Br J Haematol - 2022 - Mehta - Recommendations for laboratory testing of UK patients with acute myeloid leukaemia

Comprehensive Summary Br J Haematol 2023;200:150–159 “Recommendations for laboratory testing of UK patients with acute myeloid leukaemia” (BSH Good Practice Paper) 1. Executive Summary • Purpose – Provide UK‐wide good-practice guidance on the laboratory investigation of acute myeloid leukaemia (AML) at diagnosis, during follow-up and at relapse, with an emphasis on rapid, standardised testing that aligns with 2022 WHO/ICC disease classifications, 2022 ELN risk stratification and access to newly approved targeted therapies. • Scope – Covers sampling, morphology, flow cytometry, conventional cytogenetics, molecular genetics (single-gene, NGS panels, fusion assays), measurable residual disease (MRD) testing, turnaround times (TAT) and integrated reporting. • Key driver – Recent changes in AML classification/risk models and approval of multiple genotype-directed drugs mandate faster, more uniform laboratory workflows. • Audience – Specialist Integrated Haematological Malignancy Diagnostic Services (SIHMDS), district hospitals, haematologists, clinical scientists and commissioners. 2. Key Topics & Main Points 2.1 Updated Disease Frameworks • WHO-2022 & ICC-2022 place genetics centre-stage and remove the ≥20 % blast threshold for AML with “Defining Genetic Abnormalities” (DGA). • ELN-2022 risk model now integrates additional mutations (e.g. TP53, “secondary-type” genes) and no longer uses FLT3-ITD allelic ratio for risk assignment, although ratio retains prognostic value. 2.2 Therapeutic Implications • 10 new AML drugs licensed by FDA since 2018; 6 have NICE approval (midostaurin, arsenic trioxide, gemtuzumab ozogamicin, CPX-351, gilteritinib, venetoclax). • Timely detection of FLT3, IDH1/2, TP53, CD33 expression and myelodysplasia-related cytogenetics directly influences drug eligibility (midostaurin, gilteritinib, ivosidenib/enasidenib, CPX-351, GO). 2.3 Laboratory Workflow Highlights • Sampling – Bone-marrow (BM) aspirate + trephine recommended; peripheral blood (PB) acceptable when marrow unobtainable or in leukostasis. Mandatory storage of DNA/RNA & cell suspensions. • Morphology – Remains critical for rapid APL recognition; ≥200 nucleated BM cells (or ≥500 in trephine) must be examined. • Flow cytometry – Two-stage panels (ALOT screen then AML-specific); minimum 8-colour (moving to 10-colour); provide same-day results for urgent cases; CD33 status must be stated. • Genetics – Rapid single-target assays (FLT3, NPM1, CBF fusions) within 3 days; PML::RARA within 24 h if APL suspected; full karyotype in 7 days; AML NGS panel (14 days) covering at least 15 mandated genes; MRD assays (NPM1, CBF, PML::RARA, KMT2A) with clear TAT tiers. • Integrated reporting – SIHMDS must issue unified reports combining morphology, immunophenotype and genetics, citing the classification system used. 3. Important Details & Findings 3.1 Sampling & Logistics • BM: 2–5 ml EDTA for flow, 2–3 ml EDTA for DNA/RNA, 5 ml EDTA for fusion/MRD; heparin tube for cytogenetics (arrive ≤24 h). • PB: 10–20 ml EDTA diagnostic, 20 ml EDTA MRD; adequate when circulating blasts ≥20 %. • CSF: deliver within hours or collect into Transfix; consider FISH for known cytogenetic lesion. 3.2 Morphology • Blast definition refined; in monocytic AML promonocytes are counted as blasts. • AML-MR replaces “AML with myelodysplasia-related changes”; morphology alone no longer sufficient for this subtype. 3.3 Flow Cytometry & MRD • Urgent TAT: same working day if received <15:30; routine MRD ≤2–3 working days. • Identify APL and CBF phenotypes quickly; include BPDCN markers (CD123, CD4, CD56). • ELN-qualified MRD assays; discrepancy between morphology and MRD to be managed via integrated review. 3.4 Genetic Testing & Turn-around Targets Mandatory at diagnosis (repeat at relapse): – FLT3-ITD/TKD, NPM1 exon 11 (≤3 days) – CBFB::MYH11, RUNX1::RUNX1T1 by FISH/PCR/karyotype (≤3 days) – Complete karyotype (≤7 days) – KMT2A break-apart FISH if no DGA (≤14 days) – AML NGS panel including ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2, TP53, FLT3, IDH1/2, DNMT3A, WT1 (≤14 days) PML::RARA (RT-PCR or FISH) when APL suspected (≤24 h). Molecular MRD: routine ≤14 days; urgent (post-course 2, suspected relapse) 3–7 days. 3.5 Inherited Predisposition • Somatic NGS pipelines must flag potential germline variants in high-actionability genes (RUNX1, CEBPA, DDX41, ANKRD26, ETV6, GATA2). • Positive cases warrant skin-fibroblast confirmation and referral to Clinical Genetics; family history screening advised. 4. Consolidated Recommendations A. Sampling & Reporting 1. Obtain BM aspirate plus trephine in all suspected AML; PB acceptable only when marrow unobtainable or patient frail. 2. Collect and store DNA, RNA and cell suspensions at diagnosis/relapse for future MRD studies or trials; ensure patient consent. 3. Transport BM/PB to cytogenetics within 24 h and to molecular labs within 48 h. 4. Issue integrated SIHMDS reports combining morphology, flow cytometry and genetics, clearly stating the classification used. B. Morphology 5. Examine ≥200 nucleated BM cells (and ≥500 on trephine) to establish AML diagnosis. 6. Relay urgent morphological impressions promptly to laboratory specialists, especially if APL or CBF AML is suspected. C. Flow Cytometry 7. Provide rapid flow-cytometric results: same working day for urgent samples, ≤2–3 working days for others. 8. Use a two-stage panel (acute leukaemia screen followed by AML-specific antibodies); extend panels for BPDCN or megakaryoblastic AML when required. 9. Record CD33 expression quantitatively in every diagnostic or relapse report (essential for GO eligibility). 10. Always consider BPDCN in differential diagnosis and include CD123, CD4, CD56 when appropriate. 11. Forward diagnostic samples to a designated reference laboratory for ELN-qualified MFC-MRD testing; track MRD with the recommended 8-colour core panel or equivalent. D. Genetics 12. Perform comprehensive cytogenetic and molecular testing at both diagnosis and relapse. 13. Complete PML::RARA testing (FISH or RT-PCR) within 24 h whenever APL is suspected. 14. Deliver results for CBF fusions, FLT3-ITD and FLT3-TKD within 72 h (≤3 days). 15. Finalise conventional karyotype within 7 days of sample receipt. 16. Run an AML NGS panel (minimum gene list above) and report within 14 days; ensure robust detection of technically challenging genes (e.g. CEBPA, FLT3-ITD). 17. Use partner-agnostic KMT2A testing (break-apart FISH or RNA-NGS) when no other DGA is found. 18. Clearly flag any variant that may be germline in origin in the report and initiate confirmatory germline testing/referral as needed. 19. Provide molecular MRD monitoring for NPM1-mutated AML, CBFB::MYH11, RUNX1::RUNX1T1 and PML::RARA cases (and consider for other fusions), adhering to routine (≤14 days) and urgent (3–7 days) TATs. E. Quality & Governance 20. Flow and genetics laboratories must participate in external quality assurance schemes and maintain validated thresholds (e.g., CD33 positivity). 21. SIHMDS should keep standard operating procedures for send-away tests and ensure seamless logistics between referring centres and specialist labs. 5. Relevant Statistics / Data • 10 new AML therapeutics (2018–2021); 6 have NICE approvals. • 14-gene (minimum) NGS panel mandated; covers 9 “secondary-type” genes plus TP53, FLT3, IDH1/2, DNMT3A, WT1. • Turn-around thresholds: PML::RARA ≤24 h, FLT3/NPM1/CBF ≤3 days, karyotype ≤7 days, KMT2A FISH & NGS panel ≤14 days, WGS ≤42 days. • Flow cytometry urgent TAT: same day; routine MRD ≤2–3 days. • Children/young adults: additional MyeChild01 FISH panel for rare fusions (TAT ≤7 days). 6. Conclusions The BSH Good Practice Paper establishes nationally harmonised laboratory standards for AML, aligning diagnostic workflows with modern genomic classifications and therapy options. Rapid, integrated testing across morphology, flow cytometry and genetics is essential to: • Ensure accurate classification and risk assessment (WHO-2022, ELN-2022). • Enable timely initiation of targeted treatments (midostaurin, GO, CPX-351, gilteritinib, venetoclax, etc.). • Provide sensitive MRD monitoring to refine prognosis and guide post-remission therapy. Adherence to the outlined recommendations and turnaround times is expected to improve uniformity of care and patient outcomes across the UK.