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Introduction: Chimeric antigen receptor T (CAR T) cells are an important emerging therapy for hematologic malignancies and are currently being investigated for other cancer types as well. CAR-T cells require a retrovirus or lentivirus to introduce the chimeric antigen receptor gene into the activated T cells. Safety measures are put in place by design to eliminate any chance to create replication competent virus. Multiple plasmids carrying specific components of the virus are used to reduce the chances of creating a replication competent lentivirus. Despite these measures, safety concerns have been posed by the FDA regarding use of a lentivirus for this treatment due to the potential for replication competent lentiviruses (RCL) to arise. FDA requires monitoring of both CAR T products as well as patients undergoing therapy. It has proven to be difficult to achieve acceptable detection sensitivity of the VSV-G sequence in a background of gDNA due to the high level of homology to many areas in gDNA. Methods: We have developed a new rapid and sensitive qPCR assay targeting the vesicular stomatitis virus G glycoprotein (VSV-G), an envelope protein on lentiviral vectors. Incorporation of the envelope gene would be required to generate a replication competent virus. The intended use of the validated assay is to detect and monitor for RCL presence in human blood samples collected during clinical monitoring. A validated reference assay targeting RPPH1 will be used to quantify the amount of amplifiable genomic DNA in each extracted DNA sample. The size of the amplicons are 66 and 87 base pairs for VSV-G and RPPH1 respectively. The VSV-G assay test includes one positive and one negative control per assay run. The VSV-G positive control is composed of plasmid containing the VSV-G target sequence spiked into a background of VSV-G negative human gDNA. The negative control is composed of negative gDNA without VSV-G plasmid. Results: Improved sensitivity without non-specific amplification was achieved by optimized primer and probe selection. Additionally sensitivity and specificity were enhanced by employing a step-down PCR protocol whereby the annealing and extension temperature is lowered in stepwise fashion over the first five cycles of PCR. Genomic DNA inputs up to 200ng per reaction showed high efficiency of amplification from 10 to 1e6 copies target sequence per reaction. Conclusion: This assay will provide a rapid and sensitive method for safety monitoring of patients undergoing CAR-T therapy with therapeutic cells generated using lentiviral vectors. Citation Format: David Burke, Kristine Drafahl, Clark Fjeld, Chad Galderisi, Cindy Spittle. Enhanced sensitivity detection of replication competent lentivirus by qPCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 896.

PD-L1 expression and MSI status are useful predictive biomarkers for checkpoint inhibitors (CIs) in some tumor types. However, studies have demonstrated that the identification of all potential patients who may benefit from CIs will require the analysis of both tumor cell and tumor microenvironment (TME) biomarkers. For example, recent data suggests that tumor mutation burden (TMB) and tumor mutation signatures as well as inflammatory signatures and T cell clonality in the tumor microenvironment may serve as independent predictive biomarkers of CI response. The need for evaluation of multiple biomarkers using IHC, PCR and/or NGS using limited amounts of tissue poses a challenge in the clinical setting. Here we conduct a study to evaluate whether a panel of tumor and TME biomarkers can be accurately analyzed using low DNA and RNA input from FFPE specimens or using plasma-derived cfDNA. Multiple methods for assessing TMB, MSI status and mutation signatures were compared. The impact of sample quality on biomarker analysis was also evaluated. A set of late stage CRC and matched plasma samples were used in this study. DNA and RNA were co-extracted from FFPE sections using RecoverAll™ kit. cfDNA was extracted from plasma using the MagMAX™ Kit. DNA and RNA yields from FFPE ranged from 855-3875ng and 477-5200ng, respectively. cfDNA yields ranged from 8-38ng. Based on the KAPA DNA QC results, the FFPE samples were determined to be high quality. TMB and mutation signature analysis of FFPE samples were performed using Oncomine Tumor Mutation Load (TML) Assay, the Oncomine Comprehensive Assay v3 (OCAv3) and WES. MSI status in FFPE tissue and cfDNA was determined using the Promega MSI Analysis System. The mutation spectrum in the matched cfDNA samples was determined using the Oncomine PanCancer cfTNA Assay. Inflammatory (IFNG) signatures were assessed using the targeted RNASeq panel, Oncomine Immune Response Research Assay (OIRRA). The clonality of TILs was analyzed using NGS. TMB results generated from the targeted panels using only 20ng DNA input were comparable to WES results. Differences in TMB counts between the NGS assays are likely due to differences in genomic coverage and data filtering. MSI-H status and/or mutations in critical DNA repair or proofreading genes such as MSH6 and POLE correlated with high TMB. Gene expression profiling using OIRRA was successful for all FFPE RNA samples. In summary, this study demonstrates that comprehensive genomic analysis of predictive biomarkers for CI therapy can be performed from limited FFPE samples using co-extraction methods and a combination of targeted NGS and PCR-based assays. DNA and RNA quality have a significant impact on the accuracy of TMB analysis and gene expression profiling. In cases where no tumor tissue is available, the analysis of biomarkers such as MSI and mutation signatures may be possible using cfDNA. Citation Format: Xiaodong Wang, Zhenyu Yan, Quyen Vu, David Smith, Chad Galderisi, Cynthia S. Spittle, Jin Li. Comprehensive genomic analysis of predictive biomarkers for checkpoint inhibitor therapy from a limited amount of FFPE tissue and cfDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1702.

FLT3 ITD, a prognostic marker and drug target, is present in ~19% of AML patients. FLT3 ITD is a tandem repeat of the entire or partial exon 13-15 region. FLT3 ITDs range in size from 3bp to above 300bp and impact RTK signaling. Recent publications have shown that FLT3 ITD can be detected at single nucleotide resolution by NGS panels. However, detection of large FLT3 ITD and accurate reporting of ITD frequency remain challenging for NGS methods. False negative ITD results or inaccurate mutant/wild type allele ratio measurements could negatively alter treatment decisions for AML patients. The Illumina TruSight Myeloid panel (TMP) is a commercial NGS assay that covers 54 genes involved in myeloid malignancies. Although the TMP assay can detect SBS and small indel, medium to large size FLT3 ITD cannot be detected. Here we present an enhanced workflow to overcome these challenges. Positive controls, dynamic range, accuracy, and inter-run reproducibility were validated for the detection of SBS, indel, and FLT3 ITD using reference standards and DNA extracted from cell lines and AML patient EDTA blood and bone marrow aspirate samples. NGS libraries were prepared following Illumina's standard protocol. 275bp X2 sequencing instead of the standard 150bp X2 was run on MiSeq flowcell v3. Data analysis was performed using a MolecularMD custom pipeline. The analysis of FLT3 ITD was conducted using three methods including two off-the-shelf programs, Pindel and ITDseek, as well as Cigar, a MolecularMD developed ITD detection algorithm. FLT3 ITDs at 126 bp and 21 bp in PL-21 and MOLM-13 cell lines, respectively, were detected by TMP assay using the three algorithms. In contrast to ITDseek which only reports ITD sequence without frequency information, Pindel and Cigar report both ITD sequence and frequency. Compared to Pindel, ITD frequency reported by the custom algorithm is closer to that determined by the fragment analysis. Serial dilution of the 126bp ITD into wild type DNA showed the 0.55% ITD (from fragment analysis) can be reproducibly detected at ~0.6% by the Cigar algorithm. FLT3 ITDs ranging in size from 6bp to 300bp were detected in clinical AML samples using the enhanced TMP workflow and confirmed by a fragment analysis method. Limit of detection, reproducibility and accuracy of SBS and indel detection in other key genes by the TMP assay have also been validated. Low-level mutations, i.e. NRAS G12D at 1.7%, NRAS G13D at 2%, and IDH2 R140Q at 1.4%, were detected by TMP panel from AML samples and further confirmed with an independent assay. A long deletion (52bp) in exon 9 of CALR was also detected by the custom pipeline. In summary, the enhanced TMP workflow allows for accurate detection and reporting of SBS, indel and FLT3 ITDs including large duplications. Optimized detection of FLT3 ITDs will improve AML patient selection for targeted therapy. Citation Format: Xiaodong Wang, Zhenyu Yan, Peng Fang, Weihua Liu, Scott Glynn, Jennifer Biroschak, Chad Galderisi, Cindy Spittle, Jin Li. An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4522.

Checkpoint inhibitors have been approved for the treatment of solid tumor and hematological 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. Tumor mutation burden (TMB) has been correlated with response to checkpoint inhibitors and is emerging as a key biomarker for predicting checkpoint inhibitor response. So far, the methods used to assess tumor mutation burden have included exome sequencing and multiple laboratory-developed targeted NGS panels (e.g., FoundationOne and MSK-IMPACT). In order to fully determine the value of TMB as a predictive biomarker for immunotherapy, a standardized panel, workflow and data analysis pipeline for TMB assessment are needed. In this study we evaluated the performance of a commercially available targeted NGS panel and workflow for TMB analysis. A set of 30 FFPE tumor samples including colon, renal, gastric, endometrial, and lung tissues was analyzed. DNA and RNA were extracted using the RecoverAll Total Nucleic Acid Isolation Kit. DNA quantity and quality were assessed using Qubit and qPCR, respectively. Samples were analyzed with the ThermoFisher Oncomine™ Mutation Load Research Assay (TML), which evaluates tumor mutation load (mutations/Mb) by interrogating 409 cancer-related genes, spanning ~1.7 megabases of the genome. TMB was measured by counting somatic single-base substitutions per Mb at ≥10% allele frequency in single, non-matched, tumor DNA samples. The impact of DNA quality on the TMB score was evaluated. Deamination errors (i.e. G>A and C>T) in poor quality FFPE samples was found to cause the overestimation of TMB. Therefore, a delta Ct cutoff was established to qualify DNA samples for TMB analysis. 12 of the 30 samples were also analyzed using a comparator NGS panel covering ~1.25 megabases. The correlation of TMB results between the two panels was 0.87. Overall, TMB was lowest in RCC (9-17/Mb) compared to NSCLC and CRC (16-37/Mb). MSI status was determined using the Promega MSI Analysis System v1.2. A correlation was observed between TMB and MSI status in a subset of samples. Reproducibility of the assay was also evaluated. To identify clinically relevant mutations and genetic alteration associated with high mutation burden, the Oncomine Comprehensive assay v3 (OCAv3) was also used to analyze the sample set. Mutations in genes involved in several DNA repair pathways were found to correlate with TMB. This study demonstrated the feasibility of utilizing a commercial targeted NGS panel and data analysis pipeline for TMB evaluation in clinical FFPE tumor samples. Standardization of TMB analysis will enable the clinical validation of TMB as a predictive biomarker for therapy selection. Citation Format: Peng Fang, Zhenyu Yan, Quyen Vu, David Smith, Chad Galderisi, Cynthia S. Spittle, Jin Li. Evaluation of a commercial targeted NGS panel for tumor mutation burden assessment in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3614.

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.

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

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

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

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

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

Background: PTEN is a tumor suppressor that negatively regulates the PI3K signaling pathway by dephosphorylating PIP3, converting it to PIP2. PTEN loss of function results in activation of Akt and downstream signaling pathways, and has been associated with numerous cancers, most commonly endometrial cancer, glioblastoma, melanoma and prostate cancer. PTEN loss can be a result of multiple different aberrations which include mutations in the coding region leading to frame-shift or stop codons, genomic deletions as well as promoter methylation leading to loss of PTEN protein. Determination of PTEN status, both at the DNA and protein levels, may be important for clinical decision-making because loss of PTEN is associated with resistance to various targeted therapies, while inactivating mutations may confer sensitivity to therapeutics targeting PI3K pathway members. We have developed Sanger sequencing, immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) assays to assess PTEN mutation status in a set of FFPE patient specimens. Methods: Our Sanger sequencing assay for PTEN was developed to detect known hotspot mutations in exons 5-8, which occur in the majority of PTEN mutations reported in the COSMIC database. The IHC and CISH assays were developed using commercially available reagents. All assays were validated using a combination of plasmids, cell lines and FFPE tissues for specificity, sensitivity, reproducibility and concordance. Results: 22 patient FFPE tissue specimens from a variety of tumor types including colon, lung, pancreas, uterus and prostate were evaluated for PTEN mutation and expression status by both Sanger sequencing and IHC methods. Sequencing results were: 9 wild-type, 12 heterozygous mutant, and 1 hemizygous mutant. 7 of the 9 specimens reported as wild-type by sequencing exhibited normal protein expression levels by IHC, while 2 were PTEN negative by IHC, suggesting that mechanisms other than mutations in exons 5-8 contributed to the loss of expression of PTEN protein. These 2 specimens were further analyzed by CISH to determine copy number. Of the 13 specimens reported as mutant by sequencing, IHC protein expression results indicated that 6 were PTEN positive and 7 were PTEN negative, underscoring the observation that exon 5-8 mutation status alone is not sufficient to predict PTEN expression levels. Data on CISH analysis of the specimen that was hemizygous mutant by sequencing will be reported to confirm the copy loss. Conclusion: The data suggest that additional mutations outside the exon 5-8 region as well as promoter methylation may result in loss of PTEN protein, and that accurate clinical assessment of PTEN mutation and protein expression status in FFPE tissues requires complimentary methods including Sanger sequencing, IHC and CISH methodologies. Citation Format: Kellen Sakrison, Jennifer Wright, Eric Bruening, Stephane Wong, Cynthia Spittle, Sabita Sankar, Chad Galderisi. Clinical assessment of PTEN mutation in FFPE tissue: comparison of Sanger sequencing, immunohistochemistry and chromogenic in situ hybridization 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 39. doi:10.1158/1538-7445.AM2013-39