Lymphoid Clonality Studies (IGH, TCR)

Lymphoid clonality studies test whether a population of lymphocytes comes from a single cell (clonal) or from many different cells (polyclonal). Each B and T cell rearranges its antigen receptor genes (immunoglobulin heavy chain, IGH, and kappa and lambda light chains for B cells, and T-cell receptor beta and gamma, TRB and TRG, for T cells) into a unique sequence. A tumour that arises from one cell carries a single dominant rearrangement.

The test is done by PCR that amplifies the rearranged gene regions, followed by capillary electrophoresis (a single sharp peak suggests a clone, a bell shaped distribution suggests a polyclonal population) or by next generation sequencing, which gives the exact sequence and a more sensitive result.

It is used when the diagnosis of lymphoma is uncertain on morphology and immunohistochemistry, when a reactive process is the alternative, and when two lymphoid proliferations need comparison. DNA is extracted from fresh or formalin fixed tissue, blood, bone marrow or fluid. Results take 3 to 7 days.

With multiple primer sets, clonality testing detects about 85 to 95% of B-cell lymphomas and 90 to 95% of T-cell lymphomas. Clonality alone does not prove malignancy, since benign clonal expansions occur.

Indications.

• Suspected lymphoma with equivocal histology: atypical lymphoid infiltrates in lymph nodes, skin, gastrointestinal tract (gastric MALT lymphoma) and other tissues.
• Cutaneous T-cell lymphoma (mycosis fungoides) versus inflammatory dermatosis, using T-cell receptor gene studies together with clinical and histological findings.
• Distinguishing a clonal lymphoma from a reactive lymphoid hyperplasia, including in post-transplant lymphoproliferative disorders.
• Confirming that a relapse or a second lymphoma is related to the first (clonal relationship), by comparing the sequences.
• Staging: detection of marrow or blood involvement when morphology and flow cytometry are not clear.
• Minimal residual disease monitoring (sequencing based, in selected diseases) after treatment.
• Lymphoproliferation in patients with immunodeficiency or autoimmune disease.

Not indicated when morphology and immunophenotype already give a clear diagnosis, or as a screening test for lymphoma.

Specimen requirements.

• Fresh, frozen or formalin fixed, paraffin embedded tissue with an adequate proportion of lesional cells. DNA from old blocks is fragmented, so short amplicon assays are used.
• Peripheral blood, bone marrow aspirate or other fluid in EDTA.
• A matched control sample (normal tissue) for some sequencing based assays.
• Clinical and histological details, the immunophenotype and the question to be answered.
• Samples with few lesional cells can be microdissected.

Normal appearances.

A polyclonal result shows a broad, bell shaped peak distribution of amplified fragments (a Gaussian pattern) with no dominant peak, which means many different rearrangements. A sample with too few lymphocytes shows a few random, non-reproducible peaks (pseudoclonality).

Interpretation.

• Clonal: a dominant, reproducible peak (or sequence) of the expected size, seen in duplicate reactions, over the polyclonal background. The result is reported as "clonal rearrangement detected" in the tested gene.
• Polyclonal: no clonal product, which supports a reactive process, though it does not exclude a lymphoma, particularly when tumour cells are few.
• Oligoclonal or pseudoclonal: several small peaks in a small sample, and these are not reported as clonal.
• No amplification: DNA quality is poor, and the test is repeated or invalid.
• B-cell results: IGH (framework regions 1, 2 and 3) with kappa light chain testing increases sensitivity, because somatic hypermutation in germinal centre derived lymphomas can prevent primer binding.
• T-cell results: TRG and TRB testing in combination. TRG alone gives more false positives.
• Comparison of two samples: identical rearrangement sequences indicate a shared clone (relapse or spread), and different rearrangements suggest separate lymphomas.
• The result is always interpreted with histology, immunohistochemistry and the clinical picture, and clonality of a small, dominant population does not alone establish malignancy.

Critical values and findings needing immediate action.

• A clonal result in a suspected lymphoma, with high risk clinical features (rapid growth, airway compromise, cytopenias): urgent referral to haematology.
• Clonality in a transplant recipient with lymphoproliferation: review by the transplant team for reduction of immunosuppression and staging.
• A polyclonal or non-diagnostic result in a patient with strong clinical suspicion: repeat sampling and further tests, since the biopsy may have missed the lymphoma.
• Findings in the marrow or blood that change staging: communication to the haematology team.

Limitations and artefacts.

• Clonal does not mean malignant: benign clonal expansions occur in older people (T-cell clones), chronic infection, autoimmune disease (Sjögren syndrome, Hashimoto thyroiditis), inflammatory skin disease (lichen planus, lymphomatoid papulosis), and after vaccination.
• Pseudoclonality in very small samples, and in biopsies with few lymphocytes, gives false clonal peaks, and duplicate testing is essential.
• False negative results: low proportion of lesional cells, somatic hypermutation that blocks primers, and incomplete primer coverage.
• Degraded DNA from fixed tissue gives poor amplification, and large amplicons fail.
• Differences between laboratories in primer sets, cut offs and interpretation criteria.
• Results from capillary methods do not give sequence information, so two clones with the same size cannot be told apart.
• Sensitivity falls in cutaneous T-cell lymphoma at early stages, with only a small clone among reactive cells.
• Interpretation needs a pathologist or haematologist with experience.

Special groups.

• Older adults: small T-cell clones of uncertain significance are common, so interpretation is cautious.
• Patients with immunodeficiency, transplants or HIV: lymphoproliferative disorders can be polyclonal, oligoclonal or monoclonal, and the pattern is part of classification.
• Patients with autoimmune disease: B-cell clones in glandular tissue carry risk of marginal zone lymphoma, and follow up is planned.
• Patients with skin rashes and suspected mycosis fungoides: repeated biopsies and clonality, together with the clinical course.
• Children: reactive lymphoid proliferations are common, and a polyclonal result is the expected finding.
• Regions: clonality testing is available in a few centres, so samples are sent to reference laboratories. Immunohistochemistry and flow cytometry are tried first, and clonality testing is reserved for difficult cases. Infection related lymphoproliferation (Epstein-Barr virus, HIV) is common.

Follow up tests.

• Flow cytometry of tissue, blood or marrow for light chain restriction and aberrant immunophenotype.
• Immunohistochemistry panels for lymphoma subtyping.
• Fluorescence in situ hybridisation for translocations (MYC, BCL2, BCL6, IGH rearrangements).
• Next generation sequencing for mutations and sequence based clonality.
• Epstein-Barr virus testing in lymphoproliferation.
• Staging imaging (CT, PET-CT) and bone marrow biopsy.
• Repeat or larger biopsy of the lesion, or another site.
• Specialist haematology review.