Association of age at diagnosis and genetic mutations in patients with neuroblastoma

Abstract

Context: Neuroblastoma is diagnosed over a wide age range from birth through young adulthood, and older age at diagnosis is associated with a decline in survivability.

Objective: To identify genetic mutations that are associated with age at diagnosis in patients with metastatic neuroblastoma.

Design, setting, and patients: Whole genome sequencing was performed on DNA from diagnostic tumors and their matched germlines from 40 patients with metastatic neuroblastoma obtained between 1987 and 2009. Age groups at diagnosis included infants (0-<18 months), children (18 months-<12 years), and adolescents and young adults (≥12 years). To confirm the findings from this discovery cohort, validation testing using tumors from an additional 64 patients obtained between 1985 and 2009 also was performed. Formalin-fixed, paraffin-embedded tumor tissue was used for immunohistochemistry and fluorescence in situ hybridization. Telomere lengths were analyzed using whole genome sequencing data, quantitative polymerase chain reaction, and fluorescent in situ hybridization.

Main outcome measure: Somatic recurrent mutations in tumors from patients with neuroblastoma correlated with the age at diagnosis and telomere length.

Results: In the discovery cohort (n = 40), mutations in the ATRX gene were identified in 100% (95% CI, 50%-100%) of tumors from patients in the adolescent and young adult group (5 of 5), in 17% (95% CI, 7%-36%) of tumors from children (5 of 29), and 0% (95% CI, 0%-40%) of tumors from infants (0 of 6). In the validation cohort (n = 64), mutations in the ATRX gene were identified in 33% (95% CI, 17%-54%) of tumors from patients in the adolescent and young adult group (9 of 27), in 16% (95% CI, 6%-35%) of tumors from children (4 of 25), and in 0% (95% CI, 0%-24%) of tumors from infants (0 of 12). In both cohorts (N = 104), mutations in the ATRX gene were identified in 44% (95% CI, 28%-62%) of tumors from patients in the adolescent and young adult group (14 of 32), in 17% (95% CI, 9%-29%) of tumors from children (9 of 54), and in 0% (95% CI, 0%-17%) of tumors from infants (0 of 18). ATRX mutations were associated with an absence of the ATRX protein in the nucleus and with long telomeres.

Conclusion: ATRX mutations were associated with age at diagnosis in children and young adults with stage 4 neuroblastoma.

Trial registration: clinicaltrials.gov Identifier: NCT00588068.

Figure 1. ATRX mutations in neuroblastoma

(A) Diagram of the amino acid sequence of ATRX and the changes that result from the 5 single-nucleotide variations and 5 in-frame deletions found in the ATRX gene. (B) Photo of an ethidium bromide stained agarose gel with the PCR products for each of the 5 deletions shown in (A). D indicates the diagnostic tumor and G represents the germline DNA. (C) Sanger sequence traces of the PCR amplicons from (B) with junction breakpoints indicated. (D) Sanger sequence traces of the PCR amplicon spanning the frameshift mutation K425_E426fs.

Figure 2. Telomere analysis in neuroblastoma

(A) Histogram of the difference in telomeric reads for each of the 40 tumors in the discovery cohort. The samples with ATRX mutations are indicated by asterisks. The reference value for each tumor was the telomere length from matched normal DNA from the same patient. (B) Boxplot of normalized telomeric reads for the germline DNA (G) and the diagnostic tumor (D) from the WGS data for the 40 tumors and matched germline samples in the discovery cohort. The number of telomeric reads was normalized to the average genomic coverage for that particular sample. The upper and lower edge of the box represents the 75^th and 25^th percentile, respectively. The median is indicated as a horizontal line within the box and the vertical lines represent the lowest and highest values still within 1.5 of the interquartile range. (C) Histogram of the qPCR for telomeres in the 10 samples in the discovery cohort with ATRX mutations as well as 4 controls with wild type ATRX. Data are plotted as difference between tumor and germline for each patient. Red bars indicate those samples with increased telomere length and grey bars indicate those samples with shorter telomeres. (D–F) Images of control cells and 2 samples with mutant ATRX hybridized with the telomere FISH probe (red) and stained with DAPI to visualize the nucleus (blue). A low power overlay image is shown as well as a black and white image of the telomere FISH signal and a high magnification view highlighting the large ultrabright signal (arrows) in the ATRX mutant neuroblastoma cells. Scale bar: 5 μm for low magnification view and 1 μm for the high magnification view.

Figure 3. ATRX protein expression in neuroblastoma

(A) Immunohistochemistry for ATRX protein (brown) in a neuroblastoma tumor with wild type ATRX. Hematoxylin was used as the counterstain, and it stains the unlabeled (ATRX negative) nuclei blue. Blood vessels are clearly visible (*) and representative nuclear staining of ATRX is highlighted (arrow). (B,C) Immunohistochemistry for ATRX protein on 2 neuroblastoma samples with ATRX mutations. The vascular endothelial cells lining the blood vessel (*) are immunopositive for ATRX (arrows) but the tumor cells are negative. Scale bar: 25 μm for low magnification and 5 μm for high magnification.