ACT-Discover: identifying karyotype heterogeneity in pancreatic cancer evolution using ctDNA

Abstract

Background Liquid biopsies and the dynamic tracking of somatic mutations within circulating tumour DNA (ctDNA) can provide insight into the dynamics of cancer evolution and the intra-tumour heterogeneity that fuels treatment resistance. However, identifying and tracking dynamic changes in somatic copy number alterations (SCNAs), which have been associated with poor outcome and metastasis, using ctDNA is challenging. Pancreatic adenocarcinoma is a disease which has been considered to harbour early punctuated events in its evolution, leading to an early fitness peak, with minimal further subclonal evolution. Methods To interrogate the role of SCNAs in pancreatic adenocarcinoma cancer evolution, we applied whole-exome sequencing of 55 longitudinal cell-free DNA (cfDNA) samples taken from 24 patients (including 8 from whom a patient-derived xenograft (PDX) was derived) with metastatic disease prospectively recruited into a clinical trial. We developed a method, Aneuploidy in Circulating Tumour DNA (ACT-Discover), that leverages haplotype phasing of paired tumour biopsies or PDXs to identify SCNAs in cfDNA with greater sensitivity. Results SCNAs were observed within 28 of 47 evaluable cfDNA samples. Of these events, 30% could only be identified by harnessing the haplotype-aware approach leveraged in ACT-Discover. The exceptional purity of PDX tumours enabled near-complete phasing of genomic regions in allelic imbalance, highlighting an important auxiliary function of PDXs. Finally, although the classical model of pancreatic cancer evolution emphasises the importance of early, homogenous somatic events as a key requirement for cancer development, ACT-Discover identified substantial heterogeneity of SCNAs, including parallel focal and arm-level events, affecting different parental alleles within individual tumours. Indeed, ongoing acquisition of SCNAs was identified within tumours throughout the disease course, including within an untreated metastatic tumour. Conclusions This work demonstrates the power of haplotype phasing to study genomic variation in cfDNA samples and reveals undiscovered intra-tumour heterogeneity with important scientific and clinical implications. Implementation of ACT-Discover could lead to important insights from existing cohorts or underpin future prospective studies seeking to characterise the landscape of tumour evolution through liquid biopsy.

Refphase: Multi-sample phasing reveals haplotype-specific copy number heterogeneity

“…It is conceivable that this allelic imbalance is the result of circulating tumour DNA affecting the artificial normal sample, whose underlying data was derived from blood. This would be consistent with recent results highlighting the use of phasing to identify SCNAs in ctDNA from metastatic samples [ 54 ]. A further 24/71 false positive allelic imbalance segments were found to affect chromosome 7 in 23 different tumours, 19 of which overlapped.…”

Pancreatic cancer environment: from patient-derived models to single-cell omics

“…Hidalgo et al selected the PDX model because of its exceptional purity of tumor cells to amplify the genomic allelic imbalance genes to evaluate cfDNA samples for somatic copy number alterations (SCNAs). 33 They observed a high degree of homogeneity among driver mutations, specifically within KRAS and TP53, supporting the prevailing PC model evolution, suggesting a punctuated course characterized by early clonal events. Pathologically, PC has various types, including ductal adenocarcinoma, and special subtypes, including ductal, acinar cell, small glandular, and small cell carcinomas.…”