Heterogeneity Of Prostate Cancer Research Articles

Lineage Plasticity and Stemness Phenotypes in Prostate Cancer: Harnessing the Power of Integrated "Omics" Approaches to Explore Measurable Metrics.

Prostate cancer (PCa), the most frequent and second most lethal cancer type in men in developed countries, is a highly heterogeneous disease. PCa heterogeneity, therapy resistance, stemness, and lethal progression have been attributed to lineage plasticity, which refers to the ability of neoplastic cells to undergo phenotypic changes under microenvironmental pressures by switching between developmental cell states. What remains to be elucidated is how to identify measurements of lineage plasticity, how to implement them to inform preclinical and clinical research, and, further, how to classify patients and inform therapeutic strategies in the clinic. Recent research has highlighted the crucial role of next-generation sequencing technologies in identifying potential biomarkers associated with lineage plasticity. Here, we review the genomic, transcriptomic, and epigenetic events that have been described in PCa and highlight those with significance for lineage plasticity. We further focus on their relevance in PCa research and their benefits in PCa patient classification. Finally, we explore ways in which bioinformatic analyses can be used to determine lineage plasticity based on large omics analyses and algorithms that can shed light on upstream and downstream events. Most importantly, an integrated multiomics approach may soon allow for the identification of a lineage plasticity signature, which would revolutionize the molecular classification of PCa patients.

Dissecting the effects of METTL3 on alternative splicing in prostate cancer.

Although the role of METTL3 has been extensively studied in many cancers, its role in isoform switching in prostate cancer (PCa) has been poorly explored. To investigate its role, we applied standard RNA-sequencing and long-read direct RNA-sequencing from Oxford Nanopore to examine how METTL3 affects alternative splicing (AS) in two PCa cell lines. By dissecting genome-wide METTL3-regulated AS events, we noted that two PCa cell lines (representing two different PCa subtypes, androgen-sensitive or resistant) behave differently in exon skipping and intron retention events following METTL3 depletion, suggesting AS heterogeneity in PCa. Moreover, we revealed that METTL3-regulated AS is dependent on N6-methyladenosine (m6A) and distinct splicing factors. Analysis of the AS landscape also revealed cell type specific AS signatures for some genes (e.g., MKNK2) involved in key functions in PCa tumorigenesis. Finally, we also validated the clinical relevance of MKNK2 AS events in PCa patients and pointed to the possible regulatory mechanism related to m6A in the exon14a/b region and SRSF1. Overall, we characterize the role of METTL3 in regulating PCa-associated AS programs, expand the role of METTL3 in tumorigenesis, and suggest that MKNK2 AS events may serve as a new potential prognostic biomarker.

Loss of SYNCRIP unleashes APOBEC-driven mutagenesis, tumor heterogeneity, and AR-targeted therapy resistance in prostate cancer.

Tumor mutational burden and heterogeneity has been suggested to fuel resistance to many targeted therapies. The cytosine deaminase APOBEC proteins have been implicated in the mutational signatures of more than 70% of human cancers. However, the mechanism underlying how cancer cells hijack the APOBEC mediated mutagenesis machinery to promote tumor heterogeneity, and thereby foster therapy resistance remains unclear. We identify SYNCRIP as an endogenous molecular brake which suppresses APOBEC-driven mutagenesis in prostate cancer (PCa). Overactivated APOBEC3B, in SYNCRIP-deficient PCa cells, is a key mutator, representing the molecular source of driver mutations in some frequently mutated genes in PCa, including FOXA1, EP300. Functional screening identifies eight crucial drivers for androgen receptor (AR)-targeted therapy resistance in PCa that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3, and AR. These results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring AR-targeted therapy resistance in PCa.

Abstract A043: Identifying tumor heterogeneity and drug sensitivity in primary prostate cancer towards personalized medicine

Abstract Introduction: Prostate cancer (PCa) is a highly heterogeneous disease with multiple and distinct cancer foci within the same prostate gland. We hypothesize that heterogeneous lesions, may be linked to different functional properties e.g. differential drug response sensitivity. Here we investigate which histopathological and molecular properties are associated with ability to maintain PCa cells as ex vivo organoid cultures and possibly specific drug responses. Methods: To simultaneously assess the histopathology, genetic profile and organoid drug sensitivity, we employed mirror biopsies for FFPE and cell preparations. Four cores of prostate tissue from radical prostatectomies (N=13) were used for generating patient-derived organoids (PDOs). Targeted genomic sequencing was performed on PDOs and their parental FFPE tissues. Drug response was evaluated by ATP-based viability assay after treatment with standard-of-care (AR inhibitors), and approved compounds for other malignancies (DNA synthesis, receptor tyrosine kinase (RTK) and mTOR inhibitors). RNA sequencing was performed on the parental cores. Associations among mutations, transcriptomic profile and PDO responses were being further explored using machine learning (ML) algorithms towards drug response prediction. Results: Histologically heterogeneous cores with different Gleason Score (GS range 6 to 9) per prostate were found in 76% of cases (N=10/13). A fraction of 78% of tumor-containing cores (N=22/28) and 66% of benign cores (N=16/24) gave rise to PDOs, showing a high formation efficiency. PCa somatic mutations were found in 19/32 cores, 11 cores of which shared mutations with their matching PDOs. The transcriptomic profile of each case was overall preserved among different cores. Drug responses in PDOs revealed not only inter- but also intra-patient heterogeneity with only 2 patient cases (N=2/13) having positive correlation within the different cores of the same case (Pearson’s R =0.58, 0.72 based on z-score viability). Instead, RTK inhibitors, Crizotinib and Ponatinib, showed high efficacy in the majority of cases (p<0.01 over vehicle, N=29/32 and N=30/32, respectively). Statistically significant correlations were found among 8 genomic mutations (mainly FGFR1, JAK2 and cell cycle members) and sensitivity to 5 drug compounds. Different features were tested for ML model, with RNA-seq outperforming the rest features; on average 260-1800 genes per drugs were selected for the drug prediction. ML linear regression models showed high Pearson’s correlation (R>0.8) between predicted and observed drug response values in 13/13 patients and 10/11 compounds tested. Conclusions: The majority of primary PCa cases showed genetic and histopathological multifocality, representing a highly heterogeneous cohort. PDOs recapitulated the genetic signature of the parental tissues. Drug responses of PDOs did not follow patient-specific patterns but rather revealed high inter- and intra-patient heterogeneity. Therapy response could be predicted based on transcriptomic profile for personalized treatment decision. Citation Format: Juening Kang, Sofia Karkampouna, Katja Ovchinnikova, Panagiotis Chouvardas, George Thalmann, Marianna Kruithof-de Julio. Identifying tumor heterogeneity and drug sensitivity in primary prostate cancer towards personalized medicine [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A043.

Abstract A071: SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Currently, the development of efficacious agents against metastatic castration resistance prostate cancer is hampered by the lack of preclinical models that accurately reflect disease heterogeneity and treatment response. To overcome this barrier, we have developed a preclinical metastasis model using LuCap136 (PDM136), an androgen receptor (AR) positive patient-derived xenograft (PDX) that forms metastases in bone and soft tissues 3 weeks after intracardiac injection. With a combined PTEN −/−, TP53 −/mut, RB1 −/− genotype, PDM136 represents a subgroup of difficult-to-treat mPCRC. PDM136 is organotropic for bone. Mice usually reach the end point after 6-7 weeks due to paraplegia and/or cachexia. Metastatic cells maintain the expression of nuclear AR and epithelial marker cytokeratin 8. Surgical castration significantly inhibits metastatic progression and prolonged the survival rate, however, tumor will eventually progress to castration resistance. To our knowledge, PDM136 is the first AR-positive prostate cancer metastasis model that recapitulates the disease progress of mCRPC. To define the molecular mechanisms of castration resistance in PDM136, we performed RNA expression profiling on bone and soft tissue metastasis collected from intact and castrated mice. Differential expression analysis revealed over 500 transcripts were significantly altered after castration, with SOX2 and ASCL1 among the top up-regulated genes. Gene set enrichment analysis showed decreased AR signaling and increased neuroepithelium transformation in the castrated group. To validate the RNA-seq data, we performed IHC on proteins whose genes were identified by RNAseq including SOX2, ASCL1, as well as androgen receptor (AR), glucocorticoid receptor (GR) and neuroendocrine marker SYP. Surprisingly, we found that the expression of the five markers is organ-specific upon castration. Unsupervised clustering of the IHC stained markers from the bone of castrated mice revealed that AR and GR were undetectable in majority of foci whereas substantial numbers of foci in soft tissue metastasis expressed AR and GR at moderate intensity. Furthermore, we found that AR and GR expression were mutually exclusive in many foci from soft tissue metastasis. SOX2 and ASCL1 were mainly expressed in AR negative tumors, while SYP was expressed in a subpopulation of SOX2+ positive foci. Notably, liver metastasis had the most SYP expressing foci among all organs suggesting liver microenvironment promotes neuroendocrine differentiation. In summary, our IHC results revealed inter-tumor and organ-specific cellular heterogeneity induced by castration. PDM136 is a platform to understand the mechanism of responsible for mCRPC, but also a valuable tool for anti-mCRPC drug discovery. The co-existence of multiple phenotypes indicated that combination therapies targeting multiple pathways are essential to harness this disease. Citation Format: Anson Ku, Juan Yin, Adam G. Sowalsky, Kathleen Kelly. SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A071.

Abstract A049: Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer

Abstract Introduction: ETS gene rearrangements involving ERG, ETV1 and ETV4, as well as PTEN deletions are among the most common genomic alterations in prostate cancer. Although these events are found to be mutually exclusive, the multifocality of prostate cancer suggests that they may co-occur in some tumors. The immunohistochemistry (IHC) detection of both ERG overexpression and PTEN protein loss are reliable substitutes for fluorescence in situ hybridization (FISH) assay to probe genomic alterations. The lack of specific antibodies that can discriminate between multiple ETS factors, however, has limited the evaluation for the co-occurrence of these genomic alterations in prostate cancer. Improving our ability to identify the multifocality or inter-focal heterogeneity of tumors can enhance our understanding of biological mechanisms of tumor initiation and progression, and help stratify patients for treatment. Methods: Using monoclonal antibodies (mAbs) that we developed against ETV1 and ETV4, we examined their expression in relation to ERG and PTEN expression by IHC using two TMAs constructed from primary prostate cancer specimens of independent patient cohorts: one was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men; the other, from a single 2 mm core of individual tumors from a cohort of 152 AA and 304 CA men. Positive expression for each ETS factor and PTEN were evaluated for association with race and clinico-pathologic features that include PSA at diagnosis, Gleason score, pathologic T stage, biochemical recurrence, and metastasis. Univariate analysis of BCR free survival as a function of ERG, ETV1 and ETV4 and PTEN expression was performed for both patient cohorts. Results: ERG(+) expression alone or in combination with PTEN loss were significantly higher in CA men in both patient cohorts. When expression of ETS factors were evaluated together with PTEN status, positive expression of ETS factors and PTEN loss were significantly more prevalent among CA men in both patient cohorts ETV1 and ETV4 expression were detected at 5% or less in both patient cohorts. Cases harboring tumor foci that were positive for more than one marker evaluated frequently showed either intra-tumor or inter-tumor heterogeneity. Univariate analysis of PTEN-ETS protein expression status and BCR free survival showed that patients with loss of PTEN expression trends towards poorer BCR free survival regardless of ETS expression status. Conclusion: We showed that the IHC detection of multiple aberrantly expressed proteins such as ERG, ETV1, ETV4 and PTEN in prostate tumor specimens using highly specific mAbs can reveal the heterogeneity and multi-focality in prostate cancer, which could be used to enhance the stratification of patients for novel treatment strategies. Citation Format: Shyh-Han Tan, Cara Schafer, Denise Young, Jiji Jiang, Yingjie Song, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn. Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A049.

Abstract A057: Microscale analysis of histone modifications in rare cell populations in prostate cancer

Abstract Pre-clinical models have identified diverse epigenetic alterations in prostate cancer that associate with treatment resistance and may also be therapeutic targets. However, these assays typically require thousands of cells and are difficult to scale for patient tumor biopsies. This report seeks to adapt the Cleavage Under Targets and Tagmentation (CUT&Tag) assay for use on a microfluidic technology to eliminate cell loss associated with complex, multi-step epigenetic assays to facilitate analysis of low input primary tumor samples. Exclusive Liquid Repellant (ELR) platforms pass magnetic bead bound analytes through immiscible phases to separate and extract analytes of interest while eliminating traditional centrifugation and wash steps that lead to sample loss. This platform enables adaptation of complex molecular assays such as CUT&Tag for lossless sample manipulation. We focus on the analysis of histone modifications in rare cells utilizing antibodies for H3K27 acetylation and methylation. Comparison of prostate cancer cell lines with LNCaP, V16D, 49F, 42D, and H660s with serial dilutions was performed to evaluate the sensitivity and specificity of this integrated assay. The CUT&Tag assay was performed in standard fashion on a serial dilution of 50-100,000 cells from LNCaP and V16D calls and showed dropout of recoverable DNA at 50 cells. Sequencing of DNA at these dilutions confirmed identification of differential histone modifications consistent with previously published data. CUT&Tag was adapted to ELR through the serial patterning of each buffer in distinct wells connected by an oil interface. In order to optimize the assay for reproducibility and increased sensitivity, we optimized antibody binding/washing to minimize the protein A-Transposase inhibition. This optimization showed increased antibody binding by 36% compared to protocol defined concentrations. We have now successfully performed this assay with a sensitivity of ~100 cells with recoverable DNA for sequencing to identify heterogenous histone patterns in each cell line. CUT&Tag has improved sensitivity to detect epigenetic alterations that associate with aggressive prostate cancer. The integration of CUT&Tag with ELR improves both sensitivity and reproducibility for epigenetic analysis at low input cell numbers. This assay is now being utilized to analyze patient tumor samples and prostate cancer heterogeneity. Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Jamie M. Sperger, Marina N. Sharifi, Chao Li, Duane S. Juang, Cole S. Gilsdorf, Shuang G. Zhao, David J. Beebe, Joshua M. Lang. Microscale analysis of histone modifications in rare cell populations in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A057.

Editorial Comment.

No AccessJournal of UrologyTranslational Research Article26 May 2023Editorial CommentThis article comments on the following:Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance Udit Singhal and Simpa S. Salami Udit SinghalUdit Singhal Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan Department of Urology, Mayo Clinic, Rochester, Minnesota More articles by this author and Simpa S. SalamiSimpa S. Salami Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003507.01AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookLinked InTwitterEmail "Editorial Comment." The Journal of Urology, Publish Ahead of Print, pp. REFERENCES 1. Molecular biomarkers in localized prostate cancer: ASCO guideline. J Clin Oncol. 2020; 38:1474-1494. Crossref, Medline, Google Scholar 2. Transcriptomic heterogeneity in multifocal prostate cancer. JCI Insight. 2018; 3(21):e123468. Crossref, Medline, Google Scholar 3. Multimodal biomarkers that predict the presence of Gleason pattern 4: potential impact for active surveillance. J Urol. 2023; 210(2):000. Google Scholar 4. . EDRN Prostate MRI Biomarker Study (P-MRI); 2023. https://ClinicalTrials.gov/show/NCT03784924. Google Scholar Submitted April 19, 2022; accepted April 20, 2023; published 000. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsRelated articlesJournal of Urology1 May 2023Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Udit Singhal Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan Department of Urology, Mayo Clinic, Rochester, Minnesota More articles by this author Simpa S. Salami Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan More articles by this author Expand All Submitted April 19, 2022; accepted April 20, 2023; published 000. Advertisement PDF downloadLoading ...

Transcriptome Profiling of Prostate Cancer, Considering Risk Groups and the TMPRSS2-ERG Molecular Subtype.

Molecular heterogeneity in prostate cancer (PCa) is one of the key reasons underlying the differing likelihoods of recurrence after surgical treatment in individual patients of the same clinical category. In this study, we performed RNA-Seq profiling of 58 localized PCa and 43 locally advanced PCa tissue samples obtained as a result of radical prostatectomy on a cohort of Russian patients. Based on bioinformatics analysis, we examined features of the transcriptome profiles within the high-risk group, including within the most commonly represented molecular subtype, TMPRSS2-ERG. The most significantly affected biological processes in the samples were also identified, so that they may be further studied in the search for new potential therapeutic targets for the categories of PCa under consideration. The highest predictive potential was found with the EEF1A1P5, RPLP0P6, ZNF483, CIBAR1, HECTD2, OGN, and CLIC4 genes. We also reviewed the main transcriptome changes in the groups at intermediate risk of PCa-Gleason Score 7 (groups 2 and 3 according to the ISUP classification)-on the basis of which the LPL, MYC, and TWIST1 genes were identified as promising additional prognostic markers, the statistical significance of which was confirmed using qPCR validation.

Visualization of Intermetastatic Heterogeneity in Mixed Neuroendocrine Carcinoma-Acinar Adenocarcinoma of the Prostate by 68Ga-PSMA, 68Ga-FAPI, and 18F-FDG PET/CT.

Mixed neuroendocrine carcinoma-acinar carcinoma is an uncommon histological type of neuroendocrine prostate cancer. It has been rarely reported in de novo prostate malignancies. In this case, we present 68Ga-PSMA (prostate-specific membrane antigen), 68Ga-FAPI, and 18F-FDG PET/CT findings in the de novo form of mixed large-cell neuroendocrine carcinoma-acinar adenocarcinoma of the prostate. Different levels of radiotracer uptake were observed in different metastatic sites on 68Ga-PSMA, 68Ga-FAPI, and 18F-FDG PET/CT. This case demonstrates that the multitracer PET/CT strategy may be used for the noninvasive detection of the intermetastatic heterogeneity in metastatic neuroendocrine prostate cancer.

Multi-omics analysis reveals a macrophage-related marker gene signature for prognostic prediction, immune landscape, genomic heterogeneity, and drug choices in prostate cancer.

Macrophages are components of the innate immune system and can play an anti-tumor or pro-tumor role in the tumor microenvironment owing to their high heterogeneity and plasticity. Meanwhile, prostate cancer (PCa) is an immune-sensitive tumor, making it essential to investigate the value of macrophage-associated networks in its prognosis and treatment. Macrophage-related marker genes (MRMGs) were identified through the comprehensive analysis of single-cell sequencing data from GSE141445 and the impact of macrophages on PCa was evaluated using consensus clustering of MRMGs in the TCGA database. Subsequently, a macrophage-related marker gene prognostic signature (MRMGPS) was constructed by LASSO-Cox regression analysis and grouped based on the median risk score. The predictive ability of MRMGPS was verified by experiments, survival analysis, and nomogram in the TCGA cohort and GEO-Merged cohort. Additionally, immune landscape, genomic heterogeneity, tumor stemness, drug sensitivity, and molecular docking were conducted to explore the relationship between MRMGPS and the tumor immune microenvironment, therapeutic response, and drug selection. We identified 307 MRMGs and verified that macrophages had a strong influence on the development and progression of PCa. Furthermore, we showed that the MRMGPS constructed with 9 genes and the predictive nomogram had excellent predictive ability in both the TCGA and GEO-Merged cohorts. More importantly, we also found the close relationship between MRMGPS and the tumor immune microenvironment, therapeutic response, and drug selection by multi-omics analysis. Our study reveals the application value of MRMGPS in predicting the prognosis of PCa patients. It also provides a novel perspective and theoretical basis for immune research and drug choices for PCa.

Reversible epigenetic alterations mediate PSMA expression heterogeneity in advanced metastatic prostate cancer.

Prostate-specific membrane antigen (PSMA) is an important cell surface target in prostate cancer. There are limited data on the heterogeneity of PSMA tissue expression in metastatic castration-resistant prostate cancer (mCRPC). Furthermore, the mechanisms regulating PSMA expression (encoded by the FOLH1 gene) are not well understood. Here, we demonstrate that PSMA expression is heterogeneous across different metastatic sites and molecular subtypes of mCRPC. In a rapid autopsy cohort in which multiple metastatic sites per patient were sampled, we found that 13 of 52 (25%) cases had no detectable PSMA and 23 of 52 (44%) cases showed heterogeneous PSMA expression across individual metastases, with 33 (63%) cases harboring at least 1 PSMA-negative site. PSMA-negative tumors displayed distinct transcriptional profiles with expression of druggable targets such as MUC1. Loss of PSMA was associated with epigenetic changes of the FOLH1 locus, including gain of CpG methylation and loss of histone 3 lysine 27 (H3K27) acetylation. Treatment with histone deacetylase (HDAC) inhibitors reversed this epigenetic repression and restored PSMA expression in vitro and in vivo. Collectively, these data provide insights into the expression patterns and regulation of PSMA in mCRPC and suggest that epigenetic therapies - in particular, HDAC inhibitors - can be used to augment PSMA levels.

Landscape of prostate-specific membrane antigen heterogeneity and regulation in AR-positive and AR-negative metastatic prostate cancer.

Tumor expression of prostate-specific membrane antigen (PSMA) is lost in 15-20% of men with castration-resistant prostate cancer (CRPC), yet the underlying mechanisms remain poorly defined. In androgen receptor (AR)-positive CRPC, we observed lower PSMA expression in liver lesions versus other sites, suggesting a role of the microenvironment in modulating PSMA. PSMA suppression was associated with promoter histone 3 lysine 27 methylation and higher levels of neutral amino acid transporters, correlating with 18F-fluciclovine uptake on positron emission tomography imaging. While PSMA is regulated by AR, we identified a subset of AR-negative CRPC with high PSMA. HOXB13 and AR co-occupancy at the PSMA enhancer and knockout models point to HOXB13 as an upstream regulator of PSMA in AR-positive and AR-negative prostate cancer. These data demonstrate how PSMA expression is differentially regulated across metastatic lesions and in the context of the AR, which may inform selection for PSMA-targeted therapies and development of complementary biomarkers.

Abstract 3370: Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer

Abstract Aggressive variants of prostate cancer (AVPC) emerge from prostate adenocarcinoma following enduring therapeutic pressure. They grow independent of the androgen receptor, progress rapidly and can show traits of neuroendocrine (NE) differentiation. As progression to AVPC is not well detected by prostate specific antigen monitoring, new biomarkers are urgently needed. The aim of this study is to evaluate the detection of circulating tumor cells (CTCs) in combination with gene expression analysis of NE genes to identify AVPC patients with neuroendocrine traits. Blood samples were collected from patients suffering from AVPC (n=78, including 25 patients with histological evidence of NEPC) as well as from patients with hormone-sensitive prostate cancer (HSPC) as controls (n=12) at the University Medical Center Hamburg-Eppendorf. CTC counts were determined by CellSearch analysis. In parallel, CTCs were immuno-magnetically enriched using the AdnaTest and bulk gene expression of prostate-specific and NE genes was analyzed by semi-quantitative PCR. From a subset of patients, CTCs were additionally enriched by Parsortix to compare gene expression of CTCs with EpCAM-based or size-based CTC enrichment. Using CellSearch analysis, CTCs were found in 88.9 % of AVPC patients. CTC counts ranged between 0 – 20,000 CTCs/7.5 ml of blood with a median of 32 CTCs/7.5 ml. Similarly, 81.8 % of NEPC patients were positive for CTCs with a median count of 35 CTCs/7.5 ml (range 0-13,000). In contrast, CTCs were found in only 50 % of HSPC patients at lower concentrations (median count of 0.5 CTCs/7.5 ml, range 0-34). CTC counts were significantly elevated in AVPC and NEPC patients compared to HSPC (p = 0.002, p = 0.012), but no significant differences were found between AVPC and NEPC samples.In 93.8 % of AVPC samples, CTCs expressed prostate-specific genes such as KLK3 or PSMA. At least one of the NE transcripts was detected in 37.5 % of AVPC patients. In CTCs from NEPC patients, the positivity for prostate markers was significantly reduced to 66.6 % (p = 0.01) and the positivity for NE markers was increased to 61.1 %. A random Forrest model trained on all analyzed transcripts allowed for a distinction of HSPC and NEPC with an AUC of 79.5 %. Comparison of CTC enrichment strategies in a subset of 13 patients showed similar NE marker detection by size-based enrichment (53.8 % NE-positive) compared to the AdnaTest (61.5 % NE-positive). AVPC and NEPC patients show a high CTC burden that will allow subsequent molecular analyses, and CTC counts allowed a distinction between blood samples from HSPC and NEPC patients. Gene expression analysis revealed a high degree of inter-patient heterogeneity for neuroendocrine-specific transcripts with a reduction of prostate markers in NEPC patients. Future molecular characterization and longitudinal monitoring of CTCs will shed more light on the evolution of AVPC and NEPC. Citation Format: Lina Merkens, Sarah Greimeier, Sabine Riethdorf, Klaus Pantel, Gunhild von Amsberg, Stefan Werner. Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3370.

Abstract 3891: Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed cancer among American men, which has traditionally been treated through hormone therapy. However, after hormone therapy many patients with PCa still develop a more aggressive stage of PCa, called metastasis castration resistant prostate cancer (mCRPC). The second- generation antiandrogens, such as enzalutamide or apalutamide, are used to competitively inhibit the androgen receptor (AR) signaling and achieved great clinical success. However, most of the mCRPC patients would develop resistance to the targeted therapy drugs within 6 months to 2 years after initial administration. Consequently, understanding the molecular mechanism of antiandrogen resistance, has become a critical endeavor to provide a greater benefit to patients. mCRPC is characterized by extensive heterogeneity of genomic copy number alterations, which may lead to antiandrogen resistance. TP53 and RB1 alterations have been reported to be dramatically enriched in mCRPC neuroendocrine cancer and we have previously found that inactivation of both TP53 and RB1 confers resistance to antiandrogen through lineage plasticity, where cancer cells can transdifferentiate from a luminal lineage to a mixture of basal and neuroendocrine lineages, which is no longer dependent on AR signaling. Despite these exciting discoveries, only 10% of patients have TP53 and RB1 loss and it is estimated that approximately 40% of patients develop antiandrogen resistance through an undiscovered mechanism.To identify more genomic alterations which confer antiandrogen resistance, we performed an in vivo library screening and identified some of the frequently depleted genes as top candidates mediating antiandrogen response, including the chromatin helicase DNA-binding factor (CHD1). Strikingly, the loss of CHD1 establishes an altered chromatin landscape and enables the activation of heterogenous resistant subclones to emerge, including clones with ectopic NR3C1/GR, POU3F2/BRN2, TBX2, and NR2F1. This work provided an innovative model to explain the dramatically increased tumor heterogeneity in therapy resistant prostate cancer and suggested potential druggable targets to overcome resistance. Building on those exciting results, we are now examining the underlying mechanism how the epigenetic rewiring in resistant tumor clones confer resistance and monitoring the evolution of those heterogenous resistant subclones. Furthermore, we are revealing the collaborative function of those four resistant driver genes in promoting metastasis to various distal organs through multidisplinary approaches including single cell transcriptomics and 3D organoid modeling. The completion of this study will not only add clarity to the mechanism of antiandrogen resistance but may also lead to the development of novel biomarker and therapeutic approaches to overcome resistance. Citation Format: Xiaong li, Su Deng, Julisa Gonzalez, Carla Rodriguez Tirado, Choushi Wang, Nickolas A. Johnson, Lauren Metang, Ping Mu. Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3891.

Abstract NG10: Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets

Abstract Untreated prostate cancers rely on androgen receptor (AR) signaling for growth and survival, forming the basis for the initial efficacy of androgen deprivation therapy (ADT). Yet the disease can relapse and progress to a lethal stage termed castration-resistant prostate cancer (CRPC). Reactivation of AR signaling represents the most common driver of CRPC growth, and next-generation AR signaling inhibitors (ARSIs) are now used in combination with ADT as first-line therapy. However, ARSIs can result in selective pressure, thereby generating AR-independent tumors. The transition from AR dependence frequently accompanies a change in a phenotype resembling developmental transdifferentiation or “lineage plasticity”. Neuroendocrine prostate cancer, which lacks a defined pathologic classification, is the most studied type of lineage plasticity. However, most AR-null tumors do not exhibit neuroendocrine features and are classified as “double-negative prostate cancer”, the drivers of which are poorly defined. Lineage plasticity studies in CRPC are limited by the lack of genetically defined patient-derived models that recapitulate the disease spectrum. To address this, we developed a biobank of organoids generated from patient biopsies to study the landscape of metastatic CRPC and allow for functional validation assays. Proteins called transcription factors (TFs) are drivers of tumor lineage plasticity. To identify the key TFs that drive the growth of AR-independent tumors, we integrated epigenetic and transcriptomic data generated from CRPC models. We generated ATAC-seq (assay for transposase-accessible chromatin sequencing) and RNA-seq data from 22 metastatic human prostate cancer organoids, six patient-derived xenografts (PDXs), and 12 derived or traditional cell lines. We classified the 40 models into four subtypes and predicted key TFs of each subtype. Besides the well-characterized AR-dependent (CRPC-AR) and neuroendocrine subtypes (CRPC-NE), we identified two novel AR-negative/low groups, including a Wnt-dependent subtype (CRPC-WNT), driven by TCF/LEF TFs, and a stem cell-like (SCL) subtype (CRPC-SCL), driven by the AP-1 family of TFs. To apply the subtype classification to patient samples, we derived RNA-seq signatures from the organoids and applied them to 366 patient samples from two independent CRPC cohorts. The generated signatures recapitulated the four-subtype classification and revealed that CRPC-SCL is the second most prevalent group. Patients from CRPC-SCL are also associated with shorter time under ARSI treatment compared to CRPC-AR, indicating that the ARSI treatments were less effective for CRPC-SCL patients. Additional chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicated that AP-1 (FOSL1) collaboratively binds with TEAD and transcription coactivators, YAP and TAZ. Knocking down of AP-1 (FOSL1), YAP/TAZ decreased cell growth of CRPC-SCL and showed a decrease of chromatin accessibility at CRPC-SCL-specific open chromatin sites and down-regulation of YAP/TAZ target gene expression. In addition, the expression of AP-1 (FOSL1) decreased upon YAP/TAZ knockdown suggesting a positive feedback loop as well as YAP/TAZ as actional targets in CRPC-SCL. We used two small-molecule inhibitors, verteporfin and T-5224, that act on the YAP/TAZ/AP-1 pathway for their potential use as therapeutics for CRPC-SCL tumors, both inhibited the growth of samples from CRPC-SCL but not CRPC-AR. By overexpressing an AP-1 family gene (FOSL1) in AR-high cells, we observed an increase in chromatin accessibility at CRPC-SCL-specific open chromatin sites as well as significant up-regulation of CRPC-SCL signature genes, suggesting that AP-1 functions as a pioneering factor and master regulator for CRPC-SCL. All this work was recently published in Science (Tang, Xu et al. Science, 2022) where I am the co-first author. In summary, by using a diverse biobank of organoids, PDXs, and cell lines that recapitulate the heterogeneity of metastatic prostate cancer, we created a map of the chromatin accessibility and transcriptomic landscape of CRPC. We validated the CRPC-AR and CRPC-NE subtypes and report two novel subtypes of AR-negative/low samples, CRPC-SCL and CRPC-WNT, as well as their respective key TFs. Additional analysis revealed a model in which YAP, TAZ, TEAD, and AP-1 function together and drive oncogenic growth in CRPC-SCL samples. In addition, we proposed small inhibitors of YAP and TAZ that can potentially be used to treat CRPC-SCL patients. Overall, our results show how the stratification of CRPC patients into four subtypes using their transcriptomes can potentially inform appropriate clinical decisions. Citation Format: Fanying Tang, Duo Xu, Shangqian Wang, Chen Khuan Wong, Alexander Martinez-Fundichely, Cindy J. Lee, Sandra Cohen, Jane Park, Corinne E. Hill, Kenneth Eng, Rohan Bareja, Teng Han, Eric Minwei Liu, Ann Palladino, Wei Di, Dong Gao, Wassim Abida, Shaham Beg, Loredana Puca, Maximiliano Meneses, Elisa de Stanchina, Michael F. Berger, Anuradha Gopalan, Lukas E. Dow, Juan Miguel Mosquera, Himisha Beltran, Cora N. Sternberg, Ping Chi, Howard I. Scher, Andrea Sboner, Yu Chen, Ekta Khurana. Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG10.

Transcriptomic Heterogeneity in High-risk Prostate Cancer and Implications for Extraprostatic Disease at Presentation on Prostate-specific Membrane Antigen Positron Emission Tomography

Prostate-specific membrane antigen (PSMA) positron emission tomography (PET) has greater specificity and sensitivity for detection of extraprostatic prostate cancer (PCa) at presentation than conventional imaging. Although the long-term clinical significance of acting on these findings is unknown, it has been shown that the risk of upstaging is prognostic for long-term outcomes in men with high-risk (HR) or very high-risk (VHR) PCa. We evaluated the association between the risk of upstaging on PSMA PET and the Decipher genomic classifier score, a known prognostic biomarker in localized PCa that is being evaluated for its predictive ability to direct systemic therapy intensification. In a cohort of 4625 patients with HR or VHR PCa, the risk of upstaging on PSMA PET was significantly correlated with the Decipher score (p < 0.001). These results should be seen as hypothesis-generating and warrant further studies on the causal pathways linking PSMA findings, Decipher scores, extraprostatic disease, and long-term clinical outcomes. Patient summaryWe found significant correlation between the risk of having prostate cancer outside the prostate gland on a sensitive scan (based on prostate-specific membrane antigen [PSMA]) at initial staging and the Decipher genetic score. The results warrant further studies on the causal pathways between PSMA scan findings, Decipher scores, disease outside the prostate, and long-term outcomes.

Enzalutamide-induced signatures revealed by epigenetic plasticity using single-cell multi-omics sequencing in prostate cancer

Prostate cancer is morphologically and molecularly heterogeneous, which poses obstacles for early diagnosis and treatment. Advancements in understanding the heterogeneity of prostate cancer will help navigate through these challenges and ultimately benefit patients. In this study, we integrated single-cell sequencing for transposase-accessible chromatin and whole transcriptome in prostate cancer cell lines, aiming to decode the epigenetic plasticity upon enzalutamide (ENZ) treatment. By comparing the cell populations representing early-treatment response or resistance to the initial tumor cells, we identified seven signature gene sets; they present consistent trends of chromatin closing co-occurred with down-regulated genes during early response and chromatin opening with up-regulated genes upon maintaining drug resistance. In the molecular signatures, we found genes ZNF337, MAPK15, and ESRRG are favorable in progression-free prognosis during early response, while genes CCDC150, CCDC18, and POC1A marked poor prognosis underpinning the pre-existing drug resistance in The Cancer Genome Atlas prostate adenocarcinoma cohort. Ultimately, drug-target analyses nominated combinatory drug candidates to either enhance early-treatment response or potentially overcome ENZ resistance. Together, our integrative, single-cell multi-omics approach in pre-clinical models is effective in identifying informative signatures from complex molecular events, illustrating diverse drug responses in prostate cancer, and invoking novel combinatory drug strategies to inform clinical decision making.

Our Current Understanding of the Heterogeneity in Prostate Cancer and Renal Cell Carcinoma

Urological cancer is a collective term for cancers of the bladder, kidney, ureter, penis, prostate, and testicles. Last year, more than 444,000 people were diagnosed with urinary cancers in the United States. In this review, we talk about the complexity of prostate and kidney cancer.

Patient-derived organoids represent intra- and interindividual heterogeneity in castration-resistant prostate cancer

Patient-derived organoids represent intra- and interindividual heterogeneity in castration-resistant prostate cancer