Diagnosis Of Low-grade Urothelial Carcinoma Research Articles

CDCP1 (CUB domain containing protein 1) is a potential urine-based biomarker in the diagnosis of low-grade urothelial carcinoma.

Urine-based cytology is non-invasive and widely used for clinical diagnosis of urothelial carcinoma (UC), but its sensitivity is less than 40% for low-grade UC detection. As such, there is a need for new diagnostic and prognostic biomarkers of UC. CUB domain containing protein 1 (CDCP1) is a type I transmembrane glycoprotein highly expressed in various cancers. Using tissue array analysis, we demonstrated that CDCP1 expression in UC patients (n = 133), especially in those with low-grade UC, was significantly higher than in 16 normal persons. In addition, CDCP1 expression in urinary UC cells could also be detected by using immunocytochemistry method (n = 11). Furthermore, in 5637-CD cells, overexpression of CDCP1 affected the expression of epithelial mesenchymal transition-related markers and increased matrix metalloproteinase 2 expression and migration ability. Conversely, the knockdown of CDCP1 in T24 cells had the opposite effects. Using specific inhibitors, we demonstrated the involvement of c-Src/PKCδ signaling in the CDCP1-regulated migration of UC. In conclusion, our data suggest that CDCP1 contributes to the malignant progression of UC and may have the potential as a urine-based biomarker for detecting low-grade UC. However, a cohort study needs to be conducted.

Improvement of the Collection, Maintenance, and Analysis of Neoplastic Cells from Urine Specimens with the Use of CytoMatrix.

Urine cytology is a non-invasive test used in combination with cystoscopy for screening and follow-up of urothelial carcinoma (UC). Although cytology can be used to efficiently identify high-grade UC, it has a lower accuracy for the diagnosis of low-grade UC or patients with presence of atypical urothelial cells (AUC). For these reasons, ancillary tests have been added to urine cytology in order to improve the accuracy. However, the poor abundance of neoplastic cells in most samples and the absence of a “tissue-like” structure remains a major challenge. We used a novel synthetic support called CytoMatrix which has the property of capturing and storing cells and micro-macro aggregates within its three-dimensional structure. The urine specimens were obtained from 12 patients: 6 with suspected urothelial neoplasia (low- and high-grade) and 6 with AUC or non-neoplastic samples. The first step is the urine samples preparation, through several centrifugation passages; the second step consists in absorbing cells on the CytoMatrix, and in the subsequent formalin fixation, standard processing and paraffin embedding to prepare FFPE-CytoMatrix block. In the final step, sections are consecutively cut, stained with hematoxylin-eosin (H&E), and analyzed via UroVysion FISH and immunohistochemistry (IHC). Using our simple and reliable protocol, we can improve the quality of urine specimens, allowing a better collection, maintenance, and analysis of cells, with the advantage of using ancillary tests to support cytological diagnosis and the advantage of storing cellular material in a FFPE-CytoMatrix block.

Cytokeratin 20 immunocytochemistry on urine sediments: A potential low-cost adjunct to cytology in the diagnosis of low-grade urothelial carcinoma.

Urine cytology is the corner-stone for the diagnosis of urothelial neoplasia; however, a substantial proportion of low-grade carcinomas are reported as inconclusive owing to scant cellularity and subtle cytological features. Biomarkers applied on urine sediment smears of such patients are likely to be clinically relevant. Access to Food and Drug Administration approved urinary biomarkers in resource limited setting is poor. Detection of cytokeratin 20 (CK20) in urine sediments, although still a research tool, is a promising marker as immunocytochemistry is performed regularly in several Indian laboratories. We tested the clinical utility of CK20 immunocytochemistry as a potential low-cost adjunct to urine cytology in diagnosis of low-grade urothelial carcinoma. One hundred and fifty fresh, voided urine specimens from 42 cases of biopsy proven urothelial neoplasia (14 high grade, 28 combined low-grade [n=26]) and low malignant potential [n=2]), and 20 non-neoplastic lesions were included in the study sample. Confident diagnosis of malignancy was possible in five (17.8%) low-grade malignancies. Thirteen of 16 (81.3%) low-grade malignancies with inconclusive cytology showed positive CK20 expression. This reduced the proportion of low-grade cases with inconclusive cytology from 57.1% to 10.7% (P=.021). In addition, we could correctly classify one case of bladder lithiasis with false positive urine cytology. Discrepant CK20 staining (positive) was seen in one patient with acute cystitis. CK20 expression in non-umbrella cells is a robust marker of urinary bladder carcinoma. It has potential clinical utility for identification of low-grade urothelial malignancy with inconclusive cytological diagnosis.

Urinary tract washing specimens containing atypical urothelial tissue fragments are significantly associated with urothelial neoplasia.

We previously found that the presence of atypical urothelial tissue fragments (AUTF) was associated with an increased risk of high-grade urothelial carcinoma (HGUC) but not low-grade urothelial neoplasia (LGUN) in voided urine (VU) specimens. However, we subsequently found that patients with LGUN were more likely to have cytologic atypia in urinary washing (UW) specimens, suggesting that cytologic atypia found in UW specimens might be associated with both LGUN and HGUC. In this study, we retrospectively examined UW specimens containing AUTF to determine whether they were associated with HGUC, LGUN, or both HGUC and LGUN. 1173 UW specimens and any follow up biopsies were identified over a 10-year period and the presence of AUTF was recorded based on the original clinical diagnosis. The presence of AUTF in UW specimens was significantly associated with high-risk indeterminate diagnosis or definitive diagnosis of HGUC (P < .001). In addition, AUTF specimens were significantly associated with a neoplastic diagnosis of low-grade urothelial carcinoma (LGUC) or HGUC on subsequent biopsies when compared to specimens lacking AUTF (P = .047). The overall rate of CIS/HGUC in specimens with AUTF was 33.0 vs. 13.5% for specimens without AUTF (P = .051). Urothelial tissue fragments (UTF) found in UW specimens should be examined for the presence of cytomorphological atypia, as the presence of AUTF almost triples the risk of HGUC. As opposed to what has been found in VU specimens, AUTF in UW specimens is also associated with an increased risk of LGUN.

Cytopathologic differential diagnosis of low-grade urothelial carcinoma and reactive urothelial proliferation in bladder washings: a logistic regression analysis.

Conventional cytomorphologic assessment is the first step to establish an accurate diagnosis in urinary cytology. In cytologic preparations, the separation of low-grade urothelial carcinoma (LGUC) from reactive urothelial proliferation (RUP) can be exceedingly difficult. The bladder washing cytologies of 32 LGUC and 29 RUP were reviewed. The cytologic slides were examined for the presence or absence of the 28 cytologic features. The cytologic criteria showing statistical significance in LGUC were increased numbers of monotonous single (non-umbrella) cells, three-dimensional cellular papillary clusters without fibrovascular cores, irregular bordered clusters, atypical single cells, irregular nuclear overlap, cytoplasmic homogeneity, increased N/C ratio, pleomorphism, nuclear border irregularity, nuclear eccentricity, elongated nuclei, and hyperchromasia (p˂0.05), and the cytologic criteria showing statistical significance in RUP were inflammatory background, mixture of small and large urothelial cells, loose monolayer aggregates, and vacuolated cytoplasm (p˂0.05). When these variables were subjected to a stepwise logistic regression analysis, four features were selected to distinguish LGUC from RUP: increased numbers of monotonous single (non-umbrella) cells, increased nuclear cytoplasmic ratio, hyperchromasia, and presence of small and large urothelial cells (p=0.0001). By this logistic model of the 32 cases with proven LGUC, the stepwise logistic regression analysis correctly predicted 31 (96.9%) patients with this diagnosis, and of the 29 patients with RUP, the logistic model correctly predicted 26 (89.7%) patients as having this disease. There are several cytologic features to separate LGUC from RUP. Stepwise logistic regression analysis is a valuable tool for determining the most useful cytologic criteria to distinguish these entities.

Accuracy and interobserver variability of the cytologic diagnosis of low-grade urothelial carcinoma in instrumented urinary tract cytology specimens.

The aim of this study was to determine the accuracy of diagnosis and level of agreement of the cytologic diagnosis of low-grade urothelial carcinoma (LGUC) among experienced cytopathologists to help the development of the upcoming classification scheme ("The Paris System") of urinary cytology (UTCy). Forty ThinPrep (Cytyc, Boxborough, MA) UTCy specimens (19 with corresponding biopsy specimens diagnosed as LGUC and 21 with negative biopsy and/or cystoscopy specimens) were blindly reviewed by six cytopathologists from three institutions and diagnosed as negative for malignancy (NM), atypical urothelial cells (AUCs), and LGUC. Interobserver agreement was measured with the weighted κ statistic. Each observer's receiver operating characteristic (ROC) curves and the area under the ROC curve (AUROC) were calculated. Four to six reviewers diagnosed LGUC in five (26%) of 19 UTCy specimens, corresponding to a biopsy diagnosis of LGUC. Overall agreement was fair (κ = 0.30). After collapsing AUC with NM, the reviewers' ranges of sensitivity, specificity, positive predictive value, and negative predictive value for LGUC were 21% to 53%, 81% to 95%, 71% to 90%, and 57% to 67%, respectively. AUROC varied from 0.66 to 0.74. We found that UTCy has a low sensitivity but relatively high specificity, resulting in poor to fair accuracy for the diagnosis of LGUC; the level of agreement between reviewers was only fair to moderate.

Is a consistent cytologic diagnosis of low-grade urothelial carcinoma in instrumented urinary tract cytologic specimens possible? A comparison between cytomorphologic features of low-grade urothelial carcinoma and non-neoplastic changes shows extensive overlap, making a reliable diagnosis impossible

The ability to consistently diagnose low-grade urothelial carcinoma (LGUC) in urinary tract cytology (UTCy) specimens remains controversial, as the reported sensitivity of UTCy in the detection of LGUC is as low as 10%. To determine whether a consistent cytologic diagnosis of LGUC is possible, we assessed the presence and frequency of previously described cytomorphologic features of LGUC in UTCy from patients with LGUC and a negative control group. Biopsy-proven cases of LGUC from June 1, 2010 to January 31, 2014 were identified; UTCy obtained within 3 months prior to biopsy composed the study group (n = 98). The negative control group consisted of UTCy obtained from patients with negative cystoscopy and biopsy (n = 53). All specimens were masked and reviewed in random order to evaluate 17 cytomorphologic parameters. Univariate statistical analyses demonstrated that the prevalence of paired cells, clumpy chromatin, and cytoplasmic homogeneity was higher in the study group; however, multivariate analysis did not show these features as significant predictors of LGUC. No cytomorphologic feature was statistically significant in the LGUC group versus the negative control group. The presence of 3-dimensional papillary structures with fibrovascular cores is diagnostic of LGUC, but it is only seen in a small minority (2 of 98) cases. Our results reemphasize the fact that urinary tract cytology has a low sensitivity for the diagnosis of LGUC and suggest that, instead of striving to detect LGUC in urine specimens, we should concentrate on the clinically relevant goal of urine cytology-the detection of high-grade lesions.

Detection of MicroRNAs in Archival Cytology Urine Smears

MicroRNAs’ dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC). Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205) in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage) was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06–4.60 µg). Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears (slope = -3.4084; R-squared = 0.99; efficiency = 1.94). miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

Value of p16INK4a in the diagnosis of low‐grade urothelial carcinoma of the urinary bladder in urinary cytology

The sensitivity of urinary cytology for the diagnosis of urothelial carcinomas is low, particularly in low-grade carcinomas. The UroVysion test is a fluorescent in situ hybridization multiprobe assay that increases the sensitivity of urinary cytology. However, this test is not widely available. P16(INK4a) , a protein involved in cell cycle progression, is overexpressed in urothelial carcinoma. Immunocytochemical expression of p16(INK4a) has been examined in biopsy samples from urothelial carcinomas, but few studies have addressed this protein in urine cytology. The authors compared the results of p16(INK4a) immunoreactivity in cytology and biopsy samples from 83 cases, including low-grade urothelial carcinomas, reactive epithelial lesions, and negative cases. p16(INK4a) assessment of in urine cytology samples showed a sensitivity of 66.7% and a specificity of 82.8% in the diagnosis of low-grade urothelial carcinomas. On the basis of these results, the authors propose that immunocytochemical detection of p16(INK4a) is a reliable tool in urine cytology, both for the diagnosis of low-grade urothelial carcinomas and for follow-up purposes. More retrospective and prospective studies are required to verify these results.

Immunostaining as a Diagnostic Aid in Cytopathologic Study of Upper Urinary Tract Urothelial Carcinoma

To evaluate preoperative diagnosis of low-grade urothelial carcinoma (LGUC) and urothelial neoplasms of unknown malignant potential (UMP UN) of the upper urinary tract (UUT) and its role in disease management, especially in the context of nephron-sparing treatment possibilities.Wash and brush ureteral specimens of LGUC/UMP UN of the UUT with histopathologic correlation were retrieved at our institution for 7 years and studied along with 7 ureteral specimens from nonneoplastic ureteral lesions.Of 30 specimens from 25 LGUC/UMP UN, 5 were negative for tumor cells and 3 showed cytologic atypia. The remaining 22 contained tumor cells with characteristic features of urothelial carcinoma, including hard and soft criteria. The 4 hard criteria included branching stromal cores, dyshesive cell networks, 3-dimensional papillary clusters with stromal core and atypia associated with CK20-positive cells. The 2 soft criteria were hypercellularity and atypia in CK20-negative cells. All LGUC/UMP UN of the UUT were associated with at least 1 hard criterion or both soft criteria.Branching stromal cores, 3-dimensional papillary clusters, dyshesive cell networks and CK20-positive atypia immunostaining appear specific for LGUC/UMP UN of the UUT but are seen in few cases. Combined soft and hard criteria will increase sensitivity to 83%.

The effects of the current World Health Organization/International Society of Urologic Pathologists bladder neoplasm classification system on urine cytology results.

Historically, the diagnostic accuracy of urine cytology for the detection of urothelial carcinoma has been low, particularly for low-grade lesions. In 1998, the World Health Organization and International Society of Urologic Pathologists (WHO/ISUP) established a new classification system for urothelial neoplasms. It has been postulated that the accuracy of urine cytology for the detection of low-grade carcinomas should improve, because the current WHO/ISUP system classifies bladder lesions that lack any significant cytologic atypia as papillary urothelial neoplasms of low malignant potential (PUNLMP). Also, the accuracy of urinary cytology for the detection of high-grade lesions is expected to decrease, because the criteria for high-grade lesions is lowered, placing most of the previous WHO Grade 2 tumors into the current high-grade carcinoma category. One hundred bladder biopsies that were classified according to the previous WHO classification system were examined by one pathologist and were reclassified according to the new WHO/ISUP scheme. All biopsies had corresponding urine specimens that had been diagnosed previously by a different cytopathologists. The patients' previous WHO and current WHO/ISUP surgical diagnoses were compared with the corresponding urine cytology. In addition, cytospins obtained from 40 patients with a histologic diagnosis of low-grade urothelial carcinoma (n = 20 patients) and high-grade urothelial carcinoma (n = 20 patients) according to the WHO/ISUP classification system were reviewed to identify any outstanding cytologic features. According to the original WHO classification system, there were 26 patients with Grade 1 transitional cell carcinoma (TCC), 61 patients with Grade 2 TCC, and 13 patients with Grade 3 TCC. The corresponding cytology was positive in 11 of 26 Grade 1 tumors, 28 of 61 Grade 2 tumors, and 12 of 13 Grade 3 tumors. After the reclassification, there was 1 papilloma, 12 PUNLMP lesions, 50 low-grade urothelial carcinomas, and 37 high-grade carcinomas. The cytology was positive in 0 of 1 papillomas, 5 of 7 PUNLMP lesions, 18 of 50 low-grade carcinomas, and 28 of 37 high-grade carcinomas. In addition, morphologic uniformity and cytoplasmic homogeneity (50.0% and 45.0%, respectively) were seen more commonly in low-grade bladder tumors. The diagnostic accuracy of urine cytology for high-grade lesions decreased, as expected; however, cytologic detection of low-grade urothelial carcinomas was remarkably lower than expected. In addition, no outstanding cytologic features could be identified to make a definitive diagnosis.