What is Next-Generation Sequencing (NGS) in Hematology (Heme) and Oncology (Onc)?

Next-Generation Sequencing (NGS) in Hematology and Oncology

Next-generation sequencing (NGS) is a powerful molecular diagnostic technology that enables simultaneous assessment of multiple genomic regions, revolutionizing clinical care in hematologic malignancies and solid tumors by providing critical information for diagnosis, classification, prognostication, and treatment selection.

Definition and Basic Principles

• NGS refers to massively parallel sequencing technology that allows for high-throughput DNA or RNA sequencing, enabling the analysis of hundreds to thousands of genes simultaneously in a single test 1
• The technology involves spatial separation of individual DNA molecules, PCR amplification of predetermined genomic regions, parallel sequencing by synthesis, and computational data processing to identify genomic alterations 1
• Two main NGS approaches are used in clinical practice:
  • Amplicon-based sequencing: Uses multiple PCR reactions, is faster, more sensitive for detecting low-frequency variants, but typically limited to smaller gene panels 1
  • Hybrid capture-based sequencing: Uses hybridization to generate libraries, is more complex but better for analyzing larger genomic regions and detecting gene fusions and copy number variations 1

Clinical Applications in Hematology-Oncology

• Diagnostic applications:

  • Discriminating between related hematologic disorders (e.g., MDS vs. aplastic anemia vs. myeloproliferative disorders) 1
  • Identifying disease-defining mutations (e.g., SF3B1 mutation in ring sideroblast anemia) 1
  • Detecting gene fusions critical for diagnosis (e.g., ETV6-NTRK3 in secretory carcinoma) 1
  • Supporting diagnosis in cases with ambiguous morphology or immunophenotype 2

• Classification applications:

  • Supporting WHO classification criteria (e.g., SF3B1 mutation for MDS-RS classification) 1
  • Molecular subtyping of hematologic malignancies 1, 2
  • Identifying genomic signatures specific to disease subtypes 2

• Prognostication:

  • Detecting mutations associated with inferior prognosis (e.g., TP53, RUNX1) 1
  • Identifying mutations associated with favorable prognosis (e.g., SF3B1) 1
  • Determining tumor mutational burden (TMB) for immunotherapy response prediction 1
  • Risk stratification in acute myeloid leukemia according to ELN criteria 3

• Treatment selection:

  • Identifying patients suitable for targeted therapies (e.g., IDH1/2 inhibitors in AML) 1
  • Detecting actionable gene fusions (e.g., ALK, ROS1 in lung cancer) 1
  • Identifying resistance mutations (e.g., EGFR T790M in lung cancer) 1
  • Detecting BRCA1/2 mutations in ovarian cancer for PARP inhibitor therapy 1

• Disease monitoring:

  • Tracking clonal evolution during disease progression 1
  • Monitoring for treatment failure or resistance 1, 4
  • Detecting minimal residual disease 4

Clinical Implementation Guidelines

• Indications for NGS testing in hematologic malignancies:

  • Recommended for diagnostic workup of myelodysplastic syndromes 1
  • Valuable for acute myeloid leukemia risk stratification 2, 3
  • Useful for cases with diagnostic uncertainty or unusual clinical presentations 2
  • Beneficial for patients with treatment resistance or disease progression 1, 4

• Indications for NGS testing in solid tumors (ESMO recommendations):

  • Strongly recommended for lung adenocarcinoma to assess level I alterations 1
  • Recommended for cholangiocarcinoma to identify actionable mutations 1
  • Indicated for prostate cancer to detect level I alterations 1
  • Appropriate for carcinoma of unknown primary 1
  • Can be used in ovarian cancer to determine somatic BRCA1/2 mutations 1

Technical Considerations

• Sample requirements:

  • DNA-based NGS typically requires 250ng of high-quality DNA 1
  • RNA-based NGS is preferred for detecting gene fusions 1
  • Fresh or properly preserved tissue samples yield better results than degraded specimens 1

• Panel selection:

  • Targeted panels typically include 50-100 genes recurrently mutated in myeloid diseases 1
  • Comprehensive panels may include up to 400-500 genes for broader genomic profiling 2, 3
  • Panel design should include coverage of clinically actionable genes relevant to the disease 1, 3

• Quality control:

  • Laboratories should confirm unexpected or discordant results using alternative methods 1
  • Adequate sequencing depth is essential for detecting low-frequency variants 1
  • Proper bioinformatic pipelines are crucial for accurate variant calling and interpretation 1

Limitations and Challenges

• NGS is not universally deployed due to substantial resource requirements and complex assay design, performance, and interpretation 1
• Distinguishing between germline and somatic variants can be challenging without matched normal tissue 5
• Interpretation of variants of uncertain significance requires expertise and may evolve over time 4
• Cost considerations may limit widespread implementation in some clinical settings 1
• NGS is still rarely incorporated into clinical guidelines despite demonstrated benefits 1

Future Directions

• Integration of NGS with other diagnostic modalities for comprehensive disease characterization 4
• Development of standardized reporting formats and interpretation guidelines 4
• Expansion of targeted therapy options based on NGS-identified alterations 2, 3
• Implementation of liquid biopsy approaches for non-invasive molecular profiling and monitoring 4