A 6-Year-Old with Pancytopenia and a Suspicious Bone Marrow
A 6-year-old boy presents with 3 weeks of bruising, fatigue, and fever. CBC: hemoglobin 6.2 g/dL, platelets 22 x10^9/L, WBC 2.8 x10^9/L with 10% blasts on manual differential. Bone marrow aspirate is hemodilute; core biopsy shows 85% cellularity with sheets of small round blue cells. Flow cytometry shows a population of CD19+, CD10+, CD22+, surface immunoglobulin-negative, TdT+ cells. Cytogenetics on the marrow aspirate fails to grow. The oncology team wants to start induction chemotherapy but needs molecular risk stratification. The lab has a peripheral blood sample with 40% blasts and a bone marrow core biopsy block.
1. How do you interpret the flow cytometry, and what molecular testing would you prioritize on the available specimens?
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The flow profile (CD19+, CD10+, CD22+, sIg-negative, TdT+) is diagnostic of B-lymphoblastic leukemia/lymphoma (B-ALL). The pancytopenia with blasts and 85% cellular marrow support acute leukemia.
Molecular testing priorities (on peripheral blood with 40% blasts and/or the core biopsy block):
- BCR-ABL1 (t(9;22)) by RT-PCR or FISH - critical because it defines a high-risk group and directs tyrosine kinase inhibitor therapy. FISH on the core block or PCR on peripheral blood can detect it even when karyotype fails.
- KMT2A (MLL) rearrangement by FISH - associated with poor prognosis in infants/children.
- ETV6-RUNX1 (t(12;21)) by FISH or RT-PCR - favorable risk.
- TCF3-PBX1 (t(1;19)) and BCR-ABL1-like signature if available.
- IKZF1 deletion and CDKN2A/B deletion by FISH/MLPA - adverse risk.
- Next-generation sequencing (NGS) panel for RAS, JAK2, CRLF2, PAX5 alterations if tissue allows.
Because the karyotype failed, FISH on the core biopsy (or peripheral blood) is essential; RT-PCR on peripheral blood can detect fusion transcripts. Do not delay induction for results if the patient is unstable, but send testing first.
2. The peripheral blood RT-PCR is positive for BCR-ABL1 (p190). How does this change management, and what are the limitations of the test?
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BCR-ABL1-positive B-ALL is a high-risk subtype that requires tyrosine kinase inhibitor (TKI) therapy (e.g., imatinib or dasatinib) added to multiagent chemotherapy. It also identifies patients who may be eligible for allogeneic stem cell transplant in first remission if high-risk features persist.
Limitations of RT-PCR:
- False negatives if the breakpoint is atypical or the transcript is not covered by the primer set; FISH or NGS can complement.
- False positives from contamination or from a minor BCR-ABL1+ clone of uncertain significance; confirm with FISH or quantitative PCR.
- p190 vs p210 vs p230 transcripts differ; the p190 isoform is more common in ALL and may be missed by assays designed for CML (p210).
- Quantification requires a standardized assay (International Scale) and a baseline value for monitoring minimal residual disease (MRD).
Management: start TKI + chemotherapy, monitor BCR-ABL1 transcript levels for MRD, and consider HSCT if poor molecular response. Also send FISH for BCR-ABL1 on the core to confirm and NGS for additional lesions (IKZF1).
3. The bone marrow core biopsy block is the only tissue with adequate cellularity. What are the technical and interpretive pitfalls of FISH on a formalin-fixed paraffin-embedded (FFPE) core biopsy for this case?
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FISH on FFPE core biopsy pitfalls:
- Truncation artifact: sectioning cuts nuclei, causing signal loss and false monosomy/deletion calls. Use thick sections (4-5 µm) and score only intact nuclei; require ≥2 signals per nucleus for a valid cell.
- Fixation artifact: over- or under-fixation causes weak or absent signals; digestion time must be optimized.
- Low cellularity/necrosis: the core may have crush or necrotic areas; select viable tumor regions.
- Background autofluorescence: use appropriate filters and negative controls.
- Scoring criteria: for BCR-ABL1, use a dual-color dual-fusion probe; count ≥200 nuclei and use cutoffs (e.g., >1-2% fusion signals). For deletions (IKZF1), use ≥100 nuclei and compare with normal control.
- Clonal vs. random loss: distinguish true deletion (both signals lost in a clonal pattern) from sectioning artifact (single signal in many cells).
Interpretation: FISH on the core can confirm BCR-ABL1 and IKZF1 deletion, but karyotype remains important for complex karyotypes; consider NGS on the block if FISH is equivocal. Always correlate with flow, morphology, and PCR.