Oncotarget

Clinical Research Papers:

Ioncopy: a novel method for calling copy number alterations in amplicon sequencing data including significance assessment

Jan Budczies _, Nicole Pfarr, Albrecht Stenzinger, Denise Treue, Volker Endris, Fakher Ismaeel, Nikola Bangemann, Jens-Uwe Blohmer, Manfred Dietel, Sibylle Loibl, Frederick Klauschen, Wilko Weichert and Carsten Denkert

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Oncotarget. 2016; 7:13236-13247. https://doi.org/10.18632/oncotarget.7451

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Abstract

Jan Budczies1,2, Nicole Pfarr2,3,4, Albrecht Stenzinger2,3,5,6, Denise Treue1, Volker Endris2,3, Fakher Ismaeel7, Nikola Bangemann8, Jens-Uwe Blohmer8, Manfred Dietel1,2, Sibylle Loibl9, Frederick Klauschen1, Wilko Weichert2,3,4,5 and Carsten Denkert1,2

1 Institute of Pathology, Charité University Hospital, Berlin, Germany

2 German Cancer Consortium (DKTK), partner sites Berlin, Heidelberg and Munich, Germany

3 Institute of Pathology, University Hospital, Heidelberg, Germany

4 Institute of Pathology, Technical University Munich (TUM), Munich, Germany

5 National Center for Tumor Diseases (NCT), Heidelberg, Germany

6 Department of Pathology, Center for Integrated Diagnostics (CID), Massachusetts General Hospital/Harvard Medical School, Boston, USA

7 Department of Gynecology, Charité University Hospital, Berlin, Germany

8 Interdisciplinary Breast Center, Charité University Hospital, Berlin, Germany

9 German Breast Group (GBG), Neu-Isenburg, Germany

Correspondence to:

Jan Budczies, email:

Keywords: targeted sequencing, amplicon sequencing, semiconductor sequencing, copy number alterations, breast cancer

Received: August 26, 2015 Accepted: January 23, 2016 Published: February 17, 2016

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.


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