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T-Cell Receptor Gene Rearrangement Detection in Suspected Cases of Cutaneous T-Cell Lymphoma

      Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of extranodal non-Hodgkin’s lymphoma that present on the skin with no evidence of extracutaneous disease at the time of diagnosis (
      • Willemze R.
      • Jaffe E.
      • Burg G.
      • et al.
      WHO-EORTC classification for cutaneous lymphomas.
      ). The general consensus is that CTCLs are typically monoclonal in origin, developing from a single T cell that forms a malignant clone of T cells in the skin (
      • Kandolf Sekulovic L.
      • Cikota B.
      • Stojadinovic O.
      • et al.
      TCR y-gene rearrangement analysis in skin samples and peripheral blood of mycosis fungoides patients.
      ). Mycosis fungoides (MF) and Sézary syndrome (SS) are the two most common types of CTCL, accounting for almost 65% of CTCLs. In the early stages of disease, MF can pose a diagnostic quandary for physicians and often leads to a delay in diagnosis despite multiple skin biopsies. This occurs because the neoplastic T cells in the skin in the early stages of MF are far outnumbered by the body’s immune response through tumor-reactive T cells and other effector cells in the skin. Together with the neoplastic T cells, these resemble a nonspecific inflammatory infiltrate on microscopy, making it difficult to distinguish from other benign dermatoses such as eczema or psoriasis (
      • Mishra A.
      • Porcu P.
      Early CTCL diagnosis, a (miR)age no more?.
      ). SS often has nonspecific features histologically or they are less marked changes than are typically seen in MF, thus also posing diagnostic difficulties from skin biopsy alone (
      • Willemze R.
      • Jaffe E.
      • Burg G.
      • et al.
      WHO-EORTC classification for cutaneous lymphomas.
      ).
      T-cell receptor (TCR) gene rearrangement studies demonstrated by PCR are used to detect clonality and can be a highly reliable method to aid in diagnosing CTCL (
      • Ponti R.
      • Fierro M.T.
      • Quaglino P.
      • et al.
      TCRγ-chain rearrangement by PCR-based GeneScan: diagnostic accuracy improvement and clonal heterogeneity analysis in multiple cutaneous T-cell lymphoma samples.
      ). The technique is dependent on the fact that T cells bear a unique antigen receptor on their cell surface that serves as a specific marker for that cell and its clonal progeny. If a cell undergoes neoplastic transformation, its TCR becomes a tumor marker specific to that cell lineage (
      • Wood G.S.
      T-cell receptor and immunoglobulin gene rearrangements in diagnosing skin disease.
      ). Using PCR and high-resolution electrophoresis, a TCR clone can be demonstrated in up to 90% of skin biopsies in MF cases (
      • Bottaro M.
      • Berti E.
      • Biondi A.
      • et al.
      Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.
      ).
      This article describes how the TCR gene rearrangement analysis is carried out and interpreted, as well as its advantages, limitations, and use in the diagnosis of CTCL.

      HOW IS TCR GENE REARRANGEMENT ANALYSIS PERFORMED?

      TCRs are protein heterodimers expressed on the cell surface of T lymphocytes. They are composed of either α and β chains (the vast majority) 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). TCRs are unique to individual T lymphocytes and are formed by the rearrangement and assembly of the TCR genes during T lymphocyte development in the thymus, a process called somatic recombination. The gene segments that encode the separate distinct regions of the TCR protein exist as multiple nonidentical sets. There are, for example, 12 TCR-γ V-region gene segments.
      Figure thumbnail fx1
      During T-cell maturation, the TCR genes undergo rearrangement. Thus, in TCR-γ chain assembly, a single V region from several V gene segments is positioned next to a single J region that has also been selected from several J gene segments to make a highly exclusive rearranged V/J gene region that is eventually expressed on the TCR-γ protein. To further add to the exclusivity of the TCR, there is then random loss of the original nucleotides at the ends where the V and J segments join. This loss is replaced by the insertion of N nucleotides at the joins. The result is a unique V/N nucleotide/J structure that serves as a “fingerprint” for that particular T cell and all its clonal progeny (
      • Wood G.S.
      T-cell receptor and immunoglobulin gene rearrangements in diagnosing skin disease.
      ;
      • Wood G.S.
      • Tung R.M.
      • Haeffner A.C.
      • et al.
      Detection of clonal T-cell receptor γ gene rearrangements in early mycosis fungoides/Sézary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE.
      ).
      TCR gene rearrangement analysis with PCR can be performed on any TCR gene. The TCR-γ gene is preferred because it is rearranged early in T-cell development both in α/β and in γ/δ T cells, and T cells generally retain intact rearranged TCR-γ genes although the protein may no longer be expressed on mature T cells. The γ gene structure is also simpler, with only 12 variable segments compared with other TCR genes such as the TCR-β gene, which has over 60 variable segments. Thus, fewer sets of primers are needed to detect most of the possible gene rearrangements (
      • Kandolf Sekulovic L.
      • Cikota B.
      • Stojadinovic O.
      • et al.
      TCR y-gene rearrangement analysis in skin samples and peripheral blood of mycosis fungoides patients.
      ;
      • Wood G.S.
      T-cell receptor and immunoglobulin gene rearrangements in diagnosing skin disease.
      ).
      The process involves three main steps: (1) extraction of genomic DNA from the skin specimen; (2) PCR-based amplification of the TCR-γ gene rearrangements from the genomic DNA; and (3) detection of the PCR products, most commonly by fractionation of the resulting products by gel electrophoresis (Figure 1) (
      • Wood G.S.
      • Tung R.M.
      • Haeffner A.C.
      • et al.
      Detection of clonal T-cell receptor γ gene rearrangements in early mycosis fungoides/Sézary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE.
      ).
      Figure thumbnail gr1
      Figure 1Method of TCR-γ gene rearrangement. (a) A skin biopsy specimen is taken from active lesions. (b) The skin specimen is placed into a container, which is placed on ice for transport. (c) The genomic DNA is isolated, and PCR amplification is started. (d) The PCR products are analyzed using gel electrophoresis. (c, Concept by E.K. Martin, Svenska Dagbladet.)
      d, Adapted from http://cg.scs.carleton.ca/~morin/teaching/compbio/electro.html.
      1. Extraction of genomic DNA from the skin specimen. Genomic DNA can be isolated from both fresh and formalin-fixed paraffin-embedded tissue. Although both methods are acceptable, fresh-frozen samples are preferred because of the higher sensitivity associated with isolating genomic DNA from them. If the specimen is in formalin-fixed paraffin, then DNA is isolated from paraffin blocks by deparaffinizing specimens by incubation with xylene, 100% ethanol, and acetone. After this, a mixture including proteinase K is added to the specimen and incubated overnight at 37 C or for 3 hours at 55 C (
      • Sprouse J.
      • Werling R.
      • Hanke D.
      • et al.
      T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.
      ).
      2. PCR-based amplification of the TCR-γ gene rearrangements from the genomic DNA. Once the genomic DNA is extracted, the PCR-based amplification process is started. Amplification is performed by adding genomic DNA as the template, combined with primer, nucleotides including all four bases (adenine, thymine, cytosine, and guanine), and DNA polymerase to a test tube. Next, the test tube is placed in a thermocycler machine that raises the temperature to denature the DNA, then lowers the temperature to allow primers and DNA to anneal, and finally raises the temperature again to allow the DNA polymerase to extend the primers by adding nucleotides to the DNA template strand (
      • Sprouse J.
      • Werling R.
      • Hanke D.
      • et al.
      T-cell clonality determination using polymerase chain reaction (PCR) amplification of the T-cell receptor gamma-chain gene and capillary electrophoresis of fluorescently labeled PCR products.
      ;
      • Guitart J.
      • Kaul K.
      A new polymerase chain reaction–based method for the detection of T-cell clonality in patients with possible cutaneous T-cell lymphoma.
      ). (See the recent Research Techniques Made Simple article on PCR (
      • Garibyan L.
      • Avashia N.
      Polymerase chain reaction.
      ) for a detailed description of the process.)
      3. Detection of the PCR products. The final step involves analysis of the PCR products by gel electrophoresis to separate the PCR products according to their nucleotide sequence and size. The gel contains a urea/formamidegradient that results in partial melting of the PCR products and therefore reduces the mobility at a point in the gradient determined by the nucleotide sequence. If there are enough numbers and fragments that are of identical size and sequence, as is produced by PCR of TCR genes from the genomic DNA of a monoclonal T-cell population, an intense clonal band is visible under UV light after the gel is stained with ethidium bromide (Figure 2). If there are heterogeneous fragments amplified from polyclonal cells, then only a diffuse smear is seen (Figure 3) (
      • Bottaro M.
      • Berti E.
      • Biondi A.
      • et al.
      Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.
      ; see Figure 1).
      Figure thumbnail gr2
      Figure 2PCR/denaturing gradient gel electrophoresis (DGGE): dominant T-cell clonal patterns. Discrete sharp bands are present in lanes 1–6, 8, 9, 11, 12, and 15. These bands reflect the presence of dominant TCR-γ gene rearrangements and contrast with the diffuse smears present in and also in lanes 7, 13, and 16–20, which correspond to the absence of detectable dominant TCR-γ gene rearrangements.
      Reprinted with permission from
      • Wood G.S.
      • Tung R.M.
      • Haeffner A.C.
      • et al.
      Detection of clonal T-cell receptor γ gene rearrangements in early mycosis fungoides/Sézary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE.
      .
      Figure thumbnail gr3
      Figure 3PCR/denaturing gradient gel electrophoresis (DGGE): polyclonal T-cell pattern. Vy1-8 PCR products (lanes 1–6) and Vy9 PCR products (lanes 7–13) were subjected to DGGE, stained with ethidium bromide, and photographed using UV illumination. Lanes 1–5 and 7–12 contain diffuse smears lacking discrete bands. This is the typical result in the absence of detectable dominant TCR-γ gene rearrangements. Lane 6 shows Vy1-8 carryover–negative control containing all reagents except target DNA.
      Reprinted with permission from
      • Wood G.S.
      • Tung R.M.
      • Haeffner A.C.
      • et al.
      Detection of clonal T-cell receptor γ gene rearrangements in early mycosis fungoides/Sézary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE.
      .

      ADVANTAGES

      Prior to the use of PCR and gel electrophoresis to detect clonality in suspected cases of CTCL, the Southern blot technique was commonly used. In comparison to the above technique, the Southern blot analysis is time consuming, requires large skin samples, and can detect only 5–10% of clonal subpopulations of cells (
      • Kandolf Sekulovic L.
      • Cikota B.
      • Stojadinovic O.
      • et al.
      TCR y-gene rearrangement analysis in skin samples and peripheral blood of mycosis fungoides patients.
      ).
      PCR has several additional advantages over Southern blot analysis. It is a much more sensitive technique that can be done with standard-size punch and shave biopsy specimens; in addition, it is less expensive than Southern blot analysis, and it takes only 2–3 days to complete. Unlike Southern blotting, which typically requires radiolabeled hybridization probes, PCR does not require radioactive agents. In addition, PCR can analyze lesions with sparse lymphocytic infiltrates, as is often the case in early-stage MF. Despite the low numbers of tumor cells, amplification ensures they are detected (
      • Bottaro M.
      • Berti E.
      • Biondi A.
      • et al.
      Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.
      ).

      LIMITATIONS

      Although the advent of PCR and gel electrophoresis has increased the sensitivity of detecting TCR-γ gene rearrangement, several limitations to this technique can lead to false-negative results. The TCR-γ gene of selected T lymphocytes may not amplify owing to factors such as section thickness and cell size variations or the degradation of DNA in the histological processing of tissue (
      • Gellrich S.
      • Lukowsky A.
      • Schilling T.
      • et al.
      Microanatomical compartments of clonal and reactive T-cells in mycosis fungoides: molecular demonstration by single cell polymerase chain reaction of T-cell receptor gene rearrangements.
      ). Other limitations include poor tissue sampling (i.e., a biopsy taken from a skin sample with a small number of malignant T cells) and the possibility that the primers used may not cover all possible TCR-γ gene rearrangements. Finally, in the later stages of the disease, TCR gene deletion is possible as the cell undergoes malignant transformation leading to nonamplification of TCR-γ gene sequences (
      • Kandolf Sekulovic L.
      • Cikota B.
      • Stojadinovic O.
      • et al.
      TCR y-gene rearrangement analysis in skin samples and peripheral blood of mycosis fungoides patients.
      ). False positives can also occur because of the presence of rare benign lymphoid infiltrates, leading to “pseudoclonal” results. Another cause of false positives is cross-contamination of specimens by clonal DNA from positive controls. For these reasons, it is not possible to rely on the results of the TCR gene rearrangement analysis solely to diagnose a patient with CTCL.

      WHEN IS TCR GENE REARRANGEMENT ANALYSIS USED IN DERMATOLOGY?

      The most common use of TCR gene rearrangement analysis is to aid in the diagnosis of CTCL. In the case of SS, the leukemic form of CTCL, the presence of a T-cell clone in the skin and blood, together with certain cytomorphologic features and immunophenotypic features in the blood, is helpful in making a diagnosis and differentiating from benign inflammatory conditions that can mimic this condition. TCR gene rearrangement has not been shown to improve with resolution of disease and thus cannot be used as a marker of disease activity.
      It is important to note that a positive clonality result does not necessarily equate to malignancy, as several benign dermatoses can produce a similar pattern, including lymphomatoid papulosis, pityriasis lichenoides et varioliformis acuta, lymphomatoid drug eruptions, actinic reticuloid, and lichen planus (
      • Gellrich S.
      • Lukowsky A.
      • Schilling T.
      • et al.
      Microanatomical compartments of clonal and reactive T-cells in mycosis fungoides: molecular demonstration by single cell polymerase chain reaction of T-cell receptor gene rearrangements.
      ). It is essential that clinical, histological, and immunological data be taken into consideration when making a diagnosis of CTCL and that the TCR gene rearrangement analysis is used as an adjunct to help make the case for or against a diagnosis of CTCL, rather than as the sole diagnostic feature.

      IS TCR GENE REARRANGEMENT ANALYSIS USED FOR ANYTHING ELSE?

      TCR gene rearrangement is an important event in T-cell ontogeny, and any disruption in this cycle may result in disease. T cells have been implicated in the pathogenesis of immunodeficiency, autoimmunity, and atopy. One of the most pivotal uses of TCR gene rearrangement analysis is in the diagnosis of lymphocytic disease and in distinguishing it from reactive lymphoid proliferations. It has been used in monitoring minimal residual disease after bone marrow transplantation, aiding in the diagnosis of immunodeficiency states, and characterizing T cells at disease sites in patients with allergy or autoimmune disease (
      • Bottaro M.
      • Berti E.
      • Biondi A.
      • et al.
      Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.
      ).

      SUMMARY AND FUTURE DIRECTIONS

      TCR gene rearrangement analysis with PCR and gel electrophoresis is a widely used adjunct in the diagnosis of CTCL. Most studies on TCR gene rearrangement have focused on the diagnosis of CTCL or response to therapy. Few have addressed whether these molecular methods are a useful staging tool for predicting prognosis. The technical advantages of PCR make it the method of choice for detecting clonality, and it will continue to be the method of choice until newer methods are shown to have advantages over this technique.
      MF disseminates to blood, lymph nodes, and viscera in the advanced stages. A positive clone in the blood is detected more frequently with progressive stages of cutaneous disease, such as those with cutaneous tumors or erythroderma. Studies have shown that the detection of a clone in dermatopathic lymph nodes (i.e., early-stage MF) can be associated with a worse prognosis. T-cell clones have even been found in the blood of a significant proportion of patients with early-stage MF, and the presence of a peripheral blood clone was shown to be associated with a worse outcome (
      • Bottaro M.
      • Berti E.
      • Biondi A.
      • et al.
      Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.
      ). To date, such studies have involved small numbers of patients, and studies with a larger patient population will be needed to confirm these observations and to evaluate how best to incorporate these data in the staging of MF.
      Figure thumbnail fx2
      MF is often preceded by a chronic nonspecific dermatitis that does not have diagnostic features of MF histologically, yet harbors a dominant T-cell clone. This observation has been termed “clonal dermatitis.” Molecular studies have demonstrated that both diseases can share the same monoclonal population. Thus, clonal dermatitis may represent a subgroup of patients who are at increased risk of developing clinically overt MF. Future studies could help determine their long-term risk of MF. It is necessary to closely follow these patients to diagnose lymphoma at the earliest possible time and to institute the correct treatment (
      • Bottaro M.
      • Berti E.
      • Biondi A.
      • et al.
      Heteroduplex analysis of T-cell receptor gamma gene rearrangements for diagnosis and monitoring of cutaneous T-cell lymphomas.
      ).
      What does the future hold for this molecular technique? This highly sensitive tool enables earlier and more accurate diagnosis of CTCL. With further studies and greater understanding of the pathogenesis of cutaneous lymphomas, these tools may be used for more precise disease staging and the ability to provide better prognostic information for patients, thus allowing for more tailored treatment (
      • Wood G.S.
      T-cell receptor and immunoglobulin gene rearrangements in diagnosing skin disease.
      ;
      • Gellrich S.
      • Lukowsky A.
      • Schilling T.
      • et al.
      Microanatomical compartments of clonal and reactive T-cells in mycosis fungoides: molecular demonstration by single cell polymerase chain reaction of T-cell receptor gene rearrangements.
      ).

      CME ACCREDITATION

      This CME activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education through the Joint Sponsorship of ScientiaCME and Educational Review Systems. ScientiaCME is accredited by the ACCME to provide continuing medical education for physicians. ScientiaCME designates this educational activity for a maximum of one (1) AMA PRA Category 1 Credit. Physicians should claim only credit commensurate with the extent of their participation in the activity.
      To take the online quiz, follow the link below: http://www.classmarker.com/online-test/start/?quiz=xh352d98e54d1a8a

      SUPPLEMENTARY MATERIAL

      A PowerPoint slide presentation appropriate for journal club or other teaching exercises is available at http://dx.doi.org/10.1038/jid.2014.73

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