Prostate Cancer and Prostatic Diseases (2010) 13, 117–125 & 2010 Nature Publishing Group All rights reserved 1365-7852/10 www.nature.com/pcan ORIGINAL ARTICLE The identification of chromosomal translocation, t(4;6)(q22;q15), in prostate cancer L Shan1, L Ambroisine2, J Clark3,RJYa´n˜ez-Mun˜oz1, G Fisher2, SC Kudahetti1, J Yang1, S Kia1, X Mao1, A Fletcher3, P Flohr3, S Edwards3, G Attard3, J De-Bono3, BD Young1, CS Foster4, V Reuter5, H Moller6, TD Oliver1, DM Berney1, P Scardino7, J Cuzick2, CS Cooper3 and Y-J Lu1, on behalf of the Transatlantic Prostate Group 1Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK; 2Cancer Research UK Centre for Epidemiology, Mathematics and Statistics, Wolfson Institute of Preventive Medicine, Queen Mary University of London, London, UK; 3Male Urological Cancer Research Centre, Institute of Cancer Research, Surrey, UK; 4Division of Cellular and Molecular Pathology, University of Liverpool, Liverpool, UK; 5Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 6King’s College London, Thames Cancer Registry, London, UK and 7Department of Urology, Memorial Sloan Kettering Cancer Center, New York, NY, USA Our previous work identified a chromosomal translocation t(4;6) in prostate cancer cell lines and primary tumors. Using probes located on 4q22 and 6q15, the breakpoints identified in LNCaP cells, we performed fluorescence in situ hybridization analysis to detect this translocation in a large series of clinical localized prostate cancer samples treated conservatively. We found that t(4;6)(q22;q15) occurred in 78 of 667 cases (11.7%). The t(4;6)(q22;q15) was not independently associated with patient outcome. However, it occurs more frequently in high clinical T stage, high tumor volume specimens and in those with high baseline PSA (P ¼ 0.001, 0.001 and 0.01, respectively). The t(4;6)(q22;q15) occurred more frequently in samples with two or more TMPRSS2:ERG fusion genes caused by internal deletion than in samples without these genomic alterations, but this correlation is not statistically significant (P ¼ 0.0628). The potential role of this translocation in the development of human prostate cancer is discussed. Prostate Cancer and Prostatic Diseases (2010) 13, 117–125; doi:10.1038/pcan.2010.2; published online 23 February 2010 Keywords: chromosome translocation; genomic instability; prognosis Introduction Chromosomal translocations are recognized initiating events in some hematological malignancies and soft Prostate cancer is the most common male malignancy tissue sarcomas, and are associated with tumor progres- and the second most common cause of cancer death in sion and even response to therapies.6 Recent identifica- men in the Western countries.1 Prostate cancer is a tion of frequent fusion genes in prostate6–8 and lung9,10 heterogeneous disease with a highly variable natural cancers highlights the potential roles of recurrent history of disease development and progression.2 Many chromosomal translocations and fusion genes in solid prostate cancers diagnosed at an early stage are indolent tumors. Fusion of the ETS transcription factor genes to but a proportion progress rapidly to become life TMPRSS2 and other genes has been identified in a high threatening. However, it is difficult to predict the proportion of prostate cancer cases.7,8 The association of progression potential of early stage cancers.3 In spite of ERG fusion with poor outcome in conservatively the numerous investigations into the molecular mechan- managed patients has been reported,11 although recent ism of development of the disease,4 the nature and studies correlating genomic alterations and prostate significance of genetic changes associated with prostate cancer patient outcome in large series of clinical samples cancer development and progression are largely un- showed genetic instability to be critical in prostate cancer known. These highlight the need for genetic investiga- development and progression.12,13 In addition, recent tions into this malignancy.5 transcriptome sequencing analysis revealed many more fusion genes and chromosomal alterations occurring in prostate cancer cells, although some of them may arise in late stage during cancer progression.14 Correspondence: Dr Y-J Lu, Centre for Molecular Oncology and Using multiplex fluorescence in situ hybridization Imaging, Institute of Cancer, Barts and The London School of (FISH) we previously identified a t(4;6) chromosomal Medicine and Dentistry, Queen Mary University of London, translocation in prostate cancer cell lines and primary Charterhouse Square, London, EC1M 6BQ, UK. tumors15 and mapped the breakpoints in LNCaP E-mail: [email protected] 15 Received 20 October 2009; revised 15 December 2009; accepted cells using FISH analysis. In this study, according to 18 December 2009; published online 23 February 2010 the breakpoint locations revealed, we performed an T(4;6)(q22;q15) in prostate cancer L Shan et al 118 extensive analysis of clinical prostate cancer samples by without initial treatment except early hormone manage- FISH on tissue microarrays (TMAs) using probes located ment. Clinical outcome data were available for all these on 4q22 and 6q15. We identified the t(4;6)(q22;q15) cases. The median follow-up time was 121 months (8–203 chromosomal translocation in a number of prostate months) and more than 80% of the men were diagnosed cancer samples and correlated it with clinical features. after the age of 65. The 10-year overall survival rate was 50 and 17% of the patients died of prostate cancer.2 TMAs were constructed using a manual tissue microarrayer into Materials and methods 35 Â 22 Â 7 mm paraffin wax blocks. Up to four tumor cores of 0.6 mm diameter were taken from each prostate sample. Clinical materials and TMA construction National approval was obtained from the Northern Multi- Four batches of TMAs were made: the first batch included Research Ethics Committee for the collection of the one TMA containing 16 cases of non-prostate non- cohort and followed by local research ethics committee malignant controls from a variety of human tissue types approval at individual collaborating hospitals. (Table 1). The second batch was a TMA made from 34 BPH A pathologist (DB) examined all samples and graded samples collected from the Barts and The London Hospital each cancer specimen with Gleason scores and the TURP specimens. The third batch comprised two TMAs samples on the fourth batch of TMAs were also centrally constructed from 68 archival anonymous prostate cancer reviewed by pathologists (DB, CSF and VR) in the samples from radical prostatectomy specimens and Transatlantic Prostate Group. 6 morphologically non-malignant prostate samples ob- tained from the Barts and The London Hospital. These three batches of TMAs were constructed in 35 Â 22 Â 4mm FISH probe preparation blocks of paraffin wax using a manual tissue microarrayer Bacterial artificial chromosomes including RP11-18N21, (Beecher Instruments, Sun Prairie, WI, USA). Triplicate RP11-681L8 and RP11-240J11 on distal 4q22 (probe set I, cores of 1 mm diameter were taken from each sample. The see Figure 1a), RP11-111J1, RP11-595C20 and RP1-214H13 collection of these specimens was approved by the local on 6q14.3 proximal to the 6q15 breakpoint (probe set II, ethical committee. see Figure 1b) and RP1-44N23, RP1-154G14 and RP11- The fourth batch comprised 24 TMAs constructed from 104N3 on distal 6q15 (probe set III, see Figure 1c) were a large cohort of 808 cases of TURP prostate cancer obtained from the Wellcome Trust Sanger Institute specimens. All the patients were managed conservatively (Hinxton Hall, Cambridge, UK). The bacterial artificial chromosome DNA extraction, amplification and labeling Table 1 Scoring of non-malignant control samples on the TMAs were the same as described earlier.16 Briefly, bacterial for colocalization of probes on 4q22 and 6q14.3 artificial chromosome DNA was amplified using illustra Sample Total cells Colocalization Percentage GenomiPhi V2 DNA amplification kit (GE Healthcare positive Life Sciences, Buckinghamshire, UK) following the manufacturer’s instructions and then labeled with biotin Appendix 98 5 5.10 or digoxigenin (DIG) using the nick translation method. Breast 60 2 3.30 Cervix 1 104 4 3.80 Cervix 2 67 1 1.49 Colon 59 1 1.69 FISH analysis Foreskin 57 2 3.51 Inflamed gall bladder 54 1 1.90 The FISH analysis was performed using the method as Kidney 53 1 1.89 described earlier12 with slight modifications. Briefly, Lung 50 1 2.00 m Placenta 60 2 3.30 4 m TMA sections were cut onto SuperFrostPlus glass Prostate 65 1 1.50 slides (VWR International, Poole, UK) and baked at 65 1C Small Intestine 59 3 5.10 overnight. TMA slides were dewaxed in xylene and fixed Stomach 55 1 1.80 with boiling ethanol. The tissue sections were then Testis 50 1 2.00 brought to boiling-point in pre-treatment buffer (SPOT- Tonsil 63 2 3.17 light tissue pre-treatment kit, Zymed, South San Fran- Uterus 55 1 1.80 cisco, CA, USA) before cooling to room temperature and Abbreviation: TMA, tissue microarray. digested with pepsin solution (Digest All-3, Invitrogen, Probe set I Probe set II Probe set III 0.54Mb 0.39Mb 0.80Mb RP11-18N21 RP11-240J11 RP11-111J1 RP11-214H13 RP1-44N23 RP11-104N3 RP11-68L8 RP11-595C20 RP1-154G14 4q22.3 6q14.3 6q15 C4orf37 LOC643962 MAP3K7 BACH2 Figure 1 Maps showing the position of the bacterial artificial chromosomes (BACs) used as probes in FISH assays. (a) Probe set I includes three BACs, RP11-18N21, RP11-681L8 and RP11-240J11, corresponding to a 0.54-Mb region of distal 4q22 breakpoint. (b) Probe set II includes three BAC clones, RP11-111J1, RP11-595C20 and RP1-214H13, corresponding to a 0.39-Mb region of 6q14.3 (proximal to 6q15 breakpoint).