title: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. itakura@ucla.edu
authors.forename: Eijun
authors.initials: E
authors.lastname: Itakura
authors.forename: Rong-Rong
authors.initials: RR
authors.lastname: Huang
authors.forename: Duan-Ren
authors.initials: DR
authors.lastname: Wen
authors.forename: Eberhard
authors.initials: E
authors.lastname: Paul
authors.forename: Peter H
authors.initials: PH
authors.lastname: Wünsch
authors.forename: Alistair J
authors.initials: AJ
authors.lastname: Cochran
mesh_terms: D000951:Antigens, Neoplasm / Q000032:analysis / Q000235:genetics*; D045744:Cell Line, Tumor; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D019746:Intramolecular Oxidoreductases / Q000032:analysis / Q000235:genetics*; D058965:MART-1 Antigen; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics*; D009506:Nevus / Q000235:genetics* / Q000378:metabolism; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000235:genetics* / Q000378:metabolism; D016707:Tissue Fixation
chemical_list: D000951:Antigens, Neoplasm; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005557:Formaldehyde; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase