Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas

Abstract

Malignant astrocytomas are a deadly solid tumor in children. Limited understanding of their underlying genetic basis has contributed to modest progress in developing more effective therapies. In an effort to identify such alterations, we performed a genome-wide search for DNA copy number aberrations (CNA) in a panel of 33 tumors encompassing grade 1 through grade 4 tumors. Genomic amplifications of 10-fold or greater were restricted to grade 3 and 4 astrocytomas and included the MDM4 (1q32), PDGFRA (4q12), MET (7q21), CMYC (8q24), PVT1 (8q24), WNT5B (12p13), and IGF1R (15q26) genes. Homozygous deletions of CDKN2A (9p21), PTEN (10q26), and TP53 (17p3.1) were evident among grade 2 to 4 tumors. BRAF gene rearrangements that were indicated in three tumors prompted the discovery of KIAA1549-BRAF fusion transcripts expressed in 10 of 10 grade 1 astrocytomas and in none of the grade 2 to 4 tumors. In contrast, an oncogenic missense BRAF mutation (BRAF(V600E)) was detected in 7 of 31 grade 2 to 4 tumors but in none of the grade 1 tumors. BRAF(V600E) mutation seems to define a subset of malignant astrocytomas in children, in which there is frequent concomitant homozygous deletion of CDKN2A (five of seven cases). Taken together, these findings highlight BRAF as a frequent mutation target in pediatric astrocytomas, with distinct types of BRAF alteration occurring in grade 1 versus grade 2 to 4 tumors.

Figure 1. Copy number changes in pediatric astrocytoma

A, Heat map showing genome-wide copy number gains (red) and losses (green) in 33 pediatric astrocytomas. Individual astrocytomas are arranged left to right on the X-axis from low-to-high grade. Tumors with (+) and without (−) BRAF^V600E mutations are indicated. Chromosome landmarks are listed vertically on both sides of the map. Note the minor number of red and green blocks among grade I and grade II tumors. B, Vertical scatter plot showing individual and mean fractional genome alteration values for each malignancy grade of tumor. Gray colored data points represent cases with BRAF^V600E mutations.

Figure 2. High level amplifications and homozygous deletions in pediatric astrocytoma

Examples of genome copy number plots for tumors with high-level (>10 copies) genomic amplifications (A), and for tumors showing focal homozygous deletions of the CDKN2A locus (B).

Figure 3. BRAF fusion alterations are common in PA tumors

A, High resolution view of copy number duplications at 7q34 and juxtaposition of BRAF (left horizontal line at position 138) and KIAA1549 (right horizontal line at positioin 140) sequences in grade I and grade II tumors. B, RT-PCR analysis of KIAA1549-BRAF fusion transcripts in non-neoplastic brain (N1-N4), grade I astrocytoma (n=10), grade II astrocytoma (n=10), and one grade III astrocytoma (tumor 24) reveals selective expression of fusion transcripts in grade I tumors. RT-PCR analysis of wild-type BRAF serves as a positive control. DNA sequencing of the RT-PCR fusion products revealed expression of KIAA1549-exon-15/BRAF-exon-9 transcripts (highest mobility fragment: 4 cases), KIAA1549-exon-16/BRAF-exon-11 transcripts (intermediate fragment: 1 case), and KIAA1549-exon-16/BRAF-exon-9 transcripts (lowest mobility fragment; 5 cases). Note that one grade II astrocytoma displayed a 7q34 duplication (tumor 11), but no KIAA1549-BRAF fusion transcript was detected.

Figure 4. BRAF mutation in pediatric astrocytomas

A, Sequence traces from grade II, III, and IV astrocytomas showing presence of both BRAF wild-type alleles (T) and mutant (A) BRAF^V600E alleles in all instances where the V600E mutation has occurred. B, Average copy number variation across 7q34. For each tumor, copy number was calculated for: a ~ 2Mb region inclusive of KIAA1549 and BRAF (gray bar); a 5Mb region proximal to KIAA1549 and BRAF (left black bar); and a 5Mb distal to KIAA1549 and BRAF (right black bar). This data representation highlights tumors with duplications limited to KIAA1549 and BRAF (tumors 3, 9, 11) and tumors with duplications of a larger 7q34 chromosomal region that includes KIAA1549 and BRAF (tumors 32, 35, 36, 39, 40, 41). Tumor 27 contains a 7q34 duplication and further 7q34 amplification distal to BRAF.