PMC7280449_01
Distribution of invasive and intraductal lesions in the pancreas. Pre-operative images showing a stricture in the distal bile duct (DBD) (arrowheads in A,B) with no apparent tumors detected in the surrounding tissues. Note the abnormal density in the pancreatic parenchyma surrounding the superior mesenteric vein (SMV) (white arrow in A) visualized by an axial slice of the contrast-enhanced computed tomography, stricture of the SMV (red arrow in A) by 3D-reconstruction of the portal phase, and small dilatations in the branch duct (white arrow in B) detected by magnetic resonance imaging (B). (C) Resected specimens and corresponding hematoxylin and eosin images showing the distribution of each invasive cancer and intraductal neoplasm. Areas marked by black and green dashed lines indicate the invasive compartments and intraductal papillary mucinous neoplasm (IPMN), respectively. Note that the areas marked by black circle were high-grade dysplasias found in the main pancreatic duct (yellow arrows); asterisk, SMV. Black arrowheads indicate common bile duct (CBD). Scale bars, 1 cm. (D) Schema of tumor compartments from which DNA was isolated for genotyping: red star, invasive carcinoma; blue circle, high-grade pancreatic intraepithelial neoplasia (PanIN); yellow circle, low-grade PanIN; green square, IPMN; gray diamond shape, low-grade atypia.
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F1
Contrast-enhanced computed tomography revealed stenosis in the distal bile duct and a low-density area in the pancreatic parenchyma surrounding the superior mesenteric vein (SMV), separate from the bile duct (Figure 1A). Magnetic resonance cholangiopancreatography revealed dilatations of the pancreatic branch ducts around the stricture of the distal bile duct; however, no pancreatic or bile duct junction abnormalities were detected (Figure 1B). IPMN was located close to the distal bile duct stenosis (Figure 1C). In addition to the macroscopically identified lesions, histological examination of the apparent normal-looking pancreas showed high-grade PanIN in the main pancreatic duct and scattered low-grade branch duct lesions (Figure 1D). Several low-grade PanINs with various types of KRAS mutations (G12D, G12V, and G12R) were distributed in the resected pancreatic specimens (Table 1, Figure 2, Supplementary Table 1).
Histological evaluation of multiple tumor foci in the resected pancreas. Pathological findings of representative tumor compartments utilized for multi-region sequencing; hematoxylin and eosin staining (left panel; low- and high-powered view) and images of RNF43 and ARID1A immunohistochemistry (right panels; high-powered view with same magnification as H&E) are shown: a, pancreatic ductal adenocarcinoma (PDA) in the distal bile duct; g, high-grade pancreatic intraepithelial neoplasia (PanIN) in the main pancreatic duct (MPD); f, low-grade intraductal papillary mucinous neoplasm (IPMN) in a branch duct; h, high-grade PanIN in the MPD; n, low-grade PanIN in the MPD. Note partial loss of expression of ARID1A as indicated by the arrowhead. Scale bars, 100 mum.
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F2
Several low-grade PanINs with various types of KRAS mutations (G12D, G12V, and G12R) were distributed in the resected pancreatic specimens (Table 1, Figure 2, Supplementary Table 1). MPD, as the apparent founder tumor, and then acquired additional mutations in TGFBR1 (E499X) and RBM10 (R765X) (Table 1, Figure 2). Moreover, another high-grade PanIN in the MPD was found to be related to the founder lesion marked by KRAS G12D and RNF43 V50R mutations but had distinct promotive mutations in the ARID1A (P650fs and R1335X) and RBM10 (S288X) genes, which emerged after the development of the earliest tumor clone with a minimum set of oncogenes. The analysis revealed the partial loss of ARID1A expression that is suggestive of loss-of-function mutations, whereas the expression of RNF43 was preserved in tumor components with RNF43 V50R (Figure 2).
Tumor evolution map displaying the accumulation of mutations. The high-grade pancreatic intraepithelial neoplasia (PanIN) found in the main pancreatic duct (MPD), marked by "h" and "k," was considered to be the founder lesion that progressed to an invasive carcinoma (A-D; red stars) by acquiring a TGFBR1 mutation, whereas the branched subclone with ARID1A mutations remained as intraductal lesions.
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F3
On the basis of the disease distribution and mutation accumulation profile (Figure 3), we concluded that the precursor harboring a KRAS G12D mutation had acquired an RNF43 V50R mutation to establish a "founder clone" that subsequently acquired various additional mutation types, thereby enabling it to spread throughout the pancreas.