Retinal cell fate specification from multipotent retinal progenitors is governed by dynamic changes in chromatin structure and gene expression. Methylation at cytosines in DNA (5mC) is actively regulated for proper control of gene expression and chromatin architecture. Numerous genes display active DNA demethylation across retinal development; a process that requires oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and is controlled by the ten-eleven translocation methylcytosine dioxygenase (TET) enzymes. Using an allelic series of conditional TET enzyme mutants, we determine that DNA demethylation is required upstream of NRL and NR2E3 expression for the establishment of rod-photoreceptor fate. Using histological, behavioral, transcriptomic, and base-pair resolution DNA methylation analyses, we establish that inhibition of active DNA demethylation results in global changes in gene expression and methylation patterns that prevent photoreceptor precursors from adopting a rod-photoreceptor fate, instead producing a retina in which all photoreceptors specify as cones. Our results establish the TET enzymes and DNA demethylation as critical regulators of retinal development and cell fate specification, elucidating a novel mechanism required for the specification of rod-photoreceptors.

1053 Background: T-DXd, targeting HER2, is approved in hormone receptor positive or negative (HR+/-) HER2-low (1-2+ by immunohistochemistry [IHC]) and HR+/HER2-ultralow (0+ with membrane staining) aBC. SG, targeting TROP2, is approved in HR+/HER2 negative [ -; < 3+ by IHC] and triple negative BC (TNBC; HR-/HER2-). Given limitations in determining IHC 0-1+ status and lack of predictive biomarkers, biomarkers for sequencing these ADCs in those with HER2- aBC are needed. Recently, Thomas et al. reported the discovery of ADC TRS—a generalized model combining individual ADC target expression, proliferation, and adhesion to predict multi-ADC target/tumor type clinical benefit—as well as validation of SG and T-DXd TRS models (tuned by pan-tumor response rates) by a qRT-PCR based clinical trial assay (CTA; Strata Select ADC [SSA]) for predicting clinical benefit of first ADC (SG or T-DXd) in those with HER2- aBC (ASCO 2024 #3140; AACR 2025 #1014). Here, we evaluated ADC target expression and T-DXd/SG TRS status from SSA validation cohort HER2- BC patients stratified by clinical HR/HER2 IHC status. Methods: Adults with aBC from an observational trial (NCT03061305) with valid FFPE tumor tissue results from SSA validation cohort testing were included. aBC types were determined by HR/HER2 IHC results (by ASCO/CAP scoring), with those HER2 3+ or HER2 amplified (by Strata Select testing) considered HER2+. ADC target component expression ( HER2 or TROP2; pan-tumor scaled absolute expression) and T-DXd and SG TRS statuses (+ associated with more clinical benefit) by SSA were compared by IHC defined aBC types. Results: 230 patients with aBC from SSA validation testing were included (median age 58 yrs, 40% self-reported non-European;180 with definitive aBC type [see Table for distribution]). HER2 expression was significantly increased vs. TROP2 in HER2+ and HR+/HER2 aBC, and did not significantly differ in those with TNBC (see Table). Across all patients, 42%, 30%, 27% and 0.4% were T-DXd/SG TRS +/+, -/-, +/- and -/+, respectively. Results were similar in those with HER2 IHC 0+ (n = 37, median HER2 vs. TROP2 = 2.2 vs. 2.3, p = 0.51; 8% and 3% T-DXd/SG TRS +/- and -/+, respectively. Conclusions: Pan-tumor optimized, validated ADC TRS models support sequencing T-DXd before SG in nearly all patients with advanced HER2- BC, including those with TNBC and HER2 0+ IHC. Prospective evaluation of the CTA is warranted. ADC target expression ( HER2 or TROP2 ) and T-DXd | SG TRS status by SSA in BC patients. T-DXd/SG TRS Status BCa Type (n) HER2^ TROP2^ p-value^ +/+ -/- +/- -/- HER2+ 43 6.4 2.6 <0.0001 42% 7% 51% 0% IHC NA* 50 2.6 2.7 0.31 44% 40% 16% 0% HR+/HER2- 76 3.0 2.3 <0.0001 54% 20% 26% 0% TNBC 61 2.3 2.3 0.7 25% 52% 21% 2% Total 230 2.9 2.4 <0.0001 42% 30% 27% 0.4% ^Median, pan-tumor scaled HER2 and TROP2 target expression; Wilcoxon test. *IHC not available; HER2 not amplified.

Background Identification and characterization of circulating tumor markers, designated as “liquid biopsies,” have greatly impacted the care of cancer patients. Although more recently referring to circulating tumor DNA (ctDNA), the term liquid biopsy initially was coined to refer to any blood-borne biomarker related to malignancy, including circulating tumor cells (CTCs) in blood. In this manuscript, we review the specific state of the art of CTCs in breast cancer. Content Liquid biopsies might play a clinical role across the entire spectrum of breast cancer, from risk assessment, prevention, screening, and treatment. CTC counts have been shown to carry clear, independent prognostic information in the latter situation. However, the clinical utility of CTCs in breast cancer remains to be determined. Nonetheless, in addition to CTC enumeration, analyses of CTCs provide tumor molecular information representing the entire, often-heterogeneous disease, relatively noninvasively and longitudinally. Technological advances have allowed the interrogation of CTC-derived information, providing renewed hope for a clinical role in disease monitoring and precision oncology. Summary This narrative review examines CTCs, their clinical validity, and current prospects of clinical utility in breast cancer with the goal of improving patient outcomes.

Legends for Supplementary Figures S1-S5.

HER2-targeted therapy has improved breast cancer survival, but treatment resistance and disease prevention remain major challenges. Genes that enable HER2/Neu oncogenesis are the next intervention targets. A bioinformatics discovery platform of HER2/Neu-expressing Diversity Outbred (DO) F1 Mice was established to identify cancer-enabling genes. Quantitative Trait Loci (QTL) associated with onset ages and growth rates of spontaneous mammary tumors were sought. Twenty-six genes in 3 QTL contain sequence variations unique to the genetic backgrounds that are linked to aggressive tumors and 21 genes are associated with human breast cancer survival. Concurrent identification of TSC22D3, a transcription factor, and its target gene LILRB4, a myeloid cell checkpoint receptor, suggests an immune axis for regulation, or intervention, of disease. We also investigated TIEG1 gene that impedes tumor immunity but suppresses tumor growth. Although not an actionable target, TIEG1 study revealed genetic regulation of tumor progression, forming the basis of the genetics-based discovery platform.

Supplementary Figures S1-2, Tables S1-3. Figure S1. Comparison of the number of high confidence, non-synonymous somatic point mutations/indels and high level copy number alterations (CNAs) by tumor grade. Figure S2. Copy number plots of representative benign and borderline phyllodes tumors. Table S2: Somatic non-synonymous mutations identified in FFPE phyllodes tumors subjected to targeted next generation sequencing. Table S3: Detailed information about identified somatic non-synonymous mutations.

Precision and immuno-oncology clinical decisions are based on predictive biomarkers. Circulating biomarkers offer a minimally invasive approach to monitor intra-patient tumor heterogeneity and detect in real-time the clinically-relevant evolving clonal architecture. Although currently underutilized, single-cell DNA next generation sequencing of circulating tumor cells (CTC) is a particularly suitable method to complement tissue and circulating tumor DNA (ctDNA). Here we analyzed 113 individual CTC, 21 ctDNA, 15 white blood cells (WBC) samples and 15 tissue biopsies, from 15 CTC-positive lobular breast cancer patients. CTC were enriched with the CellSearch® system and isolated as single cells with the DEPArray™ system. CTC, WBC, and ctDNA underwent scNGS with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space to detect mutations, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). 99.1% of single cells and 95.2% of ctDNA samples were informative. Our CTC-based precision medicine reporting platform, MI-CTCSeq, detected CTC in 9 of 15 patients (60%) with FDA-approved actionable alterations including in the oncogenes PIK3CA and FGFR2 and HER2. 3 of these 9 (33%) harbored actionable alterations not shared between all 3 analyte types (tissue, CTC and ctDNA) including 3 actionable mutations found in CTC and ctDNA only, 1 in tissue and ctDNA only, and 1 in ctDNA only. Two of those ctDNA mutations were identified near the limit of detection and with a priori knowledge from tissue or CTC. Interestingly, 1 patient with plentiful CTC had no detectable ctDNA. Another patient’s tissue biopsy was inadequate for sequencing while both liquid biopsy analytes were abundant. 13 patients (87%) displayed intra-patient, inter-CTC genomic heterogeneity of driver mutations. 1 of 4 (25%) patients with CTC available in >1 timepoint displayed fluctuations in their CTC subclonal makeup between timepoints. Data from this patient’s 2 tissue biopsies, 4 ctDNA samples, and 27 individual CTC over 6 timepoints, combined to reveal in unprecedented detail inter-metastatic lesion and inter-CTC heterogeneity and evolution in response to endocrine, chemo and immunotherapy selective pressures. In a novel approach we show detection of single-cell CTC TMB and MSI. CTC TMB was highly concordant (R 0.81) with the corresponding tissue biopsies. Further, in a novel observation, we detected intra patient, inter-CTC heterogeneity of TMB and MSI, which has potential implications for immunotherapy response and development of resistance. Taken together, these data support the non-invasive biomarker interrogation and monitoring by liquid biopsy that incorporates CTC to complement tissue in informing treatment approaches. Citation Format: Andi K. Cani, Emily M. Dolce, Kevin Hu, Chia-Jen Liu, Elizabeth P. Darga, Dan Robinson, Yi-Mi Wu, Dafydd G. Thomas, Costanza Paoletti, Scott A. Tomlins, James M. Rae, Aaron M. Udager, Arul M. Chinnaiyan, Erin F. Cobain, Daniel F. Hayes. Serial monitoring of circulating tumor cells and circulating tumor DNA in metastatic lobular breast cancer identifies intra-tumor heterogeneity and precision and immuno-oncology biomarkers of therapeutic importance. [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 5595.

Supplementary Figure 1. Study overview to determine the ability of MRI/ultrasound fusion guided biopsy to resample the same prostate cancer foci using molecular markers of clonality.

Figure S1. (A) Overexpression of p53 in olfactory neuroblastoma tumor harboring an inactivating TP53 mutation. (B) Representative example of p53 expression in olfactory neuroblastoma tumor lacking TP53 mutation. Immunohistochemistry, 400x magnification. figure S2. (A) Nuclear localization of beta catenin in olfactory neuroblastoma tumor harboring an activating CTNNB1 mutation. (B) Representative example of membranous beta catenin expression in olfactory neuroblastoma tumor lacking CTNNB1 mutation. Immunohistochemistry, 400x magnification. Table S1: Immunohistochemistry protocols Table S2: Clinicopathologic features of olfactory neuroblastoma cohort Table S3: DNA sequencing statistics for profiled olfactory neuroblastoma samples Table S4: Prioritized somatic nonsynonymous mutations in olfactory neuroblastoma cases identified by next generation sequencing Table S5: FGFR3 copy number in olfactory neuroblastomas by next generation sequencing Table S6. Targeted multiplexed PCR based RNA sequencing statistics for ONB samples

Sanger sequencing validation of next generation sequencing (NGS) variant calls ; Alternative view of integrative molecular profiling heatmap ; p16 expression via immunohistochemistry ; Kaplan-Meier analysis of histologic subtypes of PeSCCA ; Comparison of prioritized molecular alterations in penile (PN) squamous cell carcinomas (SCCA) compared to other SCCAs .

Supplementary Figure S1. Clinical timelines. Clinical timelines and treatment from first diagnosis until enrollment into MI-CTC-ONCOSEQ for the 11 metastatic breast cancer patients; Supplementary Figure S2. Sanger sequencing of WGA CTC DNA and Western blot confirming the presence of A569S mutation; Supplementary Figure S3. MCF-7 (ESR1 A569S) are not estrogen independent over 5 days in hormone-free conditions; Supplementary Figure S4. The tamoxifen metabolites 4-hydroxytamoxifen and endoxifen have no increased agonistic effect in MCF-7 cells over-expressing ER-A569S; Supplementary Figure S5. Inhibition of estradiol-stimulated MCF-7 parental or A569S- overexpressing cells. Supplementary Table S1. Oncomine Comprehensive Panel (OCP) target genes by presence of the MseI restriction site; Table S2. NGS of BT-474 cells spiked into blood and purified identifies expectedTP53 mutation; Supplementary Table S3. Results of CTC purification by DEPArray; Supplementary Table S4: Mean CTC Next Generation Sequencing parameters; Supplementary Table S5. NGS-identified prioritized mutations in individual and pooled CTC samples; Supplementary Table S6. 2 x 2 contingency tables of concordance for alterations in CTC vs tissue; Supplementary Table S7. Patient #2 analysis of primary, clinical metastatic tissue, research biopsy, pt-DNA by ddPCR; Supplementary Table S8. Primer/probe sequences used in Sanger sequencing and ddPCR.

Clinicopathologic data for all profiled penile squamous cell carcinoma (PeSCCA) samples ; Sequencing data for all profiled penile squamous cell carcinoma (PeSCCA) samples ; Detailed somatic variant information from PeSCCA profiled by next generation sequenicng ; PeSCCA HPV genotyping and p16 immunohistochemistry (IHC) results .

Abstract The advent of high-throughput technologies has enabled the analysis of minute amounts of tumor-derived material purified from body fluids, termed “liquid biopsies.” Prostate cancer (PCa) management, like in many other cancer types, has benefited from liquid biopsies at several stages of the disease. Although initially describing circulating tumor cells in blood, the term “liquid biopsy” has come to more prominently include cell-free, circulating tumor DNA, as well as RNA, proteins, and other molecules. They provide tumor molecular information representing the entire, often-heterogeneous disease, relatively noninvasively and longitudinally. Blood has been the main liquid biopsy specimen in PCa, and urine has also proven beneficial. Technological advances have allowed clinical implementation of some liquid biopsies in PCa, in disease monitoring and precision oncology. This narrative review introduces the main types of blood-based PCa liquid biopsies focusing on advances in the past 5 years. Clinical adoption of liquid biopsies to detect and monitor the evolving PCa tumor biology promises to deepen our understanding of the disease and improve patient outcomes.

<p>Description of additional methods and procedures used in the study. Also includes Supplementary References.</p>

Legend to for supplementary Figure 1

Clinical decisions for precision and immuno-oncology therapies are based on predictive biomarkers commonly obtained from a single metastatic biopsy, or from archived primary tumor material. Circulating genomic biomarkers present a minimally invasive way to monitor the intra-patient tumor heterogeneity and its fluctuations in order to provide a real-time evaluation of the changing clonal architecture with potential therapeutic implications. Single-cell DNA next generation sequencing (scNGS) of circulating tumor cells (CTC) is a particularly well-suited method of unraveling and monitoring that heterogeneity to complement biomarker information obtained from tissue and cell-free circulating tumor DNA (ctDNA). In this proof-of-concept study we analyzed 123 CTC, 15 white blood cells (WBC), and ctDNA from 15 CTC-positive lobular breast cancer patients, five of whom had CTC available at both metastatic baseline and after progression on a variety of therapies chosen at their physician’s discretion. CTC were enriched with the CellSearch® system and isolated as single cells with the DEPArray™ system. Whole genome amplified CTC DNA underwent scNGS with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space to detect mutations, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). 99.1% of cells were informative, with a mean sequencing depth of 664x. Using our previously developed, CTC-based precision medicine reporting platform, MI-CTCSeq, multiple CTC in seven of 15 patients (47%) had mutations that were actionable by FDA-approved targeted therapies including in the oncogenes PIK3CA (alpelisib) and FGFR2 (erdafitinib). 13 patients (87%) displayed intra-patient, inter-CTC genomic heterogeneity of putative driver mutations. Two of five (40%) patients with CTC at both baseline and progression displayed fluctuations in their CTC subclonal makeup between timepoints. One of the two harbored a baseline ESR1 (estrogen receptor α) p.D538G activating mutation that largely disappeared at progression and was replaced by a CTC subclone with a different ESR1 activating mutation, p.Y537S. Intriguingly, this patient’s CTC also harbored an FGFR2 p.K659M mutation in an actionable “hotspot” at progression, which was absent at baseline, suggesting potential utility of serial monitoring by CTC scNGS. TMB scores and MSI status in CTC were highly concordant with those measured in clinical tissue biopsies. Taken together, these data suggest the non-invasive interrogation of the CTC genomic landscape and its serial monitoring to inform precision and immuno-oncology treatments in real time. Citation Format: Andi K. Cani, Emily M. Dolce, Elizabeth P. Darga, Kevin Hu, Chia-Jen Liu, James M. Rae, Daffyd G. Thomas, Scott A. Tomlins, Arul M. Chinnaiyan, Aaron M. Udager, Costanza Paoletti, Erin F. Cobain, Daniel F. Hayes. Serial monitoring of single-cell circulating tumor cell genomics in metastatic lobular breast cancer to identify precision and immuno-oncology biomarkers with therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1700.

The success of immune checkpoint inhibitors rests on biomarkers such as tumor mutation burden (TMB) and microsatellite instability (MSI), both FDA-approved predictors of anti PD-1/L1 therapy benefit. Tissue biopsies often collected once in the metastatic setting through an invasive procedure, or archived primary tumor tissue often collected much prior to treatment consideration, are the specimen types of choice for biomarker identification. The tissue sample originates from a limited region of one disease site, which may limit its usefulness given intra-patient tumor heterogeneity. TMB and MSI measurement by liquid biopsy, including proteins, circulating tumor cells (CTC), and cell-free circulating tumor DNA (ctDNA), is an attractive, minimally-invasive way to obtain a real-time picture of the entire disease. While TMB and MSI assessment from ctDNA have been reported, their measurement can be limited by low ctDNA tumor fraction. Single-cell next generation sequencing of CTC, on the other hand, is a particularly well-suited, but largely unexplored method of measuring TMB and MSI to complement tissue and ctDNA for better overall specificity of detection. In this proof-of-concept study, we show the ability to detect single-cell TMB and MSI. We analyzed 14 CTC and 4 ctDNA samples from 6 metastatic breast cancer patients, as well as 3 single cells and 1 cell pellet sample each from HCT-116 (MSI-High) and WiDr (MSI-Low) cell lines. CTC and cell line cells were enriched with the CellSearch® system and/or isolated with the DEPArray™ system. Whole genome amplified single-cell DNA was sequenced with the Oncomine Comprehensive Assay covering ~500 genes and 1.1Mb of genomic space. TMB and MSI scores obtained in CTC and ctDNA were compared to those measured in matched clinical tissue biopsies. Single-cell TMB scores and MSI status were assessable in all CTC tested. CTC TMB scores were highly concordant with the matched tissue samples (r=1.00), as were ctDNA TMB scores (r=0.98) in patients with assessable TMB scores in both biospecimen types compared. Importantly, TMB was detectable in CTC from one patient whose tissue sample was inadequate for clinical sequencing, and from another patient with inadequate, low tumor fraction ctDNA. Intriguingly, one patient harbored 3 TMB-high and 2 TMB-low CTC, potentially indicating intra-patient TMB heterogeneity. The known MSI-low status from clinical tumor tissue sequencing was correctly detected in CTC and ctDNA from all patients. MSI status and scores from single cells of HCT-116 and WiDr cell lines purified with the DEPArray™ system (mimicking CTC isolation), perfectly matched that of the corresponding cell pellet samples (r=1.00). Taken together, these data suggest the potential validity and continued interrogation of potential utility of CTC TMB and MSI detection to complement tissue and ctDNA in guiding checkpoint inhibitor immunotherapy. Citation Format: Andi K. Cani, Emily M. Dolce, Chia-Jen Liu, Brittany Rupp, Elizabeth P. Darga, Costanza Paoletti, Dafydd G. Thomas, Yi-Mi Wu, Dan R. Robinson, Sunitha Nagrath, Arul M. Chinnaiyan, Scott A. Tomlins, Aaron M. Udager, John M. Carethers, Erin F. Cobain, Daniel F. Hayes. Assessment of tumor mutation burden and microsatellite instability by single-cell circulating tumor cell genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 611.

Background: There is an ongoing need to develop prognostic biomarkers to improve the management of clear cell renal cell carcinoma (ccRCC).Objective: To leverage enriched pathways in ccRCC to improve risk-stratification.Design, setting, and participants: We retrospectively identified two complementary discovery cohorts of patients with ccRCC who underwent (1) radical nephrectomy (RNx) with inferior vena cava tumor thrombectomy (patients = 5, samples = 24) and (2) RNx for localized disease and developed recurrence versus no recurrence (n = 36). Patients with localized ccRCC (M0) in The Cancer Genome Atlas (TCGA, n = 386) were used for validation.Outcome measurements and statistical analysis: A differential expression gene (DEG) analysis was performed on targeted RNA next-generation sequencing data from both discovery cohorts. Using TCGA for validation, Kaplan-Meier survival analysis and multivariable Cox proportional hazard testing were utilized to investigate the prognostic impact of DEGs, cell cycle proliferation (CCP), and a novel epithelial-mesenchymal transition (EMT) score on progression-free (PFS) and disease-specific (DSS) survival.Results and limitations: In the discovery cohorts, we observed overexpression of WT1 and CCP genes in the tumor thrombus versus the primary tumor, as well as in patients with recurrence versus those without recurrence. A hallmark pathway analysis demonstrated enrichment of the EMT-and CCP-related pathways in patients with high WT1 expression in the TCGA (validation) ccRCC cohort. CCP and EMT scores were derived in the validation cohort, which was stratified into four risk groups using Youden Index cut points: CCPlow/EMTlow, CCPlow/EMThigh, CCPhigh/EMTlow, and CCPhigh/EMThigh. The CCPhigh/EMThigh risk group was associated with the worst PFS and DSS (both p < 0.001). In a multivariable analysis, CCPhigh/EMThigh was independently associated with poor PFS and DSS (hazard ratio = 4.6 and 10.3, respectively; p < 0.001).Conclusions: We demonstrate the synergistic prognostic impact of EMT in tumors with a high CCP score. Our novel EMT score has the potential to improve risk stratification and provide potential novel therapeutic targets. Patient summary: Genes involved in epithelial-mesenchymal transition provides impor- tant prognostic information for patients with clear cell renal cell carcinoma. (c) 2021 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Vitreoretinal lymphoma (VRL) is an uncommon eye malignancy, and VRLs of T cell origin are rare. They are difficult to treat, and their molecular underpinnings, including actionable genomic alterations, remain to be elucidated. At present, vitreous fluid liquid biopsies represent a valuable VRL sample for molecular analysis to study VRLs. In this study, we report the molecular diagnostic workup of a rare case of bilateral T cell VRL and characterize its genomic landscape, including identification of potentially targetable alterations. Using next-generation sequencing of vitreous-derived DNA with a pan-cancer 126-gene panel, we found a copy number gain of BRAF and copy number loss of tumor suppressor DNMT3A. To the best of our knowledge, this represents the first exploration of the T cell VRL cancer genome and supports vitreous liquid biopsy as a suitable approach for precision oncology treatments.

BACKGROUND:Despite biomarker development advances, early detection of aggressive prostate cancer (PCa) remains challenging. We previously developed a clinical-grade urine test (Michigan Prostate Score [MiPS]) for individualized aggressive PCa risk prediction. MiPS combines serum prostate-specific antigen (PSA), the TMPRSS2:ERG (T2:ERG) gene fusion, and PCA3 lncRNA in whole urine after digital rectal examination (DRE). OBJECTIVE:To improve on MiPS with a novel next-generation sequencing (NGS) multibiomarker urine assay for early detection of aggressive PCa. DESIGN, SETTING, AND PARTICIPANTS:Preclinical development and validation of a post-DRE urine RNA NGS assay (Urine Prostate Seq [UPSeq]) assessing 84 PCa transcriptomic biomarkers, including T2:ERG, PCA3, additional PCa fusions/isoforms, mRNAs, lncRNAs, and expressed mutations. Our UPSeq model was trained on 73 patients and validated on a held-out set of 36 patients representing the spectrum of disease (benign to grade group [GG] 5 PCa). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:The area under the receiver operating characteristic curve (AUC) of UPSeq was compared with PSA, MiPS, and other existing models/biomarkers for predicting GG ≥3 PCa. RESULTS AND LIMITATIONS:UPSeq demonstrated high analytical accuracy and concordance with MiPS, and was able to detect expressed germline HOXB13 and somatic SPOP mutations. In an extreme design cohort (n = 109; benign/GG 1 vs GG ≥3 PCa, stratified to exclude GG 2 cancer in order to capture signal difference between extreme ends of disease), UPSeq showed differential expression for T2:ERG.T1E4 (1.2 vs 78.8 median normalized reads, p < 0.00001) and PCA3 (1024 vs 2521, p = 0.02), additional T2:ERG splice isoforms, and other candidate biomarkers. Using machine learning, we developed a 15-transcript model on the training set (n = 73) that outperformed serum PSA and sequencing-derived MiPS in predicting GG ≥3 PCa in the held-out validation set (n = 36; AUC 0.82 vs 0.69 and 0.69, respectively). CONCLUSIONS:These results support the potential utility of our novel urine-based RNA NGS assay to supplement PSA for improved early detection of aggressive PCa. PATIENT SUMMARY:We have developed a new urine-based test for the detection of aggressive prostate cancer, which promises improvement upon current biomarker tests.