Oncogenic gene fusions in nonneoplastic precursors as evidence that bacterial infection can initiate prostate cancer.

Prostate adenocarcinoma is the second most commonly diagnosed cancer in men worldwide, and the initiating factors are unknown. Oncogenic TMPRSS2:ERG (ERG+) gene fusions are facilitated by DNA breaks and occur in up to 50% of prostate cancers. Infection-driven inflammation is implicated in the formation of ERG+ fusions, and we hypothesized that these fusions initiate in early inflammation-associated prostate cancer precursor lesions, such as proliferative inflammatory atrophy (PIA), prior to cancer development. We investigated whether bacterial prostatitis is associated with ERG+ precancerous lesions in unique cases with active bacterial infections at the time of radical prostatectomy. We identified a high frequency of ERG+ non-neoplastic-appearing glands in these cases, including ERG+ PIA transitioning to early invasive cancer. These lesions were positive for ERG protein by immunohistochemistry and ERG messenger RNA by in situ hybridization. We additionally verified TMPRSS2:ERG genomic rearrangements in precursor lesions using tricolor fluorescence in situ hybridization. Identification of rearrangement patterns combined with whole-prostate mapping in three dimensions confirmed multiple (up to eight) distinct ERG+ precancerous lesions in infected cases. We further identified the pathogen-derived genotoxin colibactin as a potential source of DNA breaks in clinical cases as well as cultured prostate cells. Overall, we provide evidence that bacterial infections can initiate driver gene alterations in prostate cancer. In addition, our observations indicate that infection-induced ERG+ fusions are an early alteration in the carcinogenic process and that PIA may serve as a direct precursor to prostate cancer.

Multiplex immunohistochemical phenotyping of T cells in primary prostate cancer.

BACKGROUND: Most prostate cancers are "immune cold" and poorly responsive to immune checkpoint inhibitors. However, the mechanisms responsible for the lack of a robust antitumor adaptive immune response in the prostate are poorly understood, which hinders the development of novel immunotherapeutic approaches. AIMS: Most inflammatory infiltrates in the prostate are centered around benign glands and stroma, which can confound the molecular characterization of the antitumor immune response. We sought to analytically validate a chromogenic-based multiplex immunohistochemistry (IHC) approach applicable to whole slide digital image analysis to quantify T cell subsets from the tumor microenvironment of primary prostatic adenocarcinomas. As an initial application, we tested the hypothesis that PTEN loss leads to an altered antitumor immune response by comparing matched regions of tumors within the same individual with and without PTEN loss. MATERIALS & METHODS: Using the HALO Image Analysis Platform (Indica Labs), we trained a classifier to quantify the densities of eight T cell phenotypes separately in the tumor epithelial and stromal subcompartments. RESULTS: The iterative chromogenic approach using 7 different antibodies on the same slide provides highly similar findings to results using individually stained slides with single antibodies. Our main findings in carcinomas (benign removed) include the following: i) CD4+ T cells are present at higher density than CD8+ T cells; ii) all T cell subsets are present at higher densities in the stromal compartment compared to the epithelial tumor compartment; iii) most CD4+ and CD8+ T cells are PD1+; iv) cancer foci with PTEN loss harbored increased numbers of T cells compared to regions without PTEN loss, in both stromal and epithelial compartments; and v) the increases in T cells in PTEN loss regions were associated with ERG gene fusion status. DISCUSSION: This modular approach can apply to any IHC-validated antibody combination and sets the groundwork for more detailed spatial analyses. CONCLUSION: Iterative chromogenic IHC can be used for whole slide analysis of prostate tissue samples and can complement transcriptomic results including those using single cell and spatial genomic approaches.

An in Situ Atlas of Mitochondrial DNA in Mammalian Tissues Reveals High Content in Stem and Proliferative Compartments.

Mitochondria regulate ATP production, metabolism, and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Because most measurements of mtDNA use homogenates of complex tissues, little is known about cell-type-specific mtDNA copy number heterogeneity in normal physiology, aging, and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, an in situ hybridization approach to generate a single-cell-resolution atlas of mtDNA content in mammalian tissues was validated. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared with differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared with cells within glomeruli and collecting duct epithelial cells. In mice, decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. This study provides a novel approach for quantifying species- and cell-type-specific mtDNA copy number and dynamics in any normal or diseased tissue that can be used for monitoring the effects of interventions in animal and human studies.

Ribosomal RNA transcription governs splicing through ribosomal protein RPL22.

Ribosome biosynthesis is a cancer vulnerability executed by targeting RNA polymerase I (Pol I) transcription. We developed advanced, specific Pol I inhibitors to identify drivers of this sensitivity. By integrating multi-omics features and drug sensitivity data from a large cancer cell panel, we discovered that RPL22 frameshift mutation conferred Pol I inhibitor sensitivity in microsatellite instable cancers. Mechanistically, RPL22 directly interacts with 28S rRNA and mRNA splice junctions, functioning as a splicing regulator. RPL22 deficiency, intensified by 28S rRNA sequestration, promoted the splicing of its paralog RPL22L1 and p53 negative regulator MDM4. Chemical and genetic inhibition of rRNA synthesis broadly remodeled mRNA splicing controlling hundreds of targets. Strikingly, RPL22-dependent alternative splicing was reversed by Pol I inhibition revealing a ribotoxic stress-initiated tumor suppressive pathway. We identify a mechanism that robustly connects rRNA synthesis activity to splicing and reveals their coordination by ribosomal protein RPL22.

Bipolar androgen therapy plus nivolumab for patients with metastatic castration-resistant prostate cancer: the COMBAT phase II trial.

Cyclic high-dose testosterone administration, known as bipolar androgen therapy (BAT), is a treatment strategy for patients with metastatic castration-resistant prostate cancer (mCRPC). Here, we report the results of a multicenter, single arm Phase 2 study (NCT03554317) enrolling 45 patients with heavily pretreated mCRPC who received BAT (testosterone cypionate, 400 mg intramuscularly every 28 days) with the addition of nivolumab (480 mg intravenously every 28 days) following three cycles of BAT monotherapy. The primary endpoint of a confirmed PSA50 response rate was met and estimated at 40% (N = 18/45, 95% CI: 25.7-55.7%, P = 0.02 one-sided against the 25% null hypothesis). Sixteen of the PSA50 responses were achieved before the addition of nivolumab. Secondary endpoints included objective response rate (ORR), median PSA progression-free survival, radiographic progression-free survival (rPFS), overall survival (OS), and safety/tolerability. The ORR was 24% (N = 10/42). Three of the objective responses occurred following the addition of nivolumab. After a median follow-up of 17.9 months, the median rPFS was 5.6 (95% CI: 5.4-6.8) months, and median OS was 24.4 (95% CI: 17.6-31.1) months. BAT/nivolumab was well tolerated, resulting in only five (11%) drug related, grade-3 adverse events. In a predefined exploratory analysis, clinical response rates correlated with increased baseline levels of intratumoral PD-1 + T cells. In paired metastatic tumor biopsies, BAT induced pro-inflammatory gene expression changes that were restricted to patients achieving a clinical response. These data suggest that BAT may augment antitumor immune responses that are further potentiated by immune checkpoint blockade.

MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression.

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type-specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by 2 orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression.

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by two orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

Supraphysiologic Testosterone Induces Ferroptosis and Activates Immune Pathways through Nucleophagy in Prostate Cancer.

The discovery that androgens play an important role in the progression of prostate cancer led to the development of androgen deprivation therapy (ADT) as a first line of treatment. However, paradoxical growth inhibition has been observed in a subset of prostate cancer upon administration of supraphysiologic levels of testosterone (SupraT), both experimentally and clinically. Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive prostate cancer cells. This was initiated by the induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activated nucleic acid sensors converge on NFκB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT resulted in increased migration of cytotoxic immune cells to tumor beds in xenograft models and patient tumors. Collectively, these findings indicate that SupraT may inhibit a subset of prostate cancer by activating nucleic acid sensors and downstream immune signaling. SIGNIFICANCE: This study demonstrates that supraphysiologic testosterone induces two parallel autophagy-mediated processes, ferritinophagy and nucleophagy, which then activate nucleic acid sensors to drive immune signaling pathways in prostate cancer.

A MYC and RAS co-activation signature in localized prostate cancer drives bone metastasis and castration resistance.

Understanding the intricacies of lethal prostate cancer poses specific challenges due to difficulties in accurate modeling of metastasis in vivo. Here we show that NPK EYFP mice (for Nkx3.1 CreERT2/+ ; Pten flox/flox ; Kras LSL-G12D/+ ; R26R-CAG-LSL-EYFP/+) develop prostate cancer with a high penetrance of metastasis to bone, thereby enabling detection and tracking of bone metastasis in vivo and ex vivo. Transcriptomic and whole-exome analyses of bone metastasis from these mice revealed distinct molecular profiles conserved between human and mouse and specific patterns of subclonal branching from the primary tumor. Integrating bulk and single-cell transcriptomic data from mouse and human datasets with functional studies in vivo unravels a unique MYC/RAS co-activation signature associated with prostate cancer metastasis. Finally, we identify a gene signature with prognostic value for time to metastasis and predictive of treatment response in human patients undergoing androgen receptor therapy across clinical cohorts, thus uncovering conserved mechanisms of metastasis with potential translational significance.

Cytopathologist-performed and ultrasound-guided fine needle aspiration cytology enhances diagnostic accuracy and avoids pitfalls: An overview of 20 years of personal experience with a selection of didactic cases.

Over the last few decades, fine needle aspiration cytology (FNA) has emerged as a SAFE (Simple, Accurate, Fast, Economical) diagnostic tool based on the morphologic evaluation of cells. The first and most important step in obtaining accurate results from FNA is to procure sufficient and representative material from the lesion and to appropriately transfer this material to the laboratory. Unfortunately, the most important aspect of this task occurs beyond the control of the cytopathologist, a key reason for obtaining unsatisfactory results with FNA. There is growing interest in the field of cytology in "cytopathologist-performed ultrasound (US)-guided FNA," which has been reported to yield accurate results. The first author has been applying FNA in his own private cytopathology practice with a radiologist and under the guidance of US for more than 20 years. This study retrospectively reviews the utility of this practice. We present a selection of didactic examples under different headings that highlight the application of FNA by a cytopathologist, accompanied by US, under the guidance of a radiologist, in the form of an "outpatient FNA clinic." The use of this technique enhances diagnostic accuracy and prevents pitfalls. The highlights of each case are also outlined as "take-home messages."