Original Article Apocrine-Differentiated Cells Are Likely to Express Uroplakin II

Int J Clin Exp Pathol 2016;9(2):1933-1939 www.ijcep.com /ISSN:1936-2625/IJCEP0014073

Original Article Apocrine-differentiated cells are likely to express uroplakin II

Shogo Tajima, Kenji Koda

Department of Pathology, Fujieda Municipal General Hospital, Shizuoka, Japan Received August 6, 2015; Accepted September 25, 2015; Epub February 1, 2016; Published February 15, 2016

Abstract: Uroplakin II antibody is exclusively specific for urothelial carcinoma. Non-urothelial carcinoma has not been reported to be immunoreactive for uroplakin II. In the present study, we hypothesized that breast carcinoma showing apocrine differentiation, such as invasive pleomorphic lobular carcinoma (IPLC) and apocrine carcinoma (AC), stains positive for Uroplakin II. We identified 6 cases of IPLC between 2000 and 2014 by searching a computerized pathological database. We randomly selected 10 cases of each classic invasive lobular carcinoma (cILC) and AC, and five cases of apocrine metaplasia (AM) that coexisted in surgically resected breast carcinoma specimen. Immu- nohistochemistry was performed for Uroplakin II, GATA3, CK7, CK20, and other representative markers positive for urothelial carcinoma. All cases of IPLC, AC, and AM, except those of cILC, showed immunoreactivity for Uroplakin II. Poorly differentiated urothelial carcinoma sometimes shows similar morphology to IPLC with the immunophenotype: CK7+, CK20-, GATA3+, Uroplakin II+. In the present study, this immunophenotype was observed in all the cases of IPLC and AC. Therefore, when studying metastatic, poorly differentiated carcinoma showing the aforementioned immunophenotype, we should consider the possibility of it being IPLC in addition to metastatic urothelial carcinoma.

Keywords: Uroplakin II, breast, apocrine differentiation, invasive pleomorphic lobular carcinoma, apocrine carcinoma

Introduction GATA3, Uroplakin II is positive for metastatic carcinoma, it should be considered as meta- Recently developed Uroplakin II antibody static urothelial carcinoma, as has been sug- (clone: BC21) was found to be exclusively spe- gested by previous studies; no breast carcino- cific to the urothelium and urothelial carcinoma has ever been reported to be positive for mas when evaluated in various normal and Uroplakin II [1, 3, 4]. neoplastic tissues [1]. It specifically stained urothelium among 37 US Food and Drug We happened to examine a case of invasive Administration normal tissue types, including 3 pleomorphic lobular carcinoma (IPLC) metastat- breast tissue samples [1]. Regarding neoplas- ic to urinary bladder as routine surgical pathol- tic tissue, 20 tumor types were immunostained ogy practice. Since it resembled a poorly diff- with it and urothelial carcinoma was exclusively erentiated urothelial carcinoma, immunohisto- positive for it, except for one of eighty-eight chemistry was performed with markers positive prostatic adenocarcinoma. However, this one for urothelial carcinoma, such as Uroplakin II, positive case was considered to be metastatic GATA3, p63, p40, and 34βE12 [4, 5]. This case urothelial carcinoma that had spread to the was found to be diffusely positive for these prostate gland [1]. markers except for p63 and p40, which showed focal positivity. GATA3 is an immensely sensitive and relatively specific marker for urothelial as well as breast IPLC is a subtype of invasive lobular carcinoma carcinoma [2]. When encountering metastatic (ILC), which exhibited relatively high-grade carcinoma that is positive for GATA3, the pos- nuclei and abundant cytoplasm compared with sibility of urothelial and breast carcinoma classic invasive lobular carcinoma (cILC); IPLC should be first considered. If in addition to is considered to behave more aggressively than Uroplakin II in apocrine-differentiated cells

Figure 1. Histological findings. A. IPLC case 2: Nuclear enlargement and eosinophilic cytoplasm observed in tumor cells (x400). B. cILC case 2: Nuclear enlargement of the tumor cells is modest compared with that of IPLC. Reduced cytoplasm in the tumor cells is observed (x400). C. AC case 2: Cytoplasmic abundance and eosinophilia of the tumor cells are more prominent than that of IPLC (x400). D. AM case 3: The cytoplasmic abundance and eosinophilia of the constituent cells are conspicuous but the nuclei are less atypical than those of IPLC and AC (x400). IPLC: invasive pleomoprhic lobular carcinoma; cILC: classic invasive lobular carcinoma; AC: apocrine carcinoma; AM: apocrine metaplasia. cILC [6]. In addition, IPLC exhibits apocrine dif- other markers expected to be positive for uro- ferentiation [7-9]. Thus, we hypothesized that thelial carcinoma are included in this study. We apocrine differentiation might be related to further analyzed cases of cILC and apocrine Uroplakin II immunoreactivity. metaplasia (AM) for comparison.

Examining metastatic carcinoma with poorly Materials and methods differentiated and high-grade morphology and GATA3 expression, breast carcinoma including We identified 58 surgically resected cases of IPLC and poorly differentiated urothelial carci- ILC between 2000 and 2014 from a computer- noma are considered for the differential diag- ized pathological database. On the basis of his- nosis. Among breast carcinomas showing apo- tological examination results, we classified the crine differentiation, apocrine carcinoma (AC) is cases exhibiting enlarged nuclei (approximately not recognized as exhibiting poorly differentiat- 4 times the size of a lymphocyte) with an irregu- ed morphology. Thus, the aim of this study was lar nuclear membrane, marked nuclear hyper- to examine Uroplakin II expression in IPLC. chromasia, prominent nucleoli, increased Moreover, immunohistochemical analyses of mitotic activity, and moderate-to-abundant

1934 Int J Clin Exp Pathol 2016;9(2):1933-1939 Uroplakin II in apocrine-differentiated cells

Table 1. Results showing immunohistochemical analysis of each were available for all the case of IPLC, cILC, AC, and AM selected cases. Each opera- Case Uroplakin II GATA3 p63 p40 CK7 CK20 34βE12 GCDFP-15 tive specimen was fixed in IPLC 10% buffered-formalin fol- 1 3+ 3+ 0 0 3+ 0 3+ 3+ lowed by paraffin embed- ding. 2 2+ 3+ 0 0 3+ 0 3+ 3+ 3 3+ 2+ 1+ 1+ 3+ 0 3+ 3+ Immunohistochemistry was 4 2+ 3+ 0 0 3+ 0 3+ 3+ performed for 4-µm-thick 5 3+ 3+ 1+ 1+ 3+ 0 3+ 3+ sections obtained from par- 6 1+ 3+ 0 0 3+ 0 3+ 3+ affin-embedded blocks using cILC primary antibodies against 1 0 3+ 0 0 3+ 0 3+ 1+ Uroplakin II, GATA3, p63, 2 0 3+ 0 0 3+ 0 3+ 2+ p40, CK7, CK20, 34βE12, and gross cystic disease flu- 3 0 3+ 0 0 3+ 0 3+ 1+ id protein-15 (GCDFP-15), 4 0 3+ 0 0 3+ 0 3+ 0 respectively. Additionally, all 5 0 3+ 0 0 3+ 0 3+ 1+ the selected cases of IPLC 6 0 3+ 0 0 3+ 0 3+ 1+ and cILC were immunosta- 7 0 3+ 1+ 1+ 3+ 0 3+ 1+ ined with E-cadherin to con- 8 0 3+ 0 0 3+ 0 3+ 0 firm the diagnosis. Immuno- 9 0 3+ 0 0 3+ 0 3+ 1+ histochemistry was perfor- 10 0 3+ 0 0 3+ 0 3+ 0 med using a BenchMark GX AC Autoimmune Stainer and an 1 2+ 3+ 0 0 3+ 0 0 3+ I-View DAB detection kit. The interpretations were scored 2 3+ 3+ 0 0 3+ 0 0 3+ as follows: 0 = less than 5% 3 1+ 2+ 0 0 3+ 0 0 3+ tumor cell positivity; +1 = 4 2+ 2+ 0 0 3+ 0 0 3+ 5-10% tumor cell positivity; 5 3+ 3+ 0 0 3+ 0 0 3+ +2 = 11-50% tumor cell posi- 6 3+ 3+ 0 0 3+ 0 0 3+ tivity; and +3 = more than 7 1+ 2+ 0 0 3+ 0 0 3+ 50% tumor cell positivity. 8 1+ 1+ 0 0 3+ 0 0 3+ 9 3+ 1+ 0 0 3+ 0 0 3+ This study was approved by the institutional review 10 3+ 3+ 0 0 3+ 0 0 3+ board. AM 1 3+ 0 0 0 3+ 0 0 3+ Results 2 3+ 1+ 0 0 3+ 0 0 3+ 3 3+ 0 0 0 3+ 0 0 3+ IPLC cases had enlarged 4 3+ 0 0 0 3+ 0 0 3+ nuclei and abundant eosino- 5 3+ 0 0 0 3+ 0 0 3+ philic cytoplasm when compared to cILC (Figure 1A, 1B). IPLC: invasive pleomoprhic lobular carcinoma; cILC: classic invasive lobular carcinoma; AC: apocrine carcinoma; AM: apocrine metaplasia. AC and AM exhibited a prominent cytoplasmic abundance and eosinophilia than IPLC eosinophilic, finely granular cytoplasm as IPLC, with characteristic cytoplasmic granularity, but in addition to the loosely cohesive growth pat- the nuclei were more atypical in AC than in AM tern and immunonegativity for E-cadherin com- (Figure 1C, 1D). monly noted in ILC [10]. Six cases (6/58; 10.3%) fulfilled the criteria of IPLC. Ten surgically Selected IPLC and cILC cases were confirmed resected cases of cILC and AC, and five cases to be immunonegative for E-cadherin. Except of AM coexisting in surgically resected speci- for E-cadherin, the result of immunohistochem- men of breast carcinoma were randomly select- ical analyses of each case is shown in Table 1 ed. Formalin-fixed, paraffin-embedded blocks and a summary of immunoreactivity for each

1935 Int J Clin Exp Pathol 2016;9(2):1933-1939 Uroplakin II in apocrine-differentiated cells

Table 2. Summary of immunoreactivity of IPLC, cILC, AC, and AM for each immunohistochemical marker used in this study Score Uroplakin II GATA3 p63 p40 CK7 CK20 34βE12 GCDFP-15 IPLC (n = 6) 3+ 3 (50%) 5 (83%) 0 0 21 (100%) 0 21 (100%) 21 (100%) 2+ 2 (33%) 1 (17%) 0 0 0 0 0 0 1+ 1 (17%) 0 2 (33%) 2 (33%) 0 0 0 0 0 0 0 4 (67%) 4 (67%) 0 21 (100%) 0 0 cILC (n = 10) 3+ 0 10 (100%) 0 0 10 (100%) 0 10 (100%) 0 2+ 0 0 0 0 0 0 0 1 (10%) 1+ 0 0 1 (10%) 1 (10%) 0 0 0 6 (60%) 0 10 (100%) 0 9 (90%) 9 (90%) 0 10 (100%) 0 3 (30%) AC (n = 10) 3+ 5 (50%) 5 (50%) 0 0 10 (100%) 0 0 10 (100%) 2+ 2 (20%) 3 (30%) 0 0 0 0 0 0 1+ 3 (30%) 2 (20%) 0 0 0 0 0 0 0 0 0 10 (100%) 10 (100%) 0 10 (100%) 10 (100%) 0 AM (n = 5) 3+ 5 (100%) 0 0 0 5 (100%) 0 0 5 (100%) 2+ 0 0 0 0 0 0 0 0 1+ 0 1 (20%) 0 0 0 0 0 0 0 0 4 (80%) 5 (100%) 5 (100%) 0 5 (100%) 5 (100%) 0 IPLC: invasive pleomoprhic lobular carcinoma; cILC: classic invasive lobular carcinoma; AC: apocrine carcinoma; AM: apocrine metaplasia. immunohistochemical marker is presented in was observed only in IPLC (Figure 2I) and cILC Table 2. (2/6: 33% and 1/10: 10%, respectively) with a score of 1+ in all the positive cases. p40 Uroplakin II immunoreactivity for IPLC, cILC, AC, showed low expression in IPLC (Figure 2J) and and AM is shown by a representative figure of cILC when compared to p63 (2/6: 33% and each tumor type (Figure 2A-D). Immunopositivity 1/10: 10%, respectively) and no expression of IPLC, AC, and AM to Uroplakin II was observed in AC and AM. CK7 was immunopositive with in all cases examined; they shared consider- a score of 3+ for all cases examined. How- able immunoreactivity for GCDFP-15 as a com- ever, CK20 was completely immunonegative. mon feature. Prominence of Uroplakin II expres- Furthermore, expression of 34βE12 was sion was in the following order: AM, IPLC, and observed exclusively in IPLC and cILC with a AC. All the cases of AM expressed Uroplakin constant 3+ score. GCDFP-15 was consistently IIwith score 3+; however, half of IPLC and AC immunoreactive for IPLC, AC, and AM cases showed Uroplakin II expression of score 3+ with with a score of 3+, while it had a variable immu- the rest of the cases exhibiting its expression of noreactivity in cILC (ranging from 0 to 2+). A score 1+ or 2+; and cILC was immunonegative score of 2+ was observed in only one case for Uroplakin II in all cases examined. GATA3 (10%) and a score of 1+, in 6 cases (60%) with immunoreactivity for IPLC, cILC, AC, and AM is the remaining 3 cases (30%) showing shown by a representative figure of each tumor immunonegativity. type (Figure 2E-H). GATA3 was expressed in all cases of IPLC and cILC with almost all the cases Discussion being evaluated as score 3+, but its expression status seemed to be lower in AC with 5 of 10 We discovered that IPLC, AC, and AM had simi- cases (50%) being evaluated as score 1+ or 2+. lar immunophenotypes considering Uroplakin II GATA3 was less likely to be expressed in AM and GCDFP-15 immunoreactivity. Uroplakin II is (1/5: 20%; score 1+). Immunopostivity for p63 one of the four isoforms (others are Uroplakin

1936 Int J Clin Exp Pathol 2016;9(2):1933-1939 Uroplakin II in apocrine-differentiated cells

Ia, Ib, and III), which enhances the permeability barrier and strength of the urothelium and is normally produced by urothelial cells [11]. Likewise, GCDFP-15 is a marker of apocrine differentiation [12]. Previous studies have shown that breast carcinoma is immunonegative for Uroplakin II [1, 3, 4], in which breast carcinoma showing apocrine differentiation was prob- ably not included. In addition, since cILC examined in this study was immunonegative for Uroplakin III, the interpretation that apocrine differentiation and Uroplakin II immunopositivity is correlated, seems to be valid.

Considering the viewpoint of differential diagnosis, the distinction between IPLC and poorly differentiated urothelial carcinoma seems to be difficult when encountering GATA3-expressing poorly differentiated metastatic carcinoma. Decrease in E-cadherin expression is noted in poorly differentiated urothelial carcinoma [13], and a loss of expression is a feature of plasmacytoid variant of urothelial carcinoma, which is similar to ILC and expresses GATA3 [5]. Even though IPLC shows apocrine differentiation, its degree of cytoplasmic abundance and eosinophilic granularity does not match that of AC and AM. However, distinction between AC and urothelial carcinoma is relatively easy at the metastatic site, since there are no reports that urothelial carcinoma has eosinophilic granular cytoplasm as prominent as that Figure 2. Immunohistochemical findings. A. IPLC case 3: Immunopositiv- observed in AC. We thus focused ity for Uroplakin II (score 3+) (x400). B. cILC case 4: Immunonegativity on the distinction between IPLC for Uroplakin II (score 0) (x400). C. AC case 4: Immunopositivity for Uro- and poorly differentiated urotheli- plakin II (score 2+) (x400). D. AM case 3: Immunopositivity for Uropla- kin II (score 3+). Note immunonegativity for normal breast epithelium al carcinoma. (x400). E. IPLC case 5: Immunopositivity for GATA3 (score 3+) (x400). F. cILC case 3: Immunopositivity for GATA3 (score 3+) (x400). G. AC case In addition to total immunoreactiv- 8: Immunopositivity for GATA3 (score 1+) (x400). H. AM case 4: Immuno- ity for 34EβE12, in some cases, negativity for GATA3 (score 0). Note immunopositivity for normal breast IPLC expressed p63 and p40. The epithelium (x400). I. IPLC case 3: Scattered Immunopositive tumor cells expression of these three markers for p63 (x400). J. IPLC case 3: Scattered Immunopositive tumor cells for p40 (x400). IPLC: invasive pleomoprhic lobular carcinoma; cILC: clas- is typically observed in urothelial sic invasive lobular carcinoma; AC: apocrine carcinoma; AM: apocrine carcinoma [4, 5]. When encoun- metaplasia. tering metastatic carcinoma, CK7

1937 Int J Clin Exp Pathol 2016;9(2):1933-1939 Uroplakin II in apocrine-differentiated cells and CK20 is often stained to identify the pri- References mary site of it [14, 15]. According to previous reports as well as our present findings, CK7 is [1] Hoang LL, Tacha DE, Qi W, Yu C, Bremer RE, expected to be commonly expressed in IPLC Chu J, Haas TS and Cheng L. A newly developed uroplakin II antibody with increased sen- and urothelial carcinoma [5]. On the other sitivity in urothelial carcinoma of the bladder. hand, CK20 is not expressed in IPLC and is Arch Pathol Lab Med 2014; 138: 943-949. often expressed in urothelial carcinoma [5]. [2] Clark BZ, Beriwal S, Dabbs DJ and Bhargava R. Thus, if CK20 immunoreactivity is observed in Semiquantitative GATA-3 immunoreactivity in metastatic carcinoma in addition to GATA3, the breast, bladder, gynecologic tract, and other primary site of the metastatic carcinoma is cytokeratin 7-positive carcinomas. Am J Clin expected to be the urinary bladder. However, Pathol 2014; 142: 64-71. CK20 is not a marker commonly immunoposi- [3] Tian W, Guner G, Miyamoto H, Cimino-Mathews A, Gonzalez-Roibon N, Argani P, Li X, Sharma R, tive for urothelial carcinoma, and its expression Subhawong AP, Rezaei K, Bivalacqua TJ, Ep- decreases when urothelial carcinoma is poorly stein JI, Bishop JA and Netto GJ. Utility of uro- differentiated [5]. On the other hand, CK7 is plakin II expression as a marker of urothelial more frequently expressed than CK20 [5]. carcinoma. Hum Pathol 2015; 46: 58-64. Immunopositivity for GATA3 is maintained in [4] Hoang LL, Tacha D, Bremer RE, Haas TS and poorly differentiated urothelial carcinoma [16]; Cheng L. Uroplakin II (UPII), GATA3, and p40 Uroplakin II is expressed in approximately 60% are Highly Sensitive Markers for the Differen- high-grade urothelial carcinoma [1, 17]. Thus, tial Diagnosis of Invasive Urothelial Carcinoma. Appl Immunohistochem Mol Morphol 2015; metastatic urothelial carcinoma showing poorly 23: 711-6. differentiated morphology with CK7+, CK20-, [5] Paner GP, Annaiah C, Gulmann C, Rao P, Ro JY, GATA3+, and Uroplakin II+ are anticipated. In Hansel DE, Shen SS, Lopez-Beltran A, Aron M, addition, metastatic IPLC is expected to show Luthringer DJ, De Peralta-Venturina M, Cho Y CK7+, CK20-, GATA3+, and Uroplakin II+ immu- and Amin MB. Immunohistochemical evalua- nophenotype. Therefore, when encountering tion of novel and traditional markers associat- metastatic carcinoma with poorly differentiated with urothelial differentiation in a spectrum of variants of urothelial carcinoma of the uri- ed morphology that show this immunophento- nary bladder. Hum Pathol 2014; 45: 1473- type, the possibility of IPLC should also be 1482. considered even though Uroplakin II is [6] Butler D and Rosa M. Pleomorphic lobular car- immunopositive. cinoma of the breast: a morphologically and clinically distinct variant of lobular carcinoma. In conclusion, this study is the first to report the Arch Pathol Lab Med 2013; 137: 1688-1692. immunoreactivity of non-urothelial carcinomas [7] Eusebi V, Magalhaes F and Azzopardi JG. Pleo- for Uroplakin II. These are breast carcinomas morphic lobular carcinoma of the breast: an showing apocrine differentiation, such as IPLC aggressive tumor showing apocrine differenti- and AC. At the metastatic site, IPLC and poorly ation. Hum Pathol 1992; 23: 655-662. [8] Bentz JS, Yassa N and Clayton F. Pleomorphic differentiated urothelial carcinoma might show lobular carcinoma of the breast: clinicopatho- similar morphologies, which indicated that logic features of 12 cases. Mod Pathol 1998; Uroplakin II immunopositivity in metastatic car- 11: 814-822. cinoma with poorly differentiated morphology [9] Vargas AC, Lakhani SR and Simpson PT. Pleo- does not directly lead to the diagnosis of poorly morphic lobular carcinoma of the breast: mo- differentiated urothelial carcinoma and the lecular pathology and clinical impact. Future possibility of IPLC should also be considered. Oncol 2009; 5: 233-243. [10] Jacobs M, Fan F and Tawfik O. Clinicopatho- Disclosure of conflict of interest logic and biomarker analysis of invasive pleomorphic lobular carcinoma as compared with None. invasive classic lobular carcinoma: an experi- ence in our institution and review of the litera- ture. Ann Diagn Pathol 2012; 16: 185-189. Address correspondence to: Dr. Shogo Tajima, [11] Wu XR, Kong XP, Pellicer A, Kreibich G and Sun Department of Pathology, Fujieda Municipal General TT. Uroplakins in urothelial biology, function, Hospital, 4-1-11 Surugadai, Fujieda, Shizuoka 426- and disease. Kidney Int 2009; 75: 1153-1165. 8677, Japan. Tel: 054-646-1111; Fax: 054-646- [12] Mazoujian G, Pinkus GS, Davis S and Haa- 1122; E-mail: [email protected] gensen DE Jr. Immunohistochemistry of a

1938 Int J Clin Exp Pathol 2016;9(2):1933-1939 Uroplakin II in apocrine-differentiated cells

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1939 Int J Clin Exp Pathol 2016;9(2):1933-1939