PURPOSE We hypothesized that circulating tumor DNA (ctDNA) molecular residual disease (MRD) analysis without prior mutational knowledge could be performed after neoadjuvant chemotherapy to assess oligometastatic colorectal cancer (CRC) treated surgically with curative intent. We also investigated urine as an alternative analyte for ctDNA MRD detection in this nongenitourinary setting. PATIENTS AND METHODS We applied AVENIO targeted next-generation sequencing to plasma, tumor, and urine samples acquired on the day of curative-intent surgery from 24 prospectively enrolled patients with oligometastatic CRC. Age-related clonal hematopoiesis was accounted for by removing variants also present in white blood cells. Plasma and urine ctDNA MRD were correlated with tumor cells detected in the surgical specimen, and adjuvant treatment strategies were proposed based on ctDNA-inferred tumor mutational burden (iTMB) and targetable alterations. RESULTS Seventy-one percent of patients were treated with neoadjuvant chemotherapy. Tumor-naive plasma ctDNA analysis detected MRD at a median level of 0.62% with 95% sensitivity and 100% specificity, and 94% and 77% sensitivity when only considering patients treated with neoadjuvant chemotherapy and putative driver mutations, respectively. In urine, ctDNA MRD detection specificity remained high at 100%, but sensitivity decreased to 64% with median levels being 11-fold lower than in plasma ( P < .0001). Personalized ctDNA MRD oncogenomic analysis revealed 81% of patients might have been candidates for adjuvant immunotherapy based on high iTMB or targeted therapy based on actionable PIK3CA mutations. CONCLUSION Tumor-naive plasma ctDNA analysis can sensitively and specifically detect MRD in patients with oligometastatic CRC after neoadjuvant chemotherapy. Urine-based ctDNA MRD detection is also feasible; however, it is less sensitive than plasma because of significantly lower levels. Oligometastatic patients with detectable MRD may benefit from additional personalized treatment based on ctDNA-derived oncogenomic profiling.

Introduction: Molecular profiling of tumor samples is becoming routine practice in the clinic, particularly for therapy selection. However, in some cases, tumor tissue is not available for testing. Liquid biopsy, which enables the analysis of circulating tumor DNA (ctDNA) shed from the tumor into the blood, can be used as a surrogate for conventional tissue based testing to detect somatic mutations. Furthermore, ctDNA profiling can have potential applications in tumor burden monitoring. Some technical challenges must be overcome to accurately and sensitively detect the often low amount of ctDNA present in plasma. The AVENIO ctDNA Analysis Kits (Targeted, Expanded and Surveillance Kits; Research Use Only) were developed to sensitively detect all 4 major mutation classes: single nucleotide variants, insertions/deletions, fusions, and copy number variations, using a hybrid capture target enrichment workflow, molecular barcoding, and next generation sequencing. We previously presented a comprehensive study of 370+ samples demonstrating analytical performance of the assays on the Illumina NextSeq 500. As ctDNA based analysis becomes more prevalent in the clinical research community, there is great desire for high performing and cost-effective assays that are easy to implement and available across multiple sequencing platforms. Methods: We used the commercially available AVENIO ctDNA Analysis Kits (Expanded, Target and Surveillance Kits; Research Use Only) pre-sequencing workflow to process a panel of cell line blends and plasma-derived ctDNA samples in order to compare the performance of the assays on multiple Illumina sequencing platforms: NextSeq 500, HiSeq 4000 and HiSeq 2500. Ninety samples were tested across all platforms. The samples contain SNVs at 0.5% minor allele frequency (MAF), deletions at 1% MAF, selected fusions at 1% MAF, and MET amplification at 2.3 copies. Analysis was done using the AVENIO ctDNA analysis pipeline for all sequencing runs across the sequencing platforms. Results: On the NextSeq 500, the AVENIO ctDNA Analysis Kits achieve sensitivities of &gt;99% for 0.5% SNVs, &gt;99% for 1% deletions, &gt;96% for 1% fusions, and &gt;99% for 2.3 copies of MET amplification, with 96% to &gt;99% specificity for all mutations. Similar performance is observed across additional Illumina platforms, HighSeq 4000 and HiSeq 2500. Across all platforms, key sequencing metrics are consistent to achieve required sequencing depths, on-target rates, and uniformity. Also, it is noted that in order to achieve required depths, each platform requires different levels of sample multiplexing. We demonstrated that the AVENIO ctDNA Analysis Kits achieve high sensitivity and specificity across multiple high throughput sequencing platforms. We also will highlight the key performance differences and considerations when performing the assay across these sequencing platforms. Citation Format: Jonathan Choi, Richard Dannebaum, Ashla Singh, Rob Foley, Jorge Dinis, Cindy Choi, Bosun Min, Jingchuan Li, Liang Feng, Fergal Casey, Janet Jin. Performance of the AVENIO ctDNA assays across multiple high-throughput next-generation sequencing platforms [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 3648.

The discovery of circulating tumor DNA (ctDNA) and the development of highly sensitive technologies have made the utility of liquid biopsies in clinical practice more feasible. Potential applications for liquid biopsies include early cancer detection, tumor genotyping in the absence of tissue biopsies, disease monitoring, and identification of drug resistance markers. However, there is a multitude of technical challenges to accurately detect ctDNA with high sensitivity and reproducibility. Various methods have been established by research centers and companies, each with its advantages and limitations. We have developed a sensitive NGS-based liquid biopsy assay, called the AVENIO ctDNA Analysis Kits (research use only), which includes three panels targeting biomarkers for solid tumors. The AVENIO ctDNA Analysis Kits include cfDNA extraction from up to 5ml plasma input, optimized reagents and workflow for NGS library preparation and target enrichment. It is accompanied with an intuitive software and data analysis package to easily obtain results. The assay sensitively detects 4 cancer relevant mutation classes - single nucleotide variations (SNVs), insertions/deletions, copy number variations and structural rearrangements. We conducted a study with 200+ samples of plasma-derived cfDNA, cell line blends containing relevant cancer mutations, and clinical samples, with which we demonstrate the analytical sensitivity of the three distinct AVENIO gene panels (covering 17, 77 and 197 genes). We demonstrate &gt;95% sensitivity for SNVs at 0.5% mutant allele frequency; &gt;95% sensitivity for 1% indels; &gt;95% sensitivity for 1% structural variants. We also demonstrate assay robustness through multiple replicates, day-to-day, and operator-to-operator reproducibility. In conclusion, the AVENIO ctDNA Analysis Kits for solid tumors encompasses a comprehensive assay and analysis workflow aimed to provide researchers with robust, reliable, highly sensitive and ease to use liquid biopsy assays to conduct the necessary studies to demonstrate the utility of circulating biomarkers in routine clinical testing. Citation Format: Jonathan Choi, Cindy Choi, Jorge Dinis, Jingchuan Li, Sean Chien, Bosun Min, Abraham Munoz, Janet Jin, Amrita Pati, Liang Feng, Johnny Wu, Ashla Singh, Fergal Casey. Performance of a robust and sensitive liquid biopsy workflow for mutation detection in plasma [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 5677. doi:10.1158/1538-7445.AM2017-5677

Background Microsatellite instability (MSI) is a hypermutable phenotype resulting from DNA mismatch repair deficiency and is observed in up to 10-15% of early-stage colorectal cancers (CRC). It manifests as abnormal lengthening or shortening of DNA repeats at specific genomic loci. MSI-High (MSI-H) CRC patients have been shown to respond favorably towards both chemotherapy and immunotherapy and have different prognosis than MSS patients and the FDA has approved Pembrolizumab as the first cancer therapy based on a class of biomarkers rather than a cancer type. With increasing options for targeted therapy in solid tumors, being able to combine MSI testing with other cancer biomarkers for approved therapies is beneficial. We show proof of concept of a pan-cancer gene panel using an NGS-based assay and bioinformatics workflow that enables classification of MSI status along with detection of SNVs, fusions, indels and CNVs from tumor tissue samples. Unlike the gold standard PCR-based test, our method does not require a matched normal sample, thus enabling MSI detection based on tumor tissue alone. Methods Our workflow combines whole genome library preparation, hybrid capture target enrichment, high-throughput sequencing and a proprietary analysis algorithm. We expanded the number of genomic loci to improve the sensitivity of MSI detection. The analysis algorithm was trained using DNA from pure and mixed cell lines, tumor tissue and normal samples with known MSI status to classify the molecular alterations between MSI-H and MSS genotypes from sequencing reads. The algorithm leverages repeat lengths in homopolymeric MSI loci in order to predict instability of individual loci followed by aggregation using a statistical framework to make the final call on MSI status. Results The above algorithm was evaluated on a cohort of 134 stage II and stage III CRC subjects who underwent curative intent surgery. MSI status for these samples was orthogonally tested using a PCR-based MSI Analysis System (31 positives, 103 negatives) and a dMMR system. Using a pre-determined threshold, the algorithm yielded 100% sensitivity and 100% specificity on the above cohort without using matched normal tissue. A second cohort evaluated includes 47 CRC patients that are enriched for MSI positive status by virtue of their selection criteria (BRAF positive). Very high concordance on MSI status with an orthogonal method was observed. Data demonstrating high concordance with a PCR_based MSI system will be available at the presentation. Conclusions Here we present an NGS-based assay and bioinformatics workflow with robust analytical performance for MSI detection in FFPE tissue samples without a paired normal. Citation Format: Amrita Pati, Hao Wang, Hamid Mirebrahim, Seng Saelee, Joshua Lefkowitz, Sean Chien, Ashla Singh, Fergal Casey, Vera Rapoport, Xiaoju Max Ma, John Lee, Alex Lovejoy, Daniel Klass, Hans-Peter Adams. Detecting microsatellite instability in ffpe tissue from crc subjects using next generation sequencing [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 4252.

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