Comparative genetic profiling of neuroblastoma at the onset vs recurrence or progression of the tumor and determination of the prognostic role of genetic alterations

Abstract

Neuroblastoma (NB) is the most prevalent extracranial solid embryonal tumor in children, capable of both spontaneous regression and extremely aggressive course. The clinical heterogeneity of NB feeds the continual interest in its molecular genetic profiles as a potential key to the variable tumor behaviors and treatment personalization. To date, a number of studies indicate unfavorable prognostic roles of p53 and RAS–MAPK pathway activation, as well as that of telomere maintenance mechanisms. The increase in mutational load observed in recurrent NB has been shown to reduce the degree of subclonal heterogeneity, leading to pronounced distinction between genetic profiles of the tumor as recorded at primary and recurrent manifestations. This study aimed to analyze the alterations in genetic profiles of NB over time from the initial verification of the diagnosis till a disease-related adverse event (relapse or progression), as well as the potential prognostic significance of such alterations. The retrospective-prospective study enrolled 46 patients with morphologically verified peripheral neurogenic tumors, receiving treatment entirely or partially at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology from July 2013 till December 2021 in accordance with the modified NB-2004 protocol. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The cohort included an observation group of 25 patients, 1 intermediate-risk case and 20 high-risk cases. The main inclusion criterion concerned the availability of tumor tissue suitable for molecular examination, obtained both at the onset of the disease and at relapse/progression. We used the multiplex ligation-dependent probe amplification assay (MLPA) for the assessment of segmental and copy number chromosome aberrations, next-generation sequencing (NGS) to identify nucleotide variants, and reverse transcription polymerase chain reaction to assess the TERT gene expression levels. The statistical analysis used data available by April 01, 2022. In the studied cohort, the analysis confirmed prognostic contribution of MYCN amplifications (p < 0.001) detected at the onset of the disease or at its progression, as well as of 1p deletions (p = 0.01) detected in primary tumors. The expression of TERT in primary NBs was shown to correlate with the clinical course of the disease in the intermediate-risk and observation group, including local recurrence/progression (n = 3; –5 (–10.5…–3.5)), systemic relapses (n = 3; –2 (–2.5…–1)) and the lack of second adverse event (n = 20; –7 (–9…–5.5)), р = 0.037. Pairwise comparison of mutational profiles at the onset of the disease and at relapse revealed extreme degree of instability and significant variations: 38.5% (10/26) of the patients had no clinically significant genetic variants in the analyzed genomic region of interest in paired samples, 19.2% (5/26) of the patients had identical oncogenic variants in primary and relapsed tumor tissues, albeit with altered variant allele fraction (2/5), loss of one of the variants identified in the primary tumor (1/5), or acquisition of additional mutations upon recurrence (2/5), in 19.2% (5/26) of the patients, oncogenic genetic variants detected in primary NB were subsequently lost, in 7.7% (2/26) of patients, a loss of an oncogenic genetic variant identified at the onset on the disease was accompanied by the emergence of new clinically significant variants, and 15.4% (4/26) of the patients presented with new mutations in relapsed tumor tissue. Genetic variants leading to RAS–MAPK pathway activation and р53 pathway inhibition in primary tumors were identified in 31% (9/29) and 20.7% (6/29) of primary tumors and 29% (9/31) and 6.5% (2/31) of relapsed tumors, respectively. Risk group stratification based solely on cytogenetic aberrations fails to account for all cases of aggressive course of NB. Advanced molecular genetic approaches including MLPA and NGS, as well as the TERT mRNA quantitation, provide expanded characterization of the tumor biology and aggressiveness. Although prognostic markers are typically found in primary tumors, for NB, the use of such markers can be hampered by extremely high intratumoral heterogeneity. The data on acquisition or loss of prognostically significant molecular genetic markers in the course of the disease is useful for long-term prognosis and selection of targets for specific therapies.