Primary bladder adenocarcinoma is a rare tumor comprising no more than 2% of all primary vesical malignancies [1–3]. in our series, the male to female ratio was 5:1 with mean age of 64.6 years. Eight of 12 cases (66.7%) originated from the posterior wall. Four tumors were seen in the anterior wall near the dome. However, they did not meet the other required criteria to be classified as urachal type adenocarcinoma. These findings were in accordance to published literature [2, 3, 7, 8].
The average size of tumor documented in our study was 4.25 cm (range 0.7-6.8 cm). Histologically, PBA is usually well to moderately differentiated and most frequently are of the enteric type, comprised of glandular structures lined by cuboidal to columnar cells with basally located vesicular nuclei and prominent nucleoli. The cytoplasm is usually apical with or without mucin vacuoles, the former representing goblet cells found in benign and malignant colonic glands. Mitoses and dirty necrosis are frequently seen [1, 3, 4, 7]. Varying degree of extracellular mucin may be seen with these tumors. The mucinous variant is comprised of abundant extracellular pools of mucin with floating tumor cells. Additionally, some of the tumors may also harbor signet ring cells with intracellular mucin, the latter often referred to as the signet ring cell variant of PBA [2, 18].
In the current study, there was a predominance of moderately differentiated PBA (75%), all of which were of the enteric type. Extracellular mucin and signet ring cells were seen in 66.7% and 41.7% cases, respectively. Case #6 had predominance of signet ring cells (>75%) and was classified as signet ring cell adenocarcinoma. Cystitis cystica/glandularis was documented in three-quarters of the cases and a villous adenoma like lesion was seen in 25% cases. Other variants, such as hepatoid and clear cell types, were not included in this study.
Secondary bladder adenocarcinoma, which is more frequent than PBA, is represented by either direct extension of tumor from surrounding organs like the rectum, colon and female genital tract or lymphatic and hematogenous spread of tumor. MCA is by far the most frequent type in SBA. It is important to distinguish PBA from MCA for disease staging (local versus metastatic), and management [2, 3, 16].
Abnormal localization of β-catenin has been well documented in colonic adenocarcinomas with mutation in APC tumor suppressor gene . The wild-type APC functions to degrade free cytoplasmic β-catenin, an important molecule in cadherin mediated cell-to-cell adhesion system. In patients with mutated or absent APC, β-catenin translocates to the nucleus of the cell and acts as a transactivating factor for the transcriptional factor Tcf-4 to regulate the expression of a number of downstream target genes that are believed play a role in oncogenesis . Wang et al. first exploited this finding in the setting of PBA and observed absence of nuclear staining in all PBA and nuclear staining in 81% MCA. This was reported to be a helpful distinction between the two entities . Subsequently, few other studies reported the utility of β-catenin staining in settings of signet ring cell adenocarcinomas of bladder and urachal adenocarcinomas [2, 14, 18]. In our study, nuclear staining was predominantly seen in MCA and CA unlike PBA. In two cases of PBA nuclear staining was seen in less than 5% of the tumor cells. These findings are similar to the previously reported study by Gopalan et al.  who reported focal nuclear staining (15% cells) in 1 of 24 cases of urachal adenocarcinomas. However, the nuclear staining was seen in the clear cell urothelial component of the tumor rather than the adenocarcinoma component itself. Overall the difference in β-catenin staining pattern between PBA and MCA was statistically significant (χ2 test, p=0.001).
The e-cadherin molecule forms an important component of the cell-cell adhesion complex in association with other proteins including catenin molecules (α-, β-, and γ) and p120 . Loss of membranous localization of e-cadherin has been associated with invasion, metastasis and aggressive behavior in many human malignancies [21, 22]. Loss of membranous staining with abnormal nuclear localization has been reported in pituitary, pancreatic, esophageal and urothelial tumors [21–24]. The mechanism of this abnormal localization is still unclear. Loss of membranous e-cadherin expression in urothelial carcinoma has been correlated with aggressive disease, increased risk of nodal metastasis and death . Thomas et al. reported loss of membranous e-cadherin expression in approximately one-third of foci of PBA demonstrating signet-ring cell morphology in comparison to colonic type PBA; however, they did not encounter abnormal nuclear localization of e-cadherin .
In the current study, none of the CA or UC controls demonstrated nuclear staining pattern. E-cadherin molecule is comprised of 3 domains – extracellular, transmembrane and intracytoplasmic. Amongst the commercially available antibody clones, 36/E clone is directed against the cytoplasmic portion of e-cadherin. Our results of e-cadherin are based on use of this clone. (Table 1) The same clone was also used by the prior studies, which reported aberrant nuclear localization of e-cadherin [21–24].
CDX-2 is a mammalian homeobox gene, encoding a nuclear transcription factor, which is implicated as a tumor suppressor . Nuclear staining is seen in normal colonic epithelial cells and colonic adenocarcinoma has been reported [3, 25]. CDX-2 expression was seen in 83.3% cases of PBA, all cases of MCA and CA which is in accordance with the prior studies [3, 14–16, 25]. Interestingly, Suh et al.  also reported absence of CDX-2 expression in half of the cases of PBA. Two cases (16.7%) of PBA in our series were negative for CDX-2.
Villin is a 93-kilodalton actin-binding protein involved in the maintenance of brush border apparatus. Its expression can be seen in gastrointestinal tract and renal epithelial cells as well as in adenocarcinomas of gastrointestinal tract, pancreas, endometrium and ovary . A limited number of studies have analyzed the role of villin in distinguishing PBA from SBA which reported overlapping staining between PBA and MCA [16–18]. We too did not find any difference in staining pattern between the two tumor groups.
The CK7 and CK20 staining profile has been used previously in distinguishing tumors from different sites. Gastrointestinal tumors, including colonic and rectal adenocarcinomas, tend to be CK20+ and CK7-. For urothelial neoplasms, the staining profile is the exact opposite. This difference was studied previously in PBA, but with limited success. Many PBA have been reported to show a CK20+ and CK7- profile, similar to colonic adenocarcinomas [3, 15, 17]. CK7 immunoexpression was seen in all cases of urothelial carcinoma and one third of PBAs. In contrast, only one (8.33%) case of SBA was positive for CK7 and all CAs were negative. CK20 immunostaining was seen in all PBAs, SBAs and colonic adenocarcinoma. Based on these findings, CK7 appeared to be of limited utility in distinguishing PBAs from secondary bladder adenocarcinomas and CK20 was not able to distinguish between the two tumor groups.
We also attempted to study the molecular aspect of PBA and MCA with a limited number of available cases (5 PBA and 4 MCA). FISH analysis using UroVysion probe sets were performed. To the best of our knowledge, our study is the first one that utilized molecular methods to attempt to distinguish PBA from MCA. In our series of nine cases, the predominant abnormality was homozygous loss of 9p21 followed by polysomy pattern and single chromosome gain (chromosomes 3, 7). These findings however did not appear to be helpful in distinguishing PBA from SBA. Kipp et al . studied FISH findings using UroVysion probe sets in bladder carcinoma variants. In their study, the most frequent abnormality documented in bladder adenocarcinoma was 9p21 homozygous loss followed by single gain only. They did not find significant difference in the chromosomal abnormalities between the different tumor types. This study, unlike ours, did not compare bladder adenocarcinoma with metastatic adenocarcinoma.