Abstract Purpose: The prostate tumor microenvironment (TME) is immunologically “cold,” limiting the efficacy of immunotherapy. Vaccine-induced immune activation has shown promise, but the spatial distribution and function of immune clones within the TME remain poorly understood. Methods: We applied spatial V(D)J sequencing, combining long- and short-read analysis of the CDR3 region, to map T and B cell clonotypes alongside gene expression signatures directly in prostate tissue while preserving spatial context. Prior data from a phase 1/2a clinical trial (NCT01784913) of the telomerase (hTERT) peptide vaccine UV1, administered in combination with radiation therapy, used TCRseq to investigate changes in the TME and T cell repertoires before and after vaccination, as well as factors distinguishing the cancer vaccine non-responders from the responders who had a very favourable outcome. Results: TCRseq data revealed distinct profiles in tissue prior to vaccination that differentiated responders from non-responders. Notably, a T cell receptor motif signature in pre-vaccination tissue was associated with patients who mounted an early immune response to the vaccine. Differences in transcriptomic profiles and T cell repertoires were observed among patients with early, late, or no immune response, linking immune contexture to clinical outcomes. Conclusions: Our findings provide high-resolution insights into immune-tumor interactions in prostate cancer, demonstrating that specific immune clonotypes underlie effective vaccine responses and may inform future immunotherapy strategies, and that spatial V(D)J analysis can be leveraged to explore the spatial contexture of the prostate cancer tumor microenvironment. Citation Format: Eirik Høye, Karishma Sajnani, Reetta Nätkin, Sini Hakkola, Antti Kiviaho, Thomas Cecchetto, Anthony Mathelier, Matti Nykter, Wolfgang Lilleby, Sini Eerola, Alfonso Urbanucci, Tapio Visakorpi, Heini Kallio. Mapping immune clonotypes in prostate cancer using spatial V(D)J to resolve cancer vaccine response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7441.

The prostate cancer tumor microenvironment (TME) contains various cell type identities. We recently described club-like epithelial cells as a constitutive part of the TME present in tissue regions with increased myeloid-derived suppressor cell infiltration. The origin, exact function, and identity of club-like cells remain unresolved. Here we used bulk, single-cell, and spatial transcriptomics to characterize club-like cells and their relation towards androgen deprivation therapy. We additionally analyzed multiplex immunofluorescence data for interactions between club-like cells and myeloid-derived suppressor cells. Furthermore, we investigated club-like expression in lung and pancreatic tumors. In the spatial transcriptomics data, club-like enriched regions comprise a mixture of club, hillock, and senescent epithelial cells. Prostate cancer tumors from men treated with androgen deprivation therapy display elevated expression of hillock and senescence, but not the club signature. A high club signature score is associated with improved overall survival in TCGA lung adenocarcinoma (LUAD, HR = 0.59, p = 5.1x10-4), while a high hillock gene signature score indicates worse overall survival in both LUAD (HR = 1.80, p = 9.3x10-5) and pancreatic ductal adenocarcinoma (PDAC, HR = 1.94, p = 1.4x10-3). In PDAC, club-like expression is associated with IL-1β+ myeloid cell interaction. We define IL-1β+ myeloid cells in the prostate single-cell data and demonstrate co-localization with club cells in the spatial transcriptomics data. Finally, we show that epithelial cells from castration-resistant prostate cancer samples have an elevated hillock signature compared to normal prostate and treatment-naïve prostate cancer, suggesting hillock expansion or phenotypic plasticity during disease progression. Our results indicate that the canonical club phenotype arises in response to inflammatory signaling, while closely related hillock cells survive androgen deprivation in prostate cancer and indicate poor overall survival in LUAD and PDAC tumors. Antti Kiviaho, Sini K. Eerola, Heini M. Kallio, Miina Hoikka, Aliisa M. Tiihonen, Iida Salonen, Alexander Giesen, Ebrahim Afyounian, Sini Hakkola, Charles T. Parker, Mikael Marttinen, Mazlina Ismail, Wout Devlies, Teuvo L. Tammela, Teemu J. Murtola, Ian G. Mills, Steven Joniau, Gerhardt Attard, Frank Claessens, Tapio Visakorpi, Alfonso Urbanucci, Kirsi J. Rautajoki, Matti Nykter. Prostate club-like cells resist treatment and carry prognostic value in lung and pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5314.

Abstract Background: The detection of cancer-associated DNA in urine samples has significant potential in urothelial cancer (UC) detection and surveillance. Every year millions of patients with urinary symptoms undergo cystoscopy, an examination of the bladder using a flexible scope. Only 10% of these invasive examinations result in a cancer diagnosis. Cystoscopy is also used in post-operative surveillance, with many UC patients requiring life-long cystoscopies. Methods: We developed a high-volume urine DNA collection kit and laboratory platform and prospectively analyzed 433 urine samples collected from diagnostic and post-operative timepoints from 167 UC patients and 95 negative controls from January 2021 to September 2023. We used a hybridization capture assay targeting the coding regions of 21 commonly mutated genes in urothelial tumors, as well as 8 frequently copy number altered loci. Samples were sequenced using Illumina NovaSeq instruments. Mutations, copy number alterations, and chromosomal rearrangements were computationally identified. Key Findings: In the diagnostic settings our assay achieved a sensitivity of 100% for pT1+ bladder cancer, 97% for pTa bladder cancer, and 91% for upper tract urothelial cancer (UTUC) (100% for pT1+ or high grade) in an all-comers UC cohort, at a specificity of 93%. Post-operative urine DNA surveillance informed by preoperative urine mutations detected 28/30 (93%) residual cancer cases found in cystoscopy or surgery, with two pTa low grade recurrences missed. Using a secondary, highly stringent urine tumor DNA detection threshold (100% specificity for UC), we show that urine DNA testing in the diagnostic setting may allow up to 68% of UC patients to be scheduled directly to operating room procedures, reducing hospital workload and expediting care. In addition, we demonstrate the feasibility of urine DNA for detecting underlying Lynch syndrome in UC patients by identifying three UTUC patients who carried germline defects in mismatch repair genes MSH2 or MSH6, indicative of Lynch syndrome. In two of these patients our assay also found a somatic second hit and high mutation load. Conclusions and Clinical Implications: These results show promise in urine DNA based cancer diagnostics even in patients with low grade tumors. The assay is designed to offer a non-invasive and cost-effective alternative for cystoscopies. Citation Format: Jussi Nikkola, Lauri Ryyppö, Juuso Vuorinen, Heini Kallio, Hanna Selin, Pyry Jämsä, Jonne Åkerla, Tarmo Pekkarinen, Antti Kaipia, Matti Nykter, Thea Veitonmäki, Matti Annala. Sensitive urothelial cancer detection via high volume urine DNA analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB105.

You have accessJournal of UrologyBladder Cancer: Epidemiology & Evaluation II (MP35)1 May 2024MP35-05 DEVELOPING NON-INVASIVE AND COST-EFFECTIVE METHODS FOR UROTHELIAL CANCER DETECTION AND SURVEILLANCE USING A URINE DNA PANEL (UROSCOUT ASSAY) Jussi Nikkola, Thea Veitonmäki, Lauri Ryyppö, Juuso Vuorinen, Heini Kallio, Hanna Selin, Pyry Jämsä, Antti Kaipia, Tarmo Pekkarinen, Jonne Åkerla, Matti Nykter, and Matti Annala Jussi NikkolaJussi Nikkola , Thea VeitonmäkiThea Veitonmäki , Lauri RyyppöLauri Ryyppö , Juuso VuorinenJuuso Vuorinen , Heini KallioHeini Kallio , Hanna SelinHanna Selin , Pyry JämsäPyry Jämsä , Antti KaipiaAntti Kaipia , Tarmo PekkarinenTarmo Pekkarinen , Jonne ÅkerlaJonne Åkerla , Matti NykterMatti Nykter , and Matti AnnalaMatti Annala View All Author Informationhttps://doi.org/10.1097/01.JU.0001009372.61513.54.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The detection of cancer-associated DNA in urine samples has significant potential in urothelial cancer detection and surveillance, but current assays tend to miss low grade tumors. This study aimed to develop a cost-effective diagnostic panel capable of identifying all clinical stages of urothelial cancer. METHODS: Up to 100 mL of urine at the time of the first surgery for urothelial carcinoma was prospectively collected from patients. We developed a hybridization capture assay targeting the coding regions of 21 commonly mutated genes in urothelial tumors, as well as 8 frequently copy number altered loci. Samples were sequenced using Illumina NovaSeq instruments. Mutations, copy number alterations, and chromosomal rearrangements were computationally identified. RESULTS: From January 2021 to September 2023, we collected urine samples from 134 patients with pathologically confirmed bladder cancer and 27 patients with upper tract urothelial carcinoma (UTUC), clinically representing a comprehensive cohort of urothelial cancer cases. Our diagnostic assay achieved high sensitivity in detecting overall bladder cancer (104/106 detected, 98%), pTa low grade bladder cancer (48/49 detected, 98%) and UTUC (21/23 detected, 91%). We also analyzed 177 post-operative urine samples from 94 patients and detected residual cancer in 18/20 (90%) samples where a time-matched cystoscopy or surgical tissue was positive, with only two pTa low grade recurrences missed. We compared urine cell pellet DNA and urine cell-free DNA, finding similar fractions of cancer DNA across these analytes but higher DNA yields (median 9.91 ng/mL vs. 2.95 ng/mL) and higher molecular weight DNA in the urine cell pellet. Three UTUC patients carried germline defects in mismatch repair genes MSH2 or MSH6, indicative of Lynch syndrome. In two of these patients our panel also found a somatic second hit and high mutation load. The assay's specificity was 93%, determined using 95 negative control samples from hematuria, kidney cancer, and healthy individuals. CONCLUSIONS: These results using the UroScout diagnostic assay show promise in urine DNA based cancer diagnostics even in patients with pTa low grade tumors. The panel is designed to offer a non-invasive and cost-effective alternative for cystoscopies. Source of Funding: Jane and Aatos Erkko Foundation, Finnish Medical Foundation, Sigrid Juselius Foundation, Finnish Urological Research Fund, Pirkanmaa Cancer Foundation, Finnish Cancer Foundation, Tampere University Hospital Competitive Research Fund © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e582 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Jussi Nikkola More articles by this author Thea Veitonmäki More articles by this author Lauri Ryyppö More articles by this author Juuso Vuorinen More articles by this author Heini Kallio More articles by this author Hanna Selin More articles by this author Pyry Jämsä More articles by this author Antti Kaipia More articles by this author Tarmo Pekkarinen More articles by this author Jonne Åkerla More articles by this author Matti Nykter More articles by this author Matti Annala More articles by this author Expand All Advertisement PDF downloadLoading ...

Prostate cancer treatment resistance is a significant challenge facing the field. Genomic and transcriptomic profiling have partially elucidated the mechanisms through which cancer cells escape treatment, but their relation toward the tumor microenvironment (TME) remains elusive. Here we present a comprehensive transcriptomic landscape of the prostate TME at multiple points in the standard treatment timeline employing single-cell RNA-sequencing and spatial transcriptomics data from 110 patients. We identify club-like cells as a key epithelial cell subtype that acts as an interface between the prostate and the immune system. Tissue areas enriched with club-like cells have depleted androgen signaling and upregulated expression of luminal progenitor cell markers. Club-like cells display a senescence-associated secretory phenotype and their presence is linked to increased polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) activity. Our results indicate that club-like cells partake in inducing myeloid inflammation previously associated with androgen deprivation therapy resistance, providing a rationale for their therapeutic targeting.### Competing Interest StatementThe authors have declared no competing interest.

Epithelial ovarian cancer (EOC) is one of the leading causes of cancer-related death in women worldwide, and is characterized by a high rate of recurrence after surgery and chemotherapy. We sought to implement a circulating tumor DNA (ctDNA)-based blood test for more accurate post-operative surveillance of this disease. We analyzed 264 plasma samples collected between June 2016 and September 2021 from 63 EOC patients using tumor-guided plasma cell-free DNA analysis to detect residual disease after treatment. Assay specificity was verified using cross-patient analysis of 1,195 control samples. ctDNA was detected in 51 of 55 (93%) samples at diagnosis, and 18 of 18 (100%) samples at progression. Positive ctDNA in the last on-treatment sample was associated with rapid progression (median 1.02 versus 3.38 yr, HR = 5.63, P < 0.001) and reduced overall survival (median 2.31 versus NR yr, HR = 8.22, P < 0.001) in patients with high-grade serous cancer. In the case of 12 patients, ctDNA assays detected progression earlier than standard surveillance, with a median lead time of 5.9 mo. To approach the physical limits of ctDNA detection, five patients were analyzed using ultra-sensitive assays interrogating 479-1,856 tumor mutations, capable of tracking ctDNA fractions down to 0.0004%. Our results demonstrate that ctDNA assays achieve high sensitivity and specificity in detecting post-operative residual disease in EOC.

Prostate cancer treatment resistance is a significant challenge facing the field. Genomic and transcriptomic profiling have partially elucidated the mechanisms through which cancer cells escape treatment, but their relation toward the tumor microenvironment (TME) remains elusive. Here we present a comprehensive transcriptomic landscape of the prostate TME at multiple points in the standard treatment timeline employing single-cell RNA-sequencing and spatial transcriptomics data from 120 patients. We identify club-like cells as a key epithelial cell subtype that acts as an interface between the prostate and the immune system. Tissue areas enriched with club-like cells have depleted androgen signaling and upregulated expression of luminal progenitor cell markers. Club-like cells display a senescence-associated secretory phenotype and their presence is linked to increased polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) activity. Our results indicate that club-like cells are associated with myeloid inflammation previously linked to androgen deprivation therapy resistance, providing a rationale for their therapeutic targeting. The prostate cancer tumour microenvironment in the context of treatment remains to be explored. Here, single-cell RNA-sequencing and spatial transcriptomics analysis of samples at multiple treatment points from 120 patients suggests that club-like cells may contribute to treatment resistance.

Abstract Background: Prostate cancer (PCa) is the second most common cancer in men. Despite its high prevalence, many patients carry an indolent form of the disease and are thus suspect to overtreatment. Conversely, some cases treated with androgen deprivation therapy can develop into castration resistant prostate cancer (CRPC), for which there is no curative treatment. Understanding why some tumors are more aggressive than others could lead to more accurate patient risk stratification. Here we characterize cancer and normal cell subpopulations within prostate tissue in their spatial context using a multimodal integrative approach. Methods: We performed spatially resolved transcriptomics (ST) on a set of primary tumor PCa, CRPC and benign prostatic hyperplasia (BPH) patient samples. In addition to ST, we produced RNA-seq, DNA-seq and assay for transposase accessible chromatin using sequencing (ATAC-seq) data, allowing for multiomic integration within and across sample categories. We performed extensive analysis of ST data, employing unsupervised clustering, spot expression signal deconvolution, differential gene expression analysis and copy number variation (CNV) inference. Main Results: The systematic analysis of spot expression profiles revealed a high degree of variation in nearby tissue regions, as we found up to three unique luminal cell populations inside a one millimeter radius in PCa. Similarly in locally recurrent CRPC, we identified cumulative CNVs in proximal luminal cell populations, with the inferred CNV profiles validated through DNA-seq. A set of marker genes was calculated for each unique cell population, with multiple PCa associated genes found to be differentially expressed. Although we observed significant variation in the luminal cell populations, the stromal gene expression was markedly similar across all samples. Conclusions: We discovered shared, similar and unique cell populations both within and across different PCa and CRPC sections. We observed various luminal cell populations with distinct gene expression profiles in samples from both progression stages. The close spatial proximity of these cell clusters suggests that ST can be used to discover and examine finely detailed populations in their original spatial environment. Citation Format: Antti Kiviaho, Heini M. Kallio, Sini K. Eerola, Elisa M. Vuorinen, Tomi Häkkinen, Sinja Taavitsainen, Ebrahim Afyounian, Teemu Tolonen, Juha Kesseli, Alfonso Urbanucci, Kirsi J. Rautajoki, Teuvo L. Tammela, Tapio Visakorpi, Matti Nykter. Spatially resolved transcriptomics points to distinct malignant cell populations within primary and castration resistant prostate cancer. [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 5644.

Fluoroquinolones are a commonly used prophylaxis in transrectal ultrasound-guided prostate biopsy (TRUS-Bx), even though fluoroquinolone-resistant Escherichia coli has been associated with infectious complications after TRUS-Bx. The present study describes fluoroquinolone resistance mechanisms and antimicrobial susceptibility among intestinal E. coli , isolated from TRUS-Bx patients in a prospective study showing very few infectious prostate biopsy adverse events. This Multi-IMPROD sub-study included a total of 336 patients who received either ciprofloxacin, levofloxacin, or fosfomycin as prophylaxis before TRUS-Bx. E. coli could be cultured from 278 fecal swab samples, and 27 (9.7%) of these showed resistance to ciprofloxacin, and 14 (5.0%) were susceptible with increased exposure (I). Chromosomal and transferable fluoroquinolone resistance mechanisms were found among ciprofloxacin non-susceptible isolates, but both qnr genes and single gyrA mutations were found also among the ciprofloxacin-susceptible E. coli population. Low-level fluoroquinolone resistance is commonly associated with ESBL production in Enterobacterales . However, ESBL and qnr genes were not associated in our material, 14 isolates were ESBL producers and only 14.3% of them had the qnr gene, although 85.7% of the ESBL producers were ciprofloxacin non-susceptible. In the Multi-IMPROD substudy, only two mild urinary tract infections were reported, indicating that the antimicrobial susceptibility or resistance pattern of E. coli does not correlate with the onset of post-biopsy adverse events. We conclude that in our clinical settings, ciprofloxacin and levofloxacin prophylaxis is effective, and no severe post-biopsy infections were detected despite the intestinal colonization of genotypically and phenotypically fluoroquinolone-resistant E. coli .

Background: Several systemic therapeutic options exist for metastatic castrate-sensitive prostate cancer (mCSPC). Circulating tumor DNA (ctDNA) can molecularly profile metastatic castration-resistant prostate cancer and can influence decision-making, but remains untested in mCSPC. Objective: To determine ctDNA abundance at de novo mCSPC diagnosis and whether ctDNA provides complementary clinically relevant information to a prostate biopsy. Design, setting, and participants: We collected plasma cell-free DNA (cfDNA) from 53 patients newly diagnosed with mCSPC and, where possible, during treatment. Targeted sequencing was performed on cfDNA and DNA from diagnostic prostate tissue. Results and limitations: The median ctDNA fraction was 11% (range 0-84%) among untreated patients but was lower (1.0%, range 0-51%) among patients after short-term (median 22 d) androgen deprivation therapy (ADT). TP53 mutations and DNA repair defects were identified in 47% and 21% of the cohort, respectively. The concordance for mutation detection in matched samples was 80%. Combined ctDNA and tissue analysis identified potential driver alterations in 94% of patients, whereas ctDNA or prostate biopsy alone was insufficient in 19 cases (36%). Limitations include the use of a narrow gene panel and undersampling of primary disease by prostate biopsy. Conclusions: ctDNA provides additional information to a prostate biopsy in men with de novo mCSPC, but ADT rapidly reduces ctDNA availability. Primary tissue and ctDNA share relevant somatic alterations, suggesting that either is suitable for molecular subtyping in de novo mCSPC. The optimal approach for biomarker development should utilize both a tissue and liquid biopsy at diagnosis, as neither captures clinically relevant somatic alterations in all patients. Patient summary: In men with advanced prostate cancer, tumor DNA shed into the bloodstream can be measured via a blood test. The information from this test provides complementary information to a prostate needle biopsy and could be used to guide management strategies. Sequencing data were deposited in the European Genome-phenome Archive (EGA) under study identifier EGAS00001003351. (C) 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Background A significant subset of prostate cancer (PC) patients with a castration-resistant form of the disease (CRPC) show primary resistance to androgen receptor (AR)-targeting drugs developed against CRPC. As one explanation could be the expression of constitutively active androgen receptor splice variants (AR-Vs), our current objectives were to study AR-V s and other AR aberrations to better understand the emergence of CRPC. Methods We analysed specimens from different stages of prostate cancer by next-generation sequencing and immunohistochemistry. Results AR mutations and copy number variations were detected only in CRPC specimens. Genomic structural rearrangements of AR were observed in 5/30 metastatic CRPC patients, but they were not associated with expression of previously known AR-V s. The predominant AR-V s detected were AR-V3 , AR-V7 and AR-V9 , with the expression levels being significantly higher in CRPC cases compared to prostatectomy samples. Out of 25 CRPC metastases that expressed any AR variant, 17 cases harboured expression of all three of these AR-V s. AR-V7 protein expression was highly heterogeneous and higher in CRPC compared to hormone-naïve tumours. Conclusions AR-V3 , AR-V7 and AR-V9 are co-expressed in CRPC metastases highlighting the fact that inhibiting AR function via regions common to all AR-Vs is likely to provide additional benefit to patients with CRPC.

ObjectivesTo determine, in a prospective, multicentre setting, the prevalence of fluoroquinolone‐resistant (FQ‐R) and extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli (E. coli) strains in men undergoing transrectal ultrasonography‐guided prostate biopsy (TRUS‐Bx) in Finland; and to survey the associated risk factors for having the previously mentioned strains.Patients and MethodsThis is a substudy of the trial investigating the role of magnetic resonance imaging (MRI) in prostate cancer diagnosis (Improved Prostate Cancer Diagnosis – Combination of Magnetic Resonance Imaging Targeted Biopsies and Biomarkers Multi‐institutional Study [multi‐IMPROD], NCT02241122). In all, 359 patients from four study centres were recruited to this prospective study. After having signed the informed consent form, these men with suspicion of prostate cancer completed a detailed questionnaire on their medical, smoking, and travelling history, as well as their recent use of antibiotics. After the bi‐parametric MRI scan, TRUS‐Bx was taken and a rectal swab sample was collected and cultured for determining the antimicrobial susceptibility profile of E. coli strains. The potential risk factors for having FQ‐R or third‐generation cephalosporin‐resistant (3GC‐R) E. coli strains were analysed using univariate and multivariate logistic regression analysis.ResultsThe percentage of FQ‐R and 3GC‐R E. coli strains amongst the study population was 13% and 8%, respectively. Amongst patients having E. coli strains, the rate of FQ‐R and 3GC‐R strains was 14% and 8%, respectively. Of the 3GC‐R E. coli strains, 62% proved to be ESBL‐producers and 88% were also FQ‐R. In multivariate analysis, international travel during the preceding year significantly increased the risk of having a FQ‐R E. coli strain (odds ratio [OR] 3.592, P = 0.001) and, unexpectedly, use of antibiotics during the previous year significantly decreased this risk (OR 0.442, P = 0.035). No significant risk factors for having 3GC‐R E. coli were identified.ConclusionThe occurrence of intestinal FQ‐R and/or 3GC‐R (potentially ESBL‐producing) E. coli strains in men undergoing TRUS‐Bx in Finland is notable. The finding is consistent with the global increase in antimicrobial resistance. International travel appears to be an indisputable risk factor for having intestinal FQ‐R E. coli strains. The contemporary antimicrobial resistance situation should be taken into account in the care of post‐TRUS‐Bx infections.

Advances in prostate cancer biology and diagnostics are dependent upon high-fidelity integration of clinical, histomorphologic, and molecular phenotypic findings. In this study, we compared fresh frozen, formalin-fixed paraffin-embedded (FFPE), and PAXgene-fixed paraffin-embedded (PFPE) tissue preparation methods in radical prostatectomy prostate tissue from 36 patients and performed a preliminary test of feasibility of using PFPE tissue in routine prostate surgical pathology diagnostic assessment. In addition to comparing histology, immunohistochemistry, and general measures of DNA and RNA integrity in each fixation method, we performed functional tests of DNA and RNA quality, including targeted Miseq RNA and DNA sequencing, and implemented methods to relate DNA and RNA yield and quality to quantified DNA and RNA picogram nuclear content in each tissue volume studied. Our results suggest that it is feasible to use PFPE tissue for routine robot-assisted laparoscopic prostatectomy surgical pathology diagnostics and immunohistochemistry, with the benefit of significantly improvedDNA and RNA quality and RNA picogram yield per nucleus as compared with FFPE tissue. For fresh frozen, FFPE, and PFPE tissues, respectively, the average Genomic Quality Numbers were 7.9, 3.2, and 6.2, average RNA Quality Numbers were 8.7, 2.6, and 6.3, average DNA picogram yields per nucleus were 0.41, 0.69, and 0.78, and average RNA picogram yields per nucleus were 1.40, 0.94, and 2.24. These findings suggest that where DNA and/or RNA analysis of tissue is required, and when tissue size is small, PFPE may provide important advantages over FFPE. The results also suggest several interesting nuances including potential avenues to improve RNA quality in FFPE tissues and confirm recent suggestions that some DNA sequence artifacts associated with FFPE can be avoided.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

We report the first combined analysis of whole-genome sequence, detailed clinical history, and transcriptome sequence of multiple prostate cancer metastases in a single patient (A21). Whole-genome and transcriptome sequence was obtained from nine anatomically separate metastases, and targeted DNA sequencing was performed in cancerous and noncancerous foci within the primary tumor specimen removed 5 yr before death. Transcriptome analysis revealed increased expression of androgen receptor (AR)-regulated genes in liver metastases that harbored an AR p.L702H mutation, suggesting a dominant effect by the mutation despite being present in only one of an estimated 16 copies per cell. The metastases harbored several alterations to the PI3K/AKT pathway, including a clonal truncal mutation in PIK3CG and present in all metastatic sites studied. The list of truncal genomic alterations shared by all metastases included homozygous deletion of TP53, hemizygous deletion of RB1 and CHD1, and amplification of FGFR1. If the patient were treated today, given this knowledge, the use of second-generation androgen-directed therapies, cessation of glucocorticoid administration, and therapeutic inhibition of the PI3K/AKT pathway or FGFR1 receptor could provide personalized benefit. Three previously unreported truncal clonal missense mutations (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) were expressed at the RNA level and assessed as druggable. The truncal status of mutations may be critical for effective actionability and merit further study. Our findings suggest that a large set of deeply analyzed cases could serve as a powerful guide to more effective prostate cancer basic science and personalized cancer medicine clinical trials.

Abstract Prostate cancer (PC) is prevalent in both indolent and lethal forms. Although many PC patients diagnosed today have organ-confined disease curable by prostatectomy or radiation therapy, 20-30% of PCs will relapse to lethal disease within 5-years of treatment. Relatively little attention has been paid to distinguishing the molecular characteristics of proven lethal metastatic PC from non-lethal cancers. A better understanding of the origins and evolution of lethal cancers should allow screening and treatment to be better tailored to the needs of each patient. To this end, we performed an integrative molecular profiling of a lethal PC from one patient (A21). High-coverage whole genome sequence, transcriptome sequence, and methylation analysis was performed on 9 anatomically separate metastases obtained by autopsy, and targeted sequencing was performed in multiple cancerous and noncancerous foci within the radical prostatectomy specimen removed 5 years prior to death. Molecular results were analyzed in relation to detailed clinical data. Integrated whole genome sequence analysis revealed convergent evolution of AR gene amplification events, and inception of p.L702H mutation in the AR present only in liver metastases. In addition, the analysis showed parallel increases in AR regulated transcripts in the liver metastases, suggesting a dominant effect by the mutation. Mutation of PI3/PI4 kinase member PIK3CG was found in all metastases but in no primary tumor foci studied. The study demonstrated the power of an integrated approach to interrogate clonal evolution of cancer suggesting that such studies are valuable on the individual level and could lead the way towards personalized treatment. In the individual studied, cessation of corticosteroid treatment and/or therapeutic manipulation of PI3K/AKT/mTOR activity theoretically could have provided a personalized benefit. These findings suggest that similar integrated analysis in large cohorts of patients with metastatic cancer could accelerate progress in establishing effective personalized cancer medicine. Citation Format: Heini M.L. Kallio, Matti Annala, Kati Kivinummi, Gunilla Högnäs, Gunes Gundem, David C. Wedge, Peter Van Loo, Holger Heyn, Michael R. Emmert-Buck, William B. Isaacs, Manel Esteller, Ultan McDermott, Matti Nykter, Tapio Visakorpi, G. Steven Bova. Clonal evolution of a lethal prostate cancer: Integrated whole genome analysis case study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2015-3883

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Our previous microarray study showed that the non-specific cytotoxic cell receptor protein 1 (Nccrp1) transcript is significantly upregulated in the gastric mucosa of carbonic anhydrase IX (CA IX)-deficient (Car9(-/-)) mice. In this paper, we aimed to characterize human NCCRP1 and to elucidate its relationship to CA IX. Recombinant NCCRP1 protein was expressed in Escherichia coli, and a novel polyclonal antiserum was raised against the purified full-length protein. Immunocytochemistry showed that NCCRP1 is expressed intracellularly, even though it has previously been described as a transmembrane protein. Using bioinformatic analyses, we identified orthologs of NCCRP1 in 35 vertebrate genomes, and up to five paralogs per genome. These paralogs are FBXO genes whose protein products are components of the E3 ubiquitin ligase complexes. NCCRP1 proteins have no signal peptides or transmembrane domains. NCCRP1 has mainly been studied in fish and was thought to be responsible for the cytolytic function of nonspecific cytotoxic cells (NCCs). Our analyses showed that in humans, NCCRP1 mRNA is expressed in tissues containing squamous epithelium, whereas it shows a more ubiquitous tissue expression pattern in mice. Neither human nor mouse NCCRP1 expression is specific to immune tissues. Silencing CA9 using siRNAs did not affect NCCRP1 levels, indicating that its expression is not directly regulated by CA9. Interestingly, silencing NCCRP1 caused a statistically significant decrease in the growth of HeLa cells. These studies provide ample evidence that the current name, "non-specific cytotoxic cell receptor protein 1," is not appropriate. We therefore propose that the gene name be changed to FBXO50.