Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas.

Transforming growth factor beta (TGF-beta) is an extracellular ligand that binds to a heterodimeric receptor, initiating signals that regulate growth, differentiation, and apoptosis. Many cancers, including pancreatic cancer, harbor defects in TGF-beta signaling and are resistant to TGF-beta-mediated growth suppression. Genetic alterations of DPC4, which encodes a DNA binding protein that is a downstream component of the pathway, most frequently occur in pancreatic and biliary carcinomas. We searched for other targets of mutation of the TGF-beta pathway in these cancers. We report somatic alterations of the TGF-beta type I receptor gene ALK-5. Homozygous deletions of ALK-5 were identified in 1 of 97 pancreatic and 1 of 12 biliary adenocarcinomas. A germ-line variant of ALK-5, presumably a polymorphism, was identified, but no somatic intragenic mutations were identified upon sequencing of all coding regions of ALK-5. Somatic alterations of the TGF-beta type II receptor gene (TGFBR2) were identified in 4 of 97 (4.1%) pancreas cancers, including a homozygous deletion in a replication error-negative cancer and three homozygous frameshift mutations of the poly(A) tract of the TGF-beta type II receptor in replication error-positive cancers. We also studied other related type I receptors of the TGF-beta superfamily. In a panel of pancreas cancers preselected for loss of heterozygosity at the ALK-1 locus, sequencing of all coding exons of the ALK-1 gene revealed no alterations. No homozygous deletions were detected in the ALK-1, ALK-2, ALK-3, or ALK-6 genes in a panel of 86 pancreatic cancer xenografts and 11 pancreatic cancer and 22 breast cancer cell lines. The rate of genetic inactivation of TGF-beta pathway members was determined in 45 pancreatic cancers. Eighty-two % of these pancreatic cancers had genetic inactivation of the DPC4, p15, ALK-5, or TGFBR2 genes. Our results indicate that the TGF-beta type I and type II receptor genes are selective targets of genetic inactivation in pancreatic and biliary cancers.

Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene.

Mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) is a component of a stress and cytokine-induced signal transduction pathway involving MAPK proteins. The MKK4 protein has been implicated in activation of JNK1 and p38 MAPK on phosphorylation by conserved kinase pathways. A recent report on the deletion and mutation of the MKK4 gene in human pancreatic, lung, breast, testicle, and colorectal cancer cell lines suggests an additional role for MKK4 in tumor suppression. Both the gene function and the infrequency of mutations might be considered atypical for many human tumor suppressor genes, and constitutional DNA was not previously available to determine whether the reported sequence variants had preceded tumor development. Here, we report that homozygous deletions are detected in 2 of 92 pancreatic adenocarcinomas (2%), 1 of 16 biliary adenocarcinomas (6%), and 1 of 22 breast carcinomas (when combined with reported sequence alterations, 3 of 22 or 14%). In addition, in a panel of 45 pancreatic carcinomas prescreened for loss of heterozygosity, one somatic missense mutation of MKK4 is observed and confirmed in the primary tumor (2%). Mapping of the homozygous deletions further indicated MKK4 to lie at the target of deletion. The finding of a somatic missense mutation in the absence of any other nucleotide polymorphisms or silent nucleotide changes continues to favor MKK4 as a mutationally targeted tumor suppressor gene. Coexistent mutations of other tumor suppressor genes in MKK4-deficient tumors suggest that MKK4 may participate in a tumor suppressive signaling pathway distinct from DPC4, p16, p53, and BRCA2.

High-resolution deletion mapping of chromosome arm 1p in pancreatic cancer identifies a major consensus region at 1p35.

Chromosomal arm 1p has long been suspected, on the basis of loss of heterozygosity (LOH) and other data, to harbor a tumor suppressor gene important in pancreatic carcinomas and other tumors. We constructed a high-resolution map of LOH at I p in a panel of pancreatic adenocarcinomas. Using 44 markers, we identified LOH on I p in 49% of 43 cancers. Breakpoints in 1p were identified in 15 of the carcinomas and could be used to ascertain consensus patterns. We found a major consensus region of LOH at 1p35 between loci D1S233 and D1S247. This region participates in the majority of LOH events on 1 p in pancreatic cancer. These data provide a roadmap for further regional mapping, homozygous deletion searches, comparison to LOH patterns seen in other tumor types, and prioritization of studies using candidate genes.

Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers.

Peutz-Jeghers syndrome (PJS) is an autosomal-dominant disorder characterized by hamartomatous polyps in the gastrointestinal tract and by pigmented macules of the lips, buccal mucosa, and digits. Less appreciated is the fact that PJS also predisposes patients to an increased risk of gastrointestinal cancer, and pancreatic cancer has been reported in many PJS patients. It was recently shown that germline mutations of the STK11/LKB1 gene are responsible for PJS. We investigated the role of STK11/LKB1 in the development of pancreatic and biliary cancer in patients with and without the PJS. In a PJS patient having a germline splice site mutation in the STK11/LKB1 gene, sequencing analysis of an intestinal polyp and pancreatic cancer from this patient revealed loss of the wild-type allele of the STK11/LKB1 gene in the cancer. Inactivation of STK11/LKB1, by homozygous deletions or somatic sequence mutations coupled with loss of heterozygosity, was also demonstrated in 4-6% of 127 sporadic pancreatic and biliary adenocarcinomas. Our results demonstrate that germline and somatic genetic alterations of the STK11/LKB1 gene may play a causal role in carcinogenesis and that the same gene contributes to the development of both sporadic and familial forms of cancer.

Novel homozygous deletions of chromosomal band 18q22 in pancreatic adenocarcinoma identified by STS marker scanning.

The identification of homozygous deletions in sporadic neoplasms has been pivotal in the positional cloning of several tumor suppressor genes. Chromosomal arm 18q harbors the DPC4, SMAD2, and DCC genes and is suspected on the basis of high frequencies of allelic loss to harbor additional tumor suppressor genes. We applied high-resolution sequence-tagged site (STS) marker scanning to a panel of 106 pancreatic adenocarcinomas to identify novel regions of homozygous deletions on 18q. Three homozygous deletions were identified. Physical mapping of these deletions showed them to be nonoverlapping, but clustered in an approximately 7- to 10-Mb region of chromosome band 18q22. Each deletion spanned physical distances of nearly 1.3 to 3 Mb. A number of transcribed genes map within these deletions. The identification of these homozygous deletions might aid in the identification of novel tumor suppressor genes on chromosomal arm 18q. Genes Chromosomes Cancer 25:370-375, 1999.

Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.

The clinical and pathological features of carcinomas of the pancreas with DNA replication errors (RER+) have not been characterized. Eighty-two xenografted carcinomas of the pancreas were screened for DNA replication errors using polymerase chain reaction amplification of microsatellite markers. Cases with microsatellite instability in at least two markers of a minimum of five tested were considered RER+. RER status was correlated with histological appearance, karyotype of the carcinomas when available, K-ras mutational status, and patient outcome. Three (3.7%) of the eighty-two carcinomas were RER+. In contrast to typical gland-forming adenocarcinomas of the pancreas, all three RER+ carcinomas were poorly differentiated and had expanding borders and a prominent syncytial growth pattern. Neither a Crohn's-like lymphoid infiltrate nor extracellular mucin production were prominent. Ductal adenocarcinomas of the pancreas typically contain a mutant K-ras gene, yet all three RER+ carcinomas had wild-type K-ras. One of the three RER+ carcinomas was karyotyped and showed a near diploid pattern. All three of the RER+ tumors were removed via Whipple resection. One of the three patients is free of disease 16 months after pancreaticoduodenectomy, one is alive and free of tumor at 52 months but developed two colon carcinomas during this period, and the third died of pancreatic cancer at 4 months. None of the three patients had a family history of colorectal carcinoma. A review of the K-ras wild-type carcinomas in a previously characterized series of pancreatic carcinomas with known K-ras mutational status identified two additional cancers with poor differentiation, a syncytial growth pattern, and pushing borders. Both of the cancers were diploid and both patients were longterm survivors (over 5 years). The inclusion of such patients in previous prognostic studies of pancreas cancer may explain the failure of histological grade to be a predictor of prognosis. These data suggest that DNA replication errors occur in a small percentage of resected carcinomas of the pancreas and that wild-type K-ras gene status and a medullary phenotype characterized by poor differentiation, and expanding pattern of invasion, and syncytial growth should suggest the possibility of DNA replication errors in carcinomas of the pancreas.