Translocation (2;14) associated with complex rearrangements of the Ig heavy chain in non-Hodgkin lymphoma.

Cytogenetic analysis of a patient with non-Hodgkin lymphoma revealed the following karyotype: 49,XXX,t(2;14)(q21;q32),+4,+8,del(13)(q14q21). Southern blot analysis with an Ig region probe showed non-productive rearrangements indicative of a translocation involving the Ig locus. However, molecular cloning of the abnormal rearrangements did not show novel sequences derived from chromosome 2 but showed that the BCL-6 gene was juxtaposed to the IgH enhancer. Three further clones with abnormal rearrangements involving the Ig locus, particularly Iggamma3, were isolated. This suggests that the mature lymphoid cells, in this patient, were capable of undergoing indiscriminate switch cleavage and religation.

Identification of amplified genes in a patient with acute myeloid leukemia and double minute chromosomes.

A case of acute myeloid leukemia (M2) with double minute chromosomes and complex karyotypic abnormalities was analyzed cytogenetically and molecularly. Comparative genomic hybridization (CGH) showed that the 8q24 region that contains the MYC oncogene was not amplified. Instead, amplification of chromosomal regions 11q23-->qter and 9p11-->pter was identified. Southern blot analysis confirmed the CGH findings and showed that the ETS1, FLI1, SRPR, NFRKB, and KCNJ5 genes located at 11q23-->24 were amplified, whereas the MLL at 11q23 was not amplified. Additionally, the IFN beta 1 and CDKN2A genes at 9p were amplified, but to a lesser degree. This is the first example of a case of acute myeloid leukemia with double minute chromosomes that has not involved amplification of either the MYC or the MLL genes.

Methylation status of the 3rd exon of the c-MYC oncogene in B-cell malignancies.

We examined the methylation status of the third exon of the MYC oncogene in 39 patients with B-cell malignancies. DNA was digested with MspI plus EcoRI or HpaII plus EcoRI and hybridised with a probe specific for the third exon of MYC. Thirty four patients showed complete methylation of the CCGG site. Four patients, one with chronic B-cell leukaemia and one with pro-lymphocytic leukaemia (PLL) and two with B-cell lymphoma showed partial hypomethylation of the CCGG site, while another patient with PLL showed complete hypomethylation of the CCGG site. These results suggest that hypomethylation of the MYC oncogene is infrequent in B-cell tumours but may be involved in the development of some cases of B-cell malignancies.

Clonally unrelated BCR-ABL-negative acute myeloblastic leukemia masquerading as blast crisis after busulphan and interferon therapy for BCR-ABL-positive chronic myeloid leukemia.

We report a patient with Philadelphia (Ph)-positive, BCR-ABL rearrangement positive, chronic myeloid leukemia (CML) with a prolonged chronic phase of 24 years who was first prescribed alpha-2 interferon 22 years after initial diagnosis. This therapy was tolerated poorly on account of thrombocytopenia, but an eventual major cytogenetic response was followed soon afterwards by transformation to terminal acute myeloid leukemia (AML). Cytogenetic studies indicated that the transformed myeloblasts were karyotypically normal and Ph negative. Although polymerase chain reaction (PCR) analysis of total leukemic mRNA remained BCR-ABL positive, other molecular studies, including Southern blotting and fluorescent in situ hybridization (FISH) analyses, showed that myeloblasts were BCR-ABL rearrangement negative. PCR-based clonality studies using an X-chromosome-linked restriction fragment polymorphism within the phosphoglycerate kinase gene (PGK1) further showed that the Ph-negative blast cells had a different clonal origin from the Ph-positive clone of chronic phase. We suggest that cases of underlying Ph-negative leukemic transformation in Ph-positive CML warrant further study and should be considered for trial of intensive remission induction therapy as appropriate for acute leukemia.

Characterization of the C-MYC amplicon in a case of acute myeloid leukemia with double minute chromosomes.

We have characterized the double minute chromosomes in a case of acute myeloid leukemia (AML). Southern blot analysis showed that the C-MYC was amplified. Further analysis with probes located both 3' and 5' of MYC indicated that the amplicon was at least 700 kb in size, extending from the papilloma virus integration site situated 500 kb 5' of MYC to the PVT gene located 280 kb 3' of MYC. This appears to be the largest MYC-containing amplicon in human leukemia.

Hypermethylation of the M27beta (DXS255) locus in chronic B-cell leukaemia.

We have investigated the methylation status of the M27beta (DXS255) locus in 21 female patients with chronic B-cell leukaemia and in 20 normal controls. DNA was digested with Pst1 and then with the methylation sensitive enzyme HpaII and probed with the M27beta probe. Eight patients (38%) showed hypermethylation of the M27beta locus which was not seen in any of the normal controls. Hypermethylation of the M27beta locus has also been found in acute myeloid leukaemia, acute lymphocytic leukaemia and lymphoma, suggesting that hypermethylation of the M27beta locus is associated with the leukaemic process.

Aberrant rearrangements of the immunoglobulin heavy chain switch region in chronic B-cell leukemia.

Analysis of the organisation of the Cmu-switch region of the immunoglobulin heavy chain locus in B-lymphocytes from 80 patients with chronic B-cell leukemia revealed 25 patients with abnormal rearrangements that could not be explained by the normal recombination events that take place in B-lymphocytes. Detailed analysis with probes spanning the Cmu -switch region and various restriction digests localised the rearrangements in two thirds of the patients to a 1300 bp region at the 5' end of the switch region while in the remaining patients the rearrangements occurred in the switch region. The consequences of these aberrant rearrangements remain to be determined, but their clustering to a defined region of the switch region suggests a "hot spot" that may be involved in the aetiology of the disease.

Genes and chromosomes in chronic B-cell leukemia.

Cytogenetic analysis of patients with chronic B-cell leukemia (B-CLL) indicates that 50% have chromosome abnormalities, while fluorescence in situ hybridization (FISH) and molecular techniques reveal an even higher incidence. Trisomy 12 and deletions or translocation of chromosome 13q14 are the most common abnormalities, but in neither case has the gene or genes involved in the abnormalities been identified. Combined FISH and immunophenotyping studies suggest that both abnormalities are secondary events in B-CLL. Other recurring chromosome abnormalities include 6q-, 11q- and 12p-, but the genes involved in these abnormalities have not been identified. Involvement of the BCL1, BCL2, and BCL3 genes has been reported, but the numbers are low and the cases tend to be atypical. Trisomy 12 in association with complex karyotypic abnormalities is associated with a poor prognosis, and FISH studies show a strong correlation between trisomy 12, atypical morphology, and advanced disease. Ten to 15% of patients have mutations of p53 which is associated with advanced disease, resistance to treatment, and poor survival.

Elevated frequency of the C2 allele of the ETS-I oncogene in elderly subjects.

We studied the frequency of an Sst I polymorphism of the ETS-I oncogene in 122 elderly subjects (mean age 78.14 years) and 115 teenagers (mean age 16.9 years). No difference in the frequency of the three genotypes (C1C1, C1C2, C2C2) was found between the two groups. However, the C2 allele occurred more frequently in the elderly subjects (chi 2 = 5.49, P < 0.02). These data suggest that the presence of the C2 allele may be associated with survival to old age.

Elevated frequency of a SstI polymorphism of the Ets-1 oncogene in non-Hodgkin's lymphoma patients.

We studied the frequency of a SstI polymorphism of the Ets-1 oncogene in 100 patients with non-Hodgkin's lymphoma, 44 patients with Hodgkin's disease, 49 patients with chronic myeloid leukemia, and 100 controls. There was no difference in the genotype frequency between the controls and patients with either Hodgkin's disease or chronic myeloid leukemia. In contrast, there was a highly significant difference in the distribution of the three genotypes between the patients with non-Hodgkin's lymphoma and the controls (X2 = 10.76, 2df, p = 0.004) with the C2 allele being more frequent in the lymphoma patients. Molecular cloning indicated that the polymorphic SstI site lay 304 bp from exon 7. This is the second association of the SstI polymorphism of the Ets-1 oncogene with a lymphoid disorder and suggests that the presence of the C2 allele is associated with a predisposition to develop a lymphoid malignancy.

Identification of three RFLPs at the HCK locus on chromosome 20.

New HindIII, RsaI and TaqI restriction fragment length polymorphisms (RFLPs) within the haemopoietic cell kinase gene in chromosome band 20q11.2 are described. These RFLPs provide a useful marker for linkage analysis in proximal 20q.

Increased frequency of the S allele of the L-myc oncogene in non-Hodgkin's lymphoma.

We studied 100 patients with non-Hodgkin's lymphoma, 44 patients with Hodgkin's disease and 100 controls for the prevalence of the EcoRI restriction fragment polymorphism of the L-myc oncogene. No difference in the frequency of the three genotypes (LL, LS, SS) was found between the patient and control groups. However, the S allele was found to occur more frequently in the non-Hodgkin's lymphoma patients (chi 2 = 4.57, P = 0.032). These data confirm an earlier report and suggest that the presence of the S allele is associated with susceptibility to non-Hodgkin's lymphoma.

Oligoclonal B-cell leukemia characterized by spontaneous cell division and telomere association.

Cytogenetic analysis of unstimulated cultures from a female patient with chronic B-cell leukemia (CLL) revealed three cytogenetically distinct clones, suggesting that the patient's leukemia was oligoclonal. Immunoglobulin heavy chain gene rearrangement studies revealed 1 germline and 4 rearranged bands, indicative of an oligoclonal leukemic population. Further evidence of oligoclonality was provided by X-linked RFLP studies. This is the first report of oligoclonality in CLL demonstrated by cytogenetic, immunoglobulin gene rearrangement, and X-chromosome inactivation studies. In addition to oligoclonality, the patient's leukemic cells exhibited telomere association, a Robertsonian translocation, and clonal evolution, suggesting an underlying genomic instability.

Cloning and sequencing of a t(14;19) breakpoint that involves the C mu switch region.

The t(14;19) is a recurring translocation found in a small number of cases of chronic B-cell leukemia (CLL). We have cloned and sequenced the breakpoint in a patient with a t(14;19) and shown that the breakpoint on chromosome 14 occurred in the C mu switch region, and that the breakpoint on chromosome 19 occurred in the 5' untranslated region of the BCL3 gene. This is in contrast to all the other reported cases with a t(14;19) in which the breakpoints on chromosome 14 occurred in the C alpha 1 or C alpha 2 switch region, and the breakpoints on chromosome 19 occurred upstream of the BCL3 gene. Our results further emphasize the importance of the switch region in the t(14;19) translocation.

Lack of 5' BCL2 rearrangements in B-cell leukemia.

We found no rearrangements in the 5' region of the BCL2 gene in DNA samples from 60 patients with chronic B-cell leukemia (CLL). This compares with the presence of these rearrangements in up to 10% of patients in other reports, and suggests that the incidence of 5' BCL2 rearrangements in CLL is considerably less than 10%.

Elevated frequency of an ETS-1 restriction fragment polymorphism in chronic B-cell leukaemia.

We studied the frequency of an SstI polymorphism in 70 patients with chronic B-cell leukaemia (CLL) and 100 normal controls. There was a highly significant difference in the distribution of the three genotypes between the CLL patients and the normal controls (chi 2 = 13.46, 2 df, P < 0.001). The C2 allele was found more frequently in CLL patients and may be a marker for a predisposition to develop CLL.

XbaI RFLP of ETS-1 oncogene in chronic B-cell leukaemia.

The influence of a polymorphic variant of the ETS-1 oncogene on the predisposition to develop chronic B-cell leukemia (CLL) was investigated. A total of 59 patients with CLL and 59 controls were examined for the frequency of an XbaI restriction fragment length polymorphism RFLP of the ETS-1 oncogene which has been reported to occur more frequently in patients with hematological malignancies than in normal controls. We found no significant difference in the allele frequency between the CLL patients and the normal controls. These data suggest that the presence of the XbaI RFLP is not associated with CLL.

Complex karyotypic evolution in B-cell chronic lymphocytic leukemia.

Cytogenetic analysis at diagnosis in a female patient with chronic B-cell leukemia showed a single abnormal clone with a 4p+ abnormality, 46,XX, -4, +der(4)t(4;?)(p16;?). Six additional clones evolved from this clone during the following 4 1/2 years and showed 3p+, 4p-, and 11q- chromosomes in addition to the 4p+ abnormality. Immunoglobulin heavy chain gene rearrangement studies showed two rearranged bands and a faint germline band. Following splenectomy, a strong germline and faint rearranged bands were seen, suggesting that the majority of cells were normal, whereas cytogenetic studies showed that the karyotypically abnormal cells were still present. The combination of cytogenetic and Ig gene rearrangement studies provides detailed information regarding the number of circulating normal and leukemic cells.

Molecular definition of interstitial deletions of chromosome 13 in leukemic cells.

Three patients with leukemia and one with a myeloproliferative disorder carried an interstitial deletion of chromosome 13, del(13)(q12q14), in leukemic cells. Proximal and distal breakpoints of the deleted segment were characterized by using DNA restriction fragment length polymorphisms of chromosome 13 supplemented by quantitative densitometry of hybridization signals to determine the copy number of individual loci. Both proximal and distal breakpoints varied between patients, and it is unlikely that a significant hybrid gene was formed by rejoining at the breakpoint junctions. The retinoblastoma gene was encompassed by the deleted segment in all four patients.

Linkage studies in tuberous sclerosis. Chromosome 9?, 11?, or maybe 14!

Published reports show linkage of tuberous sclerosis (TSC) to either chromosome 9 in some families or chromosome 11 in other families. We studied 243 individuals (82 with TSC) from 16 multigenerational TSC families. The diagnosis of TSC conformed to the criteria of Gomez. Penetrance was estimated at 0.90. DNA markers were analyzed using Southern blotting, probe hybridization, autoradiography, and genetic linkage analysis. Two-point lod scores for TSC were calculated for 43 genetic markers distributed over 11 chromosomes. Tests for homogeneity rejected the null hypothesis of homogeneity. Linkage to TSC was excluded (z less than or equal to -2, theta greater than or equal to 0.05) for 23 of these markers including 9q34 and 11q markers. One family gave z(theta max) = 1.8, theta max = 0.001 with ABO (on 9q34), and two other families attained lod scores greater than 1 for 9q34-region markers. The lod score for TSC versus chromosome 14 marker pAW101 (D14S1) was z(theta max) = 1.98, theta max = 0.15. A single large family has overall negative lod scores for markers localized to both chromosome 9 and chromosome 11. These data confirm genetic heterogeneity in TSC and suggest linkage of some families to 9q34. Furthermore, the data suggest that 14q may be an interesting area.