Urine-derived bladder cancer organoids (urinoids) as a tool for cancer longitudinal response monitoring and therapy adaptation.

BACKGROUND: Bladder cancer is one of the most common cancer types worldwide. Generally, research relies on invasive sampling strategies. METHODS: Here, we generate bladder cancer organoids directly from urine (urinoids). In this project, we establish 12 urinoid lines from 22 patients with non-muscle and muscle-invasive bladder tumours, with an efficiency of 55%. RESULTS: The histopathological features of the urinoids accurately resemble those of the original bladder tumours. Genetically, there is a high concordance of single nucleotide polymorphisms (92.56%) and insertions & deletions (91.54%) between urinoids and original tumours from patient 4. Furthermore, these urinoids show sensitivity to bladder cancer drugs, similar to their tissue-derived organoid counterparts. Genetic analysis of longitudinally generated tumoroids and urinoids from one patient receiving systemic immunotherapy, identify alterations that may guide the choice for second-line therapy. Successful treatment adaptation was subsequently demonstrated in the urinoid setting. CONCLUSION: Therefore, urinoids can advance precision medicine in bladder cancer as a non-invasive platform for tumour pathogenesis, longitudinal drug-response monitoring, and therapy adaptation.

Prognostic impact of waiting time between diagnosis and treatment in patients with cervical cancer: A nationwide population-based study.

OBJECTIVE: Prior research underlined the importance of timely oncological care as longer waiting times from diagnosis to treatment may result in poorer survival outcomes. The aim of this study was to determine the impact of waiting time from diagnosis to treatment on overall survival (OS) in patients with cervical cancer treated with curative intent. METHODS: Patients from a nationwide population-based cohort with newly diagnosed cervical cancer between 2010 and 2019 were studied. Patients who underwent surgery or (chemo)radiotherapy with curative intent were selected. Waiting time (i.e. interval between first pathologic confirmation and treatment) was modelled as continuous (i.e. linear per week), dichotomized (i.e. ≤8 versus >8 weeks), and polynomial (i.e. restricted cubic splines). The association with OS was examined using Cox regression analyses. RESULTS: Among 6895 patients with cervical cancer, 2755 treated with primary surgery and 1898 who received primary (chemo)radiotherapy were included. Mean waiting time was 8.5 (±4.2) weeks to surgery and 7.7 (±2.9) weeks to (chemo)radiotherapy. Adjusted for confounders, waiting time to surgery was not significantly associated with OS (continuous HR 0.97 [95%CI: 0.93-1.01], dichotomized HR 0.93 [95%CI: 0.68-1.27], polynomial HR not significant). Similarly, a longer waiting time to (chemo)radiotherapy was not significantly associated with poorer OS (continuous HR 0.97 [95%CI: 0.93-1.00], dichotomized HR 0.91 [95%CI: 0.75-1.09], polynomial HR not significant). CONCLUSIONS: This large population-based study demonstrates that a longer waiting time (of up to 12 weeks) from diagnosis to treatment in patients with cervical cancer treated with curatively intended surgery or (chemo)radiotherapy does not negatively impact survival.

Genomic and transcriptomic plasticity in treatment-naive ovarian cancer.

Intra-tumor heterogeneity is a hallmark of many cancers and may lead to therapy resistance or interfere with personalized treatment strategies. Here, we combined topographic mapping of somatic breakpoints and transcriptional profiling to probe intra-tumor heterogeneity of treatment-naïve stage IIIC/IV epithelial ovarian cancer. We observed that most substantial differences in genomic rearrangement landscapes occurred between metastases in the omentum and peritoneum versus tumor sites in the ovaries. Several cancer genes such as NF1, CDKN2A, and FANCD2 were affected by lesion-specific breakpoints. Furthermore, the intra-tumor variability involved different mutational hallmarks including lesion-specific kataegis (local mutation shower coinciding with genomic breakpoints), rearrangement classes, and coding mutations. In one extreme case, we identified two independent TP53 mutations in ovary tumors and omentum/peritoneum metastases, respectively. Examination of gene expression dynamics revealed up-regulation of key cancer pathways including WNT, integrin, chemokine, and Hedgehog signaling in only subsets of tumor samples from the same patient. Finally, we took advantage of the multilevel tumor analysis to understand the effects of genomic breakpoints on qualitative and quantitative gene expression changes. We show that intra-tumor gene expression differences are caused by site-specific genomic alterations, including formation of in-frame fusion genes. These data highlight the plasticity of ovarian cancer genomes, which may contribute to their strong capacity to adapt to changing environmental conditions and give rise to the high rate of recurrent disease following standard treatment regimes.

Mutations in LRRC50 predispose zebrafish and humans to seminomas.

Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.

Value of multiparametric magnetic resonance imaging for local staging of invasive urinary bladder tumours.

BACKGROUND: Initial tumour staging in bladder cancer mainly relies on the histo-pathological outcome of the transurethral bladder tumour resection (TURBT) and imaging by means of a CT-scan (CT-intravenous urography; CT-IVU). The reported risk of understaging varies from 24-50%. To further improve the the evaluation of depth of invasion of the bladder tumour the application of magnetic resonance imaging (MRI) may be useful. To substantiate the additional value of this imaging modality the present observational study was designed. STUDY DESIGN: This is a prospective observational study to analyse bladder tumour staging with multiparametric magnetic resonance imaging (mpMRI) in patients with a known bladder tumour, who are planned for radical cystectomy. STUDY POPULATION: Patients with an invasive bladder cancer who are planned for radical cystectomy. INTERVENTION: Patients were accrued during their visit to the outpatient department of urology. They underwent routine cystoscopy, laboratory tests (including serum Creatinin) and CT-IVU investigations and subsequently a mpMRI. MAIN STUDY PARAMETERS/ENDPOINTS: To demonstrate the value of mpMRI in the initial staging of bladder tumours using radiological bladder tumour stage (T-stage) based on mpMRI and pathological bladder tumour stage based on 'whole-mount' histo-pathology after radical cystectomy. RESULTS: Thirty-seven participants with known bladder tumours underwent mpMRI and subsequent cystectomy. After mpMRI 10 participants were diagnosed with non-muscle-invasive bladder cancer (NMIBC) and 27 participants with muscle-invasive bladder cancer (MIBC). In the 'whole-mount' pathology results 12 participants had NMIBC and 25 participants had MIBC. We found a sensitivity and specificity of 0.88 en 0.58 respectively, for the evaluation of MIBC. The positive and negative predictive value were 81% and 70% respectively. The diagnostic accuracy of mpMRI to differentiate between NMIBC and MIBC was 78%. CONCLUSIONS: We found a sensitivity of 88% and a specificity of 58% for mpMRI to discriminate NMIBC from MIBC.

The quality of 5-aminolevulinic acid-induced photodynamic diagnosis and transurethral resection of bladder tumors: does the urologist play a role?.

INTRODUCTION: The aim of this study is to evaluate the quality of photodynamic diagnosis (PDD) and transurethral resection of bladder tumors (TURBT) among different urologists. PATIENTS AND METHODS: The selected data consists of 194 patients, 268 5-aminolevulinic acid (5-ALA)-induced PDD procedures and 934 biopsies. Tumors were resected and biopsies were taken from suspicious areas under guidance of white light endoscopy and 5-ALA-induced fluorescence cystoscopy. The quality of PDD was determined by evaluating the mean number of tumors resected by 5 urologists and, thereafter, assessing the time to recurrence between groups. RESULTS: Urologist 1 took 37% more biopsies (p < 0.001) and diagnosed 42% more tumors (p = 0.005) and 46% more false positives (p < 0.001) from bladders compared to urologists 2, 3, 4 and 5 together. The mean time to bladder cancer recurrence for all recurrences within 0-18 months was 11.0 months for operator 1 and 8.3 months for the other urologists (p = 0.01). CONCLUSIONS: The resecting urologist appears to be an important factor for the quality of standard and PDD-assisted TURBT. Learning curve programs may be required with experienced surgeons accompanying those with less experience.

Significant Inter- and Intralaboratory Variation in Gleason Grading of Prostate Cancer: A Nationwide Study of 35,258 Patients in The Netherlands.

PURPOSE: Our aim was to analyze grading variation between pathology laboratories and between pathologists within individual laboratories using nationwide real-life data. METHODS: We retrieved synoptic (n = 13,397) and narrative (n = 29,377) needle biopsy reports from the Dutch Pathology Registry and prostate-specific antigen values from The Netherlands Cancer Registration for prostate cancer patients diagnosed between January 2017 and December 2019. We determined laboratory-specific proportions per histologic grade and unadjusted odds ratios (ORs) for International Society of Urological Pathologists Grades 1 vs. 2-5 for 40 laboratories due to treatment implications for higher grades. Pathologist-specific proportions were determined for 21 laboratories that consented to this part of analysis. The synoptic reports of 21 laboratories were used for analysis of case-mix correction for PSA, age, year of diagnosis, number of biopsies and positive cores. RESULTS: A total of 38,321 reports of 35,258 patients were included. Grade 1 ranged between 19.7% and 44.3% per laboratory (national mean = 34.1%). Out of 40 laboratories, 22 (55%) reported a significantly deviant OR, ranging from 0.48 (95% confidence interval (CI) 0.39-0.59) to 1.54 (CI 1.22-1.93). Case-mix correction was performed for 10,294 reports, altering the status of 3/21 (14%) laboratories, but increasing the observed variation (20.8% vs. 17.7%). Within 15/21 (71%) of laboratories, significant inter-pathologist variation existed. CONCLUSION: Substantial variation in prostate cancer grading was observed between and within Dutch pathology laboratories. Case-mix correction did not explain the variation. Better standardization of prostate cancer grading is warranted to optimize and harmonize treatment.

Expression of hypoxia-inducible factor-1α and -2α in whole-mount prostate histology: relation with dynamic contrast-enhanced MRI and Gleason score.

The aim of this study was to investigate the association between the immunohistochemical expression of hypoxia-inducible factor (HIF)-1α and HIF-2α and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameters Ktrans and kep in prostate cancer. Therefore, 15 patients with biopsy-confirmed prostate cancer underwent a pre-operative 3T DCE-MRI scan. Immunohistochemical analysis of HIF-1α and HIF-2α, and of CD31 for microvessel density (MVD) was performed. Tumor areas were delineated on whole-mount histopathological sections. Nuclear HIF expression was correlated with the quantitative DCE-MRI parameters Ktrans and kep, MVD and Gleason score. HIF expression was highly heterogeneous within tumors and between patients. Pronounced expression of HIF-2α was present, while HIF-1α expression was more limited. Larger tumors showed higher HIF-2α expression (p=0.041). A correlation between HIF-2α and Ktrans p5th was found (r=0.30, p=0.02), but no differences in Ktrans, kep and MVD were observed for different levels of HIF expression. HIF expression was not associated with Gleason score. In conclusion, in this whole-mount prostate cancer study, larger prostate tumors showed frequently high HIF-2α expression, suggesting that larger tumors are clinically most relevant. However, HIF-1α and HIF-2α were not correlated with DCE-MRI parameters. Given the pronounced expression of HIF-2α and independence of Gleason score, HIF expression may function as a biomarker to guide boost dose prescription.

Urodynamic effects of transrectal intraprostatic Ona botulinum toxin A injections for symptomatic benign prostatic hyperplasia.

OBJECTIVE: To investigate urodynamic, symptomatic, and histologic effects of intraprostatic injection with Ona botulinum toxin A for benign prostatic hyperplasia. METHODS: Patients >55 years with symptomatic benign prostatic hyperplasia failing medical therapy were treated. Inclusion criteria were International Prostate Symptom Score >7, prostate volume 30-50 cm(3), and urodynamic bladder outlet obstruction >Schäfer grade 2. A transrectal intraprostatic injection of 200 IU Ona botulinum toxin A was given. Filling cystometry and pressure flow studies were performed at 3, 6, and 12 months post injection. International Prostate Symptom Score, International Prostate Symptom Score quality of life, prostate-specific antigen, and prostate volume were measured up until 12 months; prostate biopsies before and after Ona botulinum toxin A injection were done for histology and cell proliferation. RESULTS: Fifteen men (mean age 64.9 years) were included. Ona botulinum toxin A injection was well tolerated with few complications. Postvoid residual improved (170 to 80 mL), but maximum flow rate and bladder outlet resistance parameters did not change during follow-up. International Prostate Symptom Score and International Prostate Symptom Score quality of life improved (22 to 13 and 5 to 2, respectively), whereas prostate-specific antigen and prostate volume remained unaltered. Cell proliferation did not decrease and in 37% and 64% of pre- and posttreatment biopsies, respectively, some degree of prostatitis was found. Ten of 15 patients eventually underwent transurethral prostate resection because of persisting symptoms. CONCLUSION: Intraprostatic Ona botulinum toxin A for symptomatic benign prostatic hyperplasia did not affect urodynamic outcomes, except for postvoid residual. Although symptom scores improved, we were not able to show change in prostate volume, prostate-specific antigen, or histologic outcomes. A placebo effect of intraprostatic Ona botulinum toxin A could not be ruled out.

Discrimination between benign and malignant prostate biopsies using three-dimensional chromatin texture analysis by confocal laser scanning microscopy.

OBJECTIVE: To evaluate the clinical usefulness of computing three-dimensional (3-D) nuclear texture features on prostate biopsy specimens to discriminate among benign, prostatic intraepithelial neoplasia (PIN), and malignant specimens. STUDY DESIGN: Twelve prostate cancer biopsy specimens were selected, diagnosed as either benign (N = 4), PIN (N = 4), or malignant (N = 4). Sections 14 microm thick were stained. 3-D image stacks of selected benign and malignant areas were obtained by confocal laser scanning microscopy (CLSM) and analyzed off-line using in-house-developed software for 3-D semiautomated segmentation and calculation of texture features. The power of the 3-D texture features to discriminate among the pooled benign (N = 1,507), PIN (N = 673), and malignant nuclei (N = 1,251) was established by multivariate linear discriminant analysis. RESULTS: A total of 68.8% of the benign nuclei, 77.2% of the PIN nuclei, and 78.5% of the malignant nuclei could be classified correctly after cross validation. CONCLUSION: Quantification of changes in the distribution of nuclear chromatin by means of 3-D texture feature computation on CLSM images allows discriminating most benign and malignant prostate nuclei, which could be useful in cases that are difficult to diagnose morphologically.

Photodynamic diagnosis (5-aminolevulinic acid) of transitional cell carcinoma after bacillus Calmette-Guérin immunotherapy and mitomycin C intravesical therapy.

BACKGROUND: Photodynamic diagnosis (PDD) is a technique that enhances the detection of occult bladder tumors during cystoscopy using a fluorescent dye. OBJECTIVE: To study the differential effects of bacillus Calmette-Guérin (BCG) and mitomycin C (MMC) intravesical therapy on the false-positive rate of PDD of bladder cancer. DESIGN, SETTING, AND PARTICIPANTS: This study included 552 procedures and 1874 biopsies. INTERVENTION: Tumors were resected and biopsies were taken from suspicious areas, under guidance of white-light endoscopy and 5-ALA (5-aminolevulinic acid)-induced fluorescence cystoscopy. MEASUREMENTS: The influence of intravesical BCG immunotherapy and intravesical MMC chemotherapy on pyuria, inflammation, and PDD specificity was examined in univariate analyses. RESULTS AND LIMITATIONS: BCG significantly results in inflammation (odds ratio [OR]: 1.53, p=0.002), leukocyturia (OR: 1.84, p=0.034), and false positives in PDD (OR: 1.49, p=0.001). However, a single BCG instillation within 3 mo before PDD is most likely not associated with increased false-positive rates (OR: 0.35, p=0.26). Leukocyturia normalizes within 6 wk after the last BCG instillation, but PDD specificity is reduced up to 3 mo. CONCLUSIONS: BCG is an important predictor for false positives in PDD (5-ALA). More than one BCG instillation within 3 mo before fluorescence cystoscopy decreases the specificity of PDD.

Expression and localization of hypoxia proteins in prostate cancer: prognostic implications after radical prostatectomy.

OBJECTIVES: To uncover novel biomarkers that will help predict which patients are at risk and may guide future treatment decisions. A significant proportion of prostate cancer patients treated with radical prostatectomy will experience a recurrence of the disease. Given the substantial role of hypoxia in prostate cancer development and treatment, this investigation focuses on the Hypoxia Inducible Factor (HIF-1alpha) pathway. METHODS: A tissue microarray was constructed of prostate cancer tissue collected from 71 patients undergoing radical prostatectomy. The expression of proteins involved in the HIF-1alpha pathway was investigated by an immunohistochemical approach and correlated to clinical features including the time to biochemical recurrence. RESULTS: Expression of GLUT1 correlated significantly (P <.05) with a shorter time to biochemical recurrence after radical prostatectomy and was independent from the Gleason grade and stage of cancer. Furthermore, our studies revealed for the first time that accumulation of prolyl-4-hydroxylases 1 especially in the nucleus, is a significant indicator for a worse prognosis (P <.001). CONCLUSIONS: This study confirms the upregulation of proteins involved in the HIF-1alpha hypoxia pathway in prostate cancer cells, indicative of a hypoxic tumor state. Importantly, we report the identification of 2 novel markers, GLUT1 and prolyl-4-hydroxylases 1, with prognostic significance for patients undergoing radical prostatectomy.

Predictors of false positives in 5-aminolevulinic acid-induced photodynamic diagnosis of bladder carcinoma: identification of patient groups that may benefit most from highly specific optical diagnostics.

OBJECTIVES: To identify patient groups associated with a high probability of false positives in photodynamic diagnosis (PDD) of bladder cancer for which the use of highly specific optical instruments could be beneficial. METHODS: This study includes the data of 306 patients. Under white light and 5-aminolevulinic acid-induced fluorescence light guidance, tumor locations were recorded, cold-cup biopsies were taken and tumors resected. Age, gender, recent transurethral resection of bladder tumor (TURBT), previous intravesical therapy, and urinary tract infections were examined for association with the false-positive rates in fluorescence cystoscopy by performing a multivariate analysis. RESULTS: Significant univariate associations were found between false positives and gender (P = .009, odds ratio [OR] = 0.51), previous intravesical therapy (P = .03, OR = 1.78), previous BCG instillations (P = .03, OR = 2.05), and TURBT in the past 90 days (P = .01, OR = 2.37). In the multivariate regression model, female gender (male; P = .005, OR = 0.41) and TURBT within 90 days before PDD (P = .01, OR = 2.38) are significant independent predictors of false-positive findings in PDD. CONCLUSIONS: Recent TURBTs and female gender are significant independent predictors of false positives in fluorescence cystoscopy.

Discrimination between benign and malignant prostate tissue using chromatin texture analysis in 3-D by confocal laser scanning microscopy.

BACKGROUND: Analysis of chromatin texture may improve both the diagnosis and the assessment of the prognosis of prostate cancer. Confocal laser scanning microscopy (CLSM) allows performing measurements in nuclei reconstructed in 3-D. The aim of this study was to evaluate the clinical usefulness of 3-D texture analysis of prostate tissue. METHODS: Image stacks of eight prostate cancer sections were obtained by CLSM of both benign and malignant areas. Texture feature values were computed for individual nuclei. The discriminative power of the texture features was established by receiver operating characteristic (ROC) analysis and linear discriminant analysis (LDA). RESULTS: Texture features were identified that could discriminate between benign and malignant nuclei. LDA correctly classified 89% of the nuclei of the pooled set of benign and malignant nuclei. CONCLUSIONS: 3-D nuclear texture features allow discrimination of most benign and malignant prostate nuclei. We estimate that the classification rates can be increased by improving the image quality.