EXPRESSION of CD10 and CA19.9 in TRANSITIONAL CELL CARCINOMA of the URINARY BLADDER: CORRELATION with TUMOR GRADE and STAGE Shimaa Sh

AAMJ, Vol. 10, N. 3, Sep, 2012, Suppl-2 ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ EXPRESSION OF CD10 AND CA19.9 IN TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER: CORRELATION WITH TUMOR GRADE AND STAGE Shimaa Sh. Samah, Hala M El Safy, Hala E- Abdel Hamied and Eman M. Ahmed Department Of Pathology, Faculty of Medicine, Al- Azhar University, Cairo, Egypt. ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ABSTRACT Background: CD10 has been suggested as a useful prognostic marker for urothelial carcinoma. Carbohydrate antigen 19-9 (CA19-9) plays important roles in the invasion and metastasis of cancers. There are limited data on the use of CA19-9 as a tumor marker in bladder carcinoma. Objective: To assess the immunohistochemical expression of CD10 and CA19.9 in transitional cell carcinoma (TCC) of the urinary bladder to determine the correlation between this expression and tumor grade and stage. Materials and Methods: Cd10 and CA19-9 immunohistochemical analysis (IHC) was used to evaluate 38 fixed, paraffin-embedded samples which included TCC of urinary bladder. Tumor tissue blocks and clinical data of the cases were collected from the files of the Pathology Department of al zahaaa Hospital, between January, 2009 and August, 2011. Results: Immunohistochemical (IHC) expression and immunoscoring of CD10 and CA19.9 had a significant correlation with WHO 2004 grade of urothelial carcinoma. There was significant correlation between CD10 immunohistochemical expressions with stage; while there was no significant correlation between CA19.9 immunohistochemical expressions with tumor stage. Conclusions: CD10 and CA19-9 immunohistochemical expression could be of value in differentiation between high and low-grade urothelial carcinoma.

Bladder cancer represents the second most frequent malignancy of the genitourinary tract following prostate cancer, and the ninth most common cause of cancer worldwide, with rising incidence (Hegele et al., 2010). Approximately 90% of malignant bladder tumors are transitional cell carcinoma (TCC). The highest incidence is in the sixth and seventh decades of life; men are affected more often than women (Mills et al., 2010).

The prognosis of TCC depends largely on the histological grade and the stage of the tumor at diagnosis (Kumar et al., 2010). Many investigators have shown a statistically significant difference in the survival between patients with noninvasive tumors, tumor confined to lamina propria, and those with muscle infiltration (Kamat et al., 2006, Shariat et al., 2006, Mills et al., 2010).

Several tumor markers including the bladder tumor antigen (BTA) (series of markers) nuclear matrix proteins (NMP22) and fibrinogen degradation products have been approved for clinical use in the setting of early detection of bladder tumors. However, these markers have limited sensitivities and higher false-positive rates (Poulakis et al., 2001).

CD10 is a cell-surface metalloprotease that can limit cellular responses to peptide hormones by hydrolyzing them. In addition to its enzymatic function, the CD10 protein has a direct role in signal transduction pathways that regulate cell growth and apoptosis and because of its structural similarity to the matrix metalloproteases in the stroma, CD10 is thought also to affect the invasion and metastatic potential of tumor cells by altering the cellular microenvironment ( Iwaya et al., 2002 and Kandemir et al., 2010). ). CD10 expression in intra- tumoral stromal cells may also contribute to tumor progression (Bahadir et al. 2009).

AAMJ, Vol. 10, N. 3, Sep, 2012, Suppl-2 ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Few studies have investigated CD10 expression in urinary bladder tissues. Its expression has been reported to occur in 43%–67% of urothelial neoplasms. While in the majority of reports, CD10 expression shows an inverse correlation with tumor grade, a positive correlation with grade has been noted in others (Bircan et al., 2006, Bahadir et al. 2009, Kandemir et al., 2010).

The carbohydrate antigen CA19.9 (Sialyl Le-a) is a blood group-related antigen and its expression requires the expression of Lewis a blood group antigen which is a cancer-associated phenomenon (Chuang and Liao 2004). It has been reported that the expression of carbohydrate antigens correlate with atypical grade and clinical stage of cancer in some carcinomas (Forones and Tanaka, 1999, Boeck et al., 2006, Goonetilleke and Siriwardena, 2007). Recently, a relationship between CA19.9 and urothelial cancers has been found (Hegele et al., 2010). However, in the literature few studies had been published in evaluating the expression of CA19.9 in transitional cell carcinoma of the bladder.

In this study, we aim to assess CD10 and CA19-9 immunohistochemical expression in transitional cell carcinoma of the urinary bladder and correlate this expression with histopathological tumor grade and stage.

MATERIAL AND METHODS

A total of 38 fixed, paraffin-embedded samples which included transitional cell carcinoma (TCC) of urinary bladder were included in this study .The specimens were collected from the surgical files of the Histopathology Department of Al-Azhar University Hospital between January, 2009 and August, 2010 , after obtaining an informed consent and approval of local ethical committee. Clinicopathological information was obtained from medical charts .The mean age of those patients was 61.4 (range, 24 to 71) years. There were 32 males and 6 females. All patients underwent transurethral resection of

Shimaa Sh. Samah et al ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ bladder tumors. Three sections of 5 micron thickness were cut from the paraffin blocks and one section were stained with hematoxylin and eosin to reevaluate the diagnosis and to assessment the histopathological staging and grading of the tumors. The TNM system was used for pathologic staging: Ta: noninvasive papillary urothelial carcinoma; T1: tumor invading subepithelial connective tissue; T2: tumor invading muscularispropria, and T3: tumor invading perivesical tissue (Sobin and Wittekind, 2002). Tumors were graded according to the WHO 2004 - grading system (Sauter et al., 2004). G1 and G2 were taken as low grade and G3 and G4 as high grade tumors (Epstein et al., 1998). The other two sections were mounted on positive charged slide and immune- stained by mouse monoclonal antibodies against CD10 and CA19.9.

Immunohistochemistry:

For immunohistochemical study; unstained positively charged slides (Biogenix) were prepared from each paraffin block for immunostaining with mouse monoclonal antibodies against:CD10 and CA19-9 [Dako, U.S.A]. Immunohistochemical reactions were carried out using Labeled Streptavidin- Biotin2 System- Horseradish Peroxidase (LSAB2 System-HRP). The LSAB2 System, HRP is based on a modified labeled Avidin-Biotin (LAB) technique in which a biotinylated secondary antibody forms a complex with peroxidase- conjugated streptavidin molecules.

The entire antibody complex is made visible by addition of an appropriate substrate chromogen reagent, which is converted by the peroxidase label to brown-colored precipitate at the site of antigen localization in tissue. The chromogen used is diaminobenzidine (DAB) produced by Dako (U.S.A).

Five microns thick sections were cut from the selectively collected paraffin blocks and mounted on positively charged slides. Slides were incubated overnight at 55°C and deparaffinized by immersion in xylene. Rehydration of the slides in a

AAMJ, Vol. 10, N. 3, Sep, 2012, Suppl-2 ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ graded series of alchoholat room temperature was performed. Then the slides were immersed in 3% hydrogen peroxide solution. Slides were washed by phosphate buffer saline (PBS; pH 7.2).The mounted sections were immersed in citrate buffer (PH 6.0) then boiled in this solution in a microwave for 10 minutes.Slides were allowed to cool at room temperature then washed in PBS. Then the slides were incubated in humidity chamber overnight with pre-diluted primary monoclonal antibodies CD10 and CA19-9 (recommended dilution 1:50). Sections were incubated with biotinylatedanti-mouse immunoglobulins for 10 min at room temperature, followed by washing in PBS for 5 minutes.Then sections were incubated with streptavidin-horseradish peroxidase for another 10 minutes, followed by washing in PBS for 5 minutes.Sections were incubated with DAB for 30 minutes at room temperature. DAB was used as a color reagent. Sections were washed in distilled water for 5 minutes then counterstained with 0.5% Mayer's hematoxylin for 1-4 minutes to enhance visualization of the tissue and cytological details.Slides were then washed with distilled water and processed in up graded alcohols.Slides were cleared in xylene for 3 minutes, and finely mount with a coverslip using DPX.

Positive and Negative Control:

As a negative control for 2 markers, a tumor tissue was processed through the above sequences but the primary antibody was omitted, instead phosphate buffer solution was added. For positive control, sections from normal liver were stained for CD10, while sections from colorectal carcinoma were considered as positive control for CA19-9.

Shimaa Sh. Samah et al ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Evaluation of Immunostaining: Regarding CD10, brown staining of the cell membrane and/or cytoplasm is considered positive. Brown staining of the cytoplasm by CA19-9 is considered positive. . The proliferative index program was used to obtain the percentage of positive immune-stained cells; a mean of 500 cell count was performed in each case at high power Number (X400) of positi afterve cells selecting the highest staining areas at low Total number of counted cells power.

% of positive cells = X 100.

Scoring System:

Each stained urothelial tumor section was analyzed for both presence and extent of staining. Each stained urothelial tumor section was analyzed for both presence and extent of staining. The extent of immunoreactivity to CD10 was scored semiquantitatively; based on the percentage of positive cells; according to (Bahadir et al.,2009): (0): < 5% positive cells, (+1) low expression: 5%-50% positive cells and (+2) strong expression : > 50% positive cells. Regarding CA19-9, the extent of staining was scored semiquantitatively; based on the percentage of positive cells; according to the following criteria (Hegele et al 2010): (0) : When coloration is negative ,(+1) week : < 25% positive cells ,(+2) moderate : 25%-50% positive cells and (+3) strong: > 50% positive cells.

Statistical Analysis:

Statistical analysis was performed using SPSS v18.00 (statistical package for social sciences) and Microsoft Excel 2007 programs. Data analysis was done using Chi-square test for Tables with frequencies. P value < 0.05 is considered statistically significant.

AAMJ, Vol. 10, N. 3, Sep, 2012, Suppl-2 ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ RESULTS A total of 38 cases of transitional cell carcinomas of the bladder were enrolled into this study. The age of the patients ranged from 24 to 71 (mean ± SD 61.4 ± 9.7). There were 32 males (84.21%) and 6 females (15.78%) with an average male to female ratio of 8:1. According to the 2004 grading system of urothelial carcinoma of the bladder 18/38 (47.36 %) of the cases were of high grade and 20/38 (52.63%) of the cases were of low grade. The distribution of cases of urothelial carcinoma of the bladder according to the pathological T- stage of urothelial carcinoma according to AJCC/UICC was as follow: Stage Ta was 26/38 ( 68.42%), Stage T1 was 7/38 (18.42%) and stage T2 was 5/38 (13.16%) .

Immunohistochemical expression of CD10:

CD10 staining was cytoplasmic, but membranous staining was also observed. 18 out of 38 (47.36%) of TCC (EC) were positive for CD10. Positive rate increased significantly in high grade tumors (Table 1), and the degree of immunostaining score of expression was also increased significantly with the histological grade of TCC (P value 0.000) . (Fig 1 A,B,C) (Table 2).

Table 1: Positive rate of CD10 according to WHO 2004 grading of TCC.

CD10 High grade Low grade P-value No. % No. % Negative (n=20) 3 16.70% 17 85.00% 0.000 Positive (n=18) 15 83.30% 3 15.00%

Table 2: Immunostaining score of CD10 according to WHO 2004 grading of urothelial carcinoma CD10 immunoscoring

0 1+ 2+ Total P value Grade High grade 3 7 8 18 0.000 Low grade 17 0 3 20 Total 20 7 11 38 Total 2020 77 1111 3838

According to stage, the positive rate of the CD10 expression was 8/26 (30.76%) in stage Ta, 5 /7 (71.42 %) in stage T1, and 5/5 ( 100 %) in stage T2, this difference being statistically significant (P value 0.007) (Table 3). The degree of immunostaining score of CD10 expression was also increased significantly with the stage of TCC (P value 0.001) (Table 4).

Table 3: Positive rate of CD10 according to tumor stage. CD10 Ta T1 T2 P-value No. % No. % No. % Negative ( 20) 18 69.23 2 28.57 0 00.00% Positive (18) 8 30.76% 5 71.42% 5 100.00% 0.007 Total 26 100.00% 7 100.00% 5 100.00%

Table 4: Immunostaining score of CD10 according to tumor stage CD10

0 1+ 2+. Total . P value Stage Ta 18 5 3 26.00 T1 2 2 3 7.00 0.001 T2 0 0 5 5.00 Total 20 7 11 38.00

The immunohistochemical analysis of the expression of CA 19.9 protein showed a significant correlation with tumor grade. Sixteen (80%) out of 20 samples of low grade tumors were positive for CA19.9 ,while 8 out of 18 high grade tumors (44%) were positive for the same marker (p= 0.023) (Table 5).

There was also a significant correlation found between immunostaining score of CA19.9 and tumor grade (p= 0.040) (Fig 2A,B,C) (Table 6).

However, the correlation between both immunohistochemical expression and immunostaining score and the AJCC/UICC stage of urothelial carcinoma were not statistically significant (p values 0.433 and 0.453 respectively) (Table 7, 8).

Table 5. positive rate of CA19-9 according to the tumor grade

CA19 .9

High grade Low grade P-value

No. % No. %

Negative 10 55.60% 4 20.00% 0.023 Positive 8 44.40% 16 80.00%

Table 6. Immunostaining score of CA 19-9 marker according tumor grade

CA 19-9 immunoscoring

0 1+ 2+ 3+ Total P value

Grade

High grade 10 1 5 2 18 0.040 Low grade 4 2 4 10 20

Total 14 3 9 12 38

Table 7. positive rate of CA19-9 expression in cases according to tumor stage

CA19-9

Ta T1 T2 P-value

No. % No. % No. %

Negative 8 69.23% 4 57.14% 2 20.00%

Positive 18 30.76% 3 42.85% 3 60.00% 0.433 100.00 5 100.00 Total 26 100.00% 7 % %

Table 8: Immunostaining score of CA 19.9 marker according to tumor stage

CA19-9

0 1+ 2+. 3+ Total. P value

Stage

Ta 8 1 8 9 26.00

T1 4 1 1 1 7.00 0.453

T2 2 1 0 2 5.00

Total 20 7 11 12 38.00

Correlation between two markers (CD10 and CA19-9 antibodies) in high and low grade tumors

There was a significant correlation between (CD10 and CA19-9 antibodies) in high grade tumors (P value 0.034) (Table 9) , as well as a significant correlation between two markers and low grade tumors (P value 0.001) (Table 10). There was inverse relationship between CD10 to CA19-9 expression in high and low grade tumors (Fig. 3).

Table 9. Positive rate of CD10 and CA19-9 in high grade tumors High grade Chi-square test CD10 CA19-9 X2 P-value No. % No. % Negative 3 16.67% 10 55.56% Positive 15 83.33% 8 44.44% 4.500 0.034 Total 18 100.00% 18 100.00%

1A 2A 2A 1A

9 9

C C CC CC 1B 2B 1B

2C 1c

Fig.,1: (A) Low grade TCC showing week membranous staining of CD10 in neoplastic epithelial cells. (B) High grade TCC showing strong membranous staining of CD10 in neoplastic epithelial cells.(C ) High grade TCC showing strong membranous and cytoplasmic staining of CD10 in neoplastic epithelial cells.

Fig.,2: (A) Low grade TCC showing strong membranous staining of CD19.9 in neoplastic epithelial cells. (B)) High grade TCC showing week membranous staining of CD19.9 in neoplastic epithelial cells.(C) High grade TCC showing week membranous staining of CD19.9 in neoplastic epithelial cells (original magnifications, 200)

Low grade Chi-square test

CD10 CA19-9

No. % No. % X2 P-value

Negative 17 85.00% 4 20.00%

Positive 3 15.00% 16 80.00% 14.436 0.001

Total 20 100.00% 20 100.00%

CD10 CDCA19 19-9

100%

80%

60% 40%

20%

Negative Positive Negative Positive

Low grade High grade

Fig., 3: Relation of CD10 to CA19-9 expression in high and low grade tumors

100

The age of the patients ranged from 24 to 71 (mean ± SD 61.4 ± 9.7). There were 32 males (84.21%) and 6 females (15.78 %) with an average male to female ratio of 7.3:1. These results agree with those mentioned by Bahadir et al. (2009). The most important element in the pathologic evaluation of urothelial cancer is the recognition of the presence and extent of invasion (Matalka et al., 2008) .High recurrence rate in TCC of the bladder is a vital problem despite the improvement of treatment options. A number of molecular markers have been reported to be of prognostic value, including those who are tumor suppressor gene p53, cell cycle regulator proteins p27 and many others. However, there are still conflicting results although some of them may be attributed to different staining protocols or patient selections (Schoenberg , 2001).

The first study assessing CD10 activity in bladder was performed by Koiso et al (1994). They found that both enzyme activity and IHC expression were higher in superficial cancers than invasive cancers and normal urothelium. They concluded that CD10 was expressed at a certain stage of differentiation in the course of neoplastic process. In 2000, Chu and Arber reported positive cytoplasmic staining in 54% (13/24) of urothelial carcinomas, while there was no reaction in non-neoplastic tissues. However, the correlation of CD10 expression with pathologic stage or histologic grade was not investigated in that study. Those results suggested that neoplastic tissues rather than non-neoplastic epithelium have a propensity for CD10 expression. Conversely, Murali and Delprado (2005) demonstrated CD10 staining in 50% (5/10) of non-neoplastic urothelium. Despite the small number of cases, they found CD10 expression in 80% of invasive carcinomas and also proved that the staining intensity for the high- grade group (including invasive carcinoma, high-grade papillary urothelial

101

AAMJ, Vol. 10, N. 3, Sep, 2012, Suppl-2 ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ carcinoma, and carcinoma in situ) was statistically higher than that of the low- grade group (including low-grade papillary urothelial carcinoma, papillary urothelial neoplasm of low malignant potential and normal urothelium).

In the current study, we demonstrated CD10 IHC expression in urothelial carcinomas of the bladder. CD10 staining of the malignant cells revealed a strong correlation not only with histologic grade but also with pathologic stage (p value 0.001, 0.007 respectively). Moreover, a significant correlation between tumor grade, stage, and immunostaining score of CD10 was found. A progressive increase in the immunostaining score of CD10 along with an increase in tumor grade suggests that upregulation in antigen expression may occur in higher grade tumors. The same was true for the stage of the tumors, pTa tumors mainly expressed +1 reaction for CD10 of cases, while pT2 expressed +2 in 100% (5/5), which was in agreement with a studies done by Bahadir et al.(2009) and Abdou (2007) and disagreed with other studies done by Kandemir et al. (2010) . Bircan et al. (2006) found an inverse correlation between CD10 expression and tumor stage, but no association with histologic grade or staining score was detected ,the authors proposed that the higher level of CD10 expression in noninvasive carcinomas appears to inhibit cell invasion.

There may be several possibilities about the role of CD10 in urothelial tumorigenesis. First, Derangements in the CD10-dependent signal transduction pathways that regulate cell growth and apoptosis (Sumitomo et al., 2000) as a result of abnormal expression of CD10 might have a role in uncontrolled cell proliferation and tumorigenesis. Second, CD10 is a cell surface metalloprotease and it is known to hydrolyze endothelin(Ship and Look, 1984) and might have a role in urothelial angiogenesis by modulating endothelin levels that facilitates cancer cell invasion and metastasis. This appears the most likely explanation for the significant correlation of CD10 with grade and stage in our study. Another possible mechanism is that the increased IHC CD10 expression with increasing 102

Shimaa Sh. Samah et al ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ grade and stage may indicate accumulation of mutated, non functional CD10 rather than its normal counterpart (Sobin and Wittekind, 2002). In this study as well as in others (Chuang and Liao, 2004 and Kajiwara et al., 2005 and Bahadir et al., 2009, there was a significant correlation between both CA19.9 immunohistochemical (expression and scoring) and the (WHO 2004) grade of urothelial carcinoma (p = 0.023, 0.040 respectively), there was a downregulation of the immunohistochemical (expression and scoring) of the CA19.9 antigen with increasing tumor grade. CA19.9 immunohistochemical expression and scoring did not correlate with the AJCC/UICC pathological T-staging of TCC in our study (p values 0.433 and 0.543 respectively). These findings are in accordance with those reported by others (Chuang and Liao (2004), Kajiwaraet al.(2005). but disagreed with other studies done by (Hegele et al., 2010).

This can be explained by the small sample size; the improper staging of the specimens due to subjective errors in assessing the stage or improper transurethral resection technique in which deeper tissues especially the muscular layer had not been taken, may contribute to under or over estimation of staging in these cases which in turn affects the number of cases in each stage.

In conclusion, this study revealed that CA19.9 and CD10 immunohistochemical expression could be of valuable significance in the differentiation between high and low-grade TCC of the bladder and consequently in determining the prognosis in such cases.

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