | [23–27] |
NRASa |
HRASa |
MSX1 | Gain of function | 4p16.2 | ARAF activation | [28, 29] |
MMP20 | NA | 11q22.2 | Activation of pathway | [30] |
KIF15 | NA | 3p21.31 | Activation of pathway | [31–33] |
High penetrance genes | CHEK2 | Loss of function | 22q12.1 | MAPK1/3 inhibition | [5, 34, 35] |
AXIN2 | | 17q24.1 | | |
BRCA2 | Loss of function | 13q13.1 | GRB2 | [36–39] |
SDHB | | 1p36.13 | | |
SMARCA4 | Loss of function | 19p13.2 | NF1a | [40–42] |
MAPK1a |
LZTR1 | Loss of function | 22q11.21 | NF1 | [23, 25, 26, 43, 44] |
NRAS inhibition |
BRCA1 | Loss of function | 17q21.31 | NF1 | [45–48] |
MAPK3 |
MAPK1 |
NBPF23 | | 1q21.1 | | |
SEZ6L2/PRRT2 | | 16p11.2 | | |
APC | Loss of function | 5q22.2 | MAP2K1 inhibition | [39, 49] |
TP53 | Loss of function | 17p13.1 | MAPK1 | [50–83] |
HRAS |
GRB2 |
KRAS |
BRAF |
SHC1 |
PTPN11 |
MAP2K1 |
MAP2K2] |
ARAF |
Additionally, NB has been reported to be associated with diseases well-known to predispose cancer, such as Li Fraumeni syndrome (
TP53 mutations) [
42], familial paraganglioma/pheochromocytoma (
SDHB mutations) [
43], and Beckwith–Wiedemann syndrome (
CDKN1C mutations or loss of expression) [
44]. It has also been associated with other rare syndromic diseases, more particularly with those characterized by the presence of germline mutations located along the RAS-MAPK pathway. This group of diseases, also known as
RASopathies, includes neurofibromatosis 1 (
NF1 mutations), Costello syndrome (
HRAS mutations), Noonan syndrome, and such Noonan-like syndromes as Noonan syndrome-like disorder with loose anagen hair and the LEOPARD syndrome (mutations in
PTPN11,
SOS1,
KRAS,
NRAS,
RAF1,
BRAF,
MEK1,
SHOC2,
MEK2,
RIT1,
and CBL) [
41,
45‐
47].