Genetic changes in intraductal breast cancer detected by comparative genomic hybridization

Ductal carcinoma in situ (DCIS) is considered a direct precursor of invasive ductal breast cancer (IDC). We combined tissue microdissection and comparative genomic hybridization to identify genetic changes in five DCIS lesions with no invasion and in two that were adjacent to IDC. Extensive genetic changes characterized pure DCIS cases with gains of 1q, 6q, 8q, and Xq as well as losses of 17p and chromosome 22 being most often involved. Except for the Xq gain, these changes are also common to IDC. Separate analysis of DCIS and IDC components in the same tumor revealed an almost identical pattern of genetic changes in one case, whereas substantial differences were found in another. We conclude that many of the common genetic changes in IDC may take place before development of invasive growth. However, a simple linear progression model may not always account for the DCIS-IDC transition.     

Multiple genetic aberrations including evidence of chromosome 11q13 rearrangement detected in pituitary adenomas by comparative genomic hybridization

OBJECT: This study was conducted to determine whether comparative genomic hybridization (CGH) is a more sensitive method for detecting genetic aberrations than other tests currently in use. METHODS: The authors used CGH to examine 40 primary and 13 recurrent adenomas obtained from 52 patients for loss and gain of genetic material. Copy number aberrations (CNAs) were detected in 25 (48%) of the 52 patients studied. The chromosomes affected were, in order of decreasing frequency, 11, 7, X, 1, 8, 13, 5, 14, 2, 6, 9, 10, 12, 3, 18, 21, 4, 16, 15, 19, 22, and Y. Endocrinologically active adenomas were more likely to contain (p = 0.009) and had a greater number (p = 0.003) of CNAs. Of 26 adenomas with CNAs, 18 showed multiple aberrations involving entire chromosomes or chromosome arms. The most frequent CNA involving a chromosome subregion, which was present in four (8%) of 53 adenomas, was the loss of all chromosome 11 material except for a preserved common segment containing 11q13. Immunoperoxidase staining did not detect cyclin D1 expression in those four cases, making cyclin D1 an unlikely target of this rearrangement. CONCLUSIONS: These findings indicate that genetic abnormalities are present in pituitary adenomas at a higher rate than previously reported, are associated with endocrinological activity, and often involve several chromosomes. Rearrangement at 11q13 may inactivate a tumor suppressor gene or activate an oncogene that is important in the initiation or progression of sporadic pituitary adenomas.     

Recurrent DNA copy number changes in 1q, 4q, 6q, 9p, 13q, 14q and 22q detected by comparative genomic hybridization in malignant mesothelioma

Comparative genomic hybridization (CGH) analyses were performed on 27 human pleural mesothelioma tumour specimens, consisting of 18 frozen tumours and nine paraffin-embedded tumours, to screen for gains and losses of DNA sequences. Copy number changes were detected in 15 of the 27 specimens with a range from one to eight per specimen. On average, more losses than gains of genetic material were observed. The loss of DNA sequences occurred most commonly in the short arm of chromosome 9 (p21-pter), in 60% of the abnormal specimens. Other losses among the abnormal specimens were frequently detected in the long arms of chromosomes 4 (q31.1-qter, 20%), 6 (q22-q24, 33%), 13 (33%),14 (q24-qter, 33%) and 22 (q13, 20%). A gain in DNA sequences was found in the long arm of chromosome 1 (cen-qter) in 33% of the abnormal specimens. Our analysis is the first genome-wide screening for gains and losses of DNA sequences using comparative genomic hybridization in malignant pleural mesothelioma tumours. The recurrent DNA sequence changes detected in this study suggest that the corresponding chromosomal areas most probably contain genes important for the initiation and progression of mesothelioma.     

Chromosomal assignment of the gene for human DNA-PKcs interacting protein (KIP) on chromosome 15q25.3-q26.1 by somatic hybrid analysis and fluorescence in situ hybridization

The aim of this study was to determine the in vitro activity of ranitidine, ebrotidine, bismuth citrate, omeprazole, and lansoprazole against 113 clinical isolates of Helicobacter pylori cultured from gastric biopsies. An agar dilution method using Mueller-Hinton agar plus 7%, horse blood, an inoculum of 10(6) cfu/spot, and incubation in a CO2 incubator for 2 to 5 days was used. The minimum inhibitory concentrations for 50 and 90% of the isolates tested, respectively, were as follows: ranitidine, 1024 and 1024 mg/l; ebrotidine, 64 and 256 mg/l; bismuth citrate, 1 and 4 mg/l; omeprazole, 16 and 16 mg/l; and lansoprazole, 1 and 1 mg/l. Ebrotidine was more active than ranitidine, and lansoprazole was the most active compound against the Helicobacter pylori isolates tested.     

Identification of complex chromosome rearrangements in the gibbon by fluorescent in situ hybridization (FISH) of a human chromosome 2q specific microlibrary, yeast artificial chromosomes, and reciprocal chromosome painting

Chromosome painting has revealed that the human chromosome homologs in lesser apes are often fragmented and translocated to a number of different hylobatid chromosomes. We investigated the fragmented human chromosome 2 homologs in gibbons to illustrate a new strategy in mapping regional and band-specific chromosomal homologies between species. Previous research showed that the DNA library specific to human chromosome 2 paints parts of four gibbon (lar species group) chromosomes (viz., 1, 10, 12, and 16) and yields five distinct hybridization signals (including two on gibbon chromosome 16). However, the exact segments of human chromosome 2 that were translocated to the various gibbon chromosomes could not be distinguished. To determine the origin of the human chromosome 2 signals, we hybridized a microlibrary for the long arm of human chromosome 2, as well as YACs specific for most of the major bands on this chromosome, to metaphases of the gibbon. For reciprocal chromosome painting, we hybridized flow-sorted gibbon chromosome probes to human chromosome 2. Each method added additional insights that helped clarify the shuffling of human chromosome 2 material in the highly reorganized gibbon genome. There was an excellent correspondence between these complementary techniques. YAC 958d2 identified the breakpoint between human chromosome 2 material present on gibbon chromosomes 10 and 16. The reciprocal chromosome painting permitted a more complete and regional assignment of homology between segments on various gibbon chromosomes to human chromosome 2. The results show that a combination of reciprocal chromosome painting, subregional microlibraries, and band-specific probes (such as YACs) can be used to identify homologies between species and to rapidly construct detailed comparative chromosome maps, especially when the karyotypes are highly rearranged.     

Identification of a novel gene, MASL1, within an amplicon at 8p23.1 detected in malignant fibrous histiocytomas by comparative genomic hybridization

We used comparative genomic hybridization to study malignant fibrous histiocytomas (MFHs) from 19 patients to detect changes in the copy number of DNA sequences, along entire chromosomes. Together with losses and gains in various chromosomal regions, distinct high-level amplifications were found at six loci (4q12-21, 8p21-pter, 8q24.1-qter, 9q12-13, 12p11.2-pter, and 15q11.2-15), suggesting that those regions may contain unknown (proto) oncogenes. We focused on the 8p amplicon, where detailed characterization allowed us to determine that the minimal common amplified region lay between markers D8S1819 and D8S550 at 8p23.1. A novel gene designated MASL1 (MFH-amplified sequences with leucine-rich tandem repeats 1) was isolated from within this narrowly defined region. Expression of the MASL1 gene was enhanced significantly in MFH tumors bearing the 8p amplicon. The primary structure of its deduced product revealed an ATP/GTP-binding site, three leucine zipper domains, and a leucine-rich tandem repeat, all of which are important structural or functional elements for interactions among proteins related to the cell cycle. These features suggest that overexpression of MASL1 might well be oncogenic with respect to MFH.     

Linkage analysis of infantile pyloric stenosis and markers from chromosome 9q11-q33: no evidence for a major gene in this candidate region

In order to investigate useful indicators of lead contamination, 108 Japanese Black calves from a lead-contaminated farm were used. The lead concentrations in blood (Ph-B), delta-aminolevulinic acid dehydratase (ALA-D) activities, and free erythrocyte protoporphyrin (FEP) concentrations were examined. A significant negative correlation was obtained between Pb-B concentrations and ALA-D activities (r = -0.621, p < 0.01). A significant positive correlation was obtained between Ph-B concentrations and FEP concentrations (r = 0.850, p < 0.01). The calves were divided by Pb-B concentrations (micrograms/100 ml) into 5 groups (A = < or = 30, B = 31 approximately 90, C = 91 approximately 150, D = 151 approximately 210, E = > or = 211) to observe the relation of ALA-D activities, FEP concentrations and ALA-D activities in group B (r = -0.706, p < 0.05). A significant positive correlation was obtained between Pb-B concentrations and FEP concentrations in groups A, B and D (A = r = 0.496, p < 0.01; B = r = 0.686, p < 0.01; D = r = 0.529, p < 0.05). These results indicate that FEP concentrations were good indicators of lead contamination.     

From amplification to gene in thyroid cancer: a high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. We now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. We used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3-6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKCepsilon), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKCepsilon as a previously unmapped candidate gene involved in thyroid tumorigenesis.     

Detection of K-ras point mutations at codon 12 in pancreatic juice for the diagnosis of pancreatic cancer by hybridization protection assay: a simple method for the determination of the types of point mutation

The present study was undertaken to detect K-ras oncogene point mutations at codon 12 in pure pancreatic juice (PPJ) by the hybridization protection assay (HPA) method for the diagnosis of pancreatic cancer (PC). This assay can be carried out within 30 min and can determine not only the presence of a mutation, but also the mutational type of K-ras at codon 12. The minimal ratio of mutant DNA detectable by the HPA was 5-10% of the total DNA. PPJ was collected through a cannula under duodenal fiberscope control from 20 patients with PC and 20 patients with chronic pancreatitis (CP). Analysis of PPJ by the HPA revealed that the incidence of K-ras point mutations at codon 12 was 55% (11/20) in patients with PC and 0% (0/20) in those with CP. Mutational types of K-ras at codon 12 in PC were aspartic acid (Asp) in nine cases, both Asp and cysteine in one case, and arginine in one case. Analysis of K-ras point mutations at codon 12 in PPJ using the HPA method seems promising as a new genetic test for the diagnosis of PC, because the HPA method is simple, and can easily determine the mutational type.