Zhenyu Yan

Abstract 1397: Oncomine focus assay: Simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in 52 oncogenes relevant to solid tumors

Introduction: The Oncomine™ Focus Assay (OFA) is aimed to simultaneously detect and report hotspot mutations, Copy Number Variants (CNVs) and gene fusions in 52 oncogenes clinically relevant to solid tumors. With minimal DNA/RNA input, OFA employs AmpliSeq™ library preparation chemistry to enrich target regions for Ion Torrent™ Next Generation Sequencing. Variants are annotated for actionability by Oncomine® Knowledgebase. Here we report an analytical validation of OFA. Methods: DNA and RNA, extracted from the FFPE processed GM12878, was used as the negative control to evaluate the specificity of OFA. A RNA sample containing multiple oncogenic gene fusions and a DNA sample containing multiple hotspot SNV and indels were used as the positive controls. Fresh DNA from cancer cell lines (HCC1143 or NCI-H2122) along with DNA from matched normal cell lines, HorizonDx NGS standards TruQ-1, TruQ-2, several engineered FFPE samples with gene fusions or copy number changes, and 43 clinical FFPE samples of a variety of solid tumor types (non-small cell lung cancer, colorectal cancer, gastrointestinal stromal tumor, ovarian cancer, pancreatic cancer, and melanoma) were used to evaluate the OFA performance. The analysis of the sequencing data was primarily performed with the OFA pipeline integrated with the Oncomine® Knowledgebase. Only the genetic alterations with clinical utility were selected as the final output from the data pipeline. The SBS and indels detected by OFA were confirmed by Sanger sequencing. Any CNVs detected were confirmed by FISH, if possible, and detected fusions were confirmed by RT-PCR or FISH. Results: No clinically relevant genetic alterations were detected from the negative control FFPE-GM12878, indicating the high specificity of the OFA. The LOD for SBS and short indel detection was 5%, as assessed by TruQ-1 and TruQ-2, each containing 9-10 variants. The assay can detect gene fusion RNA present as 0.1%-1% of the total RNA, and gene amplifications are detected with a minimum of 50% tumor cell content. A known 12X CCND1 amplifications in HCC1143 and a 13X MYC amplification in NCI-H2122 were detected. In addition, a 13X EGFR amplification and 11X CDK4 amplification were detected in a lung cancer FFPE sample and confirmed by FISH. The analytical specificity for detection of SBS and indels was 100% (25/25). The analytical specificity for detection of CNV gain and gene fusions, and the assay sensitivity are currently under assessment. Conclusions: These results demonstrate the high sensitivity and specificity of OFA. With as little as 10 ng RNA and 10 ng DNA, OFA provides biomarker analysis of patient FFPE tumor specimens focusing on the detection of genetic alterations that have been targeted by oncology drugs or supported by published clinical evidence. Citation Format: Peng Fang, Zhenyu Yan, Paul Labrousse, Weihua Liu, Jennifer Biroschak, Jennifer Wright, Selby Weeks, Cindy Spittle, Chad Galderisi, Jin Li. Oncomine focus assay: Simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in 52 oncogenes relevant to solid tumors. [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 1397.

Abstract A044: Immunotherapy biomarker assessment in RCC using IHC, gene expression profiling, and mutation burden assessment

The field of immunotherapy includes multiple approaches that harness the power of the immune system to destroy cancer cells. Checkpoint inhibitors have been approved for the treatment of solid tumor and hematologic malignancies. While significant responses have been observed in a subset of patients, outcomes are variable and there is a need to identify additional predictive biomarkers beyond PD-L1 levels as measured by IHC. A more comprehensive assessment of the tumor and its microenvironment can be accomplished through gene expression profiling, mutation burden analysis, and immune repertoire profiling. Exploratory studies such as those presented here using targeted RNA and DNA sequencing will continue to expand our understanding of tumor immune cell interactions and how this information can be used to improve patient selection for checkpoint inhibitors and other immunotherapy. In this study, a set of 30 FFPE tumor samples including CRC, RCC, and NSCLC was analyzed using an IO Biomarker IHC Panel (PD-L1, CD8, CD3, and CD163). PD-L1 staining (% and intensity) was scored for both tumor cells (TC) and immune cells (IC). T-cell and macrophage markers were scored as high, medium, and low. Samples were also analyzed using the ThermoFisher Oncomine™ Immune Response Research Assay. This targeted RNA-sequencing panel measures the expression of 391 genes involved in tumor-immune cell interactions. RNA was extracted using RecoverAll and the quantity and quality was assessed using Qubit and RT-qPCR, respectively. A significant correlation was observed between the RT-qPCR quality score and mapped sequencing reads. A subset of 7 RCC samples with the highest RNA quality and sequencing QC metrics were selected for additional analysis. A trend was observed in the RNA expression level of individual genes such as PD-L1, PD1, CD8A, TNFRSF9, and LAG3 and a composite expression score based on 10 interferon gamma-related genes. Unsupervised clustering was performed using several published gene expression signatures that have been found to correlate with immune response (IR) and/or tumor inflammation. The gene content ranged between 4-13 genes and overlapped between some but not all signatures. The 13-gene signature classified six RCC samples as “high IR” and one sample as “low IR.” Interestingly, similar results were obtained when samples were classified using the other signatures. PD-L1 IHC positive staining patterns varied widely in the “high IR” samples and ranged from 40%TC/30%IC to 0%TC/5%IC. The “low IR” sample was PD-L1 negative in TC and IC. To further understand the relationship between the tumor’s genetic profile and the tumor microenvironment, mutation burden and immune repertoire analysis was performed using NGS-based methods. The results from the two panels will be presented. This exploratory study details how targeted RNA and DNA sequencing can identify patient subsets based on a multiplexed signature rather than a single marker. The use of both molecular and tissue-based assays will lead to a more comprehensive understanding of tumor and immune biology that may uncover new biomarkers for optimal stratification of patients for personalized immunotherapy treatment. Citation Format: Peng Fang, Zhenyu Yan, Xiaodong Wang, Wes Chang, Chad Galderisi, Cindy Spittle, Jin Li. Immunotherapy biomarker assessment in RCC using IHC, gene expression profiling, and mutation burden assessment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A044.

Abstract A29: Mutation profiling of colorectal cancer ctDNA using AmpliSeq CHP2 cancer panel

Introduction: Analysis of ctDNA has potential applications in targeted therapy selection and disease monitoring in the clinical management of colorectal cancer. Here we use an amplicon based target enrichment method, AmpliSeq v2 (CHP2), to study a set of tumor/plasma pairs (n = 13) collected from stage IV colon cancer patients to determine whether amplicon based NGS can be used to profile mutations in ctDNA from CRC patients. Methods: The CHP2 is designed to survey 2800 mutations in 50 cancer-related genes. Matched tumor and plasma samples were purchased from Indivumed. DNA was extracted from 3 or 5 ml plasma using QIAamp DSP circulating NA kit and eluted in 10 μl buffer per ml plasma. The sample input used in the CHP2 reactions was between 3.8 - 55 ng ctDNA. The background noise level of hotspot nt positions was calculated from 10 healthy donor plasma samples. A custom data analysis program was developed to detect variants clearly different from background noise. ddPCR mutation assays were purchased from BioRad and performed using the QX200. ddPCR data was analyzed using QuantaSoft software. Results: The ctDNA samples can be divided into two groups based on the DNA concentration measured by Qubit (range of 0.16-13 ng/ul): 6 with >1 ng/ul and 7 with 10% (N = 7), 10% allele frequency were detected in 3 ctDNA samples (ctDNA concentration > 1ng/ul) that have co-existing mutations in KRAS and PIK3CA and/or APC gene, suggesting polyclonal evolution in multiple oncogenic pathways. Several Germline SNPs were detected in multiple genes and excluded from concordance calculation between tumor and plasma. A small subset of ctDNA samples, including 1 negative and 2 positive samples (KRAS G12D 1.4% and PIK3CA E545G 0.9%) was also analyzed by ddPCR and results were consistent with those obtained using CHP2. Conclusions: AmpliSeq CHP2 cancer panel achieved 0.06-0.2% allele frequency sensitivity and 85% (11/13) tumor/plasma concordance, which are comparable with previous reports using alternative panels. We also demonstrate the efficiency of target enrichment by AmpliSeq based multiplex PCR, which can detect as low as two mutant copies from wild type background. This high sensitivity method is ideal for clinical sample testing where the amount of plasma-derived DNA is limited. Citation Format: WeiHua Liu, Zhenyu Yan, Paul Labrousse, Peng Fang, Jennifer Biroschak, Cindy Spittle, Chad Galderisi, Jin Li. Mutation profiling of colorectal cancer ctDNA using AmpliSeq CHP2 cancer panel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A29.

Abstract 4261: Oncomine® Cancer Panel: simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in solid tumors

Introduction: The Oncomine® Cancer Panel (OCP) is designed to simultaneously detect and report hotspot mutations, Copy Number Variants (CNVs) and gene fusions in 143 genes with minimal DNA/RNA sample input. The OCP utilizes the AmpliSeq™ library preparation chemistry for the Ion Torrent™ Next Generation Sequencing (NGS) platform, in combination with data annotations by the Oncomine® Knowledgebase. Here we report analytical validation of OCP. Methods: DNA and RNA, extracted from the FFPE processed GM12878, was used as the negative control to evaluate the specificity of OCP. A RNA sample containing multiple oncogenic gene fusions, and a DNA sample containing multiple hotspot SNV and indels were used as the positive control. Fresh DNA from cancer cell lines (HCC1143 or NCI-H2122) along with DNA from matched normal cell lines, HorizonDx NGS standards TruQ-1, TruQ-2, several engineered FFPE samples with gene fusions or copy number changes, and 26 clinical FFPE samples of a variety of solid tumor types (lung, breast, colon, ovary, stomach, uterus and larynx) were used to evaluate the OCP performance. The analysis of the sequencing data was primarily performed with the OCP pipeline integrated with Oncomine® Knowledgebase from Life Technologies, supplemented by the MolecularMD proprietary pipeline. Only the genetic alterations with clinical utility were selected as the final output from the data pipeline. Of these detected by OCP, the SBS and indels were confirmed by the Illumina TruSeq, Ion Torrent AmpliSeq commercial cancer panels, or by Sanger sequencing. Any CNVs detected were confirmed by FISH, if possible, and detected fusions were confirmed by RT-PCR or FISH. Results: No clinically relevant genetic alterations were detected from the negative control FFPE-GM12878, indicating the high specificity of the OCP. High specificity was achieved, in part, using stringent filters, which removed error prone regions from analysis. The LOD for SBS and short indel detection was 5%, as assessed by TruQ-1 and TruQ-2, each containing 15 variants. While the exact LOD for gene fusion detection is currently under evaluation, the 100%, 50% and 20% fusion were detected in the RNA samples from serially diluted EML4-ALK or SL34A2-ROS1 fusion into the FFPE-GM12878 RNA. Four known gene amplifications (MYC, CCND1, MDM2, AKT1) with CNV from 5.4X to 14.5X in HCC1143, and a 15.7X MYC amplification in NCI-H2122 were detected. In addition, an EML4-ALK.E13A20 fusion was detected in a lung cancer FFPE sample, and confirmed by FISH. The analytical sensitivity and the specificity of the OCP are currently assessed with 26 clinical samples and three engineered FFPE samples. Conclusions: These results demonstrate the high sensitivity and specificity of OCP. With as little as 10 ng RNA and 20 ng DNA, OCP provides comprehensive screening of patient FFPE tumor specimens for the detection of a broad spectrum of clinically relevant genetic alterations. Citation Format: Peng Fang, Zhenyu Yan, Weihua Liu, Jennifer Biroschak, Paul Labrousse, Jennifer Wright, Cindy Spittle, Chad Galderisi, Li Jin. Oncomine® Cancer Panel: simultaneous detection of clinically relevant hotspot mutations, CNVs, and gene fusions in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4261. doi:10.1158/1538-7445.AM2015-4261

Abstract 4925: Liquid biopsy using the ion AmpliSeq v2 cancer panel

Introduction: Plasma cell-free circulating tumor DNA (ctDNA) has the potential to serve as a noninvasive surrogate to determine tumor genotype and monitor treatment response. Here we report the analytical validation of the Ion AmpliSeq v2 Cancer Panel (ACP) in plasma ctDNA samples. Methods: Mutations were assessed using the ACP, designed to survey 2800 mutations in 50 cancer-related genes. TruQ reference standards were used to determine LOD. Healthy donor and cancer patient plasma samples were used for other studies. Eight libraries were barcoded and sequenced with PGM sequencing 200 kit v2 on a 318 v2 chip. The sequencing data were analyzed with a combination of Torrent Suite 3.4.2, VarScan, our proprietary analysis pipeline and IGV. The background noise level of hotspot nt positions was calculated from multiple runs of known wild type DNA. In order to increase the sensitivity of the assay to detect low-level mutation, a custom program was developed to detect variants clearly different from background noise (95% confidence) and not reported by the two variant callers. PCR methods were used for concordance testing. Results: The performance characteristics of the AmpliSeq v2 Cancer Panel were demonstrated in 1) Minimum DNA input and LOD: With 10 ng DNA input, 43/43 and 42/43 expected mutations were detected at 2.5% or 1%, respectively. With 1 ng DNA input, 42/43 and 40/43 expected mutations were detected at 2.5% and 1%, respectively. 2) Coverage: The mean library coverage for 40 plasma libraries was 2912x with only 5 non-critical regions below 250x coverage. 3) Baseline error rate: 95% of 2290 hot-spots for SBS showed Conclusions: The ACP offers a useful tool for comprehensive somatic mutation profiling in ctDNA. The limit of detection is 1-2% mutation frequency for most mutations with slight variation across different nucleotide positions. The low concentration of ctDNA obtained from plasma samples can present a challenge. Less than 10 ng DNA input may result in allele drop out for low level mutations due to the inefficient amplification of multiplex PCR compared with PCR methods using a short length single amplicon. Higher DNA input to rescue allele drop-out is under evaluation. Citation Format: WeiHua Liu, Zhenyu Yan, Candice L. Horn, Fabio Nunes, Steven M. Bray, Philip J. Ebert, Peng Fang, Jennifer Biroschak, Cindy Spittle, Chad Galderisi, Jin Li. Liquid biopsy using the ion AmpliSeq v2 cancer panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2015-4925

Abstract A208: Mutation detection in FFPE and plasma circulating DNA with a focused ALK-EGFR-KRAS Next-Generation Sequencing panel.

Background: Mutations in ALK, EGFR and KRAS have well-established or anticipated clinical utility in predicting response to targeted therapies either approved or in development for patients with non-small cell lung cancer (NSCLC). Here we report results from a proof-of-concept study using a custom Ion AmpliSeq Next-Generation Sequencing (NGS) panel designed to sequence a range of clinically relevant exons in ALK, EGFR and KRAS genes in formalin fixed paraffin-embedded (FFPE) DNA and plasma circulating DNA. Methods: The cumulative 2.1kb regions of interest (ROI) include exons 20-25 of ALK, exons 18-22 of EGFR, and exons 2-4 of KRAS, as well as the first 3 bp of the intron at the intron-exon boundaries. The custom panel was designed by Ion AmpliSeq Designer. Sequencing data were analyzed with Torrent Suite 3.4 and MolecularMDs proprietary analysis pipeline. Results: The study demonstrated the robustness of the custom NGS assay with at least 90% of reads-on-target, an average coverage of above 5000x, and no less than 81% uniformity in the reads. In addition, the minimum coverage for each ROI in all the samples tested was no less than 1000x. The limit of detection (LOD) of the NGS assay, as determined using dilutions of cell line DNA standards, was 0.7% for single base substitution (SBS) mutations, and 1.2% for indels. In addition, the assay was able to quantify mutations with frequencies as low as ∼1% in plasma circulating DNA. The assay detected 4 KRAS SBS mutations (G12C, G12D, G12V and Q61H), 2 EGFR SBS mutations (G719A, V769M) and 1 EGFR exon 19 deletion (G746-A750del) in 10 FFPE specimens tested from colon and lung cancer patients. These variants were each confirmed using Cancer Panels (Ion Torrent AmpliSeq and Illumina TrueSeq). The assay also detected T790M, L858R, V769M and the exon 19 deletion in EGFR in 7 plasma samples obtained from NSCLC patients. Each of these variants was confirmed by Sanger sequencing, Qiagen RGQ assay, or by MolecularMDs proprietary EGFR droplet digital PCR (ddPCR) assay. A low-frequency EGFR exon 19 deletion identified by the Qiagen RGQ assay was not seen in either the NGS assay or ddPCR assay. No other potential false negatives were identified in the custom NGS assay. Conclusions: This study demonstrates the feasibility of creating a sensitive and specific ALK-EGFR-KRAS focused NGS assay that covers broader regions of the target genes than the hotspots represented in commercial panels. With a DNA input of merely 20ng of FFPE DNA, or 1-2ng of the plasma circulating DNA, the assay was able to detect both SBS and small indels in the targeted regions. Given the low DNA input requirements and the capability of plasma-based testing, this assay and other custom NGS panels may enable routine monitoring of mutation status for relevant genes in patients with various solid tumors, and may ultimately inform clinical decision-making. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A208. Citation Format: Peng Fang, Agus Darwanto, Zhenyu Yan, Weihua Liu, Kimberly Pelak, Jessica Kristof, Philip C. Mack, Sabita Sankar, Chad Galderisi, Jin Li. Mutation detection in FFPE and plasma circulating DNA with a focused ALK-EGFR-KRAS Next-Generation Sequencing panel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A208.

Abstract 3509: Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: LKB1 (liver kinase B1)/STK11 (serine-threonine kinase 11) is a tumor suppressor that encodes a serine/threonine kinase which negatively regulates the mTOR (mammalian target of rapamycin) signaling pathway. Somatic mutations in LKB1 occur most notably in lung adenocarcinoma, cervical cancers and melanoma. Inactivation of LKB1 is caused by point mutations, homozygous deletions or promoter methylation. LKB1 mutation status is a predictive marker for responsiveness to both MEK and PI3K inhibitors. We have developed next-generation sequencing (NGS), Sanger sequencing and immunohistochemistry (IHC) assays to assess LKB1 mutation and protein expression status in a set of FFPE patient specimens. Methods: For NGS, we used the Ion Torrent platform to sequence a 4.4kb region of interest (ROI) around LKB1. A combination of TorrentSuite and in-house tools were used for alignment, variant calling and prioritization of variants. Sanger sequencing was used to validate the presence and absence of variants with a frequency of at least 10% that were identified by NGS. An IHC assay was developed using a commercially available antibody. These assays were validated using a combination of cell lines, a genetically engineered mouse model and FFPE tissue for specificity, sensitivity, reproducibility and concordance. Results: 35 patient FFPE tissues from a variety of tumors including cervix, endometrium, pancreas, skin and lung were evaluated for LKB1 mutation status using NGS and IHC methods. About 96% of the coding region of LKB1 had sufficient sequencing coverage (read depth >500x) to reliably call low frequency somatic variants. Analytical validation using cell line dilutions demonstrated that our NGS assay is able to detect single base changes with frequencies as low as 2-5%, and small indels with frequencies as low as 5-10%. We identified 9 coding and splice site variants in our preliminary analysis of 18/35 of the FFPE specimens. We compared the variants in 7 of these FFPE samples to the variants identified in these samples by Sanger sequencing, and found 100% concordance between the variant calls, for NGS variants with frequencies greater than 20%. Sanger sequencing in the remaining FFPE samples is ongoing, and lower frequency NGS variants will be confirmed by other methods. We compared the validated NGS variants with the IHC results to examine the correlation of sequencing results with protein expression analysis. In our preliminary analysis of the first 18 FFPE samples, all of the samples with an IHC score of zero or one (IHC negative) had either a nonsense (n=1) or missense (n=4) variant in STK11. Conclusions: Our data suggest that accurate assessment of LKB1 status may require complementary methods including NGS and IHC methods. Citation Format: Kimberly Pelak, Jennifer Wright, Zhenyu Yan, Agus Darwanto, Weihua Liu, Peng Fang, Jin Li, Sabita Sankar, Chad Galderisi. Complementary analysis of LKB1/STK11 mutation and protein expression status using next-generation sequencing, Sanger sequencing and immunohistochemistry. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2013-3509

Abstract 3490: Highly sensitive detection of EGFR T790M on Ion Torrent PGM.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: EGFR T790M mutation leads to treatment resistance in ∼50% of NSCLC patient undergoing TKI treatment. Early detection of the emergence of this resistance mutation allows for tailoring of the treatment regimen. Detection of EGFR T790M requires higher sensitivity techniques than those needed to detect EGFR activating mutations, and quantitative measurement of T790M may also provide value in monitoring disease progression. Here we demonstrate that our proprietary amplicon next-generation sequencing (NGS) on Ion Torrent PGM provides an attractive solution for T790M detection with the advantage of high sensitivity, specificity, and quantification capability. Methods: We developed a proprietary library prep method for amplicon sequencing on the Ion Torrent PGM. Ion Torrent barcode sequences were refined in our validation to minimize barcode cross contamination. This optimization of barcodes allows for accurate mutation quantification, especially critical for variants with a frequency below 1%. This also added the benefit of higher throughput and lower cost. We employ this non-enrichment method to quantitatively measure T790M. DNA from the NCI-H1975 cell line was serially diluted into wild-type NA19240 DNA to determine the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility of the method. We also used reference FFPE DNA from HorizonDx to validate the accuracy and robustness of the assay. We further validated the test by comparing the performance of our NGS method with ddPCR using the same sample set (serially diluted H1975 into wild-type DNA controls). Results: Our data demonstrated that the LOD for T790M is 0.2%. And the method could reproducibly identify variants at these frequencies. With an input of 10ng of the HorizonDx FFPE DNA, we measured T790M frequency at 6.7%, which is concordant with the 6.5% mutation frequency reported by HorizonDx. We cross-validated our NGS assay with ddPCR, and the mutation frequencies detected by both platforms are nearly identical, and have a regression coefficient of 0.9995, with comparable LOD of 0.2%. Furthermore, our NGS assay allows us to detect other variants located in this amplicon, e.g. SNP Q787Q, with LOD at 1%. The phasing status of compound mutations can also be determined by this assay. To further improve the sensitivity, an enrichment method was developed, and the enriched T790M was sequenced by PGM. This method can semi-quantitatively measure T790M frequency with LOD of 0.03%. Conclusion: Our EGFR T790M NGS assay provides a unique option to reliably quantify T790M down to 0.2% with low DNA input. This highly sensitive and specific detection capability may enable earlier detection of emerging therapeutic resistance, particularly if FFPE results reported here extend to a more accessible specimen type, i.e. plasma circulating DNA, amenable to periodic patient monitoring. Citation Format: Agus Darwanto, Peng Fang, Zhenyu Yan, Weihua Liu, Kim Pelak, Jessica Kristof, Sabita Sankar, Cynthia Spittle, Chad Galderisi, Jin Li. Highly sensitive detection of EGFR T790M on Ion Torrent PGM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3490. doi:10.1158/1538-7445.AM2013-3490

Abstract 3229: Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods.

Introduction: The TruSeq Amplicon Cancer Panel (TSACP), a highly multiplexed targeted resequencing assay for use on the Illumina MiSeq platform, is designed for detecting the hotspot mutations in 212 Regions of Interest (ROI) from 48 cancer related genes. Here we report our validation study on the reproducibility, sensitivity and the specificity of detecting single base substitutions and small indels by the TSACP. Methods: We used well characterized cancer cell lines harboring clinically relevant variants as positive controls and HapMap samples NA12878 and NA19240 as wild type control samples. DNA from 8 cancer cell lines was serially diluted into the control DNA NA12878 for validating the Limit of Detection (LOD) of the TSACP assay. A total of 41 FFPE patient specimens representing a variety of cancer types were analyzed in a blinded fashion to evaluate the analytical sensitivity and specificity. DNA quality was assessed using a qPCR assay. With no gold standard available as a reference method to detect mutations with comparable sensitivity, concordance testing was performed using the Ion AmpliSeq Cancer Panel. Variants detected by both panels were considered as true positives. Variants that were only covered by one of the two panels were confirmed by a third method, either Sanger sequencing for variants with frequencies above 10% or a custom Ion TargetSeq Assay for variants with frequencies below 10%. Data were analyzed using MiSeq Reporter software and our proprietary analysis pipeline. In addition to reporting hotspots mutations, we also report “Critical Variants” such as non-synonymous coding mutations and splicing site mutations that fall within the ROI. Results: 95% of our ROIs were sequenced at minimum of 0.2X normalized coverage. A cell line dilution study showed that the LOD of confirmed variants is 5%. DNA extracted from 4 of the 41 FFPE specimens failed the template QC by qPCR and failed in the subsequent sequencing run. A total of 124 unique critical variants, including single base substitution, single- or multi- base (up to 21bp) deletion, one- or two- base insertion, were identified in the cancer cell lines and 37 qualified FFPE samples. The intra-assay and inter-assay reproducibility was ≥96%. Using our proprietary analysis pipeline, the analytical sensitivity and specificity for the FFPE samples were both 99%. One false negative of TSACP was identified by the Ion AmpliSeq Cancer Panel and was further confirmed by Sanger sequencing. This allele drop-off occurred as a result of the capture probe falling on a SNP. Homopolymer indels in KIT and STK11 were accurately identified with the Illumina sequencing chemistry. Conclusions: These studies demonstrate that the TSACP assay is highly specific and sensitive and is suitable for screening patient FFPE tumor specimens for a spectrum of clinically relevant somatic mutations. Citation Format: Peng Fang, Zhenyu Yan, Weihua Liu, Agus Darwanto, Kim Pelak, Kim Anoe, Cynthia Spittle, Sabita Sankar, Chad Galderisi, Jin Li. Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3229. doi:10.1158/1538-7445.AM2013-3229