Expression of miR‐100 and miR‐138 as prognostic biomarkers in non‐muscle‐invasive bladder cancer

microRNA alterations are involved in bladder cancer tumorigenesis. The aim of the current study was to evaluate the potential role of miR‐100 and miR‐138 as prognostic biomarkers in Ta/T1 non‐muscle‐invasive bladder cancer (NMIBC). We assessed a quantitative RT‐PCR analysis of miR‐100 and miR‐138 in 50 bladder tumor samples (stage Ta/T1) and four healthy adjacent tissues. Western blot analysis was used to measure protein expression of FGFR3 and cyclin D3 in order to know whether these targets can be regulated by miR‐100 and miR‐138, respectively. The statistical analysis included non‐parametric tests (Mann–Whitney U and Kruskal–Wallis) and univariate survival analysis by Kaplan–Meier method and the log‐rank test. Low expression of miR‐138 characterized recurrent tumors (p = 0.043), and higher expression levels were associated with longer recurrence‐free survival (p = 0.012). However, low miR‐100 expression correlated with longer progression‐free survival (marginal significance; p = 0.053) and cancer‐specific overall survival (p = 0.006). Additionally, higher levels of miR‐100 were associated with negative FGFR3 protein expression (p = 0.032) and higher levels of miR‐138 were associated with positive cyclin D3 protein expression (p = 0.037). Our results support miR‐138 and miR‐100 as prognostic biomarkers in patients with NMIBC.     

Prostate‐specific membrane antigen expression in tumor‐associated vasculature of breast cancers

Prostate‐specific membrane antigen (PSMA) has been found to be expressed in the tumor‐associated neovasculature of multiple solid tumor types including breast cancers. However, thus far, the number of cases studied from some tumor types has been limited. In this study, we set out to assess PSMA expression in the tumor‐associated vasculature associated with invasive breast carcinomas in a sizable cohort of patients. One hundred and six patients with AJCC stage 0‐IV breast cancer were identified. Ninety‐two of these patients had primary breast cancer [invasive breast carcinoma with or without co‐existing ductal carcinoma in situ (DCIS) (74) or DCIS alone (18)]. In addition, 14 patients with breast cancer metastases to the brain were identified. Immunohistochemical staining for PSMA and CD31 was performed on parallel representative tumor sections in each case. Tumor‐associated vascular endothelial cell PSMA immunoreactivity was semi‐quantitatively assessed based on two parameters: overall percent of endothelial positivity and staining intensity. PSMA expression for tumor‐associated vascular endothelial cells was scored 0 if there was no detectable PSMA expression, 1 if PSMA staining was detectable in 5–50%, and 2 if PSMA expression was positive in >50% of microvessels. CD 31 staining was concurrently reviewed to confirm the presence of vasculature in each case. Tumor‐associated vasculature was PSMA‐positive in 68/92 (74%) of primary breast cancers and in 14/14 (100%) of breast cancers metastatic to brain. PSMA was not detected in normal breast tissue or carcinoma cells. All but 2 cases (98%) showed absence of PSMA expression in normal breast tissue‐associated vasculature. The 10‐year overall survival was 88.7% (95% CI = 80.0%, 93.8%) in patients without brain metastases. When overall survival (OS) was stratified based on PSMA score group, patients with PSMA scores of 0, 1, and 2 had 10‐year OS of 95.8%, 96.0%, and 79.7%, respectively (p = 0.12). When PSMA scores of 0 and 1 were compared with 2, there was a statistically significant difference in OS (96.0% vs 79.7%, respectively, p = 0.05). Patients with a PSMA score of 2 had a significantly higher median tumor size compared with patients in the lower PSMA score groups (p = 0.04). Patients with higher nuclear grade were more likely to have a PSMA score of 2 compared with patients with lower nuclear grade (p < 0.0001). Patients with a PSMA score of 2 had a significantly higher median Ki‐67 proliferation index compared with patients in the lower PSMA score groups (p < 0.0001). Patients with estrogen receptor (ER)‐negative tumors were more likely to have a PSMA score of 2 compared with patients with ER‐positive tumors (p < 0.0001). Patients with progesterone receptor (PR)‐negative tumors were more likely to have a PSMA score of 2 compared with patients with PR‐positive tumors (p = 0.03). No significant association was observed between PSMA score group status and lymph node involvement (p = 0.95). Too little variability was present in Human epidermal growth factor receptor‐2 (Her2/neu) amplified tumors to correlate with PSMA score group status. To date, this is the first detailed assessment of PSMA expression in the tumor‐associated vasculature of primary and metastatic breast carcinomas. Further studies are needed to evaluate whether PSMA has diagnostic and/or potential therapeutic value.     

Dysregulation of toll‐like receptor (TLR) 2 expression on monocytes and upregulation of the frequency of T cells expressing TLR2 in patients with chronic hepatitis C virus infection

Toll‐like receptors (TLRs) initiate inflammatory responses that may play a role in disease progression in patients infected with hepatitis C virus (HCV). TLR2 and TLR4 surface expression were assessed on CD14⁺ monocytes, CD4⁺ and CD8⁺ T cells in treatment naïve patients with chronic HCV infection with fibrosis, without fibrosis, co‐infected with human immunodeficiency virus (HIV), and in healthy controls. Increased expression of TLR2 was found on monocytes in HCV‐infected patients with fibrosis (p < 0.01), co‐infected with HIV (p = 0.03), and possibly in patients without fibrosis (p = 0.07) when compared to controls. TLR2 positive CD4⁺ and CD8⁺ T cells were upregulated in patients with fibrosis only (p < 0.01). However, expression of TLR2 was not associated with T cell activation. TLR4 expression was similar in patients and healthy controls. In conclusion, TLR2 expression on monocytes and the frequency of T cells expressing TLR2 may contribute to disease progression in chronic HCV infection.     

Clinicopathological study of lip cancer: a retrospective hospital‐based study in Taiwan

To evaluate the clinicopathological characteristics, high‐risk lifestyle factors (HRLF: chronic exposure to sun, betel quid, alcohol, and tobacco), and prognostic factors of lip cancer. The hospital records of patients with pathologically confirmed lip squamous cell carcinoma (LSCC, n = 112) and lip basal cell carcinoma (LBCC, n = 21) were reviewed. Differences in clinicopathological characteristics between LSCC and LBCC, upper and lower lip, and status of second primary tumors were compared by chi‐square test and logistic regression. The prognostic factors for LSCC were analyzed by Cox regression. Compared with LBCC patients, LSCC patients were men‐predominant (p < 0.001), had younger ages at onset (p < 0.001), and higher rates of lower lips involvement (p < 0.001) and HRLFs. Patients with second primary tumors were highly associated with lower lip cancer involvement (adjusted odds ratio = 2.91, p = 0.03). Patients with lower lip cancer had more HRLFs with an increasing linear trend (p = 0.004). The poorer prognostic factors of LSCC for disease‐specific survival were advanced stage III/IV [crude hazard ratio (CHR) = 11.16, p < 0.001], tumor dimension >4 cm (CHR = 8.19, p = 0.006), lymph node involvement (CHR = 11.48, p < 0.001), and recurrence (CHR = 3.96, p = 0.01); whereas for disease‐free survival were moderately to poorly differentiated LSCC (CHR = 4.97, p = 0.002) and alcohol consumption (CHR = 3.13, p = 0.04). LSCC and lower lip cancer were highly associated with HRLFs.     

Adipose tissue derived stromal stem cell therapy in murine ConA‐derived hepatitis is dependent on myeloid‐lineage and CD4+ T‐cell suppression

Mesenchymal stromal stem cells (MSCs) are an attractive therapeutic model for regenerative medicine due to their pluripotency. MSCs are used as a treatment for several inflammatory diseases, including hepatitis. However, the detailed immunopathological impact of MSC treatment on liver disease, particularly for adipose tissue derived stromal stem cells (ADSCs), has not been described. Here, we investigated the immuno‐modulatory effect of ADSCs on hepatitis using an acute ConA C57BL/6 murine hepatitis model. i.v. administration of ADSCs simultaneously or 3 h post injection prevented and treated ConA‐induced hepatitis. Immunohistochemical analysis revealed higher numbers of CD11b⁺, Gr‐1⁺, and F4/80⁺ cells in the liver of ConA‐induced hepatitis mice was ameliorated after the administration of ADSCs. Hepatic expression of genes affected by ADSC administration indicated tissue regeneration‐related biological processes, affecting myeloid‐lineage immune‐mediating Gr‐1⁺ and CD11b⁺ cells. Pathway analysis of the genes expressed in ADSC‐treated hepatic inflammatory cells revealed the possible involvement of T cells and macrophages. TNF‐α and IFN‐γ expression was downregulated in hepatic CD4⁺ T cells isolated from hepatitis livers co‐cultured with ADSCs. Thus, the immunosuppressive effect of ADSCs in a C57BL/6 murine ConA hepatitis model was dependent primarily on the suppression of myeloid‐lineage cells and, in part, of CD4⁺ T cells.     

Coexistence of MACC1 and NM23‐H1 dysregulation and tumor budding promise early prognostic evidence for recurrence risk of early‐stage colon cancer

The tumor‐node‐metastasis (TNM) classification, the presence of a mucinous component, and signet ring cells are well‐known criteria for identifying patients at a high risk for recurrence and determining the therapeutic approach for early‐stage colon cancer (eCC). Nevertheless, recurrence can unexpectedly occur in some eCC cases after surgical resection. The aims of the present study were to evaluate the relation of dysregulated MACC1, c‐MET, and NM23‐H1 expression with the histopathological features of tumors in recurrence formation in eCC cases. A total of 100 sporadic eCC patients without poor prognosis factors were evaluated in this study. The relationship between the altered expression of MACC1, c‐MET, and NM23‐H1 and pathological microenvironmental features, including the presence of tumor budding and desmoplasia, were assessed. The primary outcomes, including 5‐year overall survival (OS) and disease‐free survival (DFS), were also measured. Compared with nonrecurrent patients, the expression level of MACC1 was 8.27‐fold higher, and NM23‐H1 was 11.36‐fold lower in patients with recurrence during the 5‐year follow‐up (p = 0.0345 and p = 0.0301, respectively). In addition, the coexistence of high MACC1 and low NM23‐H1 expression and tumor budding was associated with short OS (p < 0.001). We suggest that the combination of reduced NM23‐H1, induced MACC1, and the presence of tumor budding are promising biomarkers for the prediction of recurrence and may aid the stratification of patients with stage II colon cancer for adjuvant chemotherapy.     

Notch1 modulates mesenchymal stem cells mediated regulatory T‐cell induction

Notch1 signaling is involved in regulatory T (Treg)‐cell differentiation. We previously demonstrated that, when cocultured with CD3⁺ cells, mesenchymal stem cells (MSCs) induced a T‐cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4⁺ T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI‐I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC‐induced CD4⁺CD25^highFOXP3⁺ cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF‐β signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg‐cell induction mediated by MSCs.     

Post‐cervical conization outcomes in patients with high‐grade intraepithelial lesions

To investigate the rates of residual, recurrent and invasive disease after cervical conization in patients diagnosed with cervical intraepithelial neoplasia (CIN) grades 2/3. A retrospective study was conducted with 274 patients undergoing cervical conization due to diagnosis of CIN 2/3. Cervical conization was done through the Loop Electrosurgical Excision Procedure (LEEP) and Cold Knife Conization. Data related to personal, familial, gynecological, and obstetric antecedents, as well as surgical specimens margins were collected from medical records. The outcome after conization was evaluated, including the time of follow‐up and disease recurrence. The outcome after conization was not associated with age of menarche (p = 0.920), age of the first sexual intercourse (p = 0.533), number of parturition (p = 0.063), number of sexual partners (p = 0.328), immunosuppression (p = 0.225), smoking habit (p = 0.193), and conization type (p = 0.198). However, the outcome presented a significant association with age (p < 0.001), pregnancy numbers (p = 0.009), use of hormonal contraception methods (p = 0.016), menopause (p = 0.007), type of margins (p = 0.011), and cone histological results (p = 0.030). The routine control of all patients who had undergone cervical conization is obligate, independently of surgical margins, due to the risk of disease recurrence; the older patients and those with CIN 3 should have a more rigorous follow‐up.     

PD‐L1 expression and deficient mismatch repair in ductal adenocarcinoma of the prostate

This study aimed to investigate the expression of programmed death receptor ligand 1 (PD‐L1) and deficient mismatch repair (dMMR) in ductal adenocarcinoma of the prostate. A tissue microarray of 32 ductal and 42 grade‐matched acinar adenocarcinomas was used. Slides were stained for PD‐L1, PD‐L2, MMR proteins, CD4 and CD8. PD‐L1 expression in tumor cells was only seen in 3% (1/34) of ductal and 5% (2/42) of acinar adenocarcinomas (p = 1.0), while PD‐L1 expression in tumor‐infiltrating immune cells was seen in 29% (10/34) of ductal and 14% (6/42) of acinar adenocarcinomas (p = 0.16). dMMR, as defined by loss of one or more of the MMR proteins, was identified in 5% (4/73) of cases, including 1 ductal and 3 acinar adenocarcinomas. There was a suggested association between infiltration of CD8+ lymphocytes and ductal subtype (p = 0.04) but not between CD4+ lymphocytes and tumor type (p = 0.28). The study shows that both dMMR and PD‐L1 expression is uncommon in tumor cells of both ductal and acinar adenocarcinoma of the prostate, while PD‐L1 expression in tumor‐infiltrating immune cells is a more common finding.     

Different cytokine production and toll‐like receptor expression induced by heat‐killed invasive and carrier strains of Neisseria meningitidis

Neisseria meningitidis may cause severe invasive disease. The carriage state of the pathogen is common, and the reasons underlying why the infection becomes invasive are not fully understood. The aim of this study was to compare the differences between invasive and carrier strains in the activation of innate immunity. The monocyte expression of TLR2, TLR4, CD14, and HLA‐DR, cytokine production, and the granulocyte oxidative burst were analyzed after in vitro stimulation by heat‐killed invasive (n = 14) and carrier (n = 9) strains of N. meningitidis. The expression of the cell surface markers in monocytes, the oxidative burst, and cytokine concentrations were measured using flow cytometry. Carrier strains stimulated a higher production of inflammatory cytokines and oxidative burst in granulocytes than invasive strains (all p < 0.001), whereas invasive strains significantly up‐regulated TLR2, TLR4 (p < 0.001), and CD14 (p < 0.01) expression on monocytes. Conversely, the monocyte expression of HLA‐DR was higher after the stimulation by carrier strains (p < 0.05) in comparison to invasive strains. The LPS inhibitor polymyxin B abolished the differences between the strains. Our findings indicate different immunostimulatory potencies of invasive strains of N. meningitidis compared with carrier strains.     

Autophagy and Bcl‐2/BNIP3 death regulatory pathway in non‐small cell lung carcinomas

We recently showed that non‐small cell lung carcinomas (NSCLCs) are of dismal prognosis when encompassing accelerated autophagic activity. The regulation of this abnormally functioning degradation system and its association with hypoxia and apoptosis in lung carcinoma patients is unexplored. In this study we used 115 NSCLC tissues to examine the immunohistochemical expression of four distinct molecules – the major regulator of autophagy Beclin 1, the anti‐apoptotic and anti‐autophagic protein Bcl‐2, the pro‐apoptotic and pro‐autophagic protein BNIP3, and a marker of hypoxia and glucolysis, the glucose transporter Glut 1. Most cases showed reduced reactivity for Beclin 1 (62%) and Bcl‐2 (82%) proteins, almost half of our sample revealed strong BNIP3 expression (57%), whereas most of the carcinomas strongly expressed Glut 1 antigen (71%). Beclin 1 expression showed no association with survival. Bcl‐2 positivity was a marker of good prognosis (p = 0.04), whereas BNIP3 (p = 0.0004) and Glut 1 (p = 0.03) expression correlated with poor outcome in Stage I disease. Autophagic status was negatively associated with Bcl‐2 (p = 0.0006), but positively with Glut 1 expression (p = 0.001). In conclusion, the accelerated autophagic status in NSCLC is unrelated to Beclin 1 and BNIP3 expression, but does show significant association with Bcl‐2 reactivity. Furthermore, we showed important correlations between glucolysis and autophagy, guiding new pathways in future lung carcinoma research.     

Vascular adhesion protein‐1 as indicator of breast cancer tumor aggressiveness and invasiveness

Recent studies suggest that vascular adhesion protein‐1 (VAP‐1), a 180‐KDa homodimeric glycoprotein, may be associated with cancer‐related events including tumor cell migration, motility, invasion, or metastasis. Therefore, this study applies VAP‐1 immunohistochemical staining to demonstrate the invasiveness component of the breast cancer. The VAP‐1 staining results were compared in 148 breast cancer cases to identify possible correlations with clinical status, including age, tumor size, tumor grade, TNM stage, lymphatic invasion, metastasis, recurrence, and survival rate. Immunohistochemical staining results showed VAP‐1 negative or weak staining in normal ducts and ductal carcinoma in situ (DCIS), but these phenotypes were positively associated with a stiffened VAP‐1 that presented at the invasive front of the lesion. Our data demonstrated that VAP‐1 expression was positively associated with lymphatic invasion, distant metastasis, and patient survival in breast carcinoma. Notably, VAP‐1 expression was found to be significantly correlated with the overall survival (p < 0.0001). Multivariate Cox analysis indicated that VAP‐1 expression was a significant independent prognostic indicator of overall survival in breast carcinoma (p < 0.0001). In conclusion, this study suggests that VAP‐1 is linked to progression of tumor invasion and metastasis in breast carcinoma. VAP‐1 is shown to be a biomarker that can be predict invasive potential and clinical outcome in breast cancer.     

Aberrant deleted in liver cancer‐1 expression is associated with tumor metastasis and poor prognosis in urothelial carcinoma

This study explored the potential role of deleted in liver cancer‐1 (DLC‐1) as a prognostic indicator of cancer metastasis and survival in urothelial carcinoma (UC). Tissue microarrays were constructed from paraffin‐embedded specimens from 88 UC patients, and immunohistochemical staining was performed to investigate the association of DLC‐1 with clinicopathologic characteristics and clinical outcome. The DLC‐1 expression showed a significant positive correlation with tumor location (p = 0.041) and a significant negative correlation with advanced histological grade (p = 0.013). In tumors with low DLC‐1 expression, Bcl‐2 positivity was observed in 24.4% of cases. The DLC‐1 expression had significant negative associations with Bcl‐2 expression (p = 0.032) and with highly metastatic UC (p = 0.032). Kaplan–Meier analysis showed that DLC‐1 protein expression was negatively associated with both overall survival (OS) (p = 0.035) and with distant metastasis‐free survival (DMFS) (p = 0.041), but not with disease‐free survival. Multivariate analyses indicated that tumor size was the significant independent predictors of OS (p = 0.048); however, only DLC‐1 expression was a significant independent predictor of DMFS (p = 0.019). In conclusion, reduced DLC‐1 protein expression may be an important factor in tumor progression and a useful prognostic molecular marker in UC.     

Overexpression of cyclooxygenase‐2 in urothelial carcinoma in conjunction with tumor‐associated‐macrophage infiltration,hypoxia‐inducible factor‐1α expression,and tumor angiogenesis

This study examines whether the expression of cyclooxgenase‐2 (COX‐2) in urothelial carcinoma (UC) is associated with macrophage infiltration, hypoxia‐inducible factor‐1α (HIF‐1α) expression and angiogenesis. We investigated the expression of COX‐2 associated with HIF‐1α and performed double immunohistochemical analysis of 216 UCs for COX‐2 expression and the correlation with tumor‐associated‐macrophage (TAM) density and microvessel density (MVD) in situ. A high expression of COX‐2 was positively correlated with tumor invasiveness, histologic grade and HIF‐1α expression in UC (p<0.0001, p=0.003, p<0.0001, respectively). Quantification of double staining of COX‐2/CD34 and COX‐2/CD68 showed that a higher MVD and TAM density was found in COX‐2 high‐expression than in COX‐2 low‐expression tumor fields (p<0.0001). Adjacent to the principal of COX‐2 expression areas, MVD value and TAM density were significantly increased in HIF‐1α high‐expression specimens compared with HIF‐1α low‐expression ones (p<0.0001). Interestingly, our data revealed that high COX‐2 expression (p=0.002), high HIF‐1α expression (p<0.0001) and TAM density (p<0.0001) were all associated with high MVD value. Our results suggest that COX‐2 may produce a cooperative effect in promoting tumor progression and may be involved in the process of angiogenesis through increasing TAM infiltration or HIF‐1α regulation by hypoxia.     

Cyclooxygenase‐2 expression on urothelial and inflammatory cells of cystoscopic biopsies and urine cytology as a possible predictive marker for bladder carcinoma

Cyclooxygenase‐2 (COX‐2) is a key inducible enzyme involved in the production of prostaglandins. It contributes to human carcinogenesis by various mechanisms. The aim of the current study was to elucidate the possible involvement of COX‐2 in human bladder carcinoma by examining its expression on both urothelial and inflammatory cells in tissue biopsies and urine cytology samples of different urinary bladder lesions. A total of 65 patients were included in the study and were selected from cases admitted to Urology Department, Theodor Bilharz Research Institute (TBRI), Giza, Egypt. They represented seven control cases with almost normal‐looking bladder tissue; pure chronic cystitis (n=12); premalignant lesions (18) in the form of squamous metaplasia (n=8) or urothelial dysplasia (n=10) as well as transitional cell carcinoma (TCC) (n=18), and squamous cell carcinoma (SqCC) (n=10). Immunohistochemistry of formalin‐fixed, paraffin‐embedded tissue sections and urine cytology samples was performed for all cases using COX‐2 (H‐62): sc‐7951, a rabbit polyclonal antibody. The study revealed positive COX‐2 expression on the urothelial and inflammatory cells of cystoscopic biopsies from all cases of pure chronic cystitis, squamous metaplasia and SqCC compared with 42.8% and 71.4% of normal controls, respectively. The score of urothelial COX‐2 expression was sequentially up‐regulated from normal to chronic cystitis (either pure or associated with premalignant changes) (p<0.05) to malignant changes (p<0.05). However, the inflammatory cellular expression was down‐regulated with malignant transformation compared with chronic cystitis (p<0.05). In TCC, COX‐2 was over‐expressed on both urothelial and inflammatory cells in advanced tumors. Urine cytology samples were positive for COX‐2 in a comparable manner to that observed in cystoscopic biopsies. Accordingly, the results of the current study have provided new information in two aspects: First, is the possibility of using the differential COX‐2 expression on both inflammatory and urothelial cells as markers for premalignant or malignant transformation; second, besides cystoscopy, urine cytology was found to have a high sensitivity for COX‐2 expression and hence proved to be valuable in malignancy as a non‐invasive substitute for cystoscopy.     

S100P as a marker for poor survival and advanced stage in gallbladder carcinoma

AimsGallbladder carcinomas usually present in advanced stages and has a dismal prognosis despite modern imaging techniques and aggressive surgical intervention. Identification of biologic markers for early diagnosis and improved therapeutic strategies is thus of paramount importance. S100P has been identified in a variety of malignant neoplasms of the gastrointestinal and pancreaticobiliary systems, but it is not yet known if S100P expression is associated with clinically-relevant characteristics of gall bladder carcinoma. The aims of the present study were: 1) to investigate the relationship between S100P expression and histological type, grade, tumor-node-metastasis stage, presence of vascular invasion, perineural invasion and necrosis; and 2) to evaluate for any S100P-defined difference in the risk for tumor recurrence or death.MethodImmunostains for S100P were performed on 4 tissue microarray blocks containing 91 cases of gall bladder carcinoma.ResultThe intensity of S100P staining was significantly associated with pathological T stage 4 (p = 0. 0238). Staining intensity ≥3 in ≥25% tumor cells was associated with pathological T stage 4 (p = 0.0005). A higher S100P immunoreactivity score (IRS) was significantly associated with higher TNM stage (p = 0.0341). Age (p = 0.0485), presence of vascular invasion (p = 0.0359), pathological T stage (p = 0.0291) and TNM stage (p = 0.0153) were significantly associated with tumor recurrence. Intense S100P reactivity was associated with decreased overall survival [hazard ratio = 9.614; 95% confidence interval (CI), 1.873–49.338; p = 0.0067].ConclusionOur findings indicate that S100P over-expression is a potential prognostic marker for gall bladder carcinoma and is significantly associated with advanced tumor stage and poorer survival.