title: Ioncopy: a novel method for calling copy number alterations in amplicon sequencing data including significance assessment.
abstract: Recently, it has been demonstrated that calling of copy number alterations (CNAs) from amplicon sequencing (AS) data is feasible. Most approaches, however, require non-tumor (germline) DNA for data normalization. Here, we present the method Ioncopy for CNA detection which requires no normal controls and includes a significance assessment for each detected alteration.Ioncopy was evaluated in a cohort of 184 clinically annotated breast carcinomas. A total number of 252 amplifications were detected, of which 183 (72.6%) could be validated by a call of an additional amplicon interrogating the same gene. Moreover, a total number of 33 deletions were found, whereof 27 (81.8%) could be validated. Analyzing the 16 most frequently amplified genes, validation rates of over 89% could be achieved for 11 of these genes. 11 of the top 16 genes showed significant overexpression in the amplified tumors. 89.5% of the HER2-amplified tumors were GRB7 and STARD3 co-amplified, whereas 68.4% of the HER2-amplified tumors had additional MED1 amplifications. Correlations between CNAs measured by amplicons in HER2 exons 19, 20 and 21 were strong (all R > 0.93). AS based detection of HER2 amplifications had a sensitivity of 90.0% and a specificity of 98.8% compared to the gold standard of HER2 immunohistochemistry combined with in situ hybridization.In summary, we developed and validated a novel method for detection and significance assessment of CNAs in amplicon sequencing data. Using Ioncopy, AS offers a straightforward and efficient approach to simultaneously analyze gene amplifications and gene deletions together with simple somatic mutations in a single assay.
authors.affiliation: Institute of Pathology, Charité University Hospital, Berlin, Germany.|German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Budczies
authors.affiliation: German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany.|Institute of Pathology, University Hospital, Heidelberg, Germany.|Institute of Pathology, Technical University Munich (TUM), Munich, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Pfarr
authors.affiliation: German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany.|Institute of Pathology, University Hospital, Heidelberg, Germany.|National Center for Tumor Diseases (NCT), Heidelberg, Germany.|Department of Pathology, Center for Integrated Diagnostics (CID), Massachusetts General Hospital/Harvard Medical School, Boston, USA.
authors.forename: Albrecht
authors.initials: A
authors.lastname: Stenzinger
authors.affiliation: Institute of Pathology, Charité University Hospital, Berlin, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Treue
authors.affiliation: German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany.|Institute of Pathology, University Hospital, Heidelberg, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Endris
authors.affiliation: Department of Gynecology, Charité University Hospital, Berlin, Germany.
authors.forename: Fakher
authors.initials: F
authors.lastname: Ismaeel
authors.affiliation: Interdisciplinary Breast Center, Charité University Hospital, Berlin, Germany.
authors.forename: Nikola
authors.initials: N
authors.lastname: Bangemann
authors.affiliation: Interdisciplinary Breast Center, Charité University Hospital, Berlin, Germany.
authors.forename: Jens-Uwe
authors.initials: JU
authors.lastname: Blohmer
authors.affiliation: Institute of Pathology, Charité University Hospital, Berlin, Germany.|German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Dietel
authors.affiliation: German Breast Group (GBG), Neu-Isenburg, Germany.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Loibl
authors.affiliation: Institute of Pathology, Charité University Hospital, Berlin, Germany.
authors.forename: Frederick
authors.initials: F
authors.lastname: Klauschen
authors.affiliation: German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany.|Institute of Pathology, University Hospital, Heidelberg, Germany.|Institute of Pathology, Technical University Munich (TUM), Munich, Germany.|National Center for Tumor Diseases (NCT), Heidelberg, Germany.
authors.forename: Wilko
authors.initials: W
authors.lastname: Weichert
authors.affiliation: Institute of Pathology, Charité University Hospital, Berlin, Germany.|German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Denkert
mesh_terms: D000465:Algorithms; D001943:Breast Neoplasms / Q000235:genetics*; D056915:DNA Copy Number Variations / Q000235:genetics; D005260:Female; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: amplicon sequencing; breast cancer; copy number alterations; semiconductor sequencing; targeted sequencing