Molecular analysis of the human chromosome 5q13.3 region in patients with hairy cell leukemia and identification of tumor suppressor gene candidates.

The pathogenesis of hairy cell leukemia (HCL) remains largely unknown since no specific genetic lesion has been identified in this disease. Previous cytogenetic analysis from our group has shown that chromosome abnormalities involving the 5q13 band are common in HCL, occurring in approximately 1/3 of patients. The data suggest that the 5q13.3 band is likely to harbor a gene involved in the transformational events of this disease. We have recently found two cosmids flanking the 5q13.3 breakpoint in patients with HCL, and the distance between them is approximately 35 kb, as analyzed by fiber-FISH. The two cosmids have been located between the markers SGC34998 and WI-15505/WI-6897 by radiation hybrid mapping. Five of 11 patients with HCL had a hemizygous deletion of the two cosmids, indicating that the function of a tumor suppressor gene may be lost. With the aim of delineating the critical region of 5q13.3 loss in patients with HCL, we have constructed an integrated contig of YAC, BAC, PAC, P1, and cosmid clones that covers the region. Within this area, three expressed sequences were identified as candidates for the putative 5q13.3 tumor suppressor gene involved in the pathogenesis of HCL.

A FISH cosmid 'cocktail' for detection of 13q deletions in chronic lymphocytic leukaemia--comparison with cytogenetics and Southern hybridization.

The most frequent structural chromosome abnormality in chronic lymphocytic leukaemia (CLL) is deletion at chromosome 13q14. Studies with Southern blot hybridisation have revealed deletions in the region located telomeric of the retinoblastoma gene in more than 40% of cases. The highest frequency of homozygous deletions has been found at the D13S319 locus and it is likely that a new tumour suppressor gene is located close to this region. We have analysed deletions in the D13S319 region in 20 selected CLL patients using conventional cytogenetic analysis, fluorescence in situ hybridisation (FISH) and Southern blot hybridisation. FISH and Southern hybridisation are equally efficient in detecting deleted clones in our study. However, FISH analysis indicate that subclones with different numbers of alleles in the D13S319 region can exist simultaneously. The cytogenetic analyses confirm that clones with different chromosomal abnormalities can occur in patients with CLL and that 13q14 deletions can be limited to one of these subclones. Furthermore, the FISH analyses show that trisomy 12 and deletion of 13q14 can occur in the same cell clone. Finally, our study confirms that mitogen stimulation of peripheral blood cells from CLL patients before FISH analysis may result in a sharp increase in normal appearing cells, which can hide leukaemic clones with deletions in the D13S319 region.

Characterization of a hairy cell leukemia-associated 5q13.3 inversion breakpoint.

Previous cytogenetic analysis has indicated that chromosome anomalies involving the 5q13 band are common in hairy cell leukemia (HCL), occurring in approximately 1/3 of the patients. The data suggest that 5q13.3 is likely to harbor a gene involved in the transformational event of this disease. We selected a constitutional inv(5)(p13.1q13.3) in a patient with HCL as the starting point in an attempt to identify the relevant gene in 5q13.3. By using double color interphase fluorescence in situ hybridization (FISH) techniques, we have identified two cosmid probes from a chromosome 5-specific library that flank the 5q13.3 inversion breakpoint proximally and distally. Pulsed field gel electrophoresis (PFGE) and interphase FISH experiments suggest that the two markers are at a distance of no more than 300 kb. YAC probes covering a 21 Mb region at 5q13 were used to map the 5q13.3 inversion breakpoint and the breakpoint is located within the D5S646-D5S620 region. Two non-chimeric YACs have been identified that span the breakpoint. FISH analysis revealed that four other patients with cytogenetic aberrations of 5q carried inversions/deletions that involved the same 5q13.3 breakpoint region. The identification of a gene involved in hairy cell leukemogenesis in this region will be of major importance in the elucidation of the transformational events of HCL.

Amplification of multiple regions of chromosome 12, including 12q13-15, in chronic lymphocytic leukaemia.

Trisomy 12 is a frequent abnormality in chronic lymphocytic leukaemia (CLL). The biological importance of trisomy 12 is still poorly understood but it has been suggested that one or several genes are duplicated leading to malignant transformation. We present a case with amplification of 12q13-22 found in a clinically aggressive relapse of CLL. A smaller region, 12q13-15, was amplified most frequently and a YAC containing the MDM2 gene gave the highest number of signals. Additionally, in a subclone an amplicon containing at least 5 copies of a cosmid from 12q23-24 was detected. The case shows that small duplications of chromosome 12, not revealed by cytogenetic analysis, may occur in CLL. Also, it shows that cytogenetic clonal evolution can occur in CLL without morphological evidence of blast transformation. Our results indicate that the 12q13-15 region carries an important gene for CLL progression.

Increased frequency of chromosome abnormalities in fibroblasts from hairy cell leukemia patients.

A hereditary component is implicated in many different cancers, including hairy cell leukemia (HCL), and may involve an instability of the genome. We have previously documented recurrent clonal and non-clonal chromosomal abnormalities in hairy cells. To ascertain whether this instability of the genome is restricted to the malignant cells or if it might also include normal cells we performed cytogenetic investigations on skin fibroblasts and hairy cells from eight HCL patients and skin fibroblasts from eight referents. The frequency of chromosome abnormalities, regardless of clonality, was significantly increased in the fibroblasts from patients compared to referents. Also, five patients compared to one referent showed clonal abnormalities in their fibroblasts. Immunohistochemical investigations excluded the possibility that the fibroblast cultures were contaminated with hairy cells. Two patients had constitutional abnormalities, inv(5)(p13.1q13.3) and t(13;14), and one additional patient, possibly mosaic, showed the same abnormality, inv(9)(p21-22q22), in both fibroblasts (17/30) and blood (5/21) cells. Aberrations in patient fibroblasts also included sporadic inv(5), del(6)q, inv(19), and del(20)q, abnormalities previously shown to occur in hairy cells. A clonal expansion with trisomy 7 occurred in vitro as documented by fluorescence in situ hybridization (FISH). The only clonal abnormality occurring in a referent was -Y/-Y,+15 in an elderly male. In conclusion, a constitutional chromosomal instability may precede chromosome abnormalities and be of importance in the development of hairy cell leukemia.

A novel human CCAAT/enhancer binding protein gene, C/EBPepsilon, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor alpha/delta locus.

Members of the CsolidusEBP family of transcriptional factors have been implicated in the regulation of genes in a variety of tissues. We report here the isolation and characterization of the human C/EBPepsilon gene (CEBPE). By using low-stringency hybridization conditions and probes derived from the C/EBPalpha and C/EBPdelta genes, we have isolated overlapping genomic clones that cover almost 25 kb of the C/EBPepsilon gene locus and corresponding cDNA clones. DNA sequence analysis reveals that the gene encodes a protein highly homologous to rat CRP1. The gene was assigned to chromosome 14q11.2 by fluorescence in situ hybridization and was physically linked to the genetic marker D14S990. Based on linkage data derived from this marker, we positioned the CEBPE gene between the T-cell receptor alpha/delta locus and a cluster of four serine proteases expressed exclusively in hematopoietic cells. Expression of C/EBPepsilon was detected in Jurkat T-cell and in HL 60 promyelocytic cell lines. From a variety of normal human tissues studied, expression of mRNA was monitored only in peripheral blood mononuclear cells, tissues involved in the immune system, and ovaries. These data demonstrate that the C/EBPepsilon gene shows a restricted pattern of expression, has an intriguing chromosomal location, and suggest a possible role for the regulation of certain genes in cells of myeloid and lymphoid lineages.

Hairy cell leukemia is characterized by clonal chromosome abnormalities clustered to specific regions.

Cytogenetic analysis was performed on B-cell mitogen-stimulated cells from 36 patients with symptomatic hairy cell leukemia. Evaluable metaphases were achieved from 30 patients, and (67%) showed clonal abnormalities. Recurrent chromosomal aberrations involving chromosomes 1, 2, 5, 6, 11, 19, and 20 were found. The abnormalities were mostly deletions and inversions, whereas translocations and numerical abnormalities, except trisomy 5, were rare. Fourteen patients showed multiple clones, which mostly were unrelated and found in different combinations in individual cells. Cells with non-clonal abnormalities identical to those found in clonal changes in other patients were common. Chromosome 5 was involved in clonal aberrations in 12 of 30 (40%) patients, most commonly as trisomy 5 (n = 4), or pericentric inversions (n = 6) and interstitial deletions (n = 4) involving band 5q13. Three patients showed two and 1 patient three different clones that involved chromosome 5. In addition, 1 patient had a rare constitutional inversion of chromosome 5 with breakpoints at p13.1 and q13.3. Pericentric inversions and interstitial deletions of chromosome 2 occurred clonally in 4 patients (13%) and in single cells of another 6 patients. Deletions of chromosome 1 at band q42 was found in 5 patients, and 1 patient had a translocation between 1q42 and a supernumerary chromosome 5. Deletions of 6q and 11q were similar to those commonly found in other lymphoproliferative disorders. Trisomy 5, structural abnormalities involving the pericentromeric regions of chromosomes 5 and 2, and 1q42 abnormalities were findings distinguishing the karyotypes in hairy cell leukemia from those of other hematologic malignancies.

del(3p)(p13p21) in renal cell adenoma and del(4p)(p14) in bilateral renal cell carcinoma in two unrelated patients with von Hippel-Lindau disease.

Karyotype analyses of renal cell adenoma in one patient and bilateral renal cell carcinomas (RCC) in another unrelated patient have been performed. Both patients belonged to families with von Hippel-Lindau disease (vHL). In the adenoma, we found a clonal del(3)(p13p21) and a small clone of two cells with an additional del(14)(q13). This result indicates that the same region that is often deleted in RCC may also be deleted in a renal cortical adenoma. This finding may facilitate the localization of a tentative renal cell adenoma/carcinoma tumor suppressor locus. In the tumors from the patient with bilateral carcinomas we found a clonal del(4)(p14) on one side and on the other a del(4)(p14) together with del(14)(q13). In this case, there was no detectable 3p defect in the tumors. This result raises the question whether an alternative/additional locus on chromosome 4p may be involved in the RCC/vHL syndrome. Constitutional karyotypes were in both cases normal.

The gene for insulin-like growth factor-binding protein-1 is localized to human chromosomal region 7p14-p12.

Insulin-like growth factors (IGF) I and II are bound to high-affinity binding proteins in the blood circulation and other body fluids. These IGF-binding proteins are expressed at different concentrations in different tissues and are thought to regulate the activity of IGF I and II. Cloned cDNA for IGF-binding protein-1 (IGFBP1) has been used to verify the location of its gene to human chromosome 7 by Southern blotting to DNA from a human-mouse hybrid cell line. Further, by in situ hybridization the gene was regionally localized to 7p14-p12, and a Mendelian-inherited two-allele BglII restriction enzyme length polymorphism was identified, with the most frequent allele occurring in 53% of the chromosomes.