Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations.

Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.

Genomic alterations in distal bile duct carcinoma by comparative genomic hybridization and karyotype analysis.

We report genomic abnormalities identified in 14 human primary common bile duct carcinomas analyzed by cytogenetics or comparative genomic hybridization, or both. Combining the results of the two methods of analysis, 11 chromosomal arms were observed to be gained in whole or in part, and 9 chromosomal arms were lost in whole or in part in at least four tumors each. The most frequently lost chromosomal regions were, in decreasing order: 18q (eight tumors); 6q and 10p (seven tumors each); 8p, 12q, and 17p (six tumors each); and 7q, 12p, and 22q (four tumors each). The most frequently gained regions were 8q and 20q (six tumors each); 12p, 17q, and Xp (five tumors each); and 2q, 6p, 7p, 11q, 13q, and 19q (four tumors each). These results are similar to those we have previously reported in pancreatic cancer and suggest that carcinomas of the common bile duct and pancreas share a number of genetic changes.

Development and characterization of a cytokine-secreting pancreatic adenocarcinoma vaccine from primary tumors for use in clinical trials.

Preclinical studies with murine tumor models have demonstrated that tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of eliminating small amounts of established tumor. In particular, tumors that express the cytokine GM-CSF produce potent systemic antitumor immune responses against poorly immunogenic murine tumors. These results have encouraged the development of paracrine-cytokine secreting tumor vaccines for gene therapy of human cancer. GM-CSF recruits professional antigen-presenting cells, which in turn activate effector T cells. These findings suggest that allogeneic as well as autologous tumor cells can be used as the tumor source for developing cancer vaccines. A major obstacle to creating genetically modified human allogeneic tumor vaccines is the absence of stable cell lines required for efficient gene transfer, because most human tumors isolated from primary surgical specimens fail to proliferate in long-term culture. We have developed a method for the routine generation of in vitro cell lines from primary tumors of the pancreas. This method overcomes the common problem of stromal and fibroblast overgrowth that can inhibit the in vitro expansion of many histologic types of tumors. In addition, we have analyzed 12 of these cell lines for cytokeritin and mutated K-ras expression to demonstrate that they derive from the original epithelial tumor tissue. The lines can be genetically modified to stably express the cytokine GM-CSF. These methods should be helpful to investigators attempting to establish cell lines from other histologic tumor types for the development of allogeneic genetically modified tumor vaccines.

Consistent chromosome abnormalities in adenocarcinoma of the pancreas.

Little is known about the somatic genetic changes which characterize pancreatic adenocarcinoma. The identification of acquired genomic alterations would further our understanding of the biology of this neoplasm. We have studied 62 primary pancreatic adenocarcinomas obtained from surgical resections using classical cytogenetics and fluorescent in situ hybridization methods. Clonally abnormal karyotypes were observed in 44 neoplasms. Karyotypes were generally complex (greater than three abnormalities) and included both numerical and structural chromosome abnormalities. Many tumors contained at least one marker chromosome. The most frequent whole chromosomal gains were chromosomes 20 (eight tumors) and 7 (seven tumors). Losses were much more frequent: chromosome 18 was lost in 22 tumors followed in frequency by chromosomes 13 (16 tumors), 12 (13 tumors), 17 (13 tumors), and 6 (12 tumors). Structural abnormalities were frequent. Two hundred nine chromosome breakpoints were identified. Excluding Robertsonian translocations, the chromosomal arms most frequently involved were 1p (12); 6q (11); 7q and 17p (9 each); and 1q, 3p, 11p, and 19q (8 each). Portions of the long arm of chromosome 6 appeared to be lost in nine tumors. To determine whether the apparent losses of portions of 6q are real, four tumors with 6q deletions were hybridized with a biotin-labeled microdissection probe from 6q24-ter. Loss of one copy of this region was verified in three of four tumors. In addition, double minute chromosomes were identified in eight cases. To our knowledge, these represent the first primary specimens of pancreatic adenocarcinoma with cytogenetic evidence of gene amplification.

Chromosome analysis of nine endocrine neoplasms of the pancreas.

Endocrine neoplasms of the pancreas differ from the more common adenocarcinomas of the pancreas not only in histologic appearance, but also in clinical presentation and biologic behavior. Chromosomes were analyzed from nine fresh pancreatic endocrine neoplasms. Clonal chromosomal abnormalities were found in five; all were malignant neoplasms. One showed only a loss of the Y chromosome and another had a small triploid population of cells in addition to a normal mainline, with a karyotype of 61-66,XX, -X, -1, -2, -3, -4, +5, -6, +7, -11, -14, +17, +18, +20, +mar1,x2, +mar2,inc. Three neoplasms had near-haploid clones. One neoplasm had a composite karyotype of 31-36,X, +1, +3, +5, +7, +9, +10, +17, +18, +mar. Two were from the same patient, who had the autosomal dominant syndrome MEN-1. The same clone, described as 29,X, +add(1)(p12), +5, +7, +8, +18, +19, was found in both the primary pancreatic neoplasm and in the metastatic tumor. To our knowledge, this is the first report of a haploid clone in both a primary and metastatic solid tumor, and suggests that the near-haploid state is at least compatible with metastasis. These data, combined with the limited reports of cytogenetic data from endocrine pancreatic neoplasms, suggest that at least half of such neoplasms will have an abnormal karyotype.

Chromosome abnormalities in pancreatic adenocarcinoma.

Adenocarcinoma of the pancreas is the fifth most common cause of cancer deaths in the United States, yet few cytogenetic studies of this tumor have been reported. We analyzed 26 primary tumors to identify which chromosome abnormalities occur most frequently in this neoplasm. One carcinoma was well differentiated and mucin producing, 18 were moderately well differentiated, and seven were poorly differentiated. Only normal karyotypes were obtained from nine carcinomas. The remaining 17 carcinomas frequently had normal metaphase cells in addition to simple to highly complex karyotypes. The modal chromosome number in 20 carcinomas was diploid or near-diploid; four carcinomas had both a major near-diploid and near-triploid or near-tetraploid component, and two were near-tetraploid. Numerical abnormalities included loss of whole copies of chromosomes 6, 17, and 18, and gains of chromosome 20. Structural abnormalities were frequent, with 1p, 2p, 3p, 4q, 6q, 7q, 11q, and 17p recurrently involved. Results of this study were combined with karyotypes of 19 other primary adenocarcinomas of the pancreas reported in the literature. The combined data involving 117 breakpoints suggest that careful analysis of chromosome 20, proximal 1q, 6q, proximal 8p, and proximal 17p could be productive in defining genes involved in adenocarcinoma of the pancreas.