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Solid tumors remain a major health concern with approximately 14 million new cases and 8.2 million cancerrelated deaths per year. Currently multimodality treatment regimens are being explored to make advances against this deadly disease. Tumors are characterized by genomic instability and tailored therapies are being considered based on genetic profiling of cancers, driving precision medicine. This review outlines the various genetic changes present in solid tumors, evaluates the technologies currently used for identification of variants and compares the large panels available for clinical utility and comprehensiveness.

Introduction: A comprehensive somatic tumor profile with associated treatment selection options requires the detection of gene fusions. After evaluating the clinical utility of multiple methods of gene fusion detection, it was determined that the Archer FusionPlex Solid Tumor Panel (AFPSTP) best compliments the JAX Cancer Treatment ProfileTM (JAX-CTPTM) clinical test in terms of workflow, specimen requirements and turnaround time. Here we describe our analytical validation process for the AFPSTP assay. Methods: AFPSTP was validated using 24 samples: 5 JAX Patient Derived Xenograft (PDX) cases, 4 translocation positive controls, 2 FFPE cancer samples, 1 normal tissue sample, and 12 cell lines. Nine of the cell lines were previously identified as positive for fusion transcripts and 3 lacked detectable fusion events. The validation was executed in 5 phases: (1) confirm that AFPSTP was able to detect known fusion or lack of fusion events in characterized specimens; (2) determine inter-assay concordance; (3) determine intra-assay concordance; (4) LOD and (5) sensitivity. Results: The fusion detection results for this validation are listed in Table 1. All but one of these fusion events was previously identified. The one novel fusion was confirmed using TaqMan RT-PCR. In addition to the expected fusions, 4 false positive events were detected, 2 due to mispriming and 2 determined to be WT read through transcripts. The fusion detection inter and intra-assay concordance was found to be 100% and the sensitivity was calculated to be 91.67% at a LOD of 5%. Conclusion: This analysis outlines the clinical validation of the incorporation of AFPSTP into the JAX-CTPTM test system. Once incorporated, the AFPSTP assay will accomplish the goal of making JAX-CTPTM a more comprehensive somatic tumor profiling assay without affecting the current acceptable turnaround time or required input material. List of 15 samples that were found to be fusion positive and the corresponding detected fusion.HorizonDx EML4/ALK PositiveEML4 → ALK variant 1HorizonDx RET PositiveCCDC6 → RETHorizonDx ROS PositiveSLC34A2 → ROS1HorizonDx Triple PositiveEML4 → ALK variant 3bHorizonDx Triple PositiveSLC34A2 → ROS1HorizonDx Triple PositiveCCDC6 → RETA673 Cell LineEWSR1 → FLI1VCaP Cell LineTMPRSS2 → ERGKM-12 Cell LineTPM3 → NTRK1RPMI-2650 Cell LineBRD4 → NUTM1NCI-H716 Cell LineFGFR2 → COL14A1OCI-AML2 Cell LineMBNL1 → RAF1REH Cell LineETV6 → RUNX1MDA-MB-175-VII Cell LineTENM4 → NRG1ASPS-1 Cell LineTFE3 → ASPSCR1ASPS-1 Cell LineASPSCR1 → TFE3PDX1EML4 → ALK 3bPDX2SYN2 → PPARG Citation Format: Samantha Helm, Aleksandra Ras, Vanessa Spotlow, Kevin Kelly, Susan Mockus, Cara Statz, Guruprasad Ananda, Joan Malcolm, Gregory J. Tsongalis. Validation of the Archer FusionPlex solid tumor panel in the JAX cancer treatment profileTM. [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 3630.

Introduction In the constantly changing field of oncology precision medicine, it is exceedingly important to keep diagnostic and therapeutic assays clinically relevant. Next generation sequencing (NGS) panels in oncology are greatly impacted by new findings in clinical actionability. In order to ensure that cancer panels continue to provide the most beneficial results to patients, they must be regularly updated. In keeping with this idea, JAX has launched a new 212 oncology gene panel which focuses on genes and variants with documented actionability, referred to as ActionSeq. Methods Development of ActionSeq included the optimization of a new targeted capture assay. This process included running multiple batches of samples through the assay to determine appropriate DNA input, ligation times, PCR cycles, and pooling conditions. The fully optimized assay was then validated using 24 uncharacterized FFPE samples. The validation was executed in 5 phases: (1) confirm that assay optimizations yielded sufficient wet lab results; (2) LOD & sensitivity (3) inter-assay concordance; (4) intra-assay concordance; (5) specificity and accuracy. Results During development, the standard protocol was optimized using a 200ng input, 30 minute ligation period, 5 cycles of pre-PCR, and the pooling of 4 samples per hybridization reaction. Wet lab processing results of the first validation batch can be seen in Table 1. The inter- and intra-assay concordances were found to be ≥ 96% for variants and 100% for CNVs. The sensitivity was calculated to be 98.92% at a LOD of 3% for SNVs, 100% at a LOD of 8% for INDELs, 100% at a LOD of 6 copies for CNV amplifications, and 100% at a LOD of 0 copies for CNV deletions. The specificity and accuracy were found to be 100% for all mutation types. Conclusion Based on the success of this validation ActionSeq has been incorporated into the JAX clinical test menu. This addition accomplished the goal of providing a more clinically relevant (actionable) somatic tumor profiling assay to patients and clinicians. Table 1.This table displays the wet lab processing results for the 24 samples used to confirm the efficiency of the developmental optimizations made to the assay prior to beginning the clinical validation.SamplePost Prep ng/ulCapture Pool ng/ulAverage Base Pair SizeMean Target CoveragePercent Duplication1271.4130951123%2241.4130959423%3241.5230265922%4291.5230265221%5212.2229053516%6352.2229056013%7292.2329953017%8722.58304119614%9652.5830473214%10432.5830457113%1142230372218%12372.030361116%13272.030373518%14421.930973120%15401.930968319%16671.930967516%17481.930957320%18531.8930165519%19311.8930176322%20821.8930176117%21561.8930187719%22341.9932977118%23481.9932988616%24781.9932984115%Established Quality Control Cut Offs:Post Library Prep ng/ul:≥ 20Post Capture Pool ng/ul:≥ 0.5PC Average Base Pair Size:250-350Mean Target Coverage:≥ 500Percent Duplication:≤ 25% Citation Format: Samantha Helm, Vanessa Spotlow, Aleksandra Ras, Kevin Kelly, Guruprasad Ananda, Sara Patterson, Honey V. Reddi. Development and validation of the ActionSeqTM test 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 757. doi:10.1158/1538-7445.AM2017-757

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