Chromosome-specific Repetitive DNA Probes Research Articles

Numerical aberrations of chromosomes 8, 9, 11, and 17 in squamous cell carcinoma of the pharynx and larynx: a fluorescence in situ hybridization and DNA flow cytometric analysis of 50 cases

Squamous cell carcinoma of the pharynx and larynx (SCCPL) is a genetically complex disease and is frequently associated with nonrandom chromosomal alterations. Fifty primary SCC of the pharynx (oropharynx, n=11, and hypopharynx, n=11) and larynx ( n=28) were examined for numerical aberrations of chromosomes 8, 9, 11, and 17 with a panel of chromosome-specific repetitive DNA probes by fluorescence in situ hybridization (FISH). DNA ploidy analysis was also performed by flow cytometry (FCM). Aneusomic copy numbers of chromosomes 8, 9, 11, and 17 were discovered in 66%, 68%, 68% and 78% of tumors, respectively. FCM showed abnormal DNA content in 74% of cases (mean DNA index=1.69). Polysomy was the main finding in both DNA-aneuploid and DNA-diploid tumors (64.5% of cases). Numerical aberrations of chromosomes 8 and 11 correlated to DNA ploidy by FCM ( P<0.05). Aneusomy was present in 69.23% of DNA-diploid tumors. Marked intratumoral and intertumoral chromosomal heterogeneity was noted between individual tumors, suggesting a notable heterogeneity in aneuploid and diploid cell populations. Interphase FISH can be used to study important cytogenetic changes which occur during the development of SCC of the pharynx and larynx.

Use of FISH Analysis for Diagnosis of Renal Cell Carcinoma Subtypes

Cytogenetic and molecular genetic techniques have been used in demonstrating the chromosomal abnormalities which characterize specific subtypes of renal cell carcinoma (RCC). The aim of this study was to determine the efficiency of fluorescent in situ hybridization (FISH) technique in characterizing various subtypes of RCC based on the presence of specific chromosome abnormalities found in each RCC subtype. FISH was performed on touch imprint smears from eight renal cell carcinomas histologically confirmed by established criteria. In four tumors with histologic features of chromophobe renal cell carcinoma (ChRCC), interphase FISH was performed using centromeric probes for chromosomes 1, 2, 6, 10, 12, 17 and 21. All four ChRCC tumors showed one FISH signal corresponding to one copy number for each of these chromosomes. Two papillary RCCs included in this study showed trisomy 7 and 17, and loss of chromosome Y, using the corresponding chromosome centromeric probes. Similarly, we tested two clear cell RCCs for chromosome 3 short arm deletion with DNA probe 3p21.3. Both tumors showed loss of 3p21.3 signal. We conclude that interphase FISH performed on touch imprint smears is a relatively simple, rapid and reliable method for detecting chromosome abnormalities which are specific for various subtypes of RCC.

Detection of aneuploidy in interphase nuclei from non-small cell lung carcinomas by fluorescence in situ hybridization using chromosome-specific repetitive DNA probes

Interphase fluorescence in situ hybridization (FISH) is particularly useful for detecting chromosome changes in tumors exhibiting a low mitotic index, as is the case in many human non-small cell lung carcinomas (NSCLCs). A panel of centromeric DNA probes specific for the autosomes 6, 7, 8, 9, 12, 17, and 18 was used to analyze 17 primary NSCLCs. Evidence for aneuploidy was obtained in all specimens. Gain of part or all of chromosome 7 was especially prominent, occurring in a large population of cells in each of 14 tumors (82%). Extra centromeric copies of chromosomes 6, 12, and 17 were also common, being observed in 9 to 11 cases each. Gain of chromosome 9 was infrequent (three tumors). In two cases, most of the nuclei had only a single chromosome 9 fluorescent signal. Karyotypic findings were available for six cases and were generally consistent with the FISH data. Both methods revealed considerable heterogeneity within individual tumors. NSCLC specimens from 26 males were assayed with a Y-specific centromeric sequence; loss of the Y was observed in 13 cases (50%). These investigations demonstrate the feasibility of interphase FISH for the successful analysis of numerical chromosome changes in NSCLCs.

The use of FISH with chromosome-specific repetitive DNA probes for the follow-up of leukemia patients: Correlations and discrepancies with bone marrow cytology

The use of fluorescence in situ hybridization (FISH) for the purpose of repeated follow-up examination of bone marrow samples from 38 leukemia patients was investigated. On the basis of conventional cytogenetic analysis, patients with acute leukemia whose leukemic cells carried numerical chromosomal aberrations were selected and followed with repetitive DNA probes that specifically hybridize to one chromosome type. Repeated cytogenetic metaphase analyses would have been laborious and not sensitive or quantitative enough to follow declining numbers of aberrant cells. FISH, as an interphase cytogenetic technique, provides a rapid and simple alternative with high sensitivity. Although FISH data before and after chemotherapy were in agreement with bone marrow cytology in 30 of 38 patients, discrepancies were noticed in specific cases. These could be explained by the presence of cytogenetically distinct subclones that behave differently during treatment, the presence of differentiated leukemic cells, changes in the chromosomal constitution caused by clonal relapse, or the fact that a numerical aberration is found by conventional chromosome banding analysis while the target region to which the probe is directed is still present in the nucleus as a diploid set.

Cervical cytology by means of fluorescence in situ hybridization with a set of chromosome-specific DNA probes.

To reveal the numerical aberration of chromosome 1 and chromosome 17 in cervical neoplasia. Fluorescence in situ hybridization (FISH) with chromosome-specific repetitive DNA probes was applied on cervical smears of cervical intraepithelial neoplasia (CIN) I, CIN II, and CIN III, and of invasive carcinoma cases, to detect numerical aberrations of chromosome 1 (#1) and chromosome 17 (#17). The cases were histologically classified as CIN I (n = 9), CIN II (n = 12), CIN III [severe dysplasia (n = 14), carcinoma in situ (CIS) (n = 14)], and invasive carcinoma (IC) squamous-cell carcinoma, large-cell nonkeratinizing type (n = 12). FISH was applied on the same cervical smears of these cases after Papanicolaou's staining, and copies in marked atypical cells were counted using a fluorescence microscope. The 9.49 +/- 2.59%/9.72 +/- 1.40% (#1/#17) cells showed an aneuploid pattern in CIN I, 22.5 +/- 3.98%/15.5 +/- 3.02% (#1/#17) in CIN II, 44.3 +/- 7.18%/ 45.01 +/- 5.61% (#1/#17) in severe dysplasia, 52.66 +/- 6.32/48.9 +/- 7.55% (#1/#17) in CIS, and 66.22 +/- 3.20%/57.38 +/- 5.35% in IC. The loss of a chromosome in CIN III cases and the gain of a chromosome in CIS and IC cases should be noted (p < 0.01). Because FISH has revealed the numerical aberrations of chromosome 1 and 17 in cervical neoplasia, this is an especially useful method for biological dosimetry and cancer biology.

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry.

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome 1-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using beta-galactosidase/ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. The described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.

Detection of numerical chromosome aberrations in brain tumours by fluorescence in situ hybridization on smear preparations of small tumour biopsies.

We present a technique for the preparation and interphase cytogenetic analysis of native tumour cell nuclei from smear preparations of small biopsies of human brain tumours. The presence of tumour-specific numerical chromosome aberrations can be determined by fluorescence in situ hybridization (FISH) analysis using chromosome-specific repetitive DNA probes. The FISH analysis of smear preparations provides cytogenetic information about brain tumour samples, within 2 days, avoiding time-consuming and artefact-prone tumour cell culture.

Chromosome 2-specific DNA clones from flow-sorted chromosomes of tomato

We obtained DNA clones specific to tomato chromosome 2 from a small number of chromosomes collected by flow sorting. Suspensions of metaphase chromosomes were prepared from 3-month-old tomato cell cultures of Lycopersicon pennellii. Isolated chromosomes stained with chromomycin A3 and Hoechst 33258 were analyzed on an EPICS 753 flow cytometer using a UV laser to excite Hoechst fluorescence and a 458 nm laser to excite chromomycin A3 fluorescence. Chromosomes from well-resolved peaks on a bivariate flow karyotype were sorted directly onto membrane filters for spot-blot analysis. The filters were processed and hybridized with chromosome-specific repetitive DNA probes. In this way tomato chromosome 1 and chromosome 2 were assigned to peaks in the bivariate flow karyotypes. One thousand copies of the putative chromosome 2 were flow-sorted directly into microfuge tubes. DNA specific to chromosome 2 was amplified by a polymerase chain reaction (PCR) technique using universal 22mer degenerate oligonucleotide primers (DOP) sequences. DOP-PCR yields a smear of fragments of various sizes from 250 to 1600 bp. Amplified products were cloned into the Bluescript plasmid vector. Approximately 11% of the clones contained sequences with highly repetitive elements, and 85% contained only low-copy-number sequences. Eleven clones containing low-copy-number sequences that detect restriction fragment length polymorphisms were placed on the molecular linkage map of tomato. All showed linkage to chromosome 2.

Applications of DNA flow cytometry and fluorescence in situ hybridization using a chromosome-specific DNA probe on paraffin-embedded tissue sections of primary malignant melanomas.

We have applied DNA flow cytometric analysis to paraffin-embedded tissue sections of primary malignant melanomas. Conventionally, flow cytometric analysis of paraffin-embedded tissue sections has been done by the method of Hedley et al. We added ultrasound treatment to the method of Hedley et al. and a lower value of coefficient of variation was shown. Furthermore, a new technique, fluorescence in situ hybridization with a chromosome-specific repetitive DNA probe, was used for the analysis of chromosomal numerical aberrations in the same paraffin-embedded tissue sections. The DNA flow cytometric analysis showed that in 8 cases six primary malignant melanomas were of the aneuploid pattern and two cases of lentigo maligna (melanoma in situ) were of the diploid pattern. By fluorescence in situ hybridization, the two cases with the diploid pattern had spots/nucleus of 1.28 and 1.12, and those with the aneuploid pattern had spots/nucleus from 2.01 to 2.27. Only one nodular melanoma in an aneuploid case showed spots/nucleus of 1.71. These data indicate that fluorescence in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors and may be useful for the study of tumor cell heterogeneity.

Detection of aneuploidy in human sperm by fluorescence in situ hybridization (FISH): different frequencies in fresh and stored sperm nuclei.

Fluorescence in situ hybridization (FISH) with chromosome-specific repetitive DNA probes provides a new method for rapid detection of aneuploidy in human sperm. There is widespread interest in this technique for basic research, as well as for screening men exposed to potential aneugens. A number of laboratories have reported a wide range in the frequency of aneuploidy for chromosomes in human sperm, suggesting that FISH may not reflect the true frequency accurately. We have serendipitously discovered that the length of time that fixed frozen sperm nuclei are stored affects both the hybridization efficiency and disomy frequency of individual chromosomes. This may explain some of the variation in the aneuploidy frequencies observed among laboratories.

Rapid detection of aneuploidy in uncultured chorionic villus cells using fluorescence in situ hybridization

Rapid detection of aneuploidy using chromosome-specific repetitive DNA probes and the potential diagnostic accuracy of fluorescence in situ hybridization (FISH) on interphase cells of chorionic villus samples (CVS) are presented. Analyses demonstrated the ability to correctly identify aneuploidy using FISH in uncultured CVS. Our preliminary investigation suggests that this technique offers a significant clinical potential to circumvent problems of culture, time, and cost in cytogenetic analysis.

In Situ Detection of Supernumerary Aberrations of Chromosome-Specific Repetitive DNA Targets in Interphase Nuclei in Human Melanoma Cell Lines and Tissue Sections

The use of non-radioactive in situ hybridization (ISH) with chromosome-specific repetitive DNA probes to study genomic changes, aneuploidy, and heterogeneity during melanocytic tumor progression, relies on its applicability to non-mitotic interphase nuclei, present in cell suspensions and tissue sections. Therefore, we studied the feasibility of detecting numerical aberrations with respect to the (peri-) centromere regions of chromosomes 1 and 7 in intact nuclei of two human melanoma cell lines with different metastatic behavior in nude mice. In addition, we used paraffin sections from xenograft lesions, obtained by inoculation of these cell lines in nude mice (subcutaneous tumors and spontaneous lung metastases). Paraffin sections from the original primary cutaneous melanoma (with a subepidermal and a dermal part) and two loco-regional metastases were also studied, one of which was the source for the cell lines. These cells and tissues represent examples of materials used in different approaches to the study of melanocytic tumor progression. Regarding the targeted sequences, ISH analysis showed that both cell lines were heterogeneous and aneuploid. The results correlated well with those obtained by ISH on metaphase spreads. Differences between the lines, which could not be detected by flow-cytometric or conventional karyotyping analysis, included data suggestive of a polyploid subpopulation and an extra copy of chromosome 7 in the metastasizing cell line. The polyploid population could be detected also in the paraffin sections of the corresponding subcutaneous xenografts and lung metastases in the mice. Both areas in the patients' primary melanoma could be evaluated separately and showed similar supernumerary aberrations of the chromosome-specific targets. These abnormalities matched those found in both metastases. Our results demonstrate that ISH can be used to visualize genomic abnormalities at the single-cell level in melanocytic nuclei in their natural context, which makes it a promising tool in the histopathology of melanocytic lesions and in the study of melanocytic tumor progression.

Targeted cytogenetic analysis of gastric tumors byin situhybridization with a set of chromosome-specific dna probes

Fluorescent in situ hybridization (FISH) with biotinated chromosome-specific repetitive DNA probes was used for the cytogenetic study of ten gastric adenocarcinomas. All tumors (eight male, two female patients) were histologically moderately or poorly differentiated and nine of ten had metastasized to regional lymph nodes. The authors applied a set of satellite DNA probes, specific for chromosomes 1, 7, 17, X, and Y in order to detect numerical chromosome aberrations in freshly isolated tumor cell nuclei. Normal diploid human lymphocyte nuclei and, in a number of cases, normal gastric mucosa served as controls. Parallel with the hybridization experiments DNA flow cytometric study of acridine orange (AO)-stained tumor cells was carried out. By means of FISH the authors found seven cases to be aneuploid, the other three cases appeared diploid. This was confirmed by DNA flow cytometric analysis with AO. Furthermore, loss of the Y chromosome in a high percentage of cells was seen by FISH in six of eight tumors from male patients. In the other two male samples a possible loss was observed in a small proportion of cells (15%). In three patients from whom the authors had normal gastric mucosa the Y loss was restricted to the tumor cells. These data indicate that in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors, at least with respect to the detection of numerical chromosome abnormalities.

Human chromosome-specific repetitive DNA probes: Targeting in situ hybridization to chromosome 17 with a 42-base-pair alphoid DNA oligomer

The pericentric region of human chromosome 17 was targeted for specific in situ hybridization of the alphoid DNA subfamily enriched on this chromosome. A recombinant DNA clone containing the entire higher order chromosome 17 alphoid repeat preferentially hybridized to the pericentric region of chromosome 17, but frequently cross-hybridized to other chromosomes under normal stringency conditions. Chromosomal specificity, after in situ hybridization to metaphase spreads and interphase nuclei, was improved by using a subclone containing predominantly monomer 1 of the higher order repeat. Further improvement was achieved by synthesizing a 42-nucleotide oligomer of a divergent region of monomer 1. Southern blot analysis confirmed the improved specificity of the shorter probes. Reducing the potential of repetitive DNA probes to cross-hybridize increases the usefulness of the probes, especially when they are used for localizing individual chromosomes in interphase nuclei.

90 Detection of chromosomal aberrations in interphase nuclei from human primary breast tumors using in situ hybridization with chromosome-specific repetitive DNA-probes

90 Detection of chromosomal aberrations in interphase nuclei from human primary breast tumors using in situ hybridization with chromosome-specific repetitive DNA-probes

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes.

Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (Xqter----Xp22.2::Yq11----Yqter) contained in the Chinese hamster X human hybrid cell line 445 X 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.