Consultation on UTUC II Stockholm 2022: diagnostic and prognostic methods-what's around the corner?

PURPOSE: To map current literature and provide an overview of upcoming future diagnostic and prognostic methods for upper tract urothelial carcinoma (UTUC), including translational medical science. METHODS: A scoping review approach was applied to search the literature. Based on the published literature, and the experts own experience and opinions consensus was reached through discussions at the meeting Consultation on UTUC II in Stockholm, September 2022. RESULTS: The gene mutational profile of UTUC correlates with stage, grade, prognosis, and response to different therapeutic strategies. Analysis of pathway proteins downstream of known pathogenic mutations might be an alternative approach. Liquid biopsies of cell-free DNA may detect UTUC with a higher sensitivity and specificity than urinary cytology. Extracellular vesicles from tumour cells can be detected in urine and may be used to identify the location of the urothelial carcinoma in the urinary tract. 3D microscopy of UTUC samples may add information in the analysis of tumour stage. Chemokines and chemokine receptors were linked to overall survival and responsiveness to neoadjuvant chemotherapy in muscle-invasive bladder cancer, which is potentially also of interest in UTUC. CONCLUSION: Current diagnostic methods for UTUC have shortcomings, especially concerning prognostication, which is important for personalized treatment decisions. There are several upcoming methods that may be of interest for UTUC. Most have been studied for urothelial carcinoma of the bladder, and it is important to keep in mind that UTUC is a different entity and not all methods are adaptable or applicable to UTUC.

Genomic profile - a possible diagnostic and prognostic marker in upper tract urothelial carcinoma.

OBJECTIVES: To investigate gene alterations as diagnostic and prognostic markers in upper tract urothelial carcinoma (UTUC). PATIENTS AND METHODS: Patients with UTUC who underwent nephroureterectomy between 2005 and 2012 were followed until November 2020. DNA was extracted from paraffin-embedded tumour tissue. Next-generation sequencing using a 388-gene panel was performed. First a blinded analysis using principal component analysis and hierarchical clustering was used to search for patterns of mutations. Then a comparative analysis using analysis of variance (ANOVA) was used to search for mutations enriched in groups of various grades, stages, and survival. In addition, careful manual annotation was used to identify pathogenic mutations over-represented in tumours of high grade/stage and/or poor survival. RESULTS: A total of 39 patients were included. All tumour stages and grades were represented in the cohort. The median follow-up was 10.6 years. In all, 11 patients died from UTUC during the follow-up. Tumour mutational burden showed a statistically significant correlation with stage, grade, and stage + grade. Grade 1, Grade 2, and Grade 3 tumours had different mutational patterns. Patients who died from UTUC had pathogenic mutations in specific genes e.g. tumour protein p53 (TP53) and HRas proto-oncogene, GTPase (HRAS). Patients with Ta Grade 1 tumours with a known pathogenic fibroblast growth factor receptor 3 (FGFR3) mutation did not die from UTUC. CONCLUSION: The genetic analysis was highly concordant with histopathological features and added prognostic information in some cases. Thus, results from genomic profiling may contribute to the choice of treatment and follow-up regimens in the future.

Predicting invasiveness and disease-specific survival in upper tract urothelial carcinoma: identifying relevant clinical tumour characteristics.

PURPOSE: The aim of this prospective study was to identify the tumour characteristics that are associated with invasiveness and those that are relevant for disease-specific survival (DSS) in upper tract urothelial carcinoma, UTUC. METHODS: From a prospective consecutive cohort of patients with suspicion of UTUC, those who were diagnosed with UTUC using URS prior to rNU between 2005 and 2012 were included. Tumour characteristics were analysed for prediction of invasiveness and association with DSS. Stages were categorised as superficial (pTa-1 and CIS only) or invasive (≥  pT2). Tumours were graded according to WHO 1999 classification. DSS was analysed regarding possible association with stage, grade, size, multifocality, location, ploidy and rate of proliferation. Associations were tested using Fisher's exact test, Pearson Chi-square or Cox's regression. Kaplan-Meier survival curves were constructed. RESULTS: Forty-five consecutive patients were included, and 43 of them were included in the final analyses because their rNU specimens were available for reassessment. The only tumour characteristics that were significantly associated with stage were tumour grade (P < 0.001), DNA ploidy (P = 0.045) and rate of proliferation (P = 0.004). No association with stage was noted for size, multifocality or location. Grade, stage and rate of proliferation were associated with DSS. CONCLUSIONS: Grade, DNA ploidy and S-phase fraction were the only tumour characteristics associated with stage in our study. However, DNA ploidy was not associated with DSS. The prognostic factors that we identified were tumour grade, stage, and S-phase fraction.

Volumetric imaging: a potential tool to stage upper tract urothelial carcinoma.

PURPOSE: To investigate whether volumetric imaging of tumor vasculature can be used to phenotypically characterize advanced upper tract urothelial carcinoma, and if this technique can distinguish aggressive invasive tumors from non-aggressive superficial ones. METHODS: In a pilot study, two TaG1 and two T3G3 formalin-fixed paraffin-embedded (FFPE) tumor samples were examined using the DIPCO pipeline (Tanaka et al. in Nature Biomed Eng 1(10):796-806. https://doi.org/10.1038/s41551-017-0139-0 , 2017). Briefly, punch biopsies of FFPE tumors were deparaffinized, cleared, immunolabeled with the vessel marker CD34 and imaged with a light-sheet microscope. Thereafter, the three-dimensional (3D) vasculature of the tumors was analyzed and characterized using a specialized image processing software. RESULTS: We found that T3G3 tumors had increased CD34 density kurtosis and skewness compared to TaG1 tumors. This suggests that analysis of the 3D vasculature can distinguish between high-grade invasive and low-grade superficial tumors. CONCLUSIONS: Volumetric imaging of tumor samples may represent novel methodology that can complement conventional histopathology. Volumetric imaging enabled us to differentiate between invasive and non-invasive upper tract urothelial carcinoma. The method is of particular interest in diagnostic work-up of patients with upper tract urothelial carcinoma as previous findings indicate that volumetric imaging of vascular patterns could be used to differentiate superficial and invasive urothelial carcinoma, irrespective of if the tumor sample was deep or superficial. However, further and more extensive studies are required before this method can be applied clinically.

Diagnostic accuracy of upper tract urothelial carcinoma: how samples are collected matters.

OBJECTIVES: The aims of this study were to determine the accuracy and reliability of cytology, histopathology and ploidy of specimens obtained at ureterorenoscopy, to evaluate the importance of how samples are collected and to determine whether cytology is an alternative to histology of biopsies. METHODS: This prospective study investigated the accuracy of grading of endoscopically taken cytology and histopathology samples from 45 consecutive patients by comparing these with subsequent nephroureterectomy specimens. Histopathology grading was done according to WHO 1999 and 2004 classifications. Ploidy was determined using photospectrometry. RESULTS: Forty-five patients were included. Both cytology and histopathology identified almost all cancers (91% and 94%, respectively) in collected samples. In cytology as well as in histopathology, agreement in grade between barbotage and nephroureterectomy specimens was statistically significant for both 1999 and 2004 WHO classifications. All cancers in the endoscopic biopsies were identified as pathological, although the grading was not correct in all cases. A statistically significant correlation was found between grade and ploidy in grade 1 and grade 3 nephroureterectomy specimens. CONCLUSIONS: Specimens collected at ureterorenoscopy (biopsies for histology, barbotages for cytology and analysis of ploidy) proved to be relevant and useful. Barbotage cytology identified 91% of all cancers, a high rate compared to techniques used in other studies, and was also sensitive in detecting low-grade tumours. Barbotage cytology and biopsy histology were equally efficient in detecting cancer. The authors recommend that both barbotage and biopsy be performed in addition to complete ureterorenoscopy. Moreover, if there is no visible lesion, cytology is the only reliable option.

Diagnostic accuracy of computed tomography urography and visual assessment during ureterorenoscopy in upper tract urothelial carcinoma.

OBJECTIVE: To investigate diagnostic accuracy of multiphase computed tomography urography (MCTU) and visual assessment at ureterorenoscopy (URS) for detection of upper tract urothelial carcinoma (UTUC). PATIENTS AND METHODS: Consecutive patients referred to our tertiary care centre were included in a prospective study covering the period 2005 to 2012. The patients underwent initial imaging and URS with focal samples taken. Cytopathological results served as reference standard. RESULTS: We investigated 174 renal units (RUs; renal pelvis and adjacent ureter) in 148 patients. UTUC was found in 104 RUs. MCTU had an accuracy of 0.74, sensitivity of 0.89, specificity of 0.51, positive predictive value of 0.73, and negative predictive value of 0.75; corresponding values for URS were 0.84, 0.84, 0.85, 0.89, and 0.78, respectively. MCTU had significantly higher sensitivity and accuracy compared with other imaging techniques (P<0.05). Compared with MCTU, URS had similar sensitivity but significantly greater specificity and accuracy. CONCLUSION: Both MCTU and URS are important tools in the diagnostic evaluation of UTUC although neither of those techniques achieves 100% accuracy. MCTU should be chosen as the radiographical method if there are no contraindications and URS should always be combined with focal cytology and biopsies of suspicious lesions. To enhance diagnostic precision both MCTU and URS with focal samples should be included in the diagnostic procedure. The present results add impact to current diagnostic guidelines.

Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism.

Previously, we have shown that Wnt-5a strongly regulates dopaminergic neuron differentiation by inducing phosphorylation of Dishevelled (Dvl). Here, we identify additional components of the Wnt-5a-Dvl pathway in dopaminergic cells. Using in vitro gain-of-function and loss-of-function approaches, we reveal that casein kinase 1 (CK1) delta and CK1epsilon are crucial for Dvl phosphorylation by non-canonical Wnts. We show that in response to Wnt-5a, CK1epsilon binds Dvl and is subsequently phosphorylated. Moreover, in response to Wnt-5a or CK1epsilon, the distribution of Dvl changed from punctate to an even appearance within the cytoplasm. The opposite effect was induced by a CK1epsilon kinase-dead mutant or by CK1 inhibitors. As expected, Wnt-5a blocked the Wnt-3a-induced activation of beta-catenin. However, both Wnt-3a and Wnt-5a activated Dvl2 by a CK1-dependent mechanism in a cooperative manner. Finally, we show that CK1 kinase activity is necessary for Wnt-5a-induced differentiation of primary dopaminergic precursors. Thus, our data identify CK1 as a component of Wnt-5a-induced signalling machinery that regulates dopaminergic differentiation, and suggest that CK1delta/epsilon-mediated phosphorylation of Dvl is a common step in both canonical and non-canonical Wnt signalling.