Genomic Identification of Significant Targets in Cancer Analysis Identifies Significant Recurrent Events in Specific PLGG Subtypes.
The percentage of the genome altered by copy-number alterations (CNAs) in diffuse PLGGs was significantly lower than among previously profiled adult low- and high-grade gliomas (P < 10−6, Mann–Whitney test, Fig. 1A) (13, 14). Few (12/44; 27%) of these tumors harbored alterations affecting more than 90% of the length of a chromosome arm (Fig. 1B), compared with an 83–97% rate among adult low- and high-grade tumors (15). One of the PLGG samples exhibited chromothripsis on chromosome 8 (chr8) (highlighted in Fig. 2A, PLGG27). The most significantly recurrent arm-level CNAs were gains of chromosomes 7 (11% of tumors), 8 (7%), and 5q (5%) and loss of 1p (2%) (Fig. 1C). These events have all been described in pediatric high-grade gliomas and adult gliomas with varying frequencies (15, 16).
CNAs among diffuse PLGGs. (A) Fraction of the genome altered by CNAs is lower among diffuse PLGGs compared with adult LGGs and high-grade gliomas (P < 10−6). Blue bars indicate medians. (B) Amplifications (red) and deletions (blue) among 44 diffuse PLGGs (x axis) across the genome (y axis) ordered by copy-number status. Significance (x axis) of (C) arm-level and (D) focal deletions (Left, blue) and amplifications (Right, red) across the genome (y axes). Putative gene targets within the peak regions are indicated where known.
DA2 samples with focal 8q gains identified by aCGH share a common centromeric breakpoint within MYBL1. (A) aCGH data for the five DA2 samples with 8q gain, magnified on the right. Red: copy-number gain; green: loss. The highlighted sample (PLGG27, blue box) exhibits chromothripsis of chr8 but shows no other copy-number changes on other chromosomes. (B) FISH probes corresponding to sequences immediately distal (red) and proximal (green) of MYBL1 confirm that the single-copy gain identified by aCGH is a duplication involving oneMYBL1 allele. The D8Z2 centromere enumeration probe (aqua) was used as a control. (C) Schematic representation of the proto-oncogene MYB family and breakpoints observed in our cohort in relation to the viral oncogene v-MYB.
We found 6 significantly recurrent regions of focal deletion and 17 significantly recurrent regions of focal amplification (Fig. 1D and Table S2). One deleted region on 9p21.3 contained cyclin-dependent kinase inhibitor 2A and 2B (CDKN2A and CDKN2B), known tumor suppressors that had previously been reported in diffuse PLGGs (17); a second region was immediately adjacent to this one. A third region (6q26) contained 252 genes, including the proto-oncogene MYB. Two regions (10q21.3 and 8p22) contained single genes with no known relation to cancer or neural development, catenin (cadherin-associated protein), alpha 3 (CTNNA3) and zeta sarcoglycan (SGCZ), respectively. The sixth region (13q31.3) contained 48 genes and was adjacent to the known tumor suppressor RB1. We did not identify any focal deletions of other known tumor suppressors involved in adult or pediatric brain tumors such as neurofibromin 1 (NF1), phosphatase and tensin homolog(PTEN), or cyclin-dependent kinase inhibitor 1C (CDKN1C).
One of the 17 focally gained regions contained BRAF. However, the canonical BRAF-KIAA1549 duplication-fusion was detected in only four samples: two GGs and two LGG-NOS. This is in contrast to pilocytic astrocytomas, among which >80% of tumors harbor a BRAF duplication (18) (P < 0.0001, Fisher’s exact test). We also determined BRAF V600E mutation status in 24 tumors with sufficient DNA for sequencing. We found mutations in 54% of the diffuse PLGGs (Fig. 1A and Table S1), consistent with previously published rates for diffuse PLGGs (8).
A second focally gained region (3q26.33) contained the stem cell and glial transcription factor sex determining region Y-box 2 (SOX2), which is amplified in adult glioblastomas (19). Two additional regions (2q12.1 and 5q14.3) contained factors that control telencephalic neural progenitor proliferation and differentiation: POU class 3 homeobox 3 (POU3F3) (also known as BRN1) and microRNA 9-2 (20, 21). A fifth region (1q21.3) contained myeloid cell leukemia sequence 1 (MCL1), a known oncogene amplified in several cancer types (22). Twelve regions either contained over 150 genes or did not contain genes with known roles in cancer or neural development. We did not observe any high-level amplification of receptor tyrosine kinases (e.g., EGFR,PDGFRA), which are observed frequently in both adult and pediatric high-grade gliomas (19, 23).
The most statistically significant recurrent focal aberration (q = 3.37 × 10−6) was a gain on chromosome 8q involving the transcription factor MYBL1. Although MYBL1 is not a known oncogene, it is closely related to the proto-oncogene MYB. In contrast to prior reports (11), no amplifications or gains of the proto-oncogene MYBwere identified in our study set. All of the focal 8q gains occurred in DA2s (P = 0.0057, Fisher’s exact test), comprising 28% (5/18) of this histologic subtype. In contrast, MYBL1 was not in a significant amplification peak across 3,131 cancers comprising multiple other cancer types that we had previously analyzed (22) or, specifically, among adult low- or high-grade gliomas (15).
All five DA2 samples with 8q focal gains exhibited a common centromeric breakpoint within MYBL1 after exon 9, including the sample with chromothripsis of chr8 (Fig. 2A). To confirm the MYBL1 centromeric breakpoint, we performed fluorescence in situ hybridization (FISH) using a probe slightly telomeric to the breakpoint on all eight DA2 samples with sufficient tissue available (Fig. 2B and Fig. S2). All DA2 samples with 8q gain (3/3) demonstrated duplication of one allele in more than 60% of the nuclei in each tumor whereas none of the other DA2 samples showed duplication (0/5) (P = 0.018, Fisher’s exact test).
The tight clustering of these breakpoint sites, and particularly their location immediately preceding the C-terminal negative regulatory domains of MYBL1 and MYB, suggested a mechanism for rearrangement and creation of functional, truncated genes reminiscent of the viral oncogene v-MYB (Fig. 2C). Indeed, we also identified a homologous breakpoint between exons 10 and 11 of MYB on 6q in one angiocentric glioma with a focal 6q deletion (Fig. S3) similar to that previously reported in a single angiocentric glioma with a 6q deletion (11). An additional angiocentric glioma (PLGG45), not included in our initial diffuse PLGG cohort and genomic identification of significant targets in cancer (GISTIC) analysis, also exhibited a deletion in 6q at the same location in MYB as seen in PLGG29 (Fig. S3).
Whole-Genome Sequencing of a DA2 with 8q Focal Gain Defines a Tandem Duplication–Truncation of MYBL1.
To further characterize the MYBL1 amplicon and its genetic context, we performed 90× whole-genome sequencing of a DA2 sample with MYBL1 gain but no other CNAs (PLGG24, Table S3). Whole-genome sequencing of PLGG24 determined the centromeric breakpoint of the 8q amplicon to single-base resolution between exons 9 and 10 of MYBL1 (Fig. 3A). The telomeric sequence was located in an intergenic region 38 kb from matrix metallopeptidase 16 (MMP16). We validated the breakpoint locations in this sample using PCR on native genomic DNA from the same tumor (Fig. S2 C and D). Taken together, our data define a tandem duplication–truncation of MYBL1.
Characterization of the MYBL1-truncating rearrangement in a DA2 sample (PLGG24). (A) Circos plot (Left) of 90× whole-genome sequence data showing a single-copy-number gain on 8q (red, inner heatmap ring) and corresponding intrachromosomal rearrangement (green) involving MYBL1 and an intergenic region outside MMP16. A schematic diagram represents the 8q tandem duplication (Lower Right). All nonsynonymous mutations and insertions and deletions are also listed (Upper Right). (B) Truncated MYBL1 transcripts (MYBL1-trunc1 andMYBL1-trunc2) identified by 3′-RACE. (C) RT-PCR demonstrating MYBL1-trunc2 expression in two additional PLGG samples with MYBL1 gain.
Apart from this event, whole-genome sequencing of PLGG24 revealed a sparsely altered genome. No other CNAs or fusion events were identified, and the BRAF V600E mutation was not present. Three nonsynonymous mutations in exons were identified (Table S4); none of these have been reported in association with cancer. The genome-wide mutation rate (1.48/Mb) and the number of nonsynonymous mutations in exons (three per genome) were low compared with pediatric and adult high-grade astrocytomas (mutation count means: 15 and 47.3 per genome, respectively) (16). The low rate of CNAs, mutations, and translocations in this sample highlight the potential biological impact of MYBL1 duplication–truncation.
Tandem Duplication–Truncation of MYBL1 Results in Expression of Oncogenic Transcripts.
To determine whether the MYBL1 duplication–truncation resulted in expression of fused transcripts, we performed 3′-rapid amplification of cDNA ends (3′-RACE) on cDNA generated from RNA of PLGG24. We identified two populations of MYBL1 transcripts, both of which contained MYBL1 exons 1–9 but also acquired short noncanonical sequences fused to the 3′ ends, leading to premature translation stops (Fig. 3B). The first transcript (MYBL1-trunc1) contained an additional 15 bp of intronic sequence, and the second transcript (MYBL1-trunc2) acquired 36 bp from the intergenic region near MMP16. MYBL1-trunc1 was retrieved more frequently (74%) than MYBL1-trunc2 (26%); the wild-type transcript was not observed. We then performed RT-PCR to detect MYBL1-trunc1 and MYBL1-trunc2 in the two other PLGGs with 8q focal amplification for which we had sufficient RNA (PLGG25 and PLGG28). We detected MYBL1-trunc2 in both of these samples (Fig. 3C), indicating that this transcript is recurrently expressed in DA2s.
To assess the oncogenic potential of these transcripts, we transduced 3T3 cells with lentiviruses containingMYBL1-trunc1, MYBL1-trunc2, full-length MYBL1-wt, or GFP control constructs and plated them in soft agar. Both aberrantly truncated MYBL1 sequences produced soft agar colony growth indicative of transformation (Fig. 4A). No evidence of colony formation was noted with full-length MYBL1-wt or GFP control. Both MYBL1-trunc1– and MYBL1-trunc2–transformed 3T3 cells were also able to form tumors with malignant histology in Nude mice, whereas cells transduced with full-length MYBL1-wt constructs showed no evidence of tumor formation (Fig. 4 B–D). These results suggest that the truncation of MYBL1 is oncogenic.
(A) Anchorage-independent growth of 3T3 cells transduced with MYBL1-wt, MYBL1-trunc1, or MYBL1-trunc2 retroviruses, relative to GFP controls. (B) In vivo tumor formation after injection of 3T3 cells transduced withMYBL1-wt (n = 5), MYBL1-trunc1 (n = 5), or MYBL1-trunc2 (n = 5) (arrows) into the flanks of Nude mice. Tumor volume was calculated 6 wk post injection (C), and tumors were subjected to histologic analysis by H&E staining (D).