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ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 4  |  Page : 1149-1154

The diagnostic role of vimentin in differentiation between muscularis propria and muscularis mucosa in urinary bladder carcinoma


Department of Pathology, Faculty of Medicine, Menoufia University, Shibin El Kom, Egypt

Date of Submission06-Sep-2016
Date of Acceptance25-Nov-2016
Date of Web Publication04-Apr-2018

Correspondence Address:
Noha H Ghanem
Department of Pathology, Faculty of Medicine, Menoufia University, Shibin El Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.229226

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  Abstract 


Objective
The aim of this study was to evaluate the diagnostic value of vimentin expression for differentiation between muscularis mucosa (MM) and muscularis propria (MP) in urinary bladder carcinoma cases.
Background
The single most important prognostic factor in urothelial carcinoma is the pathological stage, which includes the anatomic depth of invasion on which major therapeutic decisions are made. Correct assessment of invasion necessitates discrimination between MM and MP. Vimentin is the most widely distributed intermediate filament and is expressed in virtually all mesenchymal tissues.
Materials and methods
This study was carried out on 70 urinary bladder specimens, including 59 cases of primary urinary bladder carcinoma and 11 cystitis specimens. The specimens were submitted to vimentin immunostaining under the streptavidin–biotin amplified system.
Results
MM was identified histologically in 40 cases, whereas MP was morphologically designated in all cases (70 cases). Vimentin was negative in MP and showed positive expression in 32/40 (80%) cases of MM with a statistically significant difference (P = 0.000), 80% of sensitivity, and 100% of specificity.
Conclusion
Negative vimentin expression in MP could be a useful tool for identification of MP with 80% of sensitivity and 100% of specificity in urinary bladder carcinoma cases.

Keywords: muscularis mucosa, muscularis propria, urinary bladder carcinoma, vimentin


How to cite this article:
Kandil MA, Abdou AG, El-Kady NM, Ghanem NH. The diagnostic role of vimentin in differentiation between muscularis propria and muscularis mucosa in urinary bladder carcinoma. Menoufia Med J 2017;30:1149-54

How to cite this URL:
Kandil MA, Abdou AG, El-Kady NM, Ghanem NH. The diagnostic role of vimentin in differentiation between muscularis propria and muscularis mucosa in urinary bladder carcinoma. Menoufia Med J [serial online] 2017 [cited 2018 May 21];30:1149-54. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1149/229226




  Introduction Top


Urinary bladder carcinoma is one of the most common malignancies in Egypt and all over the world. In Egypt, it represents the third most common malignancy as it accounts for 6.94% of total malignancies in both sexes according to the National Population-Based Cancer Registry Program (2008–2011)[1], 8.7% according to the National Cancer Registry (2014)[2], and 7% according to the Ain-Shams Cancer Registry (2001–2010)[3].

In the USA, the incidence is 36.4 in men and 9.0 in women per 100 000 (2008–2012) with an estimated 76 960 new cases and 16 390 deaths in 2016[4].

The single most important prognostic factor in urothelial carcinoma is the pathological stage, which includes the anatomic depth of invasion on which major therapeutic decisions are made[5]. Similar to noninvasive tumors, a carcinoma confined to the lamina propria (LP) is usually treated conservatively, whereas one extending into the muscularis propria (MP) almost always dictates more aggressive management[6]. Although this might appear relatively straightforward, there are several well-known pitfalls in the pathological staging of bladder carcinoma [6–8] that one should be aware of to avoid both understaging and overstaging of bladder carcinoma. This overstaging caused by misdiagnosis of muscularis mucosa (MM) as MP leads to unnecessary aggressive management (including cystectomy) of a tumor that would otherwise be treated conservatively (intravesical bacillus calmette–guerin therapy). Understaging will lead to improper treatment with an increased rate of death[6],[9].

Differentiation between MM and MP can be made on the basis of histological form, histological relations, or antigenic properties. MM is usually dispersed, has an interrupted layer, and is usually associated with vascular plexus,[10] whereas MP forms regular bundles[11]. However, hyperplastic MM could be appreciated in 17–53% of specimens simulating thick bundles of MP[11],[12]. The vascular plexus is absent in MP, but it can be absent in MM as well, especially in transurethral resection of urinary bladder tumor (TURBT) specimens. Moreover, TURBT specimens are not rare to be of low quality and low quantity, leading to disorientation of the specimens. There are topographical variations of the MP muscle bundles in bladder trigone and at the insertion of the ureter[11],[13]. The trigonal MP extends more superficially and often shows gradual diminution of the bundles' size as they extend to an almost suburothelial location.

Adipose tissues cannot be used as landmarks either because they can be seen in between MP muscle bundles or because they extend superficially to the LP[14]. The immunological profile seems to be a reliable tool for the differentiation of MM and MP. However, until today, data are very limited and no widely accepted antigen has yet been identified as a sensitive marker.

Vimentin is the most widely distributed intermediate filament (IF) and is expressed in virtually all mesenchymal tissues[15]. Studies show that the smooth muscle cells of the MP rarely and weakly express vimentin, whereas smooth muscle cells of the MM have moderate or strong expression[16].

Therefore, our study aimed at evaluation of the diagnostic value of vimentin expression for differentiation between MM and MP.


  Materials and Methods Top


The protocol was approved by the Ethics committee of the faculty and an informed written consent was obtained from all participants. This is a retrospective study conducted on 70 urinary bladder specimens (59 cases of primary urinary bladder carcinoma received as cystectomy specimens and 11 cystitis cases received as TURBT specimens) after obtaining approval from the ethics committee. The specimens were retrieved from the archives of the Pathology Department, Faculty of Medicine, Menoufia University, spanning the period between January 2014 and January 2016. They were selected on the basis of the presence of muscle layers either in neoplastic or non-neoplastic cases and the ability to differentiate MP from MM. Cases with overlapping features were excluded.

Several sections of 4 μm thickness were cut from each block. One was stained with routine hematoxylin and eosin to confirm the diagnosis and to evaluate the histopathologic characteristics of malignant cases including the type, grade, and muscle invasion. Further evaluation and distinction between MM and MP were made in each case. Other sections were cut on poly-l-lysine-coated slides to be immunostained with vimentin.

Evaluation of specimens (malignant and cystitis groups) to differentiate muscularis mucosa from muscularis propria

MM is characterized by thin slender bundles arranged in a single layer of interrupted, wispy dispersed, or continuous but often discontinuous smooth muscle fibers[7],[17].

Hyperplastic patterns of MM could be (a) aggregates of hyperplastic MM with haphazard outlines distinct from that of MP and (b) hyperplastic compact MM with parallel muscle fibers and regular outline arranged singly or in small groups that occasionally strongly resembled an MP muscle but distinguishable from it on the basis of the location in the LP[11]. Commonly the LP vascular plexus is present in every section, most often in association with the MM muscle[10].

On the contrary, MP is characterized by coarse muscle bundles with rounded contours. The MP most commonly has a relatively regular interface with the LP. A distinctive pattern was noted in the trigone where occasionally there was a gradual diminution of the size of the MP muscle bundles as they extended to almost a suburothelial location[11].

Immunohistochemistry

Immunostaining was carried out using the streptavidin–biotin amplified system with the Universal Dakocytomation-labeled streptavidin–biotin-2 system, using horseradish peroxidase (LSAB-2 System, HRP Kit, Catalog No. k0679, Dako, Carpinteria, CA, USA). The slides were deparaffinized using xylene and then rehydrated in decreasing concentrations of ethanol. Antigen retrieval using microwave heating (20 min; 10 mmol/citrate buffer, pH 6.0) after inhibition of endogeneous peroxidase activity (hydrogen peroxidase for 15 min) was performed. The primary antibody, mouse monoclonal antibody against vimentin (V9) (MS-129-P; Thermo Scientific, Waltham, MA USA), received as a 1 ml concentrate and diluted by PBS in a dilution 1:50, was applied on the slides and the slides were incubated overnight at room temperature in a humidity chamber. Finally, detection of the bound antibody was accomplished using a modified labeled avidin–biotin reagent for 20 min, followed by a PBS wash. A 0.1% solution of diaminobenzidine was used for 5 min as a chromogen. Slides were counterstained with Mayer's hematoxylin for 5–10 min. Negative control slides were prepared by omitting the primary antibodies from the staining procedure. Tissue sections prepared from leiomyosarcoma were used as a positive control for vimentin.

Immunostaining interpretation

Vimentin expression was evaluated in non-neoplastic and malignant groups.

Any cytoplasmic staining in any number of cells is required to assign positivity. Vimentin expression was evaluated in both MM and MP[16].

Statistical analysis

Data were collected, tabulated, and statistically analyzed using a personal computer with statistical package for the social sciences (SPSS, version 22; SPSS Inc., Chicago, Illinois, USA).

Statistical calculations were made to assess the diagnostic values of qualitative data, such as intensity of expression.

Accuracy = true-positive+true-negative cases/total number of examined cases.

Sensitivity = true-positive cases/true-positive+ false- negative cases×100.

Specificity = true-negative/true-negative+false-positive cases×100.

Positive predictive value = true-positive cases/true- positive+false-positive cases×100.

Negative predictive value = true-negative cases/true -negative+false-negative cases×100.


  Results Top


Histologic differentiation between muscularis mucosa and muscularis propria

Histopathological examination identified MM in all cystitis cases (11/11) and in 29/59 malignant cases, with a total number of 40 cases. MM was masked by invading tumor cells in the remaining 30 cases of the malignant group (59 cases); hence, it was difficult to be identified.

MP was found in 70 cases (11 cystitis and 59 malignant).

The clinicopathological characteristics of the cystitis group

The patients' ages ranged between 50 and 65 years, with a mean ± SD of 56.36 ± 4.84 years and a median of 55 years. Eight (72.7%) patients were male and three (27.3%) patients were female, with a male to female ratio of 2:7. Four types of cystitis were detected: chronic nonspecific in four (36.3%) cases, polypoid in three (27.3%) cases, bilharzial in two (18.2%) cases, and metaplastic in two (18.2%) cases.

Clinicopathological characteristics of the malignant group

The patients' ages ranged between 45 and 65 years, with a mean age ± SD of 55.47 ± 6.95 and a median of 57 years. Thirty-nine (66.1%) patients were male and 20 (33.9%) were female, with a male to female ratio of 1:95.

Grossly, malignant tumors presented as mass in 45/59 (76.3%) cases, ulcer in 14/59 (23.7%) cases.

Twenty-three of 59 (38.98%) cases were transitional cell carcinoma and 36 of 59 (61.1%) were squamous cell carcinoma.

Squamous cell carcinoma grading revealed 19/59 (32.2%) cases of grade 3, 14/59 (23.7%) of grade 2, and 3/59 (5.1%) of grade 1. However, transitional cell carcinoma grading revealed 16/59 (27.1%) of high grade and 7/59 (11.9%) of low grade.

Thirty-nine (66.1%) cases presented at advanced stage as 19/59 (32.2%) cases belonged to stage III and 20/59 (33.9%) were of stage IV; 7/59 (11.9%) cases were diagnosed as stage I and 13 (22%) cases were diagnosed as stage II.

Lymph nodes were positive in 25/59 (42.4%) cases and negative in 34/59 (57.6%) cases. Bilharziasis was present in 34/59 (57.6%) cases and absent in 25/59 (42.4%) cases. MP invasion was seen in 52/59 (88.1%) cases and was absent in 7/59 (11.9%) cases.

Immunohistochemical results of vimentin expression in muscularis mucosa and muscularis propria

MM showed cytoplasmic expression of vimentin in 35/40 (87.5%) cases [Figure 1]. Five cases were negative [5/40 (12.5%)] [Table 1].
Table 1: Comparison between muscularis mucosa and muscularis propria as regarding vimentin expression

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Figure 1: Muscularis mucosa show moderate expression of vimentin.

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All MP of examined cases were negative for vimentin [Figure 2]. This absent vimentin expression in the MP was limited to smooth muscle bundles. However, moderate and strong expression was seen in the endomysial and endothelial cells interspersed between the smooth muscle fibers [Figure 2].
Figure 2: Muscularis propria was negative to vimentin except few endomysial and endothelial cells interspersed between the smooth muscle fibers.

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The diagnostic validity of vimentin in differentiation between muscularis mucosa and muscularis propria

Use of vimentin expression for identification of MM revealed 80% sensitivity, 100% specificity, 100% positive predictive value, 89.74% negative predictive value, and 92.73% accuracy [Table 2].
Table 2: The diagnostic validity of vimentin expression for differentiation between muscularis mucosa and muscularis pro

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  Discussion Top


Vimentin is a protein that in humans is encoded by the VIM gene. Vimentin is a type-III (IF)[18] protein having a molecular weight of 57 kDa that is expressed in mesenchymal cells. IF, along with tubulin-based microtubules and actin-based microfilaments, comprises the cytoskeleton, with vimentin and desmin being the major constituents of the IF networks in smooth muscles. All IF proteins are expressed in a highly developmentally regulated manner; vimentin is the major cytoskeletal component of mesenchymal cells[19]. Because of this, vimentin is often used as a marker of mesenchymally derived cells or cells undergoing an epithelial-to-mesenchymal transition, during both normal development and metastatic progression[19].

Vimentin plays a significant role in supporting and anchoring the position of the organelles in the cytosol. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally[20].

Vimentin expression in muscle cells varies with the development stage, being expressed in early stages and disappearing in the later stages[21]. Three studies had suggested the role of vimentin in differentiation between MM and MP [16],[22],[23].

In the present study, vimentin showed 80% sensitivity and 100% specificity according to the present study. Loss of expression of vimentin in MP according to our results was not expected as vimentin is positive in virtually all cells, but this absence of expression agreed with other studies [16],[22],[23]. This absence may be explained by the dynamic nature of vimentin in the form of spatial rearrangement and biochemical changes in smooth muscle cells in response to external activation, which is important when offering flexibility to the cell. This could also be explained by vimentin expression in muscle cells varying with the development stage, being expressed in early stages and disappearing in later stages[21] In pathological muscle diseases, strong and diffuse vimentin immunoreactivity reappeared in the fibers[24].

However, the Council and Hameed[16] had demonstrated positive vimentin expression in one MP. This could be explained by re-expression of vimentin in muscles occurring during regeneration as confirmed by Gallanti et al.[24], who found re-expression of vimentin in regenerating muscles. In pathological muscle diseases, strong and diffuse vimentin immunoreactivity reappeared in the fibers[24]. Studying smooth muscle regeneration and maturation in rats bladders in vivo revealed early appearance of vimentin and α-actin within the first 10 days. Distinct desmin expression was observed later, when the first smooth muscle cells were recognized[25].

Our result disagrees with that of Poletajew et al.[23], who found that there is a clinical difference in vimentin expression, but it was not statistically significant. It can be argued that the very limited number in their study (only 11 cases) made it statistically unreliable[23].

The third study was not designed for vimentin but for cadherin expression in the urinary bladder, and differential expression of vimentin was noticed by chance with no further evaluation of vimentin in the study[22].

Vimentin had been used as a prognostic marker indicating epithelial–mesenchymal transition and bad prognosis, but this is beyond our study[26],[27].

Differentiation between MM and MP had been attempted in several studies using different markers. According to Poletajew et al.[23], 12 markers had been used, including actin, caldesmon, type-IV collagen, cytokeratin, desmin, elastin, fibronectin, filamin, laminin, miotilin, smoothelin, and vimentin. The latter study demonstrated stronger expression of desmin, filamin, type-IV collagen, and smoothelin within MP compared with MM, and the reverse was noticed for vimentin[23]. Desmin equally stained MM and MP without significant difference, as per Kamel et al.[28].


  Conclusion Top


Negative vimentin expression in MP can be a useful tool for identification of MP, with 80% sensitivity and 100% specificity in urinary bladder carcinoma cases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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