Wolf in sheep's clothing - Oral proliferative verrucous leukoplakia: Progression with longitudinal molecular insights

BackgroundOral proliferative verrucous leukoplakia (OPVL) is a chronic form of oral leukoplakia that progresses to a multifocal disease with confluent, exophytic and proliferative features. The clinical differential diagnosis for OPVL includes frictional keratosis, leukoplakia, chronic hyperplastic candidiasis, squamous papilloma, verrucous hyperplasia, verrucous carcinoma and squamous cell carcinoma. In this study, we aimed to delineate the dynamic changes in molecular signature during OPVL progression. We compare to a cohort of oral cavity keratinizing squamous cell carcinoma (OSCC) patients covering the spectrum of verrucous carcinoma to invasive squamous cell carcinoma including cytologically bland cuniculatum variant.MethodsSamples from a large OPVL lesion that exhibited a histopathologic continuum of OPVL progression.ResultsCanonical hotspot TERT promoter mutations were identified in all patients. TERT C228T was dominant and mutually exclusive with TERT C250T. In patients with TERT C250T, there was concurrent PI3 point mutation. TP53 mutations were also consistently found (8/10). At the protein level, p53 was abnormal, with loss of function and gain of function.ConclusionsOPVL is a pathology that shows proximity to the gene expression profile of OSCC, highlighting signatures in common that can be important targets for drug treatment, as well as in the development of diagnostic and prognostic strategies for this disease.     

Fascin stain as a potential marker of invasiveness in carcinomas of the urinary bladder: a retrospective study with biopsy and cytology correlation

The evaluation of invasion in urothelial carcinomas of the urinary bladder cannot be determined on cytology and can be particularly challenging in biopsy cases with limited sampling. Recent studies of bladder resection specimens suggest that fascin overexpression may be a marker of aggressive urothelial carcinomas and can help facilitate the assessment of invasion. In this study, we evaluated urine cytology and corresponding biopsy specimens with proven invasive urothelial carcinoma for fascin expression by immunohistochemistry. Thirty‐five patients diagnosed with positive urine cytology and biopsy‐proven invasive urothelial carcinoma between January 2003 and February 2009 were identified. We found increased fascin expression in 100% (35/35) of SurePathTM&!trade; urine cytology preparations as well as 100% (35/35) of corresponding biopsy cases with invasive urothelial carcinoma. On urine cytology, cytoplasmic fascin staining was moderate to intense in malignant tumor cell clusters and single cells and not observed in benign urothelial cells. Staining in biopsy cases was generally intense and cytoplasmic and present in both the invasive (100%) and noninvasive (31%) components of the lesion. These findings uphold the association of increased fascin expression in invasive urothelial carcinomas of the urinary bladder. We furthermore demonstrate that fascin staining can be performed successfully on SurePathTM&!trade; urine cytology preparations in which increased fascin expression correlates with invasion on biopsy. While not a definitive marker of invasion, as it is observed in in situ carcinoma, we conclude that the utilization of fascin immunohistochemistry on urine cytology might serve as a useful adjunct in predicting invasiveness in subsequent biopsies. Diagn. Cytopathol. 2010. © 2010 Wiley‐Liss, Inc.     

Urine: beyond cytology for detection of malignancy

In the present review we discuss various ancillary modalities for detection of malignancies in urine samples, with an emphasis on urothelial carcinomas. Flow cytometry, bladder tumor antigen (BTA), nuclear matrix protein (NMP), matrix metalloproteinase (MMP), human chorionic gonadotrophic (HCG), telomerase, and other techniques are discussed. DNA FCM is a relatively costly and sophisticated technique. It has a practical application in the diagnosis of bladder cancer among subjects at high risk and is of value in monitoring the course of the disease and anticipating recurrence following conservative treatment. The BTA test is a simple, rapid, and inexpensive adjunct to cystoscopy and the results of the test are equivalent or superior to those of voided urinary cytology. NMP-22 immunoassay is a useful diagnostic test for predicting recurrence of urothelial malignancy. It is also a cost-effective and sensitive screening test for detecting tumor in patients with urothelial carcinoma. Beta-HCG estimation in urine samples appears to be an efficient diagnostic marker for the assessment of distant metastasis in bladder carcinoma rather than a screening test. Other ancillary techniques such as detection of expression of cytokeratin 20 by RT-PCR, MMP-9 estimation, and fluorescent in situ hybridization and telomerase activity are rarely applied clinically in routine urinary samples and are not cost-effective.     

Expression and function of SIRT6 in muscle invasive urothelial carcinoma of the bladder

SIRT6, a member of the class III histone deacetylase, has been shown to inhibit glycolysis and promote DNA double strand break repairs. Despite of its proposed tumor suppressor role, no significant differences in SIRT6 mRNA levels among normal bladder urothelium, non-muscle invasive, and muscle invasive urothelial carcinoma were noted in the two largest bladder cancer gene expression datasets available in Oncomine^TM. We therefore studied the expression and function of SIRT6 in muscle invasive urothelial carcinoma of the bladder. Immunohistochemistry studies of SIRT6 on radical cystectomy samples showed a dramatic decline of SIRT6 expression when bladder cancer progressed from T2 to T4. Functional study with bladder cancer cell lines confirmed its role in inhibiting glycolysis and cell proliferation. Reducing SIRT6 with siRNA, however, did not sensitize bladder cancer cells to drug induced DNA damage. The differential expression patterns of SIRT6 amongst different T stages of muscle invasive bladder cancers indicate less reliance on glycolysis when urothelial carcinoma invades deeper through the bladder and into the adjacent tissues.     

Frequent FGFR3 mutations in urothelial papilloma

Activating point mutations in the FGFR3 gene occur frequently in low-grade and low-stage bladder carcinomas, whereas they are rare in high-grade carcinomas. This study investigates the incidence of FGFR3 mutations in 12 urothelial papillomas and 79 pTaG1 tumours which were regraded according to the 1998 WHO/ISUP classification system, resulting in 62 papillary urothelial neoplasms of low malignant potential (PUNs-LMP) and 17 low-grade papillary urothelial carcinomas (LG-PUCs). FGFR3 mutation analysis of 21 ovarian Brenner tumours was also performed. Seventy-seven cases were detected with a mutation in the FGFR3 gene. The mutations were exclusively found in bladder neoplasms. In urothelial papilloma, generally considered a benign lesion, 9/12 (75%) mutations were found. This report is the first to describe a genetic defect in urothelial papilloma. A comparable percentage of mutations was found in PUNs-LMP (85%) and LG-PUCs (88%). No mutations were found in matched normal DNA from bladder tumour patients. The mean follow-up was 5.78 years (range 0.21-17.60 years). Five patients developed high-grade papillary urothelial carcinoma from 2.5 to 12 years after first diagnosis. Two patients died of bladder cancer. The mean number of recurrences (recurrence rate) per year was 0.03, 0.21, and 0.46, respectively, for papilloma, PUN-LMP, and LG-PUC. Urothelial papilloma is a rare lesion with a benign natural behaviour compared with PUN-LMP and LG-PUC of the bladder, but from a molecular perspective, papillomas should be classified together with all well-differentiated urothelial neoplasms.     

High expression of Notch ligand Jagged2 is associated with the metastasis and recurrence in urothelial carcinoma of bladder

Background: The Notch signaling pathway is closely related with human organ development and tumorgenisis. Jagged2 is among the most popular topic in Notch studies currently. Recent studies found its vital role in tumor metastasis in breast cancer; however, its expression profile and its prognostic value in urothelial carcinoma of bladder have not been investigated. Methods: Immunohistochemistry was used to detect the expression of Jagged2 in 120 bladder urothelial carcinoma. Moreover, the expression of Jagged2 was analyzed by Western blot in 60 bladder urothelial carcinoma and 20 normal epithelial tissues. MTT assay and flow cytometry and transwell assay were used to examine the proliferative and invasive ability of bladder cancer cells with the treatment of GSIXX (the inhibitor of Jagged2). Prognostic value of Jagged2 expression and its correlation with tumor metastasis and recurrence were evaluated, and the proliferative and invasive ability and cell cycle process of the bladder cancer cells were detected as well. Results: There was a significantly higher Jagged2 expressions in bladder urothelial carcinoma and highly invasive bladder T24 cells than those in bladder normal tissues and the superficial bladder BIU-87 cells. Jagged2 expression was positively correlated with histological grade, p T stage, recurrence, and metastasis. With the increasing concentration of GSIXX, we found that not only the cell proliferation and invasion activity decreased significantly, but also the cell cycle was blocked at G₂/M stage. Conclusions: Jagged2 expression status was closely correlated with important histopathologic characteristics (grades and stages) and the recurrence and metastasis of bladder urothelial carcinomas. Furthermore, Jagged2 played an important function on the bladder cancer cells’ proliferation by regulating the cancer cell cycle from G₁/S to G₂/M and probably promoted the invasion and metastasis of bladder cancer.     

The molecular-morphologic correlates of bladder cancer

Environmental exposure to carcinogens trigger molecular events that lay the background for the subsequent development of urothelial carcinoma from the basal and intermediate layers of urothelial lining. Clinical classes of non-muscle invasive bladder cancer and muscle invasive bladder cancer are broadly divided into basal and luminal subtypes each with their molecular, morphologic and outcome characteristics. Morphology of cancer represents the sum total of genetic and epigenetic changes. The current review highlights the molecular – morphologic correlates identifiable in our daily clinical practice, the knowledge of which will help develop a deeper understanding of 1) early evolving lesions, 2) grade heterogeneity, 3) importance of recognizing subtypes of urothelial carcinoma and 4) basis of treatment resistance of urothelial carcinoma.     

Malignant glandular lesions and glandular differentiation in invasive/noninvasive urothelial carcinoma of the urinary bladder

Although the lumen of the urinary bladder is covered with only urothelial epithelium, malign glandular lesions (eg, nonurachal adenocarcinoma) and benign lesions (eg, cystitis cystica and cystitis glandularis) can also rarely occur in this site due to its characteristic embryologic development. Glandular differentiation is uncommon in urothelial carcinomas and is even less common in noninvasive urothelial cancers. In addition, in situ urothelial carcinomas are more likely to progress in the presence of glandular differentiation toward high-grade urothelial carcinomas and/or aggressive urothelial carcinomas. Pure nonurachal adenocarcinomas and mixed carcinomas (urothelial carcinoma and adenocarcinoma) are very rare, and their pathogenesis is not clear. Most of the nonurachal adenocarcinomas are thought to arise on the grounds of cystitis glandularus with intestinal metaplasia. Here, I present 2 cases with noninvasive urothelial carcinoma with substantial glandular differentiation showing progression to signet ring cell carcinoma and invasive urothelial carcinoma, one case with mixed carcinoma (urothelial carcinoma and adenocarcinoma) and another case with pure adenocarcinoma developing from cystitis glandularis with intestinal metaplasia, and discuss malign glandular lesions in the bladder and invasive/noninvasive urothelial carcinomas with glandular differentiation.     

Measuring the dimension of invasive component in pT1 urothelial carcinoma in transurethral resection specimens can predict time to recurrence

Recurrences of nonmuscle-invasive urothelial bladder cancer are very common following resection. Predictive histopathologic variables in transurethral resection of bladder tumor (TURBT) specimens are of particular importance especially in determining the behavior of lamina propria-invasive tumors (high grade T1 stage). A total of 110 patients who underwent TURBT for urothelial carcinoma (1997-2005) from a single institution were retrospectively reviewed. Amount of tumor invasion by urothelial carcinoma was assessed in terms of percentage, focality (focal vs multifocal), and dimension (DI, aggregate length of invasion). Of 110 patients, 39 (35%) were found to have invasive high-grade urothelial carcinoma, including 9 females. Mean age was 70 years (range, 56-94 years). Twenty-three patients with high-grade T1 urothelial carcinoma had available follow-up information. Recurrence rate in these 23 patients was 96% (22/23). Nearly all of the recurences (221/22, 95%) occurred within 1 year of the initial TURBT. There was an inverse correlation of DI with time to recurrence (P < .05; correlation coefficient, −0.47). Urothelial carcinoma with a greater DI (>0.5 cm) had a mean time to recurrence of less than 6 months. Percentage of tumor invasion and focality was not associated with recurrence. The aggregate length of invasion may be a prognostic variable for high-risk nonmuscle-invasive bladder cancer. Measuring “aggregate length of invasive tumor,” if further validated in larger studies, could provide a practical alternative in substaging pT1 tumors in TURBT specimens.     

Non-radioactive measurement of telomerase activity in human bladder cancer,bladder washings,and in urine

Early diagnosis is still the most important prerequisite for successful cancer treatment and this holds true for bladder cancer. Urine cytology is commonly used as a non-invasive screening procedure for the detection of bladder carcinoma, but this method is labour-intensive and often generates false-negative results. The ribonucleoprotein telomerase appears to be a promising new cancer marker, since its activity has been reported to correlate with indefinite growth. The aim of this study was to investigate whether telomerase activity can be detected in bladder cancer and in corresponding bladder washings. For this purpose, a sensitive non-radioactive TRAP (telomeric repeat amplification protocol) detection system was developed. With this technique, telomerase activity was found in 95 per cent of the carcinomas (n=20), in 70 per cent of the corresponding bladder washings, but in none of the urine samples obtained from patients with bladder carcinoma. No telomerase activity was detectable in normal urothelium or in samples from dysplastic urothelium. The data obtained from bladder washings show that superficial carcinoma cells released into the bladder still harbour telomerase activity. The absence of telomerase activity in voided urine is thus most likely due to degradation or inactivation under these conditions. The high rate of telomerase activity in bladder carcinoma indicates that the activation of telomerase is a common step in the tumourigenesis of bladder cancer. © 1998 John Wiley & Sons, Ltd.     

Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer

Many molecular alterations are known to occur in urothelial carcinoma of the bladder, but their significance for tumor progression is poorly understood. Deletions of chromosome 8p are frequently found in several tumor types and are often associated with progressive disease. In all, 99 bladder tumors were screened for deletions at 8p using loss of heterozygosity (LOH) and multicolor fluorescence in situ hybridization FISH analyses. Allelic loss on chromosome 8p in at least one marker was found in 25/99 (25%) tumors. There was a significant correlation of 8p deletions with invasive tumor growth and a highly significant association with papillary growth pattern in patients with invasive disease. cDNA array analyses revealed that secreted Frizzled-related protein 1 (sFRP1), an antagonist of Frizzled receptors and Wnt pathway activation on chromosome 8p12-11.1, is frequently downregulated in bladder cancer. To investigate sFRP1 as a candidate for a putative progression-related gene on 8p, urothelial cell lines and primary urothelial carcinomas were screened for sFRP1 expression using quantitative real-time PCR, Northern blot, immunofluorescence and immunohistochemistry (IHC). Of the investigated bladder cancers, 38% showed loss of sFRP1 expression by quantitative RT-PCR. Evaluation of the protein expression by IHC using tissue microarrays containing 776 bladder cancers revealed loss or strong reduction of sFRP1 expression in 66% of cases. SFRP1 loss was associated with higher tumor stage and grade and shorter overall survival. In addition, loss of sFRP1 was an independent indicator of poor survival in patients with papillary but not with muscle invasive bladder cancer. There were neither mutations in the coding region of sFRP1 nor homozygous deletions at 8p12-11.21. However, promoter methylation was detected using methylation-specific PCR in 29% of cases. In conclusion, we could show a close correlation of chromosome 8p deletions and progression of papillary bladder tumors. The sFRP1 gene on chromosome 8p12-11.1 could be a candidate gene for the predicted, progression-related tumor suppressor gene in bladder cancer and could contribute to urothelial carcinogenesis.     

Myeloid leukemia in a urine specimen: A case report and review of the literature

Urinary tract cytology has a long history of utilization for the diagnosis and follow‐up of tumors involving the urothelial tract. As expected, the most common tumor encountered in exfoliative urine cytology is urothelial carcinoma. While the sensitivity of urinary tract cytology for the diagnosis of low‐grade urothelial carcinomas is low, its sensitivity and accuracy for high grade urothelial carcinomas is much higher. However, nonurothelial malignancies, such as hematopoietic malignancies, can also be encountered in urine specimens. Leukemic cells in urine can be diagnosed readily by cytological examination in cases where more invasive procedures are difficult to perform. Additionally, cell block sections can be utilized to determine the immunocytochemical profile of the tumor cells to confirm the diagnosis. Herein we report a case of a 75‐year‐old man with a past medical history of acute myeloid leukemia (AML), who presented with congested heart failure and painless macroscopic hematuria. AML relapse was diagnosed. Cytological examination of the urine using a ThinPrep® smear, cytospin preparation, and immunohistochemical stains performed on the cell block sections were examined. Findings were consistent with leukemic cells of myeloid origin in the bladder washing specimen. Diagn. Cytopathol. 2014;42:700–704. © 2013 Wiley Periodicals, Inc.     

Squamous lesions of the bladder

Squamous lesions of the bladder encompass a broad range of benign and malignant lesions. Many of the squamous lesions restricted to the urothelial surface are generally indolent, such as squamous metaplasia, squamous papilloma and condyloma acuminatum. However, some superficial lesions such as verrucous squamous hyperplasia and squamous cell carcinoma in situ may precede the development of invasive squamous cell carcinoma. Squamous lesions present within the wall of the bladder are often malignant and include classic squamous cell carcinoma and the verrucous squamous cell carcinoma and basaloid squamous cell carcinoma variants. One exception, however, is pseudoepitheliomatous (pseudocarcinomatous) hyperplasia, which is a benign mimicker of invasive squamous cell carcinoma. Our review summarizes the available data on clinical presentation, histopathologic features, differential diagnosis and ancillary tests of the squamous lesions of the bladder.     

Morphometric differences between urothelial cells in voided urine of patients with grade I and grade II bladder tumours

The morphometric differences between the urothelial cells in the voided urine of 24 patients with grade I and grade II bladder tumours were measured. In both wet-fixed Papanicolaou-stained smears as in air-dried Giemsa-stained preparations, the cells in the grade I tumours are larger and have more pronounced anisocytosis and a smaller nucleus to cytoplasm area (N/C) ratio. Although absolute dimensions of cells and nuclei in the two preparatory technique differ significantly, the average N/C ratios are similar. In both methods almost all grade I tumours have average N/C cell size ratios of less than 0.6 and grade II tumours more than 0.6. Morphometrically urothelial cells could not be distinguished from cells exfoliated from grade I bladder tumours. The results indicate that it is feasible to classify bladder tumours using the morphometric values of the exfoliated urothelial cells alone.