N. Bienz, D. L. Cardy, M. J. Leyland and M. A. Hulten. (1993)
"Trisomy 12 in B-cell chronic lymphocytic leukaemia: an evaluation of 33 patients by direct fluorescence in situ hybridization (FISH)."
An adaptation of the standard fluorescence in situ hybridization (FISH) technique allowing rapid analysis (4 h) has been used to study the prevalence of trisomy 12 in 33 patients with B-CLL, of whom 54% have been shown to have this abnormality. The presence of trisomy 12 has been compared with clinical parameters, and there may be a relationship between the prevalence of trisomy 12 in B-CLL and duration of disease.
Br J Haematol 85:819-822
We performed cytogenetic analyses of peripheral blood lymphocytes from 82 Midwestern B-cell chronic lymphocytic leukemia (B-CLL) patients. The cells were cultured with mitogens for 3-4 days. At least 15 metaphase cells were analyzed in 79 (96%) cases. Fifty (63%) of the 79 patients had clonal chromosomal alterations. Structural modifications of the long arm of chromosome 13 at or near band 13q14 were the most frequent abnormalities, identified in 23 (46%) of the patients with clonal abnormalities. In several patients, the abnormality involving band 13q14 was the sole chromosomal alteration. There was a high incidence of complex karyotypes. Nine patients had multiple subclones that appeared to result from clonal evolution; seven patients had cytogenetically unrelated clones; three patients had both subclones and cytogenetically unrelated clones. Nonclonal abnormalities were also prominent. Our study confirms the high incidence of clonal abnormalities involving chromosome arm 13q and documents the clustering of abnormalities at band 13q14 in B-CLL. The evidence for clonal evolution and the presence of multiple unrelated clones in these patients suggest that B-CLL may not be a karyotypically stable disease.
Genes Chromosom Cancer 4:273-80
Roughly 25% of human B-cell chronic lymphocytic leukaemias (CLL) are characterized by a chromosomal lesion involving 13q14. This region contains the retinoblastoma gene (RB1). We have used a variety of techniques to determine whether RB1 or some other locus is the critical region in 11 cases of low grade B-cell malignancy (mainly CLL), all with deletions or translocations involving 13q14. In all cases, except the one with minimal disease, there was deletion or a structural lesion in the region of D13S25, with at least 4 cases showing homozygous disruption. We conclude that D13S25 lies close to a tumour suppressor locus whose inactivation contributes to the initiation or progression of low grade B-cell malignancy. This locus is located at least 530 kilobases telomeric to RB1.
Nat Genet 3:67-72
Structural rearrangements involving chromosome 13 are frequently seen in B-cell chronic lymphocytic leukaemia. The presence of reciprocal translocations involving 13q14 in 10-15% of cases pinpoints the location of a gene important in leukaemogenesis. In order to characterise the exact location of the 13q14 breakpoint, somatic cell hybrids were constructed between mouse 3T3 cells and leukaemic cells from 5 patients with translocations involving chromosome 13. Hybrid pairs were isolated which carried either of the two derivative chromosomes carrying subsections of 13 and the position of the breakpoint investigated using a series of probes along the length of the chromosome. In all cases breakpoint region associated with rhabdomyosarcoma tumours and proximal to the D13S31 locus which lies in 13q14.3. In three translocations the RB1 gene was deleted as a result of the translocation but in at least one other case the BCLL breakpoint did not involve the RB1 gene, which consistently cosegregated in hybrids carrying other proximal markers. The D13S25 probe, which lies between RB1 and D13S31, however, was deleted in the translocation retaining RB1. It appears therefore that deletion of a gene(s) in this 2Mbp region is a critical event in some cases of BCLL tumorigenesis.
Oncogene 8:1415-9
We have previously shown that 30% of patients with B-cell chronic lymphocytic leukemia (B-CLL) have hemizygous deletions of the retinoblastoma (RB1) gene at 13q14. RB1 gene deletions may thus participate in malignant transformation of B-CLL, but it is also possible that a neighboring gene on 13q is the relevant one. To answer this question the remaining RB1 allele of eight clones with hemizygous deletions was studied by reverse transcription-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and immunofluorescense techniques. Cells from 10 patients without RB1 gene deletions were also studied by these methods. Lack of RB1 mRNA and RB protein expression was seen in leukemia cells from one of the patients. All other cases were found to be normal with regard to immunofluorescense, RT-PCR, and SSCP analysis, indicating at least one functional RB1 allele and supporting the importance of another gene in the 13q14 deletions. We then performed extended Southern blot analyses of the 13q region, using probes for 10 different loci. In 14 of 31 CLL clones (45%), deletions of a region telomeric to the RB1 gene (D13S25) were observed. In 4 of the cases the deletions were homozygous. Hemizygous deletions of the RB1 gene were observed in 11 of these patients and in none of the patients without D13S25 deletions. These data thus indicate that a gene(s) telomeric to RB1 is involved in the malignant transformation of CLL clones and that deletions of this region are a common event in this disease.
Proc Natl Acad Sci U S A 90:8697-701
The incidence of trisomy 12 was studied by conventional chromosome analysis in 111 patients referred as B-cell chronic lymphocytic leukaemia (B-CLL). Fluorescent in situ hybridization (FISH) was also applied in 34 of those patients with either a normal karyotype or no analysable mitoses. By karyotyping, trisomy 12 was present in 11.7% (13/111), whereas additional FISH increased the incidence to 14.4% (16/111). When subdividing our cases in either typical CLL (n = 90), fulfilling the FAB classification criteria, or atypical CLL (n = 21), with one or more variations from those criteria, the incidence of +12 by metaphase analysis was 3% and 48%, respectively. Additional FISH increased the incidence to 4% and 57%. The most common aberration in atypical CLL was FMC7 positivity (n = 11), followed by CD5 negativity (n = 8), strong surface immunoglobulin staining (n = 7) and atypical morphology (n = 6). Trisomy 12 could only be demonstrated in a small proportion of neoplastic cells in all positive cases. By FISH and/or karyotyping, all available samples at diagnosis of the disease were positive.
Br J Haematol 87:523-528
Chromosomal abnormalities are detected by conventional cytogenetic or FISH analysis in 50% of chronic lymphocytic leukaemias (CLL). Trisomy 12 and del 13q14 account for 70% of these abnormalities. The incidence of these two abnormalities was studied in CLL patients by Southern blot analysis using a highly purified B-cell malignant population (CD5 > 95%, CD3 < 5%). Probes for the D13S25 marker on chromosome 13 band q14 and for the RBTN3 gene on chromosome 12 band p12-13, were used. Deletion of the D13S25 was detected in 17/42 patients (43%) in a homozygous (9.5%) or heterozygous (30%) configuration. Deletion of the D13S25 marker appears to be a clonal and early event in CLL development since it is detected in > 95% of the malignant clonal population. Conversely, trisomy 12 is rarely a clonal event (5/33 patients, 15%) and a varying proportion of cells carrying this abnormality can be demonstrated in 30% of CLL patients (10/33 patients).
Br J Haematol 90:476-478
Chronic lymphocytic leukemia (CLL) has consistent 13q abnormalities detected by conventional cytogenetics. Using interphase cytogenetics we show deletion of a 1Mb 13q12.3 locus, encompassing the BRCA2 gene in 80% of 35 CLL cases studied. Homozygous deletion of BRCA2, located within the minimum deletion consensus, was detected in a significant population of cells in 60% of the cases. Deletion of the previously described 13q14 locus (analyzed with RB1 and D13S25 probes) was seen in 63% of the cases. Homozygous deletion of RB1 was seen in one case. Seven of the cases (32%) with D13S25 deletion had a population of cells with homozygous deletion. Deletions at the 13q12 and 13q14 loci result from distinct events since they were not contiguous. These data provide evidence for the existence of a new tumor supressor locus in B-cell CLL located at 13q12.3. BRCA2, located within the minimum deletion consensus, is a candidate for the gene whose somatic inactivation could play a role in the initiation or progression of b-cell CLL.
Blood (in press)
Chromosomal deletions of band 13q14 occur recurrently in BCR/ABL negative chronic
myeloproliferative disorders (CMPD), including myelosclerosis with myeloid metaplasia (MMM), polycythemia vera (PV), essential thrombocythemia (ET), juvenile chronic myeloid leukemia (JCML), and the so-called BCR/ABL- chronic myeloid leukemia (CML). The RBI tumor suppressor locus, mapping to 13q14, has long since been hypothesized as the important gene. In this report, we have determined the frequency of 13q14 deletions at the molecular level in a large panel of BCR/ABL- CMPD at different disease stages and performed a detailed genetic analysis of gross rearrangements/deletions and point mutations of the RBI gene in these disorders. Our data show that molecular deletions of 13q14 are detected in a relatively large fraction of BCR/ABL- CMPD (38%), that they appear to be more frequent in MMM than in other BCR/ABL- CMPD, and that they may be present at diagnosis or occur during blastic evolution of the neoplasia. The RBI gene displayed a germline configuration in all BCR/ABL- CMPD tested, suggesting that 13q14 deletions in these disorders affect a tumor suppressor locus distinct from RBI.
Genes-Chromosomes-Cancer 14:106-11