Pancreatic ductal adenocarcinoma (PDAC) is classified as one of the most lethal malignancies, characterized by a poor prognosis and very low survival rates. Up to 10% of all PDAC cases occur
in families with two (at least one of the cases debuting <50 years of age) or more affected first-degree relatives (FDRs), which is defined as familial pancreatic cancer (FPC). The familial aggregation implies genetic contribution to the disease by germline variants or mutations that co-segregate with FPC within the affected families. Unfortunately, currently known mutations in susceptibility genes including BRCA1, BRCA2, PALB2, ATM, STK11, PRSS1, SPINK1, etc. explain less than 20 % of all FPC cases, leaving most of the genetic basis of the disease unclear. Although highly advanced techniques for analyzing DNA sequence variations are available nowadays, the genetic architecture of FPC has been scarcely investigated, perhaps due to the rarity of the disease. By taking advantage of a recently established Danish cohort of FPC predisposed families, this PhD project aims to perform genetic studies on FPC, from assessment of the overall genetic contribution to application of nextgeneration sequencing (NGS) techniques on FPC patients and their first-degree relatives (FDRs) – aiming to identify rare DNA sequence variations or mutations underlying the observed familial clustering of the disease. Findings from the study could help revealing the genes and their functional pathways that are underpinning the development and progression of FPC.
The PhD project started with profiling of the Danish national cohort of FPC families including FPC patients and their predisposed relatives. Our analysis of clinical features showed that FPC is characterized by high lethality, with a low 5-year survival rate of 2.5% in the cohort and a high familial clustering with 19% of occurrence among FDRs in the respective families, thus implying a strong genetic component in FPC. Based on data of FPC patients and their FDRs, we were able to estimate an intraclass correlation coefficient (ICC) of 0.25, corresponding to a narrow sense additive heritability estimate of 0.51 in the FPC family cohort. The estimated heritability suggests a strong
genetic component in FPC etiology, which encourages molecular genetic studies, utilizing highthroughput NGS analysis, to uncover the underlying genetic variants.
First-degree relatives of FPC patients are at elevated risk of developing the disease due to sharing of their genetic materials within families. Using DNA extracted from whole blood from 61
FDRs of FPC patients in 27 families, we performed whole-genome sequencing (WGS) to identify rare genetic variants that may contribute to the high susceptibility to the disease in FPC families.
Through analysis of heterozygous premature truncating variants (PTV), we identified cancer-related genes and cancer-driver genes harboring multiple germline mutations, which are over-represented by functional pathways related to transport of small molecules, innate immune system, ion channel transport, and stimuli-sensing channels. Association analysis using gnomAD as external control samples detected 20 significant genes including PALD1, LRP1B, COL4A2, CYLC2, ZFYVE9, BRD3, AHDC1, etc. Functional annotation showed that the significant genes were enriched by gene clusters encoding for extracellular matrix and associated proteins. The detected rare genetic mutations
characterize FDRs of FPC patients as genetically predisposed individuals subject to risk assessment to promote early diagnosis and intervention.
The PhD project further conducted WGS on DNA samples extracted from benign formalinfixed paraffin-embedded (FFPE) tissues from 35 FPC patients in 27 FPC predisposed families in the
Danish national cohort. The WGS identified 821 genes hosting PTVs, among them 40 genes harboring 2 or more PTVs. Significant overlaps of the 40 genes were found (p<1e-22) with known cancer genes, cancer driver genes and genes found in previous studies on cancer, including ATM, POLE, BRCA2, TYR03, PABPC1 and SSC5D. The 821 PTV genes are significantly over-represented in biological pathways in cancer development and progression including: extracellular matrix organization, signaling by RHO GTPases and RHO GTPase cycle. The identified pathways may contribute to the development and progression of FPC, with our findings strongly underlining the genetic heterogeneity of the disease.
Overall, WGS on FPC patients and their FDRs detected a multitude of rare mutations displaying a high degree of allelic and locus heterogeneity in patients and predisposed family members, with hosting genes of detected variants being significantly overrepresented by cancer driver genes and cancer-related genes that mediate cell proliferation, migration, and invasion. The genetic heterogeneity is functionally characterized by significant enrichment of multiple biological pathways in cancer etiology. The high genetic heterogeneity in FPC calls for NGS-based panel testing strategies for efficient risk assessment and personalized handling of individuals at risk. The WGS results from the PhD study provide novel genes harboring potential mutational hotspots for future validation and replication.