Retention of Interstitial Genes Between TMPRSS2 and ERG Is Associated with Low-Risk Prostate Cancer Stephen J

Published OnlineFirst November 10, 2017; DOI: 10.1158/0008-5472.CAN-17-0529 Cancer Tumor and Stem Cell Biology Research

Retention of Interstitial Genes between TMPRSS2 and ERG Is Associated with Low-Risk Prostate Cancer Stephen J. Murphy1, Farhad Kosari1, R. Jeffrey Karnes2, Aqsa Nasir1, Sarah H. Johnson1, Athanasios G. Gaitatzes1,3, James B. Smadbeck1, Laureano J. Rangel4, George Vasmatzis1, and John C. Cheville1,5

Abstract

TMPRSS2-ERG gene fusions occur in over 50% of prostate interstitial sequences occurred more frequently in very low-risk cancers, but their impact on clinical outcomes is not well tumors. These tumors also frequently displayed ERG gene understood. Retention of interstitial genes between TMPRSS2 fusions involving alternative 50-partners to TMPRSS2,specifi- and ERG hasbeenreportedtoinfluence tumor progression in cally SLC45A3 and NDRG1 and other ETS family genes, which an animal model. In this study, we analyzed the status of retained interstitial TMPRSS2/ERG sequences. Lastly, tumors TMPRSS2-ERG fusion genes and interstitial genes in tumors displaying TMPRSS2-ERG fusions that retained interstitial from a large cohort of men treated surgically for prostate cancer, genes were less likely to be associated with biochemical recur- associating alterations with biochemical progression. Through rence (P ¼ 0.028). Our results point to more favorable clinical whole-genome mate pair sequencing, we mapped and classified outcomes in patients with ETS family fusion-positive prostate rearrangements driving ETS family gene fusions in 133 cases of cancers, which retain potential tumor-suppressor genes in the very low-, low-, intermediate-, and high-risk prostate cancer interstitial regions between TMPRSS2 and ERG. Identifying from radical prostatectomy specimens. TMPRSS2-ERG gene these patients at biopsy might improve patient management, fusions were observed in 44% of cases, and over 90% of these particularly with regard to active surveillance. Cancer Res; 77(22); fusions occurred in ERG exons 3 or 4. ERG fusions retaining 1–11. 2017 AACR.

Introduction of patients with prostate cancer has remained limited. In the largest cohorts of prostate cancer patients tested to date involving While large advances have been made in the understanding of a combined total of over 5,500 cases, ERG overexpression was not the biology of prostate cancer, there remain significant gaps in our prognostic for biochemical recurrence (BCR) or disease-specific understanding of prostate cancer initiation and progression, and mortality following radical prostatectomy (3–6). In addition, the this affects the management of prostate cancer patients. Clinically, presence of TMPRSS-ERG fusion has not been predictive of biomarkers are needed to better stratify patients that have prostate improved response to radiotherapy, although a link between ETS cancer with a low risk of progression from those at higher risk. This fusions and altered DNA repair mechanisms has been proposed is critically important as many patients are choosing active sur- (7, 8). However, clinical associations have been predicted with veillance with presumptive low-risk disease based on limited ETS fusion status and response to hormonal therapies (3, 9). In sampling of their prostate cancer. Even though more than a several cohorts of active surveillance patients, men whose tumors decade has passed since the discovery of TMPRSS2-ERG gene were ERG fusion positive at diagnosis were more than twice as fusion (1, 2), the impact of this fusion in the clinical management likely to require treatment compared with men with ERG-negative tumors. (10). The ETS-family of transcription factors currently comprises 29 1Biomarker Discovery Program, Center of Individualized Medicine, Mayo Clinic, unique genes in humans, including ERG(21q22), ETV1(7p21), 2 Rochester, Minnesota. Department of Urology, Mayo Clinic, Rochester, Minne- and ETV4(17q21) (11). Conserved ETS DNA-binding domains sota. 3Genomics Systems Unit, Mayo Clinic, Rochester, Minnesota. 4Department define the family members, with additional basic helix-loop-helix of Health Sciences Research, Mayo Clinic, Rochester, Minnesota. 5Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. pointed domains, transcriptional activation and/or inactivation domain variably present, instilling specific cellular functions (12). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Overexpression of ETS family proteins in prostate cancer is commonly initiated through the positioning of androgen-responsive S.J. Murphy and F. Kosari contributed equally to this article. promoters in frame with ETS-family member genes, resulting in Corresponding Authors: J.C. Cheville, Mayo Clinic, 200 First St., SE, Rochester, disease-driven oncogenic functions. The androgen-responsive MN 55905. Phone: 507-284-3867; Fax: 507-284-1599; E-mail: gene, TMPRSS2 (21q22), is principally observed fused near its [email protected]; or G. Vasmatzis, [email protected] first exon, placing its promoter in frame with foremost 50-exons of doi: 10.1158/0008-5472.CAN-17-0529 ETS-family transcription factors, retaining their characteristic 0 2017 American Association for Cancer Research. functional domains (1). Although less numerous, additional 5

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fusion partners include other prostate-responsive genes, stitial sequence between TMPRSS2 and ERG is more commonly SLC45A3, NDRG1, HERV-K 22q11.23, and C15orf21, as well as found in low-risk prostate cancer. a strongly expressed housekeeping gene (HNRNPA2B1) with no prostate specificity or androgen-responsiveness (13, 14). Materials and Methods The prevalence of ETS-family fusions in prostate cancer suggests Microarray expression data a significant role in prostate cancer development (1, 15, 16). The Expression analyses used an Affymetrix (U133PLUS2) micro- elucidation of the oncogenic activation of members of the ETS- array dataset from a prior study (29) that included expression family of transcription factors was envisioned to have significant profiles of laser capture microdissected-derived collections of clinical impact in the treatment of prostate cancer and provided prostate tumor cells from Mayo patients including 28 cases of rational therapeutic targets (2). However, although recurrent gene Gleason score 6 (low-risk) and 36 cases of Gleason score 7 and fusions involving ALK, ROS, MET, and ABL have been tremen- higher prostate cancer (intermediate- and high-risk) as well as dously successful therapeutic targets in other cancers, fusions lymph node metastases. Normalized expression values were involving ETS-family of transcription factors have proven noto- computed by the "gcrma" R/Bioconductor package (https:// riously difficult to target (2, 17). ETS members are associated with www.bioconductor.org/). The ERG status of tumors was based the regulation of cell growth, proliferation, differentiation, and on the log intensity values of "213541_s_at" probeset above or apoptosis, through activation or repression of target genes (18). 2 below a threshold of 6. Associations of fusion and BCR status with However, in vivo studies recapitulating ERG or ETV1 expression in gene expression levels were calculated by group t tests and mice fell short of generating carcinoma, resulting only in the corrected for multiple hypotheses testing using the "qvalue" development of prostatic intraepithelial neoplasia (PIN; refs. 12, package in R. Reported values are P values after multiple hypoth- 19, 20). Thus although early acquisition of the fusion suggests a esis correction (q values). role in prostate cancer initiation, ETS fusions could potentially be primers to tumorigenesis, with other subsequent driver mutations, such as PTEN loss (21), dictating cancer progression. With Tissue collection and processing no consistent association of TMPRSS2-ERG gene fusion with Consecutive cases selected from the Mayo Clinic prostate clinicopathologic features nor cancer outcome, the prognostic cancer–frozen repository from radical prostatectomy specimens role of TMPRSS2-ERG gene fusions in prostate cancer remains were initially grouped according to Gleason grade group (30), limited (22). with 78 cases of grade group 1 (Gleason 3þ3), 15 cases of grade Although significant biological differences have been observed group 2 (Gleason 3þ4), 13 cases of grade group 3 (Gleason 4þ3), among different ETS family fusions, the clinical impact is still 8 cases of grade group 4 (Gleason 4þ4), and 17 cases of grade emerging (3). The structures of TMPRSS2-ERG fusions have been group 5 (Gleason scores 9 and 10). Grade group 1 was addition- demonstrated to impart differing biological functions in vitro, ally subgrouped according to tumor volume as very low-risk which could potentially influence clinical responses (23, 24). The (clinically insignificant confined grade group 1 Gleason score 6 3 mechanism of the gene fusion may additionally influence prog- cancer with tumor volume less than 0.7 cm ; INS GS6; 53 cases) nosis (25, 26). Two mechanisms of TMPRSS2-ERG fusion have and low-risk larger volume grade group 1 Gleason score 6 (tumor 3 been described that while resulting in structurally identical fusion volume greater than 0.7 cm ; LV GS6; 26 cases). Grade groups 2 products, proceed through either an interstitial deletion or an and 3 were combined to an intermediate-risk group Gleason score insertional recombination, with the key difference lying in the 7 (GS7; 29 cases) and grade groups 4 and 5 similarly grouped presence or absence of genes contained in the approximately 3 Mb together as high-risk Gleason score 8 and higher cancers (GS8þ; interstitial region (27). A recent report by Linn and colleagues 25 cases). Processing of radical prostatectomy specimens occurred investigated the potential role of these interstitial genes using as previously described (31). All specimens were handled in the mouse models (28). Seventeen known protein encoding genes lie fresh state and analyzed initially with a frozen microtome tech- between TMPRSS2 and ERG, with BACE2, ETS2, HMGN1, and nique. The prostate was inked, serially sectioned in the horizontal MX1 having potential function in tumorigenesis, and their in vitro plane, and right apex, left apex, and bladder base margins (per- studies in cell lines indicated a potential role for ETS2 as a tumor- pendicular) were examined microscopically followed by standard suppressor gene in prostate cancer (28). In addition, loss of just a sections from the inferior, mid, and superior posterior prostate single copy of ETS2 resulted in the development of larger high- and right and left vesicles. In cases of insignificant Gleason score 6 grade PIN lesions, compared with controls, and Lin and collea- cancers, the entire posterior prostate was examined with addi- gues suggested that loss of these interstitial genes could result in tional sections of the right and left anterior prostate. Clinicopath- more aggressive prostate cancer. Of note, the limitation of mouse ologic data from this patient cohort have been previously models expressing TMPRSS2-ERG gene fusions to progress described (21) and are provided in Supplementary Table S1. The beyond PIN necessitated a PTEN-null model to drive tumor study was approved by an Institutional Review Board. progression (28). In order to better stratify prostate cancer patients, we need to Isolation of DNA and mate pair sequencing increase our understanding of the mechanisms underlying Tumor was collected by laser capture microdissection (Arcturus tumor progression. The advent of high-throughput next-gen- instrument) from 10-mm unstained fresh-frozen sections and eration sequencing (NGS) has dramatically propelled our DNA amplified directly from cells, as previously described knowledge of somatic variation present in tumors. Here, we (32, 33). In Gleason score 7, the Gleason patterns 3 and 4 were utilize mate pair NGS of 133 prostate cancers, ranging from collected and analyzed separately. Indexed libraries were prepared very low- to high-risk disease, to characterize the presence, using the Illumina Mate-Pair (MP) Kit following the manufac- diversity, and structure of all ETS-family rearrangements. Spe- turer's instructions and sequenced as two libraries per lane on the cifically, we tested the hypothesis that the retention of inter- Illumina HiSeq2000 platform (34). Data were processed using

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ERG Fusions in Low Risk Prostate Cancer

previously described binary indexing mapping algorithms devel- large volume Gleason score 6 (LV GS6); 26 (20%) cases, inter- oped in our group (35). The read-to-reference-genome-mapping mediate-risk Gleason score 7 (GS7); 29 (22%) cases and high-risk algorithm was modified to map both mate pair reads across the Gleason score 8 and higher (GS8þ); 25 (19%) cases, as described whole genome. Discordant mapping mate pair reads covered by at in Materials and Methods and in Supplementary Table S1. The least five associates were identified for further analysis. Concor- tiling of the genome with larger fragments (3 kb) through mate dant mapping mate pair reads were used to determine frequency pair sequencing (MPseq) enabled efficient genome-wide profiling coverage levels across the genome (35). All raw data mate pair of chromosomal rearrangements. The profile of all ETS family reads mapping to chromosome 21 for each case studied are rearrangements, focusing specifically on TMPRSS2-ERG fusion deposited in the NCBI Sequence Read Archive (SRA) database, and interstitial deletions between the two genes, was investigated which can be accessed at the following link: https://trace.ncbi. in all cases. nlm.nih.gov/Traces/sra/sra.cgi?study=SRP119412. As expected, TMPRSS2-ERG fusions were observed at high frequency in our population, present in 45% (60 of 133 cases) Statistical analyses of the study cohort. In the very low-risk, low-risk, and interme- A c2 test was used to identify associations of fusion status and diate-risk groups, fusions were observed in 43%, 59%, and 52% of Gleason score. Altogether, there were a total of 99 samples in three cases, respectively (Fig. 2A). In the intermediate group, identical fusion categories including tumors with no fusion (NEG, n ¼ 46), TMPRSS2-ERG fusions were observed in adjacent Gleason pat- tumors with ERG fusion but retaining interstitial regions (RET, n ¼ terns 3 and 4 of the same tumor, which were isolated individually. 20), and tumors with ERG fusion and deleted interstitial regions The high-risk group had a much-reduced ERG fusion–positive (DEL, n ¼ 33). Gleason scores were categorized as either GS6 population at 24%. This high-risk group was previously reported (including "INS GS6" and "LV GS6") or GS7 and higher. Using to have high frequency in PTEN deletion events, many in the "pwr.chisq.test" function in the "pwr" package, we determined absence of TMPRSS2-ERG fusions (21). Figure 2B illustrates the there was 90.5% power to detect a medium effect size of 0.35 or spectrum of TMPRSS2-ERG gene fusion junctions present in our higher at 0.05 significant levels. Reported P value was based on the study. Fusions of TMPRSS2 exon 1 or 2 to ERG exons 3 and 4 were c2 analysis. predominant and observed similarly between the different risk The three groups of samples were also analyzed for associations groups. Ninety percent of the fusions involved exon 3 or 4 of ERG. with BCR. The powerCT function in the "powerSurvEpi" R pack- Similarly, 85% of fusions originated from exon 1 or 2 from 0 age indicated that given the number of samples in RET and DEL, TMPRSS2. The high selection of fusions at the foremost-5 exons there was a 73% power to detect a relative risk of 3.74 or higher of ERG retained the oncogenic functional domains. Similarly, 0 with a type I error of 0.05. Using RET group as the baseline, the fusions with the foremost-5 exons of TMPRSS2 enable gain of associations of DEL and NEG groups with the BRC were computed promoter functions with minimal interference of TMPRSS2 struc- by using the coxph function in the R "survival" package. Reported tural domains. Although the variation in fusion structure is results are the P values by the likelihood ratio tests. significant, the low occurrence of fusions at later exons of both TMPRSS2 and ERG potentially indicates lesser oncogenic poten- fi Results tial, tting the early presentation of these alterations with key functions in tumor initiation. Interestingly, the majority of Interstitial gene expression and BCR fusions varying from ERG exon 3/4 fusions were from the very The expression level of interstitial genes between TMPRSS2 and low-risk (insignificant) prostate cancer (5 of 6 cases). ERG with BCR, defined as PSA 0.4 post–radical prostatectomy, was evaluated in microarray data on a set of 64 radical prosta- TMPRSS2-ERG fusion and interstitial deletion fi tectomy specimens. Cases were initially strati ed for ERG expres- In addition to using discordant mapping reads to define chro- sion and the expression levels of interstitial genes plotted mosomal breakpoints, MPseq utilizes concordant reads to define (Fig. 1A). Although no details of the mechanism of ERG fusion copy-number variations across the genome. Frequency coverage were available in this case study, a drop in expression was evident across chromosome 21 for each case is presented in Supplemen- for the majority of genes in the ERG-positive group. Most signif- tary Fig. S1 and Supplementary Table S2. We utilized the copy- q icant drops in expression levels were associated with IGSF5 ( number data to investigate interstitial chromosomal sequence < 9 q < 5 value 10 ) and FAM3B ( value 10 ). However, mean ETS2 loss between TMPRSS2 and ERG on chromosome 21 in different levels were not decreased in the ERG-positive group, indicative of Gleason score groupings and the presence or absence of ERG fl potential additional mechanistic regulatory factors in uencing fusions (Fig. 2C). No difference in the distribution of TMPRSS2- fi gene expression through promoter-speci c effects. In addition, no ERG fusion with interstitial deletion was seen between Gleason fi signi cant association of interstitial gene expression loss with BCR score 6 cancer and Gleason score 7 and higher (P ¼ 0.30). was observed in the ERG-positive cases (Fig. 1B). Thus, to evaluate However, TMPRSS2-ERG–positive cases in the absence of a dele- further associations of clinical outcome, the precise mechanism of tion were significantly enriched in the very low-risk and low-risk TMPRSS2-ERG gene fusions was pursued in an independent Gleason score 6 groups (P ¼ 0.0006; Fig. 2C). The pattern of exon prostate cancer cohort. fusions did not appear to change significantly between the groups; however, the size of samples in different groups was too small for Spectrum of TMPRSS2-ERG fusions rigorous statistical considerations (Fig. 2D). The mechanism of TMPRSS2-ERG gene fusion was investigated in an independent dataset of 133 prostate cancers from radical Mechanisms of TMPRSS2-ERG fusions prostatectomy specimens of varying Gleason grade and tumor TMPRSS2-ERG fusion events proceed by two distinct mechan- volumes. Cases were split into four groups: very low-risk (clinical isms: direct fusions or complex rearrangement events. In our insignificant Gleason score 6; INS GS6) 53 (40%) cases, low-risk series, direct fusions encompassed the most common mechanism

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Figure 1. Interstitial gene expressions from microarray data. Box plots represent

log2 expression (y axis) of interstitial genes between ERG and TMPRSS2 (x axis) in ERG fusion–negative (gray) and positive (white) prostate cancer cases (A) and in ERG fusion–positive cases without (gray) or with (white) BCR (B). Thick black bars represent median expression levels. Genes with signiﬁcant differential expression , q < 0.05.

involving 36 (63%) of the ERG-positive cases. Figure 3A illustrates very low-risk Gleason score 6 case, and a Gleason score 7 case, a direct deletion-fusion in a representative case, together with the involved complex rearrangements involving four and three dif- characteristic copy loss of interstitial genes between TMPRSS2 and ferent chromosomes, respectively, but ultimately retained the ERG on chromosome 21. The remaining 21 cases (37%) had interstitial regions between TMPRSS2 and ERG (Supplementary complex rearrangements involving TMPRSS2-ERG potentially Fig. S2). The next three cases in Table 1, PR007, PR013, and occurring via concurrent fusion and rearrangement. Figure 3B PR042, involved direct TMPRSS2-ERG fusions with additional exemplifies a complex rearrangement with loss of the interstitial independent nonrelated events hitting either the TMPRSS2 or gene region for a very low-risk Gleason score 6 case in which a 50 ERG genes, each of these three fusions occurred via a standard kb region within the DSCAM gene is retained between the interstitial deletion mechanism. TMPRSS2-ERG fusion and the rest of the interstitial sequence The next seven cases listed in Table 1 involved complex non- lost. Figure 3C exemplifies a complex rearrangement where the direct (50)TMPRSS2-ERG(30) fusions (Table 1). For these cases, no interstitial gene region is retained for a very low-risk Gleason score direct paired sequencing reads were detected connecting 6 case in which the interstitial regions translocate to within TMPRSS2 and ERG, but combinations of rearrangements were UMODL1, an adjacent gene on chromosome 21. The complex discovered for each case bringing the two genes together for TMPRSS2-ERG fusion cases were divided into three classes (Table productive fusions (Fig. 3B). Two of these seven cases resulted 1). The first 19 cases presented with complex events in the in loss of the interstitial sequence. presence of standard direct (50)TMPRSS2-ERG(30) fusion events. The majority of the complex TMPRSS2-ERG fusion events Three of these events resulted in loss of interstitial regions between (15 of 25, 60%) occurred in the very low-risk group. Overall, TMPRSS2 and ERG, but contained links from the 30 region of 76% (19 of 25) occurred in the very low- and low-risk Gleason TMPRSS2 and the 50 region of ERG at the breakpoints to other score 6 cancers compared with 16% (4 of 25) in Gleason score sites. The remaining 16 cases presented with complex balanced 7 and 8% (2 of 25) in Gleason score 8 and higher. rearrangements that retained the interstitial region sequences (in one case, there was a copy-number gain in this region). Several of TMPRSS2-independent ERG rearrangements these cases were characterized by complex translocations involv- In addition to the cases with TMPRSS2-ERG fusion, eight cases ing up to three different chromosomes (Supplementary Fig. S2). A contained ERG fusions with other androgen-driven genes,

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Figure 2. Spectrum of TMPRSS2-ERG gene fusions in prostate cancer. A, Percentage of cases in very low-risk (GS6 INS; gray), low-risk (GS6 LV; checkered), intermediate-risk (GS7; black), high-risk (GS8þ; hashed), or from all prostate cancer cases (black and white) with TMPRSS2-ERG gene fusions. B, Break down of cases from each group according to exon fusion position. T# and E# refer to speciﬁc exons in TMPRSS2 and ERG genes, respectively. C, Percentage of cases of Gleason score 6 (GS6; gray) or Gleason score 7 and above (GS7þ; black) that are ERG positive through deletion or no deletion of interstitial region between TMPRSS2 and ERG genes. D, Distribution of ERG-positive cases with or without interstitial deletion according to ERG exon fusion position.

SLC45A3 in ﬁve cases, and NDRG1 in three cases (Table 1, bottom TMPRSS2 and ERG fusions (Table 2). ETV1 was affected in ﬁve section). All of these cases were Gleason score 6 cancers, with six additional cases, whereas only two were in the correct orientation very low- and two low-risk tumors. Similar to the TMPRSS2-ERG to produce a fusion product. In a very low-risk Gleason score 6 fusions, the majority of these cases (6 of 8) fused with exon 3 or 4 case, the previously reported androgen-driven gene HNRNPA2B1 of ERG, with two additional fusions at exons 5 and 6 (Fig. 3D). Six (13, 14) was fused to the promoter of ETV1 (Table 2, PR135). In of the fusions also occurred at foremost 50-exons of SLC45A3 or another very low-risk cancer case (Table 2, PR60), ETV1 was NDRG1. joined with a novel partner PCBP2 on chromosome 12. Other Four additional cases presented with TMPRSS2 fusions to other novel 50 drivers were observed for ETV family members, ETV3, ETS family genes, two to ETV1 and one each to ETV4 and ETV5. ETV4, ETV6, SPDEF, and FLJ1, involving EMC1, FASN, VMP1, The ETV1 and ETV4 fusions were in very low-risk Gleason score 6 PACSIN1, and ZZZ3, respectively. All but PACSIN1 were signif- cancers and the ETV5 in a Gleason score 7 cancer (Table 1). Thus, icantly expressed in prostate tissue from expression data (data although the number of cases is relatively small, like the complex not shown). Previously reported androgen-driven fusions TMPRSS2-ERG fusions, these alternative TMPRSS2 and ERG HMGN2P46-ETV4 and SLC45A3-ELK4 (13, 14) were each seen fusions were more common in the low-risk prostate cancers. in a single case. SLC45A3 was also observed fused to two previously unreported non-ETS family genes: ZBTB7B and AADACL2. Other ETS family and androgen-driven fusions Nonproductive fusions with ETS family members were also seen Additional cases contained breakpoints affecting other ETS in a number of cases with predicted incorrect orientation for family members, occurring independently to the common expression (Table 2). Evaluation of productive fusions with a large

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Figure 3. TMPRSS2-ERG fusion mechanisms. A, Direct interstitial deletion TMPRSS2- ERG fusion in very low-risk insignificant Gleason score 6 case, PR057. B, Complex interstitial deletion TMPRSS2-ERG fusion in very low-risk insignificant Gleason score 6 case, PR058. C, Complex no interstitial deletion TMPRSS2-ERG fusion in very low-grade insignificant Gleason score 6 case, PR014. Normal genome structure represented above rearranged genome region for each case, with specific genome positions and exons indicated. Coverage across chromosome 21 for each case is illustrated in lower left corner of each example with the presence or absence of interstitial gene regions between TMPRSS2 and ERG. D, SLC45A3-and NDRG1-driven ERG fusions. Positions of fusion breakpoints (vertical gray arrows) with case name and ERG exon fusion position (E#) indicated in the SLC45A3/ELK4 1q32.1 (top) and NDRG1 8q24.22 (bottom) gene regions, illustrated as horizontal dark arrows, with exon positions indicated as vertical lines.

panel of androgen-driven/prostate-specific genes yielded just six S1). Of these cases, 49 represented the control ERG-negative additional events, with one of the most functionally significant group with no ETS fusions, 34 and 22 contained TMPRSS2-ERG involving a KLK3-TP53 fusion in a case in the Gleason score 8 and fusions with interstitial region deletion or retention, respectively, higher category, which might have affected PSA levels for that with 12 cases excluded due to no clinical follow-up on BCR, the patient. Finally, as previously reported (13, 14), the overlap of presence of alternative ETS fusions to ERG, or the presence of additional ETV fusion events with TMPRSS2-ERG fusions was interstitial region gains/homozygous losses (Supplementary minimal. Table S1). There was no significant difference in the BCR rate between ERG fusion–negative and –positive cases (Fig. 4A, P ¼ Associations of BCR and interstitial gene deletion 0.15). However, in univariate analysis cancers with ERG fusion ERG translocation with no loss of the interstitial sequence without interstitial deletion were associated with a significantly between TMPRSS2 and ERG, including translocation to alterna- lower BCR incidence than cancers with ERG fusion and interstitial tive driver genes to TMPRSS2, was more frequent in the Gleason deletion (P value ¼ 0.028; Fig. 4B) and also compared with the score 6 cancers, with an increased association with very low- and ERG-negative tumors (P value ¼ 0.014). Cancers with an ERG low-risk disease (Fig. 2C). The clinicopathologic features of the fusion with deletion had risk profiles similar to the cancers MPseq cases were available for 118 (89%; Supplementary Table without the fusion (Fig. 4C).

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. www.aacrjournals.org Table 1. TMPRSS2 and ERG gene fusion events Case Group ERG fusion TMPRSS2 events ERG events ETS expression Fusion mechanism Downloaded from Complex TMPRSS2-ERG 11 GS7 T3-E3 Duplication event ERG Retention fusion events 14 GS6 INS T1-E4 T1-UMODL1(21q) E4/UMODL1(21q) ERG Retention 20 GS6 INS T1-E4 T5-ZMIZ1(10q), C10orf11(10q)/T2 E4/ng(10q) ERG Retention 21 GS7 T1-E4 T1-PXDN(2p)Bal, FOXP1(3p)/T4 FOXP1Pr(3p)/E5 ERG Retention 28 GS7 T1-E4 E5-GRIN3A(9q)(GP4) ERG Gain

29 GS6 INS T1-E4 T2-E4C, DSCAM(21q)/T5 ERG Deletion Published OnlineFirstNovember10,2017;DOI:10.1158/0008-5472.CAN-17-0529 44 GS6 LV T1-E3 T1-ZNF589(3p) ERG Retention 47 GS6 INS T2-E3 T4/ng(2p) ng(2p)/E4 ERG Retention cancerres.aacrjournals.org 68 GS6 INS T2-E5 Complex rearrangement between ERG Retention TMPRSS2 and ERG 80 GS6 LV T1-E4 E3-T2 HMGN1(21q)-E4, E4/RTN1(14q) ERG Retention 84 GS6 INS T1-E4 C6orf106(6p)-T6,T6/HSP90AA1(14q) E4/ANPEP(4q) ERG Retention 87 GS6 INS T1-E1 EP/PARM1(4q) ERG Retention 92 GS6 INS T1-E5 C8orf37AS1(8q)-T1,T(Pr)/TTC3(21q) IGSF5pA/E5 ERG Retention 96 GS6 INS T2-E4 Associated internal E5/T3 ERG Retention rearrangement event 99 GS6 INS T3-E4 T4/ng(3q) PTPRM(18p)/E4 ERG Retention 108 GS8þ T1-E3 T11/CAMTA1(1p) DSCAM(21q)-E4, E3/RERE(1p) ERG Deletion 130 GS8þ T1-E3 T1-BACE2(21q) E3-BACE2(21q) ERG Retention 132 GS6 INS T1-E3 T1-BDP1(5q), T4/ng(5q) ERG Deletion

Research. 13 GS7 T2-E4 COMPLEX Inversion around ERG ERG Deletion 42 GS6 INS T1-E3 E4/ng(16q12.2) Bal ERG Deletion Complex nondirect 35 GS7 T6-E3 C Complex fusion ERG Retention additional events predicted 48 GS6 LV T1-E3 C PRDM15-T2, T1-ng(21q22.2) ng(21q22.2)/E4 ERG Retention 58 GS6 INS T1-E4 C T1-DSCAM DSCAM-E5 ERG Deletion 72 GS6 INS T1-E C CHODL(21q)/T1 ERG Retention 97 GS6 INS T1-E1 C T2-E1 E1/TRABD2B(1p33) ERG Deletion 74 GS6 LV T4-E4 C T4/SLC1A3(5p) E4-CAPSL(5p) nd Retention 95 GS6 IND T5-E3 C Complex inverted fusion no nd Retention additional events detected SLC45A3 events 19 GS6 INS S1-E3 SLC45A3(1q)-E3 ERG Retention 43 GS6 LV S1-E6 T4-PDE4D(5q) SLC45A3(1q)-E6, E5/ELK4(1q) ERG Retention R uin nLwRs rsaeCancer Prostate Risk Low in Fusions ERG 66 GS6 INS S1-E3 SLC45A3(1q)-E3 ERG Retention acrRs 72)Nvme 5 2017 15, November 77(22) Res; Cancer 70 GS6 INS S5-E4 T2-ELK4(1q),ELK4/SLC45A3-T12 SLC45A3(1q)-E4, E12/ng(9p) ERG/ELK4 Retention 89 GS6 INS S1-E4 SLC45A3(1q)-E4 ERG Retention NDRG1 events 17 GS6 INS N3-E4 NDRG1(8q)-E4 ERG Retention 79 GS6 LV N5-E5 NDRG1(8q)-E5 ERG Retention 133 GS6 INS N1-E4 NDRG1(8q)-E4, CLDN14(21q)/E4 ERG Retention ETV events 62 GS7 T2-V1 T2-ETV5(3q), ETV5 Retention T4-DGKG(3q) 69 GS6 INS T1-V2 T1-ETV1(7p), ICA1(7p)-T1 ETV1 Retention 91 GS6 INS T1-V1 T1-ETV4(17q), T4/ng(9q) ETV4 Retention 93 GS6 INS T2-V3 T2-ETV1(7p), T4-ANKRD10(13q) ETV1 Retention NOTE: Case groups, very low-risk Gleason score 6, GS6 INS; low-risk Gleason score 6, LV GS6; intermediate-risk Gleason score 7, GS7; and high-risk Gleason score 8 and higher, GS8þ. Fusion genes: T, TMPRSS2;E,ERG;S, SLC45A3;N,NDRG1;V,ETV1, -4, or -5; C, complex event. Number following gene shorthand indicates fusing exon. Productive and nonproductive aligned fusions separated by "-" or "/," respectively. Chromosomal cytobands are indicated in parenthesis after the gene names in the events columns. OF7 Published OnlineFirst November 10, 2017; DOI: 10.1158/0008-5472.CAN-17-0529

Murphy et al.

Table 2. Additional ETS gene family and androgen-driven gene fusions Productive Nonproductive Other androgen Predicted ERG fusion Case Group fusions fusions driven expression status 6 GS7 ETV1/ETV1 nd 7 GS7 KLKP1-SRPK2 SRPK2 T1-E4 13 GS7 EMC1-ETV3Pr C ETV3 T2-E4 14 GS6 INS ELK3/ATF71P T1-E4 15 GS6 INS FASN-ETV4 ETV4 nd 17 GS6 INS NDRG1/TMEM132B ZBTB16-MBOAT2 MBOAT2 N3-E4 21 GS7 NDRG1/ng T1-E4 23 GS7 APPBP2-CUEDC1 CUEDC1 T1-E3 31 GS7 VMP1-ETV6 C ETV6 T1-E4 32 GS7 HMGN2P46-LGSN LGSN nd 34 GS7 SLC45A3-ZBTB7B ETV6/ng ZBTB7B nd 35 GS7 ETV6/YBX3 C T6-E3 45 GS6 LV ZZZ3-FLI1 FLJ1 T1-E3 52 GS7 FLJ1/ng T1-E1 54 GS6 LV ETV4/GOSR2 T1-E4 60 GS6 INS PCBP2-ETV1 MAP3K12/ETV1 ETV1 nd 63 GS7 ELF5/ng nd 65 GS6 INS SLC45A3-ELK4 ETV1/ng ELK4 nd 69 GS6 INS TMPRSS2-ETV1 ETV1/ICA1pA ETV1 nd 73 GS6 INS SLC45A3-AADACL2 SLC45A3/LIPH AADACL2 nd 77 GS6 LV TSC22D1-CLYBL CLYBL T2-E3 88 GS6 INS ETV6/ABCC9 nd 89 GS6 INS ELK4Pr/FGGY nd 97 GS6 INS ETV6-CD163 ETV6 T1-E1 C 105 GS8þ ETV1/FOXA1 KLK3-TP53 TP53 nd 116 GS8þ GABPA/TXLNG2P T1-E3 118 GS8þ HMGN2P46-ETV4, ETV4 SPDEF nd PACSIN1-SPDEF 122 GS8þ ETV3L/CCT3 ELF2/ng nd 128 GS8þ DHCR24-GLIS1 GLIS1 nd 129 GS7 ETV3/ng nd 133 GS6 INS SLC30A4pA-NDRG1 NDRG1/DCTN6 NDRG1 N1-E4 135 GS6 INS HNRNPA2B1-ETV1 ETV1 nd NOTE: Case groups, very low-risk Gleason score 6, GS6 INS; low-risk Gleason score 6, LV GS6; intermediate-risk Gleason score 7, GS7; and high-risk Gleason score 8 and higher, GS8þ. Productive and nonproductive aligned fusions separated by "-" or "/," respectively. ETS family genes indicated in bold text. C, complex fusion events; nd, no fusion detected. TMPRSS2 (T) and ERG (E) gene fusions indicated with exon position.

Discussion stitial genes in prostate cancer would be expected to additionally The mechanism of TMPRSS2-ERG gene fusion on chromosome affect expression levels even from a single retained allele. Signif- 21q22, through either deletion of interstitial regions or insertion- icantly reduced expression of a number of interstitial genes, al chromosomal rearrangements, has been hypothesized to influ- specifically IGSF5 and FAM3B, was present in the ERG fusion ence the clinical outcome of prostate cancer tumors (24, 27). group (Fig. 1A); however, the role of interstitial genes in prostate PTEN-null murine models encompassing TMPRSS2-ERG fusion cancer remains to be determined. As no copy number data were through interstitial loss were recently reported to develop poorly available for these tumors, no subcategorization according to differentiated carcinomas that did not occur in mice with fusions interstitial gene loss was possible. retaining the interstitial genes, implicating ETS2 as a tumor- Mate pair sequencing afforded us the ability to precisely map all suppressor gene (28). Subsequent in vitro studies supported a rearrangements affecting the TMPRSS2 and ERG gene loci and role for ETS2 as a tumor-suppressor gene in prostate cancer cell other ETS family genes. We used these data to analyze association lines. Growth inhibition and apoptosis following downregula- of different TMPRSS2-ERG fusion mechanisms with BCR in an tion of ETS2 expression in human prostate cancer cells has also independent set of 133 prostate cancers, ranging from very low- been reported (36). Although ERG and ETS2 are both members of risk, insignificant Gleason score 6 to high-risk Gleason score 9 the ETS family of transcription factors, ERG contains an additional prostate cancers. TMPRSS2-ERG gene fusions were observed in activation domain at its C-terminus (12). It is therefore compel- 44% of the cases, in line with a recent study on 1,577 prostate ling to hypothesize that somatically induced expression of ERG, cancers from 8 independent cohorts, which reported 46% occur- or other ETS factors such as ETV1, may interfere with the functions rence (range, 38%–64%; ref. 5). Eight cases (6%) presented with of ETS2, which is expressed in normal prostate tissue (28). Our breakpoints in the ETV1 gene, but only half were predicted to be analysis of interstitial gene levels in RNA expression data from 64 productive fusions. In total, 14 cases (11%) presented with cases of prostate cancer failed to demonstrate any association predicted productive fusions on other ETS genes including ETV3 between ERG and ETS2 expression levels (Fig. 1A). As the majority (1 case), ETV4 (3 cases), ETV5 (1 case), ETV6 (2 cases), ELK4 (2 of the ERG-positive cases would be expected to have occurred cases), and FLJ1 (1 case) also consistent with other larger cohort through interstitial deletion, it is clear that the gene expression is a studies (3–5, 37–39). The number of ERG rearrangements retain- poor surrogate for allelic loss. Pathway regulation of these inter- ing the intervening sequences was 38% (26/68), with TMPRSS2 as

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ERG Fusions in Low Risk Prostate Cancer

Figure 4. ERG fusion and BCR. Survival curves of prostate cancer cases free of BCR with years from surgery (radical prostatectomy) illustrated for ERG- positive (black line) and -negative (gray line) cases (A) and with ERG-positive cases split into those with interstitial deletion (ERGþ/IntDel; solid line) or without interstitial deletion (ERGþ/ IntRet; hatched line; B). Number of cases in each group indicated (n). C, Distribution of very low-risk insigniﬁcant (GS6 IND; solid gray shading), low-risk large volume (GS6 LV; checkered gray shading), Gleason score 7 (GS7; solid black shading), and 8þ (GS8þ; hatched gray shading) cases with ERG fusion status and loss or no loss of interstitial genes.

the 50-partner in 73% (19/26), also consistent with a study by Despite the limited number of cases, there was a statistically Esgueva and colleagues on 540 prostate cancers (38%, with 71% significant difference in a univariate analysis of time to BCR (P TMPRSS2; ref. 37). The frequency of SLC45A3- and NDGR1-ERG value ¼ 0.028). Only four of 22 ERG-positive patients (18%) with gene fusions (6%) was also consistent with the study by Esgueva. retained sequences developed BCR compared with 35% (12 of However, we believe this is the first study to identify these cases 34) with deletion. There was no significant difference in recur- selectively presenting in very low-risk cancers. rence between cancers with TMPRSS2-ERG fusion with interstitial ERG and other ETS family member fusion sites had bias toward deletion and cancers without ETS fusions (P value ¼ 0.90). This the foremost 50-exons, as did the TMPRSS2, SLC45A3, and result exemplifies the complex heterogeneity in prostate cancer, NRGD1 driver's fusion sites. Interestingly, the majority of fusions with multiple additional factors contributing to prognosis both in differing from this norm were more commonly present in very fusion-negative and -positive groups. Although a meta-analysis by low-risk Gleason score 6 cancer. Thus, retention or loss of func- Petterssen and colleagues (4) did not demonstrate a worse out- tional domains of ETS 30-genes and the degree of inclusion of come for men whose prostate cancer had an ERG gene fusions domains from the 50-driver gene would be expected to affect occurring by deletion, they additionally acknowledged caution functionality of fusion proteins. Recently, the retention of specific from this observation from limited analysis with small numbers domains with differentially fused TMPRSS2-ERG fusion proteins, of cases. Esgueva and colleagues similarly reported on the mech- specifically SPOP binding sites within exon 3 of ERG, was dem- anism of ERG fusions; however, statistical analysis revealed no onstrated to alter the half-life of the expressed fusion proteins significant association between assessed gene rearrangements and (24). The frequency of breakpoints occurring within 21q22.2 clinical features such as Gleason grade, stage, or BCR (37). (ERG) and 21q22.13 (TMPRSS2) compared with the rest of However, higher numbers of ERG rearrangements through inter- chromosome 21 (Supplementary Fig. S3) supports a driver mech- stitial deletion (28%) presented with BCR compared with non- anism and selection for these fusions in prostate cancer. The deletion (17%). Data on the association of interstitial loss and frequent occurrence in very small volume Gleason score 6 cancers BCR available from a very recent copy-number analysis dataset also supports the predicted early occurrence of these fusions from 284 clinically significant prostate cancers (39) revealed (1, 15, 16). slightly better outcome in ERG-expressing tumors with interstitial Our dataset predicted a better outcome for prostate tumors retention (Supplementary Fig. S4). Although these studies failed with TMPRSS2-ERG fusions that retained the interstitial genes. to yield significant separation of the groups, in contrast to our

www.aacrjournals.org Cancer Res; 77(22) November 15, 2017 OF9

Murphy et al.

dataset, these studies focused on nonindolent cancers. Our study complexity often associated with TMPRSS2-ERG fusions. Specif- greatly benefited from our unique access to a significant number ically, the technique enabled precise localization of the interstitial of fresh-frozen very-low volume prostate cancers and our laser sequences through insertion in adjacent areas of the genome. Our capture microdissection expertise. The fact that ERG fusion is data supported the notion that the retention of interstitial genes is considered an early event in prostate cancer progression and that more frequently associated with very low- and low-risk prostate additional driver somatic mutations, such PTEN loss, are neces- cancers and that determining the status of the interstitial genes sary to develop poorly differentiated carcinomas in ERG fusion– may be useful in the risk stratification of patients with newly positive prostate cancer murine models indicates these early ERG diagnosed prostate cancer. rearranged tumors as boiling pots, primed for these additional mutations. Hence, the retention of a potential tumor-suppressor Disclosure of Potential Conflicts of Interest gene in the interstitial region could be considered a damper on G. Vasmatzis is CEO at WholeGenome LLC. No potential conflicts of interest progression, which could retain many prostate tumors in the low- were disclosed by the other authors. risk state and thus be enriched in this population. As such, interstitial retention alone may not be a marker for clinical Authors' Contributions significant prostate cancer, but combined with the detected Conception and design: S.J. Murphy, F. Kosari, R.J. Karnes, G. Vasmatzis, absence of other alterations, such as PTEN loss, could better J.C. Cheville Development of methodology: S.J. Murphy, G. Vasmatzis, J.C. Cheville predict prognosis. Acquisition of data (provided animals, acquired and managed patients, One limitation of this study, despite being the largest study provided facilities, etc.): S.J. Murphy, F. Kosari, R.J. Karnes, A. Nasir, L.J. Rangel, of its kind to date, was the relatively small sample size for G. Vasmatzis, J.C. Cheville statistical analysis. The reported association with the risk of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, BCR in ERG fusion–positive tumors with different interstitial computational analysis): S.J. Murphy, F. Kosari, R.J. Karnes, S.H. Johnson, deletions status was significant in a univariate but not in a A.G. Gaitatzes, J.B. Smadbeck, L.J. Rangel, G. Vasmatzis, J.C. Cheville Writing, review, and/or revision of the manuscript: S.J. Murphy, F. Kosari, multivariate analysis. This may be the result of the close associ- R.J. Karnes, S.H. Johnson, L.J. Rangel, G. Vasmatzis, J.C. Cheville ation of retention of interstitial genes and the Gleason score, which Administrative, technical, or material support (i.e., reporting or organizing was the best predictor of BCR in our study. Nevertheless, our data, constructing databases): R.J. Karnes, A. Nasir, S.H. Johnson, J.C. Cheville finding is important not only because it adds to our understanding Study supervision: J.C. Cheville of the initial events that influence the course of prostate cancer progression, but also because of its translational potential. A Acknowledgments number of studies from our laboratory and also from other We would like to thank Bruce Eckloff and Bob Sikkink from the Mayo Genomics Sequencing Core for the Mate Pair sequencing. investigators have reported that the genomic DNA rearrangements are often shared between disparate Gleason patterns in the same tumor (33, 40–42). Therefore, knowledge of the exact fusion Grant Support mechanism in any Gleason pattern in a tumor can be telling about This work was supported by James and Dorothy Nelson Benefactor Funds and Mayo Clinic Center for Individualized Medicine (CIM). the probability of BCR. One scenario where this information The costs of publication of this article were defrayed in part by the would be valuable is in estimating the PSA progression risk in payment of page charges. This article must therefore be hereby marked patients with GS6 biopsies given the estimated 30% rate of missing advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate high-grade areas by the biopsy needle during the biopsy procedures this fact. in these patients. In conclusion, MPseq provided in depth whole-genome assess- Received February 20, 2017; revised June 27, 2017; accepted August 15, 2017; ment of rearrangements within each tumor and detailed the published OnlineFirst November 10, 2017.

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www.aacrjournals.org Cancer Res; 77(22) November 15, 2017 OF11

Retention of Interstitial Genes between TMPRSS2 and ERG Is Associated with Low-Risk Prostate Cancer

Stephen J. Murphy, Farhad Kosari, R. Jeffrey Karnes, et al.

Cancer Res Published OnlineFirst November 10, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-0529

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