Fascin determination in urothelial carcinomas of the urinary bladder: a marker of invasiveness.
Abstract: Context.--Invasion and the depth of invasion affect significantly the prognosis in urothelial carcinomas. The histopathologic evaluation of invasion may be problematic in some cases. Application of new immunohistochemical markers may facilitate the assessment of invasion. Fascin, one of these markers, is an actin-bundling protein involved in tumor cell migration. Fascin expression is increased in various carcinomas. Prior to this research, to our knowledge, only one study exists regarding fascin expression in urothelial carcinomas.

Objective.--To characterize the expression of fascin in additional cases of urothelial carcinoma and to verify statistically a relationship between fascin overexpression and invasiveness in these tumors.

Design.--We examined fascin immunoreactivity in 116 specimens of urothelial carcinomas obtained from 116 patients including 96 men and 20 women. Fifty-eight cases were ranked as low-grade carcinomas, pTa stage, and 58 cases were ranked as high-grade carcinomas--11 were ranked as stage pTa, 21 were ranked as pT1, and 26 were ranked as pT2 carcinomas. Fascin immunoreactivity was assessed semiquantitatively in tumor cells. In each case, we ascribed 3 immunoreactivity scores, one for extent, one for intensity, and a combined immunoreactivity score.

Results.--The combined immunoreactivity score was significantly higher in invasive carcinomas. In addition, strong staining was observed exclusively in invasive carcinomas. None of the pTa tumors demonstrated intense staining, including those ranked at the higher grade.

Conclusions.--Our results point to an association between fascin immunostaining and urothelial carcinoma invasiveness and suggest that fascin overexpression may be a marker of aggressive urothelial carcinomas.
Subject: Tumors (Research)
Tumors (Diagnosis)
Tumors (Prognosis)
Cell research
Authors: Karasavvidou, Foteini
Barbanis, Sotirios
Pappa, Dimitra
Moutzouris, George
Tzortzis, Vasilios
Melekos, Michael D.
Koukoulis, George
Pub Date: 12/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: Dec, 2008 Source Volume: 132 Source Issue: 12
Topic: Event Code: 310 Science & research Canadian Subject Form: Tumours; Tumours; Tumours
Accession Number: 230246888
Full Text: Fascin is a 55-kDa globular protein that belongs to a unique family of actin-bundling proteins. Three forms of fascin exist in vertebrates: fascin 1, which is expressed by mesenchymal tissues and in the nervous system; fascin 2, which is expressed by retinal photoreceptor cells; and fascin 3, which is testis specific. (1-4) Fascin 1 (also known as fascin) contributes to the formation of various actin-based structures, including the cellular surface protrusions that mediate cell movement. (5) In vitro studies showed that elevated levels of fascin increased the speed of cell migration and emphasized the relationship between fascin overexpression and motility of transformed cells. (5)

Fascin expression is increased in human carcinomas compared with nonneoplastic epithelia. Several investigators have reported that fascin overexpression may correlate with invasion and metastasis and that fascin may be a new prognostic marker in certain carcinomas. (6-13)

The biologic behavior of urothelial carcinomas is associated with the depth of invasion. To our knowledge, only one previous study exists regarding fascin expression in urothelial carcinomas. (14) One of the aims of our study was to investigate the relationship between fascin expression and invasion of urothelial carcinomas.

MATERIALS AND METHODS

Patients and Surgical Specimens

We assessed the immunostaining of fascin in 116 specimens of urothelial carcinomas from 116 patients who underwent transurethral bladder resections between 2001 and 2003. The specimens were retrieved from the files of the Department of Pathology of the University Hospital of Thessaly. The cases were selected to represent various grades and stages. Selection criteria included good presentation of morphology and antigenicity, that is, cauterized or quantitatively inadequate material was avoided. Strictly speaking these were not consecutive cases. Of 116 patients, the 96 men and 20 women had a mean age of 67.13 years, ranging from 39 to 88 years. No patients reported a history of previous treatment.

The tumors were papillary carcinomas and included 58 cases of low-grade carcinomas, pTa stage, and 58 cases of high-grade carcinomas--11 were stage pTa carcinomas, and the remainder were invasive carcinomas (21 pT1 and 26 pT2). A significant component of nonneoplastic urothelium was noted in 17 specimens.

The samples were fixed in 10% buffered formalin solution and embedded in paraffin blocks. The tumors were graded according to the 2004 World Health Organization classification of urinary tract tumors (15) and staged according to the American Joint Committee on Cancer System of 2002. (16)

Immunohistochemical Procedures

Sections (4 [micro]m) from selected paraffin blocks of each case were obtained. Deparaffinization, rehydration, and antigen unmasking were achieved by boiling sections in Trilogy reagent (Cell Marque, Rocklin, Calif) for a total of 1 hour in a commercially available steamer. After quenching endogenous peroxidase, slides were incubated for 25 minutes with a monoclonal anti-fascin (55k-2) antibody (1:100; Cell Marque) at room temperature. Staining was developed with substrate chromogen solution (EnVision, DAKO, Glostrup, Denmark) and diaminobenzidine for 10 minutes. Slides were counterstained with Harris hematoxylin for 1 minute, dehydrated, and mounted with DPX solution.

Assessment of Immunohistochemical Staining

All slides were initially evaluated by 2 pathologists (F.K., S.B.), blindly and independently. Two aspects of immunoreactivity were evaluated semiquantitatively--extent and intensity. The immunostaining was cytoplasmic. Extent and intensity of cytoplasmic staining varied. These 2 aspects of immunostaining--extent and intensity--were evaluated for statistical analysis. To facilitate the statistical analysis we determined jointly consensus immunoreactivity scores as described in the following. The extent of immunostaining was categorized into 4 groups according to the percentage of immunostained neoplastic cells: less than 25% + 25% to 50% (2+), 50% to 75% (3+), and more than 75% (4+). Predominant intensity of immunostaining of tumor cells was categorized according to the staining of endothelial cells used as internal controls into weak (1+), moderate (2+), or intense (3+ ) categories. The weak staining category was assessed to be less than that of endothelial cells, the moderate staining category was determined to be equal to that of endothelial cells, and the intense staining category exhibited more than that of endothelial cells. For each case, a combined immunoreactivity score was calculated by multiplying the score for extent by the score for intensity. Therefore, the combined immunoreactivity score ranged from 0 to 12.

Continuous parameters were used to compare the 3 separate groups pTa, pT1, and pT2 using the Kruskal-Wallis test (Table 1), whereas continuous parameters were used to compare the 2 groups pTa and p(T1 + T2) using the Mann-Whitney U test with Bonferroni correction (Table 2). An effect was considered significant when P < .05. Statistical analysis was performed using SPSS v. 13.0 (SPSS Inc, Chicago, Ill).

RESULTS

Urothelial pT1 carcinomas and pT2 carcinomas exhibited a statistically significant increase in all 3 parameters used to assess fascin immunoreactivity (combined immunoreactivity score, intensity, extent) when compared with pTa carcinomas (Tables 1 and 2). When pT1 and pT2 carcinomas were compared, no significant differences were detected in any of the parameters used to indicate immunoreactivity. When pT1 and pT2 carcinomas were combined and compared with pTa carcinomas, obvious and significant differences existed in all 3 parameters used to assess fascin immunostaining. Indeed, an intense level of fascin staining was observed exclusively in invasive carcinomas (Figures 1 through 3) and was also detectable in the noninvasive component (Figure 4). Papillary carcinomas without histologically detectable invasion (pTa) did not exhibit any cases with intense immunostaining (Figure 5). This was also true for high-grade pTa carcinomas.

We also noted that immunoreactivity decreased in foci with a marked cauterization artifact. Nonneoplastic urothelium did not demonstrate immunostaining. Reactive urothelium in the presence of inflammation exhibited sparse and weak staining, but intense staining was not detected.

COMMENT

Fascin expression is normally elevated and readily detectable in cells that require a fortified cytoskeleton to maintain dendritic processes, that is, neurons and antigenpresenting dendritic cells, or to support extended surfaces, that is, endothelial cells.17 Fascin is not immunohistochemically detectable in most normal epithelial cells, although it is known to be involved in adherens junctions. Increased fascin expression in neoplastic epithelial tissues, that is, various carcinomas, may be immunohistochemically detectable. Fascin overexpression is also suggested to disrupt epithelial junctions and it may provide an increased potential for invasion and metastasis. Upregulation of fascin has been documented in neoplastic cells of ovarian, colonic, breast, pulmonary, pancreatic, gastric, renal, and skin carcinomas.6-13 Fascin overexpression appears to characterize a subset of biologically aggressive breast carcinomas.6 Moreover, it is an independent prognostic factor in

stage I pulmonary carcinomas8 and in stage III to IV colonic adenocarcinomas18 associated with increased metastatic potential. Based on the previous information and detailed data in the relevant literature, 2 tentative preliminary conclusions are supported: (1) fascin overexpression is associated with carcinomatous change and it may be related to tumor progression and (2) evaluation of fascin overexpression by immunostaining seems justifiable.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

A study by Tong et al (14) was the first to demonstrate fascin overexpression in urothelial carcinomas. In general, their findings are similar to ours and encapsulation of their key observation that a progressive increase of fascin-1 expression from superficial to deeply (into muscle) invasive urothelial carcinomas of the urinary bladder" exists could apply to our findings. We also confirmed their conclusion that fascin expression was not related to tumor grade. However, Tong et al used a different antibody with modifications of the immunohistochemical protocol and they evaluated immunoreactivity differently. The analysis of our data validates statistically the observations from both bodies of research that increased fascin staining correlates with invasion in bladder carcinomas.

The pertinent literature includes many established or provisional progression markers of urothelial carcinoma. Therefore, one could argue that no purpose exists in adding another candidate immunomarker for tumor progression, especially when genomic arrays appear to promise accurate and independent prognostic/predictive evaluation.

First, all progression markers in urothelial carcinomas are not pathobiologically identical. Some reflect loss of TP53 function and others indicate cell-cycle deregulation. (19,20) Among the latter, previous reports include p16, p27, p21, cyclin D1, RB1, surviving/BIRC5, and Aurora A markers. (19-23) Cell-cycle deregulation results in an increased growth fraction, measurable as the MIB-1 labeling index, and in some studies was shown to reflect tumor progression. (24) Most of these biomarkers reflect evolution through currently explored carcinogenetic pathways. Therefore, although they may correlate with tumor stage, they cannot be viewed as markers of invasiveness. In bladder urothelial carcinomas, studies focused on invasion-related markers including stromelysin 3, cathepsins D and E, MMP-2, TIMP, CD44, E- and N-cadherins, motility-related protein 1, and gelsolin. (25-34) A systematic review of these markers is beyond the scope of this discussion. However, gelsolin should be emphasized because it is a neighbor and a coworker of fascin in lamellipodia of motile cells. (35) In lamellipodia, a functionally important actin mesh exists, in which fascin facilitates the assembly and gelsolin the disassembly of actin. (35) In an interesting study, gelsolin was found to be a "strong prognostic indicator of tumor progression" in bladder urothelial carcinomas. (34) Moreover, gelsolin overexpression was associated with invasiveness and tumor recurrence. (34) Therefore, it is not a surprise that fascin is a marker of invasiveness. A larger scale study with a stricter and longer follow-up design could determine if fascin exceeds the prognostic/predictive value of its counterpart, gelsolin. Based on this discussion, it can be concluded that the large number of candidate biomarkers should not prevent the analysis of new ones, especially within a defined pathobiologic context. In regard to the methodologic breakthroughs of genomic arrays, it should be recognized that fascin is expressed in many mesenchymal cells, such as myofibroblasts and endothelial cells, such that total fascin overexpression could measure the extent of desmoplastic reaction or neoangiogenesis instead of genuine fascin upregulation in tumor cells.

In general, a marker of invasiveness could be helpful in overcoming certain histopathologic practical obstacles in urothelial carcinomas. However, the role of fascin as a surrogate marker of invasion has to be determined by additional work that should include prospective clinicopathologic analyses with meticulous statistical evaluation in a large number of cases.

References

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Foteini Karasavvidou, MD; Sotirios Barbanis, MD, PhD; Dimitra Pappa, MD; George Moutzouris, MD, PhD; Vasilios Tzortzis, MD, PhD; Michael D. Melekos, MD, PhD; George Koukoulis, MD, PhD

Accepted for publication May 19, 2008.

From the Departments of Pathology (Drs Karasavvidou, Barbanis, Pappa, and Koukoulis) and Urology (Drs Moutzouris, Tzortzis, and Melekos), University Hospital, Thessaly, Greece.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Foteini Karasavvidou, MD, Department of Pathology, University Hospital of Larissa, 411 10 Larissa, Greece (e-mail: fotkarasa@ yahoo.gr).
Table 1. Comparison of Fascin Expression Among Groups (pTa, pT1,
pT2) of Urothelial Carcinomas *

                                          Groups (pTNM)

                                     pTa
Results                            (n = 69)        (n = 21) ([dagger])

CIS, mean [+ or -] SD         2.23 [+ or -] 1.06   6.67 [+ or -] 4.50
Median (min-max)                   2 (0-6)              6 (0-12)
Intensity, mean [+ or -] SD   1.09 [+ or -] 0.28   2.05 [+ or -] 1.07
Median (min-max)                   1 (1-2)               2 (0-3)
Extent, mean [+ or -] SD      2.07 [+ or -] 0.90   2.67 [+ or -] 1.39
Median (min-max)                   2 (0-4)               3 (0-4)

                                Groups (pTNM)

                                   (n = 26)
Results                       ([double dagger])     P Value ([section])

CIS, mean [+ or -] SD         7.92 [+ or -] 4.10           .0001
Median (min-max)                   9 (0-12)
Intensity, mean [+ or -] SD   2.42 [+ or -] 1.03           .001
Median (min-max)                    3 (0-3)
Extent, mean [+ or -] SD      2.88 [+ or -] 1.34           .001
Median (min-max)                    3 (0-4)

* CIS indicates combined immunoreactivity score.

([dagger]) Comparison with pTa group (CIS: P = .001, intensity: P =
.001, extent: P = .04) using Mann-Whitney U test after Bonferroni
correction.

([double dagger]) Comparison with pTa group (CIS: P =.001,
intensity: P = .001, extent: P = .001) and with pT1 group (CIS: P =
.99, intensity: P = .33, extent: P = .99) using Mann-Whitney U test
after Bonferroni correction.

([section]) Overall comparison using the Kruskal-Wallis test.

Table 2. Comparison of Fascin Expression Between 2 Groups [pTa and
p(T1 + T2)] of Urothelial Carcinomas *

                                          Groups (pTNM)

                                      pTa               p(T1 + T2)
Results                            (n = 69)              (n = 47)

CIS, mean [+ or -] SD         2.23 [+ or -] 1.06    7.36 [+ or -] 4.28
Median (min-max)                    2 (0-6)              8 (0-12)
Intensity, mean [+ or -] SD   1.09 [+ or -] 0.28    2.26 [+ or -] 1.05
Median (min-max)                    1 (1-2)               3 (0-3)
Extent, mean [+ or -] SD      2.07 [+ or -] 0.90    2.79 [+ or -] 1.35
Median (min-max)                    2 (0-4)               3 (0-4)

Results                       P Value ([dagger])

CIS, mean [+ or -] SD                .001
Median (min-max)
Intensity, mean [+ or -] SD          .001
Median (min-max)
Extent, mean [+ or -] SD             .001
Median (min-max)

* CIS indicates combined immunoreactivity score.

([dagger]) Comparison between groups using Mann-Whitney U test.
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