Research Techniques Made Simple: T-Cell Receptor Gene Rearrangement

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Research Techniques Made SimpleT-Cell Receptor
Gene Rearrangement

• Pooja Chitgopeker and Debjani Sahni
•  
• Department of Dermatology, Boston University and
  Boston Medical Center, Boston, Massachusetts, USA

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Introduction

• Primary cutaneous T-cell lymphomas (CTCLs) are a
  heterogeneous group of non-Hodgkins lymphoma
  that present on the skin with no evidence of
  extracutaneous disease at the time of diagnosis
• Mycosis fungoides (MF) and Sezary syndrome (SS)
  are the two most common types of CTCL
• General consensus is that CTCLs are monoclonal in
  origin
• T-cell receptor (TCR) gene rearrangement studies
  demonstrated with PCR are used to detect
  clonality and aid in the diagnosis of primary CTCL

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What Is TCR Gene Rearrangement?

• Each normal T cell bears a unique antigen
  receptor on its cell surface
• This serves as a specific marker for that cell
  and its clonal progeny
• If the cell becomes malignant, then this TCR will
  become a tumor marker specific to that cell
  lineage
• TCR clone can be demonstrated in up to 90 of
  skin biopsies in MF cases using PCR and gel
  electrophoresis

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T-Cell Receptor Structure

• TCRs are composed of either a and ß chains or ?
  and d chains
• Each of these TCR chains is composed of several
  distinct regions, called variable (V), joining
  (J), diversity (D), and constant (C)
• During T-lymphocyte development in the thymus,
  various regions from V, D, and J gene regions of
  the TCR gene join to make a unique final TCR
  protein
• TCR-? is preferred as it is a much simpler gene
  compared to the other TCR genes and it is
  rearranged early in T-cell development

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How Is TCR Gene Rearrangement Performed?
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How Is TCR Gene Rearrangement Performed?

1. Skin biopsy is taken from active lesions
2. The biopsy specimen is placed in a small test
   tube and transported over ice
3. The genomic DNA is extracted from the tissue and
   PCR-based amplification is started
4. PCR products are analyzed using gel
   electrophoresis here we are looking for the
   presence of a monoclonal band to indicate a
   positive result

Wood et al. Detection of clonal T-cell receptor
? gene rearrangements in early mycosis
fungoides/Sezary syndrome by polymerase chain
reaction and denaturing gradient gel
electrophoresis (PCR/DGGE). Journal of
Investigative Dermatology. 1994103(1)34-41.
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Advantages
Prior to PCR and gel electrophoresis, the Southern blot technique was used. Compared to Southern blot, PCR
1. Is less time-consuming
2. Requires only a standard-size punch and shave biopsy
3. Takes only 2-3 days to complete
4. Does not require radioactive agents
5. Can analyze lesions with sparse lymphocytic infiltrates
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Limitations
TCR-? gene may not amplify due to
1. Loss of genes or cells/cell size variation and section thickness
2. Degradation of DNA while processing the tissue
False-negative results from the test may be produced due to
4. Biopsy taken from skin with a small number of malignant T cells
5. TCR gene deletion is possible as the cell undergoes malignant transformation
6. Primers may not cover all the possible TCR-? gene rearrangements
False-positive results from the test may be produced due to
7. High sensitivity of PCR thus, it can detect a dominant clone in other cases of histologically proven chronic dermatitis
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When Is It Used?

• Most commonly aiding in the diagnosis of CTCL
• In SS, presence of a T-cell clone in the skin and
  blood along with certain other cytomorphological
  and immunophenotypic features in the blood aid in
  diagnosis and monitor and evaluate disease
• Identification of lineage evolution and monitor
  and evaluate disease
• Other uses aid in diagnosis of immunodeficiency
  and characterize T cells at disease sites in
  patients with allergy or autoimmune disease

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Summary and Future Directions

• TCR gene rearrangement analysis with PCR and gel
  electrophoresis is a useful adjunct in the
  diagnosis of CTCL when used with clinical,
  histopathological, and immunological studies
• T-cell clones have been found in early-stage MF.
  Relevance for use in additional staging
  methodneed further studies to assess
• Studies are needed to determine the long-term
  risk of MM/SS among patients with other
  nonspecific dermatitis who have positive clonal
  TCR-? gene rearrangement