NGS Lung Cancer Applications: Tissue and ctDNA Reporting

Next-generation sequencing (NGS) has transformed lung cancer diagnostics by enabling comprehensive genetic profiling. This technology helps identify key molecular alterations that guide targeted therapies and improve patient outcomes. Advancements in both tissue-based and liquid biopsy approaches now allow clinicians to make informed treatment decisions based on a tumor’s unique genetic landscape.

Common Molecular Alterations

Lung cancer is driven by diverse genetic changes that influence tumor behavior and therapeutic response. Among these, mutations in the epidermal growth factor receptor (EGFR) gene occur in approximately 10-15% of non-small cell lung cancer (NSCLC) cases in Western populations and up to 50% in East Asian patients (Lindeman et al., 2018). Exon 19 deletions and the L858R point mutation in exon 21 promote tumor cell proliferation by activating downstream signaling pathways. Targeted therapies such as osimertinib have shown significant efficacy, with the FLAURA trial reporting a median progression-free survival of 18.9 months compared to 10.2 months with first-generation EGFR inhibitors (Soria et al., 2018). However, resistance mechanisms like the T790M mutation and MET amplification often emerge, necessitating ongoing molecular monitoring.

Rearrangements in the anaplastic lymphoma kinase (ALK) gene are present in approximately 3-5% of NSCLC cases, predominantly in younger, non-smoking individuals with adenocarcinoma histology (Shaw et al., 2013). These fusions, most commonly involving EML4-ALK, result in constitutive kinase activation. ALK inhibitors such as alectinib and lorlatinib have significantly improved outcomes, with the ALEX trial demonstrating a median progression-free survival of 34.8 months for alectinib compared to 10.9 months for crizotinib (Peters et al., 2020). However, resistance mutations such as G1202R can limit long-term efficacy.

KRAS mutations, particularly the G12C variant, represent another major oncogenic driver, occurring in approximately 25-30% of NSCLC cases (Skoulidis et al., 2021). Historically considered undruggable, KRAS G12C inhibitors such as sotorasib and adagrasib have provided new therapeutic options. The CodeBreaK 100 trial reported an objective response rate of 37.1% with sotorasib in previously treated patients (Hong et al., 2020). Resistance mechanisms, including secondary KRAS mutations and bypass signaling through SHP2 and PI3K pathways, remain challenges requiring combination treatment approaches.

Other actionable targets in NSCLC include MET exon 14 skipping mutations, RET fusions, and HER2 mutations. MET exon 14 alterations, found in 3-4% of lung adenocarcinomas, impair degradation of the MET receptor, sustaining oncogenic signaling. Capmatinib and tepotinib have shown response rates exceeding 40% in treatment-naïve patients (Wolf et al., 2020). RET fusions, present in 1-2% of NSCLC cases, respond well to selpercatinib and pralsetinib (Gavreto et al., 2020). HER2 mutations, particularly exon 20 insertions, have been effectively targeted with trastuzumab deruxtecan (Li et al., 2022).

Main NGS Platforms

The landscape of NGS platforms has expanded, offering diverse options for comprehensive molecular profiling in lung cancer. Each platform varies in sequencing chemistry, read length, and bioinformatics capabilities, influencing its suitability for clinical and research applications. Illumina, Thermo Fisher Scientific, and BGI Genomics dominate the field, optimizing their platforms for detecting key oncogenic drivers and resistance mechanisms.

Illumina’s sequencing-by-synthesis (SBS) technology underpins widely used platforms such as NovaSeq, NextSeq, and MiSeq. These instruments provide high-throughput, short-read sequencing with exceptional accuracy, making them well-suited for identifying point mutations, small insertions and deletions (indels), and copy number variations. The TruSight Oncology 500 assay enables simultaneous detection of over 500 cancer-related genes, including EGFR, ALK, and MET alterations. High sensitivity and specificity make this platform particularly effective for detecting low-frequency variants in heterogeneous tumor samples.

Thermo Fisher Scientific’s Ion Torrent technology offers an alternative approach, leveraging semiconductor sequencing to detect nucleotide changes in real time. The Ion GeneStudio S5 system, paired with the Oncomine Precision Assay, enables targeted sequencing of clinically relevant alterations in NSCLC. Unlike Illumina’s short-read platforms, Ion Torrent provides faster turnaround times, often delivering results within 24 to 48 hours. Its ability to sequence homopolymer regions with greater accuracy makes it useful for detecting complex insertions and deletions, such as EGFR exon 20 insertions.

BGI Genomics utilizes DNA nanoball (DNB) sequencing technology, which enhances signal fidelity by reducing amplification errors. The MGISEQ-2000 and DNBSEQ-T7 platforms support large-scale sequencing applications with high accuracy and cost efficiency. Although less common in North America and Europe, BGI’s platforms have gained traction in Asia. Their ability to generate high-depth sequencing data with reduced error rates enhances their utility in detecting low-frequency variants in liquid biopsy samples.

Tissue-Based Approaches

Histologically confirmed lung cancer specimens remain the foundation for molecular profiling, providing high-quality DNA and RNA for NGS. Formalin-fixed paraffin-embedded (FFPE) tissue is the most commonly used sample type but presents challenges such as DNA fragmentation and cross-linking. Optimized extraction protocols and quality control measures, including DNA integrity assessment via the DV200 metric, ensure sufficient material for sequencing.

Comprehensive genomic profiling (CGP) of tumor tissue enables the simultaneous detection of point mutations, indels, copy number alterations, and gene fusions. Hybrid capture-based NGS panels, such as FoundationOne CDx, provide broad coverage of clinically relevant genes with high sensitivity for low-allele frequency mutations. Amplicon-based approaches, exemplified by the Archer FusionPlex assay, excel in identifying gene rearrangements, particularly in ALK, RET, and ROS1 fusions, which may be missed by fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC). The choice of assay depends on tumor purity, available tissue quantity, and the specific genomic alterations under investigation.

Tumor heterogeneity poses a challenge, as spatial variations in genetic composition can lead to underrepresentation of subclonal mutations. Macrodissection techniques, in which tumor-rich regions are manually selected from histological slides, help enrich malignant cells and improve variant detection. Deep sequencing with high read depth enhances sensitivity, particularly for detecting resistance mutations. Despite these refinements, biopsy specimens may not fully capture the evolving mutational landscape, necessitating repeat sampling in cases of disease progression.

Circulating Tumor DNA Testing

Circulating tumor DNA (ctDNA) testing has emerged as a minimally invasive method for detecting genomic alterations in lung cancer. Tumor-derived DNA fragments are released into the bloodstream through apoptosis and necrosis, providing insight into the evolving genetic landscape. Unlike tissue-based sequencing, which captures a single snapshot of a tumor’s genomic profile, ctDNA testing reflects both spatial and temporal tumor heterogeneity, making it useful for monitoring disease progression and therapy resistance.

The sensitivity of ctDNA assays depends on tumor burden, shedding rate, and assay design. Digital PCR methods, such as droplet digital PCR (ddPCR), offer high specificity for detecting known mutations at very low allele frequencies but lack broader genomic profiling capabilities. NGS-based liquid biopsy assays, such as Guardant360 and FoundationOne Liquid CDx, enable simultaneous detection of multiple oncogenic drivers and resistance mutations from a single blood draw. These assays use hybrid capture or amplicon-based enrichment strategies to maximize sensitivity, with detection thresholds as low as 0.1% variant allele frequency, enhancing their utility in early-stage disease and minimal residual disease assessment.

Interpreting Findings

Interpreting NGS results in lung cancer requires a nuanced understanding of variant classification, allele frequency, and clinical relevance. Genomic alterations detected through tissue or ctDNA testing must be categorized based on their oncogenic potential and therapeutic implications. Pathogenic mutations in driver genes such as EGFR, ALK, and KRAS typically guide treatment selection, whereas variants of uncertain significance (VUS) pose challenges in clinical decision-making. The American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) provide standardized guidelines for variant classification, incorporating mutational hotspots, functional studies, and population frequency data to determine pathogenicity.

Beyond individual alterations, comprehensive genomic profiling identifies co-occurring mutations that can modify drug sensitivity and resistance patterns. For example, concurrent TP53 mutations in EGFR-mutant NSCLC have been associated with poorer responses to tyrosine kinase inhibitors. Tumor mutational burden (TMB) and microsatellite instability (MSI) assessments provide additional prognostic and predictive information, particularly for immunotherapy eligibility. Reporting frameworks such as OncoKB and clinical trial databases like My Cancer Genome assist oncologists in translating genomic findings into actionable treatment strategies.