OBJECTIVE\nTo investigate whether messenger ribonucleic acid (mRNA) expression of TMPRSS2-ERG fusion gene, a suggested prostate cancer (PCa) biomarker, was specific to cancerous lesions alone and to study the expression of SPINK1 and PCA3 mRNAs in the same cohort to also explore the proposed mutual exclusivity of TMPRSS2-ERG and SPINK1 expression.\nMETHODS\nLevels of 2 TMPRSS2-ERG transcripts, PCA3, and SPINK1 mRNAs were measured with highly standardized reverse transcription quantitative polymerase chain reaction assays in cystoprostatectomy specimens from 19 patients with invasive bladder cancer and 174 radical prostatectomy (RP) samples (88 histologically benign prostate [HBP] tissues and 86 from cancerous lesions) from 87 patients with clinically localized PCa.\nRESULTS\nExpression of TMPRSS2-ERG transcripts was detected in 45 of 88 (51%) HBP tissues from RP specimens and more frequently (57 of 86, 66%) found in cancerous lesions. In contrast, TMPRSS2-ERG expression was detected in only 2 of 19 (11%) cystoprostatectomy specimens, both with incidental PCa foci elsewhere in the gland. Similar trends of changes in the expression of PCA3 and SPINK1 were present in HBP tissue from RP compared with cystoprostatectomy specimens.\nCONCLUSION\nAlthough the expression of TMPRSS2-ERG, SPINK1, and PCA3 mRNA is higher or more frequently found in cancerous lesions, HBP tissues from patients with clinically localized PCa manifest molecular, mRNA level changes that are absent in cystoprostatectomy specimens lacking incidental PCa foci or infrequent in cystoprostatectomy specimens containing incidental PCa. If this finding is replicated, these molecular assays could be used to inform men with negative biopsy results about the likelihood of cancerous lesions in unsampled regions and hence the need for repeat biopsy.