KRAS Screening

KRAS mutation analysis was performed in the Genetics and Molecular Pathology Department at Monash Health, using the clinically validated KRAS StripAssay™ (ViennaLab Diagnostics) in accordance with manufacturer protocols and standard clinical practice. Where possible, DNA was extracted from fresh frozen EUS-FNA biopsies sourced from the multi-centre VPCB using the AllPrep DNA/RNA Universal Kit (Qiagen), although archival formalin-fixed paraffin embedded (FFPE) tissue was used where fresh frozen biopsy tissue was not available. The isolation of gDNA from FFPE tissue was performed on 5 x 10 micron-thick sections using the ReliaPrep FFPE gDNA Miniprep System (Promega). Prospective tissue testing on diagnostic biopsies was preferred, although archival or previously stored specimens from the VPCB could be used where fresh tissue was not feasible or available. DNA samples were quantified using the Qubit Fluorometer (Life Technologies) and quality assessed by TapeStation (Agilent Technologies). At the time of consent to sample collection for the VPCB, patients could elect for their treating physician to be contacted in the event of a significant genomic finding. KRAS wild-type results were notified to treating physicians who were able to offer referral for screening for the study if they deemed it clinically appropriate.