Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1)

Abstract

Purpose: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact.

Materials and methods: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571).

Results: Genomic ALK amplification (ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no-ALKa [n = 860] 51% [95% CI, 47 to 54], [P < .001]), particularly in cases with metastatic disease. ALK mutations (ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA (P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome.

Conclusion: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

Trial registration: ClinicalTrials.gov NCT01704716.

Conflict of interest statement

Walentyna BalwierzHonoraria: Shire, Gilead Sciences, Novartis, AmgenConsulting or Advisory Role: Amgen, Novartis, Roche, TakedaTravel, Accommodations, Expenses: Jazz Pharmaceuticals, Shire, Roche, Servier Martin ElliottConsulting or Advisory Role: Bayer Dominique Valteau-CouanetConsulting or Advisory Role: EUSA PharmaResearch Funding: Orphelia PharmaPatents, Royalties, Other Intellectual Property: Royalties from Apeiron to SIOPENTravel, Accommodations, Expenses: EUSA Pharma, Jazz Pharmaceuticals Deborah A. TweddleHonoraria: Eusa PharmaTravel, Accommodations, Expenses: EUSA Pharma Ruth LadensteinHonoraria: Apeiron Biologics, Boehringer Ingelheim, EUSA PharmaConsulting or Advisory Role: Apeiron Biologics, Boehringer Ingelheim, EUSA PharmaResearch Funding: Apeiron Biologics, EUSA PharmaPatents, Royalties, Other Intellectual Property: Apeiron Biologics, EUSA PharmaExpert Testimony: Apeiron Biologics, EUSA PharmaTravel, Accommodations, Expenses: Apeiron Biologics, EUSA Pharma Gudrun SchleiermacherHonoraria: BMSResearch Funding: Bristol Myers Squibb, Pfizer, MSDavenir, RocheTravel, Accommodations, Expenses: RocheNo other potential conflicts of interest were reported.

Figures

FIG 1.

Flow diagram of patient inclusion. A total of 3,334 patients with HR-NB were enrolled in the HR-NBL1 trial from 188 centers. Among these, 2,350 patients were not included in this study, either because no contributive tumor material was available, or because these was no FU data, or both. Thus, 1,092 patients from 132 centers were included in this study. aClonal level: > 20% MAF. bSubclonal level: MAF 0.1%-20%. FU, follow-up; HR-NB, high-risk neuroblastoma; MAF, mutated allele fraction.

FIG 2.

Genetic alterations of ALK in patients with HR-NB. (A) Copy-number profile of case 536. Genomic coamplification of MYCN and ALK is observed on chromosome 2, encompassing the regions between position 15,440,477 and 16,822,999 and between 29,113,790 and 30,309,749 bp (human genome assembly hg19; UCSC Genome Browser Home). (B) Frequency distribution of mutated ALK alleles at the studied chromosome regions, encompassing the AA positions F1174, L1190, L1196, R1245, D1270, G1272, M1273, A1274, R1275, and Y1278 detected, in 762 samples. ALK mutations involved the common mutational hotspots (F1174, F1245, and R1275) in 12.5% (97 out of 772) of cases, at a clonal level (MAF 20%-93%) in 72 cases, and at a subclonal level (MAF < 20%) in 25 cases. At the F1174 hotspot (chr2: 29,443,695-29,443,697), alterations were observed in 44 cases: 42 cases harbored a mutation leading to the AA change F1174L, one case with F1174I, and one case with F1174S, with MAFs ranging from 0.12% to 78%. At the R1275 hotspot (chr2: 29,432,849-29,430,139), mutations were detected in 43 cases: 38 cases harbored a mutation leading to the AA change R1275Q and five cases with R1275L, with the MAFs ranging from 0.2% to 93%. Ten cases showed ALK mutations at the F1245 hotspot (chr2: 29,436,858-29,436,860) within exon 24. Three samples showed the F1245L mutation, three cases carried the F1245C mutation, three showed the F1245I mutation, and one showed mutation F1245V mutation (Fig 1 and Appendix Table A1). Other ALK mutations were detected at residues I1170, L1190 (two cases), L1196, D1270, G1272, M1273, A1274, and Y1278 within the explored regions, leading to a nonsynonymous AA change with a predicted functional impact. All these mutations were clonal (MAF > 20%) except for M1273I (MAF 0.2%) and I1170 (MAF 2.8%). AA, amino acid; aCGH, array comparative genomic hybridization; bp, base pair; HR-NB, high-risk neuroblastoma; MAF, mutated allele fraction; UCSC, University of California, Santa Cruz.

FIG 3.

Survival in the ALK analysis cohort. (A) OS according to ALK amplification status in 901 patients: presence of ALK amplification (n = 41), 5-year OS 28% (95% CI, 15 to 42) versus absence of ALK amplification (n = 860), 5-year OS 51% (95% CI, 47 to 54); P < .0001. (B) OS according to ALK mutation status in 762 patients: presence of an ALK mutation (n = 106), 5-year OS 41% (95% CI, 31 to 51) versus absence of an ALK mutation (n = 656), 5-year OS 49% (95% CI, 45 to 53); P = NS. (C) OS according to ALK clonal or subclonal mutation status in 762 patients: no mutation (n = 656), 5-year OS 49% (95% CI, 45 to 53); clonal mutations (n = 76), 5-year OS 34% (95% CI, 23 to 45); and subclonal mutations (n = 30), 5-year OS 59% (95% CI, 39 to 74), respectively; P = .018. (D) OS according to the presence of any ALK alterations in 611 patients with known ALK amplification and ALK mutation status: presence of an ALK alteration (n = 146), 5-year OS 37% (95% CI, 29 to 45); versus absence of ALK alterations (n = 465), 5-year OS 51% (95% CI, 46 to 55); P = .005. (E) OS according to the type of ALK alteration in the cohort of 571 patients with known ALK amplification and ALK mutation status: no alteration (n = 465), 5-year OS 51% (95% CI, 46 to 55); clonal mutations (n = 65), 5-year OS 33% (95% CI, 21 to 44); subclonal mutations (n = 12), 5-year OS 48% (95% CI, 26 to 67); and ALK amplification (n = 19), 5-year OS 26% (95% CI, 10 to 47), respectively; P = .001. (F) OS according to ALK alterations (ALK amplification or clonal ALK mutation) in patients who received immunotherapy (n = 141): To evaluate the impact of ALK alterations (ALK amplification or clonal ALK mutation) in patients who received dinutuximab beta, OS was calculated from the start of dinutuximab beta treatment and evaluated using the same approaches as described in the Materials and Methods section. ALK alteration (ALK amplification or clonal ALK mutation, n = 29, 5-year OS 48% [95% CI, 28 to 65]) versus no ALK alteration (n = 112) 67% (95% CI, 56 to 75); P = .034. Patient details: Appendix Table A3. HR, hazard ratio; NS, not significant; OS, overall survival; ref, reference.

FIG A1.

Treatment flowchart of the HR-NBL1 Protocol (ClinicalTrials.gov: NCT01704716, EudraCT: 2006-001489-17) over the whole period. aInfants and children with a body weight below 12 kg will be dosed at 0.67 mg/kg/d. In infants weighing ≤ 5 kg, a further 1/3 dose reduction is advised. AUC, area under the curve; BUMEL, busulfan and melphalan; CAV, cyclophosphamide plus doxorubicin or vincristine; CEM, carboplatin, etoposide, and melphalan; CH14.18/CHO, human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 produced in Chinese hamster ovary (CHO) cells; COJEC, chemotherapy schedule COJEC defined below; GFR, glomerular filtration rate; IL-2, interleukin-2; IV, intravenous; P or E, cisplatin or etoposide; R1, randomization 1; R2, randomization 2; R3, randomization 3; R4, randomization 4; RT, radiotherapy; SCR, stringent complete response; TP, time period; TVD, topotecan-vincristine-doxorubicin.

FIG A2.

Comparison of patients in the ALK analysis cohort and patients not in the ALK analysis cohort. (A and B) EFS and OS of the ALK analysis cohort and patients not in the ALK cohort. (A) No statistically significant difference in EFS and (B) OS was observed between patients included in the ALK analysis cohort (n = 1,092, from 132 centers; red line), patients not included in this study from the same centers (n = 1,665, blue line) and patients not included in this study from centers not participating in this study (n = 577, green line) (5-year EFS: 40% [95% CI, 37 to 43] v 37% [95% CI, 35 to 40] v 33% [95% CI, 29 to 37]; 5-year OS: 49% [95% CI, 46 to 53] v 48% [95% CI, 46 to 51] v 44% [95% CI, 40 to 59]; P = NS). (C) Recruitment, by year (x-axis), in the ALK analysis cohort (% of patients: y-axis; absolute numbers: in the blue bars). The % and number of patients not included in the ALK analysis cohort from centers participating, and from nonparticipating centers, are indicated in orange and gray, respectively. EFS, event-free survival; NS, not significant; OS, overall survival.

FIG A3.

Double event of ALK amplification and ALK mutation detected in one case (case 15). The SNP array shows an amplified region in chromosome 2 encompassing the ALK gene. Sanger sequencing profile shows R1275Q mutation (MAF = 93.3%) in the same case. HD, high definition; MAF, mutated allele fraction; SNP, single-nucleotide polymorphism.

FIG A4.

MAF of subclonal ALK mutations detected by TDS and confirmed by a second independent TDS experiment. Red spots representing the MAF for each ALK mutation are plotted on the x-axis (first TDS experiment) and y-axis (second TDS experiment), with a strong correlation between the two independent experiments (r2 = 0.9924, P < .0001). Blue spots represent subclonal ALK mutations with a very low MAF (< 0.1%) not confirmed in an independent experiment and not retained in the analysis (n = 6). MAF, mutated allele fraction; TDS, targeted deep sequencing.

FIG A5.

Survival in the ALK analysis cohort (n = 1,092 patients) according to known prognostic factors. (A) EFS and OS in the ALK analysis cohort population (n = 1,092 patients). Five-year EFS (blue line) 40% (95% CI, 37 to 43); 5-year OS (red line) 49% (95% CI, 46 to 53). (B) OS according to age. Five-year OS in patients < 1 year of age at diagnosis (red line) 50% (95% CI, 37 to 61); in patients 1-1.5 years of age at diagnosis (blue line) 58% (95% CI, 49 to 66); in patients 1.5-5 years of age at diagnosis (green line) 50% (95% CI, 46 to 53); and in patients > 5 years of age at diagnosis (purple line) 43% (95% CI, 35 to 50); P = NS (pseudo-value regression). (C) OS according to number of involved MCs. Five-year OS in patients with localized disease (red line) 67% (95% CI, 58 to 75), in patients with involvement of one MC (blue line) 65% (95% CI, 55 to 73), two MCs (green line) 52% (95% CI, 46 to 58), or over two MCs (purple line) 41% (95% CI, 36 to 46); P < .001. (D) OS according to stage. Five-year OS in patients with localized disease (red line) 67% (95% CI, 58 to 75), in patients with stage 4 disease (blue line) 47% (95% CI, 44 to 50), or stage 4s disease (green line) 54% (95% CI, 25 to 76); P < .001. (E) OS according to MYCN amplification in stage 4 disease. Five-year OS in patients with MNA (blue line) 46% (95% CI, 41 to 51), in patients without MNA (red line) 48% (95% CI, 44 to 53), NS (pseudo-value regression). (F) OS according to treatment period, before (< March 2010) or after (> March 2010) the definition of HDC by BUMEL and immunotherapy maintenance as standard treatment. A significant improvement survival because of BUMEL and GD2 standard therapy is observed. Five-year OS in patients having been treated before March 2010 (red line) 46% (95% CI, 41 to 51) versus after March 2010 (blue line) 51% (95% CI, 47 to 56); P = .039.- BUMEL, busulfan and melphalan; cHR, crude hazard ratio; EFS, event-free survival; HDC, high-dose chemotherapy; HR, hazard ratio; MC, metastatic compartment; MNA, MYCN-amplified; NS, not significant; OS, overall survival; ref, reference.