title: Development and validation of a microRNA based diagnostic assay for primary tumor site classification of liver core biopsies.
abstract: Identification of the primary tumor site in patients with metastatic cancer is clinically important, but remains a challenge. Hence, efforts have been made towards establishing new diagnostic tools. Molecular profiling is a promising diagnostic approach, but tissue heterogeneity and inadequacy may negatively affect the accuracy and usability of molecular classifiers. We have developed and validated a microRNA-based classifier, which predicts the primary tumor site of liver biopsies, containing a limited number of tumor cells. Concurrently we explored the influence of surrounding normal tissue on classification. MicroRNA profiling was performed using quantitative Real-Time PCR on formalin-fixed paraffin-embedded samples. 278 primary tumors and liver metastases, representing nine primary tumor classes, as well as normal liver samples were used as a training set. A statistical model was applied to adjust for normal liver tissue contamination. Performance was estimated by cross-validation, followed by independent validation on 55 liver core biopsies with a tumor content as low as 10%. A microRNA classifier developed, using the statistical contamination model, showed an overall classification accuracy of 74.5% upon independent validation. Two-thirds of the samples were classified with high-confidence, with an accuracy of 92% on high-confidence predictions. A classifier trained without adjusting for liver tissue contamination, showed a classification accuracy of 38.2%. Our results indicate that surrounding normal tissue from the biopsy site may critically influence molecular classification. A significant improvement in classification accuracy was obtained when the influence of normal tissue was limited by application of a statistical contamination model.
authors.affiliation: Department of Oncology, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark; Center for Genomic Medicine, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. Electronic address: Katharina.Anne.Perell@regionh.dk.
authors.forename: Katharina
authors.initials: K
authors.lastname: Perell
authors.affiliation: Department of Mathematical Sciences, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Vincent
authors.affiliation: Department of Pathology, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Ben
authors.initials: B
authors.lastname: Vainer
authors.affiliation: Department of Pathology, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Bodil Laub
authors.initials: BL
authors.lastname: Petersen
authors.affiliation: Department of Pathology, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Federspiel
authors.affiliation: Department of Oncology, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Anne Kirstine
authors.initials: AK
authors.lastname: Møller
authors.affiliation: Center for Genomic Medicine, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Madsen
authors.affiliation: Department of Mathematical Sciences, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Niels Richard
authors.initials: NR
authors.lastname: Hansen
authors.affiliation: Center for Genomic Medicine, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Lennart
authors.initials: L
authors.lastname: Friis-Hansen
authors.affiliation: Center for Genomic Medicine, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Finn Cilius
authors.initials: FC
authors.lastname: Nielsen
authors.affiliation: Department of Oncology, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
authors.forename: Gedske
authors.initials: G
authors.lastname: Daugaard
mesh_terms: D001706:Biopsy; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008113:Liver Neoplasms / Q000145:classification / Q000175:diagnosis / Q000378:metabolism / Q000556:secondary; D035683:MicroRNAs / Q000096:biosynthesis*; D012334:RNA, Neoplasm / Q000096:biosynthesis*; D012680:Sensitivity and Specificity
chemical_list: D035683:MicroRNAs; D012334:RNA, Neoplasm
keywords: Classification; Liver biopsy; Metastases; Surrounding tissue; Tissue contamination; microRNA