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Genomic Catastrophes Frequently Arise in Esophageal Adenocarcinoma and Drive Tumorigenesis

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Genomic Catastrophes Frequently Arise in Esophageal Adenocarcinoma and Drive Tumorigenesis

Katia Nones et al. Nat Commun.

Abstract

Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n=40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.

Figures

Figure 1
Figure 1. Mutational signatures found in EAC
(a) Five mutational signatures were detected in EAC. Each signature is represented by the proportion of somatic substitutions (C>A, C>G, C>T, T>A, T>C and T>G). Substitutions are displayed in a trinucleotide context (including information about the bases immediately 3′ and 5′ to the mutated base) resulting in 96 potential contexts. (b) Contribution of mutational signatures operative in individual tumours. Each bar represents a tumour and the y axis represents the contribution of each signature within tumours, shown as number of mutations per Mb. The BRCA, the unknown and APOBEC signatures were most prevalent only in one tumour each (BRCA contributed ~57% of OESO_1636 mutations and unknown signature represents 67% of OESO_0303 mutations). The APOBEC signature, previously described in EAC and other tumour types, contributes to more than 50% of the mutations in tumour OESO_1154, with small contributions in other samples. The age signature, previously described in EAC, is the second major operative mutational processes in this cohort. The signature characterized by T>G at TTsites is the most prominent within this cohort, representing ≥40% of mutations in 10 of 22 tumours (Supplementary Fig. 1).
Figure 2
Figure 2. Evidence of chromothripsis in EAC tumours
(a) Circos plot of tumour OESO_1527 containing copy number and BAF in the outer rings and somatic structural variants (SVs) are represented by lines in the inner ring. Colour of the lines represents SV type as indicated in the legend. Circos plot shows a high concentration of SVs on chromosome 9. (b) Zoomed-in view of events on chromosome 9 of tumour OESO_1527 showing evidence of chromosome shattering (chromothripsis). From top to bottom, graphs show SVs, copy number, logR ratio and BAF. There are changes in copy number state, concentration of a high number of SVs and retention of heterozygosity. (c) The genome distribution of somatic SVs for tumour OESO_3213. Circos plot containing copy number and SVs shows a concentration of events on chromosome 8. (d) Zoomed-in view of events on chromosome 8 in OESO_3213 showing SVs, copy number, logR ratio and B-allele frequency. (e) Inferred double-minute chromosome (DM) harbouring MYC oncogene. Blue blocks show fragments of chromosome 8 inferred to form DM and green block shows position of FISH probe. (f) Agarose gel showing PCR verification of SV events inferred to contribute to the DM. T, tumour, N, adjacent normal esophagus, numbers indicate SVs as shown in e. (g) FISH confirms copy number alterations in d by showing MYC amplification as multiple scattered signals and two copies of the chromosome 8 in a representative tumour cell nucleus. (Green—fluorescently labelled BAC RP11-367L7—MYC gene region; Red—centromeric region of chromosome 8—CHR8-10-RE). Scale bar, 10 μm. Supplementary Figures 11 and 12 are full images of gel and FISH presented in (f) and (g), respectively.
Figure 3
Figure 3. Evidence of a chromothriptic event involving four chromosomes
(a) Overview of the distribution of events in the genome of tumour OESO_0384. Circos plot of tumour OESO_0384 containing copy number and B-allele frequency (outer rings) and somatic structural variants (SVs) are represented by lines in the inner ring. Colour of the lines represents SV type as indicated in the legend. (b) Detailed view of chromosomes 12, 13, 17 and 20 involved in the complex localized event. (c) Inferred DNA double-minute chromosome (DM) involving regions of chromosomes 12, 13 and 20. Red and green bars indicate location of FISH probes shown in e. (d) Agarose gel showing PCR verification of SV events inferred to be part of the DM in c. T, tumor, N, adjacent normal oesophagus, numbers indicate SVs as labelled in c. (e,f) FISH analysis demonstrated amplification of the two regions tested in representative tumour cell nuclei, plus the frequent colocalization of signals indicating fusion of regions of chromosomes 13 and 20 (see arrows for examples). Green—RP11-122N18 (chr13:78,416,550-78,591,831) to the EDNRB gene region, Red—RP11-192K14 (chr20:52,178,644-52,324,774). (g) FISH images show amplification and frequent colocalization of signals (see arrows for examples) indicating that chromosome regions of chr 13 and chr 12 are part of the inferred DM. FISH Green—RP11-122N18 (chr13:78,416,550-78,591,831) to EDNRB gene region and Red—RP11-77H17- (chr12:69,154,590-69,318,752) to MDM2 gene region. Scale bar, 10 μm. Supplementary figures 13 to 16 are full images of Figure 3(d) to (g).
Figure 4
Figure 4. Chromothripsis and breakage-fusion-bridge (BFB) evidence in an EAC tumour
OESO_3845 contained a high number of SVs concentrated in two chromosomes, 12 and 18. (a) Circos plot shows overall distribution of SVs in the genome. Circos plot containing copy number and B-allele frequency in the outer rings and somatic structural variants (SVs) are represented by lines in the inner ring. Colour of the lines represents SV type as indicated in the legend. (b) Zoomed-in view of events on chromosome 12. Graph shows from the top, SVs, copy number, logR ratio and B-allele frequency. Copy number profile suggests loss of telomeric p arm and SV events suggest several cycles of BFB on chromosome 12, with 20 inversions mapped to the amplified region (24,387,412 to 28,333,288 bp). (c) Zoomed-in view of events on chromosome 18. Graph shows SVs, copy number, logR ratio and B-allele frequency. SV events and copy number data suggests shattering of chromosome 18 with switches in copy number state, concentration of a high number of SVs and retention of heterozygosity characteristic of a chromothriptic event.

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