Hubert Zirngibl

Molecular Analysis of Microdissected Tumors and Preneoplastic Intraductal Lesions in Pancreatic Carcinoma

Little or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16(INK4), DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at the p16(INK4) locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at the DPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions of p16(INK4) and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16(INK4) compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs.

Prognostic value of microscopic peritoneal dissemination: comparison between colon and gastric cancer.

PURPOSE: We evaluated the incidence and prognostic relevance of microscopic intraperitoneal tumor cell dissemination of colon cancer in comparison with dissemination of gastric cancer as a rational for additive intraperitoneal therapy. METHODS: Peritoneal washouts of 90 patients with colon and 111 patients with gastric cancer were investigated prospectively. Sixty patients with benign diseases and 8 patients with histologically proven gross visible peritoneal carcinomatosis served as controls. Intraoperatively, 100 ml of warm NaCl 0.9 percent were instilled and 20 ml were reaspirated. In all patients hematoxylin and eosin staining (conventional cytology) was performed. Additionally, in 36 patients with colon cancer and 47 patients with gastric cancer, immunostaining with the HEA-125 antibody (immunocytology) was prepared. The results of cytology were assessed for an association with TNM category and cancer grade, based on all patients, and with patient survival, among the R0 resected patients. RESULTS: In conventional cytology 35.5 percent (32/90) of patients with colon cancer and 42.3 percent (47/111) of patients with gastric cancer had a positive cytology. In immunocytology 47.2 percent (17/36) of patients with colon cancer and 46.8 percent (22/47) of patients with gastric cancer were positive. In colon cancer, positive conventional cytology was associated with pT and M category (P=0.044 andP=0.0002), whereas immunocytology was only associated with M category (P=0.007). No association was found between nodal status and immunocytology in colon cancer and with the grading. There was a statistically significant correlation between pT M category and conventional and immunocytology in gastric cancer (P<0.0015/P=0.007 andP<0.001/P=0.009, respectively). Positive immunocytology was additionally associated with pN category (P=0.05). In a univariate analysis of R0 resected patients (no residual tumor), positive immunocytology was significantly related to an unfavorable prognosis in patients with gastric cancer only (n=30). Mean survival time was significantly increased in patients with gastric cancer with negative cytology compared with positive cytology (1,205 (standard error of the mean, 91)vs. 771 (standard error of the mean, 147) days;P=0.007) but not in patients with colon cancer (1,215 (standard error of the mean, 95)vs. 1,346 (standard error of the mean, 106) days;P=0.55). CONCLUSIONS: Because microscopic peritoneal dissemination influences survival time after R0 resections only in patients with gastric but not with colon cancer, our results may provide a basis for a decision on additive, prophylactic (intraperitoneal) therapy in gastric but not colon cancer.

Genomic instability in colorectal carcinomas : Comparison of different evaluation methods and their biological significance

In order to demonstrate the relationship between microsatellite instability and other types of genomic instability, a series of 56 sporadic colorectal carcinomas was investigated by flow cytometrical ploidy analysis, oligonucleotide fingerprinting, and microsatellite polymerase chain reaction (PCR). Stabilization of the p53 gene product was analysed by immunohistochemistry and proliferative activity was determined flow cytometrically and by silver staining of nucleolar organizer regions (AgNORs). Of the 56 carcinomas, 11 (19 per cent) exhibited microsatellite instability; 33 of the cases were aneuploid (59 per cent) and 29 (52 per cent) showed alterations of the oligonucleotide fingerprints. There was a significant correlation of microsatellite instability with localization of these tumours proximal to the splenic flexure, diploid DNA content, and less frequent p53 stabilization. A solid growth pattern, mucinous differentiation, and a Crohns‐like lymphoid infiltrate were also characteristic for those tumours. The results demonstrate for the first time a significantly lower proliferative activity in tumours with microsatellite instability. Data obtained from DNA flow cytometry or from oligonucleotide fingerprinting did not correlate with such tumour characteristics. It is proposed that the use of microsatellite PCR facilitates specifically the detection of a group of colorectal cancers which may differ in pathogenesis and perhaps prognosis.