Additional file 1 of DNA methylation classifier to diagnose pancreatic ductal adenocarcinoma metastases from different anatomical sites
Creators: name: Calina, Teodor G.
name: Perez, Eilís
name: Grafenhorst, Elena
name: Benhamida, Jamal
name: Schallenberg, Simon
name: Popescu, Adrian
name: Koch, Ines
name: Janik, Tobias
name: Chen, BaoQing
name: Ihlow, Jana
name: Roessler, Stephanie
name: Goeppert, Benjamin
name: Sinn, Bruno
name: Bahra, Marcus
name: Calin, George A.
name: Taube, Eliane T.
name: Pelzer, Uwe
name: Neumann, Christopher C. M.
name: Horst, David
name: Knutsen, Erik
name: Capper, David
name: Dragomir, Mihnea P.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Environmental Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Cancer
subject: Plant Biology

description: Supplementary Material 1: Figure S1. Upgrading the classifier. A Confusion matrix with the results of the anomaly detection layer for the biological validation samples (n = 3579). B Comparison of the probability score of the correct class between fresh frozen and FFPE tissue in the validation cohort. C Confusion matrix with the results of the anomaly detection layer for the technical validation samples (n = 15). D Confusion matrix with the classifier results of the technical validation samples - EPICv2 (n = 15). Figure S2. Characterization of the positive control samples. A Examples of H&amp;E and IHC staining of peritoneal carcinomatosis from PAAD, B PAAD lung metastasis, C PAAD lymph node metastasis, and D PAAD liver metastasis. E Overview of the patient characteristics. Figure S3. t-SNE analysis of the reference and positive control samples. The two-dimensional representation of the reference cohort and positive control samples (n = 415) using the t-SNE method based on DNA methylation profiles. The color code of the samples represents: A the origin of the study set, B material type, C array type, D metastases origin, E IHC profile suggestive for, and F tumor purity. Figure S4. Off label use of the classifier. A The two-dimensional plot representation using the t-SNE method, based on the DNA methylation profiles of the positive control group (n = 16) together with reference samples (n = 399) and anomaly detection samples (10 different carcinomas, n = 787). BRCA—breast invasive carcinoma, ESCA—esophageal carcinoma, LUAD—lung adenocarcinoma, STAD–stomach adenocarcinoma, LIHC–liver hepatocellular carcinoma, COAD–colon adenocarcinoma, READ–rectal adenocarcinoma, UCEC–uterine corpus endometrial carcinoma, CESC–cervix squamous cell carcinoma and endocervical adenocarcinoma, PRAD–prostate adenocarcinoma. B Confusion matrix with the results of the anomaly detection layer for the negative control samples (n = 124). Figure S5. Factors influencing the classifier results. Correlation between the probability score of the correct class and the A CD3, B CD20 immune infiltrate, C Ki-67 proliferation rate, and D tumor purity. Comparison of the probability score of the correct class between E PAAD metastasis locations, F morphology, G tumor grade, H p53 expression pattern, I SMAD4 expression pattern, J GATA6 level, K CK7/20 expression profile, L ANXA1/10 score, and M IHC score. N Representative H&amp;E staining and IHC characterization of a primary PAAD and matched PAAD met.PC organoid. O Copy number plot for primary PAAD organoid and for PAAD met.PC organoid model. The plots show the chromosomal alterations at the respective CpG sites, deletions are below and gains above the baseline located at 0. Figure S6. DNA methylation-associated organotropism of pancreatic ductal adenocarcinoma metastases. A Summary copy number plot for PAAD met.Liv. (n = 21). B Summary copy number plot for primary PAAD (n = 16). C Summary copy number plot for PAAD met.PC (n = 11). The plots show the frequency of chromosomal alterations at the respective CpG sites, deletions are below and gains above the baseline located at 0. Additionally, 14 genes with known roles in PAAD are highlighted. Figure S7. Pathway analysis for differentially methylated probes of enhancers and promoters using methylGSA. A Pathways reaching an overlap of over 10% between discovered genes and pathway genes for comparing differentially methylated probes between PAAD met.Liv. and PAAD met.PC B Pathways reaching an overlap of over 10% between discovered genes and pathway genes for comparing differentially methylated probes between primary PAAD and PAAD met.Liv.. Purple marks pathways also discovered by using Enrichr. Table S1. Characteristics of the internal set of brain metastases. Table S2. Tumor purity estimated by the pathologist and number of slides used for the DNA extraction. Table S4. Patient characteristics of the positive control samples. Table S5. Histological and immunohistochemical characteristics of the positive control samples. Table S6. Histological and immunohistochemical characteristics of the PAAD met.Liv. samples.