Large Cell and Small Cell Neuroendocrine Bladder Carcinoma Immunohistochemical and Outcome Study in a Single Institution

Anatomic Pathology / NEUROENDOCRINE BLADDER CARCINOMA Large Cell and Small Cell Neuroendocrine Bladder Carcinoma Immunohistochemical and Outcome Study in a Single Institution Francisco Alijo Serrano, MD, 1 Nora Sánchez-Mora, MD, PhD, 1,2 José Angel Arranz, MD, PhD, 3 Carlos Hernández, MD, PhD, 4 and Emilio Álvarez-Fernández, MD, PhD 1 Key Words: Small cell bladder carcinoma; Immunohistochemical profile; Survival Abstract We studied 44 cases of small cell bladder carcinoma (SCBC) and 2 cases of large cell neuroendocrine bladder carcinoma (LCNBC) to determine the immunohistochemical profile and biologic behavior. Thyroid transcription factor (TTF)-1, cytokeratin (CK)20, chromogranin A (CgA), synaptophysin, neuron-specific enolase (NSE), and Leu- 7 studies were performed. TTF-1+ cases were stained for surfactant protein A (SP-A). The immunohistochemical profile for 44 SCBC cases was as follows: TTF-1+, 11 (25%); CK20+, 3 (7%); CgA+, 13 (30%); synaptophysin+, 22 (50%); NSE+, 35 (80%); and Leu-7+, 30 (68%), and for 2 LCNBC cases was as follows: TTF-1+, 2 (100%); CgA+, (50%); synaptophysin+, 1 (50%); NSE+, 2 (100%); and Leu- 7+, 2 (100%). All cases with TTF-1 expression were negative for SP-A, except 1 case. This case was a mixed SCBC with TTF-1 expression in the urothelial component, which also expressed SP-A. Immunohistochemical markers were not associated with survival. The prognosis of SCBC is relatively better than its pulmonary counterpart. LCNBC seems to be a rarely recognized entity. TTF-1 expression is not limited to small cell lung carcinoma. Extrapulmonary small cell carcinoma is a rare but wellcharacterized tumor occurring at sites as diverse as the gastrointestinal tract, thymus, larynx, salivary gland, skin, breast, prostate, and cervix. 1-7 Small cell bladder carcinoma (SCBC) is very uncommon, accounting for fewer than 1% of all urinary bladder carcinomas, 8,9 and has a poor prognosis, as does its pulmonary counterpart. 8,10-12 This neoplasm is associated with tobacco smoking and frequently seems associated with other carcinomatous components, such as urothelial carcinoma, adenocarcinoma, sarcomatoid carcinoma, or mixtures of these components. 12,13 In addition to SCBC, primary large cell neuroendocrine bladder carcinoma (LCNBC) has been recognized more recently. There are only 8 cases reported in the literature. 14 Immunohistochemical markers commonly used to demonstrate neuroendocrine differentiation, such as chromogranin, synaptophysin, and neuron-specific enolase (NSE), are variably expressed by SCBC. 15 The expression of thyroid transcription factor (TTF)-1 by SCBC has been reported by Jones et al 16 and Soriano et al 17 in series of 44 and 10 cases, respectively. No previous report has been found concerning the expression of surfactant protein A (SP-A). A Merkel cell carcinoma like immunophenotype with positive expression of cytokeratin (CK)20 had been reported in extrapulmonary small cell carcinoma. 18-20 Some reports suggested that Merkel cell carcinoma like CK20+ salivary gland small cell carcinomas have a better prognosis than CK20 tumors. 18,19 The aim of our work was to study the immunohistochemical expression of TTF-1, SP-A, CK20, chromogranin A (CgA), synaptophysin, NSE, and Leu-7 in a series of 46 cases: 44 cases of SCBC and 2 cases of LCNBC from a single institution to further elucidate the immunohistochemical Am J Clin Pathol 2007;128:733-739 733 733 733

Alijo Serrano et al / NEUROENDOCRINE BLADDER CARCINOMA profile and the biologic behavior according to the expression of these markers. Materials and Methods Clinical data for 44 patients with SCBC and 2 with LCNBC were reviewed for clinical information and follow-up status. All tumors were classified according to criteria established by the World Health Organization classification system. 21 The 2002 TNM classification system was used for pathologic staging. Archival materials from these 46 cases (accessioned from January 1990 to December 2005) were retrieved from the surgical pathology files of the Gregorio Marañón University General Hospital, Madrid, Spain. For light microscopy, 4-µm sections were cut from paraffin blocks and stained with H&E. Histologic slides were reviewed at least 3 times by all 3 pathologist participants in the study (F.A.S., N.S.-M., and E.Á.-F.). All tumors fulfilled the criteria established for small cell carcinoma according to the World Health Organization classification system. Additional sections were obtained for immunohistochemical staining, which was performed on an automatic immunostainer (TechMate 500, DAKO, Carpinteria, CA). All cases were stained for TTF-1, CK20, CgA, synaptophysin, NSE, and Leu- 7. Cases with TTF-1 expression were also stained for SP-A. Immunohistochemical stains for TTF-1 were carried out using monoclonal mouse anti TTF-1 antibody (clone SPT24, 1:100 dilution; Novocastra, Newcastle upon Tyne, England), CK20 (clone Ks20.8, 1:50 dilution; Novocastra), CgA (clone LK2H10, 1:2 dilution; DAKO, Carpinteria, CA), synaptophysin (polyclonal, 1:8 dilution; Biomeda, Foster City, CA), NSE (clone BBS/NC/VI-H14, DAKO); Leu-7 (clone HNK-1, 1:20 dilution; Becton Dickinson, San Jose, CA); and SP-A (clone PE10, 1:80 dilution; DAKO). Statistical Analysis Individual information and baseline characteristics were summarized using descriptive statistics. Comparison of proportions was performed with the χ 2 according to the Yates test or Fisher exact test (for cross-tabulations with an expected value in any cell δ5). Test results were considered significant if 2-tailed P values were less than.05. Survival was measured from the time of diagnosis to the date of death or the latest follow-up. Overall survival was estimated using the Kaplan- Meier method. Statistical analyses were performed using SPSS software (version 11.5, SPSS, Chicago, IL). Results Table 1 summarizes relevant clinical data for the cases studied. Of the patients, 41 (89%) were men and 5 (11%) were women. The average age was 69.7 years (range, 40-89 years; SD, 11.9 years). Tobacco use was reported by 33 patients (72%) and denied by 13 (28%). Tobacco consumption was more frequent in men (P =.001). All cases were treated by surgery (27 by transurethral resection and 19 by radical cystoprostatectomy). In addition to surgery, 20 (43%) of 46 cases received chemotherapy, 12 (26%) received radiotherapy, and 3 (7%) received palliative treatment. Of the 46 tumors, 28 (61%) were located in the posterior vesicle wall, 12 (26%) in the lateral walls, and 6 (13%) in the anterior wall. Histologically, 28 (61%) of 46 tumors showed a homogeneous, pure microcytic pattern Image 1A. In 16 cases (35%), an additional urothelial component was also observed, and 2 cases (4%) fulfilled the histologic criteria for LCNBC characterized by large, polygonal cells with a low nuclear/cytoplasmic ratio. Coarse chromatin and frequent nucleoli, mitotic activity in excess of 10 mitoses per 10 high-power fields, and multiple areas of necrosis were also seen Image 1B. Pathologic staging was I in 4 (9%) of 46 cases, II in 21 (46%), III in 12 (26%), and IV in 9 (20%). Among the 46 cases, 13 (28%) showed positive staining for TTF-1 (11/44 SCBC; 2/2 LCNBC) Image 1C. These cases were 9 pure small cell carcinomas, 2 small cell carcinomas with a urothelial carcinoma component, and 2 LCNBCs. All cases with TTF-1 expression were negative for SP-A, except 1 case (8%). This case was a small cell carcinoma with TTF-1 expression in the urothelial component, which also expressed SP-A Image 1D and Image 1E. CK20 expression was observed in 3 cases (7%). The positive stain was diffuse in the cytoplasm. Neuroendocrine marker expression was as follows: NSE+, 37 (80%; 35/44 SCBC; 2/2 LCNBC); CgA+, 14 (30%; 13/44 SCBC; 1/2 LCNBC); synaptophysin+, 23 (50%; 22/44 SCBC; 1/2 LCNBC); and Leu-7+, 32 (70%; 30/44 SCBC; 2/2 LCNBC) Table 2. None of the immunohistochemical markers used in our study showed significant statistical correlation with any of the clinical parameters analyzed. Follow-up information was available for all cases. The mean follow-up was 20.1 months (δ = 27.5; range, 1-120 months). The 3-year survival rate was 27.5%. The mean survival for the whole series was 40 months (95% confidence interval [CI], 24-56 months). Of the 46 patients, 29 (63%) died of disease, 7 (15%) were alive with tumor (local relapse: 2 patients with urothelial carcinoma, 4 with metastatic disease, and 1 with lung adenocarcinoma), and 10 (22%) were alive without evidence of disease after 5 years of follow-up. The 3-year survival rate by stage was as follows: I, 100%; II, 58%; III, 47%; and IV, 4%. The overall 5-year survival rate by stage was as follows: I, 100%; II, 39% (mean, 51 months; 95% CI, 22-80 months); III, 46.8% (mean, 33 months; 95% CI, 19-46 months); and IV, 4% (mean, 16 734 Am J Clin Pathol 2007;128:733-739 Downloaded 734 from https://academic.oup.com/ajcp/article-abstract/128/5/733/1760252

Anatomic Pathology / ORIGINAL ARTICLE Table 1 Clinical and Pathologic Characteristics of 46 Patients With Small Cell Bladder Carcinoma Case No/Sex/Age (y) TNM Stage Histologic Type Treatment Survival (mo)/status 1/M/61 II Microcytic, mixed RCP 9/AWD 2/F/60 I Microcytic, pure TUR 4/AWD 3/M/80 II Microcytic, mixed TUR 4/AWD 4/M/85 II Microcytic, pure TUR 3/DOD 5/M/58 I Microcytic, mixed TUR, RT, CH 3/AD 6/M/65 IV Microcytic, pure TUR 12/DOD 7/M/78 III Microcytic, pure RCP 20/AD 8/F/77 IV Microcytic, mixed TUR, RT 2/DOD 9/M/75 III Microcytic, pure TUR, CH 30/DOD 10/M/71 II Microcytic, pure TUR, RT, CH 16/AD (urothelial carcinoma) 11/M/40 II LCNBC RCP, CH 13/AWD 12/M/76 III Microcytic, pure RCP, RT, CH 31/AWD 13/M/58 IV Microcytic, mixed RCP 13/DOD 14/M/73 II Microcytic, pure TUR 1/DOD 15/M/46 III Microcytic, pure RCP, CH 29/AWD 16/M/61 II Microcytic, pure TUR, RT 7/DOD 17/M/58 IV Microcytic, mixed RCP, CH 9/DOD 18/M/67 IV Microcytic, mixed RCP, CH 21/DOD 19/M/75 IV Microcytic, pure TUR, RT 3/DOD 20/M/70 III Microcytic, mixed RCP 3/DOD 21/M/77 I Microcytic, pure RCP, CH 37/AWD 22/F/78 II Microcytic, mixed TUR 5/DOD 23/M/82 III Microcytic, mixed RCP, CH 17/DOD 24/M/70 IV Microcytic, pure TUR, CH 31/DOD 25/M/68 III Microcytic, pure RCP, CH 44/AWD 26/M/80 IV Microcytic, pure TUR, palliative 8/DOD 27/M/77 IV Microcytic, pure RCP, RT 16/DOD 28/F/43 IV LCNBC RCP, RT 12/DOD 29/M/75 IV Microcytic, pure TUR, RT, CH 27/DOD 30/M/85 I Microcytic, mixed TUR 57/AD (urothelial carcinoma) 31/M/79 IV Microcytic, pure TUR, RT 8/DOD 32/M/75 IV Microcytic, pure TUR 2/DOD 33/F/40 II Microcytic, mixed RCP, CH 87/AD 34/M/65 II Microcytic, pure TUR 12/DOD 35/M/80 II Microcytic, mixed TUR, RT 47/DOD 36/M/73 II Microcytic, mixed TUR, CH 98/AD (lung carcinoma) 37/M/69 IV Microcytic, pure RCP, CH 2/DOD 38/M/74 IV Microcytic, pure TUR, CH 6/DOD 39/M/78 IV Microcytic, pure TUR, palliative 24/DOD 40/M/54 IV Microcytic, pure RCP, CH 120/AWD 41/M/79 IV Microcytic, pure TUR 1/DOD 42/M/79 III Microcytic, mixed TUR, RT 12/DOD 43/M/65 IV Microcytic, pure RCP, CH 2/DOD 44/M/59 IV Microcytic, pure RCP, CH 14/DOD 45/M/89 IV Microcytic, pure TUR, palliative 1/AD 46/M/82 II Microcytic, mixed TUR 1/AWD AD, alive with disease; AWD, alive without disease; CH, chemotherapy; DOD, died of disease; LCNBC, large cell neuroendocrine bladder carcinoma; RCP, radical cystoprostatectomy; RT, radiotherapy; TUR, transurethral resection. months; 95% CI, 5-27 months). Among the 9 patients with stage IV disease, 1 with metastatic disease at diagnosis underwent radical cystoprostatectomy after complete remission to chemotherapy. This patient was alive and free of disease more than 10 years following cystectomy. The clinicopathologic variables studied were age, sex, smoking history, histologic tumor type, use of chemotherapy and radiation therapy, and immunohistochemical markers (CgA, synaptophysin, NSE, Leu-7, and TTF-1). We found no statistically significant differences associated with survival. The TNM stage was the only prognostic factor with a statically significant relationship to survival (log rank, 0.002). Discussion Small cell carcinoma is an uncommon tumor in the bladder. The prognosis for SCBC is generally poor. 8,10-12 However, a relatively better survival for patients with bladder cancer than that of the pulmonary counterpart has been reported. There are only 19 reports of SCBC series that include at least 10 patients. 7,8,11-17,22-31 According to these reports, the 5-year survival rate for all stages is variable, ranging between 8% and 40% Table 3. When comparing the survival rate of our series with the only published report in which the cases were properly staged, 23 a certain similarity of Am J Clin Pathol 2007;128:733-739 735 735 735

Alijo Serrano et al / NEUROENDOCRINE BLADDER CARCINOMA A B C D E Image 1 A, Section of small cell bladder carcinoma showing rosette formation (H&E, original magnification 20). B, Large cell neuroendocrine bladder carcinoma showing palisading at the periphery of the nest of tumor cells with central necrosis. Large tumor cells have abundant cytoplasm with large nuclei, vesicular nuclear chromatin, and prominent nucleoli. No glandular or squamous differentiation is seen (H&E, original magnification 20). C, Immunostaining for thyroid transcription factor (TTF)-1 in small cell bladder carcinoma (original magnification 20). D, Immunostaining for surfactant protein A in the urothelial component (original magnification 10). E, Immunostaining for TTF-1 in a case with mixed small cell papillary urothelial bladder carcinoma showing TTF-1 nuclear expression in the urothelial component (original magnification 20). survival can be found. The 5-year rates for patients with stage II, III, and IV disease were 63.6%, 15.4%, and 10.5%, respectively. In our series, the survival rate with stage I tumors was 100%; for stage II, 39%; for stage III, 47%; and for stage IV, 4%. The outcome is relatively better for SCBC than for small cell lung carcinoma. One possible explanation is that tumor volume may be smaller in SCBC than in small cell lung cancer of limited stage at the time of diagnosis.27 It is important to mention that there are some reported cases of stage IV SCBC that showed a long-term survival31 like one 736 Am J Clin Pathol 2007;128:733-739 Downloaded 736 from https://academic.oup.com/ajcp/article-abstract/128/5/733/1760252 of our cases. This has not been previously described in small cell lung carcinoma. Primary LCNBC represents a rare and new histologic tumor type. We found 2 cases of pure primary LCNBC. The clinical behavior trends are similar to those of SCBC in the only 8 cases previously reported14 and both cases reported by us. The diagnosis of SCBC can be made on morphologic grounds alone, even if neuroendocrine differentiation cannot be demonstrated.21 In addition, a novel immunohistochemical marker, TTF-1, has been demonstrated in this tumor. Cheuk et

Anatomic Pathology / ORIGINAL ARTICLE Table 2 Immunohistochemical Profile of Small Cell Bladder Carcinoma in Different Studies * Neuron-Specific Thyroid Transcription Study No. of Cases Enolase Chromogranin A Synaptophysin Leu-7 Factor-1 Present study, 2006 46 37 (80) 14 (30) 23 (50) 32 (70) 13 (28) Abrahams et al, 31 2005 51 25 30 70 Jones et al, 16 2005 44 39 Soriano et al, 17 2004 10 100 50 40 20 Helpap, 25 2002 20 100 100 Nabi et al, 26 2001 11 0 36.4 76 Trias et al, 22 2001 23 86 30 Iczkowski et al, 28 1999 46 93 65 76 Abbas et al, 7 1995 135 90 40.6 45.5 34.3 Grignon et al, 11 1992 22 100 89 Blomjous et al, 8 1989 18 83.3 22.2 66.6 27.7 Mills et al, 30 1987 12 83.3 25 58.3 * Data are given as number (percentage) or percentage of positive cases. Table 3 Small Cell Bladder Carcinoma: A Review of Case Series With 10 or More Cases No. of Source of Overall Survival (%)/ Study Cases Cases * Follow-up Survival Quek et al, 14 2005 25 1 30/2 y; 10/5 y Median, 13 mo Abrahams et al, 31 2005 51 1 40/5 y Median, 23 mo Wang et al, 13 2005 50 5 45/2 y; 16/5 y Jones et al, 16 2005 44 5 9/5 y Choong et al, 23 2005 44 1 Stage II, 63.6/5 y; stage III, Mean, 1.7 y 15.4/5 y; stage IV, 10.5/5 y Bex et al, 24 2005 25 1 Median, 8 mo (range, 2-84 mo); limited disease, 12 mo (range, 4-84 mo); extensive disease, 5 mo (range, 2-17 mo) Mangar et al, 15 2004 14 Cases referred 24/2 y Median, 5 mo to 1 hospital Cheng et al, 12 2004 64 5 23/3 y; 16/5 y Soriano et al, 17 2004 10 1 Mean, 8.7 mo Helpap, 25 2002 20 Own and Mean, 6.9 mo consultation cases Nabi et al, 26 2001 11 1 Mean, 16.5 mo (range, 6-30 mo) Trias et al, 22 2001 23 2 18.2/6.2 mo Lohrisch et al, 27 1999 14 Cases referred All groups, 36/5 y; limited Mean, 47 mo (95% confidence interval, to 1 hospital stage, 44/5 y 18.5-76.1); median, 41 mo Iczkowski et al, 28 1999 46 5 28/5 y Mean, 17 mo (range, 1-143 mo) Abbas et al, 7 1995 135 Compiled all cases 25/2 y; 8/5 y Mean, 19.6 mo (range, 0-18 y) published until 1995 Holmang et al, 29 1995 25 1 Autopsy, 3 cases; extensive disease, 4 cases; limited disease, 18 cases; 13 died of progressive disease (mean, 7.3 mo); 5 alive without disease (median, 10 y) Grignon et al, 11 1992 22 1 Mean, 9.8 mo (range, 2-21 mo); median, 10 mo Blomjous et al, 8 1989 18 4 14 (78%) died of tumor (mean, 9.5 mo); 3 alive at 22, 23, and 38 mo; 1 alive at 5 y Mills et al, 30 1987 12 4 Median, 4 mo (range, 2.5-12 mo) * The number of hospitals is given unless otherwise indicated. al 32 and Agoff et al 33 reported TTF-1 expression in 1 of 3 and 2 of 4 cases of SCBC, respectively, and Jones et al, 16 in a series of 44 cases of SCBC, demonstrated TTF-1 expression in 17 cases (39%). In our study, TTF-1 expression was seen in 27% (12/44) of the cases of SCBC. TTF-1 immunoreactivity showed no correlation with any of the clinical parameters analyzed. It is important to mention that expression of TTF-1 and SP-A was also observed in the non small cell component of 1 case. This finding has not been reported previously. Unexpected expression of SP-A has been reported in other studies, including 3 cases of thyroid carcinoma, 6 of prostatic carcinoma, 34 and 6 of breast carcinoma 35 but not in primary bladder carcinoma. All these cases were reported in pulmonary metastases from the tumors. This raised doubt about whether the positive expression of the SP-A is a result of its synthesis by tumor cells or of phagocytosis of the SP-A by tumor cells. Only 1 case of extrapulmonary primary colorectal carcinoma expressing Am J Clin Pathol 2007;128:733-739 737 737 737

Alijo Serrano et al / NEUROENDOCRINE BLADDER CARCINOMA SP-A has been reported previously. 36 The possible explanation for this could be the relationship of the biochemical structure of SP-A with a number of cellular proteins that share homology with its molecule, like calcium-dependent lectins and type-iv collagen. 37,38 This coexpression of TTF-1 and SP- A must be considered when evaluating carcinomas of unknown origin appearing clinically as disseminated disease. One pure and one mixed small cell carcinoma recurred as purely papillary carcinoma. The recurrent tumors did not show neuroendocrine markers or TTF-1. In addition to studying the expression of TTF-1 in SCBC, we also examined the expression of CgA, synaptophysin, NSE, Leu-7, and SP-A in this group of tumors. None showed any relationship with survival. The only prognostic factor for survival was the TNM classification. CK20 expression has also been shown to be a useful marker in distinguishing Merkel cell carcinoma from small cell carcinoma. However frequent CK20 positivity was observed in salivary gland small cell carcinomas, suggesting that may be a subtype of Merkel cell carcinoma with clinical evolution that is less aggressive than other small cell carcinomas. 18,19 CK20 expression also had been reported in vaginal small cell carcinoma in 1 case. 20 Because the prognosis for SCBC is relatively better than that of its pulmonary counterpart, we studied CK20 expression as a possible marker with clinical significance. Our results showed expression in only 3 cases, and the pattern of staining was diffuse cytoplasmic and without prognostic significance. Therefore CK20 expression did not prove to be a diagnostic or prognostic marker for SCBC in our study. The prognosis of SCBC seems to be relatively better than that of its pulmonary counterpart, especially in stage I tumors. 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