Next-generation Sequencing Liquid Biopsy Research Articles

Abstract 5038: Validation of a comprehensive next-generation sequencing liquid biopsy assay for clinical diagnostics and clinical trial applications

Abstract Liquid biopsy next generation sequencing (NGS) provides a powerful non-invasive approach to detect solid tumor derived somatic variants in a massively parallel manner. Liquid biopsy NGS has rapidly become an invaluable choice of testing technologies for clinical utility where invasive tissue biopsy is inaccessible and/or inadequate for molecular characterization. We present here a targeted NGS gene panel assay designed and validated specifically for liquid biopsy characterization. The assay was designed to test cell free DNA and cell free RNA from blood plasma for detection of single nucleotide variants (SNV), insertion/deletion (Indel), copy number variants (CNV), and gene fusions present in 52 most commonly mutated genes in cancers. The validation was performed to assess assay accuracy, specificity, sensitivity, repeatability, and reproducibility, with including pre-characterized reference controls, retrospective plasma samples from cancer patients, and prospectively-collected clinic blood specimen. Triplicates were included and testing was performed at different times and by different operators to assess the assay precision. Over 100 clinic blood specimens were collected in Streck cfDNA tubes from multiple trial centers, including treatment naïve phase III-IV patients with solid tumor (lung cancer, breast cancer, brain cancer, prostate cancer, kidney cancer, melanoma, etc) as well as heathy donors, processed within 7 days for plasma separation and total nucleotide acid (TNA) extraction, and assessed by the NGS assay for mutation detection. The identified mutations and wild-types were verified with orthogonal ddPCR. Assay sensitivity as low as 0.1% for SNV and Indel, 1.3 fold change for CNV, and 10 copies for gene fusion were observed, with near-perfect assay specificity (>99.99%) at these sensitivity levels. In conclusion, a targeted NGS liquid biopsy assay was analytically validated under medical oversight, with a demonstrated rigor of the test by its high sensitivity/specificity and robust reproducibility. The validated assay has been utilized for clinical diagnostics and to support research and development efforts in clinical trials. Citation Format: Xin-Xing Tan, Tricia Peters, Meredith Berry, Kimberly Barber, Kristyn Jeter, Brigitte Lovell, Paula Elizondo, Yanglong Mou, Brad Thomas, Heng Xie, Jiannan Guo. Validation of a comprehensive next-generation sequencing liquid biopsy assay for clinical diagnostics and clinical trial applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5038.

Abstract 3671: Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants

Abstract For in vitro Diagnostic use only. Not available in all regions including the United States. Introduction Minimally invasive blood-based liquid biopsy using cell-free total nucleic acid (cfTNA) and next generation sequencing (NGS) has substantially evolved in the field of clinical diagnostics for oncology, for detection of molecular therapeutic targets in non-small cell lung cancer (NSCLC). Here we describe analytical validation results for cfTNA using the CE-IVD Oncomine™ Dx Express Test (ODxET) assay, a qualitative in vitro diagnostic test that uses targeted NGS technology, the Ion Torrent Genexus™ Dx Integrated Sequencer to detect SNV, indel and copy number gain present in 42 genes and fusions in 7 genes from cfTNA isolated from NSCLC plasma samples. Methods Plasma samples from NSCLC patients were screened for variants of interest on several important gene loci. Screening involved isolation of cfTNA from K2-EDTA-derived plasma using the Genexus™ Cell-Free Total Nucleic Acid Purification kit on an automated purification system and sequencing was performed on the Genexus™ Dx integrated sequencer using the ODxET assay. Plasma samples having clinically relevant variants from the set of screened plasma samples were used to perform the validation studies. Results Analytical sensitivity and specificity of NSCLC plasma samples were characterized through determination of limit of detection (LOD) and limit of blank (LOB), respectively. The LOD level tested for DNA SNVs, and indels at 5 ng (minimum) input level ranged from 0.62% to 1.82% allelic frequency (AF), while 30 ng (maximum) input shows the LOD level ranging from 0.23% to 0.42% AF. The LOD for the tested RNA fusions ranged from 4.2 to 19.6 molecular counts for 5ng input and 4.9 to 8.0 molecular counts for 30ng input. Furthermore, LOB for the ODxET assay was determined by testing wild type (WT) cfTNA extracted from 30 healthy donor blood plasma samples, which were confirmed to be negative at all variant locations. The analytical accuracy study shows the false positive rate at 0.20% for SNVs, 0% for indel locations and fusion targets. In addition, the analytical reproducibility study shows 99.64% intra-run repeatability call rate for DNA variants and 98.75% for RNA variants. Conclusion In conclusion, we establish that the ODxET assay on Ion Torrent Genexus Dx Integrated Sequencer, is fast and efficient while demonstrating high sensitivity, specificity, and reproducibility for testing NSCLC liquid biopsy samples. Citation Format: Madhuri Jasti, Swasti Raut, Diarra Hassell, Nader Ezzedine, Daniela Garcia, Stephen Wunsch, Nick Siepert, Elliott Martinez, Emilia Ostrowska, Jeff Schagemann, Jian Gu, Thilanka Jayaweera, Luming He, Angie Cheng. Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3671.

Abstract 5018: Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications

Abstract “For research use only. Not for diagnostic procedures.” Introduction: Liquid biopsy is a non-invasive alternative to tissue biopsies for cancer profiling using cell-free total nucleic acids (cfTNA; including cell free DNA (cfDNA) and cell free RNA (cfRNA)), the biological analytes derived from tumor cells floating in blood. However, it is crucial to optimize the whole blood fractionation workflows, since limited access to a suitable centrifuge at blood collection sites poses a challenge to this critical fractionation step, causing delays in processing the blood, leading to decreased sensitivity of variant detection and increased occurrence of false positives. Current study shows a systematic comparison of the effects of 8 different blood fractionation conditions for spin speeds, temperatures and duration on the levels of cfTNA derived from K2-EDTA-derived blood from multiple healthy and non-small cell lung cancer (NSCLC) donors, using next generation sequencing (NGS). Methods: CfTNA from K2-EDTA derived plasma was isolated using the MagMAXTM Cell-Free Total Nucleic Acid kit and the Genexus™ Cell free total nucleic acid kit, utilizing the KingFisher TM Flex and GenexusTMpurification systems. Yield and integrity of cfTNA isolated was evaluated by Qubit™, Agilent Tapestation and quantitative real time PCR (qRT-PCR). Furthermore, the impact of these test conditions on quality control specifications of DNA and RNA was assessed by performing sequencing on an Ion TorrentTM GenexusTM sequencing instrument using the AmpliSeqTM HD target amplification assay, that reports sequence variations in DNA for 42 cancer related genes and RNA in 7 cancer related genes. Results: Results showed comparable performance for all the 8 blood fractionation conditions tested, for yield and cfDNA profiles compared to control condition, indicating that there is no significant impact of these conditions on either yield or integrity of the cfDNA isolated. In addition, NGS data also showed that on target reads and median molecular coverage for DNA are comparable for all the conditions tested. However, sequencing data demonstrated that one test condition showed significantly better performance for RNA quality control specifications in terms of RNA mapped reads and molecular coverage for process controls used in the assay, compared to all other conditions tested. Conclusion: Data suggests that one of the whole blood fractionation conditions tested impact the cfRNA recovery from plasma without negatively affecting the yield, and integrity of isolated cfDNA and shows increased sensitivity for RNA process controls in NGS. Citation Format: MADHU JASTI, Sergio Hernandez, Nick Siepert, Lokesh Kodandaramaswamy, Thilanka Jayaweera, Jeoffrey Schageman, Angie Cheng, Susanne Lopez, Miao Der Chen. Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5018.

Abstract 5030: Evaluation of different blood collection tubes for liquid biopsy NGS applications

Abstract Intro: Liquid biopsy is fast becoming a standard practice in oncology research due to recent advancements in Next Generation Sequencing (NGS). To detect genetic variants, whole blood samples are collected in blood collection tubes (BCTs) and fractionated to obtain plasma which is then used to isolate cell free nucleic acid (cfNA) for analysis. While the limited availability of cfNA is a challenge in liquid biopsy, storage condition and duration between blood collection and fractionation have a significant impact on the quantity and quality of the cfNA due to degradation and genomic DNA and RNA contamination. There are several BCTs available on the market offering stabilization of cfNA profiles to overcome the above challenges. Methods: Here we compare the NGS performance of cfNA derived from three commonly used BCTs on the market, BD Vacutainer™ K2EDTA Tubes, Streck™ Cell-Free DNA, and PAXgene® Blood ccfDNA tubes. Matched whole blood samples collected in the above-mentioned tube types were obtained from commercial vendors which were derived from 38 healthy donors as well as 6 cancer research samples. Whole blood samples were fractionated as per manufacturer recommendations and cfNA was isolated and quantified using Genexus™ Cell-Free Total Nucleic Acid Purification Kit on Genexus™ purification system. Isolated cfNA was then sequenced using Ion Ampliseq™ HD target amplification assay on Genexus™ Integrated sequencer. Results: Data shows that K2EDTA collected samples contain slightly higher amounts of cfNA compared to PAXgene® and Streck™ samples. When the cfDNA profiles were analyzed by Agilent™ BioAnalyzer™, K2EDTA derived cfDNA showed varying degrees of genomic DNA contamination which can inflate the cfDNA concentration. However, both Streck™ and PAXgene® derived cfDNA showed little to no detectable genomic DNA contamination. As a result, a higher percentage of PAXgene® and Streck™ samples did not meet the minimum input requirement (5ng) for the sequencing assay. Sequencing data for DNA showed that K2EDTA samples had higher total reads and median molecular coverage compared to the other two BCT types. However, all samples had passing QC metrics, and mean read length, on target reads, and uniformity were comparable between the three tube types. When DNA variant detection was evaluated using cancer research samples, no significant difference was observed in performance between K2EDTA and PAXgene® samples. Interestingly, sequencing data for RNA shows that PAXgene® samples had slightly higher mapped reads on average, and significantly higher internal process control coverage compared to both K2EDTA and Streck™ samples. Whereas Streck™ samples had significantly lower internal process control coverage. Conclusion: In summary, this study demonstrates the advantages and disadvantages of using different BCTs for liquid biopsy applications, in terms of cfNA yields, isolated DNA profiles, and sequencing performance. Citation Format: Nicholas Siepert, Emilia Ostrowska, Luming Qu He, Madhuri Jasti, Kris Lea, Thilanka Jayaweera, Angie Cheng. Evaluation of different blood collection tubes for liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5030.

Abstract 6132: Exploring genomic complexity in lung cancer at the Tijuana/California border: A real-world perspective through liquid biopsy comprehensive genomic profiling

Abstract Objective: This study explores the application of liquid biopsy with next-generation sequencing (NGS) in profiling the genomic landscape of lung cancer patients from the Tijuana/California Border region. Specifically, we employ blood-based comprehensive genomic profiling (CGP) to assess the complexity of the genomic profile in these patients. Materials and Methods: From April 2019 to November 2023, 37 lung cancer patients from Tijuana underwent profiling using the FoundationOne®Liquid CDx (F1LCDx®) assay, focusing on circulating tumor DNA (ctDNA). The assay, which spans 324 cancer-related genes and genomic signatures, provides insights into tumor fraction (TF) and blood-based tumor mutational burden (bTMB). Variants were classified based on the ESMO Scale of Clinical Actionability for molecular Targets (ESCAT). Results: A notable 88.6% of patients exhibited at least one detectable alteration in plasma. The most frequently mutated genes were TP53 (51.3%), EGFR (16.2%), DNMT3A (13.5%), and KRAS (10.8%). Tumor mutational burden (TMB) was assessable in 9 out of 37 patients, with values ranging from 0 to 10 mut/Mb. According to ESCAT classification, 63.1% of samples revealed potentially actionable alterations (Tier I-II), while an additional 24.1% harbored alterations for which approved drugs are available in other cancer types (Tier III). Conclusion: Liquid biopsy NGS emerges as a viable and valuable strategy for guiding personalized therapy. Given restricted accessibility to new drugs or clinical trials in Mexico, the use of blood-based CGP may enhance the identification of druggable alterations, thereby increasing the likelihood of accessing targeted drugs or enrolling in clinical trials. Citation Format: Jorge Alberto Guadarrama-Orozco, Jennifer Ramirez-Puente, Elizabeth Dominguez-Gonzalez. Exploring genomic complexity in lung cancer at the Tijuana/California border: A real-world perspective through liquid biopsy comprehensive genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6132.

Explorative cost-effectiveness analysis of colorectal cancer recurrence detection with next-generation sequencing liquid biopsy in Spain, France, and Germany.

Next-generation sequencing liquid biopsy (NGS-LB) for colorectal cancer (CRC) detection and surveillance remains an expensive technology as economies of scale have not yet been realized. Nevertheless, the cost of sequencing has decreased while sensitivity has increased, raising the question of whether cost-effectiveness (CE) has already been achieved from the perspective of European healthcare systems. This health economic (HE) modeling study explores the CE of NGS-LB for CRC based on direct treatment costs compared to standard care without liquid biopsy in Spain, France, and Germany. A structured literature search was used to collect evidence from 2009 to 2020 on the stage-dependent quality of life (quality-adjusted life-years, QALY), efficacy, and total direct treatment costs (TDC) of NGS-LB. A decision-analytic Markov model was developed. Over the remaining lifetime, cumulative life expectancy (LE), TDC, and QALYs were calculated for 60-year-old men and women in CRC stage III with different assumed effects of NGS-LB of 1% or 3% on improved survival and reduced stage progression, respectively. The use of NGS-LB increases LE by 0.19 years in Spanish men (France: 0.19 years, Germany: 0.13 years) and by 0.21 years in Spanish women (France: 0.21 years, Germany: 0.14 years), respectively. The 3% discounted cost per QALY gained was 35,571.95 € for Spanish men (France: 31,705.15 €, Germany: 37,537.68 €) and 35,435.71 € for Spanish women (France: 31,295.57 €, Germany: 38,137.08 €) in the scenario with 3% improved survival and reduced disease progression. Compared to the other two countries, Germany has by far the highest TDC, which can amount to >80k euros in the last treatment year. In this explorative HE modeling study, NGS-LB achieves generally accepted CE levels in CRC treatment from the health system perspective in three major European economies under assumptions of small improvements in cancer recurrence and survival. Confirmation of these findings through clinical trials is encouraged.

Circulating tumor DNA and radiological tumor volume identify patients at risk for relapse with resected, early-stage non-small-cell lung cancer

Predicting relapse and overall survival (OS) in early-stage non-small-cell lung cancer (NSCLC) patients remains challenging. Therefore, we hypothesized that detection of circulating tumor DNA (ctDNA) can identify patients with increased risk of relapse and that integrating radiological tumor volume measurement along with ctDNA detectability improves prediction of outcome. We analyzed 366 serial plasma samples from 85 patients who underwent surgical resections and assessed ctDNA using a next-generation sequencing liquid biopsy assay, and measured tumor volume using a computed tomography-based three-dimensional annotation. Our results showed that patients with detectable ctDNA at baseline or after treatment and patients who did not clear ctDNA after treatment had a significantly worse clinical outcome. Integrating radiological analysis allowed the stratification in risk groups prognostic of clinical outcome as confirmed in an independent cohort of 32 patients. Our findings suggest ctDNA and radiological monitoring could be valuable tools for guiding follow-up care and treatment decisions for early-stage NSCLC patients.

Pan-Tumor Analytical Validation and Osimertinib Clinical Validation in EGFR Mutant Non–Small-Cell Lung Cancer, Supporting the First Next-Generation Sequencing Liquid Biopsy in Vitro Diagnostic

Comprehensive genotyping is necessary to identify therapy options for patients with advanced cancer; however, many cancers are not tested, partly because of tissue limitations. Next-generation sequencing (NGS) liquid biopsies overcome some limitations, but clinical validity is not established and adoption is limited. Herein, clinical bridging studies used pretreatment plasma samples and data from FLAURA (NCT02296125; n=441) and AURA3 (NCT02151981; n=450) pivotal studies to demonstrate clinical validity of Guardant360 CDx (NGS LBx) to identify patients with advanced EGFR mutant non-small-cell lung cancer who may benefit from osimertinib. The primary end point was progression-free survival (PFS). Patients with EGFR mutation as identified by NGS LBx had significant PFS benefit with first-line osimertinib over standard of care (15.2 versus 9.6 months; hazard ratio, 0.41; P<0.0001) and with later-line osimertinib over chemotherapy (8.3 versus 4.2 months; hazard ratio, 0.34; P<0.0001). PFS benefits were similar to the original trial cohorts selected by tissue-based EGFR testing. Analytical validation included accuracy, precision, limit of detection, and specificity. Analytical validity was established for EGFR mutation detection and pan-tumor profiling. Panel-wide limit of detection was 0.1% to 0.5%, with 98% to 100% per-sample specificity. Patients with EGFR mutant non-small-cell lung cancer by NGS LBx had improved PFS with osimertinib, confirming clinical validity. Analytical validity was established for guideline-recommended therapeutic targets across solid tumors. The resulting US Food and Drug Administration approval of NGS LBx demonstrated safety and effectiveness for its intended use and is expected to improve adherence to guideline-recommended targeted therapy use.

Liquid Biopsy Versus Tissue Biopsy to Determine Front Line Therapy in Metastatic Non-Small Cell Lung Cancer (NSCLC)

In the last decade, non–small-cell lung cancer (NSCLC) treatment has improved with the approval of multiple therapies to target specific genetic alterations. Though, next generation sequencing (NGS) has traditionally been conducted from tissue biopsy samples, developing data supports the use of plasma-based circulating tumor DNA (ctDNA), also known as “liquid biopsy,” to complement tissue biopsy approaches in guiding front-line therapy. This study is a retrospective analysis of 170 new NSCLC patients treated at 2 cancer centers within a 5-year period who received both tissue and liquid biopsy NGS as standard of care. Based on a treatment schema defined by testing sufficiency, biomarker detection, and turnaround time (TAT), physicians based the majority of their treatments on liquid biopsy results (73.5%) versus tissue biopsy (25.9%). Liquid biopsy NGS returned results on average 26.8 days faster than tissue and reported higher testing success. For guideline-recommended biomarkers, liquid biopsy was 94.8% to 100% concordant with tissue. In comparing testing modalities, a liquid-first approach identified guideline-recommended biomarkers in 76.5% of patients versus 54.9% in a tissue-first approach. There was no significant difference in time-to-treatment, or survival outcomes (overall survival and progression free survival) based on liquid versus tissue biopsy findings. This research demonstrates that liquid biopsy NGS is an effective tool to capture actionable genetic alterations in NSCLC. Due to its high concordance to tissue, faster TAT, and similarity in outcomes and time-to-treatment, liquid biopsy can be used either as a first-line test or concordantly with tissue biopsy to guide treatment decisions in NSCLC.

260MO Liquid biopsy NGS test identifies actionable DNA mutations from CSF specimens of lung cancer patients

Lung cancer is one of the top causes of cancer death. Liquid biopsies have emerged as a non-invasive method to identify patients who may respond to therapy, enabling dynamic monitoring of minimal residual disease and drug resistance. While plasma specimens have commonly been used for liquid biopsies, we validated the use of cerebrospinal fluid (CSF) for liquid biopsy next-generation sequencing (NGS) testing.

EP16.02-013 Retrospective Analysis of BRCA1/2 Alterations in Advanced NSCLC Using An Amplicon-based NGS Liquid Biopsy Assay

Homologous recombination deficiency (HRD), characterized by tumor genomic instability, frequently occurs due to alterations in BRCA1/2. HRD has been described in non-small cell lung cancer (NSCLC), but its biological and clinical relevance in NSCLC remains unclear. Recent evidence suggests genomic instability due to HRD may alter tumor immunogenicity and predict response to immune checkpoint inhibitors (ICI), highlighting the utility of screening pathogenic BRCA1/2 alterations in NSCLC. Here, we examine the use of an ultrasensitive next-generation sequencing (NGS) liquid biopsy assay in the molecular analysis of pathogenic BRCA1/2 alterations in metastatic lung adenocarcinomas.

1099P Front-line liquid biopsy for early molecular assessment and treatment of hospitalized lung cancer patients

Molecular characterization of tumor samples has become pivotal in the management of patients affected by non-small cell lung cancer (NSCLC), although this process is often time-consuming. Some lung neoplasms are initially detected after hospitalization through the First Aid Department and the performance status of new patients might worsen while molecular analyses are processed. The primary aim of this ongoing study is to evaluate the performance of “up-front” next-generation sequencing (NGS) through liquid biopsy of hospitalized patients with newly detected lung neoplasm in parallel with conventional diagnosis. We aim to enroll 30 consecutive patients, irrespective of smoking history, immediately after detection of symptomatic, locally advanced or metastatic lung neoplasm. Liquid biopsy from peripheral blood is performed at baseline, in parallel with conventional biopsy, when feasible. Additionally, liquid biopsies are repeated during treatment and at progression in patients with any molecular alteration at baseline. Oncomine™Lung cfTNA Research Assay or Oncomine™Lung cfDNA panels are employed for processing plasma samples in NGS. Between January and May 2022, we recruited 18 hospitalized patients. Liquid biopsy NGS identified five EGFR activating mutations, one ALK rearrangement, five KRAS mutations and one ERBB2 mutation. Median time to results of liquid biopsy was 8 days (5-15) compared to 17 (8-34) by conventional biopsy. Liquid biopsy and conventional biopsy were consistent in all cases apart from one patient who was wild type at liquid biopsy and harbored EGFR exon 19 deletion at tissue biopsy. Four patients with actionable oncogenic drivers at liquid biopsy (one ALK rearrangement, two EGFR mutations and one ERBB2 mutation) could not undergo conventional biopsies due to clinical contraindications. Front-line liquid biopsy might improve the management of symptomatic, hospitalized patients with lung cancer, potentially leading to early start of targeted therapy. Enrollment and longitudinal monitoring of oncogenic drivers with liquid biopsy are ongoing.

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre

AimsPresently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary). Materials and methodsThis was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit. ResultsThe results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation. ConclusionThis study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

Abstract 5323: Analytic and clinical validation of a new pan-cancer NGS liquid biopsy test for the detection of copy number variations, fusions/exon skipping, somatic variants and indels

Abstract Liquid biopsy tests are an integral part of the molecular diagnostic workup of patients with multiple cancer types. We have extended validation studies of a new liquid biopsy test panel that uses an amplicon-based targeted next-generation sequencing (NGS) panel with a semiconductor-based system, the Ion GeneStudio S5 Prime. The workflow requires very low input (20 ng) of cell-free nucleic acids extracted from human plasma and has been demonstrated to yield results in less than 72 hours from sample receipt at the laboratory. This report details the analytic performance of the test panel for detecting copy number variations (CNVs), fusions and exon skipping variants in plasma. These data add to previously reported validation data for the detection of somatic nucleotide variants (SNV) and indels [97.7% - 100% concordant with orthogonal droplet digital PCR (ddPCR) tests]. The updated study sample sets included consented clinical and normal healthy, donor specimens, and reference cell lines to detect fusions and exon skipping [HCC78 (ROS1), CRL-5935 (EML4-ALK), KM12 (NTRK1) and HTB-178 (MET exon 14 skipping)], and CNVs [CRL-5928 (HER2 gain), SK-BR-3 (HER2, CDK6, EGFR and MYC gains), HCC-827 (EGFR gain) and CRL-5909 (MET gain)]. The limit of detection (LOD) for fusion/skipping was initially established at a molecular coverage of 42 copies and was then verified by detection of three additional fusion/skipping variants. The LOD for CNVs was initially established at 1.40 fold-change and was then verified with the detection of three additional CNVs. Verification was conducted with specimens near the initially established LOD. In the precision studies, all expected fusion/skipping variants were detected in all runs and all days of testing (n=18/18) generating a hit rate of 100%. An average CV of 20.5% (range 8.7% to 34.8%) for repeatability and 20.8% (range 15.7% to 30.5%) for reproducibility was established. Twenty eight of 29 CNVs were detected, generating a hit rate of 96.6%. An average CV of 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for reproducibility was established for detected CNVs. We conclude that the validated test panel meets the criteria for being rapid, highly sensitive and specific, and has utility for the reporting of clinically relevant variants of interest in multiple cancer types. Citation Format: Audrey Audetat, Cherie Tschida, Sarah Kreston, Leisa Jackson, Hestia Mellert, Adam Stephen, Gary A. Pestano. Analytic and clinical validation of a new pan-cancer NGS liquid biopsy test for the detection of copy number variations, fusions/exon skipping, somatic variants and indels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5323.

Incidental discovery of acute myeloid leukemia during liquid biopsy of a lung cancer patient

Liquid biopsy is considered an alternative to standard next-generation sequencing (NGS) of solid tumor samples when biopsy tissue is inadequate for testing or when testing of a peripheral blood sample is preferred. A common assumption of liquid biopsies is that the NGS data obtained on circulating cell-free DNA is a high-fidelity reflection of what would be found by solid tumor testing. Here, we describe a case that challenges this widely held assumption. A patient diagnosed with lung carcinoma showed pathogenic IDH1 and TP53 mutations by liquid biopsy NGS at an outside laboratory. Subsequent in-house NGS of a metastatic lymph node fine-needle aspiration (FNA) sample revealed two pathogenic EGFR mutations. Morphologic and immunophenotypic assessment of the patient's blood sample identified acute myeloid leukemia, with in-house NGS confirming and identifying pathogenic IDH1, TP53, and BCOR mutations, respectively. This case, together with a few similar reports, demonstrates that caution is needed when interpreting liquid biopsy NGS results, especially if they are inconsistent with the presumptive diagnosis. Our case suggests that routine parallel sequencing of peripheral white blood cells would substantially increase the fidelity of the obtained liquid biopsy results.

Comprehensive molecular profiling of advanced cancers in a real-world setting using an ultrasensitive amplicon-based next-generation sequencing (NGS) liquid biopsy assay.

3062 Background: Molecular profiling of circulating tumor DNA (ctDNA) by NGS has demonstrated the value of liquid biopsy in informing treatment decisions and monitoring disease progression in cancer patients. Here we report results from the real-world clinical application of an ultrasensitive amplicon-based NGS liquid biopsy assay for advanced cancers. Methods: Plasma cell-free DNA (cfDNA) from 1,338 consecutive samples (51.3% lung, 15.6% breast, 8.4% colorectal, 26.8% from 18 other cancer types; 86.3% of cancer samples were metastatic) underwent real-world testing in a Singapore-based, CAP-accredited, CLIA-certified laboratory from Jan 2018 – Nov 2020. Genomic alterations were analyzed using an amplicon-based NGS assay that detects alterations in 80 cancer-related genes, microsatellite instability (MSI) and cancer-causing viruses, with previously validated cfDNA detection limits of 0.1% variant allele frequency (VAF) for SNPs and indels, 5% tumor fraction, and 2 IU/mL plasma, respectively. Results: ctDNA was successfully detected in 70.0% of cancer samples (76.3% from metastatic and 24.7% from localized tumors) for a total of 977 unique variants. Across ctDNA-positive samples the median VAF was 1.4%, and 8.5% of all reported variants had VAFs between 0.01 – 0.09%. The most frequently altered genes were TP53 (48.8%), EGFR (36.9%), KRAS (17.4%), PIK3CA (15.3%), and APC (9.5%). In ctDNA-positive lung cancer, 74.6% of samples (66.5% EGFR+) harbored ≥1 actionable target, with 29.9% of patients treated with a first- or second-generation EGFR tyrosine kinase inhibitor harboring an EGFR T790M resistance mutation. Among breast cancers, ESR1 or PIK3CA alterations predicting drug resistance/response were detected in 44.2% of HR+ cases. In colorectal cancer, alterations implicated in anti-EGFR resistance ( NRAS, KRAS, BRAF, ERBB2) were found in 50.0% of samples. Profiling of cfDNA also enabled cancer monitoring, even among cancers with low genetic biomarker prevalence. 50% of nasopharyngeal cancer samples were Epstein-Barr virus-positive while 35.7% of liver cancer samples were Hepatitis B virus-positive, enabling an additional yield of 14.3% and 7.1% respectively, over ctDNA detection. cfDNA profiling of cancers of unknown primary origins informed potential tumor origins via the detection of tumor type-specific alterations such as mutations in AR, ESR1, GNAS, EGFR genes, viral biomarkers, and MSI. Conclusions: The use of a comprehensive pan-cancer liquid biopsy panel with rational inclusion of actionable and driver genes resulted in detection of ctDNA in the vast majority of clinical cases processed routinely. These findings support the utility of ultrasensitive amplicon-based NGS liquid biopsy assays in probing cancer drug sensitivity/resistance, tumor burden monitoring, and molecular characterization of tumors.

Next-Generation Sequencing with Liquid Biopsies from Treatment-Naïve Non-Small Cell Lung Carcinoma Patients.

Simple SummaryScreening for genomic alterations in treatment-naïve non-small cell lung carcinoma (NSCLC) is mainly done by tissue biopsy (TB), an invasive approach. However, it may not be possible to obtain a TB, the patient does not consent to it and/or the extracted nucleic acids are of poor quantity and/or quality for further genomic analyses, so a liquid biopsy (LB) is the only option to detect molecular target(s) for first-line treatment in these patients. However, a LB at diagnosis is still not often used in clinical centers since a TB is currently the gold standard approach for histological diagnosis, assessment of the PD-L1 status on tumor cells and evaluation of the molecular alterations. A number of different approaches are already available for the assessment of genetic abnormalities with LB, but next-generation sequencing (NGS) is the most promising. This review provides an overview of the main studies currently using LB NGS at diagnosis for NSCLC. We discuss its advantages and limitations in comparison with a TB and the perspectives for the future.Recently, the liquid biopsy (LB), a non-invasive and easy to repeat approach, has started to compete with the tissue biopsy (TB) for detection of targets for administration of therapeutic strategies for patients with advanced stages of lung cancer at tumor progression. A LB at diagnosis of late stage non-small cell lung carcinoma (NSCLC) is also being performed. It may be asked if a LB can be complementary (according to the clinical presentation or systematics) or even an alternative to a TB for treatment-naïve advanced NSCLC patients. Nucleic acid analysis with a TB by next-generation sequencing (NGS) is gradually replacing targeted sequencing methods for assessment of genomic alterations in lung cancer patients with tumor progression, but also at baseline. However, LB is still not often used in daily practice for NGS. This review addresses different aspects relating to the use of LB for NGS at diagnosis in advanced NSCLC, including its advantages and limitations.

A Highly Sensitive Next-Generation Sequencing-Based Genotyping Platform for EGFR Mutations in Plasma from Non-Small Cell Lung Cancer Patients.

Simple SummaryIn this study, Sel-CapTM, a next-generation sequencing (NGS)-based genotyping platform, showed high sensitivity for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma samples collected from 185 patients with non-small cell lung cancer (NSCLC). In the early-stage NSCLC, Sel-Cap liquid biopsy was able to detect more than half the EGFR mutations, which were detected in tumor tissue (sensitivity: 50% and 78% for Ex19del and L858R respectively, with tumor results as the references), while the conventional NGS could not detect any. Sel-Cap liquid biopsy was particularly sensitive for resistant mutation T790M (sensitivity: 88%). In addition, we conducted a retrospective study to monitor T790M using Sel-Cap in 34 patients who progressed on first-line tyrosine kinase inhibitors (EGFR-TKIs). The study suggested that the first appearance of T790M in plasma, ranging from at treatment baseline to over three years post-EGFR-TKI initiation, may be useful for prediction of disease progression (around 5 months in advance).Sel-CapTM, a digital enrichment next-generation sequencing (NGS)-based cancer panel, was assessed for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma for non-small cell lung cancer (NSCLC), and for application in monitoring EGFR resistance mutation T790M in plasma following first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Using Sel-Cap, we genotyped plasma samples collected from 185 patients for mutations Ex19del, L858R, and T790M, and compared results to those of PNAclampTM tumor biopsy (reference method, a peptide nucleic acid-mediated polymerase chain reaction clamping) and two other NGS liquid biopsies. Over two-thirds of activating mutations (Ex19del and L858R), previously confirmed by PNAclamp, were detected by Sel-Cap, which is 4–5 times more sensitive than NGS liquid biopsy. Sel-Cap showed particularly high sensitivity for T790M (88%) and for early-stage plasma samples. The relationship between initial T790M detection in plasma and progression-free survival (PFS) following first-line EGFR-TKIs was evaluated in 34 patients. Patients with T790M detected at treatment initiation (±3 months) had significantly shorter PFS than patients where T790M was first detected >3 months post treatment initiation (median PFS: 5.9 vs. 26.5 months; p < 0.0001). However, time from T790M detection to disease progression was not significantly different between the two groups (median around 5 months). In conclusion, Sel-Cap is a highly sensitive platform for EGFR mutations in plasma, and the timing of the first appearance of T790M in plasma, determined via highly sensitive liquid biopsies, may be useful for prediction of disease progression of NSCLC, around 5 months in advance.

Clinical performance of a comprehensive novel liquid biopsy test for identifying non-small cell lung cancer (NSCLC) patients for treatment with osimertinib.

9553 Background: Genotyping is required to identify cancer patients (pts) eligible for targeted therapy; however, many do not receive biomarker testing, in part due to limitations associated with tissue-only genotyping practices and the growing list of biomarkers recommended to be tested. Liquid biopsy overcomes many of these limitations but is not yet fully adopted. We report here the clinical performance of a comprehensive liquid biopsy test based on next generation sequencing (NGS) of circulating tumor DNA (ctDNA) for the identification of NSCLC patients with EGFR exon 19 deletions (ex19del) or L858R mutations ( EGFRm) or EGFR T790M, eligible for treatment with osimertinib. Methods: 441 (79%) of 556 pts randomized in FLAURA (NCT02296125; first-line osimertinib vs comparator EGFR TKI in EGFRm NSCLC) and 300 (72%) of 419 pts from AURA3 (NCT012151981; osimertinib vs chemotherapy in NSCLC pts with T790M at progression on EGFR TKI) were retrospectively tested with Guardant360 (G360), a 74-gene ctDNA NGS assay assessing single nucleotide variants, insertion-deletions, amplifications, and fusions in genes relevant to targeted therapy selection as well as microsatellite instability. Progression-free survival (PFS) of pts with EGFRm or T790M detected by G360 was compared to pts detected by the cobas EGFR Mutation Test (cobas) using tissue or plasma with an unadjusted cox model. Results: Treatment with osimertinib was associated with a significant PFS benefit relative to control therapy in NSCLC pts with EGFRm (FLAURA) and T790M (AURA3) detected using G360 (Table). Observed clinical benefit for pts with EGFRm or T790M detected by G360 was similar to that for pts with EGFRm or T790M identified by cobas using tissue or plasma specimens. Conclusions: This analysis demonstrates that G360 accurately identifies pts for osimertinib therapy while simultaneously providing comprehensive genotyping for other therapeutic molecular targets. The application of NGS liquid biopsy has the potential to increase rates of pts genotyped and access to precision medicine. [Table: see text]

A phase II randomized study of telaglenastat, a glutaminase (GLS) inhibitor, versus placebo, in combination with pembrolizumab (Pembro) and chemotherapy as first-line treatment for KEAP1/NRF2-mutated non-squamous metastatic non-small cell lung cancer (mNSCLC).

TPS9627 Background: Mutational activation of the KEAP1/NRF2 pathway occurs in &gt;20% of NSCLC patients (pts). KEAP1/NRF2 activation protects tumor cells from diverse forms of oxidative stress and promotes tumor growth and survival. In pts w/ advanced NSCLC, mutation of the KEAP1/NRF2 pathway is associated w/ dramatically reduced survival and poor outcomes following standard-of-care therapy. These tumors have increased dependence on GLS-mediated conversion of glutamine to glutamate due to upregulation of NRF2 target genes involved in glutamine metabolism. Telaglenastat (CB-839), an investigational, first-in-class, potent, oral GLS inhibitor, has demonstrated preclinical activity in KEAP1/NRF2-mutated NSCLC cell lines and xenograft models. This study will evaluate the safety and efficacy of telaglenastat + standard-of-care pembro and chemotherapy as 1L therapy for KEAP1/NRF2-mutated non-squamous mNSCLC (NCT04265534). Methods: This phase II, randomized, multicenter, double-blind study will enroll ~120 pts with histologically or cytologically documented stage IV non-squamous NSCLC w/ KEAP1 or NRF2 mutation, no prior systemic therapy for mNSCLC, measurable disease (RECIST v1.1), ECOG PS 0-1, and no EGFR, ALK, ROS, or other actionable mutation w/ available approved therapy in 1L setting. KEAP1 or NRF2 mutations will be determined by next generation sequencing (NGS), and study-provided liquid biopsy NGS will be available. Pts will be randomized 1:1 to receive telaglenastat (800 mg BID PO) or placebo, in combination with pembro, carboplatin, and pemetrexed at standard doses on day 1 of each 21-day cycle. Pts will be stratified by STK11/LKB1 mutational status and M stage of cancer (M1a-b vs M1c). The study will include an initial safety run-in period (n=12; 1 cycle). Co-primary endpoints are safety and investigator-assessed progression-free survival (RECIST v1.1). Secondary endpoints include overall response rate, duration of response, overall survival, and efficacy analysis in the subgroup of pts w/ biochemical confirmation of KEAP1/NRF2 pathway activation. Findings of this novel NGS biomarker-selected study will inform the efficacy and safety profile of telaglenastat + standard-of-care chemoimmunotherapy for 1L treatment of KEAP1/NRF2-mutated, non-squamous mNSCLC. Clinical trial information: NCT04265534 .