RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi

Abstract

Melanoma antigen recognized by T cells 1 (MART-1) and tyrosinase-related protein-2 (TRP-2) are two useful markers for immunohistochemical detection of melanocytic tumors. However, these markers may be passively acquired (phagocytosed) rather than actively synthesized. Reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) can amplify even small amounts of specific mRNA in cells and therefore confirm the cellular source of a marker. We developed a one-step RT in situ PCR procedure in which Thermus thermophilus DNA polymerase synthesizes and amplifies cDNA from mRNA in a single reaction mixture. To examine its practicability and feasibility with formalin-fixed, paraffin-embedded (FFPE) tissue, we compared the results of one-step RT in situ PCR with those of immunohistochemistry (IHC). MART-1 mRNA was identified in the cytoplasm of lesional cells from 23/26 primary melanomas (92%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). MART-1 epitope was detected by IHC in 23/24 primary melanomas (96%), 9/9 metastatic melanomas (100%) and 5/6 nevi (83%). TRP-2 mRNA was identified in the cytoplasm of lesional cells from 17/26 primary melanomas (65%), 6/9 metastatic melanomas (67%) and 4/6 nevi (67%). TRP-2 epitope was detected by IHC in 20/ 24 primary melanomas (83%), 9/9 metastatic melanomas (100%) and 4/6 nevi (67%). Both techniques detected MART-1 and TRP-2 in FFPE melanoma cell lines. Neither marker was detected in squamous cell carcinomas or basal cell carcinomas by RT in situ PCR or IHC. We conclude that the RT in situ PCR technique can be successfully applied to FFPE tissue to determine the cellular sources of gene expression observed by conventional PCR approaches.

Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2

“…This is illustrated by the generation of serological reagents such as mAb T311 to tyrosinase and mAbs A103 or M2-7C10 to Melan-A/MART-1, which revealed high congruency between molecular and immunohistochemical data 15;16;26–28 . Interestingly, one of the few previous immunohistochemical studies of TRP-2 employing a polyclonal reagent revealed discrepancies between mRNA and protein expression for TRP-2 unlike Melan-A/MART-1, which was analyzed in the same study 14 . Diminished specificity is the inherent trade-off for good sensitivity for polyclonal reagents and polyclonal reagents to TRP-2 appear to be no exception.…”

“…Two of the studies were done on cryosections rather than on archival paraffin material 22;24 . Only one previous report assessed the presence of TRP-2 in a selected number of archival pigmented lesions 14 . All previous analyses used solely polyclonal reagents, which were rabbit derived and provided by a collaborating lab or a commercially available goat reagent 14;22–24 .…”

“…Only one previous report assessed the presence of TRP-2 in a selected number of archival pigmented lesions 14 . All previous analyses used solely polyclonal reagents, which were rabbit derived and provided by a collaborating lab or a commercially available goat reagent 14;22–24 . Another study addressed the expression of TRP-2 by in situ hybridization 25 .…”

“…No major decrease in immunoreactivity was present in the dermal component, as seen with tyrosinase 27;30;32–34 . Though nevi were previously analyzed with other non-commercial polyclonal reagents, no exact description of the TRP-2 presence in nevocytes was given 14;23 . In primary cutaneous melanoma, the high percentage (84%) of TRP-2 positive lesions is comparable to the expression of Melan-A, tyrosinase, or gp100 27;30;32–34 .…”

“…Although ample knowledge has been acquired in surgical pathology as well as in tumor immunology about the presence of gp100, Melan-A, tyrosinase, and MITF, little is known about TRP-2, especially its distribution pattern in melanomas and related tumors as well as its potential presence in other non-melanocytic tissues and tumors 14 . In the present study we identified a monoclonal antibody to TRP-2, verified its specificity, and analyzed the expression pattern of TRP-2 in panels of normal and tumor tissues.…”