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Carcinogenesis, 2015, Vol. 36, No. 11, 1333–1340 doi:10.1093/carcin/bgv137 Advance Access publication September 21, 2015 Original Manuscript original manuscript Overexpression of enhancer of zeste homolog 2 (EZH2) characterizes an aggressive subset of prostate cancers and predicts patient prognosis independently from pre- and postoperatively assessed clinicopathological parameters Nathaniel Melling1,2,†, Erik Thomsen1,†, Maria Christina Tsourlakis1, Martina Kluth1, Claudia Hube-Magg1, Sarah Minner1, Christina Koop1, Markus Graefen3, Hans Heinzer3, Corinna Wittmer1, Guido Sauter1, Waldemar Wilczak1, Hartwig Huland3, Ronald Simon1,*, Thorsten Schlomm3,4, Stefan Steurer1 and Till Krech1 Institute of Pathology, 2General, Visceral and Thoracic Surgery Department and Clinic, 3Martini-Clinic, Prostate Cancer Center and 4Department of Urology, Section for translational Prostate Cancer Research, University Medical Center HamburgEppendorf 20246, Hamburg, Germany 1 *To whom correspondence should be addressed. Tel: +49 40 7410 57214; Fax: +49 40 7410 55997; Email: [email protected] † These authors contributed equally to this work. Abstract Enhancer of zeste homolog 2 (EZH2) plays an important role in tumor development and progression by interacting with histone and nonhistone proteins. In the current study, we analyzed prevalence and prognostic impact of EZH2 in prostate cancer. EZH2 expression was analyzed by immunohistochemistry on a tissue microarray containing more than 12 400 prostate cancer specimens. Results were compared to tumor phenotype, biochemical recurrence and molecular subtypes defined by ERG status as well as genomic deletions of 3p, 5q, 6q and PTEN. EZH2 immunostaining was detectable in 56.6% of 10 168 interpretable cancers and considered strong in 1.1%, moderate in 12.2% and weak in 43.3% of cases. High EZH2 expression was strongly associated with high Gleason grade (P < 0.0001), advanced pathological tumor stage (P < 0.0001), positive nodal status (P < 0.0001), elevated preoperative PSA level (P = 0.0066), early PSA recurrence (P < 0.0001) and increased cell proliferation P < 0.0001). High-level EZH2 staining was also associated with TMPRSS2:ERG rearrangement and ERG expression in prostate cancers (P < 0.0001) and was linked to deletions of PTEN, 6q15, 5q21 and 3p13 (P < 0.0001 each) particularly in ERG-negative cancers. The prognostic impact of EZH2 was independent of established pre- and postoperatively assessed clinicopathological parameters. EZH2 has strong prognostic impact in prostate cancer and might contribute to the development of a fraction of genetically instable and particularly aggressive prostate cancers. EZH2 analysis might therefore be of clinical value for risk stratification of prostate cancer. Introduction Prostate cancer is the most prevalent cancer in men in Western societies (1) Although most prostate cancers have a rather indolent clinical course, this disease still represents the third most common cause of cancer-related death in men. A reliable distinction between the indolent and the aggressive forms of the disease is Received: July 8, 2015; Revised: September 9, 2015; Accepted: September 13, 2015 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]. 1333 1334 | Carcinogenesis, 2015, Vol. 36, No. 11 Abbreviations EZH2 ERG E26 PSA PRC2 TMA enhancer of zeste homolog 2 v-ets avian erythroblastosis virus oncogene homolog prostate-specific antigen polycomb repressive complex 2 tissue microarray highly desirable to improve therapy decisions. Despite recent advances, the only established pretreatment prognostic parameters currently include Gleason grade and tumor extent on biopsies, preoperative prostate-specific antigen (PSA) and clinical stage. Because these data are statistically powerful but not sufficient for optimal individual treatment decisions, it can be hoped that a better understanding of disease biology will eventually lead to the identification of clinically applicable molecular markers that enable a more reliable prediction of prostate cancer aggressiveness. Enhancer of zeste homolog 2 (EZH2) is the catalytic part of the Polycomb repressive complex 2 (PRC2), which regulates genes involved in development and cell cycle progression (2). It catalyzes the trimethylation of Histone 3 on lysine 27 (H3K27me3) and thus induces chromatin compaction and transcriptional repression (2). This regulatory mechanism applies for the transcriptional control of various cancer related genes, including for example the cell cycle regulator p16 and the cell adhesion molecule E-Cadherin (3,4). In addition, EZH2 also interacts with various nonhistone proteins such as STAT3, estrogen receptor and beta catenin and thus indirectly serves as a transcriptional activator of their target genes (5,6). It is thus not surprising that overexpression of EZH2 has been implicated in initiation and progression of various cancer entities such as breast cancer, gastric cancer, bladder cancer and prostate cancer and has been found to be associated with adverse clinicopathologic features in all of these (7–10). Consequently, anti EZH2 therapies are currently under development (11–14). In prostate cancer studies on 64–259 primary tumors suggest that EZH2 overexpression may be linked to adverse features of this disease and may represent a potent predictor of patient prognosis (8,9,15–17). These promising findings encouraged us to study the putative prognostic value of EZH2 in a large cohort including more than 12 400 prostate cancers that have been assembled in a tissue microarray (TMA) format. The database attached to our TMA contains pathological and clinical follow-up data, as well as data on key molecular alterations, such as ERG fusion and genomic deletion of FOXP1, 3p13, 5q21 and 6q15. Materials and methods Patients Radical prostatectomy specimens were available from 12 427 patients, undergoing surgery between 1992 and 2012 at the Department of Urology and the Martini Clinics at the University Medical Center HamburgEppendorf. Follow-up data were available for a total of 12 344 patients with a median follow-up of 36 months (range: 1–241 months; Table 1). PSA values were measured following surgery and PSA recurrence was defined as a time point when postoperative PSA was at least 0.2 ng/ml and increasing at subsequent measurements. All prostate specimens were analyzed according to a standard procedure, including a complete embedding of the entire prostate for histological analysis (18). The TMA manufacturing process was described earlier in detail (19). In short, one 0.6mm core was taken from a representative tissue block from each patient. The tissues were distributed among 27 TMA blocks, each containing 144–522 tumor samples. For internal controls, each TMA block also contained various control tissues, including normal prostate tissue. The usage of archived diagnostic left-over tissues for manufacturing of TMAs and their analysis for research purposes as well as patient data analysis has been approved by the local ethics committee (Ethics commission Hamburg, WF-049/09 and PV3652). All work has been carried out in compliance with the Helsinki Declaration. Usage of patient data and routinely archived formalin fixed left-over patient tissue samples for research purposes by the attending physician is approved by local laws and does not require written consent (HmbKHG, §12,1). The molecular database attached to this TMA contained results on ERG expression in 10 711 (20), ERG break apart FISH analysis in 7122 (expanded from (21)) and deletion status of CHD1 (5q21) in 7932 (expanded from (22)), MAP3K7 (6q15) in 6069 (expanded from (23)), PTEN (10q23) in 6704 (expanded from (24)) and FOXP1 (3p13) in 7081 (expanded from (25)) cancers. Immunohistochemical data on Ki67-labelling index (Ki67-LI) and HDAC1 were available from 7010 cancers (expanded from (26)) and from 9744 cancers (27). Immunohistochemistry Freshly cut TMA sections were immunostained on 1 day and in one experiment. Slides were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave at 121°C in pH 9 Tris-EDTA-Citrate buffer. Primary antibody specific for EZH2 clone 6A10 (mouse monoclonal antibody, Abnova, Taipeh, Taiwan; cat#MAB9542; dilution 1:150) was applied at 37°C for 60 min. Bound antibody was then visualized using the EnVision Kit (Dako, Glostrup, Denmark) according to the manufacturer´s directions. Staining was predominantly nuclear and no staining was found in normal tissue. EZH2 staining was typically found in either all (100%) or none (0%) of the tumor cells in a given cancer spot. Staining intensity of all cases was thus semiquantitatively assessed in four categories: negative, weak, moderate and strong. The percentage of positive tumor cells (typically 100%) was not separately recorded. Statistics For statistical analysis, the JMP 10.0.2 software (SAS Institute Inc., NC) was used. Contingency tables were calculated to study association between EZH2 expression and clinicopathological variable, and the Chi-square (Likelihood) test was used to find significant relationships. Kaplan–Meier curves were generated for PSA recurrence free survival. The log-Rank test was applied to test the significance of differences between stratified survival functions. Cox proportional hazards regression analysis was performed to test the statistical independence and significance between pathological, molecular, and clinical variables. Results Technical issues A total of 10 168 (81.5%) of tumor samples were interpretable in our TMA analysis. Reason for noninformative cases (2259 spots; 18.1%) included lack of tissue samples or absence of unequivocal cancer tissue in the TMA spot. EZH2 immunohistochemistry In normal prostatic glands, no EZH2 staining was observed. In cancers, EZH2 immunostaining was localized to the nucleus. Positive staining was seen in 5755 of our 10 168 (56.6%) interpretable tumors and was considered weak in 43.3%, moderate in 12.2% and strong in 1.1% of cancers. Representative images of positive and negative EZH2 immunostainings are given in Figure 1. Elevated EZH2 expression was significantly linked to advanced pathological tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), lymph node metastases (P < 0.0001) and positive surgical margin when all tumors were jointly analyzed (Table 2). A weaker but still significant association was seen for high preoperative PSA levels (P = 0.0066; Table 2). In subgroup analyses all of these associations were stronger in ERG negative than in ERG-positive cancers (Supplementary Tables 1 and 2, available at Carcinogenesis Online). Association with TMPRSS2:ERG fusion status and ERG protein expression To evaluate whether EZH2 expression is associated with ERG status in prostate cancers, we used data from previous studies N.Melling et al. | 1335 Table 1. Pathological and clinical data of the arrayed prostate cancers No. of patients (%) Follow-up (months) n Mean Median Age (years) ≤50 51–59 60–69 ≥70 Pretreatment PSA (ng/ml) <4 4–10 10–20 >20 pT stage (AJCC 2002) pT2 pT3a pT3b pT4 Gleason grade ≤3 + 3 3 + 4 4 + 3 ≥4 + 4 pN stage pN0 pN+ Surgical margin Negative Positive Study cohort on TMA (n = 12 427) Biochemical relapse among categories 11 665 (93.9%) 48.9 36.4 2769 (23.7%) — — 334 (2.7%) 3061 (24.8%) 7188 (58.2%) 1761 (14.3%) 81 (24.3%) 705 (23%) 1610 (22.4%) 370 (21%) 1585 (12.9%) 7480 (60.9%) 2412 (19.6%) 812 (6.6%) 242 (15.3%) 1355 (18.1%) 737 (30.6%) 397 (48.9%) 8187 (66.2%) 2660 (21.5%) 1465 (11.8%) 63 (0.5%) 1095 (13.4%) 817 (30.7%) 796 (54.3%) 51 (81%) 2983 (24.1%) 6945 (56.2%) 1848 (15%) 584 (4.7%) 368 (12.3%) 1289 (18.6%) 788 (42.6%) 311 (53.3%) 6970 (91%) 693 (9%) 1636 (23.5%) 393 (56.7%) 9990 (81.9%) 2211 (18.1%) 1848 (18.5%) 853 (38.6%) Numbers do not always add up to 12 427 in the different categories because of cases with missing data. Percentage in the column ‘Study cohort on TMA’ refers to the fraction of samples across each category. Percentage in column ‘Biochemical relapse among categories’ refers to the fraction of samples with biochemical relapse within each parameter in the different categories. AJCC, American Joint Committee on Cancer. (expanded from (20,21)). Data on TMPRSS2:ERG fusion status obtained by FISH were available from 5977 and by immunohistochemistry from 8943 tumors with evaluable EZH2 immunostaining. Data on both ERG FISH and IHC were available from 5744 cancers, and an identical result (ERG IHC positive and break by FISH or ERG IHC negative and missing break by FISH) was found in 5485 of 5744 (95.5%) cancers. EZH2 staining was more frequent in TMPRSS2:ERG rearranged and ERG-positive prostate cancers. Positive EZH2 immunostaining was seen in 65.2% (ERG IHC) and 68.3% (ERG FISH) of ERG-positive cancers, and in 52.4 and 58.3% of cancers without ERG staining and ERG rearrangement, respectively (P < 0.0001 each; Figure 2). This difference in overall EZH2 staining was largely due to more weak staining cases in ERG-positive cancers than in ERG-negative cancers, while moderate and strong EZH2 staining showed comparable frequencies in both subgroups (Figure 2). Association to other key genomic deletions Earlier studies had provided evidence for distinct molecular subgroups of prostate cancers defined by TMPRSS2:ERG fusions and several genomic deletions. Others and we had described previously a strong link of PTEN and 3p13 deletions to ERG positivity and of 5q21 and 6q15 deletions to ERG negativity (22,23,25). To examine, whether EZH2 expression might be particularly associated with one of these genomic deletions, EZH2 data were compared to preexisting findings on PTEN (10q23), FOXP1 (3p13), MAP3K7 (6q15) and CHD1 (5q21) deletions. Elevated EZH2 expression levels were strongly and consistently linked to all of these deletions if all cancers were jointly analyzed, as well as in the subset of ERG-negative cancers (P < 0.0001 each, Figure 3a and b). These associations were clearly attenuated but still significant in ERG-positive cancers (Figure 3c). Association to tumor cell proliferation (Ki67-LI) EZH2 overexpression was significantly linked to increased cell proliferation as measured by Ki67-LI in all cancers and in subgroup analyses by Gleason grade and ERG status (P < 0.0001 each). ERG negative and Gleason grade ≥4 + 4 cancers with strong EZH2 expression displayed a particularly high proliferative fraction (P < 0.0001 each). Again this association was attenuated in ERG-positive cancers. All comparisons with the Ki67-LI are summarized in Supplementary Table 3, available at Carcinogenesis Online. Association with PSA recurrence Follow-up data were available from 9149 patients with interpretable EZH2 immunostaining on the TMA. Tumors with moderate or strong EZH2 immunostaining showed a significantly shortened PSA recurrence-free interval if all cancers were jointly analyzed and also in the subgroup analyses of ERGfusion negative and positive cancers (P < 0.0001, Figure 4a–c). Because EZH2 activity has been suggested to depend on the 1336 | Carcinogenesis, 2015, Vol. 36, No. 11 Figure 1. Representative pictures of EZH2 immunostaining in prostate cancer with (a) negative, (b) weak, (c) moderate and (d) strong staining. Table 2. Association between EZH2 immunostaining results and prostate cancer phenotype in all cancers (N) Parameter EZH2 (%) Evaluable Negative Weak Moderate Strong P All cancers 10 168 43.4 Tumor stage pT2 6574 47.2 pT3a 2250 36.7 pT3b-pT4 1303 35.0 Gleason grade ≤3 + 3 2346 55.3 3 + 4 5717 41.8 4 + 3 1580 34.7 ≥4 + 4 472 30.1 Lymph node metastasis N0 5727 41.3 N+ 594 32.0 Preoperative PSA level (ng/ml) <4 1251 44.8 4–10 6052 43.6 10–20 2043 42.0 >20 713 41.2 Surgical margin Negative 8045 44.8 Positive 1934 38.1 43.3 12.2 1.1 43.1 45.2 41.6 9.2 16.6 20.1 0.5 1.5 3.3 <0.0001 38.9 46.5 40.6 37.1 5.6 11.2 21.8 26.3 0.1 0.5 2.9 6.6 <0.0001 44.1 40.2 13.5 23.4 1.2 4.4 <0.0001 40.8 43.9 43.8 42.4 12.7 11.7 13.1 14.4 1.8 0.8 1.1 2.0 0.0066 43.1 43.8 11.2 16.1 0.8 2.0 <0.0001 with negative, weak and moderate HDAC1 expression (HDAC1 low). For the combined analysis, we further defined the EZH2/ HDAC1 status per tumor as follows: Tumors with low expression of both EZH2 and HDAC1 (both low), tumors with low expression of one protein but high expression of the other (one low/one high) and tumors with high expression of both proteins (both high). This analysis revealed an increasingly unfavorable outcome in patients with increasing levels of both proteins (P < 0.0001, Figure 4d). Multivariate analysis activity of histone deacetylases (HDACs) (9), we performed an additional analysis after combining EZH2 and HDAC1 immunostaining results, which we have previously analyzed on the same TMA (27). In order to reduce data complexity we grouped EZH2 into subsets with moderate or strong expression (EZH2 high) and those with negative or weak expression (EZH2 low). Accordingly, we regrouped our previous HDAC1 data into subsets with strong HDAC1 expression (HDAC1 high) and those Four different types of multivariate analyses were performed evaluating the clinical relevance of EZH2 expression in different scenarios (Supplementary Table 4, available at Carcinogenesis Online). Scenario 1 evaluated all postoperatively available parameters including pathological tumor stage, pathological lymph node status (pN), surgical margin status, preoperative PSA value and pathological Gleason grade obtained after the morphological evaluation of the entire resected prostate. In scenario 2, all postoperatively available parameters with exception of nodal status were included. The rational for this approach was that the indication and extent of lymph node dissection is not standardized in the surgical therapy of prostate cancer and that excluding pN in multivariate analysis can markedly increase case numbers. Two additional scenarios had the purpose to model the preoperative situation as much as possible. Scenario 3 included EZH2 expression, preoperative PSA, clinical tumor stage (cT stage) and Gleason grade obtained on the prostatectomy specimen. Since postoperative determination of a tumors Gleason grade is ‘better’ than the preoperatively determined Gleason grade (subjected to sampling errors and consequently under-grading in more than one third of cases (28)), another multivariate analysis was added. In scenario 4, the preoperative Gleason grade obtained on the original biopsy was combined with preoperative PSA, cT stage and EZH2 expression. EZH2 expression proved to be an independent prognostic N.Melling et al. | 1337 Figure 2. Association between EZH2 immunostaining results and the ERG status determined by IHC and FISH analysis. Breakage indicates rearrangement of the ERG gene according to FISH analysis. parameter in all scenarios when all tumors were analyzed (P < 0.0001 each). These associations also held true in subgroup analyses by ERG status, albeit attenuated in ERG-positive cancers (Supplementary Table 4, available at Carcinogenesis Online). Discussion The results of this study demonstrate that overexpression of EZH2 protein is a strong and independent predictor of PSA recurrence in prostate cancer, and that this effect is more substantial in ERG negative than in ERG-positive carcinomas. Detectable EZH2 expression was found in cancerous prostate epithelium but absent in normal epithelium in our study, suggesting a role for increased EZH2 expression in prostate cancer development. This finding is in agreement with an earlier study of Varambally et al. (9) reporting increasing EZH2 levels from normal epithelium to prostate intra-epithelial neoplasia (PIN) and prostatic carcinoma. A total of 56.6% of prostate cancers showed detectable EZH2 expression in our study. Earlier studies analyzing TMAs with 64–259 primary prostate cancers reported about 8–90% EZH2 cancers with relevant EZH2 staining. However, these studies employed various different antibodies and scoring criteria to distinguish between lower and higher levels of EZH2 expression, hampering a direct comparison with our data (8,9,15–17,29,30). The particularly high levels of EZH2 expression in high grade and advanced prostate cancers suggest a substantial contribution of EZH2 expression to tumor progression. In line with our findings, significant associations of EZH2 expression with tumor stage, grade, nodal metastases, increased proliferation and unfavorable outcome have been suggested in several earlier studies analyzing up to 292 prostate cancers for EZH2 expression (8,9,15,17). Known reasons for a tumor-promoting role of EZH2 overexpression in prostate and other cancers include its documented impact on the activity of pathways governing cellular functions that are relevant for cancer such as cell growth, adhesion, motility and apoptosis (reviewed in (31)). Interestingly, EZH2 overexpression was strongly linked to the presence of ERG fusion in our study, the most frequent molecular alteration in prostate cancer. More than half of all prostate cancers, particularly those of young patients, carry gene fusions linking the androgen-regulated TMPRSS2 gene with the transcription factor ERG (20,32). These genomic rearrangements result in an androgen-driven overexpression of ERG in affected cells (33) and, as a consequence, altered expression of more than 1600 genes in prostate epithelial cells (34). The association of EZH2 overexpression and TMPRSS2-ERG fusions is most probably caused by transactivation of the EZH2 promoter by ERG (35–37). Notably, it has been suggested that ERG and EZH2 may synergistically promote prostate cancer progression based on their pivotal role in chromatin remodeling (37,38). Aberrations in chromatin remodeling proteins are often found in prostate and other cancers. These include alterations of histone modifiers, such as EZH2, and chromatin remodeling complexes that move, eject or restructure nucleosomes (39–41). ERG has been shown to alter chromatin structure by opening cryptic transcription factor binding sites (38). Next to TMPRSS2-ERG fusions, chromosomal deletions represent the second most frequent type of genomic aberrations occurring at frequencies up to 40% in prostate cancer (42,43). In particular, deletions of PTEN (20%), 6q (20%), 5q (10%) and 3p (10%) belong to the most prevalent genomic alterations in this disease, which are strongly linked to either positive (PTEN, 3p) or negative ERG status (6q, 5q) and are associated with poor patient prognosis (22–25). Remarkably, all of these genomic deletions, some of which are inversely linked to each other, were associated with high levels of EZH2 expression, suggesting a role for EZH2 overexpression in the induction of chromosomal instability. In fact, it is known that perturbations of chromatin structure caused by alterations of polycomb group proteins such as EZH2 can result in genomic instability due to transcriptional deregulation of genes involved in maintenance of genomic integrity (44) For example EZH2 overexpression in breast cancer cells induces extra centrosomes and chromosomal instability by inhibiting the cell cycle checkpoint protein BRCA1 (45) and by impairing formation of RAD51 repair foci at sites of DNA strand breaks (46). Irrespective of the mechanisms by which EZH2 provokes tumor aggressiveness the data from our study demonstrate, that EZH2 expression is one of the strongest prognostic features in prostate cancer described as to yet. Even though the relationship with tumor phenotype and prognosis was somewhat less impressive in ERG-positive than in ERG-negative tumors, EZH2 was a strong and independent prognostic feature in both subgroups, which makes it a promising candidate for a routine application. This is all the more true as EZH2 predicted 1338 | Carcinogenesis, 2015, Vol. 36, No. 11 Figure 3. Association between positive EZH2 immunostaining results and deletions of PTEN, 5q21 (CHD1), 6q15 (MAP3K7) and 3p13 (FOXP1) in (a) all cancers as well as the subsets of (b) ERG-negative and (c) ERG-positive cancers according to ERG-IHC analysis. prognosis irrespective of whether pre- or postoperative established prognostic features were included in multivariate analysis. It is also noteworthy, that our method of analyzing EZH2 in minute tissue cores largely mimics the analysis of core needle biopsies that would be available in a preoperative scenario. A clinical benefit of EZH2 measurement in biopsies is also supported by two studies analyzing EZH2 expression by IHC in core needle biopsies from 72 and 209 prostate cancer patients and reporting significant associations of EZH2 expression with clinical outcome (16,30). EZH2 has gained substantial interest as a target for epigenetic anticancer therapy. Recent preclinical studies described significant effects of EZH2 in experimental systems derived from cancers of the lung (12), ovary (11), as well as in melanomas (14) and hematological malignancies (13). Clinical phase I/II studies using the EZH2 inhibitor E7438 have recently been initiated in diffuse large B-cell lymphoma patients (NCT01897571). Of note, it has been suggested that EZH2mediated gene silencing may depend on the activity of histone deacetylases (HDACs) (9)—another promising therapy target in prostate cancer (47,48)—and that a combinatorial epigenetic therapy targeting both EZH2 and HDACs may enhance the antitumor activity in leukemia (49). We have earlier demonstrated a strong prognostic role of HDAC1 expression in prostate cancer (27). In the context of combining treatment targets, it is interesting that our combined analysis of HDAC1 and EZH2 in prostate cancer revealed an increasingly unfavorable outcome in patients with increasing levels of both proteins. Simultaneous EZH2 and HDAC1 targeting may, thus, be instrumental in prostate cancer. N.Melling et al. | 1339 Figure 4. Association between EZH2 expression and biochemical recurrence (BCR) in (a) all cancers, (b) ERG fusion negative cancers, (c) ERG fusion positive cancers and (d) combined impact of EZH2 and HDAC1 expression on BCR in all cancers. Supplementary material Supplementary Tables 1–4 can be found at http://carcin.oxfordjournals.org/ Funding Project CancerTelSys (grant number 01ZX1302) in the E:med program of the German Federal Ministry of Education and Research (BMBF). Acknowledgements We thank Julia Schumann, Sünje Seekamp and Inge Brandt for excellent technical assistance. N.M., E.T., C.H.M., G.S., R.S. and T.K. conceived and designed the study, analyzed the data and drafted the manuscript. C.B. and D.M. performed most of the key immunohistochemical analyses. G.S. and R.S. were involved in the original conception of the study. M.C.T., M.K., N.M., C.H.M. and S.M. provided data. N.M., E.T., M.C.T., S.M., C.K., C.W., G.S., S.S., W.W. and T.K. participated in tissue processing, pathological diagnosis and immunohistochemical analysis. C.K., M.G., H.H., H.H., G.S., R.S. and T.S. provided materials, clinical follow-up data and technical assistance. All authors have read and approved the manuscript. 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