TMPRSS2-ERG fusion transcripts in matched urine and needle rinse material after biopsy for the detection of prostate cancer.
Gene rearrangements commonly found in hematologic malignancies were rarely reported in solid tumors until the identification of a fusion gene in PCa, involving TMPRSS2 and members of the ETS transcription factor family(ETV1, ETV2, ETV4, and ERG) (7). A number of studies that used fluorescence in situ hybridization and quantitative reverse-transcription PCR (qRT-PCR) confirmed the presence of a fusion gene in prostate tissue in 40%-80% of PCa (14-17). The highest rates were observed with the use of nested qRT-PCR, with TMPRSS2-ERG representing around 90% of the fusions (15, 16). Because a diagnostic strategy based on prostate tissue is inadequate for clinical application, noninvasive methods, similar to those for PCA3 (18), were developed to detect the fusion transcripts in urine (19-23) voided after digital rectal examination (DRE). In contrast with a very high diagnostic specificity (>90%) and a very good predictive value for positive biopsy, the overall diagnostic sensitivity of the test was reported to be only around 50%, although it could be improved by combining results with PSA, DRE (21), and PCA3 (20, 23). One drawback of those studies was the inability to match findings in urine with those in prostate tissue in the same patient. With a fusion gene prevalence as high as 80%, the presence of false-negative results with the urine test is possible, making the fusion gene assay not sufficiently reliable for use as a screening test for PCa detection.
The purpose of the prospective study described here was (a) to determine the prevalence rate of the fusion gene and the type of fusion with urine and biopsy needle rinse material (BRM), and (b) to optimize various factors, including sample collection and preanalytical and analytical procedures, that may improve the diagnostic sensitivity of the fusion gene assay.
Patients and Methods
Eighty consecutive patients with a serum total PSA concentration >4 [micro]g/L and/or abnormal DRE were referred to our department for prostate biopsy and were enrolled prospectively between September 2011 and May 2012. The study was undertaken in accordance with the recommendation of the hospital ethics committee, and all patients provided informed consent. Of the 80 patients, 13 had a history of previous prostate biopsy. A DRE was performed first, followed by transrectal ultrasonography (TRUS) examination. The transrectal probe was then removed, and patients were asked to void in a 100-mL polypropylene collection cup. Immediately after the urine void, the probe was reintroduced transrectally for guiding prostate biopsy (Achieve[R] biopsy gun with a 25-cm 18G needle). Ten to 12 cores were sampled in most patients and removed from the biopsy needle in a cup containing buffered formalin. At the end of the biopsy procedure, the needle was repeatedly rinsed in a 15-mL polypropylene tube containing 5 mL RNA Protect Cell (Qiagen). Patients who had not been able to void before biopsy were asked again to provide a urine sample. We analyzed each biopsy core separately. In case of cancer involvement, we determined cancer size and Gleason score. A microfocal cancer (MFC) was defined by a single core involved with [less than or equal to] 3 mm of Gleason score 3 + 3 cancer. After collection, urine samples were immediately cooled on ice. All samples were processed within 2 h by centrifugation at 4[degrees]C and 3000gfor 10 min. We used the whole urine sample for analysis. Urinary sediments were washed with cold PBS. Pelleted cells from all samples were disrupted in a buffer that lysed the cells and stabilized the mRNA (Qiagen). Lysates were stored at-20[degrees]C until use. Whole-urine samples collected from women served as negative controls.
PREPARATION OF cDNA
Total RNA was isolated by use of an RNeasy Micro kit (Qiagen) according to the manufacturer's instructions and was digested by DNase in a polypropylene tube (Qiagen) before reverse transcription. We performed the cDNA synthesis in duplicate in a 20-[micro]L reaction volume containing 10 [micro]L total RNA, 1 U Sensiscript Reverse Transcriptase (Qiagen), 2 [micro]L 10 X RT buffer, 10 [micro]mol/L random hexamer primers (Life Technologies), 0.5 mmol/L of each dNTP, and 10 U RNase inhibitor (Promega). The tubes were incubated at 37[degrees]C for 1 h, and then stored at-20[degrees]C until use.
We used the primers and hydrolysis probes (Eurofins) described by Clark et al. (21) with the same PCR cycling conditions. We used 5 [micro]L cDNA product as template for amplification with LightCycler Taqman Master (Roche) following the manufacturer's recommendations. GAPDH gene (NM_002046.3) amplification was performed as control for extraction and reverse transcription procedures. PSA gene (M27274.1) amplification served as control of the presence of prostate cells in the biological samples. The assays obtained with cDNA prepared from serial dilutions of a culture of VCaP cells are provided in Supplemental Table 1, which accompanies the online version of this article at http://www.clinchem.org/content/vol59/issue1. Samples with <200 VCaP cells, indicating poor recovery of prostate cells in urine, were excluded from further analysis. The dynamic range of the fusion gene assay is depicted in online Supplemental Fig. 1. After resolution by electrophoresis in a 3% agarose gel stained with ethidium bromide, amplified PCR products were sampled from the gel, purified on a MinElute column (Qiagen), and sequenced. The length of the different types of fusion PCR products is shown in online Supplemental Table 2. Samples that did not produce any band on the electrophoresis gel underwent amplification with 8 [micro]L cDNA product.
Qualitative variables are described as n (%) and quantitative variables as mean (SD). Data were compared by use of Fisher exact test and the unpaired Student f-test, as appropriate. Paired urine samples were compared with the nonparametric Wilcoxon test. We used a 5% significance level in all comparisons. For all statistical analyses, we used the Statview Statistical package, version 5.0 (SAS Institute).
The characteristics of the 80 patients are summarized in Table 1. Prostate biopsyshowed cancer in 45 patients (56%). Clinical characteristics of patients diagnosed with PCa were not statistically different from those with benign biopsy except for age (Table 1). Serum PSA was higher in patients diagnosed with PCa, but the difference was not significant (P = 0.063). Of the 45 patients diagnosed with PCa, biopsy Gleason score was 3 + 3 in 25 (55.6%)and >3 + 4 in 20 (44.4%). Of those 20 patients, 16 were Gleason score 3 + 4, 2 were Gleason 4 + 3, 1 was Gleason 4 + 4, and 1 was Gleason 3 + 5. Insufficient or absent biological material led to the exclusion of 3 patients with Gleason score [greater than or equal to] 3 + 4(2 were Gleason 3 + 4 and 1 was Gleason 4 + 4), leaving 77 patients included for analysis.
OVERALL FUSION GENE DETECTION
The data of fusion gene detection obtained with the different types of samples are summarized in Table 2. Among patients with benign biopsy, 1 patient with a history of adenocarcinoma detected on transurethral resection of the prostate was found to be positive for fusion gene. The diagnostic sensitivity, specificity, and positive and negative predictive values for the prediction of cancer on biopsy were 69% (95% CI 53%85%), 83%, 85%, and 67% (50%-83%) with urine assays, respectively; 62% (47%-78%), 93%, 92%, and 65% (50%-80%) with BRM; and 89% (73%-99%), 77%, 86%, and 81% with paired samples.
FUSION GENE VARIANTS
The variant resulting from the fusion between exon 1 of TMPRSS2 and exon 4 of ERG was found in 29 patients. This transcript was associated with other variants (including TMPRSS2 exon 2/ERG exon 4 fusion, TMPRSS2 exon 1/ERG exon 2 fusion, and TMPRSS2 exon 2/ERG exon 2 fusion) in 6 patients. Two patients had a predominant TMPRSS2 exon 1/ERG exon 2 fusion transcript, and 1 patient had a fusion composed of TMPRSS2 exon1/catenin-[alpha]3 (NM_013266.2) exon 12/ ERG exon 4.
FUSION GENE STATUS ACCORDING TO GLEASON SCORE AND BIOPSY SIGNIFICANCE
To stratify the risk for PCa, fusion gene status was analyzed according to Gleason score and the presence of MFC (Table 3). The diagnostic sensitivity for fusion gene detection was lower when biopsies showed MFC, whatever the material used, alone or combined, although the difference was significant only with BRM (P = 0.014). In the subgroup of patients with Gleason score >3 + 3, the diagnostic sensitivity for fusion gene detection was lower with urine alone, although the difference was not statistically significant.
COMPARISON BETWEEN URINE SPECIMENS SAMPLED BEFORE AND AFTER BIOPSY
Of the 61 patients for whom urine was collected (Table 1), 5 were excluded, leaving 56 patients for analysis. Of these 56 patients, prebiopsy urine only was available in 24, postbiopsy urine only in 27, and both samples in 5. The detection rate of fusion gene was 74% (95% CI 54%-93%) in prebiopsy urine and 72% (52%-93%) in postbiopsy urine. Paired samples collected in 5 patients allowed for a comparison of cell recovery rate between the two types of samples (Table 4). The number of prostate cells was higher in postbiopsy urine (P = 0.043). The difference in the number of cells positive for fusion detection, however, was not significant (P = 0.0679).
EFFECT OF PREANALYTICAL TREATMENT ON FUSION GENE STATUS
A higher number of noninformative samples (8 of 27 vs 2 of 42;P = 0.011) were found in patients with Gleason score > 3 + 3. This finding, together with the lower diagnostic sensitivity of the urine assay in this subgroup, led us to hypothesize that cells with an aggressive tumor pattern could possibly show a greater frailty to preanalytical procedures. Therefore, we compared the influence of centrifugation force on 5 samples by dividing them into 2 halves, 1 centrifuged at 900g for 8 min and 1 at 3000g for 10 min (Table 5). In patients with Gleason score 3 + 3, the yield of prostate cells was higher when centrifugation was performed at 3000g. In comparison, the yield observed with centrifugation speed at 3000g was much lower in 1 patient with Gleason score > 3 + 3 cancer, suggesting important cell lysis during centrifugation.
Our results show that fusion gene is associated with PCa at a very high frequency. By combining the results obtained with paired urine and BRM, we detected the presence of TMPRSS2-ERG fusion gene in 89% of the patients with cancer on prostate biopsy and confirmed the good predictive value of the fusion gene assay for positive prostate biopsy. With urine and BRM combined, our detection rate reached 95% in patients with no evidence of MFC. To our knowledge, this method is the first to show such results. However, to be of any value as a screening test, the urine assay should show higher diagnostic sensitivities. Such a fusion gene assay may, however, stand as an additional detection tool that would allow more accurate diagnosis in some cases. Given the 100% specific association between TMPRSS2-ERG fusion and PCa, a positive assay should be given more credence than negative biopsies. In our study, the detection of a fusion gene in 5 patients without cancer on biopsy may suggest that cancer was present but missed by biopsies. Concordant with these findings is the identification by Nguyen et al. (24) of 2 patients with negative biopsy and positive fusion assay, with 1 case confirmed as PCa in a subsequent biopsy. Our result should therefore be interpreted as the early detection of cancers probably too small in their development to be sampled by biopsy. As reported before in autopsy studies, random prostate biopsies may miss approximately 40% of cancers according to their true prevalence (3), whatever the core number, location, or route taken. Selecting those patients at risk of harboring cancers potentially missed by biopsies is of utmost importance to propose appropriate PSA follow-up and possible rebiopsy.
Another interest of the fusion gene assay may be its potential value as a characterization tool. The presence of only 1 biopsy core involved with cancer on biopsy does not eliminate the presence of other contiguous or distant foci. The presence of such secondary foci may be predicted by fusion gene detection assay. It has been shown that different tumor foci may express different fusion variants (16, 25). Therefore, patients with 2 or more different fusion variants are highly suspect of harboring > 1 tumor focus, and possibly bilateral disease. On the other hand, patients with MFC on biopsy were reported to harbor insignificant cancer in 2 of 3 cases (26). Interestingly, the diagnostic sensitivity of fusion gene detection was lower in those patients, suggesting its potential value as a staging tool.
The low diagnostic sensitivity of the urine assay observed in patients with higher Gleason scores (62%) was somewhat disappointing. Thus, our attention focused on identifying the protocol steps that might be improved to obtain higher detection rates. We first investigated the most efficient way to mobilize prostate cells in the urine. Rostad et al. (22) demonstrated the importance of prostatic massage by DRE before urine collection for improving the detection of fusion transcripts (69% vs 24%). In our study, we chose to collect urine after DRE and used the TRUS probe to perform a more vigorous prostatic massage: only 2 urine samples containing < 200 cells on the basis of PSA expression were excluded from analysis. However, we acknowledge that this threshold does not guarantee the presence of sufficient tumor cells to be detected in the urine sample. In this regard, Hessels et al. (20) suggested that the high false-negative rate of PCA3 and TMPRSS2ERG might be due to the fact that very few cancer cells were released into the urine after DRE. Moreover, Tinzl et al. (27) hypothesized that these false-negative samples may represent a subgroup of PCa having a lower tendency to invade the prostate ductal system, and therefore shedding fewer cells in the urine. The mobilization of cancer cells located at the anterior site of the prostate may also be questionable. Our results suggested that performing biopsies before sampling urine may facilitate the shedding of prostate cells into urine. However, although urine samples collected after biopsy did contain substantially more cells, this did not translate into any significant increase in fusion transcript detection rates.
The second point that drew our attention was related to the preanalytical step. To work with the highest amount of RNA possible, we chose to analyze the whole micturition, which precluded stabilization of RNA by a reagent similar to that used for BRM. Therefore, we rigorously respected a delay of <2 h between sampling and treatment of the urine. This inconvenience does not exist with the Gen-Probe technology (23), but the volume of urine is then limited to 2.5 mL. Our finding that centrifugation speed was a factor that may have contributed to the loss of sample quality--especially for high-Gleason-score cancers--led us to change the operating conditions in the course of the study. This brought heterogeneity to our results, especially regarding quantification, but resulted in an improved rate of detection of fusion transcripts in samples from patients with high-Gleason-score cancers. Concordant with this result is the observation from Rostad et al. (22) that fusion transcripts could be detected not only in the pellets after urine centrifugation, but also in the supernatant of some samples. Therefore, if fusion transcripts are partly released in the urine owing to partial lysis of frail tumor cells, their quantification may not be accurate, rendering incorrect the analysis of correlation with clinico-pathological parameters. On the other hand, centrifugation at low speed may decrease the detection rate of MFC. We are currently testing the association of a first centrifugation of the urine sample at 900g followed by a second centrifugation at 3000g of the supernatant and pooling both pellet lysates. The observation with the fusion transcripts holds true for other genes. The only assay that is not affected by this drawback is based on GenProbe technology.
The third step we carefully examined concerned the analytical conditions. Our main goal was to optimize the conditions of extraction, RT, and PCR by working with the highest ratio possible of amount of nucleic acid to reaction volume (see online Supplemental Fig. 2). Furthermore, we used a Sensiscript reverse transcriptase specifically designed to transcribe small amounts of RNA. Under these conditions, the analytical sensitivity of the assay was 8 pg RNA in the RT reaction (corresponding to <3 VCaP cells). Nguyen et al. (24) used a whole transcriptome amplification step and a qPCR with SYBR green. With their methodology, they detected 10 pg VCaP RNA in the presence of 100 ng fusion-negative urine RNA. Our experimental protocol is similar to that described for BCR-ABL transcripts by use of the LightCycler t(9;22) quantification kit (Roche) (28).
Our study has some limitations, the most important being the absence of available pre-and postbiopsy urine samples for all patients, which brought heterogeneity to our results. Another limitation is the absence of radical prostatectomy samples as reference for precise cancer characterization.
In conclusion, our results show that the expression of TMPRSS2-ERG fusion gene occurred in at least 80% of prostate cancers. We also confirmed that detection could be improved by performing assays in both urine and BRM. Quantification of transcripts to predict clinically important disease needs to be further investigated.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: No sponsor was declared.
Acknowledgments: We are grateful to Dr. H. Baaddi, INSERM U955, Henri Mondor hospital, Assistance Publique-Hcopitaux de Paris, Creteil, France, for the gift of VCaP cell line.
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Phuong-Nhi Bories,  * Patrick Younes,  Marc Zerbib,  Lydie Denjean,  Theodora Popovici,  Luc Cynober, [1,4] and Nicolas Barry Delongchamps 
 Department of Clinical Chemistry,  Department of Radiology, and  Department of Urology, Hopital Cochin, Assistance Publique-Hopitaux de Paris, Paris, France; 4 Laboratory of Biological Nutrition, EA 4466, Faculte de Pharmacie, University Paris Descartes, Paris, France.
Received June 28, 2012; accepted September 10, 2012.
Previously published online at DOI: 10.1373/clinchem.2012.192260
 Nonstandard abbreviations: PCa, prostate cancer; PSA, prostate-specific antigen; qRT-PCR, quantitative reverse-transcription PCR; DRE, digital rectal examination; BRM, biopsy needle rinse material; TRUS, transrectal ultrasonography; MFC, microfocal cancer.
 Human genes: PCA3, prostate cancer antigen 3 (non-protein coding); TMPRSS2, transmembrane protease, serine 2; ERG, v-ets erythroblastosis virus E26 oncogene homolog (avian); ETV, ets variant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
* Address correspondence to this author at: Laboratoire de Biochimie, Groupe hospitalier Cochin-Broca-Hotel-Dieu, Hopital Cochin, 27 rue du Faubourg SaintJacques, 75679 Paris cedex 14, France. Fax +33-1-58-41-15-85; e-mail firstname.lastname@example.org.
Table 1. Clinical parameters for patients undergoing prostate biopsy. Negative Positive Parameter biopsy biopsy n 35 45 Mean age, years (SD) (a) 62.0 (5.6) 66.9 (8.0) Serum total PSA, [micro]g/L (b) Mean (SD) 7.99 (3.74) 11.35 (1.55) Range 1.40-20.0 1.46-43.0 Abnormal DRE, n (%) 3 (8.6) 9 (20) Ethnic origin, n (%) White 34 41 Black 1 4 Previous biopsy, n (%) 6(17.1) 8(17.8) Number of biopsy cores Mean (SD) 11.2(1.3) 10.8 (1.7) Range 10-15 6-19 Mean total length of cores, mm (SD) 193.3 (22.8) 186.0(43.8) Mean core length, mm (SD) 17.4(2.0) 17.2 (2.4) Sampled specimens, n BRM 32 44 Urine 26 35 Matched specimens, n 23 34 Specimens with insufficient yield, n BRM 4 7 Urine 2 3 Parameter All n 80 Mean age, years (SD) (a) 64.7 (7.4) Serum total PSA, [micro]g/L (b) Mean (SD) 9.95 (7.89) Range 1.40-43.0 Abnormal DRE, n (%) 12 (15) Ethnic origin, n (%) White 80 Black 5 Previous biopsy, n (%) 14(17.5) Number of biopsy cores Mean (SD) 10.9(1.6) Range 6-19 Mean total length of cores, mm (SD) 189.2 (36.1) Mean core length, mm (SD) 17.3 (2.2) Sampled specimens, n BRM 76 Urine 61 Matched specimens, n 57 Specimens with insufficient yield, n BRM 11 Urine 5 (a) t Test, P = 0.003. (b) t Test, P = 0.0633. Table 2. Fusion gene status of patients undergoing prostate biopsy. Specimens according Positive for fusion to pathology n gene, n (%) Benign biopsy All specimens 35 5 (14) BRM 28 2 (7) Urine 24 4 (17) Paired BRM and urine 17 4 (24) Cancer biopsy All specimens 42 32 (76) BRM 37 23 (62) Urine 32 22 (69) Paired BRM and urine 27 24 (89) Table 3. Fusion gene status according to Gleason score and biopsy significance. Positive for fusion Gleason score n gene, n (%) 3 + 3 All specimens 25 20 (80) BRM 23 13 (57) Urine 19 14 (74) Paired BRM and urine 17 15 (88) [greater than or equal to] 3 + 4 All specimens 17 12 (71) BRM 14 10 (71) Urine 13 8 (62) Paired BRM and urine 10 9 (90) Biopsy significance MFC (a) All specimens 10 6 (60) BRM (b) 9 2 (22) Urine 8 5 (63) Paired BRM and urine 7 5 (71) Macrofocal cancer All specimens 32 26 (81) BRM (b) 28 21 (75) Urine 24 17 (71) Paired BRM and urine 20 19 (95) (a) Microfocal cancer is defined as a single core involved with [less than or equal to] 3 mm of Gleason score 6 cancer. Cancer with more adverse pathological features considered to be macrofocal cancer. (b) Fisher test, P = 0.014. Table 4. Comparison of recovery rate of prostate cells in urine specimens collected before and after biopsy. Prebiopsy urine (b) Postbiopsy urine Biopsy Prostate cores, cells, Tumor Prostate Tumor mm (a) n (c) cells, n cells, n cells, n 2/6 2500 5 20 000 5 8/65 23 23 50 50 6/56 700 70 2000 280 6/37 900 5 3000 50 4/53 800 5 15 000 230 (a) Number of cores with cancer/total length of tissue with cancer. (b) Prebiopsy urine samples were collected after an attentive examination including DRE and TRUS probe. (c) cDNA obtained from urine specimens was amplified for PSA and fusion genes by PCR. The results are expressed as equivalent number of VCaP cells using a standard curve as given in online Supplemental Table 1. Table 5. Influence of centrifugation speed on recovery rate of prostate cells in urine. (a) 900g for 8 min 3000g for 10 min Biopsy Prostate cores, cells, Tumor Prostate Tumor mm (b) n (c) cells, n cells, n cells, n 0/0/0 10 000 Not detected 35 000 10 3 + 3/1/1 700 Not detected 3500 Not detected 3 + 3/2/6 25 000 400 25 000 700 3 + 3/7/26 1500 15 1500 15 3 + 5/9/88 15 000 400 2000 30 (a) After homogenization, the urine samples were divided into 2 tubes and centrifuged at 2 different speeds. (b) Gleason score/number of cores with cancer/total length of tissue with cancer. (c) cDNA obtained from urine specimens was amplified for PSA and fusion genes by PCR. The results are expressed as equivalent number of VCaP cells using a standard curve as given in online Supplemental Table 1.
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|Title Annotation:||Cancer Diagnostics|
|Author:||Bories, Phuong-Nhi; Younes, Patrick; Zerbib, Marc; Denjean, Lydie; Popovici, Theodora; Cynober, Luc;|
|Date:||Jan 1, 2013|
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