The 5-Hydroxymethylcytosine Landscape of Prostate Cancer

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. SIGNIFICANCE: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.publishedVersionPeer reviewe

Circulating tumour DNA as a comprehensive multi-omic tool for profiling advanced prostate cancer

Life-prolonging targeted therapies exploiting genomic vulnerabilities are now standard-of-care in metastatic castration-resistant prostate cancer (mCRPC). However, consensus on optimal patient selection—including choice of molecular profiling—remains unestablished. Plasma cell-free DNA (cfDNA) is an established minimally-invasive tool for real-time profiling and may facilitate genomic stratification of mCRPC. In this thesis, next-generation sequencing was applied to cfDNA and tumour tissue to define the population frequencies and clinicogenomic properties of therapeutically-relevant mCRPC molecular subtypes: activating AKT1/PIK3CA mutations, mismatch-repair deficiency (MMRd), and DNA repair defects in BRCA2/CDK12/ATM. Targeted ctDNA sequencing detected AKT1/PIK3CA mutations in 6% of mCRPC. Clonal prevalence, consistent temporal redetection across serial cfDNA, and mutant-allele imbalance indicate PI3K pathway dependency in select patients. MMRd (4%) was identifiable in ctDNA via multiple correlated metrics of genome-scarring, and was associated with mutation-enabled oncogene activation, heightened subclonal diversity, and poor response to AR-targeted treatment. Defects in BRCA2/CDK12/ATM (15%) involved biallelic inactivation in most patients, suggesting driver etiology. Tumour suppressors were controlled via deleterious rearrangements in BRCA2-defective samples but via (negative-feedback) oncogene amplification in context of CDK12 defects. BRCA2/CDK12/ATM defects were re-detected across 94% of serial ctDNA and in all archival primary tissues, indicating origination prior to metastatic dissemination. BRCA2 and CDK12 (but not ATM) was associated with poor clinical outcomes. Optimized targeted cfDNA testing necessitates understanding of the dynamic clonal composition of bulk ctDNA. We performed deep whole-genome sequencing (WGS) of serial plasma and synchronous metastases, revealing ctDNA populational diversity involving variable whole-genome duplication and shifts in mutational processes. Although tissue and ctDNA harboured concordant clonally expanded driver alterations, most individual metastases contributed only a minor share of total ctDNA. By comparing serial ctDNA before and after progression on AR pathway inhibitors, we reveal population restructuring converging solely on AR augmentation as the dominant genomic driver of acquired treatment resistance. Finally we leverage ctDNA nucleosome footprints to infer metastatic tissue mRNA abundance, including treatment-induced changes in AR transcription factor signaling. In this context, my thesis informs future precision oncology clinical trial design and ctDNA testing strategies, and establishes serial ctDNA WGS as a discovery tool for understanding complex metastatic tumour biology.Science, Faculty ofGraduat

Genomic features of lung-recurrent hormone sensitive prostate cancer

PURPOSE Pulmonary involvement is rare in metastatic hormone-sensitive prostate cancer (mHSPC) that recurs after treatment for localized disease. Guidelines recommend intensive systemic therapy, similar to patients with liver metastases, but some lung-recurrent mHSPC may have good outcomes. Genomic features of lung metastases may clarify disease aggression, but are poorly understood since lung biopsy is rarely performed. We present a comparative assessment of genomic drivers and heterogeneity in metachronous prostate tumors and lung metastases. METHODS We leveraged a prospective functional imaging study of 208 biochemically recurrent prostate cancers to identify 10 patients with lung-recurrent mHSPC. Histologic diagnosis was attained via thoracic surgery or fine-needle lung biopsy. We retrieved clinical data and performed multiregion sampling of primary tumors and metastases. Targeted and/or whole-exome sequencing was applied to 46 primary and 32 metastatic foci. RESULTS Unusually for mHSPC, all patients remained alive despite a median follow-up of 11.5 years. Several patients experienced long-term freedom from systemic treatment. The genomic landscape of lung-recurrent mHSPC was typical of curable prostate cancer with frequent PTEN, SPOP, and chromosome 8p alterations, and there were no deleterious TP53 and DNA damage repair gene mutations that characterize aggressive prostate cancer. Despite a long median time to recurrence (76.8 months), copy number alterations and clonal mutations were highly conserved between metastatic and primary foci, consistent with intrapatient homogeneity and limited genomic evolution. CONCLUSION In this retrospective hypothesis-generating study, we observed indolent genomic etiology in selected lung-recurrent mHSPC, cautioning against grouping these patients together with liver or bone-predominant mHSPC. Although our data do not generalize to all patients with lung metastases, the results encourage prospective efforts to stratify lung-recurrent mHSPC by genomic features. (C) 2022 by American Society of Clinical Oncolog

Genomic features of lung-recurrent hormone-sensitive prostate cancer

PURPOSE Pulmonary involvement is rare in metastatic hormone-sensitive prostate cancer (mHSPC) that recurs after treatment for localized disease. Guidelines recommend intensive systemic therapy, similar to patients with liver metastases, but some lung-recurrent mHSPC may have good outcomes. Genomic features of lung metastases may clarify disease aggression, but are poorly understood since lung biopsy is rarely performed. We present a comparative assessment of genomic drivers and heterogeneity in metachronous prostate tumors and lung metastases. METHODS We leveraged a prospective functional imaging study of 208 biochemically recurrent prostate cancers to identify 10 patients with lung-recurrent mHSPC. Histologic diagnosis was attained via thoracic surgery or fine-needle lung biopsy. We retrieved clinical data and performed multiregion sampling of primary tumors and metastases. Targeted and/or whole-exome sequencing was applied to 46 primary and 32 metastatic foci. RESULTS Unusually for mHSPC, all patients remained alive despite a median follow-up of 11.5 years. Several patients experienced long-term freedom from systemic treatment. The genomic landscape of lung-recurrent mHSPC was typical of curable prostate cancer with frequent PTEN, SPOP, and chromosome 8p alterations, and there were no deleterious TP53 and DNA damage repair gene mutations that characterize aggressive prostate cancer. Despite a long median time to recurrence (76.8 months), copy number alterations and clonal mutations were highly conserved between metastatic and primary foci, consistent with intrapatient homogeneity and limited genomic evolution. CONCLUSION In this retrospective hypothesis-generating study, we observed indolent genomic etiology in selected lung-recurrent mHSPC, cautioning against grouping these patients together with liver or bone-predominant mHSPC. Although our data do not generalize to all patients with lung metastases, the results encourage prospective efforts to stratify lung-recurrent mHSPC by genomic features. (C) 2022 by American Society of Clinical Oncolog

Morphologic and genomic characterization of urothelial to sarcomatoid transition in muscle-invasive bladder cancer.

INTRODUCTION The sarcomatoid morphology of muscle-invasive bladder cancer (MIBC) is associated with unfavorable prognosis. However, the genomic, transcriptomic, and proteomic relationship between conventional urothelial and synchronous sarcomatoid morphology is poorly defined. METHODS We compiled a cohort of 21 MIBC patients with components of conventional urothelial and adjacent sarcomatoid morphology within the same tumor focus. We performed comprehensive pathologic and immunohistochemical characterization and in 4 selected cases, subjected both morphologic components to targeted DNA sequencing and whole transcriptome analysis. RESULTS Synchronous sarcomatoid and urothelial morphology from the same MIBC foci shared truncal somatic mutations, indicating a common ancestral clone. However, additional mutations or copy number alterations restricted to the either component suggested divergent evolution at the genomic level. This was confirmed at the transcriptome level since while the urothelial component exhibited a basal-like subtype (TCGA2014: cluster III, LundTax: basal/squamous-like), the sarcomatoid morphology was predominantly cluster IV (claudin-low). Protein expression was consistent with a basal-like phenotype in both morphologies in 18/21 of cases. However, most cases had evidence of active epithelial-to-mesenchymal transition (E-Cad ↓ and Zeb1 or TWIST1 ↑) from urothelial toward the sarcomatoid morphology. Drug response signatures nominated different targets for each morphology and proposed agents under clinical investigation in liposarcoma or other sarcoma. PD-L1 expression was higher in the sarcomatoid than the urothelial component. CONCLUSIONS Conventional urothelial and adjacent sarcomatoid morphologies of MIBC arise from the same common ancestor and share a basal-like phenotype. However, divergence between the morphologies at the genome, transcriptome, and proteome level suggests differential sensitivity to therapy