When is a Next-Generation Sequencing (NGS) panel recommended as a diagnostic tool?

When to Use NGS Panels as a Diagnostic Tool

NGS panels are strongly recommended for advanced non-squamous non-small cell lung cancer, cholangiocarcinoma, and prostate cancer to identify actionable driver mutations that guide targeted therapy selection, as well as for hereditary disorders with genetic heterogeneity where multiple genes could explain the clinical syndrome. 1, 2

Cancer Diagnostics: Primary Indications

Lung Adenocarcinoma (Advanced Non-Squamous NSCLC)

• All patients with advanced adenocarcinoma require comprehensive NGS testing to detect actionable mutations in EGFR, KRAS, BRAF, HER2, MET, and gene fusions in ALK, ROS1, RET, NTRK1-3, and NRG1 1, 2
• NGS is strongly endorsed over serial single-gene testing because sequential testing depletes scarce tissue material and misses potentially actionable targets 1
• The panel must combine DNA-based NGS for mutations with RNA-based NGS for fusion detection, as RNA methods are superior for identifying gene rearrangements 1, 2
• Testing should be completed before first-line therapy initiation to ensure patients receive the most appropriate targeted treatment 2

Squamous Cell Lung Cancer

• NGS is not routinely recommended for squamous histology 1
• Consider testing only in exceptional cases: age <50 years, never smokers, or former light smokers where adenocarcinoma component may be missed on small biopsy 1, 2

Other Solid Tumors with NGS Indications

• Cholangiocarcinoma: Multigene NGS recommended to assess level I alterations (high-evidence actionable targets), using RNA-based methods for fusion detection 1
• Prostate cancer: Multigene NGS recommended to detect actionable alterations 1
• Ovarian cancer: NGS can determine somatic BRCA1/2 mutations to guide PARP inhibitor therapy 1
• Colorectal cancer: NGS may substitute for PCR-based testing if cost-neutral 1

Tumors Where NGS is NOT Currently Recommended

• Breast cancer, gastric cancer, pancreatic cancer, and hepatocellular carcinoma have no current indication for routine tumor NGS in daily practice 1
• These recommendations reflect the absence of sufficient level I actionable alterations that would justify the cost from a public health perspective 1

Hereditary Disorders: When NGS Excels

Genetic Heterogeneity Scenarios

• NGS panels are particularly valuable when multiple genetic loci could explain a clinical syndrome, making them more cost-effective than sequential single-gene testing 3, 4
• Hereditary tumor syndromes with increasing genetic complexity (breast/ovarian cancer syndromes, Lynch syndrome, polyposis syndromes) require multi-gene panel analysis of germline mutations 5

Movement Disorders and Neurogenetic Conditions

• NGS testing achieves diagnostic yields of 10.1-15.7% for Parkinson's disease (familial and early-onset), 11.7-37.5% for dystonia, 12.1-61.8% for ataxia/spastic paraplegia 6
• Particularly useful in unusual presentations or positive family history suggesting heritable disease 6

Muscular Dystrophies

• Whole exome sequencing (WES) effectively detects sarcoglycanopathy and calpainopathy mutations by capturing all protein-coding regions 3
• For conditions with genetic heterogeneity like limb-girdle muscular dystrophies, WES simultaneously evaluates all relevant genes rather than requiring multiple targeted tests 3

Specialty-Specific Diagnostic Yields

• Highest diagnostic yields: dermatology (60%), ophthalmology (42%) 7
• Lowest yields: gastrointestinal diseases and pulmonary diseases (10% each) 7

Hematologic Malignancies

Acute and Chronic Myeloid Diseases

• Multi-gene panel diagnostics are essential for meeting WHO classification criteria and European LeukemiaNet prognostic systems for acute myeloid leukemia 5

Mature B-Cell Lymphomas

• NGS enhances diagnostic accuracy by detecting WHO-classified markers: BRAF mutations in hairy cell leukemia (100% detection), MYD88/CXCR4 mutations in lymphoplasmacytic lymphoma 8
• In cases with non-interpretable cytogenetics, NGS detects pathogenic variants in 61% of patients, compensating for inconclusive findings 8
• When morphological assessment is limited, NGS identifies relevant mutations in 70% of cases, significantly outperforming cytogenetics (30%) 8

Critical Implementation Requirements

Technical Specifications

• Sensitivity must be ≥1% variant allele frequency for mutation detection 1, 2
• Complete coverage of all specified exons with validated bioinformatics pipelines for accurate variant calling 2
• Quality control is essential—NGS is primer-dependent, so the panel composition determines which abnormalities can be detected 1
• Some panels detect mutations, gene rearrangements, and copy number variations, but these capabilities are not uniform across all commercial or institutional assays 1

Sample Type Considerations

• DNA is more stable than RNA but less sensitive for gene fusions and intronic alterations (e.g., MET exon 14 skipping) 1
• Complementary DNA and RNA NGS panels may be required to cover all clinically relevant alterations with sufficient sensitivity 1
• NGS can be performed on formalin-fixed paraffin-embedded specimens 1

Turnaround Time

• Results must be available before first-line therapy initiation to guide treatment decisions 2
• Challenges include complexity of bioinformatics, cost, and turnaround time for comprehensive whole-genome/exome sequencing 1
• Targeted NGS using cancer-related gene panels allows faster unbiased variant detection compared to whole-genome approaches 1

Resistance Testing and Disease Progression

• At progression on targeted therapy, repeat molecular testing is essential to identify resistance mechanisms 2
• This can be accomplished through re-biopsy or cell-free DNA (cfDNA) testing in liquid biopsy 1, 2
• Circulating tumor DNA provides an alternative source when tissue re-biopsy is not feasible 1

Common Pitfalls and Limitations

Variant Interpretation Challenges

• NGS typically identifies more than one candidate variant, making interpretation challenging 4
• Variants of uncertain significance (VUS) create interpretation difficulties—one academic center reduced VUS reporting from 50% (2012) to 22% (2017) as knowledge improved 7
• Coverage and accuracy are <100%, resulting in both missed variants and false positive findings 4

Cost-Effectiveness Considerations

• From a payer perspective, evidence of cost-effectiveness for multigene sequencing in daily practice is weak 1
• Implementation requires significant investments in sequencing and bioinformatics workflows 1
• NGS results could lead to expensive drugs being recommended outside approved indications 1

Quality Control Requirements

• Multiple variant calling tools and rigorous filtering are essential to remove false positives 1, 3
• Confirmation of relevant genetic variants should be performed using Sanger sequencing before clinical decisions 3
• Testing should be performed in CLIA/CAP-certified laboratories with review by qualified clinical molecular geneticists 3

Detection Limitations

• Complex structural variants or mutations in non-coding regions may not be detected by exome sequencing and might require whole genome sequencing 3
• Different bioinformatics pipelines between laboratories can result in discordant results 3

What NGS Cannot Replace

• Multi-cancer early detection tests (e.g., Grail, Galleri) are NOT recommended for routine use in asymptomatic populations by ESMO or ASCO 9
• High cost may limit widespread implementation despite theoretical benefits 9