Bone Marrow Cytogenetics

Cytogenetic analysis detects acquired chromosomal abnormalities in marrow, blood or tissue cells. Conventional karyotyping arrests dividing cells in metaphase, bands the chromosomes (G-banding) and gives a genome wide view of numerical and structural change at a resolution of about 5 to 10 megabases.

Fluorescence in situ hybridisation (FISH) uses labelled DNA probes on metaphase or interphase nuclei and finds targeted rearrangements, deletions and gains, including cryptic ones, in non-dividing cells. Chromosomal microarray detects copy number change and copy neutral loss of heterozygosity without cell culture. The result classifies the disease, estimates prognosis, selects treatment and provides a marker for monitoring.

Clonal abnormalities are found in roughly half of adult acute myeloid leukaemia and myelodysplastic syndromes, in most acute lymphoblastic leukaemia, and in over 95% of chronic myeloid leukaemia. Myeloma and chronic lymphocytic leukaemia proliferate poorly in culture, so karyotyping often misses their abnormalities, and interphase FISH finds them in most cases.

Clone definition. At least 2 cells with the same structural abnormality or chromosome gain, or at least 3 cells with the same chromosome loss. At least 20 metaphases should be analysed when available.

Indications.

• Suspected or proven acute leukaemia, myelodysplastic syndrome, myeloproliferative neoplasm
• chronic myeloid leukaemia (diagnosis and treatment failure)
• eosinophilia with suspected clonal disease
• myeloma (FISH)
• chronic lymphocytic leukaemia (FISH before each line of treatment)
• lymphoma with fresh tissue
• unexplained persistent cytopenia
• suspected therapy related myeloid neoplasm
• chimerism after sex mismatched allogeneic transplant

Specimen.

• Marrow aspirate, 1 to 3 mL, with the first pull preferred, in preservative free sodium heparin. EDTA is unsuitable for culture.
• Blood in sodium heparin when circulating blasts or abnormal lymphocytes are plentiful.
• Fresh lymph node or tissue in culture medium.
• Room temperature transport, delivery within 24 to 48 hours, and no freezing. Clotted samples fail.
• Myeloma FISH needs plasma cells enriched by CD138 selection, because they are few in unselected marrow.

Culture and analysis. Unstimulated culture for 24 to 72 hours suits myeloid disease. Mitogen stimulation is needed for chronic lymphocytic leukaemia (CpG oligonucleotide with interleukin-2) and T-cell disease. Cells are harvested, banded and analysed, and the clone is described in the nomenclature for human chromosomes.

Turnaround. Rapid FISH for PML::RARA, BCR::ABL1 or KMT2A takes 24 to 48 hours. Karyotype takes 7 to 14 days. Microarray takes 7 to 14 days.

Acute myeloid leukaemia (usual risk grouping).

• Favourable: t(15;17) PML::RARA, t(8;21) RUNX1::RUNX1T1, inv(16) or t(16;16) CBFB::MYH11.
• Intermediate: normal karyotype, t(9;11) MLLT3::KMT2A, and abnormalities not classed as favourable or adverse.
• Adverse: complex karyotype (3 or more unrelated abnormalities), monosomal karyotype, -5 or del(5q), -7, abnormalities of 17p, inv(3) or t(3;3) (GATA2, MECOM), t(6;9) DEK::NUP214, t(9;22) BCR::ABL1, and KMT2A rearrangements other than t(9;11).
• Therapy related disease: -5 or -7 and complex karyotypes follow alkylating agents after about 5 to 7 years, and KMT2A or RUNX1 rearrangements follow topoisomerase II inhibitors after about 1 to 3 years.

Acute lymphoblastic leukaemia.

• Favourable in children: high hyperdiploidy (over 50 chromosomes) and ETV6::RUNX1 t(12;21), which is cryptic on karyotype and needs FISH or PCR.
• Adverse: KMT2A rearrangements (t(4;11), common in infants), hypodiploidy (under 44 chromosomes), near haploidy, iAMP21, TCF3::HLF t(17;19). BCR::ABL1 t(9;22) was adverse, and outcome has improved with tyrosine kinase inhibitors.
• Burkitt leukaemia and lymphoma: MYC rearrangement, usually t(8;14), occasionally t(2;8) or t(8;22).

Chronic myeloid leukaemia. t(9;22)(q34;q11.2) producing BCR::ABL1 in over 95%, with variant translocations in a few. Additional abnormalities (+8, second Philadelphia chromosome, i(17q), +19) indicate progression. Complete cytogenetic response means no Philadelphia positive metaphases. Quantitative PCR has largely replaced karyotype for monitoring, but karyotype is still needed at diagnosis and when treatment fails.

Myelodysplastic syndromes (revised international prognostic scoring system groups).

• Very good: -Y, del(11q).
• Good: normal, del(5q), del(12p), del(20q), double abnormality including del(5q).
• Intermediate: del(7q), +8, +19, i(17q), other single or double abnormalities.
• Poor: -7, inv(3), t(3q), del(3q), double abnormality including -7 or del(7q), complex with 3 abnormalities.
• Very poor: complex with over 3 abnormalities.

Isolated del(5q) predicts response to lenalidomide. Loss of Y alone in an older man does not establish myelodysplasia.

Myeloma (FISH on plasma cells). High risk: del(17p), t(4;14), t(14;16), t(14;20), and gain or amplification of 1q. t(11;14) is common and predicts sensitivity to venetoclax. Hyperdiploidy (trisomies of odd numbered chromosomes) carries lower risk.

Chronic lymphocytic leukaemia (FISH). Sole del(13q) is favourable, trisomy 12 is intermediate, del(11q) is adverse, and del(17p) with TP53 mutation is adverse and predicts a poor response to chemoimmunotherapy. Clonal evolution makes repeat testing necessary before each new line of treatment.

Eosinophilia with clonal disease. FIP1L1::PDGFRA (a cryptic 4q12 deletion detected by FISH or PCR) and PDGFRB rearrangements (t(5;12)) respond to imatinib. FGFR1 rearrangements (8p11) are aggressive and respond poorly to usual therapy.

Lymphoma. t(14;18) IGH::BCL2 in follicular lymphoma, t(11;14) CCND1::IGH in mantle cell lymphoma, and MYC rearrangement with BCL2 or BCL6 rearrangement (double hit) in high grade B-cell lymphoma.

Critical findings needing immediate action.

• PML::RARA or morphology of promyelocytic leukaemia needs same day haematology contact. Treatment with all-trans retinoic acid is started on suspicion, without waiting for the result.
• BCR::ABL1 in acute leukaemia, KMT2A rearrangement in an infant, or TCF3::HLF, since each changes treatment urgently.
• TP53 loss or del(17p) in chronic lymphocytic leukaemia, myeloma or acute myeloid leukaemia.

Pitfalls.

• Culture failure with no analysable metaphases occurs in a minority of samples, so FISH or microarray is then needed.
• A normal karyotype can reflect normal dividing cells and does not exclude a clone, particularly in slowly proliferating disease.
• Cryptic abnormalities (ETV6::RUNX1, NUP98::NSD1, FIP1L1::PDGFRA) are missed by karyotype.
• FISH detects only what the probe targets, and each probe has a laboratory specific cut off for positivity.
• Small clones fall under the detection limit of karyotyping.
• Clonal haematopoiesis in older adults can produce small clones without overt disease, so interpret with morphology, flow cytometry and molecular findings.
• Haemodilute samples and a low blast count reduce yield.
• Chromosome quality limits resolution in some samples.

Special groups.

• Children: risk adapted therapy depends on cytogenetics at diagnosis.
• Infants: KMT2A rearrangements predominate and carry a poor outlook.
• Down syndrome: constitutional trisomy 21 is present in blood, and blast acquired abnormalities need a separate analysis.
• Suspected germline predisposition (RUNX1, GATA2, DDX41 and others): a skin fibroblast culture or germline sequencing is needed to separate acquired from constitutional change.
• Post-transplant: sex mismatched donors allow chimerism assessment by XX and XY FISH.
• Older adults: complex and monosomal karyotypes are more frequent.

Follow up tests. Targeted FISH panels, molecular panels (NPM1, FLT3, CEBPA, IDH1, IDH2, TP53), RT-PCR for BCR::ABL1 and PML::RARA, chromosomal microarray, next generation sequencing, flow cytometry, bone marrow morphology, germline testing, human leucocyte antigen typing in transplant candidates.