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Purpose: The growth-related oncogene alpha (GROalpha) is a secreted interleukin-like molecule that interacts with the CXCR2 G-protein-coupled receptor. We found that the mRNA and protein products of GROalpha are more highly expressed in neoplastic than normal colon epithelium, and we studied potential mechanisms by which GROalpha may contribute to tumor initiation or growth., Experimental Design: Cell lines that constitutively overexpress GROalpha were tested for growth rate, focus formation, and tumor formation in a xenograft model. GROalpha expression was determined by Affymetrix GeneChip (241 microdissected colon samples), real-time PCR (n = 32), and immunohistochemistry. Primary colon cancer samples were also employed to determine copy number changes and loss of heterozygosity related to the GROalpha and fibulin-1 genes., Results: In cell cultures, GROalpha transfection transformed NIH 3T3 cells, whereas inhibition of GROalpha by inhibitory RNA was associated with apoptosis, decreased growth rate, and marked up-regulation of the matrix protein fibulin-1. Forced expression of GROalpha was associated with decreased expression of fibulin-1. Expression of GROalpha mRNA was higher in primary adenocarcinomas (n = 132), adenomas (n = 32), and metastases (n = 52) than in normal colon epithelium (P < 0.001). These results were confirmed by real-time PCR and by immunohistochemistry. Samples of primary and metastatic colon cancer showed underexpression of fibulin-1 when compared with normal samples. There were no consistent changes in gene copy number of GROalpha or fibulin-1, implying a transcriptional basis for these findings., Conclusion: Elevated expression of GROalpha is frequent in adenocarcinoma of the colon and is associated with down-regulation of the matrix protein fibulin-1 in experimental models and in clinical samples. GROalpha overexpression abrogates contact inhibition in cell culture models, whereas inhibition of GROalpha expression is associated with apoptosis. Importantly, coexpression of fibulin-1 with GROalpha abrogates key aspects of the transformed phenotype, including tumor formation in a murine xenograft model. Targeting GRO proteins may provide new opportunities for treatment of colon cancer.

Objectives: Gastric intestinal metaplasia (GIM) associated with H. pylori (HP) has been considered a premalignant lesion. However, GIM phenotype associated with HP infection and gastric cancer is unclear. The expression of COX-2 in relation to GIM phenotype is also unknown., Methods: We evaluated cellular phenotype and COX-2 expression in the GIM from HP-positive and -negative patients from Japan in the absence of gastric cancer (n = 31) by using a colon epithelium specific monoclonal antibody (mAb Das-1) and anti-COX-2 antibody. COX-2 expression was also examined in patients with gastric cancer (n = 34), both in the cancer and in the GIM areas away from the cancer field., Results: Sixty-eight percent of HP-positive GIM reacted with mAb Das-1, whereas the reactivity in the HP-negative GIM was only 25% (P < 0.001). The COX-2 expression was present in 32% of HP-positive GIM and in only 9% of HP-negative GIM (P < 0.001). In the cancer group, COX-2 expression was localized both in the cancer area (94%) and in the GIM (82%) away from the cancer. Each of the COX-2-positive tissue was also positive to mAb Das-1., Conclusion: HP infection is highly associated with the development of colonic-phenotype of GIM, and about half of them expressed COX-2. COX-2 expression was frequent in both gastric cancer and the GIM adjacent to the cancer. The results suggest that the presence of mAb Das-1 and COX-2 reactivity in the GIM identify the subgroup of patients who may be at risk for gastric cancer and may need close surveillance.

Background: Some forms of gastric intestinal metaplasia (GIM) may be precancerous but the cellular phenotype that predisposes to gastric carcinogenesis is not well characterised. Mucin staining, as a means of differentiating GIM, is difficult. A monoclonal antibody, mAb Das-1 (initially called 7E(12)H(12)), whose staining is phenotypically specific to colon epithelium, was used to investigate this issue., Methods: Using mAb Das-1, by a sensitive immunoperoxidase assay, we examined histologically confirmed GIM specimens from two countries, the USA and Japan. A total of 150 patients comprised three groups: group A, GIM (fields away from the cancer area) from patients with gastric carcinoma (n=60); group B, GIM with chronic gastritis (without gastric carcinoma) (n=72); and group C, chronic gastritis without GIM (n=18)., Results: Fifty six of 60 (93%) patients with GIM (both goblet and non-goblet metaplastic cells) from group A reacted intensely with mAb Das-1. Cancer areas from the same 56 patients also reacted. In contrast, 25/72 (35%) samples of GIM from patients in group B reacted with mAb Das-1 (group A v B, p<0.0001). None of the samples from group C reacted with the mAb., Conclusions: Reactivity of mAb Das-1 is clinically useful to simplify and differentiate the phenotypes of GIM. The colonic phenotype of GIM, as identified by mAb Das-1, is strongly associated with gastric carcinoma.

Objective: Using a novel monoclonal antibody (mAb Das-1) that specifically reacts with colon epithelium, we examined if there is a phenotypic change of small intestinal enterocytes toward colonocytes in small intestinal neoplastic tissue., Methods: Tissue sections of the small intestine consisting of adenomas (n = 20, five with histories of familial polyposis), adenocarcinomas (eight primary and one metastatic from colon). carcinoids (n = 2), and hyperplastic polyps (n = 3) were examined by a sensitive immunoperoxidase assay using mAb Das-1 (IgM isotype). Normal jejunal (n = 10) and colonic (n = 10) biopsy specimens were also included as additional controls., Results: mAb Das-1 reacted with normal colonic epithelium but not with jejunal mucosa. However, mAb Das-1 reacted strongly with each of the five adenomas (100%) from patients with histories of familial polyposis, but only five of 15 (33%) of the adenomas from nonfamilial polyposis patients, and each of the eight (100%) adenocarcinomas of the small intestine (p < 0.001). The reactivity with the adenomas from nonfamilial polyposis patients was very focal, whereas in the adenomas with familial polyposis the reactivity was more extensive. Each of the eight carcinomas reacted strongly with mAb Das-1. Adjacent normal small intestinal mucosa did not react. Hyperplastic polyps and the carcinoids did not react with mAb Das-1., Conclusion: These data demonstrate a phenotypic change in small intestinal epithelium toward the colonic phenotype, particularly in familial polyposis and in adenocarcinomas. mAb Das-1 may be clinically useful in identifying small intestinal adenomas with "high risk" for malignancy, such as in familial polyposis.

In situ carcinomas must penetrate their own basement membrane to be classified as invasive, and subsequently infiltrate surrounding connective tissue and cross vascular basement membranes to metastasize hematogenously. Accordingly, in many studies, integral basement membrane components, including type IV collagen, laminin, and heparan sulfate proteoglycan, have been localized in a spectrum of tumors to gain insight into their role in neoplasia. A number of recently identified extracellular matrix molecules and isoforms of the aforementioned proteins have been localized to the basement membrane zone, illustrating another level of biochemical heterogeneity in these structures. As the complexity of these matrices becomes more apparent, their roles in maintaining homeostasis and in tumor biology falls into question. Of the new group of collagens localized to the basement membrane zone, type XV was the first to be characterized (Cell Tissue Res, 286:493-505, 1996). This nonfibrillar collagen has a nearly ubiquitous distribution in normal human tissues via a strong association with basement membrane zones, suggesting that it functions to adhere basement membrane to the underlying stroma. To begin investigation of this protein in malignant tumors, we have localized type XV in human colonic adenocarcinomas and compared its distribution with that of type IV collagen and laminin. Collagens XV and IV and laminin were found in all normal and colonic epithelial, muscle, fat, neural, and vascular basement membrane zones, as shown previously. In moderately differentiated, invasive adenocarcinomas, laminin and type IV collagen were sometimes observed as continuous, linear deposits around some of the malignant glands, but more often they were seen in either discontinuous deposits or were completely absent. In contrast, type XV collagen was characterized as virtually absent from the basement membrane zones of malignant glandular elements in moderately differentiated tumors. Nevertheless there were also similarities; all 3 proteins were usually present in the stroma and adjacent vascular basement membrane zones surrounding invasive glands. The loss of type XV collagen from these malignant epithelial basement membrane zones and its increased interstitial expression suggests a role for this protein in the invasive process and the possibility that it may provide a sensitive indicator of tumor invasion.

Purpose: Urethral adenocarcinoma is a rare malignancy whose origin remains controversial. The monoclonal antibody mAbDas1 (formerly 7E12H12) was developed against a unique colonic epithelial epitope and is reactive in areas of intestinal metaplasia. Recently the antibody was shown to react in cystitis glandularis as well as adenocarcinoma of the bladder, suggesting that cystitis glandularis may be the precursor of bladder adenocarcinoma. We examined urethral adenocarcinomas and benign urethral specimens using mAbDas1 to determine whether it could provide insight into their histogenesis., Materials and Methods: Archival tissue from 12 cases of primary female urethral adenocarcinoma and urethral specimens of inflamed urethral mucosa, urethritis glandularis and transitional cell carcinoma was studied. Immunohistochemical analysis of formalin fixed, paraffin embedded archival tissue was done using the monoclonal antibody mAbDas1. Tumors were also evaluated with a prostate specific antigen (PSA) polyclonal antibody as previous studies have noted PSA reactivity in these tumors., Results: Of the 12 cases 9 were columnar/mucinous adenocarcinoma, 2 clear cell adenocarcinoma and 1 a cribriform pattern resembling adenocarcinoma of the prostate. All columnar/mucinous adenocarcinomas reacted positively (6 strongly and 3 focally) with the mAbDas1 antibody but did not react with the PSA antibody. The tumor with a cribriform pattern reacted strongly with PSA but did not react with mAbDas1. The 2 clear cell adenocarcinomas did not react with either antibody. The benign urethral specimens demonstrated strong reactivity to the mAbDas1 antibody in areas of urethritis glandularis but normal and inflamed urethral mucosa and transitional cell carcinoma did not react., Conclusions: Primary adenocarcinoma of the female urethra arises from more than 1 tissue of origin. Columnar/mucinous adenocarcinomas of the female urethra and urethritis glandularis demonstrate consistent reactivity with the mAbDas1 antibody, suggesting that these tumors arise from glandular metaplasia analogous to the potential histogenesis previously demonstrated in the bladder. PSA reactivity occurred in 1 tumor with a cribriform pattern and likely represents origin from Skene's glands. Clear cell adenocarcinomas did not react with either antibody, suggesting a third possible pathway in the development of this rare subset of adenocarcinomas.

Objective: To determine if the monoclonal antibody 7E12H12, which reacts with a 40 kDa protein in normal human enterocytes and has been shown to be a marker for intestinal metaplasia and adenocarcinoma arising in the bladder, could assist in distinguishing prostatic, urachal and vesical adenocarcinoma, using a sensitive immunohistochemical assay., Materials and Methods: Fifteen primary prostatic adenocarcinomas and five adenocarcinomas of the urinary bladder were selected for a retrospective evaluation. The monoclonal antibody 7E12H12 (IgM isotype) was used in an immunoperoxidase assay to survey formalin-fixed, paraffin-embedded archival tissue specimens., Results: All vesical adenocarcinomas reacted positively with the antibody, regardless of grade; none of the 15 prostatic specimens reacted positively in either the benign or malignant glandular epithelium., Conclusion: The monoclonal antibody 7E12H12 can differentiate primary adenocarcinoma of the bladder from secondary adenocarcinoma arising in the prostate and may be a useful tool in diagnostic pathology.

Purpose: Primary adenocarcinoma of the bladder is a rare neoplasm whose histogenesis is poorly understood. Current data support the concept that adenocarcinoma of the bladder and urachus evolves from zones of intestinal metaplasia that become dysplastic and invasive. To address this hypothesis further we determined the immunoreactivity of benign and malignant epithelial tissue from the bladder and urachus with a monoclonal antibody that is reactive with colonic epithelium to evaluate the presence of a common reactive epitope., Materials and Methods: The monoclonal antibody 7E12H12 (IgM isotype), developed against a colonic epithelial protein, was used in an immunoperoxidase assay to survey formalin fixed, paraffin embedded archival tissue specimens. A total of 26 specimens obtained by endoscopic biopsy or extirpative surgery, including benign and malignant bladder and urachal epithelial abnormalities, was chosen for retrospective evaluation., Results: All adenocarcinoma reacted positively regardless of the histological variant, differentiation, or bladder or urachal origin. In contrast, transitional cell and squamous cell carcinomas were nonreactive. Also, the pattern of reactivity in tissues that contained benign epithelial proliferations suggested a stepwise transition with no reactivity in normal urothelium or Brunn's epithelial nests, rare staining of cystitis cystica, and uniformly positive reactivity in cystitis glandularis and frank colonic intestinal metaplasia of the bladder and urachus., Conclusions: The shared, aberrant phenotypic expression of a unique colonic epitope in benign epithelial metaplasia, and adenocarcinoma of the bladder and urachus suggests a common underlying pathway toward adenocarcinoma in cystic and urachal adenocarcinoma. The implications for diagnostic pathology are discussed.

Objective: To determine if there is a reactive epitope common to colonic epithelium and Barrett epithelium, a premalignant metaplastic columnar-lined esophagus, usually arising as a complication of chronic reflux esophagitis., Design: A monoclonal antibody, 7E12H12 (IgM isotype), developed against a colonic epithelial protein was used in a sensitive immunoperoxidase assay using formalin-fixed, paraffin-embedded tissue. One hundred sixteen tissue specimens from the esophagus, stomach, duodenum, and jejunum were examined. Twenty-two biopsy specimens were taken from 22 patients with benign Barrett epithelium, and 12 specimens were obtained from 12 patients with adenocarcinoma of the esophagus arising in Barrett epithelium. The remaining 85 tissue specimens were obtained from various parts of the upper gastrointestinal tract of patients with or without disease states., Results: 21 of 22 (95%) Barrett epithelium specimens and all 12 adenocarcinomas arising from Barrett epithelium reacted with the 7E12H12 monoclonal antibody. Other tissues from esophagus, gastroesophageal junction, stomach, duodenum, or jejunum did not react. Squamous cell carcinoma of the esophagus also did not react., Conclusions: Barrett epithelium shares phenotypic expression of colonic epithelium. The 7E12H12 monoclonal antibody may provide insight into the origin and cellular lineage of Barrett epithelium.