key: cord-0000201-nv228vte
authors: Lu, Qing; Nunez, Esperanza; Lin, Chunrun; Christensen, Kimberly; Downs, Tracy; Carson, Dennis A.; Wang-Rodriguez, Jessica; Liu, Yu-Tsueng
title: A sensitive array-based assay for identifying multiple TMPRSS2:ERG fusion gene variants
date: 2008-09-15
journal: Nucleic Acids Res
DOI: 10.1093/nar/gkn585
sha: 1a7676018fd73150a122c2afb1362324dbab4918
doc_id: 201
cord_uid: nv228vte

Studies of gene fusions in solid tumors are not as extensive as in hematological malignancies due to several technical and analytical problems associated with tumor heterogeneity. Nevertheless, there is a growing interest in the role of fusion genes in common epithelial tumors after the discovery of recurrent TMPRSS2:ETS fusions in prostate cancer. Among all of the reported fusion partners in the ETS gene family, TMPRSS2:ERG is the most prevalent one. Here, we present a simple and sensitive microarray-based assay that is able to simultaneously determine multiple fusion variants with a single RT–PCR in impure RNA specimens. The assay detected TMPRSS2:ERG fusion transcripts with a detection sensitivity of <10 cells in the presence of more than 3000 times excess normal RNA, and in primary prostate tumors having no >1% of cancer cells. The ability to detect multiple transcript variants in a single assay is critically dependent on both the primer and probe designs. The assay should facilitate clinical and basic studies for fusion gene screening in clinical specimens, as it can be readily adapted to include multiple gene loci.

Chromosome rearrangements are a characteristic feature of cancer. More than 350 gene fusions, as a consequence of chromosome aberrations, have been identified (1) . While gene fusions are common in hematological malignancies, their presence in solid tumors is not as well studied due to several technical and analytic problems related to tumor heterogeneity (1) . Only very limited gene fusion events were discovered in solid tumors, mostly in sarcomas, until the recent discovery of TMPRSS2:ETS fusion genes in prostate cancer (2) . This finding has since changed the general view that gene fusions play only a minor role in the pathogenesis of epithelial tumors. Therefore, there is renewed interest in searching for fusion genes in solid tumors, due to their potential impact on basic research and clinical application as has been demonstrated in chronic myelogenous leukemia (CML) (3, 4) .

The recurrent gene fusion event in prostate cancer involves an androgen controlled gene, TMPRSS2, and members (ERG, ETV1 and ETV4) of the ETS transcription factor family (2, 5, 6) . Among these fusion genes, TMPRSS2:ERG is the most prevalent and the only member detected in the majority of reports. This fusion transcript results from $3 Mb interstitial deletion between these two loci at chromosome 21q22. It was found in approximately half (15-78%) of all prostate cancers (2, (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . As an androgen-related transcription factor controlling cell proliferation, TMPRSS2:ERG has been associated with disease pathogenesis and is a promising biomarker for prostate cancer progression, prognostication and early detection (18) (19) (20) (21) . While the presence of TMPRSS2:ETS fusion genes is highly prostate cancerspecific, its significance as a prognostic biomarker is still controversial partly because many of the clinical studies have been relatively small scale. Therefore, it is important to develop a simple and robust assay for identifying various TMPRSS2:ETS and potential fusion genes in other solid tumors. However, this could be challenging due to high heterogeneity in prostate cancer and other solid tumors, compared to leukemias and lymphomas (22) .

Several approaches that have been used previously for hematological malignancies have been applied to detect TMPRSS2:ERG exon fusion variants. These include fluorescent in situ hybridization (FISH) (2, 12, 14, 17, 23) , RT-PCR and sequencing (2, 7, 9, 13) , quantitative PCR (qPCR) (2, 8, 24) and array-based comparative genome hybridization (array CGH) (10) (11) (12) . FISH may be the most commonly used method, but it has relatively low resolution, and therefore, cannot accurately determine different fusion variants. Array CGH has a higher resolution but is costly and often fails when there is normal cell contamination.

RT-PCR and qPCR are relatively easy to perform. However, to assess multiple potential fusion variants requires multiple sets of primers and probes, and a corresponding large quantity of RNA templates. Moreover, sequencing RT-PCR products is laborious and difficult to adapt in routine clinical laboratories. Here, we describe an exon array-based detection system, combined with a RT-PCR reaction, that accurately determines multiple TMPRSS2:ERG fusion transcripts in specimens with only a minor population of tumor cells. The method adopts several features of the Virochip (25) protocol to establish a specific, sensitive and semi-quantitative assay that is very useful for analyzing highly heterogeneous solid tumors.

The cell lines described in the article were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as recommended. Frozen unpurified prostate tissues were obtained during routine surgery, and classified pathologically by one of us. The total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The primary tumor samples were purified by Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA).

The exon and junction probes are 30-mer oligonucleotides synthesized by Integrated DNA Technologies (Coralville, IA, USA) or Illumina/Invitrogen (San Diego, CA, USA) and printed on poly-L-lysine slides at 50 mM along with Human Cot-1 DNA (Invitrogen), which is enriched for repetitive sequences, and herring sperm DNA (Promega, Madison, WI, USA), which was used as nonspecific control. The printing procedure has been described and essentially follows the manual of the DeRisi arrayer with silicon microcontact printing pins (Parallel Synthesis Technologies, Inc. Santa Clara, CA, USA) (25) (26) (27) . Arrays were postprocessed with succinic anhydride-based method for blocking before hybridization as previously described (27) . The protocols related to array printing and hybridization in this article generally can be found in the following link: http://cat.ucsf.edu/equipment/arrayer/index.html.

The RT-PCR reaction was performed with an OneStep RT-PCR kit (Qiagen) essentially following the manufacturer's protocol, except that the final reaction volume was scaled down to 20 ml. The forward (GTT TCC CAG TCA CGA TCC AGG AGG CGG AGG CGG A) and reverse primers (GTT TCC CAG TCA CGA TCG GCG TTG TAG CTG GGG GTG AG) are located at exon 6 of ERG and exon 1 of TMPRSS2 respectively, as described (2, 9) . The 5 0 -ends of both primers have the sequence of primer B (GTT TCC CAG TCA CGA TC) for the subsequent step of PCR labeling with a single primer B as described previously (25) .

The procedure is a modification of the previously reported 'Round ABC' protocol (25) . Briefly, the RT-PCR reaction was assembled at 48C in a PCR workstation and transferred to a thermocycler with the block preheated to 508C. The initial reverse transcription was performed at 508C for 30 min and followed by 958C for 15 min to activate HotStarTaq DNA polymerase as well as to inactivate the reverse transcriptases (Round A). The PCR conditions were 35 cycles at 928C for 30 s, 558C for 30 s and 688C for 1.5 min with a final extension step at 688C for 5 min (Round B). One microliter of unpurified product was subsequently used as a template for another 20 cycles of amplification to label the amplicons via a previously described 'Round C' PCR protocol (948C for 30 s, 408C for 30 s, 508C for 30 s and 728C for 1 min) with primer B and a 4:1 mixture of aminoallyl dUTP (Ambion, Austin, TX, USA) and dTTP for probe labeling (25) . The labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns (Zymo Research, Orange, CA, USA), eluted in 9 ml of sodium bicarbonate (pH 9.0) and coupled with 1 ml of DMSO dissolved Cy3 NHS esters (GE Healthcare, Piscataway, NJ, USA) for 30-60 min. The Cy3-labeled amplicons were purified with DNA Clean-up and Concentrator-5 columns and eluted with 10 ml of 10 mM Tris-HCl (pH 8.0). Then, the Cy3-labeled amplicons were diluted in water and combined with 3.6 ml of 20Â SSC, 0.5 ml of HEPES (pH 7.0) and finally 0.5 ml of 10% SDS to reach final volume of 25 ml. The mixed solution was heated for 2 min at 958C, cooled to room temperature and hybridized to the exon mapping arrays at 638C overnight essentially as previously described (25) (26) (27) . The hybridized arrays were washed and scanned with a GenePix 4000B scanner (Molecular Device, Sunnyvale, CA, USA) and analyzed by GenePix Pro 6.0 software.

Frozen prostate cancer samples were sectioned onto slides. Cell nuclei were isolated in situ with ice-cold cytoskeleton buffer (CSK: 300 mM sucrose, 100 mM NaCl, 10 mM PIPES, 3 mM MgCL 2 , 1 mM EGTA and 0.5% Triton X-100) (28) . The slides were fixed by dipping in ice-cold methanol for 3 min, followed by ice-cold acetone. After air drying the slides, they were allowed to age for at least 1 week in ethanol.

DNA-FISH was carried out according to a method for single copy loci detection (28) . The protocol was adapted with few modifications, using 50-mer oligonucleotides specific to the loci of interest and labeled with a desired hapten. Two probes were used for FISH for breakapart assay. A probe located at the promoter region of TMPRSS2 was labeled with biotin (Bio/GACTCCA GGAGCGCTCCCCAGAATCCCCTTCCTTAACCCA AACTCGAGCC). The other probe at exon 2 of ERG was labeled with 5 0 -6-carboxyfluorescein (56FAM) (56FAM/ GATCTTTGGAGACCCGAGGAAAGCCGTGTTGA CCAAAAGCAAGACAAATG). Detection of probes was achieved by using antibodies conjugated to quantum dots (Qdot) against the hapten label. Conditions were optimized to use a combination of two antibodies (1 : 200) obtained from Invitrogen-Molecular Probes TM (Qdot 655 Ã sheep anti-Bio primary antibody conjugate; Qdot 525 Ã Goat anti-FITC whole IgG primary antibody conjugate). Image acquisition was done with a Zeiss Axioplan 2e microscope (Carl Zeiss, Inc.). All pictures in the corresponding three channels were deconvolved and optical sections merged to produce 2D pictures using Axiovision 4.0 software (Carl Zeiss, Inc.) and Image J (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997-2006.)

To develop a multiplexing assay that is highly sensitive in clinical samples of high complexity, we adopted our Virochip system (25) . The key protocol, Round ABC, designed for unbiased amplification (29) , is crucial for identifying various fusion variants in this application.

Through literature review, we found that most of the TMPRSS2:ERG fusion junctions are between exons 1 or 2 of the TMPRSS2 and exons 2-5 of the ERG (2, 7, 9, 13) . Such constraints perhaps are related to whether a functional ERG protein can be made from the gene fusions (9) . Therefore, we initially used a pair of primers at exon 1 of the TMPRSS2 and exon 6 of the ERG for RT-PCR. As shown in Figure 1A , PCR products were only generated when there was a gene fusion, since the two primers are located at different genes. Subsequently, the PCR products were labeled and hybridized to an exon array for mapping the exons near the fusion junction. Printed on the array are 30-mer oligonucleotide probes derived from exons 1-3 of the TMPRSS2 and exons 1-5 of the ERG (Table 1) . Each selected sequence is represented by two complementary probes (F: forward and R: reverse complement) since sometimes PCR-labeled amplicons may bind to only one strand of the probe, based on empirical observations (25) . We observed that probes with reverse complementary (R) orientation worked consistently with our RT-PCR labeling protocol.

A prostate cancer cell line, VCaP, (30) with a TMPRSS2 and ERG fusion (2) was used for initial feasibility testing. The total RNA was subjected to RT-PCR with a pair of primers located at exon 6 of ERG and exon 1 of TMPRSS2 (2, 9) . The unpurified product was labeled and hybridized on the microarray ( Figure 1B ). Only spots corresponding to exon 1 of TMPRSS2 and exons 4-5 of ERG developed strong signals. This result indicates the fusion junction is at the exon 1 of TMPRSS2 and exon 4 of ERG, which is consistent with a previous report (2) .

To mimic a typical clinical situation, in which small population of cancer cells are present among normal host cells in a primary tumor, we spiked decreasing amounts of total RNA extracted from VCaP cells into an excess of HeLa RNA, which does not have the fusion transcripts. The detection limit is 32 pg of VCaP RNA in the presence of 100 ng of HeLa RNA (Figure 2 ). This translates into only 1-3 cancer cells in the presence of 3000 times more normal cells. The level of sensitivity is superior to previous methods for detecting fusion transcripts (24) .

To test the ability of the exon mapping array to detect and characterize TMPRSS2:ERG fusion transcripts in clinical samples, we isolated total RNA from frozen unpurified primary prostate tissues obtained during surgery. T1F  GGGCGGGGAGCGCCGCCTGGAGCGCGGCAG  T2F  ACATTCCAGATACCTATCATTACTCGATGC  T3F  GGTCACCACCAGCTATTGGACCTTACTATG  T1/2F  TGGAGCGCGGCAGGTCATATTGAACATTCC  G1F  AGGGACATGAGAGAAGAGGAGCGGCGCTCA  G2F  AGACCCGAGGAAAGCCGTGTTGACCAAAAG  G3F  GCTGGTAGATGGGCTGGCTTACTGAAGGAC  G4F  TTATCAGTTGTGAGTGAGGACCAGTCGTTG  G5F  CTCTCCTGATGAATGCAGTGTGGCCAAAGG  T1R  CTGCCGCGCTCCAGGCGGCGCTCCCCGCCC  T2R  GCATCGAGTAATGATAGGTATCTGGAATGT  T3R  CATAGTAAGGTCCAATAGCTGGTGGTGACC  T1/2R  GGAATGTTCAATATGACCTGCCGCGCTCCA  G1R  TGAGCGCCGCTCCTCTTCTCTCATGTCCCT  G2R  CTTTTGGTCAACACGGCTTTCCTCGGGTCT  G3R  GTCCTTCAGTAAGCCAGCCCATCTACCAGC  G4R CAACGACTGGTCCTCACTCACAACTGATAA G5R CCTTTGGCCACACTGCATTCATCAGGAGAG T, TMPRSS2; G, ERG. F, forward probe; R, reverse complement probe.

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).

We used FISH analysis to independently confirm our array approach. Our FISH procedure (28) employed 50-mer probes that were labeled with small haptens for target hybridization in conjunction with individual hapten-specific, quantum dot conjugated antibodies for signal detection. The resolution of this method is $50 kb. We designed two probes for the FISH assays, one at the promoter region of TMPRSS2 (green in Figure 5 ) and the other at exon 2 of ERG (red in Figure 5 ). We observed heterogeneity of the FISH patterns in some primary prostate cancer samples ( Figure 5 ). It is more difficult to find interstitial deletions between TMPRSS2 and ERG in tumor samples containing low percentages of cancer cells by FISH. Therefore, we used samples that contained 480% cancer cells without detectable fusion genes to confirm the results of the arrays. The FISH experiments revealed no genomic deletions at this location for all the selected samples (#5, #7, #9, #19 and #20) that were similarly nondeleted by array hybridization.

We have established a simple assay that can concurrently profile variants of TMPRSS2:ERG fusion transcripts by combining a single RT-PCR with an exon array. The modified 'Round ABC' protocol, which was TMPRRS2e1R  TMPRRS2e1/2R  TMPRRS2e2R  TMPRRS2e3R  ERGe1R  ERGe2R  ERGe3R  ERGe4R  ERGe5R  ERGe6R  T1G1R  T1G2R  T1G3R  T1G4R  T1G5R  T1G6R  T2G1R  T2G2R  T2G3R  T2G4R  T2G5R  T2G6R   #1  #15  #17  #4  #13 #10 #18 Figure 4 . Cluster analysis of seven prostate cancer samples having fusion transcripts. The signal intensity of each feature is divided by the intensity of a nonspecific control (herring sperm DNA) to normalize the data for cluster analysis. The result is shown in Table 3 . The samples having similar fusion transcript variants were clustered together by the program. 1  30  7  T1-G4; T2-G4  2  2 0  5  3  5 0  5  4  20  6  T1-G4  5  8 0  9  6  1  6  7  9 0  8  8  2 0  4  9  8 0  8  10  1  6  T1-G2  11  2  6  12  70  7  13  20  9  T1-G4  14  1  6  15  70  8  T1-G4; T2-G4  16  20  8  17  80  8  T1-G4; T2-G4  18  50  7  T1-G2; T1-G3; T1-G4  19  80  7  20 80 7 Figure 5 . Heterogeneity of FISH patterns of interstitial deletion between TMPRSS2 and ERG in a primary prostate tumor. An unpaired green dot (TMPRSS2 probe, indicated by arrows) suggests an interstitial deletion. Nonrandom variation of FISH patterns is shown by the fact that most of the green and red signals (two different but nearby probes) are paired in each panel. This variation is expected on a heterogeneous aneuploid cancer cell population, which often makes it difficult to distinguish meaningful events from random background aberrations.

originally designed for genomic amplification (29) and has been widely adopted for chromatin immunoprecipitation (ChIP) and whole-genome DNA microarrays (ChIP-chip) (32, 33) and Virochip (25, 34) experiments, is a simple and relatively unbiased amplification procedure to semiquantitatively measure the fusion variants in a complex sample. Previously, the same simple procedure was used to obtain 83% (25 kb/30 kb) of the SARS coronavirus genome with total nucleic acids isolated from a viral culture (25) . The inclusion within the array of probes derived from individual exons and potential fusion junctions simplifies the breakpoint mapping and increases the confidence of data interpretation (Figures 3 and 4) . In contrast to some reports that used multiple primers targeted to every potential fusion junction in hematological malignancies (35) (36) (37) (38) , we used a single set of primers for target amplification (Figure 1 ). The fusion junctions were subsequently decoded by array. This design significantly reduces the problems associated with primer dimers in the multiplex PCR reaction, and creates more room for future assays to include additional fusion genes. Furthermore, most searches for fusion genes have focused on blood cancers, because the cells can be purified before analysis. The application of the previous methodologies is less useful for highly complex solid tumors that are inevitably admixed with normal cells. For example, a previously reported MLLFusionChip could not be applied to samples with cancer cells of 55-10% in 1 mg of total RNA (39) .

The current assay should facilitate a thorough compilation of the gene fusion variants in primary prostate specimens, which may be useful for stratifying the aggressiveness of prostate cancer (13) . In this regard, fusion variants of EWS with another member of the ETS family, FLI1, have been shown to be an independent predictor of disease progression in Ewing's sarcoma (40, 41) . It will be of interest to compare in transfected cells the biological activities of the different TMPRSS2:ERG variants from patients with contrasting clinical outcomes (41) .

While some studies have suggested that the presence of TMPRSS2:ERG fusions is associated with more aggressive disease or higher Gleason tumor grade, other investigators did not reach the same conclusion (12, 14, 17, 20, 23, 42) . We also did not find such an association in a small series of samples. However, all of these results are defective due to small sample size. The technology described here should make possible a larger scale investigation to find whether there is a correlation between the aggressiveness of the disease and the presence of specific fusion genes.

It is crucial to have true cancer-specific biomarkers for early cancer detection as well as for minimal residual disease monitoring, which has been extensively demonstrated in hematologic maligancies (43) . Such biomarkers could help to avoid under-or over-treatment. Thus, there is past interest (24, 44) in applying TMPRSS2:ERG fusion assays for such application since PSA and many other markers in development are not truly prostate cancer-specific (45) . A recent study reported a TMPRSS2:ERG assay with a sensitivity of detecting 1600 VCaP cells (24) . However, this level of sensitivity might not be sufficient for broad clinical application, especially with small biopsy specimens or urine samples. We were able to achieve an assay sensitivity of 532 pg of total RNA derived from VCaP cells, an equivalent to 1-3 cells (Figure 2 ). Because our assay is simple and amenable to automation, it is readily adaptable to clinical studies. While it has been challenging to adapt microarray-based technology to the clinic, some tests (e.g. AmpliChip CYP450 and MammaPrint) have been approved by FDA (46) .

The same strategy can be applied to detect other less prevalent fusion transcripts (TMPRSS2:ETV1 and TMPRSS2:ETV4) in prostate cancer (2, 5, 6) . In addition, the exon array approach can also be applied to other fusion genes, such as BCR-ABL in CML and clonal Ig/TCR rearrangements in lymphocytic malignancies. While this methodology development was motivated by the clinical need, it is generally applicable to other research requirements that are analogous to the situation for detecting fusion genes in the single cell level when a large excess of normal cells are present. For example, a developmental biologist may use a similar approach to screen mutants that have a desirable gene fusion when direct gene targeting is not feasible.

There are some shortcomings of using RNA transcripts as prostate cancer biomarkers, despite our ability to achieve very sensitive detection of TMPRSS2:ERG fusion variants. First, RNA is unstable and difficult to process in routine clinical assays. Second, commonly used drugs that inhibit androgen growth pathways, including GnRH agonists and testosterone antagonists, may diminish the production of the TMPRSS2:ERG mRNA fusion transcript, thereby producing false-negative results in patients on hormonal therapy with evolving androgenindependent tumors. Indeed, it has been reported that TMPRSS2:ERG mRNA fusion transcripts are not expressed in androgen-independent tumors in spite of the presence of interstitial deletions in between TMPRSS2 and ERG at chromosome 21q22 (10) . While FISH is useful for identifying genomic rearrangements, it has relatively lower resolution and is difficult to use in highly heterogeneous samples with small percentages of tumor cells. We have recently developed a technology, designated Primer Approximation Multiplex PCR (PAMP) for identifying breakpoints in genomic DNA without the need to purify cancer cells from normal tissues (26) . We are optimizing this assay for detecting the breakpoints between TMPRSS2 and ERG loci for primary prostate tumors to overcome any potential problems associated with RNA based biomarkers. In addition, the DNAbased assay will provide information about whether multiple fusion transcripts in a sample are derived from alternative splicing or tumor heterogeneity. The best approach may ultimately be to combine DNA and RNA based assays in a common format.

Institutes of Health (CA119335 to UCSD NanoTumor Center of Excellence for Cancer Nanotechnology, CA133634 to Y. T. Liu, NS034934 to

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M.G. Rosenfeld); NCI Cancer Center (2 P30 CA023100-23 to DNA Sequencing Shared Resource, UCSD Cancer Center) partially. Funding for open access charge: CA133634.Conflict of interest statement. None declared.