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.

Targeted next‐generation sequencing enables reliable detection of HER2 (ERBB2) status in breast cancer and provides ancillary information of clinical relevance

“…Both reported a high concordance rate (>95 and 97%, respectively) between copy number data obtained by FISH and targeted NGS data. Applying the bioinformatics tool Ioncopy, Budczies et al (2016) reported similar results with fresh tissue and a specific gene panel for targeted NGS that is different from the one employed here. Our results demonstrate that our NGS-based method could be used in an up-front diagnostic setting with a specificity of 100%.…”

Genetic alterations in hepatocellular carcinoma: An update

“…Currently, oncologists are testing novel, molecularly targeted agents for treating HCC. Therefore, in an era of precision cancer medicine, monitoring clinically relevant genetic alterations is important for stratifying patients for targeted therapies[9]. …”

Pan‐cancer analysis of copy number changes in programmed death‐ligand 1 (PD‐L1, CD274) – associations with gene expression, mutational load, and survival

“…However, the mechanisms that govern expression levels of the PD‐ligands, such as PD‐L1, are currently not well understood. A straight‐forward mechanism that received surprisingly little attention until now are copy number changes (Albertson, ; Conrad et al, ; Zack et al, ; Zarrei et al, ) of the coding regions, which could in principle be easily detected by FISH or massive parallel sequencing approaches employed for routine diagnostics (Budczies et al, ; Endris et al, ; Stenzinger et al, ). Since the work by Green et al () on Hodgkin lymphoma, amplification of PD‐L1 in tumors has so far only been described for basal‐like and triple negative breast cancer (Ali et al, ; Barrett et al, ; Sabatier et al, ), gastric cancer (Bass et al, ), and a subgroup of lung cancer (Goldmann et al, ; Ikeda et al, ).…”