Abstract A1-17: Genomic analysis of metachronous pancreatic ductal adenocarcinoma
Authors: Connor Ashton A. , Chan-Seng-Yue Michelle , Denroche Robert E. , Borgida Ayelet , Liang Sheng-Ben , Notta Faiyaz , Stein Lincoln , Roehrl Michael H. , McPherson John , Gallinger Steven 
Published: November 2015

Abstract
Background: Improving outcomes for pancreatic ductal adenocarcinoma (PDAC) requires more complete understanding of pancreatic carcinogenesis, including tumor heterogeneity and mutational evolution. Such studies in PDAC are hampered by presentation at advanced stages and rarely operable recurrences. We present a case of metachronous PDAC managed by serial resections and interrogated by whole genome sequencing of all three lesions.
Methods: Tumors underwent either laser capture microscopy or flow cytometry of fresh frozen tissue. Germline DNA was obtained from normal tissue. Genomic DNA was isolated, and sequencing libraries were prepared. Whole genome sequencing was performed on the Illumina HiSeq2500 system. Pipeline analysis included quality control, alignment to the reference genome (hg19), variant calling and annotation. Structural variants were called by the union of two tools, DELLY and CREST. Copy number variants were called using an in-house tool. Single nucleotide polymorphisms and short insertions and deletions were identified by the intersection of two tools, Strelka and MuTect, and annotated using ANNOVAR. All structural, copy number and non-silent variants were manually reviewed in the Integrated Genome Viewer. Secondary analyses were done in R. Representative sections of the three tumors also underwent formalin fixation, paraffin embedding, sectioning at five micron thickness and hematoxylin and eosin staining for microscopic analysis.
Results: The first tumor was located in the head of the pancreas and resected by Whipple operation, the second and third in the body and tail, respectively, and resected by completion pancreatectomy 14 months later. The patient received chemotherapy in the interim.
Histologically, all 3 tumors were moderately differentiated adenocarcinomas with focal mucinous areas and extensive lymphovascular and perineural invasion. There were no distinguishing morphological features.
For the 3 lesions, we achieved average sequence coverage of 52.3x, 54.3x, and 70.7x, respectively.
A total of 69 structural variants and 62 copy number variants were identified, with concordance of 75% and 92% in all three lesions.
A total of 61 non-silent exonic variants were identified, with concordance of 33% in all three lesions. A KRAS (NM_004985) c.G34C missense and TP53 (NM_001126115) c.G418A missense were present in all three, whereas a SMAD4 (NM_005359) c.118delC frameshift was present in only the second and third tumors.
Hierarchical clustering by non-silent variants, copy number variants, and an intersect of all variants showed the tumors in the head and tail to be more closely related to one another than to the body lesion, whereas clustering by structural variants showed little difference between the three.
Conclusions: The highly similar structural and copy number profiles across the three lesions suggests that these tumors arose from a common precursor, and that large chromosomal changes occurred early in its mutational evolution.
The poor concordance of simple somatic mutations imply that these evolved gradually and are responsible for tumor heterogeneity and possibly acquired phenotypes, include therapeutic resistance.
This study also demonstrates that isolated, intra-parenchymal metastases (as opposed to de novo tumors within a “field defect”) are possible with PDAC, which has been suggested in case reports but never definitively proven, further implying that these patients may benefit from re-operation despite disease relapse.
Further analysis will include mathematical modeling of mutation rates and validation in three additional metachronous cases identified in our biospecimen repository and in specimens obtained by our rapid autopsy program.
Citation Format: Ashton A. Connor, Michelle Chan-Seng-Yue, Robert E. Denroche, Ayelet Borgida, Sheng-Ben Liang, Faiyaz Notta, Lincoln Stein, Michael H. Roehrl, John McPherson, Steven Gallinger. Genomic analysis of metachronous pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-17.