What are the uses of Myeloid Next-Generation Sequencing (NGS)?

Myeloid Next-Generation Sequencing: Clinical Applications

Myeloid NGS panels should be used for detecting mutations that guide diagnosis, prognosis, treatment selection, and monitoring of treatment response in patients with myeloid malignancies including AML, MDS, MPN, and CML.

Primary Diagnostic Applications

Establishing Clonality and Diagnosis

• NGS is critical for confirming clonality in diagnostically challenging cases of MDS or CMML, particularly when cytogenetics are normal 1
• The American Society of Hematology recommends using NGS to discriminate between related hematologic disorders such as MDS vs. aplastic anemia vs. myeloproliferative disorders 2
• NGS identifies disease-defining mutations such as SF3B1 in ring sideroblast anemia 2
• In MDS cases, only 47% show abnormal cytogenetics, whereas NGS detects pathogenic mutations in 74% of cases, making it superior for establishing diagnosis 1
• Among patients with suspected myeloid malignancies, 54.5% harbor at least one clinically significant mutation: 77% in AML, 48% in MDS, and 45% in MPN 3

Fusion Detection

• NGS can detect gene fusions critical for diagnosis with high sensitivity and specificity 2
• RNA-based NGS is preferred for detecting gene fusions according to the College of American Pathologists 2
• NGS shows high concordance with cytogenetics for fusion detection, providing opportunity for testing streamlining 4

Treatment Selection and Targeted Therapy

FLT3 Mutation Detection in AML

• NGS is essential for identifying FLT3 mutations in newly diagnosed AML patients to determine eligibility for midostaurin (RYDAPT) therapy 5
• FLT3 mutation-positive AML patients should receive midostaurin 50 mg twice daily on days 8-21 of induction and consolidation chemotherapy 5
• NGS panels can reliably detect FLT3-ITD mutations, which are technically challenging 3

BCR-ABL1 Kinase Domain Mutations in CML

• NGS should be used to detect low-level BCR-ABL1 kinase domain mutations in CML patients with inadequate response to TKI therapy 6
• NGS is more effective than conventional Sanger sequencing for detecting low-level mutations (detection threshold 3-5% variant allele frequency vs. 15-20% for Sanger) 6
• The NCCN recommends NGS with myeloid mutation panel for CML patients with no identifiable BCR-ABL1 mutations by standard methods 6
• Prospective monitoring demonstrates that TKI-resistant low-level mutations are invariably selected if patients are not switched to appropriate alternative TKI 6

Resistance Mutation Profiling

• NGS detects resistance mutations in genes other than BCR-ABL1 that may confer TKI resistance or portend disease progression 6
• NGS identifies asciminib-specific resistance mutations (P223S, A337V, P465S, V468F, I502L) and compound mutations involving T315I 6

Prognostic Risk Stratification

High-Risk Mutation Detection

• TP53 mutations are associated with particularly poor outcomes and higher relapse risk after stem cell transplantation 6, 7
• RAS-pathway mutations combined with complex karyotype indicate very poor prognosis even after transplantation 6
• Spliceosome mutations (SF3B1, SRSF2, U2AF1) are frequently detected in AML arising from MDS/MPN (39%) compared to de novo AML (7-14%) 1
• Molecular genetic testing for mutations such as TP53, RUNX1, and SF3B1 provides additional prognostic information beyond cytogenetics 7

Integration with Risk Scoring

• NGS results should be integrated with IPSS-R scoring for comprehensive risk stratification in MDS 7
• Mutation analysis complements cytogenetic abnormalities and bone marrow blast percentage in determining prognosis 7

Disease Monitoring and Measurable Residual Disease

• NGS enables serial monitoring to assess effectiveness of therapy and detect emerging resistance mutations 8
• Surveillance using NGS can identify clonal evolution and predict impending relapse, enabling preemptive treatment 6
• Regular NGS assessment shapes patient care and is considered vital for managing complex hematological disorders 9

Technical Implementation Considerations

Panel Design Requirements

• Targeted panels should include coverage of clinically actionable genes relevant to myeloid diseases 2
• DNA-based NGS typically requires 250ng of high-quality DNA 2
• Panels must include technically challenging genes such as CEBPA, CALR, and FLT3 3
• A 48-gene panel can achieve 99.6% sensitivity and 100% specificity for variants with allele frequencies ≥5% 3

Critical Interpretation Caveats

• Expert interpretation is essential to distinguish relevant somatic disease-associated mutations from polymorphisms, rare germline variants, and clonal hematopoiesis of indeterminate potential (CHIP) variants 9
• Laboratories should confirm unexpected or discordant NGS results using alternative methods 2
• Adequate sequencing depth is essential for detecting low-frequency variants 2

Clinical Workflow Integration

• NGS should be implemented as a frontline diagnostic test alongside bone marrow examination and cytogenetics for suspected myeloid malignancies 4
• The high concordance between NGS and cytogenetics provides opportunity for testing reduction and streamlining 4
• NGS testing is mandatory for AML diagnosis according to NCCN guidelines when integrated with morphology and cytogenetics 7
• Results should guide treatment decisions including hypomethylating agent selection, TKI choice, and stem cell transplantation timing 7