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1.  Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule 
The Journal of Cell Biology  1994;125(2):437-446.
The epithelial glycoprotein 40 (EGP40, also known as GA733-2, ESA, KSA, and the 17-1A antigen), encoded by the GA-733-2 gene, is expressed on the baso-lateral cell surface in most human simple epithelia. The protein is also expressed in the vast majority of carcinomas and has attracted attention as a tumor marker. The function of the protein is unknown. We demonstrate here that EGP40 is an epithelium-specific intercellular adhesion molecule. The molecule mediates, in a Ca(2+)- independent manner, a homophilic cell-cell adhesion of murine cells transfected with the complete EGP40 cDNA. Two murine cell lines were tested for the effects of EGP40 expression: fibroblastic L cells and dedifferentiated mammary carcinoma L153S cells. The expression of the EGP40 protein causes morphological changes in cultures of transfected cells--increasing intercellular adhesion of the transfectants--and has a clear effect on cell aggregating behavior in suspension aggregation assays. EGP40 directs sorting in mixed cell populations, in particular, causes segregation of the transfectants from the corresponding parental cells. EGP40 expression suppresses invasive colony growth of L cells in EHS-matrigel providing tight adhesions between cells in growing colonies. EGP40 can thus be considered a new member of the intercellular adhesion molecules. In its biological behavior EGP40 resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs), although no immunoglobulin-like repeats are present in the EGP40 molecule. Certain structural similarities in general organization of the molecule exist between EGP40 and the lin-12/Notch proteins. A possible role of this adhesion molecule in formation of architecture of epithelial tissues is discussed. To reflect the function of the molecule the name Ep-CAM for EGP40 seems appropriate.
PMCID: PMC2120036  PMID: 8163559
2.  Invasive front of colorectal cancer: Dynamic interface of pro-/anti-tumor factors 
Tumor-host interaction at the invasive front of colorectal cancer represents a critical interface encompassing a dynamic process of de-differentiation of colorectal carcinoma cells known as epithelial mesenchymal transition (EMT). EMT can be identified histologically by the presence of “tumor budding”, a feature which can be highly specific for tumors showing an infiltrating tumor growth pattern. Importantly, tumor budding and tumor border configuration have generated considerable interest as additional prognostic factors and are also recognized as such by the International Union Against Cancer. Evidence seems to suggest that the presence of tumor budding or an infiltrating growth pattern is inversely correlated with the presence of immune and inflammatory responses at the invasive tumor front. In fact, several tumor-associated antigens such as CD3, CD4, CD8, CD20, Granzyme B, FOXP3 and other immunological or inflammatory cell types have been identified as potentially prognostic in patients with this disease. Evidence seems to suggest that the balance between pro-tumor (including budding and infiltrating growth pattern) and anti-tumor (immune response or certain inflammatory cell types) factors at the invasive front of colorectal cancer may be decisive in determining tumor progression and the clinical outcome of patients with colorectal cancer. On one hand, the infiltrating tumor border configuration and tumor budding promote progression and dissemination of tumor cells by penetrating the vascular and lymphatic vessels. On the other, the host attempts to fend off this attack by mounting an immune response to protect vascular and lymphatic channels from invasion by tumor buds. Whereas standard pathology reporting of breast and prostate cancer involves additional prognostic features, such as the BRE and Gleason scores, the ratio of pro- and anti-tumor factors could be a promising approach for the future development of a prognostic score for patients with colorectal cancer which could complement tumor node metastasis staging to improve the clinical management of patients with this disease.
PMCID: PMC2795176  PMID: 20014453
Colorectal cancer; Prognosis; Tumor invasive front; Tumor budding; Tumor growth pattern; Tumor infiltrating lymphocytes; Tumor immunity; Microsatellite instability
3.  High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression 
BMC Cancer  2009;9:385.
Egr-1 (early growth response-1 transcription factor) has been proposed to be involved in invasion and metastasis processes of human bladder cancer, but Egr-1 protein expression levels in human bladder cancer have not been investigated. In the present study we investigated the expression levels of Egr-1 protein in early stages of human bladder cancer and correlated it to later progression.
Expression of Egr-1 protein in human bladder cancer was examined by immunohistochemistry, on a tissue microarray constructed from tumors from 289 patients with non-muscle invasive urothelial bladder cancer.
The frequency of tumor cells with nuclear Egr-1 immunolabelling correlated to bladder cancer stage, grade and to later progression to muscle-invasive bladder cancer (T2-4). Stage T1 tumors exhibited significantly higher frequencies of tumor cells with nuclear Egr-1 immunolabelling than Ta tumors (P = 0.001). Furthermore, Kaplan-Meier survival analysis showed that a high frequency of tumor cells with nuclear Egr-1 immunolabelling was significantly associated with a higher risk of progression to stage T2-4 (log-rank test, P = 0.035). Tumor cells with nuclear Egr-1 immunolabelling were found to localize at the tumor front in some of the tumor biopsies.
The results from this study support a potential involvement of Egr-1 in the progression from non-muscle invasive bladder cancers to muscle invasive bladder cancer.
PMCID: PMC2774864  PMID: 19878561
4.  Long-term prognostic value of the combination of EORTC risk group calculator and molecular markers in non-muscle-invasive bladder cancer patients treated with intravesical Bacille Calmette-Guérin 
Urology Annals  2011;3(3):119-126.
Background and Objectives:
To evaluate the long-term prognostic value of the combination of the EORTC risk calculator and proapoptotic, antiapoptotic, proliferation, and invasiveness molecular markers in predicting the outcome of intermediate- and high-risk non-muscle-invasive bladder cancer (NMIBC) treated with intravesical Bacille Calmette-Guérin (BCG) therapy.
Materials and Methods:
This study included 42 patients accrued prospectively presenting with intermediate- to high-risk NMIBC (high-grade T1 tumors or multiple rapidly recurrent tumors refractory to intravesical chemotherapy) treated with transurethral resection (TUR) and BCG. TUR samples were analyzed for the molecular markers p53, p21 waf1/cip, Bcl-2, CyclinD1, and metallothionein 9 (MMP9) using immunohistochemistry. Frequency of positivity, measured as a percentage, was assessed alone or in combination with EORTC risk calculator, for interaction with outcome in terms of recurrence and progression using univariate analysis and Kaplan-Meier survival curves.
Median follow-up was 88 months (mean, 99; range, 14-212 months). The overall recurrence rate was 61.9% and progression rate was 21.4%. In univariate analysis, CyclinD1 and EORTC risk groups were significantly associated with recurrence (P value 0.03 and 0.02, respectively), although none of the markers showed a correlation to progression. In combining EORTC risk groups to markers expression status, high-risk group associated with positive MMP9, Bcl-2, CyclinD1, or p21 was significantly correlated to tumor recurrence (log rank P values <0.001, 0.03, 0.02, and 0.006, respectively) and when associated with positive MMP9 or p21, it was significantly correlated to progression (log rank P values 0.01 and 0.04, respectively).
Molecular markers have a long-term prognostic value when combined with EORTC scoring system and they may be used to improve the predictive accuracy of currently existing scoring system. Larger series are needed to confirm these findings.
PMCID: PMC3183702  PMID: 21976923
BCG; Bcl-2; bladder cancer; cyclinD1; EORTC; high risk; long-term response; metallothionein 9; p53; p21; waf1/cip
5.  Immunohistochemical expression of EGP40, a tumor marker, in different grades of oral squamous cell carcinoma 
The aim of this study was to see the distribution and pattern of staining of epithelial glycoprotein 40 EGP-40 (also known as GA733-2, ESA, KSA, 17-1A antigen) in the different grades of OSCC.
Materials and Methods:
30 biopsy reports retrieved from the files of the Department of Oral Pathology and Microbiology, College of Dental Surgery, Manipal were used. These comprised of 10 microslides each of 3 different histological grades of Oral Squamous Cell Carcinoma, namely Well Differentiated, Moderately Differentiated and Poorly Differentiated carcinomas. Immunoperoxidase staining for IgG, was performed by the unlabelled antibody peroxidase-antiperoxidase complex (PAP) method. The criteria used to define an antigen positive area were: Homogenous /Patchy staining of the section and Cytoplasmic/ Membranous staining of the tumor cells.
The expression of EGP 40 in different grades of OSCC showed an inverse relationship to differentiation and a direct relationship with the proliferation of the tumor cells and its expression became more pronounced as the grade worsened i.e. from well to poorly differentiated OSCC.
In the present study, the surface antigen EGP40 (Ep-CAM) was detected in the different grades of OSCC with its expression becoming more pronounced as the grade worsened i.e. from well to poorly differentiated OSCC. However, further studies are required to understand the dualistic role of EGP40 (Ep-CAM) in mediating cell to cell adhesion preventing cell scattering and its heterogeneous expression in promoting tumor invasion and metastasis and also to determine its exact role and significance at a practically applicable level.
PMCID: PMC3125653  PMID: 21731275
EGP40; ESA; GA733-2; KSA; OSCC; 17-1A
6.  Low molecular weight cyclin E is associated with p27-resistant, high-grade, high-stage and invasive bladder cancer 
Cell Cycle  2012;11(7):1468-1476.
Expression of low molecular weight (LMW) isoforms of cyclin E is a strong predictor of poor outcome in patients with breast cancer. The purpose of this study was to examine the expression of full-length and LMW cyclin E in bladder cancer cell lines and patient tumors. We used western blotting, immunoprecipitation and kinase assays to examine the expression and activity of key cell cycle-regulatory proteins in various human bladder cell lines, both tumorigenic and non-tumorigenic. We also analyzed cyclin E expression, kinase activity and immune complex binding partners in 43 tissue samples from grade 2 and 3 transitional cell carcinomas. Cyclin E was overexpressed and LMW isoforms were present only in bladder cancer cells. Overexpression of LMW isoforms of cyclin E and increased cyclin E kinase activity were both significantly associated with tumorigenicity of the bladder cell lines (p = 0.005 and 0.022, respectively). Binding of the cyclin-dependent kinase inhibitors p21 and p27 to LMW cyclin E did not inhibit the kinase activity of cyclin E and cyclin-dependent kinase 2 in primary tumor samples overexpressing LMW cyclin E. Full-length and LMW cyclin E were significantly overexpressed in grade 3 tumors compared with grade 2 tumors (p = 0.004). Finally, LMW cyclin E levels were significantly associated with a non-papillary growth pattern (p = 0.031) and invasiveness (p = 0.021) of the bladder tumors and poor overall survival (p = 0.06). These results suggest that LMW cyclin E can be used as a new prognostic marker for bladder cancer.
PMCID: PMC3350882  PMID: 22441703
cyclin E; p27; Cdk2 kinase; bladder cancer; cell cycle
7.  Estrogen receptor-β expression and pharmacological targeting in bladder cancer 
Oncology Reports  2013;30(1):131-138.
A role for estrogen signaling in urothelial carcinoma of the bladder (UCB) is suggested to be associated with more advanced disease with worse outcomes in women. Estrogen receptor β (ERβ) is the predominant receptor in bladder tissues. We aimed to ascertain whether ERβ correlates with clinicopathological predictors of aggressive bladder cancer and worse survival outcomes. ERβ was measured by immunohistochemistry in malignant and adjacent benign bladder tissues in patients (N=72) with UCB who underwent radical cystectomy. ERβ expression was tested for statistical association with clinicopathological variables and patient survival. ERβ expression was determined in bladder cancer cell lines, and the effects of the selective estrogen modulator tamoxifen and the ERβ agonist diarylpropionitrile on cell growth were determined. The ERβ level was significantly higher in malignant vs. benign urothelium (P<0.001) and was strongly associated with aggressive tumor histology characterized by lymphovascular (P=0.008) and perineural (P=0.006) invasion, and clinical histories of pelvic irradiation (P=0.005), hydronephrosis (P=0.022) and no intravesical chemotherapy (P=0.038). All patients with a high (>70%) percentage of ERβ positivity in tissue with >3-month follow-up developed recurrent disease (P=0.009). Higher ERβ level was predictive of worse recurrence-free and overall survival following cystectomy, after adjustment for tumor stage, and remained significantly associated with recurrence-free survival in the multivariable analysis including tumor stage, nodal stage and lymphovascular invasion. Activation of ERβ in bladder cancer cell lines led to significant increases in proliferation, while pharmacological inhibition with tamoxifen blocked cell growth. Our study supports a role for ERβ in aggressive UCB. Pharmacological targeting of ERβ warrants further investigation as a therapeutic strategy in UCB.
PMCID: PMC3729232  PMID: 23612777
epigenetic; hormonal; urothelial; transitional cell
8.  The Management of Non-Invasive Bladder Tumours with Doxorubicin Intravesical Instillation after Transurethral Resection 
Evaluation of the intravesical instillation of doxorubicin for its effect on disease recurrence for patients with non-invasive bladder tumour.
The study was performed at Al Assad University Hospital in Lattakia, Syria and included patients with non-invasive bladder tumours who were managed with transurethral resection and induction and maintenance therapy with intravesical doxorubicin. They were followed up by cystoscopy every 3 months for 2 years and every 6 months thereafter with special emphasis on recurrence rates.
The study included 85 patients with non-invasive bladder tumours: 23 with non-invasive papillary carcinoma (Stage Ta), 62 with tumour invading subepithelial connective tissue (Stage T1). Twelve patients had well differentiated tumours (Grade 1), 48 had moderately differentiated (Grade 2), 25 had poorly differentiated (Grade 3) tumours. The total recurrence rate was 23%. The rates of recurrence were 56% in Grade 3 and 0% in Grade 1. The recurrence rate was 41% in patients with large tumours versus 17% in those with small tumours; 44% in those with multiple tumours compared to 18% in those with solitary tumours; 30% of Stage Ta tumours recurred and 21% of Stage T1 tumours.
In short term follow-up, our rate of recurrence was 23%. Adjuvant intravesical doxorubicin was shown to reduce the recurrence of superficial bladder cancer. Tumour grade, size and number were shown to be prognostic factors for recurrence.
PMCID: PMC3074761  PMID: 21509275
Intravesical Instillations; Doxorubicin; Non-Invasive bladder cancer
9.  The Impact of the Receptor of Hyaluronan-Mediated Motility (RHAMM) on Human Urothelial Transitional Cell Cancer of the Bladder 
PLoS ONE  2013;8(9):e75681.
Hyaluronan (HA) is a carbohydrate of the extracellular matrix with tumor promoting effects in a variety of cancers. The present study addressed the role of HA matrix for progression and prognosis of human bladder cancer by studying the expression and function of HA-related genes.
Tissue samples of 120 patients with different stages of transitional cell bladder cancer, who underwent surgical treatment for bladder cancer at the University Hospital of Essen were analysed. mRNA-expression levels of HA synthases (HAS1-3) and HA-receptors (RHAMM and CD44) were evaluated by real time RT-PCR in comparison to healthy bladder tissue as control. In uni- and multivariate cox proportional hazard survival regression analysis, the impact of the gene expression levels on survival was assessed. In vitro knock-down of RHAMM, CD44 and HAS isoenzymes was achieved by siRNA and lentiviral shRNA in J82 bladder cancer cells. Transfected cells were analysed in vitro with regard to proliferation, cell cycle and apoptosis. J82 cells after knock-down of RHAMM were xenografted into male nu/nu athymic mice to monitor tumor progression in vivo.
In invasive tumor stages RHAMM-, HAS1 and HAS2 mRNA-expression levels were elevated whereas HAS3v1 was reduced as compared to non-invasive tumors. Subsequently, Kaplan-Meier analysis revealed reduced bladder cancer specific survival in patients with high RHAMM mRNA and low HAS3v1 expression. Elevated RHAMM in invasive tumors was confirmed by RHAMM immunohistochemistry. Furthermore, multivariate analysis revealed that only RHAMM expression was associated with poor prognosis independent from other survival factors (HR=2.389, 95% CI 1.227-4.651, p=0.01). Lentiviral RHAMM knock-down revealed reduced J82 cell proliferation in vitro and reduced xenograft tumor growth in vivo.
The data suggest that RHAMM plays a crucial role in mediating progression of muscle-invasive bladder cancer and recommends RHAMM for further evaluation as a prognostic marker or therapeutic target in bladder cancer therapy.
PMCID: PMC3775893  PMID: 24069434
10.  Dual Peroxisome Proliferator–Activated Receptor α/δ Agonist GFT505 Improves Hepatic and Peripheral Insulin Sensitivity in Abdominally Obese Subjects 
Diabetes Care  2013;36(10):2923-2930.
The development of new insulin sensitizers is an unmet need for the treatment of type 2 diabetes. We investigated the effect of GFT505, a dual peroxisome proliferator–activated receptor (PPAR)-α/δ agonist, on peripheral and hepatic insulin sensitivity.
Twenty-two abdominally obese insulin-resistant males (homeostasis model assessment of insulin resistance >3) were randomly assigned in a randomized crossover study to subsequent 8-week treatment periods with GFT505 (80 mg/day) or placebo, followed by a two-step hyperinsulinemic-euglycemic insulin clamp with a glucose tracer to calculate endogenous glucose production (EGP). The primary end point was the improvement in glucose infusion rate (GIR). Gene expression analysis was performed on skeletal muscle biopsy specimens.
GFT505 improved peripheral insulin sensitivity, with a 21% (P = 0.048) increase of the GIR at the second insulin infusion period. GFT505 also enhanced hepatic insulin sensitivity, with a 44% (P = 0.006) increase of insulin suppression of EGP at the first insulin infusion period. Insulin-suppressed plasma free fatty acid concentrations were significantly reduced on GFT505 treatment (0.21 ± 0.07 vs. 0.27 ± 0.11 mmol/L; P = 0.006). Neither PPARα nor PPARδ target genes were induced in skeletal muscle, suggesting a liver-targeted action of GFT505. GFT505 significantly reduced fasting plasma triglycerides (−21%; P = 0.003) and LDL cholesterol (−13%; P = 0.0006), as well as liver enzyme concentrations (γ-glutamyltranspeptidase: −30.4%, P = 0.003; alanine aminotransferase: −20.5%, P = 0.004). There was no safety concern or any indication of PPARγ activation with GFT505.
The dual PPARα/δ agonist GFT505 is a liver-targeted insulin-sensitizer that is a promising drug candidate for the treatment of type 2 diabetes and nonalcoholic fatty liver disease.
PMCID: PMC3781493  PMID: 23715754
11.  Predictors of Intravesical Therapy Use in Non-Muscle Invasive Bladder Cancer: Results from the Surveillance, Epidemiology, And End-Results Program’s 2003 Patterns of Care Project 
The Journal of Urology  2008;180(2):520-524.
In response to variations in cancer care, organizations have developed clinical guidelines. In the case of non-muscle invasive bladder cancer (also known as superficial bladder cancer), two similar sets of guidelines were released in the late 1990’s that provide care recommendations. The purpose of this study was to examine patterns of intravesical therapy utilization in non-muscle invasive bladder cancer in 2003, to determine if disparities in the quality of cancer care remained.
Data from the Surveillance, Epidemiology, and End-Results (SEER) Program’s 2003 bladder cancer patterns of care project were used. Subjects newly diagnosed with non-muscle invasive bladder cancer in 2003 were included. Clinical and sociodemographic data were obtained from the SEER program and detailed medical record review. Statistical analyses were performed to identify independent predictors of intravesical therapy use in the entire cohort and in a subset of high-risk patients.
685 patients were included in the study. 216 (31.5%) patients received intravesical therapy. In addition to higher tumor stage and grade, intravesical therapy use was independently associated with race/ethnicity and geographic region. In the subset of 350 high-risk patients, 42% received intravesical therapy. Stage, grade, race/ethnicity and geographic region were independently associated with intravesical therapy use in this sub-cohort.
These data suggest there is underutilization of intravesical therapy, even in patients with high-risk non-muscle invasive bladder cancer, and disparities in the quality of care exist. Barriers to utilization of this cancer treatment must be identified, particularly in higher-risk individuals, and providers must become more aware of existing clinical guidelines.
PMCID: PMC3327445  PMID: 18550088
non-muscle invasive bladder cancer; intravesical therapy; disparities; quality of care; clinical guidelines
12.  Role of the VEGF ligand to receptor ratio in the progression of mismatch repair-proficient colorectal cancer 
BMC Cancer  2010;10:93.
The VEGF family of ligands and receptors are intimately involved in tumor angiogenesis, lymphangiogenesis and metastasis. The evaluation of VEGF ligand/receptor ratios may provide a more profound understanding of the involvement of these proteins in colorectal tumour progression. The aim of this study was to elucidate the role of the VEGF ligand/receptor ratios on tumour progression and metastasis in patients with mismatch repair-proficient colorectal cancer.
Immunohistochemistry for VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2 and VEGF3 was carried out on 387 mismatch repair-proficient colorectal cancers using a tissue microarray. Evaluation of immunoreactivity was performed semi-quantitatively and the ligand/receptor expression ratio was obtained.
An increased VEGF-A/VEGFR1 ratio, VEGF-A and VEGFR1 was linked to the presence of peritumoral lymphocytic inflammation at the invasive front (p = 0.032; p = 0.005; p = 0.032, respectively). VEGFR1 expression was related to poorer outcome in multivariable analysis with pT stage, pN stage, vascular invasion, and post-operative therapy. A higher ratio of VEGF-A/VEGFR2 was linked to advanced TNM stage (p = 0.005) while VEGF-A and VEGFR2 were elevated in tumours with an infiltrating tumour growth pattern (p = 0.006; p = 0.014; p = 0.006). No effect of VEGF-A/VEGFR2, VEGF-A or VEGFR2 on survival time was noted.
Our findings highlight an involvement of VEGF-A, VEGR1 and VEGFR2 in events occurring at the invasive tumour front and a potential prognostic role of VEGFR1 expression in mismatch repair-proficient colorectal cancers. The VEGF-A ligand to VEGFR1 or VEGFR2 ratio may represent an alternative evaluation system for identifying patients with poorer clinical outcome.
PMCID: PMC2841667  PMID: 20222950
13.  Correlative studies on uPA mRNA and uPAR mRNA expression with vascular endothelial growth factor, microvessel density, progression and survival time of patients with gastric cancer 
AIM: To investigate the correlations between the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and vascular endothelial growth factor (VEGF) protein and clinicopathologic features, microvessel density (MVD) and survival time.
METHODS: In situ hybridization and immuno-histochemistry techniques were used to study the expressions of uPA mRNA, uPAR mRNA, VEGF and CD34 protein in 105 gastric carcinoma specimens.
RESULTS: Expressions of uPA mRNA, uPAR mRNA and VEGF protein were observed in 61 (58.1%) cases, 70 (66.7%) cases and 67 (63.8%) cases, respectively. The uPA mRNA and uPAR mRNA positive expression rates in infiltrating-type cases (73.7%, 75.4%), stage III-IV (72.1%, 75.4%), vessel invasion (63.2%, 69.9%), lymphatic metastasis (67.1%, 74.4%) and distant metastasis (88.1%, 85.7%) were significantly higher than those of the expanding-type (χ2 = 15.57, P = 0.001; χ2 = 6.91, P = 0.046), stage I-II (χ2 = 19.22, P = 0.001; χ2 = 16.75, P = 0.001), non-vessel invasion (χ2 = 11.92, P = 0.006; χ2 = 14.15, P = 0.002), non-lymphatic metastasis (χ2 = 28.41, P = 0.001; χ2 = 22.5, P = 0.005) and non-distant metastasis (χ2 = 12.32, P = 0.004; χ2 = 17.42, P = 0.002; χ2 = 11.25, P = 0.012; χ2 = 18.12, P = 0.002).The VEGF positive expression rates in infiltrating-type cases (75.4%), stage III-IV (88.5%), vessel invasion (82.9%), lymphatic metastasis (84.3%) and distant metastasis (95.2%) were significantly higher than those of the expanding-type (χ2 = 9.61, P = 0.021), stage I-II (χ2 = 16.66, P = 0.001), non-vessel invasion (χ2 = 29.38, P = 0.001), non-lymphatic metastasis (χ2 = 18.68, P = 0.005), and non-distant metastasis (χ2 = 22.72, P = 0.007; χ2 = 21.62, P = 0.004). The mean MVD in the specimens positive for the uPA mRNA, uPAR mRNA and VEGF protein was markedly higher than those with negative expression groups. Moreover, a positive relation between MVD and uPA mRNA (rs = 0.199, P = 0.042), uPAR mRNA (rs = 0.278, P = 0.035), and VEGF (rs = 0.398, P = 0.048) expressions was observed. The mean survival time in cases with positive uPA mRNA, uPAR mRNA and VEGF protein expression or MVD value ≥ 54.9 was significantly shorter than those in cases with negative expression or MVD value < 54.9.
CONCLUSION: uPA and uPAR expressions are correlated with enhanced VEGF-induced tumor angiogenesis and may play a role in invasion and nodal metastasis of gastric carcinoma, thereby serving as prognostic markers of gastric cancer.
PMCID: PMC4087704  PMID: 16810742
Stomach neoplasm; Urokinase-type plasminogen activator; Urokinase-type plasminogen activator receptor; Vascular endothelial growth factor; Prognosis
14.  CD8+ lymphocyte infiltration is an independent favorable prognostic indicator in basal-like breast cancer 
Tumor infiltrating lymphocytes may indicate an immune response to cancer development, but their significance remains controversial in breast cancer. We conducted this study to assess CD8+ (cytotoxic T) lymphocyte infiltration in a large cohort of invasive early stage breast cancers, and to evaluate its prognostic effect in different breast cancer intrinsic subtypes.
Immunohistochemistry for CD8 staining was performed on tissue microarrays from 3992 breast cancer patients. CD8+ tumor infiltrating lymphocytes were counted as intratumoral when in direct contact with tumor cells, and as stromal in adjacent locations. Kaplan-Meier functions and Cox proportional hazards regression models were applied to examine the associations between tumor infiltrating lymphocytes and breast cancer specific survival.
Among 3403 cases for which immunohistochemical results were obtained, CD8+ tumor infiltrating lymphocytes were identified in an intratumoral pattern in 32% and stromal pattern in 61% of the cases. In the whole cohort, the presence of intratumoral tumor-infiltrating lymphocytes was significantly correlated with young age, high grade, estrogen receptor negativity, human epidermal growth factor receptor-2 positivity and core basal intrinsic subtype, and was associated with superior breast cancer specific survival. Multivariate analysis indicated that the favorable prognostic effect of CD8+ tumor infiltrating lymphocytes was significant only in the core basal intrinsic subgroup (Hazard ratio, HR = 0.35, 95% CI = 0.23-0.54). No association with improved survival was present in those triple negative breast cancers that lack expression of basal markers (HR = 0.99, 95% CI = 0.48-2.04) nor in the other intrinsic subtypes.
CD8+ tumor infiltrating lymphocytes are an independent prognostic factor associated with better patient survival in basal-like breast cancer, but not in non-basal triple negative breast cancers nor in other intrinsic molecular subtypes.
PMCID: PMC3446382  PMID: 22420471
15.  Cancer Immunoediting from Immunosurveillance to Tumor Escape in Microvillus-Formed Niche: A Study of Syngeneic Orthotopic Rat Bladder Cancer Model in Comparison with Human Bladder Cancer1 
Neoplasia (New York, N.Y.)  2010;12(6):434-442.
Cancer cells can develop an attenuated immunogenicity and/or create an immunosuppressive microenvironment to prevent tumor eradication by host immune system, the so-called “cancer immunoediting” hypothesis. The aim of the present study was to find evidence for this hypothesis by using a rat orthotopic bladder cancer model. Fisher rats were inoculated with AY-27 cells (a Fisher rat bladder cancer cell line). Cultured cancer cells, rat and human bladder cancer tissues, and publicly available microarray data from human bladder cancer were analyzed by means of bioinformatics and morphology. Results showed that 12 of 24 differentially expressed pathways were concordant in connection to cell cycle and proliferation between rats and humans (both non-muscle-invasive and muscle-invasive tumors) and that 11 of the 24 pathways, including major histocompatibility complex, were related to host immunosurveillance with activations of T cells and natural killer cells in rats. The altered pathways and morphogenesis of this rat model corresponded more closely with those of human muscle-invasive rather than non-muscle-invasive tumors. A unique ultrastructure displaying microvillus-formed niches was found in small areas within the tumor of both rats and humans. These niches were interconnected with desmosomes between cancer cells and without infiltration of lymphocytes. The expression of E-cadherin, selectins, PGP9.5, vascular endothelial growth factor, caspase-3, CD133, Oct-4, nestin, CD3, and CD45RA was lower in the tumor than in the adjacent normal epithelium. We suggest that the microvillus-formed niche that harbors a few implanted cancer cells might be the compartment that prevents the tumor eradication by the host immune system.
PMCID: PMC2887496  PMID: 20563246
16.  Low circulating serum levels of second mitochondria-derived activator of caspase (Smac/DIABLO) in patients with bladder cancer 
International Journal of Oncology  2012;40(4):1246-1250.
Smac/DIABLO promotes apoptosis by antagonizing inhibitor of apoptosis proteins. The expression of Smac/DIABLO in tissues has been reported in various cancers; however, little is known about circulating levels of Smac/DIABLO. The present study was designed to first determine if Smac/DIABLO can be detected in the serum and then assess whether the circulating levels of Smac/DIABLO are of prognostic significance in patients with bladder cancer. The levels of Smac/DIABLO in the sera of 173 patients with bladder cancer and 36 normal donors were determined by using an enzyme-linked immunosorbent assay. The mean serum level of Smac/DIABLO in patients with bladder cancer was approximately 2-fold lower than that in normal donors. The mean level of serum Smac/DIABLO in patients with muscle-invasive bladder cancer was lower than that in patients with non-muscle invasive cancer. In addition, the mean serum Smac/DIABLO level in patients with T4 muscle-invasive bladder cancer was lower than that in patients with T2 and T3 cancers. The mean serum level of Smac/DIABLO in patients with Grade 3 bladder cancer was lower than that in patients with Grade 1 and Grade 2 cancers. Analysis by Kaplan-Meier revealed that patients with Ta and T1 non-muscle invasive bladder cancer with high level of serum Smac/DIABLO (more than mean value) had a longer postoperative tumor-free interval than those with low level (less than mean value) in the 3-year follow-up. Furthermore, patients with T2–T4 muscle-invasive bladder cancer with high serum Smac/DIABLO level (more than mean value) had a higher postoperative disease-free rate when compared with patients with low level (less than mean value) in the 5-year follow-up. The present study is the first to analyze circulating levels of Smac/DIABLO in the serum. The findings demonstrate that the mean serum level of Smac/DIABLO was downregulated in patients with bladder cancer compared to control healthy individuals, especially high grade muscle-invasive bladder cancer. Noteworthy, lower serum level of Smac/DIABLO predicted early recurrence in patients with bladder cancer. Overall, the findings suggest that measuring the levels of Smac/DIABLO in the serum may be considered a prognostic parameter in patients with bladder cancer. Furthermore, Smac/DIABLO may be a molecular therapeutic target in bladder cancer.
PMCID: PMC3584575  PMID: 22218530
Smac/DIABLO; bladder cancer; prognosis
17.  The association between the recurrence of solitary non-muscle invasive bladder cancer and tumor infiltrating lymphocytes 
Croatian Medical Journal  2012;53(6):598-604.
To evaluate whether tumor infiltrating lymphocytes (TIL) in biopsy specimens are associated with the clinical outcome of non-muscle invasive bladder cancer.
We retrieved tumor specimens from 115 patients with solitary papillary non-muscle invasive bladder cancer treated between 1996 and 2006 and constructed tissue microarrays. Patients were divided in two groups: those with recurrent disease (N = 69) and those without recurrent disease (N = 46) during the follow up of minimum 5 years. All patients were treated with initial transurethral resection and none received adjuvant therapy. Immunhistochemical staining was performed with anti-CD3, CD4, CD8, and Granzyme B (GrB). The CD4+:CD8+ and GrB+:CD8 ratios were determined.
Tumor infiltrating lymphocytes were predominantly observed within cancer stroma, and only rare individual cells were observed intraepithelially. The group without recurrent disease had lower levels of CD3+ and CD8+ lymphocytes than the group with recurrent disease (P = 0.0001, P = 0.0002, respectively). The CD4+:GrB+ and GrB+:CD8+ ratios were significantly higher in patients without recurrent disease (P = 0.0002, P = 0.039, respectively).
This study revealed a possible connection between TIL number and bladder cancer recurrence. TIL subset ratio showed different patterns in recurrent and non-recurrent tumors, which is why it could become a useful a prognostic clinical index if our findings are confirmed in randomized trials.
PMCID: PMC3541585  PMID: 23275325
18.  Genetic Susceptibility to Distinct Bladder Cancer Subphenotypes 
European urology  2009;57(2):283-292.
Clinical, pathologic, and molecular evidence indicate that bladder cancer is heterogeneous with pathologic/molecular features that define distinct subphenotypes with different prognoses. It is conceivable that specific patterns of genetic susceptibility are associated with particular subphenotypes.
To examine evidence for the contribution of germline genetic variation to bladder cancer heterogeneity.
Design, setting, and participants
The Spanish Bladder Cancer/EPICURO Study is a case-control study based in 18 hospitals located in five areas in Spain. Cases were patients with a newly diagnosed, histologically confirmed, urothelial cell carcinoma of the bladder from 1998 to 2001. Case diagnoses were reviewed and uniformly classified by pathologists following the World Health Organisation/International Society of Urological Pathology 1999 criteria. Controls were hospital-matched patients (n = 1149).
A total of 1526 candidate variants in 423 candidate genes were analysed. Three distinct subphenotypes were defined according to stage and grade: low-grade nonmuscle invasive (n = 586), high-grade nonmuscle invasive (n = 219), and muscle invasive (n = 246). The association between each variant and subphenotype was assessed by polytomous risk models adjusting for potential confounders. Heterogeneity in genetic susceptibility among subphenotypes was also tested.
Results and limitations
Two established bladder cancer susceptibility genotypes, NAT2 slow-acetylation and GSTM1-null, exhibited similar associations among the subphenotypes, as did VEGF-rs25648, which was previously identified in our study. Other variants conferred risks for specific tumour subphenotypes such as PMS2-rs6463524 and CD4-rs3213427 (respective heterogeneity p values of 0.006 and 0.004), which were associated with muscle-invasive tumours (per-allele odds ratios [95% confidence interval] of 0.56 [0.41–0.77] and 0.71 [0.57–0.88], respectively) but not with non–muscle-invasive tumours. Heterogeneity p values were not robust in multiple testing according to their false-discovery rate.
These exploratory analyses suggest that genetic susceptibility loci might be related to the molecular/pathologic diversity of bladder cancer. Validation through large-scale replication studies and the study of additional genes and single nucleotide polymorphisms are required.
PMCID: PMC3220186  PMID: 19692168
Urinary bladder cancer; Genetic polymorphism; Heterogeneity; Tumour subphenotypes; Pathologic characteristics
19.  Diphtheria Toxin-Epidermal Growth Factor Fusion Protein DAB389EGF for the treatment of bladder cancer 
The novel fusion protein, DAB389EGF, is comprised of both the catalytic and translocation domains of diphtheria toxin that are fused to the human epidermal growth factor, providing a targeting and a toxicity component. We tested DAB389EGF for anti-tumor activity in both in vitro and in vivo urinary bladder cancer models.
Experimental Design
Human bladder cancer lines were treated with DAB389EGF and assessed for growth inhibition and clonogenic suppression. Using 6–8 week old female athymic nude mice implanted orthotopically with HTB9 cells, DAB389EGF was administered intravesically twice weekly for two weeks. The response of the luciferase expressing HTB9 cells was monitored via bioluminescence as the primary endpoint..
Treatment response with DAB389EGF was specific and robust, with an IC50 ranging from 0.5 to 15ng/ml in 8 tested bladder cancer cell lines, but greater than 50ng/ml in the EGFR-negative H520 control cell line. Simulating short duration intravesical therapy used clinically, a 2 hour treatment exposure of DAB389EGF (10ng/ml) produced clonogenic suppression in three selected bladder cancer cell lines. In vivo, luciferase activity was suppressed in 5 of 6 mice treated with DAB389EGF (70 μl (1ng/μL) per mouse), as compared to only 1 of 6 mice treated with a control DT fusion protein. Histologic assessment of tumor clearance correlated with the bioluminescent changes observed with DAB389EGF treatment. Immunocompetent mice treated with intravesical DAB389EGF did not demonstrate any non-specific systemic toxicity.
The intravesical delivery of targeted-toxin fusion proteins is a novel treatment approach for non-muscle-invasive urinary bladder cancer. With appropriate targeting, the treatments are effective and well tolerated in vivo.
PMCID: PMC3537889  PMID: 23172881
20.  HYAL-1 Hyaluronidase: A Potential Prognostic Indicator for Progression to Muscle Invasion and Recurrence in Bladder Cancer 
European urology  2009;57(1):86-94.
For bladder cancer (BCa) patients undergoing bladder-sparing treatments, molecular markers may aid in accurately predicting progression to muscle invasion and recurrence. Hyaluronic acid (HA) is a glycosaminoglycan that promotes tumor metastasis. Hyaluronoglucosaminidase 1 (HYAL-1)–type hyaluronidase (HAase) promotes tumor growth, invasion, and angiogenesis. Urinary HA and HAase levels are diagnostic markers for BCa.
We evaluated whether HA and HYAL-1 can predict progression to muscle invasion and recurrence among patients with non–muscle-invasive BCa.
Design, setting, and participants
Based on tissue availability, tissue microarrays were prepared from a cohort of 178 BCa specimens (144 non–muscle invasive, 34 muscle invasive). Follow-up information was available on 111 patients with non–muscle-invasive BCa (mean follow-up: 69.5 mo); 58 patients recurred and 25 progressed to muscle invasion (mean time to progress: 22.3 mo).
HA and HYAL-1 expression was evaluated by immunohistochemistry and graded for intensity and area of staining. Association of HA and HYAL-1 staining with BCa recurrence and muscle invasion was evaluated by univariate and multivariate models.
Results and limitations
HA and HYAL-1 expression correlated with tumor grade, stage, and multifocality (p < 0.05). In non–muscle-invasive BCa specimens, HYAL-1 staining was higher (234.3 ± 52.2; 200.6 ± 61.4) if patients experienced progression to muscle invasion or recurrence when compared with no progression or recurrence (164.1 ± 48.2; 172.1 ± 57; p < 0.001). HA staining correlated with muscle invasion (p < 0.001). In univariate analysis, age (p = 0.014), multifocality (p = 0.023), and HYAL-1 staining (p < 0.001) correlated with muscle invasion, whereas only HYAL-1 correlated with recurrence (p = 0.013). In multivariate analysis, significantly associated with muscle invasion (p < 0.001; 76.8% accuracy) and recurrence (p = 0.01; 67.8% accuracy).
HYAL-1 is a potential prognostic marker for predicting progression to muscle invasion and recurrence.
PMCID: PMC2828527  PMID: 19345473
Bladder cancer; Hyaluronic acid; Hyaluronidase; HYAL-1; non-muscle invasive bladder cancer; Prognostic markers; Tissue microarray
21.  Ethnic differences in glucose disposal, hepatic insulin sensitivity, and endogenous glucose production among African American and European American women 
Metabolism  2011;61(5):634-640.
Intravenous glucose tolerance tests (IVGTT) have demonstrated lower whole-body insulin sensitivity (SI) among African Americans (AA) compared to European Americans (EA). Whole-body SI represents both insulin-stimulated glucose disposal, primarily by skeletal muscle, and insulin's suppression of endogenous glucose production (EGP) by liver. A mathematical model was recently introduced that allows for distinction between disposal and hepatic insulin sensitivity. The purpose of this study was to examine specific indexes of insulin sensitivity among AA and EA women to determine whether lower whole-body insulin sensitivity in AA may be attributed to insulin action at muscle, liver, or both.
Participants were 53 non-diabetic, premenopausal AA and EA women. Profiles of EGP and indexes of Disposal SI and Hepatic SI were calculated by mathematical modeling and incorporation of a stable isotope tracer (6,6-2H2glucose) into the IVGTT. Body composition was assessed by dual energy X-ray absorptiometry.
After adjustment for percent fat, both Disposal SI and Hepatic SI were lower among AA (p=0.009 for both). Time profiles for serum insulin and EGP revealed higher peak insulin response and corresponding lower EGP among AA women compared to EA.
Indexes from a recently-introduced mathematical model suggest that lower whole-body insulin sensitivity among non-diabetic AA women is due to both hepatic and peripheral components. Despite lower Hepatic SI, AA displayed lower EGP, resulting from higher post-challenge insulin levels. Future research is needed to determine the physiological basis of lower insulin senstivity among AA and its implications for type 2 diabetes risk.
PMCID: PMC3288425  PMID: 22071009
mathematical modeling; intravenous glucose tolerance test; hepatic glucose production
22.  Expression of RFC/SLC19A1 is Associated with Tumor Type in Bladder Cancer Patients 
PLoS ONE  2011;6(7):e21820.
Urinary bladder cancer (UBC) ranks ninth in worldwide cancer. In Egypt, the pattern of bladder cancer is unique in that both the transitional and squamous cell types prevail. Despite much research on the topic, it is still difficult to predict tumor progression, optimal therapy and clinical outcome. The reduced folate carrier (RFC/SLC19A1) is the major transport system for folates in mammalian cells and tissues. RFC is also the primary means of cellular uptake for antifolate cancer chemotherapeutic drugs, however, membrane transport of antifolates by RFC is considered as limiting to antitumor activity. The purpose of this study was to compare the mRNA expression level of RFC/SLC19A1 in urothelial and non-urothelial variants of bladder carcinomas. Quantification of RFC mRNA in the mucosa of 41 untreated bladder cancer patients was performed using RT-qPCR. RFC mRNA steady-state levels were ∼9-fold higher (N = 39; P<0.0001) in bladder tumor specimens relative to normal bladder mRNA. RFC upregulation was strongly correlated with tumor type (urothelial vs. non-urothelial; p<0.05) where median RFC mRNA expression was significantly (p<0.05) higher in the urothelial (∼14-fold) compared to the non-urothelial (∼4-fold) variant. This may account for the variation in response to antifolate-containing regimens used in the treatment of either type. RFC mRNA levels were not associated with tumor grade (I, II and III) or stage (muscle-invasive vs. non-muscle invasive) implying that RFC cannot be used for prognostic purposes in bladder carcinomas and its increased expression is an early event in human bladder tumors pathogenesis. Further, RFC can be considered as a potential marker for predicting response to antifolate chemotherapy in urothelial carcinomas.
PMCID: PMC3132752  PMID: 21760912
23.  Multivariate Analysis of the Prognostic Significance of Resection Weight after Transurethral Resection of Bladder Tumor for Non-Muscle-Invasive Bladder Cancer 
Korean Journal of Urology  2012;53(7):457-462.
Tumor size and multiplicity are known to be important prognostic factors in non-muscle-invasive bladder cancer (NMIBC). However, evaluation of accurate tumor size is subjective and difficult. Furthermore, there are limitations to the objectification of tumor volume in the case of multiple lesions. In this study, we investigated the relation between resection weight after transurethral resection of bladder tumor (TURBT) and the prognosis of NMIBC.
Materials and Methods
This was a retrospective analysis of 406 patients diagnosed with pTa or pT1 bladder tumors after TURBT between September 1999 and May 2010. The patient's age, sex, underlying diseases, cancer stage, grade, multiplicity, tumor size, lymphovascular invasion, and resection weight were analyzed in relation to cancer progression and recurrence. The resection weight was weighted after formaldehyde fixation.
The mean follow-up time was 76.9 months (range, 12 to 167 months) in 406 patients diagnosed as having NMIBC. Mean resection weight was 4.5 g (range, 0.1 to 35.0 g). The cancer recurred in 99 patients (24.4%), and disease progression was noted in 30 patients (7.4%). Resection weight was categorized as greater than or less than 2 g by use of receiver operator characteristic curves. Cancer grade (p=0.022) and multiplicity (p=0.043) were significantly related to cancer recurrence in the analysis with Cox's multivariate proportional hazard model. Cancer grade (p=0.001) and resection weight (p=0.018) were related to disease progression.
Resection weight after TURBT was significantly related to progression of NMIBC. Resection weight was an independent factor of progression. Further management should be considered if the resection weight exceeds 2 g.
PMCID: PMC3406190  PMID: 22866215
Prognosis; Tumor burden; Urinary bladder neoplasms
24.  Tumor budding is a significant indicator of a poor prognosis in lung squamous cell carcinoma patients 
Molecular Medicine Reports  2012;6(5):937-943.
Lung cancer is a leading cause of cancer mortality worldwide and patients occasionally develop local recurrence or distant metastasis soon after curative resection. Reports of new therapeutic strategies for lung squamous cell carcinoma (SqCC) are extremely rare, while selective anticancer therapy has been reported for lung adenocarcinoma. The aim of this study was to identify clinicopathological prognostic factors for SqCC. We analyzed tumor budding and infiltrative patterns (INF) in 103 cases of surgically-resected SqCC. Tumor infiltrative patterns were classified into three groups (INFa, b and c) and INFc was infiltrative growth at the tumor invasive front. The cases with an INFc component [INFc(+)]were significantly associated with venous invasion (P=0.014) and the scirrhous stromal type (P<0.001). The overall survival rate of patients with INFc(+) was significantly lower than that of patients without the INFc component [INFc(−); P=0.003]. Tumor budding was defined as a single cancer cell or a small nest of up to four cancer cells within stromal tissue. The cases with tumor budding [Bud(+)] were significantly associated with lymph node metastasis (P=0.001), lymphatic invasion (P=0.002), INFc(+) (P<0.001) and the scirrhous stromal type (P=0.014). Patients with the Bud(+) type had a lower overall survival rate than patients with the Bud(−) type (P<0.001). Multivariate analysis demonstrated that tumor budding [hazard ratio (HR), 2.766; 95% confidence interval (CI), 1.497–5.109] and lymph node metastasis (HR, 1.937; 95% CI, 1.097–3.419) were independent predictors of mortality. In conclusion, tumor budding is a significant indicator of a high malignant potential and poor prognosis in SqCC of the lung.
PMCID: PMC3493086  PMID: 22940760
lung cancer; squamous cell carcinoma; patient prognosis; tumor budding
25.  Clinicopathologic Factors Affecting Recurrence after Curative Surgery for Stage I Colorectal Cancer 
The objective of the current study was to identify the clinicopathological risk factors affecting recurrence after a curative resection for stage I colorectal cancer.
We retrospectively studied 434 patients who underwent a curative resection for stage I colorectal cancer between January 1999 and December 2004. Postoperative oral chemotherapy was performed in 189 patients (45.3%). The following prognostic factors were correlated with recurrence: age, gender, preoperative carcinoembryonic antigen level, location of tumor, T stage, size of tumor, histologic differentiation, growth pattern, and lymphovascular invasion. The median follow-up duration was 65 months.
The overall recurrence rate was 4.6% (20/434). The median time to recurrence was 33 months. Two-thirds of the recurrence occurred more than two years after surgery. Risk factors associated with recurrence were rectal cancer (P = 0.009), T2 stage (P = 0.010), and infiltrative growth pattern (P = 0.020). A Cox proportional hazards regression analysis demonstrated that the infiltrative growth pattern was an independent predictor for recurrence. Tumor cell budding was observed in all pathologic reviews with recurrence.
Long-term follow-up is necessary for stage I colorectal patients with high risk factors like rectal cancer, T2 stage, and infiltrative growth pattern.
PMCID: PMC3296942  PMID: 22413082
Colorectal neoplasms; Recurrence; Risk factors

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