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Immunohistochemical Studies to Examine the Diagnostic and Prognostic Implications of Epithelial-to-Mesenchymal Transition in Patients with Urothelial Carcinoma of the Bladder

  • R. Singh
  • A. Mandhani
  • V. Agrawal
  • M. GargEmail author
Medicine
  • 52 Downloads
Part of the following topical collections:
  1. Topical Collection on Medicine

Abstract

In the present study, quantitative expression of epithelial-to-mesenchymal transition (EMT)–associated markers was investigated immunohistochemically and their diagnostic and prognostic significance was evaluated in patients diagnosed with non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Immunohistochemical (IHC) staining was performed in a cohort of 65 formalin-fixed and paraffin-embedded human urinary bladder tumor tissues. Tissues’ sections were obtained from archives of the Department of Pathology at SGPGIMS, India. Epithelial marker (E-cadherin), mesenchymal markers (N-cadherin and vimentin), and EMT-activating transcription factors (ATFs) (Snail and Slug) were examined for their cellular localization (membranous/cytoplasmic/nuclear) and quantitative expressions in terms of IHC score. Expression of the aforesaid markers was statistically correlated with various clinicohistopathological variables. These variables were obtained from histopathology reports and subsequent follow-up and OPD visits of patients. Impact of these markers was assessed on recurrence-free survival (RFS) in NMIBC cases and progression-free survival (PFS) in MIBC cases. The data was analyzed using SPSS 20.0 software. Focal loss of membranous E-cadherin showed statistical relevance with hematuria and tumor grade (p < 0.001, p = 0.005: independent sample t test) in MIBC patients. Novel membranous expression of N-cadherin exhibited relevance with hematuria (p = 0.011, one sample t test) in NMIBC, while membranous expression of vimentin showed correlation with tumor grade and age (p < 0.001, p < 0.001, one sample t test) in MIBC cases. Nuclear immunopositivity of Snail showed statistical association with tumor grade in both NMIBC and MIBC cases (p < 0.001, Moses non-parametric test; p < 0.001, one sample t test) and with hematuria in MIBC cases (p = 0.007, independent sample t test). Additionally, nuclear immunopositivity of Slug showed statistical association with tumor type in NMIBC and tumor grade in MIBC cases (p < 0.001, Moses non-parametric test; p < 0.001, one sample t test). Kaplan-Meier along with logrank statistics examined association between EMT profile and RFS in NMIBC cases (p < 0.001). Significant association of EMT biomarkers with clinicohistopathological outcomes may aid urologists to correlate its dynamic functions in urothelial tumorigenesis.

Keywords

Bladder cancer Diagnostics Histopathology Metastasis Tumor markers 

Introduction

Bladder cancer is the second most common malignancy of the genitourinary tract [1]. Ninety percent of the bladder cancers are histologically classified as urothelial carcinoma of bladder (UCB). Of these, 80% are diagnosed as non-muscle-invasive bladder cancer (NMIBC) that arises through papillary pathway and can be successfully treated [2]. Overall survival of patients diagnosed with NMIBC is 70 to 80% and they exhibit very high recurrence rates (50 to 90%). There is a lack of consensus among clinicians on treatment modality based on radical removal or conservative approach for high-grade NMIBC. About 25 to 30% of patients are presented with more advanced muscle-invasive bladder cancer (MIBC)/metastatic diseases. Despite improved surgical procedures, 50% of such patients experience disease progression and eventual death [3]. Deciphering the molecular events during different courses of disease progression may explain clinical heterogeneity.

Essential functions of epithelial-to-mesenchymal transition (EMT) are reported during embryonic development and wound healing. Recently, EMT is examined as a key feature in the pathogenesis of epithelial cancers including UCB. Pathophysiologically, it is characterized by loss of homotypic adhesion and cell polarity, transformation of epithelial cells into spindle-shaped mesenchymal cells, and increased cell invasion and migration. At molecular level, EMT is defined by the loss of epithelial markers like E-cadherin, novel expression of mesenchymal markers like N-cadherin and vimentin, and upregulation of EMT-activating transcription factors (EMT-ATFs) including Zeb, Twist, Snail, and Slug [3, 4, 5, 6]. Zinc finger transcription factors, Snail and Slug, repress E-cadherin by binding to its promoter, upregulate mesenchymal proteins, and thereby promote EMT [7]. Experimental studies examine the pathological expression of putative markers of EMT and its association with increased motility and tumor invasion potential during development of UCB [8, 9].

Given the expertise required and cost of standard diagnostic techniques, quantitative investigation of EMT biomarkers of clinical importance may provide better understanding on potential functions of EMT in cancer initiation and invasion. Studying EMT profile may help urologists to predict the risk associated with tumor recurrence/progression and administer the right treatment to give the maximum benefit to the patients. The present study is taken up to examine the quantitative expression and cellular localization of putative EMT biomarkers immunohistochemically and their prognostic relevance in patients of histologically proven bladder cancer.

Materials and Methods

Clinical Samples

Patients were examined with the symptoms of hematuria as a major sign followed by urinary frequency or irritative symptoms. Assessment for primary tumors included bimanual examination under anesthesia before and after endoscopic surgery (biopsy or transurethral resection) or histologic verification for the absence or presence of tumor. Tumor stage and grade were identified using tumor-node-metastasis (TNM) classification according to WHO-ISUP, 2004 guidelines [10]. Imaging techniques were used for evaluation of lymph nodes. Imaging of the chest, abdominal ultrasound, and computerized tomography of the abdomen whenever required were performed to detect common metastatic sites. A cohort of 65 formalin-fixed paraffin-embedded tumor tissues of patients diagnosed with UCB were examined in the present study after taking their informed consent. Tissues’ blocks/sections were retrieved from the archives of the Department of Pathology at Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India, after obtaining ethical clearance from the Institutional Bioethical Cell between 2014 and 2016 (Institutional Ethical Committee (IEC) no. 2014-166-CP-80). Surgical procedure employed was either transurethral resection of bladder tumor (TURBT) in 84.6% cases or radical cystectomy in 15.4% cases. According to histopathology reports, 7 out of 10 radical cystectomy patients were examined to have lymph node positivity. Nevertheless, patients who underwent TURBT were assessed for clinical staging (based on radiological examinations) to diagnose lymph node positivity. Lymphadenopathy (lymph node enlargement) was reported in only 3 out of 55 patients in transurethral resected tumors. This was followed by pathological staging which ruled out the possibility of lymph node involvement. Clinicohistopathological variables of the patients were obtained from histopathology reports and subsequent follow-up and OPD visits of patients (Table 1).
Table 1

Clinicohistopathological profile of patients diagnosed with urothelial carcinoma of bladder

Clinicopathological variables

Number [n (%)]

Total no. of patients

65 (100)

Age (years) median, range

60, 38–82

 ❖n < 60

26 (40)

 ❖n ≥ 60

39 (60)

Gender

 ❖Males

61 (93.8)

 ❖Females

4 (6.2)

Hematuria

 ❖Present

54 (83.07)

 ❖Absent

11 (16.93)

Smoking/tobacco chewing status

 

 ❖Smokers

34 (52.3)

 ❖Non-smokers

19 (29.2)

 ❖No information

12 (18.5)

Tumor type

 ❖UCB

65 (100)

 ❖SCC/others

0 (0)

Tumor grade

 ❖Low

18 (27.7)

 ❖High

47 (72.3)

Tumor stage

 ❖Ta-T1 (low/NMIBC)

35 (53.8)

 ➢Low grade

18 (51.4)

 ➢High grade

17 (48.6)

 ❖T2-T4 (high/MIBC)

30 (46.2)

 ➢Low grade

0 (0)

 ➢High grade

30 (100)

Tumor type

 ❖Primary

34 (52.3)

 ❖Recurrent

31 (47.7)

Surgical procedure

 ❖TURBT

55 (84.6)

 ❖Radical cystectomy

10 (15.4)

 ➢N0

3/10 (30)

 ➢N1

7/10 (70)

Metastasis

 ❖M0

3/10 (30)

 ❖M1

7/10 (70)

Follow-up available for 56/65 patients

 ❖Progression

17/56 (30.3)

 ❖Death

17/56 (30.3)

 ❖Death due to cancer

6/17 (35.2)

NMIBC, non-muscle-invasive bladder cancer; MIBC, muscle-invasive bladder cancer; TURBT, transurethral resection of bladder tumor; UCB, urothelial carcinoma of bladder; SCC, squamous cell carcinoma

Antibodies

Primary antibodies used in the study include antibodies to E-cadherin (EP700Y; 246R-15; Cell Marque, Rocklin, CA) (1:100 dilution), N-cadherin (6G11; M3613; Dako, Carpinteria, CA) (1:50 dilution), vimentin (SP20; 347R-15; Cell Marque) (1:50 dilution), Snail (Ab180714; Abcam, Cambridge, CA) (1:100 dilution), and Slug (H-140; sc-15,391; Santa Cruz Biotechnology, Santa Cruz, CA) (1:50 dilution). Immunohistochemical (IHC) staining for marker proteins was performed using UltraVision Quanto detection system, horseradish peroxidase, and diaminobenzidine (Thermo Fisher Scientific Life Sciences), according to the manufacturer’s instructions.

Immunohistochemical Staining

A 3-μ-thick tumor tissue sections from paraffin-embedded tumor blocks were cut using a microtome and taken onto poly-l-lysine-coated glass slides. Sections were fixed at 50°–70 °C for 3–4 h. Tissue sections were then deparaffinized using 100% xylene for 5 min and washed with absolute, 70%, 50%, and 20% alcohol for 5 min, each followed by washing the slides in running water. Subsequently, sections were subjected for antigen retrieval by carrying out two cycles of 15 min each at 98 °C in either citrate (low pH) buffer or Tris-EDTA (Tris ethylenediaminetetraacetic acid) (high pH) buffer. Buffer and its pH conditions depend on the type of primary antibody used. After washing two to three times, tissue sections were treated with 3% hydrogen peroxide in methanol for 30 min to block the activity of endogenous peroxidase. This is followed by protein blocking using skimmed milk powder in 1% wash buffer. Tissue sections were incubated with 100 μl of the primary antibodies in dark for about an hour followed by addition of 40 μl of the enhancer (secondary antibody conjugated horseradish peroxidase) for 10 min at room temperature. Browning of the product was detected after addition of diaminobenzidine and hydrogen peroxide as chromogen and substrate respectively. Counter staining was done with Mayers’ hematoxylin for 1 min followed by its mounting using dibutyl phthalate xylene (DPX) solution.

Immunohistochemical Evaluation

Immunohistochemical evaluation of biomarkers for their expression and cellular localization was done using a Nikon Eclipse 50i light microscope (Nikon, Tokyo, Japan). About 1000 cells were considered in consecutive fields at a magnification of × 400. Immunoreactivity of the tumor tissue was analyzed on the basis of IHC score. IHC score was tabulated by multiplying the percentage of positive cells and staining intensity. Staining intensity was scored on the scale of 0 to 3, where 0 represents no expression, 1 weak expression, 2 moderate expression, and 3 strong expression of marker proteins.

IHC score of more than 200 was considered as strong expression and less than 200 as weak expression for E-cadherin, Snail, and Slug. Any immunohistochemical expression of N-cadherin and/or vimentin in tumor cells represents their novel expression (IHC score of 0 means no expression and > 0 represents novel expression).

Statistical Analysis

Quantitative expression of EMT biomarkers was statistically examined with clinicohistopathological features using non-parametric Mann-Whitney U test and Moses test, independent t test, and one sample t test (SPSS version 20.0 software; IBM). Spearman correlation test was used to deduce inter-correlation among aforesaid EMT markers. Kaplan-Meier method with logrank test was done to examine the association between EMT profile and recurrence-free survival (RFS)/progression-free survival (PFS). Time period between the date of first surgery as an entry date and the date of tumor recurrence post TURBT or last follow-up was used to identify the period of assessment for RFS. Time period between the date of surgery and the date of tumor progression/last follow-up/death of patient was examined to assess PFS. Experiments were done in triplicate and data are presented as mean ± standard error of the mean. The p values equal or less than 0.05 were considered statistically significant.

Results

Dynamics of EMT Cascade and Phenotypic Transition in UCB

EMT phenotype is associated with the changes in the epithelial morphology of cells. Tightly adherent cuboidal-shaped cells growing as discrete colonies with apicobasal polarity undergo transition to cells with poor adherence property and mesenchymal and stellate morphology. Immunohistochemical staining was performed to examine the putative EMT phenotype in a cohort of 65 UCB patients and the results were correlated with the established clinicohistopathological predictors to evaluate their association with aggressiveness of disease (Table 2). Figure 1 panels 1 to f present graphical representation of percent immunohistochemical (IHC) expression of EMT markers (E-cadherin, N-cadherin, vimentin, Snail, and Slug) in 65 tumors classified according to tumor stage, tumor grade, tumor type, hematuria, smoking/tobacco chewing status, and age in years.
Table 2

Expression analysis and immunohistochemical (IHC) score of epithelial-to- mesenchymal transition (EMT) associated markers in sixty five patients with urothelial carcinoma of bladder

Clinicopathological parameters

E-Cadherin

N-Cadherin

Vimentin

Snail

Slug

IHC score ≤ 200 (n = 10)

IHC score > 1 (n = 11)

IHC score > 1 (n = 9)

IHC score > 200 (n = 20)

IHC score > 200 (n = 12)

Percent

p

Percent

p

Percent

p

Percent

p

Percent

p

Grade

 

0.8

 

0.00**

 

0.5

 

0.9

 

0.73

 Low grade

2/10 (20%)

 

5/11 (45.5%)

 

4/9 (44.4%)

 

9/20 (45%)

 

5/12 (41.6%)

 

 High grade

8/10 (80%)

 

6/11 (54.5%)

 

5/9 (55.6%)

 

11/20 (55%)

 

7/12 (58.4%)

 

Stage

 

0.6

 

0.3

 

0.5

 

0.78

 

0.61

 NMIBC

3/10 30%)

 

6/11 (54.5%)

 

4/9 (44.4%)

 

12/20 (60%)

 

7/12 (58.4%)

 

 MIBC

7/10 (70%)

 

5/11 (45.5%)

 

5/9 (55.6%)

 

8/20 (40%)

 

5/12 (41.6%)

 

Age 58 (38–78)

 

0.28

 

0.7

 

0.78

 

0.029**

 

0.37

 < 60

6/10 (60%)

 

4/11 (36.4%)

 

2/9 (22.2%)

 

7/20 (35%)

 

4/12 (33.3%)

 

 ≥ 60

4/10 (40%)

 

7/11 (63.6%)

 

7/9 (77.8%)

 

13/20 (65%)

 

8/12 (66.7%)

 

Type of tumor

 

0.41

 

0.8

 

0.00**

 

0.55

 

0.87

 Primary

5/10 (50%)

 

8/11 (72.7%)

 

7/9 (77.8%)

 

10/20 (50%)

 

7/12 (58.4%)

 

 Recurrent

5/10 (50%)

 

3/11 (27.3%)

 

2/9 (22.2%)

 

10/20 (50%)

 

5/12 (41.6%)

 

Hematuria

 

0.017*

 

0.33

 

0.3

 

0.046**

 

0.008***

 Present

7/10 (70%)

 

10/11 (90.9%)

 

6/9 (66.6%)

 

16/20 (80%)

 

10/12 (83.3%)

 

 Absent

3/10 (30%)

 

1/11 (9.1%)

 

3/9 (33.3%)

 

4/20 (20%)

 

2/12 (16.7%)

 

Smoking/tobacco chewing

 

0.69

 

0.26

 

0.00**

 

1

 

0.00**

 Yes

4/10 (40%)

 

6/11 (54.5%)

 

4/9 (44.4%)

 

11/20 (55%)

 

10/12 (83.3%)

 

 No

3/10 (30%)

 

5/11 (45.5%)

 

4/9 (44.4%)

 

4/20 (20%)

 

2/12 (16.7%)

 

 Unknown

3/10 (30%)

 

0/11 (0%)

 

1/9 (11.2%)

 

5/20 (25%)

 

0/12 (0%)

 

IHC score, immunohistochemical score; NMIBC, non-muscle-invasive bladder cancer; MIBC, muscle-invasive bladder cancer; *Mann-Whitney U test; **Moses non-parametric test; ***Independent sample t test; p ≤ 0.05 considered to be significant as shown in italics

Fig. 1

Graphical representation of percent immunohistochemical (IHC) expression of epithelial-to-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, vimentin, Snail, and Slug) in 65 urinary bladder tumors. Percent IHC staining/expression analysis in tumors classified according to a tumor stage, b tumor grade, c tumor type, d hematuria, e smoking status, and f age in years. LS, low-stage/non-muscle-invasive bladder cancer; HS, high-stage/muscle-invasive bladder cancer; LG, low grade; HG, high grade; P, primer; R, recurrent; +, present; -, absent; S, smoking; NS, no smoking; A≥60, age more than or equal to sixty years; A < 60: Age less than sixty years

E-Cadherin Expression

Immunostaining of membranous E-cadherin was although observed to be uniform and strong with almost 100% of the cells being positive, however, focal loss in its membranous expression (IHC score ≤ 200) was seen in 8.5% (3/35) NMIBC cases and 23.3% (7/30) MIBC cases (Figs. 2a, 3a, 4a). Nuclear/cytoplasmic expression of E-cadherin was not observed. E-Cadherin loss showed statistical association with tumor grade (p = 0.005, one sample t test) and hematuria (p < 0.001, independent sample t test) in MIBC cases. No statistical association between loss in expression of E-cadherin and clinicohistopathological parameters was reported in the NMIBC cases.
Fig. 2

Immunohistochemical (IHC) staining for EMT markers at the magnification of × 400. a Positive control for E-cadherin membranous expression in the normal urothelium. b Positive internal control for N-cadherin showing positive stromal area and negative epithelial/urothelial layer. c Positive internal control for vimentin showing positive stromal area and negative epithelial/urothelial layer. d Negative control for Snail. e Negative control for Slug. EMT, epithelial-to-mesenchymal transition

Fig. 3

Immunohistochemical (IHC) staining for EMT markers in non-muscle-invasive bladder cancer cases. a Hundred percent cells positive for E-cadherin membranous staining. b About 70% cells showing N-cadherin membranous immunopositivity. c About 1–2% cells showing vimentin membranous immunopositivity. d About 80–90% cells showing Snail nuclear expression; e About 90% cells showing Slug nuclear expression and IHC positivity. EMT, epithelial-to-mesenchymal transition

Fig. 4

Immunohistochemical (IHC) staining in muscle-invasive bladder cases. a Loss of E-cadherin expression showing < 100% positivity. b About 70–80% cells positive for N-cadherin staining. c About 4–5% cells showing vimentin immunopositivity. d Hundred percent immunopositivity for Snail staining. e Hundred percent immunopositivity for Slug staining. EMT, epithelial-to-mesenchymal transition

N-Cadherin Expression

IHC staining exhibited novel membranous expression of N-cadherin (IHC score > 0) in 17.1% (6/35) NMIBC and 16.6% (5/30) MIBC cases (Figs. 2b, 3b, 4b). Internal controls for N-cadherin were examined for the absence of any expression in urothelial area; however, strong staining was noted in stromal area (Fig. 2b). Maximum IHC score observed for N-cadherin was 240, where tumor areas exhibited moderate to strong intensity. Novel membranous expression of N-cadherin showed statistical association with hematuria (p = 0.011, one sample t test) in NMIBC cases. However, none of the clinicohistopathological variable exhibited any statistical correlation in MIBC patients.

Vimentin Expression

Internal control for vimentin showed strong stromal expression and no immunostaining in urothelial area (Fig. 2c). Novel membranous expression of vimentin was rarely noted throughout the tumor area; however, its limited focal localization (IHC score > 0) was observed in 11.4% (4/35) NMIBC and 16.6% (5/30) MIBC cases (Figs. 3c, 4c). Maximum IHC score for vimentin focal localization towards the invasive front of infiltrating tumor (MIBC) was observed as 30. Statistical analysis reported significance of novel membranous immunoexpression of vimentin with tumor grade and age of MIBC patients (p < 0.001, p < 0.001, one sample t test).

Snail Expression

Immunohistochemical analysis witnessed an apparent enrichment in strong (20/65 (30.7%)) versus weak (45/65 (69.3%)) nuclear expression of Snail in urothelial area in the cohort of 65 samples. Snail showed non-specific cytoplasmic staining; however, only nuclear staining was taken into account during analysis. Adjoining normal urothelium occasionally exhibited weak immunoreactivity to Snail (IHC score less than 100). Strong nuclear expression (IHC score > 200) of Snail was observed in 34.2% (12/35) NMIBC cases and 23.3% (7/30) MIBC cases (Figs. 2d, 3d, 4d). Negative control for Snail was taken to compare its expression with the tumor specimens (Fig. 2d). Statistical association was observed between nuclear gain in Snail expression and tumor grade both in NMIBC and MIBC cases (p < 0.001, Moses non-parametric test; p < 0.001, one sample t test) respectively. Statistical relevance was noted between nuclear immunoexpression of Snail and hematuria in MIBC patients (p = 0.007, independent sample t test).

Slug Expression

Slug showed apparent enrichment in strong (12/65 (18.5%)) versus weak (53/65 (81.5%)) nuclear expression in urothelium in the cohort of 65 samples. Non-specific cytoplasmic staining of Slug was not considered during analysis. Adjoining normal urothelium demonstrated weak nuclear immunoreactivity for Slug (IHC score less than 100). Strong nuclear expression of Slug (IHC score > 200), pivotal for EMT cascade, was observed in 20% (7/35) NMIBC and 16.6% (5/30) MIBC cases (Figs. 2e, 3e, 4e). Negative control was taken for Slug to compare its expression with the tumor specimens (Fig. 2e). Nuclear gain in Slug immunoexpression showed significance with tumor type in NMIBC cases (p < 0.001, Moses non-parametric test) and with tumor grade in MIBC cases (p < 0.001, one sample t test).

Correlation Among the Expression of EMT Markers

Statistically relevant correlation was reported between focal loss of membranous E-cadherin and strong nuclear immunoexpression of Slug (p = 0.009, Spearman’s correlation coefficient). The rest of the aforementioned markers did not demonstrate any correlation.

Association Between EMT Profile and Survival of Patients

Follow-up data was available and compiled for 56 out of 65 patients. An average time point of 24 months was taken for RFS and PFS analysis. Out of 56, 17 patients (30.3%) were presented with disease progression and 17 patients (30.3%) died during the follow-up. Out of the 17 deaths, 6 died due to cancer whereas the remaining 11 died due to other reasons, e.g., cardiac arrest. Kaplan-Meier along with logrank statistics determined an association between EMT profile and RFS in NMIBC patients (p < 0.001) (Fig. 5a). The present study fails to deduce any relevance between EMT profile and PFS of MIBC patients (Fig. 5b).
Fig. 5

Kaplan-Meier survival curves with respect to the EMT profile in bladder cancer patients. a Recurrence-free survival (RFS) in non-muscle-invasive bladder cancer patients. b Progression-free survival (PFS) in muscle-invasive bladder cancer patients

Discussion

Decisions regarding treatment modalities are currently based on TNM staging. It provides information on morphology of tumor cells but fail to provide risk assessment for RFS/PFS probability of patients. Burning of the tissue during cauterization makes it difficult to identify the involvement of either stromal or superficial muscular layer in invasion and thus complicates the tumor staging/TNM classification. Owing to the limitations of classical clinicohistopathological staging of tumor, examination of molecular markers of clinical relevance is important for accurate prediction of recurrence, progression, and clinical/therapeutic outcome [11]. Detailed understanding on the complex behavior of EMT in bladder tumors is essential to yield them as diagnostic and prognostic markers/tools in clinical setting [12, 13, 14, 15]. The current study evaluates panel of five putative EMT markers immunohistochemically and their association with histopathological variables in NMIBC and MIBC patients. EMT profile (the presence or absence of molecular alterations) was examined for its association with survival probabilities (RFS/PFS) of the patients.

Diminution/loss of membranous E-cadherin is established as a well-known primary event in epithelial tumorigenesis [16, 17]. Based on immunostaining results, the present study fails to demonstrate any significant association of E-cadherin loss in NMIBC patients. Nevertheless, its focal loss has been examined in 23.3% of MIBC patients. In accordance with the study by Hu et al., strong association is reported between E-cadherin loss and grade in advanced stage tumors [18]. Results contradict earlier studies, where the prognostic significance of E-cadherin loss was examined with tumor invasiveness and nodal metastasis [19, 20]. Stated loss of E-cadherin in tumors from hematuric muscle-invasive patients defines EMT as an early event in bladder muscle invasiveness. This observation hypothesizes its function in triggering biological pathway that culminates into hematuria.

N-Cadherin and vimentin immunoexpressions are reported in urothelial tumor cells with stellate and fibroblast morphology. Occasional stromal positivity of these proteins is noted in tumor cells which indicate their role in tumor cell motility and aggressiveness. Novel immunostaining of membranous N-cadherin and vimentin represents the defining feature of EMT in bladder tumorigenesis [9, 21, 22, 23]. Consistent with our previous report, novel/focal expression of N-cadherin in epithelial/urothelial area has been observed to be significantly associated with tumor grade (p = 0.001) after combining all the stages together [9]. This finding defines infiltrative feature of tumor cells due to the morphological transformation of epithelial cells into spindle-shaped cells and thereby poor prognosis [21, 23, 24]. Given the established invasive functions of vimentin, its statistical significance with tumor grade (p < 0.001) in MIBC patients identifies these tumors at high risk to invade and progress. Study by Baumgart et al. reported strong relevance of vimentin with tumor stage and grade but failed to deduce any significance of N-cadherin [21]. Current results report an association between vimentin immunopositivity and smoking/tobacco chewing status of patients (p = 0.001) after combining all the stages together. These results are in concordance with the study by Liang et al. who examined the effects of tobacco smoke-triggered EMT alterations in mouse bladder tissue [25].

Dynamics of EMT-ATFs including Snail and Slug point towards its pivotal role in aggravating the disorganization of the epithelial cells by repressing membranous E-cadherin [26]. The present study fails to demonstrate the concurrent E-cadherin loss and nuclear gain of Snail and Slug in same tumors and therefore, unlike previous findings, it does not support the involvement of Snail/Slug in cadherin switch [7]. Statistical association between hematuria and nuclear immunopositivity of Snail (p = 0.007) in muscle-invasive patients conforms to the hypothesis that Snail triggers the biological pathway important for hematuria. In concordance with recent study, Snail expression has been observed to be an independent predictor of tumor grade in patients with NMIBC and MIBC [27]. In contrast to the previous studies, Snail does not exhibit positive correlation with tumor stage, grade, recurrence and lymph node metastasis, and PFS [28]. Wu et al. identified elevated Slug in invasive or metastatic bladder cancer and its critical role in EMT via control of cadherin switch [7]. Strong association of nuclear gain of Slug with tumor type (p < 0.001) in non-muscle-invasive and tumor grade (p < 0.001) in muscle-invasive patients predicts its role in tumor recurrence in low-stage tumors and in the development of muscle invasion. Current findings on inter-correlation between E-cadherin loss and strong nuclear expression of Slug hypothesize the fact that the two events although influence one another but may not occur at same time frame and may get interrupted by number of cross talks [21].

Expressions of E-cadherin, N-cadherin and vimentin, and Snail and Slug have been evaluated as significant prognostic factors in predicting RFS in non-muscle-invasive patients [29, 30].

Immunohistochemical analysis of EMT associated biomarkers provides insight into the molecular pathogenesis of UCB and offers the potential to accurately predict survival outcomes. Nevertheless, multi-institutional studies involving phased biomarker analysis with large number of clinical specimen are still needed to validate the accuracy of current prediction models.

Notes

Acknowledgements

One of the co-authors, RS is thankful to University Grants Commission (UGC), Govt. of India for providing research fellowship.

Funding Information

This study received research grant (SR/SO/HS-0113/2010) from the Department of Science and Technology (DST), Govt. of India.

Compliance with Ethical Standards

This study does not involve any clinical trial on human participants.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of LucknowLucknowIndia
  2. 2.Uro-oncology and Minimally Invasive SurgeryFortis Escorts Kidney & UrologyNew DelhiIndia
  3. 3.Department of PathologySanjay Gandhi Post Graduate Institute of Medical SciencesLucknowIndia

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