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The genetics of nephroblastoma (Wilms tumor) occurring in adults is largely unknown, as studies have largely been limited to isolated case reports. We, therefore, studied 14 adult Wilms tumors for genetic alterations, using expanded targeted sequencing on 11 cases. The patients ranged from 17 to 46 years of age (mean and median, 31 y), and there were 8 males and 6 females. Five Wilms tumors harbored BRAF V600E mutations. All of these had better-differentiated areas identical to metanephric adenoma, as has previously been described. In 3 such cases, microdissection studies revealed that the BRAF V600E mutation was present in both the metanephric adenoma and Wilms tumor areas; however, additional genetic alterations (including TERT promoter mutations in 2 cases, ASLX1/ATR mutations in 1 other case) were limited to the Wilms tumor component. These findings suggest that the Wilms tumor developed from the metanephric adenoma. Other adult Wilms tumors harbored genetic alterations previously reported in the more common pediatric Wilms tumors, including WT1 mutations (2 cases), ASLX1 mutations (3 additional cases), NSD2 mutation (1 additional case), and 11p loss (3 cases). In summary, a significant subset of adult Wilms tumors (specifically those of epithelial type with differentiated areas) harbor targetable BRAF V600E mutations and appear to arise from metanephric adenomas as a consequence of additional acquired genetic alterations. Other adult Wilms tumors often harbor genetic alterations found in their more common pediatric counterparts, suggesting at least some similarities in their pathogenesis.

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 chromogenicin situhybridization (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.

Lipid uptake occurs through caveolae, plasma membrane invaginations formed by caveolins (CAV) and caveolae-associated protein 1 (CAVIN1). Genetic alterations of CAV1N1 and CAV1 modify lipid metabolism and underpin lipodystrophy syndromes. Lipids contribute to tumorigenesis by providing fuel to cancer metabolism and supporting growth and signaling. Tumor stroma promotes tumor proliferation, invasion, and metastasis, but how stromal lipids influence these processes remain to be defined. Here, we show that stromal CAVIN1 regulates lipid abundance in the prostate cancer microenvironment and suppresses metastasis. We show that depletion of CAVIN1 in prostate stromal cells markedly reduces their lipid droplet accumulation and increases inflammation. Stromal cells lacking CAVIN1 enhance prostate cancer cell migration and invasion. Remarkably, they increase lipid uptake and M2 inflammatory macrophage infiltration in the primary tumors and metastasis to distant sites. Our data support the concept that stromal cells contribute to prostate cancer aggressiveness by modulating lipid content and inflammation in the tumor microenvironment.Implications:This study showed that stromal CAVIN1 suppresses prostate cancer metastasis by modulating tumor microenvironment, lipid content, and inflammatory response.

Obesity and inactivity have been associated with advanced-stage prostate cancer, and poor prostate cancer outcomes, though the underlying mechanism(s) is unknown. To determine whether telomere shortening, which has been associated with lethal prostate cancer, may be a potential underlying mechanism, we prospectively evaluated the association between measures of adiposity, physical activity, and telomere length in 596 participants in the Health Professionals Follow-up Study, who were surgically treated for prostate cancer. Using tissue microarrays, we measured telomere length in cancer and benign cells using a telomere-specific FISH assay. Adiposity and activity were assessed via questionnaire within 2 years of diagnosis. Adjusting for age, pathologic stage, and grade, the median and SD of the per cell telomere signals were determined for each man for stromal cells and cancer cells by adiposity and activity categories. Overweight/obese men (54%) were similar to normal weight men on most factors, but had higher Gleason sum and lower activity levels. Overweight/obese men had 7.4% shorter telomeres in stromal cells than normal weight men (P = 0.06). The least active men had shorter telomeres in stromal cells than more active men (Ptrend = 0.002). Men who were overweight/obese and the least active had the shortest telomeres in stromal cells (20.7% shorter; P = 0.0005) compared with normal weight men who were the most active. Cancer cell telomere length and telomere length variability did not differ by measures of adiposity or activity. Telomere shortening in prostate cells may be one mechanism through which lifestyle influences prostate cancer risk and outcomes. Cancer Prev Res; 8(8); 737–42. ©2015 AACR.

Supplemental Table 1 and 2. Association of pre-diagnostic BMI, waist circumference, weight change from age 21, total physical activity and vigorous physical activity with telomere length (Supplemental Table 1) and telomere variability (Supplemental Table 2) in cancer cells, stromal cells, basal cells, and luminal epithelial cells in men surgically treated for clinically localized prostate cancer, HPFS.

Figure S1 shows cell growth and lipid distribution data. Figure S2 shows lipid distribution and invasion data. Figure S3 shows additional transcriptomic data. Figure S4 and S5 shows additional orthotopic data.

Odds ratios (OR) and 95% confidence intervals (CI) for the association between body mass index (BMI) category and telomere variability* in prostate cancer cells overall and stratified by prostate cancer aggressiveness at surgery among men surgically treated for prostate cancer at Johns Hopkins Hospital, 1992 to 2016.

Supplementary Figure 1: Radiotherapy suppresses tumor growth. Supplementary Figure 2: Characterization of the TME of B16/F10 tumors post-RT. Supplementary Figure 3: Stereotactic radiation does not change Treg in draining lymph nodes nor in spleens of treated mice. Supplementary Figure 4: Persistently increased Treg Post-RT. Supplementary Figure 5: Stereotactic radiation increases the suppressive markers of Treg in the RENCA tumor model. Supplementary Figure 6: Stereotactic radiation increases the suppressive markers of Treg in the MC38 tumor model. Supplementary Figure 7: Stereotactic radiation does not change the expression of the suppressive markers of Treg in DLNs. Supplementary Figure 8: Stereotactic radiation does not change the expression of the suppressive markers of Treg in spleens. Supplementary Figure 9: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the B16/F10 model. Supplementary Figure 10: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the RENCA model. Supplementary Figure 11: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the MC38 model. Supplementary Figure 12: Expression of 4-1BB on TIL-CD8+ cells. Supplementary Figure 13: The effect of TGF-beta blockade on different T cell subsets. Supplementary Figure 14: TGF-beta expression in the tumor microenvironment. Supplementary Figure 15: The effect of Fingolimod (FTY720) on peripheral blood lymphocytes counts. Supplementary Figure 16: Chemokine/cytokine expression of a selected panel in the tumor microenvironment post-radiation.

Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992–1004. ©2017 AACR.

Table S1. Sequences of oligonucleotides for CRISPR, telomere assays, and RT-qPCR. Table S2. Mean telomere lengths assessed from TRF southern blots of LAPC-4 EV and ATRXKO clones, and CWR22Rv1 EV and ATRXKO clones. Table S3. Microarray results of top 10 differentially expressed genes ranked by FDR adjusted P-value, comparing parental ATRX WT cell lines (LAPC-4 and CWR22Rv1) versus ATRXKO cell lines (LAPC-4 ATRXKO1-3 and CWR22Rv1 ATRXKO1-3). Table S4. Microarray results of top 10 differentially expressed genes with FDR adjusted P-value < 0.005 ranked by absolute fold-change, comparing LAPC-4 ATRXKO1-3 clones versus CWR22Rv1 ATRXKO1-3 clones.

Table S1: Weight of the tumors;Figure S1: Correlation of in vivo NIR imaging and ex vivo biodistribution analysis; Figure S2: Evaluation of injected J591-conjugates; Figure S3: Assessment of tumor vascular permeability; Figure S4: Passive dye uptake in PSMA-positive and PSMA-negative tumors; Figure S5: Ex vivo tumor image analysis of LMD/PSMA xenograft model; Figure S6: Ex vivo biodistribution analysis of J591-conjugates in LMD tumor model; Figure S7: Ex vivo tumor image analysis of PC3/PSMA xenograft model; Figure S8: PSMA expression and J591 conjugate distribution

In our prior studies, obesity was associated with shorter telomeres in prostate cancer-associated stromal (CAS) cells, and shorter CAS telomeres were associated with an increased risk of prostate cancer death. To determine whether the association between obesity and shorter CAS telomeres is replicable, we conducted a pooled analysis of 790 men who were surgically treated for prostate cancer, whose tissue samples were arrayed on five tissue microarray (TMA) sets. Telomere signal was measured using a quantitative telomere-specific FISH assay and normalized to 4′,6-diamidino-2-phenylindole for 351 CAS cells (mean) per man; men were assigned their median value. Weight and height at surgery, collected via questionnaire or medical record, were used to calculate body mass index (BMI; kg/m2) and categorize men as normal (T2), obese men had a 3-fold increased odds of short CAS telomeres (OR: 3.06; 95% confidence interval: 1.07–8.75; Ptrend = 0.045) when compared with normal weight men. Telomere shortening in prostate stromal cells may be one mechanism through which lifestyle influences lethal prostate carcinogenesis.Prevention Relevance:This study investigates a potential mechanism underlying the association between obesity and prostate cancer death. Among men with aggressive prostate cancer, obesity was associated with shorter telomeres prostate cancer associated stromal cells, and shorter CAS telomeres have been associated with an increased risk of prostate cancer death.

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRXKO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRXKO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRXKO TERCmut cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance.Implications:These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.

Figure S1. LAPC-4 and CWR22RV1 are telomerase-positive ALT-negative cell lines. Figure S2. ATRXKOs were generated in LAPC-4 and CWR22Rv1 cells. Figure S3. Representative telomere restriction fragment (TRF) southern blots and signal intensity traces. Figure S4. TERT and TERC expression levels. Figure S5. CRISPR cas9 genome editing strategies targeting the TERC locus. Figure S6. RT-qPCR validation of genes differentially expressed in microarray.

Supplementary Materials and Methods

Background: TH1 cytokines, such as IFNγ and TNFα, and potentially innate cytokines, such as IL6, can potentiate the immune response to tumor. Cytokines, such as IL1β, IL8, and IL10, may suppress anticancer immunity. Thus, we prospectively evaluated the association between peripheral-cytokine concentrations and prostate cancer.Methods: We conducted an age-race matched case–control study (268 pairs) of incident prostate cancer in CLUE-II. We measured plasma IFNγ, IL10, IL12p70, IL1β, IL6, IL8, and TNFα concentrations using an ultrasensitive multiplex kit. ORs and 95% confidence intervals (CI) were calculated using conditional logistic regression.Results: The OR of prostate cancer decreased across quartiles of IFNγ (highest vs. lowest quartiles: OR, 0.49; 95% CI, 0.30–0.81; Ptrend = 0.006), TNFα (OR, 0.56; 95% CI, 0.33–0.96; Ptrend = 0.01), and IL6 (OR, 0.46; 95% CI, 0.26–0.79; Ptrend = 0.007). Higher TNFα (OR, 0.28; 95% CI, 0.09–0.85; Ptrend = 0.01) and IL6 (OR, 0.20; 95% CI, 0.06–0.67; Ptrend = 0.003) concentrations were associated with lower Gleason sum ≥7 disease risk. Other cytokines were not as clearly associated with risk.Conclusions: Men with a prediagnostic circulating TH1 profile and higher IL6 may have a lower risk of prostate cancer, including aggressive disease. Whether this profile reflects (i) an intraprostatic immune environment in benign tissue that protects against prostate cancer, (ii) the immune milieu in response to a prostate adenocarcinoma that inhibits tumor growth and detectability, and/or (iii) a systemic immune profile that mediates the influence of modifiable factors on risk, warrants additional study.Impact: Identifying specific inflammatory cytokines associated with prostate cancer may lead to improved prevention and treatment strategies. Cancer Epidemiol Biomarkers Prev; 23(11); 2561–7. ©2014 AACR.

Supplementary methods

Supplementary Table S4: DAVID clustering of 1660 Illumina probes up-regulated at least 2-fold in PD-L1+ melanomas

Supplemental Figure 1. (A) ALT-positive PanNET patients with lymph node metastasis (median survival, 131 months) had a tendency of better survival than ALT-negative PanNET patients with nodal metastasis (54 months, HR=0.31, 95% CI=0.08-1.19; p=0.088). (B) ALT-positive PanNET patients with distant metastasis (overall 5-year survival rate, 75.0%) trended toward better survival than ALT-negative patients (25.0%), but was not statistically significant (p=0.118).

Supplementary Table S1, Supplementary Table S3, Supplementary Table S5. Supplementary Table S1: Genes selected for multiplex qRT-PCR array Supplementary Table S3: Functionally annotated gene categories from DAVID analysis of whole genome microarray results comparing PD-L1+ to PD-L1(-) melanomas. Supplementary Table S5: Genes over-expressed in PD-L1+ vs. PD-L1(-) melanomas, assessed by qRT-PCR.

Purpose: Alternative lengthening of telomeres (ALT), a telomerase-independent telomere maintenance mechanism, is strongly associated with ATRX and DAXX alterations and occurs frequently in pancreatic neuroendocrine tumors (PanNET).Experimental Design: In a Korean cohort of 269 surgically resected primary PanNETs and 19 sporadic microadenomas, ALT status and nuclear ATRX and DAXX protein expression were assessed and compared with clinicopathologic factors.Results: In PanNETs, ALT or loss of ATRX/DAXX nuclear expression was observed in 20.8% and 19.3%, respectively, whereas microadenomas were not altered. ALT-positive PanNETs displayed a significantly higher grade, size, and pT classification (all, P < 0.001). ALT also strongly correlated with lymphovascular (P < 0.001) and perineural invasion (P = 0.001) and the presence of lymph node (P < 0.001) and distant metastases (P = 0.002). Furthermore, patients with ALT-positive primary PanNETs had a shorter recurrence-free survival [HR = 3.38; 95% confidence interval (CI), 1.83–6.27; P < 0.001]. Interestingly, when limiting to patients with distant metastases, those with ALT-positive primary tumors had significantly better overall survival (HR = 0.23; 95% CI, 0.08–0.68; P = 0.008). Similarly, tumors with loss of ATRX/DAXX expression were significantly associated with ALT (P < 0.001), aggressive clinical behavior, and reduced recurrence-free survival (P < 0.001). However, similar to ALT, when limiting to patients with distant metastases, loss of ATRX/DAXX expression was associated with better overall survival (P = 0.003).Conclusions: Both primary ALT-positive and ATRX/DAXX-negative PanNETs are independently associated with aggressive clinicopathologic behavior and displayed reduced recurrence-free survival. In contrast, ALT activation and loss of ATRX/DAXX are both associated with better overall survival in patients with metastases. Therefore, these biomarkers may be used as prognostic markers depending on the context of the disease. Clin Cancer Res; 23(6); 1598–606. ©2016 AACR.

Correlation between ALT status and ATRX and DAXX protein expression.

Purpose: Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1+ versus PD-L1(−) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1+ melanoma microenvironment coordinately contributing to immunosuppression.Experimental Design: Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1+ versus PD-L1(−) melanomas.Results: Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1+ melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1+ melanomas, involved in CD8+ T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells.Conclusions: These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti–PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies. Clin Cancer Res; 21(17); 3969–76. ©2015 AACR.

Figure S1, Figure S2, Figure S3. Supplementary Figure S1: Overlay of PD-L1 and LAG-3 IHC demonstrates close geographic association of these cell-surface markers in a melanoma lymph node metastasis. Supplementary Figure S2: Comparison of immunohistochemistry (IHC) and in situ hybridization (ISH) for LAG-3 expression in a melanoma lymph node metastasis. Supplementary Figure S3: IL-32-g activates CD3+ T cells and induces their expression of PD-L1.

Risk of prostate cancer excluding cases diagnosed within 2 and 5 years comparing highest quartilea of cytokine concentration with lowest quartile_a of concentration, CLUE II.

(A) ALT-positive PanNET patients with lymph node metastasis (median survival, 131 months) had a tendency of better survival than ALT-negative PanNET patients with nodal metastasis (54 months, HR=0.31, 95% CI=0.08-1.19; p=0.088). (B) ALT-positive PanNET patients with distant metastasis (overall 5-year survival rate, 75.0%) trended toward better survival than ALT-negative patients (25.0%), but was not statistically significant (p=0.118).

Subsets of high-grade gliomas, including glioblastoma (GBM), are known to utilize the alternative lengthening of telomeres (ALT) pathway for telomere length maintenance. However, the telomere maintenance profile of one subtype of GBM-giant cell GBM-has not been extensively studied. Here, we investigated the prevalence of ALT, as well as ATRX and SMARCAL1 protein loss, in a cohort of classic giant cell GBM and GBM with giant cell features. To determine the presence of ALT, a telomere-specific fluorescence in situ hybridization assay was performed on 15 cases of classic giant cell GBM, 28 additional GBMs found to have giant cell features, and 1 anaplastic astrocytoma with giant cell features. ATRX, SMARCAL1, and IDH1 protein status were assessed in a proportion of cases by immunohistochemistry and were compared to clinical-pathologic and molecular characteristics. In the overall cohort of 44 cases, 19 (43%) showed evidence of ALT. Intriguingly, of the ALT-positive cases, only 9 (47.4%) displayed loss of the ALT suppressor ATRX by immunohistochemistry. Since inactivating mutations in SMARCAL1 have been identified in ATRX wild-type ALT-positive gliomas, we developed an immunohistochemistry assay for SMARCAL1 protein expression using genetically validated controls. Of the 19 ALT-positive cases, 6 (31.5%) showed loss or mis-localization of SMARCAL1 by immunohistochemistry. Of these cases, four retained ATRX protein expression, while two cases also displayed ATRX loss. Additionally, we assessed five cases from which multiple temporal samples were available and ALT status was concordant between both tumor biopsies. In summary, we have identified a subset of giant cell GBM that utilize the ALT telomere maintenance mechanism. Importantly, in addition to ATRX loss, ALT-positive tumors harboring SMARCAL1 alterations are prevalent in giant cell GBM.

Heterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1R132H/WT) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1R132H/WT in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1R132H/WT-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1R132H/WT transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G2–M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non–cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1R132H/WT upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies. Implications: The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant–mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.

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.

PurposeCurrent 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.Experimental DesignWe optimized our method allowing for semi-automated telomere length determination in single cells in fixed tissue, and tested the telomere biomarker in tissue microarrays from 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.ResultsCompared 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% 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 intact tumors (RR=6.74, 95% CI 1.46-37.6, p=0.015).ConclusionsThe telomere biomarker is robust and associated with poor outcome independent of current pathologic indicators in surgically-treated men.Translational RelevanceCurrent prognostic biomarkers in localized prostate cancer are inadequate imperfect predictors; therefore, new biomarkers are needed to improve the prognostic classification and management of these patients. In a five-cohort study, we confirmed that the tissue-based telomere biomarker – the combination of more variable telomere length among prostate cancer cells and shorter telomere length in prostate cancer-associated stromal cells – was associated with progression to metastasis and prostate cancer death independent of currently used prognostic indicators after prostatectomy for clinically-localized disease. Importantly, the telomere biomarker was associated with poor outcome in men with intermediate risk disease, as well as in men with intact PTEN tumors. Thus, this tissue-based telomere biomarker has the translational potential to improve treatment and surveillance decision-making.

Epigenetic alterations are increasingly recognized as important contributors to the development and progression of pancreatic ductal adenocarcinoma. 5-hydroxymethylcytosine (5hmC) is an epigenetic DNA mark generated through the ten-eleven translocation (TET) enzyme-mediated pathway and is closely linked to gene activation. However, the timing of alterations in epigenetic regulation in the progression of pancreatic neoplasia is not well understood. In this study, we hypothesized that aberrant expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and subsequent global 5hmC alteration are linked to early tumorigenesis in the pancreas. Therefore, we evaluated alterations of 5hmC and TET1 levels using immunohistochemistry in pancreatic neoplasms (n = 380) and normal ducts (n = 118). The study cohort included representation of the full spectrum of precancerous lesions from low- and high-grade pancreatic intraepithelial neoplasia (n = 95), intraductal papillary mucinous neoplasms (all subtypes, n = 129), intraductal oncocytic papillary neoplasms (n = 12), and mucinous cystic neoplasms (n = 144). 5hmC and TET1 were significantly downregulated in all types of precancerous lesion and associated invasive pancreatic ductal adenocarcinomas compared with normal ductal epithelium (all p < 0.001), and expression of 5hmC positively correlated with expression of TET1. Importantly, downregulation of both 5hmC and TET1 was observed in most low-grade precancerous lesions. There were no clear associations between 5hmC levels and clinicopathological factors, thereby suggesting a common epigenetic abnormality across precancerous lesions. We conclude that downregulation of 5hmC and TET1 is an early event in pancreatic tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cancer-associated fibroblasts (CAFs) expressing FAP promote tumor growth, invasion and immune suppression. FAP is a molecular imaging target in cancer and its protease activity is a potential anti-cancer drug target. While FAP expression was associated with biochemical recurrence after prostatectomy using tissue microarrays (TMAs)(DOI:10.1158/2767-9764.CRC-21-0183), its expression has not been comprehensively correlated with morphological features of disease aggressiveness beyond grade. 48 cases of localized prostatic adenocarcinoma were used for a multiplex immunohistochemical assay that included FAP, CD45, CD163, p63 and Keratin 8. Each of these markers were individually optimized by single-plex staining and were then performed as an iterative IHC assay (PMID:35188986) and quantification was performed using the HALO image analysis platform (Indica Labs). We developed a second multiplex assay including FAP, vimentin, smooth muscle actin, desmin, CD31, S100, keratin 8 and p63. FAP IHC staining was analytically validated using a series of positive and negative cell lines. In normal appearing (non-inflamed) prostate regions, FAP expression was negative. By contrast, upregulation of FAP was observed in the stromal compartment in regions of chronic inflammation and some proliferative inflammatory atrophy (PIA) lesions. In tumor regions, only 1 case was totally negative for FAP. Except for 2 cases, in which there was focal weak tumor cell staining, all FAP expression was present in the tumor stromal compartment (TSC). The vast majority of FAP expression was associated with Vim+/Des- fibroblasts, which at times were also positive for SMA (Vim+/Des-/SMA+), indicating they are myofibroblasts. There was very infrequent FAP expression associated with M2 macrophages, endothelial cells, nerves and vimentin-negative smooth muscle cells, indicating that the majority of FAP expression occurs in fibroblasts. Preliminary quantitative data using visual estimations of the fraction of TSC expressing FAP (range, 0-80%, mean=35.8%, median=30%, SD=23.7) was not associated with the overall GS/grade group, but was associated with non-organ confined disease. Furthermore, there was a trend towards increased FAP expression in cases showing intraductal carcinoma and large cribriform formation. Ongoing studies are in process to obtain quantitative data by image analysis and to correlate FAP expression with the presence of overall inflammatory cell density and the density of M2 macrophages. Additionally, we will characterize the cases in terms of molecular features including TMPRSS2-ERG status and PTEN genomic status. Future use of these panels will facilitate studies of associations of FAP with clinical outcome, MRI and PET imaging, response to specific treatments and to help characterize FAP as a predictor of response to emerging FAP inhibitors and FAP-targeted prodrugs. Citation Format: Fernanda Caramella Pereira, Alan K. Meeker, Kenneth Pienta, Qizhi Zheng, Tracy Jones, Srinivasan Yegnasubramanian, Jessica L. Hicks, Sujayita Roy, Angelo M. De Marzo, W. Nathaniel Brennen. Multiplex IHC assay to explore regional heterogeneity of FAP and inflammatory cell density in localized prostatic adenocarcinoma. [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 4631.

The role of truncated androgen receptor splice variant-7 (AR-V7) in prostate cancer biology is an unresolved question. Is it simply a marker of resistance to 2nd-generation androgen receptor signaling inhibitors (ARSi) like abiraterone acetate (Abi) and enzalutamide (Enza) or a functional driver of lethal resistance via its ligand-independent transcriptional activity? To resolve this question, the correlation between resistance to ARSi and genetic chances and expression of full length AR (AR-FL) vs. AR-V7 were evaluated in a series of independent patient-derived xenografts (PDXs). While all PDXs lack PTEN expression, there is no consistent requirement for mutation in TP53, RB1, BRCA2, PIK3CA, or MSH2, or expression of SOX2 or ERG and ARSi resistance. Elevated expression of AR-FL alone is sufficient for Abi but not Enza resistance, even if AR-FL is gain-of-function (GOF) mutated. Enza resistance is consistently correlated with enhanced AR-V7 expression. In vitro and in vivo growth responses of Abi-/Enza-resistant LNCaP-95 cells in which CRISPR-Cas9 was used to knockout AR-FL or AR-V7 alone or in combination were evaluated. Combining these growth responses with RNAseq analysis demonstrates that both AR-FL- and AR-V7-dependent transcriptional complementation are needed for Abi/Enza resistance.

Lipid uptake occurs through caveolae, plasma membrane invaginations formed by caveolins (CAV) and caveolae-associated protein 1 (CAVIN1). Genetic alterations of CAV1N1 and CAV1 modify lipid metabolism and underpin lipodystrophy syndromes. Lipids contribute to tumorigenesis by providing fuel to cancer metabolism and supporting growth and signaling. Tumor stroma promotes tumor proliferation, invasion, and metastasis, but how stromal lipids influence these processes remain to be defined. Here, we show that stromal CAVIN1 regulates lipid abundance in the prostate cancer microenvironment and suppresses metastasis. We show that depletion of CAVIN1 in prostate stromal cells markedly reduces their lipid droplet accumulation and increases inflammation. Stromal cells lacking CAVIN1 enhance prostate cancer cell migration and invasion. Remarkably, they increase lipid uptake and M2 inflammatory macrophage infiltration in the primary tumors and metastasis to distant sites. Our data support the concept that stromal cells contribute to prostate cancer aggressiveness by modulating lipid content and inflammation in the tumor microenvironment. Implications: This study showed that stromal CAVIN1 suppresses prostate cancer metastasis by modulating tumor microenvironment, lipid content, and inflammatory response.

BACKGROUND: Small cell neuroendocrine carcinoma (SCNC) of the prostate is an aggressive subtype with frequent TP53 mutation and RB1 inactivation, however the molecular phenotype remains an area of investigation. Here, we compared telomere lengths in prostatic SCNC and usual-type prostatic adenocarcinoma (AdCa). METHODS: We studied 32 cases of prostatic SCNC (including 11 cases with concurrent AdCa) and 347 cases of usual-type AdCa on tissue microarrays. Telomere lengths in tumor cells were qualitatively compared to that in normal cells using a telomere-specific fluorescence in situ hybridization assay. ERG, PTEN and TP53 status were assessed in a proportion of cases using genetically validated immunohistochemistry protocols. Clinical-pathologic and molecular characteristics of cases were compared between telomere groups using the chi-square test. RESULTS: A significantly higher proportion of prostatic SCNC cases (50%, 16/32) showed normal/long telomeres compared to AdCa cases (11%, 39/347; p

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

Subsets of Neurofibromatosis Type 1 (NF1)-associated solid tumors have been shown to display high frequencies of ATRX mutations and presence of alternative lengthening of telomeres (ALT). In addition, a potential biologic vulnerability of ALT-positive cancer cells is ATR inhibition, a regulator of homologous recombination. We studied the phenotype of combined NF1 and ATRX deficiency in malignant solid tumors. For these experiments, cell lines derived from NF1-deficient sporadic glioblastomas (U251, SF188), a NF1-associated ATRX mutant glioblastoma cell line (JHH-NF1-GBM1), a 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 (AZD6738 and VE-822), in vitro, with or without a MEK inhibitor (AZD6244, selumetinib) 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 (AZD6738 and VE-822) in vitro and in vivo. In addition, ATR inhibitors sensitized U251 cells to temozolomide, but not MEK inhibition (AZD6244), irrespective of ATRX level manipulation; whereas, the JHH-NF1-GBM1 cell line (ATRX loss/ALT-positive) demonstrated sensitivity to ATR inhibition (AZD6738), but not temozolomide. Similar effects were noted using the MPNST cell line NF90.8 after combined ATRX knockdown and TERC KO; however, not in the MPNST cell line ST88-14. Taken together, our study supports the feasibility of targeting the ATR pathway in subsets of NF1-deficient and associated tumors. Tumors with pre-existing ALT, or that subsequently develop ALT after ATRX downregulation, are particularly vulnerable to this therapeutic approach.

Telomeres, the repetitive DNA elements at chromosome ends, are pivotal for maintenance of genome integrity. Previous studies from our group and others have highlighted the translational potential of tissue-based telomere length measurements to address the clinical challenge of improving diagnosis, individualized risk stratification, and accurate prognostication of different diseases. Here, we describe a high-throughput method that quantitates cell type-specific telomere lengths at a single cell level in archival tissues from patient cohorts for research on prognosis. This approach is based on telomere-specific fluorescence in situ hybridization (FISH) combined with multiplex immunostaining for cell type-specific antibodies, followed by semi-automated slide scanning and multi-channel acquisition of fluorescent images using the TissueFAXS Plus microscopy workstation and TissueQuest software (TissueGnostics). Here, we demonstrate that this method is sufficiently robust and reproducible to detect biologically significant differences in telomere lengths in archived tissues either on whole slides or sampled across tissue microarrays, which is essential when assessing prognosis in large patient cohorts.

High-grade glioma, including anaplastic astrocytoma and glioblastoma (GBM) patients, have a poor prognosis due to the lack of effective treatments. Therefore, the development of new therapeutic strategies to treat these gliomas is urgently required. Given that high-grade gliomas frequently harbor mutations in the SNF2 family chromatin remodeler ATRX, we performed a screen to identify FDA-approved drugs that are toxic to ATRX-deficient cells. Our findings reveal that multi-targeted receptor tyrosine kinase (RTK) and platelet-derived growth factor receptor (PDGFR) inhibitors cause higher cellular toxicity in high-grade glioma ATRX-deficient cells. Furthermore, we demonstrate that a combinatorial treatment of RTKi with temozolomide (TMZ)–the current stand-ard of care treatment for GBM patients–causes pronounced toxicity in ATRX-deficient high-grade glioma cells. Our findings suggest that combinatorial treatments with TMZ and RTKi may increase the therapeutic window of opportunity in patients who suffer high-grade gliomas with ATRX mu-tations. Thus, we recommend incorporating the ATRX status into the analyses of clinical trials with RTKi and PDGFRi., This work was supported by grants from Danish National Research Foundation (DNRF115), Danish Cancer Society (KBVU-2017_R167-A11063), European Research Council (ERC-2015-STG- 679068), Nordea-fonden (02-2017-1749) and the Spanish Ministry of Science and Innovation (PID2020- 119329RB-I00). David Pladevall-Morera was supported with a PhD scholarship from the Lundbeck Foundation (R218-2016-415) and funding from Dansk Kræftforskningsfond. María Castejón-Griñán holds an Incorporación fellowship from the Junta de Andalucía. Paula Aguilera was supported with a Juan de la Cierva formación fellowship from the MICINN and an Incorporación fellowship from the Junta de Andalucía. Toyota Fonden and Læge Sofus Carl Emil Friis og hustru Olga Doris Fonden funded the acquisition of the high-content microscope used in this study.

A key hallmark of cancer, unlimited replication, requires cancer cells to evade both replicative senescence and potentially lethal chromosomal instability induced by telomere dysfunction. The majority of cancers overcome these critical barriers by upregulating telomerase, a telomere-specific reverse transcriptase. However, a subset of cancers maintains telomere lengths by the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. The presence of ALT is strongly associated with recurrent cancer-specific somatic inactivating mutations in the ATRX-DAXX chromatin-remodeling complex. Here, we generate an ALT-positive adenocarcinoma cell line following functional inactivation of ATRX and telomerase in a telomerase-positive adenocarcinoma cell line. Inactivating mutations in ATRX were introduced using CRISPR-cas9 nickase into two prostate cancer cell lines, LAPC-4 (derived from a lymph node metastasis) and CWR22Rv1 (sourced from a xenograft established from a primary prostate cancer). In LAPC-4, but not CWR22Rv1, abolishing ATRX was sufficient to induce multiple ALT-associated hallmarks, including the presence of ALT-associated promyelocytic leukemia bodies (APB), extrachromosomal telomere C-circles, and dramatic telomere length heterogeneity. However, telomerase activity was still present in these ATRXKO cells. Telomerase activity was subsequently crippled in these LAPC-4 ATRXKO cells by introducing mutations in the TERC locus, the essential RNA component of telomerase. These LAPC-4 ATRXKO TERCmut cells continued to proliferate long-term and retained ALT-associated hallmarks, thereby demonstrating their reliance on the ALT mechanism for telomere maintenance. Implications: These prostate cancer cell line models provide a unique system to explore the distinct molecular alterations that occur upon induction of ALT, and may be useful tools to screen for ALT-specific therapies.

The characterization of tissues using multiple different primary antibodies detected by secondary antibodies, each possessing a different colored fluorophore (multiplex immunofluorescence), is a powerful technique but often impaired by endogenous autofluorescence present in the specimen. Our current research involves the use of multiplex immunofluorescence to identify specific cell phenotypes within the tumor microenvironment in archival formalin-fixed paraffin-embedded human prostate cancer tissue specimens. These specimens frequently possess high levels of autofluorescence, in part due to the biological age of the tissues and long storage times. This autofluorescence interferes with and, in the worst cases, completely obscures the desired immunofluorescent signals, thus impeding analyses by decreasing signal-to-noise. Here, we demonstrate that a recently published protocol for photochemical bleaching significantly decreases autofluorescence (80% average decrease of the brightest autofluorescent signals), across the visible spectrum, in fixed, archival prostate tissue specimens from aged men, that have been sectioned onto glass slides and stored for several months. Importantly, the method is compatible with subsequent immunofluorescence staining and yields markedly improved signal-to-noise. Inclusion of this method should facilitate studies employing multiplex immunofluorescence in sections cut from archival fixed human prostate tissues.

Prostate cancer (PCa) is a heterogeneous group of tumors with variable clinical courses. In order to improve patient outcomes, it is critical to clinically separate aggressive PCa (AG) from non-aggressive PCa (NAG). Although recent genomic studies have identified a spectrum of molecular abnormalities associated with aggressive PCa, it is still challenging to separate AG from NAG. To better understand the functional consequences of PCa progression and the unique features of the AG subtype, we studied the proteomic signatures of primary AG, NAG and metastatic PCa. 39 PCa and 10 benign prostate controls in a discovery cohort and 57 PCa in a validation cohort were analyzed using a data-independent acquisition (DIA) SWATH–MS platform. Proteins with the highest variances (top 500 proteins) were annotated for the pathway enrichment analysis. Functional analysis of differentially expressed proteins in NAG and AG was performed. Data was further validated using a validation cohort; and was also compared with a TCGA mRNA expression dataset and confirmed by immunohistochemistry (IHC) using PCa tissue microarray (TMA). 4,415 proteins were identified in the tumor and benign control tissues, including 158 up-regulated and 116 down-regulated proteins in AG tumors. A functional analysis of tumor-associated proteins revealed reduced expressions of several proteinases, including dipeptidyl peptidase 4 (DPP4), carboxypeptidase E (CPE) and prostate specific antigen (KLK3) in AG and metastatic PCa. A targeted analysis further identified that the reduced expression of DPP4 was associated with the accumulation of DPP4 substrates and the reduced ratio of DPP4 cleaved peptide to intact substrate peptide. Findings were further validated using an independently-collected tumor cohort, correlated with a TCGA mRNA dataset, and confirmed by immunohistochemical stains of PCa tumor microarray (TMA). Our study is the first large-scale proteomics analysis of PCa tissue using a DIA SWATH-MS platform. It provides not only an interrogative proteomic signature of PCa subtypes, but also indicates the critical roles played by certain proteinases during tumor progression. The spectrum map and protein profile generated in the study can be used to investigate potential biological mechanisms involved in PCa and for the development of a clinical assay to distinguish aggressive from indolent PCa.

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.