Next-Generation Assessment of Human Epidermal Growth Factor Receptor 2 (ERBB2) Amplification Status

Establishing ERBB2 [human epidermal growth factor receptor 2 (HER2)] amplification status in breast and gastric carcinomas is essential to treatment selection. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) constitute the current standard for assessment. With further advancements in genomic medicine, new clinically relevant biomarkers are rapidly emerging and options for targeted therapy are increasing in patients with advanced disease, driving the need for comprehensive molecular profiling. Next-generation sequencing (NGS) is an attractive approach for up-front comprehensive assessment, including ERBB2 status, but the concordance with traditional methods of HER2 assessment is not well established. The Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay, a hybrid capture-based NGS assay interrogating the coding regions of 410 cancer-related genes, was performed on manually macrodissected unstained sections from formalin-fixed, paraffin-embedded breast (n = 213) and gastroesophageal (n = 39) tumors submitted for clinical mutation profiling. ERBB2 status was assessed using a custom bioinformatics pipeline, and NGS results were compared to IHC and FISH. NGS ERBB2 amplification calls had an overall concordance of 98.4% (248/252) with the combined IHC/FISH results in this validation set. Discrepancies occurred in the context of low tumor content and HER2 heterogeneity. ERBB2 amplification status can be reliably determined by hybridization capture-based NGS methods, allowing efficient concurrent testing for other potentially actionable genomic alterations, particularly in limited material. Establishing ERBB2 [human epidermal growth factor receptor 2 (HER2)] amplification status in breast and gastric carcinomas is essential to treatment selection. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) constitute the current standard for assessment. With further advancements in genomic medicine, new clinically relevant biomarkers are rapidly emerging and options for targeted therapy are increasing in patients with advanced disease, driving the need for comprehensive molecular profiling. Next-generation sequencing (NGS) is an attractive approach for up-front comprehensive assessment, including ERBB2 status, but the concordance with traditional methods of HER2 assessment is not well established. The Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay, a hybrid capture-based NGS assay interrogating the coding regions of 410 cancer-related genes, was performed on manually macrodissected unstained sections from formalin-fixed, paraffin-embedded breast (n = 213) and gastroesophageal (n = 39) tumors submitted for clinical mutation profiling. ERBB2 status was assessed using a custom bioinformatics pipeline, and NGS results were compared to IHC and FISH. NGS ERBB2 amplification calls had an overall concordance of 98.4% (248/252) with the combined IHC/FISH results in this validation set. Discrepancies occurred in the context of low tumor content and HER2 heterogeneity. ERBB2 amplification status can be reliably determined by hybridization capture-based NGS methods, allowing efficient concurrent testing for other potentially actionable genomic alterations, particularly in limited material. CME Accreditation Statement: This activity (“JMD 2017 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2017 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“JMD 2017 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2017 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Human epidermal growth factor receptor 2 (HER2) is a 185-kDa transmembrane tyrosine kinase receptor encoded by the ERBB2 gene (chromosome 17q12) that is a biomarker and therapeutic target in patients with breast and gastric cancers. Approximately 18% to 25%1Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science. 1987; 235: 177-182Crossref PubMed Scopus (9911) Google Scholar, 2Owens M.A. Horten B.C. Da Silva M.M. HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues.Clin Breast Cancer. 2004; 5: 63-69Abstract Full Text PDF PubMed Scopus (548) Google Scholar, 3Yaziji H. Goldstein L.C. Barry T.S. Werling R. Hwang H. Ellis G.K. Gralow J.R. Livingston R.B. Gown A.M. 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Members of the Cancer Biomarker Reporting Committee, College of American PathologistsTemplate for reporting results of HER2 (ERBB2) biomarker testing of specimens from patients with adenocarcinoma of the stomach or esophagogastric junction.Arch Pathol Lab Med. 2015; 139: 618-620Crossref PubMed Scopus (20) Google Scholar IHC detects the expression of the HER2 protein in a tumor, whereas FISH and chromogenic in situ hybridization/silver in situ hybridization detect amplification of the ERBB2 gene. With rapid advances in genomic medicine, new molecular alterations of clinical relevance are emerging, driving the need for broad comprehensive genotyping. With the advent of next-generation sequencing methods (NGS), the capability to concurrently derive copy number alteration (CNA) data from the same assays used to detect sequence alterations provides a cost- and tissue-efficient alternative to current single-gene assessment methods. To date, ERBB2 assessment by NGS methods has not been clinically validated, and the concordance with traditional methods is not well established. The current study details our experience in the development and validation of an algorithm for assessing ERBB2 amplification status using a hybrid capture-based NGS assay [Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets assay (MSK-IMPACT)],34Cheng D.T. Mitchell T.N. Zehir A. Shah R.H. Benayed R. Syed A. Chandramohan R. Liu Z.Y. Won H.H. Scott S.N. Brannon A.R. O'Reilly C. Sadowska J. Casanova J. Yannes A. Hechtman J.F. Yao J. Song W. Ross D.S. Oultache A. Dogan S. Borsu L. Hameed M. Nafa K. Arcila M.E. Ladanyi M. Berger M.F. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology.J Mol Diagn. 2015; 17: 251-264Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar and establishes the concordance with traditional methods. After obtaining institutional review board approval, the digital records of the Molecular Diagnostics service were searched for breast and GE specimens submitted for molecular analysis by the MSK-IMPACT assay,34Cheng D.T. Mitchell T.N. Zehir A. Shah R.H. Benayed R. Syed A. Chandramohan R. Liu Z.Y. Won H.H. Scott S.N. Brannon A.R. O'Reilly C. Sadowska J. Casanova J. Yannes A. Hechtman J.F. Yao J. Song W. Ross D.S. Oultache A. Dogan S. Borsu L. Hameed M. Nafa K. Arcila M.E. Ladanyi M. Berger M.F. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology.J Mol Diagn. 2015; 17: 251-264Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar as part of an institutional clinical cancer genomics initiative.35Hyman D.M. Solit D.B. Arcila M.E. Cheng D.T. Sabbatini P. Baselga J. Berger M.F. Ladanyi M. Precision medicine at Memorial Sloan Kettering Cancer Center: clinical next-generation sequencing enabling next-generation targeted therapy trials.Drug Discov Today. 2015; 20: 1422-1428Crossref PubMed Scopus (117) Google Scholar DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue after macrodissection, if necessary. Tumor percentages were recorded for each case as an estimate of tumor purity based on semiquantitative analysis of the hematoxylin and eosin–stained section by a pathologist. Specimens containing at least 10% tumor were suitable for analysis. Only cases with concurrent HER2 analysis previously performed by IHC [PATHWAY anti-HER-2/neu (4B5) (Ventana, Tucson, AZ); HercepTest (Dako, Carpinteria, CA)] and/or FISH [HER2 IQFISH pharmDx (Dako); PathVysion HER-2 DNA Probe Kit (Vysis, Downers Grove, IL)] using a Federal Drug Administration–approved method were included in this validation study. The IHC and FISH HER2 test results were reported in accordance with the guideline recommendations for breast cancer31Wolff A.C. Hammond M.E. Hicks D.G. Dowsett M. McShane L.M. Allison K.H. Allred D.C. Bartlett J.M. Bilous M. Fitzgibbons P. Hanna W. Jenkins R.B. Mangu P.B. Paik S. Perez E.A. Press M.F. Spears P.A. Vance G.H. Viale G. Hayes D.F. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.J Clin Oncol. 2013; 31: 3997-4013Crossref PubMed Scopus (2816) Google Scholar and gastric malignancies.32Ajani J.A. Bentrem D.J. Besh S. D'Amico T.A. Das P. Denlinger C. Fakih M.G. Fuchs C.S. Gerdes H. Glasgow R.E. Hayman J.A. Hofstetter W.L. Ilson D.H. Keswani R.N. Kleinberg L.R. Korn W.M. Lockhart A.C. Meredith K. Mulcahy M.F. Orringer M.B. Posey J.A. Sasson A.R. Scott W.J. Strong V.E. Varghese Jr., T.K. Warren G. Washington M.K. Willett C. Wright C.D. McMillian N.R. Sundar H. National Comprehensive Cancer NetworkGastric cancer, version 2.2013: featured updates to the NCCN Guidelines.J Natl Compr Canc Netw. 2013; 11: 531-546Crossref PubMed Scopus (400) Google Scholar, 33Bartley A.N. Christ J. Fitzgibbons P.L. Hamilton S.R. Kakar S. Shah M.A. Tang L.H. Troxell M.L. Members of the Cancer Biomarker Reporting Committee, College of American PathologistsTemplate for reporting results of HER2 (ERBB2) biomarker testing of specimens from patients with adenocarcinoma of the stomach or esophagogastric junction.Arch Pathol Lab Med. 2015; 139: 618-620Crossref PubMed Scopus (20) Google Scholar IHC and/or FISH results were both used to classify each tumor as HER2 amplified or nonamplified in accordance with the standard practice at our institution; IHC was performed first on all specimens, and only equivocal cases were reflexed to FISH. If there was a discordance between the IHC and the NGS result, FISH was performed when material was available. Details of the MSK-IMPACT assay have been previously published.34Cheng D.T. Mitchell T.N. Zehir A. Shah R.H. Benayed R. Syed A. Chandramohan R. Liu Z.Y. Won H.H. Scott S.N. Brannon A.R. O'Reilly C. Sadowska J. Casanova J. Yannes A. Hechtman J.F. Yao J. Song W. Ross D.S. Oultache A. Dogan S. Borsu L. Hameed M. Nafa K. Arcila M.E. Ladanyi M. Berger M.F. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology.J Mol Diagn. 2015; 17: 251-264Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar Briefly, MSK-IMPACT is a comprehensive molecular profiling assay that involves hybridization capture and deep sequencing of all exons and selected introns of up to 410 oncogenes and tumor-suppressor genes, allowing the detection of point mutations, small and large insertions or deletions, and rearrangements. In addition to capturing all coding regions of the genes, the assay also captures >1000 intergenic and intronic single-nucleotide polymorphisms (tiling probes), interspersed homogenously across the genome, aiding the accurate assessment of genome-wide copy number. In total, the probes target approximately 1.2 megabases of the human genome. Genomic DNA was extracted from FFPE tumor tissue after manual macrodissection to ensure at least 10% tumor content. DNA input ranged from 100 to 200 ng, as measured by fluorometric methods. Matching peripheral blood (EDTA tube) from each patient was also extracted for use as a normal control. Sequence libraries were prepared (Kapa Biosystems, Wilmington, MA) through a series of enzymatic steps, including shearing of double-stranded DNA, end repair, A-base addition, ligation of bar-coded sequence adaptors, and low-cycle PCR amplification. Multiple bar-coded sequence libraries were pooled and captured using our custom-designed biotinylated probes (Roche NimbleGen, Madison, WI). Captured DNA fragments were sequenced on an Illumina HiSeq2500 (Illumina, San Diego, CA) as 100-bp paired-end reads that were then subjected to the bioinformatics analysis pipeline. Sequencing reads were aligned to the human genome (hg19) using Burrows-Wheeler Aligner software version 0.7.5a (http://arxiv.org/abs/1303.3997, last accessed October 1, 2013). ABRA36Mose L.E. Wilkerson M.D. Hayes D.N. Perou C.M. Parker J.S. ABRA: improved coding indel detection via assembly-based realignment.Bioinformatics. 2014; 30: 2813-2815Crossref PubMed Scopus (101) Google Scholar was used for read realignment around indels, and Genome Analysis Toolkit37McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14776) Google Scholar was used for base quality score recalibration. Duplicate reads were marked and not used in the downstream analyses. The copy number pipeline builds on the existing data management and analysis systems previously described for the MSK-IMPACT mutation profiling assay.34Cheng D.T. Mitchell T.N. Zehir A. Shah R.H. Benayed R. Syed A. Chandramohan R. Liu Z.Y. Won H.H. Scott S.N. Brannon A.R. O'Reilly C. Sadowska J. Casanova J. Yannes A. Hechtman J.F. Yao J. Song W. Ross D.S. Oultache A. Dogan S. Borsu L. Hameed M. Nafa K. Arcila M.E. Ladanyi M. Berger M.F. Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization capture-based next-generation sequencing clinical assay for solid tumor molecular oncology.J Mol Diagn. 2015; 17: 251-264Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar The first step in the pipeline is to calculate the coverage of each captured target region (exons and tiling probes). After BAM files are generated, we calculate coverage using the Genome Analysis Toolkit Depth of Coverage tool, discarding reads with mapping quality <20.37McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14776) Google Scholar Coverage values are normalized for the overall coverage of the sample, square root transformed, and adjusted for the GC content of each target region using Loess normalization. This normalization removes the GC content dependency of the coverage values (Figure 1, A and B). Captured regions with coverage values in the lowest fifth percentile of coverage in >20% of the normal samples are excluded from the analysis. These regions usually correspond to genes with presence of pseudo copies and have a high rate of false-positive results. Detection of CNAs requires a diploid normal genome as a comparator. For this purpose and to eliminate the sample type-dependent coverage differences, we use a series of FFPE normal samples captured and sequenced with the MSK-IMPACT assay. Normalized coverage values from each region in the tumor sample are divided by the normalized coverage values from each of the normal controls and are log-transformed. Using the log-ratios between a tumor and each normal control, we calculate the sum of square value as a proxy for the level of signal/noise ratio. The normal with the lowest sum-squared log-ratio value is chosen as the comparator for further downstream analysis. Log-ratio values are then further segmented using the circular binary segmentation algorithm (Figure 1B).38Olshen A.B. Venkatraman E.S. Lucito R. Wigler M. Circular binary segmentation for the analysis of array-based DNA copy number data.Biostatistics. 2004; 5: 557-572Crossref PubMed Scopus (1612) Google Scholar Segmented values are grouped into clusters with a minimum separation t