T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides.

BACKGROUND: The diagnosis of mycosis fungoides (MF) is often difficult because of significant clinical and histopathologic overlap with inflammatory dermatoses. T-cell receptor (TCR)gamma chain rearrangement by polymerase chain reaction (PCR) (TCR-PCR) is a helpful adjuvant tool in this setting, but several of the inflammatory dermatoses in the differential diagnosis of MF may contain a clonal T-cell proliferation. OBJECTIVE: We examined whether analysis for T-cell clonality and comparison of the clones with the standardized BIOMED-2 PCR multiplex primers for the TCRgamma chain from two anatomically distinct skin sites improves diagnostic accuracy. METHODS: We examined two biopsy specimens each from 10 patients with unequivocal MF, from 18 patients with inflammatory dermatoses, and from 18 patients who could initially not be definitively given a diagnosis based on clinical and histopathologic criteria. RESULTS: Eight of 10 patients with unequivocal MF had an identical clone in both biopsy specimens. Two of 18 patients with inflammatory dermatoses were found to have a clone in one of the biopsy specimens. On further follow-up of the 18 patients with morphologically nondiagnostic biopsy specimens, 13 of 18 were later confirmed to have MF and 5 of 18 had inflammatory dermatoses. Eleven of 13 patients with MF had an identical clone in both biopsy specimens; two of 13 had a polyclonal amplification pattern in both biopsy specimens. Four of 5 patients with inflammatory dermatoses had no clone in either biopsy specimen. One patient with an inflammatory dermatosis had an identical clone in both specimens. The sensitivity of TCR-PCR analysis to evaluate for an identical clone at different anatomic skin sites (dual TCR-PCR) is 82.6% and the specificity is 95.7%. LIMITATIONS: The number of patients in the study group was limited. CONCLUSION: These data suggest that dual TCR-PCR is a very promising technique with high specificity in distinguishing MF from inflammatory dermatoses.

Identification of endogenous HLA-A2-restricted reactivity against shared melanoma antigens in patients using the quantitative real-time polymerase chain reaction.

This study was conducted to determine whether reactivity to melanoma cells of pretreatment peripheral blood mononuclear cells (PBMCs) from patients with metastatic melanoma correlated with subsequent response to treatment with interleukin-2 (IL-2). The sensitivity of the quantitative real-time polymerase chain reaction (PCR) assay was optimized, including the total number of cells used (3 x 10(6) in 1 mL), the responder-to-stimulator cell ratio (5:1), the optimal time to incubate PBMCs with tumor (2 h), the appropriate tumor stimulators (melanoma cell lines differing only in the expression of histocompatibility leukocyte antigen [HLA-A2]), the duration of recovery in the culture of PBMCs after cryopreservation (18-24 h), and the medium used (Iscove, 10% human AB serum). Using this optimized assay to detect HLA-A2-restricted antitumor reactivity in the pretreatment PBMCs from patients with melanoma, positive reactive responses were detected in 7 of 28 patients with an objective clinical response to IL-2 therapy compared with 6 of 21 positive reactive responses in nonresponding patients. None of 12 healthy donors were positive in this study. Thus, there was no significant difference in the reactivity of pretreatment PBMCs when responders were compared with nonresponders, although the melanoma patients had an increased incidence of response compared with healthy donors (p = 0.05). The PBMCs from 11 of the 13 melanoma patients with pretreatment HLA-A2-restricted antimelanoma reactivity were tested against a panel of transfectants expressing known shared melanoma antigens. Anti-MART-1 reactivity was detected in the pretreatment PBMCs of three patients. It thus appears that some melanoma patients are immunologically primed to antigens expressed on the tumor surface, although the HLA-A2-restricted antimelanoma activity detected in this real-time PCR assay was not predictive of patients' responses to IL-2 therapy.