Diagnostic Roles of Immunohistochemical Markers CK20, CD44, AMACR, and p53 in Urothelial Carcinoma In Situ.

Background and Objectives: This study aimed to evaluate the diagnostic roles of various immunohistochemical (IHC) markers in urothelial carcinoma in situ (uCIS) through a meta-analysis and review of diagnostic test accuracy. Materials and Methods: The IHC markers CK20, CD44, AMACR, and p53 were evaluated in the present study. We analyzed the expression rates of the IHC markers and compared their diagnostic accuracies. Results: The estimated expression rates were 0.803 (95% confidence interval [CI]: 0.726-0.862), 0.142 (95% CI: 0.033-0.449), 0.824 (95% CI: 0.720-0.895), and 0.600 (95% CI: 0.510-0.683) for CK20, CD44, AMACR, and p53, respectively. In the comparison between uCIS and reactive/normal urothelium, the expression of CK20, AMACR, and p53 in uCIS was significantly higher than in reactive/normal urothelium. CD44 showed significantly lower expression in uCIS than in the reactive/normal urothelium. Among the markers, AMACR had the highest sensitivity, specificity, and diagnostic odds ratio. The AUC on SROC was the highest for CK20. Conclusions: In conclusion, IHC markers, such as CK20, CD44, AMACR, and p53, can be useful in differentiating uCIS from reactive/normal urothelium.

Penile extramammary Paget disease associated with urothelial carcinoma in situ: Case report and literature review.

BACKGROUND: Extramammary Paget disease (EMPD) is an uncommon disease affecting older men and women. Clinically, it appears as a plaque lesion with an erythematous or leukoplakic background in regions with abundant apocrine glands such as female external genitalia, perineum, scrotum, and penis. METHODS: We are presenting an 85-year-old patient with recurrent erythematous plaque lesions involving the penis and known to have urothelial carcinoma (UC) in situ of the bladder. A literature review of EMPD secondary to UC has been conducted through PubMed and Google Scholar search engines. RESULTS: The histopathologic examination revealed a proliferation of Paget cells within the surface squamous epithelium. The lesional cells displayed vesicular nuclei, clear cytoplasm, and a positive staining for CK7, CK20, HER2, GATA3 as well as p40, and negative staining for SOX10, CK5/6, and CDX2. The literature review revealed 18 more cases of EMPD associated with UC, including 4 with non-invasive UC. Most of them were treated surgically, but the disease recurred in nine cases and death of disease was reported in at least four patients, all of them associated with invasive UC. CONCLUSION: The prognosis of penile EMPD seems to be dictated by the stage of the underlying UC.

Immunohistochemistry in hollow urinary tract.

There is no ideal marker or established immunohistochemistry panel to confirm urothelial differentiation. Immunohistochemistry must be always indicated in concrete differential diagnostic consideration. In this review article, immunohistochemistry will be discussed in the three different settings - distinction of benign and malignant changes of urothelium, the most frequent pitfalls and non-urothelial neoplasms of urinary tract.

Precursor lesions of the urinary bladder.

The classification of neoplastic precursor lesions in the urinary tract has evolved slowly with the gradual accumulation of clinicopathological data. Current nomenclature was codified most recently by the 2016 WHO classification, which is based on primary data with clinical outcome, consensus group statements and considerations of practical utility in routine diagnosis. This review discusses precursor lesions of urothelial, squamous and glandular lineage. For urothelial neoplasia, both flat lesions with atypia and early 'difficult-to-classify' proliferations are considered. Subtypes of squamous metaplasia, florid non-invasive squamous proliferations and frank squamous dysplasia are also addressed. Finally, rare glandular precursors of adenocarcinoma are reviewed, to include intestinal metaplasia, glandular dysplasia and villous adenoma. For each category, morphology (including differential diagnostic considerations), immunohistochemistry and any known molecular correlates are detailed. The goal is to provide a concise, practical up-to-date overview of this complex topic.

Differential diagnosis of urothelial carcinoma in situ from non-neoplastic urothelia: Analysis of CK20, CD44, P53 and Ki67.

BACKGROUND: Flat urothelial lesions comprise a spectrum of morphologic changes ranging from reactive atypia to carcinoma in situ (CIS). Urothelial dysplasia and CIS are associated with the recurrence and progression of urothelial carcinoma. Distinguishing CIS and dysplasia from reactive atypia based on histolopathogical features alone is often difficult. Using different immunohistochemical markers such as Cytokeratin 20 (CK20), CD44, p53, and Ki-67 is recommended for differential diagnosis. The aim of this study was to evaluate the immunohistochemical pattern of these antibodies to differentiate different flat urothelial lesions. METHODS: In this cross- sectional study, three groups of bladder biopsy specimens were evaluated: 20 samples with reactive urothelial lesions, 20 histologically diagnosed as CIS, and 20 morphologically normal samples. Immunohistochemical staining of CK20, p53, CD44 and Ki-67 markers was performed on paraffin-embedded blocks. The groups were compared using chi square test, and the diagnostic value of the markers were evaluated with sensitivity, specificity, positive and negative predictive values. RESULTS: CK20 was full thickness positive in 15 (75%) CIS samples and negative in all samples of the normal and reactive groups (p<0.001); CD44 was positive in 2 (10%) cases of the CIS group and in 17 (85%) of the reactive group; this marker was negative in all the normal samples (p<0.001). P53 was positive in 12 (60%) samples of the CIS group and negative in all samples of the normal and reactive groups (p<0.001). Ki67 was positive in 13 (65%) samples of the CIS group and 1 (5%) sample of the reactive group. This marker was negative in all samples of the normal group (p<0.001). CONCLUSION: The results of this study revealed that CK20, CD44, P53 and Ki67 are useful in distinguishing CIS from reactive and normal samples. However, they should be used in a panel including at least three markers. Correlation with the morphologic features is necessary.

Digital volumetric assessment of CIS and tumor mass compliments conventional histopathological assessment in muscle-invasive urothelial bladder cancer.

Carcinoma in situ (CIS) of the bladder is a known parameter regarding the prognosis and recurrence tendency of urothelial carcinomas. Nevertheless, there is little evidence whether the amount of CIS or other precursor lesions, as well as the quantified tumor mass of muscle-invasive urothelial carcinoma, has an influence on the survival or recurrence rate of affected patients. From 80 patients with muscle invasive urothelial bladder cancer and radical cystectomy, 23 samples each were obtained as part of a whole organ mapping in a single institution study, in which the precursor lesions and tumor area were digitally measured and further correlated to pathological standard parameters, patient survival, molecular luminal and basal subtypes, and immune infiltration. Significant correlations were found between tumor mass and surface lining CIS amount for pT-stage, lymphovascular invasion, and perineural infiltration. Furthermore, an increased tumor mass as well as an increased amount of CIS combined with an increased tumor mass showed a significantly reduced survival rate in multivariable analysis (HR = 2.75; P = 0.019 vs. HR = 3.54; P = 0.002) as well as a significantly increased recurrence. No correlations could be found with molecular subtypes and immune infiltration. The exact measurement of the tumor mass with and without the CIS surface area, whether manually or, more specifically, digitally, could be incorporated into routine diagnostics and implemented as an independent predictor for patient post-surgical outcomes. It can therefore serve as an additional predictor for risk stratification and, if necessary, intensified follow-up care or therapy.

Myxoid Pseudotumor Changes Affecting the Distal Ureter Associated With Urothelial Carcinoma In Situ.

Myxoid pseudotumor is a pseudoneoplastic fibroblastic proliferation that has been described in the perinephric and renal sinus fat tissue. It is characterized by the presence of a myxoid matrix, intermingled with the adipocytes, and a hypocellular population of spindle-shaped and stellate cells. We report a myxoid pseudotumor involving the distal ureter, which broadens the spectrum of possible localizations of this lesion around the urinary tract. It occurred in an 80-year-old patient who underwent a nephroureterectomy indicated after an incidental radiological finding of a thickening of the distal left ureter wall which suggested a ureteral neoplasm. He had two voided urine and one ureteroscopic sample cytologies diagnosed as high-grade urothelial carcinoma, as well as a retrograde ureteroscopy ureteral biopsy which was diagnosed as urothelial carcinoma in situ. This emphasizes the problem of the possible misdiagnosis of myxoid pseudotumor as a ureteral infiltrative carcinoma due to the radiological findings being badly interpreted, compounded by the preoperative cytohistologic data on malignancy. A diffuse urothelial carcinoma in situ was seen in our specimen without infiltrative or papillary tumors. This would not support an obstructive pathogenetic mechanism as has been hypothesized for myxoid pseudotumor.

Urothelial carcinoma in situ with "overriding" features can evade detection by mimicking umbrella cells.

Urothelial carcinoma in situ (uCIS) is typically recognized by overtly malignant cells with characteristic nuclear features; multiple histologic patterns have been described. A rare "overriding" pattern, in which uCIS tumor cells extend on top of normal urothelium, has previously been mentioned in the literature, but not well described. Herein, we report 3 cases of uCIS with "overriding" features. Detailed morphologic evaluation revealed somewhat subtle cytologic atypia: variably enlarged hyperchromatic nuclei and scattered mitotic figures but with abundant cytoplasm and limited to superficial urothelium. Immunohistochemical (IHC) analysis showed a distinctive diffuse positive aberrant p53 pattern, limited to the atypical surface urothelial cells; these cells also showed CK20+, CD44-, and increased Ki-67. In 2 cases, there was a history of urothelial carcinoma and adjacent conventional uCIS. In the third case, the "overriding" pattern was the first presentation of urothelial carcinoma; therefore, next-generation sequencing molecular testing was also performed, revealing pathogenic mutations in TERTp, TP53, and CDKN1a to further support neoplasia. Notably, the "overriding" pattern mimicked umbrella cells, which normally line surface urothelium, can have abundant cytoplasm and more variation in nuclear and cell size and shape, and show CK20+ IHC. We therefore also evaluated umbrella cell IHC patterns in adjacent benign/reactive urothelium, which showed CK20+, CD44-, p53 wild-type, and very low Ki-67 (3/3). We also reviewed 32 cases of normal/reactive urothelium: all showed p53 wild-type IHC in the umbrella cell layer (32/32). In conclusion, caution is warranted to avoid overdiagnosis of usual umbrella cells as CIS; however, "overriding" uCIS should be recognized, may have morphologic features that fall short of the diagnostic threshold of conventional CIS, and requires further study.

Urine cytology findings in patients with biopsy-confirmed urothelial carcinoma in situ with plasmacytoid features.

BACKGROUND: Urine cytology is an important screening tool in the diagnosis of high-grade urothelial carcinoma. Diagnosis in urine samples follows criteria outlined by The Paris System for Reporting Urinary Cytology (TPS). However, cytologic characteristics of the recently described urothelial carcinoma in situ with plasmacytoid features (P-CIS) have not been described, and it is unknown whether they conform to TPS criteria for high-grade urothelial carcinoma. This study was aimed at better characterizing possibly unique cytologic features of P-CIS. METHODS: The authors collected urine cytology specimens from patients with subsequent bladder biopsy-proven P-CIS. Specimens were re-reviewed according to the TPS criteria. The proposed cytologic features of P-CIS (eccentric, enlarged, and hyperchromatic nuclei) were evaluated; this included the reproducibility of 3 cytopathologists for the proposed cytologic features. RESULTS: Seventy-four urine specimens from 18 patients with P-CIS-diagnosed bladder biopsies were identified. The TPS diagnoses of the 74 urine cytology specimens were as follows: negative for high-grade urothelial carcinoma (n = 26), atypical urothelial cells (n = 26), suspicious for high-grade urothelial carcinoma (n = 12), and high-grade urothelial carcinoma (n = 10). Only 7 urine specimens met the proposed cytologic criteria for P-CIS, and they had TPS diagnoses of negative for high-grade urothelial carcinoma (n = 1), atypical urothelial cells (n = 3), and high-grade urothelial carcinoma (n = 3). The κ interobserver agreement ranged from poor to fair. CONCLUSION: The features of P-CIS on urine cytology are subtle and infrequently reproducible and often do not meet the TPS criteria for diagnosis as high-grade urothelial carcinoma. In specimens that do not meet TPS criteria for high-grade urothelial carcinoma, P-CIS cytology in isolation would be best classified as atypical urothelial cells.

CK20 versus AMACR and p53 immunostains in evaluation of Urothelial Carcinoma in Situ and Reactive Atypia.

Ancillary testing with immunohistochemistry has shown recent promise in the workup of equivocal bladder lesions. We read with interest the recent findings of Alston et al., who assessed the diagnostic utility of alpha-methylacyl-CoA racemase (AMACR) in comparison to cytokeratin 20 (CK20) in evaluation of atypia in challenging flat urothelial lesions in the differential between carcinoma in situ (CIS) and reactive atypia. AMACR was reported to be a somewhat more specific but less sensitive marker for CIS than CK20, though showing weaker intensity. Spurred by their report, with the knowledge that we had consistently and consecutively performed AMACR, CK20, and p53 on flat urothelial lesions challenging enough to reach intradepartmental consensus, we performed a retrospective review. Similarly, we found that AMACR was less sensitive (80%) and more specific (100%) than CK20, with the same caveat of less staining intensity. Additionally, our p53 review identified a significant rate (~ 27%) of equivocal/non-informative findings. Taken together, our experience in this consecutive cohort confirms the impression of Alston et al. regarding the utility and challenges of AMACR use, while highlighting challenges with p53, which we plan to use more sparingly prospectively.

Immunohistochemical staining patterns of Ki-67 and p53 in florid reactive urothelial atypia and urothelial carcinoma in situ demonstrate significant overlap.

Flat urothelial lesions with atypia may pose significant diagnostic challenges. Given frequent increased proliferation rates in florid reactive urothelial atypia and limited studies on the interpretation of p53 stains in the urothelium (following current standard guidelines for correlation with P53 mutation status), we sought to further study the discriminatory value of Ki-67 and p53 for florid reactive urothelial atypia versus urothelial carcinoma in situ (CIS). Bladder specimens diagnosed as reactive urothelial atypia (n = 40) and CIS (n = 40) were assessed by immunohistochemical staining with antibodies for Ki-67, p53, CD44, and CK20. Immunoreactivity was scored based on percent cells positive for Ki-67 and pattern of reactivity with p53 (aberrant: diffuse strong positive or negative; normal: patchy/wild type). CD44 and CK20 reactivity patterns served as adjunctive internal validation controls for reactive urothelial atypia and CIS, as previously described. In reactive urothelial atypia, Ki-67 ranged from 0% to 90% (mean, 34% ± 26) with 30 cases (75%) having >10%. In CIS, Ki-67 ranged from 5% to 95% (mean, 50% ± 25) with 17 cases (43%) having >50%. In all 40 cases (100%) of reactive urothelial atypia, p53 expression had a wild-type pattern. In CIS, aberrant p53 expression was identified in 15 cases (37%): 3 cases (7%) were p53 negative (i.e. null phenotype) and 12 cases (30%) showed strong and diffuse nuclear reactivity (in >85% of cells). The remaining 25 cases (63%) of CIS had a p53 wild-type pattern of expression. Cytoplasmic CK20 immunoreactivity in umbrella cells was seen in 34 cases (85%) of reactive urothelial atypia, and 6 cases (15%) were negative. In addition, 35 cases (88%) of reactive urothelial atypia demonstrated full-thickness CD44 expression, while 5 cases (12%) had expression confined to the basal/parabasal layers of the urothelium. Strong and diffuse CK20 positivity was present in 39 cases (98%) of CIS, and patchy positivity was detected in 1 case (2%). None of the CIS cases overexpressed CD44: 16 cases (40%) showed focal expression in the nonneoplastic basal cell layer; 24 cases (60%) demonstrated no staining. In summary, Ki-67 has poor discriminatory value for reactive urothelial atypia versus CIS and adds little to the classic CK20/CD44 immunophenotype. While p53 sensitivity for CIS is relatively low (30%) and interpretation as either wild type or negative may be challenging in a small subset of cases, strong and diffuse nuclear reactivity was 100% specific in the distinction from florid reactive urothelial atypia in this cohort.

Diagnosis of urothelial carcinoma in situ using blue light cystoscopy and the utility of immunohistochemistry in blue light-positive lesions diagnosed as atypical.

Carcinoma in situ (CIS) is difficult to visualize with white light cystoscopy (WLC), whereas blue light cystoscopy (BLC) using photosensitizing agents improves detection rates. We retrospectively reviewed transurethral biopsies of bladder tumors in which both WLC and BLC evaluations were performed (n = 135 samples from 79 patients). Biopsies were classified based on the presence/absence of fluorescence under BLC and the final pathological report (CIS/benign/atypical). Forty-one (30%) cases were diagnosed as CIS; of those, 38 (93%) were BLC(+), including 23 that were WLC(-). Conversely, 51 (38%) lesions were BLC(+) but classified as non-CIS. Eleven BLC(+) cases were diagnosed as "atypical." These cases were anonymized and reviewed by 7 pathologists for concordance and then immunostained for CK20, p53, and Ki-67. Immunohistochemistry results were interpreted as consistent with CIS if there was full-thickness staining of CK20, more than 50% p53-positive cells, and more than 50% Ki-67-positive cells. Review of BLC(+)/atypical cases showed a mean agreement of 79%, and none of the cases showed staining pattern consistent with CIS. Therefore, all 11 cases of BLC(+)/atypical were considered non-CIS for the final analysis. All patients with BLC(+)/atypical lesions had a history of intravesical Bacillus Calmette-Guerin and/or mitomycin. Using final pathology as the reference, sensitivity, specificity, and negative predictive value of BLC were 93% (confidence interval [CI], 80.1%-98.5%), 46% (CI, 35.4%-56.3%), and 94% (CI, 82.5%-97.8%), respectively. The low specificity of BLC leads to BLC(+) lesions with atypical diagnosis. Morphological classification of these lesions is fairly consistent among different pathologists. Immunohistochemistry for p53/CK20/Ki-67 in this setting is only helpful to potentially avoid overcalling CIS.

Enhancer of zeste homolog 2 (EZH2) expression in bladder cancer.

BACKGROUND: Studies evaluating enhancer of zeste homolog 2 (EZH2) expression and oncologic outcomes in bladder cancer have been discrepant. EZH2 expression in noninvasive bladder cancer is not well studied. We thus set out to address the discrepancy in previous reports, and to study expression of EZH2 in noninvasive bladder cancer and its associations, in a large cystectomy cohort. MATERIALS AND METHODS: EZH2 expression was evaluated in tissue microarray material (invasive and noninvasive cancer). Associations between EZH2 expression and oncologic outcomes, tumor stage, and disease type were determined. Receiver operating characteristic analysis was performed for EZH2 expression in the diagnosis of invasive carcinoma and flat carcinoma in situ (CIS) compared to benign urothelium. RESULTS: EZH2 expression was most common in CIS, followed by invasive carcinoma, noninvasive papillary urothelial carcinoma, and benign urothelium, in decreasing order (P<0.05 all comparisons, linear model). The receiver operating characteristic analysis demonstrated an area under the curve of 0.92 for CIS and 0.83 for invasive carcinoma, both compared to benign urothelium. At a cutoff of≥4, this corresponded to sensitivities of 89% and 73%, and specificities of 82% and 82%, for CIS and invasive carcinoma, respectively. The EZH2 expression was not associated with oncologic outcomes, including recurrence-free survival and death from bladder cancer. The EZH2 expression status (positive or negative) of noninvasive and invasive carcinomas taken from the same bladder correlated (P = 0.05, Fisher exact). CONCLUSION: That EZH2 status of noninvasive and invasive cancer correlated in individual patients suggests that EZH2 may be a marker of lineage. EZH2 may offer diagnostic utility, particularly in flat urothelial CIS vs. benign urothelium. The present study supports that EZH2 expression in bladder cancer is not predictive of oncologic outcome.

Nuclear size measurement for distinguishing urothelial carcinomas from reactive urothelium on tissue sections.

BACKGROUND: Pathological diagnosis of urothelial carcinoma (UC) is primarily based on cytological atypia. It has previously been shown that high-grade (HG) UC, particularly UC in situ cells (CIS), can be over five times the size of a lymphocyte. However, this has not been demonstrated in comparison to reactive urothelium. The objective of this study was to empirically compare the difference in nuclear size of UC cells with reactive urothelial cells. METHODS: Using CellSens imaging software, we measured urothelial nuclear length (l) and width (w) on digital images of H&E sections. The area (a) of a nucleus was calculated based on the oval shape of most urothelial cells. Lymphocytes were measured to calculate normalized urothelial linear and area ratios. RESULTS: A total of 1085 urothelial cell nuclei from 60 cases were measured from reactive urothelium, low grade (LG) UC, HG UC and CIS. CIS nuclei were found to have an a 2.75 times larger than reactive nuclei (p < 0.001). A nuclear size cut-off of 11 um for l and 7 um for w was found to be sensitive [98.09 % (95 % CI: 95.60-99.38 %) and 89.31 % (95 % CI: 83.6-91.82 %) for l and w, respectively] and specific [92.60 % (95 % CI: 87.13-95.82 %) and 85.71 % (95 % CI: 79.49-90.63 %) for l and w, respectively] for distinguishing CIS from reactive atypia. CONCLUSIONS: Nuclear morphometry can be used to differentiate CIS from reactive atypia. A l over 11 um and a w over 7 um and is highly sensitive and specific for CIS compared to reactive urothelium. This difference in nuclear size may be used as a tool for differentiating the flat urothelial lesions from reactive urothelium in daily practice.

Management of carcinoma in situ of the bladder: best practice and recent developments.

Management of carcinoma in situ of the bladder remains a complex and challenging endeavor due to its high rate of recurrence and progression. Although it is typically grouped with other nonmuscle invasive bladder cancers, its higher grade and aggressiveness make it a unique clinical entity. Intravesical bacillus Calmette-Guérin is the standard first-line treatment given its superiority to other agents. However, high rates of bacillus Calmette-Guérin failure highlight the need for additional therapies. Radical cystectomy has traditional been the standard second-line therapy, but additional intravesical therapies may be more appealing for non-surgical candidates and patients refusing cystectomy. The subject of this review is the treatment strategies and available therapies currently available for carcinoma in situ of the bladder. It discusses alternative intravesical treatment options for patients whose condition has failed to respond to bacillus Calmette-Guérin therapy and who are unfit or unwilling to undergo cystectomy.

Diagnosis and management of urothelial carcinoma in situ of the lower urinary tract: a systematic review.

CONTEXT: Urothelial carcinoma in situ (CIS) has a high propensity for progression. It is usually reported within the heterogeneous context of non-muscle-invasive bladder cancer (NMIBC) but warrants special consideration. OBJECTIVE: To review the contemporary literature on the diagnosis and management of CIS. EVIDENCE ACQUISITION: A systematic search using broad terms to capture the diagnosis and treatment of CIS was performed according to Preferred Reporting Items for Systematic Reviews and Meta-analysis criteria. Full-text original articles, reviews, and editorials from 1966 to 2014 in English were included. References from selected articles, relevant guidelines, and conference abstracts were searched. Abstracts were excluded. EVIDENCE SYNTHESIS: A total of 1887 articles were identified, of which 120 were used in this review. Most reports were retrospective and heterogeneous in caseload. There is a lack of standardised classification of CIS. Many studies consider CIS in the context of NMIBC without a clear separation of the subset with CIS. Recent prospective phase 2 and 3 studies have improved the evidence base. CONCLUSIONS: We are beginning to understand that CIS has a spectrum of biologic potential. Bacillus Calmette-Guérin immunotherapy appears superior to other intravesical agents and may alter the natural history of CIS. New imaging modalities, agents, and treatment strategies have emerged in recent years with the aim of better identification of CIS, more bladder-preserving treatments, and prevention of surgical overtreatment. PATIENT SUMMARY: Improvements in imaging techniques combined with new bladder-preserving treatments will continue to have an impact on the outcomes of bladder carcinoma in situ.