Genomic ALK alterations in primary and relapsed neuroblastoma

Abstract

Background: Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse.

Methods: ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively.

Results: At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients <18 months. At relapse, ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome.

Conclusions: The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.

Fig. 1. Consort diagram of the study.

Flow diagram of patient inclusion in this study. Patients that are listed in the category “ALK alteration status” represent the overlap of the cohorts for which ALK mutation and for which ALK amplification status was available. ddPCR, digital droplet PCR.

Fig. 2. ALK alterations in neuroblastoma at diagnosis.

a ALK mutation frequencies in neuroblastoma obtained at diagnosis. b Age at diagnosis of patients with ALK-mutated versus non-mutated neuroblastoma obtained at diagnosis. c ALK amplification frequencies in neuroblastoma obtained at diagnosis. d Age at diagnosis of patients with ALK-amplified versus non-amplified neuroblastoma obtained at diagnosis. Boxplots show the median, first and third quartile (boxes), with whiskers indicating the minimum and maximum of the data within 1.5× the interquartile range. P values for a and c were calculated using Fisher’s exact test or Chi-squared test where appropriate, and for b and d using Wilcoxon–Mann–Whitney test. HR, high-risk; NHR, non-high-risk; ampl, amplified; m, months; St4, stage 4; loc, localized disease.

Fig. 3. ALK alterations in neuroblastoma at relapse.

a ALK mutation frequencies in relapsed neuroblastoma. b Age at diagnosis of patients with ALK-mutated versus non-mutated relapsed neuroblastoma. c ALK amplification frequencies in relapsed neuroblastoma. d Age at diagnosis of patients with ALK-amplified versus non-amplified relapsed neuroblastoma. Boxplots show the median, first, and third quartile (boxes), with whiskers indicating the minimum and maximum of the data within 1.5× the interquartile range. P values for a and c were calculated using Fisher’s exact test or Chi-squared test where appropriate, and for b and d using Wilcoxon–Mann–Whitney test. HR, high-risk; NHR, non-high-risk; ampl, amplified; m, months; St4, stage 4; loc, localized disease.

Fig. 4. Comparison of ALK alteration frequencies in neuroblastoma obtained at diagnosis and at relapse.

a ALK mutation frequencies in neuroblastoma obtained at diagnosis versus relapse in the entire cohort (left) and in the subgroup of patients of whom paired samples were available (right). b ALK mutation frequencies in neuroblastoma obtained at diagnosis versus relapse in high-risk tumors (HR) in the entire cohort (left) and in the subgroup of patients of whom paired samples were available (right). c ALK mutation frequencies in neuroblastoma obtained at diagnosis versus relapse in non-high-risk tumors (NHR) in the entire cohort (left) and in the subgroup of patients of whom paired samples were available (right). d ALK amplification frequencies in neuroblastoma obtained at diagnosis versus relapse in the entire cohort (left) and in the subgroup of patients of whom paired samples were available (right). e ALK alteration frequencies in stage 4 neuroblastoma of patients <18 months versus ≥18 months at diagnosis in the cohort of tumors obtained at diagnosis (left) and at relapse (right). f Schematic representation of the ALK tyrosine kinase domain (TKD) and the amino acid positions and frequencies of mutations detected in neuroblastoma samples at diagnosis and at relapse. g Frequencies of mutation types at position F1174 at diagnosis and at relapse. h Frequencies of mutation types at position F1245 at diagnosis and at relapse. i Frequencies of mutation types at position R1275 at diagnosis and at relapse. Of note, mutation type of one mutation detected in a tumor at relapse had remained unknown. P values were calculated using Fisher’s exact test.

Fig. 5. Impact of ALK alterations on patient survival.

a OS of patients with ALK-mutated tumors versus patients with ALK wildtype tumors at diagnosis (5-year OS, 66% versus 76%). b OS of high-risk patients with tumors harboring ALK mutations at allelic fractions >20% versus ≤ 20% versus ALK wildtype (5-year OS, 28% versus 57% versus 54%). c EFS of high-risk patients with tumors harboring ALK mutations at allelic fractions >20% versus ≤ 20% versus ALK wildtype (5-year EFS, 17% versus 57% versus 33%). d OS of patients with ALK-amplified tumors versus ALK non-amplified tumors at diagnosis (5-year OS, 22% versus 60%). e OS of patients with ALK-amplified versus non-amplified tumors at relapse (5-year OS, 0% versus 57%). f OS of patients with ALK-altered versus ALK-non-altered tumors at relapse (5-year OS, 37% versus 61%). P values were calculated by log-rank and, in case of non-proportional hazards, Gehan-Breslow test. OS, overall survival; y, years; ampl, amplification; AF, allelic fraction.

Fig. 6. Dynamics of ALK alterations over the course of disease.

a Comparison of allelic fractions of ALK mutations detected at diagnosis and relapse by NGS versus ddPCR. In two patients (P2 and P13), two different ALK mutations were detected in the same tumor samples. X indicates that no material was available, check marks indicate that mutations were detected by dideoxy-sequencing only, thus leaving their allelic fraction unknown. b Longitudinal monitoring of allelic fractions (as determined by ddPCR) of two different ALK mutations (R1275Q and F1174L) over the course of disease in an individual patient. The allelic fraction of the HRAS mutation occurring at week 275 was determined by panel NGS. ALK inhibitory treatment and the clinical course of disease are shown at the top, with arrowheads indicating progression or relapse of disease. Timepoints and localizations of biopsies are indicated by arrows; biopsies of metastasis were taken from the same maxillary metastasis, except the last biopsy, which was derived from bone marrow. c Schematic diagram for the clonal evolution of cancer cell populations reconstructed from whole-exome sequencing data in patient P13. T1–T6 represent the consecutive biopsies that were taken from the patient. Gray boxes highlight clones that were private to the respective biopsy. ALK and HRAS mutations are indicated as they appeared in chronological order. d Clonal composition of each biopsy. The ancestral clone C0 is illustrated on the left to indicate its presence in every biopsy. AF, allelic fraction; NGS, next generation sequencing; ddPCR, digital droplet PCR; mut, mutation; D, diagnosis; R, relapse; met, metastasis; PT, primary tumor; BM, bone marrow.