Many of these tumors had a substantial fraction of normal stromal cells. Total RNA (5-50 ng) from prostate cancers (n = 20) and nonmalignant hyperplastic prostate tissues (n = 10) were subjected to RT-PCR labeling and array hybridization. The results showed that 7/20 cancers but 0/10 nonmalignant samples had TMPRSS2:ERG fusion genes. To confirm the presence of the gene fusions, direct sequencing was performed for the seven samples. The sequencing data validated the exon fusion findings revealed by the array assays. Similar to other reports (7, 12, 13) , some samples clearly showed two or more bands on the agarose gel when the PCR products were subjected to electrophoresis, corresponding to two or more fusion transcripts in the same specimens. The multiple fusion transcripts in a single prostate cancer sample may reflect tumor heterogeneity or alternative splicing events. In order to map multiple fusion junctions in a single assay, we redesigned the exon array to include junction probes between exons 1 and 2 of the TMPRSS2 gene and exons 1-6 of the ERG gene ( Table 2 ). The modified probe set showed very clearly that the patient sample #15 had two fusion transcripts and also revealed the relative ratios of the two fusion transcripts through their respective signal intensities ( Figure 3 ). In this case, the two fusion transcripts are between exon 4 of the ERG fused to either exon 1 (T1G4) or exon 2 (T2G4) of the TMPRSS2. The signal intensity of T2G4 junction probe is weaker than that of the T1G4 junction probe ( Figure 3A) , consistent with the intensities of the probes within the exons. These two fusion transcripts are very likely due to alternative splicing. Figure 4 summarizes the cluster analysis (31) of the seven arrays; the results are shown in Table 3 . Multiple fusion variants were found in 4/7 positive samples. Table 3 also lists the percentages of cancer cells in the tumors, the Gleason tumor grades and the detected variants of TMPRSS2:ERG fusion transcripts. In this small sample set, there is no clear association between tumor Figure 2 . Assay sensitivity. The VCaP total RNA was serially diluted in a solution containing HeLa RNA to mimic the heterogeneous cell population in primary tumors or human body fluids. The total amount of RNA for each reaction is 100 ng. The laser power (PMT 600, 100% output) was adjusted to maximize the sensitivity of detection. Therefore, the intensity of the each expected feature (T1, G4, G5) is at the saturated level. The signal disappeared when the VCaP RNA was diluted from 1:3125 (32 pg) to 1:15625 (6.4 pg). T1G1F  CCTGGAGCGCGGCAGCCCCCGAGGGACATG  T1G2F  CCTGGAGCGCGGCAGGTTATTCCAGGATCT  T1G3F  CCTGGAGCGCGGCAGCCGTCAGGTTCTGAA  T1G4F  CCTGGAGCGCGGCAGGAAGCCTTATCAGTT  T1G5F  CCTGGAGCGCGGCAGATGCCACCCCCAAAC  T1G6F  CCTGGAGCGCGGCAGATCCTACGCTATGGA  T2G1F  ATGGCTTTGAACTCACCCCCGAGGGACATG  T2G2F  ATGGCTTTGAACTCAGTTATTCCAGGATCT  T2G3F  ATGGCTTTGAACTCACCGTCAGGTTCTGAA  T2G4F  ATGGCTTTGAACTCAGAAGCCTTATCAGTT  T2G5F  ATGGCTTTGAACTCAATGCCACCCCCAAAC  T2G6F  ATGGCTTTGAACTCAATCCTACGCTATGGA  T1G1R  CATGTCCCTCGGGGGCTGCCGCGCTCCAGG  T1G2R  AGATCCTGGAATAACCTGCCGCGCTCCAGG  T1G3R  TTCAGAACCTGACGGCTGCCGCGCTCCAGG  T1G4R  AACTGATAAGGCTTCCTGCCGCGCTCCAGG  T1G5R  GTTTGGGGGTGGCATCTGCCGCGCTCCAGG  T1G6R  TCCATAGCGTAGGATCTGCCGCGCTCCAGG  T2G1R  CATGTCCCTCGGGGGTGAGTTCAAAGCCAT  T2G2R  AGATCCTGGAATAACTGAGTTCAAAGCCAT  T2G3R  TTCAGAACCTGACGGTGAGTTCAAAGCCAT  T2G4R  AACTGATAAGGCTTCTGAGTTCAAAGCCAT  T2G5R  GTTTGGGGGTGGCATTGAGTTCAAAGCCAT  T2G6R  TCCATAGCGTAGGATTGAGTTCAAAGCCAT T, TMPRSS2; G, ERG. F, forward probe; R, reverse complement probe. grade and the presence of fusion transcripts. A relatively larger study also showed that the presence of fusion transcript was associated with tumor stage but not tumor grade (12) . Consistent with the VCaP titration study (Figure 2 ), the clinical assay can detect the fusion transcript when only 1% tumor cells is present in the prostate tissue (sample 10).