Practical Bioinformatic DNA-Sequencing Pipeline for Detecting Oncogene Amplification and EGFRvIII Mutational Status in Clinical Glioblastoma Samples  Michael L. Miller, Jessica Tome-Garcia, Aneta Waluszko, Tatyana Sidorenko, Chitra Kumar, Fei Ye, Nadejda M. Tsankova  The Journal of Molecular Diagnostics  Volume 21, Issue 3, Pages 514-524 (May 2019) DOI: 10.1016/j.jmoldx.2019.02.001 Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Diverse reference data set with sequencing pipeline consistency over time. A: Distribution of tissue sources, sequenced by the molecular pathology laboratory during the first half of 2015, used to generate reference values. The reference values allow measurement of the deviation from each amplicon's expected coverage. B: Comparison of gene coverage in positive sensitivity controls, generated from mixed cell line DNA in independent sequencing experiments over time, demonstrating consistency in the sequencing pipeline. Normalized positive sensitivity control data extracted from the reference data set sequencing runs is compared with identically processed positive sensitivity control data from independent sequencing experiments, performed at different times. CNS, central nervous system; GI, gastrointestinal; LN, lymph node; NOS, not otherwise specified. The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Validation of next-generation sequencing (NGS)–based strategy for detecting epidermal growth factor receptor (EGFR) amplification. A: Graphic representation of EGFR amplicons (black and red boxes) used in the data analysis and their relationship to the EGFR gene (numbered exons shown as blue boxes). Amplicons 1 and 2 (red) are lost in EGFR variant III (EGFRvIII). B: Assessment of linear sensitivity of NGS data for detecting EGFR amplification. Genomic DNA from a cell line with EGFR amplification (MDA-MB-468) is serially diluted with DNA derived from cells that lack EGFR amplification (NCI-H460 cells). A positive linear correlation between normalized EGFR NGS coverage (y axis) and relative amount of EGFR DNA is seen up to approximately 50% dilution (dashed line), above which linearity is lost [linear regression with all data]. Percentages of cell line dilution and tumor cellularity are directly proportional. C: Relative EGFR coverage z scores (normalized to reference population) in both reference (ref.) and all central nervous system (CNS) cases, further subdivided into primary (1°) CNS lesions and glioblastomas (GBMs). For the reference population, a box-and-whisker plot is shown along with the first and 99th percentiles indicated with blue dots. A cutoff of 5 SDs above the mean, corresponding to the 99th percentile in the reference population, demarcates two distinct populations within the CNS group. The proportion of EGFR-amplified samples per group is shown above the dot plot. C, E, and F: Green dots indicate EGFR-amplified samples; red dots, discrepant sample positive by CISH only. D: Representative chromogenic in situ hybridization (CISH) micrographs of tissue from nonamplified (top image) and EGFR-amplified (bottom image) tumors (black pigment indicates DNA EGFR probe; red pigment, DNA CEP7 probe). E: Comparison of NGS-based detection of EGFR amplification to CISH in a subset of glioblastoma samples for which CISH data were available. All cases that show amplification by NGS also show amplification by CISH (green indicates samples with amplification and black indicates samples without amplification). In a single case, CISH reveals very focal amplification, in <1% of tumor cells, which is not detected by NGS (red point). F: EGFR amplification in glioblastomas stratified by tumor cellularity. The proportion of EGFR-amplified samples per group is shown above the dot plot. Dotted horizontal lines in B, C, E, and F indicate positive z score cutoff; green horizontal lines, the mean of the EGFR-amplified samples; black horizontal lines, the mean of the non-amplified samples. Original magnification, ×600 (D). The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Discovery of focal gene amplifications across the Ion AmpliSeq panel in primary and metastatic central nervous system (CNS) lesions. Heatmap depicts relative gene coverage for all CNS lesions (z score normalized to the reference population). Focal gene amplification is defined by a z score ≥ 5 (bottom panel). The most frequently amplified gene is EGFR, followed by PDGFRA, KIT, MET, and AKT1. In addition to glioblastoma, EGFR amplification is detected in one low-grade glioma and in two anaplastic astrocytomas, which were reclassified to glioblastoma based on independent molecular studies (each of the three samples indicated by an asterisk), as well as in one metastatic lung carcinoma (dagger). Hotspot single-nucleotide variants (SNVs) detected clinically using the Ion AmpliSeq panel are also shown (mutational status for IDH1/2 and BRAF are shown on top, alongside histologic grade; remaining SNVs are shown on bottom, integrated with amplification status). n = 117 CNS lesions. The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Inferring epidermal growth factor receptor variant III (EGFRvIII) in central nervous system (CNS) lesions using normalized read-coverage dropout. A: Representative tracks (from integrated genome viewer) highlight samples without EGFR amplification (top), with amplification but without EGFRvIII (middle), and with both amplification and EGFRvIII (bottom). Boxed areas in the top panel are enlarged below. Relative signal loss at exons 3 and 7 (red asterisks) in the sample with EGFRvIII is shown in the leftmost enlargements, with red exons indicating regions lost in the mutation. B: Comparison of quantitative next-generation sequencing (NGS) approach to long-range PCR (LR-PCR) for detection of EGFRvIII in a subset of samples for which frozen tissue was available to extract nondegraded genomic material. Except for one sample per comparison (red), all samples with NGS-detected EGFRvIII were also positive (pos.) by LR-PCR (green), whereas all samples without EGFRvIII by NGS were also negative (neg.) by LR-PCR (black). B and D: Green dots indicate samples with EGFRvIII detected by NGS (or by both NGS and LR-PCR when data available); red dots, discordant samples. C: Representative electrophoretic fractionation of DNA products from LR-PCR highlights two representative samples with genetically distinct EGFRvIII deletions (lanes 1 and 2) and one representative sample without the mutation (lane 3). Molecular weight ladder (L) with band sizes indicated in kilobases. D: Comparison of normalized read coverage at EGFR used to determine EGFRvIII mutant status (Cv3) in glioblastomas (GBMs) and the reference population (ref.). Values 10 SDs above the mean (dotted horizontal lines in B and D) were considered positive for the mutation as this cutoff revealed two distinct populations. For the reference population, a box-and-whisker plot is shown along with the first and 99th percentiles indicated with blue dots. Refer to Supplemental Figure S4A for remaining CNS data. E: Heatmap illustrating EGFR amplicon coverage and strategy for detecting EGFRvIII. Given the loss of exons 2 to 7 in EGFRvIII, the mutation's presence is inferred by the loss of reads corresponding to this region (amplicons 1 and 2) relative to the remaining EGFR amplicons downstream of exon 7 (amplicons 3 to 8) (EGFRvIII detection defined as z score ≥ 10). For comparison, EGFR amplification is redemonstrated on the lowest heatmap panel, also highlighting the two anaplastic astrocytomas reclassified as glioblastoma (asterisks), as well as the metastatic lung carcinoma (dagger). The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Gene coverage analysis yields low technical variation in clinical samples. Heatmap depicting coefficient of variation in sequencing coverage among paired clinical samples, with darker blue indicating greater variation between paired values. True replicates, which correspond to the same specimen sequenced on two occasions (pairs A to D), exhibit markedly less variation than randomly paired clinical samples (pairs E to L). The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Validation of next-generation sequencing (NGS)–based strategy for detecting EGFR amplification. Comparison of NGS-based detection of EGFR amplification to chromogenic in situ hybridization (CISH) in a subset of central nervous system samples for which CISH data were available. For glioblastomas only, refer to Figure 2E. Green dots indicate those positive (pos.) by both methods, whereas red dots indicate discordant or negative (neg.) results. Horizontal bars indicate means; dashed horizontal line, positive z score cutoff. The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Validation of next-generation sequencing (NGS)–based strategy for detecting PDGFRA amplification. A: Relative PDGFRA read coverage (normalized to reference population, indicated by blue dots), expressed as z score, in both reference (ref.) and all central nervous system (CNS) cases, with the latter further subdivided into primary (1°) CNS lesions and glioblastomas (GBMs). For the reference population, a box-and-whisker plot is shown along with the first and 99th percentiles indicated with blue dots. Similar to the EGFR amplification analysis shown in Figure 2, a cutoff of 5 SDs above the mean—corresponding to the 99th percentile in the ref. population—demarcates two distinct populations within the CNS group. The proportion of PDGFRA-amplified samples per group is shown above the dot plot, with green indicating those positive by NGS. B: PDGFRA amplification in GBMs stratified by tumor cellularity. The proportion of PDGFRA-amplified samples per group is shown above the dot plot. Green horizontal bars indicate the mean for samples positive by NGS; black horizontal bars, the mean for NGS-negative samples. A and B: Dotted horizontal lines indicate positive z score cutoff. C: Representative micrographs of fluorescence in situ hybridization performed on four different tumors to validate NGS-based detection of PDGFRA amplification. More than two red signals within a tumor cell indicates PDGFRA amplification. In the bottom right corner, a tumor sample with EGFR amplification but not PDGFRA amplification is shown as a negative control (red fluorophore indicates DNA PDGFRA probe). Arrows highlight cells with PDGRA amplification or those without amplification in the case of the negative control (bottom right panel). Original magnification, ×1000 (C). The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S4 Detection of EGFRvIII in central nervous system (CNS) tumors using next-generation sequencing (NGS) and relationship to tumor cellularity. A: Representation of normalized read coverage (z score) at EGFR used to determine EGFRvIII mutant status (Cv3). Values 10 SDs above the mean (indicated by dashed horizontal line) are considered positive for mutant EGFRvIII because this cutoff revealed two distinct populations within all CNS, primary (1°) CNS, and glioblastoma (GBM) samples. For the reference (ref.) population, a box-and-whisker plot is shown along with the first and 99th percentiles indicated with blue dots. B: EGFRvIII in GBMs stratified by tumor cellularity, with the portion of samples positive by NGS per group shown. Green horizontal bars indicate the mean for samples positive by NGS; black horizontal bars, the mean for NGS-negative samples. A and B: Green dots indicate samples positive by NGS (or both methods when data are available); red dots, discordant results. The Journal of Molecular Diagnostics 2019 21, 514-524DOI: (10.1016/j.jmoldx.2019.02.001) Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions