Longer prostate stromal cell telomere length is associated with increased risk of death from other cancers.

Background: Telomeres are located at chromosomal termini and function to maintain genomic integrity. Telomere dysfunction is a well-recognized contributor to aging and age-related diseases, such as prostate cancer. Since telomere length is highly heritable, we postulate that stromal cell telomere length in the tissue of a particular solid organ may generally reflect constitutive stromal cell telomere length in other solid organs throughout the body. Even with telomere loss occurring with each round of cell replication, in general, telomere length in prostate stromal cells in mid-life would still be correlated with the telomere length in stromal cells in other organs. Thus, we hypothesize that prostate stromal cell telomere length and/or telomere length variability is a potential indicator of the likelihood of developing future solid cancers, beyond prostate cancer, and especially lethal cancer. Methods: To explore this hypothesis, we conducted a cohort study analysis of 1,175 men who were surgically treated for prostate cancer and were followed for death, including from causes other than their prostate cancer. Results: In this cohort study with a median follow-up of 19 years, we observed that longer prostate stromal cell telomere length measured in tissue microarray spots containing prostate cancer was associated with an increased risk of death from other solid cancers. Variability in telomere length among these prostate stromal cells was possibly positively associated with risk of death from other solid cancers. Conclusion: Studying the link between stromal cell telomere length and cancer mortality may be important for guiding the development of cancer interception and prevention strategies.

The TERT Promoter is Polycomb-Repressed in Neuroblastoma Cells with Long Telomeres.

Acquiring a telomere maintenance mechanism is a hallmark of high-risk neuroblastoma and commonly occurs by expressing telomerase (TERT). Telomerase-negative neuroblastoma has long telomeres and utilizes the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. Conversely, no discernable telomere maintenance mechanism is detected in a fraction of neuroblastoma with long telomeres. Here, we show, unlike most cancers, DNA of the TERT promoter is broadly hypomethylated in neuroblastoma. In telomerase-positive neuroblastoma cells, the hypomethylated DNA promoter is approximately 1.5 kb. The TERT locus shows active chromatin marks with low enrichment for the repressive mark, H3K27me3. MYCN, a commonly amplified oncogene in neuroblstoma, binds to the promoter and induces TERT expression. Strikingly, in neuroblastoma with long telomeres, the hypomethylated region spans the entire TERT locus, including multiple nearby genes with enrichment for the repressive H3K27me3 chromatin mark. Furthermore, subtelomeric regions showed enrichment of repressive chromatin marks in neuroblastomas with long telomeres relative to those with short telomeres. These repressive marks were even more evident at the genic loci, suggesting a telomere position effect (TPE). Inhibiting H3K27 methylation by three different EZH2 inhibitors induced the expression of TERT in cell lines with long telomeres and H3K27me3 marks in the promoter region. EZH2 inhibition facilitated MYCN binding to the TERT promoter in neuroblastoma cells with long telomeres. Taken together, these data suggest that epigenetic regulation of TERT expression differs in neuroblastoma depending on the telomere maintenance status, and H3K27 methylation is important in repressing TERT expression in neuroblastoma with long telomeres. SIGNIFICANCE: The epigenetic landscape of the TERT locus is unique in neuroblastoma. The DNA at the TERT locus, unlike other cancer cells and similar to normal cells, are hypomethylated in telomerase-positive neuroblastoma cells. The TERT locus is repressed by polycomb repressive complex-2 complex in neuroblastoma cells that have long telomeres and do not express TERT. Long telomeres in neuroblastoma cells are also associated with repressive chromatin states at the chromosomal termini, suggesting TPE.

Alternative lengthening of telomeres: mechanism and the pathogenesis of cancer.

Telomere maintenance and elongation allows cells to gain replicative immortality and evade cellular senescence during cancer development. While most cancers use telomerase to maintain telomere lengths, a subset of cancers engage the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. ALT is present in 5%-10% of all cancers, although the prevalence is dramatically higher in certain cancer types, including complex karyotype sarcomas, isocitrate dehydrogenase-mutant astrocytoma (WHO grade II-IV), pancreatic neuroendocrine tumours, neuroblastoma and chromophobe hepatocellular carcinomas. ALT is maintained through a homology-directed DNA repair mechanism. Resembling break-induced replication, this aberrant process results in dramatic cell-to-cell telomere length heterogeneity, widespread chromosomal instability and chronic replication stress. Additionally, ALT-positive cancers frequently harbour inactivating mutations in either chromatin remodelling proteins (ATRX, DAXX and H3F3A) or DNA damage repair factors (SMARCAL1 and SLX4IP). ALT can readily be detected in tissue by assessing the presence of unique molecular characteristics, such as large ultrabright nuclear telomeric foci or partially single-stranded telomeric DNA circles (C-circles). Importantly, ALT has been validated as a robust diagnostic and prognostic biomarker for certain cancer types and may even be exploited as a therapeutic target via small molecular inhibitors and/or synthetic lethality approaches.

Chromogenic detection of telomere lengths in situ aids the identification of precancerous lesions in the prostate.

BACKGROUND: Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. METHODS: A robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). RESULTS: This new assay (telomere chromogenic in situ hybridization ["Telo-CISH"]) produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. CONCLUSIONS: In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Prognostic and therapeutic potential of senescent stromal fibroblasts in prostate cancer.

The stromal component of the tumour microenvironment in primary and metastatic prostate cancer can influence and promote disease progression. Within the prostatic stroma, fibroblasts are one of the most prevalent cell types associated with precancerous and cancerous lesions; they have a vital role in the structural composition, organization and integrity of the extracellular matrix. Fibroblasts within the tumour microenvironment can undergo cellular senescence, which is a stable arrest of cell growth and a phenomenon that is emerging as a recognized hallmark of cancer. Supporting the idea that cellular senescence has a pro-tumorigenic role, a subset of senescent cells exhibits a senescence-associated secretory phenotype (SASP), which, along with increased inflammation, can promote prostate cancer cell growth and survival. These cellular characteristics make targeting senescent cells and/or modulating SASP attractive as a potential preventive or therapeutic option for prostate cancer.

Predicting recurrence in pancreatic neuroendocrine tumours: role of ARX and alternative lengthening of telomeres (ALT).

BACKGROUND: While many pancreatic neuroendocrine tumours (PanNET) show indolent behaviour, predicting the biological behaviour of small nonfunctional PanNETs remains a challenge. Nonfunctional PanNETs with an epigenome and transcriptome that resemble islet alpha cells (ARX-positive) are more aggressive than neoplasms that resemble islet beta cells (PDX1-positive). In this study, we explore the ability of immunohistochemistry for ARX and PDX1 and telomere-specific fluorescence in situ hybridisation (FISH) for alternative lengthening of telomeres (ALT) to predict recurrence. METHODS: Two hundred fifty-six patients with PanNETs were identified, and immunohistochemistry for ARX and PDX1 was performed. Positive staining was defined as strong nuclear staining in >5% of tumour cells. FISH for ALT was performed in a subset of cases. RESULTS: ARX reactivity correlated with worse disease-free survival (DFS) (P = 0.011), while there was no correlation between PDX1 reactivity and DFS (P = 0.52). ALT-positive tumours (n = 63, 31.8%) showed a significantly lower DFS (P < 0.0001) than ALT-negative tumours (n = 135, 68.2%). ARX reactivity correlated with ALT positivity (P < 0.0001). Among nonfunctional tumours, recurrence was noted in 18.5% (30/162) of ARX-positive tumours and 7.5% (5/67) of ARX-negative tumours. Among WHO grade 1 and 2 PanNETs with ≤2 cm tumour size, 14% (6/43) of ARX-positive tumours recurred compared to 0 of 33 ARX-negative tumours and 33.3% (3/9) ALT-positive tumours showed recurrence versus 4.4% (2/45) ALT-negative tumours. CONCLUSION: Immunohistochemistry for ARX and ALT FISH status may aid in distinguishing biologically indolent cases from aggressive small low-grade PanNETs, and help to identify patients who may preferentially benefit from surgical intervention.

Chromogenic detection of telomere lengths in situ aids the identification of precancerous lesions in the prostate.

Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. Here, a robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). This new assay ("Telo-CISH") produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.

Reprint of: The Diagnostic and Prognostic Utility of Incorporating DAXX, ATRX, and Alternative Lengthening of Telomeres (ALT) to the Evaluation of Pancreatic Neuroendocrine Tumors (PanNETs).

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of neoplasms with increasing incidence and an ill-defined pathobiology. Although many PanNETs are indolent and remain stable for years, a subset may behave aggressively and metastasize widely. Thus, the increasing and frequent detection of PanNETs presents a treatment dilemma. Current prognostic systems are susceptible to interpretation errors, sampling issues, and do not accurately reflect the clinical behavior of these neoplasms. Hence, additional biomarkers are needed to improve the prognostic stratification of patients diagnosed with a PanNET. Recent studies have identified alterations in death domain-associated protein 6 (DAXX) and alpha-thalassemia/mental retardation X-linked (ATRX), as well as alternative lengthening of telomeres (ALT), as promising prognostic biomarkers. This review summarizes the identification, clinical utility, and specific nuances in testing for DAXX/ATRX by immunohistochemistry and ALT by telomere-specific fluorescence in situ hybridization in PanNETs. Furthermore, a discussion on diagnostic indications for DAXX, ATRX, and ALT status is provided to include the distinction between PanNETs and pancreatic neuroendocrine carcinomas (PanNECs), and determining pancreatic origin for metastatic neuroendocrine tumors in the setting of an unknown primary.

Genomic Characterization of Prostatic Basal Cell Carcinoma.

Basal cell carcinoma (BCC) of the prostate is a rare tumor. Compared with the more common acinar adenocarcinoma (AAC) of the prostate, BCCs show features of basal cell differentiation and are thought to be biologically distinct from AAC. The spectrum of molecular alterations of BCC has not been comprehensively described, and genomic studies are lacking. Herein, whole genome sequencing was performed on archival formalin-fixed, paraffin-embedded specimens of two cases with BCC. Prostatic BCCs were characterized by an overall low copy number and mutational burden. Recurrent copy number loss of chromosome 16 was observed. In addition, putative driver gene alterations in KIT, DENND3, PTPRU, MGA, and CYLD were identified. Mechanistically, depletion of the CYLD protein resulted in increased proliferation of prostatic basal cells in vitro. Collectively, these studies show that prostatic BCC displays distinct genomic alterations from AAC and highlight a potential role for loss of chromosome 16 in the pathogenesis of this rare tumor type.

Prognostic and Predictive Biomarkers for Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine tumors (PanNETs) represent a clinically challenging disease because these tumors vary in clinical presentation, natural history, and prognosis. Novel prognostic biomarkers are needed to improve patient stratification and treatment options. Several putative prognostic and/or predictive biomarkers (eg, alternative lengthening of telomeres, alpha-thalassemia/mental retardation, X-linked (ATRX)/Death Domain Associated Protein (DAXX) loss) have been independently validated. Additionally, recent transcriptomic and epigenetic studies focusing on endocrine differentiation have identified PanNET subtypes that display similarities to either α-cells or ß-cells and differ in clinical outcomes. Thus, future prospective studies that incorporate genomic and epigenetic biomarkers are warranted and have translational potential for individualized therapeutic and surveillance strategies.

The prostate tissue-based telomere biomarker as a prognostic tool for metastasis and death from prostate cancer after prostatectomy.

Current biomarkers are inadequate prognostic predictors in localized prostate cancer making treatment decision-making challenging. Previously, we observed that the combination of more variable telomere length among prostate cancer cells and shorter telomere length in prostate cancer-associated stromal cells - the telomere biomarker - is strongly associated with progression to metastasis and prostate cancer death after prostatectomy independent of currently used pathologic indicators. Here, we optimized our method allowing for semi-automated telomere length determination in single cells in fixed tissue, and tested the telomere biomarker in five cohort studies of men surgically treated for clinically localized disease (N = 2,255). We estimated the relative risk (RR) of progression to metastasis (N = 311) and prostate cancer death (N = 85) using models appropriate to each study's design adjusting for age, prostatectomy stage, and tumor grade, which then we meta-analyzed using inverse variance weights. Compared with men who had less variable telomere length among prostate cancer cells and longer telomere length in prostate cancer-associated stromal cells, men with the combination of more variable and shorter telomere length had 3.76 times the risk of prostate cancer death (95% confidence interval [CI] 1.37-10.3, p = 0.01) and had 2.23 times the risk of progression to metastasis (95% CI 0.99-5.02, p = 0.05). The telomere biomarker was associated with prostate cancer death in men with intermediate risk disease (grade groups 2/3: RR = 9.18, 95% CI 1.14-74.0, p = 0.037) and with PTEN protein intact tumors (RR = 6.74, 95% CI 1.46-37.6, p = 0.015). In summary, the telomere biomarker is robust and associated with poor outcome independent of current pathologic indicators in surgically treated men.

The diagnostic and prognostic utility of incorporating DAXX, ATRX, and alternative lengthening of telomeres to the evaluation of pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of neoplasms with increasing incidence and an ill-defined pathobiology. Although many PanNETs are indolent and remain stable for years, a subset may behave aggressively and metastasize widely. Thus, the increasing and frequent detection of PanNETs presents a treatment dilemma. Current prognostic systems are susceptible to interpretation errors, sampling issues, and do not accurately reflect the clinical behavior of these neoplasms. Hence, additional biomarkers are needed to improve the prognostic stratification of patients diagnosed with a PanNET. Recent studies have identified alterations in death domain-associated protein 6 (DAXX) and alpha-thalassemia/mental retardation X-linked (ATRX), as well as alternative lengthening of telomeres (ALT), as promising prognostic biomarkers. This review summarizes the identification, clinical utility, and specific nuances in testing for DAXX/ATRX by immunohistochemistry and ALT by telomere-specific fluorescence in situ hybridization in PanNETs. Furthermore, a discussion on diagnostic indications for DAXX, ATRX, and ALT status is provided to include the distinction between PanNETs and pancreatic neuroendocrine carcinomas (PanNECs), and determining pancreatic origin for metastatic neuroendocrine tumors in the setting of an unknown primary.

Therapeutic Vulnerability to ATR Inhibition in Concurrent NF1 and ATRX-Deficient/ALT-Positive High-Grade Solid Tumors.

Subsets of Neurofibromatosis Type 1 (NF1)-associated solid tumors have been shown to display high frequencies of ATRX mutations and the presence of alternative lengthening of telomeres (ALT). We studied the phenotype of combined NF1 and ATRX deficiency in malignant solid tumors. Cell lines derived from NF1-deficient sporadic glioblastomas (U251, SF188), an NF1-associated ATRX mutant glioblastoma cell line (JHH-NF1-GBM1), an NF1-derived sarcoma cell line (JHH-CRC65), and two NF1-deficient MPNST cell lines (ST88-14, NF90.8) were utilized. Cancer cells were treated with ATR inhibitors, with or without a MEK inhibitor or temozolomide. In contrast to the glioma cell line SF188, combined ATRX knockout (KO) and TERC KO led to ALT-like properties and sensitized U251 glioma cells to ATR inhibition in vitro and in vivo. In addition, ATR inhibitors sensitized U251 cells to temozolomide, but not MEK inhibition, irrespective of ATRX level manipulation; whereas, the JHH-NF1-GBM1 cell line demonstrated sensitivity to ATR inhibition, but not temozolomide. Similar effects were noted using the MPNST cell line NF90.8 after combined ATRX knockdown and TERC KO; however, not in ST88-14. Taken together, our study supports the feasibility of targeting the ATR pathway in subsets of NF1-deficient and associated tumors.

Novel forms of prostate cancer chemoresistance to successful androgen deprivation therapy demand new approaches: Rationale for targeting BET proteins.

In patients with prostate cancer, the duration of remission after treatment with androgen deprivation therapies (ADTs) varies dramatically. Clinical experience has demonstrated difficulties in predicting individual risk for progression due to chemoresistance. Drug combinations that inhibit androgen biosynthesis (e.g., abiraterone acetate) and androgen signaling (e.g., enzalutamide or apalutamide) have proven so effective that new forms of ADT resistance are emerging. In particular, prostate cancers with a neuroendocrine transcriptional signature, which demonstrate greater plasticity, and potentially, increased predisposition to metastasize, are becoming more prevalent. Notably, these subtypes had in fact been relatively rare before the widespread success of novel ADT regimens. Therefore, better understanding of these resistance mechanisms and potential alternative treatments are necessary to improve progression-free survival for patients treated with ADT. Targeting the bromodomain and extra-terminal (BET) protein family, specifically BRD4, with newer investigational agents may represent one such option. Several families of chromatin modifiers appear to be involved in ADT resistance and targeting these pathways could also offer novel approaches. However, the limited transcriptional and genomic information on ADT resistance mechanisms, and a serious lack of patient diversity in clinical trials, demand profiling of a much broader clinical and demographic range of patients, before robust conclusions can be drawn and a clear direction established.

Mutational Landscape and Outcomes of Conjunctival Melanoma in 101 Patients.

PURPOSE: To evaluate targetable mutations and molecular genetic pathways in conjunctival melanoma with clinical correlation. DESIGN: Observational case series. PARTICIPANTS: Patients with conjunctival melanoma. MAIN OUTCOME MEASURES: Mutational profile of the tumor by next-generation sequencing (NGS), alternative lengthening of telomeres (ALT) by fluorescence in situ hybridization (FISH), and ATRX immunohistochemistry. Outcomes at 2 years and 5 years of tumor-related metastasis and death were recorded. RESULTS: Of the 101 patients, mean age at presentation was 60 years, 52% were male, and 88% were White. The NGS panels initially targeted BRAF only (n = 6, 6%), BRAF/NRAS (n = 17, 17%), and BRAF/NRAS/NF1 (n = 10, 10%). Sixty-eight tumors were tested with the expanded 592-gene panel. Next-generation sequencing identified high-frequency mutations in NF1 (29/74, 39%), BRAF (31/101, 31%), NRAS (25/95, 26%), and ATRX (17/68, 25%). Of those with an ATRX mutation, 12 (71%) had an additional NF1 mutation. A subset analysis of 21 melanomas showed that the ATRX mutation was associated with loss of ATRX protein expression and ALT. Loss of ATRX expression and ALT were present in both intraepithelial and invasive tumors, suggesting that an ATRX mutation is an early event in conjunctival melanoma progression. The NF1 and ATRX mutations were associated with tarsal (vs. nontarsal) tumors (NF1: 28% vs. 9%, P = 0.035, ATRX: 41% vs. 14%, P = 0.021) and orbital (vs. nonorbital) tumors (ATRX: 24% vs. 2%, P = 0.007). ATRXMUT (vs. ATRXWT) tumors were associated with a lower 2-year rate of metastasis (0% vs. 24%, P = 0.005). NRASMUT (vs. NRASWT) tumors were associated with a greater 2-year rate of metastasis (28% vs. 14%, P = 0.07) and death (16% vs. 4%, P = 0.04), with a 5-fold increased risk of death (relative risk, 5.45 [95% confidence interval, 1.11-26.71], P = 0.039). CONCLUSIONS: This study confirms the high frequency of previously documented BRAF and NRAS mutations and recently reported ATRX and NF1 mutations in conjunctival melanoma. An NRAS mutation implied increased risk for metastasis and death. Loss of ATRX and ALT may be early events in conjunctival melanoma development.

Novel plasma exosome biomarkers for prostate cancer progression in co-morbid metabolic disease.

Comorbid Type 2 diabetes (T2D), a metabolic complication of obesity, associates with worse cancer outcomes for prostate, breast, head and neck, colorectal and several other solid tumors. However, the molecular mechanisms remain poorly understood. Emerging evidence shows that exosomes carry miRNAs in blood that encode the metabolic status of originating tissues and deliver their cargo to target tissues to modulate expression of critical genes. Exosomal communication potentially connects abnormal metabolism to cancer progression. Here, we hypothesized that T2D plasma exosomes induce epithelial-mesenchymal transition (EMT) and immune checkpoints in prostate cancer cells. We demonstrate that plasma exosomes from subjects with T2D induce EMT features in prostate cancer cells and upregulate the checkpoint genes CD274 and CD155. We demonstrate that specific exosomal miRNAs that are differentially abundant in plasma of T2D adults compared to nondiabetic controls (miR374a-5p, miR-93-5p and let-7b-3p) are delivered to cancer cells, thereby regulating critical target genes. We build on our previous reports showing BRD4 controls migration and dissemination of castration-resistant prostate cancer, and transcription of key EMT genes, to show that T2D exosomes require BRD4 to drive EMT and immune ligand expression. We validate our findings with gene set enrichment analysis of human prostate tumor tissue in TGCA genomic data. These results suggest novel, non-invasive approaches to evaluate and potentially block progression of prostate and other cancers in patients with comorbid T2D.

Clinical implications of cell-of-origin epigenetic characteristics in non-functional pancreatic neuroendocrine tumors.

Primary non-functional pancreatic neuroendocrine tumors (NF-PanNETs) are a heterogeneous group of neuroendocrine neoplasms that display highly variable clinical behavior. Therefore, NF-PanNETs often present clinical teams with a dilemma: the uncertain metastatic potential of the tumor has to be weighed against the morbidity associated with surgical resection. Thus, rather than utilizing current radiologic thresholds, there is an urgent need for improved prognostic biomarkers. Recent studies aimed at understanding the epigenetic underpinnings of NF-PanNETs have led to the identification of tumor subgroups based on histone modification and DNA methylation patterns. These molecular profiles tend to resemble the cellular origins of PanNETs. Subsequent retrospective analyses have demonstrated that these molecular signatures are of prognostic value and, importantly, may be useful in the preoperative setting. These studies have highlighted that sporadic NF-PanNETs displaying biomarkers associated with disease progression and poor prognosis, such as alternative lengthening of telomeres, inactivating alpha thalassemia/mental retardation X-linked (ATRX) or death domain-associated protein (DAXX) gene mutations, or copy number variations, more often display alpha cell characteristics. Conversely, NF-PanNETs with beta cell characteristics often lack these unfavorable biomarkers. Alternative lengthening of telomeres, transcription factor protein expression, and possibly DNA methylation can be assessed in endoscopic ultrasound-guided tumor biopsies. Prospective studies focusing on cell-of-origin and epigenetic profile-driven decision making prior to surgery are likely to be routinely implemented into clinical practice in the near future. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

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.