Concurrent translocation of BCL2 and MYC with a single immunoglobulin locus in high-grade B-cell lymphomas.

B-cell leukaemia or lymphoma with a combination of t(8;14)(q24;q32) of Burkitt leukaemia/lymphoma and t(14;18)(q32;q21) of follicular lymphoma may present clinically as de novo acute lymphoblastic leukaemia or transformation of follicular lymphoma to aggressive histology diffuse lymphoma. A number of cell lines have been reported with a complex t(8;14;18) with fusion of MYC, IGH and BCL2 on the same derivative 8 chromosome. The objective of this study was to determine the frequency and chromosomal features of this der(8)t(8;14;18) in a series of acute leukaemias and malignant lymphomas. A database of 1350 leukaemia and lymphoma karyotypes was searched for cases with structural alterations affecting both 8q24 and 18q21. A total of 55 cases were identified, of which eight revealed a complex der(8)t(8;14;18) with an MYC-IGH-BCL2 rearrangement resulting from translocation of BCL2 and MYC with a single disrupted IGH allele. Molecular cytogenetic investigation is essential to identify cases of high-grade leukaemia/lymphoma with concurrent translocations affecting the BCL2 and MYC loci.

Polyomavirus large T antigen-dependent DNA amplification.

DNA amplification is a readily measurable indicator for genome destabilization. Contrary to normal senescing cells, those of most immortal or transformed cell lines are karyotypically unstable and permissive for amplification. Permissivity for amplification can be generated by gene products of several DNA tumor viruses whereby their interaction with the tumorsuppressor protein p53 is important. p53 is the major protein involved in check point control of DNA damage. Polyomavirus large T antigen is also involved in immortalization and transformation of cells but it does not interact with p53. We, therefore, examined whether this protein could still make the non-permissive cell line REF52 permissive for gene amplification. To this end REF52 cell lines were constructed which conditionally expressed the wild type polyomavirus large T antigen or a mutant form unable to bind the retinoblastoma protein. Using the inhibitor of de novo pyrimidine biosynthesis, phosphonoacetyl-L-aspartate (PALA), as selective agent we found that PALA resistant cells arise with a frequency of about 5 x 10(-5) and that the interaction of polyomavirus large T protein with the retinoblastoma protein or another related pocket protein is important for this to occur. PALA resistant cells have an increased number of chromosomes and dicentric chromosomes which are considered as starting point for DNA structures characteristic for amplified DNA. Such structures were indeed found with the help of fluorescence in situ hybridization. PALA resistant cells appear normal with respect to p53. Our data indicate that PALA induces a G1 block which can be partially overcome by polyomavirus large T protein by its interaction with E2F-pocket protein complexes providing further evidence that these complexes are downstream targets of p53.

Mutational hot spots within the carboxy terminal region of the LMP1 oncogene of Epstein-Barr virus are frequent in lymphoproliferative disorders.

We have recently identified in Epstein-Barr virus (EBV) positive Hodgkin's disease (HD) a variant of the latent membrane protein 1 (LMP1) oncogene characterized by four point mutations and a 30 base pair deletion. These findings led us to test whether such mutants were also present in other lymphoproliferative disorders (LPD). We analysed 98 EBV DNA positive cases (67 LPD, 15 benign conditions, 16 lymphoblastoid cell lines) by PCR for deletions within the LMP1 gene. DNA sequencing of the region coding for the carboxy terminal protein domain was performed on 24 cases. In 13 cases the same combination of 4 point mutations at positions 168,320, 168,308, 168,295 and 168,225 was identified. Of these cases, 12 had an additional point mutation at position 168,357 and eight at position 168,355, and nine had a 30 base pair deletion including nucleotides 168,285 to 168,256. These deletion mutants were identified in HD, angioimmunoblastic lymphadenopathy, B-immunoblastic lymphoma, peripheral T-cell lymphoma, and two lymphoblastoid cell lines. Our findings reveal a high frequency of non-random point mutations at preferential sites within the 3' (carboxy terminal) region of the LMP1 oncogene. The association of these mutational hot spots with LPD suggests that they are involved in EBV related lymphomagenesis and that they define a clinically relevant EBV strain.

Establishment and comprehensive analysis of a new human transformed follicular lymphoma B cell line, Tat-1.

A spontaneously EBV transformed follicular lymphoma (FL) cell line, Tat-1, was established from the lymph node biopsy specimen of a patient with B cell FL, grade 1 in transformation to high grade disease. Tat-1 cells expressed lymphoid markers and developed tumor masses in immunodeficient mice. Bcl-2, Bcl-X(L), Bax and p53 protein expression was revealed by Western blotting. Flow cytometric analysis confirmed P-gp expression. Cytogenetically, the Tat-1 cell line showed identical chromosomal alterations to that of the initial biopsy specimen, among which the most notable were the t(14;18) typical of FL and additional abnormalities involving chromosomes 1, 8 and 13. Multicolor FISH analysis delineated all abnormalities, including a t(1p;8q), a der(8)(8q24::14q32::18q21) and a der(13)(13q32::8q24::14q32::18q21). Further FISH investigations using a locus-specific probe cocktail containing c-myc, IgH and bcl-2 revealed fusion of these three loci on the derivatives 8 and 13, in addition to the derivative 14 IgH/bcl-2 fusion and an extra copy of c-myc on derivative chromosome 1. These results demonstrate an additional example of the deregulation of bcl-2 and c-myc expression through recombination with a single IgH enhancer region. The unusual molecular features of the Tat-1 cell line render it a unique tool for studies focused on cytogenetic alterations, expression of multidrug resistance phenotype and expression of anti-apoptotic proteins in FL.

Comparative genomic hybridization: a new approach to screening for intrauterine complete or mosaic aneuploidy.

In the practice of clinical genetics chromosomal aneuploidy in both mosaic and nonmosaic forms has long been recognized as a cause of abnormal prenatal and postnatal development. Traditionally, cytogenetic analysis of cultured lymphocytes has been used as a standard test for detection of constitutional aneuploidies. As lymphocytes represent only one lineage, chromosomal mosaicism expressed in other tissues often remains undetected. The purpose of this study was to assess the utilization of molecular cytogenetic analysis for detection of chromosomal aneuploidy in placental tissues. Using placentas from 100 pregnancies with viable nonmalformed livebirths, both trophoblast and chorionic stroma were analyzed using comparative genomic hybridization (CGH). In all cases with an indication of chromosomal imbalance by CGH, fluorescence in situ hybridization (FISH) analysis was performed to confirm the presence of aneuploidy. To differentiate between constitutional aneuploidy and confined placental mosaicism (CPM), amniotic membrane was analyzed by CGH and FISH techniques. Our results demonstrated five placentas with CPM for chromosomes 2, 4, 12, 13, and 18, respectively, and two constitutional nonmosaic aneuploidies (47,XXX and 47,XXY). Molecular cytogenetic studies of human placental tissues enables easy analysis of both embryonic (amnion) and extraembryonic (chorion) cell lineages. Detection at birth of chromosomal defects affecting intrauterine placental and fetal development is important because these chromosomal defects may continue to have an influence on postnatal development.

Detection of a novel t(6;15)(q21;q21) in a pediatric Wilms tumor.

We report a novel cytogenetic finding in a favorable histology Wilms tumor occurring in a 4-month-old boy. Karyotypic analysis demonstrated a t(6;15)(q21;q21) in all tumor cells examined. This was confirmed using fluorescence in situ hybridization analysis. Molecular analysis of this rearrangement may provide clues to understanding the pathobiology of Wilms tumor.

Comparative genomic hybridization: a new tool for reproductive pathology.

OBJECTIVE: To demonstrate the effectiveness of comparative genomic hybridization (CGH) for analysis of reproductive pathology specimens in clinical cytogenetics laboratories. DESIGN: A total of 856 CGH analyses were performed on various placental and fetal tissues derived from 368 specimens of spontaneous abortions and on placentas from 219 pregnancies with live-born infants. The live-born infants were clinically evaluated as normally developed, with either a normal birth weight or with intrauterine growth restriction; some live-born infants had an abnormal prenatal triple screen with normal cytogenetic results on amniotic fluid cell cultures. RESULTS: Comparative genomic hybridization analysis was successfully performed on 856 samples from spontaneously aborted specimens and term placentas. Failure of analysis occurred in 1.6% of samples and was due to an insufficient amount of tissue for DNA extraction. Comparative genomic hybridization identified aneuploidy in 53% of spontaneous abortion samples and 3.1% of term placentas. CONCLUSIONS: Comparative genomic hybridization analysis is a useful clinical tool for detection of aneuploidy in placental and fetal tissues. It provides a genome-wide screen while eliminating tissue culture failures, culture artifacts, and maternal cell contamination. We present practical guidelines for interpreting CGH profiles derived from human reproductive specimens.

High-resolution FISH of the entire integrated Epstein-Barr virus genome on extended human DNA.

Here we report a high-resolution fluorescence in situ hybridization (FISH) analysis of the integrated Epstein-Barr virus (EBV) genome in chromosomes, decondensed interphase nuclear chromatin, and linearly extended chromatin fibers. We analyzed the EBV DNA integrated into the human genome in the well-characterized Burkitt's lymphoma cell line Namalwa, which contains two complete EBV genomes. The integration occurs via the terminal repeats of the virus and was always detectable at chromosome band 1p35. Using the biotinylated BamHIW fragment of the viral DNA, we observed distinct pairs of signals or small nuclear RNA "tracks" within interphase nuclei. FISH to stretched DNA fibers has a higher resolving power and; therefore, enables analysis of the structural organization of DNA. Application of this methodology to linearly extended chromatin of Namalwa cells using different EBV fragments allowed us to visualize the ordered arrangement of the integrated virus. Based on the predicted span of 0.34 nm per base pair for relaxed DNA, length measurements of 30 images showed a good correlation between the mean physical length of hybridized EBV DNA of 52.8 microns (158 kb) without the terminal repeats, and the EBV genomic length of 172 kb, including the terminal repeats. This DNA mapping procedure represents a useful tool for studying the structural organization of integrated viral genomes, and its application will have implications for the understanding of integration processes.

Screening of human placentas for chromosomal mosaicism using comparative genomic hybridization.

Detection of confined placental mosaicism (CPM) in term placental tissues is usually accomplished by conventional cytogenetic analysis of cultured chorionic stroma and direct preparations from trophoblast or, more recently, by fluorescence in situ hybridization (FISH) on interphase nuclei. In this study, we describe the use of comparative genomic hybridization (CGH) for detection of chromosomal aneuploidy in term placentas and evaluate the sensitivity of this novel approach for CPM diagnosis in multiple placental samples acquired from five pregnancies prenatally diagnosed with CPM7 and CPM16. Each sample of placental villi was separated enzymatically into trophoblast and chorionic stroma, and the level of aneuploidy (three signals/nuclei) in each tissue was determined by FISH analysis, using centromeric DNA probes specific for chromosome 7 (D7Z1/Z2) or 16 (D16Z2). Aneuploidy levels ranged from 5.2-96.1% in the 11 tissues with CPM7 and 9.8-93% in the 29 tissues with CPM16. Subsequently, CGH analysis of DNA from the trophoblast and chorionic stroma of the same tissue sites detected the trisomic clone in all placental tissues with aneuploidy (16%, as determined by FISH analysis). Our results demonstrate the sensitivity of CGH analysis for detection of chromosomal aneuploidy mosaicism and support our contention that the CGH technique is the most effective cytogenetic method for screening term placentas for the presence of CPM.

Non-random integration of Epstein-Barr virus in lymphoblastoid cell lines.

In order to examine the role of Epstein-Barr virus (EBV) in the immortalization of human B lymphocytes and in the pathogenesis of lymphoid malignancies, we investigated whether the EBV integration into the human genome is randomly distributed or whether the virus integrates preferentially at certain sites. Twelve in vitro immortalized human lymphoblastoid cell lines (LCLs), two in vivo infected LCLs, and one Burkitt's lymphoma cell line (EB2) were examined by non-radioactive in situ hybridization (ISH) with a biotinylated EBV probe. Recurrent hybridization sites were detected in all 15 cell lines. The chromosomes frequently carrying the EBV genome were chromosomes 1, 2, 4, and 5. In more than 70 chromosomal bands, a greater number of integration sites than expected was found (p < 0.05). Approximately half of these bands were involved in the majority of the cell lines (for example, 1p31, 1q43, 2p22, 3q28, 4q13, 5p14, 5q12, and 11p15) whereby band 5p14 was involved in all LCLs analyzed. Virtually no viral integrations were found on the sex chromosomes (X, Y). The majority of the EBV integrations was found in G-band-positive material (p < 0.0001). Thus, our findings clearly show that EBV integrates into the human genome in a non-random manner.