The Evolutionary Origins of Recurrent Pancreatic Cancer

Abstract

Surgery is the only curative option for stage I/II pancreatic cancer; nonetheless, most patients will experience a recurrence after surgery and die of their disease. To identify novel opportunities for management of recurrent pancreatic cancer, we performed whole-exome or targeted sequencing of 10 resected primary cancers and matched intrapancreatic recurrences or distant metastases. We identified that recurrent disease after adjuvant or first-line platinum therapy corresponds to an increased mutational burden. Recurrent disease is enriched for genetic alterations predicted to activate MAPK/ERK and PI3K-AKT signaling and develops from a monophyletic or polyphyletic origin. Treatment-induced genetic bottlenecks lead to a modified genetic landscape and subclonal heterogeneity for driver gene alterations in part due to intermetastatic seeding. In 1 patient what was believed to be recurrent disease was an independent (second) primary tumor. These findings suggest routine post-treatment sampling may have value in the management of recurrent pancreatic cancer. SIGNIFICANCE: The biological features or clinical vulnerabilities of recurrent pancreatic cancer after pancreaticoduodenectomy are unknown. Using whole-exome sequencing we find that recurrent disease has a distinct genomic landscape, intermetastatic genetic heterogeneity, diverse clonal origins, and higher mutational burden than found for treatment-naïve disease.See related commentary by Bednar and Pasca di Magliano, p. 762.This article is highlighted in the In This Issue feature, p. 747.

Figure 1.. Recurrent pancreatic cancer after adjuvant therapy has distinct genetic features compared to the primary tumor.

A. Schematic illustrating the temporal and spatially distinct sample types collected for this study. In all patients the primary tumor is designated PT1. The number of samples of recurrent disease varied per patient, ranging from PT2-PT4 up to PT2-PT11. B. Pancreatic tail remnant and attached spleen removed at autopsy of patient PAM41. Arrowheads indicate the resection margin and dashed circle indicate the location and dimensions of the recurrent tumor. C. Integration of mutation type, sample location, radiation therapy, chemotherapy, and mutational signature for each sample. D. Comparison of the prevalence of distinct mutational signatures in primary carcinomas (P) versus recurrent disease (R) per mutational signature indicates a statistically significant increase in mutations characteristic of the DNA damage repair signature only (two-sided χ2-squared test, p<0.0001). E. Comparison of DNA damage repair signature in patients who did (n=4) and did not (n=6) receive a platinum agent as adjuvant or first line therapy. All comparisons by two-sided Mann-Whitney U test.

Fig. 2.. Genetic Features of Recurrent Pancreatic Cancer.

A. Oncoprint of somatic mutations and copy number alterations present in the pre and post-treatment samples analyzed for each patient (clonal mutations). B. Oncoprint of somatic mutations and copy number alterations present in one or more samples of the recurrent PDA only, but not in the matched originally resected primary tumor (subclonal mutations). The full list of somatic alterations identified in both the primary and recurrent disease are listed in Tables S6 and S7. C. Frequency of diploid (D), tetraploid (T) and allelic imbalance (AI) at the KRAS locus in primary tumors versus recurrent disease. D. Proportion of genetic events leading to allelic imbalance in recurrent pancreatic cancer. Complete data are shown in Table S8. E. Schematic illustration of convergent genetic events that increase mutant KRAS dosage in the recurrent disease of PAM37. Abbreviations used are WGD, whole genome duplication and CN LOH, copy neutral loss of heterozygosity.

Figure 3:. Somatic alterations in recurrent pancreatic cancer alterations reflect expansion of subclones pre-existent in the primary tumor.

A. Representative density cloud plots of the primary tumor (PT1) and one matched sample of recurrent disease for nine different patients. Subclonal expansion of cells containing a PIK3CA E524K mutation (PAM39), a KRAS G12V and AKT D323H mutation (PAM40), a NOTCH1 frameshift mutation (PAM41), KMT2C frameshift mutation (PAM45) and CHD8 nonsense and missense mutations (PAM46) in the recurrent disease are seen. The cancer cell fractions of representative clonal driver genes for all cases (i.e. KRAS, TP53, SMAD4 and/or GNAS) are shown for reference. B-D. Droplet digital PCR analysis of mutant allele abundance in the primary tumor and one matched sample of recurrent disease in three different patients. In all three patients subclones were preexistent at low allele frequencies in the primary tumor. Each dot represents one droplet, and color bars at top right of each plot indicate relative intensity of the VIC labeled mutant (x axis) and FAM labeled wild type allele (y axis) fluorescent labels.

Figure 4:. Metachronous pancreatic cancer simulating a local recurrence.

A. Clinical timeline of events in this patient. Liver and peritoneal metastases were radiographically evident at 18 months and a mass in the pancreatic remnant was noted at autopsy. B. Phylogenetic analysis of whole exome sequencing data generated for this patient indicates the metachronous pancreatic cancer contains a unique repertoire of somatic driver gene alterations including a KRAS-G12D mutation compared to the first primary (PT1) that contains a G12V mutation. C. Representative density cloud plot of the first primary tumor (PT1) and one sample of the metachronous primary tumor (PT3). The majority of somatic alterations in each primary are mutually exclusive from each other.

Figure 5:. Recurrent pancreatic cancer originates from two distinct evolutionary origins.

A. Phylogenetic analysis of the relationships of the primary tumor to the local recurrence and liver metastases in patient PAM40. All recurrent disease is the result of clonal expansion of a single pre-existent subclone notable for an AKT1 and KRAS mutations (See Fig 2B). The primary tumor (PT1) is the outgroup in the tree. A subclonal TRIP12 mutation is also seen in a single liver metastasis. B. Color code of sample origins in PAM40. C. Phylogenetic analysis of the relationships of the primary tumor to the local recurrence and liver metastases in patient PAM39. In this patient the recurrent disease is the result of more than one clonal expansion. The preexistent PIK3CA mutation has expanded in the lineage that gave rise to samples PT2 and PT9. D. Color code of sample origins in PAM39. E,F. Jaccard indices for each pairwise comparison in PAM40 (monophyletic recurrence) versus PAM39 (polyphyletic recurrence). G. Comparison of the average Jaccard index for primary tumors and their matched recurrences in patients with monophyletic recurrences versus those with polyphyletic recurrences. Monophyletic recurrences are significantly different from their matched primary tumor indicating passage through a genetic bottleneck, whereas no difference is found between the primary tumor and matched recurrences in patients with polyphyletic recurrences. (comparisons by a Student’s two-sided T test). Monophyletic (“common origin”) recurrences are associated with an improved disease-free survival (H) but not overall survival (I) in this small cohort (comparisons by log-rank test of Kaplan Meier survival curves).

Figure 6:. Examples of intermetastatic seeding in recurrent pancreatic cancer.

For each patient the previously resected primary tumor is indicated by PT1 to the left of each patient schematic. +M, positive surgical margin; +LN, positive lymph nodes. In the body maps for all three patients (panels A,C,E), red lines reflect migration from the primary tumor and blue lines indicate migration from a site of recurrent disease. Solid lines indicate high confidence migration patterns and dashed lines indicate low confidence migration patterns inferred by MACHINA (panels B,D,F). Each patient had at least one high confidence migration event from one site of recurrent disease to another. In patient PAM45, two migration patterns were equally likely to have occurred (F) although both predict that the primary tumor seeded the local recurrence and the perirectal metastasis seeded the abdominal metastases.

Figure 7:. Origins of local recurrences after surgical resection.

For each patient the previously resected primary tumor is indicated by PT1 to the left of each patient schematic. -M, negative surgical margin; +LN, positive lymph nodes, -LN, negative lymph nodes. In the body maps for all three patients (panels A,C,E), red lines reflect migration from the primary tumor and blue lines indicate migration from a site of recurrent disease. Solid lines indicate high confidence migration patterns and dashed lines indicate low confidence migration patterns inferred by MACHINA (panels B,D,F). In PAM41 (A,B) and PAM43 (C,D) the local recurrence is seeded by the originally resected primary tumor, whereas in PAM42 (E,F) it is seeded by a lung metastasis. In patients PAM41 (A,B) and PAM42 (E,F) three or more migration patterns were equally likely to have occurred.