Biomedical subjects
D Pinkel
Publications and source records attributed to D Pinkel.
Karyotypic heterogeneity and its relation to labeling index in interphase breast tumor cells.
We have used fluorescence in situ hybridization (FISH) with chromosome-specific probes and immunofluorescent detection of in vivo bromodeoxyuridine (BrdUrd) incorporation to evaluate simultaneously numerical chromosome aberrations and proliferative activity of breast cancers. The number of distinct hybridization domains specific for repetitive pericentromeric sequences on chromosomes 1, 7, 11, 15, 17, and X was used as an indicator of copy number of these chromosomes in interphase tumor cells from 23 human breast cancers. Every tumor analyzed showed a heterogeneous distribution of copy number for at least one chromosome type. The copy number distribution for different chromosomes within a tumor frequently showed differing patterns. Major cell populations showing monosomy were relatively rare, occurring only in five cases for chromosome 17, once for chromosome 1, and once for chromosome 15. Flow cytometric analysis of DNA ploidy correlated well with FISH analysis, although flow cytometry failed to detect aneuploidy when only a few chromosomes were affected. To determine whether cell populations with different chromosomal copy numbers have identical proliferation characteristics in vivo, BrdUrd incorporation and centromeric copy number were detected simultaneously. Comparison of the chromosome copy number distribution in BrdUrd-positive cells vs. the distribution of the entire cell population showed different distributions in seven of the 20 cases analyzed. This study demonstrates the common occurrence of chromosome copy number heterogeneity and suggests that a cell phenotype (proliferation) may be associated with genotypic subpopulations.
Deficiency of p53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability.
By crossing mice that carry a null allele of p53 with transgenic mice that develop mammary adenocarcinomas under the influence of a Wnt-1 transgene, we have studied the consequences of p53 deficiency in mammary gland neoplasia. In Wnt-1 transgenic mice homozygous for the p53 null allele, tumors appear at an earlier age than in animals heterozygous or wild-type at the p53 locus. About half of the tumors arising in p53 heterozygotes exhibit loss of the normal p53 allele, implying selection for p53-deficient cells. Mammary tumors lacking p53 display less fibrotic histopathology and increased genomic instability with aneuploidy, amplifications, and deletions, as detected by karyotype analysis and comparative genomic hybridization. In one tumor, the amplified region of chromosome 7 had an ectopically expressed int-2/FGF3 proto-oncogene, a gene known to cooperate with Wnt-1 in the production of mammary tumors. These findings favor a model in which p53 deficiency relaxes normal restraints on chromosomal number and organization during tumorigenesis.
Treatment of acute lymphoblastic leukemia in a second remission.
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A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis.
The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously.
Computer image analysis of comparative genomic hybridization.
We describe and evaluate the image-processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818, 1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes. The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented.
Semiautomated DNA probe mapping using digital imaging microscopy: II. System performance.
This paper describes an evaluation of a semiautomated, multicolor image-analysis system to map cloned probes along metaphase chromosomes. Mapping with this system consists of fluorescence in situ hybridization (FISH) for probe localization, automatic acquisition of multicolor images showing total chromosomal DNA and probe location(s), and automatic determination of the fractional locations of the probes along the chromosomes relative to the short arm telomere (FLpter). The system was evaluated by mapping ten phage and ten cosmid probes previously mapped to chromosome 3 with other procedures. The standard deviations of FLpter measurements averaged 3.4 Mb and 2.6 Mb for phage and cosmid probes, respectively. With this variation, the order of two probes mapped in separate hybridizations could be determined with 95% confidence when their separation was greater than 2.5 Mb. In all cases, the probe locations and order were consistent with previous mapping data. FLpter values were converted to band locations using measurements of the band locations made using digital imaging microscopy. This proved superior to conversions made using ISCN ideograms.
Aneuploidy in late-step spermatids of mice detected by two-chromosome fluorescence in situ hybridization.
A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late-step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick-translated with biotin- or digoxigenin-labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in approximately 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late-step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, approximately 3/10,000 spermatids had fluorescence phenotypes indicative of X-X or 8-8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of "bridging biomarkers" between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin.
Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization.
Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of approximately 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of approximately 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring.
Prevention and treatment of meningeal leukemia in children.
The prevention of meningeal leukemia has long been a keystone in its cure. The need was recognized when it became apparent in the 1950s and 1960s that meningeal relapse heralded hematologic relapse and a fatal course and that its incidence increased as systemic chemotherapy became more effective in controlling hematologic and visceral leukemia. Evasion of a biologic safety net, the blood-CSF barrier, is required to prevent meningeal leukemia. Three methods are used: meningeal radiotherapy, intrathecal administration of antileukemia drugs, and high-dosage intravenous antileukemia drugs. Recent and current clinical studies reflect a continuing dialogue about which methods are preferable and under what circumstances. For prevention of meningeal leukemia, extended intrathecal therapy and intensive systemic chemotherapy appear to be as effective as radiotherapy for most patients. For treatment of overt meningeal leukemia, meningeal radiotherapy may be necessary. However, its administration compromises subsequent systemic chemotherapy so that delay may be advisable to allow intensive systemic chemotherapy for control of concurrent hematologic and visceral leukemia, whether clinically evident or not. For patients with meningeal leukemia at diagnosis, cranial irradiation may be delayed or possibly omitted if evidence of disease is minimal and intrathecal and systemic chemotherapy are intensive. For those who develop meningeal leukemia while on therapy or after its completion, cranial or craniospinal irradiation is probably required as well as intensive intrathecal and systemic chemotherapy. Hopefully, current and future studies will dispel the uncertainties and better quantitate risks and benefits of alternative methods. Whatever method is used, careful attention to technical details is required to assure optimal efficacy at the least possible expense in immediate toxicity and late sequelae.
Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization.
Comparative genomic hybridization was applied to 5 breast cancer cell lines and 33 primary tumors to discover and map regions of the genome with increased DNA-sequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copy-number affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression. Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.
Physical mapping of chromosome 17 cosmids by fluorescence in situ hybridization and digital image analysis.
We used fluorescence in situ hybridization and digital image analysis to localize cosmids along human chromosome 17. Seventy-one cosmids were selected at random from a chromosome 17 library constructed from a partial Sau3AI digest of flow-sorted chromosomes from a mouse-human hybrid cell line. Sixty-three of these (89%) gave a signal only on chromosome 17. The 40 cosmids producing the most distinct hybridization signals in metaphase and interphase cells were precisely mapped using digital image analysis. An additional 20 cosmids, previously mapped by linkage analysis, were also mapped. The order of these probes determined by metaphase mapping was consistent with the order determined by linkage analysis.
Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors.
Comparative genomic hybridization (CGH) is a powerful new method for molecular cytogenetic analysis of cancer. In a single hybridization, CGH provides an overview of DNA sequence copy number changes (losses, deletions, gains, amplifications) in a tumor specimen and maps these changes on normal chromosomes. CGH is based on the in situ hybridization of differentially labeled total genomic tumor DNA and normal reference DNA to normal human metaphase chromosomes. After hybridization and fluorescent staining of the bound DNAs, copy number variations among the different sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target metaphase chromosomes. CGH is in particular useful for analysis of DNA sequence copy number changes in common solid tumors where high-quality metaphase preparations are often difficult to make, and where complex karyotypes with numerous markers, double minutes, and homogeneously stained chromosomal regions are common. CGH only detects changes that are present in a substantial proportion of tumor cells (i.e., clonal aberrations). It does not reveal translocations, inversions, and other aberrations that do not change copy number. At present, CGH is a research tool that complements previous methods for genetic analysis. CGH will advance our understanding of the genetic progression of cancer and highlight important genomic regions for further study. Direct clinical applications of CGH are possible, but will require further development and validation of the technique. We describe here our recent optimized procedures for CGH, including DNA labeling, hybridization, fluorescence microscopy, digital image analysis, data interpretation, and quality control, emphasizing those steps that are most critical. We will also assess sensitivity and resolution limits of CGH as well as discuss possible future technical improvements.
Role of bone marrow transplantation for acute lymphoid leukemia.
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Visualizing tumour amplification.
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Defining the steps in a multistep mouse model for mammary carcinogenesis.
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Molecular cytogenetics of human breast cancer.
Our studies of human breast cancer using CGH and FISH with precisely mapped probes have revealed a surprising amount of intratumor and intertumor heterogeneity in human breast cancers. However, some regions of abnormal copy number are found frequently. We believe that the regions of frequent abnormality harbor novel cancer genes, and that identification of these is important for diagnosis, prognostication, and the development of new therapies.
Detection of a human chromosomal translocation t(8;9) in a baby with multiple malformations using two-color fluorescence in situ hybridization.
A human chromosomal translocation t(8;9) was detected using two-color fluorescence in situ hybridization with probes capable of staining the entire lengths of each of these chromosomes. The chromosome 8 probe was labeled with biotin and detected with Texas red, while the chromosome 9 probe was labeled with AAF and detected with FITC. In normal metaphase spreads, two metaphases from the proband, two red, one green and one part red and part green derivative chromosome were seen. The bicolor chromosome corresponded to translocation of a chromosome 8 segment to the distal part of the q region of one chromosome 9, as originally indicated by banding analysis. In interphase nuclei of the proband, four domains with bright fluorescence were recognized in many nuclei. Two were red, one was green, and the fourth had portions of both colors, indicating the presence of the translocation.