Germline Mutations in Pancreatic Adenocarcinoma
Condition: Germline Mutation Abnormality; Pancreatic Cancer; Predisposition, Genetic; Hereditary Cancer
MeSH terms: Adenocarcinoma; Genetic Predisposition to Disease
Study type: Observational

Summary: 
      This study will assess the hereditary component of pancreatic cancer in the largest series of
      patients up to date through the parallel analysis of 62 cancer-associated genes. The
      investigators will obtain germline DNA from blood samples that have been collected from 2000
      to 2019 from patients with pancreatic cancer. The investigators plan to analyze germline DNA
      for mutations and single nucleotide polymorphisms (SNPs) in genes that have been previously
      linked to a predisposition towards cancer.

      The outcome can provide useful insight on the overall understanding of pancreatic
      pathogenesis while possible associations with age of diagnosis, tumor stage and other cancer
      types might arise. In addition to that, it can lead to the characterization of new variants
      or even new genes that predispose to pancreatic cancer.

      Confirmed deleterious mutations in established cancer genes can provide valuable clinical
      information that can lead to effective, individualized patient management. Furthermore,
      family relatives of the individuals found to carry mutations can also benefit from
      established screening protocols for various cancer types, such as frequent colonoscopies in
      the case of an MMR mutation predisposing for Lynch syndrome, or preventative surgeries in the
      case of a deleterious BRCA1 or BRCA2 mutation. In addition to that, specific therapies that
      have been previously shown to be effective in breast or ovarian cancer patients with BRCA1 &
      BRCA2 mutations, such as platinum-based chemotherapy and PARP inhibitors can be also
      effective in mutations carriers with pancreatic cancer.
    

Detailed description: 
      Background Pancreatic ductal adenocarcinoma has poor prognosis mainly highlighted by the low
      5-year survival rate, which is estimated to be around 6%. Although pancreatic cancer
      incidence is approximately 0.5% in the general population, it has a high-mortality rate. It
      is being registered as the fourth leading cause of cancer-related deaths in the United
      States. Poor prognosis is mainly attributed to the lack of efficient treatment options.

      With the majority of pancreatic adenocarcinoma cases being sporadic, familial clustering
      (defined by the presence of at least two first degree relatives with pancreatic cancer) is
      observed in ~20% of the cases, whereas a clear genetic cause is identified in approximately
      half of these cases. Pancreatic cancer is known to occur in a range of cancer predisposing
      syndromes, such as hereditary breast and ovarian cancer, Peutz-Jeghers, familial adenomatous
      polyposis, Lynch, Li-Fraumeni and familial atypical multiple mole syndromes, while there have
      been clear associations with loss-of-function mutations in non-syndromic cancer genes, such
      as PALB2 and ATM. In addition, individuals carrying mutations in PRSS1 gene that predisposes
      for hereditary pancreatitis are also considered to be at high-risk for pancreatic cancer. It
      is therefore clear that pancreatic cancer is not related with a single cancer predisposition
      gene, but with numerous known and probably yet to be discovered, cancer susceptibility genes.

      On the other hand, the accurate fraction of genetic predisposition in pancreatic cancer
      hasn't been determined. Previous studies have evaluated the prevalence of loss-of-function
      mutations in known cancer predisposition genes in pancreatic cancer patients through the use
      of multi-gene panels. The mutation yield ranged from 3.8% to 9.7% in genes that were
      previously found relevant to pancreatic cancer predisposition.

      Through these studies, it has been highlighted that family history and stage of pancreatic
      cancer or age at diagnosis is not always a predictor of an underlying genetic factor, while
      phenotypic heterogeneity is usually associated with mutation status. The search for novel
      predisposition genes in pancreatic cancer patients without family history is ongoing through
      candidate genes analysis or through whole exome sequencing.

      Research methodology Clinicopathological characteristics

      Genomic DNA from pancreatic cancer patients will be retrospectively and prospectively
      collected from the Hellenic Cooperative Oncology Group (HeCOG) Tumor Repository, through
      years 2000-2019. Written informed consent has already been obtained from all patients for the
      use and storage of their biologic material along with their approval for their participation
      in research studies. The study will be in agreement with the 1975 Helsinki statement, revised
      in 1983.

      Clinicopathologic characteristics of patients with pancreatic cancer will be retrieved form
      their medical record. Diagnosis will be confirmed through pathology reports, which will also
      provide information about grade, proliferation rate, histological subtype and lymphovascular
      invasion. From the patient medical record, tumor size, nodal involvement as well as
      metastatic lesions (TNM staging) will be reported. Importantly, detailed family history will
      be collected. The collection of these information will be in compliance with the regulations
      of the Bioethics committees of participating hospitals. Clinicopathological characteristics
      will be correlated with the different genomic abnormalities identified by sequencing.

      Germline DNA extraction

      Blood samples will be collected from patients with pancreatic cancer. Germline DNA from white
      blood cells will be isolated using a commercially available blood DNA extraction kit.
      Germline DNA will then be subjected to mechanical fragmentation via sonication to reach a DNA
      size of approximately 200-250 bp for downstream next-generation sequencing (NGS) analysis.

      Next-Generation Sequencing

      Targeted capture enrichment NGS relies on in-solution hybridization of DNA libraries with
      TACS (Targeted Capture Enrichment Sequences) for the identification of germline mutations in
      cancer patients. First, TACS that target mutations associated with hereditary cancer (in a
      total of 62 cancer predisposing genes) are designed and prepared by PCR followed by
      biotinylation. Biotinylated TACS will then be immobilized on streptavidin coated magnetic
      beads for subsequent hybridisation with the DNA libraries. DNA libraries are prepared from
      patient derived-germline DNA that fulfill quality control criteria (DNA yield, DNA integrity
      and fragment size) according to established protocols. Library preparation involves the 3'
      end and 5' end adaptor ligation and each DNA library will be barcoded using unique
      oligonucleotide DNA sequences to allow subsequent discrimination after sequencing. Subsequent
      to barcoding, samples will undergo hybridization using custom made 5'-biotynylated TACS
      followed by PCR amplification. Finally, enriched DNA libraries will be normalised and pooled
      on a NGS platform prior to paired-end sequencing using manufacturer's protocols.

      Bioinformatic and Data Analysis Sequence data from leucocyte (buffy coat) DNA will be
      demultiplexed and aligned to the human genome (hg19) using appropriate aligner algorithms
      (BWA) to generate a BAM file. A combination of variant calling algorithms will be used to
      call germline mutations. Local realignment will be performed for accurate detection of small
      insertions and deletions and appropriate algorithms to detect rearrangements. Bioinformatics
      tools for copy number alterations (CNAs) calling such as ASCAT-2, CNVkit or ANACONDA will be
      used for the assessment of focal gene amplification or deletion events. Read depth-based
      analysis will also be taken into consideration for assessment of gene-level CNAs. Appropriate
      variant annotators will be used to annotate the identified alterations to each gene. Germline
      variant databases such as ClinVar will be used for the classification of variants according
      to the ACMG guidelines.
    

Intervention name(s): Mutations
Intervention type(s): Genetic
Observational model: Cohort
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        Genomic DNA from patients with pancreatic cancer will be retrospectively and prospectively
        collected from the Hellenic Cooperative Oncology Group (HeCOG) Tumor Repository, through
        years 2000-2019. Written informed consent has already been obtained from all patients for
        the use and storage of their biologic material along with their approval for their
        participation in research studies. The study will be in agreement with the 1975 Helsinki
        statement, revised in 1983.
      
Biospecimen description: 
      Germline DNA from white blood cells will be isolated using a commercially available blood DNA
      extraction kit. Remaining germline DNA will be safely stored in HeCOG's repository.