Tissue-based Next-generation Sequencing Research Articles

1481O - Clinical development of molecular-targeted therapies for non-small cell lung cancer through nationwide genome screening in Japan (LC-SCRUM-Japan)

1481O - Clinical development of molecular-targeted therapies for non-small cell lung cancer through nationwide genome screening in Japan (LC-SCRUM-Japan)

Abstract 2275: Detection of IDH1 mutations in plasma cell-free circulating tumor DNA (ctDNA) from patients with cholangiocarcinoma

Abstract Somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene are detected in 13-15% of cholangiocarcinoma (CC) cases overall and up to ~25% of intrahepatic CC cases. Ivosidenib (AG-120) is a first-in-class, oral, potent, reversible, targeted inhibitor of the mutant IDH1 (mIDH1) protein, and AG-881 is an oral, potent inhibitor of both mIDH1 and mIDH2. Phase 1 studies for both molecules in patients with mIDH1/2 solid tumors, including CC, have completed enrollment, and analyses are ongoing (AG120-C-002 [NCT02073994]; AG881-C-002 [NCT02481154). Patients with mIDH1 CC were enrolled into the clinical studies based on local tissue testing for the IDH1 mutation. In this work, we examined the feasibility of mIDH1 detection in ctDNA from patients with mIDH1 CC, and explored the correlation with detection by tissue-based next-generation sequencing (NGS) assays and with plasma levels of the oncometabolite D-2-hydroxyglutarate (2-HG). Baseline plasma samples were collected from a total of 41 patients, including 28 from AG120-C-002 and 13 from AG881-C-002. A BEAMing digital PCR platform (Sysmex) was used for the detection and quantification of five mIDH1 alleles, including R132C, R132H, R132L, R132S, and R132G, with 0.02% analytical sensitivity (0.04% for R132H). Tumor tissues were available from 35 of 41 patients and analyzed using the FoundationOne and/or Personalis ACE cancer research targeted panel. Additionally, baseline plasma levels of 2-HG were measured and correlated with plasma mIDH1 ctDNA. Detection of mIDH1 in plasma ctDNA was concordant with IDH1 mutation status by central testing of tissue in 32 of 35 patients (91.4%), with 31 mIDH1-positive results from both plasma and tissue (88.6%). One patient was mIDH1-negative in both plasma and tumor. In all 31 mIDH1-positive cases, the mIDH1 allele detected was concordant. In general, mIDH1 variant allele frequency (VAF) was lower in plasma than in tumor tissue (mean VAF 2.9% in plasma, 26.2% in tumor). Additionally, the level of plasma ctDNA mIDH1 VAF was significantly correlated with baseline plasma 2-HG concentration (Spearman R=0.535, p=0.0009). Correlation with clinical outcome is currently being explored and will be presented. Taken together, our results demonstrate the feasibility of IDH1-R132 mutation detection in plasma from CC patients, with a 91.4% concordance rate with detection in tumor tissue. These results provide a rationale for exploring liquid biopsy-based testing methods when the feasibility of repeated biopsies or sample exhaustion limits the ability to detect actionable mutations through tissue-based NGS panels, which can be a major challenge for trial participation in this indication. Confirmatory liquid biopsy studies will be conducted with baseline and longitudinal samples collected from the ongoing phase 3 study of ivosidenib in mIDH1 CC (NCT02989857). Citation Format: Elia Aguado-Fraile, Sung Choe, Camelia Gliser, Lori Steelman, Liewen Jiang, Bin Fan, Kha Le, Maeve A. Lowery, Susan Pandya, Bin Wu. Detection of IDH1 mutations in plasma cell-free circulating tumor DNA (ctDNA) from patients with cholangiocarcinoma [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 2275.

Correlation of circulating tumor DNA (ctDNA), tissue-based genomic profiling and clinical efficacy in the biomarker directed Ph1b trial in metastatic bladder cancer (BISCAY).

4553 Background: BISCAY is a biomarker-directed Ph1b multi-arm platform study exploring the combination of targeted therapies with anti-PD-L1, Durvalumab, in advanced urothelial cancer. Methods: Next generation sequencing (NGS) of tumour tissue samples from > 380 patients(pts) was performed using the FoundationOne assay alongside IHC for PD-L1. ct DNA from pts enrolled in trial modules at treatment initiation was profiled using the Guardant Health OMNI platform assessing a panel of 500 genes. For a subset of pts, serial plasma samples were also analysed to monitor early signs of response vs. resistance and changes in ct DNA dynamics using a bespoke NGS panel of 10 genes. Results: To date 149 pts have been actively enrolled across 7 different biomarker selected and unselected treatment modules. Across all screened pts the most prevalent genomic alterations in tumour tissue were TERT promoter (65%), TP53 (59%), KMT2D 21%, KDM6A 21%, with the most common CNV CDKN2A/ B loss (32 %). All enrolled pts tested had detectable ctDNA in plasma. Similar genomic alterations, both frequency and type, were detected in both plasma ctDNA and tumour tissue with high concordance for module specific biomarkers used for patient allocation (80% (8/10) for ATM, BRCA1 and 2). Alterations in putative biomarkers predictive of response to anti-PD-L1, such as HRR/MMR alterations and high bTMB levels ( > 20mut/Mb) were observed in22% and 40% patient plasma samples, respectively. Correlations between biomarkers across modules treatment efficacy have been explored. Conclusions: All pts with advanced bladder cancer enrolled on BISCAY who were plasma profiled had detectable ctDNA; frequencies of genomic alterations (in both tumour tissue and plasma) were comparable to prior published data sets. ctDNA may be an attractive alternative to tissue-based NGS, providing comprehensive dynamic snapshots of genomic landscapes at the start and during therapy, and warrants further prospective investigation in trials.

The use of liquid and tissue biopsy genomic testing in lung cancer: A single-institution experience.

e20692 Background: Liquid biopsy is an evolving minimally invasive technique to detect cell-free DNA (cfDNA) and cfRNA mutations by next generation sequencing (NGS) in plasma. In our Thoracic Oncology Program, we compared plasma liquid biopsy and tissue-based NGS assay results. Methods: Circulogene Theranostics Personalized Gene Profile (CGP), a 50-gene plasma NGS panel with proprietary direct-on-specimen enrichment technology, and tissue Caris Molecular Intelligence (CMI) NGS cfDNA and cfRNA including microsatellite instability (MSI)/microsatellite stability (MSS) and total mutational burden (TMB) results were retrospectively compiled and compared upon diagnosis. Results: 106 non-surgical lung cancer patients (median age 65 years, range 27-88; 66 men, 40 women) underwent CGP testing. 49 patients had sufficient tissue for comparative CMI. MSI detected in 20.4% (10/49) by CGP; no tissue MSI was found by CMI (0/44). 3 out of 4 (75%) MSI detected by CGP had high TMB ≥ 10 mut/Mb by CMI. 75% MSS patients by CGP had low TMB (12/16). Comparative plasma versus tissue mutations findings: TP53 mutations 60.3% (64/106) CGP and 69.3% (34/49) CMI. CGP TP53 mutated patients, high TMB 70.5 % (20/34) by CMI; EGFR mutations 13.2% (14/106) CGP and 14.2% (7/49) CMI; KRAS mutations 2.8 % (3/106) by CGP versus 28.5% (19/49); one ROS1 by CGP missed by CMI and one ALK by CGP insufficient tissue CMI. A higher frequency of BRAF 16.9% (18/106), PIK3CA 28.3% (30/106), PTEN 22.6% (24/106), and MET 7.5% (8/106) alterations was identified by plasma/CGP than comparative tissue/CMI 6.1% (3/49), 2% (1/49), 6.1% (3/49), and 0% (0/49) respectively. Conclusions: Our findings indicate that Circulogene liquid biopsy NGS detected common mutations, including actionable variants in lung cancer, providing expanded and complementary tumor molecular biology and therapeutic information to tissue NGS.

Characterization of genomic alterations as biomarkers of immune checkpoint inhibitor (ICI) response in metastatic urothelial carcinoma (mUC).

400 Background: ICIs have expanded therapeutic options for mUC patients (pts); however, biomarkers such as PD-L1 have not served as reliable predictors of treatment efficacy. High tumor mutation burden (TMB) has been previously described as a potential biomarker for predicting ICI response in several indications, but its utility is still being explored in mUC. Here, we compare the genomic landscapes and clinical outcomes of mUC pts following ICI therapy using an investigational solid tissue-based next-generation sequencing assay to assess TMB and identify genetic correlates of ICI response. Methods: 20 pts with mUC treated with ICIs at Duke Cancer Institute were identified. Tumor samples were retrospectively evaluated with a Personal Genome Diagnostics Assay for somatic variants across > 500 genes, as well as TMB and microsatellite status. Tumor samples were also stained for PD-L1 status using the Dako 22C3 IHC assay. Deidentified clinical information was extracted from the medical record and tumor response was evaluated using RECIST 1.1 criteria. Results: Pts were grouped by overall response following ICI therapy as either responders (“R”, n = 6) or non-responders (“NR”, n = 13). Pts exhibited a wide range of TMB scores (0.7 to 30.4 mutations/Mb), with a mean TMB score of 9.60 vs. 3.87 mut/Mb in R vs NR groups, respectively; however, this difference was not statistically significant ( p = 0.284). 18 pts were evaluated for PD-L1 status, with only 2 positive samples (one in each group). Rs had significantly more mutations in histone methylation genes (KDM6A, KMT2C, and KMT2D), (67% vs 15% in NRs, p = 0.0039). FGFR3 mutations were also more frequent in R vs NR (67% vs 5%, p = 0.0339). Finally, there was a higher frequency of mutations in TP53 and BRCA1 in the NRs. Conclusions: In this mUC cohort, neither TMB nor PD-L1 correlated with response to ICI therapy. Histone modifying genes and FGFR3 mutations were more frequent in responders, whereas BRCA1 and TP53 mutations were enriched in non-responders, warranting future prospective studies to understand underlying mechanisms of ICI response and resistance in mUC.

Abstract 4272: A novel approach to differentiation of somatic vs. germline variants in liquid biopsies using a betabinomial model

Abstract Background: A recent large study found that over 2% of advanced cancer patients have unidentified germline alterations found incidentally during next-generation sequencing (NGS) for targetable somatic alterations. However, tissue-based NGS cannot definitively distinguish germline from somatic mutations without comparison to normal tissue. Because somatic variants typically occur in plasma at allele fractions 1-2 orders of magnitude lower than germline, liquid biopsy often enables differentiation of incidental germline mutations. Not uncommonly, high tumor burden or allelic imbalance from copy number variation or loss of heterozygosity complicates simplistic differentiation based on assumption of germline 50% allele frequency. We developed a novel statistical method for discriminating between somatic and germline variants in deep sequencing data, and applied to data generated from the Guardant 360 targeted cfDNA assay. Our model dynamically incorporates prior knowledge of germline SNP variants and observed allele frequencies within a sample, and gains power in larger panels like the GuardantOmni 500-gene assay. Methods: Our method uses common heterozygous SNPs to model local germline allele count behavior, and calls variants somatic if they deviate significantly from the observed germline mutant allele fraction (MAF). A betabinomial model is well suited to this problem, because it models both the mean and variance of mutant allele counts at common SNPs. This is important since simpler methods like fixed MAF cutoffs or Poisson models may not represent the variance in molecule counts appropriately. Results: We evaluated our method against variants from 361 clinical samples that were manually annotated by reviewers with expertise in cancer genomics. Of the 11,679 heterozygous variants considered in these samples, 7,221 (62%) had two or more common SNPs in the same gene, so that a local germline heterozygous MAF could be estimated and the betabinomial model could be applied. Using the manual annotation as ground truth, we created ROC curves for a classifier based on the betabinomial model with varying p-value cutoffs, as well as a classifier based on MAF cutoffs. The AUC for the betabinomial model was 0.996, as compared with an AUC of 0.985 for MAF cutoffs. The ROC curve allowed us to choose a betabinomial p-value cutoff that maintained a similar level of specificity as a 15% MAF cutoff (99.2% for MAF cutoff, 99% for betabinomial), but with significantly increased sensitivity (89% for MAF cutoff, 97% for betabinomial). Conclusions: We have developed a novel method for differentiation of somatic versus germline variants in a single plasma sample. Identifying incidental germline alterations in advanced cancer patients may identify new opportunities to apply targeted therapy such as PARP inhibitors for BRCA1/2, and may also serve to alert physicians and their patients to familial risk. Citation Format: Tracy Nance, Elena Helman, Carlo Artieri, Jennifer Yen, Thomas P. Slavin, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. A novel approach to differentiation of somatic vs. germline variants in liquid biopsies using a betabinomial model [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 4272.

Rapid, ultra low coverage copy number profiling of cell-free DNA as a precision oncology screening strategy.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization.

P2.03b-093 Validation and Performance of a Standardized ctDNA NGS Assay across Two Laboratories: Topic: Biomarkers

Molecular profiling of tumors using circulating tumor DNA (ctDNA) in the blood of cancer patients, as a liquid biopsy, is rapidly becoming established as a useful source of information to aid clinical decision-making when a solid tumor biopsy is not available, or is limited in amount or quality. DNA alterations are often found in a small fraction of the total cell free DNA in plasma, and their detection requires specially designed assays that are sensitive and reproducible. Individual hotspot mutations can be assayed using technologies such as droplet/digital PCR, but multiplexing such assays is limited by the small amount of clinical material. This can be addressed by assays based on next generation sequencing (NGS), to create sensitive panels for ctDNA analysis. For clinical application, it is essential that such NGS assays be standardized and reproducible, both intra-and inter-laboratory. Standardization for tissue-based NGS assays has only recently been implemented, after much discussion. We describe a strategy for validation and standardization of a high sensitivity NGS-based ctDNA assay between two laboratories, based in the US and UK. The enhanced TAm-Seq® assay (eTAm-Seq™) uses efficient library preparations and bespoke algorithms to identify cancer mutations within a panel of 34 genes, covering cancer hotspots as well as entire coding regions of selected genes. To ensure this complex process is standardised and controlled, a high level ISO and CLIA quality management system is implemented. To perform analytical validation of this assay, we used reference standards and plasma controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We compared performance of the assay between two laboratories, finding a high rate of concordance and reproducibility. Using DNA quantities typical of those found in up to 4ml of plasma from cancer patients, our assay provides high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Standard dilution curves of well-characterized reference samples show that the accuracy of the eTAm-Seq® assay is predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. Additional data with associated clinical data will be presented at the meeting. Our data demonstrates eTAm-SeqTM assy's robustness and performance in two labs, supporting its use as a basis for clinical applications globally, allowing a high degree of standardization and comparability for molecular profiling of tumors using liquid biopsy.

A pilot study evaluating concordance between blood-based and patient-matched tumor molecular testing within pancreatic cancer patients participating in the Know Your Tumor (KYT) initiative.

Recent improvements in next-generation sequencing (NGS) technology have enabled detection of biomarkers in cell-free DNA in blood and may ultimately replace invasive tissue biopsies. However, a better understanding of the performance of blood-based NGS assays is needed prior to routine clinical use. As part of an IRB-approved molecular profiling registry trial of pancreatic ductal adenocarcinoma (PDA) patients, we facilitated blood-based NGS testing of 34 patients from multiple community-based and high-volume academic oncology practices. 23 of these patients also underwent traditional tumor tissue-based NGS testing. cfDNA was not detected in 9/34 (26%) patients. Overall concordance between blood and tumor tissue NGS assays was low, with only 25% sensitivity of blood-based NGS for tumor tissue NGS. Mutations in KRAS, the major PDA oncogene, were only detected in 10/34 (29%) blood samples, compared to 20/23 (87%) tumor tissue biopsies. The presence of mutations in circulating DNA was associated with reduced overall survival (54% in mutation-positive versus 90% in mutation-negative). Our results suggest that in the setting of previously treated, advanced PDA, liquid biopsies are not yet an adequate substitute for tissue biopsies. Further refinement in defining the optimal patient population and timing of blood sampling may improve the value of a blood-based test.

Abstract B140: Genomic profiling of over 5,000 consecutive cancer patients with a CLIA-certified cell-free DNA NGS test: Analytic and clinical validity and utility

Abstract Background: Analysis of cell-free circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) enables non-invasive real-time testing. The Guardant360® (G360) ctDNA panel includes all NCCN-recommended solid tumor genomic targets and sequences complete exons of 68 genes for single nucleotide variants (SNVs), and fusions, amplifications and indels for a subset. We conducted updated validity studies for this larger panel. Methods: Analytical sensitivity was assessed via serial dilution studies of cell-free DNA (cfDNA) with known alterations spiked into healthy cfDNA background. SNVs detected with G360 were compared to whole-exome sequencing at an outside lab for analytic specificity. Tissue NGS for fusions, SNVs, indels, and/or amplifications was compared to G360 results. Consecutive G360 tests over 12 months were analyzed for assay success rate, yield, mutant allele fractions (MAF), and actionability. Results: Serial dilution studies confirmed the previously reported 0.1% lower limit of detection. The accuracy and analytic specificity studies were 98.6% concordant, with 3 putative false positives. Sanger sequencing by a 3rd lab for these discordant calls confirmed the G360 calls as true positives, verifying the previously reported &amp;gt;99.9999% specificity. Retrospective review of outside tumor test reports of 56 patients with varied genomic alterations found 90% concordance with G360 (77/86 variants: 4/4 fusions, 9/9 indels, 13/17 amps, 51/56 SNVs). Of 5,491 tests, 99.5% were successfully reported with somatic alterations found in 76% [median MAF 0.42% (range 0.1-92.8%)]. Lung, breast and colon cancers were most common (see Table). Overall, 10% of patients had &amp;gt;1 on-label matched therapy identified, 54% off-label, and 77% had clinical trial options. On-label matched therapy rates rose for lung adenocarcinoma (15%), breast (16%) and colon (16%) cancers (Table 1). Conclusion: The updated G360 test achieves single-molecule sensitivity to 0.1% MAF and analytical specificity of &amp;gt;99.9999%, as reported previously. Clinical concordance of plasma to tissue-based NGS across all four types of genomic alterations was high, and actionability rose modestly, especially in lung, breast and colon cancer. Clinical utility included biopsy avoidance, rescue of tissue samples with insufficient DNA and tissue-false negatives due to tumor evolution and heterogeneity. Table 1Cancer Type% of 4,888 unique patients% with ctDNA alterations identified% with on-label therapy options identified% with off-label therapy options identified% with clinical trial options identifiedMost commonly involved gene2nd most commonly involved gene3rd most commonly involved geneLung - ALL33%82%10%52%78%TP53 (55%)KRAS (18%)EGFR (18%)Lung adenocarcinoma11%82%15%56%78%TP53 (51%)EGFR (23%)KRAS (19%)Breast15%74%16%58%79%TP53 (43%)PIK3CA (28%)ERBB2/ESR1 (10%) / (9%)CRC12%82%16%72%88%TP53 (61%)APC (38%)KRAS (38%)PDAC7%70%3%57%72%TP53 (47%)KRAS (42%)PIK3CA (6%)Ovarian3%81%1%42%81%TP53 (69%)PIK3CA (15%)KRAS (11%)CUP3%85%0%64%83%TP53 (40%)KRAS (24%)PIK3CA (14%)All100%76%10%54%77%TP53 (50%)KRAS (17%)PIK3CA (14%) Citation Format: Kimberly C. Banks, Stefanie A. W. Mortimer, Oliver A. Zill, Richard B. Lanman, Helmy Eltoukhy, AmirAli Talasaz. Genomic profiling of over 5,000 consecutive cancer patients with a CLIA-certified cell-free DNA NGS test: Analytic and clinical validity and utility. [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 B140.