Analytical Validation of a Next-Generation Sequencing Assay to Monitor Immune Responses in Solid Tumors Jeffrey M. Conroy, Sarabjot Pabla, Sean T. Glenn,

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Analytical Validation of a Next-Generation Sequencing Assay to Monitor Immune Responses in Solid Tumors Jeffrey M. Conroy, Sarabjot Pabla, Sean T. Glenn, Blake Burgher, Mary Nesline, Antonios Papanicolau-Sengos, Jonathan Andreas, Vincent Giamo, Felicia L. Lenzo, Fiona C.L. Hyland, Angela Omilian, Wiam Bshara, Moachun Qin, Ji He, Igor Puzanov, Marc S. Ernstoff, Mark Gardner, Lorenzo Galluzzi, Carl Morrison The Journal of Molecular Diagnostics Volume 20, Issue 1, Pages 95-109 (January 2018) DOI: 10.1016/j.jmoldx.2017.10.001 Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Overview of the assay. A: The assay relies on a next-generation sequencing (NGS) platform to simultaneously measure gene expression (GEX) of 54 transcripts related to tumor-infiltrating lymphocytes (TILs) and other immunologic functions in the tumor microenvironment, as well as neoplastic mutational burden (MuB). B: The workflow consists of a first evaluation of slides by a pathologist, optional macrodissection of tumor tissue, single-step RNA and DNA extraction, followed by target amplification and NGS of both RNA and DNA libraries in a unique sequencing run. NGS data are processed through the bioinformatics pipeline with run quality control, reporting, and pathologist sign-out in a custom laboratory information system. C: Performance is evaluated for several preanalytical and analytical parameters by a series of reproducibility studies and by experiments that demonstrate sensitivity, accuracy, and specificity across a broad range of tumor types. CTL, cytotoxic T lymphocyte; FF, fresh-frozen; FFPE, formalin-fixed, paraffin-embedded; gDNA, genomic DNA; LIS, laboratory information system; PD-L1, programmed death-ligand 1; Prep, preparation. The Journal of Molecular Diagnostics 2018 20, 95-109DOI: (10.1016/j.jmoldx.2017.10.001) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 RNA-sequencing preanalytical validation. A: Gene expression scatter plots of log10 normalized reads per million (nRPM) for target transcripts demonstrate comparable results for RNA extracted from 12 matched internally and externally sectioned unstained slides (left) and 13 formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen (FF) samples (right). B: Comparison of RNA-sequencing results obtained with specimens artificially built to contain increasing percentage of normal (left) or necrotic (right) tissue demonstrates a high degree of correlation (r = Pearson product-moment correlation coefficient). C: Comparison of RNA-sequencing results obtained with increasing RNA inputs (left) and genomic DNA (gDNA) contamination (right; 33% and 50%). D: Scatter plot of nRPM for batch size of samples per run. n = 4 and 16 samples (D, left); n = 8 and 16 samples (D, right). The Journal of Molecular Diagnostics 2018 20, 95-109DOI: (10.1016/j.jmoldx.2017.10.001) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 DNA-sequencing preanalytical validation. A: Reduced neoplastic content results in fewer detected mutations, requiring >50% neoplastic cells for accurate clinical interpretation of high or not high mutational burden (MuB) (dashed line indicates a threshold). B: MuB calls are maintained across a wide range of relative abundance of necrotic tissue, including 100%. C: MuB calls are not influenced by the number of samples included per run. D: Reduced sequencing coverage results in fewer detected mutations, calling for >850,000 bases of DNA-sequencing with ≥20× coverage for accurate results. The Journal of Molecular Diagnostics 2018 20, 95-109DOI: (10.1016/j.jmoldx.2017.10.001) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Mutational burden (MuB) and depth of coverage. Eight DNA samples with variable read depth (gray bars) and mutations detected per megabase of coding sequence (black line). The Journal of Molecular Diagnostics 2018 20, 95-109DOI: (10.1016/j.jmoldx.2017.10.001) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Correlation between RNA-sequencing (seq) and immunohistochemistry (IHC). Formalin-fixed, paraffin-embedded (FFPE) blocks from 57 tumors of varying histologic characteristics were assembled in a tissue microarray (TMA), sectioned, and processed for the immunohistochemical assessment of programmed death-ligand 1 (PD-L1) and CD8A expression. A different block for the same set of samples was used for targeted RNA-seq. A: Representative image of PD-L1 expression as assessed by IHC with low (left) and high (right) tumor proportion score (TPS) and correlation plot of RNA-seq results [log2-transformed normalized CD274 reads per million (nRPM)] and PD-L1 IHC results. B: Representative image of T-cell infiltration as assessed by CD8A IHC with low and high T-cell infiltration and correlation plot of RNA-seq results (log2-transformed nRPM) and CD8 IHC results (number of CD8A+ T cells/mm2). The Journal of Molecular Diagnostics 2018 20, 95-109DOI: (10.1016/j.jmoldx.2017.10.001) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Gene expression reproducibility studies. Pairwise correlation across three unique specimens (specimen three run as biological replicate), two technicians (T), 2 days (D), four independent runs (R), and multiple barcodes for the 54 validated transcripts. The Journal of Molecular Diagnostics 2018 20, 95-109DOI: (10.1016/j.jmoldx.2017.10.001) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Trend lines of run level controls. A and B: No template control (NTC) compared with average sample mapped reads for all RNA-sequencing (A) and DNA-sequencing (B) runs. C and D: Positive (PC) and negative (NC) gene expression (GEX; C) and mutational burden (MuB; D) run controls compared with samples and NTC across all runs. The yellow solid lines and flanking dashed and solid lines represent means ± 1, 2, and 3 SDs. The Journal of Molecular Diagnostics 2018 20, 95-109DOI: (10.1016/j.jmoldx.2017.10.001) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions