Original article
Clinicopathological implications to micropapillary bladder urothelial carcinoma of the presence of sialyl Lewis X-decorated mucin 1 in stroma-facing membranes

https://doi.org/10.1016/j.urolonc.2017.06.004Get rights and content

Highlights

  • MPUC preferentially exhibits stroma-facing membrane expression of MUC1 and sLeX.

  • MUC1 is a potential scaffold protein for sLeX carbohydrates in MPUC.

  • Stroma-facing membrane MUC1 expression is predictive of worse outcomes.

Abstract

Objectives

Bladder urothelial carcinoma (UC) comprises more than 90% of all bladder cancers. Among several UC variants, micropapillary UC (MPUC) is a rare one with high potential for lymphovascular invasion and subsequent lymph node metastasis. Histologically, MPUC is characterized by the presence of small papillary carcinoma cell clusters surrounded by lacunar spaces. Immunohistochemically, the outer circumference of these clusters, that is, the stroma-facing membrane of carcinoma cells, is reportedly almost invariably positive for mucin 1 (MUC1) protein and to a lesser extent for sialyl Lewis X (sLeX) carbohydrates; however, the clinicopathological implications of these expression patterns have not been fully investigated.

Materials and methods

We performed immunohistochemical analysis of MPUC (n = 11) and conventional UC (n = 57) for MUC1 and sLeX to determine whether these factors immunolocalized. Dual immunofluorescence staining was also carried out to assess MUC1 and sLeX colocalization. We also performed Western blot analysis of Chinese hamster ovary cells misexpressing both recombinant epitope-tagged MUC1 and glycosyltransferases enabling sLeX biosynthesis.

Results

MPUC samples preferentially exhibited both MUC1 protein and sLeX carbohydrate expression on the stroma-facing membrane of carcinoma cells. Based on univariate analysis, MUC1 expression in that pattern was positively correlated with tumor extension, lymphovascular invasion, lymph node metastasis, disease stage, and relatively poor patient prognosis. A comparable sLeX expression pattern also correlated positively with tumor extension and nodal metastasis. Based on multivariate analysis, localization of MUC1 and sLeX on the stroma-facing side of the membrane was positively correlated with lymph node metastasis.

Conclusions

Overall, our immunofluorescence findings as well as immunoprecipitation analyses of Chinese hamster ovary cell transfectants strongly suggest that MUC1 is a potential scaffold protein for sLeX carbohydrates in MPUC. Both MUC1 and sLeX may cooperatively contribute to MPUC histogenesis and clinicopathological characteristics.

Introduction

Bladder cancer is one of the most common cancers worldwide, and more than 90% of cases are histologically classified as urothelial carcinoma (UC) [1], [2]. Several UC histologic variants have been recognized, and among them, micropapillary UC (MPUC), first described by Amin et al. [3], is a rare form with high propensity for lymphovascular invasion and subsequent lymph node metastasis [1], [4], [5]. Reflecting its biological aggressiveness, the clinical stage of MPUC at initial presentation is usually more advanced than that of conventional UC [1], [3], [6].

Similar to micropapillary adenocarcinomas arising at other anatomical sites, MPUC is histologically characterized by cleft formation around carcinoma cell clusters: specifically, carcinoma cells form small papillary clusters without fibrovascular cores, and these clusters are present within lacunar spaces formed in tumor stroma [3], [5]. Immunohistochemically, the outer circumference of the clusters, namely, the stroma-facing side of carcinoma cell membranes, is almost invariably mucin 1 (MUC1)-positive [7], [8].

MUC1, also known as epithelial membrane antigen, is a species of membrane-tethered glycoprotein composed of 2 noncovalently bound subunits: a larger N-terminal subunit consisting of a long extracellular domain and a smaller C-terminal subunit containing a short extracellular domain, a transmembrane region, and a cytoplasmic tail [9], [10], [11]. The long extracellular domain of the former contains a serine/threonine-rich region termed “variable number tandem repeats” (VNTR), which is usually extensively O-glycosylated with a series of Lewis blood group-related carbohydrate antigens, such as sialyl Lewis A (sLeA) and sialyl Lewis X (sLeX) [11], [12], [13].

We previously demonstrated that 14 of 78 (17.9%) bladder UC cases expressed 6-sulfated sLeX [2]. In that report, we also noted that, in addition to 6-sulfated sLeX, nonsulfated sLeX was present in all MPUC cases examined (n = 5), and similar to the expression pattern of MUC1, sLeX was expressed along the outer circumference of small papillary carcinoma cell clusters formed in MPUC [2]. However, the clinicopathological implications of these expression patterns have not been fully investigated.

This was undertaken to determine whether the presence of MUC1 and sLeX in stroma-facing membranes of MPUC correlates with patients׳ clinicopathological characteristics. We also performed immunofluorescence and immunoprecipitation analyses to determine whether MUC1 can serve as a scaffold protein for sLeX carbohydrates.

Section snippets

Tissue samples and clinicopathological information

Formalin-fixed, paraffin-embedded tissue blocks of surgical specimens of either MPUC (n = 11; 10 males and 1 female; mean age = 68.9 years) or conventional UC (n = 57; 46 males and 11 females; mean age = 72.2 years) bladder cancer were retrieved from the pathology archives at University of Fukui Hospital. All tissue slides were reviewed by 2 experienced pathologists (Y.S. and M.K.) to confirm the initial diagnosis. Among 11 MPUC cases, 5 were initially diagnosed as conventional UC, and these

Stroma-facing membranes of MPUC carcinoma cells preferentially express MUC1 and sLeX

To assess potential immunolocalization of MUC1 protein and sLeX carbohydrate in MPUC, we first carried out immunohistochemistry for both molecules. As shown in Fig. 1 (upper middle panel), MUC1 expression was mostly restricted to the outer circumference of small papillary carcinoma cell clusters in all MPUC cases examined (n = 11). Samples of conventional UC exhibited diffuse cytoplasmic MUC1 expression in 54 of 57 (94.7%) cases, and none showed the stroma-facing membrane pattern characteristic

Discussion

In the present study, we preferentially observed MUC1 expression, as well as that of sLeX, on the stroma-facing side of membranes of MPUC carcinoma cells. These factors colocalized with each other, and additional immunoprecipitation analysis of MUC1- and sLeX-overexpressing CHO cells suggested that MUC1 could serve as an sLeX scaffold protein in MPUC.

We recently reported preferential expression of 6-sulfated sLeX recognized by MECA-79 monoclonal antibody on apical membranes of small-sized

Conclusion

Here we show that stroma-facing membrane expression of both MUC1 and sLeX is preferentially observed in MPUC, and that MUC1 is a potential sLeX carbohydrate scaffold protein in MPUC. We also show that this expression pattern of MUC1 and to a lesser extent sLeX is predictive of worse clinical outcomes. These findings overall suggest that both may cooperate to contribute to MPUC histogenesis or clinicopathological characteristics.

Acknowledgments

We thank Mr. Hisataka Kato and Ms. Noriko Maruta for technical assistance, Dr. Taro Tamura for assistance with statistical analysis, and Dr. Elise Lamar for critical reading of the article. This work was supported in part by a Grant-in-Aid for Young Scientists 15K19049 (to H.H.) and a Grant-in-Aid for Scientific Research 15K08343 (to M.K.) from the Japan Society for the Promotion of Science, Japan.

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These authors contributed equally to this work.

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