Grace Q. Zhao

Abstract A035: CRC MRD detection using Accu-ActTMNGS technology

Circulating tumor DNA (ctDNA) is a promising biomarker for detecting minimal residual disease (MRD) and for monitoring treatment of patients with colorectal cancers (CRC). Any technology used for this purpose, however, will face extreme performance demands. In order to build a high-performance multiplex next-generation sequencing (NGS) platform suitable for cancer MRD using ctDNA, we developed Accu-Act TM , an NGS-based assay capable of detecting low-frequency variants in plasma ctDNA with high precision. In our protocol, rolling-circle amplification is used to circularize denatured double-stranded cell-free DNA (cfDNA) and convert it into long tandem repeats, thus enabling consensus-based concatemer error correction. We demonstrated Accu-Act TM ’s sensitivity and specificity by testing it on cfDNA samples with known variant frequencies and cfDNA collected from healthy individuals (n = 100). Our results showed that the sensitivity of Accu-Act was 0.1% with an error rate of 1 in 1 million for 20ng of input cfDNA. Concordance analysis was performed using Accu-Act, a 61-gene assay, on 152 tumor/plasma pairings of preoperative samples derived from patients with CRC (stage I-IV). Depending on stage, we report 66-92% patient detection rate. Post-surgery ctDNA profiling was performed on 52 patients (stage I-IV) enrolled in our prospective MRD study. The results showed that 26% of patients had detectable postoperative ctDNA, among whom 72% had disease progression within two years. Only one out of the 41 patients without detectable postoperative ctDNA went on to relapse, and one patient died of a lung infection. Our study showed that ctDNA is a promising prognostic biomarker for CRC relapse after R0 resection (HR (95%CI) 33.00 (4.05 - 270), P TM NGS-based ctDNA assay has high accuracy and is suitable for MRD in CRC patients. Accu-Act TM should make a significant contribution in the development of personalized cancer treatment. Citation Format: Xinxing Li, Grace Zhao, Xianwen Zhang, Yanping Sun, Yi Wang, Canping Ruan, Paul Tang, Malek Faham, Shengrong Lin, Kang Ying, Zhiqian Hu. CRC MRD detection using Accu-Act TM NGS technology [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 A035.

Abstract 2742: Cross-platform comparison of four leading technologies for detectingEGFRmutations in circulating tumor DNA from plasma of patients with non-small cell lung carcinoma

Analysis of circulating tumor DNA (ctDNA) is emerging as a powerful tool for guiding targeted therapy and monitoring tumor evolution in patients with non-small cell lung cancer (NSCLC), particularly when fresh tissue biopsy is not available. This study compared the ability of four leading technology platforms to detect epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, T790M and G719X) in ctDNA from NSCLC patients. The platforms included two amplification refractory mutation systems (cobas-ARMS and ADx-ARMS), a droplet digital polymerase chain reaction platform (ddPCR) and a next-generation sequencing platform (Firefly NGS). Fifteen EGFR mutations across twenty NSCLC patients were identified. We observed superior sensitivity and specificity of cobas-ARMS, ddPCR and Firefly NGS platforms, while ADx-ARMS was only suitable for the qualitative detection of EGFR mutations with allele frequency higher than 1% in plasma samples. We observed high concordance between the plasma and tissue EGFR mutational profiles for three driver mutations that are known targets of the first generation EGFR-TKI therapy (L858R, E19-dels, and G719X). Discrepancies between plasma and tissue EGFR mutational profiling could be attributed to spatial and temporal tumor heterogeneity. This pilot study illustrates the promise of ctDNA analysis in the context of treatment evaluation and drug resistance detection, and results will be validated in follow-up studies. Citation Format: Ting Xu, Xiaozheng Kang, Xiaofang You, Dai Liang, Dequan Tian, Wanpu Yan, Yongbo Yang, Hongchao Xiong, Zhen Liang, Grace Q. Zhao, Shengrong Lin, Ke-Neng Chen, Guobing Xu. Cross-platform comparison of four leading technologies for detecting EGFR mutations in circulating tumor DNA from plasma of patients with non-small cell lung carcinoma [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 2742. doi:10.1158/1538-7445.AM2017-2742

Abstract 1375: An ultra-sensitive cell free DNA liquid biopsy assay for cancer treatment monitoring

In order to take advantage of the narrow time window for optimal treatment efficacy, highly sensitive disease monitoring is critical in the successful management of cancer. Currently, treatment efficacy is assessed by using a combination of protein cancer biomarkers and imaging. However, both methods present limitations with regard to specificity or sensitivity due to their dependency on tumor size. Recently, a number of studies have suggested that monitoring cell free DNA (cfDNA) may provide a more specific alternative for tracking cancer treatment with greatly improved sensitivity. Here we introduce a novel next-generation sequencing based mutation detection system aimed at improving the sensitivity, reliability, and clinical utility of cancer treatment monitoring. Our system, comprised of Nebula, a whole genome amplification technology that is capable of amplifying nanogram quantities of cfDNA >1000-fold, and Firefly, a proprietary technology combining molecular biology and computational algorithm for error-suppression, has reduced the rate of random sequencing errors to 10-6. As a result, we are able to detect 1.5 variant copies from 10ng of input cfDNA with a detection rate of 46%. We have validated the Nebula-Firefly assay on a patient cohort with either colorectal (CRC) or lung cancer. Here we report the successful detection of drug resistant mutations and various genomic alterations associated with minimal residual detection (MRD) in sample cfDNA. These initial findings have led to the exploration of Nebula-Firefly as the technological backbone for a noninvasive, scalable approach for the early detection, treatment, and monitoring of cancer. Citation Format: Grace Zhao, Li Weng, Paul Tang, Johnny Sun, Yi Huang, Lingchen Guo, Hongyan Wang, Xiaozheng Kang, Wei Shen, Kang Ying, Shengrong Lin. An ultra-sensitive cell free DNA liquid biopsy assay for cancer treatment monitoring. [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 1375.

Abstract 2788: Comprehensive high-depth target sequencing in circulating tumor DNAs of patients with inflammatory and non-inflammation breast cancers

Inflammatory breast cancer (IBC) is an extremely aggressive form of locally advanced breast cancer that affects about 5% of breast cancer patients. The prognosis of IBC patients is remarkably poor, with a three-year survival rate of approximately 30% compared to 60% for patients with non-IBC breast cancers. These facts highlight the importance of accurate characterization, early detection, and timely treatment of IBC patients. Thus, it is important to develop novel and clinically applicable non-invasive biomarkers to characterize the unique presentation of IBC. In this study, we searched for somatic mutations in the circulating tumor DNAs (ctDNAs) that could be used to non-invasively characterize IBC patients and inform their clinical management. Using ctDNAs extracted from plasma of 10 pairs of IBC and non-IBC patients that were matched on major demographic and clinical variables, we conducted a high-depth target next-generation sequencing study that interrogated a comprehensive panel of 127 TCGA (The Cancer Genome Atlas)-reported cancer-related genes with >7000 uniquely designed and validated probes. Overall, we obtained >500x coverage in >80% of the interrogated regions, and >100x coverage in >97% of the regions. We found that C>T mutations predominated in well-reported mutated genes such as TP53, PIK3CA, EGFR, and CDH1. Compared to non-IBC patients, IBC patients appeared to have a higher percentage of mutations in PIK3CA but a lower percentage in TP53. Interestingly, about 78% of mutated genes that were only detected in IBC patients encode zinc finger-related proteins, a family of transcriptional factors that have been implicated in IBC development. In comparison, about 43% of genes that were detected only in non-IBC patients encode proteins important to cell division regulation. Furthermore, network-based stratification (NBS) analysis of the mutation profile revealed clusters of IBC relative to non-IBC samples, indicating the potential of mutation profiling in identifying molecularly distinct subtypes of IBC patients. Preliminary longitudinal analysis of ctDNAs from three patients with multiple plasma samples indicated that de novo mutations in important genes including PIK3CA, RB1, and KRAS appeared in patient blood after chemotherapy and/or targeted therapy treatments. Moreover, the emergence of some of these mutations was temporally correlated with the responses of patients to the treatments they received. Overall, this study provides novel evidence that ctDNA mutation status may help to non-invasively characterize IBC tumors, and might also serve as a novel non-invasive marker to monitor treatment efficacy and prognosis of breast cancer patients. Future studies with larger sample sizes are warranted to confirm our findings and identify additional clinically useful markers for the characterization and management of IBC and non-IBC patients. Citation Format: Hushan Yang, Xue Zhong, Qiang Wei, Zhaomei Mu, Zhong Ye, Yinzhi Lai, Huei-Wen Lin, Rebecca Jaslow, Tiffany Avery, Laura Austin, Zhaohui Sun, Shengrong Lin, Grace Zhao, Ling Fang Tang, Ronald E. Myers, Juan P. Palazzo, Laura Biederman, Bingshan Li, Massimo Cristofanilli. Comprehensive high-depth target sequencing in circulating tumor DNAs of patients with inflammatory and non-inflammation breast cancers. [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 2788. doi:10.1158/1538-7445.AM2015-2788