What is the initial molecular study to send from a bone marrow aspirate (BMAT) for an older patient presenting with pancytopenia?

Initial Molecular Study for Pancytopenia in Older Patients

Cytogenetics (conventional karyotyping) is the mandatory initial molecular study to send from bone marrow aspirate in older patients with pancytopenia, as it provides essential diagnostic and prognostic information for myelodysplastic syndromes (MDS), the most common cause in this age group. 1

Required Initial Molecular/Cytogenetic Testing

Conventional Cytogenetics (Karyotype)

• Conventional cytogenetic analysis with evaluation of at least 20 metaphases is mandatory for all patients with suspected MDS or unexplained pancytopenia 1
• An abnormal clone requires identification in at least 2 of 20 cells carrying the same karyotypic abnormality 1
• Cytogenetics are of major prognostic importance and essential for IPSS-R risk stratification in MDS, which is the primary concern in older patients with pancytopenia 1
• Specific MDS-associated karyotypes (del(5q), del(20q), +8, -7/del(7q)) serve as decisive diagnostic criteria even with minimal dysplasia 1

Complementary FISH Analysis

• FISH should be performed when conventional cytogenetics fails or to detect cryptic abnormalities 1
• If no or insufficient metaphases are obtained, FISH analysis for monosomy 5/deletion 5q, monosomy 7/deletion 7q, trisomy 8, TP53 deletion, and 20q deletion must be undertaken 1
• FISH is particularly important as karyotype analysis may miss clinically significant cryptic aberrations 1

Additional Molecular Studies to Consider

FLT3 Mutation Testing

• FLT3 gene mutations (ITD and TKD) should be performed immediately if acute myeloid leukemia (AML) is suspected to allow timely initiation of FLT3 inhibitors 1
• This is critical given that older patients with pancytopenia may present with AML rather than MDS 1

NPM1 and CEBPA Mutations

• NPM1 and double CEBPA mutations should be assessed at diagnosis given their prognostic significance in AML 1
• These mutations can override morphological uncertainties in diagnosis 1

Prognostic Molecular Markers

• TP53, RUNX1, and ASXL1 mutations classify patients to adverse ELN risk groups and testing is advised 1
• However, mutations in MDS-associated genes should not be used alone to support a diagnosis of MDS in the absence of morphological criteria 2

Critical Workflow Algorithm

Step 1: Conventional Karyotype First

• Always attempt conventional cytogenetics as the primary molecular study 1
• This provides both diagnostic information (specific MDS-associated abnormalities) and prognostic stratification 1

Step 2: If Karyotype Fails or Is Normal

• Proceed to FISH for common MDS abnormalities (5q-, 7/7q-, +8, 20q-, TP53) 1
• Consider optical genome mapping (OGM) as an alternative when conventional cytogenetics yields insufficient metaphases 1

Step 3: If AML Is Suspected (>5% Blasts)

• Add immediate FLT3 mutation testing 1
• Include NPM1, CEBPA, and prognostic markers (TP53, RUNX1, ASXL1) 1

Step 4: Consider NGS Panel

• Next-generation sequencing panels can provide additional diagnostic and prognostic information 1, 3
• NGS contributed substantially to diagnostic evaluation in 77% of cytopenia cases in real-world settings 3
• However, NGS should complement, not replace, cytogenetics and morphology 2

Common Pitfalls to Avoid

Do Not Rely on Molecular Testing Alone

• The presence of somatic mutations without morphological dysplasia or cytogenetic abnormalities does not establish a diagnosis of MDS 2
• Idiopathic pancytopenia patients with mutations did not develop MDS over median follow-up of 444 days 2
• This represents Clonal Cytopenia of Undetermined Significance (CCUS) rather than MDS 3

Do Not Skip Cytogenetics

• Cytogenetics cannot be replaced by molecular testing for initial evaluation 1
• Specific cytogenetic abnormalities are WHO diagnostic criteria for MDS 1
• Karyotype is essential for IPSS-R prognostic scoring, which guides treatment decisions 1

Do Not Forget Non-Malignant Causes

• In older patients, megaloblastic anemia remains a common reversible cause of pancytopenia (74% in one series) 4
• Serum B12, RBC folate, and serum erythropoietin should be obtained before initiating therapy 1
• Flow cytometry for PNH clone should be considered, especially in younger patients or those with hypoplastic marrows 1

Age-Specific Considerations

• MDS incidence increases dramatically with age: 30.2 per 100,000 in ages 70-79 years and 59.8 per 100,000 in those ≥80 years 1
• Older patients have higher rates of adverse cytogenetics and lower tolerance for intensive therapy 1
• The median age at MDS diagnosis is 70-75 years, making it the primary diagnostic consideration in older patients with pancytopenia 1