Presentation on theme: "Research Techniques Made Simple: T-Cell Receptor Gene Rearrangement Pooja Chitgopeker and Debjani Sahni Department of Dermatology, Boston University and."— Presentation transcript:
Research Techniques Made Simple: T-Cell Receptor Gene Rearrangement Pooja Chitgopeker and Debjani Sahni Department of Dermatology, Boston University and Boston Medical Center, Boston, Massachusetts, USA
Introduction Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of non-Hodgkin’s 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
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
T-Cell Receptor Structure TCRs are composed of either α and β chains or γ and δ 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-γ is preferred as it is a much simpler gene compared to the other TCR genes and it is rearranged early in T-cell development
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 γ gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE). Journal of Investigative Dermatology. 1994;103(1):34-41.
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
Limitations TCR-γ 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-γ 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
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
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 method— need 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-γ gene rearrangement