Ann Mongan

Abstract B17: Validation of targeted gene expression profiling panel for identifying biomarker signatures of immunotherapy responders

Cancer immunotherapy has led to an unprecedented, long-lasting response in populations susceptible to the therapies. Despite the therapeutic potential, identifying biomarkers and stratifying populations that are likely to respond has been a challenge. Gene expression profiling has previously been successfully used to stratify individuals based on survival and treatment characteristics, but there exist limitations with the prevalent technologies. In particular, full transcriptome gene expression estimates use limited biological material to measure the concentrations of tens of thousands uninformative genes and often lack the depth required to accurately measure expression levels of lowly-expressed genes. These genes may be critical to the identification of a signature associated with immunotherapy responders. To efficiently measure the expression of the key genes potentially informative of an immunotherapy response, we developed a high-throughput targeted gene expression solution measured by our RNA Ion Oncomine Immune Response Profiling panel* containing 395 genes. This panel provides information about the expression of genes involved in tumor checkpoint inhibition (including CTLA4, PD-1, PD-L1, OX-40, 4-1BB, TIM3, LAG3) and other targets such as CSF1R, and IDO1, as well as additional markers of T cell signaling pathway, interferon signaling, and markers of tumor infiltrating lymphocytes (TIL). We used publicly available TCGA data to demonstrate the need and develop a solution for a new normalization procedure that allows for accurate comparisons of samples within various cancer types. Furthermore, we verified a linear and unbiased estimate of fold change in our assays across mixing concentrations of a cell-line titration experiment. Finally, by achieving a high correlation (r > .99) of technical replicates, along with robust expression estimation even at low input amounts (10 ng RNA), our panel offers a valuable solution for biomarker research in cancer immunotherapy. *For research use only. Not for use in diagnostic procedures. Citation Format: Aleksandr Pankov, Yongming Sun, Yuan-Chieh Ku, Warren Tom, Jianping Zheng, Timothy Looney, Janice Au-Yong, Fiona Hyland, Ann Mongan. Validation of targeted gene expression profiling panel for identifying biomarker signatures of immunotherapy responders. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B17.

Abstract 1631: Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM

T-cell repertoire (TCR) sequencing by next-generation sequencing (NGS) is a valuable tool for building a deeper understanding of the adaptive immune system. As immunotherapies, particularly T-cell dependent therapies, show increasing potential in treating cancer, the ability to gain a detailed, unbiased view of the TCR becomes imperative for biomarker discovery, immune response to treatment, and study of tumor microenvironments. A key question the field seeks to understand is the relationship between circulating T-cells and infiltrating T-cells at the tumor site. Here, we present a novel approach for TCR sequencing using the Ion S5 ™ sequencer which leverages simplified library construction workflows and offers a more complete characterization of the entire V(D)J region of TCRB. This method can leverage mRNA as input, minimizing requirements in starting materials and focusing sequencing to productive TCRB arrangements. This approach targets the constant (C) and the FR1 regions, minimizing the potential for primer bias and greatly increasing the phylogenetic information content compared to techniques that exclusively characterize the CDR3 domain. Our results show that the observed circulating T-cell repertoire size is approximately 2 orders of magnitude higher than the infiltrating T-cell repertoire. Accordingly, while it is difficult to fully capture the complete repertoire of circulating T-cells due to its vast diversity, we show that it is possible to reliably capture the complete infiltrating T-cell repertoire with as high as 10 samples on the Ion 530 ™ chip. Replicate sequencing runs of infiltrating T-cells offers correlation of ~0.9, indicating that the results were reproducible, and the samples were sequenced to appropriate depth. In summary, we believe that this workflow will allow researchers to more routinely characterize the infiltrating T-cell repertoire and offers the field a better understanding of the impact of repertoire diversity on tumor elimination. Citation Format: Geoffrey Lowman, Elizabeth Linch, Lauren Miller, Denise Topacio-Hall, Timothy Looney, Alex Pankov, Yongming Sun, Xinzhan Peng, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the circulating and infiltrating T-cell repertoire on the Ion S5TM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1631. doi:10.1158/1538-7445.AM2017-1631

Abstract 3567: Sequencing the human TCRβ repertoire on the Ion S5™ System

Next-generation Sequencing (NGS) is proving an important tool in increasing understanding of the human immune system, and thereby cancer immunology. αβ-T cells are the primary constituents of human cell-mediated adaptive immunity. The antigen specificity of each αβ-T cell is encoded in the 500-600 bp transcript encompassing the variable portion of the rearranged TCRα and TCRβ subunits, which can be read via NGS in a process termed repertoire sequencing. Until now, the main challenge the field faces is the lack of a technology that can provide a contiguous read of 600 bp to minimize the complexity of designing bias-prone primers and informatics challenges of stitching short reads. Here we leverage the long read capability of Ion 530™ chip to comprehensively sequence all three CDR domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire profiling studies. Initial testing with Leukocyte total RNA demonstrates that this multiplex PCR assay produced repertoires that were much more similar to data derived from 5’RACE protocol than the commonly used BIOMED2 primer set. This result suggested that the use of long reads minimizes bias by allowing targeting of less variable regions. To further assess the performance of the assay, we designed a model system of 30 plasmid controls containing common human T-cell CDR3 sequences. Each plasmid was amplified individually and sequenced to confirm the detection of a single clonal population. Analytical sensitivity of the assay and accuracy of the accompanied analysis solution were further evaluated by spiking in plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results showed the assay offers linearity over 5 orders of magnitude of decreasing input concentration. In summary, we have demonstrated a NGS workflow for TCRβ sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less than 48 hours. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Denise S. Topacio-Hall, Tim Looney, Yongming Sun, Lauren Miller, Elizabeth Linch, Geoffrey Lowman, Lifeng Lin, Mark Andersen, Fiona Hyland, Ann Mongan. Sequencing the human TCRβ repertoire on the Ion S5™ System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2017-3567

Abstract 5364: A targeted NGS solution to evaluate gene expression signature of the tumor microenvironment from 40 NSCLC FFPE and matched fresh frozen samples

Cancer cells and their surrounding non-malignant cells, including immune cells, signaling molecules, stromal and extracellular matrix, create the tumor microenvironment (TME). The composition of this TME plays important roles in tumor progression, evading growth suppressors and activating metastasis. However, the regulatory mechanism and function of each constituent remains poorly understood. With several checkpoint blockade therapy studies, the presence of PD-L1 has been reported to be a promising marker to predict positive response. Current IHC methods to measure PD-L1 are subjective and highly variable. A higher-throughput and standardized solution that can systematically measure gene expression of cells present in the TME has emerged to be a more desirable alternative. Here, we applied the OncomineTM Immune Response Research Assay to measure the expression of 395 genes in non-small cell lung cancer (NSCLC) samples from 40 matched FFPE and fresh frozen sample types. This assay leverages NGS technology to sequence and count reads derived from the original transcript. With an input requirement of 10 ng of total RNA, libraries were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM System. Results showed that, despite small input amount, the expression profiles of FFPE and fresh frozen samples are highly correlated with an average correlation greater than 0.9. We selected 22 genes out of the panel to validate expression with qPCR using FFPE samples. These genes were selected to cover a range of low, medium, and high expressors per our NGS data. Again, we observed a strong correlation (R ~ 0.9) between NGS and qPCR data. Approximately 80% of the 40 samples show moderate to high expression of CD8+ T cell cytokines, IFNG and TNFa. We further found that the expression of CD8A and CD8B are highly correlated with CD4, suggesting the co-presence of both cytotoxic and helper T cells. High correlation among CD4, FOXP3, TGFB1, and IL2RA (CD25) also suggests that their expression can be used as markers for the presence of Treg cells. We conducted a differential expression analysis between a group of samples (n=8) with high percentage of surrounding and infiltrating lymphocytes and another group (n=5) with low stromal content but devoid of infiltrating lymphocytes. Interestingly, we found a large number of genes which annotated as markers for infiltrating lymphocytes (CTSS, CXCR4, CD37, SRGN, FCER1G, SAMHD1, and GZMA) are significantly up-regulated in samples with high percentage of surrounding and infiltrating lymphocytes. In summary, this study highlights the robustness of using a targeted panel to understand the composition and regulatory mechanism of the TME and tumor immune response. Citation Format: Yuan-Chieh Ku, Warren Tom, Yongming Sun, Alex Pankov, Tim Looney, Fiona Hyland, Janice Au-Young, Ann Mongan. A targeted NGS solution to evaluate gene expression signature of the tumor microenvironment from 40 NSCLC FFPE and matched fresh frozen samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5364. doi:10.1158/1538-7445.AM2017-5364

Abstract 31: A new research solution for circulating cell free DNA with short amplicons allow accurate detection of 70 COSMIC mutations at 1% detection limit

Non-invasive determination of the resistance or recurrence of tumor development will allow for a safer, more cost effective method for cancer research. As circulating tumor DNA makes up only a small fraction of cell-free DNA (cfDNA) recovered from blood plasma, targeted sequencing offers an ideal tool for mutation detection. Furthermore, as cfDNA is more fragmented than genomic DNA, it presents new technical challenges. In particular, reports in the literature have shown that the majority of cfDNA fragment sizes fall between 120-200 bp, with a median length of 170 bp. Consistent with these reports, we observed an amplicon size dependent improvement in coverage with input samples containing high amounts of fragmented DNA (FFPE or cfDNA). To optimize for cfDNA selection, we have released an Ion AmpliSeq™ designer that offers amplicons of 100-140 bp in size. In this study we conducted a head-to-head comparison between short (100-140 bp) and long (135-175 bp) designs for the same set of targets. These two panels evaluate 1000 COSMIC hotspot mutations from 73 frequently mutated genes. We applied a protocol using magnetic beads to isolate cfDNA from one control (Coriel GM23485) and 3 cfDNA samples. We completed the molecular characterization of the isolated cfDNA using the multiplexing capabilities of Ion AmpliSeq™ library preparation and Ion PGM™ sequencer. Our results showed the short amplicon design led to an improvement of library yield that correlated with the amounts of fragmented DNA contained in the cfDNA samples. The shorter amplicon design also offered improved depth coverage and uniformity for fragmented DNA, while maintaining high performance with genomic DNA. Variant analysis of GM23485 with 70 known mutations at 1% allelic frequency showed >95% positive predictive value with no false negative. In short, the bead-based cfDNA isolation protocol and short amplicon designs offer a simple sample preparation workflow to facilitate rapid mutation detection for cfDNA in cancer research. Citation Format: Ann Mongan, Richard Chien, Antonio Martinez-Alcantara, Fiona Hyland. A new research solution for circulating cell free DNA with short amplicons allow accurate detection of 70 COSMIC mutations at 1% detection limit. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 31.

Abstract 3622: Complete workflow for detection of low frequency somatic mutations from cell-free DNA using Ion Torrent™ platforms

Research detecting of somatic mutations in circulating cell-free DNA (cfDNA) using research blood samples from subjects previously diagnosed with cancer provides a potential non-invasive approach to monitor cancer status and evaluate cancer evolution in the future. However, most of the existing mutation detection methods show insufficient sensitivity to detect cfDNA mutations since only small amount of mutant gene fragments, derived from tumor cells, is present in a large amount of normal circulating DNA background. We demonstrated a complete workflow that includes blood collection, cfDNA isolation, library preparation, sequencing, and data analysis to enable detection of rare DNA variants in blood plasma samples. Blood samples were collected using Streck™ DNA tubes followed by plasma preparation and cfDNA isolation using MagMAX™ Cell-Free DNA Isolation Kit. Library preparation was performed using Oncomine™ lung cfDNA kit. Barcoded libraries were pooled and sequenced on Ion Torrent™ Next Generation Sequencing Platforms. Sequencing data was analyzed in Torrent Suite™ using variantCaller-cfDNA plugin. ∼150 biomarkers relevant to non-small cell lung cancer were interrogated in one sequencing run. We demonstrated detection sensitivity at 0.1% frequency using engineered mutants that were spiked into control DNA samples. The workflow was tested on a set of research samples from matched tumor FFPE and blood plasma collected from research subjects with non-small cell lung cancer (NSCLC). About 1 mL of plasma was processed using the workflow described above. RecoverAll™ Multi-Sample RNA/DNA Isolation Workflow was used to isolate DNA from FFPE samples, followed by library preparation, sequencing and data analysis using the same workflow described above. Summary of variant calls from matched cfDNA and FFPE tumor samples are presented here. Results indicate high sensitivity of the workflow and expected levels of concordance between variants detected in the two types of research samples. In this study, we developed a highly sensitive and reliable research workflow to detect rare somatic mutations in circulating cfDNA samples. Significant overlapping of mutations discovered in FFPE tumor and cfDNA samples suggests that this workflow may be used to monitor tumor dynamics in NSCLC and potentially other tumors in the future. Disclaimer: For research use only. Not for use in diagnostic procedures. Citation Format: Jian Gu, Dumitru Brinza, Ann Mongan, Richard Chien, Dalia Dhingra, Fiona Hyland, Kelli Bramlett. Complete workflow for detection of low frequency somatic mutations from cell-free DNA using Ion Torrent™ platforms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3622.

Abstract 3941: Novel biomarkers and multiplexed NGS to stratify FFPE NSCLC by tumor infiltrating lymphocytes and histopathology phenotypes

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA There is increasing evidence supporting the association of tumor infiltrating lymphocytes (TIL) and drug sensitivity of solid tumors. In particular, primary and meta-analyses have reported a positive correlation between TIL level and outcome in advanced non-small cell lung cancer (NSCLC) treated with checkpoint inhibitors such as PD-1 and PD-L1. Recent trials with immunotherapies have started including TIL assessment in the study protocol in recognition of this metric as a predictive and prognostic biomarker. TIL levels are typically quantified by visual assessment of HE Schalper KA, 2015). Furthermore, as investigators are often also interested in measurements of additional biomarkers such as IFNg as well as the drug targets, a gene panel approach offers a convenient solution to objectively quantify expression levels of these informative markers. Here we report the discovery and verification of a unique gene expression signature that is capable of stratifying FFPE samples of NSCLC tumors by TIL levels and histopathology phenotypes (adenocarcinoma vs. squamous cell carcinoma). Gene expression was measured by an RNA Ion AmpliSeq Gene Expression research panel* containing 200 assays. Each research sample was measured with replicates at library generation step and sequencing step. Technical replicates were found to have >0.99 correlation among each other. Assays on the panels were also found to be robust with respect to low input amount (1-10 ng RNA). *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Ann Mongan, Sophie Rozenzhak, Geoffrey Bien, David Chi, Hiroyoshi Nishikawa, Fiona Hyland, Jim Godsey. Novel biomarkers and multiplexed NGS to stratify FFPE NSCLC by tumor infiltrating lymphocytes and histopathology phenotypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3941.

Abstract 3959: Detection of somatic mutations at 0.1% frequency from cfDNA in peripheral blood with a multiplex next-generation sequencing assay

Background: Effective blood screening for tracking of recurrence and resistance of tumors may improve outcomes in the future. Research studies suggest that virtually all tumors carry somatic DNA mutations, and these may serve as biomarkers that also can be tracked in blood. One of the sources containing tumor DNA in blood is circulating cell-free DNA (cfDNA). Tumor DNA comes from different tumor clones, and its abundance in plasma can be very low at critical stages such as early recurrence or development of resistance. Hence, there is great interest in being able to detect mutation biomarkers at very low frequency from cfDNA for detection and characterization of tumor clones. Method: We present a research use only analysis workflow for peripheral monitoring that enables detection of low frequency DNA variants in blood. We developed an analysis algorithm that models errors accumulated during amplification and sequencing, and accurately reconstructs sequence of original DNA molecules based on multiple next generation sequencing reads. The reads contain genomic sequence and an adaptor that allows identification of reads originated from the same DNA molecule. We then developed a variant calling method that uses accurately reconstructed sequences to enable sensitive and specific detection of somatic mutations to 0.1% allele ratio. We demonstrate the analysis in control and archived cfDNA research samples. We used a next generation sequencing assay that allows interrogation of ∼150 biomarkers relevant in lung from COSMIC and Oncomine™ databases, and de-novo variant detection at ∼1,700 genomic positions in 11 genes implicated in non-small cell lung cancer (NSCLC).The assay delivers >95% on target reads and highly uniform amplification across targeted cfDNA molecules. Results: We tested the limits of variant detection in controlled dilution series and in cfDNA. First, we diluted engineered AcroMetrix™ Oncology Hotspot Control plasmids into background GM24385 genomic DNA down to 0.1% frequency, and then fragmented into fragments with average size of 170bp. The Acrometrix sample contains ∼45 common tumor mutations interrogated by our assay. Next, we used 0.1% Horizon9s (HD780) cfDNA reference sample that contains 8 mutations at our hotspot positions including two large insertion and deletion variants of size >10bp. Finally, we performed analytical verification of variant detection performance in cfDNA using a dilution series of normal blood samples. We achieved >95% sensitivity with >20ng input DNA and >90% sensitivity with ∼20ng input DNA and Conclusions: This new lung cfDNA analysis workflow may facilitate researchers to study relevant NSCLC biomarkers at 0.1% frequency in cfDNA. Analysis is compatible with lower frequency variant detection, but will require higher input DNA amount and higher sequencing coverage. Citation Format: Dumitru Brinza, Ann Mongan, Richard Chen, Dalia Dhingra, Jian Gu, Janice Au-Young, Fiona Hyland, Kelli Bramlett. Detection of somatic mutations at 0.1% frequency from cfDNA in peripheral blood with a multiplex next-generation sequencing assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3959.