Detection Of Chromosomal Aberrations Research Articles

Split-signal FISH for detection of chromosome aberrations

Chromosome aberrations are frequently observed in hematopoietic malignancies. These aberrations can deregulate expression of an oncogene, resulting in aberrant expression or overexpression, or they can form leukemia-specific chimeric fusion proteins. Detection of chromosome aberrations is an important tool for classification of the malignancy and for the definition of risk groups, which need different treatment protocols. We developed rapid and sensitive split-signal fluorescent in situ hybridization (FISH) assays for frequently occuring chromosome aberrations. The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. In normal karyotypes, two co-localized green/red signals are visible, but a translocation results in a split of one of the co-localized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses of two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity.

Incidence of Chromosomal Aberrations in Embryos From a Fertile Group of Patients

Preimplantation Genetic Diagnosis (PGD) has been used primarily for the detection of chromosomal aberrations in embryos of patients with recurrent miscarriages or failed implantation. Unlike other patient populations where PGD has been applied, who suffer either from certain degree of infertility and/or genetic defects, our PGD program primarily treats patients for family balancing, as well as aneuploidy screening. The aim of the study was to investigate the incidence of chromosomal abnormalities in this group of fertile patients.

Standardised fluorescence in situ hybridisation in cytological and histological specimens

Fluorescence in situ hybridisation (FISH) has gained major impact in the detection of chromosomal aberrations. The application of FISH, however, is hampered due to complicated protocols. We therefore designed a FISH protocol that allows the fast and reliable application on cytological and histological specimens. Cytological and histological specimens of bone marrow, lymph nodes, liver and breast were analysed with 14 sets of centromere or locus-specific probes. A pretreatment of enzymatic digestion and microwave heating with the same incubation times for all specimens and probes used was performed. Hybridisation efficiency was proved by calculating the thresholds for monosomy, trisomy and translocations. Values in cytological specimens reached 9.0, 3.9 and 4.6%, respectively. In histological sections, values of 6.4% were seen for aneusomy and 3.1% for translocations. However, values for monosomy reached 20.5% in bone marrow and 61.8% in liver tissues due to cutting artifacts. In bone marrow with acute myeloid leukaemias, lymph nodes with follicular and mantle cell lymphomas, breast carcinomas and liver cell carcinomas, FISH confirmed aberrations already found using conventional cytogenetics. The protocol shown here is easy to perform and can be used with cytological and histological specimens. Moreover, with "hands on" time of less than 2 h, FISH can also be used for daily routine purposes.

Detection of chromosomal aberrations on paraffin tissue sections of benign thyroid tumours by fluorescence-in situ-hybridization (FISH)

Detection of chromosomal aberrations on paraffin tissue sections of benign thyroid tumours by fluorescence-in situ-hybridization (FISH)

Characterization of 3p, 5p, and 3q in two nasopharyngeal carcinoma cell lines, using region-specific multiplex fluorescence in situ hybridization probes

Amplification of chromosome arms 3q and 5p and deletion of 3p were frequently detected in nasopharyngeal carcinoma (NPC) with comparative genomic hybridization and loss of heterozygosity studies. To identify the minimal amplified or deleted regions in these arms, structural aberrations in chromosome arms 3p, 3q, and 5p in two NPC cell lines, CNE1 and SUNE1, were studied with multiplex-color FISH (M-FISH) and chromosome region-specific probes (CRP). All CRPs, which were generated from microdissected DNA, were specific and strong in intensity, and sensitive enough to detect chromosome aberrations including translocations, deletions, and amplifications of target regions. In these two NPC cell lines, minimal regions of deletion and amplification were found at 3p12 and 3q26∼q27, respectively. On 5p, most of the regions were amplified as intact copies. Interregion translocations of these three arms were also observed. The amplification on 3q26∼q27 provided useful hints for further screening the minimal amplification at RP11-115J24 (3q26.2), containing candidate oncogene eIF-5A2. M-FISH with CRPs is thus not only useful in revealing a comprehensive picture of structural aberrations in target chromosomes, but also in narrowing down the minimal region for screening cancer-related genes.

Detection of chromosome aberrations during prenatal genetic testing for single gene disorders

Detection of chromosome aberrations during prenatal genetic testing for single gene disorders

Preimplantation genetic diagnosis for aneuploidy screening in repeated implantation failure

Chromosomal abnormalities are thought to be responsible for implantation failure, and among chromosomal abnormalities in normally developing embryos, aneuploidy is the most frequent. Genetic testing of preimplantation embryos for chromosomal aneuploidy allows selection of chromosomally normal embryos, and early detection of chromosomal aberration will increase the chance of conceiving. Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS), performed by polar body or blastomere analysis, is used in infertile patients treated with assisted reproduction technologies, especially in those with a poor prognosis, e.g. repeated IVF failure, advanced maternal age, or recurrent spontaneous abortion. The aim of this paper is to clarify the impact of PGD-AS in repeated implantation failure. In this review, the data collected so far regarding PGD-AS in this patient group will be discussed in depth.

Specificity of interphase fluorescence in situ hybridization for detection of chromosome aberrations in tumor pathology

Interphase fluorescence in situ hybridization (IFISH) is an interesting and intriguing cytogenetic approach in the study of tumor chromosomal abnormalities when metaphases are not available. This technique can be applied to different types of tumor nuclei, including imprinted nuclei (IM), nuclei obtained from conventional cytogenetic procedures (PB), frozen nuclei, paraffin-embedded nuclei (PE), and nuclei extracted from paraffin-embedded sections (EX). IFISH is a high-sensitivity approach in tumor studies that can give evidence of genetic aberrations present in a small percentage of cells that are likely to escape detection if only molecular techniques are applied. Despite its high sensitivity and versatility, IFISH is an indirect cytogenetic method and needs controls to have adequate specificity. This study includes present data obtained in IFISH experiments using different types of probes (α-satellite and YAC clones) hybridized on different types of normal control nuclei, such as PB, IM, EX, and PE nuclei, to define the threshold level for monosomy and trisomy of different chromosomal regions. My findings demonstrate that the cut-off values depend both on the types of probes and on the types of target nuclei. Therefore, even if IFISH is a versatile, high-sensitivity technique for detecting chromosomal abnormalities, the lack of accurate controls may result in the misdiagnosis of some abnormalities.

Detection of chromosome aberrations during prenatal genetic testing for single gene disorders

Detection of chromosome aberrations during prenatal genetic testing for single gene disorders

Evaluation of the relative sensitivity of chromosome painting (FISH) as an indicator of radiation-induced damage in human lymphocytes.

Fluorescence in situ hybridization with whole chromosome libraries, also known as chromosome "painting", is an easy and rapid method for detection of chromosome aberrations. To evaluate the sensitivity of this in radiation dosimetry we have made comparisons with G-banding analysis and also with physicochemical measurements of radiation induced DNA damage (DNA strand breaks and 8-hydroxydeoxyguanosine formation). Heparinised human blood was irradiated at room temperature with a range (0-10 Gy) of gamma irradiation from a cobalt 60 source. Chromosome spreads prepared from phytohaemagglutinin stimulated "whole blood" lymphocyte cultures were hybridized in situ with the whole chromosome 1 library, coded, and scored for aberrant cells. Dose response curves plotted as percent abnormal cells obtained by the two cytogenetic methods were similar and it would appear that chromosome "painting" compared favourably with G-banding for the detection of aberrations. The measurement of DNA strand breaks by a fluorimetric alkaline unwinding assay showed similar sensitivity to chromosome "painting" whereas the formation of 8-hydroxydeoxyguanosine did not correlate with aberration frequencies.

P53‐Positive Tumor‐Distant Squamous Epithelia of the Head and Neck Reveal Selective Loss of Chromosome 17

The progression of clinically manifest premalignant lesions in the head and neck region to primary or second primary cancer is characterized by numerical and structural chromosomal aberrations. However, many tumors arise from histologically inconspicuous mucosal sites. The objective was to investigate whether chromosomal aberrations can be detected in tumor-distant mucosa and whether they can help predict the risk of second primary malignancy. A retrospective series of 72 clinically healthy, arbitrarily taken mucosal samples from 53 patients with squamous cell carcinoma of the head and neck was studied. A previous analysis of the p53 protein status had revealed both p53-positive and p53-negative samples. The samples were analyzed by fluorescence in situ hybridization (FISH) using centromeric probes specific for the chromosomes 1, 10, 17, and 18. Tumor-distant mucosa generally showed increased numerical chromosomal aberrations, which consisted mainly of monosomies. In the group of patients with p53-positive epithelia, all aberrations including gains of chromosomes (trisomies, tetrasomies) were more frequent. Monosomy for chromosome 17 was most significantly and selectively enhanced in this group (Wilcoxon Scores [rank sum] test, P =.0002). Detectable chromosomal aberrations occur in clinically healthy epithelia in patients at risk for secondary head and neck cancer. Specifically, unbalanced chromosome 17 monosomy in conjunction with p53 protein overexpression may constitute a valuable biomarker for progressive "field cancerization."

Split-signal FISH for detection of chromosome aberrations in acute lymphoblastic leukemia

Chromosome aberrations are frequently observed in precursor-B-acute lymphoblastic leukemias (ALL) and T-cell acute lymphoblastic leukemias (T-ALL). These translocations can form leukemia-specific chimeric fusion proteins or they can deregulate expression of an (onco)gene, resulting in aberrant expression or overexpression. Detection of chromosome aberrations is an important tool for risk classification. We developed rapid and sensitive split-signal fluorescent in situ hybridization (FISH) assays for six of the most frequent chromosome aberrations in precursor-B-ALL and T-ALL. The split-signal FISH approach uses two differentially labeled probes, located in one gene at opposite sites of the breakpoint region. Probe sets were developed for the genes TCF3 (E2A) at 19p13, MLL at 11q23, ETV6 at 12p13, BCR at 22q11, SIL-TAL1 at 1q32 and TLX3 (HOX11L2) at 5q35. In normal karyotypes, two colocalized green/red signals are visible, but a translocation results in a split of one of the colocalized signals. Split-signal FISH has three main advantages over the classical fusion-signal FISH approach, which uses two labeled probes located in two genes. First, the detection of a chromosome aberration is independent of the involved partner gene. Second, split-signal FISH allows the identification of the partner gene or chromosome region if metaphase spreads are present, and finally it reduces false-positivity.

Detection of chromosomal aberrations in spermatocytic and classical seminoma

SALL4 is one of the master transcriptional factors that are crucial in maintaining the pluripotency of embryonic stem cells (ESCs). While the expression of SALL4 is normally restricted to ESCs and somatic stem cells, we found that it is aberrantly expressed in ALK-positive anaplastic large cell lymphoma (ALK + ALCL), a type of lymphoid malignancy carrying a mature T-cell immunophenotype. shRNA knockdown of SALL4 in ALK + ALCL cell lines resulted in apoptosis and cell-cycle arrest, and significantly decreased colony formation on soft agar. These changes correlated with the downregulation of several anti-apoptotic proteins and facilitators of cell-cycle progression. Based on the differential response to a SALL4 reporter construct, we were able to separate two distinct cell subsets in Karpas 299 (an ALK + ALCL cell line), namely SALL4high and SALL4low. Importantly, the biological effects induced by SALL4 knock-down in Karpas 299 were restricted to the purified SALL4high cells, and this finding further supports the concept that SALL4 is biologically important in ALK + ALCL. Lastly, the expression of SALL4 was not dependent on the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK)/STAT3 signaling axis, the key oncogenic driver in ALK + ALCL. To conclude, for the first time, our study has revealed the oncogenic contributions of an ESC protein in the pathogenesis of ALK + ALCL.

Detection of chromosomal aberrations in prostate cancer by fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) is a powerful tool for quantitative analysis of chromosomes and genes and can be applied in a variety of specimens, including cell cultures, isolated nuclei from fresh and fixed tissues, and histological tissue sections. For detection of numerical chromosome aberrations, we examined prostatic cancer samples at our department. In addition, we also observed primary and secondary aberrations taking part in the initiation and progression of tumours. FISH using chromosome-specific alpha-satellite DNA probes for chromosomes 7, 8, 9, 10, 17, X and Y was performed on 19 prostatic cancer and 19 benign prostatic hyperplasia (BPH) samples obtained from transurethral resection (TUR) and archival paraffin-embedded blocks. Numerical aberrations were observed in 41% of the tumours studied. A range of aberrant copy numbers of chromosome 9 (68%), 7 (63%), 8 (58%), 17 (37%), Y (32%) and 10 (26%) was observed. We did not observe significant aberrations in BPH samples. In prostate cancer patients, chromosomes 7 (47%), 8 (58%) and 9 (63%) were monosomic by FISH. Monosomy 8 and 9 were significant differences (p>0.05) between prostate cancer and BPH patients. FISH analysis could be observed an one of strongest methods of analysis in detecting numerical aberrations of individual chromosomes with application to paraffin-block samples, metaphase and, interphase nuclei. To our knowledge, this analysis is firstly studied in Turkish patients. Therefore, results of this analysis may be important for Turkish patients.

Application of comparative genomic hybridization technique for detection of chromosomal aberrations in benign cystic teratoma

Benign cystic teratoma, also known as dermoid cyst, is the most common of all ovarian neoplasms accounting for 10%–20% of all ovarian cysts. Most have a normal 46,XX karyotype. Less than 2% can undergo malignant transformation. By using the comparative genomic hybridization technique on nine benign ovarian cysts we were able to show two cases with chromosomal aberrations not seen before in dermoid cysts but known to be involved in malignant ovarian tumors. We speculate that these are the tumors carrying the potential for malignant transformation.

Detection of chromosomal aberrations in lung tissue and peripheral blood lymphocytes using interphase fluorescence in situ hybridization (FISH).

Detection of chromosomal aberrations in lung tissue and peripheral blood lymphocytes using interphase fluorescence in situ hybridization (FISH).

Factors influencing the prenatal detection of structural congenital heart diseases.

To assess the factors influencing the prenatal detection rate of structural congenital heart diseases (CHDs). A retrospective study was conducted at a major obstetric hospital in Australia between 1 January 1996 and 30 June 1999. The medical records of all fetuses and infants born with CHD, except those with isolated patent ductus arteriosus or secundum atrial septal defect, were reviewed. Only pregnancies that had prenatal ultrasound scan assessments for morphological surveys were included. The following factors that may influence the detection rate were assessed: complexity of the lesions; experience of the sonographers (performance in tertiary versus non-tertiary institutions); presence of other structural or chromosomal anomalies; and maternal body mass index (BMI). The incidence of structural CHD in this series, excluding cases referred from other hospitals, was 7.0 per 1000 (179/25 529). Of the 179 pregnancies with CHD, 151 had prenatal ultrasound scans and were included in the study. The overall detection rate for CHDs in this series was 40.4%. The detection rate for isolated septal defects was poor (13.7%). The detection rates were significantly higher for complex lesions (54%), for lesions with concomitant septal defects (66.7%), and for lesions with abnormal four-chamber views (62.9%). The detection rate was also higher if the scan was performed in the tertiary institution, and if there were other chromosomal or structural anomalies. Maternal BMI did not affect the detection rate in the current series. Stepwise logistic regression analysis showed that three independent variables affecting the detection rate were complexity of the cardiac lesion, experience of the operator, and the detection of chromosomal anomalies. A high detection rate for major CHDs can be achieved in a screening setting but there is still room for improvement in scanning skills in the four-chamber view and great-artery analysis in both tertiary and local centers.

CD40L stimulation enhances the ability of conventional metaphase cytogenetics to detect chromosome aberrations in B-cell chronic lymphocytic leukaemia cells.

Conventional metaphase cytogenetics underestimates the frequency of specific chromosome aberrations in B-cell chronic lymphocytic leukaemia (B-CLL) as a result of the very low proliferative activity of these cells in vitro. New molecular approaches, such as fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH), may circumvent this problem, at least in part, but these techniques are either strongly dependent on the knowledge of candidate regions or detect only unbalanced aberrations. In the present study, we analysed 27 B-CLL peripheral blood samples by metaphase cytogenetics after CD40 ligand (CD40L)-induced cell cycle stimulation. In comparison with the simultaneous use of B-cell mitogens such as 12-O-tetradecanoylphorbol-13-acetate (TPA), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), CD40L stimulation of B-CLL cells induced a threefold increase in metaphases amenable to analysis by conventional cytogenetics. The analysis of these metaphases confirmed all genetic abnormalities detected by FISH. Moreover, CD40L-enhanced cytogenetics revealed complex karyotypic aberrations in 11 out of 27 patients (41%). In one case, a balanced translocation t(11;16)(p15;p13.1), so far unreported in B-CLL, was detected. Taken together, the results of our study show the potential of CD40L-enhanced metaphase cytogenetics to detect more and new chromosome aberrations in B-CLL.

Chromosomal aberrations in peripheral lymphocytes of children as biomarkers of environmental exposure and life style

The original purpose of our study was to determine if the detection of chromosomal aberrations in peripheral lymphocytes of children might be used as a biomarker of environmental pollution and life style. We compared the results of cytogenetic analyses performed in children and adolescents in the periods 1984–1993 and 1994–1999, in a total of 3402 subjects. The frequency of aberrant cells (AB.C.) markedly decreased in the period 1994–1999 compared with the period 1984–1993. The decreases in AB.C. were significant in the age groups 7–15 and 16–19 years: 1.63% AB.C. versus 1.14% AB.C. and 2.02% AB.C. versus 1.08% AB.C., respectively ( P<0.01). No difference in the frequency of AB.C. was observed in newborns. Based on our experience, we believe that monitoring the spontaneous level of chromosomal aberrations in children over 5 year periods may be used to examine the general changes in environmental pollution in larger geographic areas.

Detection of Chromosomal Aberrations in Well-Differentiated Hepatocellular Carcinoma by Bright-Field In Situ Hybridization

Differentiation between well-differentiated hepatocellular carcinoma (HCC) and nonmalignant lesions with increased cellular proliferation may be difficult in needle biopsies. Based on recurrent chromosome aberrations known for HCC, we developed a nonfluorescent in situ hybridization technique that allows combination with morphological analysis in bright-field microscopy. Fourteen biopsies of HCC and 31 samples of regenerative nodules (n = 10), chronic hepatitis (n = 10), fibrosis or cirrhosis of unknown origin (n = 5), focal nodular hyperplasia (n = 2), primary biliary cirrhosis (n = 2), steatosis (n = 1), and adenomatous hyperplasia (n = 1) were analyzed with probes specific for the centromeric regions of chromosomes 1, 6, 7, and 8. After microwave pretreatment and in situ hybridization, signals were detected using a tyramine-based system and AEC as substrate. Evaluation of signals was done by conventional bright-field microscopy. Using this approach, aberrant counts were seen for at least one chromosome in 12/14 cases of HCC. In contrast, none of the nonmalignant lesions revealed aberrant counts for any of the chromosomes analyzed. In conclusion, this new combination of in situ hybridization and tyramine amplification allows fast and reliable evaluation of chromosome aberrations in a histomorphological context similar to paraffin immunohistochemistry. Registration of imbalances contributes to a reliable differentiation between malignant and nonmalignant lesions of the liver.