NGS and MRD — Clinical Importance in AML
NGS and MRD — Clinical Importance in AML
Part I: Next-Generation Sequencing (NGS)
What NGS Does
NGS (next-generation sequencing) is a massively parallel DNA/RNA sequencing platform that can interrogate hundreds to thousands of genomic loci simultaneously from a single clinical sample. In AML workup, an NGS panel is run on bone marrow at diagnosis to:
- Identify somatic mutations — driver and cooperating lesions in the leukemic clone
- Classify the disease — WHO/ICC 2022 classification requires molecular data; morphology alone is insufficient
- Assign ELN risk — favorable / intermediate / adverse risk stratification is mutation-driven
- Guide therapy selection — targetable mutations (FLT3, IDH1/2, NPM1) dictate whether targeted agents are added; non-targetable mutations (ASXL1, SRSF2, TP53) confirm the need for HMA-based regimens
- Establish MRD targets — the diagnostic VAF of each mutation becomes the baseline for longitudinal monitoring
Ishamma's NGS (Oncomine Myeloid Assay GX V2)
Lab: Agilus Diagnostics, Gurugram | Sample date: 2025-11-22 | QC: 3345x coverage, 99.54% uniformity — high-quality run
| Gene | Change | VAF | Clinical Significance |
|---|---|---|---|
| RUNX1 | p.(Asp93GlyfsTer32) | 4% | Adverse-risk AML per ELN/WHO 2022; preferred MRD target |
| ASXL1 | p.(Trp1411Ter) | 28% | Epigenetic deregulator; inferior OS; confirms secondary-type AML; CHIP-prone |
| BCOR | p.(Arg810Ter) | 17% | BCL6 corepressor LOF; founding/co-founding clone |
| SRSF2 | p.(Pro95His) | 31% | Canonical CHIP hotspot; splicing dysregulation; adverse prognostic |
| STAG2 | p.(Met1102TyrfsTer34) | 16% | Cohesin LOF; genomic instability; X-linked |
| NRAS | p.(Gly12Asp) | 3% | Subclonal MAPK activator; unstable — not suitable for MRD |
RNA fusion panel: Negative — no gene rearrangements detected.
Net diagnostic output from NGS:
- Classified as AML with myelodysplasia-related gene mutations (WHO/ICC 2022) — requires multiple MDS-related gene mutations, which Ishamma has (ASXL1, BCOR, SRSF2, STAG2)
- Adverse ELN risk — driven by RUNX1 + ASXL1 + SRSF2
- No targetable mutations (no FLT3-ITD/TKD, no IDH1/2, no NPM1) — confirms Aza-Ven as the appropriate backbone
- Provides the mutation inventory from which MRD targets are selected
Part II: Measurable Residual Disease (MRD)
What MRD Is
MRD is the detection of residual leukemic cells below the threshold of conventional morphology (<5% blast threshold by microscopy) or standard cytogenetics. High-sensitivity assays — including NGS, digital PCR (dPCR), and multiparameter flow cytometry (MFC) — can detect 1 leukemic cell in 10,000–1,000,000 normal cells (10⁻⁴ to 10⁻⁶ sensitivity).
Why MRD Matters
| Scenario | Implication |
|---|---|
| Morphologic CR, MRD-negative | Deepest achievable response; best prognosis; supports dose de-escalation or cycle spacing |
| Morphologic CR, MRD-positive | Residual disease persists; 2–4× higher relapse risk; warrants closer monitoring or therapy intensification |
| MRD rising (previously negative) | Molecular relapse — precedes overt blast relapse by weeks to months; enables preemptive regimen change |
| MRD stable (persistent low positive) | Clone present but controlled; watch-and-wait vs. therapy adjustment depends on trajectory |
NGS as MRD Platform
NGS-based MRD tracking re-sequences the same loci identified at diagnosis, measuring whether the VAF of each mutation has fallen (response), is undetectable (remission), or is rising (relapse). Compared to PCR, NGS-MRD is:
- Broader — can track multiple mutations simultaneously (composite panel)
- Mutation-agnostic — does not require a pre-designed probe per mutation (as PCR does)
- Sensitive at ≥0.1–1% VAF with high-depth sequencing (error-corrected NGS approaches reach 0.01%)
Part III: NGS → MRD Continuum in Ishamma's Case
Step 1 — Diagnostic NGS (Nov 2025)
The Oncomine panel identified the 6-mutation clonal architecture. This step is done.
Step 2 — Select MRD Targets
Not all mutations are equal as MRD markers (see Mrd Targets Explained for full rationale):
| Mutation | MRD Role | Reason |
|---|---|---|
| RUNX1 | Primary target | Frameshift — leukemia-specific, not CHIP-associated, Tier I |
| STAG2 | Supplementary | Less CHIP-prone than ASXL1/SRSF2; supports composite assessment |
| BCOR | Supplementary | Co-founding clone; low VAF persistence may reflect residual CHIP |
| ASXL1, SRSF2 | Not solo targets | Canonical CHIP mutations — persist even after leukemia eradication |
| NRAS | Excluded | Subclonal, unstable — not trackable |
Recommended composite panel: RUNX1 primary + BCOR + STAG2. All three undetectable = high-confidence MRD-negative.
Step 3 — Serial MRD Monitoring
The Day 21 BMBx (Bone Marrow Biopsy 2025 12 31) confirmed morphologic response (cellularity 60% → 25–30%), but morphology cannot determine whether the leukemic clone is contracting at a molecular level.
Standard NGS-MRD cadence on Aza-Ven for non-transplant AML:
- After Cycle 2–3: First molecular assessment — establishes whether RUNX1 VAF has cleared or persists
- After Cycle 4–6: Second assessment — confirms trajectory (deepening response vs. plateau)
- Every 3–6 months thereafter: Ongoing surveillance; key decision point at each cycle reassessment
Why MRD Monitoring Matters Even Without Transplant Eligibility
At 81 years, allo-HSCT is not feasible. MRD monitoring still serves 4 non-transplant functions:
- Treatment duration — MRD-negative on Aza-Ven supports considering cycle spacing or dose de-escalation to reduce cumulative cytopenias and improve QoL
- Dose justification — VEN has already been reduced (10d → 5d) and AZA capped at 5d. MRD data provides objective justification for further reductions vs. requiring intensification
- Early relapse detection — RUNX1 VAF rise will precede blast relapse on CBC/morphology by weeks to months; intervention is more effective before overt relapse
- Distinguishing CHIP from active disease — At 81, ASXL1 (28%) and SRSF2 (31%) will likely persist in bone marrow indefinitely regardless of leukemia control; RUNX1 clearance is the clean signal for true remission
Current Status Summary
| Parameter | Status |
|---|---|
| Diagnostic NGS | Done (Nov 2025, Oncomine — high quality) |
| Morphologic response | Confirmed (Day 21 BMBx, Dec 2025) |
| MRD target defined | RUNX1 (primary), per Agilus recommendation |
| NGS-MRD post-treatment | Not done — critical gap |
| Recommended timing | After Cycle 2–3 (was Feb–Mar 2026; now overdue at Cycle 4+) |
NGS-MRD with RUNX1 VAF tracking is overdue. Recommend raising with Bijay Prabhakaran Nair at next visit. Test can be performed from peripheral blood (liquid biopsy) or bone marrow aspirate. Lab: Agilus Diagnostics (same lab that ran the diagnostic panel — ensures assay continuity).
Related Pages
- Aml — Full NGS panel, molecular profile, treatment cycles
- Mrd Targets Explained — Detailed MRD target rationale and CHIP caveat
- Bone Marrow Biopsy 2025 12 31 — Day 21 morphologic response (no molecular MRD)
- Azacitidine / Venetoclax — Treatment context
- Bijay Prabhakaran Nair — Treating oncologist
Filed from query, 2026-05-03.