Mad-related genes in the human.

Resistance to the growth inhibitory effects of TGF-beta is common in human cancers. However, the mechanism(s) by which tumour cells become resistant to TGF-beta are generally unknown. We have identified five novel human genes related to a Drosophila gene called Mad which is thought to transduce signals from TGF-beta family members. One of these genes was found to be somatically mutated in two of eighteen colorectal cancers, and three of the other genes were located at chromosomal positions previously suspected to harbor tumour suppressor genes. These data suggest that this gene family may prove to be important in the suppression of neoplasia, imparting the growth inhibitory effects of TGF-beta-like ligands.

DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1.

About 90 percent of human pancreatic carcinomas show allelic loss at chromosome 18q. To identify candidate tumor suppressor genes on 18q, a panel of pancreatic carcinomas were analyzed for convergent sites of homozygous deletion. Twenty-five of 84 tumors had homozygous deletions at 18q21.1, a site that excludes DCC (a candidate suppressor gene for colorectal cancer) and includes DPC4, a gene similar in sequence to a Drosophila melanogaster gene (Mad) implicated in a transforming growth factor-beta (TGF-beta)-like signaling pathway. Potentially inactivating mutations in DPC4 were identified in six of 27 pancreatic carcinomas that did not have homozygous deletions at 18q21.1. These results identify DPC4 as a candidate tumor suppressor gene whose inactivation may play a role in pancreatic and possibly other human cancers.

An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12.

We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2). Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs. The YAC contig comprises 16 clones that together span the entire BRCA2 region. The PAC contig comprises 22 clones that together span the DPC region. Seventy cosmid clones were localized within and near the DPC region. Thirty-five sequence-tagged sites were defined and localized within the map. The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region.

Homozygous deletion map at 18q21.1 in pancreatic cancer.

Absolute genetic differences between neoplastic and nonneoplastic cells can be discerned at sites of homozygous deletions. These deletions are of critical interest because they might be useful in the identification of defective biochemical pathways in neoplastic cells, and subsequently for the development of new treatment strategies in human cancer. We identified an area at 18q21.1 involved by homozygous deletions in 30% of pancreatic carcinomas. To characterize the homozygous deletions, we constructed a detailed physical map of nearly 2 Mb, containing yeast artificial chromosomes, P1-derived artificial chromosomes, cosmids and 24 sequence-tagged sites. The homozygously deleted are contained a new candidate tumor-suppressor gene (DPC4). To date, 23 (64%) of 35 pancreatic carcinomas carry at least one homozygous deletion at a published locus. The study of the total gene content of these loci, facilitated by the sequence-tagged site markers and maps of these regions, should help to reveal the absolute biochemical differences between neoplastic and nonneoplastic cells for a common human tumor.

Tumor-suppressive pathways in pancreatic carcinoma.

During tumorigenesis, positive selection is exerted upon those tumor cells that alter rate-limiting regulatory pathways. A corollary of this principle is that mutation of one gene abrogates the need for alteration of another gene in the same pathway and also that the coexistence in a single tumor of mutations in different genes implies their involvement in distinct tumor-suppressive pathways. We studied 42 pancreatic adenocarcinomas for genetic alterations in the K-ras oncogene and the p16, p53, and DPC4 tumor suppressor genes. All of them had the K-ras gene mutated. Thirty-eight % of the tumors had four altered genes, another 38% had three altered genes, 15% had two altered genes, and 8% of the tumors had one altered gene. Interestingly, we noted a high concordance of DPC4 and p16 inactivations (P = 0.007), suggesting that the genetic inactivation of p16 increases the selective advantage of subsequent mutation in DPC4. No statistically significant association was identified between the alteration of these cancer genes and pathological or clinical parameters. This type of multigenic analysis in human tumors may serve to substantiate experimental tumor models and thus increase our understanding of the truly physiologically relevant tumor-suppressive pathways that are abrogated during human tumorigenesis.

Increased risk of incident pancreatic cancer among first-degree relatives of patients with familial pancreatic cancer.

It has been estimated that familial aggregation and genetic susceptibility play a role in as many as 10% of patients with pancreatic cancer (PC). The quantified prospective risk of PC among first-degree relatives of PC patients has not been investigated. Families enrolled in the National Familial Pancreas Tumor Registry (NFPTR) prior to September 1, 1998 were followed to estimate the risk and incidence of PC among first-degree relatives of patients with PC. Analyses were performed separately on kindreds with at least two first-degree relatives with PC (familial pancreatic carcinoma (PC); n = 150) at the time the kindred was enrolled in the NFPTR and on kindreds without a pair of affected first-degree relatives (sporadic PC; n = 191). A subanalysis was performed on familial PC kindreds containing three or more affected members at the time of enrollment in the NFPTR (n = 52). Risk was estimated by comparing observed new cases of PC during the observation period with expected numbers based on the United States population-based Surveillance, Epidemiology and End Results program data. Incidence was estimated using person-years risk analyses. During the observational period, six incident PCs developed in the first-degree relatives: two in the sporadic PC kindreds, and four in the familial PC kindreds. The PC risk in the sporadic PC kindreds was not significantly greater than expected [observed/expected = 6.5 (95% CI = 0.78-23.3)] with an incidence rate of 24.5/10(5)/ year. There was a significantly increased 18-fold risk (95% CI = 4.74-44.5) of PC among first-degree relatives in familial PC kindreds, with an incidence of 76.0/10(5)/year. In the subset of familial PC kindreds with three or more affected family members at the time of enrollment, there was a 57-fold (95% CI = 12.4-175) increased risk of PC and an incidence of 301.4/10(5)/year compared with the Surveillance, Epidemiology and End Result age-adjusted incidence of PC in the U.S. (8.8/10(5)/year). When stratified by age, the risk was largely confined to relatives over the age of 60. This study is the first analysis of incident PC occurring in familial PC kindreds. The risk and incidence of PC is exceptionally high among at-risk first-degree relatives in familial PC kindreds in which at least three first-degree relatives have already been diagnosed with PC. Familial PC kindreds are a reasonable high-risk group for PC screening and chemoprevention research.

Molecular cytogenetic alterations associated with rapid tumor cell proliferation in advanced urinary bladder cancer.

Invasive urinary bladder carcinomas are characterized by a high number of cytogenetic alterations which are thought to pinpoint the location of critical genes, some of which may be involved in cell cycle control. To identify genomic alterations that may affect such genes the proliferative activity (Ki67 labeling index) was assessed in 93 invasively growing bladder carcinomas analyzed by comparative genomic hybridization. Only a few changes were significantly associated with rapid tumor cell proliferation, including 3p+ (p=0.0357), 6p+ (p=0.003), 8q+ (p=0.0273), and 11q- (p=0.0329). Among these alterations 6p+ is of particular interest because high level 6p22 amplifications occur frequently in bladder cancer. The particular strong association between 6p+ and a high tumor cell proliferation being independent of grade and stage suggests that a putative oncogene on 6p22 involved in cell cycle regulation.

Interferon DNA polymorphism in chronic leukemia.

The interferon (IFN) system (alpha, beta and gamma IFNs) is closely related to the first line of defenses against viral and tumoral diseases. Chronic leukemic and chronic lymphoproliferative patients respond in variable degrees to therapy with exogenous IFN. Remission after treatment with IFN-alpha in hairy cell leukemia (HCL) and in chronic myelogenous leukemia (CML) have been reported by other authors. In order to determine whether there are differences in IFN-alpha and beta genes between healthy and chronic leukemic individuals and among the different chronic leukemic patients, restriction fragment length polymorphism (RFLP) analyses was performed in a panel of patients with HCL, CML and chronic lymphocytic leukemia (CLL), and in a sample of healthy individuals. A significant difference in the allelic frequencies for the IFN-beta and Sst I enzyme in Chronic leukemias, mainly of myeloid origin, compared with the healthy individuals, was found.

INK4 genes in pancreatic carcinoma.

The INK4 family includes the structurally and functionally related p15, p16 and p18 genes. in vitro they arrest the cell in G1 phase, thus far in an apparently similar manner. It is not clear yet how their functions relate in vivo, whether these genes have distinct or redundant functions. The INK4 genes are thought to be candidates for tumor suppressor genes. The p16 gene has been found to be inactivated in many tumor types, usually by homozygous deletion. In most tumors it was noted that the deletions involve the neighboring p15 gene as well, thus it was not clear whether these inactivation events targeted p16, p15, or both genes. p16 was also found to be inactivated by intragenic mutations in 40% of pancreatic carcinomas; these cancers provide a unique opportunity to test whether p16 and p15 must both be inactivated during tumorigenesis. If p15 were a second target at chromosome 9p, it would be predicted to be inactivated in at least some of these tumors. In addition to studies of the p15 gene in these pancreatic cancers, we assayed for mutations in the p18 gene, which offered a third, independent site to infer possible redundant functions of the INK4 proteins. Sequence analysis indicated that p15 and p18 were not targets of inactivation in pancreatic carcinoma. p16, therefore, plays a different role in vivo, since inactivation of only this gene had proven to confer a selective growth advantage to evolving clones of pancreatic tumor cells. The preference for homozygous deletions as a means to inactivate p16 remains unexplained.

Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.

Homozygous deletions have been central to the discovery of several tumor-suppressor genes, but their finding has often been either serendipitous or the result of a directed search. A recently described technique [Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, 946-951] held out the potential to efficiently discover such events in an unbiased manner. Here we present the application of the representational difference analysis (RDA) to the study of cancer. We cloned two DNA fragments that identified a homozygous deletion in a human pancreatic adenocarcinoma, mapping to a 1-centimorgan region at chromosome 13q12.3 flanked by the markers D13S171 and D13S260. Interestingly, this lies within the 6-centimorgan region recently identified as the BRCA2 locus of heritable breast cancer susceptibility. This suggests that the same gene may be involved in multiple tumor types and that its function is that of a tumor suppressor rather than that of a dominant oncogene.

Isolation of YAC insert sequences by representational difference analysis.

We present a method for the isolation of YAC insert sequences by representational difference analysis (RDA). To achieve maximal representation of the sequences, the amplicons were generated from a Mbol digestion product. RDA was performed using a 970 kb insert YAC clone. After two rounds of re-association and selective amplification 92% of the difference product represented sequences derived from the YAC insert. Twenty insert-specific sequence-tagged sites were readily defined. The difference product was also successfully used to isolate microsatellite markers, to identify clones from a human PAC library and as a chromosome painting probe in fluorescence in situ hybridization.

Somatic deletions in hereditary breast cancers implicate 13q21 as a putative novel breast cancer susceptibility locus.

A significant proportion of familial breast cancers cannot be explained by mutations in the BRCA1 or BRCA2 genes. We applied a strategy to identify predisposition loci for breast cancer by using mathematical models to identify early somatic genetic deletions in tumor tissues followed by targeted linkage analysis. Comparative genomic hybridization was used to study 61 breast tumors from 37 breast cancer families with no identified BRCA1 or BRCA2 mutations. Branching and phylogenetic tree models predicted that loss of 13q was one of the earliest genetic events in hereditary cancers. In a Swedish family with five breast cancer cases, all analyzed tumors showed distinct 13q deletions, with the minimal region of loss at 13q21-q22. Genotyping revealed segregation of a shared 13q21 germ-line haplotype in the family. Targeted linkage analysis was carried out in a set of 77 Finnish, Icelandic, and Swedish breast cancer families with no detected BRCA1 and BRCA2 mutations. A maximum parametric two-point logarithm of odds score of 2.76 was obtained for a marker at 13q21 (D13S1308, theta = 0.10). The multipoint logarithm of odds score under heterogeneity was 3.46. The results were further evaluated by simulation to assess the probability of obtaining significant evidence in favor of linkage by chance as well as to take into account the possible influence of the BRCA2 locus, located at a recombination fraction of 0.25 from the new locus. The simulation substantiated the evidence of linkage at D13S1308 (P < 0.0017). The results warrant studies of this putative breast cancer predisposition locus in other populations.