Downregulation of MCM2 contributes to the reduced growth potential of epithelial progenitor cells in chronic nasal inflammation.

BACKGROUND: Recent studies have shown that human nasal epithelial progenitor cells (hNEPCs) are characterized by poor proliferation capacities during chronic nasal inflammation. OBJECTIVE: We sought to investigate the key molecular functions and candidates that contribute to the reduced growth potential of hNEPCs in chronically inflamed nasal mucosa. METHODS: Nasal biopsy specimens were obtained from 28 patients with nasal polyps (NPs) and 13 healthy controls. hNEPCs from nasal samples were cultured for 3 consecutive passages, and their molecular and functional profiles were analyzed by RNA sequencing. The minichromosome maintenance protein (MCM) family gene MCM2 was validated in hNEPCs and tissue samples from patients with NPs and control subjects by cell cycle, quantitative PCR, and Western blot analyses; small interfering RNA-mediated knockdown assay; and immunofluorescent staining. RESULTS: Compared with control hNEPCs, NP-derived hNEPCs showed (1) reduced growth kinetics, as evidenced by the colony-forming efficiency and doubling time; (2) inhibited cell cycle progression, as evidenced by gene ontology and/or pathway and cell cycle analyses; and (3) downregulated expression of MCM2, the key protein of the MCM complex, which is critical for DNA replication at the G1/S checkpoint. Moreover, hNEPCs with MCM2 knockdown showed a decreased proliferation rate, and the MCM2 protein level in basal cells was significantly lower in abnormally remodeled nasal epithelium than in normal epithelium. CONCLUSION: These results demonstrate inhibited cell cycle progression and MCM2 downregulation in basal or progenitor nasal epithelial cells from NP tissue, which may contribute to the decreased growth potential of hNEPCs in chronically inflamed upper airways.

Diagnosis of urinary bladder urothelial carcinoma by immunocytology with p53, MCM5, MCM2 and Ki-67 antibodies using cell blocks derived from urine.

OBJECTIVE: Immunocytochemistry has attained a marginal role in urology so far. Combining the morphological and immunophenotypical changes of the urothelial cells retrieved from urine is a logical approach. The study aimed to analyse the diagnostic potential of immunocytological staining in the detection of high-grade and low-grade urothelial carcinoma. METHODS: Freshly voided urine was collected from 152 consecutive individuals, cytology classes were determined and cell blocks produced. A total of 77 patients were diagnosed with urothelial carcinoma and 75 patients had various benign urological conditions. Immunocytochemistry was performed using four antibodies: p53, MCM2, MCM5 and Ki-67. A diagnostic power to detect low grade and high-grade urothelial carcinoma was analysed for each antibody and their combinations with cytology. RESULTS: There were no significant differences between patients with low-grade tumours and control group. Antibodies p53 and Ki-67 slightly improved the sensitivity of urinary cytology while maintaining its specificity. The best negative predictive value was demonstrated in combinations of cytology and MCM5 (88.9%) and cytology, p53 and MCM5 (90.6%). In the diagnosis of high-grade tumours, all antibodies apart from MCM2 yielded better sensitivity and specificity than cytology alone (receiver operating characteristic curves: p53 = 0.853, MCM5 = 0.931, and Ki-67 = 0.895). Combined with cytology, the sensitivities went even higher for the cost of lower specificity. The best diagnostic performance was observed in the combination of MCM5 and Ki-67 (sensitivity = 96.2%; specificity = 80%). CONCLUSIONS: Immunocytochemistry with p53, MCM5 and Ki-67 antibodies can improve the diagnostic power of urinary cytology in the detection and follow-up of urinary bladder urothelial carcinoma.

ProEx C as Diagnostic Marker for Detection of Urothelial Carcinoma in Urinary Samples: A Review.

The gold standard for the detection of urothelial carcinoma is represented by urethro-cystoscopy and biopsy. Both procedures are invasive and expensive and therefore cytology is often used as first approach to investigate on a possible neoplasia, being a safe and cost-effective diagnostic modality of evaluation. Because cytology alone is not highly sensitive for detection of low grade urothelial carcinoma and recurrence of the disease, several adjunct markers and urine based tests for urothelial carcinoma have been developed, which can help in the final diagnosis. In particular, ProEx C is an immunohistochemical cocktail containing antibodies direct against topoisomerase IIα (TOP2A) and minichromosome maintenance 2 (MCM2) proteins. It proved to be a valid biomarker especially in detecting squamous intraepithelial lesions in cervical liquid-based samples and in discerning these lesions from their mimickers, as well as in ovarian, endometrial, vulvar, primary and metastatic melanomas, breast, pancreatic and renal cell carcinomas. This brief review covers the effective utility of ProEx C as adjunct tool in assessing the urothelial lesions in urine cytology, also providing prognostic and therapeutic information to help in clinical decisions.

Characterization and clinical validation of MCM2 and TOP2A monoclonal antibodies in the BD ProEx™ C assay: An immunoassay which detects aberrant S-phase induction in cervical tissue.

BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. RESULTS: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. CONCLUSIONS: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.

Expression of ProEx C in primary and metastatic urothelial carcinoma.

BACKGROUND: ProEx C is an antibody cocktail targeting the expression of topoisomerase IIα and minichromosome maintenance protein-2. Both these proteins are over-expressed in the cell nucleus during aberrant S-phase induction of neoplastic cells, which leads to cell proliferation. The aim of this study was to determine whether ProEx C expression can detect primary and metastatic urothelial carcinoma (UC). METHODS: Thirty one fine needle aspiration cell blocks (CB) with metastatic UC were identified. Immunohistochemical staining for ProEx C and thrombomodulin was performed. Additionally, staining for Pro Ex C was also performed in tissue microarrays (TMA) of 46 cases of primary UC and carcinomas from colon (80), stomach (31), pancreas (33), liver (92), ovary (24), endometrium (25), breast (60), lung (27), kidney (32), and prostate (44), as well as melanoma (22). Nuclear staining of ProEx C and membrane staining of thrombomodulin in at least 10% tumor cells was considered a positive result. RESULTS: Both ProEx C and thrombomodulin have similar sensitivity for metastatic UC (84% vs. 77%, p=0.75; whereas ProEx C yielded a higher sensitivity of 93% for primary UC than thrombomodulin (72%, p=0.01). In addition to UC, ProEx C is also expressed in most of the malignant neoplasms tested in our TMA study, and has the highest sensitivity in colon and stomach carcinomas (94%). CONCLUSION: ProEx C has high sensitivity for UC. However, it is also expressed in carcinomas of colon, stomach, breast, and lung carcinomas and may not be a useful marker for workup of metastatic UC.