TECHNICAL Assessment of T-cell Clonality via T-cell Receptor-  Rearrangements in Cutaneous T-cell–Dominant Infiltrates Using Polymerase Chain Reaction and Single-stranded DNA Conformational Polymorphism Assay Michael Chen, BA,* April Deng, MD, PhD,‡ A. Neil Crowson, MD,§ Mythily Srinivasan, PhD,  Kurtis H. Yearsley, PhD,† Scott Jewell, PhD,† Carl Morrison, MD,† Susan Long, BA,† Robert Werling, MD,† and Cynthia Magro, MD† Abstract: Discerning the pathologic significance of cutaneous T- cell infiltrates can pose a diagnostic challenge for dermatopatholo- gists. Reactive conditions such as drug-associated lymphomatoid hy- persensitivity and lymphomatoid lupus erythematosus can demon- strate lymphoid atypia and a phenotype resembling cutaneous T-cell lymphoma (CTCL). Further, lymphoid dyscrasias such as pityriasis lichenoides chronica, large plaque parapsoriasis, and atypical pig- mentary purpura confuse the picture because they not only mimic CTCL but also represent prelymphomatous states with inherent ma- lignant potential. Although the emergence of a dominant clone has been considered a clue indicative of a T-cell dyscrasia, there are re- ports concerning the identification of monoclonality in biopsies of reactive lymphoid infiltrates. We have conducted a modified single- stranded DNA conformational polymorphism (SSCP) assay using paraffin-embedded, formalin-fixed tissue on 92 T-cell–rich biopsies to determine the relative specificity and sensitivity of this methodol- ogy. In addition, laser capture microdissection (LCM) was performed on 22 of the 92 samples to isolate the area of interest and to compare its specificity and sensitivity with those SSCP assays performed with- out LCM. We found that monoclonality or oligoclonality is 86% spe- cific for preneoplastic and neoplastic states, whereas the finding of polyclonality appears to be relatively specific for a reactive process. Some cases of reversible T-cell dyscrasia produced a molecular pro- file mimicking lymphoma or prelymphomatous states by virtue of monoclonality or oligoclonality. Although LCM appears to improve the sensitivity for detecting preneoplastic conditions, the relative specificity appears to be the same as that encountered with routine SSCP. Key Words: T-cell clonality, cutaneous T-cell–rich infiltrates (Appl Immunohistochem Mol Morphol 2004;12:373–379) A lthough molecular analysis to establish clonality was originally confined to frozen tissue using a Southern blot technique, 1 studies are now routinely performed on paraffin- embedded, formalin-fixed tissue using the polymerase chain reaction (PCR) assay. Although demonstration of monoclonal- ity by PCR has been held to be a finding characteristic of cu- taneous T-cell lymphoma (CTCL), 1–4 cutaneous disorders other than CTCL may also exhibit T-cell monoclonality by PCR. In particular, lesions of parapsoriasis, lymphomatoid lu- pus profundus, 5 lymphomatoid papulosis (LyP), 6 pityriasis li- chenoides chronica (PLC), 7,8 idiopathic and drug-associated atypical pigmentary purpura, 9,10 and drug-associated lympho- matoid hypersensitivity 10–12 have all been shown to manifest monoclonality by T-cell receptor (TCR)- solution phase PCR analysis. These conditions represent either preneoplastic or re- active conditions in the setting of iatrogenic or endogenous immune dysregulation or both. In contrast to the monoclonal- ity seen in CTCL, one would expect that preneoplastic lesions would manifest oligoclonality and that reactive conditions would demonstrate polyclonality. 11,13 In this study, PCR and single-stranded DNA conformational polymorphism (SSCP) assay for TCR- gene rearrangement were used to analyze T- cell clonality in formalin-fixed, paraffin-embedded tissues. Our goal was to assess its sensitivity and specificity as a diag- nostic adjunct in the separation of lymphoma, preneoplastic pseudolymphomatous reactive states, and nonlymphomatoid reactive infiltrates. In addition, we assessed the potential for laser capture microdissection (LCM) to enhance the specificity and sensitivity of the assay. We used the PCR/SSCP assay on Manuscript received August 25, 2003; accepted November 18, 2003. From the *College of Medicine and Public Health and †Department of Pathol- ogy, Ohio State University, Columbus, Ohio; the ‡Dermatology Depart- ment, Maryland University Medical System, Baltimore, Maryland; the §Departments of Dermatology and Pathology, University of Oklahoma and Regional Medical Laboratory, St. John Medical Center, Tulsa, Okla- homa; and the  Department of Oral Pathology, Indiana University School of Dentistry, Indianapolis, Indiana. Reprints: Cynthia Magro, MD, Department of Pathology, Ohio State Univer- sity, N305 Doan Hall, 410 West Tenth Avenue, Columbus, OH 43210 (e-mail: magro-1@medctr.osu.edu). Copyright © 2004 by Lippincott Williams & Wilkins Appl Immunohistochem Mol Morphol • Volume 12, Number 4, December 2004 373