Background

B lymphocytes have multifaceted functions in the tumour microenvironment, and their prognostic role in human cancers is controversial. Here we aimed to identify tumour microenvironmental factors that influence the prognostic effects of B cells.

Methods

We conducted a gene expression analysis of 3585 patients for whom the clinical outcome information was available. We further investigated the clinical relevance for predicting immunotherapy response.

Results

We identified a novel B cell-related gene (BCR) signature consisting of nine cytokine signalling genes whose high expression could diminish the beneficial impact of B cells on patient prognosis. In triple-negative breast cancer, higher B cell abundance was associated with favourable survival only when the BCR signature was low (HR = 0.68, p = 0.0046). By contrast, B cell abundance had no impact on prognosis when the BCR signature was high (HR = 0.93, p = 0.80). This pattern was consistently observed across multiple cancer types including lung, colorectal, and melanoma. Further, the BCR signature predicted response to immune checkpoint blockade in metastatic melanoma and compared favourably with the established markers.

Conclusions

The prognostic impact of tumour-infiltrating B cells depends on the status of cytokine signalling genes, which together could predict response to cancer immunotherapy.

Genomic alterations resulting in loss of control over the cell cycle is a fundamental hallmark of human malignancies. Whilst pan-cancer studies have broadly assessed tumour genomics and their impact on oncogenic pathways, analyses taking the baseline signalling levels in normal tissue into account are lacking. To this end, we aimed to reclassify the cell cycle activity of tumours in terms of their tissue of origin and determine if any common DNA mutations, chromosome arm-level changes or signalling pathways contribute to an increase in baseline corrected cell cycle activity. Combining normal tissue and pan-cancer data from over 13,000 samples we demonstrate that tumours of gynaecological origin show the highest levels of corrected cell cycle activity, partially owing to hormonal signalling and gene expression changes. We also show that normal and tumour tissues can be separated into groups (quadrants) of low/high cell cycle activity and propose the hypothesis of an upper limit on these activity levels in tumours.

Purpose

Although numerous biology-driven subtypes have been described previously in metastatic castration-resistant prostate cancer (mCRPC), unsupervised molecular subtyping based on gene expression has been less studied, especially using large cohorts. Thus, we sought to identify the intrinsic molecular subtypes of mCRPC and assess molecular and clinical correlates in the largest combined cohort of mCRPC samples with gene expression data available to date.

Experimental design

We combined and batch-effect corrected gene expression data from four mCRPC cohorts from the Fred Hutchinson Cancer Research Center (N = 157), a small-cell neuroendocrine (NE) prostate cancer (SCNC)-enriched cohort from Weill Cornell Medicine (N = 49), and cohorts from the Stand Up 2 Cancer/Prostate Cancer Foundation East Coast Dream Team (N = 266) and the West Coast Dream Team (N = 162).

Results

Hierarchical clustering of RNA-sequencing data from these 634 mCRPC samples identified two distinct adenocarcinoma subtypes, one of which (adeno-immune) was characterized by higher gene expression of immune pathways, higher CIBERSORTx immune scores, diminished ASI benefit, and non-lymph node metastasis tropism compared with an adeno-classic subtype. We also identified two distinct subtypes with enrichment for an NE phenotype, including an NE-liver subgroup characterized by liver metastasis tropism, PTEN loss, and APC and SPOP mutations compared with an NE-classic subgroup.

Conclusions

Our results emphasize the heterogeneity of mCRPC beyond currently accepted molecular phenotypes, and suggest that future studies should consider incorporating transcriptome-wide profiling to better understand how these differences impact treatment responses and outcomes.

The impact of variations in the three-dimensional structure of the genome has been recognized, but solid cancer tissue studies are limited. Here, we performed integrated deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5-hydroxymethylcytosine (5hmC) sequencing and RNA sequencing across a cohort of 80 biopsy samples from patients with metastatic castration-resistant prostate cancer. Dramatic differences were present in gene expression, 5-methylcytosine/5hmC methylation and in structural variation versus mutation rate between A and B (open and closed) chromatin compartments. A subset of tumors exhibited depleted regional chromatin contacts at the AR locus, linked to extrachromosomal circular DNA (ecDNA) and worse response to AR signaling inhibitors. We also identified topological subtypes associated with stark differences in methylation structure, gene expression and prognosis. Our data suggested that DNA interactions may predispose to structural variant formation, exemplified by the recurrent TMPRSS2-ERG fusion. This comprehensive integrated sequencing effort represents a unique clinical tumor resource.

BACKGROUNDAndrogen receptor signaling inhibitors (ARSIs) have improved outcomes for patients with metastatic castration-resistant prostate cancer (mCRPC), but their clinical benefit is limited by treatment resistance.METHODSTo investigate the mechanisms of ARSI resistance, we analyzed the whole-genome (n = 45) and transcriptome (n = 31) sequencing data generated from paired metastatic biopsies obtained before initiation of first-line ARSI therapy for mCRPC and after radiographic disease progression. We investigated the effects of genetic and pharmacologic modulation of SSTR1 in 22Rv1 cells, a representative mCRPC cell line.RESULTSWe confirmed the predominant role of tumor genetic alterations converging on augmenting androgen receptor (AR) signaling and the increased transcriptional heterogeneity and lineage plasticity during the emergence of ARSI resistance. We further identified amplifications involving a putative enhancer downstream of the AR and transcriptional downregulation of SSTR1, encoding somatostatin receptor 1, in ARSI-resistant tumors. We found that patients with SSTR1-low mCRPC tumors derived less benefit from subsequent ARSI therapy in a retrospective cohort. We showed that SSTR1 was antiproliferative in 22Rv1 cells and that the FDA-approved drug pasireotide suppressed 22Rv1 cell proliferation.CONCLUSIONOur findings expand the knowledge of ARSI resistance and point out actionable next steps, exemplified by potentially targeting SSTR1, to improve patient outcomes.FUNDINGNational Cancer Institute (NCI), NIH; Prostate Cancer Foundation; Conquer Cancer, American Society of Clinical Oncology Foundation; UCSF Benioff Initiative for Prostate Cancer Research; Netherlands Cancer Institute.

Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen signaling. Androgen deprivation therapy and second-generation androgen receptor (AR)-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by AR and neuroendocrine (NE) markers. Molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterized treatment-emergent mCRPC by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing from 210 tumors. AR-/NE- tumors were clinically and molecularly distinct from other mCRPC subtypes, with the shortest survival, amplification of the chromatin remodeler CHD7, and PTEN loss. Methylation changes in CHD7 candidate enhancers were linked to elevated CHD7 expression in AR-/NE+ tumors. Genome-wide methylation analysis nominated Krüppel-like factor 5 (KLF5) as a driver of the AR-/NE- phenotype, and KLF5 activity was linked to RB1 loss. These observations reveal the aggressiveness of AR-/NE- mCRPC and could facilitate the identification of therapeutic targets in this highly aggressive disease.

Significance

Comprehensive characterization of the five subtypes of metastatic castration-resistant prostate cancer identified transcription factors that drive each subtype and showed that the double-negative subtype has the worst prognosis.

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.