Differential expression of hMLH1 and hMSH2 is related to bladder cancer grade,stage and prognosis but not microsatellite instability

Defects in the DNA mismatch repair proteins result in microsatellite instability and malignancy in hereditary non-polyposis colorectal carcinoma (HNPCC). However, the role of mismatch repair (MMR) proteins and microsatellite instability (MSI) in transitional cell carcinoma of the bladder is less clear. In our study, the expression of 2 MMR proteins and the frequency of MSI in Transitional cell carcinoma of the bladder (TCC) were investigated. One hundred eleven patients with TCC of the bladder were studied, with complete clinicopathological data (median follow up of 5 years, range 5-16 years). Immunohistochemistry was used to detect the expression levels of hMLH1 and hMSH2. Microsatellite analysis for 14 loci (10 loci from the Bethesda consensus panel and the repeats in the TGFbetaR2, BAX, hMSH3 and hMSH6 genes) was performed on 84 tumors. Reduced expression of either MMR protein was seen in 26 of 111 tumors (23%). Reduced expression was seen more commonly in muscle invasive (p<0.03) and high grade TCC (p<0.03) than in superficial, low grade tumors. By 5 years, reduced expression of either MMR protein was associated with fewer recurrences of superficial tumors (p=0.015) and fewer relapses in all tumors (p=0.03), compared to tumors with normal expression. Nine tumors had reduced expression of both MMR proteins, analysis which suggests a synergistic reduction in expression (p=0.001). MMR expression was related to patient age, younger patients being more likely to have reduced MMR expression than older patients (p<0.01). MSI was seen at multiple loci in 1 tumor (1%) and at a single locus in 6 tumors (7%). MSI was not associated with MMR expression. Our findings indicate that reduced expression of the MMR proteins may have an important contribution in the development of a subset of TCCs and suggest a potential role for MMR expression as prognostic indicators.     

Mononucleotide markers of microsatellite instability in carcinomas of the urinary bladder.

AIMS: To determine the presence of microsatellite instability (MSI) and to assess the expression of the human mismatch repair (MMR) gene products hMLH1 and hMSH2 in primary transitional cell carcinomas (TCCs) of the urinary bladder in relation to clinico-pathological parameters. METHODS: Seventy-two cases of primary TCC were screened for the presence of alterations in MSI markers by molecular techniques and evaluated immunohistochemically for the expression of hMLH1 and hMSH2 proteins. Clinical data were available in 70 cases. The percentage of MSI rose to 16.6%. RESULTS: Reduced (<20%) hMLH1 expression was closely related to the presence of MSI (p=0.0004). Neither MMR proteins nor MSI was associated with grade, stage, papillary status. Clinical outcome analysed as a function of MSI did not show significant differences in terms of both disease-free and overall survival. Reduced hMLH1 expression was a significant predictor of shorter disease-free survival in univariate and multivariate analysis. CONCLUSIONS: The presence of MSI is not related to classical clinico-pathological parameters in TCCs, nor does it appear to be of prognostic significance. hMLH1 was an important indicator for recurrence.     

上皮细胞钙粘蛋白(E-cadherin) 在膀胱移行细胞癌中的表达及其意义

 目的　观察上皮细胞钙粘蛋白(E-cad)表达与膀胱移行细 胞癌(TCC)分级、分期等生物学行为的关系。方法　采用恒冷切片免疫组 织化学SP法,对28例TCC和10例正常膀胱粘膜新鲜冰冻标本进行E-cad表达的检测。结果　对照组10例正常膀胱粘膜E-cad均正常表达,实验组28例TCC,仅50%(14/ 28)正常表达,二组差异显著(P<0.01)。TCC不同分级、分期间E-cad表达有显著性差 异(P<0.05),G3比G1+G2表达低,浸润性TCC比浅表性TCC表达低。初发与复发、 单发与 多发TCC之间比较,E-cad表达均未见显著性差异(P>0.05)。本组未见E-cad表达完全 缺失的TCC。结论　正常膀胱粘膜组织E-cad保留表达,TCCE-cad表达降 低,E-cad表达与TCC分级、分期呈负相关;E-cad表达与TCC的数目,与肿瘤的初发或复发 无关。     

Differential expression of the mismatch repair gene hMSH2 in malignant prostate tissue is associated with cancer recurrence

BACKGROUND: Mismatch repair (MMR) genes are responsible for coordinated correction of misincorporated nucleotides formed during DNA replication. Inactivating mutations in MMR genes have been described in sporadic cancers and a hereditary cancer predisposition syndrome. Mismatch repair deficiency causes instability at microsatellites and increased mutation rates. Although microsatellite instability (MSI) has been described in high-grade and lymph node positive prostate carcinoma specimens, an analysis comparing hMSH2 expression, MSI, and outcome in clinically organ confined prostate carcinoma has not been reported. METHODS: Immunohistochemical analysis of benign and malignant prostate tissue from 101 patients was performed using a monoclonal antibody specific for the hMSH2 protein. Expression was correlated with MSI using dinucleotide repeat markers and laser-captured microdissected DNA from normal and tumor cells. hMSH2 protein expression and MSI were assessed with respect to pathologic stage, Gleason score, and time to detectable serum prostate specific antigen (PSA) after prostatectomy in patients with clinically localized prostate carcinoma. RESULTS: In normal glands, hMSH2 staining was minimal to low and confined to the basal cell layer. In 32% of benign prostatic hyperplasia cases, hMSH2 staining was increased in the basal and luminal cell layers whereas 71% of cancer specimens had uniform moderate to high staining. Microsatellite instability was detected in 60% of absent to low staining and 26% of moderate to high staining prostate carcinoma specimens. Differential staining in benign versus malignant prostate tissues was statistically significant (P < 0.001) as was the correlation between absent to low hMSH2 staining and presence of MSI (P = 0.028). Decreased risk for PSA recurrence after radical prostatectomy correlated with absent to low hMSH2 staining in malignant prostate tissue but was only marginally significant (P = 0.05 for 24 month recurrence and P = 0.08 for overall time to PSA recurrence). CONCLUSIONS: The results of the current study demonstrate differential hMSH2 expression in benign and malignant prostate tissue. Moreover, hMSH2 expression is altered in a subset of clinically localized prostate carcinoma specimens independent of pathologic stage and Gleason pattern. A statistically significant correlation between hMSH2 immunohistochemical staining intensity and MSI also was identified in prostate carcinoma specimens. Furthermore, the time to cancer recurrence as determined by detectable serum PSA after prostatectomy was associated with hMSH2 staining intensity. Taken together, our results suggest that hMSH2 gene expression in prostate carcinoma may be a useful prognostic marker for outcome in men with clinically organ confined prostate carcinoma.     

H-ras mutations in rat urinary bladder carcinomas induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide and sodium saccharin, sodium ascorbate, or related salts

Male F344 rats were fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazoly]formamide for 6 weeks and then fed 3% or 5% sodium saccharin, 5% sodium ascorbate, 3.12% calcium saccharin, 1.34% sodium chloride, 5.2% calcium saccharin plus 1.34% sodium chloride, or basal diet alone for 72 weeks. Protein and DNA were extracted from 89 bladder tumors [87 transitional cell carcinomas (TCC), 1 papilloma, and 1 sarcoma] from 86 rats p21 expression was examined by Western blotting using a monoclonal antibody against p21 (NCC-RAS-004). H-ras mutations in exons 1 and 2 were examined by direct sequencing of DNA amplified by polymerase chain reaction. Sequencing results demonstrated mutations at codon 61 (CAA to CGA in 15 TCCs; CAA to CTA in 2 TCCs), at codon 12 (GGA to TGG in 1 TCC), and at codon 13 (GGC to GTC in 3 TCCs). Mutations at codon 61 were confirmed by faster mobility of the p21 band in Western blots. The level of p21 expression varied among samples, but many TCCs appeared to express more p21 than controls. The overall incidence of H-ras mutations was 24.4% (21 of 86 rats). The type of chemical used for the promoting phase had essentially no effect on H-ras mutation, suggesting that the effects observed were related to FANFT administration. The frequency of H-ras mutation in each group was negatively related to the incidence of carcinoma (r = -0.85; P less than 0.01). Two groups of tumors (with or without the mutated ras gene) were compared for tumor size (reflected by the bladder weight), histological grading, and the presence of invasion. The size of tumors with mutated ras was significantly smaller than those without mutated ras. There was no difference in the histological grading between the two groups. Although not statistically significant, histological invasion was more frequently observed in tumors with mutated ras (14.3%) than in tumors without mutation (3.1%).     

Immunohistochemical findings of nitric oxide synthase expression in urothelial transitional cell carcinoma including dysplasia

Several in vitro studies have shown that nitric oxide (NO) produced by NO synthase (NOS), such as inducible NOS (iNOS) play an important role in tumor biology. We immunohistochemically examined the expression of iNOS and p53 proteins in patients with transitional cell carcinoma (TCC) of the urinary tract, including adjacent dysplastic lesions to determine the significance of the tumor behavior. Of total 94 tumors, in the present study, 41 (43.6%) tumors exhibited homogeneous immunostaining (diffuse strong positivity in tumor cells, >60%) and 53 (56.4%) tumors heterogeneous staining (variable positivity in tumor cells, 20-60%). No TCCs exhibited negative iNOS immunostaining was found. Thirty (31.9%) of 94 TCCs were positive with anti-p53 antibody, including 23 of homogeneous and 7 of heterogeneous staining. Of 23 TCCs with homogeneous p53 immunostaining, 11 tumors exhibited homogeneous iNOS immunoreaction. In the present study, dysplastic lesions adjacent to carcinomas were detected in 64 cases including 36 TCCs with homogeneous iNOS expression. All dysplastic lesions adjacent to the 36 TCCs with homogeneous iNOS immunostaining exhibited homogeneous iNOS immunostaining. No significant association between iNOS immunoreactivity and any clinicopathological factors as well as p53 immuno-reactivity were found. These in vivo findings provide evidence for frequent iNOS protein expression in TCC. In addition, our observations indicate that overexpression of iNOS expression may be one of the early events in the carcinogenesis of TCC.     

Missense mutation of the hMSH6 and p53 genes in sporadic urothelial transitional cell carcinoma

Functional defects in DNA mismatch repair genes have been shown to be associated mainly with hereditary human malignancies. We examined genomic DNA from 88 sporadic transitional cell carcinomas (TCCs) of the urinary tract for mutations in hMSH6 gene by polymerase chain reaction and direct sequencing analysis. Mutational status of p53 gene was also studied as a potential target of genetic instability secondary to hMSH6 dysfunction. A total of 5 cases (5.7%: 5/88) displayed hMSH6 mutations all consisted of transition and located in exon 4, including three cases with missense mutation and two without change of corresponding amino acid. These three tumors with hMSH6 missense mutation had no microsatellite instability with five microsatellite markers tested. p53 gene mutations were detected in 22 cases (25.0%: 22/88). No tumors with p53 mutation had any hMSH6 missense mutations. Compared to the cases without hMSH6 alterations, the three patients with hMSH6 alterations had more frequent additional primary cancer (P<0.05). These findings provide the first in vivo evidence for the type of alterations and frequency of possible involvement of the hMSH6 mutations in sporadic type urothelial TCCs.     

Expression of thymidine phosphorylase as an indicator of poor prognosis for patients with transitional cell carcinoma of the bladder

BACKGROUND: Thymidine phosphorylase (TP), which is identical to platelet-derived endothelial cell growth factor (PD-ECGF), stimulates chemotaxis of endothelial cells and is involved in the angiogenesis of human solid tumors. METHODS: The activity and expression of TP were examined in human transitional cell carcinomas (TCCs) of the bladder, and their association with clinicopathologic findings was determined. The activity of the enzyme in 37 TCCs and 12 adjacent nonneoplastic tissues was measured spectrophotometrically. The expression of TP was also examined by immunoblotting. Immunohistochemical analysis was performed on 108 TCCs. RESULTS: TP activity in the carcinomas was higher than that in adjacent normal tissues (P = 0.002). TP activity in Grade 3 tumors or those classified as pT2-4 was higher than in Grade 1 and 2 tumors (P = 0.017) or those classified as pT1 (P = 0.007). The level of expression of TP detected by immunoblotting correlated well with TP activity. Immunohistochemical analyses showed that 62 of 108 cases (57.4%) were TP positive. There was a significant correlation between TP expression and histologic grade, infiltration pattern, local invasion, and lymph node metastasis. TP expression as a prognostic variable was studied using the Cox proportional hazards model. TP overexpression was an independent prognostic factor, as were lymph node metastasis and local invasion. CONCLUSIONS: These findings suggest that TP activity and its level of expression influence the progression of TCC and the prognoses of patients with this disease.     

Altered expression of beta-catenin in renal cell cancer and transitional cell cancer with the absence of beta-catenin gene mutations.

Loss of normal beta-catenin expression and the beta-catenin gene mutations have been shown to contribute to the malignant character of various cancers. Using PCR-single-strand conformation polymorphism and DNA direct sequencing, we examined the presence of genetic alterations within the third exon of beta-catenin, which are frequently observed in other tumors, in transitional cell cancer (TCC) and renal cell cancer (RCC) cell lines, and in tumor specimens. The degrees of expression and intracellular distribution of beta-catenin were detected by immunohistochemical staining in 77 primary and 12 metastatic RCCs and in 81 primary TCCs. Western blot analysis was also applied to confirm the degree of beta-catenin expression in the cell lines and some tumor samples. We failed to reveal any genetic alterations, at least in the third exon of the beta-catenin gene, in RCC and TCC. Reduced membranous immunoreactivity of beta-catenin was observed in portions of RCC (15.5%) and TCC (24.7%) and was correlated with advanced stages and nodal involvement in RCC and with advanced stages and multiple tumors in TCC. Within the power limitations of this small study, beta-catenin abnormal expression was not correlated with recurrence or survival in either RCC or TCC. Interstitial deletions and mutations in the third exon of beta-catenin do not play a significant role in RCC or TCC tumorigenesis. Down-regulation of normal beta-catenin expression might contribute to the malignant character of RCC and TCC and result in tumor progression. However, this event is not an independent prognostic factor for recurrence or tumor specific survival.     

粘着斑激酶在膀胱移行细胞癌中表达的意义

目的 :探讨粘着斑激酶 (FAK)在膀胱移行细胞癌 (TCC)中的表达与肿瘤分化、浸润的关系 ,以及与p53、bcl 2、ki 67表达的关系。方法 :采用免疫组织化学方法 ,测定FAK在 81例膀胱TCC中的表达情况。结果 :FAK在膀胱癌组织中的表达明显高于癌旁黏膜 ,浸润性膀胱癌明显高于非浸润性 ,p53或ki 67阳性膀胱癌明显高于阴性组。结论 :FAK可能是一种肿瘤转化相关酶 ,又是一种肿瘤演变相关酶 ;它的表达量可作为膀胱癌发生发展过程中有价值的指标。     

Expression of matrix metalloproteinases in human transitional cell carcinoma of the urinary bladder

Matrix metalloproteinases (MMPs) are associated with invasion and metastasis of several human malignant tumors. Especially so MMP-7, as it is mainly produced by the actual cancer cells. However, the expression of MMP-7 in transitional cell carcinoma (TCC) of the urinary bladder has not been previously investigated. We examined expressions of MMP-7 and MMP-2 in TCC, and compared them with clinicopathological characteristics in TCCs. Tumor samples from 20 patients with TCC of the urinary bladder who had surgery performed at the Osaka City University Medical School Hospital were immunohistochemically stained. Expression of MMP-2 was significantly stronger in advanced stage and high grade tumors than in low stage and low grade tumors. MMP-7 was also expressed in TCC, having a tendency to be more strongly expressed in high than in low grade tumors. However, there was no significant difference of MMP-7 expression between advanced and low stages. This increased expression of MMP-7 in high grade TCC of the urinary bladder may be associated with tumor invasion and metastasis.     

Relation between cyclooxygenase-2 expression and tumor invasiveness and patient survival in transitional cell carcinoma of the urinary bladder.

BACKGROUND: Expression of the inducible form of cyclooxygenase (COX)-2 is known to correlate with development of transitional cell carcinoma (TCC) of the human urinary bladder. However, the clinical significance of COX-2 expression with respect to clinicopathologic findings and patient survival is unknown. METHODS: COX-2 expression was examined immunohistochemically in tumor tissues obtained from 108 patients who underwent radical cystectomy for TCCs, without knowledge of clinicopathologic findings. Correlation between COX-2 expression and clinicopathologic findings and patient survival was determined. RESULTS: COX-2 expression was detected in 34 of 108 (31%) tumors but in none of 10 normal uroepithelial samples. Univariate logistic regression analysis showed a significant correlation between COX-2 expression and local invasion, infiltration pattern, lymphatic invasion, and venous invasion. However, multivariate logistic regression analysis revealed that only local invasion correlated significantly with COX-2 expression (P = 0.047). Cox proportional hazards regression analysis showed that both local invasion (P = 0.008) and lymph node metastasis (P = 0.001) were independent prognostic factors; however, COX-2 expression (P = 0.16) was not. CONCLUSIONS: The authors showed that COX-2 overexpression plays a role in development and invasion of TCCs, but not prognosis of patients with TCC. COX-2 inhibitors may be useful for chemoprevention of TCCs and treatment of invasive disease.     

Mutations of hMLH1 and hMSH2 in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with microsatellite instability and abnormalities of mismatch repair protein expression.

PURPOSE: The relationship between germ-line mutations of hMSH2 and hMLH1, microsatellite instability (MSI), and loss of DNA mismatch repair (MMR) gene expression were studied to formulate an effective selection protocol for patients with suspected hereditary nonpolyposis colorectal cancer who should be offered genetic testing. PATIENTS AND METHODS: Patients eligible for germ-line analysis of hMLH1 and hMSH2 were selected. Tumor specimens were obtained to assess MSI and loss of MMR gene expression. RESULTS: Among 37 patients who participated in the study, two hMSH2 and two hMLH1 missense mutations (11%) were detected, none of which was found in a panel of 60 healthy volunteers. High MSI was found in five tumors (19%) and low MSI in 10 tumors (39%); 12 tumors (46%) were microsatellite stable. Four tumors demonstrated loss of hMLH1, and three tumors demonstrated loss of hMSH2 protein expression. CONCLUSION: No relationship was found between MMR gene mutations and MSI; low or no MSI was found in the four patients with germ-line mutations, and none of the five patients with high MSI demonstrated abnormalities of MMR genes. On the contrary, loss of hMLH1 or hMSH2 expression was found in the tumors from three of the four patients demonstrating germ-line mutations. These data suggest that germ-line mutations of the MMR gene can occur in people with MSI-negative tumors. Sensitive clinical criteria and the study of MMR gene expression may be useful to identify this subset of patients.     

环氧化酶-2 在膀胱癌组织及尿脱落细胞中的表达和意义

 目的 探讨COX-2在膀胱癌组织中的表达，了解尿脱落细胞COX-2表达在膀胱癌早期诊断中的价值。方法 应用免疫组化技术检测48例膀胱移行细胞癌组织、免疫细胞化学技术检测40例膀胱移行细胞癌患者和30例非肿瘤患者尿脱落细胞COX-2的表达。结果 膀胱移行细胞癌组织COX-2阳性表达率为72．9％，对照组正常膀胱黏膜无表达。COX-2的表达与膀胱癌临床分期显著相关(P<0．05)，不同病理分级膀胱癌的表达差别无显著性意义。非肿瘤患者尿脱落细胞无COx-2表达，膀胱癌尿脱落细胞COX-2免疫细胞化学检测阳性率为67．5％，明显高于常规尿细胞学的37．5％(P<0．05)，尤其对于G1级和Ta～T1期的低级、早期肿瘤，尿脱落细胞COX-2免疫细胞化学检测与常规尿细胞学检查相比，具有显著性意义(P<0．05)。结论 COX-2在膀胱移行细胞癌的发生发展中起重要作用，与肿瘤的浸润、转移相关。尿脱落细胞COX-2表达检测特异性高，可作为早期诊断膀胱癌的一种标志物。     

Tissue factor expression correlates with disease-specific survival in patients with node-negative muscle-invasive bladder cancer

Tissue factor (TF), a transmembrane glycoprotein responsible for initiating the extrinsic pathway of blood coagulation plays a key role in cancer growth, metastasis and angiogenesis. Various studies have demonstrated the prognostic potential of TF expression in several cancers. However, its role in bladder cancer is unclear. This study evaluated the prognostic potential of TF expression in muscle-invasive bladder tumors from patients treated with radical cystectomies. Immunohistochemical staining using a monoclonal antibody (mAb) anti-TF was carried out on sections of tissue microarray blocks containing cores of muscle-invasive bladder tumors (4 cores/tumor) from 218 patients. The intensity of the staining was evaluated on a scale from 0 to 3 by two independent observers who were both unaware of the clinicopathological characteristics of the samples. TF was expressed in 77.6% of tumors, independently from baseline characteristics (age, gender, stage and grade) as assessed using the chi(2) and Student t tests. During follow-up (median: 2.6 years), 45.4% of the patients died from the progression of their cancer. Kaplan-Meier survival showed that among the 103 patients with node-negative (N0) transitional cell carcinoma (TCC), those with TF-positive tumors had shorter bladder cancer-specific survival (p = 0.0276). Moreover, multivariate Cox regression analysis showed they had a 3.15-fold greater risk of dying from bladder cancer (95% CI: 1.1-9.0; p = 0.032). In conclusion, TF expression was an independent predictor of disease-specific survival in N0 muscle-invasive TCCs treated by radical cystectomy and therefore, might help identify patients at higher risk of disease progression. These patients could potentially benefit from adjuvant chemotherapy.     

Clinical implications of the expression of epidermal growth factor receptors in human transitional cell carcinoma.

To evaluate the distribution and density of epidermal growth factor (EGF) receptors (EGF-Rs) on urothelium, immunohistological studies using a monoclonal antibody to the binding portion of the human EGF-R were performed on frozen specimens of normal urothelium (N = 20), urothelium from patients with nonurothelial urological malignancies (N = 15) and inflammatory diseases (N = 8), low grade superficial transitional cell carcinomas (TCC) (N = 13), high grade superficial or invasive TCC (N = 28), and endoscopically normal appearing urothelium from patients with low grade superficial (N = 5) or high grade (N = 21) TCC elsewhere in the bladder (or ipsilateral renal pelvis/ureter). EGF-Rs are found only on the basal layer of epithelial cells (with scattered representation on intermediate cells) in 95% of normal urothelial specimens and 100% of pathological specimens without urothelial malignancy. Alternatively, 92.3% of specimens of low grade superficial TCC and 100% of high grade TCCs had EGF-Rs richly expressed on the superficial as well as the deeper layers of urothelium. This "malignant" distribution of EGF-Rs was also found on all specimens of endoscopically normal appearing urothelium in patients with TCC elsewhere. The density of EGF-Rs correlated closely with tumor grade on both "premalignant" and frankly neoplastic urothelium. We conclude that the expression of EGF-Rs on urothelium favors the interaction of premalignant and malignant tissue with urinary EGF. To determine if altering the physiochemical environment of urine could interfere with this interaction, the effects of pH on the binding of and growth responses to EGF were assessed on four human TCC cell lines. Scatchard plots demonstrated that varying pH from 5.0 to 7.5 did not significantly change the total number of receptors, but EGF-R affinity was reduced approximately 20-fold as pH decreased from 7.5 to 5 in each TCC target. Similarly, significant growth stimulation by EGF at pH 7.5 was abrogated at pH less than or equal to 7.0 while growth rates in the absence of EGF remained unchanged at lower pHs. It thus appears that urinary acidification may hold promise in the management and prevention of recurrent bladder cancer.