Chromatin remodeling restraints oncogenic functions in prostate cancer

Chromatin remodeling restraints oncogenic functions in prostate cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chromatin remodeling restraints oncogenic functions in prostate cancer Chiara Lanzuolo, Valentina Rosti, Cristiano Petrini, Giovanni Lembo, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5219856/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Primary prostate cancer (PCa) is characterized by multifocal growth and a highly variable clinical course, which is not effectively predicted by prognostic screenings. Innovative strategies for the stratification of primary prostate cancers are still needed. Using prostate biopsies, we analyzed the epigenome of 17 chemo-naïve patients with putative PCa for genome-wide mapping of heterochromatic and euchromatic domains, as well as their three-dimensional (3D) compartmentalization in the cell nucleus. We identified two subgroups of cancer patients with different degrees of chromatin 3D architecture and transcriptome alterations: the LDD (Low Degree of Decompartmentalization) and HDD (High Degree of Decompartmentalization) groups. HDD subtype exhibits an extensive chromatin reorganization that restrains tumor potential, by repressing pathways related to extracellular matrix remodeling and phenotypic plasticity. We derived an 18-genes transcriptional signature that distinguishes HDD from LDD subtype and we confirmed its prognostic relevance across multiple cohorts covering more than 900 prostate cancer patients in total. We propose this transcriptional signature derived from chromatin compartmentalization analysis as a novel prognostic tool that could be adopted at the time of the diagnostic prostate biopsy. Biological sciences/Genetics/Epigenetics/Gene silencing Health sciences/Biomarkers/Prognostic markers Chromatin remodeling PCa subtypes heterochromatin biopsies prostate cancer prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Prostate cancer (PCa) is the second most common tumor type among males and accounts for 10% of cancer-related deaths 1 . While the majority of PCa cases present as indolent and slow-growing, about 20% of prostate tumors progress to metastatic and lethal forms. Currently, PCa diagnosis relies on histological evaluation of multiple biopsies, usually conducted following elevated prostate-specific antigen (PSA) levels or abnormal digital rectal examination (DRE) 2 . Despite the use of tumor grading systems, the clinical variability and frequent multifocality of PCa complicate the accurate prediction of cancer outcomes, hindering treatment guidance 3 . Consequently, many patients with silent PCa are over-treated 4 . Although the identification of driver coding mutations in solid tumors has significantly advanced the field of clinical oncology 5 , the translation of PCa genetic biomarkers into clinical practice has only marginally improved prognosis accuracy 6 . This limitation underscores the need for novel biomarkers that can more precisely guide clinical decision-making in PCa management. Emerging evidence suggest that epigenetic information, which regulates transcriptional plasticity, plays a critical role in tumor evolution 7 . Epigenetic dysfunctions have been shown to initiate carcinogenesis even in the absence of driver mutations 8 , offering a new perspective on cancer research. 9 The three-dimensional (3D) genome architecture, which is hierarchically organized on multiple, highly regulated structural levels in the nucleus, has emerged as crucial for regulating cellular programs 9 , 10 . Notably, the physical separation between euchromatin, the more accessible and transcriptionally active part of the chromatin, and heterochromatin, which is compacted and gene-poor, is a hallmark of healthy cells. Morphological changes in nuclear organization, referred to as nuclear atypia, still represent a gold standard for diagnosis and staging of various cancers, including prostate cancer 11 . A recent study on metastatic prostate cancer patients reported multilevel alterations in chromatin structures associated with poor survival outcomes 12 . However, this work was performed on advanced PCa stages, thus providing limited information for earlier diagnostic samples. The same consideration applies to previous transcriptional studies performed on surgically removed prostates 13 – 15 . In this study, we aimed to comprehensively characterize chromatin compartment organization in PCa using our recently developed 4f-SAMMY-seq 16 . We analyzed fresh needle biopsies from PCa patients at their initial clinical presentation. We identified patient-specific changes in chromatin compartments and we extrapolated the underlying genomic dysregulation. Our findings reveal that large-scale chromatin remodeling and transcriptional repression are associated with a protective, antitumoral effect. Based on these insights, we derived a novel gene expression signature to classify this specific PCa subtype. We validated the predictive power of this 18-gene signature across multiple patient cohorts, encompassing more than 900 individuals. This signature demonstrates significant potential as a tool for early prognosis prediction in PCa and could be readily implemented in clinical practice. Questa firma dimostra un potenziale significativo come strumento per la previsione precoce della prognosi nel PCa e potrebbe essere facilmente implementata nella pratica clinica Results To characterize chromatin architecture in prostate cancer patients at the time of diagnosis, we collected fresh needle biopsies from chemo-naïve patients undergoing the standard diagnostic procedure. Each patient was punctured to collect 14 needle biopsy cores for histopathological examination (diagnostic biopsies) and 1 to be processed for our research project (research biopsy – Fig. 1a). Each research biopsy was divided into two parts: one-third was used for histopathological and immuno-histological analyses. At the same time, the rest of the tissue sample was enzymatically digested to obtain a cell suspension further split for epigenomic (4f-SAMMY-seq), transcriptomic (RNA-seq) and cellular composition (flow-cytometry) analyses. Our cohort includes seven non-neoplastic controls (CTR), i.e. patients that turned out to be negative for cancer, and ten primary prostate cancer patients (PCa) with histologically confirmed tumor and Gleason Score (GS) between 3+3 and 5+5 (Fig. 1b). We also report in Fig. 1b the percentage of diagnostic biopsy cores positive for cancer cells (PPC), their distribution in a prostate gland diagram (DPC), and the puncture site of the research biopsy used for this study. All the research biopsies of prostate cancer patients were obtained from punctures adjacent to one or more positive diagnostic biopsy cores (Fig. 1b). In addition to the Gleason Score assigned to the patient in the clinical report (GS-P), we recorded the Gleason Score assigned to the closest diagnostic biopsy (GS-CDB) and the score assigned to the research dedicated biopsy (GS-RDB, Extended Data Fig. 1a). We noted that the histopathological examination on research biopsies did not confirm the presence of tumor cells in four out of ten cases (Extended Data Fig. 1a). However, it must be noted that the Gleason Score assignment was done on a third of the tissue biopsy and that all research biopsy samples express diagnostic PCa tumoral markers like PCA3, HPN and GOLM1 17–19 (Extended Data Fig. 1b). Therefore, we retained all ten PCa samples in our experimental analyses. Our cohort of PCa patients is relatively homogeneous in terms of age (median 72 years; interquartile range, abbreviated as IQR, 62-89 years) and serum PSA level (median 16 ng/mL, IQR: 7.1-45.5) but heterogeneous in terms of clinical features as percentage of cancer positive biopsy cores (median: 21.4%, IQR: 21.4-39.2), clinical stage (T1c: 60%, T2a-T2b: 20%, T3: 20%) and index lesions on Magnetic Resonance Imaging (MRI) (40% with Prostate Imaging Reporting and Data System (PI-RADS) ≥3 and 60% with PI-RADS 1/2) (Extended Data Table 1). Survival outcomes are not available due to the long clinical course of primary PCa and the short follow-up time since the biopsies were collected (median time from biopsy less than four years). We also examined tissue samples of our cohort by confocal microscopy. Non-neoplastic controls and PCa patients' cells showed similar nuclear areas but a significant difference in nuclear circularity, with PCa patients’ cells showing nuclei deformations (Extended Data Fig. 1c, d). Staining of the nuclear lamina with the Lamin A/C antibody highlighted a slight depletion of the signal in the nuclear interior, corresponding to a parallel decrease in the Hoechst staining (Extended Data Fig. 1e). Although descriptive, these data suggest a cancer-specific remodeling of the nuclear and genome organization in PCa cells. When dissociating the needle biopsy tissue samples (size around 2 cm in length, Extended Data Fig. 2a, see Methods), we achieved a viable cell yield in the range of 30,000-80,000 cells per sample (Extended Data Fig. 2b). As a further control, we examined the cell type composition in a set of 13 prostate biopsies by flow cytometry (Extended Data Fig. 2c-f). We didn't detect significant differences in the relative proportion of epithelial (EpCAM/CD326+, CD45-), leukocyte (EpCAM/CD326-, CD45+) or stromal cells (EpCAM/CD326-, CD45-) between non-neoplastic controls and PCa samples (Extended Data Fig. 2g). All samples showed significant fraction of stromal cells (average 68,8% controls and 70,9% PCa patients), while we found an average of 18,5% and 15,2% of epithelial cells in control and PCa biopsies, respectively. We applied 4f-SAMMY-seq to examine genome architecture in prostate biopsies 16 . This method relies on the sequential isolation and high-throughput sequencing of distinct chromatin fractions separated according to their accessibility and solubility properties, which are expected to correlate with chromatin epigenetic and transcriptional status (Extended Data Fig. 3a). The more soluble fractions (S2S, S2L) are associated with gene activation markers, and the less soluble fractions (S3, S4) are enriched for constitutive heterochromatin. To confirm the applicability of this technique on fresh prostate biopsies, we first sequenced 4f-SAMMY fractions of non-neoplastic control samples. We examined the DNA sonication and short fragments (S2S) selection to obtain high-quality libraries (Extended Data Fig. 3b). We verified that we obtained sequencing reads with comparable quality across samples (Extended Data Fig. 3c). The sequencing reads distribution profiles for individual fractions from CTR samples show a high level of concordance (Extended Data Fig. 3d), with the highest correlation values for S2L fractions (median: 0.94). To study euchromatic and heterochromatic domains, we defined the solubility profile as the ratio of high-throughput sequencing reads distribution in S2L over S3 fractions (see Methods), where higher and lower values correspond to regions enriched for open and closed chromatin, respectively. We compared the consensus solubility profiles across all CTR and PCa specimens with epigenomic marks in normal prostate tissue for post-translational histone modifications (histone marks) associated with active (H3K27ac, H3K36me3) and inactive (H3K9me3) chromatin states 20 (Fig. 1c). We observed a high similarity of solubility profiles among CTR samples and a clear concordance with the location of euchromatin marks (H3K27ac and H3K36me3), along with a negative correlation with the heterochromatin mark profile (H3K9me3). These observations were also confirmed in a genome-wide quantification of correlations for individual samples (Fig. 1d). Instead, in PCa biopsies we observed that the solubility profile is more heterogeneous and its correlation with histone marks is more variable with respect to CTR samples (Fig. 1c, d). Visual inspection of individual solubility profiles showed a patient-specific degree of chromatin alterations (Extended Data Fig. 3e). Moreover, we noticed clear differences among PCa samples, with five out of ten samples losing the regular chromatin compartmentalization, as also highlighted by their correlation with histone marks (Fig. 1d). These differences are remarkably not attributable to the cell composition of the samples (Extended Data Fig. 2g). Thus, 4f-SAMMY-seq data describe a conserved genome architecture among control biopsies with a separation between euchromatin and heterochromatin. On the other hand, diagnostic early-stage PCa biopsies show a patient-specific chromatin solubility dysregulation that mirrors PCa’s highly heterogenous nature. To further analyze the chromatin 3D organization, we used the sequencing data of all the 4f-SAMMY-seq fractions to identify active and inactive compartments, named "A" and "B", respectively, as per the convention adopted for Hi-C derived compartments 16 (Fig. 2a, b, see Methods). However, unlike Hi-C, the chromatin compartments inferred from 4f-SAMMY-seq are defined based on their chromatin accessibility and solubility properties 16 . CTR samples showed a consistent pattern across all seven individuals with 27% of the entire genome having conserved active A compartment and 39% having conserved inactive B compartment across all CTRs (Fig. 2b, c). Instead, PCa samples showed patient-specific chromatin compartment alterations (Fig. 2b, c). We quantified the genomic regions with a different compartment assignment in each PCa sample with respect to the consensus among CTR samples. We labelled these compartment switches as "A to B" or "B to A" for the regions that in CTR samples were A or B, respectively. Unsupervised clustering based on compartment similarity identified two subgroups of cancer patients (Fig. 2d) that, considering the amount of compartment alterations with respect to CTR samples, we named LDD (Low Degree of Decompartmentalization; average 5% over the entire genome for "A to B" and 5% for "B to A" compartment switches) and HDD (High Degree of Decompartmentalization; average 14% over the entire genome "A to B" and 24% "B to A"). We also noted a consistent intra-group similarity for LDD samples with 0.78 mean Jaccard Index (JI) (variance 0.001). On the other hand, HDD samples show more heterogeneity in chromatin compartmentalization (JI 0.42 mean and 0.012 variance) (Fig. 2d). In line with patient-specific genome remodeling, we did not find regions with compartment changes concordant across all LDD and HDD tumoral samples compared to CTR consensus. However, we found recurrent "B to A" regions, indicating higher chromatin accessibility compared to controls, concordant across all HDD patients and covering 2% of the entire genome (538 genes) (Extended Data Fig. 4a). Gene ontology analysis highlighted an enrichment of genes associated with Protein Deubiquitination and Regulation of Programmed Cell Death (such as FAS) (Extended Data Fig. 4b). Instead, the HDD-specific "A to B" compartment switch regions, which correspond to reduced chromatin accessibility compared to controls, (0.6% of the genome - 59 genes) included genes involved in PI 3-kinase activities (such as PIK3R5 and PIK3R6) and lipid catabolic processes (such as PNLIP and STS), both of which are known drivers of prostate cancer progression 21–23 . It should be remarked that the LDD vs HDD groups stratification of PCa patients would not be immediately evident based on known clinically relevant pathophysiological features such as PSA level, Gleason Score, age at diagnosis and number of positive cores (Extended Data Fig. 4c). Furthermore, the analysis of the global transcriptome on prostate samples did not separate so clearly the HDD and LDD subtypes (Extended Data Fig. 4d). We excluded the association of HDD and LDD subtypes with cell composition and immune cell infiltration, estimated by flow-cytometry (Extended Data Fig. 4e) and RNA-seq data deconvolution (see Methods, Extended Data Fig. 4f, g). We also estimated the copy number alterations (CNA) distribution across all samples based on 4f-SAMMY-seq reads coverage (see Methods). The inferred copy-number states are notably similar for both LDD and HDD subtypes (Extended Data Fig. 4h), suggesting that structural alterations do not determine the described subtypes. We further investigated epigenetic differences between LDD and HDD groups. We compared the histone mark enrichment peaks from normal prostate tissue with the 4f-SAMMY-seq solubility profiles of CTR, LDD and HDD samples. As expected, we found that the solubility profile of CTR samples is higher in ChIP-seq peaks for open histone marks (H3K27ac and H3K36me3) and lower in constitutive heterochromatin (H3K9me3 peaks) (Fig. 2e). The HDD group showed instead an inverted trend, thus confirming a large-scale chromatin architecture remodeling. Interestingly, the LDD subgroup, despite the limited alterations in chromatin compartmentalization (Fig. 2b, c), showed an intermediate solubility pattern between the other two groups, especially for H3K27ac and H3K9me3 domains. We next examined the functional effects of chromatin compartment reorganization by matched RNA-seq analysis. Comparing transcription profiles between CTR (n=7) and PCa (n=10) samples uncovered 66 differentially expressed genes (DEGs): 22 up-regulated and 44 down-regulated in PCa with respect to CTR (Fig. 3a). The limited number of DEGs is likely due to the heterogeneity of expression across tumor patients' biopsies. Then, we considered LDD and HDD subtypes separately. We identified only 30 DEGs in LDD comparison to CTR samples: 9 up- and 21 down-regulated. Instead, we found 162 up- and 410 down-regulated genes in HDD compared to CTR samples (Fig. 3a). This suggests that the higher degree of chromatin compartment remodeling is associated with a more extensive gene expression dysregulation. We found 5 DEGs up-regulated and 4 DEGs down-regulated in common among all comparisons of different PCa (Fig. 3b). Seven genes already described in the literature as prostate cancer biomarkers (HPN, BICD1, GOLM1, TARP) and two down-regulated tumor suppressor genes (SERPINB5 and GATA6) 19,24–28 . To further dissect the molecular differences between LDD and HDD subtypes, we performed an enrichment analysis for Gene Ontology (GO) annotations on the list of DEGs in HDD vs CTR. We didn't perform this analysis on LDD vs CTR samples due to the small number of DEGs. Nevertheless, in LDD DEGs, we found the upregulation of two critical genes, PDLIM5 and CAMKK2, involved in cell migration and invasion 29,30 . In the other comparison, we discovered that HDD up-regulated genes are enriched for the "negative regulation of cell motility" class. whereas down-regulated ones include GO classes related to stroma remodeling (Fig. 3c), suggesting decreased functions usually associated with tumor progression. These findings were also confirmed through GeneSet Enrichment Analysis (GSEA) 31 (Extended Data Fig. 5a). We then applied a complementary approach for the functional analysis of transcriptome profiles for both LDD and HDD comparison to CTR samples. We used PROGENy 32 , a curated signaling pathway compendium derived from perturbation experiments. This compendium includes weights quantifying the response of each gene to pathway perturbations, thus allowing a weighted analysis of transcriptional response to specific signaling cues (see Methods, Extended Data Fig. 5b). We found a positive enrichment for Androgen pathway activity in both LDD and HDD subgroups. Notably, we found a positive enrichment for TGFbeta activity in LDD, whereas it has negative enrichment in the HDD subgroup. These results demonstrate that the LDD and HDD subgroups are different in their chromatin architecture and gene expression, thus representing previously undescribed PCa patient subtypes. The HDD chromatin reorganization is also reflected in more prominent chromatin remodeling and transcription repression that confer antitumoral effect. Then, we explored the possible connections between epigenetic remodeling and functions, analyzing compartment changes and transcription at the individual patient level. By considering patient-specific DEGs and compartment switches (see methods), we observed that the majority of DEGs (90% for LDD; 70% for HDD) are located in domains not involved in a compartment transition (Fig. 3d). This suggests that, as extensively described in other models 33–35 , changes in chromatin architecture are not enough to trigger transcriptional dysfunction, but they establish conditions that enable transcriptional deregulation. A higher proportion of DEGs (30%) were found in compartment switch regions for HDD. Within these genes, we identified a trend where up-regulated genes were associated with "B to A" switch regions, while down-regulated genes were associated with "A to B" compartment switch regions (Fig. 3d, e). GO analysis revealed significant terms only for the latter class (973 downregulated DEGs in HDD “A to B” transitions), with “basal cell of prostate epithelium” emerging as the most enriched class, alongside terms related to “hypoxia” and “glycolysis” (Fig. 3f), confirming an acquired silenced state of tumorigenic genes 36 . Transcription factor enrichment analysis identified the Polycomb protein Suz12 (Fig. 3f), a subunit of the well-known Polycomb repressive Complex 2 (PRC2), as a key regulator of the identified genes. Interestingly, 58 among the 139 enriched Suz12 targets were described overexpressed in diverse phases of PCa progression, and 8 (SEMA5A, RET, PDGFRA, TWIST2, CDH13, NTN1, EGFR, DUOX2) were associated with positive regulation of cell motility (term of biological processes gene-set), suggesting that in HDD the acquired “heterochromatin-like” state plays a role in constraining cancer phenotypic plasticity. To identify a signature distinguishing HDD vs LDD subtypes, we examined the differentially expressed genes in comparing HDD vs LDD transcriptomic profiles. We identified 101 DEGs: 77 down- and 24 up-regulated genes in HDD with respect to LDD (Fig. 4a). As seen in the HDD vs CTR comparison, we found differential expression of Epithelial-Mesenchymal Transition (EMT) genes (GSEA analysis – Fig. 4b and Extended Data Fig. 5c) as well as differential activity of Androgen and TGFbeta pathways (PROGENy analysis - Extended Data Fig. 5d). Then, we asked if our signature of 101 genes could provide prognostic information on patients. Thus, we queried the cancer genome atlas (TCGA) for the RNA-seq data of 499 prostate adenocarcinomas (PRAD) 37 . We defined a quantitative score named PCI (prostate Compartmentalization Index) to summarize the HDD vs LDD transcriptional signature activity for each patient in this cohort (see Methods). We sorted TCGA samples based on the PCI and defined HDD-like and LDD-like patients based on the PCI score indicating a more prevalent HDD (PCI > 0) or LDD (PCI <0) signature, respectively (Fig. 4c). Intriguingly, survival analysis for biochemical recurrence-free status (BCR) indicated that patients belonging to the HDD-like cluster show a better prognosis (Fig. 4d), confirming the “protective” chromatin compartment alterations described above. This is a remarkable result because the signature was established using biopsies taken at the time of diagnosis, yet it can predict prognosis in more advanced, surgically removed tumor samples from the TCGA. TCGA samples, extracted from prostatectomies are expected to have generally higher cellularity and tumor purity, with respect to needle biopsies. We noted a potential association between HDD-like and LDD-like groups and the TCGA sample’s tumor purity scores (Fig. 4c). Nevertheless, multivariate Cox regression analysis, including the tumor purity score in the model confirmed that the HDD-like classification is a valid independent prognostic predictor of biochemical recurrence (Fig. 4e). The HDD-like phenotype is associated with a better outcome (HR=0.5, CI=0.33-0.88, p-value=0.01) on top of the reference clinical features such as pathological (TNM) stage, age at diagnosis and Gleason Score. We further refined the signature to retain only the genes with expression more significantly associated with good or bad prognosis, finally resulting in the 18 genes refined signature (Extended Data Fig. 6a, b, see Methods). As expected, the reduced 18 genes signature improves the stratification of the TCGA cohort used to refine the signature itself (p-value < 0.0001; Extended Data Fig. 6c), also independently of tumor purity levels (Extended Data Fig. 6d, e). We then validated the clinical relevance of our refined 18 gene signature across multiple independent prostate cancer patients’ cohorts. We obtained a significant prognostic stratification trend, where HDD-like patients show a better prognosis (Fig. 5a, b, c). These independent cohorts include transcriptomic profiles obtained with RNA-seq and microarrays, thus attesting the applicability of our signature to heterogenous datasets. Overall, our results confirm the validity of chromatin-informed patient stratification and the derived gene expression signature to discriminate patients for prognosis. Discussion Prostate cancer diagnosis, which relies on the histological examination of multiple biopsies, has revealed extensive heterogeneity and multifocality of PCa 2 . This procedure cannot predict the 20% of prostate cancer cases that progress to metastases, resulting in a high mortality rate. Importantly, most transcriptome-based analyses, which might capture the phenotypic plasticity, are unable to provide early insights into clinical progression. Thus, there is an urgent need for new biomarkers to refine the stratification of prostate cancer patients. The 3D genome architecture in the cell nucleus is crucial for the epigenetic regulation of transcription, with its reorganization frequently linked to neoplastic conditions 12 , 38 . In this work, using 4f-SAMMY-seq and transcriptome analysis on a limited number of cells (10,000–50,000 cells), we analyzed the epigenome structure and function of PCa patients-derived biopsies to infer their plasticity (Fig. 1 ). We identified two novel subtypes of PCa patients presenting different degrees of chromatin compartments and transcriptional alterations: the HDD and LDD subtypes (Fig. 2 ). Samples with HDD features display a marked chromatin remodeling that promotes transcriptional repression of cancer progression pathways, suggesting that these chromatin changes may counteract the tumor evolution (Fig. 3 ). Specifically, by intersecting patient-specific 4f-SAMMY-seq and RNA-seq datasets, we found that in HDD, down-regulated genes located in “A to B” transitions are directly involved in prostate cancer progression, with “basal cell of prostate epithelium” being the most enriched class (Fig. 3 f). Since basal cells possess stemness-like properties and are prevalent in advanced, castration-resistant, and metastatic prostate cancers 36 , this geneset down-regulation further suggests that the epigenetic state of HDD patients may restrict the phenotypic plasticity of cancer cells. Furthermore, transcription factor enrichment analysis identified Suz12 as a key regulator of over 10% of all HDD down-regulated genes in “A to B” regions, suggesting that PRC2, a key complex in prostate cancer progression 39 , may mediate this transcriptional repression. Comparing HDD and LDD subtypes, we derived a specific expression signature that characterizes the HDD phenotypic state (Fig. 4 ). We confirmed that the HDD signature is significantly associated with a more favorable prognosis using four independent cohorts covering more than 900 patients with follow-up clinical annotations (Figs. 4 – 5 ). The epigenetic remodeling of epithelial tumor cells is coupled with the loss of cell identity homeostasis through multiple steps, driven by molecular changes that depend on intrinsic and extrinsic signals 40 . The tumor microenvironment plays a central role in this process, constantly adapting its stroma cell composition and transcriptional programs to favor or counteract tumor progression 41 , 42 . This is particularly evident for indolent cancers such as the PCa, whose slow progression favors the crosstalk between cancer and neighboring cells. Prostate stromal tissue, constituted by fibroblasts, endothelial cells, smooth muscle cells (SMCs), and immune cells, plays a crucial role in prostate tumorigenesis 43 – 45 . Intriguingly, transcriptional programs can have divergent trajectories in epithelial or stromal cells. This is the case of the androgen receptor (AR) pathway, whose overexpression in epithelial cells is associated with bad prognosis 46 whereas in the stroma it has a protective role by constraining cancer growth 44 . One of the significant pathways upregulated in HDD samples is the androgen receptor and, working on bulk prostatic tissue comprising 70–80% of stromal cells, we speculate that this detected AR up-regulation may be ascribed to the stroma. Further corroborating this hypothesis, the LDD PCa group correlates with a worse prognosis and its signature includes overexpression of TGFbeta that can induce a resistance to androgen deprivation therapy when expressed in the stroma 45 . To develop a molecular signature exploitable in the clinic, we selected genes with prognostic value, ultimately restricting our chromatin-based RNA signature to 18 genes validated across multiple independent cohorts (Fig. 5 ). Notably, when comparing our HDD signature with the 29 previously identified prostate cancer (PCa) signatures listed in the PCa Database (PCaDB; http://bioinfo.jialab-ucr.org/PCaDB/ ), only 3 out of the 18 genes were shared, namely ACAP3 e ATG16L2 47 and SCRIB 48 . This highlights the uniqueness and potential specificity of our gene signature for early stratification of prostate cancer patients. In summary, this study we introduced a novel experimental approach to investigate the epigenomic landscape and 3D chromatin compartmentalization in prostate biopsies. We identified two novel patient subgroups based on their epigenome profiles. The PCa subtype associated with a favorable prognosis exhibits heterochromatin reorganization that represses tumorigenic pathways. We found that the transcriptional signature derived by this chromatin-informed patient stratification constitutes a novel independent prognostic classifier. We validated the signature across multiple independent cohorts, thus confirming its potential for translatability to assess prognosis at the time of diagnosis. Methods Prostate tissues cohort, ethics approval and consent to participate Our cohort includes chemo-naïve patients followed in the Urology Division of Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico (Milan) who underwent the transrectal ultrasound-guided systematic sampling of prostate tissue (TRUS biopsy). This diagnostic procedure was performed as part of routine clinical management following the detection of abnormal digital rectal examination or an elevated PSA blood level. According to the current standard for the detection of PCa, 14 ultrasound-guided biopsy cores (diagnostic biopsies), seven from each side of the prostate gland, were collected from each patient for the clinical diagnosis. During the same procedure, one additional biopsy core to be used for our research project (research biopsy), was taken from a site directly adjacent to one of the diagnostic biopsies. The institutional ethics committee board of IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico authorized this study (authorization n 1063). All specimens were obtained after the patients had provided written informed consent following the ethical principles of biomedical research on biospecimens. To ensure optimal recovery of living cells, fresh surgical prostate biopsy specimens were placed in ice-cold saline buffer and directly transported to the laboratory within 1 hour from sample collection. Tissue samples were then immediately processed to preserve the intranuclear genomic and protein architectures of cell nuclei. Samples selected for epigenome studies consist of 17 fresh biopsies divided on the bases of the histology and the spatial distribution of the positive cores in two different groups: 10 PCa biopsies from patients with histologically confirmed prostate cancer and 7 from patients who had no cancer in any biopsy core. All the clinical data were provided by the Urology division preserving the confidentiality of patient personal data. Tissues processing The biopsy specimen was stored at 4-8°C for maximum 3 hours before dissociation, avoiding its freezing. Given the little size of needle biopsy cores (typically 20-30 mg) we empirically determined the digestion condition to achieve the optimal recovery of living cells. Briefly, the biopsy tissue was transferred in a 2 ml microcentrifuge tube and rinse twice with 1 ml of ice-cold sterile PBS 1X. Then, the tissue was cut into small pieces (~1 mm) with autoclaved surgical scissors directly in the microcentrifuge tube. The resulting minced tissue was enzymatically digested by adding 1 ml of prewarm HBSS (Gibco, 14025) containing 200 units of collagenase type I (Life Technologies, 17018-029) per ~10 mg of tissue plus 67 µg DNase I (Sigma-Aldrich, 10104159001). Tissue dissociation was carried out in a water bath at 37 °C shaking vigorously for 10 seconds every 5 minutes. To prevent tissue over-digestion, the best incubation time for each experiment was established by monitoring cell viability, cell debris and aggregates every 10 minutes (usually ranges from 1 to 1.5 hour). After completed digestion cells were washed once by topping up to 2 ml with RPMI + 10% FBS and spun down at 300 g. The cell pellet was resuspended in RPMI + 10% FBS and dispersed by passing through a 75µm cells strainer, followed by an additional wash of the filter with RPMI + 10% FBS. Finally, the cells were spun down at 300 g and resuspended in 1 ml of ice-cold PBS for counting with a hemocytometer. Histological and immunofluorescence evaluation One third portion of each research biopsy tissue specimen was embedded in Killik (Bio-Optica, 05-9801), immediately frozen in precooled isopentane (MilliporeSigma, 277258) and stored at -80°. OCT- embedded biopsy cores (one per patient) were serially sectioned with 10 μm thickness in a cryostat at -20°C. Ten slides per patient, containing multiple sections representing distinct regions of the same tissue were prepared in parallel and stored at -80°C. Hematoxylin and Eosin staining (H&E) was performed using H&E Staining Kit (Abcam, ab245880). The H&E-stained slides were reviewed by an expert genitourinary pathologist to assign the Gleason Score according to the International Society of Urinary Pathology grading system. The same pathologist evaluated all specimens (diagnostic and research biopsies) presented in this work. For immunofluorescence, frozen tissue sections were briefly thawed at room temperature (RT), placed in precooled acetone for 20 minutes at -20°C and washed 3 times in PBS 1X for 2 minutes at RT. To permeabilize tissues the sections were incubated with 0.5% Triton X-100 in PBS 1X for 10 minutes at RT with mild agitation. After three washes in PBS 1X for 2 minutes at RT, coverslips were blocked by incubating with 5% Donkey serum, 3% BSA in PBS 1X for 1 hour at RT. After three washes in PBS 1X for 2 minutes at RT, coverslips were incubated with 1:500 Lamin A/C primary antibody (Santa Cruz sc-6215) 1:500 in blocking solution overnight at 4°C in a humidified chamber, isolating the tissue section by a hydrophobic pen. The following day, sections were washed 3 times in PBS 1X for 2 minutes at RT and incubated with fluorescence-conjugated secondary antibody (Alexa FluorTM 488-conjugated Donkey Anti-goat IgG Invitrogen, A-31571), 1:500 3% BSA in PBS 1X for 1 hour at RT in a dark humidified chamber. After three washes in PBS 1X for 2 minutes at RT, the coverslips were incubated with Hoechst solution (Thermo Fisher Scientific H3570, 1:500) for 10 minutes at RT in the dark, rinsed in PBS 1X and mounted on microscope slides with ProLong antifade mounting media (Thermo Fisher Scientific, P36930). Images were acquired using a Leica TCS SP5 confocal microscope with a HCX Plan Apo ×63/1.40 objective, with a 5-zooming factor. The NIS-Elements v.5.30 software (Nikon-Lim) was employed to analyze nuclei physical parameters including area and circularity. Hoechst staining facilitated nucleus identification and delineation of the region of interest. Over 5 field of views (FOVs) were acquired and analyzed per sample, totaling the examination of more than 70 nuclei within each sample of the cohort (seven non-neoplastic controls, CTR and ten primary prostate cancer patients, PCa). The mean nuclear area or circularity was calculated for each sample and plotted alongside other samples within the same group on the charts. The analysis of Hoechst and Lamin A/C distribution across nuclei was conducted using Fiji software. Leveraging the plot profile plugin, a 10 µm fixed line was implemented to trace and capture the fluorescence intensity distribution across each nucleus. To standardize comparisons between diverse samples, a normalization step was performed. This involved calibrating the fluorescence intensity of individual pixels along the line to the mean intensity derived from the entire set of pixel lines. For each sample the mean distribution values for both Hoechst and Lamin A/C were calculated and graphically represented alongside other samples within the same group. Flow cytometry Analysis To quantify the relative amounts of cell populations in each biopsy, 10,000 cells from the digestion step were stained, acquired on a BD FACSCantoII Flow Cytometer and analyzed with FlowJo software in the INGM FACS facility. To avoid unspecific binding, antibodies were incubated with PBS-BSA 1% for 30 minutes at 4°C. TO-PRO®-3 stain (Thermofisher, T3605) was used to assess cell viability. Tissue resident leukocytes were identified as CD326-/CD45+ (EpCAM-CD326 FITC, B347197; CD45 Pacific Blu Biolegend, 982306); epithelia cells as CD326+/CD45-, and stromal cells as CD326-/CD45-. 4f-SAMMY-seq isolation of chromatin fractions Chromatin fractionation on tissue-derived single cell suspension was performed with minor adaptations to the protocol described in 16 . Cells were counted, washed in cold PBS and resuspended in 600ul cold cytoskeleton triton buffer, CSK-Tr: 10 mM PIPES pH 6,8; 100 mM NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1X Protease Inhibitor Cocktail (Roche, 04693116001); 1 mM PMSF (Sigma-Aldrich, 93482); 1 mM DTT; 0,5% Triton X-100. After 10 minutes on a rotator at 4°C, samples were centrifugated for 3 minutes at 900g at 4°C and supernatant was collected as S1 fraction. Pellets were washed for 10 minutes on the rotator at 4°C with an additional volume of the same buffer followed by centrifugation for 3 minutes at 900g at 4°C. Chromatin was then digested by using 25 units of DNase I (Invitrogen, AM2222) in 100ul of cytoskeleton CSK buffer: 10 mM PIPES pH 6.8; 100 mM NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1mM PMSF; 1X Protease Inhibitor Cocktail for 60 minutes at 37°C. To stop digestion, ammonium sulphate was added to samples to a final concentration of 250 mM and, after 5 minutes on ice, samples were pelleted at 900g for 3 minutes at 4°C and the supernatant was collected as S2 fraction. After a wash of 10 minutes on a rotator at 4°C with 200ul of CSK buffer followed by centrifugation for 3 minutes at 3000g at 4°C, the pellet was further extracted 10 minutes on a rotator at 4°C in 100ul of CSK-NaCl buffer: 10 mM PIPES pH 6.8; 2 M NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1mM PMSF; 1X Protease Inhibitor Cocktail, centrifuged at 2300 g 3 minutes at 4°C and the supernatant was collected as S3 fraction. Pellets were washed twice for 10 minutes on the rotator at 4°C followed by centrifugation at 3000 g 3 minutes at 4°C with 200ul of CSK-NaCl buffer. Finally, pellets were solubilized in 100ul of 8M urea for 10 minutes at room temperature and labelled as S4 fraction. Fractions were stored at –80°C until DNA extraction. 4f-SAMMY-seq DNA extraction, library preparation and sequencing Chromatin fractions (S2, S3 and S4) were diluted 1:2 in 1X TE buffer (10mM TrisHCl pH 8.0, 1 mM EDTA) and incubated with 3 U of RNAse cocktail (Ambion, AM2286) at 37° for 90 minutes, followed by 40μg of Proteinase K (Invitrogen, AM2548) at 55° for 150 minutes. Genomic DNA was then isolated using phenol/chloroform (Sigma-Aldrich, 77617) extraction, followed by a back extraction of phenol/chloroform with additional volume of TE1X. DNA was precipitated in 2 volumes of cold ethanol, 0.3M sodium acetate and 20ug glycogen (Ambion AM9510) for 1 hour on dry ice or overnight at -20°C. Dry pellets were resuspended in 50 ul (S2) or 15 ul (S3 and S4) of nuclease-free water and incubated at 4°C overnight. On the next day, S2 was further purified using PCR DNA Purification Kit (Qiagen, 28106) and separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) with the ratio 0.90/0.95 to obtain smaller fragments conserved as S2S ( 300bp) fractions. Both were resuspended in 20 ul of nuclease-free water and then reduced to 15ul using a centrifugal vacuum concentrator. S2L, S3 and S4 fractions were sonicated in a Covaris M220 focused-ultrasonicator using screw cap microTUBEs (Covaris, 004078) to obtain a smear of DNA fragments peaking at 150-200 bp (water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50, 150 seconds for S2L and 175 seconds for S3 and S4). Fractions were quantified using Qubit 4 fluorometer with Qubit dsDNA HS Assay Kit (Invitrogen, Q32854). Libraries were generated from each sample using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and Unique Dual Index NEBNextMultiplex Oligos for Illumina (NEB, E6440S). Libraries were then qualitatively and quantitatively checked on and run on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent, 5067-4626). Libraries with distinct adapter indexes were then multiplexed and, after cluster generation on FlowCell, sequenced for 50 bases in single or paired-end reads mode on an IlluminaNovaSeq 6000 instrument at the IEO Genomic Unit in Milan. RNA extraction, library preparation and sequencing Ten thousand cells retrieved from the digestion step were stabilized in 200µl of 1Thioglycerol/Homogenization Solution of the Maxwell® RSC miRNA Tissue Kit (Promega, AS1460) and stored frozen at -80°C for total RNA automated purification using Maxwell® RSC 48 Instrument (Promega, AS8500). Total RNA was quantified by Qubit 4 fluorometer with Qubit RNA HS Assay Kit (Invitrogen, Q32852) and assessed by Agilent 2100 Bioanalyzer using Agilent RNA 6000 Pico Kit (Agilent, 5067-1513) to inspect RNA integrity. For each sample, 1 ng of total RNA was used to construct strand specific RNA-seq libraries with SMARTer Stranded Total RNA-Seq Kit - Pico Input (Takara, 634487). The yield and quality of the libraries were evaluated on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent, 5067-4626). RNA-seq libraries were sequenced on the Illumina NextSeq™ 550 system at the sequencing facilities of Humanitas or Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico in Milan in paired-ends mode. ChIP-seq public datasets The ChIP-seq data of histone post translational modifications (histone marks) for non-neoplastic prostate gland tissue were downloaded from the following datasets available on ENCODE data portal 20 : ENCSR763IDK (H3K27ac), ENCSR768PFZ (H3K36me3) and ENCSR133QBG (H3K9me3). These data were downloaded as raw high-throughput sequencing reads (FASTQ) and analyzed as described in the next sections. ChIP-seq and 4f-SAMMY-seq high-throughput sequencing reads data analyses High-throughput sequencing reads were trimmed using Trimmomatic (v0.39) 51 with the following parameters for 4f-SAMMY-seq and ChIP-seq: 2 for seed_mismatch, 30 for palindrome_threshold, 10 for simple_threshold, 3 for leading, 3 for trailing and 4:15 for sliding window. The sequence minimum length threshold of 35 was applied to all datasets. We used the Trimmomatic “TruSeq3-SE.fa” (for single-end reads) and “TruSeq3-PE-2.fa” (for paired-end reads) as clip files. After trimming, reads were aligned using BWA (v0.7.17-r1188) 52 setting –k parameter as 2 and using as reference genome the UCSC hg38 genome (only canonical chromosomes were used) and the output saved in BAM file format. We uniformly used and aligned only a single read per DNA fragment for both single-end and paired-end sequencing samples. The PCR duplicates were marked with Picard (v2.22; https://github.com/broadinstitute/picard) MarkDuplicates option, then filtered using Samtools (v1.9) 53 . In addition, we filtered all the reads with mapping quality lower than 1. Each sequencing lane was analyzed separately and then merged at the end of the process. Reads distribution profiles analysis To compute reads distribution profiles (genomic tracks) for individual fractions of 4f-SAMMY-seq, we used Deeptools (v3.4.3) 54 bamCoverage function. For these analyses the genome was binned at 50bp, the reads extended up to 250 bp and Deeptools RPKM normalization method was used. We considered a genome size of 2,701,495,761 bp (value suggested in the Deeptools manual https://deeptools.readthedocs.io/en/latest/content/feature/effectiveGenomeSize.html) and we excluded regions known to be problematic in terms of sequencing reads coverage using the blacklist from the ENCODE portal (https://www.encodeproject.org/files/ENCFF356LFX/). To compute the genomic tracks for ChIP-seq IP over INPUT enrichment profiles (log 2 normalized ratio) we used the SPP R package (v1.16.0) 55 and R statistical environment (v3.5.2). The reads were imported from the BAM files using the “read.bam.tags” function, then filtered using “remove.local.tag.anomalies” and finally the comparisons were performed using the function “get.smoothed.enrichment.mle” setting “tag.shift = 0” and “background.density.scaling = TRUE”. The resulting enrichment signal corresponds to a log2 normalized ratio between the pair of sequencing samples. For computing the relative comparisons between two 4f-SAMMY-seq fractions (relative enrichment, i.e. log 2 normalized ratio) we used the same procedure described above for ChIP-seq IP over INPUT enrichment profiles. We defined the "solubility profile" as the relative enrichment ratio of 4f-SAMMY-seq sequencing reads distribution along the genome for S2L vs S3 fractions. We used S3 as baseline in this ratio as it is the second most consistent fraction among controls (Extended Data Fig. 3d). To compute correlations between genomic tracks, we used R (v3.5.2) base function "cor" with “method = Spearman”. The genomic tracks were imported in R using the rtracklayer (v1.42.2) 56 library. Then the original genomic track files with 50bp resolution were re-binned by averaging data at 150kb resolution using the function “tileGenome” and the correlation was computed per chromosome. The correlation values obtained for each chromosome were then summarized in one value describing the genome-wide sample correlations through a weighted mean, where the weight of each chromosome corresponds to its length. Open histone marks (H3K4me1, H3K36me3) peaks were called with MACS (v2.2.9.1) using a broad-cutoff of 0.1 57 . H3K9me3 peaks were defined using the EDD (v1.1.19) software 58 with parameters (binsize = 150 Kb and gap penalty = 25) processing the filtered bam files obtained as described above. The “required_fraction_of_informative_bins” parameter was set to 0.98. The blacklisted regions were defined as for the reads distribution profile analyses described above. The enrichment boxplots shown in Fig. 2e was produced retrieving for each histone mark peak the genomic bins that fall in that genomic region. For each group of patients (CTR, LDD and HDD groups) and for each genomic bin we plot the mean 4f-SAMMY-seq solubility profile across the group. The values used were the quantile normalized tracks (across all samples) S2LvsS3 produced using SPP (see “High-throughput sequencing reads analyses”). Genomic tracks obtained by SPP or Deeptools were saved in bigWig file format. Genomic tracks visualization The visualization of genomic tracks was performed with Gviz R library (v1.26.5) 59 . The track profile was calculated using the function “DataTrack” (the input bigWig file was imported using the function “import” of the rtracklayer library) and plot using the function “plotTracks”. Extended Data Fig. 3e has been created plotting each track as type "polygon" and all the tracks have been set using a window of 500 except for ChIP-seq of H3K27ac where the window has been set to 5000. It is worth remarking that the "window" parameter in the "DataTrack" function is referring to the number of intervals in which the displayed region is divided to plot the profile. As such, a larger number indicate a finer grain definition of the profile. As H3K27ac is known to have sharp peaks, we aimed to plot a finer grain profile. For Fig. 1c the window was set to 1000 and for all the tracks except for ChIP-seq of H3K27ac where we used 5000, i.e. the same value used for the Extended Data Fig. 3e. CTR and PCa samples track overlayed with confidence interval were drawn using at the same the time the types “a” and “confint”. The same settings of histone mark tracks of Fig. 1c were used for Fig. 2b. Chromatin compartment analysis Chromatin compartments were calculated using a revised version of the CALDER algorithm (version 1.0 60 ), as implemented in the original 4f-SAMMY-seq pipeline for the sub-compartment identification 16 . Namely, for each chromosome: i) we calculated the four 4f-SAMMY-seq fractions (S2S, S2L, S3, S4) reads distribution profiles, binned at 150kb and normalized with RPKM (see "Reads distribution profiles analysis" section); ii) for each genomic bin, defined as a vector containing the four RPKM values (one for each fraction), we calculated the Euclidean distance (dist, R stats package, method="euclidean") with all the other bins of the same chromosome. These steps produced an NxN matrix (hereinafter referred to as biochemical similarity matrix), where N is the number of bins for the considered chromosome. Starting from the biochemical similarity matrix, we performed the CALDER procedure as described in 16 , to derive the eigenvector and to reconstruct chromatin compartmentalization. Specifically, we called sub-compartments but limiting their segmentation to the highest level, thus obtaining only 2 compartments corresponding to "A" and "B" compartments (Fig. 2a, b). The consensus of compartment calls across healthy controls has been produced labeling each genomic bin (150kb) according to its more frequent compartment across samples: e.g. if a bin is labeled as A in at least 4 out of 7 CTR samples, that bin will be defined as A in the consensus of controls, and vice versa for bins labelled as B in at least 4 controls (Fig. 2c). The definition of compartment shifts was based on the concordant or discordant compartment classification for each 150kb genomic bin (Extended Data Fig. 4a). The compartment domains shared across patients as shown in Extended Data Fig. 4a were reported using the library upsetR. The clustering of patients in Fig. 2d were based on Jaccard Indexes (JI) of compartments concordance for each pair of patients. The resulting matrix of pairwise JI was grouped by hierarchical clustering based on the Euclidean distance (among rows or columns) and complete linkage using the pheatmap function in the pheatmap R library. Cell type deconvolution For cell type deconvolution analysis, we fist normalized for each sample raw count to TPM (Transcript per Million) by using R 3.6.1 environment and applying the following steps. First, read counts for each gene were normalized (divided) by the length of the gene in kilobases (RPK, Reads per kilobase). Then RPK values in a sample are summed and divided by 1.000.000 to obtain the scaling factor. Finally, RPK values for each gene are divided by the scaling factor. For the deconvolution of the 3 macro populations (immune, epithelia, stroma cells) we defined a custom signature matrix based on single cell expression data of prostate tissue from the Human Proten Atlas (https://www.proteinatlas.org/; V. 21.1) 61–63 . Following the cluster classification of Human Protein Atlas we considered as epithelia the subpopulations annotated as prostatic glandular cells, basal prostatic cells or urothelial cells. We considered as stroma the populations annotated as muscle cells, fibroblast or endothelial cells. We considered as immune cells infiltrate the populations annotated as t-cells or macrophages. Finally, we selected as representative genes for each population only those with a TPM > 10 for the specific population and a TPM < 2 in all the two remaining populations. We finally got a macropopulation signature matrix of 702 genes. We run the deconvolution analysis exploiting this signature matrix on our bulk data by using the relative mode of CIBERSORTX (V.1.0) 64 (Extended Data Fig. 4f). To compute the immunity score on our biopsies we used the software CIBERSORTX (V.1.0) 64 applying the previously computed TPM matrix of our bulk RNA (mixture file) on the LM22.txt provided in the CIBERSORTX repository. The LM22 leukocyte signature is based on a matrix of 547 genes discriminating 22 human hematopoietic cell types isolated from PBMC. We enabled batch correction by using the B-mode correction option. We next run the analysis in absolute mode by applying 500 permutations. Each sample in the mixture file gets a score that reflects the absolute proportion of each cell types in LM22 mixture file. We finally computed the absolute leukocyte score: i.e. the sum for each sample of all the 22 scores of leukocytes populations. We compared the absolute leukocyte score for each biopsy across the different groups (CTR, LDD, HDD) (Extended Data Fig. 4g). Copy Number Analysis (CNA) For copy number analysis we used paired-end 4f-SAMMY-seq reads by treating fractions from the same sample as independent replicates. Data was processed with the pipeline nf-core/sarek v3.4.0 65,66 of the nf-core collection of workflows 67 , using whole genome sequencing settings, and segmentation was called with CNVkit v0.9.10 68 . Transcriptomic analyses For RNA-seq data, the overall quality of the sequenced reads was assessed using FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) (V. 0.11.8), then reads were trimmed with Trimmomatic software 51 (version 0.39) removing the adapters (ILLUMINACLIP:Picov2smart-PE.fa), primer dimers, and low quality bases at the beginning and at the end of the reads (trimmomatic PE phred33 LEADING:3 TRAILING:3 SLIDINGWINDOWD:4:15 MINLEN:36). STAR 69 (V. 2.7.0f_0328) was used to index (STAR --runMode genomeGenerate) the Human Genome (GENCODE Release 39, GRCh38 primary assembly genome 70 ) and to align sequenced reads in paired-end mode (--readFiles R1.FASTQ R2.FASTQ) on the indexed reference genome. Multimapping reads and PCR duplicate were marked in the final output (--bamRemoveDuplicatesType Unique) and unaligned reads stored in a different file (--outReadsUnmapped Fastx). The read counts on genes were calculated using as a reference a GTF file with RefSeq annotation downloaded from UCSC (http://genome.ucsc.edu) stored in the following directory:(http://hgdownload.soe.ucsc.edu/goldenPath/archive/hg38/ncbiRefSeq/109.20190905/hg38.109.20190905.ncbiRefSeq.gtf.gz). This file was further processed to remove non canonical and mitochondrial chromosomes, selected only curated genes (NM, NR) and finally split in protein coding (NM) and noncoding (NR) files. Reads count was performed with HTSeq-count (V. 0.13.5) on bam files (previously generated by STAR) using as a feature the union of all exons in a gene. The type of library was specified with “-s reverse” parameter. The reads that align to more than one position in the reference genome were discarded (htseq-count --non-unique none). The full matrix with raw read counts for each sample were loaded in R 3.6.1 and normalized using DESeq2 71 median of ratios. Differential expression analysis was performed with DESeq2V. 1.26 72 ) using Wald test and the Benjamini and Hochberg correction for multiple tests, to compute p-values and adjusted p-values, respectively. Enrichment analyses Up-regulated genes (with adjusted p-value 1) and Down-regulated genes (with adjusted p-value < 0.05, and Log 2 Fold Change < -1) were used separately to query the gene-list enrichment tool Enrichr (https://maayanlab.cloud/Enrichr/) 73–75 over the main Gene Ontology classes (Biological Process - BP, Molecular Function - MF, Cellular Component - CC) updated to 2023 taking as final enriched terms only those with Benjamini-Hochberg adjusted p-value < 0.05. Genes within the recurrent compartment switch “A to B” and “B to A” were also queried over the main Gene Ontology classes taking also in this case the enriched terms with Benjamini-Hochberg adjusted p-value < 0.05. GSEA analysis 31 was performed in Preranked mode. Starting from raw p-values of all genes we generated a ranked matrix sorted by the score resulting from the following formula: -Log 10 (p-value) multiplied by the sign of the Log 2 Fold Change. For the gene set references we downloaded H (Hallmark) and C5 (Ontology) GMT files from MSigDB. Finally, we performed the analysis using the parameters "Number of permutations": 1000; "Collapse": No; "Enrichment Statistic": classic; "Max size": 500; "Min size":15. Resulting geneset with a family-wise error rate (FWER) < 0.05 are finally sorted using the Normalized Enrichment Score (NES). We then inferred pathway activity for the comparison of our tumor subtypes by using the R package decoupleR 76 (V. 2.4). We used the get_progeny (organism=human,top=100) function to get pathways from PROGENy (V.1.20) 32 a curated collection of signaling pathways derived from perturbation experiments, where each gene has a specific weight describing its positive or negative response to a given pathway stimulation. The top 100 responsive genes in the compendium ranked by p-value were used for each pathway. Starting from Wald statistic values previously computed for each gene with DESeq2 (LDDvsCTR, HDDvsCTR and HDDvsLDD comparisons) we run the function decouple with parameters statistics=c(“mlm”,”ulm”,”wsum”) and consensus_score=TRUE, to estimate a consensus score across the top performer methods (according to benchmark done by decoupleR developers) in pathway activity prediction. Multivariate linear model (mlm), Univariate linear model (ulm), Weighted Sum (wsum) are recommended by developers of decoupleR since these methods better estimate pathway activity considering the weights associated with pathway related genes. Patient specific DEGs and Compartment switches Significant Down-regulated genes have been identified using DESeq2 with the same workflow described above (adjusted p-value 1), comparing each individual LDD and HDD patients against the group of 7 CTRs. Patients-specific compartment switches ("A to B" and "B to A") were defined with respect to the consensus of compartment calls across CTRs. To define number of DEGs included in switch regions for each patient, patient-specific down-regulated DEGs were intersected to patient-specific A to B regions while up-regulated DEGs were intersected to patient-specific B to A regions. Then the gene lists resulting from all the patient-specific intersections of DEGs and compartment switches were merged and the reultin union gene list was used as input for functional classes enrichment analysis with Enrichr (https://maayanlab.cloud/Enrichr/). TCGA Data TCGA-PRAD RNA-Seq expression data were downloaded by using gdc-client tool on gdc_manifest.2022-08-08.txt metafile input downloaded from the GDC portal repository (https://portal.gdc.cancer.gov/; V.34.0; Release July, 27, 2022) by selecting from Web interface the TCGA project, prostate and RNA-seq count. The main clinical parameters (Gleason Score, T stage, N stage and age) and info about the samples (primary tumor, normal biopsy and metastatic) were selected from the same Web section of GDC portal repository by downloading clinical and sample-sheet files. ABSOLUTE tumor purity score 77 was downloaded from https://gdc.cancer.gov/about-data/publications/pancanatlas. The file is TCGA_mastercalls.abs_tables_JSedit.fixed.txt (Downloaded October ,13, 2022) 78 . Excluding primary normal biopsies, we obtained from the TCGA repository described above the 500 FPKMUpperQuartile normalized expression counts from prostate primary tumors. The Biochemical Recurrence Free (BCR) status for each patient was obtained from PCaDB (http://bioinfo.jialab-ucr.org/PCaDB/; Release May, 10 2021; eSet V.1.3), downloading the TCGA-PRAD_eSet.RDS object and applying the pData function from Biobase package. We then excluded the only sample with a Gleason Score pattern of 2+4 never described in literature. Despite we performed expression analyses assigning a PCI score on the entire cohort of 499 samples, the BCR data available are 466. PCI score (Prostate Compartmentalization Index) Starting from our 101 DEGs of the HDD vs LDD comparison we defined two gene lists with 24 HDD Up-regulated and 77 HDD Down-regulated genes. We subset the TCGA matrix extracting only those genes matching with our full list of 101 genes. We performed a log transformation of TCGA gene expression matrix: Log 2 (matrix +1). The log transformed expression of each gene across the 499 samples was standardized into a z-score. For each sample we computed 2 median values on the z-score transformed matrix: the median of the 24 HDD Up-regulated genes (HDD_UP) and the median of the 77 HDD Down-regulated (HDD_DOWN) genes. Finally, we computed the PCI score defined as the difference of median values: PCI=median(HDD_UP) - median(HDD_DOWN). The resulting PCI score with a positive value will indicate that a sample has HDD-like transcriptomic features, whereas a PCI negative value will classify a sample as LDD-like. Survival Analyses Univariate and multivariate regression analysis were performed in R using survival and survminer packages. First, we performed a log-rank test to compare survival Kaplan-Meier curves (in terms of BCR STATUS AND BCR TIME) between HDD-like and LDD-like samples. Then we performed a multivariate Cox proportional-hazard model including the following covariates: HDD-LDD status (previously assigned according to the PCI score) of the samples, pathological stage, age at diagnosis, Gleason Score and Tumor purity score. For the multivariate Cox regression analysis, the Gleason Score was included as numeric feature by summing the primary and secondary numeric grades, as reported for TCGA samples. Signature Refinement To refine the 101 gene signature, we applied univariate cox regression analysis on each single gene expression values across the TCGA samples, including in the model the BCR STATUS and BCR TIME. The p-values obtained for each gene are then adjusted with Benjamini-Hochberg. Then we selected only those genes whose expression is associated with Hazar Ratio (HR) 1 with a adj.p-value < 0.05. We obtained 21 candidate prognostic genes. Unsupervised clustering of these genes can cluster 2 main groups of samples with HDD-like and LDD-like features with high significance in the prognostic status of the two groups (Extended Data Fig. 6a). We further refined the signature by removing 3 genes (NPIPB3, CCNE2, FMO5) that were noisier in the unsupervised clustering heatmap: these are the only genes that cluster in a discordant manner compared to their original association to HDD or LDD phenotype in our dataset (Extended Data Fig. S6a). Finally, we used the resulting refined panel of 18 genes to recompute and validate our PCI score on other independent cohorts of transcriptomic profiles of prostate cancer patients. Validation Through the PCaDB (http://bioinfo.jialab-ucr.org/PCaDB/ Release May, 10 2021; eSet V.1.3) we downloaded normalized expression matrices and BCR data of three independent datasets of prostate cancer: Emory (GSE54460) 79 , Stockholm (GSE70769) 80 , Belfast (GSE116918) 81 . For each of them we classified samples computing the PCI score based on the refined 18 genes signature. Declarations Data availability The high-throughput sequencing data generated for this study are available in the database ArrayExpress with accession number ‘E-MTAB-14226’, at the link: https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-14226?key=3d98766f-3ac6-4c07-a4a0-1022bc521bb0. The bioinformatic pipeline along with a step-by-step tutorial explaining how to process 4f-SAMMY-seq data is available on FigShare (https:// doi.org/10.6084/m9.figshare.25437121.v1) and also linked to the Github repository for updates (https://doi.org/10.6084/m9.figshare.25438195). Authorship VR designed and performed experiments as well as data analysis. CP and GL designed performed data analysis. FG and RQ performed immunofluorescence (IF) and AF performed the IF quantification. MM performed CNA analysis. ES and EDPS participated in data analysis. MC acquired and analysed FACS data. VV supervised sequencing experiments. EM, GA, FR and EDL provided patient samples and participated to the data interpretation. MMa performed histopathology evaluation. VR, CP and GL wrote the manuscript. FF and CL conceived the study and wrote the manuscript. All authors approved the submitted version. Conflict of interests A patent application is being filed for the signature presented in this manuscript by VR, CP, GL, FF and CL. Acknowledgements We thank Maria Vivo, Beatrice Bodega, Marina Lusic, Mattia Forcato, Gioacchino Natoli, Martin Schaefer and all members of our laboratories for critical feedback on earlier versions of the manuscript. We thank Claudio Tripodo (IFOM and University of Palermo) and Giorgia Zadra (CNR) for feedback on early phases of the project. For the sequencing we acknowledge support from the Genomics Unit of the European Institute of Oncology (IEO), Department of Experimental Oncology, Humanitas Sequencing facility and Istituto Nazionale Genetica Molecolare (INGM) (Marco Ghilotti). We thank Ilaria Rancati (IFOM cell culture facility) for support with Maxwell instrument. This work was supported by Interomics Flagship Project (CNR) and MFAG (grant #18535) to CL; AIRC Start-Up (grant #16841) to FF; FRRB INTERSTRAT-CAD (grant #CP2_14/2018) to FF; AIRC fellowships n. 26942 to FG and No. 22351 to ES; PIR01_00011 “IBISCo”, PON 2014-2020 to MM. Schematic representations of Figs 1a, b and Extended Data Fig. 3a have been created using BioRender.com. References The Global Cancer Observatory (GCO) https://gco.iarc.fr/en Mottet N et al (2017) EAU-ESTRO-SIOG Guidelines on Prostate Cancer. 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EBioMedicine 2:1133–1144 Jain S et al (2018) Validation of a Metastatic Assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy. Ann Oncol 29:215–222 Tables Table 1. Clinicopathological characteristics of the study participants undergoing prostate biopsy Patient Histology (GS-P) Age at time of biopsy (years) PSA level (ng/ml) Positive cores (%) Clinical stage PI-RADS 49 D’Amico risk classification 50 CTR 28 Neg 62 3.7 - - - - CTR 31 Neg 65 7.18 - - - - CTR 32 Neg 67 3.73 - - - - CTR 91 Neg 67 8.15 - - - - CTR 92 Neg 78 4.6 - - - - CTR 94 Neg 72 8.9 - - - - CTR 98 Neg 72 20.9 - - - - PCa 93 3+4 73 8.9 14.2 T1c 2 1 (Intermediate risk) PCa 73 3+4 65 19 21.4 T1c 5 2 (Intermediate risk) PCa 75 4+3 82 13 21.4 T1c 2 2 (Intermediate risk) PCa 87 4+3 72 6.5 28.5 T1c 2 1 (Intermediate risk) Pca 35 4+4 71 4.88 21.4 T1c 4 4 (High risk) PCa 2 4+5 89 51 42.8 T2b 2 9 (High risk) PCa 100 4+5 87 29 21.4 T2a 5 8 (High risk) PCa 4 4+5 74 487 42.8 T3 4 12 (High risk) PCa 88 4+5 72 186 42.8 T3 2 12 (High risk) PCa 33 5+5 78 4 21.4 T1c 5 8 (High risk) GS-P: Gleason Score for patients’ diagnosis PSA: Prostate Specific Antigen PI-RADS: Prostate Imaging Reporting and Data System Additional Declarations Yes there is potential Competing Interest. The authors declare that the PCa signature described in this study is currently under patenting, thus constituting a potential competing interest. Supplementary Files Extendeddata.docx Cite Share Download PDF Status: Published Journal Publication published 16 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":193930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromatin architecture alterations in prostate cancer patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eOverview of the experimental design. Fresh biopsies were dissected into 2 portions: one third was flash frozen and embedded in OCT for histopathological and immunochemical analyses (1), while the rest was enzymatically digested into single cell suspensions that were further divided for flow cytometry-based phenotype assay (2), 4f-SAMMY-seq and RNA-seq (3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Study cohort biopsies: representative images of Hematoxylin and Eosin staining (H\u0026amp;E) at magnification x20 prostate biopsies of for non-neoplastic patients (CTR) and prostate cancer patients (PCa). In the schematic prostate diagram, the circles mark sites of diagnostic biopsies, the star indicates the research biopsy position. Positive cores (black circles) and Percentage of Positive Cores (PPC) are also reported. GS-P indicates diagnostic Gleason Score assigned to each patient, while GS-CDB indicates the Gleason Score assigned to the biopsy adjacent to the research biopsy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003e Epigenome profiles: distribution along chromosome 3 of ChIP-seq enrichment profiles for open (H3K27ac and H3K36me3) and closed (H3K9me3) chromatin marks, along with 4f-SAMMY-seq consensus solubility profiles of 7 CTR (green) and of 10 PCa (purple) samples. The solid line shows the mean, and the shadowed areas marks the confidence interval: +/-1.96 standard error in the solubility profiles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003e Violin plots for genome-wide Spearman correlation values (y-axis) for 4f-SAMMY-seq solubility profiles of CTR (green, n=7) and PCa (purple, n=10) samples with respect to ChIP-seq enrichment profiles in normal prostate gland for three distinct histone marks (H3K36me3, H3K27ac, H3K9me3). Each data point in the violin plots shows the correlation value of individual patients computed with genomic profiles binned at 150kb.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5219856/v1/f00446a5db83fd4e8af88603.png"},{"id":67369206,"identity":"826b1b78-8a9d-4d06-9030-658e2b862803","added_by":"auto","created_at":"2024-10-24 07:47:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProstate cancer subtypes identified by epigenomic profiles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003ePair-wise correlation matrices of reads distribution profiles for representative samples CTR31 (left) and PCa87 (right), computed at 150kb genomic bins resolution on chromosome 18 long arm. On the side of each matrix the respective first eigenvector is reported and colored to mark the position of active (\"A\" compartment with positive eigenvalues) and inactive regions (\"B\" compartment with negative eigen values). On the center of the panel the two bars show the A and B compartments along the chromosome according to a color code: dark grey for active (A) and light grey for inactive (B) compartments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Schematic representation of chromatin compartments along with chromatin marks on representative chromosome 18. From top to bottom: ChIP-seq enrichment profiles of H3K36me3 (dark red), H3K27ac (red) and H3K9me3 (light blue); A and B compartments segmentation for CTR and PCa patients (labels on the left), grouped in LDD and HDD subgroups (labels on the right).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. \u003c/strong\u003eStacked barplot illustrating the count (number of genomic bins, bottom x-axis) and the relative distribution over the entire genome (percentage, top x-axis) of genomic regions with conserved compartment class (\"A\" - black or \"B\"- grey) and regions shifting compartment (\"A to B\" or \"B to A\" - different shades of grey) for each PCa sample. The discordant chromatin compartment classification is calculated with respect to the consensus of CTR (on the top, see Methods).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003e Matrix reporting the pair-wise Jaccard Index derived from the compartment similarity among PCa samples. The patients within the matrix are arranged by unsupervised hierarchical clustering. The light-purple cluster (PCa33, PCa35, PCa93, PCa88 and PCa100) contains samples of the LDD group. The dark purple cluster (PCa75, PCa4, PCa2, PCa73, PCa87) includes HDD samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee.\u003c/strong\u003e Boxplot of solubility profiles average values (y-axis) for individual 150kb genomic bins overlapping the ChIP-seq enrichment peaks of open chromatin (H3K36me3, H3K27ac) and closed chromatin (H3K9me3) histone marks (labels on the x-axis). For each group of patients (CTR, LDD and HDD groups) and for each genomic bin we plot the mean 4f-SAMMY-seq solubility profile across the group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5219856/v1/38583fcdad75c6f59a6ead90.png"},{"id":67367385,"identity":"8f4a8d12-02d1-46cf-9b93-e316622bb558","added_by":"auto","created_at":"2024-10-24 07:31:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":110086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional features discriminating prostate cancer subtypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Volcano plot showing Differentially Expressed Genes (DEGs) in the comparison between PCa and CTR samples (left), LDD tumors subtype and CTR samples (center), HDD tumors subtype and CTR samples (right). Blue dots mark significantly down-regulated genes with adjusted p-value \u0026lt; 0.05 and log\u003csub\u003e2\u003c/sub\u003e Fold-Change \u0026lt; -1; red dots mark significantly up-regulated genes with adjusted p-value \u0026lt; 0.05 and log\u003csub\u003e2\u003c/sub\u003e Fold-Change \u0026gt; 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Proportional Venn diagram of up-regulated genes (left) and down-regulated genes (right) comparing all PCa tissues vs CTR samples, LDD tumors subtype vs CTR samples and HDD tumors subtype vs CTR samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. \u003c/strong\u003eFunctional classes enrichment analysis of HDD vs CTR DEGs using GO Cellular Component, Molecular Function and Biological Process annotations. Bar plots representing significantly enriched terms in down-regulated genes (blue) and up-regulated (red) genes, with -log\u003csub\u003e10\u003c/sub\u003e adjusted p-values reported (horizontal axis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u003c/strong\u003e Stacked bar plot depicting the percentage of DEGs in the comparison LDD vs CTR (left) and HDD vs CTR (right) across concordant (no switch - magenta) and discordant (switch - turquoise) compartments classification with respect to CTR compartments. The barplot shows the average across individual patients (n=5 for each group) and whiskers mark ± 1 Standard Error of the Mean (SEM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee.\u003c/strong\u003e Stacked bar plot illustrating the relative distribution of up-regulated and down-regulated genes (x axis) at the individual patient level, for HDD vs CTR comparison falling in the compartments switching from A in the CTR to B in HDD (light grey) and from B in the CTR to A in HDD (dark grey), as defined in Figure 3d. The barplot shows the average across individual patients (n=5 for each group) and whiskers mark ± 1 SEM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef. \u003c/strong\u003eFunctional classes enrichment analysis using the intersection between significant down-regulated (blue edge of the dot) genes included in A to B switches (light grey dot) in HDD patients. Significant enriched gene-set libraries from left: ENCODE and ChEA Consensus TFs, MsigDB Hallmark, HuBMAP ASCTplusB. The size of the dots (Count) is the number of our query genes overlapping with genes of the enriched terms. The dashed line represents the significance threshold used for the enrichment terms selection (adjusted p-value \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5219856/v1/41119a65bfc6ebb5a66701a8.png"},{"id":67367390,"identity":"c4679bd9-b2e4-4c7f-bfa0-734c031c0ea3","added_by":"auto","created_at":"2024-10-24 07:31:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":169232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrognostic stratification of patients driven by\u0026nbsp;chromatin-derived signature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Volcano plots showing Differentially Expressed Genes (DEGs) in the comparison between HDD and LDD subtypes. The adjusted p-value \u0026lt; 0.05 and log\u003csub\u003e2\u003c/sub\u003e Fold Change value \u0026gt; 1 or log\u003csub\u003e2\u003c/sub\u003e Fold Change value \u0026lt; −1 and are the cutoff values for significant up-regulated (red, n=24) and down-regulated (blue, n=77) genes, respectively. Non-significant differential expression is marked with grey color.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. \u003c/strong\u003eSelected Gene Set Enrichment Analysis (GSEA) profiles resulting from the analysis of HDD vs LDD pre-ranked expression scores with Hallmark gene sets from the Molecular Signatures Database (MSigDB) collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u003c/strong\u003e Heatmap showing RNA-seq expression values (scaled by row) across 499 prostate cancer tissues from TCGA cohort (see Methods). The sample (on columns) are sorted in ascending order according to the PCI score. Rows reports the 101 DEGs from HDD vs LDD comparison and are grouped by hierarchical clustering over Euclidean distance. The top section side bar displays groups of transcriptomic profiles categorized as LDD-like (light pink, PCI score \u0026lt; 0) and HDD-like (deep pink, PCI score \u0026gt; 0), along with the Gleason Score and tumor purity associated with each sample. A colored side bar on the left marks up-regulated (red) and down-regulated (blue) DEGs in HDD vs LDD comparison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed. \u003c/strong\u003eKaplan-Meier plot showing Biochemical Recurrence Free (BCR) status outcome of TCGA patients stratified according to their transcriptomic similarity with HDD and LDD group. The deep pink line indicates HDD-like TCGA samples, whereas the light pink line indicates LDD-like TCGA samples. P-value was obtained from the Log rank test (p-value = 0.041).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee. \u003c/strong\u003eMultivariate cox regression analysis using cox proportional hazards model (Hazard Ratio, HR) for Biochemical Recurrence Free (BCR). The following covariate parameters with p-value from Wald test have been used in the model: PCI-based classification, Clinical Stage (N, T), Age, Tumor Purity, Gleason Score.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5219856/v1/ea7d8e5360eb6ef2c66a58bc.png"},{"id":67367387,"identity":"e4037903-f356-4ac6-a4ae-0a3a1f6c5b42","added_by":"auto","created_at":"2024-10-24 07:31:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDD transcriptional signature is associated with favorable prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-c\u003c/strong\u003e. Kaplan-Meier plots based on the 18 genes refined on 100 samples from Emory cohort (GSE54460 dataset) (panel A, p-value = 0.018), on 248 sample from Belfast cohort (GSE116918 dataset) (panel B, p-value = 0.006), and on 92 samples from Stockholm cohort (GSE70769 dataset) (panel C, p-value= 0.0017). Deep pink lines indicate HDD-like samples while light pink lines indicate LDD-like samples.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5219856/v1/d268ee88ed0fb75fbfe7b98f.png"},{"id":93748195,"identity":"5c054a41-f9da-4b64-8d50-f028e67f80fd","added_by":"auto","created_at":"2025-10-17 07:10:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1832450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5219856/v1/24ca4c3d-db79-43fd-aeda-52e2a0822281.pdf"},{"id":67367391,"identity":"b506f322-13ba-4446-9e52-228241aa2010","added_by":"auto","created_at":"2024-10-24 07:31:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4768434,"visible":true,"origin":"","legend":"","description":"","filename":"Extendeddata.docx","url":"https://assets-eu.researchsquare.com/files/rs-5219856/v1/7fcb0739cad2172322bf5bcd.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe authors declare that the PCa signature described in this study is currently under patenting, thus constituting a potential competing interest.","formattedTitle":"Chromatin remodeling restraints oncogenic functions in prostate cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer (PCa) is the second most common tumor type among males and accounts for 10% of cancer-related deaths \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. While the majority of PCa cases present as indolent and slow-growing, about 20% of prostate tumors progress to metastatic and lethal forms. Currently, PCa diagnosis relies on histological evaluation of multiple biopsies, usually conducted following elevated prostate-specific antigen (PSA) levels or abnormal digital rectal examination (DRE) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite the use of tumor grading systems, the clinical variability and frequent multifocality of PCa complicate the accurate prediction of cancer outcomes, hindering treatment guidance \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Consequently, many patients with silent PCa are over-treated \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Although the identification of driver coding mutations in solid tumors has significantly advanced the field of clinical oncology \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, the translation of PCa genetic biomarkers into clinical practice has only marginally improved prognosis accuracy \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This limitation underscores the need for novel biomarkers that can more precisely guide clinical decision-making in PCa management.\u003c/p\u003e \u003cp\u003eEmerging evidence suggest that epigenetic information, which regulates transcriptional plasticity, plays a critical role in tumor evolution \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Epigenetic dysfunctions have been shown to initiate carcinogenesis even in the absence of driver mutations \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, offering a new perspective on cancer research. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe three-dimensional (3D) genome architecture, which is hierarchically organized on multiple, highly regulated structural levels in the nucleus, has emerged as crucial for regulating cellular programs \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Notably, the physical separation between euchromatin, the more accessible and transcriptionally active part of the chromatin, and heterochromatin, which is compacted and gene-poor, is a hallmark of healthy cells. Morphological changes in nuclear organization, referred to as nuclear atypia, still represent a gold standard for diagnosis and staging of various cancers, including prostate cancer \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A recent study on metastatic prostate cancer patients reported multilevel alterations in chromatin structures associated with poor survival outcomes \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, this work was performed on advanced PCa stages, thus providing limited information for earlier diagnostic samples. The same consideration applies to previous transcriptional studies performed on surgically removed prostates \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to comprehensively characterize chromatin compartment organization in PCa using our recently developed 4f-SAMMY-seq \u003csup\u003e16\u003c/sup\u003e. We analyzed fresh needle biopsies from PCa patients at their initial clinical presentation. We identified patient-specific changes in chromatin compartments and we extrapolated the underlying genomic dysregulation. Our findings reveal that large-scale chromatin remodeling and transcriptional repression are associated with a protective, antitumoral effect. Based on these insights, we derived a novel gene expression signature to classify this specific PCa subtype. We validated the predictive power of this 18-gene signature across multiple patient cohorts, encompassing more than 900 individuals. This signature demonstrates significant potential as a tool for early prognosis prediction in PCa and could be readily implemented in clinical practice. Questa firma dimostra un potenziale significativo come strumento per la previsione precoce della prognosi nel PCa e potrebbe essere facilmente implementata nella pratica clinica\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo characterize chromatin architecture in prostate cancer patients at the time of diagnosis, we collected fresh needle biopsies from chemo-na\u0026iuml;ve patients undergoing the standard diagnostic procedure. Each patient was punctured to collect 14 needle biopsy cores for histopathological examination (diagnostic biopsies) and 1 to be processed for our research project (research biopsy \u0026ndash; Fig. 1a). Each research biopsy was divided into two parts: one-third was used for histopathological and immuno-histological analyses. At the same time, the rest of the tissue sample was enzymatically digested to obtain a cell suspension further split for epigenomic (4f-SAMMY-seq), transcriptomic (RNA-seq) and cellular composition (flow-cytometry) analyses.\u003c/p\u003e\n\u003cp\u003eOur cohort includes seven non-neoplastic controls (CTR), \u003cem\u003ei.e.\u003c/em\u003e patients that turned out to be negative for cancer, and ten primary prostate cancer patients (PCa) with histologically confirmed tumor and Gleason Score (GS) between 3+3 and 5+5 (Fig. 1b). We also report in Fig. 1b the percentage of diagnostic biopsy cores positive for cancer cells (PPC), their distribution in a prostate gland diagram (DPC), and the puncture site of the research biopsy used for this study. All the research biopsies of prostate cancer patients were obtained from punctures adjacent to one or more positive diagnostic biopsy cores (Fig. 1b). In addition to the Gleason Score assigned to the patient in the clinical report (GS-P), we recorded the Gleason Score assigned to the closest diagnostic biopsy (GS-CDB) and the score assigned to the research dedicated biopsy (GS-RDB, Extended Data Fig. 1a). We noted that the histopathological examination on research biopsies did not confirm the presence of tumor cells in four out of ten cases (Extended Data Fig. 1a). However, it must be noted that the Gleason Score assignment was done on a third of the tissue biopsy and that all research biopsy samples express diagnostic PCa tumoral markers like PCA3, HPN and GOLM1\u003csup\u003e17\u0026ndash;19\u003c/sup\u003e (Extended Data Fig. 1b). Therefore, we retained all ten PCa samples in our experimental analyses.\u003c/p\u003e\n\u003cp\u003eOur cohort of PCa patients is relatively homogeneous in terms of age (median 72 years; interquartile range, abbreviated as IQR, 62-89 years) and serum PSA level (median 16 ng/mL, IQR: 7.1-45.5) but heterogeneous in terms of clinical features as percentage of cancer positive biopsy cores (median: 21.4%, IQR: 21.4-39.2), clinical stage (T1c: 60%, T2a-T2b: 20%, T3: 20%) and index lesions on Magnetic Resonance Imaging (MRI) (40% with Prostate Imaging Reporting and Data System (PI-RADS) \u0026ge;3 and 60% with PI-RADS 1/2) (Extended Data Table 1). Survival outcomes are not available due to the long clinical course of primary PCa and the short follow-up time since the biopsies were collected (median time from biopsy less than four years).\u003c/p\u003e\n\u003cp\u003eWe also examined tissue samples of our cohort by confocal microscopy. Non-neoplastic controls and PCa patients\u0026apos; cells showed similar nuclear areas but a significant difference in nuclear circularity, with PCa patients\u0026rsquo; cells showing nuclei deformations (Extended Data Fig. 1c, d). Staining of the nuclear lamina with the Lamin A/C antibody highlighted a\u0026nbsp;slight depletion\u0026nbsp;of the signal in the nuclear interior, corresponding to a parallel decrease in the Hoechst staining (Extended Data Fig. 1e). Although descriptive, these data suggest a cancer-specific remodeling of the nuclear and genome organization in PCa cells.\u003c/p\u003e\n\u003cp\u003eWhen dissociating the needle biopsy tissue samples (size around 2 cm in length,\u0026nbsp;Extended Data Fig.\u0026nbsp;2a, see Methods), we achieved a viable cell yield in the range of 30,000-80,000 cells per sample (Extended Data Fig. 2b).\u003c/p\u003e\n\u003cp\u003eAs a further control, we examined the cell type composition in a set of 13 prostate biopsies by flow cytometry\u0026nbsp;(Extended Data Fig. 2c-f). We didn\u0026apos;t detect\u0026nbsp;significant differences in the relative proportion of epithelial (EpCAM/CD326+, CD45-), leukocyte (EpCAM/CD326-, CD45+) or stromal cells (EpCAM/CD326-, CD45-)\u0026nbsp;between non-neoplastic controls and PCa samples (Extended Data Fig. 2g). All samples showed significant fraction of stromal cells (average 68,8% controls and 70,9% PCa patients), while we found an average of 18,5% and 15,2% of epithelial cells in control and PCa biopsies, respectively.\u003c/p\u003e\n\u003cp\u003eWe applied 4f-SAMMY-seq to examine genome architecture in prostate biopsies\u003csup\u003e\u0026nbsp;16\u003c/sup\u003e. This method relies on the sequential isolation and high-throughput sequencing of distinct chromatin fractions separated according to their accessibility and solubility properties, which are expected to correlate with chromatin epigenetic and transcriptional status (Extended Data Fig. 3a). The more soluble fractions (S2S, S2L) are associated with gene activation markers, and the less soluble fractions (S3, S4) are enriched for constitutive heterochromatin. \u003cs\u003e\u0026nbsp;\u003c/s\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the applicability of this technique on fresh prostate biopsies, we first sequenced 4f-SAMMY fractions of non-neoplastic control samples. We examined the DNA sonication and short fragments (S2S) selection to obtain high-quality libraries (Extended Data Fig. 3b). We verified that we obtained\u0026nbsp;sequencing reads with comparable quality across samples (Extended Data Fig.\u0026nbsp;3c).\u0026nbsp;The sequencing reads distribution profiles for individual fractions from CTR samples show a high level of concordance (Extended Data Fig. 3d), with the highest correlation values for S2L fractions (median: 0.94).\u003c/p\u003e\n\u003cp\u003eTo study euchromatic and heterochromatic domains, we defined the solubility profile as the ratio of high-throughput sequencing reads distribution in S2L over S3 fractions (see Methods), where higher and lower values correspond to regions enriched for open and closed chromatin, respectively. We compared the consensus solubility profiles across all CTR and PCa specimens with epigenomic marks in normal prostate tissue for post-translational histone modifications (histone marks) associated with active (H3K27ac, H3K36me3) and inactive (H3K9me3) chromatin states \u003csup\u003e20\u003c/sup\u003e (Fig. 1c). We observed a high similarity of solubility profiles among CTR samples and a clear concordance with the location of euchromatin marks (H3K27ac and H3K36me3), along with a negative correlation with the heterochromatin mark profile (H3K9me3). These observations were also confirmed in a genome-wide quantification of correlations for individual samples (Fig. 1d).\u003c/p\u003e\n\u003cp\u003eInstead, in PCa biopsies we observed that the solubility profile is more heterogeneous and its correlation with histone marks is more variable with respect to CTR samples\u0026nbsp;(Fig. 1c, d). Visual inspection of individual solubility profiles showed a patient-specific degree of chromatin alterations (Extended Data Fig. 3e). Moreover, we noticed\u0026nbsp;clear differences among PCa samples, with five out of ten samples losing the regular chromatin compartmentalization, as also highlighted by their correlation with histone marks (Fig. 1d). These differences are remarkably not attributable to the cell composition of the samples (Extended Data Fig. 2g).\u003c/p\u003e\n\u003cp\u003eThus, 4f-SAMMY-seq data describe a conserved genome architecture among control biopsies with a separation between euchromatin and heterochromatin. On the other hand, diagnostic early-stage PCa biopsies show a patient-specific chromatin solubility dysregulation that mirrors PCa\u0026rsquo;s highly heterogenous nature.\u003c/p\u003e\n\u003cp\u003eTo further analyze the chromatin 3D organization, we used the sequencing data of all the 4f-SAMMY-seq fractions to identify active and inactive compartments,\u0026nbsp;named \u0026quot;A\u0026quot; and \u0026quot;B\u0026quot;, respectively, as per the convention adopted for Hi-C derived compartments\u003csup\u003e\u0026nbsp;16\u003c/sup\u003e (Fig. 2a, b, see Methods). However, unlike Hi-C, the chromatin compartments inferred from 4f-SAMMY-seq are defined based on their chromatin accessibility and solubility properties\u003csup\u003e\u0026nbsp;16\u003c/sup\u003e. CTR samples showed a consistent pattern across all seven individuals with 27% of the entire genome having conserved active A compartment and 39% having conserved inactive B compartment across all CTRs (Fig. 2b, c). Instead, PCa samples showed patient-specific chromatin compartment alterations (Fig. 2b, c). We quantified the genomic regions with a different compartment assignment in each PCa sample with respect to the consensus among CTR samples. We labelled these compartment switches as \u0026quot;A to B\u0026quot; or \u0026quot;B to A\u0026quot; for the regions that in CTR samples were A or B, respectively. Unsupervised clustering based on compartment similarity identified two subgroups of cancer patients (Fig. 2d) that, considering the amount of compartment alterations with respect to CTR samples, we named LDD (Low Degree of Decompartmentalization; average 5% over the entire genome for \u0026quot;A to B\u0026quot; and 5% for \u0026quot;B to A\u0026quot; compartment switches) and HDD (High Degree of Decompartmentalization; average 14% over the entire genome \u0026quot;A to B\u0026quot; and 24% \u0026quot;B to A\u0026quot;). We also noted a consistent intra-group similarity for LDD samples with 0.78 mean Jaccard Index (JI) (variance 0.001). On the other hand, HDD samples show more heterogeneity in chromatin compartmentalization (JI 0.42 mean and 0.012 variance) (Fig. 2d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn line with patient-specific genome remodeling, we did not find regions with compartment changes concordant across all LDD and HDD tumoral samples compared to CTR consensus. However, we found recurrent \u0026quot;B to A\u0026quot; regions, indicating higher chromatin accessibility compared to controls, concordant across all HDD patients and covering 2% of the entire genome (538 genes) (Extended Data Fig. 4a). Gene ontology analysis highlighted an enrichment of genes associated with Protein Deubiquitination and Regulation of Programmed Cell Death (such as FAS) (Extended Data Fig. 4b). Instead, the HDD-specific \u0026quot;A to B\u0026quot; compartment switch regions, which correspond to reduced chromatin accessibility compared to controls, (0.6% of the genome - 59 genes) included genes involved in PI 3-kinase activities (such as PIK3R5 and PIK3R6) and lipid catabolic processes (such as PNLIP and STS), both of which are known drivers of prostate cancer progression \u003csup\u003e21\u0026ndash;23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIt should be remarked that the LDD vs HDD groups stratification of PCa patients would not be immediately evident based on known clinically relevant pathophysiological features such as PSA level, Gleason Score, age at diagnosis and number of positive cores (Extended Data Fig.\u0026nbsp;4c).\u0026nbsp;Furthermore, the analysis of the global transcriptome on prostate samples did not separate so clearly the HDD and LDD subtypes (Extended Data Fig.\u0026nbsp;4d).\u0026nbsp;We excluded the association of\u0026nbsp;HDD and LDD\u0026nbsp;subtypes with cell composition and\u0026nbsp;immune cell infiltration, estimated by flow-cytometry (Extended Data Fig.\u0026nbsp;4e) and RNA-seq data deconvolution (see Methods,\u0026nbsp;Extended Data Fig.\u0026nbsp;4f, g). We also estimated the copy number alterations (CNA) distribution across all samples based on 4f-SAMMY-seq reads coverage (see Methods). The inferred copy-number states are notably similar for both LDD and HDD subtypes (Extended Data Fig.\u0026nbsp;4h), suggesting that structural alterations do not determine the described subtypes.\u003c/p\u003e\n\u003cp\u003eWe further investigated epigenetic differences between LDD and HDD groups. We compared the histone mark enrichment peaks from normal prostate tissue with the 4f-SAMMY-seq solubility profiles of CTR, LDD and HDD samples. As expected, we found that the solubility profile of CTR samples is higher in ChIP-seq peaks for open histone marks (H3K27ac and H3K36me3) and lower in constitutive heterochromatin (H3K9me3 peaks) (Fig. 2e). The HDD group showed instead an inverted trend, thus confirming a large-scale chromatin architecture remodeling. Interestingly, the LDD subgroup, despite the limited alterations in chromatin compartmentalization (Fig. 2b, c), showed an intermediate solubility pattern between the other two groups, especially for H3K27ac and H3K9me3 domains.\u003c/p\u003e\n\u003cp\u003eWe next examined the functional effects of chromatin compartment reorganization by matched RNA-seq analysis. Comparing transcription profiles between CTR (n=7) and PCa (n=10) samples uncovered 66 differentially expressed genes (DEGs): 22 up-regulated and 44 down-regulated in PCa with respect to CTR (Fig. 3a). The limited number of DEGs is likely due to the heterogeneity of expression across tumor patients\u0026apos; biopsies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThen, we considered\u0026nbsp;LDD and HDD subtypes separately. We identified only 30 DEGs in LDD comparison to CTR samples: 9 up- and 21 down-regulated. Instead, we found 162 up- and 410 down-regulated genes in HDD compared to CTR samples (Fig. 3a). This suggests that the higher degree of chromatin compartment remodeling is associated with a more extensive gene expression dysregulation.\u0026nbsp;We found 5 DEGs up-regulated and 4 DEGs down-regulated in common among all comparisons of different PCa (Fig. 3b). Seven genes already described in the literature as prostate cancer biomarkers (HPN, BICD1, GOLM1, TARP) and two down-regulated tumor suppressor genes (SERPINB5 and GATA6)\u003csup\u003e19,24\u0026ndash;28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo further dissect the molecular differences between LDD and HDD subtypes, we performed an enrichment analysis for Gene Ontology (GO) annotations on the list of DEGs in HDD vs CTR. We didn\u0026apos;t perform this analysis on LDD vs CTR samples due to the small number of DEGs. Nevertheless, in LDD DEGs, we found the upregulation of two critical genes, PDLIM5 and CAMKK2, involved in cell migration and invasion \u003csup\u003e29,30\u003c/sup\u003e. In the other comparison, we discovered that HDD up-regulated genes are enriched for the \u0026quot;negative regulation of cell motility\u0026quot; class. whereas down-regulated ones include GO classes related to stroma remodeling (Fig. 3c), suggesting decreased functions usually associated with tumor progression. These findings were also confirmed through GeneSet Enrichment Analysis (GSEA) \u003csup\u003e31\u003c/sup\u003e (Extended Data Fig. 5a).\u003c/p\u003e\n\u003cp\u003eWe then applied a complementary approach for the functional analysis of transcriptome profiles for both LDD and HDD comparison to CTR samples. We used PROGENy \u003csup\u003e32\u003c/sup\u003e, a curated signaling pathway compendium derived from perturbation experiments. This compendium includes weights quantifying the response of each gene to pathway perturbations, thus allowing a weighted analysis of transcriptional response to specific signaling cues (see Methods, Extended Data Fig. 5b). We found a positive enrichment for Androgen pathway activity in both LDD and HDD subgroups. Notably, we found a positive enrichment for TGFbeta activity in LDD, whereas it has negative enrichment in the HDD subgroup.\u003c/p\u003e\n\u003cp\u003eThese results demonstrate that the LDD and HDD subgroups are different in their chromatin architecture and gene expression, thus representing previously undescribed PCa patient subtypes. The HDD\u0026nbsp;chromatin reorganization is also reflected in\u0026nbsp;more prominent chromatin remodeling and transcription repression\u0026nbsp;that confer antitumoral effect.\u003c/p\u003e\n\u003cp\u003eThen, we explored the possible connections between epigenetic remodeling and functions, analyzing compartment changes and transcription at the individual patient level. By considering patient-specific DEGs and compartment switches (see methods), we observed that the majority of DEGs (90% for LDD; 70% for HDD) are located in domains not involved in a compartment transition (Fig. 3d). This suggests that, as extensively described in other models \u003csup\u003e33\u0026ndash;35\u003c/sup\u003e, changes in chromatin architecture are not enough to trigger transcriptional dysfunction, but they establish conditions that enable transcriptional deregulation. A higher proportion of DEGs (30%) were found in compartment switch regions for HDD. Within these genes, we identified a trend where up-regulated genes were associated with \u0026quot;B to A\u0026quot; switch regions, while down-regulated genes were associated with \u0026quot;A to B\u0026quot; compartment switch regions (Fig. 3d, e). GO analysis revealed significant terms only for the latter class (973 downregulated DEGs in HDD \u0026ldquo;A to B\u0026rdquo; transitions), with \u0026ldquo;basal cell of prostate epithelium\u0026rdquo; emerging as the most enriched class, alongside terms related to \u0026ldquo;hypoxia\u0026rdquo; and \u0026ldquo;glycolysis\u0026rdquo; (Fig. 3f), confirming an acquired silenced state of tumorigenic genes \u003csup\u003e36\u003c/sup\u003e. Transcription factor enrichment analysis identified the Polycomb protein Suz12 (Fig. 3f), a subunit of the well-known Polycomb repressive Complex 2 (PRC2), as a key regulator of the identified genes. Interestingly, 58 among the 139 enriched Suz12 targets were described overexpressed in diverse phases of PCa progression, and 8 (SEMA5A, RET, PDGFRA, TWIST2, CDH13, NTN1, EGFR, DUOX2) were associated with positive regulation of cell motility (term of biological processes gene-set), suggesting that in HDD the acquired \u0026ldquo;heterochromatin-like\u0026rdquo; state plays a role in constraining cancer phenotypic plasticity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo identify a signature distinguishing HDD vs LDD subtypes, we examined the differentially expressed genes in comparing HDD vs LDD transcriptomic profiles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe identified 101 DEGs: 77 down- and 24 up-regulated genes in HDD with respect to LDD (Fig. 4a). As seen in the\u0026nbsp;HDD vs CTR\u0026nbsp;comparison, we found differential expression of Epithelial-Mesenchymal Transition (EMT) genes (GSEA analysis \u0026ndash;\u0026nbsp;Fig. 4b\u0026nbsp;and\u0026nbsp;Extended Data Fig.\u0026nbsp;5c) as well as differential activity of Androgen and TGFbeta pathways (PROGENy analysis -\u0026nbsp;Extended Data Fig.\u0026nbsp;5d).\u003c/p\u003e\n\u003cp\u003eThen, we asked if our signature of 101 genes could provide prognostic information on patients. Thus, we\u0026nbsp;queried the cancer genome atlas (TCGA) for the RNA-seq data of 499 prostate adenocarcinomas (PRAD)\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e. We defined a quantitative score named PCI (prostate Compartmentalization Index) to summarize the HDD vs LDD transcriptional signature activity for each patient in this cohort (see Methods). We sorted TCGA samples based on the PCI and defined HDD-like and LDD-like patients based on the PCI score indicating a more prevalent HDD (PCI \u0026gt; 0) or LDD (PCI \u0026lt;0) signature, respectively (Fig. 4c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIntriguingly, survival analysis for\u0026nbsp;biochemical recurrence-free status (BCR)\u0026nbsp;indicated that\u0026nbsp;patients belonging to the HDD-like cluster\u0026nbsp;show a better prognosis (Fig. 4d), confirming the \u0026ldquo;protective\u0026rdquo; chromatin compartment alterations described above. This is a remarkable result because the signature was established using biopsies taken at the time of diagnosis, yet it can predict prognosis in more advanced, surgically removed tumor samples from the TCGA.\u003c/p\u003e\n\u003cp\u003eTCGA samples, extracted from prostatectomies are expected to have generally higher cellularity and tumor purity, with respect to needle biopsies. We noted a potential association between\u0026nbsp;HDD-like and LDD-like groups and the TCGA sample\u0026rsquo;s tumor purity scores (Fig. 4c). Nevertheless, multivariate Cox regression analysis, including the tumor purity score in the model confirmed that the HDD-like classification is a valid independent prognostic predictor of biochemical recurrence (Fig. 4e). The HDD-like phenotype is associated with a better outcome (HR=0.5, CI=0.33-0.88, p-value=0.01) on top of the reference clinical features such as pathological (TNM) stage, age at diagnosis and Gleason Score.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe further refined the signature to retain only the genes with expression more significantly associated with good or bad prognosis, finally resulting in the 18 genes refined signature (Extended Data Fig. 6a, b, see Methods). As expected, the reduced 18 genes signature improves the stratification of the TCGA cohort used to refine the signature itself (p-value \u0026lt; 0.0001; Extended Data Fig. 6c), also independently of tumor purity levels (Extended Data Fig.\u0026nbsp;6d, e).\u003c/p\u003e\n\u003cp\u003eWe then validated the clinical relevance of our refined 18 gene signature across multiple independent prostate cancer patients\u0026rsquo; cohorts. We obtained a significant prognostic stratification trend, where HDD-like patients show a better prognosis (Fig. 5a, b, c). These independent cohorts include transcriptomic profiles obtained with RNA-seq and microarrays, thus attesting the applicability of our signature to heterogenous datasets. Overall, our results confirm the validity of chromatin-informed patient stratification and the derived gene expression signature to discriminate patients for prognosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eProstate cancer diagnosis, which relies on the histological examination of multiple biopsies, has revealed extensive heterogeneity and multifocality of PCa \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This procedure cannot predict the 20% of prostate cancer cases that progress to metastases, resulting in a high mortality rate. Importantly, most transcriptome-based analyses, which might capture the phenotypic plasticity, are unable to provide early insights into clinical progression. Thus, there is an urgent need for new biomarkers to refine the stratification of prostate cancer patients.\u003c/p\u003e \u003cp\u003eThe 3D genome architecture in the cell nucleus is crucial for the epigenetic regulation of transcription, with its reorganization frequently linked to neoplastic conditions \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In this work, using 4f-SAMMY-seq and transcriptome analysis on a limited number of cells (10,000\u0026ndash;50,000 cells), we analyzed the epigenome structure and function of PCa patients-derived biopsies to infer their plasticity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe identified two novel subtypes of PCa patients presenting different degrees of chromatin compartments and transcriptional alterations: the HDD and LDD subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Samples with HDD features display a marked chromatin remodeling that promotes transcriptional repression of cancer progression pathways, suggesting that these chromatin changes may counteract the tumor evolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Specifically, by intersecting patient-specific 4f-SAMMY-seq and RNA-seq datasets, we found that in HDD, down-regulated genes located in \u0026ldquo;A to B\u0026rdquo; transitions are directly involved in prostate cancer progression, with \u0026ldquo;basal cell of prostate epithelium\u0026rdquo; being the most enriched class (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Since basal cells possess stemness-like properties and are prevalent in advanced, castration-resistant, and metastatic prostate cancers \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, this geneset down-regulation further suggests that the epigenetic state of HDD patients may restrict the phenotypic plasticity of cancer cells. Furthermore, transcription factor enrichment analysis identified Suz12 as a key regulator of over 10% of all HDD down-regulated genes in \u0026ldquo;A to B\u0026rdquo; regions, suggesting that PRC2, a key complex in prostate cancer progression \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, may mediate this transcriptional repression.\u003c/p\u003e \u003cp\u003eComparing HDD and LDD subtypes, we derived a specific expression signature that characterizes the HDD phenotypic state (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We confirmed that the HDD signature is significantly associated with a more favorable prognosis using four independent cohorts covering more than 900 patients with follow-up clinical annotations (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe epigenetic remodeling of epithelial tumor cells is coupled with the loss of cell identity homeostasis through multiple steps, driven by molecular changes that depend on intrinsic and extrinsic signals \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The tumor microenvironment plays a central role in this process, constantly adapting its stroma cell composition and transcriptional programs to favor or counteract tumor progression \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This is particularly evident for indolent cancers such as the PCa, whose slow progression favors the crosstalk between cancer and neighboring cells. Prostate stromal tissue, constituted by fibroblasts, endothelial cells, smooth muscle cells (SMCs), and immune cells, plays a crucial role in prostate tumorigenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Intriguingly, transcriptional programs can have divergent trajectories in epithelial or stromal cells. This is the case of the androgen receptor (AR) pathway, whose overexpression in epithelial cells is associated with bad prognosis \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e whereas in the stroma it has a protective role by constraining cancer growth \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. One of the significant pathways upregulated in HDD samples is the androgen receptor and, working on bulk prostatic tissue comprising 70\u0026ndash;80% of stromal cells, we speculate that this detected AR up-regulation may be ascribed to the stroma. Further corroborating this hypothesis, the LDD PCa group correlates with a worse prognosis and its signature includes overexpression of TGFbeta that can induce a resistance to androgen deprivation therapy when expressed in the stroma \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo develop a molecular signature exploitable in the clinic, we selected genes with prognostic value, ultimately restricting our chromatin-based RNA signature to 18 genes validated across multiple independent cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Notably, when comparing our HDD signature with the 29 previously identified prostate cancer (PCa) signatures listed in the PCa Database (PCaDB; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinfo.jialab-ucr.org/PCaDB/\u003c/span\u003e\u003cspan address=\"http://bioinfo.jialab-ucr.org/PCaDB/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), only 3 out of the 18 genes were shared, namely ACAP3 e ATG16L2 \u003csup\u003e47\u003c/sup\u003e and SCRIB \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This highlights the uniqueness and potential specificity of our gene signature for early stratification of prostate cancer patients.\u003c/p\u003e \u003cp\u003eIn summary, this study we introduced a novel experimental approach to investigate the epigenomic landscape and 3D chromatin compartmentalization in prostate biopsies. We identified two novel patient subgroups based on their epigenome profiles. The PCa subtype associated with a favorable prognosis exhibits heterochromatin reorganization that represses tumorigenic pathways. We found that the transcriptional signature derived by this chromatin-informed patient stratification constitutes a novel independent prognostic classifier. We validated the signature across multiple independent cohorts, thus confirming its potential for translatability to assess prognosis at the time of diagnosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eProstate tissues cohort, ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur cohort includes chemo-naïve patients followed in the Urology Division of Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico (Milan) who underwent the transrectal ultrasound-guided systematic sampling of prostate tissue (TRUS biopsy). This diagnostic procedure was performed as part of routine clinical management following the detection of abnormal digital rectal examination or an elevated PSA blood level. According to the current standard for the detection of PCa, 14 ultrasound-guided biopsy cores (diagnostic biopsies), seven from each side of the prostate gland, were collected from each patient for the clinical diagnosis. During the same procedure, one additional biopsy core to be used for our research project (research biopsy), was taken from a site directly adjacent to one of the diagnostic biopsies. The institutional ethics committee board of IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico authorized this study (authorization n 1063). All specimens were obtained after the patients had provided written informed consent following the ethical principles of biomedical research on biospecimens. To ensure optimal recovery of living cells, fresh surgical prostate biopsy specimens were placed in ice-cold saline buffer and directly transported to the laboratory within 1 hour from sample collection. Tissue samples were then immediately processed to preserve the intranuclear genomic and protein architectures of\u0026nbsp;cell nuclei. Samples selected for epigenome studies consist of 17 fresh biopsies divided on the bases of the histology and the spatial distribution of the positive cores in two different groups: 10 PCa biopsies\u0026nbsp;from patients with histologically confirmed prostate cancer and 7\u0026nbsp;from patients who had no cancer in any biopsy core. All the clinical data were provided by the\u0026nbsp;Urology division preserving the confidentiality of patient personal data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissues processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biopsy specimen was stored at 4-8°C for maximum 3 hours before dissociation, avoiding its freezing. Given the little size of needle biopsy cores (typically 20-30 mg) we empirically determined the digestion condition to achieve the optimal recovery of living cells. Briefly, the biopsy tissue was transferred in a 2 ml microcentrifuge tube and rinse twice with 1 ml of ice-cold sterile PBS 1X. Then, the tissue was cut into small pieces (~1 mm) with autoclaved surgical scissors directly in the microcentrifuge tube. The resulting minced tissue was enzymatically digested by adding 1 ml of prewarm HBSS (Gibco, 14025) containing 200 units of collagenase type I (Life Technologies, 17018-029) per ~10 mg of tissue plus 67 µg DNase I (Sigma-Aldrich, 10104159001). Tissue dissociation was carried out in a water bath at 37 °C shaking vigorously for 10 seconds every 5 minutes. To prevent tissue over-digestion, the best incubation time for each experiment was established by monitoring cell viability, cell debris and aggregates every 10 minutes (usually ranges from 1 to 1.5 hour). After completed digestion cells were washed once by topping up to 2 ml with RPMI + 10% FBS and spun down at 300 g. The cell pellet was resuspended in RPMI + 10% FBS and dispersed by passing through a 75µm cells strainer, followed by an additional wash of the filter with RPMI + 10% FBS. Finally, the cells were spun down at 300 g and resuspended in 1 ml of ice-cold PBS for counting with a hemocytometer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological and immunofluorescence evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne third portion of each\u0026nbsp;research biopsy tissue specimen was embedded in Killik (Bio-Optica, 05-9801), immediately frozen in precooled isopentane (MilliporeSigma, 277258) and stored at -80°. OCT- embedded biopsy cores (one per patient) were serially sectioned\u0026nbsp;with\u0026nbsp;10 μm thickness\u0026nbsp;in a cryostat at -20°C. Ten\u0026nbsp;slides per patient, containing multiple sections representing distinct regions of the same tissue were prepared in parallel and stored at -80°C. Hematoxylin and\u0026nbsp;Eosin staining (H\u0026amp;E)\u0026nbsp;was performed using H\u0026amp;E Staining Kit (Abcam, ab245880). The\u0026nbsp;H\u0026amp;E-stained\u0026nbsp;slides were reviewed by an expert genitourinary pathologist to assign\u0026nbsp;the Gleason\u0026nbsp;Score according to the International Society of Urinary Pathology grading system. The same pathologist evaluated all specimens (diagnostic and research\u0026nbsp;biopsies) presented in this work.\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence, frozen tissue sections were briefly thawed at room temperature (RT), placed in precooled acetone for 20 minutes at -20°C and washed 3 times in PBS 1X for 2 minutes at RT. To permeabilize tissues the sections were incubated with 0.5% Triton X-100 in PBS 1X for 10 minutes at RT with mild agitation. After three washes in PBS 1X for 2 minutes at RT, coverslips were blocked by incubating with 5% Donkey serum, 3% BSA in PBS 1X for 1 hour at RT. After three washes in PBS 1X for 2 minutes at RT, coverslips were incubated with 1:500 Lamin A/C primary antibody (Santa Cruz sc-6215) 1:500 in blocking solution overnight at 4°C in a humidified chamber, isolating the tissue section by a hydrophobic pen. The following day, sections were washed 3 times in PBS 1X for 2 minutes at RT and incubated with fluorescence-conjugated secondary antibody (Alexa FluorTM 488-conjugated Donkey Anti-goat IgG Invitrogen, A-31571), 1:500 3% BSA in PBS 1X for 1 hour at RT in a dark humidified chamber. After three washes in PBS 1X for 2 minutes at RT, the coverslips were incubated with Hoechst solution (Thermo Fisher Scientific H3570, 1:500) for 10 minutes at RT in the dark, rinsed in PBS 1X and mounted on microscope slides with ProLong antifade mounting media (Thermo Fisher Scientific, P36930).\u003c/p\u003e\n\u003cp\u003eImages were acquired using a Leica TCS SP5 confocal microscope with a HCX Plan Apo ×63/1.40 objective, with a 5-zooming factor. The NIS-Elements v.5.30 software (Nikon-Lim) was employed to analyze nuclei physical parameters including area and circularity. Hoechst staining facilitated nucleus identification and delineation of the region of interest. Over 5 field of views (FOVs) were acquired and analyzed per sample, totaling the examination of more than 70 nuclei within each sample of the cohort (seven non-neoplastic controls, CTR\u0026nbsp;and ten primary prostate cancer patients, PCa). The mean nuclear area or circularity was calculated for each sample and plotted alongside other samples within the same group on the charts. The analysis of Hoechst and Lamin A/C distribution across nuclei was conducted using Fiji software. Leveraging the plot profile plugin, a 10 µm fixed line was implemented to trace and capture the fluorescence intensity distribution across each nucleus. To standardize comparisons between diverse samples, a normalization step was performed. This involved calibrating the fluorescence intensity of individual pixels along the line to the mean intensity derived from the entire set of pixel lines. For each sample the mean distribution values for both Hoechst and Lamin A/C were calculated and graphically represented alongside other samples within the same group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify the relative amounts of cell populations in each biopsy, 10,000 cells from the digestion step were stained, acquired on a BD FACSCantoII Flow Cytometer and analyzed with FlowJo software in the INGM FACS facility. To avoid unspecific binding, antibodies were incubated with PBS-BSA 1% for 30 minutes at 4°C. TO-PRO®-3 stain (Thermofisher, T3605) was used to assess cell viability. Tissue resident leukocytes were identified as CD326-/CD45+ (EpCAM-CD326 FITC, B347197; CD45 Pacific Blu Biolegend, 982306); epithelia cells as CD326+/CD45-, and stromal cells as CD326-/CD45-.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4f-SAMMY-seq isolation of chromatin fractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatin fractionation on tissue-derived single cell suspension was performed with minor adaptations to the protocol described in\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e. Cells were counted, washed in cold PBS and resuspended in 600ul cold cytoskeleton triton buffer, CSK-Tr: 10 mM PIPES pH 6,8; 100 mM NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1X Protease Inhibitor Cocktail (Roche, 04693116001); 1 mM PMSF (Sigma-Aldrich, 93482); 1 mM DTT; 0,5% Triton X-100. After 10 minutes on a rotator at 4°C, samples were centrifugated for 3 minutes at 900g at 4°C and supernatant was collected as S1 fraction. Pellets were washed for 10 minutes on the rotator at 4°C with an additional volume of the same buffer followed by centrifugation for 3 minutes at 900g at 4°C. Chromatin was then digested by using 25 units of DNase I (Invitrogen, AM2222) in 100ul of cytoskeleton CSK buffer: 10 mM PIPES pH 6.8; 100 mM NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1mM PMSF; 1X Protease Inhibitor Cocktail for 60 minutes at 37°C. To stop digestion, ammonium sulphate was added to samples to a final concentration of 250 mM and, after 5 minutes on ice, samples were pelleted at 900g for 3 minutes at 4°C and the supernatant was collected as S2 fraction. After a wash of 10 minutes on a rotator at 4°C with 200ul of CSK buffer followed by centrifugation for 3 minutes at 3000g at 4°C, the pellet was further extracted 10 minutes on a rotator at 4°C in 100ul of CSK-NaCl buffer: 10 mM PIPES pH 6.8; 2 M NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1mM PMSF; 1X Protease Inhibitor Cocktail, centrifuged at 2300 g 3 minutes at 4°C and the supernatant was collected as S3 fraction. Pellets were washed twice for 10 minutes on the rotator at 4°C followed by centrifugation at 3000 g 3 minutes at 4°C with 200ul of CSK-NaCl buffer. Finally, pellets were solubilized in 100ul of 8M urea for 10 minutes at room temperature and labelled as S4 fraction. Fractions were stored at –80°C until DNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4f-SAMMY-seq DNA extraction, library preparation and sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatin fractions (S2, S3 and S4) were diluted 1:2 in 1X TE buffer (10mM TrisHCl pH 8.0, 1 mM EDTA) and incubated with 3 U of RNAse cocktail (Ambion, AM2286) at 37° for 90 minutes, followed by 40μg of Proteinase K (Invitrogen, AM2548) at 55° for 150 minutes. Genomic DNA was then isolated using phenol/chloroform (Sigma-Aldrich, 77617) extraction, followed by a back extraction of phenol/chloroform with additional volume of TE1X. DNA was precipitated in 2 volumes of cold ethanol, 0.3M sodium acetate and 20ug glycogen (Ambion AM9510) for 1 hour on dry ice or overnight at -20°C. Dry pellets were resuspended in 50 ul (S2) or 15 ul (S3 and S4) of nuclease-free water and incubated at 4°C overnight. On the next day, S2 was further purified using PCR DNA Purification Kit (Qiagen, 28106) and separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) with the ratio 0.90/0.95 to obtain smaller fragments conserved as S2S (\u0026lt; 300 bp) and larger fragments labelled as S2L (\u0026gt; 300bp) fractions. Both were resuspended in 20 ul of nuclease-free water and then reduced to 15ul using a centrifugal vacuum concentrator. S2L, S3 and S4 fractions were sonicated in a Covaris M220 focused-ultrasonicator using screw cap microTUBEs (Covaris, 004078) to obtain a smear of DNA fragments peaking at 150-200 bp (water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50, 150 seconds for S2L and 175 seconds for S3 and S4). Fractions were quantified using Qubit 4 fluorometer with Qubit dsDNA HS Assay Kit (Invitrogen, Q32854). Libraries were generated from each sample using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and Unique Dual Index NEBNextMultiplex Oligos for Illumina (NEB, E6440S). Libraries were then qualitatively and quantitatively checked on and run on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent, 5067-4626). Libraries with distinct adapter indexes were then multiplexed and, after cluster generation on FlowCell, sequenced for 50 bases in single or paired-end reads mode on an IlluminaNovaSeq 6000 instrument at the IEO Genomic Unit in Milan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction, library preparation and sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTen thousand cells retrieved from the digestion step were stabilized in 200µl of 1Thioglycerol/Homogenization Solution of the Maxwell® RSC miRNA Tissue Kit (Promega, AS1460) and stored frozen at -80°C for total RNA automated purification using Maxwell® RSC 48 Instrument (Promega, AS8500). Total RNA was quantified by Qubit 4 fluorometer with Qubit RNA HS Assay Kit (Invitrogen, Q32852) and assessed by Agilent 2100 Bioanalyzer using Agilent RNA 6000 Pico Kit (Agilent, 5067-1513) to inspect RNA integrity. For each sample, 1 ng of total RNA was used to construct strand specific RNA-seq libraries with SMARTer Stranded Total RNA-Seq Kit - Pico Input (Takara, 634487). The yield and quality of the libraries were evaluated on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent, 5067-4626). RNA-seq libraries were sequenced on the Illumina NextSeq™ 550 system at the sequencing facilities of Humanitas or Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico in Milan in paired-ends mode.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP-seq public datasets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ChIP-seq data of histone post translational modifications (histone marks) for non-neoplastic prostate gland tissue were downloaded from the following datasets available on ENCODE data portal \u003csup\u003e20\u003c/sup\u003e: ENCSR763IDK (H3K27ac), ENCSR768PFZ (H3K36me3) and ENCSR133QBG (H3K9me3). These data were downloaded as raw high-throughput sequencing reads (FASTQ) and analyzed as described in the next sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP-seq and 4f-SAMMY-seq high-throughput sequencing reads data analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-throughput sequencing reads were trimmed using Trimmomatic (v0.39)\u0026nbsp;\u003csup\u003e51\u003c/sup\u003e with the following parameters for 4f-SAMMY-seq and ChIP-seq: 2 for seed_mismatch, 30 for palindrome_threshold, 10 for simple_threshold, 3 for leading, 3 for trailing and 4:15 for sliding window. The sequence minimum length threshold of 35 was applied to all datasets. We used the Trimmomatic “TruSeq3-SE.fa” (for single-end reads) and “TruSeq3-PE-2.fa” (for paired-end reads) as clip files. After trimming, reads were aligned using BWA (v0.7.17-r1188)\u0026nbsp;\u003csup\u003e52\u003c/sup\u003e setting –k parameter as 2 and using as reference genome the UCSC hg38 genome (only canonical chromosomes were used) and the output saved in BAM file format. We uniformly used and aligned only a single read per DNA fragment for both single-end and paired-end sequencing samples. The PCR duplicates were marked with Picard (v2.22; https://github.com/broadinstitute/picard) MarkDuplicates option, then filtered using Samtools (v1.9)\u0026nbsp;\u003csup\u003e53\u003c/sup\u003e. In addition, we filtered all the reads with mapping quality lower than 1. Each sequencing lane was analyzed separately and then merged at the end of the process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReads distribution profiles analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compute reads distribution profiles (genomic tracks) for individual fractions of 4f-SAMMY-seq, we used Deeptools (v3.4.3)\u0026nbsp;\u003csup\u003e54\u003c/sup\u003e bamCoverage function. For these analyses the genome was binned at 50bp, the reads extended up to 250 bp and Deeptools RPKM normalization method was used. We considered a genome size of 2,701,495,761 bp (value suggested in the Deeptools manual\u0026nbsp;https://deeptools.readthedocs.io/en/latest/content/feature/effectiveGenomeSize.html) and we excluded regions known to be problematic in terms of sequencing reads coverage using the blacklist from the ENCODE portal (https://www.encodeproject.org/files/ENCFF356LFX/).\u003c/p\u003e\n\u003cp\u003eTo compute the genomic tracks for ChIP-seq IP over INPUT enrichment profiles (log\u003csub\u003e2\u003c/sub\u003e normalized ratio) we used the SPP R package (v1.16.0)\u0026nbsp;\u003csup\u003e55\u003c/sup\u003e\u0026nbsp; and R statistical environment (v3.5.2). The reads were imported from the BAM files using the “read.bam.tags” function, then filtered using “remove.local.tag.anomalies” and finally the comparisons were performed using the function “get.smoothed.enrichment.mle” setting “tag.shift = 0” and “background.density.scaling = TRUE”. The resulting enrichment signal corresponds to a log2 normalized ratio between the pair of sequencing samples.\u003c/p\u003e\n\u003cp\u003eFor computing the relative comparisons between two 4f-SAMMY-seq fractions (relative enrichment, \u003cem\u003ei.e.\u003c/em\u003e log\u003csub\u003e2\u003c/sub\u003e normalized ratio) we used the same procedure described above for ChIP-seq IP over INPUT enrichment profiles. We\u0026nbsp;defined the\u0026nbsp;\"solubility profile\" as the relative enrichment ratio of 4f-SAMMY-seq sequencing reads distribution along the genome for S2L vs S3 fractions. We used S3 as baseline in this ratio as it is the second most consistent fraction among controls (Extended Data Fig. 3d).\u003c/p\u003e\n\u003cp\u003eTo compute correlations between genomic tracks, we used R (v3.5.2) base function \"cor\" with “method = Spearman”. The genomic tracks were imported in R using the rtracklayer (v1.42.2)\u0026nbsp;\u003csup\u003e56\u003c/sup\u003e library. Then the original genomic track files with 50bp resolution were re-binned by averaging data at 150kb resolution using the function “tileGenome” and the correlation was computed per chromosome. The correlation values obtained for each chromosome were then summarized in one value describing the genome-wide sample correlations through a weighted mean, where the weight of each chromosome corresponds to its length.\u003c/p\u003e\n\u003cp\u003eOpen histone marks (H3K4me1, H3K36me3) peaks were called with MACS (v2.2.9.1) using a broad-cutoff of 0.1\u0026nbsp;\u003csup\u003e57\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eH3K9me3 peaks were defined using the EDD (v1.1.19)\u0026nbsp;software\u0026nbsp;\u003csup\u003e58\u003c/sup\u003e with parameters (binsize = 150 Kb and gap penalty = 25) processing the filtered bam files obtained as described above. The “required_fraction_of_informative_bins” parameter was set to 0.98. The blacklisted regions were defined as for the reads distribution profile analyses described above.\u003c/p\u003e\n\u003cp\u003eThe enrichment boxplots shown in Fig. 2e was produced retrieving for each histone mark peak the genomic bins that fall in that genomic region. For each group of patients (CTR, LDD and HDD groups) and for each genomic bin we plot the mean 4f-SAMMY-seq solubility profile across the group. The values used were the quantile normalized tracks (across all samples) S2LvsS3 produced using SPP (see “High-throughput sequencing reads analyses”). Genomic tracks obtained by SPP or Deeptools were saved in bigWig file format.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic tracks visualization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe visualization of genomic tracks was performed with Gviz R library (v1.26.5)\u0026nbsp;\u003csup\u003e59\u003c/sup\u003e. The track profile was calculated using the function “DataTrack” (the input bigWig file was imported using the function “import” of the rtracklayer library) and plot using the function “plotTracks”.\u0026nbsp;Extended Data Fig.\u0026nbsp;3e has been created plotting each track as type \"polygon\" and all the tracks have been set using a window of 500 except for ChIP-seq of H3K27ac where the window has been set to 5000. It is worth remarking that the \"window\" parameter in the \"DataTrack\" function is referring to the number of intervals in which the displayed region is divided to plot the profile. As such, a larger number indicate a finer grain definition of the profile. As H3K27ac is known to have sharp peaks, we aimed to plot a finer grain profile. For Fig. 1c the window was set to 1000 and for all the tracks except for ChIP-seq of H3K27ac where we used 5000, \u003cem\u003ei.e.\u003c/em\u003e the same value used for the\u0026nbsp;Extended Data Fig.\u0026nbsp;3e. CTR and PCa samples track overlayed with confidence interval were drawn using at the same the time the types “a” and “confint”. The same settings of histone mark tracks of Fig. 1c were used for Fig. 2b.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin compartment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatin compartments were calculated using a revised version of the CALDER algorithm (version 1.0\u0026nbsp;\u003csup\u003e60\u003c/sup\u003e), as implemented in the original 4f-SAMMY-seq pipeline for the sub-compartment identification\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e. Namely, for each chromosome: i) we calculated the four 4f-SAMMY-seq fractions (S2S, S2L, S3, S4) reads distribution profiles, binned at 150kb and normalized with RPKM (see \"Reads distribution profiles analysis\" section); ii) for each genomic bin, defined as a vector containing the four RPKM values (one for each fraction), we calculated the Euclidean distance (dist, R stats package, method=\"euclidean\") with all the other bins of the same chromosome. These steps produced an NxN matrix (hereinafter referred to as biochemical similarity matrix), where N is the number of bins for the considered chromosome. Starting from the biochemical similarity matrix, we performed the CALDER procedure as described in\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e, to derive the eigenvector and to reconstruct chromatin compartmentalization. Specifically, we called sub-compartments but limiting their segmentation to the highest level, thus obtaining only 2 compartments corresponding to \"A\" and \"B\" compartments (Fig. 2a, b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe consensus of compartment calls across healthy controls has been produced labeling each genomic bin (150kb) according to its more frequent compartment across samples: e.g. if a bin is labeled as A in at least 4 out of 7 CTR samples, that bin will be defined as A in the consensus of controls, and vice versa for bins labelled as B in at least 4 controls (Fig. 2c).\u0026nbsp;The definition of compartment shifts was based on the concordant or discordant compartment classification for each 150kb genomic bin (Extended Data Fig.\u0026nbsp;4a).\u0026nbsp;The compartment domains shared across patients as shown in\u0026nbsp;Extended Data Fig.\u0026nbsp;4a were reported using the library upsetR.\u003c/p\u003e\n\u003cp\u003eThe clustering of patients in Fig. 2d were based on Jaccard Indexes (JI)\u0026nbsp;of compartments concordance for each pair of patients. The resulting matrix of pairwise JI was grouped by hierarchical clustering based on the Euclidean distance (among rows or columns) and complete linkage using the pheatmap function in the pheatmap R library.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell type deconvolution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cell type deconvolution analysis, we fist normalized for each sample raw count to TPM (Transcript per Million) by using R 3.6.1 environment and applying the following steps. First, read counts for each gene were normalized (divided) by the length of the gene in kilobases (RPK, Reads per kilobase). Then RPK values in a sample are summed and divided by 1.000.000 to obtain the scaling factor. Finally, RPK values for each gene are divided by the scaling factor.\u003c/p\u003e\n\u003cp\u003eFor the deconvolution of the 3 macro populations (immune, epithelia, stroma cells) we defined a custom signature matrix based on single cell expression data of prostate tissue from the Human Proten Atlas (https://www.proteinatlas.org/; V. 21.1) \u003csup\u003e61–63\u003c/sup\u003e. Following the cluster classification of Human Protein Atlas we considered as epithelia the subpopulations annotated as prostatic glandular cells, basal prostatic cells or urothelial cells. We considered as stroma the populations annotated as muscle cells, fibroblast or endothelial cells. We considered as immune cells infiltrate the populations annotated as t-cells or macrophages. Finally, we selected as representative genes for each population only those with a TPM \u0026gt; 10 for the specific population and a TPM \u0026lt; 2 in all the two remaining populations. We finally got a macropopulation signature matrix of 702 genes. We run the deconvolution analysis exploiting this signature matrix on our bulk data by using the relative mode of CIBERSORTX\u0026nbsp;(V.1.0) \u003csup\u003e64\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;4f).\u003c/p\u003e\n\u003cp\u003eTo compute the immunity score on our biopsies we used the software CIBERSORTX (V.1.0) \u003csup\u003e64\u003c/sup\u003e applying the previously computed TPM matrix of our bulk RNA (mixture file) on the LM22.txt provided in the CIBERSORTX repository. The LM22 leukocyte signature is based on a matrix of 547 genes discriminating 22 human hematopoietic cell types isolated from PBMC. We enabled batch correction by using the B-mode correction option. We next run the analysis in absolute mode by applying 500 permutations. Each sample in the mixture file gets a score that reflects the absolute proportion of each cell types in LM22 mixture file. We finally computed the absolute leukocyte score: i.e. the sum for each sample of all the 22 scores of leukocytes populations. We compared the absolute leukocyte score for each biopsy across the different groups (CTR, LDD, HDD) (Extended Data Fig.\u0026nbsp;4g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCopy Number Analysis (CNA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor copy number analysis we used paired-end 4f-SAMMY-seq reads by treating fractions from the same sample as independent replicates. Data was processed with the pipeline nf-core/sarek v3.4.0\u0026nbsp;\u003csup\u003e65,66\u003c/sup\u003e of the nf-core collection of workflows\u0026nbsp;\u003csup\u003e67\u003c/sup\u003e, using whole genome sequencing settings, and segmentation was called with CNVkit v0.9.10\u0026nbsp;\u003csup\u003e68\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptomic analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor RNA-seq data, the overall quality of the sequenced reads was assessed using FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) (V. 0.11.8), then reads were trimmed with Trimmomatic software \u003csup\u003e51\u003c/sup\u003e (version 0.39) removing the adapters (ILLUMINACLIP:Picov2smart-PE.fa), primer dimers, and low quality bases at the beginning and at the end of the reads (trimmomatic PE phred33 LEADING:3 TRAILING:3 SLIDINGWINDOWD:4:15 MINLEN:36). STAR \u003csup\u003e69\u003c/sup\u003e (V. 2.7.0f_0328) was used to index (STAR --runMode genomeGenerate) the Human Genome (GENCODE Release 39, GRCh38 primary assembly genome \u003csup\u003e70\u003c/sup\u003e) and to align sequenced reads in paired-end mode (--readFiles R1.FASTQ R2.FASTQ) on the indexed reference genome. Multimapping reads and PCR duplicate were marked in the final output (--bamRemoveDuplicatesType Unique) and unaligned reads stored in a different file (--outReadsUnmapped Fastx).\u003c/p\u003e\n\u003cp\u003eThe read counts on genes were calculated using as a reference a GTF file with RefSeq annotation downloaded from UCSC (http://genome.ucsc.edu)\u0026nbsp;stored in the following directory:(http://hgdownload.soe.ucsc.edu/goldenPath/archive/hg38/ncbiRefSeq/109.20190905/hg38.109.20190905.ncbiRefSeq.gtf.gz). This\u0026nbsp;file was further processed to remove non canonical and mitochondrial chromosomes, selected only curated genes (NM, NR) and finally split in protein coding (NM) and noncoding (NR) files. Reads count was performed with HTSeq-count (V. 0.13.5) on bam files (previously generated by STAR) using as a feature the union of all exons in a gene. The type of library was specified with “-s reverse” parameter. The reads that align to more than one position in the reference genome were discarded (htseq-count --non-unique none). The full matrix with raw read counts for each sample were loaded in R 3.6.1 and normalized using DESeq2 \u003csup\u003e71\u003c/sup\u003e median of ratios. Differential expression analysis was performed with DESeq2V. 1.26 \u003csup\u003e72\u003c/sup\u003e) using Wald test and the Benjamini and Hochberg correction for multiple tests, to compute p-values and adjusted p-values, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnrichment analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUp-regulated genes (with adjusted p-value \u0026lt; 0.05 and Log\u003csub\u003e2\u003c/sub\u003e Fold Change \u0026gt; 1) and Down-regulated genes (with adjusted p-value \u0026lt; 0.05, and Log\u003csub\u003e2\u003c/sub\u003e Fold Change \u0026lt; -1) were used separately to query the gene-list enrichment tool Enrichr (https://maayanlab.cloud/Enrichr/) \u003csup\u003e73–75\u003c/sup\u003e over the main Gene Ontology classes (Biological Process - BP, Molecular Function - MF, Cellular Component - CC) updated to 2023 taking as final enriched terms only those with Benjamini-Hochberg adjusted p-value \u0026lt; 0.05. Genes within the recurrent\u0026nbsp;compartment switch “A to B” and “B to A” were also queried over the main Gene Ontology classes taking also in this case the enriched terms with Benjamini-Hochberg adjusted p-value \u0026lt; 0.05.\u0026nbsp;GSEA analysis \u003csup\u003e31\u003c/sup\u003e was performed in Preranked mode. Starting from raw p-values of all genes we generated a ranked matrix sorted by the score resulting from the following formula: -Log\u003csub\u003e10\u003c/sub\u003e(p-value) multiplied by the sign of the Log\u003csub\u003e2\u003c/sub\u003e Fold Change. For the gene set references we downloaded H (Hallmark) and C5 (Ontology) GMT files from MSigDB. Finally, we performed the analysis using the parameters \"Number of permutations\": 1000; \"Collapse\": No; \"Enrichment Statistic\": classic; \"Max size\": 500; \"Min size\":15. Resulting geneset with a family-wise error rate (FWER) \u0026lt; 0.05 are finally sorted using the Normalized Enrichment Score (NES).\u003c/p\u003e\n\u003cp\u003eWe then inferred pathway activity for the comparison of our tumor subtypes by using the R package decoupleR \u003csup\u003e76\u003c/sup\u003e (V. 2.4). We used the get_progeny (organism=human,top=100) function to get pathways from PROGENy (V.1.20) \u003csup\u003e32\u003c/sup\u003e a curated collection of signaling pathways derived from perturbation experiments,\u0026nbsp;where each gene has a specific weight describing its positive or negative response to a given pathway stimulation. The top 100 responsive genes in the compendium ranked by p-value were used for each pathway. Starting from Wald statistic values previously computed for each gene with DESeq2 (LDDvsCTR, HDDvsCTR and HDDvsLDD comparisons) we run the function decouple with parameters statistics=c(“mlm”,”ulm”,”wsum”) and consensus_score=TRUE, to estimate a consensus score across the top performer methods (according to benchmark done by decoupleR developers) in pathway activity prediction. Multivariate linear model (mlm), Univariate linear model (ulm), Weighted Sum (wsum) are recommended by developers of decoupleR since these methods better estimate pathway activity considering the weights associated with pathway related genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient specific DEGs and Compartment switches\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant Down-regulated genes have been identified using DESeq2 with the same workflow described above (adjusted p-value \u0026lt; 0.05 and absolute Fold Change value \u0026gt; 1), comparing each individual LDD and HDD patients against the group of 7 CTRs. Patients-specific compartment switches (\"A to B\" and \"B to A\") were defined with respect to the\u0026nbsp;consensus of compartment calls across\u0026nbsp;CTRs.\u003c/p\u003e\n\u003cp\u003eTo define number of DEGs included in switch regions for each patient, patient-specific down-regulated DEGs were intersected to patient-specific A to B regions while up-regulated DEGs were intersected to patient-specific B to A regions. Then the gene lists resulting from all the patient-specific intersections of DEGs and compartment switches were merged and the reultin union gene list was used as input for functional classes enrichment analysis with\u0026nbsp;Enrichr (https://maayanlab.cloud/Enrichr/).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTCGA Data\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTCGA-PRAD RNA-Seq expression data were downloaded by using gdc-client tool on gdc_manifest.2022-08-08.txt metafile input downloaded from the GDC portal repository (https://portal.gdc.cancer.gov/; V.34.0; Release July, 27, 2022) by selecting from Web interface the TCGA project, prostate and RNA-seq count. The main clinical parameters (Gleason Score, T stage, N stage and age) and info about the samples (primary tumor, normal biopsy and metastatic) were selected from the same Web section of GDC portal repository by downloading clinical and sample-sheet files. ABSOLUTE tumor purity score \u003csup\u003e77\u003c/sup\u003e was downloaded from https://gdc.cancer.gov/about-data/publications/pancanatlas. The file is TCGA_mastercalls.abs_tables_JSedit.fixed.txt\u0026nbsp;(Downloaded October ,13, 2022)\u0026nbsp;\u003csup\u003e78\u003c/sup\u003e. \u0026nbsp;Excluding primary normal biopsies, we obtained from the TCGA repository described above the 500 FPKMUpperQuartile normalized expression counts from prostate primary tumors. The Biochemical Recurrence Free (BCR) status for each patient was obtained from PCaDB (http://bioinfo.jialab-ucr.org/PCaDB/; Release May, 10 2021; eSet V.1.3), downloading the TCGA-PRAD_eSet.RDS object and applying the pData function from Biobase package. We then excluded the only sample with a Gleason Score pattern of 2+4 never described in literature. Despite we performed expression analyses assigning a PCI score on the entire cohort of 499 samples, the BCR data available are 466.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCI score (Prostate Compartmentalization Index)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStarting from our 101 DEGs of the HDD vs LDD comparison we defined two gene lists with 24 HDD Up-regulated\u0026nbsp;and 77 HDD Down-regulated genes. We subset the TCGA matrix extracting only those genes matching with our full list of 101 genes. We performed a log transformation of TCGA gene expression matrix: Log\u003csub\u003e2\u003c/sub\u003e(matrix +1). The log transformed expression of each gene across the 499 samples was standardized into a z-score. For each sample we computed 2 median values on the z-score transformed matrix: the median of the 24 HDD Up-regulated genes (HDD_UP) and the median of the 77 HDD Down-regulated (HDD_DOWN) genes. Finally, we computed the PCI score defined as the difference of median values:\u0026nbsp;PCI=median(HDD_UP) - median(HDD_DOWN). The resulting PCI score with a positive value will indicate that a sample has HDD-like transcriptomic features, whereas a PCI negative value will classify a sample as LDD-like.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnivariate and multivariate regression analysis were performed in R using survival and survminer packages. First, we performed a log-rank test to compare survival Kaplan-Meier curves (in terms of BCR STATUS AND BCR TIME) between HDD-like and LDD-like samples. Then we performed a multivariate Cox proportional-hazard model including the following covariates: HDD-LDD status (previously assigned according to the PCI score) of the samples, pathological stage, age at diagnosis, Gleason Score and Tumor purity score. For the multivariate Cox regression analysis, the Gleason Score was included as numeric feature by summing the primary and secondary numeric grades, as reported for TCGA samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSignature Refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo refine the 101 gene signature, we applied univariate cox regression analysis on each single gene expression values across the TCGA samples, including in the model the BCR STATUS and BCR TIME. The p-values obtained for each gene are then adjusted with Benjamini-Hochberg. Then we selected only those genes whose expression is associated with Hazar Ratio (HR) \u0026lt; 1 or HR \u0026gt; 1 with a adj.p-value \u0026lt; 0.05. We obtained 21 candidate prognostic genes. Unsupervised clustering of these genes can cluster 2 main groups of samples with HDD-like and LDD-like features with high significance in the prognostic status of the two groups (Extended Data Fig.\u0026nbsp;6a). We further refined the signature by removing 3 genes (NPIPB3, CCNE2, FMO5) that were noisier in the unsupervised clustering heatmap: these are the only genes that cluster in a discordant manner compared to their original association to HDD or LDD phenotype in our dataset (Extended Data Fig.\u0026nbsp;S6a). Finally, we used the resulting refined panel of 18 genes to recompute and validate our PCI score on other independent cohorts of transcriptomic profiles of prostate cancer patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough the PCaDB (http://bioinfo.jialab-ucr.org/PCaDB/ Release May, 10 2021; eSet V.1.3) we downloaded normalized expression matrices and BCR data of three independent datasets of prostate cancer: Emory (GSE54460) \u003csup\u003e79\u003c/sup\u003e, Stockholm (GSE70769) \u003csup\u003e80\u003c/sup\u003e, Belfast (GSE116918) \u003csup\u003e81\u003c/sup\u003e. For each of them we classified samples computing the PCI score based on the refined 18 genes signature.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe high-throughput sequencing data generated for this study are available in the database ArrayExpress with accession number \u0026lsquo;E-MTAB-14226\u0026rsquo;, at the link: https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-14226?key=3d98766f-3ac6-4c07-a4a0-1022bc521bb0. \u0026nbsp;The bioinformatic pipeline along with a step-by-step tutorial explaining how to process 4f-SAMMY-seq data is available on FigShare (https:// doi.org/10.6084/m9.figshare.25437121.v1) and also linked to the Github repository for updates (https://doi.org/10.6084/m9.figshare.25438195).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVR designed and performed experiments as well as data analysis. CP and GL designed performed data analysis. FG and RQ performed immunofluorescence (IF) and AF performed the IF quantification. MM performed CNA analysis. ES and EDPS participated in data analysis. MC acquired and analysed FACS data. VV supervised sequencing experiments. EM, GA, FR and EDL provided patient samples and participated to the data interpretation. MMa performed histopathology evaluation. VR, CP and GL wrote the manuscript. FF and CL conceived the study and wrote the manuscript. All authors approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA patent application is being filed for the signature presented in this manuscript by VR, CP, GL, FF and CL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Maria Vivo, Beatrice Bodega, Marina Lusic, Mattia Forcato, Gioacchino Natoli, Martin Schaefer and all members of our laboratories for critical feedback on earlier versions of the manuscript. We thank Claudio Tripodo (IFOM and University of Palermo) and Giorgia Zadra (CNR) for feedback on early phases of the project. For the sequencing we acknowledge support from the Genomics Unit of the European Institute of Oncology (IEO), Department of Experimental Oncology, Humanitas Sequencing facility and Istituto Nazionale Genetica Molecolare (INGM) (Marco Ghilotti). We thank Ilaria Rancati (IFOM cell culture facility) for support with Maxwell instrument. This work was supported by Interomics Flagship Project (CNR) and MFAG (grant #18535) to CL; AIRC Start-Up (grant #16841) to FF; FRRB INTERSTRAT-CAD (grant #CP2_14/2018) to FF; AIRC fellowships n. 26942 to FG and No. 22351 to ES; PIR01_00011 \u0026ldquo;IBISCo\u0026rdquo;, PON 2014-2020 to MM. Schematic representations of Figs 1a, b and Extended Data Fig. 3a have been created using BioRender.com.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThe Global Cancer Observatory (GCO) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gco.iarc.fr/en\u003c/span\u003e\u003cspan address=\"https://gco.iarc.fr/en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMottet N et al (2017) EAU-ESTRO-SIOG Guidelines on Prostate Cancer. 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F1000Res 9:63Sarek\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEwels PA et al (2020) The nf-core framework for community-curated bioinformatics pipelines. \u003cem\u003eNature biotechnology\u003c/em\u003e vol. 38 276\u0026ndash;278 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41587-020-0439-x\u003c/span\u003e\u003cspan address=\"10.1038/s41587-020-0439-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalevich E, Shain AH, Botton T, Bastian BC (2016) CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing. PLoS Comput Biol 12:e1004873\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDobin A et al (2013) STAR: ultrafast universal RNA-seq aligner. 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BMC Bioinformatics 14:128\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuleshov MV et al (2016) Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res 44:W90\u0026ndash;W97\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadia-I-Mompel P et al (2022) decoupleR: ensemble of computational methods to infer biological activities from omics data. Bioinf Adv 2:vbac016\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarter SL et al (2012) Absolute quantification of somatic DNA alterations in human cancer. Nat Biotechnol 30:413\u0026ndash;421\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor AM et al (2018) Genomic and Functional Approaches to Understanding Cancer Aneuploidy. Cancer Cell 33:676\u0026ndash;689e3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLong Q et al (2014) Global transcriptome analysis of formalin-fixed prostate cancer specimens identifies biomarkers of disease recurrence. Cancer Res 74:3228\u0026ndash;3237\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoss-Adams H et al (2015) Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study. EBioMedicine 2:1133\u0026ndash;1144\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain S et al (2018) Validation of a Metastatic Assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy. Ann Oncol 29:215\u0026ndash;222\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Clinicopathological characteristics of the study participants undergoing prostate biopsy\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eHistology (GS-P)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eAge at time of biopsy (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003ePSA level (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003ePositive cores (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eClinical stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003ePI-RADS \u003csup\u003e49\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003eD\u0026rsquo;Amico risk classification \u003csup\u003e50\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eCTR 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eCTR 31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e7.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eCTR 32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e3.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eCTR 91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e8.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eCTR 92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eCTR 94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eCTR 98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e3+4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e14.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT1c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e1 (Intermediate risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e3+4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e21.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT1c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e2 (Intermediate risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4+3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e21.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT1c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e2 (Intermediate risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4+3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e28.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT1c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n 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12.4803%;\"\u003e\n \u003cp\u003ePCa 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4+5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e42.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT2b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e9 (High risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4+5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e21.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT2a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e8 (High risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4+5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e487\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e42.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e12 (High risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4+5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e42.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e12 (High risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003ePCa 33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e5+5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.9005%;\"\u003e\n \u003cp\u003e21.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.4803%;\"\u003e\n \u003cp\u003eT1c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.00474%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.4329%;\"\u003e\n \u003cp\u003e8 (High risk)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eGS-P: Gleason Score for patients\u0026rsquo; diagnosis\u003c/p\u003e\n\u003cp\u003ePSA: Prostate Specific Antigen\u003c/p\u003e\n\u003cp\u003ePI-RADS: Prostate Imaging Reporting and Data System\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chromatin remodeling, PCa subtypes, heterochromatin, biopsies, prostate cancer prognosis","lastPublishedDoi":"10.21203/rs.3.rs-5219856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5219856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrimary prostate cancer (PCa) is characterized by multifocal growth and a highly variable clinical course, which is not effectively predicted by prognostic screenings. Innovative strategies for the stratification of primary prostate cancers are still needed. Using prostate biopsies, we analyzed the epigenome of 17 chemo-na\u0026iuml;ve patients with putative PCa for genome-wide mapping of heterochromatic and euchromatic domains, as well as their three-dimensional (3D) compartmentalization in the cell nucleus. We identified two subgroups of cancer patients with different degrees of chromatin 3D architecture and transcriptome alterations: the LDD (Low Degree of Decompartmentalization) and HDD (High Degree of Decompartmentalization) groups. HDD subtype exhibits an extensive chromatin reorganization that restrains tumor potential, by repressing pathways related to extracellular matrix remodeling and phenotypic plasticity. We derived an 18-genes transcriptional signature that distinguishes HDD from LDD subtype and we confirmed its prognostic relevance across multiple cohorts covering more than 900 prostate cancer patients in total. We propose this transcriptional signature derived from chromatin compartmentalization analysis as a novel prognostic tool that could be adopted at the time of the diagnostic prostate biopsy.\u003c/p\u003e","manuscriptTitle":"Chromatin remodeling restraints oncogenic functions in prostate cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-24 07:31:01","doi":"10.21203/rs.3.rs-5219856/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c83e23b6-de77-4213-aab5-a97e25a043c2","owner":[],"postedDate":"October 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39293230,"name":"Biological sciences/Genetics/Epigenetics/Gene silencing"},{"id":39293231,"name":"Health sciences/Biomarkers/Prognostic markers"}],"tags":[],"updatedAt":"2025-10-17T07:10:04+00:00","versionOfRecord":{"articleIdentity":"rs-5219856","link":"https://doi.org/10.1038/s41467-025-64213-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-10-16 04:00:00","publishedOnDateReadable":"October 16th, 2025"},"versionCreatedAt":"2024-10-24 07:31:01","video":"","vorDoi":"10.1038/s41467-025-64213-4","vorDoiUrl":"https://doi.org/10.1038/s41467-025-64213-4","workflowStages":[]},"version":"v1","identity":"rs-5219856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5219856","identity":"rs-5219856","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}