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BackgroundUsing broad range cell-free DNA sequencing (BRcfDNA-Seq), a nontargeted next-generation sequencing (NGS) methodology, we previously identified a novel class of approximately 50 nt ultrashort single-stranded cell-free DNA (uscfDNA) in plasma that is distinctly different from 167 bp mononucleosomal cell-free DNA (mncfDNA). We hypothesize that uscfDNA possesses characteristics that are useful for disease detection.MethodsUsing BRcfDNA-Seq, we examined both cfDNA populations in the plasma of 18 noncancer controls and 14 patients with late-stage nonsmall cell lung carcinoma (NSCLC). In comparison to mncfDNA, we assessed whether functional element (FE) peaks, fragmentomics, end-motifs, and G-Quadruplex (G-Quad) signatures could be useful features of uscfDNA for NSCLC determination.ResultsIn noncancer participants, compared to mncfDNA, uscfDNA fragments showed a 45.2-fold increased tendency to form FE peaks (enriched in promoter, intronic, and exonic regions), demonstrated a distinct end-motif-frequency profile, and presented with a 4.9-fold increase in G-Quad signatures. Within NSCLC participants, only the uscfDNA population had discoverable FE peak candidates. Additionally, uscfDNA showcased different end-motif-frequency candidates distinct from mncfDNA. Although both cfDNA populations showed increased fragmentation in NSCLC, the G-Quad signatures were more discriminatory in uscfDNA. Compilation of cfDNA features using principal component analysis revealed that the first 5 principal components of both cfDNA subtypes had a cumulative explained variance of >80%.ConclusionsThese observations indicate that the distinct biological processes of uscfDNA and that FE peaks, fragmentomics, end-motifs, and G-Quad signatures are uscfDNA features with promising biomarker potential. These findings further justify its exploration as a distinct class of biomarker to augment pre-existing liquid biopsy approaches.

Lung adenocarcinoma is the most common type of lung cancer. Known risk variants explain only a small fraction of lung adenocarcinoma heritability. Here, we conducted a two-stage genome-wide association study of lung adenocarcinoma of East Asian ancestry (21,658 cases and 150,676 controls; 54.5% never-smokers) and identified 12 novel susceptibility variants, bringing the total number to 28 at 25 independent loci. Transcriptome-wide association analyses together with colocalization studies using a Taiwanese lung expression quantitative trait loci dataset (n = 115) identified novel candidate genes, including FADS1 at 11q12 and ELF5 at 11p13. In a multi-ancestry meta-analysis of East Asian and European studies, four loci were identified at 2p11, 4q32, 16q23, and 18q12. At the same time, most of our findings in East Asian populations showed no evidence of association in European populations. In our studies drawn from East Asian populations, a polygenic risk score based on the 25 loci had a stronger association in never-smokers vs. individuals with a history of smoking (Pinteraction = 0.0058). These findings provide new insights into the etiology of lung adenocarcinoma in individuals from East Asian populations, which could be important in developing translational applications.

Epidermal growth factor receptor exon 20 insertion mutations (EGFRexon20ins) are detected in approximately 2% of patients with non-small cell lung cancer (NSCLC). Due to a lack of effective therapy, the prognosis of these patients is typically poor. Sunvozertinib (DZD9008) was designed as an oral, potent, irreversible, and selective EGFR tyrosine kinase inhibitor, showing activity against EGFRexon20ins and other mutations. In both cell lines and xenograft models, sunvozertinib shows potent antitumor activity. In the two ongoing phase I clinical studies, sunvozertinib was tolerated up to 400 mg once daily. The most common drug-related adverse events included diarrhea and skin rash. Antitumor efficacy was observed at the doses of 100 mg and above in patients with EGFRexon20ins NSCLC across different subtypes, with prior amivantamab treatment as well as with baseline brain metastasis. The median duration of response has not been reached.SignificanceWe report the discovery and early clinical development of sunvozertinib, a potential treatment option for the unmet medical need of EGFRexon20ins NSCLC. This article is highlighted in the In This Issue feature, p. 1599.

Plasma cell-free DNA is being widely explored as a biomarker for clinical screening. Currently, methods are optimized for the extraction and detection of double-stranded mononucleosomal cell-free DNA of ∼160bp in length. We introduce uscfDNA-seq, a single-stranded cell-free DNA next-generation sequencing pipeline, which bypasses previous limitations to reveal a population of ultrashort single-stranded cell-free DNA in human plasma. This species has a modal size of 50nt and is distinctly separate from mononucleosomal cell-free DNA. Treatment with single-stranded and double-stranded specific nucleases suggests that ultrashort cell-free DNA is primarily single-stranded. It is distributed evenly across chromosomes and has a similar distribution profile over functional elements as the genome, albeit with an enrichment over promoters, exons, and introns, which may be suggestive of a terminal state of genome degradation. The examination of this cfDNA species could reveal new features of cell death pathways or it can be used for cell-free DNA biomarker discovery.

Receptor tyrosine-protein kinase ERBB3 (HER3) is expressed in most EGFR-mutated lung cancers but is not a known mechanism of resistance to EGFR inhibitors. HER3-DXd is an antibody-drug conjugate consisting of a HER3 antibody attached to a topoisomerase I inhibitor payload via a tetrapeptide-based cleavable linker. This phase I, dose escalation/expansion study included patients with locally advanced or metastatic EGFR-mutated non-small cell lung cancer (NSCLC) with prior EGFR tyrosine kinase inhibitor (TKI) therapy. Among 57 patients receiving HER3-DXd 5.6 mg/kg intravenously once every 3 weeks, the confirmed objective response rate by blinded independent central review (Response Evaluation Criteria in Solid Tumors v1.1) was 39% [95% confidence interval (CI), 26.0-52.4], and median progression-free survival was 8.2 (95% CI, 4.4-8.3) months. Responses were observed in patients with known and unknown EGFR TKI resistance mechanisms. Clinical activity was observed across a broad range of HER3 membrane expression. The most common grade ≥3 treatment-emergent adverse events were hematologic toxicities. HER3-DXd has clinical activity in EGFR TKI-resistant cancers independent of resistance mechanisms, providing an approach to treat a broad range of drug-resistant cancers. SIGNIFICANCE: In metastatic EGFR-mutated NSCLC, after disease progression on EGFR TKI therapy, treatment approaches include genotype-directed therapy targeting a known resistance mechanism or chemotherapy. HER3-DXd demonstrated clinical activity spanning known and unknown EGFR TKI resistance mechanisms. HER3-DXd could present a future treatment option agnostic to the EGFR TKI resistance mechanism.See related commentary by Lim et al., p. 16.This article is highlighted in the In This Issue feature, p. 1.

IntroductionThe TIGER-3 (NCT02322281) study was initiated to compare the efficacy and safety of rociletinib, a third-generation EGFR tyrosine kinase inhibitor (TKI) that targets EGFR T790M and common EGFR-activating mutations, versus chemotherapy in patients with NSCLC who progressed on first- or second-generation EGFR TKIs.MethodsPatients with advanced or metastatic EGFR-mutated NSCLC with disease progression on standard therapy (previous EGFR TKI and platinum-based chemotherapy) were randomized to oral rociletinib (500 or 625 mg twice daily) or single-agent chemotherapy (pemetrexed, gemcitabine, docetaxel, or paclitaxel).ResultsEnrollment was halted when rociletinib development was discontinued in 2016. Of 149 enrolled patients, 75 were randomized to rociletinib (n = 53: 500 mg twice daily; n = 22: 625 mg twice daily) and 74 to chemotherapy. The median investigator-assessed progression-free survival (PFS) was 4.1 months (95% confidence interval [CI]: 2.6-5.4) in the rociletinib 500-mg group and 5.5 months (95% CI: 1.8-8.1) in the 625-mg group versus 2.5 months (95% CI: 1.4-2.9) in the chemotherapy group. An improved PFS was observed in patients with T790M-positive NSCLC treated with rociletinib (n = 25; 500 mg and 625 mg twice daily) versus chemotherapy (n = 20; 6.8 versus 2.7 mo; hazard ratio = 0.55, 95% CI: 0.28-1.07, p = 0.074). Grade 3 or higher hyperglycemia (24.0%), corrected QT prolongation (6.7%), diarrhea (2.7%), and vomiting (1.3%) were more frequent with rociletinib than chemotherapy (0%, 0%, 1.4%, and 0%, respectively).ConclusionsRociletinib had a more favorable median PFS versus chemotherapy but had higher rates of hyperglycemia and corrected QT prolongation in patients with advanced EGFR-mutated NSCLC who progressed on previous EGFR TKI. Incomplete enrollment prevented evaluation of the primary efficacy end point.

Electric field-induced release and measurement (EFIRM) is a novel, plate-based, liquid biopsy platform capable of detecting circulating tumor DNA containing EGFR mutations directly from saliva and plasma in both early- and late-stage patients with non-small-cell lung cancer. We investigated the properties of the target molecule for EFIRM and determined that the platform preferentially detects single-stranded DNA molecules. We then investigated the properties of the EFIRM assay and determined the linearity, linear range, precision, and limit of detection for six different EGFR variants (the four most common g.Exon19del variants), p.T790M, and p.L858R). The limit of detection was in single-digit copy number for the latter two mutations, and the limit of detection for Exon19del was 5000 copies. Following these investigations, technical validations were performed for four separate EFIRM liquid biopsy assays, qualitative and quantitative assays for both saliva and plasma. We conclude that EFIRM liquid biopsy is an assay platform that interrogates a biomarker not targeted by any other extant platform (namely, circulating single-stranded DNA molecules). The assay has acceptable performance characteristics in both quantitative and qualitative assays on both saliva and plasma.

Mutations identified in the epidermal growth factor receptor (EGFR) predict sensitivity to EGFR-targeted therapy for non-small cell lung carcinoma (NSCLC). We previously reported that Electric Field-Induced Release and Measurement (EFIRM)-based liquid biopsy could detect EGFR ctDNA with >94% concordance with tissue-based genotyping. A side-by-side comparison of concordance of EFIRM and droplet digital PCR (ddPCR) for the detection of the two front-line actionable EFGR mutations was performed with paired plasma and saliva samples from 13 NSCLC patients. Deep sequencing analysis based on single-strand DNA library preparation was employed to determine the size distributions of EGFR L858R ctDNA in plasma and saliva samples. EFIRM detected both EGFR mutations with 100% sensitivity in both plasma and saliva samples, whereas ddPCR detected EGFR mutations with sensitivities of 84.6% and 15.4%, respectively. In saliva samples, the majority of EGFR L858R ctDNA fragments detected were

Liquid biopsy is a blood test that detects evidence of cancer cells or tumor DNA in the circulation. Despite complicated collection methods and the requirement for technique-dependent platforms, it has generated substantial interest due, in part, to its potential to detect driver oncogenes such as epidermal growth factor receptor (EGFR) mutants in lung cancer. This technology is advancing rapidly and is being incorporated into numerous EGFR tyrosine kinase inhibitor (EGFR-TKI) development programs. It appears ready for integration into clinical care. Recent studies have demonstrated that biological fluids such as saliva and urine can also be used for detecting EGFR mutant DNA through application other user-friendly techniques. This review focuses on the clinical application of liquid biopsies to lung cancer genotyping, including EGFR and other targets of genotype-directed therapy and compares multiple platforms used for liquid biopsy.

Advances in target therapies for lung cancer have enabled detection of gene mutations, specifically those of EGFR. Assays largely depend on the acquisition of tumor tissue biopsy, which is invasive and may not reflect the genomic profile of the tumor at treatment due to tumor heterogeneity or changes that occur during treatment through acquired resistance. Liquid biopsy, a blood test that detects evidence of cancer cells or tumor DNA, has generated considerable interest for its ability to detect EGFR mutations. However, its clinical application is limited by complicated collection methods and the need for technique-dependent platforms. Recently, simpler techniques for EGFR mutant detection in urine or saliva samples have been developed. This review focuses on advances in liquid biopsy and discusses its potential for clinical implementation in lung cancer.

The advances in targeted therapies for lung cancer are based on the evaluation of specific gene mutations especially the epidermal growth factor receptor (EGFR). The assays largely depend on the acquisition of tumor tissue via biopsy before the initiation of therapy or after the onset of acquired resistance. However, the limitations of tissue biopsy including tumor heterogeneity and insufficient tissues for molecular testing are impotent clinical obstacles for mutation analysis and lung cancer treatment. Due to the invasive procedure of tissue biopsy and the progressive development of drug-resistant EGFR mutations, the effective initial detection and continuous monitoring of EGFR mutations are still unmet requirements. Circulating tumor DNA (ctDNA) detection is a promising biomarker for noninvasive assessment of cancer burden. Recent advancement of sensitive techniques in detecting EGFR mutations using ctDNA enables a broad range of clinical applications, including early detection of disease, prediction of treatment responses, and disease progression. This review not only introduces the biology and clinical implementations of ctDNA but also includes the updating information of recent advancement of techniques for detecting EGFR mutation using ctDNA in lung cancer.

RationaleConstitutive activation of the epidermal growth factor receptor (EGFR) is prevalent in epithelial cancers, particularly in non-small cell lung carcinoma (NSCLC). Mutations identified in EGFR predict the sensitivity to EGFR-targeted therapy. Detection of these mutations is mainly based on tissue biopsy, which is invasive, expensive, and time consuming.ObjectivesNoninvasive, real-time, inexpensive detection and monitoring of EGFR mutations in patients with NSCLC is highly desirable.MethodsWe developed a novel core technology, electric field-induced release and measurement (EFIRM), which relies on a multiplexible electrochemical sensor that can detect EGFR mutations directly in bodily fluids.Measurements and main resultsWe established EFIRM for the detection of the EGFR mutations in vitro and correlated the results with tumor size from xenografted mice. In clinical application, we demonstrated that EFIRM could detect EGFR mutations in the saliva and plasma of 22 patients with NSCLC. Finally, a blinded test was performed on saliva samples from 40 patients with NSCLC. The receiver operating characteristic analysis indicated that EFIRM detected the exon 19 deletion with an area under the curve of 0.94 and the L858R mutation with an area under the curve of 0.96.ConclusionsOur data indicate that EFIRM is effective, accurate, rapid, user-friendly, and cost effective for the detection of EGFR mutations in the saliva of patients with NSCLC. We termed this saliva-based EGFR mutation detection (SABER).