Leukemia and Lymphoma Markers and Clonality

Why molecular markers matter

In acute leukemia and lymphoma, specific genetic changes define disease types, predict outcome, select targeted drugs, and provide markers for measurable residual disease (MRD). Many are detected by FISH or karyotype as well as PCR or NGS.

Acute promyelocytic leukemia

APL is defined by t(15;17)(q24;q21), producing PML::RARA, in about 98% of cases ¹. The fusion protein blocks differentiation at the promyelocyte stage.

APL is an emergency: patients often have disseminated intravascular coagulation (DIC), and hemorrhage, including into the brain, causes early deaths ¹. The fusion also makes APL exquisitely sensitive to all-trans retinoic acid (ATRA) and arsenic trioxide ¹, which together cure most patients without chemotherapy. ATRA is started on clinical suspicion, and labs confirm PML::RARA quickly by FISH or RT-PCR. Quantitative PML::RARA RT-PCR is then used for MRD monitoring.

Acute myeloid leukemia

AML genetics drive risk classification. Under ELN 2022 ²:

Favorable

• t(8;21) → RUNX1::RUNX1T1
• inv(16) or t(16;16) → CBFB::MYH11 (these two are the “core-binding factor” leukemias)
• NPM1 mutation without FLT3-ITD
• In-frame bZIP CEBPA mutation

Intermediate

• FLT3-ITD, with or without NPM1, regardless of allelic ratio ²
• t(9;11) → MLLT3::KMT2A
• Changes not classed as favorable or adverse

FLT3 mutations - internal tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations such as D835 - also select FLT3 inhibitors. The LeukoStrat CDx FLT3 assay is the FDA companion diagnostic for midostaurin, gilteritinib, and quizartinib ³. FLT3-ITD is detected by PCR and fragment analysis: the duplication makes a longer product than the wild type.

Acute lymphoblastic leukemia

• BCR::ABL1 (Ph+ ALL), usually the p190 e1a2 transcript, treated with TKIs plus chemotherapy.
• ETV6::RUNX1 (from t(12;21)), common in childhood ALL, carries a favorable prognosis.
• KMT2A rearrangements, common in infant ALL, carry a poor prognosis.
• MRD after induction, measured by flow cytometry, IG/TCR PCR, or NGS, is one of the strongest predictors of outcome.

Lymphoma translocations

Several B-cell lymphomas join an oncogene to the immunoglobulin heavy chain locus (IGH, at 14q32), placing it under the control of strong B-cell enhancers:

The BIOMED-2 primer sets included PCR for t(11;14) and t(14;18) alongside clonality targets ⁴, though FISH is more commonly used for translocations now.

Clonality testing

Each B cell rearranges its immunoglobulin genes (IGH, IGK, IGL) and each T cell its T-cell receptor genes (TRB, TRG, TRD) uniquely, joining V, D, and J segments with random nucleotides in between. A reactive population has thousands of different rearrangements; a lymphoma descends from one cell and shares one.

Clonality PCR amplifies across the rearranged junctions with consensus primers, then sizes the products by capillary electrophoresis (“GeneScanning”) or heteroduplex analysis. The standardized BIOMED-2 assays use 107 primers in 18 multiplex tubes covering IGH, IGK, IGL, TRB, TRG, and TRD ⁴.

• Polyclonal: a bell-shaped (Gaussian) spread of peaks across the size range.
• Monoclonal: one (or two, for two alleles) dominant peak above the background, reproducible in duplicate.

Clonality supports a diagnosis of lymphoma but doesn’t prove it: some reactive conditions produce small clones, and lineage can be misleading (some T-cell lymphomas have IG rearrangements). Results are always interpreted with histology, immunophenotype, and the clinical picture. Next-generation sequencing of IG/TCR rearrangements is increasingly used, and it also gives a patient-specific sequence for MRD tracking.

What to take from this

APL with t(15;17) PML::RARA is an emergency because of DIC, and it’s cured by ATRA plus arsenic. In AML, the core-binding factor fusions, NPM1 without FLT3-ITD, and bZIP CEBPA are favorable under ELN 2022, while FLT3-ITD is intermediate and selects FLT3 inhibitors. ALL markers include BCR::ABL1 p190, ETV6::RUNX1, and KMT2A. Follicular, mantle cell, and Burkitt lymphomas carry IGH translocations with BCL2, CCND1, and MYC. Clonality PCR distinguishes a monoclonal peak from a polyclonal spread and supports, but doesn’t make, a lymphoma diagnosis.