Janice Au-Young

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 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.