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Purpose: Men with localized prostate cancer may receive either photon-based intensity modulated radiation therapy (IMRT) or proton beam therapy (PBT). The PARTIQoL trial (NCT01617161) demonstrates the feasibility of performing a large, multicenter phase 3 randomized trial comparing IMRT with PBT for localized prostate cancer. Here, we report baseline features of patients enrolled on this trial and present strategies to improve feasibility of other similar trials., Methods and Materials: Patients with low- or intermediate-risk prostate cancer were randomly assigned to either PBT or IMRT with stratification by institution, age, use of rectal spacer, and fractionation schedule (conventional fractionation: 79.2 Gy in 44 fractions vs moderate hypofractionation: 70.0 Gy in 28 fractions). The primary endpoint is a change from baseline bowel health using the Expanded Prostate Index Composite score 24 months after radiation therapy. Secondary objectives include treatment-related differences in urinary and erectile functions, adverse events, and efficacy endpoints., Results: Between July 2012 and November 2021, 450 patients were successfully accrued. Patients were randomly assigned to either PBT (N = 226) or to IMRT (N = 224); 13 were ineligible or withdrew before treatment. The median age of 437 analyzed patients was 68 years (range, 46-89 years). A total of 41% of patients had low-risk and 59% had intermediate-risk disease. In total, 49% of patients were treated with conventional fractionation and 51% with moderately hypofractionation. 48% of patients used a rectal spacer. For patients receiving PBT, pencil beam scanning was used in 48%. PBT and IMRT arms were balanced for baseline variables., Conclusions: Despite significant challenges, the PARTIQoL trial demonstrated that, with targeted recruitment approaches, multicenter collaboration, payer engagement, and protocol updates to incorporate contemporary techniques, it is feasible to perform a large phase 3 randomized clinical trial to assess whether PBT improves outcomes. We will separately report primary results and continue to monitor participants for longer follow-up and secondary endpoints., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Competing Interests: Scott Delacroix, Jared Foster, Francesca Khani, Joseph Liao, Abdul Tawab-Amiri Pamela West, Alex Wyatt: No conflicts. David McConkey: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Grants and contracts – Department of Defense, Mark Foundation, Commonwealth Foundation. Brian Baumann: Consulting fees – Boston Scientific, Varian, Novartis, Blue Earth; leadership or fiduciary role – Board of Directors, National Association for Proton Therapy. Stephanie Cooper Greenberg: Patient advocate, Johns Hopkins Greenberg Bladder Cancer Institute and NCI’s Bladder Cancer Task Force. David DeGraff: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review Grants and contracts – Bristol Myers Squibb, Department of Defense; receipt of material – Astra-Zeneca. Jason Efstathiou: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review Consulting fees – Blue Earth Diagnostics, Boston Scientific, AstraZeneca, Genentech, Astellas/Pfizer, Lantheus; payment or honoraria – IBA, Elekta, UpToDate; advisory boards – Merck, Roivant Pharma, Myovant Sciences, Janssen, Bayer Healthcare, Progenics Pharmaceuticals, Pfizer, Astellas, Gilead, Lantheus, Blue Earth Diagnostics, Angiodynamics; committee leadership – Vice-Chair, GU Committee, NRG; Board member (unpaid) – Massachusetts Prostate Cancer Coalition (MPCC) American College of Radiation Oncology (ACRO), Radiation Oncology Institute (ROI). Susan Groshen: Financial support from NCI/GUSC for attending a 2-day workshop that led to the CTPM. Edward Kadel: Patent, “PD-L1 PROMOTER METHYLATION IN CANCER”; stock or stock options, 89bio, Apple, Amazon, Alphabet, Eli Lilly & Co, Inspire Medical Systems, Merck & Co, Roche, SPDR S&P Biotech ETF, Teladoc; employee of Genentech/Roche. William Kim: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Grants or contracts – NIH R01-CA241810, Merck; consulting fees – Focal Medical (spouse); advisory board – OncoRev; stock or stock options – Abbvie, Amgen, Apellis, BeiGene, BMS, Lilly, ImmunityBio, Moderna, Natera, NovoNordisk, Revolution Medicines, Tango Therapeutics, Zentalis; other financial or non-financial interests – Focal Medical, ownership interest (spouse). Seth Lerner: Co-Editor-in-Chief of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Advisory board – FerGene, Pfizer. BMS, Incyte; consultant – Vaxiion, Verity, Protara, C2i Genomics, Ferring, Aura Bioscience, Surge Therapeutics; clinical trials – Genentech (SWOG), Vaxiion, Endo, FKD, JBL (SWOG), Merck (Alliance), Aura Bioscience, Surge Therapeutics, QED Therapeutics; honoraria – UroToday, co-editor bladder cancer section UpToDate, Grand Rounds Urology; compensated editorial responsibilities – IOS Press. Trevor Levin: Grants or contracts – NCI SBIR 5R44CA200174-05; patents issued or pending – WO/2017/056451, WO/2020/023630, WO/2020/243587, WO/2022/216865A1; leadership or fiduciary role – Convergent Genomics (Executive and Board roles); stock or stock options – Convergent Genomics. Matthew Milowski: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Consulting: Loxo/Lilly; payments or honoraria, Medscape, Research to Practice; other financial or non-financial interests: Co-Editor in Chief, Clinical Genitourinary Cancer, Elsevier. Joshua Meeks: grants or contracts, VA, NIH, Department of Defense; consulting fees, Merck, AstraZeneca, Incyte, Janssen, BMS, UroGen, Prokarium, Imvax, Pfizer, Seagen/Astellas, Ferring; payments, Olympus, AUA, UroToday, OncLive; advisory boards, Astra-Zeneca, Medscape. David Miyamoto: grant support from NIH and the Radiation Oncology Institute. Kent Mouw: consulting fees, EMD Serono, Pfizer, UroGen, Riva Therapeutics. Eugene Pietzak: participation in a Data Safety Monitoring Committee or Advisory Board, QED Pharmaceuticals, Merck, Janssen, Urogen; society leadership, Chair of the Program Committtee, 2024 BCAN Think Tank. David Solit: consulting fees from Pfizer, Fog Pharma, PaigeAI, BridgeBio, Scorpion Therapeutics, FORE Therapeutics, Function Oncology, Pyramid, and Elsie Biotechnologies, Inc.; participation on a Data Safety Monitoring Board for Rain Therapeutics; stock or stock options from Scorpion Therapeutics, FORE Therapeutics, Function Oncology, Pyramid, Elsie Biotechnologies, Inc. Debashish Sundi: grants or contracts from Janssen, consulting fees from Janssen. Sara Wobker: royalties – Wolters Kluwer; honoraria – College of American Pathologists, American Society of Clinical Pathology, USCAP; travel reimbursement – Kidney Cancer Society. Andrea Apolo: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Peter Black: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Consulting fees – AbbVie, AstraZeneca, Astellas, Bayer, BMS, Combat, EMD-Serono, Ferring, Janssen, Merck, Nonagen, Nanobot, NanOlogy, Pfizer, Photocure, Prokarium, Sumitomo, TerSera, Tolmar, Verity; payments – Janssen, TerSera, Bayer, Pfizer; patents issued or pending – Veracyte.

Purpose: Radium-223 improves overall survival (OS) and reduces skeletal events in patients with bone metastatic castration-resistant prostate cancer (CRPC), but relevant biomarkers are lacking. We evaluated automated bone scan index (aBSI) and circulating tumor cell (CTC) analyses as potential biomarkers of prognosis and activity., Patients and Methods: Patients with bone metastatic CRPC were enrolled on a prospective single-arm study of standard radium-223. 99m Tc-MDP bone scan images at baseline, 2 months, and 6 months were quantitated using aBSI. CTCs at baseline, 1 month, and 2 months were enumerated and assessed for RNA expression of prostate cancer-specific genes using microfluidic enrichment followed by droplet digital polymerase chain reaction., Results: The median OS was 21.3 months in 22 patients. Lower baseline aBSI and minimal change in aBSI (<+0.7) from baseline to 2 months were each associated with better OS ( P = .00341 and P = .0139, respectively). The higher baseline CTC count of ≥5 CTC/7.5 mL was associated with worse OS (median, 10.1 v 32.9 months; P = .00568). CTCs declined at 2 months in four of 15 patients with detectable baseline CTCs. Among individual genes in CTCs, baseline expression of the splice variant AR-V7 was significantly associated with worse OS (hazard ratio, 5.20 [95% CI, 1.657 to 16.31]; P = .00195). Baseline detectable AR-V7 , higher aBSI, and CTC count ≥5 CTC/7.5 mL continued to have a significant independent negative impact on OS after controlling for prostate-specific antigen or alkaline phosphatase., Conclusion: Quantitative bone scan assessment with aBSI and CTC analyses are prognostic markers in patients treated with radium-223. AR-V7 expression in CTCs is a particularly promising prognostic biomarker and warrants validation in larger cohorts.

Purpose: There is an urgent need for biomarkers of radiation response in organ-sparing therapies. Bladder preservation with trimodality therapy (TMT), consisting of transurethral tumor resection followed by chemoradiation, is an alternative to radical cystectomy for muscle-invasive bladder cancer (MIBC), but molecular determinants of response are poorly understood., Experimental Design: We characterized genomic and transcriptomic features correlated with long-term response in a single institution cohort of patients with MIBC homogeneously treated with TMT. Pretreatment tumors from 76 patients with MIBC underwent whole-exome sequencing; 67 underwent matched transcriptomic profiling. Molecular features were correlated with clinical outcomes including modified bladder-intact event-free survival (mBI-EFS), a composite endpoint that reflects long-term cancer control with bladder preservation., Results: With a median follow-up of 74.6 months in alive patients, 37 patients had favorable long-term response to TMT while 39 had unfavorable long-term response. Tumor mutational burden was not associated with outcomes after TMT. DNA damage response gene alterations were associated with improved locoregional control and mBI-EFS. Of these alterations, somatic ERCC2 mutations stood out as significantly associated with favorable long-term outcomes; patients with ERCC2 mutations had significantly improved mBI-EFS [HR, 0.15; 95% confidence interval (CI), 0.06-0.37; P = 0.030] and improved BI-EFS, an endpoint that includes all-cause mortality (HR, 0.33; 95% CI, 0.15-0.68; P = 0.044). ERCC2 mutant bladder cancer cell lines were significantly more sensitive to concurrent cisplatin and radiation treatment in vitro than isogenic ERCC2 wild-type cells., Conclusions: Our data identify ERCC2 mutation as a candidate biomarker associated with sensitivity and long-term response to chemoradiation in MIBC. These findings warrant validation in independent cohorts., (©2023 The Authors; Published by the American Association for Cancer Research.)

Ataxia-telangiectasia mutated (ATM) plays a central role in the cellular response to DNA damage and ATM alterations are common in several tumor types including bladder cancer. However, the specific impact of ATM alterations on therapy response in bladder cancer is uncertain. Here, we combine preclinical modeling and clinical analyses to comprehensively define the impact of ATM alterations on bladder cancer. We show that ATM loss is sufficient to increase sensitivity to DNA-damaging agents including cisplatin and radiation. Furthermore, ATM loss drives sensitivity to DNA repair-targeted agents including poly(ADP-ribose) polymerase (PARP) and Ataxia telangiectasia and Rad3 related (ATR) inhibitors. ATM loss alters the immune microenvironment and improves anti-PD1 response in preclinical bladder models but is not associated with improved anti-PD1/PD-L1 response in clinical cohorts. Last, we show that ATM expression by immunohistochemistry is strongly correlated with response to chemoradiotherapy. Together, these data define a potential role for ATM as a predictive biomarker in bladder cancer.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs., Competing Interests: Declaration of interests Massachusetts General Hospital (MGH) has applied for patents regarding the CTC-iChip technology and CTC detection signatures. M.T., S.M., and D.A.H. are cofounders and have equity in Tell-Bio, which is not related to this work. The interests of these authors were reviewed and managed by MGH and Mass General Brigham (MGB) in accordance with their conflict of interest policies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Absorbable hydrogel spacer injected between prostate and rectum is gaining popularity for rectal sparing. The spacer alters patient anatomy and thus requires new auto-contouring models., Purpose: To report the development and comprehensive evaluation of two deep-learning models for patients injected with a radio-transparent (model I) versus radiopaque (model II) spacer., Methods and Materials: Model I was trained and cross-validated by 135 cases with transparent spacer and tested on 24 cases. Using refined training methods, model II was trained and cross-validated by the same dataset, but with the Hounsfield Unit distribution in the spacer overridden by that obtained from ten cases with opaque spacer. Model II was tested on 64 cases. The models auto-contour eight regions of interest (ROIs): spacer, prostate, proximal seminal vesicles (SVs), left and right femurs, bladder, rectum, and penile bulb. Qualitatively, each auto contour (AC), as well as the composite set, was assessed against manual contour (MC), by a radiation oncologist using a 1 (accepted directly or after minor editing), 2 (accepted after moderate editing), 3 (accepted after major editing), and 4 (rejected) scoring scale. The efficiency gain was characterized by the mean score as nearly complete [1-1.75], substantial (1.75-2.5], meaningful (2.5-3.25], and no (3.25-4.00]. Quantitatively, the geometric similarity between AC and MC was evaluated by dice similarity coefficient (DSC) and mean distance to agreement (MDA), using tolerance recommended by AAPM TG-132 Report. The results by the two models were compared to examine the outcome of the refined training methods. The large number of testing cases for model II allowed further investigation of inter-observer variability in clinical dataset. The correlation between score and DSC/MDA was studied on the ROIs with 10 or more counts of each acceptable score (1, 2, 3)., Results: For model I/model II: the mean score was 3.63/1.30 for transparent/opaque spacer, 2.71/2.16 for prostate, 3.25/2.44 for proximal SVs, 1.13/1.02 for both femurs, 2.25/1.25 for bladder, 3.00/2.06 for rectum, 3.38/2.42 for penile bulb, and 2.79/2.20 for the composite set; the mean DSC was 0.52/0.84 for spacer, 0.84/0.85 for prostate, 0.60/0.62 for proximal SVs, 0.94/0.96 for left femur, 0.95/0.96 for right femur, 0.91/0.95 for bladder, 0.81/0.84 for rectum, and 0.65/0.65 for penile bulb; and the mean MDA was 2.9/0.9 mm for spacer, 1.9/1.7 mm for prostate, 2.4/2.3 mm for proximal SVs, 0.8/0.5 mm for left femur, 0.7/0.5 mm for right femur, 1.5/0.9 mm for bladder, 2.3/1.9 mm for rectum, and 2.2/2.2 mm for penile bulb. Model II showed significantly improved scores for all ROIs, and metrics for spacer, femurs, bladder, and rectum. Significant inter-observer variability was only found for prostate. Highly linear correlation between the score and DSC was found for the two qualified ROIs (prostate and rectum)., Conclusions: The overall efficiency gain was meaningful for model I and substantial for model II. The ROIs meeting the clinical deployment criteria (mean score below 3.25, DSC above 0.8, and MDA below 2.5 mm) included prostate, both femurs, bladder and rectum for both models, and spacer for model II., (© 2023 American Association of Physicists in Medicine.)

Background: Previous randomised controlled trials comparing bladder preservation with radical cystectomy for muscle-invasive bladder cancer closed due to insufficient accrual. Given that no further trials are foreseen, we aimed to use propensity scores to compare trimodality therapy (maximal transurethral resection of bladder tumour followed by concurrent chemoradiation) with radical cystectomy., Methods: This retrospective analysis included 722 patients with clinical stage T2-T4N0M0 muscle-invasive urothelial carcinoma of the bladder (440 underwent radical cystectomy, 282 received trimodality therapy) who would have been eligible for both approaches, treated at three university centres in the USA and Canada between Jan 1, 2005, and Dec 31, 2017. All patients had solitary tumours less than 7 cm, no or unilateral hydronephrosis, and no extensive or multifocal carcinoma in situ. The 440 cases of radical cystectomy represent 29% of all radical cystectomies performed during the study period at the contributing institutions. The primary endpoint was metastasis-free survival. Secondary endpoints included overall survival, cancer-specific survival, and disease-free survival. Differences in survival outcomes by treatment were analysed using propensity scores incorporated in propensity score matching (PSM) using logistic regression and 3:1 matching with replacement and inverse probability treatment weighting (IPTW)., Findings: In the PSM analysis, the 3:1 matched cohort comprised 1119 patients (837 radical cystectomy, 282 trimodality therapy). After matching, age (71·4 years [IQR 66·0-77·1] for radical cystectomy vs 71·6 years [64·0-78·9] for trimodality therapy), sex (213 [25%] vs 68 [24%] female; 624 [75%] vs 214 [76%] male), cT2 stage (755 [90%] vs 255 [90%]), presence of hydronephrosis (97 [12%] vs 27 [10%]), and receipt of neoadjuvant or adjuvant chemotherapy (492 [59%] vs 159 [56%]) were similar between groups. Median follow-up was 4·38 years (IQR 1·6-6·7) versus 4·88 years (2·8-7·7), respectively. 5-year metastasis-free survival was 74% (95% CI 70-78) for radical cystectomy and 75% (70-80) for trimodality therapy with IPTW and 74% (70-77) and 74% (68-79) with PSM. There was no difference in metastasis-free survival either with IPTW (subdistribution hazard ratio [SHR] 0·89 [95% CI 0·67-1·20]; p=0·40) or PSM (SHR 0·93 [0·71-1·24]; p=0·64). 5-year cancer-specific survival for radical cystectomy versus trimodality therapy was 81% (95% CI 77-85) versus 84% (79-89) with IPTW and 83% (80-86) versus 85% (80-89) with PSM. 5-year disease-free survival was 73% (95% CI 69-77) versus 74% (69-79) with IPTW and 76% (72-80) versus 76% (71-81) with PSM. There were no differences in cancer-specific survival (IPTW: SHR 0·72 [95% CI 0·50-1·04]; p=0·071; PSM: SHR 0·73 [0·52-1·02]; p=0·057) and disease-free survival (IPTW: SHR 0·87 [0·65-1·16]; p=0·35; PSM: SHR 0·88 [0·67-1·16]; p=0·37) between radical cystectomy and trimodality therapy. Overall survival favoured trimodality therapy (IPTW: 66% [95% CI 61-71] vs 73% [68-78]; hazard ratio [HR] 0·70 [95% CI 0·53-0·92]; p=0·010; PSM: 72% [69-75] vs 77% [72-81]; HR 0·75 [0·58-0·97]; p=0·0078). Outcomes for radical cystectomy and trimodality therapy were not statistically different among centres for cancer-specific survival and metastasis-free survival (p=0·22-0·90). Salvage cystectomy was done in 38 (13%) trimodality therapy patients. Pathological stage in the 440 radical cystectomy patients was pT2 in 124 (28%), pT3-4 in 194 (44%), and 114 (26%) node positive. The median number of nodes removed was 39, the soft tissue positive margin rate was 1% (n=5), and the perioperative mortality rate was 2·5% (n=11)., Interpretation: This multi-institutional study provides the best evidence to date showing similar oncological outcomes between radical cystectomy and trimodality therapy for select patients with muscle-invasive bladder cancer. These results support that trimodality therapy, in the setting of multidisciplinary shared decision making, should be offered to all suitable candidates with muscle-invasive bladder cancer and not only to patients with significant comorbidities for whom surgery is not an option., Funding: Sinai Health Foundation, Princess Margaret Cancer Foundation, Massachusetts General Hospital., Competing Interests: Declaration of interests ARZ reports participation on a data safety monitoring board or advisory board for Janssen, Verity Pharmaceuticals, Ferring, mIR Scientific, Tolmar, and Theralase; and consulting fees from Janssen, Verity Pharmaceuticals, Ferring, mIR Scientific, Tolmar, and Theralase. NEF reports grants or contracts from Janssen, Sanofi, Astellas, Nucleix, Bayer, and Progenix; consulting fees from Amgen, Sanofi, Janssen, AbbVie, Astellas, Ferring, and Bayer; stock or stock options in Verity Pharmaceuticals and POINT Biopharma; and is Chief Medical Officer for Verity Pharmaceuticals and Chief Medical Officer for POINT Biopharma. GSK reports grants or contracts from Janssen; and participation on a data safety monitoring board or advisory board for BMS, Pfizer, EMD Serono, Theralase, Verity Pharmaceuticals, Merck, Janssen, AstraZeneca, and Ferring. MASJ reports an honorarium from Pfizer; support for attending meetings or travel from the European Association of Urology; and is the Chair of the Board of Directors of the International Kidney Cancer Coalition. SSS reports grants or contracts from Bayer, Janssen, and Seagen; consulting fees from Astellas, AstraZeneca, Bayer, BMS, Eisai, EMD Serona, Hoffmann-LaRoche, Immunomedics, Ipsen, Janssen, Merck, Pfizer, and Seagen; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Pfizer, EMD Serono, Seagen, and Bayer; and support for attending meetings or travel from EMD Serono. MDM reports participation on a data safety monitoring board or advisory board for Merck, Janssen, Eisai, and Exelixis. JAE reports consulting fees from Blue Earth Diagnostics, Boston Scientific, AstraZeneca, and Genentech; and participation on a data safety monitoring board or advisory board for Merck, Roviant Pharma, Myovant Sciences, Janssen, Bayer Healthcare, Progenics Pharmaceuticals, Pfizer, Gilead, and Lantheus. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Liquid biopsies such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have great potential to serve as prognostic and predictive biomarkers in urologic cancers. The possibility of using liquid biopsies for real-time noninvasive and dynamic monitoring of response to therapy has been an active area of investigation. In this brief review, we outline the evidence for the potential clinical utility of CTC and ctDNA analyses in prostate, urothelial, and renal cancers., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2022. Published by Elsevier Inc.)

Importance: Bladder-preserving trimodality therapy can be an effective alternative to radical cystectomy for treatment of muscle-invasive bladder cancer (MIBC), but biomarkers are needed to guide optimal patient selection. The DNA repair protein MRE11 is a candidate response biomarker that has not been validated in prospective cohorts using standardized measurement approaches., Objective: To evaluate MRE11 expression as a prognostic biomarker in MIBC patients receiving trimodality therapy using automated quantitative image analysis., Design, Setting, and Participants: This prognostic study analyzed patients with MIBC pooled from 6 prospective phase I/II, II, or III trials of trimodality therapy (Radiation Therapy Oncology Group [RTOG] 8802, 8903, 9506, 9706, 9906, and 0233) across 37 participating institutions in North America from 1988 to 2007. Eligible patients had nonmetastatic MIBC and were enrolled in 1 of the 6 trimodality therapy clinical trials. Analyses were completed August 2020., Exposures: Trimodality therapy with transurethral bladder tumor resection and cisplatin-based chemoradiation therapy., Main Outcomes and Measures: MRE11 expression and association with disease-specific (bladder cancer) mortality (DSM), defined as death from bladder cancer. Pretreatment tumor tissues were processed for immunofluorescence with anti-MRE11 antibody and analyzed using automated quantitative image analysis to calculate a normalized score for MRE11 based on nuclear-to-cytoplasmic (NC) signal ratio., Results: Of 465 patients from 6 trials, 168 patients had available tissue, of which 135 were analyzable for MRE11 expression (median age of 65 years [minimum-maximum, 34-90 years]; 111 [82.2%] men). Median (minimum-maximum) follow-up for alive patients was 5.0 (0.6-11.7) years. Median (Q1-Q3) MRE11 NC signal ratio was 2.41 (1.49-3.34). Patients with an MRE11 NC ratio above 1.49 (ie, above first quartile) had a significantly lower DSM (HR, 0.50; 95% CI, 0.26-0.93; P = .03). The 4-year DSM was 41.0% (95% CI, 23.2%-58.0%) for patients with an MRE11 NC signal ratio of 1.49 or lower vs 21.0% (95% CI, 13.4%-29.8%) for a ratio above 1.49. MRE11 NC signal ratio was not significantly associated with overall survival (HR, 0.84; 95% CI, 0.49-1.44)., Conclusions and Relevance: Higher MRE11 NC signal ratios were associated with better DSM after trimodality therapy. Lower MRE11 NC signal ratios identified a poor prognosis subgroup that may benefit from intensification of therapy.

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state-dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors., Significance: Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies., (©2022 American Association for Cancer Research.)

Purpose: Conventional rectal spacers (nonI-SPs) are low-contrast on computed tomography (CT), often necessitating magnetic resonance imaging for accurate delineation. A new formulation of spacers (I-SPs) incorporates iodine to improve radiopacity and CT visualization. We characterized placement, stability, and plan quality of I-SPs compared to nonI-SPs., Methods and Materials: Patients with intact prostate cancer (n = 50) treated with I-SPs and photons were compared to randomly selected patients (n = 50) with nonI-SPs (photon or proton therapy). The I-SP was contoured on the planning CT and cone beam CTs at 3 timepoints: first, middle, and final treatment (n = 200 scans). I-SPs Hounsfield units (HU), volume, surface area (SA), centroid position relative to prostate centroid, and distance between prostate/rectum centroids were compared on the planning CTs between each cohort. I-SP changes were evaluated on cone beam CTs over courses of treatment. Dosimetric evaluations of plan quality and robustness were performed. I-SP was tested in a phantom to characterize its relative linear stopping power for protons., Results: I-SPs yielded a distinct visible contrast on planning CTs compared to nonI-SPs (HU 138 vs 12, P < .001), allowing delineation on CT alone. The delineated volume and SA of I-SPs were smaller than nonI-SPs (volume 8.9 vs 10.6 mL, P < .001; SA 28 vs 35 cm 2 , P < .001), yet relative spacer position and prostate-rectal separation were similar (P = .79). No significant change in HU, volume, SA, or relative position of the I-SPs hydrogel occurred over courses of treatment (all P > .1). Dosimetric analysis concluded there were no significant changes in plan quality or robustness for I-SPs compared to nonI-SPs. The I-SP relative linear stopping power was 1.018, necessitating HU override for proton planning., Conclusions: I-SPs provide a manifest CT contrast, allowing for delineation on planning CT alone with no magnetic resonance imaging necessary. I-SPs radiopacity, size, and relative position remained stable over courses of treatment from 28 to 44 fractions. No changes in plan quality or robustness were seen comparing I-SPs and nonI-SPs., (Copyright © 2021 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.)

Context: Genomic stratification can impact prostate cancer (PC) care through diagnostic, prognostic, and predictive biomarkers that aid in clinical decision-making. The temporal and spatial genomic heterogeneity of PC together with the challenges of acquiring metastatic tissue biopsies hinder implementation of tissue-based molecular profiling in routine clinical practice. Blood-based liquid biopsies are an attractive, minimally invasive alternative., Objective: To review the clinical value of blood-based liquid biopsy assays in PC and identify potential applications to accelerate the development of precision medicine., Evidence Acquisition: A systematic review of PubMed/MEDLINE was performed to identify relevant literature on blood-based circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and extracellular vesicles (EVs) in PC., Evidence Synthesis: Liquid biopsy has emerged as a practical tool to profile tumor dynamics over time, elucidating features that evolve (genome, epigenome, transcriptome, and proteome) with tumor progression. Liquid biopsy tests encompass analysis of DNA, RNA, and proteins that can be detected in CTCs, ctDNA, or EVs. Blood-based liquid biopsies have demonstrated promise in the context of localized tumors (diagnostic signatures, risk stratification, and disease monitoring) and advanced disease (response/resistance biomarkers and prognostic markers)., Conclusions: Liquid biopsies have value as a source of prognostic, predictive, and response biomarkers in PC. Most clinical applications have been developed in the advanced metastatic setting, where CTC and ctDNA yields are significantly higher. However, standardization of assays and analytical/clinical validation is necessary prior to clinical implementation., Patient Summary: Traces of tumors can be isolated from blood samples from patients with prostate cancer either as whole cells or as DNA fragments. These traces provide information on tumor features. These minimally invasive tests can guide diagnosis and treatment selection., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Immunotherapy drugs have recently been approved by the Food and Drug Administration for the treatment of several genitourinary malignancies, including bladder cancer, renal cancer, and prostate cancer. Preclinical data and early clinical trial results suggest that immune checkpoint inhibitors can act synergistically with radiation therapy to enhance tumor cell killing at local irradiated sites and in some cases at distant sites through an abscopal effect. Because radiation therapy is commonly used in the treatment of genitourinary malignancies, there is great interest in testing the combination of immunotherapy with radiation therapy in these cancers to further improve treatment efficacy. In this review, we discuss the current evidence and biological rationale for combining immunotherapy with radiation therapy, as well as emerging data from ongoing and planned clinical trials testing the efficacy and tolerability of this combination in the treatment of genitourinary malignancies. We also outline outstanding questions regarding sequencing, dose fractionation, and biomarkers that remain to be addressed for the optimal delivery of this promising treatment approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ukleja, Kusaka and Miyamoto.)

Muscle-invasive bladder cancer can be treated with either radical cystectomy or bladder preservation approaches, and there is a need for reliable biomarkers to guide the optimal choice of therapy. The recent elucidation of the genomic landscape and biological drivers of bladder cancer has enabled the identification of tumor molecular features that may be helpful in driving clinical decision-making. Here, we summarize recent efforts to develop molecular biomarkers that could be leveraged to guide therapeutic decisions, post-treatment monitoring, and the optimal use of bladder preservation approaches for the effective treatment of muscle-invasive bladder cancer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Muscle-invasive bladder cancer (MIBC) is a sex-biased cancer with a higher incidence in men but worse outcomes in women. The root cause behind these observations remains unclear. To investigate whether sex-specific tumor biology could explain the differences in clinical behavior of MIBC, we analyzed the transcriptome profiles from transurethral resected bladder tumors of 1000 patients. Female tumors expressed higher levels of basal- and immune-associated genes, while male tumors expressed higher levels of luminal markers. Using molecular subtyping, we found that the rates of the basal/squamous subtype were higher in females than in males. Males were enriched with tumors of the luminal papillary (LumP) and neuroendocrine-like subtypes. Male MIBC tumors had higher androgen response activity across all luminal subtypes and male patients with LumP tumors were younger. Taken together, these data confirm differences in molecular subtypes based on sex. The role of the androgen response pathway in explaining subtype differences between men and women should be studied further. PATIENT SUMMARY: We explored the sex-specific biology of bladder cancer in 1000 patients and found that women had more aggressive cancer with higher immune activity. Men tended toward less aggressive tumors that showed male hormone signaling, suggesting that male hormones may influence the type of bladder cancer that a patient develops., (Copyright © 2020 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Introduction : Wnt signaling is important for normal development, cell proliferation, and cell differentiation. However, aberrations in the pathway can lead to tumorigenesis and cancer progression. Recent genome-wide studies have demonstrated the frequent occurrence of Wnt pathway alterations in prostate cancer. Although alterations in the canonical Wnt pathway in prostate cancer may have an impact on prognosis, recent studies suggest that the noncanonical Wnt pathway also plays an important role in disease progression and treatment resistance. Areas covered : We review the literature with regard to the potential prognostic significance of noncanonical Wnt signaling in prostate cancer. After a brief overview of the canonical and noncanonical Wnt pathways, we discuss the preclinical and clinical evidence for activation of Wnt signaling in prostate cancer. We focus on clinical evidence for noncanonical Wnt pathway components to serve as potential prognostic biomarkers. Expert opinion : Although many therapeutic options are available for men with prostate cancer, there remains an unmet need for prognostic and predictive biomarkers to precisely guide clinical management. Early evidence suggests that components of the noncanonical Wnt pathway may serve as prognostic biomarkers. However, prospective validation studies are necessary before these biomarkers can be routinely applied in the clinic.

Background: Bladder-sparing trimodality therapy (TMT) is an alternative to radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC), and biomarkers to inform therapy selection are needed., Objective: To evaluate the prognostic value of immune and stromal signatures in MIBC treated with TMT., Design, Setting, and Participants: We used a clinical-grade platform to perform transcriptome-wide gene expression profiling of primary tumors from 136 MIBC patients treated with TMT at a single institution. We observed 60 overall survival events at 5yr, and median follow-up time for patients without an event was 5.0yr (interquartile range 3.1, 5.0). Expression data from another cohort of 223 MIBC patients treated with neoadjuvant chemotherapy (NAC) and RC were also analyzed., Outcome Measurements and Statistical Analysis: Molecular subtype, immune, and stromal signatures were evaluated for associations with disease-specific survival (DSS) and overall survival (OS) in TMT patients, and in patients treated with NAC and RC., Results and Limitations: Gene expression profiling of TMT cases identified luminal (N=40), luminal-infiltrated (N=26), basal (N=54), and claudin-low (N=16) subtypes. Signatures of T-cell activation and interferon gamma signaling were associated with improved DSS in the TMT cohort (hazard ratio 0.30 [0.14-0.65], p=0.002 for T cells), but not in the NAC and RC cohort. Conversely, a stromal signature was associated with worse DSS in the NAC and RC cohort (p=0.006), but not in the TMT cohort. This study is limited by its retrospective nature., Conclusions: Higher immune infiltration in MIBC is associated with improved DSS after TMT, whereas higher stromal infiltration is associated with shorter DSS after NAC and RC. Additional studies should be conducted to determine whether gene expression profiling can predict treatment response., Patient Summary: We used gene expression profiling to study the association between tumor microenvironment and outcomes following bladder preservation therapy for invasive bladder cancer. We found that outcomes varied with immune and stromal signatures within the tumor. We conclude that gene expression profiling has potential to guide treatment decisions in bladder cancer., (Copyright © 2019 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Purpose: Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping studies have found that 5% to 15% of muscle-invasive bladder cancer (MIBC) have transcriptomic patterns consistent with NE bladder cancer in the absence of NE histology. The clinical implications of this NE-like subtype have not been explored in depth., Experimental Design: Transcriptome-wide expression profiles were generated for MIBC collected from 7 institutions and clinical-use of Decipher Bladder. Using unsupervised clustering, we generated a clustering solution on a prospective training cohort (PTC; n = 175), developed single-sample classifiers to predict NE tumors, and evaluated the resultant models on a testing radical cystectomy (RC) cohort ( n = 225). A random forest model was finalized and applied to 5 validation cohorts ( n = 1302). Uni- and multivariable survival analyses were used to characterize clinical outcomes., Results: In the training cohort (PTC), hierarchical clustering using an 84-gene panel showed a cluster of 8 patients (4.6%) with highly heterogeneous expression of NE markers in the absence of basal or luminal marker expression. NE-like tumors were identified in 1% to 6.6% of cases in validation cohorts. Patients with NE-like tumors had significantly worse 1-year progression-free survival (65% NE-like vs. 82% overall; P = 0.046) and, after adjusting for clinical and pathologic factors, had a 6.4-fold increased risk of all-cause mortality ( P = 0.001). IHC confirmed the neuronal character of these tumors., Conclusions: A single-patient classifier was developed that identifies patients with histologic urothelial cancer harboring a NE transcriptomic profile. These tumors represent a high-risk subgroup of MIBC, which may require different treatment., (©2019 American Association for Cancer Research.)

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression., Competing Interests: Conflict of interest statement: Massachusetts General Hospital has filed for patent protection for the circulating tumor cell inertial focusing (iChip) technology., (Copyright © 2019 the Author(s). Published by PNAS.)

Although muscle-invasive bladder cancer is commonly treated with radical cystectomy, a standard alternative is bladder preservation therapy, consisting of maximum transurethral bladder tumour resection followed by radiotherapy with concurrent chemotherapy. Although no successfully completed randomised comparisons are available, the two treatment paradigms seem to have similar long-term outcomes; however, clinicopathologic parameters can be insufficient to provide clear guidance in the selection of one treatment over the other. Recent advances in the molecular understanding of bladder cancer have led to the identification of new predictive biomarkers that ultimately might help guide the tailored selection of therapy on the basis of the intrinsic biology of the tumour. In this Review, we discuss the existing evidence for molecular alterations and genomic signatures as prognostic or predictive biomarkers for bladder preservation therapy. If validated in prospective clinical trials, such biomarkers could enable the identification of subgroups of patients who are more likely to benefit from one treatment over another, and guide the use of combination therapies that include other modalities, such as immunotherapy, which might act synergistically with radiotherapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The multiplicity of new therapies for breast cancer presents a challenge for treatment selection. We describe a 17-gene digital signature of breast circulating tumor cell (CTC)-derived transcripts enriched from blood, enabling high-sensitivity early monitoring of response. In a prospective cohort of localized breast cancer, an elevated CTC score after three cycles of neoadjuvant therapy is associated with residual disease at surgery ( P = 0.047). In a second prospective cohort with metastatic breast cancer, baseline CTC score correlates with overall survival ( P = 0.02), as does persistent CTC signal after 4 weeks of treatment ( P = 0.01). In the subset with estrogen receptor (ER)-positive disease, failure to suppress ER signaling within CTCs after 3 weeks of endocrine therapy predicts early progression ( P = 0.008). Drug-refractory ER signaling within CTCs overlaps partially with presence of ESR1 mutations, pointing to diverse mechanisms of acquired endocrine drug resistance. Thus, CTC-derived digital RNA signatures enable noninvasive pharmacodynamic measurements to inform therapy in breast cancer. Significance: Digital analysis of RNA from CTCs interrogates treatment responses of both localized and metastatic breast cancer. Quantifying CTC-derived ER signaling during treatment identifies patients failing to respond to ER suppression despite having functional ESR1. Thus, noninvasive scoring of CTC-RNA signatures may help guide therapeutic choices in localized and advanced breast cancer. Cancer Discov; 8(10); 1286-99. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195 ., (©2018 American Association for Cancer Research.)

Background: Cancer patients treated with pelvic radiation therapy (RT) often experience sexual health-related side effects during and following treatment. A clinical needs assessment was used to evaluate sexual health needs and to determine how needs differed between patients receiving and who had completed RT., Methods and Materials: A questionnaire was used to evaluate sexual health needs among patients treated with pelvic RT. All answers were rated using a 4-point Likert scale. Convenience sampling was used, and patients were stratified by whether they were on-treatment or in follow-up. Charts were reviewed for demographic, diagnostic, and treatment information. Pearson's χ 2 test and logistic regression analysis were used to analyze the associations between sexual health-related topics and clinical variables., Results: A total of 107 of 109 (98%) invited patients completed the questionnaire (46 females, 61 males; 52 undergoing RT, 54 completed RT). Most (75%) reported some degree of change in sexual health from the effects of cancer and/or treatment; 22% and 28% reported "quite a bit" or "very much" change, respectively. Sixty-nine percent reported that they experienced some degree of distress from sexual health changes (28% reported "very much" or "quite a bit" of distress). Seventy-six percent "agreed" or "strongly agreed" that they were interested in access to a multidisciplinary sexual health clinic (MSHC). Compared with patients currently receiving RT, patients in follow-up were significantly more likely to report worsening degrees of "change" (P = .008) and "distress" (P = .04) and to express interest in having access to an MSHC (P = .03)., Conclusion: The majority of patients receiving pelvic RT reported a change in sexual health with associated distress, with more reports among those in follow-up. Patients undergoing pelvic RT expressed a high interest in attending a radiation oncology MSHC. Our findings emphasize the important role radiation oncologists can play in the quality of life of our patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived ( n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies., Competing Interests: Conflict of interest statement: Massachusetts General Hospital and the authors have applied for patent protection for the CTC-iChip technology and the molecular signatures of melanoma cells., (Copyright © 2018 the Author(s). Published by PNAS.)

Blood-based biomarkers are critical in metastatic prostate cancer, where characteristic bone metastases are not readily sampled, and they may enable risk stratification in localized disease. We established a sensitive and high-throughput strategy for analyzing prostate circulating tumor cells (CTC) using microfluidic cell enrichment followed by digital quantitation of prostate-derived transcripts. In a prospective study of 27 patients with metastatic castration-resistant prostate cancer treated with first-line abiraterone, pretreatment elevation of the digital CTC M score identifies a high-risk population with poor overall survival (HR = 6.0; P = 0.01) and short radiographic progression-free survival (HR = 3.2; P = 0.046). Expression of HOXB13 in CTCs identifies 6 of 6 patients with ≤12-month survival, with a subset also expressing the ARV7 splice variant. In a second cohort of 34 men with localized prostate cancer, an elevated preoperative CTC L score predicts microscopic dissemination to seminal vesicles and/or lymph nodes ( P < 0.001). Thus, digital quantitation of CTC-specific transcripts enables noninvasive monitoring that may guide treatment selection in both metastatic and localized prostate cancer. Significance: There is an unmet need for biomarkers to guide prostate cancer therapies, for curative treatment of localized cancer and for application of molecularly targeted agents in metastatic disease. Digital quantitation of prostate CTC-derived transcripts in blood specimens is predictive of abiraterone response in metastatic cancer and of early dissemination in localized cancer. Cancer Discov; 8(3); 288-303. ©2018 AACR. See related commentary by Heitzer and Speicher, p. 269 This article is highlighted in the In This Issue feature, p. 253 ., (©2018 American Association for Cancer Research.)

Advances in our molecular understanding of cancer biology have paved the way to an expanding compendium of molecularly-targeted therapies, accompanied by the urgent need for biomarkers that enable the precise selection of the most appropriate therapies for individual cancer patients. Circulating biomarkers such as circulating tumor cells (CTCs) are poised to fill this need, since they are "liquid biopsies" that can be performed non-invasively and serially, and may capture the spectrum of spatial and temporal tumor heterogeneity better than conventional tissue biopsies. Increasing evidence suggests that moving beyond the enumeration of CTCs towards more sophisticated molecular analyses can provide actionable data that may predict and potentially improve clinical outcomes. In this review, we discuss the potential of molecular CTC analyses to serve as prognostic and predictive biomarkers to guide cancer therapy and early cancer detection. As technologies to capture and analyze CTCs continue to increase in sophistication, we anticipate that the potential clinical applications of CTCs will grow exponentially in the coming years., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.

Metastasis-competent circulating tumour cells (CTCs) experience oxidative stress in the bloodstream, but their survival mechanisms are not well defined. Here, comparing single-cell RNA-Seq profiles of CTCs from breast, prostate and lung cancers, we observe consistent induction of β-globin (HBB), but not its partner α-globin (HBA). The tumour-specific origin of HBB is confirmed by sequence polymorphisms within human xenograft-derived CTCs in mouse models. Increased intracellular reactive oxygen species (ROS) in cultured breast CTCs triggers HBB induction, mediated through the transcriptional regulator KLF4. Depletion of HBB in CTC-derived cultures has minimal effects on primary tumour growth, but it greatly increases apoptosis following ROS exposure, and dramatically reduces CTC-derived lung metastases. These effects are reversed by the anti-oxidant N-Acetyl Cysteine. Conversely, overexpression of HBB is sufficient to suppress intracellular ROS within CTCs. Altogether, these observations suggest that β-globin is selectively deregulated in cancer cells, mediating a cytoprotective effect during blood-borne metastasis.

Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent., Competing Interests: The authors declare no conflict of interest.

Purpose: The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR-targeted therapies. There are currently no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues., Experimental Design: We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes. This automated ISH assay was applied to tumor tissue from two distinct clinical cohorts that we hypothesized would differ in AR-V7 status., Results: We detected AR-V7 in all tumor samples from men with metastatic castration-resistant prostate cancer with tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide; n = 12). We detected AR-V7 in just one tumor from men who had undergone prostatectomy for localized adenocarcinoma (n = 30; Gleason 4 + 5 = 9 in the AR-V7-positive sample). Given the apparent distinction between the above groups by AR-V7 signal, we analyzed pretreatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naïve metastatic disease. Patients with metastases but without detectable AR-V7 RNA at baseline had significantly longer overall survival (log-rank P = 0.044) and a trend toward superior progression-free survival (log-rank P = 0.055)., Conclusions: Within an institutional cohort, the RNA ISH assay identified AR-V7 within FFPE tissue and may have prognostic value in metastatic castration-sensitive prostate cancer. These preliminary findings warrant further study in larger cohorts. Clin Cancer Res; 23(2); 363-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

TGF-β secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferation arrest during TGF-β-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with the suppression of multiple nuclear envelope proteins implicated in mitotic regulation and is phenocopied by modulating the expression of LaminB1. While TGF-β-induced mitotic defects in proliferating cells are reversible upon its withdrawal, the acquired genomic abnormalities persist, leading to increased tumorigenic phenotypes. In metastatic breast cancer patients, increased mesenchymal marker expression within single circulating tumor cells is correlated with genomic instability. These observations identify a mechanism whereby microenvironment-derived signals trigger heritable genetic changes within cancer cells, contributing to tumor evolution., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The rapidly growing array of therapeutic options in cancer requires informative biomarkers to guide the rational selection and precision application of appropriate therapies. Circulating biomarkers such as circulating tumor cells have immense potential as noninvasive, serial 'liquid biopsies' that may be more representative of the complete spectrum of a patient's individual malignancy than spatially and temporally restricted tumor biopsies. In this review, we discuss the current state-of-the-art in the isolation and molecular characterization of circulating tumor cells as well as their utility in a wide range of clinical applications such as prognostics, treatment monitoring and identification of novel therapeutic targets and resistance mechanisms to enable real-time adjustments in the clinical management of cancer.

The recent expansion of therapeutic options for the treatment of metastatic prostate cancer highlights the need for precision medicine approaches to enable the rational selection of appropriate therapies for individual patients. In this context, circulating biomarkers in the peripheral blood are attractive as readily accessible tools for predicting and monitoring therapeutic response. In the case of circulating tumor cells and circulating tumor DNA, they may also serve as a noninvasive means of assessing molecular aberrations in tumors at multiple time points before and during therapy. These so-called "liquid biopsies" can provide a snapshot view of tumor molecular architecture and may enable clinicians to monitor the molecular status of tumors as they evolve during treatment, thus allowing for individualized precision therapeutic decisions for patients over time. In this review, we outline recent progress in the field of circulating biomarkers in metastatic prostate cancer and evaluate their potential for enabling this vision of real-time precision medicine., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To compare clinical outcomes of image-based versus non-image-based interstitial brachytherapy (IBBT) for vaginal cancer., Methods and Materials: Of 72 patients with vaginal cancer treated with brachytherapy (BT), 47 had image guidance (CT=31, MRI=16) and 25 did not. Kaplan-Meier (KM) estimates were generated for any recurrence, local control (LC), disease-free interval (DFI), and overall survival (OS) and Cox models were used to assess prognostic factors., Results: Median age was 66 and median follow-up time was 24months. Median cumulative EQD2 dose was 80.8Gy in the non-IBBT group and 77Gy in the IBBT group. For non-IBBT versus IBBT, the 2-year KM LC was 71% vs. 93% (p=0.03); DFI was 54% vs. 86% (p=0.04); and OS 52% vs. 82% (p=0.35). On multivariate analysis, IBBT was associated with better DFI (HR 0.24, 95% CI 0.07-0.73). Having any 2 or more of chemotherapy, high-dose-rate (HDR) BT or IBBT (temporally correlated variables) significantly reduced risk of relapse (HR=0.33, 95% CI=0.13-0.83), compared to having none of these factors., Conclusion: Over time, the use of chemotherapy, HDR, and IBBT has increased in vaginal cancer. The combination of these factors resulted in the highest rates of disease control. Image-guided brachytherapy for vaginal cancer patients maximizes disease control., Competing Interests: Statement All authors declare that they have no conflicts of interest., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Mechanical properties of single cells have been shown to relate to cell phenotype and malignancy. However, until recently, it has been difficult to directly correlate each cell's biophysical characteristics to its molecular traits. Here, we present a cell sorting technique for use with a suspended microchannel resonator (SMR), which can measure biophysical characteristics of a single cell based on the sensor's record of its buoyant mass as well as its precise position while it traverses through a constricted microfluidic channel. The measurement provides information regarding the amount of time a cell takes to pass through a constriction (passage time), as related to the cell's deformability and surface friction, as well as the particular manner in which it passes through. In the method presented here, cells of interest are determined based on passage time, and are collected off-chip for downstream immunofluorescence imaging. The biophysical single-cell SMR measurement can then be correlated to the molecular expression of the collected cell. This proof-of-principle is demonstrated by sorting and collecting tumor cells from cell line-spiked blood samples as well as a metastatic prostate cancer patient blood sample, identifying them by their surface protein expression and relating them to distinct SMR signal trajectories.

Recent advances in microfluidic approaches have enabled the efficient isolation and detailed molecular characterization of circulating tumor cells (CTCs) in the peripheral blood of patients with cancer. Single-cell molecular analyses of CTCs reveal a tremendous degree of intracellular heterogeneity in CTC populations, reflective of heterogeneity across different patients as well as the underlying heterogeneity of tumors within each individual patient. These studies have enabled the identification of heterogeneous drug resistance mechanisms that can coexist in treatment refractory tumors. CTC analyses also enable serial noninvasive monitoring in patients and can capture the emergence of tumor heterogeneity over time, whether due to tumor evolution through genetic instability or through cellular plasticity. The presence and extent of intratumoral heterogeneity as revealed through the study of CTCs have important clinical implications for understanding and predicting the development of treatment resistance in a variety of solid tumors and for formulating appropriate therapeutic strategies in the effective treatment of cancer., (© 2016 Miyamoto et al; Published by Cold Spring Harbor Laboratory Press.)

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.

Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure., (Copyright © 2015, American Association for the Advancement of Science.)

Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30-40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The availability of new therapeutic options for the treatment of metastatic castration-resistant prostate cancer (mCRPC) has heightened the importance of monitoring and assessing treatment response. Accordingly, there is an unmet clinical need for reliable biomarkers that can be used to guide therapy. Circulating tumour cells (CTCs) are rare cells that are shed from primary and metastatic tumour deposits into the peripheral circulation, and represent a means of performing noninvasive tumour sampling. Indeed, enumeration of CTCs before and after therapy has shown that CTC burden correlates with prognosis in patients with mCRPC. Moreover, studies have demonstrated the potential of molecular analysis of CTCs in monitoring and predicting response to therapy in patients. This Review describes the challenges associated with monitoring treatment response in mCRPC, and the advancements in CTC-analysis technologies applied to such assessments and, ultimately, guiding prostate cancer treatment.

Objective: To identify factors associated with weight gain at 1 year from initiation of androgen deprivation therapy (ADT)., Methods: A retrospective review assessed weight change among 118 men with nonmetastatic prostate cancer treated with ADT for at least 6 months. Outcome associations were tested using 2-tailed t tests and linear regression., Results: Men in our cohort had significant weight gain (+1.32 kg, P=.0005) in the 1 year after ADT initiation. Three risk factors for weight gain on ADT were identified as follows: age<65 years (2.72 kg gained, P=.001), body mass index (BMI)<30 (1.98 kg gained, P=.00002), and nondiabetic status (1.56 kg gained, P=.0003). Multivariable regression found both age<65 years (beta=4.01, P=.02) and BMI<30 (beta=3.57, P=.03) to be independently predictive of weight gain, whereas nondiabetic status was nonsignificantly predictive of weight gain (beta=2.14, P=.29). Weight change was further stratified by the total number of risk factors present (risk score): scores of 0, 1, 2, and 3 risk factors corresponded to weight changes of -1.10, +0.41, +1.34, and +3.79 kg, respectively (P-trend=.0005)., Conclusion: Age<65 years and BMI<30 were both independently associated with weight gain 1 year after starting ADT. Increasing weight gain was also strongly associated with increasing number of baseline risk factors present. Despite traditional concerns about ADT in unhealthy men, these data suggest younger, healthier patients may be at higher risk for gaining weight on ADT and should be counseled accordingly., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: It is not known whether the addition of chemotherapy to radiation therapy improves outcomes in primary vaginal cancer. Here, we review clinical outcomes in patients with primary vaginal cancer treated with radiation therapy (RT) or concurrent chemoradiation therapy (CRT)., Methods: Seventy-one patients with primary vaginal cancer treated with definitive RT with or without concurrent chemotherapy at a single institution were identified and their records reviewed. A total of 51 patients were treated with RT alone; 20 patients were treated with CRT. Recurrences were analyzed. Overall survival (OS) and disease-free survival (DFS) rates were estimated using the Kaplan-Meier method. Cox regression analysis was performed., Results: The median age at diagnosis was 61 years (range, 18-92 years) and the median follow-up time among survivors was 3.0 years. Kaplan-Meier estimates for OS and DFS differed significantly between the RT and CRT groups (3-yr OS = 56% vs. 79%, log-rank p = 0.037; 3-yr DFS = 43% vs. 73%, log-rank p = 0.011). Twenty-three patients (45%) in the RT group had a relapse at any site compared to 3 (15%) in the CRT group (p = 0.027). With regard to the sites of first relapse, 10 patients (14%) had local only, 4 (6%) had local and regional, 9 (13%) had regional only, 1 (1%) had regional and distant, and 2 (3%) had distant only relapse. On univariate analysis, the use of concurrent chemotherapy, FIGO stage, tumor size, and date of diagnosis were significant predictors of DFS. On multivariate analysis, the use of concurrent chemotherapy remained a significant predictor of DFS (hazard ratio 0.31 (95% CI, 0.10-0.97; p = 0.04))., Conclusions: Vaginal cancer results in poor outcomes. Adequate radiation dose is essential to ensure curative management. Concurrent chemotherapy should be considered for vaginal cancer patients.

Background: Cabozantinib is an oral MET/VEGFR2 inhibitor. A recent phase II study of cabozantinib (100 mg daily) showed improved bone scans in subjects with metastatic castration-resistant prostate cancer (mCRPC), but adverse events (AE) caused frequent dose reductions. This study was designed to determine the efficacy and tolerability of cabozantinib at lower starting doses., Experimental Design: An adaptive design was used to determine the lowest active daily dose among 60, 40, and 20 mg. The primary endpoint was week 6 bone scan response, defined as ≥30% decrease in bone scan lesion area. The secondary endpoint was change in circulating tumor cells (CTC)., Results: Among 11 evaluable subjects enrolled at 40 mg, there were 9 partial responses (PR), 1 complete response, and 1 stable disease (SD). Of 10 subjects subsequently enrolled at 20 mg, there were 1 PR, 5 SDs, and 4 with progressive disease. Among 13 subjects enrolled on the 40 mg expansion cohort, there were 6 PRs and 7 SDs. No subjects required dose reduction or treatment interruption at 6 or 12 weeks; 3 subjects at dose level 0 discontinued due to AEs by 12 weeks. At 40 mg, median treatment duration was 27 weeks. 58% of subjects with ≥5 CTCs/7.5 mL at baseline converted to <5., Conclusions: Cabozantinib 40 mg daily was associated with a high rate of bone scan response. Cabozantinib 40 mg daily was associated with better tolerability than previously reported for cabozantinib 100 mg daily. These observations informed the design of phase III studies of cabozantinib in mCRPC., (©2013 AACR)