(Top) PASEA workflow for the detection of ctDNA KRAS mutant allele. First, blood samples are collected from patients with advanced pancreatic cancer. Next, cfDNA is extracted from the blood plasma and treated with PASEA. Then, the PASEA enriched mutant allele fragments are analysed using low-cost Sanger sequencing after PCR amplification to identify the presence of the KRAS mutant allele and its mutation subtype. ctDNA, circulating tumour DNA; PASEA, programmable enzyme-based selective exponential amplification; RPA, recombinase polymerase amplification. SNV, Single nucleotide variation; WT, wild type. (Bottom) Cross-sectional imaging revealed a heterogeneous mass in the distal pancreatic tail infiltrating the tail of the pancreas and the hilum of the spleen. (B) Distal pancreatectomy and splenectomy specimen showing a large mass (arrow) in the tail of the pancreas invading the spleen (H&E, original magnifications×40). (C) Well-circumscribed fibrotic mass with inflammatory infiltrate (arrow) composed of lymphocytes, histiocytes, plasma cells and histiocytes with emperipolesis (see inset) (H&E, original magnifications×40). Inset: Hallmark histological finding of Rosai-Dorfman disease: Large RDD histiocytes showing pale, watery-clear cytoplasm, central round nucleus, prominent nucleolus with emperipolesis (small arrow) (H&E, original magnifications×400). (D) Fibrosis (small arrow) and inflammatory infiltrate (arrow) composed of lymphocytes, plasma cells, neutrophils, emperipolesis, and histiocytes (see inset) (H&E, original magnifications×40). Inset: large histiocytes (bold arrow) (H&E, original magnifications×400). Immunohistochemical staining for hallmark histiocytes in Rosai-Dorfman disease (E) S-100 immunostain highlighting the nuclei (large arrow) and cytoplasm of lesional histiocytes, along with lymphocyte emperipolesis (small arrow) (original magnifications×40). (F) Cyclin D1 immunostaining for lesional histiocytes with nuclear staining (original magnifications×40).