| Abstract|| |
Purpose: The aim of this study was to investigate the usability of MUC1 and p53 for differential diagnosis of noninvasive papillary urothelial neoplasias, especially for distinguishing papillary urothelial neoplasm of low malignant potential (PUNLMP) from low-grade papillary urothelial carcinoma (LGPUC) when the histologic signs are not obvious. Materials and Methods: Seventeen biopsy specimens of the patients with PUNLMP, 20 with LGPUC and 13 with high-grade papillary urothelial carcinoma (HGPUC) were stained for MUC1 and p53 protein by immunohistochemical methods. Histological grading was performed according to an algorithm, which allows histological parameters used in 2004 WHO/ISUP 1998. Results: We had obvious statistical difference for aberrant expression pattern of MUC1 between PUNLMP and LGPUC-HGPUC (P = 0.007). Positivity of MUC1 expression in cytoplasm of basal cells was more observed in HGPUC and LGPUC, whereas PUNLMP was more often showing apical and superficial positivity of MUC1 expression (P = 0.001 and 0.011). Nuclear p53 protein in HGPUC was obviously more frequent than that in LGPUC and PUNLMP (P < 0.001). Measures showed statistical difference among aberrant MUC1 expression, p53 overexpression, and tumor grade (P < 0.001). Conclusions: MUC1 and p53 may be helpful immunohistochemical markers for distinguishing PUNLMP from LGPUC and HGPUC, when the histologic signs are not obvious.
Keywords: Bladder neoplasm, MUC1, p53
|How to cite this article:|
Kaymaz E, Ozer E, Unverdi H, Hucumenoglu S. Evaluation of MUC1 and P53 expressions in noninvasive papillary urothelial neoplasms of bladder, their relationship with tumor grade and role in the differential diagnosis. Indian J Pathol Microbiol 2017;60:510-4
|How to cite this URL:|
Kaymaz E, Ozer E, Unverdi H, Hucumenoglu S. Evaluation of MUC1 and P53 expressions in noninvasive papillary urothelial neoplasms of bladder, their relationship with tumor grade and role in the differential diagnosis. Indian J Pathol Microbiol [serial online] 2017 [cited 2019 Aug 24];60:510-4. Available from: http://www.ijpmonline.org/text.asp?2017/60/4/510/222959
| Introduction|| |
Since 2004, the WHO/ISUP 1998 classification  has been used for the grading of bladder neoplasms, the histological grade of bladder tumors provides information about tumor progression and recurrence. The classification of noninvasive papillary urothelial neoplasia of bladder presents a challenge, especially between papillary urothelial neoplasm of low malignant potential (PUNLMP) and low-grade papillary urothelial carcinoma (LGPUC). Yet, this distinction is important for urologists because of the different recurrence ratio. According to a novel comprehensive study; the incidence rates of recurrence for PUNLMP, LGPUC, and high-grade papillary urothelial carcinoma (HGPUC) are 17.9%, 35.0%, and 34.0%, whereas progression rates were 1.9%, 6.5%, and 28.8%. In this study, we immunohistochemically analyzed the MUC1 and p53 proteins in noninvasive papillary urothelial tumors of the bladder.
MUC1 which is also known as polymorphic epithelial mucin, is related to cancer development by mutation of glycosylation, aberrant location in the cell, and the overexpression , Although MUC1 overexpression is seen in invasive urothelial carcinoma more than noninvasive tumors, it is suggested that MUC1 has a role in the differential diagnosis of noninvasive urothelial neoplasms.,
p53 is the most widely known genetic mutation in human cancers and also in bladder urothelial carcinoma. The bladder tumor which does not show expression of the p53 protein is more restricted to the organ and is less severe and according to some authors, p53 plays a role in progression PUNLMP to LGPUC.,
In this study, we aimed to show the role of especially MUC1 in the differential diagnosis of noninvasive tumors when the histologic parameters are not obvious. We have already immunohistochemically applied p53, which is already known to be the site of use in the classification of urothelial tumors, to check the usability of MUC1 considering MUC1 plays a role in tumorigenesis by bringing cell death standstill through p53.
| Materials and Methods|| |
Fifty-five Transurethral Resection (TUR) and endoscopic biopsy materials from between 2009 and 2013 from Ankara Education and Investigation Hospital Pathology Department were performed retrospectively and prospectively. Seventeen of them were PUNLMP (mean age: 61), 20 of them were LGPUC (mean age: 63), and 13 of them were HGPUC (mean age: 73). The case of papilloma (n = 1) and normal bladder urothelial (n = 4, with/without inflammation) tissue is used as a negative control group for each biomarker.
We used a Nikon Eclipse E600 light microscope, including ×4, ×10, ×40, and ×100 objectives for microscopic evaluations of both immunohistochemical and H and E sections. The diagnosis was made with H and E stained sections by three expert pathologists according to 2004 WHO/ISUP 1998 classification. Furthermore, an algorithm that allows histological parameters used in 2004 WHO/ISUP 1998 by Shim et al. [Table 1] has taken into consideration for more objective results. After a revision according to this scoring system, we concluded that the cases comply with the previous diagnosis.
Immunohistochemical staining for each biomarker was performed automatically in a Leica BOND-MAX closed system equipment as follows: 2-μm-thick sections were prepared from formalin-fixed paraffin-embedded TMA tissue blocks and were dried in a 60°C oven overnight. The sections were placed in a Bond Max Automated Immunohistochemistry Vision Biosystem according to the following protocol. First, tissues were deparaffinized and pretreated with the Epitope Retrieval Solution 2 (EDTA-buffer pH 8.8) at 98°C for 20 min. After washing steps, peroxidase blocking was carried out for 10 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed, and then, incubated with the primary antibody for 30 min. Subsequently, tissues were incubated with polymer for 10 min and developed with DAB-Chromogen for 10 min For MUC1, the selected Kit was clone: SPM492 with 1:150 dilution by Thermo Scientific. The p53 Kit was DO-7 with 1:100 dilution by Scytek Laboratories. The equipment used 100-micron kit for each immunohistochemical staining.
For p53, 100 cells were searched for evaluation, and only nuclear staining was considered positive. We took into consideration a study by Lee et al. for determining of p53. p53 were determined semi-quantitatively based on the fraction of tumor cells showing positive nuclear staining. We had five groups according to the percentage of positive nuclear accumulation; Grade 0: 0%–10%; Grade 2: 11%–25%; Grade 3: 26%–50%; Grade 4: 51%–75%; and Grade 5: 76%–100%.
For MUC1; manual sections of five areas of each patient slide were chosen at random. We retained five morphologic parameters according to accumulation pattern and distribution pattern: Superficial or basal and apical, cytoplasmic or circumferential cytoplasm membrane. In addition, we classified these five parameters into two groups: Normal (apical/superficial) and an aberrant (basal/cytoplasmic/cytoplasm-membrane) expression pattern for a more convenient comparison with p53 status.
The results were evaluated using the SPSS version 2 software program. Student's t-test was used for investigating the relationship of variables and Pearson's test or Fisher's exact test for categorical variables. The P < 0.05 was considered statistically significant.
| Results|| |
The main morphologic findings about MUC1 and p53 are illustrated in [Figure 1].
|Figure 1: Main morphologic findings of MUC1 and p53 (a) apical and superficial staining of MUC1 in papillary urothelial neoplasm of low malignant potential (b) Grade 1 (20%) p53 expression in papillary urothelial neoplasm of low malignant potential (c) apical and cytoplasm-cytoplasmic membrane MUC1 expression, in low-grade papillary urothelial carcinoma (d and e) Grade 2 and 3 (40%–60%) p53 expression in low-grade papillary urothelial carcinoma (f) basal-cytoplasm and cytoplasm-membrane positivity of MUC1 in low-grade papillary urothelial carcinoma (g) 90% staining of p53 (Grade 4) in low-grade papillary urothelial carcinoma|
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In our study, we evaluated the distribution pattern of MUC1 in each cases. Superficial immunohistochemical staining was not statistically significant among PUNLMP, LGPUC, and HGPUC specimens. For basal positivity, there were statistically different measures between PUNLMP and HGPUC (P = 0.004) specimens. However, it was not statistically significant between PUNLMP and LGPUC (P = 0. 4) specimens, and between LGPUC and HGPUC (P = 0.154) specimens. Apical staining showed no statistically significant difference between PUNLMP, noninvasive LGPUC, and noninvasive HGPUC. Cytoplasm positivity was statistically different between HGPUC and PUNLMP (P = 0.001) specimens, and LGPUC-PUNLMP specimens (P = 0.011), but was not different between HGPUC-LGPUC specimens. Circumferentially, cytoplasm-membrane positivity was observed to be more obvious in HGPUC specimens compared to LGPUC and PUNLMP specimens, but there was no statistical difference [Table 2]. We can say for MUC1; in noninvasive papillary urothelial neoplasia, an aberrant expression pattern was positively correlated with tumor grade which was statistically significant (P = 0.007).
|Table 2: Micropapillary urothelial carcinoma main staining distribution and staining features|
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When we classify the patterns of MUC1 in different distributions into two groups of normal and aberrant staining patterns; the normal patterns of MUC1 were mostly apical in the cytoplasm of superficial cells [Figure 1]b as seen in nontumoral bladder mucosa. 12 cases of 17 PUNLMP (70.6%) specimens had normal MUC1 expression pattern. 5 cases of them had aberrant expression pattern (29.4%). 13 cases of the 20 LGPUC (65%) specimens had aberrant immunohistochemical staining; mostly whole cytoplasm/circumferential cytoplasm membrane [Figure 1]e. A total of 11 HGPUC specimens had aberrant expression pattern of MUC1, mainly in basal cells and in cytoplasm or circumferentially cytoplasm-membrane [Figure 1]j, with a ratio of 84.6%.
The relationship between the aberrant staining patterns of MUC1 was statistically significant as tumor grade increased in noninvasive papillary urothelial neoplasia cases.
The percentage of p53 expression was very low in both normal bladder mucosa and PUNLMP specimens, at 0%–10% (Grade 0–1). That is, 58% of the cases were classified as Grade 1 in p53. In LGPUC, immunostaining of p53 protein was observed heterogeneously (35%: Grade 1; 25%: Grade 3). Furthermore, p53 expression was mostly Grade 5, with a percentage of 61.5% in HGPUC specimens. It was possible to say p53 immunohistochemical grade was positively correlated with tumor grade, and this relationship was statistically significant (P< 0.001) [Table 3].
In our study, normal and aberrant staining of MUC1 and p53 grade was compared. Of the cases, in which p53 was found to be grade 0, 92% of the cases had a normal staining pattern of MUC1. As the p53 grade increases, MUC1 is more aberrantly stained. In other words, 88.9% of cases with grade 3 p53 staining pattern and 100% of cases with grade 4 p53 staining showed aberrant MUC1 expression. In cases with grade 1 and grade 2 p53 staining, MUC1 expression showed a more heterogeneous staining. That is, according to grade as the percentage of p53 staining increases, the MUC1 aberrant staining pattern is also increased, which is statistically significant (P< 0.001) [Figure 2].
We evaluated the relationship between tumor grade and age. We had statistical differences between age and tumor grade (P< 0.05). This finding supported the effect of chronicity on tumor development.
| Discussion|| |
For urinary neoplasias, currently, the most commonly experienced problem concerns the classification of noninvasive low-grade papillary urothelial neoplasia of the bladder, especially between PUNLMP and LGPUC. The only criteria for distinguishing these tumors are the degree of anaplasia or dysplasia, but it is not always histologically easy. This distinction is not always histologically easy and can sometimes be subjective. This distinction is important for urologists because of patient follow-up for tumor recurrence and progression. As we mentioned each group of tumors have different recurrence and progression rate. Using an algorithm suggested by Shim et al. that allows histological parameters used in 2004 WHO/ISUP 1998, can help to obtain more objective.
It is now known that as with so many cancer types bladder cancer displays p53 mutation. Gülçiftçi et al. suggested that the histological grade of bladder tumors is correlated with p53 overexpression, similar to Amirghofran et al. In our study, staining grade of p53 was getting higher PUNLMP to HGPUC. According to this finding, it is possible to say when the histological criteria are not obvious immunohistochemical evaluation of p53 can be used for differential diagnosis of PUNLMP and LGPUC. Kalantari and Ahmadnia  showed noninvasive urothelial tumors have a different expression ratio of p53, which is very low in PUNLMP. For this reason, they also noticed that p53 mutation does not play a primary role in the pathogenesis of urothelial tumors but contributes to the progression from PUNLMP to LGPUC. Although we observed p53 overexpression in noninvasive papillary urothelial tumors, it was very low in PUNLMP. Furthermore, immunohistochemically, we did not gain expression of p53 protein in normal bladder tissue. Thus, we can say that p53 plays a significant role in tumorigenesis in bladder tumors but that it is not the only step in carcinogenesis as Kalantari  suggested.
Prognostic significance of p53 in noninvasive urothelial tumors was first described by Sarkis et al. Koyuncuer  showed that the alteration of the p53 expression in patients with bladder cancer is a prognostic factor.
Even though studies have been carried out investigating the role of MUC1 in tumorigenesis, the number of the studies made about the relationship of urothelial neoplasms and MUC1 are limited. The most extensive study about MUC1 was made by Lau and Weiss  in bladder, endometrium, breast, lung, cervix, ovary, kidney, esophagus, stomach, pancreas, and gallbladder carcinomas. The normal expression pattern of MUC1 is superficial and apical as Rahn et al. showed. They suggested that the apical location of MUC1 is determinant of an intact pathway and is related to good prognosis. However, the aberrant expression pattern demonstrates the defect of MUC1 pathway and is related to bad prognosis. Lau et al. has also observed apical expression pattern in well-differentiated bladder carcinoma and cytoplasm-membrane expression in poorly differentiated carcinoma. Nassar et al. showed immunohistochemical expression of MUC1 at umbrella cells in normal bladder mucosa and more diffuse pattern, mainly in basal cells in urothelial cancer. Furthermore, more recently Jankovic Velickovic et al. noted in the normal urothelium, a limited expression of MUC1, predominantly to the apical membranes of the umbrella cells. MUC1 positivity was also mentioned for invasive urothelial carcinoma. In our study, we also showed that MUC1 has a normal expression pattern in nontumoral bladder epithelium and PUNLMP and gets aberrant expression pattern correlated with tumor grade, especially in cases of HGPUC and also LGPUC. Garbar et al. showed MUC1 detection and distribution in noninvasive papillary urothelial neoplasms. They suggested cytoplasmic and cytoplasm-membrane expression pattern of MUC1 can be used for distinguishing PUNLMP from LGPUC when the histologic signs are not obvious. Abd Elazeez et al. evaluated expression pattern of MUC1 similar to the methods of Garbar and Mascaux, and additionally, they evaluated the expression rate. In our study, we showed that the basal and cytoplasmic expression pattern of MUC1 was statistically different between PUNLMP and LGPUC–HGPUC. The basal expression pattern in particular can be used for distinguishing PUNLMP from LGPUC.
As mentioned by Wei et al., MUC1 is located to the apical borders of epithelial cells and under epithelial stress MUC1 is targeted to nucleus and mitochondria by heregulin-induced repair program. Nuclear and mitochondrial MUC1 both promotes the p53-dependent growth arrest response and suppresses the p53-dependent apoptotic response. With transformation and loss of polarity, MUC1 is constitutively expressed in the nucleus and mitochondria and plays a role in tumorigenesis by bringing cell death standstill through p53. Furthermore, there are studies on expression of MUC1 and p53 in many human carcinomas such as gastric, colorectal, breast, and pancreatic, there is no study showing co-expression of these biomarkers in bladder carcinomas.
We aimed to show the relationship between p53 and MUC1 in bladder carcinoma considering the pathway of cell cycle about MUC1 and p53, based on the previous studies made on co-expression of MUC1 and p53 in other malignancies. Our measures were statistically different between a high p53 expression rate and the aberrant expression pattern of MUC1, and these expression patterns of both biomarkers were also associated with high-grade urothelial tumors.
| Conclusions|| |
When the histologic criteria are not obvious for the differential diagnosis of PUNLMP and LGPUC in particular or to discriminate high-grade urothelial neoplasm, cytoplasmic and basal expression pattern (also known as aberrant expression pattern) of MUC1 can be used. In addition, immunostaining of p53 can help to distinguish PUNLMP from LGPUC because of different expression rates. We observe statistically significant measures among aberrant expression pattern of MUC1 and a high expression rate of p53 protein and tumor grade. This suggested that MUC1 plays a role in tumorigenesis through p53 by bringing cell death standstill, and supported the usability of MUC1. However, large-scale studies are necessary to demonstrate the role of MUC1 as a marker for differential diagnosis in noninvasive papillary urothelial tumors of bladder.
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Conflicts of interest
There are no conflicts of interest.
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Dr. Esin Kaymaz
Department of Pathology, Bülent Ecevit University Hospital, Zonguldak, 67000
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]