Identification of ALK Mutation in Neuroblastoma on the Point of Molecular Heterogeneity

Introduction

Recent advances in targeted therapies are becoming increasingly important for the treatment of patients with cancer, particularly for pediatric patients. Patients with higher-stage and high-risk neuroblastoma (NB) have a worse prognosis than low-risk patients as the 5-year overall survival rates in high-risk and low-risk patients are 40% to 50% and >90%, respectively.^1,2

NB is a heterogeneous disease characterized by various chromosomal rearrangements, such as MYCN gene amplification (24% of all cases), partial deletions in chromosomes 1p and 11q, partial additions in chromosome 17q, and triploidy. ³ Management of relapse in high-risk and refractory NB cases is difficult; therefore, novel treatment strategies are needed. Anti-GD2 immunotherapy and therapies directed against tumor-specific molecular alterations, such as anaplastic lymphoma kinase (ALK), increase survival of patients with high-risk NB. ² Combination of chemotherapy, radiotherapy, and stem cell transplantation is other treatment options. Isotretinoin combined with anti-GD2 immunotherapy and targeted kinase inhibition is a potential treatment strategy for patients with high-risk NB.

Different kinases—such as ALK, aurora kinase, and RET receptor tyrosine kinase—are potential therapeutic targets for various cancers. ⁴ ALK acts as an oncogenic driver in cancer; therefore, ALK gene is one of the major targets for inhibition in NB. ALK encodes a tyrosine kinase receptor belonging to the insulin receptor superfamily. It is expressed in the developing central and peripheral neural tissues, where it plays a supporting role in embryonic nervous system development; however, ALK expression decreases after birth. Neuroblastoma likely develops due to malignant transformation of partially committed sympathoadrenal neuroblasts, during fetal or early childhood development. ⁵

ALK gene is found at chromosome band 2p23; it has 29 exons. The extracellular ligand-binding, transmembrane, intracellular juxta membrane, and kinase domains of this gene comprise 1620 amino acids. The tyrosine kinase domain exists between 1116 and 1392 amino acids.^6,7 ALK is activated in cancer via different mechanisms. Anaplastic large-cell lymphoma, inflammatory myofibroblastic tumors, nonsmall cell lung cancer, and breast, colon, and renal cancers show the t(2;5) (p23;q35) chromosomal translocation. This translocation produces a fusion protein called nucleophosmin which acts as an oncogene. In NB, various point mutations or gene amplifications are observed.^6–9 Genetic alterations in ALK often lead to constitutive activation of the tyrosine kinase domain. In wild-type ALK, the kinase domain exists in an auto-inhibited conformation; the ALK receptor dimerizes and the tyrosine kinase domain is phosphorylated. This activates the MAPK, PI3K, or JAK/STAT pathways.

ALK mutations occur in the germline in 50% of familial NB cases, and only in 15% of somatic NBs. Mutations in germline cases are inherited in an autosomal dominant manner.^7,8 Patients with NB having both MYCN amplification and ALK mutations show poor prognosis. Patients receiving crizotinib or second-generation ALK inhibitors, such as alectinib and lorlatinib as targeted therapy, might receive survival benefits after selection of the ALK mutation type using next-generation sequencing (NGS). ⁹

In NB, mutations at 3 key positions, namely F1174, F1245, and R1275, account for approximately 85% of ALK mutations. R1275Q is the most common type of mutation observed in about 45% of familial cases and a third of sporadic cases, whereas F1174 and F1245 mutants are exclusively found in sporadic NB cases, at frequencies of approximately 30% and 12%, respectively.^7–9 Interestingly, the Y1278S mutation observed in patients with NB harbors gain-of-function activity. ³ The aim of this study is to investigate ALK mutation heterogeneity in NB cases in Turkey by NGS.

Materials and Methods

Ethical approval was obtained from the Local Human Non-invasive Studies Ethics Committee of our university (approval no.: 2018/05-21; date: 15.02.2018).

Results

Next-Generation Sequencing ALK Mutation Results

Pathogenic ALK mutations were detected in 13 patients (12.04%). ALK mutations were observed in intermediate- and high-risk patients (P  =  .028). Four patients with both somatic and germline R1275Q mutations belonged to the same family. One patient harbored a nonfamilial R1175Q mutation. The F1174L mutation was observed in 4 cases: one case had the F1174C mutation and another had the F1174Q mutation. One patient harbored an I1171T mutation. One patient had multiple mutations, including L1226F, S1189F, V1135A, and G1125S. In 65.74% of the 108 cases, the I1461V, K1491R, and D1529E mutations with high allele frequencies were observed that are not of clinical importance. In 44 cases, double samples were studied to explore molecular heterogeneity. Synchronous samples from different areas of the tumor were studied in 26 cases. Metachronous tumors were studied at different time points in 18 cases. There was no case of ALK mutation in only one patient. The same mutation was observed in all 44 double samples. Only in one case, 4 more ALK mutations without clinical importance was observed. The mutation sites and frequencies in relation to patient characteristics are shown in Table 1. In the survival analysis, we did not observe any association between ALK mutations or loci and clinical outcomes (including progression-free survival and overall survival). The NGS datasets generated and analyzed in this study are available from the corresponding author upon request.

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Table 1.

Patient Characteristics of the cases with ALK Mutation.

No.	Age (in months)/sex	Risk	ALK mutation	MYCN amplification	11q23 deletion	DNA ploidy	Shimada classification
NB 704	8/Female	Intermediate	R1275Q somatic  +  germline	Negative	Negative	Triploidy	Unfavorable
NB768	14/Female	Intermediate	R1275Q somatic  +  germline	Negative	Negative	Tetraploidy	Favorable
NB1235	120/Male	Intermediate	R1275Q, somatic  +  germline	Negative	Positive	Triploidy	Favorable
NB595	8/Female	Intermediate	R1275Q, somatic  +  germline	Negative	Negative	Triploidy	Unfavorable
NL 63	6/Male	High	R1275Q, somatic	Positive	Negative	Diploidy	Unfavorable
NL224	156/Female	High	F1174Q somatic	Negative	Positive	Diploidy	Favorable
NL84	31/Male	High	F1174L somatic	Negative	Negative	Diploidy	Unfavorable
NL247	13/Male	High	F1174L somatic	Positive	Positive	Diploidy	Favorable
NL220	43/Female	High	F1174L somatic	Positive	Positive	Hipoploidy	Unfavorable
NB1387	12/Male	High	F1174L somatic	Positive	Positive	Diploidy	Unfavorable
NB1303	60/Female	High	F1174C somatic	Negative	Positive	Diploidy	Unfavorable
NL154	36/Female	Intermediate	I1171T somatic	Negative	Negative	Diploidy	Unfavorable
NL241	132/Female	High	L1226F, S1189F, V1135A, G1125S somatic US	Negative	Positive	Diploidy	Unfavorable

Abbreviations: NB, neuroblastoma; ALK, anaplastic lymphoma kinase; MYCN, neuroblastoma-derived V-Myc Avian myelocytomatosis viral related oncogene; US, undetermined significance.

Among cell line samples, Kelly and SHSY5Y cells showed F1174L point mutations while LAN5 and CHP134 cells not. All 4 cell lines harbored an I1461V benign ALK mutation, which is common in NBs.

Discussion

In this study, we performed ALK gene sequencing using NGS in paired samples of NB tumor tissues to explore molecular heterogeneity. The samples were obtained from different parts of the tumor at the same time, and from primary diagnostic and tumor tissues during relapse of the same patient at different times. We did not observe molecular heterogeneity in ALK mutations between the 2 samples in either group.

ALK is only phosphorylated in developing central and peripheral nerve tissues. These mutations are overexpressed in cancer.^9,14 Although ALK oncogene activation increases in many human cancers, such as lung carcinoma and fusion proteins (NPM-ALK fusion in anaplastic large cell lymphoma; EML4-ALK in nonsmall cell lung cancer) through chromosomal translocation events, ¹⁵ more than 20 point mutations in the conserved regions of the tyrosine kinase domain have been identified in NB. ⁶ Fusion proteins lead to ligand-independent self-dimerization, autophosphorylation, and transphosphorylation of ALK. Point mutations cause autophosphorylation. ALK has been identified as a potential pharmacological target in NB when ALK knockdown results in growth inhibition in all NB cell lines with ALK mutations and some with wild-type ALK. ¹⁶

The INRG classification has led to 16 statistically distinct risk groups based on clinical and molecular features, which has made prognosis more accurate for patients and helped guide physicians in treatment regimens. In Turkey, the NB protocol based on the INRG is used as a national protocol, supported by the Turkish Pediatric Oncology Group Association. Detection of ALK and other genes by NGS for targeted therapy decisions has not yet been included in the protocol. However, it is currently used to determine new therapeutic approaches in relapsed and refractory cases. Neuroblastoma cases, including those with the R1275Q mutation in ALK, are sensitive to first-generation ALK inhibitors, whereas those with F1174 mutations are resistant. Patients with F1174 mutations are sensitive to second-generation ALK inhibitors. ¹⁷ ALK amplification is not an indication for targeted therapy in NB. In one of the 2 cases where ALK amplification was detected ALK F1174 mutation was also present. The other case that only had ALK amplification was not considered for targeted therapy.

ALK mutations were found in 12.4% of cases. Donohue et al analyzed 641 cases using whole-genome sequencing or hotspot ALK mutation profiling and found ALK mutations in 16% of cases. Approximately 10% of all NB harbor somatic ALK mutations at diagnosis (with reports of even higher incidence at relapse), 85% of which are accounted for by single nucleotide variants at 3 loci: R1275 (43%-49%), F1174 (30%-35%), and F1245 (12%). ⁷ ALK mutations are present in 8% to 14% of all NB at diagnosis, increasing to 26% to 43% at relapse. Allinson et al reported 2 cases of loss of ALK mutations during relapse (ALK F1174Land ALK R1257Q).^18–20

In the present study, patients with somatic F1174 mutations were treated with lorlatinib. The patients were clinically good for one year. Follow-up was continued. One case with F1174L mutations showed 16% of the same mutation in peripheral blood. However, this was considered to be circulating DNA instead of germline mutations. This does not apply to homozygous or heterozygous mutations. We interpreted this germline mutation was from circulating DNA but could not confirm it.

Characterization of the different point mutations in ALK observed in NB patients has led to the segregation of mutations into 3 classes: ligand-independent, ligand-dependent, and kinase-dead forms of receptor. ³ The tyrosine at positions 1158, 1278, 1162, and 1163 is important for autophagy. In the present study, mutations in the kinase domain were recorded. The first 4 cases shown in Table 1 are from the same family. There are cousins. There was one more case in the same family history of death from NB in which we could not reach the tumor tissue or blood. The R1275Q point mutation was detected as expected.^21–23 The ALK-mutated NB cases in our series were not found to be related to MYCN amplification. MYCN was not amplified in familial cases with germline R1275Q mutations. We think that this is the reason our results did not associate ALK with MYCN amplification. Most cases of ALK mutations have been reported to be amplified by MYCN. ²⁴ When we exclude familial cases the ratio of MYCN amplification is 4/9 (44.44%). This is higher than NB population but the number of cases is too small to perform meaningful statistical analysis.

The weakness and limitations of the study are that the total number of cases in which ALK mutations were studied was less than that in the literature. Another weakness is the selection bias caused by the demand from clinicians since we are only studying ALK on their demand. This may be the main reason that our mutation profile differs from other cohorts. We plan to add ALK to routine clinical diagnosis to prevent selection bias. The strength of this study is in the number of the samples. Another strength of our study is that we performed 2 sample analyses for many cases. We report F1174L mutation in germline. To our knowledge, this is the first study to evaluate the differential impact of various ALK mutation loci on NB in Turkey.

Conclusion

In this study, we aimed to identify the most common ALK mutations in NB but did not observe any somatic heterogeneity in ALK mutation status in different spatial and temporal samples of patients with NB. We concluded that it is better to study NGS in the initial biopsy of a patient, regardless of whether it is primary or metastatic. The surgical material after neoadjuvant chemotherapy was unsatisfactory. These samples included large areas of treatment effects such as necrosis, fibrosis, hemorrhage, Schwannian, and gangliocytic differentiation. The relapse of tumor tissue, if satisfactory areas of blastic tumors are present, may be used. Bone marrow biopsy is not useful for obtaining high-quality DNA samples. Bone marrow aspiration followed by MACS or FACS selection of GD-2 positive cells by anti-GD-2 antibody might be attempted if more tumor tissue is available for NGS.