Abstract
Pancreatic ductal adenocarcinoma (PDAC) with microsatellite instability (MSI)/defective mismatch repair (dMMR) is the only subtype of pancreatic cancer with potential response to immunotherapy. Here, we report the histo-molecular characterization of MSI/dMMR PDAC with immunohistochemistry, MSI-based PCR, and next-generation sequencing. Five paradigmatic cases have been identified. The main results include the first report in pancreatic cancer of MSI/dMMR intra-tumor heterogeneity, the presence of microsatellite-stable metastases from MSI/dMMR primary and recurrent B2M gene inactivation, which may confer resistance to immunotherapy. In addition to the classic PDAC drivers, ARID1A was the most common mutated gene in the cohort. Intra-tumor heterogeneity, B2M inactivation, and metastatic sites should be carefully considered in MSI/dMMR PDAC, which should also be investigated in routine diagnostic practice with specific molecular analysis. The chromatin remodeler ARID1A represents another potential driver gene in this context.
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Acknowledgements
This study was supported by Associazione Italiana Ricerca sul Cancro (AIRC 5x1000 n. 12182); Fondazione Cariverona: Oncology Biobank Project “Antonio Schiavi” (prot. 203885/2017); Fondazione Italiana Malattie Pancreas (FIMP-Ministero Salute J38D19000690001); and Italian Ministry of Health (RF CO-2019-12369662: CUP: B39C21000370001).
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CL: study conception and design; CL, DC, PN, VNA, AS: histological examination; CL, AM, GP, PM, RTL, AS: immunohistochemical analysis; CL, AM, LP, CS, RTL, AS: molecular analysis; CL, GMal, GMar, AP, RS, MM, AS: clinic-pathologic data; all authors: data elaboration and interpretation; CL, AM, RTL, AS: paper writing; all authors: final editing and approval of the present version.
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This study was approved by the Verona Ethics Committee, date of approval: 04–08-2020, project 2610-CESC, code: MN-2019.
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Supplementary Information
Supplementary Figure 1.
The different components of the primary tumor of case #1, including low grade IPMN (A), high grade IPMN (B), ductal adenocarcinoma (C), and colloid carcinoma (D) (Hematoxylin-eosin, 20x magnification). (PNG 687 kb)
Supplementary Figure 2.
A representative histological / immunohistochemical image of the primary tumor of case #1. The histology clearly shows an IPMN with an associated adenocarcinoma. All tumor components show immunohistochemical loss of MSH2 and MSH6, and retained expression of MLH1 and PMS2 (10x magnification; A: hematoxylin-eosin; B: MSH2 loss; C: MLH1 retained expression; D: PMS2 retained expression; E: MSH6 loss). (PNG 664 kb)
Supplementary Figure 3.
A representative image of case #2. The histology shows a medullary carcinoma. At immunohistochemistry, tumor cells exhibited MSH2, and MSH6 loss, and retained expression of MLH1 and PMS2 (10x magnification; A: hematoxylin-eosin; B: MSH2 loss; C: MLH1 retained expression; D: PMS2 retained expression; E: MSH6 loss). (PNG 656 kb)
Supplementary Figure 4.
Graphical representation of two heterozygous frameshift mutations, each affecting a different copy of the B2M gene, in a lymph node metastasis of medullary carcinoma of the pancreas (case no. 2). The mutational data supports biallelic inactivation of the gene. Of note, the primary tumor also harbored B2M inactivation, but this was due to homozygous mutation. (PNG 106 kb)
Supplementary Figure 5.
A representative histological / immunohistochemical image of the primary tumor of case #3. The histology shows a biphasic neoplasm, displaying medullary (A), and ductal (B) features (hematoxylin-eosin, A: 10x magnification, B: 20x magnification). To illustrate the immunohistochemical profile, an area with both components (medullary on the left, ductal on the right) was selected: MSH6 loss of expression is limited to the medullary component (C, 10x magnification, with retained expression in intra-tumor lymphocytes); conversely MSH2 (D, 10x magnification), MLH1 (E, 10x magnification), and PMS2 (F,10x magnification) show retained expression in both components. (PNG 1647 kb)
Supplementary Figure 6.
Graphical representation of two heterozygous mutations, each affecting a different copy of the B2M gene (exon 1) in nodal metastases of a mixed ductal/medullary carcinoma of the pancreas (case no. 3). A) One mutation causes a frameshift and thus a truncated protein. The second mutation causes an amino acid change at codon 8 from Ala to Asp, which was further investigated. B) Analysis of the conservation of amino acids of B2M exon 1 among primates. Codon 8 is one of the most conserved residues, residing in a stretch of hydrophobic amino acids (purple color). The sequence from residues 1 to 20 is a signal peptide that targets B2M gene product to the endoplasmic reticulum, where it joins the other components of the major histocompatibility complex I. C) Analysis of the change in hydrophobicity (hydropathy) of the leader peptide due to the mutation of Ala at codon 8 to Asp. Dots represent hydropathy scores of single residues, lines illustrate local hydropathy computed on a window of three amino acids, accounting for interactions between side chains. The hydrophobicity loss of signal peptides has been shown to inhibit translocation of proteins. D) Prediction of the recognition of B2M exon1 as a signal peptide upon mutation of Ala 8 to Asp, performed with the SignalP 5.0 software. The prediction score drops from 0.99 to 0.80, showing a damaging effect of the amino acid substitution. (PNG 158 kb)
Supplementary Figure 7.
A representative histological/immunohistochemical image of case #5. Histologically, this is a conventional pancreatic ductal adenocarcinoma with a very small, metastatic lymph node (A, Hematoxylin-eosin, 4x magnification). At immunohistochemistry, MSH2 (B, 10x magnification), and MSH6 (C, 10x magnification) show retained expression, while MLH1 (D, 10x magnification), and PMS2 (E, 10x magnification) show loss of expression. (PNG 526 kb)
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Luchini, C., Mafficini, A., Chatterjee, D. et al. Histo-molecular characterization of pancreatic cancer with microsatellite instability: intra-tumor heterogeneity, B2M inactivation, and the importance of metastatic sites. Virchows Arch 480, 1261–1268 (2022). https://doi.org/10.1007/s00428-021-03205-3
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DOI: https://doi.org/10.1007/s00428-021-03205-3