The patterns and dynamics of genomic instability in metastatic pancreatic cancer

Abstract

Pancreatic cancer is an aggressive malignancy with a five-year mortality of 97-98%, usually due to widespread metastatic disease. Previous studies indicate that this disease has a complex genomic landscape, with frequent copy number changes and point mutations, but genomic rearrangements have not been characterized in detail. Despite the clinical importance of metastasis, there remain fundamental questions about the clonal structures of metastatic tumours, including phylogenetic relationships among metastases, the scale of ongoing parallel evolution in metastatic and primary sites, and how the tumour disseminates. Here we harness advances in DNA sequencing to annotate genomic rearrangements in 13 patients with pancreatic cancer and explore clonal relationships among metastases. We find that pancreatic cancer acquires rearrangements indicative of telomere dysfunction and abnormal cell-cycle control, namely dysregulated G1-to-S-phase transition with intact G2-M checkpoint. These initiate amplification of cancer genes and occur predominantly in early cancer development rather than the later stages of the disease. Genomic instability frequently persists after cancer dissemination, resulting in ongoing, parallel and even convergent evolution among different metastases. We find evidence that there is genetic heterogeneity among metastasis-initiating cells, that seeding metastasis may require driver mutations beyond those required for primary tumours, and that phylogenetic trees across metastases show organ-specific branches. These data attest to the richness of genetic variation in cancer, brought about by the tandem forces of genomic instability and evolutionary selection.

Figure 1

Patterns of somatically acquired genomic rearrangements in pancreatic cancer. (A) Histogram showing the distribution of the number and types of rearrangement observed in 13 patients with pancreatic cancer. (B) Circle plots showing the genomic landscape of rearrangements in three representative samples. Chromosome ideograms are shown around the outer ring with copy number plots on the inner ring. Individual rearrangements are shown as arcs joining the two genomic loci, each coloured according to the type of rearrangement. (C) Example of a so-called ‘fold-back inversion’. Correctly mapping paired reads (orange) show much greater density on the right half of the figure than the left, suggesting that the copy number is higher here. The change in copy number is demarcated by anomalously mapping paired reads (green), aligning ~2kb apart on the genome and in inverted orientation. The only genomic structure which can explain this pattern is a rearrangement in which the abnormal chromosome is ‘folded back’ on itself leading to duplicated genomic segments in head-to-head (inverted) orientation. (D) The distribution of types of rearrangement was significantly different between breast cancer and pancreatic cancer (p<0.0001).

Figure 2

Phylogenetic relationships of different metastases within a patient. (A) PCR genotyping of three rearrangements across DNA from the index metastasis sequenced, other metastases from the same patient, the primary tumour and germline tissue. Somatic rearrangements may be present in all cancer samples but not the germline (omnipresent); present in some but not all metastases (partially shared); or present just in the index metastasis sequenced (private). (B) Inter-individual differences in the proportions of rearrangements that are omnipresent across metastases, partially shared by some but not all lesions or are private to the index metastasis sequenced. (C) Patterns across six broad categories of rearrangement in the proportions of variants that are omnipresent across metastases, partially shared by some but not all lesions or are private to the index metastasis sequenced. The numbers of rearrangements in each category are shown at the top. The difference in proportions between fold-back inversions and the other categories was statistically significant (p=0.003). (D) Genotyping of 57 rearrangements in PD3640 shows a coherent, nested structure, with 42 found in all metastases and the primary tumour, 7 found uniquely in the index tumour and 8 partially shared by some but not all metastases. (E) The nested structure of rearrangements defines a phylogenetic tree of relationships among the metastases and primary tumour. The length of heavy black lines is proportional to the genetic distance between nodes. Dotted lines delineate the departure points of other, unsequenced lesions from the lineage between the germline genome and that of the index metastasis.

Figure 3

Phylogenetic relationships among different metastases and the primary tumour. (A) Results of PCR genotyping for 23 rearrangements across 19 metastases and the primary tumour from patient PD3637. (B) Phylogenetic tree showing the relatedness of different metastases and the primary tumour. Note the early divergence of the primary tumour from all metastases. (C) Genotyping results for PD3638, as well as (D) PD3639, (E) PD3641, (F) PD3643 and (G) PD3642. (H) Circle plot showing that the rearrangements generating the amplicon of KRAS on chromosome 12 in PD3642 were only found in the index metastasis sequenced, and none of the other metastases or the primary tumour.

Figure 4

Organ-specific signatures of metastasis. (A) Results of PCR genotyping for 38 rearrangements across the three index metastases and five other metastases from patient PD3827. (B) Overlapping out-of-frame deletions of exon 6 of PARK2 were mutually exclusive to either the four lung metastases or the four abdominal metastases (C) A phylogenetic tree of relationships for metastases from patient PD3827, showing a clade of abdominal metastases and a further evolved clade of lung metastases. The length of heavy black lines is proportional to the genetic distance between nodes. Dotted lines delineate the departure points of other, unsequenced lesions from the lineage between the germline genome and that of the index metastasis. (D) Results of PCR genotyping for PD3828. (E) Phylogenetic tree of relationships for metastases from PD3828. (F) Model for the clonal evolution of metastases derived from the patterns of phylogenetic relationships observed. Molecular time proceeds from left to right, and is associated with subclonal evolution and expansion within the developing primary tumour. Eventually a subclone within the primary tumour acquires the capacity to metastasise (pink), but this subclone continues to acquire genetic lesions (darkening shades of brown) such that different metastases may be founded from different clones. Within the developing metastases, clonal evolution continues, and these newly developed subclones can themselves seed tertiary metastases.