To determine whether DNA analysis can be performed using the supernatants of body fluids after centrifugation at 2,000 rpm for 10 minutes, peritoneal or pleural effusions or bile were examined for K-ras mutations in 34 cases of pancreatic, colorectal, gastric, esophageal, or hepatocellular carcinoma and 15 noncancer cases. The polymerase chain reaction products for K-ras gene codons 2 to 97 of exons 1 and 2 were generated with 41 (93%) of 44 body cavity fluid and 5 (100%) of 5 bile samples. By the single strand conformation polymorphism method, point mutations were detected in the ascites supernatants of 8 (89%) of 9 cases of pancreatic carcinoma. In the remaining case, no point mutation was demonstrated because few malignant cells were present in the ascites fluid. Furthermore, K-ras point mutations were observed in the ascites supernatants of 2 cases of colorectal carcinoma and 1 case of gastric carcinoma. The DNA analysis of the supernatant of ascites fluid showed a K-ras point mutation in 3 cases of false-negative cytologic diagnosis (2 cases of pancreatic carcinoma and 1 case of colorectal carcinoma). Direct sequencing confirmed identical point mutations in the supernatants, whole cell pellets, malignant cells from the cytologic smears of ascites fluid, and cancer tissues. This novel method allows simultaneous testing for genetic abnormalities in supernatants of body fluid, after removing cells for cytologic diagnosis.
To identify the characteristics of ulcerative colitis (UC)-associated carcinomas, 8 lesions, high-grade dysplasias and invasive carcinomas, were implanted into severely combined immunodeficient (SCID) mice and/or cultured in vitro. Intramucosal neoplasias consisting of high-grade dysplasia showed extremely slow proliferation after implantation (2/3 cases) and in vitro culture failed (4 cases). However, invasive carcinomas demonstrated rapid growth both after SCID mouse implantation and in vitro (4/4 cases). From two cases of invasive carcinomas, 6 cell lines were established, and these are the first to be described in the literature. In addition to variation in immunohistochemically determined phenotypic expression regarding α α α α-fetoprotein, chromogranin A and estrogen receptors, the established cell lines showed varying differentiation (moderately or poorly n patients with long-standing ulcerative colitis (UC), colorectal dysplasias and carcinomas frequently develop, and a chronic inflammation-carcinoma sequence has been noted. [1][2][3][4][5] UC-associated tumors have several characteristics, including multiplicity and varying gross appearance of flat, depressed or villous intramucosal lesions with no clear border. 4,6) Regarding genetic alterations, although p53 alteration occurs rather often with loss of heterozygosity (LOH), a low prevalence of APC and ras mutations in UC-associated tumors has been described, along with a high LOH frequency of chromosomes 3 and 18 and p16, compared to sporadic colorectal carcinomas.7-10) Establishment of cell lines of UC-associated tumors should facilitate assessment of differences from the adenoma-carcinoma sequence or the de novo cancer pathway. In the present study, UC-associated dysplasias and invasive carcinomas were implanted into severely combined immunodeficient (SCID) mice and cultured in vitro. Genetic and histopathological features of the established cell lines were then compared with those of sporadic colorectal carcinomas, previously generated in our laboratory. Materials and MethodsSource of tumor cells. Neoplastic lesions, including 4 highgrade dysplasias and 4 invasive carcinomas were obtained from surgically resected rectal tissue in 8 cases (21-73 years old; 5 males, 3 females) with UC (Table 1). Total duration of illness was 6 to 18 years. For comparison, cell lines KE-43, KE-43C4 (subclone of KE43) and KE-24, which were established from invasive sporadic carcinoma lesions (ascending and sigmoid colons; 76-year-old male and 54-year-old female, respectively) in our laboratory were used (Table 1). This work was conducted after receiving the standard informed consent from patients and was approved by our Medical School and University Hospital Ethics Committee (No. B01-20). Implantation of tumor cells into SCID mice and in vitro cell culture.After sterilization of surgically obtained tissue with Isogen (Meiji Seika, Tokyo), tumor tissues were cut and used for implantation under the backskin of 6-week-old, female SCID mice (CB-17/ICR-SCID, Nippon Clea, Os...
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