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T-Cell Receptor and Immunoglobulin Gene Rearrangements in Diagnosing Skin Disease
Gary S. Wood, MD
Arch Dermatol. 2001;137:1503-1506.
INTRODUCTION
During the past few decades, there have been major advances in our understanding of the human immune system and its associated lymphoproliferative disorders, including those involving the skin. These advances have been facilitated by the development of new genomic technologies that have led not only to more accurate classification and diagnosis of these diseases but also to fresh insights into their pathogenesis and improved means of staging disease, monitoring response to therapy, and detecting early relapse. Rather than exhaustively summarizing this field, the purpose of this review is to provide an overview of key advances, emphasizing their current and future relevance to general dermatologists. Most of the issues raised in this article are discussed and referenced in greater detail in a recent comprehensive textbook chapter.1
RELEVANT GENOMIC TECHNOLOGIES AND THE CONCEPT OF CLONALITY
The most important advance in the molecular immunological features of lymphomas has been the recognition that each normal T and B cell bears a unique antigen receptor on its cell surface that serves as a specific marker for that cell and all of its clonal progeny. If the cell should undergo malignant transformation, then this same structure becomes a tumor-specific marker, as well. For B cells, this marker is the immunoglobulin (Ig) molecule. For T cells, it is the T-cell receptor (TCR).2-6
The basic structure of Ig and TCR molecules is the same. They both consist of protein heterodimers expressed on the cell surface. Immunoglobulin molecules are composed of heavy and light chains. T-cell receptors are composed of either  and  chains (about 95% of mature T cells) or  and  chains. Each of these Ig and TCR chains is, in turn, composed of several distinct regions (termed variable [V], joining [J], constant, and sometimes diversity). The gene segments encoding each of these separate regions of the Ig and TCR proteins exist in multiple nonidentical sets. For example, there are approximately 12 TCR V region gene segments.
During the development of an individual T cell, its TCR genes undergo rearrangement such that a single V region is brought into apposition with a single J region, and only this single rearranged V/J gene region is expressed in the final TCR protein. The selection of only one V and one J region from among many imparts a partial uniqueness to this TCR protein. Its uniqueness is further enhanced by the random loss of original (germline) nucleotides at the ends where the V and J regions join and the insertion of so-called N nucleotides to patch this gap. Thus, the resulting V/N nucleotide/J structure acquires a unique nucleotide sequence across this region that serves as an identifying "signature" or "fingerprint" for that cell and all of its clonal progeny. The same principle applies to each Ig and TCR gene and its corresponding transcript and protein. Therefore, these structures can serve as tumor clone–specific markers for B- and T-cell lymphomas.
Initially, these clone-specific Ig and TCR gene rearrangements were detected using Southern blot analysis. For various technical reasons, Southern blotting has been replaced largely by gene amplification techniques involving the polymerase chain reaction (PCR). Polymerase chain reaction–based methods have several advantages over conventional Southern blotting, including suitability for smaller tissue samples, enhanced sensitivity, more rapid results, avoidance of radioactive labeling, and decreased cost.
Many different PCR-based antigen receptor gene rearrangement assays have been developed for the analysis of cutaneous lymphoid infiltrates. Among those most widely used for studying T-cell clonality is analysis of TCR gene rearrangements using denaturing gradient gel electrophoresis. The TCR gene is targeted because its relatively simple structure requires fewer sets of PCR primers to detect most possible gene rearrangements and because T cells generally retain intact rearranged TCR genes even though most no longer express TCR proteins once they become mature. For B-cell clonality, many widely used assays involve amplification of Ig heavy chain (IgH) gene rearrangements.
The application and interpretation of these PCR clonality assays require recognition of their special features and limitations. It is impossible to judge the significance of a positive result (ie, the detection of a clonal gene rearrangement, implying the presence of a clonal lymphoid population) without first knowing the clonal detection threshold of the particular assay being used. If the assay is too sensitive, then many potentially irrelevant positive results can be detected because many inflammatory lymphoid infiltrates contain low levels of clonal lymphoid populations. In fact, this feature has made such assays useful for studying clonally restricted but reactive T and B cells in a wide variety of inflammatory skin diseases. However, for the practical purpose of diagnosing lymphomas, it appears that a detection threshold of about 1% is useful. There are few situations in which a truly inflammatory or reactive lymphoid infiltrate will have clonal T- or B-cell populations above this level. The PCR/denaturing gradient gel electrophoresis and the PCR/IgH assays in widest use generally satisfy this requirement.
False-negative results can occur for several reasons: (1) the primers used may not detect all possible gene rearrangements, (2) the particular gene targeted might not be rearranged in certain cases, (3) the relevant gene rearrangement may have been deleted from the tumor's genome, and (4) the tumor clone's concentration may fall below the detection threshold due to poor sampling or sparse lesional infiltrates. False-positive results can also occur for several reasons: (1) certain diseases that are not considered to be lymphomas can often exhibit clonal lymphoid populations (eg, the parapsoriasis group and others, as discussed later); (2) in some cases, rare benign lymphoid subsets can be selectively amplified by PCR primers, thereby giving rise to "pseudoclonal" results; and (3) specimens can become cross-contaminated with clonal DNA from other cases or positive controls.
These potential pitfalls make it important that clonality assays be performed by individuals with an extensive knowledge of not only the molecular methods used but also the clinicopathologic features of all possible diseases in the differential diagnosis. In general, the most accurate interpretation results from a careful integration of clinical, pathological, immunopathological, and molecular findings. For this reason, multidisciplinary programs specializing in the diagnosis and management of skin lymphomas and related diseases have arisen at regional medical centers throughout much of the world.7
ADVANCES IN PATHOGENESIS
Although primary cutaneous lymphomas are regarded as being localized in the skin from a clinicopathologic perspective, it is becoming increasingly apparent that these lymphomas are actually systemic illnesses in which tumor cells accumulate in the skin but also involve other tissues in a subclinical manner. For example, studies8 of mycosis fungoides using highly sensitive tumor-specific gene rearrangement assays capable of detecting 1 of 100 000 tumor cells have shown that even patients with early stage IA disease can have tumor cells within their blood, lymph nodes, and bone marrow. This indicates that early mycosis fungoides is a systemic disease, at least in a biological if not a clinical sense. This is consistent with the view that mycosis fungoides is a lymphoma of T cells belonging to the skin-associated lymphoid tissue. These cells circulate throughout the body but traffic selectively through the skin by means of special homing receptors that bind to molecules preferentially expressed by cutaneous blood vessels. The systemic nature of mycosis fungoides may help explain why topically directed therapies are rarely curative, and forms one rationale for the development of novel systemic treatments.
It has long been appreciated that patients with mycosis fungoides are at an increased risk for the development of associated lymphoproliferative disorders, such as lymphomatoid papulosis, large T-cell lymphoma, and Hodgkin disease. A comparative analysis9 of TCR gene rearrangements in patients with more than one of these diseases has shown that these disorders share the same clonal TCR gene rearrangements when they occur in the same individual. This indicates that these secondary diseases are associated clinically with mycosis fungoides because they arise as subclones of the original mycosis fungoides T-cell clone. The related question of how these diseases differ genetically from mycosis fungoides is the subject of investigation, and this investigation will probably lead to further advances in understanding their pathogenesis.
Longitudinal studies have shown that early mycosis fungoides can be preceded by chronic dermatitis that harbors a dominant T-cell clone (so-called clonal dermatitis). Molecular studies3, 10 have shown that both diseases can share the same monoclonal T-cell population. In conjunction with other evidence, this suggests that mycosis fungoides can arise from a background of chronic inflammation via the gradual selection of one dominant T-cell clone that becomes increasingly malignant over time, probably as a result of sequential somatic mutations.
A similar situation exists in the relationship between cutaneous lymphoid hyperplasia and cutaneous B-cell lymphoma. Some of these lymphomas have been preceded by cutaneous lymphoid hyperplasia harboring the same dominant B-cell clone (so-called clonal cutaneous lymphoid hyperplasia).11 Thus, the principle has emerged that cutaneous lymphomas do not necessarily arise de novo but can instead develop gradually from different types of chronic inflammatory processes.1
ADVANCES IN CLASSIFICATION
Although there are several alternative classifications for lymphomas in general, the Dutch Lymphoma Group has proposed a useful classification that pertains specifically to primary cutaneous lymphomas.12 It covers most of these tumors, and reflects 2 important molecular immunological features: all tumors included in this classification are clonal disorders, and all are divided into T- or B-cell types.
In addition to the overt skin lymphomas, molecular testing has disclosed several other skin diseases that are recognized to be lymphoproliferative disorders based on their dominant clonal T- or B-cell populations. Among others, these include all members of the parapsoriasis group (large- and small-plaque parapsoriasis, lymphomatoid papulosis, and acute and chronic forms of pityriasis lichenoides), clonal cutaneous lymphoid hyperplasia, and clonal dermatitis.
ADVANCES IN DIAGNOSIS
Analysis of TCR and IgH gene rearrangements provides a powerful diagnostic tool that complements other diagnostic testing, such as routine histopathological and immunopathological testing. Holistic integration of this information allows diagnoses to be made earlier and with more confidence, especially in those cases lacking classic morphological features. For example, clinically nodular skin lesions composed of atypical lymphoid infiltrates that exhibit abnormal patterns of antigen expression and contain molecular evidence of dominant clonality are usually regarded as lymphomas, even when this diagnosis cannot be made on morphological grounds alone.
Similarly, cutaneous patches containing superficial T-cell infiltrates with deletion of certain antigens and the presence of dominant clonality are often diagnosed as mycosis fungoides, even when the histopathological features are not fully diagnostic. Furthermore, the molecular detection of a dominant B-cell clone, combined with the presence of a few atypical B cells within a predominantly T-cell infiltrate, has allowed the recognition of an otherwise difficult-to-diagnose tumor known as the "T-cell–rich B-cell lymphoma."
The earlier recognition of these lymphomas, made possible by ancillary immunopathological and molecular biological assays, has spared afflicted patients the delay, inconvenience, morbidity, and cost of additional diagnostic testing, and has allowed the more rapid institution of specific therapy.
ADVANCES IN STAGING AND MONITORING
Once a diagnosis of lymphoma has been established, TCR or IgH gene rearrangement assays can also be used to determine the disease stage of patients and to monitor their response to therapy. Using methods with a clonal detection threshold of 1% or more, it is likely that detection of occult extracutaneous involvement will prove clinically significant. For example, initial studies13 have demonstrated that occult involvement of lymph nodes by mycosis fungoides is prognostically relevant.
Because of their enhanced sensitivity relative to routine histological testing, molecular assays can more accurately define remission and detect early relapse. In my experience, patients who stop treatment when representative skin biopsy specimens are nonspecific histologically but still positive by molecular analysis tend to relapse rapidly (unpublished data, 2000).
THE FUTURE
Although this review has focused on molecular analysis of TCR and IgH gene rearrangements, many other genetic features can be assessed using molecular methods. These include chromosomal translocations such as the t(2;5) that is present in some cases of pediatric cutaneous CD30+ large-cell lymphoma.14 This translocation results in the inappropriate expression of a tyrosine kinase, known as anaplastic lymphoma kinase, that is postulated to confer a growth advantage to the neoplastic lymphoid clone. Other genetic alterations may be more important for tumor progression than tumor initiation. For example, some patients with mycosis fungoides undergo transformation of their tumor clone into a large-cell lymphoma. These transformed cases sometimes contain mutations in the cell cycle–associated p53 gene that are not found in typical mycosis fungoides.15 This suggests a role for p53 in the progression of some skin lymphomas. In addition to providing insights into the pathogenesis of cutaneous lymphoproliferative disorders, knowledge about alterations in genes like ALK and p53 may one day form the basis for novel treatments targeted to specific gene defects.
Another genetic feature with relevance to treatment that can be studied is a lymphoma's pattern of cytokine gene expression. For example, T-cell lymphomas with a TH2 pattern (interleukins 4, 5, and 10) are likely to be good candidates for therapy with agents such as interleukin 2 and interleukin 12, which antagonize TH2 lymphoid cells.16 Several cutaneous T-cell lymphomas, including mycosis fungoides and Sézary syndrome, fall into this category. Clinical trials based on this approach are under way.
The recent advent of novel molecular technologies such as microarrays and gene chips has ushered in a new chapter in the genetic analysis of skin lymphomas. These methods allow the simultaneous determination of the expression of hundreds or thousands of genes. This has afforded us the ability to study the coordinated expression of entire families and networks of genes. This new type of information has already defined previously unrecognized prognostic subgroups among otherwise similar lymphomas and promises to provide further insights into lymphoma pathogenesis and tumor-specific therapy.
RELEVANCE TO THE GENERAL DERMATOLOGIST
What does all this mean to the general dermatologist? First, it means that we have been able to use modern immunological methods to redefine a wide array of long-recognized but poorly understood skin diseases. By putting these disorders in their true biological context, we have laid the foundation for their eventual cure. Second, it means that we have sensitive new tools available that allow us to make diagnoses earlier and with greater confidence, determine the disease stage of patients more accurately, and monitor patients' response to therapy more precisely. No longer must we rely solely on the venerable but dated technology of light microscopy to accomplish these tasks. Third, it means that we are rapidly expanding our understanding of the pathogenesis of cutaneous lymphomas. This should lead to significant advances in our ability to prognosticate and to improve outcome based on the rational selection of therapy tailored to the unique genetic features of each specific case. Last, but certainly not least, it means that state-of-the-art diagnosis and management of cutaneous lymphoproliferative disorders is a complex process. As a consequence, multidisciplinary teams of skin lymphoma specialists have been established at regional medical centers that offer the latest in diagnostic technology and novel treatment.7 General dermatologists should become familiar with such programs in their area and take full advantage of their consultative resources.
AUTHOR INFORMATION
Accepted for publication July 18, 2001.
This study was supported by grant K24 02136 from the National Institutes of Health, Bethesda, Md, and by Merit Review funding from the Department of Veterans Affairs, Washington, DC.
Corresponding author: Gary S. Wood, MD, Department of Medicine (Dermatology), University of Wisconsin, 1 S Park St, Floor 7, Madison, WI 53715.
From the Department of Medicine (Dermatology), University of Wisconsin, and the Middleton Veterans Affairs Medical Center, Madison.
REFERENCES
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1. Wood GS. The benign and malignant cutaneous lymphoproliferative disorders including mycosis fungoides. In: Knowles DM, ed. Neoplastic Hematopathology. 2nd ed. Baltimore, Md: Williams & Wilkins; 2001:1183-1233.
2. Wood GS. Cutaneous lymphoproliferative disorders: strategies for molecular biological analysis and their major findings. Springer Semin Immunopathol. 1992;13:387-399.
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3. Wood GS, Tung RM, Haeffner AC, et al. Detection of clonal T-cell receptor gene rearrangements in early mycosis fungoides/Sézary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE). J Invest Dermatol. 1994;103:34-41.
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4. Wood GS, Dummer R, Haeffner A, Crooks CF. Molecular biologic techniques for the diagnosis of CTCL. In: Burg G, Kerl H, Thiers B, eds. Dermatologic Clinics of North America: Cutaneous Lymphomas. Vol 12. Philadelphia, Pa: WB Saunders Co; 1994:231-241.
5. Wood GS, Uluer AZ. Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE): sensitivity, band pattern analysis and methodologic optimization. Am J Dermatopathol. 1999;21:547-551.
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6. Nihal M, Mikkola D, Wood GS. Detection of clonally restricted immunoglobulin heavy chain gene rearrangements in normal and lesional skin: analysis of the B cell component of the skin-associated lymphoid tissue and implications for the molecular diagnosis of cutaneous B cell lymphomas. J Mol Diagn. 2000;2:5-10.
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7. Parry EJ, Stevens SR, Gilliam AC, et al. Management of the cutaneous lymphoproliferative disorders: a multidisciplinary approach. Arch Dermatol. 1999;135:907-911.
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8. Veelken H, Wood GS, Sklar J. Molecular staging of cutaneous T-cell lymphoma: evidence for systemic involvement in early disease. J Invest Dermatol. 1995;104:889-894.
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9. Wood GS, Crooks CF, Uluer AZ. Lymphomatoid papulosis and associated cutaneous lymphoproliferative disorders exhibit a common clonal origin. J Invest Dermatol. 1995;105:51-55.
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10. Siddiqui J, Hardman DL, Misra M, Wood GS. Clonal dermatitis: a potential precursor of CTCL with varied clinical manifestations [abstract]. J Invest Dermatol. 1997;108:609.
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11. Wood GS, Ngan B-Y, Tung R, et al. Clonal rearrangements of immunoglobulin genes and progression to B-cell lymphoma in cutaneous lymphoid hyperplasia. Am J Pathol. 1989;135:13-19.
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12. Willemze R, Kerl H, Sterry W, et al. EORTC classification for primary cutaneous lymphomas: a proposal from the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer. Blood. 1997;90:354-371.
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13. Kern DE, Kidd PG, Moe R, Hanke D, Olerud JE. Analysis of T-cell receptor gene rearrangement in lymph nodes of patients with mycosis fungoides: prognostic implications. Arch Dermatol. 1998;134:158-164.
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14. Gould JW, Eppes RB, Gilliam AC, et al. Solitary primary cutaneous CD30+ large cell lymphoma of natural killer cell phenotype bearing the t(2;5)(p23;q35) and presenting in a child. Am J Dermatopathol. 2000;22:422-428.
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15. Marrogi AJ, Khan MA, Vonderheid EC, Wood GS, McBurney E. p53 tumor suppressor gene mutations in transformed cutaneous T-cell lymphoma: a study of 12 cases. J Cutan Pathol. 1999;26:369-378.
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16. Rook AH, Wood GS, Yoo E, et al. Interleukin-12 therapy induces lesion regression and cytotoxic T-cell responses in cutaneous T-cell lymphoma. Blood. 1999;94:902-908.
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