NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype.

Cervical cancer is associated with human papilloma virus infection. However, this infection is insufficient to induce transformation and progression. Loss of heterozygosity analyses suggest the presence of a tumor suppressor gene (TSG) on chromosome 6p21.3-p25. Here we report the cloning NOL7, its mapping to chromosome band 6p23, and localization of the protein to the nucleolus. Fluorescence in situ hybridization analysis demonstrated an allelic loss of an NOL7 in cultured tumor cells and human tumor samples. Transfection of NOL7 into cervical carcinoma cells inhibited their growth in mouse xenografts, confirming its in vivo tumor suppressor activity. The induction of tumor dormancy correlated with an angiogenic switch caused by a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These data suggest that NOL7 may function as a TSG in part by modulating the expression of the angiogenic phenotype.

AML1 gene over-expression in childhood acute lymphoblastic leukemia.

The present study was conducted on a series of 41 Egyptian children with newly diagnosed acute lymphoblastic leukemia (ALL) to investigate TEL and AML1 abnormalities. The TEL-AML1 fusion was observed in six patients both by RT-PCR and FISH analyses, with a frequency of 22.2% among the B-lineage group, whereas TEL deletion was seen by FISH analysis in seven patients (17.1%). By FISH analysis, nine patients (22%) showed evidence of extra AML1 copies. In five of these patients the extra copies were due to non-constitutional trisomy 21, whereas in the remaining four cases they were due to tandem AML1 copies on der(21), as evidenced by metaphase FISH. Unexpectedly however, enhanced AML1 expression levels were seen by real-time quantitative RT-PCR in 18 out of the 41 ALL patients (43.9%). This high level of AML1 expression could be an important factor contributing to the pathogenesis and progression of childhood ALL. One key mechanism for over-expression seems to be the extra copies of AML1, but other mechanisms may involve an alteration of the activity of the AML1 promoter. Here, we also report two novel findings. The first is an intragenic deletion of TEL exon 7 in a case of T cell ALL. This deletion creates a frame-shift and results in a truncated protein lacking the C-terminus that includes the ETS domain. This shorter TEL is presumably unable to bind DNA. The second finding is a rearrangement of AML1 in a case of T cell ALL due to t(4;21)(q31;q22). This is the first reported chromosomal translocation where AML1is rearranged in childhood T cell ALL.

Establishment of the B cell precursor acute lymphoblastic leukemia cell line MUTZ-5 carrying a (12:13) translocation.

Continuous leukemia-lymphoma cell lines are important research tools, in particular as starting material for the cloning of recurrent translocations. In 1998, we established the continuous leukemia cell line MUTZ-5 and its two simultaneous sister cell lines MUTZ-6 and MUTZ-7. The primary specimen was obtained from the peripheral blood of a 26-year-old man with B cell precursor acute lymphoblastic leukemia at relapse carrying a t(12;13). The immunoprofile of MUTZ-5 corresponds to that of a precursor B cell. The immunoglobulin heavy chain gene was found to be rearranged. Despite receptor expression, none of the cytokines examined enhanced proliferation; several cytokines had significant inhibitory effects. Giemsa-banding cytogenetics showed the following karyotype which was identical in all three sister cell lines: 45<2n>X, -Y, t(12;13)(p12;q13-14). The karyotype and DNA fingerprinting confirmed the malignant nature and the authenticity of the cell line, excluding cross-contamination with other cells. MUTZ-5 represents a new unique leukemia B cell line; its scientific significance lies in the t(12;13).

Genomic organization and refined mapping of the human nuclear corepressor 2 (NCOR2)/ silencing mediator of retinoid and thyroid hormone receptor (SMRT) gene on chromosome 12q24.3.

The human nuclear co-repressor 2 (N-CoR2) gene (NCOR2, previously called silencing mediator for retinoid and thyroid hormone receptor SMRT) is recruited to nuclear and non-nuclear receptors in a large repressing complex containing also N-CoR1, mSin3 and HDACs. This large complex represses transcription in absence of ligand. Herein we report the high- resolution and refined mapping of NCOR2 at the boundary of sub-bands 12q24.23 and 12q24.31, and its intron/exon structure. The gene contains 45 exons. This information should allow further study of potential NCOR2 genomic alteration in some subsets of malignancies.

6p abnormalities and TNF-alpha over-expression in retinoblastoma cell line.

Retinoblastoma is the most common primary intra-ocular tumor in childhood. It has been established that recurrent cytogenetic abnormalities are the hallmark of mostly all malignant tumors. Recurrent atypical cytogenetic abnormalities of the short arm of chromosome 6 were reported in several cases of retinoblastoma, mainly in association with reciprocal translocations. In the present study, alterations of chromosome 6 associated with retinoblastoma were studies in the Y-79 and the WERI-Rb-1 cell lines established from highly malignant retinoblastomas. Fluorescence in situ hybridization (FISH) technique was used to identify the chromosomal breakpoint on 6p in this cell line. To perform this detection, yeast artificial chromosome (YAC) clones from p21 to p22 bands were used and the breakpoint was localized on 6p21.3. Previous studies had suggested that activation of some genes on 6p could be dependent on a translocation mechanism. Expression of a candidate gene localized near the chromosomal breakpoint was measured by immunocytochemistry and flow cytometry techniques. An enhancement of the tumor necrosis factor-alpha (TNF-alpha) protein expression in Y79 cells was detected by this approach. The relationship between TNF-alpha over-expression and the malignancy of retinoblastoma is discussed.

Fluorescence in situ hybridization as a tool in molecular diagnostics of melanoma.

Structural and numerical chromosomal abnormalities have been observed in all types of malignancies. These events are associated with neoplastic pathogenesis and progression (1). For a long time, conventional cytogenetics analysis was the prevalent approach in assessing chromosomal rearrangements. Many data were collected initially from hematologic malignancies because of ease of analysis, despite the fact that they represent only 10% of all malignancies (2). Cytogenetic data on solid tumors were hampered by the technical difficulties in obtaining dividing malignant cells. During the last two decades, these problems were solved and data have been quickly acquired. Karyotyping of malignant melanoma cell lines reveals more than 95% of the cases presenting aneuploidy (numerical chromosome abnormalities: near triploidy) (3), and all the cell lines present structural abnormalities (balanced and unbalanced translocations, inversions and deletions) (see, e.g., German Collection of Microorganisms and Cell Cultures [Dr. H. Drexler] at http://www.dsmz.de (4,5). Cytogenetic analysis of malignant melanoma cells allowed the identification of nonrandom karyotypic changes involving chromosomes 1, 6, and 7 and, to some extent, chromosomes 9 and 10.

Frequent deletions at 11q23 and 13q14 in B cell prolymphocytic leukemia (B-PLL).

Deletions of the long arm of chromosomes 11 and 13 are the most frequent structural chromosome aberrations in various types of lymphoproliferative disorders. However, these regions have not been studied so far in B cell prolymphocytic leukemia (B-PLL). We have investigated the incidence of 13q deletions in 18 B-PLL cases by fluorescence in situ hybridization (FISH), using molecular probes for the RB1 and D13S25 loci. Chromosome 11q deletions were evaluated by FISH using the yeast artificial chromosome (YAC) clone 755b11 from the chromosome 11q22.3-q23.1 region, which has been previously shown to be deleted in 20% of cases of chronic lymphocytic leukemia. Chromosome 11q23 deletions were found in 7/18 (39%) cases of B-PLL. Monoallelic loss of RB1, D13S25 and BRCA2 was present in 10/18 (55%), 6/18 (33%) and 3/18 (16%) of the cases, respectively. All the cases with D13S25 and BRCA2 deletion showed RB1 loss. Deletions of 13q14 and 11q23 are frequent chromosome aberrations in B-PLL and, in contrast to CLL, there is a preferential loss of RB1 with respect to the D13S25 locus suggesting that allelic loss of the RB1 gene may play a role in the pathogenesis of B-PLL.

FISH analysis for BCL-1 rearrangements and trisomy 12 helps the diagnosis of atypical B cell leukaemias.

We have investigated the diagnostic value of fluorescence in situ hybridisation (FISH) to detect t(11;14) and trisomy 12 in 53 cases with a B cell leukaemia difficult to classify on clinical and laboratory grounds. These cases were initially diagnosed by morphology and immunophenotype and in 33 of them, on tissue histology, as follows: chronic lymphocytic leukaemia (CLL), 20, 18 of them with atypical features; B cell prolymphocytic leukaemia (B-PLL), two; mantle-cell lymphoma (MCL), 15; splenic lymphoma with villous lymphocytes (SLVL), five; lymphoplasmacytic lymphoma, six; follicular lymphoma, one and, four cases remained unclassifiable. FISH demonstrated BCL-1 rearrangement in the circulating cells from 15 cases classified as: MCL (10), atypical CLL (three) and B-PLL (two). A definitive diagnosis of MCL was made on review of the spleen histology in one out of the three atypical CLL with BCL-1 rearrangement. Trisomy 12 was detected in eight cases which included four atypical CLL, one typical CLL, two MCL and one unspecified B cell lymphoma by histology and morphology. One of the MCL had both trisomy 12 and BCL-1 rearrangement and the other was CD5+, CD23+ and had a CLL score of 3, suggesting the latter diagnosis. Our findings demonstrate that FISH analysis is useful to clarify the nature of the disease in patients presenting with a B cell leukaemia in which the diagnosis is difficult by conventional methods. FISH established with certainty the diagnosis of MCL by showing BCL-1 rearrangement in over two-thirds of cases in which this was suspected, including blastoid forms, and confirmed the diagnosis of most cases of atypical CLL.

Myeloid- and lymphoid-specific breakpoint cluster regions in chromosome band 13q14 in acute leukemia.

Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (BCP-, TCP-ALL), in differentiated and undifferentiated acute myeloblastic leukemia (AML), and in chronic myeloid leukemia (CML) at progression to blast crisis. The nature of these translocations and their pathologic consequences remain unknown. To begin to define the gene(s) involved on chromosome 13, we have performed fluorescence in situ hybridization (FISH) using a panel of YACs from the region, on a series of 10 cases of acute leukemia with t(12;13)(p12;q14) and 1 case each with "variant" translocations including t(12;13)(q21;q14), t(10;13)(q24;q14) and t(9;13)(p21;q14). In 8/13 cases/cell lines, the 13q14 break fell within a single 1.4 Mb CEPH MegaYAC. This YAC fell immediately telomeric of the forkhead (FKHR) gene, which is disrupted in the t(2;13)(q35;q14) seen in pediatric alveolar rhabdomyosarcoma. Seven of the 8 cases with breaks in this YAC were AML. In 4/13 cases, the 13q14 break fell within a 1.7-Mb YAC located about 3 Mb telomeric of the retinoblastoma (RB1) gene: all 4 cases were ALL. One case of myelodysplastic syndrome exhibited a break within 13q12, adjacent to the BRCA2 gene. These data indicate the presence of myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia.

B cell prolymphocytic leukaemia (B-PLL) with complex karyotype and concurrent abnormalities of the p53 and c-MYC gene.

We report the cytogenetic, molecular and biological characterization of a case of B-PLL with a complex karyotype and concurrent abnormalities on the p53 and c-MYC genes. Conventional cytogenetics suggested that both 17q arms were translocated to chromosomes 1q and 14p, respectively, whereas both 17p arms were not identified. In addition, a Burkitt's-like variant translocation t(2;8) was found. Study of loss of heterozygosity at 17p13 and p53 direct sequencing demonstrated the presence of only one copy of the p53 gene. A 27 bp deletion in exon 8 that resulted in the expression of a p53 protein lacking nine amino acids from the DNA binding region was also found. To confirm the presence of one copy of the p53 gene and localize it, fluorescent in situ hybridization (FISH) studies using a p53 gene probe was performed. Only one signal of p53 was visualized. Moreover, the DAPI profile of the chromosome containing the hybridization spot for the p53 probe did correspond to the cytogenetic marker identified as der(14)t(14;17). Whole chromosome 14 paint, centromere-specific for chromosome 17 and p53 gene probes were cohybridized to the preparations. This demonstrated that the der(14) contained the 17 centromere and distally the p53 gene suggesting that the der(14) contained the short arm of chromosome 17 with the breakpoint occurring in the long arm. FISH studies confirmed the involvement of c-MYC and KAPPA in the t(2;8) translocation. To our knowledge, this is the first case of B-PLL with inactivation of the p53 gene by mutation together with a Burkitt's-like t(2;8) translocation involving the c-MYC gene. The cooperation of these genes may have conferred a growth advantage which was critical in the development of this aggressive form of B-PLL.

The incidence of trisomy 3 in splenic lymphoma with villous lymphocytes: a study by FISH.

Splenic lymphoma with villous lymphocytes (SLVL) is a low-grade B-cell lymphoproliferative disorder characterized by splenomegaly and circulating villous lymphocytes. The relationship between SLVL and splenic marginal zone lymphoma (SMZL), a disorder with identical splenic histology to SLVL, is not clear. Previous studies have failed to show a consistent karyotypic abnormality in SLVL whereas trisomy 3 has been reported in patients with SMZL. The presence of trisomy 3 in SMZL and its absence in SLVL has been viewed as evidence that these are different diseases. However, it is possible that the frequency of trisomy 3 in SLVL has been underestimated because previous studies have relied on conventional cytogenetics. We have therefore used interphase fluorescence in situ hybridization (FISH) to re-assess the frequency of trisomy 3 in SLVL. We studied 70 patients, who were stratified into four groups according to the percentage of circulating villous lymphocytes. Trisomy 3 was found overall in 17% of patients. In particular, trisomy 3 was detected in 13% of cases with >50% of villous lymphocytes and which were considered typical of SLVL. In conclusion, we have demonstrated that some patients with SLVL have circulating cells with trisomy 3, which does not support the view that SLVL and SMZL are different diseases on the basis of the incidence of trisomy 3.

Cytogenetic abnormalities in multiple myeloma.

There is an increasing understanding that chromosomal abnormalities play a major role in the pathogenesis of multiple myeloma. Furthermore, they seem to predict the clinical outcome of patients according to the specific abnormalities detected. It is likely that in the future, knowledge of the cytogenetic composition will be an integral part of the evaluation of myeloma patients.

Molecular cloning of translocation t(1;14)(q21;q32) defines a novel gene (BCL9) at chromosome 1q21.

Abnormalities of chromosome 1q21 are common in B-cell malignancies and have been associated with a poor response to therapy. The nature of the involved gene(s) on chromosome 1q21 remains unknown. A cell line (CEMO-1) has recently been established from a patient with precursor-B-cell acute lymphoblastic leukemia (ALL), which exhibited a t(1;14)(q21;q32). To identify the gene involved in this translocation, we have cloned both rearranged IGHJ alleles using long-distance inverse polymerase chain reaction (LDI-PCR). Two IGHJ fragments were amplified from CEMO-1 DNA and sequenced. One allele showed novel sequences upstream of JH5 with no homology to either IGH or any other sequences on the databases. Using a single-copy Xho I fragment immediately 5' of JH5, PAC clones were isolated and mapped to chromosome 1q21 on normal metaphases by fluorescence in situ hybridization (FISH), confirming that this allele represented the t(1;14)(q21;q32) breakpoint. Sequence analysis of the 1q21 Xho I fragment showed identity with an expressed sequence tag (EST), and this probe was therefore used to probe Northern blots. Two transcripts of 6.3 kb and 4.2 kb expressed at low level in mRNA from all tissues were detected: a third transcript of 1.6 kb was expressed only in thymus, spleen, and small intestine. Full-length BCL9 cDNA clones were obtained from a normal human fetal brain cDNA library supplemented by 5' and 3' RACE. Sequence analysis predicted a protein of 1394 amino acids containing 18% proline, 11% glycine, 11% serine, and 6% methionine, but no recognizable protein motifs or significant homologies to any other known proteins. The CEMO-1 1q21 breakpoint fell within the 3' UTR of the BCL9 gene. Low-level expression of BCL9 was detected in Epstein-Barr virus-transformed normal B cells by Northern blot; in contrast, abundant BCL9 expression was observed in CEMO-1, indicating that deregulated expression of this gene was one pathological consequence of the translocation. Screening of a panel of 39 B-cell malignancies with 1q abnormalities by Southern blot showed one additional case with a breakpoint in the 3' UTR of BCL9, indicating that this was a recurrent breakpoint. FISH analysis using an 850-kb YAC spanning BCL9 identified a further case with t(1;22)(q21;q11) causing juxtaposition of BCL9 to the IGlambda locus. Other breakpoints were heterogeneous, falling both centromeric (10 cases) and telomeric (10 cases) of the BCL9 gene. These data suggest that BCL9 may be the target of translocation in some B-cell malignancies with abnormalities of 1q21 and that deregulated BCL9 expression may be important in their pathogenesis.

ATM is usually rearranged in T-cell prolymphocytic leukaemia.

T-prolymphocytic leukaemia (T-PLL) is a rare, sporadic leukaemia similar to a mature T-cell leukaemia seen in some patients with Ataxia Telangiectasia (A-T), a recessive multisystem disorder caused by mutations of the ATM gene at chromosome 11q23. ATM sequence mutations have been reported in 46% of T-PLL cases, but some cases also have karyotypic abnormalities at 11q, including 11q23. This led us to investigate the structure of the ATM locus in a panel of eight cases, two of which had 11q23 abnormalities. As expected, nucleotide changes were detected in some samples. Two remission samples were wild type. To test for structural lesions, DNA fibres were hybridized with a contig of four labelled cosmids spanning the ATM locus. In all samples there were structural lesions and in four samples both alleles were affected. This provides strong evidence for our suggestion that ATM acts as a tumour suppressor during T-PLL tumorigenesis. Some additional role for ATM during T-PLL tumorigenesis is possible since nucleotide changes were present in addition to structural lesions disrupting both alleles. The mechanism of inactivation appeared to be unusual because multiple structural lesions on one allele were often observed.

Establishing a KSHV+ cell line (BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice.

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) sequences are present in primary effusion lymphomas (PEL). KSHV+ cell lines have been established from such lymphomas. Here we report the first description of the establishment of a KSHV+, EBV- cell line (BCP-1) from the peripheral blood of a patient with PEL. Using this cell line and a KSHV+, EBV+ PEL cell line (HBL-6) previously established from ascitic fluid, we investigated whether in nonobese diabetic/severe combined immunodeficiency disease (Nod/SCID) mice tumors representing PEL can be established. When injected intravenously (IV) into Nod/SCID mice, BCP-1 and HBL-6 infiltrated organs, with only occasional macroscopic tumor formation. Intraperitoneal injections (ip) led to the development of ascites and diffuse infiltration of organs, without obviously solid lymphoma formation, resembling the diffuse nature of human PEL. To investigate a possible mechanism for the peculiar phenotype of PEL, we examine the presence of adhesion molecules and homing markers on PEL cells before and after growing in mice. Both BCP-1 and HBL-6 cells lack expression of important cytoadhesion molecules including CD11a and CD18 (LFA1 alpha and beta chains), CD29, CD31, CD44, CD54 (ICAM-1), and CD62L and E (L and E selectins).

The ataxia telangiectasia gene in familial and sporadic cancer.

The ataxia telangiectasia (A-T) gene, ATM, predisposes affected homozygotes to a wide range of malignancies. It has been suggested that this is a consequence of the genomic instability associated with the syndrome. The elevated risk of malignancy is not, however, observed among A-T heterozygotes (except, apparently, regarding breast cancer). In this report we describe results from the study of the rare sporadic disease, T cell prolymphocytic leukaemia (T-PLL). In all individuals tested, we observed that at least one ATM allele was disrupted by rearrangement, that in many cases both alleles were disrupted and that there were additional mutations, predominantly missense, that clustered toward the 3' end of the gene corresponding to the protein's phosphatidylinositol 3-kinase (PIK)-related domain. We conclude that the ATM gene can act as a tumour suppressor in the development of sporadic T-PLL. Our finding of a surfeit of mutations within ATM may reflect the involvement of the gene at more than one step in tumorigenesis. In particular, we suggest that the clustering of missense mutations may pertain to the late-onset character of both sporadic and A-T-related T-PLL, since the closest homologue of Atm protein is the yeast TEL1 protein that maintains telomere length. ATM inactivation may not be the initiating event in T-PLL tumorigenesis: prior mutation of another gene--perhaps TCL1 activation--may be obligate. This would explain the recessive character of T-PLL risk in A-T.

The BCL7 gene family: deletion of BCL7B in Williams syndrome.

The BCL7A gene, which maps to human chromosome 12q24.13, was cloned through its direct involvement with MYC and IGH in a three-way translocation in a Burkitt lymphoma cell line. Here, we describe the identification of two related human genes, BCL7B and BCL7C, which share 90% identity to the amino-terminal 51 amino acids of human BCL7A, as well as 41% identity in the same region to Drosophila melanogaster, Caenorhabditis elegans, and Brugia malayi EST sequences. This degree of relatedness in the amino-terminal domain suggests we have defined a new gene family of unknown function. There was little sequence conservation between the family members outside this conserved domain and no identified protein motifs could be deduced. Human BCL7B and BCL7C mapped to chromosome 7q11.23, and 16p11, respectively. No chromosomal rearrangements affecting BCL7B or BCL7C were detected in lymphoid malignancies. BCL7B did, however, map within the region of 7q11.23 which is commonly deleted in the congenital disorder, Williams syndrome.

Rapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction.

Clonal rearrangements of the Ig heavy chain (IGH) locus consisting of either intrachromosomal (VDJ) rearrangements or interchromosomal translocations are a consistent feature of all B-cell malignancies and may be used both diagnostically and to monitor response to therapy. Many of these rearrangements are targeted to the IGHJ segments, but only some can be amplified with regular polymerase chain reaction (PCR) techniques. To permit PCR amplification of potentially all IGHJ rearrangements, we have devised a method incorporating self-ligation of restriction endonuclease-digested DNA fragments with long-distance PCR (long-distance, inverse PCR [LDI-PCR]). We show here, using only 4 nested oligonucleotide primers, the successful amplification and DNA sequencing of all IGHJ rearrangements up to 5.4 kb in length from a panel of 13 cases and cell lines of various types of B-cell malignancy. In all cases, both VDJ and DJ IGH rearrangements and translocation breakpoints were amplified. Six cases exhibited t(14;18)(q32;q21). All translocation breakpoints were cloned and sequenced. Three cases exhibited a rearrangement to the BCL2 major breakpoint region (MBR). However, 2 other cases exhibited rearrangements between the MBR and the minor cluster region (mcr). These 2 cases broke within 44 bp of each other, confirming the presence of an additional 3' BCL2 breakpoint cluster region. The final case fell immediately 3' of the 3' UTR of the BCL2 gene adjacent to an Alu repeat. No other BCL2 breakpoints within this region have been reported. Four cases exhibited t(11;14)(q13;q32). All 3 cases with translocations targeted to the IGHJ segments were successfully amplified and sequenced, including 1 case in which the BCL1 translocation could not be detected by DNA blot using the currently available probes. All three translocation breakpointsfell outside the BCL1 major translocation cluster between 20 and 40 kb telomeric and showed no clustering. Two of the three fell within or adjacent to Alu repeat regions. LDI-PCR is a simple and robust technique that allows PCR amplification of nearly all IGHJ rearrangements.

Cytogenetic analysis of meiotic segregation in sperm from two males heterozygous for reciprocal translocations using PRINS and humster techniques.

The meiotic segregation patterns of 2 reciprocal translocations t(7;9)(q33;p21) and t(7;18)(q35;q11) were analyzed in sperm of 2 heterozygote carriers. Both sperm karyotyping and in situ PRINS labeling of sperm nuclei were performed on a sperm sample from each subject. Using the humster technique, 54 and 72 sperm chromosome complements were successfully analyzed for the t(7;9) and the t(7;18) respectively. The frequencies of alternate, adjacent 1, adjacent 2 and 3:1 segregations were 44.44%, 37.04%, 12.96% and 5.56% for the t(7;9) and 33.33%, 43.05%, 19.45% and 4.17% for the t(7;18). The PRINS procedure allowed the rapid screening of large samples of spermatozoa. However, alternate and adjacent 1 segregants were not discriminated because of the generation of centromeric signals. The segregation pattern was determined on 10,658 spermatozoa for the t(7;9) and 10,462 for the t(7;18). The distributions of segregants were similar to those obtained by sperm karyotyping. These data were pooled with results from 37 reciprocal translocations previously studied by sperm karyotyping and 6 recently investigated by FISH. The analysis of these compiled data demonstrates the particularity of the production of imbalances in male gametes; independent of the predisposition for a type of imbalance at term, there is a preferential production of adjacent 1 imbalance in sperm.

The impact of molecular cytogenetics on chronic lymphoid leukaemia.

Chronic lymphoid leukaemias are clonal expansions of B and T cells with mature membrane phenotype. Cytogenetic study of these cases usually requires mitogenic stimulation and can often be hindered by a lack of response of the tumour cells to mitogen, poor quality metaphases, complex markers and proliferation of normal cells. In situ hybridisation with fluorescence-labelled chromosome-specific centromeric DNA probe, single or low copy sequences and whole chromosome paints which hybridise to complementary sequences allow the detection of numerical and structural abnormalities on metaphase and interphase cells with much greater efficiency. Comparative genomic hybridisation uses whole genomic tumour DNA as probe which is hybridised to normal metaphases. It is particularly useful for detecting chromosomal changes without being dependent on the dividing tumour cells. The application of these techniques to the investigation of chronic lymphoid leukaemias is reviewed with emphasis on the work done in our laboratory on trisomy 12 and the tumour suppressor region 13q14 in chronic lymphocytic leukaemia, translocation t(11;14) (q13;q32) in mantle cell lymphoma and other chronic B cell leukaemias, inv(14) (q11q32), i(8q) and complex markers in T prolymphocytic leukaemia.