Comparative analysis of non-coding and coding DNA mutations in flat urothelial lesions: biological implications and insights.

Recent research in urothelial carcinoma (UC) has focused on coding mutations, leaving the significance of non-coding mutations unexplored. This study aims to evaluate non-coding DNA mutation frequencies compared to coding regions in normal urothelium and flat lesions, exploring their implications for tumor biology. Using targeted next-generation sequencing with UC-related gene panel, we analyzed non-coding and coding DNA mutation frequencies across 119 samples of flat urothelium encompassing various lesion types. Mutation patterns were examined based on the presence of associated flat or papillary tumors, and we investigated the correlation between mutation rates in target genes and genetic mutations within genomic regions. Intronic mutations (IMs) displayed variability across lesions, with normal urothelium (NU) exhibiting the highest frequency (43%) and urothelial carcinoma in situ (CIS) the lowest (9%). We observed similar sets of frequently mutated genes in both intronic and exonic regions, distinct from promoter region mutations. Although IMs paralleled exonic mutations in NU, reactive atypia, and atypia of unknown significance (AUS), they were less prevalent in dysplasia (DYS) and CIS. In contrast to CIS-associated AUS and DYS lesions, AUS-DYS lesions associated with papillary tumors exclusively exhibited recurrent intronic mutations involving FGFR3 and ERCC2, aligning with mutation patterns seen in exonic regions. ERCC2 intronic mutations correlated with the mutation rates of the gene panel. Our findings suggest that intronic mutations significantly contribute to tumor heterogeneity in urothelial lesions and may potentially be linked to genomic instability, warranting further investigation.

Current Knowledge and Prospects for Renal Hemangioblastoma and Renal Cell Carcinoma with Hemangioblastoma-like Features.

Tumors exhibiting histopathological findings similar to those of hemangioblastoma of the central nervous system (CNS-HB) rarely develop in the kidneys. Currently, renal hemangioblastoma (RHB) is considered analogous to CNS-HB; however, they differ in gross appearance, as well as immunohistochemical and molecular findings. In contrast, some renal cell carcinomas reportedly comprise distinct, clear cell renal cell carcinoma (CCRCC)- and hemangioblastoma (HB)-like areas. Initially, renal cell carcinomas with HB-like features (RCC-HBs) were considered a morphological variant of CCRCC owing to their diverse histological findings. However, the immunohistochemical and molecular findings of RCC-HBs suggest that RCC-HB is distinct from CCRCC. Additionally, one of the RCC-HBs had a focal leiomyomatous stroma and TSC2 variant, suggesting that RCC-HB and RCC with fibromyomatous stroma (RCC-FMS) might belong to the same disease entity. Therefore, we comprehensively reviewed the clinical, pathological, and molecular features of RHB, RCC-HB, and the related tumors and discussed the similarities, differences, and relationships between them. We believe that our review would serve as a foundation for further investigation on elucidating the relationship between CNS-HB, RHB, RCC-HB, and RCC-FMS.

Targeted Next-Generation Sequencing of Flat Urothelial Lesions Reveals Putative Pathobiological Pathways, Potential Biomarkers, and Rational Therapeutic Targets.

Flat urothelial lesions are controversial diagnostic and prognostic urologic entities whose importance relies mainly on their ability to progress to muscle-invasive tumors via urothelial carcinoma in situ (CIS). However, the carcinogenetic progression of preneoplastic flat urothelial lesions is not well established. Moreover, predictive biomarkers and therapeutic targets of the highly recurrent and aggressive urothelial CIS lesion are lacking. Using a targeted next-generation sequencing (NGS) panel of 17 genes directly involved in bladder cancer pathogenesis, we investigated alterations of genes and pathways with clinical and carcinogenic implications on 119 samples of flat urothelium, including normal urothelium (n = 7), reactive atypia (n = 10), atypia of unknown significance ( n = 34), dysplasia ( n = 23), and CIS (n = 45). The majority of the flat lesions were tumor-associated but grossly/microscopically or temporally separated from the main tumor. Mutations were compared across flat lesions and concerning the concomitant urothelial tumor. Associations between genomic mutations and recurrence after intravesical bacillus Calmette-Guerin treatment were estimated with Cox regression analysis. TERT promoter mutations were highly prevalent in intraurothelial lesions but not in the normal or reactive urothelium, suggesting that it is a critical driver mutation in urothelial tumorigenesis. We found that synchronous atypia of unknown significance-dysplasia-CIS lesions without concomitant papillary urothelial carcinomas had a similar genomic profile that differed from atypia of unknown significance-dysplasia lesions associated with papillary urothelial carcinomas, which harbored significantly more FGFR3, ARID1A, and PIK3CA mutations. KRAS G12C and ERBB2 S310F/Y mutations were exclusively detected in CIS and were associated with recurrence after bacillus Calmette-Guerin treatment (P = .0006 and P = .01, respectively). This targeted NGS study revealed critical mutations involved in the carcinogenetic progression of flat lesions with putative pathobiological pathways. Importantly, KRAS G12C and ERBB2 S310F/Y mutations were identified as potential prognostic and therapeutic biomarkers for urothelial carcinoma.

Use of Artificial Intelligence for the Interpretable Prediction of the Pathologic Diagnosis and Molecular Abnormalities of Flat Urothelial Lesions.

Flat urothelial lesions are important because of their potential for carcinogenesis and development into invasive urothelial carcinomas. However, it is difficult for pathologists to detect early flat urothelial changes and accurately diagnose flat urothelial lesions. To predict the pathologic diagnosis and molecular abnormalities of flat urothelial lesions from pathologic images, artificial intelligence with an interpretable method was used. Next-generation sequencing on 110 hematoxylin and eosin-stained slides of normal urothelium and flat urothelial lesions, including atypical urothelium, dysplasia, and carcinoma in situ, detected 17 types of molecular abnormalities. To generate an interpretable prediction, a new method for segmenting urothelium and a new pathologic criteria-based artificial intelligence (PCB-AI) model was developed. κ Statistics and accuracy measurements were used to evaluate the ability of the model to predict the pathologic diagnosis. The likelihood ratio test was performed to evaluate the logistic regression models for predicting molecular abnormalities. The diagnostic prediction of the PCB-AI model was almost in perfect agreement with the pathologists' diagnoses (weighted κ = 0.98). PCB-AI significantly predicted some molecular abnormalities in an interpretable manner, including abnormalities of TP53 (P = 0.02), RB1 (P = 0.04), and ERCC2 (P = 0.04). Thus, this study developed a new method of obtaining accurate urothelial segmentation, interpretable prediction of pathologic diagnosis, and interpretable prediction of molecular abnormalities.

Clinicopathological and Molecular Characteristics of Macroscopically Yellowish-Colored Chromophobe Renal Cell Carcinoma Compared to Non-Yellowish-Colored Chromophobe Renal Cell Carcinoma.

Each histological variant of renal cell tumors has a unique color. The yellowish color of clear cell renal cell carcinoma (CCRCC) is explained by the presence of intracytoplasmic lipid and glycogen accumulation. Color changes in CCRCC are correlated with clinicopathological and metabolic changes, as well as biological behavior. We analyzed and compared the clinical, histopathological, and immunohistochemical features and gene expression profiles, in lipid metabolism of yellowish-colored ChRCC (ChRCC-Y), non-yellowish-colored ChRCC (ChRCC-N), and CCRCC. Of 14 ChRCCs, we retrieved 6 ChRCC-Ys. Patients with ChRCC-Y are younger than those with ChRCC-N, and the tumor is not predominant in males. ChRCC-Ys are smaller than ChRRC-Ns. Three ChRCC-Ys exhibited individual discrete tubule formation. No ChRCC-Ns exhibited individual discrete tubule formation. Two of 6 ChRCC-Ys showed relatively diffuse adipophilin positivity. No ChRCC-Ns demonstrated diffuse positivity for adipophilin. The expression of SCD, FDFT1, and E2F1 showed a tendency to be lower in ChRCC-Y than in ChRCC-N. The expression of PDGFB showed a tendency to be higher in ChRCC-Y than in ChRCC-N. This study demonstrated ChRCC-Y did not indicate an increase in lipid and cholesterol metabolism and that ChRCC-Y did not have the common molecular alteration of CCRCC. The absence of such metabolic acceleration in ChRCC-Y might support the biological indolent behavior. Furthermore, we revealed that macroscopic color changes might be correlated with various clinicopathological features and immunohistochemical and molecular changes from different perspectives. We believe further characterization of RCC, including tumor heterogeneity, is needed to improve the management of patients with RCC.

Detection of HPV infection in urothelial carcinoma using RNAscope: Clinicopathological characterization.

BACKGROUND: Human papillomavirus (HPV) is a well-established mucosotropic carcinogen, but its impact on urothelial neoplasm is unclear. We aimed to clarify the clinical and pathological features of HPV-related urothelial carcinoma (UC). METHODS: Tissue samples of 228 cases of UC were obtained from the bladder, upper and lower urinary tract, and metastatic sites to construct a tissue microarray. The samples were analyzed for the presence of HPV by a highly sensitive and specific mRNA in situ hybridization (RISH) technique (RNAscope) with a probe that can detect 18 varieties of high-risk HPV. We also conducted immunohistochemistry (IHC) for a major HPV capsid antibody and DNA-PCR. RESULTS: The HPV detection rates varied among the methods; probably due to low HPV copy numbers in UC tissues and the insufficient specificity and sensitivity of the IHC and PCR assays. The RISH method had the highest accuracy and identified HPV infection in 12 (5.2%) of the cases. The histopathological analysis of the HPV-positive UC showed six cases of usual type UC, five cases of UC with squamous differentiation (UC_SqD), and one case of micropapillary UC. The HPV detection rate was six-fold higher in the cases of UC_SqD than in the other variants of UC (odds ratio [OR] =8.9, p = 0.002). In addition, HPV infection showed a significant association with tumor grade (OR =9.8, p = 0.03) and stage (OR =4.7, p = 0.03) of UC. Moreover, the metastatic rate was higher in HPV-positive than in negative UC (OR =3.4). CONCLUSION: These data indicate that although the incidence of HPV infection in UC is low, it is significantly associated with squamous differentiation and poor prognosis. Furthermore, our observations show that RNAscope is an ideal method for HPV detection in UC compared with the other standard approaches such as IHC and PCR assays.

Paneth-like cells in renal cell carcinomas and in cysts associated with acquired cystic kidney disease: Clinicopathologic analysis, comparative study and description of precursor lesions.

Paneth-like cells (PLCs) are different from Paneth cells (PCs) and contain Paneth-like granules, which have been reported in non-neoplastic conditions and in neoplasms of various organs. PLCs have been reported in clear cell renal cell carcinoma (CCRCC), but not in non-CCRCC, including acquired cystic disease-associated renal cell carcinoma (ACD-RCC). We analyzed clinicopathological features of 24 acquired cystic disease-associated renal cell carcinoma (ACD-RCC) with PLCs (ACD-RCCP+) and compared with those of 23 ACD-RCCs without PLCs (ACD-RCCP-). Approximately half of ACD-RCCs had PLCs and that almost all kidneys harboring ACD-RCC had cysts with PLCs. The fact that many ACD-RCCs and the cysts had PLCs is further evidence that the cyst with vacuoles and complex architecture might be a precursor lesion for ACD-RCC. The presence of PLCs may provide additional morphologic clue for distinguishing ACD-RCC from PRCC in challenging differential diagnostic workup in acquired cystic disease of the kidney setting.

Measurement and visualization of cell membrane surface charge in fixed cultured cells related with cell morphology.

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells.