Primary enteric-type adenocarcinoma of the urinary bladder is relatively uncommon. We present our experience with 109 pure, non-urachal cases—the largest series to date. This work was undertaken with the aim of describing the immunohistochemical features of adenocarcinoma of the urinary bladder associated with schistosomiasis, illustrating their histologic and immunohistochemical similarities to colorectal carcinomas. Partial or total cystectomy specimens from a cohort of Egyptian and American patients with the diagnosis of primary adenocarcinoma of the urinary bladder (109 cases) were reviewed. Paraffin sections of each tumour were stained using the labelled streptavidin–biotin method using antibodies cytokeratin 20, cytokeratin 7, CDX2, MLH1, and villin. Clinical follow-up was available for at least 36 months. An enteric (colonic) morphology was seen in most tumours; some with signet ring cells or mucinous elements. Five tumours were composed predominantly of signet ring cells and two demonstrated a pure mucinous morphology. In cases where adjacent normal mucosa was present, 23% showed either colonic metaplasia or intestinal-type cystitis glandularis. Furthermore, 24% of enteric-type adenocarcinomas had associated villous or tubulovillous adenomas with or without dysplasia. Cytokeratin 20 was expressed by 90%, cytokeratin 7 by 17%, CDX2 by 100% and villin and MLH1 by 76% and 48% of tumours respectively. The majority of tumours presented with an advanced stage and followed an aggressive clinical course.
Journal of Advanced Research (2010) 1, 151–156 Cairo University Journal of Advanced Research ORIGINAL ARTICLE Primary enteric-type adenocarcinomas of the urinary bladder are histogenetically analogous to colorectal carcinomas: Immunohistochemical evaluation of 109 cases Saad S Eissa a,∗ , Norman Block b , Hussein M Khaled c , Sohair H Shoman a , Mehdi Nassiri d , Mehrdad Nadji d a Department of Pathology, National Cancer Institute, University of Cairo, Cairo, Egypt Urology Department, University of Miami, Jackson Memorial Hospital, Miami, FL, USA c Department of Medical Oncology, National Cancer Institute, University of Cairo, Cairo, Egypt d Pathology Department, University of Miami, Jackson Memorial Hospital, Miami, FL, USA b Available online March 2010 KEYWORDS Urinary bladder; Adenocarcinoma; Immunohistochemistry; Carcinogenesis ∗ Abstract Primary enteric-type adenocarcinoma of the urinary bladder is relatively uncommon We present our experience with 109 pure, non-urachal cases—the largest series to date This work was undertaken with the aim of describing the immunohistochemical features of adenocarcinoma of the urinary bladder associated with schistosomiasis, illustrating their histologic and immunohistochemical similarities to colorectal carcinomas Partial or total cystectomy specimens from a cohort of Egyptian and American patients with the diagnosis of primary adenocarcinoma of the urinary bladder (109 cases) were reviewed Paraffin sections of each tumour were stained using the labelled streptavidin–biotin method using antibodies cytokeratin 20, cytokeratin 7, CDX2, MLH1, and villin Clinical follow-up was available for at least 36 months An enteric (colonic) morphology was seen in most tumours; some with signet ring cells or mucinous elements Five tumours were composed predominantly of signet ring cells and two demonstrated a pure mucinous morphology In cases where adjacent normal mucosa was present, 23% showed either colonic metaplasia or intestinal-type cystitis glandularis Furthermore, 24% of enteric-type adenocarcinomas had associated villous or tubulovillous adenomas with or without dysplasia Cytokeratin 20 was expressed by 90%, cytokeratin by 17%, CDX2 by 100% and villin and MLH1 by 76% and 48% of tumours respectively The majority of tumours presented with an advanced stage and followed an aggressive clinical course In conclusion, primary non-urachal enteric-type adenocarcinoma of the urinary bladder is morphologically and immunophenotypically similar – if not identical – to colonic adenocarcinoma The frequent association of enteric carcinomas of the urinary bladder with intestinal metaplasia and/or colonic-type adenomas with dysplasia suggests possible carcinogenetic pathways similar to that observed in colorectal carcinomas © 2010 Cairo University All rights reserved Corresponding author Tel.: +20 26332133/24538399;fax: +202 3644720 E-mail address: drsaadeissa@hotmail.com (S.S Eissa) 2090-1232 © 2010 Cairo University Production and hosting by Elsevier All rights reserved Peer review under responsibility of Cairo University Production and hosting by Elsevier doi:10.1016/j.jare.2010.03.006 152 S.S Eissa et al Introduction Primary non-urachal enteric adenocarcinomas of the urinary bladder are rare and usually associated with chronic irritation of the bladder mucosa on a background of intestinal metaplasia and/or intestinal-type cystitis glandularis [1–6] The histologic similarity of such adenocarcinomas to colorectal carcinoma is striking Numerous immunohistochemical stains have been reliably performed on paraffin embedded tissue sections and used as markers of tumour differentiation as well as markers of carcinogenic mechanisms In this study we present a review of the morphology and immunophenotype of 109 such cases, mostly associated with schistosomiasis In our analysis special attention was paid to the presence of intestinal metaplasia and/or preneoplastic changes of the urinary bladder mucosa The results were compared with the phenotype of colonic carcinoma previously reported Patient and methods One hundred and nine partial or radical cystectomy specimens from the National Cancer Institute, Cairo, Egypt (101 cases), and the University of Miami, Jackson Memorial Hospital, Miami, FL, USA (8 cases) were examined in this study Biographic data and clinical information including clinical presentation, type of therapy and follow-up were reviewed The histopathology of tumour and pathologic stage were recorded in all samples Special attention was paid to the status of non-involved bladder mucosa for the presence of intestinal metaplasia and/or cystitis glandularis Two representative paraffin blocks from each case were selected for immunohistochemical studies Paraffin sections, m thick, were stained using the standard labelled streptavidin–biotin method using antibodies against cytokeratin 20, cytokeratin 7, CDX2, MLH1 and villin The commercial sources and working dilutions of the antibodies are summarised in Table Heat-induced antigen retrieval was used with citrate buffer in a vegetable steamer DAB, in the presence of hydrogen peroxide, was used as the chromogen All buffers and detection reagents were from Dako (Carpinteria, CA, USA) For CDX2 and MLH1, a cytoplasmic counterstain was utilised (Fast Green) The presence, pattern and intensity of immunohistochemical reactions were evaluated in tumour cells and in non-neoplastic epithelium Results Of the 134 patients with the diagnosis of adenocarcinoma, 20 patients, who did not have adequate clinical information to exclude the possibility of urachal or metastatic colonic adenocarcinoma were eliminated Similarly, two patients with clear cell (mesonephric) adenocarcinoma and three with extension of prostatic adenocarci- Table Table Clinical stage and survival of 109 patients with primary intestinal-type adenocarcinoma of the urinary bladder (minimum of 36 month follow-up) Stage T1 T2 T3 T4 Total 11 (10%) 20 (18%) 76 (70%) (0.2%) 109 NEDa AWDa DODa 9 17 10 28 0 31 35 40 34 a NED: alive, no evidence of disease; AWD: alive with disease; DOD: dead of disease noma to the bladder were excluded from the study The remaining 109 tumours occurred predominantly in trigone, lateral or posterolateral walls None of the tumours involved the bladder dome The study cohort consisted of 68 male and 41 female patients (male/female ratio, 1.7:1.0), aged 25–78 (mean, 50.3 years) The most frequent presenting symptoms were haematuria and urinary irritation In most patients, partial or radical cystectomy was performed followed by radiation therapy Follow-up data for a minimum of 36 months was available The clinical stage of disease and the follow-up data are summarised in Table Briefly, 78 patients (72%) presented with advanced disease (T3 and T4), 61 of whom (78%) were either deceased from the disease or alive with recurrent cancer during the follow-up period All five patients with the predominant signet ring cell type adenocarcinoma were dead of disease in less than 15 months following the initial diagnosis Tumours grossly appeared as exophytic, papillary, solid, or infiltrative (Fig 1A) Ulceration was grossly present in 49 cases A linitis plastica-like diffuse transmural infiltration was seen in two cases Histologically, most tumours were composed of pseudostratified columnar epithelium forming glands that resembled colonic adenocarcinoma (Fig 1B) The histologic subtypes of tumours are summarised in Table Focal mucinous morphology, in which small nests of tumour cells floated in abundant extracellular mucin, was seen in 13 cases (Fig 1C) Two tumours were of the purely mucinous type Focal signet ring cell morphology was present in 15 cases There were also five cases composed predominantly of signet ring cells (Fig 1D); two with a linitis plastica-like transmural infiltration There were two cases showing true mixed patterns One was a combination of small cell carcinoma and signet ring cell carcinoma, each comprising about 50% of the tumour mass The other was a combined tumour (collision) between an intestinaltype adenocarcinoma (70% of the tumour mass) and a transitional cell carcinoma Calcified schistosoma eggs were identified in more than 90% of the Egyptian cases, either in the midst of the tumour or in uninvolved bladder wall No specific histologic differences were observed between the cases retrieved from the Egyptian and American series The sources and working dilutions of antibodies Antibody Source Working dilution Cytokeratin Cytokeratin 20 CDX-2 MLH1 Villin Dako (Carpinteria, CA, USA) Dako (Carpinteria, CA, USA) Biogenex (San Ramon, CA, USA) Zymed (South San Francisco, CA, USA) GeneTex (San Antonio, TX, USA) 1:200 1:25 1:100 1:20 1:100 Enteric adenocarcinoma of bladder 153 Figure (A) Macroscopic view of a primary intestinal-type adenocarcinoma of the urinary bladder (B) Gland forming adenocarcinoma of the urinary bladder Morphologically this tumour resembles a colonic adenocarcinoma Note a calcified schistosoma egg (arrow) (H&E, 100×) (C) Intestinal adenocarcinoma of the urinary bladder, mucinous type (H&E, 50×) (D) Signet ring cell type of intestinal adenocarcinoma of the urinary bladder (H&E, 200×) Of the 52 cases with adjacent mucosa not involved by tumour, 12 (23%) showed enteric (colonic) metaplasia of the surface mucosa, or an intestinal-type cystitis glandularis Further, in 24% of cases, villous or tubulovillous adenomas with varying grades of dysplasia and/or intramucosal carcinoma were present adjacent to the invasive adenocarcinoma (Fig 2A) Immunohistochemically, 100 tumours (92%) were positive for cytokeratin 20; the reactions were predominantly strong and diffuse Figure (A) Villous adenoma of the urinary bladder associated with areas of intramucosal adenocarcinoma on the left (H&E, 50×) (B) Diffuse cytokeratin 20 expression papillary areas of intestinal adenocarcinoma of the urinary bladder (immunoperoxidase, 100×) (C) Mucinous type intestinal adenocarcinoma of the urinary bladder Positive reaction for cytokeratin 20 in floating tumour cells (immunoperoxidase, 50×) (D) Expression of cytokeratin by the urothelial (right) and not by the intestinal (left) cystitis glandularis (immunoperoxidase, 100×) 154 S.S Eissa et al Figure (A) Tubulovillous adenoma of the urinary bladder with strong nuclear staining for CDX2 (immunoperoxidase, 50×) (B) Expression of CDX2 in a high-grade enteric adenocarcinoma of the urinary bladder (immunoperoxidase, 50×) (C) Villin expression is limited to the brush borders of some tumour cells in a low-grade enteric adenocarcinoma of the urinary bladder (immunoperoxidase, 100×) (D) Cytoplasmic localisation of villin in a high-grade adenocarcinoma (immunoperoxidase, 50×) (E) MLH1 expression is preserved in a villous adenoma of the urinary bladder (immunoperoxidase, 50×) (F) Marked reduction in expression of MLH1 in high-grade enteric adenocarcinoma of the urinary bladder (immunoperoxidase, 50×) Table Morphologic variants of intestinal adenocarcinoma of the urinary bladder Histologic pattern Number Enteric (colonic) type Focally mucinous type Focally signet ring type 100 13 15 Pure mucinous type Pure signet ring type Mixed cell type Enteric/transitional Signet ring/small cell 1 Total 109 (Fig 2B) Tumour cells in the areas with mucinous or signet cell morphology also reacted positively for cytokeratin 20 (Fig 2C), as did epithelial cells in intestinal metaplasia, cystitis glandularis, and tubulovillous adenomas Seventeen tumours were also focally positive for cytokeratin Areas of intestinal metaplasia and intestinal-type cystitis glandularis were negative for cytokeratin 7, whereas uninvolved transitional epithelium or urothelial type cystitis glandularis were always positive (Fig 2D) Nuclear positivity for CDX2 was observed in all enteric adenocarcinomas, tubulovillous adenomas, and intestinal metaplasia (Fig 3A and B) Of the 54 tumours that were tested for villin expression, 41 (76%) were positive in both adenocarcinoma and in adenomatous areas The brush border localisation of villin in adenomas and low-grade adenocarcinomas (Fig 3C) was different from intracytoplasmic reaction in high-grade adenocarcinomas (Fig 3D) The pattern and intensity of MLH1 expression was variable from case to case (Fig 3E) Overall, Enteric adenocarcinoma of bladder 22% of adenocarcinomas showed a reduction in the nuclear staining for MLH1 (Fig 3F) Discussion Primary adenocarcinomas of the urinary bladder are uncommon, comprising about 2% of all bladder cancers [1] The classification of adenocarcinomas of the urinary bladder has been the subject of many publications in the past Some authors have included all urothelial carcinomas with recognisable glandular differentiation in the adenocarcinoma category, while others have recognised only those without an associated transitional carcinoma component as primary adenocarcinomas of the urinary bladder [1–3,9–11] Pure non-transitional adenocarcinomas of the bladder include the enteric-(intestinal) type and the rare clear cell (mesonephric) adenocarcinomas Primary enteric-type adenocarcinomas of the urinary bladder can be further divided into urachal and non-urachal varieties The urachal type occurs in the dome or anterior wall and commonly exhibits a mucinous histomorphology [7] Non-urachal primary adenocarcinomas of the bladder usually occur in the trigone, posterior, or postero-lateral walls [4–6,8] The pure non-urachal enteric-type primary adenocarcinomas of the urinary bladder are relatively rare and, to our knowledge, this is the largest series (109 cases) of this tumour reported These tumours usually occur in association with bladder exstrophy or schistosoma associated bladder cancer Histologically, they may exhibit an enteric (intestinal/colonic), signet ring cell, or mucinous morphology When compared to transitional cell carcinomas, enteric adenocarcinomas of the urinary bladder behave more aggressively, with the signet ring cell type being associated with an extremely poor prognosis [12–14] The possibility of urachal or colonic origin was clinically excluded in our patients The majority of tumours showed a classic enteric (colonic) morphology, some with focal signet ring cell or mucinous features Two cases were predominantly mucinous type and five were composed mostly of signet ring cells Associated colonic metaplasia, colonic-type cystitis glandularis, and tubulovillous adenomas – with or without dysplasia – were observed in about one quarter of cases where adequate tissue was available for evaluation as well as reported by others [15,16] The expression profile of cytokeratin 20, cytokeratin 7, CDX2, villin and MLH1 closely resembled that of colorectal adenocarcinomas Areas with signet ring cell or mucinous morphology exhibited the same immunohistochemical profile as colonic carcinomas This similar immunohistochemical profile of adenocarcinoma of the urinary bladder and colon may be explained by their common embryogenic origin from endoderm The expression of CDX2, villin, and mismatch repair gene products in colorectal carcinomas has been studied previously CDX2 is a homeobox intestinal differentiation factor that regulates the expression of a number of key intestinal enzymes Most gastrointestinal tract adenocarcinomas, particularly those of colonic origin, express CDX2 Similar to CDX2, villin is also expressed by the majority of colonic carcinomas This actin-binding protein is localised in the microvilli of normal epithelial cells in the small and large intestines [17] In well-differentiated colonic adenocarcinomas, villin expression is seen in the brush border of neoplastic cells; a pattern that was also observed in our group of enteric adenocarcinomas of the urinary bladder Microsatellite instability is a genetic alteration caused by defects in mismatch repair genes The most commonly affected genes – orig- 155 inally described in colorectal cancers – are MLH1 and MSH2 [18] MLH1 and MSH2 abnormalities are found in up to 20% of sporadic colorectal carcinomas [18] The traditional PCR-based assays for tissue detection of mismatch repair genes have for the most part been replaced by simple immunohistochemical identification of their protein products [19] In addition to colorectal tumours, abnormality of mismatch repair genes has been shown in other malignancies, including those of urothelial origin In one study, up to 46% of bladder carcinomas were shown to have reduced expression of MLH1 by immunohistochemistry [20] It is generally believed that most cases of primary intestinal-type adenocarcinoma of the urinary bladder arise either from intestinal metaplasia or enteric-type cystitis glandularis [3–6,9] This was true in our group of patients, many of whom exhibited such an association The morphologic and immunophenotypic similarity of enteric adenocarcinomas of the urinary bladder and primary colorectal adenocarcinomas is further strengthened by the presence of premalignant stages such as adenomas with dysplasia or in situ and intramucosal carcinoma Villous adenomas of the urinary bladder have been reported before, and it has been postulated that most represent the noninvasive phase of intestinal-type adenocarcinomas [21–23] The presence of tubulovillous adenomas with varying grades of dysplasia or early invasive carcinoma in our cases supports that impression This observation further suggests that intestinal-type adenocarcinomas of the bladder may follow a carcinogenic pathway similar to that observed in colorectal carcinomas Whether or not the same multistep progression of gene abnormality of colorectal cancer can be detected in intestinal adenocarcinoma of the urinary bladder is yet to be determined In summary, enteric adenocarcinoma of the urinary bladder is histologically similar to colonic adenocarcinoma All morphologic variants may be associated with intestinal metaplasia, enteric-type cystitis glandularis, and 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Primary adenocarcinomas of the urinary bladder are uncommon, comprising about 2% of all bladder cancers [1] The classification of adenocarcinomas of the urinary bladder has been the subject of. .. as primary adenocarcinomas of the urinary bladder [1–3,9–11] Pure non-transitional adenocarcinomas of the bladder include the enteric-(intestinal) type and the rare clear cell (mesonephric) adenocarcinomas. .. adenocarcinomas of the urinary bladder are relatively rare and, to our knowledge, this is the largest series (109 cases) of this tumour reported These tumours usually occur in association with bladder