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Purpose: To investigate the use of plasma and urine DNA mutation analysis for predicting neoadjuvant chemotherapy (NAC) response and oncological outcome in patients with muscle-invasive bladder cancer. Experimental Design: Whole-exome sequencing of tumor and germline DNA was performed for 92 patients treated with NAC followed by radical cystectomy (RC). A custom NGS-panel capturing approximately 50 mutations per patient was designed and used to track mutated tumor DNA in plasma and urine. A total of 447 plasma samples, 281 urine supernatants, and 123 urine pellets collected before, during, and after treatment were analyzed. Patients were enrolled from 2013 to 2019, with a median follow-up time of 41.3 months after RC. Results: We identified tumor DNA before NAC in 89% of urine supernatants, 85% of urine pellets, and 43% of plasma samples. Tumor DNA levels were higher in urine supernatants and urine pellets compared with plasma samples (P < 0.001). In plasma, detection of circulating tumor DNA (ctDNA) before NAC was associated with a lower NAC response rate (P < 0.001). Detection of tumor DNA after NAC was associated with lower response rates in plasma, urine supernatant, and urine pellet (P < 0.001, P = 0.03, P = 0.002). Tumor DNA dynamics during NAC was predictive of NAC response and outcome in urine supernatant and plasma (P = 0.006 and P = 0.002). A combined measure from plasma and urine supernatant tumor DNA dynamics stratified patients by outcome (P = 0.003). Conclusions: Analysis of tumor DNA in plasma and urine samples both separately and combined has a potential to predict treatment response and outcome.

BACKGROUND: An improved risk assessment of patients with bladder cancer (BC) is important to optimize clinical management. OBJECTIVE: To identify whether immune cell subpopulations and cancer cell-intrinsic features are associated with outcome and response to first-line chemotherapy in BC. DESIGN, SETTING, AND PARTICIPANTS: Primary tumor tissue from 785 patients with BC (stage Ta-T4b) were stained using multiplex immunofluorescence (CD3, CD8, FOXP3, CD20, CD68, CD163, and MHC-I) and immunohistochemistry (pancytokeratin, CK5/6, GATA3, programmed death 1 [PD-1], and programmed death ligand 1 [PD-L1]). A digital image analysis quantified staining results within the carcinoma cell and stromal part of the tumor. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary endpoints were progression-free survival, recurrence-free survival, and response to first-line chemotherapy. Optimal cutoff values for investigated markers were estimated using maximally selected rank statistics and receiver operating characteristic for each primary endpoint. Time-to-event analyses were performed using Cox regression analyses. RESULTS AND LIMITATIONS: Several immune subpopulations were independently associated with clinical outcomes. Especially, high PD-1 and PD-L1 expression was independently associated with an increased risk of recurrence and progression in non-muscle-invasive tumors, but with a lower risk of recurrence in muscle-invasive tumors. Furthermore, we observed a lower likelihood of response to first-line chemotherapy in patients with basal differentiation features. Finally, a model combining clinical risk factors with our most evident prognosticator improved prediction accuracy compared with clinical risk factors alone for progression in non-muscle-invasive BC and recurrence in muscle-invasive BC. The use of tissue microarrays and a long inclusion period are limitations to this study. CONCLUSIONS: Immune cell subpopulations and cancer cell-intrinsic features are associated with different clinical outcomes in BC. PATIENT SUMMARY: Immune cells play an important role in cancer development and treatment outcomes. Infiltration with specific immune cells and the presence of markers associated with immune evasion in the tumor predict clinical outcomes in bladder cancer.

What is this summary about? This is a summary of a paper published in a medical journal that describes the results of a study called CheckMate 274. This study looked at a new treatment for muscle-invasive urothelial cancer, a type of cancer found in the urinary tract that has spread from the inner lining of the urinary tract or bladder and into the surrounding muscle wall where it can then spread to other parts of the body. The standard treatment for muscle-invasive urothelial cancer is surgery to remove affected parts of the urinary tract. However, cancer returns in more than half of people after this surgery. Adjuvant therapy is given to people after surgery with muscle-invasive urothelial cancer with a goal to reduce the risk of the cancer coming back; however, at the time this study started, there was no standard adjuvant treatment. What happened in the study? In the CheckMate 274 study, researchers compared nivolumab with a placebo as an adjuvant treatment for people with muscle-invasive urothelial cancer. The aim of the study was to understand how well nivolumab worked to reduce the chance of the cancer returning after surgery. The study also looked at what side effects (unwanted or unexpected results or conditions that are possibly related to the use of a medication) people had with treatment. What do the results mean? The results showed that people who received nivolumab versus placebo: Survived longer before the cancer was detected again, including people who had programmed death ligand-1 (shortened to PD-L1) on their cancer cells. Survived longer before a secondary cancer outside of the urinary tract was detected. Experienced no differences in health-related quality of life (the impact of the treatment on a person's mental and physical health). Had similar side effects to the people who received nivolumab in other studies. Clinical Trial Registration: NCT02632409 ( ClinicalTrials.gov )

Influence of radiotherapy technique on incidental dose to the whole ventricular system in focal irradiation of nongerminomatous germcell tumours

Background: Standard treatment of localized muscle invasive bladder cancer (MIBC) is neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC); however, only 40-50% respond to NAC and approx. 50% experience relapse. Evaluation of treatment efficacy and early detection of relapse are therefore major clinical challenges. Methods: We present a clinical update of a previously described cohort of 68 patients who received NAC prior to RC (NAC cohort; Christensen et al. JCO 2019; median follow-up (FU) of 58 months) together with evaluation of a retrospectively collected cohort of 120 patients who did not receive NAC (no-NAC cohort; median FU of 71 months). Circulating tumor DNA (ctDNA) was analyzed before NAC (NAC cohort, n=63), prior to RC (NAC cohort, n=67; no-NAC cohort, n=115) and after RC (NAC cohort, n=66; no-NAC cohort, n=37) using Signatera™. RNA-seq was performed on 176 tumors. Results: Updated clinical FU for the NAC cohort showed that ctDNA-positive patients had significantly worse recurrence-free survival (RFS) compared to ctDNA-negative patients (before NAC: HR=16, 95%CI=3.6-70.5, p=0.0002; during surveillance after RC: HR=27.6, 95%CI=7.9-96.9, p Conclusion: Presence of ctDNA was associated with worse prognosis for both NAC and no-NAC treated patients. Transcriptomic analysis of primary tumors showed that anti-tumor immune responses may be associated with a particularly good outcome whereas EMT may be promoting more aggressive disease. Citation Format: Sia Viborg Lindskrog, George Laliotis, Karin Birkenkamp-Demtröder, Iver Nordentoft, Philippe Lamy, Elshaddai Z. White, Natalia Pajak, Tine G. Andreasen, Punashi Dutta, Meenakshi Malhotra, Shruti Sharma, Mark Calhoun, Adam ElNaggar, Minetta C. Liu, Mads Agerbæk, Jørgen B. Jensen, Lars Dyrskjøt. Utility of circulating tumor DNA and transcriptomic profiling in predicting outcome in muscle invasive bladder cancer patients. [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 5600.

BACKGROUND: The role of adjuvant treatment in high-risk muscle-invasive urothelial carcinoma after radical surgery is not clear. METHODS: In a phase 3, multicenter, double-blind, randomized, controlled trial, we assigned patients with muscle-invasive urothelial carcinoma who had undergone radical surgery to receive, in a 1:1 ratio, either nivolumab (240 mg intravenously) or placebo every 2 weeks for up to 1 year. Neoadjuvant cisplatin-based chemotherapy before trial entry was allowed. The primary end points were disease-free survival among all the patients (intention-to-treat population) and among patients with a tumor programmed death ligand 1 (PD-L1) expression level of 1% or more. Survival free from recurrence outside the urothelial tract was a secondary end point. RESULTS: A total of 353 patients were assigned to receive nivolumab and 356 to receive placebo. The median disease-free survival in the intention-to-treat population was 20.8 months (95% confidence interval [CI], 16.5 to 27.6) with nivolumab and 10.8 months (95% CI, 8.3 to 13.9) with placebo. The percentage of patients who were alive and disease-free at 6 months was 74.9% with nivolumab and 60.3% with placebo (hazard ratio for disease recurrence or death, 0.70; 98.22% CI, 0.55 to 0.90; P

563 Background: Muscle-invasive bladder cancer (MIBC) accounts for ~25–30% of all bladder cancer diagnoses. With neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) as standard-of-care, the 5-year survival rate ranges from 40%–60%. Bladder-sparing protocols (BSP) have emerged as a feasible alternative to RC for MIBC treatment, however better tools are needed. In this study, we evaluated the prognostic value of circulating tumor DNA (ctDNA) in predicting recurrence in patients who achieved pathological complete response (pCR). Methods: We analyzed a previously described cohort of 68 patients (656 plasma samples; Christensen et al., JCO 2019) with MIBC who received NAC prior to cystectomy. Patients had an updated median follow-up of 58.94 months (range: 7.19-81.77) post-cystectomy. ctDNA was analyzed at baseline (before NAC; N=64), and prior to cystectomy (N=65) using a commercially available assay (Signatera, Natera, Inc.). Additionally, exploratory RNA-Seq was performed on tumors from 59 patients (samples with >5M total counts were utilized). Pathway analysis was used to compare ctDNA-positive and ctDNA-negative patients who failed to achieve pCR. Results: Of the 64 patients with ctDNA results available at baseline, 59.4% (38/64) tested ctDNA-negative, and of these 84.2% (32/38) achieved pCR. Furthermore, 40.6% (26/64) tested ctDNA-positive, and only 34.6% (9/26) achieved pCR. Likewise, prior to cystectomy, 83.9% (52/62) of patients were ctDNA-negative, and 80.7% (42/52) achieved pCR, while none of the ctDNA-positive patients achieved pCR (positive predictive value 100%; negative predictive value 80.8%). Based on both ctDNA timepoints, the probability of ctDNA-negative patients to achieve pCR was significantly higher than ctDNA-positive patients ( p

A higher incidence of cognitive impairment (CI) has previously been reported among orchiectomized testicular cancer patients (TCPs), but little is known about the underlying pathophysiology. The present study assessed CI in newly orchiectomized TCPs and explored the structural brain networks, endocrine status, and selected genotypes. Forty TCPs and 22 healthy controls (HCs) underwent neuropsychological testing and magnetic resonance imaging, and provided a blood sample. CI was defined as a z-score ≤ -2 on one neuropsychological test or ≤ -1.5 on two neuropsychological tests, and structural brain networks were investigated using graph theory. Associations of cognitive performance with brain networks, endocrine status (including testosterone levels and androgen receptor CAG repeat length), and genotypes (APOE, BDNF, COMT) were explored. Compared with HCs, TCPs performed poorer on 6 out of 15 neuropsychological tests, of which three tests remained statistically significant when adjusted for relevant between-group differences (p < 0.05). TCPs also demonstrated more CI than HCs (65% vs. 36%; p = 0.04). While global brain network analysis revealed no between-group differences, regional analysis indicated differences in node degree and betweenness centrality in several regions (p < 0.05), which was inconsistently associated with cognitive performance. In TCPs, CAG repeat length was positively correlated with delayed memory performance (r = 0.36; p = 0.02). A COMT group × genotype interaction effect was found for overall cognitive performance in TCPs, with risk carriers performing worse (p = 0.01). No effects were found for APOE, BDNF, or testosterone levels. In conclusion, our results support previous findings of a high incidence of CI in newly orchiectomized TCPs and provide novel insights into possible mechanisms.

BACKGROUND: Neoadjuvant cisplatin-based chemotherapy is standard care prior to radical cystectomy in patients with muscle-invasive bladder cancer (MIBC). OBJECTIVE: To assess efficacy and safety of two commonly used neoadjuvant schedules with different total doses and dose-intensities of gemcitabine and cisplatin (GC). METHODS: Data were collected retrospectively from all patients treated between 2010 and 2018 with neoadjuvant chemotherapy according to clinical routine at seven centres in Sweden and Denmark. Patients in Sweden received three cycles of a 4-week schedule (GC-4w: cisplatin 70 mg/m2 day 1, gemcitabine 1000 mg/m2 days 1, 8, 15, q 28 days) and in Denmark four cycles of a 3-week schedule (GC-3w: cisplatin 70 mg/m2 day 1, gemcitabine 1000 mg/m2 days 1, 8, q 21 days). Primary endpoint was pathological response at cystectomy (pT0N0 and

Purpose: To evaluate neurotoxicity in testicular cancer survivors (TCSs) years after treatment and secondly the influence of neurotoxicity on quality-of-life (QoL). Methods: We identified 2234 TCSs who completed the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity questionnaire. QoL was evaluated with the European Organization for Research and Treatment of Cancer QLQ-C30. Patients were grouped according to treatment strategy: surveillance (N = 1113), infradiaphragmatic radiotherapy (N = 301), cisplatin-etoposide-bleomycin (BEP) (N = 759), and more than one line of treatment (MTOL) (N = 61). Association of treatment modality with long-term neurotoxicity was analyzed with ordinal logistic regression. Secondly, associations between neurotoxicity and QoL were analyzed in BEP-treated patients. Analyses were age-adjusted and repeated with additional adjustment for comorbidity, smoking, and alcohol consumption. Results: After a median follow-up of 18.4 years, treatment with BEP and MTOL was associated with overall increased risk of neurotoxicity (odds ratio 2.4–4.7 depending on treatment intensity, P < 0.001) as well as subscales (peripheral neuropathy, ototoxicity, and dysfunction associated with neuropathy, all P < 0.001). Radiotherapy and surveillance were not associated with neurotoxicity. In patients treated with BEP, neurotoxicity was highly associated with all indicators of worse QoL outcomes (P-trend: 1.5 × 10−17 to 1.1 × 10−28) after almost 20 years of follow-up. Conclusions: Treatment with BEP was associated with long-term neurotoxicity, which was highly associated with decreased QoL. Strategies to ameliorate or prevent neurotoxicity should be investigated. Implications for Cancer Survivors: Treatment with chemotherapy for testicular cancer induces long-term neuro- and ototoxicity which may have severe influence on quality-of-life years after treatment cessation.

Overtreatment with cisplatin-based chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC), and currently none of the reported biomarkers for predicting response have been implemented in the clinic. Here we perform a comprehensive multi-omics analysis (genomics, transcriptomics, epigenomics and proteomics) of 300 MIBC patients treated with chemotherapy (neoadjuvant or first-line) to identify molecular changes associated with treatment response. DNA-based associations with response converge on genomic instability driven by a high number of chromosomal alterations, indels, signature 5 mutations and/or BRCA2 mutations. Expression data identifies the basal/squamous gene expression subtype to be associated with poor response. Immune cell infiltration and high PD-1 protein expression are associated with treatment response. Through integration of genomic and transcriptomic data, we demonstrate patient stratification to groups of low and high likelihood of cisplatin-based response. This could pave the way for future patient selection following validation in prospective clinical trials., There are currently only a few biomarkers to predict the response of muscle invasive bladder cancer to therapy. Here, the authors analyse 300 tumors using exome and RNA sequencing and find that tumors with a high degree of genomic instability and a non-basal/squamous gene expression subtype are most likely to respond to treatment.

Introduction: Patients with testicular cancer (TC) are mainly young and survival rates are high. MRI has several times been proposed to replace CT in follow-up of this patient group to reduce image-related radiation exposure. However, current evidence is scarce for the use of MRI in this context. Aims: First, to retrospectively evaluate the ability of MRI of the retroperitoneum and pelvis to detect relapse in patients with TC stage I. Second, to present a relevant MRI protocol of the retroperitoneum and pelvis with diffusion weighted imaging (DWI). Material and methods: A retrospective analysis of written radiology reports compared to clinical data from clinical practice from 2010 to 2018. The cohort consists of 2487 MRIs of the retroperitoneum and pelvis in 759 patients with TC stage I (524 seminoma (69.0%), 235 non-seminoma (31.0%)), including 102 patients (13.4%) with confirmed relapse. Confirmed relapse was defined when treatment was initiated for metastatic TC. Results: Ninety-five patients had a relapse in the MRI scan field during follow-up. MRI of the retroperitoneum and pelvis showed a high sensitivity of 93.8% and a high specificity of 97.4% for detecting TC relapse. The sensitivity for detecting relapse ≥10 mm in short axis lymph node diameter was 100%. The negative predictive value was 99.7%, the positive predictive value was 59.9% and the accuracy was 97.3%. Conclusions: MRI of the retroperitoneum and pelvis constitutes a safe alternative to CT in follow-up of patients with TC stage I with both a high sensitivity and a high specificity. We present a robust MRI protocol with DWI and estimate that MRI follow-up of TC stage I can be easily implemented in most modern radiology departments. Registration: Conducted with permission from the Danish Data Protection Agency (1-16-02-323-16) and the Danish Health Authority.

Purpose: Long-term cancer survivors may develop psychological late effects. The aim of the present study was to determine prevalence of high level of stress in testicular cancer survivors (TCS) compared with the general population and prevalence of high level of stress among TCS stratified by type of treatment (surveillance, bleomycin, etoposide and cisplatin (BEP), or abdominal radiotherapy (RT)). Methods: In this large, nationwide and population-based, cross-sectional study, a total of 2252 TCS filled in a questionnaire between 2014–2016 covering psychological stress (Perceived Stress Scale (PSS)), sociodemographic factors, and physical health variables. Results were compared with a reference population. The reference population consisted of 61,927 men without prior or present cancer and sampled at random from the central population. High level of stress was defined as a PSS score ≥ 16, equivalent to the highest scoring quintile in the reference population. Logistic regression models adjusted for relevant covariates were used to estimate prevalence ratios of high level of stress. Results: Distribution of TCS was: surveillance, n = 1134; BEP, n = 807; and RT, n = 311 (median time since diagnosis was 19 years). TCS were more likely to have high level of stress compared to the reference population with a prevalence ratio of 1.56 (95% CI, 1.40–1.73). Individually, surveillance, BEP and RT groups had higher level of stress compared to the reference population. Conclusions: TCS are more likely to have high level of stress. Screening programs for psychological stress should be considered as part of the follow-up program. Implications for cancer survivors: A higher level of stress is observed in TCS irrespective of treatment.

Purpose Concerns of imaging-related radiation in young patients with high survival rates have increased the use of magnetic resonance imaging (MRI) in testicular cancer (TC) stage I. However, computed tomography (CT) is still preferred for metastatic TC. The purpose of this study was to compare whole-body MRI incl. diffusion-weighted whole-body imaging with background body signal suppression (DWIBS) with contrast-enhanced, thoracoabdominal CT in metastatic TC.Methods A prospective, non-inferiority study of 84 consecutive patients (median age 33 years) with newly diagnosed metastatic TC (February 2018 - January 2021). Exclusion criteria were age Results On patient level, MRI had 98% sensitivity and 75% specificity compared to CT. On lesion level within each modality, MRI had 99% sensitivity and 78% specificity, whereas CT had 98% sensitivity and 88% specificity. MRI sensitivity was non-inferior to CT (difference 0.57% (95% CI -1.4-2.5%)). The interobserver agreement was substantial between CT and MRI. Conclusion MRI with DWIBS was non-inferior to contrast-enhanced CT in detecting metastatic TC disease. Trial registration www.clinicaltrials.gov NCT03436901, finished July 1st 2021.

Signe Sørup,1 Bianka Darvalics,1 Azza Ahmed Khalil,2 Marianne Nordsmark,2 Mette Hæe,2 Frede Donskov,3 Mads Agerbæk,2 Leo Russo,4 Dina Oksen,5 Emmanuelle Boutmy,5 Patrice Verpillat,5 Deirdre Cronin-Fenton1 1Department of Clinical Epidemiology, Department of Clinical Medicine, Aarhus University & Aarhus University Hospital, Aarhus, Denmark; 2Department of Oncology, Aarhus University Hospital, Aarhus, Denmark; 3Department of Clinical Medicine, Aarhus University, Aarhus, Denmark; 4Worldwide Medical and Safety, Pfizer, Collegeville, PA, USA; 5Global Epidemiology, Merck Healthcare KGaA, Darmstadt, GermanyCorrespondence: Signe SørupDepartment of Clinical Epidemiology, Aarhus University & Aarhus University Hospital, Olof Palmes Allé 43-45, DK-8200 Aarhus N, DenmarkTel +45 871 68398Email SigneSorup@clin.au.dkPurpose: Few studies have described real-world treatment patterns and survival before the widespread use of immune checkpoint inhibitors (ICIs). We aimed to describe anti-cancer treatment including the use of programmed cell death-1 and ligand-1 (PD-1/PD-L1) ICIs and overall survival (OS) in advanced cancer patients as a benchmarking real-world standard before widespread use of ICIs.Patients and Methods: Using nationwide Danish medical registries, we assembled cohorts of Danish patients with advanced non-small cell lung cancer (NSCLC) (n=12,283), urothelial carcinoma (n=2504), epithelial ovarian cancer (n=1466), gastric adenocarcinoma (n=1457), and renal cell carcinoma (RCC) (n=1261) diagnosed between 1/1/2013 and 31/12/2017. We describe anti-cancer treatment and OS using proportions, medians, and Kaplan–Meier methods.Results: Between 9% (ovarian cancer) and 25% (gastric adenocarcinoma) of patients did not receive anti-cancer treatment. The remaining patients received surgery, radiation therapy, and/or medical therapy. Chemotherapy was the most frequent medical therapy in all cohorts except for RCC (tyrosine kinase inhibitors). PD-L1/PD-1 ICIs were used in 7– 8% of the NSCLC and RCC cohorts—mainly as second or higher line treatments. OS was longest in patients starting treatment with surgery (eg 25.6 months [95%-confidence interval (CI)=21.9– 29.4] for NSCLC and 21.4 months [95%-CI=19.8– 23.5] for urothelial carcinoma) and shortest for radiation therapy (eg 3.9 months [95%-CI=3.6– 4.2] for NSCLC and 12.6 months [95%-CI=9.2– 17.5] for urothelial carcinoma). NSCLC patients starting with medical therapy had OS between these limits. Median OS for NSCLC patients starting treatment with PD-L1/PD-1 ICIs was 21.4 months (95%-CI=13.9-not estimable).Conclusion: Most patients with advanced NSCLC, urothelial carcinoma, epithelial ovarian cancer, gastric adenocarcinoma and RCC had poor OS in an era where only a minority received PD-L1/PD-1 ICIs. This information on treatment patterns and survival is important as a benchmarking real-world standard before widespread use of ICIs.Keywords: cancer, treatment, survival, immunotherapy, programmed cell death-1 immune checkpoint inhibitors, programmed cell death-ligand-1 immune checkpoint inhibitors

Objective Previous research has indicated cognitive decline (CD) among testicular cancer patients (TCPs), even in the absence of chemotherapy, but little is known about the underlying pathophysiology. The present study assessed changes in cognitive functions and structural brain connectomes in TCPs and explored the associations between cognitive changes and endocrine status and hypothesized risk genotypes. Methods Thirty‐eight newly orchiectomized TCPs and 21 healthy controls (HCs) comparable to TCPs in terms of age and years of education underwent neuropsychological testing, structural MRI, and a biological assessment at baseline and 6 months later. Cognitive change was assessed with a neuropsychological test battery and determined using a standardized regression‐based approach, with substantial change defined as z‐scores ≤−1.64 or ≥1.64. MRI scans and graph theory were used to evaluate changes in structural brain connectomes. The associations of cognitive changes with testosterone levels, androgen receptor gene (AR) CAG repeat length, and genotypes (APOE, COMT, and BDNF) were explored. Results Compared with HCs, TCPs showed higher rates of substantial decline on processing speed and visuospatial ability and higher rates of substantial improvement on verbal recall and visuospatial learning (p, The present study confirms previous findings of domain‐specific cognitive decline in testicular cancer patients following orchiectomy, but also points to domain‐specific improvements. The results do not indicate changes in brain connectomes or endocrine status to be the main drivers of cognitive changes.

Background Standard treatment in patients with localized muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy (NAC) followed by radical cystectomy (CX). Importantly, there is currently no clinically validated way to estimate response to NAC before CX, where a pathological assessment of tumor down-staging is used as a proxy for response. Identification and tracking of circulating tumor DNA (ctDNA) has been shown to reflect prognosis and treatment response in several cancers, including bladder cancer. Here, we aim to investigate the capability of a combined plasma, urine supernatant and urine pellet mutation analysis by deep targeted sequencing for detecting residual tumor and treatment response before CX. Methods In total, 92 patients with localized MIBC treated with NAC followed by CX were included in the study. DNA extracted from primary tumors and leukocytes were subjected to whole exome sequencing for identification of approx. 50 mutations per patient that were used to design a custom NGS panel. Urine supernatants (n=281), urine pellets (n=114) and plasma samples (n=167), collected before CX, were subjected to deep targeted sequencing employing unique molecular identifiers (UMI). Mutations were assessed using the Shearwater algorithm and samples were further assessed using Fisher’s Method and bootstrapping of random mutations. In-silico analyses identified the limit of detection to be approx. 0.1%. For 56 patients, plasma mutation data (n=288 samples) were obtained from a previously published study by our group. Results Tumor derived DNA levels in paired urine supernatants and pellets were correlated (rho=0.8), however with higher levels in pellets when adjusting for the extracted amount of DNA (p Conclusions Tumor derived DNA was more frequently observed in urine samples and at higher levels compared to plasma samples. Interestingly, we show that tumor derived DNA dynamics in urine supernatants might better reflect the local tumor environment and plasma-based tumor derived DNA better reflects outcome following surgery, i.e. disease dissemination. Citation Format: Emil Christensen, Iver Nordentoft, Sara K. Elbæk, Karin Birkenkamp-Demtröder, Ann Taber, Michael Knudsen, Philippe Lamy, Mads Agerbæk, Jørgen B. Jensen, Lars Dyrskjøt. Treatment response and outcome prediction guided by deep targeted sequencing of urine and plasma samples in patients with muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1275.

Background: Neoadjuvant chemotherapy (NAC) followed by radical cystectomy (CX), is gold standard treatment in localized muscle-invasive bladder cancer (MIBC). About 45% of patients with MIBC develop metastatic relapse within 2 years after CX. The response rate to chemotherapy and immune checkpoint inhibitors (ICI) is relatively low, and biomarker tests for monitoring response are needed. Furthermore, biomarkers for early detection of minimal residual disease (MRD) after CX is needed to enable earlier treatment initiation. Tumor-informed detection of mutations in cell-free DNA (cfDNA) from peripheral blood has shown promising results in its ability to monitor MRD. However, the low tumor fraction after surgery and limited input material obtained from a typical plasma sample limits the probability of detecting low metastatic burden scenarios. Here we implemented and applied locally a whole-genome sequencing (WGS) approach to circulating tumor DNA (ctDNA) monitoring for improving ctDNA detection. Methods: A total of 140 MIBC patients undergoing NAC and CX were enrolled, including a test cohort (n=19) and a validation cohort (n=120). cfDNA was extracted from ~1mL plasma (n=1100) and procured from longitudinal plasma sampling during NAC (response measure), pre-cystectomy (response measure), post-surgery (relapse monitoring) and during immunotherapy (ICI treatment). WGS was applied to tumor/germline pairs (coverage >30x/20x) and plasma cfDNA (>20x) facilitating detection of genome wide genomic alterations and quantification of ctDNA using the MRDetect method. Results: We developed a personalized tumor-informed WGS model by integrating genome-wide mutation and copy number variation data coupled with advanced signal processing and AI-based error suppression. Patient-specific somatic variant patterns were then used for detecting and measuring the ctDNA levels in low-input blood samples by WGS. The assay sensitivity allowed for detection of tumor fractions down to 8*10-5. Furthermore, in our test cohort of 19 patients, we detected ctDNA after CX in 7 of 8 patients with clinical relapse (88% sensitivity) and detected no ctDNA in 11 of 11 patients with no clinical relapse (100% specificity). We observed a positive lead-time for MRD-based recurrence detection compared to CT-based reccurence detection (9 months on average). The full dataset is currently being processed and will be presented at the AACR 2022 meeting. Conclusions: For precision oncology, we need to develop quantitative and non-invasive methodologies to help tailor the treatments to individual patients and monitor them for further clinical decision-making. The results indicate the clinical potential of personalized genome-wide mutation integration as an ultra-sensitive, non-invasive method for MRD detection and treatment response monitoring which could aid in clinical management of patients with bladder cancer. Citation Format: Iver Nordentoft, Karin Birkenkamp-Demtröder, Emil Christensen, Sunil Deochand, Dillon Maloney, Danielle Afterman, Tomer Lauterman, Noah Friedman, Imane Bourzgui, Nidhi Ramaraj, Zohar Donenhirsh, Ronel Veksler, Sia Viborg, Mads Agerbæk, Jørgen Bjerggaard Jensen, Jonathan Rosenfeld, Ravi Kandasamy, Iman Tavassoly, Boris Oklander, Asaf Zviran, Lars Dyrskjøt. Genome-wide circulating tumor DNA for monitoring treatment response and metastatic relapse in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 540.

Background: Survival outcomes are poor for patients with metastatic urothelial carcinoma who receive standard, first-line, platinum-based chemotherapy. We assessed the overall survival of patients who received durvalumab (a PD-L1 inhibitor), with or without tremelimumab (a CTLA-4 inhibitor), as a first-line treatment for metastatic urothelial carcinoma.Methods: DANUBE is an open-label, randomised, controlled, phase 3 trial in patients with untreated, unresectable, locally advanced or metastatic urothelial carcinoma, conducted at 224 academic research centres, hospitals, and oncology clinics in 23 countries. Eligible patients were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0 or 1. We randomly assigned patients (1:1:1) to receive durvalumab monotherapy (1500 mg) administered intravenously every 4 weeks; durvalumab (1500 mg) plus tremelimumab (75 mg) administered intravenously every 4 weeks for up to four doses, followed by durvalumab maintenance (1500 mg) every 4 weeks; or standard-of-care chemotherapy (gemcitabine plus cisplatin or gemcitabine plus carboplatin, depending on cisplatin eligibility) administered intravenously for up to six cycles. Randomisation was done through an interactive voice–web response system, with stratification by cisplatin eligibility, PD-L1 status, and presence or absence of liver metastases, lung metastases, or both. The coprimary endpoints were overall survival compared between the durvalumab monotherapy versus chemotherapy groups in the population of patients with high PD-L1 expression (the high PD-L1 population) and between the durvalumab plus tremelimumab versus chemotherapy groups in the intention-to-treat population (all randomly assigned patients). The study has completed enrolment and the final analysis of overall survival is reported. The trial is registered with ClinicalTrials.gov, NCT02516241, and the EU Clinical Trials Register, EudraCT number 2015-001633-24.Findings: Between Nov 24, 2015, and March 21, 2017, we randomly assigned 1032 patients to receive durvalumab (n=346), durvalumab plus tremelimumab (n=342), or chemotherapy (n=344). At data cutoff (Jan 27, 2020), median follow-up for survival was 41·2 months (IQR 37·9–43·2) for all patients. In the high PD-L1 population, median overall survival was 14·4 months (95% CI 10·4–17·3) in the durvalumab monotherapy group (n=209) versus 12·1 months (10·4–15·0) in the chemotherapy group (n=207; hazard ratio 0·89, 95% CI 0·71–1·11; p=0·30). In the intention-to-treat population, median overall survival was 15·1 months (13·1–18·0) in the durvalumab plus tremelimumab group versus 12·1 months (10·9–14·0) in the chemotherapy group (0·85, 95% CI 0·72–1·02; p=0·075). In the safety population, grade 3 or 4 treatment-related adverse events occurred in 47 (14%) of 345 patients in the durvalumab group, 93 (27%) of 340 patients in the durvalumab plus tremelimumab group, and in 188 (60%) of 313 patients in the chemotherapy group. The most common grade 3 or 4 treatment-related adverse event was increased lipase in the durvalumab group (seven [2%] of 345 patients) and in the durvalumab plus tremelimumab group (16 [5%] of 340 patients), and neutropenia in the chemotherapy group (66 [21%] of 313 patients). Serious treatment-related adverse events occurred in 30 (9%) of 345 patients in the durvalumab group, 78 (23%) of 340 patients in the durvalumab plus tremelimumab group, and 50 (16%) of 313 patients in the chemotherapy group. Deaths due to study drug toxicity were reported in two (1%) patients in the durvalumab group (acute hepatic failure and hepatitis), two (1%) patients in the durvalumab plus tremelimumab group (septic shock and pneumonitis), and one (Interpretation: This study did not meet either of its coprimary endpoints. Further research to identify the patients with previously untreated metastatic urothelial carcinoma who benefit from treatment with immune checkpoint inhibitors, either alone or in combination regimens, is warranted.Funding: AstraZeneca.

Background: The present study (VINGEM) is the first randomised trial comparing vinflunine/gemcitabine (VG) to standard carboplatin/gemcitabine (CG) in patients with advanced urothelial carcinoma (aUC) ineligible for treatment with cisplatin. Patients and methods: Patients with aUC, creatinine clearance 30–60 ml/min, performance status ≤1 and no prior chemotherapy for metastatic disease were randomised to the experimental arm (vinflunine 280 or 250 mg/m2 day 1, gemcitabine 1000 mg/m2 days 1 and 8, q21 days) or the control arm (carboplatin AUC 4.5 day 1, gemcitabine 1000 mg/m2 days 1 and 8, q21 days). Primary end-point was progression-free survival (PFS). Results: Sixty-two patients were randomised; a total of 59 patients were treated (29 VG, 30 CG). There was no significant difference in PFS between the treatment arms: median 6.2 months for VG versus 6.3 months for CG (hazard ratio [HR]: 0.75, 95% confidence interval [CI]: 0.44–1.28; P = 0.293). Median overall survival was 12.5 months for VG versus 10.6 months for CG. The overall response rate (ORR) was higher in the VG arm than in the CG arm (63% versus 40%) but was not statistically significant in the intention-to-treat analysis. Furthermore, VG showed a high complete response (CR) rate, 22% versus 3% in CG. In the per-protocol group, both ORR and CR were significantly higher for VG than for CG. The most common adverse events (AEs) were fatigue, haematological toxicities, gastrointestinal disorders and nausea/vomiting. Common grade III/IV AEs were neutropenia (VG 62%, CG 43%), thrombocytopenia (VG 7%, CG 37%) and febrile neutropenia (VG 31%, CG 7%). Conclusions: The combination of VG did not improve PFS compared with standard treatment with CG in patients unfit for cisplatin due to renal impairment. The response rate of VG indicates, however, an active regimen and warrants further studies. Clinicaltrials.gov number: NCT02665039.

PURPOSE To analyze the risk of cardiovascular disease (CVD) after treatment of male germ cell cancer (GCC). METHODS Clinical data were extracted from the Danish Testicular Cancer database. For each patient, 10 men matched on date of birth were identified in the Danish normal population by risk-set sampling. Cardiovascular risk factors, CVD, and associated deaths were identified in Danish registries. The association between treatment and outcomes was analyzed by separate Cox models for each outcome. Cancer treatment was included as a time-varying covariate. RESULTS We included 5,185 patients with GCC and 51,850 men in the normal population. Median follow-up was 15.8 years. Treatment with bleomycin-etoposide-cisplatin (BEP; n = 1,819) was associated with increased risks of hypertension and hypercholesterolemia. Hazard ratios (HRs) of CVD < 1 year after initiation of BEP treatment were as follows: myocardial infarction (HR, 6.3; 95% CI, 2.9 to 13.9), cerebrovascular accident (HR, 6.0; 95% CI, 2.6 to 14.1), and venous thromboembolism (HR, 24.7; 95% CI, 14.0 to 43.6). One year after BEP treatment, the risk of CVD decreased to normal levels, but after 10 years, increasing risks were found for myocardial infarction (HR, 1.4; 95% CI, 1.0 to 2.0) and cardiovascular death (HR, 1.6; 95% CI, 1.0 to 2.5). Radiotherapy (n = 780) increased the risk of diabetes at long-term follow-up (HR, 1.4; 95% CI, 1.0 to 2.0) but not that of other outcomes. With surveillance (n = 3,332), cardiovascular risk factors, CVD, and cardiovascular death data were comparable to that of the normal population. CONCLUSION Treatment with BEP was associated with highly increased risks of CVD < 1 year after treatment start and mildly increased risks after 10 years of follow-up. Radiotherapy increased the risk of diabetes but not incident CVD. The risk of CVD in patients followed in a surveillance program was comparable to that of the normal population.

Of the patients undergoing radical cystectomy, 20-80% experience relapse. Minimally invasive methods for early detection of metastatic relapse after cystectomy and for monitoring ongoing therapy are urgently needed to improve individualised follow-up and treatment. Therefore, we evaluated the use of circulating tumour DNA (ctDNA) in plasma and urine to detect metastatic relapse after cystectomy and measure treatment efficacy. We exome sequenced tumour and germline DNA from patients with muscle-invasive bladder cancer and monitored ctDNA in 370 liquid biopsies throughout the disease courses by 84 personalised digital droplet polymerase chain reaction assays targeting 61 genes. Patients were prospectively recruited between 2013 and 2017. Patients with metastatic relapse had significantly higher ctDNA levels compared with disease-free patients (p

e16527 Background: Bladder cancer (BC) is the 9th most commonly diagnosed cancer worldwide and each year responsible for 165,000 deaths. Neoadjuvant combination chemotherapy, followed by radical cystectomy, is used for the management of localized muscle-invasive bladder cancer. One of the critical challenges in this therapeutic regimen is monitoring the tumor load to assess therapeutic efficacy – this is typically performed by assessing pathological downstaging in the cystectomy specimen. A high frequency of patients presents with T0N0 at cystectomy (no indication of residual disease), and consequently, it is vital to investigate organ preservation approaches to identify those patients who may qualify for bladder preservation. For precision oncology, we need to develop quantitative and non-invasive diagnostic methodologies to help the oncologist tailor the treatments to individual patients and monitor them for further clinical decision-making. Methods: Cell-free DNA (cfDNA) mutation detection has shown significant promise in its ability to monitor minimal residual disease and disease relapse by detection of cancer mutations in the peripheral blood. However, the combination of low tumor fraction and limited input material obtained from a typical plasma sample restricts the probability of detecting low metastatic burden in cfDNA through current deep targeted sequencing methods. Results: Here we present results from applying whole-genome sequencing (WGS) of cfDNA. We integrate a genome-wide mutation and copy number monitoring approach coupled with advanced signal processing and Artificial Intelligence (AI) for measuring the tumor load from low-input blood samples (̃1mL of plasma) with ultra-sensitive detection. The increased sensitivity allowed clinical detection of tumor fraction down to 8*10-5 and recurrence detection sensitivity achieving > 65% at the first two months post-surgery. The WGS cfDNA approach is being evaluated on a patient cohort of more than 50 bladder cancer patients with longitudinal plasma sampling during neoadjuvant chemotherapy (response measure), pre-cystectomy (complete response measure), and post-surgery (relapse monitoring). Conclusions: The results indicate the clinical potential of genome-wide mutation integration as an ultra-sensitive, non-invasive diagnostic method for bladder cancer clinical management and bladder organ preservation.

PURPOSE Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence ( P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging ( P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.

391 Background: The standard of care (SOC) for patients (pts) with MIUC is radical surgery ± cisplatin-based neoadjuvant chemotherapy (chemo), but many pts are cisplatin-ineligible. There is no conclusive evidence supporting adjuvant chemo in pts who did not receive neoadjuvant chemo and in those with residual disease after neoadjuvant cisplatin. This phase 3 trial of adjuvant nivolumab (NIVO) vs placebo (PBO) in pts with MIUC after radical surgery ± neoadjuvant cisplatin (CheckMate 274) aims to address an unmet need in these pts. We report the initial results. Methods: This is a phase 3, randomized, double-blind, multicenter trial of NIVO vs PBO in pts with high-risk MIUC (bladder, ureter, or renal pelvis) after radical surgery. Pts were randomized 1:1 to NIVO 240 mg Q2W or PBO for ≤ 1 year of adjuvant treatment. Pts had radical surgery within 120 days ± neoadjuvant cisplatin or were ineligible/declined cisplatin-based chemo, evidence of UC at high risk of recurrence per pathologic staging, were disease-free by imaging, and ECOG PS ≤ 1. Primary endpoints: disease-free survival (DFS) in all randomized pts (ITT population) and in pts with tumor PD-L1 expression ≥ 1%. DFS was stratified by nodal status, prior neoadjuvant cisplatin, and PD-L1 status. Non–urothelial tract recurrence-free survival (NUTRFS) in ITT pts and in pts with PD-L ≥ 1% is a secondary endpoint. Safety is an exploratory endpoint. Results: In total, 353 pts were randomized to NIVO (PD-L1 ≥ 1%, n = 140) and 356 pts to PBO (PD-L1 ≥ 1%, n = 142). The primary endpoint of DFS was met in ITT pts (median follow-up, 20.9 mo for NIVO; 19.5 mo for PBO) and in pts with PD-L1 ≥ 1%. DFS and NUTRFS were improved with NIVO vs PBO in both populations (Table). DFS improvement with NIVO was generally consistent across subgroups. Grade 3–4 treatment-related adverse events (TRAEs) occurred in 17.9% and 7.2% of pts in the NIVO and PBO arms, respectively. Conclusions: NIVO demonstrated a statistically significant and clinically meaningful improvement in DFS vs PBO for MIUC after radical surgery, both in ITT pts and pts with PD-L1 ≥ 1%. AEs were manageable and consistent with previous reports. These results support adjuvant NIVO as a new SOC for pts with MIUC with high risk for recurrence despite neoadjuvant chemo or those ineligible for and/or declining cisplatin-based chemo. Clinical trial information: NCT02632409 . Research Sponsor: Bristol Myers Squibb[Table: see text]

INTRODUCTION Intracranial malignant germ cell tumours (iGCT) are rare brain tumours mainly diagnosed in children and younger adults. MATERIAL AND METHODS A retrospective analysis was performed by chart review of patients treated for iGCT in the northern and central region of Denmark. Teratoma only patients were not included in the study. RESULTS 20 patients with iGCT were diagnosed from 2008–2019 in Western Denmark. The cumulative incidence was 1.05 per 100.000. The yearly incidence was 0.1 per 100.000. Mean age at diagnosis was 18 years (range 8–36 years), 17 were males and 3 were females. 13 patients presented with germinoma and 7 patients with non germinomateous germ cell tumours (NGGCT). Three patients had disseminated disease, two with germinoma and one with NGGCT. All patients had received radiotherapy and 18 patients were treated with multidrug chemotherapy including platinum and etoposide before irradiation. Two patients experienced recurrent disease, both non disseminated at diagnosis, one patient with germinoma and one patient with NGGCT. Both received salvage treatment including high dose chemotherapy with stem cell transplantation and reirradiation. Two NGGCT patients died, one patient after development of an anaplastic astrocytoma in the radiation field five years after radiotherapy and one patient after intracranial hemorraghe 18 months after salvage treatment for recurrent disease. Overall survival was 90%, 100% for GCT and 71% for NGGCT. CONCLUSION The outcome of patients with iGCT in Western Denmark was comparable to the literature. A nationwide study of epidemiology and outcome of iGCT in Denmark is planned.

Background/Objective: Lapatinib is a potent HER1 and HER2 inhibitor. Gemcitabine-cisplatin (GC) is a standard chemotherapy regimen for advanced/metastatic bladder cancer. This phase I study examined the safety of lapatinib in combination with GC in patients with bladder cancer. The primary aim was to determine the maximum tolerated dose (MTD) of lapatinib in combination with GC. Methods: A 3 + 3 dose escalation protocol was used with lapatinib at 750, 1,000 and then 1,250 mg. It was dosed daily with gemcitabine (1,000 mg/m2 on days 1, 8 and 15) and cisplatin (70 mg/m2 on day 2) every 28 days. In all, 18 patients with a median age of 63 years (range 50-77) were included; 3/6, 3/5 and 6/7 patients received lapatinib at 750, 1,000 and 1,250 mg, combined with GC, in cohorts 1, 2 and 3, respectively. Results: No dose-limiting toxicities (DLTs) were observed in cohort 1 or 2 (3 patients each); in cohort 3 (2 × 3 patients), 1 of the 6 patients presented DLTs (grade 4, treatment-related febrile neutropenia and renal failure). Twelve patients received 6 cycles. Conclusions: Lapatinib at 750-1,250 mg combined with GC appears safe and tolerable. The MTD of lapatinib combined with GC in bladder cancer was 1,250 mg.

BACKGROUND: Resection of residual masses after chemotherapy plays a crucial role in management of patients with germ cell tumours (GCTs). The extent of surgery is controversial and we present the experiences from Aarhus University Hospital over a 20-year period. The aim was to evaluate survival, complications, working ability and quality of life (QOL) following a limited surgical procedure performed to resect residual masses in non-seminomatous testicular cancer patients after chemotherapy.MATERIAL AND METHODS: A consecutive patient cohort of 109 patients having surgery between 1993 and 2013 was investigated. Hospital records were reviewed and complications were graded according to the Clavien-Dindo classification. QOL data were assessed in a cross-sectional analysis using the European Organisation for Research and Treatment of Cancer (EORTC), QLQ-C30 version 3.0. Patients were matched 1:1 with controls to evaluate the influence of surgical resection on the QOL.RESULTS: With a median follow-up of 10.3 years, 11 relapses in retroperitoneum were recorded in 10 patients (9%), and four patients (5%) died of disease progression. The majority of relapses in patients considered having no evidence of disease (NED) after primary retroperitoneal surgery occurred 10 + years after treatment and was outside the field of the elective lymph node dissection. Twenty-seven (44%) grade I, 15 (24%) grade II, 7 (11%) grade IIIa and 13 (21%) grade IIIb complications were recorded. No grade IV and V complications were observed. Twenty-three patients (20%) reported loss of antegrade ejaculation. We found no significant differences between patients and controls regarding QOL.CONCLUSION: The survival outcome and complication rate are favourable and are comparable to studies involving full and modified template lymph node dissection. We find that limited resection constitutes an applicable and safe procedure. Limited surgical resection did not influence the patients' long-term QOL. Longer follow-up might be considered.

The cohort was set up in order to analyze late effects in long-term testicular cancer survivors (TCS) and to contribute to the design of future follow-up programs addressing and potentially preventing late effects. Data for this cross-sectional study were collected between January 1, 2014, and December 31, 2016, among living Danish TCS and 60% agreed to participate in the cohort (N = 2,572). Mean time since testicular cancer (TC) diagnosis was 18 years (range 7-33) and mean age of participants was 53 years (range 25-95). Data consist of results of a questionnaire with patient reported outcomes which covers a broad range of items on late-effects. The study also included data obtained through linkages to Danish registries, a biobank, and clinical data from hospital files and pathology reports originating from the Danish Testicular Cancer Database (DaTeCa). The treatment during the observation period has been nearly the same for all stages of TC and is in agreement with today's standard treatment, this allows for interesting analysis with a wide timespan. We have extensive data on non-responders and are able to validate our study findings. Data from a Danish reference population (N = 162,283) allow us to compare our findings with a Danish background population. The cohort can easily be extended to access more outcomes, or include new TCS. A limitation of the present study is the cross-sectional design and despite the large sample size, The Danish Testicular Cancer Late Treatment Effects Cohort (DaTeCa-LATE) lacks statistical power to study very rare late effects. Since it was voluntary to participate in the study we have some selection bias, for instance, we lack responders who were not in a paired relationship, but we would still argue that this cohort of TCSs is representative for TCSs in Denmark.Collaboration and data access: Researches interested in collaboration with the DaTeCa-LATE study group please contact Professor Gedske Daugaard kirsten.gedske.daugaard@regionh.dk.

Background Cisplatin-based chemotherapy may have neurotoxic effects within the central nervous system. The aims of this study were 1) to longitudinally investigate the impact of cisplatin-based chemotherapy on whole-brain networks in testicular cancer patients undergoing treatment and 2) to explore whether possible changes are related to decline in cognitive functioning. Methods Sixty-four newly orchiectomized TC patients underwent structural magnetic resonance imaging (T1-weighted and diffusion-weighted imaging) and cognitive testing at baseline prior to further treatment and again at a six-month follow-up. At follow-up, 22 participants had received cisplatin-based chemotherapy (CT) while 42 were in active surveillance (S). Brain structural networks were constructed for each participant, and network properties were investigated using graph theory and longitudinally compared across groups. Cognitive functioning was evaluated using standardized neuropsychological tests. All statistical tests were two-sided. Results Compared with the S group, the CT group demonstrated altered global and local brain network properties from baseline to follow-up as evidenced by decreases in important brain network properties such as small-worldness (P = .04), network clustering (P = .04), and local efficiency (P = .02). In the CT group, poorer overall cognitive performance was associated with decreased small-worldness (r = -0.46, P = .04) and local efficiency (r = -0.51, P = .02), and verbal fluency was associated with decreased local efficiency (r = -0.55, P = .008). Conclusions Brain structural networks may be disrupted following treatment with cisplatin-based chemotherapy. Impaired brain networks may underlie poorer performance over time on both specific and nonspecific cognitive functions in patients undergoing chemotherapy. To the best of our knowledge, this is the first study to longitudinally investigate changes in structural brain networks in a cancer population, providing novel insights regarding the neurobiological mechanisms of cancer-related cognitive impairment.

Objective The aim of this study was to determine the prevalence of cognitive impairment (CI) in newly diagnosed and orchiectomized testicular cancer (TC) patients prior to systemic treatment, and to explore biological and psychological correlates. Methods Sixty-six TC patients were compared with 25 healthy men on neuropsychological tests and a measure of cognitive complaints. CI status and a global composite score (representing overall neuropsychological performance) were calculated for each participant. Possible psychological (depression, anxiety, stress, and post-traumatic stress symptoms) and biological (cortisol, IL-6, TNF-α, and CRP) correlates and predictors of patients' cognitive functioning were explored. Results TC patients had lower scores on 6 out of 11 neuropsychological outcomes (p

The evaluation of a national screening programme for contralateral CIS testis in patients with unilateral testicular cancer showed that metachronous germ-cell cancers developed despite screening for CIS. Compared with unscreened patients, no significant difference was found in risk for metachronous cancer. Single-site biopsy including modern immunohistochemistry does not identify all cases of CIS. Background Screening programmes for contralateral carcinomain situ (CIS) testis in patients with unilateral germ-cell cancer (GCC) have never been evaluated. We investigated the effect of screening for contralateral CIS in a large nation-wide, population-based study. Patients and methods A contralateral single-site biopsy was offered to 4130 patients in whom GCC had been diagnosed in 1984–2007 (screened cohort); 462 patients in whom GCC was diagnosed in 1984–1988 comprised the unscreened cohort. Cases with CIS were offered radiotherapy. Initially CIS-negative biopsies in patients with metachronous GCC were revised according to today's standards. Risk for metachronous GCC was estimated using cumulative incidence and the Cox proportional hazards model. Results In the screened cohort, contralateral CIS was found in 181 (4.4%) patients. The cumulative incidence of metachronous GCC after 20 years was 1.9% in the screened cohort and 3.1% in the unscreened cohort (P = 0.097), hazard ratio (HR) for the unscreened cohort: 1.59 (P = 0.144). Expert revision with contemporary methodology of CIS-negative biopsy samples from patients with metachronous cancer revealed CIS in 17 out of 45 (38%) cases. Decreased risks for metachronous GCC were related to older age at diagnosis (HR 0.52 per 10 years,P Conclusions Our evaluation of a national population-based screening programme for contralateral CIS in patients with testicular cancer showed no significant difference in the risk for metachronous GCC between a screened and an unscreened cohort. Single-site biopsy including modern immunohistochemistry does not identify all cases of CIS.

Vinflunine is to date the only registered agent for second-line treatment of metastatic urothelial cell carcinoma (UCC) in Europe. However, the effect is modest. Pemetrexed has demonstrated some single-agent activity in this disease entity. In order to improve treatment possibilities for UCC patients, a phase I trial (VINTREX) was undertaken to assess the safety of vinflunine and pemetrexed in metastatic UCC patients. A dose escalation design was planned to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of a vinflunine/pemetrexed combination. Pemetrexed was added to vinflunine dosed at 280 mg/m2 on day 1 of a 21-day cycle. Three levels of pemetrexed were planned starting at 400 mg/m2. Four patients were enrolled with a mean age of 66 years and with a mean number of prior GC-cycles of 6,8. Two DLT's were observed at the lowest dose-level in cohort 1. One patient experienced grade 4 thrombocytopenia and a second demonstrated hepatobiliary toxicity grade 3 with an increase in alanine aminotransaminase. Most common grade 3 and 4 adverse events were anemia, thrombocytopenia and neutropenia. Three out of four patients received 3 cycles of pemetrexed and vinflunine, all had progressive disease. Based on these observations and due to protocol design, the study was interrupted at dose level 1 for safety reasons. The combined therapy of vinflunine (Javlor®, Pierre Fabre Pharma) and pemetrexed (Alimta®, Eli Lilly) is poorly tolerated in metastatic UCC patients. The combination cannot be recommended for further investigations in metastatic UCC. Vinflunine is to date the only registered agent for second-line treatment of metastatic urothelial cell carcinoma (UCC) in Europe. However, the effect is modest. Pemetrexed has demonstrated some single-agent activity in this disease entity. In order to improve treatment possibilities for UCC patients, a phase I trial (VINTREX) was undertaken to assess the safety of vinflunine and pemetrexed in metastatic UCC patients. A dose escalation design was planned to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of a vinflunine/pemetrexed combination. Pemetrexed was added to vinflunine dosed at 280 mg/m2 on day 1 of a 21-day cycle. Three levels of pemetrexed were planned starting at 400 mg/m2. Four patients were enrolled with a mean age of 66 years and with a mean number of prior GC-cycles of 6,8. Two DLT's were observed at the lowest dose-level in cohort 1. One patient experienced grade 4 thrombocytopenia and a second demonstrated hepatobiliary toxicity grade 3 with an increase in alanine aminotransaminase. Most common grade 3 and 4 adverse events were anemia, thrombocytopenia and neutropenia. Three out of four patients received 3 cycles of pemetrexed and vinflunine, all had progressive disease. Based on these observations and due to protocol design, the study was interrupted at dose level 1 for safety reasons. The combined therapy of vinflunine (Javlor®, Pierre Fabre Pharma) and pemetrexed (Alimta®, Eli Lilly) is poorly tolerated in metastatic UCC patients. The combination cannot be recommended for further investigations in metastatic UCC.

BACKGROUND:First-line treatment for patients with disseminated germ cell cancer (GCC) is bleomycin, etoposide, and cisplatin (BEP). A prognostic classification of patients receiving chemotherapy was published by the International Germ Cell Cancer Collaborative Group (IGCCCG) in 1997, but only a small proportion of the patients received BEP.OBJECTIVE:To estimate survival probabilities after BEP, evaluate the IGCCCG prognostic classification, and propose new prognostic factors for outcome.DESIGN, SETTING, AND PARTICIPANTS:Of a Danish population-based cohort of GCC patients (1984-2007), 1889 received first-line BEP, with median follow-up of 15 yr. Covariates evaluated as prognostic factors were age, year of treatment, primary site, non-pulmonary visceral metastases, pulmonary metastases, and tumor markers.OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:Outcomes measured were 5-yr progression-free survival (PFS), 5-yr disease-specific survival (DSS), and 5-yr overall survival (OS) as calculated using the Kaplan-Meier method and the Cox proportional hazards model.RESULTS AND LIMITATIONS:The 5-yr PFS, DSS, and OS were 87%, 95%, and 93%, respectively, for patients with seminomatous GCC (SGCC) and good prognosis. For nonseminomatous GCC (NSGCC) with good, intermediate, and poor prognosis, the 5-yr probabilities were 90%, 76%, and 55% for PFS; 97%, 87%, and 66% for DSS; and 95%, 85%, and 64% for OS, respectively. For SGCC patients, new adverse prognostic factors not included in the IGCCCG classification were higher age and lactate dehydrogenase ≥1.5 times the upper limit of normal. For NSGCC patients, higher age and pulmonary metastases were additional adverse prognostic factors. Treatment in earlier years was associated with higher mortality. Limitations include the small number of patients in the prognostic groups, and the inability to adjust for performance status and comorbidity.CONCLUSIONS:Our study reveals improved survival for disseminated GCC throughout the study period. We propose new prognostic factors for outcome for validation in larger cohorts of patients.PATIENT SUMMARY:In this study of testicular cancer patients, we evaluated prognostic factors for outcome and calculated survival after standard chemotherapy. We find that survival has improved over the years and we propose new prognostic factors for outcome for validation in larger patient cohorts.

Importance: Patients given systemic treatment for testicular germ cell cancer (GCC) are at increased risk for a second malignant neoplasm (SMN). Previous studies on SMN and causes of death lacked information on the exact treatment applied or were based on patients receiving former treatment options.Objective: To evaluate the treatment-specific risks for SMN and death in a nationwide population-based cohort of patients with GCC treated with current standard regimens.Design, Setting, and Participants: This study examined a Danish nationwide cohort of 5190 men with GCC who entered the Danish Testicular Cancer database between January 1, 1984, and December 31, 2007. Treatment results were compared with a randomly sampled, age-stratified, population-based control group. Cases of gonadal and extragonadal primary were included in the nationwide cohort. The treatments were surveillance only; retroperitoneal radiotherapy (RT); bleomycin, etoposide, and cisplatin (BEP); or more than 1 line of treatment (MTOL).Main Outcomes and Measures: Cumulative incidence and hazard ratios (HRs) for SMN and death calculated by the Cox proportional hazards model were compared with those of age-matched controls.Results: The study population comprised 2804 patients with seminoma and 2386 with nonseminoma. The median follow-up was 14.4 years (interquartile range, 8.6-20.5 years). The 20-year cumulative incidence of SMN with death as a competing risk was 7.8% (surveillance), 7.6% (BEP), 13.5% (RT), 9.2% (MTOL), and 7.0% (controls). We found no increased risk for SMN after surveillance, while the HRs were 1.7 (95% CI, 1.4-2.0), 1.8 (95% CI, 1.5-2.3), and 3.7 (95% CI, 2.5-5.5), respectively, after BEP, RT, and MTOL. Mortality owing to non-GCC causes was decreased after surveillance, but increased by 1.3 times after BEP and RT and by 2.6 times after MTOL. Excess mortality due to SMN was found after BEP (HR, 1.6; 95% CI, 1.2-2.2), RT (HR, 2.1; 95% CI, 1.5-2.9), and MTOL (HR, 5.8; 95% CI, 3.6-9.6).Conclusions and Relevance: We found no increased risk for SMN or death among patients undergoing surveillance only. The risks for SMN and death due to SMN were increased after BEP alone, RT alone, and MTOL. Approaches to define patients who might benefit from less intensive treatment are needed.

Background Disease surveillance in patients with bladder cancer is important for early diagnosis of progression and metastasis and for optimised treatment. Objective To develop urine and plasma assays for disease surveillance for patients with FGFR3 and PIK3CA tumour mutations. Design, setting, and participants Droplet digital polymerase chain reaction (ddPCR) assays were developed and tumour DNA from two patient cohorts was screened for FGFR3 and PIK3CA hotspot mutations. One cohort included 363 patients with non–muscle-invasive bladder cancer (NMIBC). The other cohort included 468 patients with bladder cancer undergoing radical cystectomy (Cx). Urine supernatants (NMIBC n =216, Cx n =27) and plasma samples (NMIBC n =39, Cx n =27) from patients harbouring mutations were subsequently screened using ddPCR assays. Outcome measurements and statistical analysis Progression-free survival, recurrence-free survival, and overall survival were measured. Fisher's exact test, the Wilcoxon rank-sum test and Cox regression analysis were applied. Results and limitations In total, 36% of the NMIBC patients (129/363) and 11% of the Cx patients (44/403) harboured at least one FGFR3 or PIK3CA mutation. Screening of DNA from serial urine supernatants from the NMIBC cohort revealed that high levels of tumour DNA (tDNA) were associated with later disease progression in NMIBC ( p =0.003). Furthermore, high levels of tDNA in plasma samples were associated with recurrence in the Cx cohort ( p =0.016). A positive correlation between tDNA levels in urine and plasma was observed (correlation coefficient 0.6). The retrospective study design and low volumes of plasma available for analysis were limitations of the study. Conclusions Increased levels of FGFR3 and PIK3CA mutated DNA in urine and plasma are indicative of later progression and metastasis in bladder cancer. Patient summary Urine and plasma from patients with bladder cancer may be monitored for diagnosis of progression and metastasis using mutation assays.

Background: The interplay between carcinoma cells and their microenvironment plays an important part in cancer development, progression, metastasis and treatment efficacy. In bladder cancer, however, little is currently known about how the tumor microenvironment affects disease outcome and treatment. To study this, we analyzed tumor tissue from a clinically well characterized cohort of 608 patients with muscle invasive bladder cancer (MIBC). Methods: Multiplex Immunofluorescence (IF) was used to assess the tumor microenvironment in 608 patients with MIBC. Digital pathology was used to quantify the immune context (CD8, CD3, FOXP3) within carcinoma cells and in the adjacent stroma. Immune cell densities were correlated to clinical characteristics, survival outcome and chemotherapy response (n=187). To explore chemotherapy response mechanisms further, a total of 107 patients with metastatic disease are also being characterized by multi-omics analysis (exome-, transcriptome-, and methylome-analysis). Patients receiving chemotherapy were included based on the following criteria: 1) treatment naive primary tumors, 2) received a minimum of 4 cycles of cisplatin based chemotherapy, 3) ECOG Performance Status Results: In total, 384 of 608 patients had metastatic disease. Analysis of the immune cell compositions revealed a immune profile consisting of high levels of CTLs and T helper cells, and a low level of Tregs significantly associated with a better outcome (p Conclusions: Results from the multiplex IF showed that immune profiles (CTLs, T helper cells, Tregs) correlated with recurrence and survival outcome. However, they did not predict chemotherapy response. A full and integrated molecular characterization of our clinically well annotated metastatic subgroup (n=107) will be presented at the AACR 2019 meeting. Citation Format: Ann Taber, Philippe Lamy, Iver Nodentoft, Emil Christensen, Frederik F. Prip, Sia V. Lindskrog, Karin Birkenkamp-Demtröder, Mads Agerbæk, Jørgen B. Jensen, Lars Dyrskjøt. Comprehensive molecular characterization of treatment naive muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 459.

Background Circulating tumor DNA (ctDNA) analysis is an emerging field for diagnosis, monitoring of treatment efficacy and detection of disease recurrence in various cancers. In bladder cancer, previous work has demonstrated prognostic value of ctDNA for early stage cancers and early detection of recurrence following radical cystectomy (CX), however more extensive investigation of the potential impact of ctDNA analysis on clinical decision making remains to be carried out. Methods A total of 68 patients with localized advanced bladder cancer to be treated with neoadjuvant chemotherapy (NAC) and CX were prospectively included. We performed RNA-Seq to identify RNA subtypes and whole exome sequencing to identify somatic mutations for ctDNA analysis. Based on allele frequency and sequence context, 16 somatic mutations were selected for each patient for multiplex PCR and subsequent ultra-deep sequencing (median coverage of 114,000x). The sample-level analytical sensitivity was determined to be >95% at 0.01% ctDNA concentration level when at least 2/16 mutations were detected. The protocol was applied to detect ctDNA in 618 blood samples procured at diagnosis, during NAC, before CX and during surveillance following CX. Results The presence of ctDNA was highly prognostic already at diagnosis before initiation of NAC (HR=31.7, p=0.001). Analysis of ctDNA dynamics during NAC revealed that clearance of ctDNA was associated with pathological downstaging (≤TaN0 at CX) and reduced disease recurrence. Conversely, continually detectable ctDNA during NAC was associated with lack of pathological downstaging and later disease recurrence (p=0.023). Our results suggest that ctDNA dynamics during NAC may offer superior predictive value compared to clinical parameters and tumor centric molecular markers such as DNA damage response mutations and RNA subtypes. Following CX, ctDNA analysis identified 92% of metastatic relapse (100% specificity) during disease monitoring (100% positive- and 98% negative predictive value). The positive lead-time over radiographic imaging was 100 days on average (range: -17-245 days). Conclusions ctDNA analysis of blood samples procured throughout the disease course of patients with bladder cancer demonstrates novel evidence of early risk stratification and monitoring of treatment efficacy, which adds to the already strong evidence of ctDNA-based recurrence detection. Collectively, this paves the way for novel ctDNA-based clinical trials. Citation Format: Emil Christensen, Karin Birkenkamp-Demtröder, Himanshu Sethi, Svetlana Shchegrova, Raheleh Salari, Iver Nordentoft, Hsin-Ta Wu, Michael Knudsen, Philippe Lamy, Sia V. Lindskrog, Ann Taber, Mustafa Balcioglu, Søren Vang, Zoe Assaf, Shruti Sharma, Antony S. Tin, Ramya Srinivasan, Dina Hafez, Thomas Reinert, Samantha Navarro, Alexander Olson, Rosie Ram, Scott Dashner, Matthew Rabinowitz, Paul Billings, Styrmir Sigurjonsson, Claus L. Andersen, Ryan Swenerton, Alexey Aleshin, Bernhard G. Zimmermann, Mads Agerbæk, Cheng-Ho J. Lin, Jørgen B. Jensen, Lars Dyrskjøt. Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 913.

Lessons Learned First trial to report safety and activity of the microtubule inhibitor vinflunine plus the tyrosine kinase inhibitor sorafenib in post-platinum metastatic urothelial cancer (mUC) patients. A recommended phase II dose was identified for the treatment combination of vinflunine plus sorafenib, with main adverse events including fatigue, febrile neutropenia, neutropenia, hypertension, and hyponatremia. An overall response rate of 41% to second-line vinflunine plus sorafenib treatment in patients with platinum-resistant mUC was confirmed. Background Platinum-progressive metastatic urothelial carcinoma (mUC) is a clinical challenge. The tyrosine kinase inhibitor sorafenib has demonstrated varied activity in mUC. This trial was designed to examine safety and activity of vinflunine plus sorafenib in mUC. Methods In addition to standard dose of vinflunine (320 or 280 mg/m2), patients received sorafenib (400, 600, or 800 mg/day), in a 3 + 3 dose-escalation phase I design. Results Twenty-two patients (median age 62.5 years) were included. Five patients received vinflunine 320 mg/m2 and 17 received 280 mg/m2. The maximum tolerated dose (MTD) of sorafenib with vinflunine 280 mg/m2 was 600 mg, and with vinflunine 320 mg/m2 it was not determined, owing to toxicity. Adverse events (AEs) grades 3 + 4 consisted of neutropenia (6 patients), febrile neutropenia (5), and hyponatremia (5). The overall response rate (ORR) in the efficacy-evaluable patients was 41% (7 of 17), all partial responses evaluated by RECIST version 1.1. Median overall survival (OS) was 7.0 months (1.8–41.7). Conclusion The defined recommended phase II dose (RPTD) was vinflunine 280 mg/m2 plus sorafenib 400 mg. Sorafenib was too toxic in combination with vinflunine 320 mg/m2. The ORR of 41% to this second-line combination treatment of mUC is noteworthy and supports further trials.

What's known on the subject? and What does the study add? Several studies have shown that defects in DNA-damage response are associated with good survival after chemotherapy and radiotherapy. Furthermore, loss of cell cycle regulators may be prognostic indicators of poor survival after cystectomy. However, the potential clinical impact of previous findings is hampered by insufficient validation of significant results in suitable cystectomy and radiotherapy cohorts. Here we use a large cohort of patients receiving radiotherapy to successfully validate the importance of MRE11 as a predictive marker of disease-specific survival (DSS). Furthermore, using two independent patient cohorts we show for the first time that TIP60 is a predictive marker of DSS after cystectomy. We show that combined use of TIP60 and MRE11 may hold the potential to guide treatment decisions. OBJECTIVE • To determine the association between the proteins: tat-interactive protein 60 kDa (TIP60), p16, meiotic recombination 11 homolog (MRE11), phosphorylated ataxia telangiectasia mutated (ATM), retinoblastoma protein (Rb), Ki67, and p53 and clinical outcome in invasive lymph node-negative bladder cancer. PATIENTS AND METHODS • Protein expression was measured by immunohistochemistry in cancer specimens from two independent cohorts of patients with bladder cancer treated with cystectomy (162 patients and 273) and one cohort of patients receiving radiotherapy (148). • Disease-specific survival (DSS) was used as the outcome measure, and patients with no disease-specific death were followed for a minimum of 36 months. RESULTS • TIP60 was significantly correlated with DSS in both cystectomy cohorts (hazard ratio [HR] 0.42, 95% confidence interval [CI] 0.26–0.68, P < 0.001 and HR 0.45, 95% CI 0.28–0.72, P= 0.001). • MRE11 was significantly correlated with DSS in the cohort receiving radiotherapy (HR 0.64, 95% CI 0.47–0.86, P= 0.005). • P16 was significantly correlated with DSS in all three cohorts (HR 0.46, 95% CI 0.30–0.75, P= 0.032; HR 0.60, 95% CI 0.37–0.97, P= 0.032; HR 0.52, 95% CI 0.28–0.96, P= 0.001). • Rb was significantly correlated with DSS in one cystectomy cohort (HR 1.71, 95% CI 1.13–2.75, P= 0.017). • Ki67, p53, and pATM were not significantly correlated with DSS in any of the cohorts. CONCLUSIONS • TIP60 protein expression was a predictive marker for DSS after cystectomy in two independent cohorts. This novel marker was the strongest predictive factor in multivariate analysis in patients receiving cystectomy. • MRE11 was shown to be a predictive marker for DSS after radiotherapy. • We have shown that TIP60 and MRE11 hold the potential to guide patients with invasive bladder cancer to either cystectomy or radiotherapy. This study was based on retrospective material and consequently we suggest that these markers should be validated in a prospective study.

BACKGROUND: Comprehensive data on late relapse (LR) and very LR (VLR) in patients with clinical stage I (CS-1) testicular cancer followed on surveillance are missing. These data are essential for planning optimal follow-up.OBJECTIVE: Assess incidence and outcome of LR (>2 yr) and VLR (>5 yr) in a large cohort of CS-1 surveillance patients, and examine differences in the clinical characteristics of patients with early relapse (ER), LR, and VLR.DESIGN, SETTING, AND PARTICIPANTS: CS-1 surveillance patients diagnosed between 1984 and 2007 were identified from the retrospective Danish Testicular Cancer (DaTeCa) database.OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We estimated survival and relapse probabilities and compared the results using log-rank tests and Cox regression analyses. We compared differences in patient characteristics by using χ(2), Fisher exact, and Mann-Whitney tests.RESULTS AND LIMITATIONS: Our study included 3366 (2000 seminoma and 1366 nonseminoma) patients. Median follow-up was 15 yr. Five-year conditional risk of LR was 5.0% and 2.1% for seminoma and nonseminoma patients, respectively. There were no significant differences in disease-specific or overall survival when comparing the LR(VLR) and ER patients by log-rank, but Cox regression adjusted for age showed a significant effect of time to relapse on survival for seminoma patients. Apart from significantly more ER nonseminoma patients with elevated human chorionic gonadotropin at relapse, there were no significant differences in patient characteristics at orchiectomy or relapse. Limitations include retrospective design and exclusion of patients who had been offered adjuvant therapy.CONCLUSIONS: The risk of VLR is minimal, and the patients carry a good prognosis. Patient characteristics of CS-1 surveillance patients with LR(VLR) do not differ significantly from patients with ER.PATIENT SUMMARY: We compared stage I testicular cancer surveillance patients with early relapse (ER) versus late relapse (LR; >2 yr). LR patients as a group did no worse than ER patients, although increased time to relapse was negatively associated with survival for seminoma patients.

Evidence suggests that testicular cancer (TC) and its treatment are associated with cognitive impairment. However, the underlying neural substrate and biological mechanisms are poorly understood. This study aimed to investigate changes in cognition and brain grey matter (GM) morphology in TC patients undergoing treatment, and to explore associations with immune markers, endocrine markers, and genotype. Sixty-five patients with stage I-III TC underwent assessment after surgery but prior to further treatment and again 6 months after. Twenty-two patients received chemotherapy (+CT), while 43 did not (−CT). Assessments included neuropsychological testing, whole-brain magnetic resonance imaging, and blood samples. Twenty-five healthy controls (HCs) underwent neuropsychological testing with a matching time interval. A regression-based approach was used to determine cognitive changes and longitudinal voxel-based morphometry (VBM) was performed to investigate changes in GM density in the TC groups. Compared with the HCs, both TC groups showed higher rates of cognitive decline (p

Background: At least half of the patients diagnosed with non–muscle-invasive bladdercancer (NMIBC) experience recurrence and approximately 15% will develop progression tomuscle invasive or metastatic disease. Biomarkers for disease surveillance are urgentlyneeded.Objective: Development of assays for surveillance using genomic variants in cell-freetumour DNA from plasma and urine.Design, setting, and participants: Retrospective pilot study of 377 samples from 12 patientswith recurrent or progressive/metastatic disease. Three next-generation sequencing methodswere applied and somatic variants in DNA from tumour, plasma, and urine weresubsequently monitored by personalised assays using droplet digital polymerase chainreaction (ddPCR). Samples were collected from 1994 to 2015, with up to 20 yr of follow-up.Outcome measurements and statistical analysis: Progression to muscle-invasive or metastaticbladder cancer; t test for ddPCR data.Results and limitations: We developed from one to six personalised assays per patient.Patients with progressive disease showed significantly higher levels of tumour DNA inplasma and urine before disease progression, compared with patients with recurrent disease(p = 0.032 and 1.3 106, respectively). Interestingly, tumour DNA was detected in plasmaand urine in patients with noninvasive disease, being no longer detectable in disease-freepatients. A significant level of heterogeneity was observed for each patient; this could be dueto tumour heterogeneity or assay performance.Conclusions: Cell-free tumour DNA can be detected in plasma and urine, even in patientswith noninvasive disease, with high levels of tumour DNA detectable before progression,especially in urine samples. Personalised assays of genomic variants may be useful fordisease monitoring.Patient summary: Tumour DNA can be detected in blood and urine in early and advancedstages of bladder cancer. Measurement of these highly tumour-specific biomarkers mayrepresent a novel diagnostic tool to indicate the presence of residual disease or to discoveraggressive forms of bladder cancer early in the disease course. Background: At least half of the patients diagnosed with non–muscle-invasive bladdercancer (NMIBC) experience recurrence and approximately 15% will develop progression tomuscle invasive or metastatic disease. Biomarkers for disease surveillance are urgentlyneeded.Objective: Development of assays for surveillance using genomic variants in cell-freetumour DNA from plasma and urine.Design, setting, and participants: Retrospective pilot study of 377 samples from 12 patientswith recurrent or progressive/metastatic disease. Three next-generation sequencing methodswere applied and somatic variants in DNA from tumour, plasma, and urine weresubsequently monitored by personalised assays using droplet digital polymerase chainreaction (ddPCR). Samples were collected from 1994 to 2015, with up to 20 yr of follow-up.Outcome measurements and statistical analysis: Progression to muscle-invasive or metastaticbladder cancer; t test for ddPCR data.Results and limitations: We developed from one to six personalised assays per patient.Patients with progressive disease showed significantly higher levels of tumour DNA inplasma and urine before disease progression, compared with patients with recurrent disease(p = 0.032 and 1.3 106, respectively). Interestingly, tumour DNA was detected in plasmaand urine in patients with noninvasive disease, being no longer detectable in disease-freepatients. A significant level of heterogeneity was observed for each patient; this could be dueto tumour heterogeneity or assay performance.Conclusions: Cell-free tumour DNA can be detected in plasma and urine, even in patientswith noninvasive disease, with high levels of tumour DNA detectable before progression,especially in urine samples. Personalised assays of genomic variants may be useful fordisease monitoring.Patient summary: Tumour DNA can be detected in blood and urine in early and advancedstages of bladder cancer. Measurement of these highly tumour-specific biomarkers mayrepresent a novel diagnostic tool to indicate the presence of residual disease or to discoveraggressive forms of bladder cancer early in the disease course.

Primary urethral cancer (PUC) is a very rare disease. This case report illustrates a successful treatment approach of a 67-year-old woman with a urethral adenocarcinoma selected for an organ preserving treatment with external beam radiotherapy (EBRT) and interstitial brachytherapy (BT) boost, using the GEC-ESTRO target concept originally designed for locally advanced cervical cancer (LACC). Treatment included EBRT with 45 Gy in 25 fractions followed by image guided adaptive interstitial BT (IGABT) with a pulsed-dose-rate (PDR) BT boost with 30 Gy in 50 hourly pulses. The D 90 for CTV HR was 79.1 Gy in EQD23. At 24 months follow-up, the patient was recurrence free and without treatment related side effects.

Background. The bladder is a tumour site well suited for adaptive radiotherapy (ART) due to large inter-fractional changes, but it also displays considerable intra-fractional motion. The aim of this study was to assess target coverage with a clinically applied method for plan selection ART and to estimate population-based and patient-specific intra-fractional margins, also relevant for a future re-optimisation strategy.Material and methods. Nine patients treated in a clinical phase II ART trial of daily plan selection for bladder cancer were included. In the library plans, 5 mm isotropic margins were added to account for intra-fractional changes. Pre-treatment and weekly repeat magnetic resonance imaging (MRI) series were acquired in which a full three-dimensional (3D) volume was scanned every second min for 10 min (a total of 366 scans in 61 series). Initially, the bladder clinical target volume (CTV) was delineated in all scans. The t = 0 min scan was then rigidly registered to the planning computed tomography (CT) and plan selections were simulated using the CTV_0 (at t = 0 min). To assess intra-fractional motion, coverage of the CTV_10 (at t = 10 min) was quantified using the applied PTV. Population-based margins were calculated using the van Herk margin recipe while patient-specific margins were calculated using a linear model.Results. For 49% of the cases, the CTV_10 extended more than 5 mm outside the CTV_0. However, in 58 of the 61 cases (97%) CTV_10 was covered by the selected PTV. Population-based margins of 14 mm Sup/Ant, 9 mm Post and 5 mm Inf/Lat were sufficient to cover the bladder. Using patient-specific margins, the overlap between PTV and bowel-cavity was reduced from 137 cm3 with the plan selection strategy to 24 cm3.Conclusion. In this phase II ART trial, 5 mm isotropic margin for intra-fractional motion was sufficient even though considerable intra-fractional motion was observed. In online re-optimised ART, population-based margin can be applied although patient-specific margins are preferable.

Minimally invasive methods for assessment of tumor burden, early detection of disease relapse and for monitoring therapeutic efficacy are needed to improve individualized follow-up and treatment for patients diagnosed with bladder cancer. The ability to predict pathologic complete response after neoadjuvant chemotherapy (NAC) through detection of ctDNA in plasma may enable strategies for bladder preservation. The aim of the study was to use patient-specific mutations to identify residual disease, metastatic relapse and to monitor treatment response in longitudinally collected plasma samples. For this, 50 patients diagnosed with locally advanced muscle-invasive bladder cancer (MIBC) and scheduled for chemotherapy were prospectively recruited between 2013 and 2017. In total, 42 patients received four cycles of cisplatin-based NAC prior to cystectomy and 82% showed response (pathologic downstaging). Eight patients received 2-6 cycles of cisplatin-based first-line chemotherapy due to diagnosis of T4b or lymph node metastasis prior to cystectomy (3CR, 2PR, 2PD, 1 ongoing). So far, 8/50 patients (16%) experienced disease relapse and three patients had metastatic progression. The mean follow-up time after radical cystectomy (RC) was 320 days (65-973); the mean follow-up time for disease-free patients was 397 days (119-778). Whole-exome sequencing (106x mean target coverage) of tumor and germline DNA was performed from a time point before systemic treatment. MUTECT2 identified a mean of 33 high (6-121), 340 (67-2838) moderate and 223 (29-2955) low-impact SNVs or insertion-deletions (InDels) per tumor. High-impact mutations in known cancer genes such as TP53 (60%), KDM6A (34%), ARID1A (32%), RB1 (28%), BRCA2 (26%), FGFR3 (22%) and ERCC2 (20%) were identified with no significant difference between responders vs nonresponders. Signatures 2, 13 (APOBEC), and 3 (BRCA) were frequently identified, with signature 13 being more prevalent in nonresponders (p=0.05). Personalized, multiplex-PCR assay-panels were individually designed, targeting each patient's tumor-specific mutations in plasma. Targeted sequencing was performed using cfDNA from 4-16 longitudinally collected plasma from each patient taken pre- and post-systemic therapy and at scheduled control visits after RC. In a blinded fashion, ctDNA was analyzed and results were compared to pathologic response and radiographic imaging data for each patient. Conclusion: Preliminary results indicated that analysis of plasma cfDNA is an appropriate method to detect metastatic relapse after RC and to monitor treatment efficacy. Here we perform a larger ctDNA study, applying direct sequencing of plasma cfDNA using highly sensitive NGS methods to determine clinical applicability of the method. Data collection is ongoing and all details will be presented at the AACR 2018 meeting. Citation Format: Karin Birkenkamp-Demtröder, Emil Christensen, Shruti Sharma, Ann Taber, Mads Agerbæk, Himanshu Sethi, Hsin-Ta Wu, Svetlana Shchegrova, Philippe Lamy, Iver Nordentoft, Ryan Swenerton, Samantha Navarro, Tony Tin, Ramya Srinivasan, John So, Spenser Alexander, Solomon Moshkevich, Bernhard Zimmermann, Cheng-Ho Jimmy Lin, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Sequencing of plasma cfDNA from patients with locally advanced bladder cancer for surveillance and therapeutic efficacy monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3653.

The study was undertaken to identify pre-treatment clinical and histopathological factors of importance for response and survival after cisplatin-based combination chemotherapy, in patients with locally advanced or metastatic transitional cell carcinoma of the urothelium.Clinical, laboratory and histopathological data from 178 consecutive patients, representing all patients treated between 1991 and 2001 in a single institution, were collected. Correlations between these data and response and survival after chemotherapy were analysed using univariate and multivariate analyses.Absence of visceral metastasis was the only parameter with independent correlation to the response to chemotherapy. Two of the analysed parameters were independently associated with increased survival: good performance status (PSor =1) and absence of visceral metastases. Stratification of the patient material according to number of these risk-factors present showed strong association with survival.It was possible to predict survival from pre-treatment clinical parameters and consequently it is possible to select groups with a high and low probability of obtaining long term survival following cisplatin-containing chemotherapy.