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The aim of this study was to validate the application of a commercially available multiplex reverse transcription polymerase chain reaction (RT-PCR) assay [Hemavision-7 System] for the seven most common leukemia translocations for routine molecular diagnostic hematopathology practice. A total of 98 samples, comprising four groups, were evaluated: Group 1, 16 diagnostic samples molecularly positive by our existing laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 2, 51 diagnostic samples negative by our laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 3, 21 prospectively analyzed diagnostic cases, without prior molecular studies; and Group 4, 10 minimal residual disease (MRD) samples. Analysis of the two previously studied cohorts (Groups 1 and 2) confirmed the diagnostic sensitivity and specificity of the multiplex assay with regard to these two translocations. Additionally, however, in the "negative" Group (Group 2) the assay revealed three unanticipated translocations (CBFbeta-MYH11, BCR-ABL, and MLL-AF4), two of which were confirmed on cytogenetics. Analysis of the prospective cohort demonstrated that the assay was cost-effective and amenable to standard laboratory practice, with an identically sensitive MRD detection rate to that of our laboratory-developed tests. Virtually all of the results obtained were consistent with the phenotype and karyotype by conventional methods. This study illustrates the utility of a kit-based multiplex RT-PCR assay for the molecular diagnosis and monitoring of leukemias and reinforces the complementary roles of molecular testing and cytogenetics in diagnostic hematopathology.

We analyzed the der(11) and der(4) genomic breakpoint junctions of a t(4;11) in the leukemia of a patient previously administered etoposide and dactinomycin by molecular and biochemical approaches to gain insights about the translocation mechanism and the relevant drug exposure. The genomic breakpoint junctions were amplified by PCR. Cleavage of DNA substrates containing the normal homologues of the MLL and AF-4 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha and etoposide, etoposide catechol, etoposide quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint junctions both involved MLL intron 6 and AF-4 intron 3. Recombination was precise at the sequence level except for the overall gain of a single templated nucleotide. The translocation breakpoints in MLL and AF-4 were DNA topoisomerase II cleavage sites. Etoposide and its metabolites, but not dactinomycin, enhanced cleavage at these sites. Assuming that DNA topoisomerase II was the mediator of the breakage, processing of the staggered nicks induced by DNA topoisomerase II, including exonucleolytic deletion and template-directed polymerization, would have been required before ligation of the ends to generate the observed genomic breakpoint junctions. These data are inconsistent with a translocation mechanism involving interchromosomal recombination by simple exchange of DNA topoisomerase II subunits and DNA-strand transfer; however, consistent with reciprocal DNA topoisomerase II cleavage events in MLL and AF-4 in which both breaks became stable, the DNA ends were processed and underwent ligation. Etoposide and/or its metabolites, but not dactinomycin, likely were the relevant exposures in this patient.

The partner gene of MLL was identified in a patient with treatment-related acute myeloid leukemia in which the karyotype suggested t(3;11)(q25;q23). Prior therapy included the DNA topoisomerase II inhibitors, teniposide and doxorubicin. Southern blot analysis indicated that the MLL gene was involved in the translocation. cDNA panhandle polymerase chain reaction (PCR) was used, which does not require partner gene-specific primers, to identify the chimeric transcript. Reverse-transcription of first-strand cDNAs with oligonucleotides containing known MLL sequence at the 5' ends and random hexamers at the 3' ends generated templates with an intra-strand loop for PCR. In-frame fusions of either MLL exon 7 or exon 8 with the GMPS (GUANOSINE 5'-MONOPHOSPHATE SYNTHETASE) gene from chromosome band 3q24 were detected. The fusion transcript was alternatively spliced. Guanosine monophosphate synthetase is essential for de novo purine synthesis. GMPS is the first partner gene of MLL on chromosome 3q and the first gene of this type in leukemia-associated translocations. (Blood. 2000;96:4360-4362)

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

Leukemias with MLL gene translocations are a complication of primary cancer treatment with DNA topoisomerase II inhibitors. How early translocations appear during primary cancer treatment has not been investigated. We tracked the leukemic clone with an MLL gene translocation during neuroblastoma therapy in a child who developed acute myeloid leukemia. The karyotype of the leukemic clone showed del(11)(q23). We used panhandle PCR-based methods to isolate the breakpoint junction involving MLL and an unknown partner gene. Marrow DNA from neuroblastoma diagnosis and DNA and RNA from serial preleukemic marrows were examined for the translocation. The karyotypic del(11)(q23) was a cryptic t(11;17). GAS7, a growth arrest-specific gene at chromosome band 17p13, was the partner gene of MLL. Two different MLL-GAS7 fusion transcripts were expressed. The translocation was already detectable by 1.5 months after the start of neuroblastoma treatment. The translocation was not detectable in the marrow at neuroblastoma diagnosis or in peripheral blood lymphocyte DNAs of six normal subjects. GAS7 is a new partner gene of MLL in treatment-related acute myeloid leukemia. MLL gene translocations can be present early during anticancer treatment at low cumulative doses of DNA topoisomerase II inhibitors. Although MLL has many partner genes and most have not been characterized, panhandle PCR strategies afford new means for detecting MLL gene translocations early during therapy when the partner gene is unknown.

Background: AF-4 is a common partner gene of MLL. AF-4 breakpoints occur in introns, but most AF-4 introns are uncharacterized., Methods and Results: We cloned AF-4 intron 4 and examined the frequency of breakpoints in this intron. The 5.8-kb intron is rich in repeat sequences and was the site of translocation in 3 of 17 leukemias with t(4;11). We cloned the der (11) and der (4) breakpoints and isolated the fusion transcripts in the cell line MV4-11 and in a de novo acute lymphoblastic leukemia (ALL). Both translocations joined MLL intron 6 and AF-4 intron 4. In MV4-11, 249 bases from AF-4 were present in both derivative chromosomes, indicating duplication. In the de novo ALL, duplication of 446 bases from MLL and AF-4 occurred. Reciprocal fusion transcripts were expressed., Conclusions: Intronic sequence of AF-4 is useful for molecular diagnosis of t(4;11). Duplicated intronic regions suggest staggered chromosomal breakage.

We used single-strand conformation polymorphism (SSCP) analysis of p53 exons 4-8 to screen for possible mutations in 25 pediatric de novo leukemias with translocations of the MLL gene at chromosome band 11q23. Of the 25 patients, 21 were infants. Fifteen cases were acute myeloid leukemia (AML), eight were acute lymphoblastic leukemia (ALL), and two cases were biphenotypic. Nineteen cases were studied at diagnosis and six at time of relapse. p53 mutations were absent in all 19 cases studied at the time of diagnosis. The only mutation was a TGC-->TTC transversion (cys-->phe) at codon 141 in exon 5 in a case of infant ALL at relapse that occurred by subclone evolution after MLL gene translocation. We previously showed that p53 mutations are also absent in pediatric treatment-related leukemias with MLL gene translocations. The absence of p53 mutations at initial transformation may suggest that the anti-apoptotic effect of mutant p53 is not important in leukemias with MLL gene translocations. Alternatively, exogenous DNA damage may be the common feature in treatment-related and de novo cases. Since MLL gene translocations may occur through DNA repair and wild-type p53 is central to DNA repair, the absence of p53 mutations raises the possibility that wild-type p53, not mutant p53, may be important in the genesis of leukemias with these translocations.

We used a new approach called panhandle polymerase chain reaction (PCR) to clone an MLL genomic translocation breakpoint in a case of acute lymphoblastic leukemia of infancy in which karyotype analysis was technically unsuccessful and did not show the translocation partner. Panhandle PCR amplified known MLL sequence 5' of the breakpoint and 3' sequence from the unknown partner gene from a DNA template with an intrastrand loop schematically shaped like a pan with a handle. The 7-kb panhandle PCR product contained the translocation breakpoint in MLL intron 8. The partner DNA included unique nonrepetitive sequences, Alu and mammalian apparent LTR-retrotransposon (MaLR) repetitive sequences, and a region of homology to expressed sequence tags. MaLR sequences have not been found before near leukemia-associated translocation breakpoints. The nonrepetitive sequences were not homologous to known partner genes of MLL. Screening of somatic cell hybrid and radiation hybrid lines by PCR and fluorescence in situ hybridization analysis of normal metaphase chromosomes mapped the partner DNA to chromosome band 4q21. Reverse transcriptase-PCR identified an MLL-AF-4 chimeric mRNA, indicating that panhandle PCR identified a fusion of MLL with a previously uncharacterized AF-4 intronic sequence. Panhandle PCR facilitates cloning translocation breakpoints and identifying unknown partner genes.

Panhandle PCR amplifies genomic DNA with known 5' and unknown 3' sequences from a template with an intrastrand loop schematically shaped like a pan with a handle. We used panhandle PCR to clone MLL genomic breakpoints in two pediatric treatment-related leukemias. The karyotype in a case of treatment-related acute lymphoblastic leukemia showed the t(4;11)(q21;q23). Panhandle PCR amplified the translocation breakpoint at position 2158 in intron 6 in the 5' MLL breakpoint cluster region (bcr). The karyotype in a case of treatment-related acute myeloid leukemia was normal, but Southern blot analysis showed a single MLL gene rearrangement. Panhandle PCR amplified the breakpoint at position 1493 in MLL intron 6. Screening of somatic cell hybrid and radiation hybrid DNAs by PCR and reverse transcriptase-PCR analysis of the leukemic cells indicated that panhandle PCR identified a fusion of MLL intron 6 with a previously uncharacterized sequence in MLL intron 1, consistent with a partial duplication. In both cases, the breakpoints in the MLL bcr were in Alu repeats, and there were Alu repeats in proximity to the breakpoints in the partner DNAs, suggesting that Alu sequences were relevant to these rearrangements. This study shows that panhandle PCR is an effective method for cloning MLL genomic breakpoints in treatment-related leukemias. Analysis of additional pediatric cases will determine whether breakpoint distribution deviates from the predilection for 3' distribution in the bcr that has been found in adult cases.

In patients with chronic myelogenous leukemia (CML), the Philadelphia chromosome may be associated with a number of other cytogenetic lesions. However, t(11;14)(q13;q32), found mainly in B-cell lymphoproliferative disorders, has not been previously reported in Ph-positive CML. We describe a patient with hematologically typical chronic phase CML in whom both cytogenetic lesions were found at diagnosis.

Translocations involving chromosome 8 at band q24 and one of the Ig loci on chromosomes 14q32, 22q11, and 2p11 are the hallmark of Burkitt's lymphoma (BL). It has been previously observed that the exact localization of the breakpoints at chromosome 8q24 can vary significantly from patient to patient, scattering over a distance of more than 300 kb upstream of c-myc and about 300 kb downstream of c-myc. To generate probes for fluorescence in situ hybridization (FISH) that detect most c-myc translocations, we screened a yeast artificial chromosome (YAC) library from normal human lymphocytes by colony hybridization, using three markers surrounding the c-myc gene as probes. We obtained 10 YAC clones ranging in size between 500 and 200 kb. Two nonchimeric clones were used for FISH on several BL cell lines and patient samples with different breakpoints at 8q24. Our results show that the YAC clones detected translocations scattered along approximately 200 kb in both metaphase chromosomes and interphase nuclei. The sensitivity, rapidity, and feasibility in nondividing cells render FISH an important diagnostic tool. Furthermore, the use of large DNA fragments such as YACs greatly simplifies the detection of translocations with widely scattered breakpoints such as these seen in BL.

Reciprocal chromosome translocations involving 11q23 are frequently associated with acute leukemias, with the t(4;11) translocation predominating among acute lymphoblastic leukemias, and the t(9;11), t(11;19) and t(6;11) translocations most common among acute myeloid leukemias. In each of these translocations the ALL-1 gene, located at 11q23 and constituting the human homologue of Drosophila trithorax, fuses to a specific gene on the partner chromosome to produce a chimeric protein. Here we report the cloning and the characterization of the partner gene from chromosome 6 (AF-6). AF-6 is expressed in a variety of cell types and encodes a protein of 1612 amino acids. The protein contains short stretches rich in prolines, charged amino acids, serines, or glutamines. In addition, the AF-6 protein contains the GLGF motif shared with several proteins of vertebrates and invertebrates thought to be involved in signal transduction at special cell-cell junctions.

Chromosomal region 11q23 is frequently rearranged in acute lymphocytic leukemias (ALLs) and in acute myeloid leukemias (AMLs), mostly in reciprocal exchanges with various translocation partners. The most common of these translocations is t(4;11)(q21;q23). It is present in approximately 10% of ALL patients, most frequently in very young children. We have recently cloned a region of chromosome 11, the ALL-1 locus, found to be rearranged in malignant cells from patients with the t(4;11), t(9;11), t(11;19), t(1;11), t(6;11), t(10;11), and del(11q23) chromosomal abnormalities. Here we report the cloning and characterization of chromosomal breakpoints from leukemic cells with t(4;11) aberrations. The breakpoints cluster in regions of 7-8 kilobases on both chromosomes 4 and 11. The presence of heptamer- and nonamer-like sequences at the sites of breakage suggests that the VDJ recombinase utilized for immunoglobulin gene rearrangement is also directly involved in these translocations. We also show that leukemic cells with t(4;11) express altered RNAs transcribed from the derivative chromosomes 11 and 4.

The 11q23 chromosome band is frequently associated with chromosomal aberrations in human leukemias. We have previously cloned a DNA fragment derived from chromosome 11 which could be used as a probe to detect rearrangements in DNAs from the leukemic cells of patients with the t(4;11), t(9;11), and t(11;19) translocations. In this study we now show that the same probe detects DNA rearrangements in malignant cells from patients with the t(1;11), t(6;11), t(10;11), and del (11q23) chromosomal abnormalities. A second probe obtained from a region located centrometric to the breakpoint cluster detects major and minor transcripts of 12.5 and 11.5 kilobases, respectively, in all cell lines examined. The same probe identifies an altered 11-kilobase RNA in all three independent cell lines with the t(4;11)(q21;q23) chromosome translocation.

A variety of chromosomal translocations occur in pediatric T-cell acute lymphoblastic leukemia (T-ALL) in which a cellular oncogene or growth-related gene is translocated to the alpha/delta locus of the T-cell receptor gene. The t(8;14)(q24;q11) has been described at the cytogenetic and molecular level, but the disease associated with this translocation has not been defined clinically. Fifteen pediatric cases of leukemia/lymphoma with a t(8;14)(q24;q11) chromosomal translocation were collected from previous publications and institutional records. The estimated prevalence of this abnormality among all cases of ALL was 1%. The t(8;14)(q24;q11) disease was characterized by male predominance (10/15), a median age of 5.5 years (range 1.8-17 years), high white blood cell count (median 95 x 10(9)/l), central nervous system infiltration (4/11), bulky extramedullary leukemia (10/11), and T-cell immunophenotype (12/15). The median event-free survival was 4 months, and the median survival, 11 months. Seven cell lines with t(8;14)(q24;q11) were established from six of the cases; four were T-lymphoblastic, one was T-lymphoblastic, but expressed myeloid-related antigens, and two were predominantly myeloid. t(8;14)(q24;q11) leukemia/lymphoma and other ALLs involving 13(q11) have in common a high tumor burden, early spread to extramedullary sites, a propensity to form T-lymphoblastic or T-myeloid cell lines and, usually, an aggressive clinical course.

Chromosomal region 11q23 participates in a number of reciprocal translocations with specific regions of chromosomes 4, 9, 19, and others. These translocations are associated with acute lymphocytic leukemia and acute myelomonocytic, monocytic, and myelogenous leukemia. From a yeast artificial chromosome containing human DNA derived from 11q23 we cloned a DNA fragment which can be used as a probe to detect rearrangements in leukemic cells from the majority of patients with the t(4;11), t(9;11), and t(11;19) translocations. The breakpoints cluster in a small DNA region of less than 5.8 kilobases.

We have analyzed the molecular features of a t(11;14)(q23;q32) chromosome translocation of a cell line established from a B-cell lymphoma. Somatic hybrid cells carrying the 11q- and/or 14q+ chromosome(s) were produced in order to map the breakpoints. Southern blot analyses of DNAs from these hybrid cell lines together with various probes from the IGH locus on chromosome 14 and the ETS-1 and CD3 genes on chromosome 11 showed that the breakpoints of the translocation occurred between the constant regions of the C phi gamma and C gamma 2 genes on chromosome 14 and between the CD3 and ETS-1 genes on chromosome 11. The t(11;14)(q23;q32) translocation does not seem to involve the same mechanism that is responsible for translocations occurring at the immunoglobulin heavy chain joining segment (JH).

Chromosome studies are helping to identify oncogenes, both known and previously unknown, involved in the pathogenesis of human lymphocytic tumors; and mechanisms by which the function of these genes is critically altered. Most extensively studied have been the chromosome translocations involving the myc gene in Burkitt's lymphomas and the bcl-2 gene in low-grade lymphomas, where "activation" of the oncogene results from association with a transcriptionally active immunoglobulin gene. Other putative oncogenes, similarly involved in translocations with immunoglobulin genes (in B-cell tumors) or T-cell receptor genes (in T-cell tumors), are currently being investigated, as well as alternative mechanisms of myc gene activation in these neoplasms. Limited clinical applications of these studies have already been forthcoming, and they should eventually lead to improvements in diagnosis, prognosis, and even therapy.

Molecular analysis of somatic cell hybrids derived from T cells carrying a t(7;14)(q35;q32) chromosomal translocation from a patient with ataxia telangiectasia and T cell leukemia indicates that the breakpoint on chromosome 14 is proximal to the IgH locus and to the D14S1 locus, while the breakpoint on chromosome 7 involves the T cell receptor beta chain locus immediately 5' to J beta 1.5 on chromosome 7. The separation of V beta and C beta observed in somatic cell hybrids defined the orientation of the T cell receptor beta chain locus on chromosome 7 where the V beta genes are centromeric and the C beta genes are telomeric. A novel chromosomal alteration, undetected cytogenetically, was revealed as being an inversion with duplication of the distal band of chromosome 14q32. The importance of the 14q32 region in the leukemogenic process is discussed.

We have cloned the translocation-associated and homologous normal MYC alleles from SKW-3, a leukemia T-cell line with the t(8;14)(q24;q11) translocation, and determined the sequence of the MYC oncogene first exon and flanking 5' putative regulatory regions. S1 nuclease protection experiments utilizing a MYC first exon probe demonstrated transcriptional deregulation of the MYC gene associated with the T-cell receptor alpha locus on the 8q+ chromosome of SKW-3 cells. Nucleotide sequence analysis of the translocation-associated (8q+) MYC allele identified a single base substitution within the upstream flanking region; the homologous nontranslocated allele contained an additional substitution and a two-base deletion. None of the deletions or substitutions localized to putative 5' regulatory regions. The MYC first exon sequence was germ line in both alleles. These results demonstrate that alterations within the putative 5' MYC regulatory regions are not necessarily involved in MYC deregulation in T-cell leukemias, and they show that juxtaposition of the T-cell receptor alpha locus to a germ-line MYC oncogene results in MYC deregulation.

The t(11;14) (q13;q32) chromosome translocation has been reported in diffuse small and large cell lymphomas and in chronic lymphocytic leukaemia (B-CLL) and multiple myeloma. Because chromosome band 14q32 is involved in this translocation, as well as in the t(8;14) (q24;q32) translocation of the Burkitt tumour, interruption of the immunoglobulin heavy-chain locus was postulated for this rearrangement. We have cloned the chromosomal joinings between chromosomes 11 and 14 and also between chromosomes 14 and 18, in B-cell tumours carrying translocations involving these chromosomes, and suggested the existence of two translocated loci, bcl-1 and bcl-2, normally located on chromosomes 11 (band q13) and 18 (band q21) respectively, involved in the pathogenesis of human B-cell neoplasms. The results indicate that in the leukaemic cells from two different cases of CLL, the breakpoints on chromosome 11 are within 8 nucleotides of each other and on chromosome 14 involve the J4-DNA segment. Because we detected a 7mer-9mer signal-like sequence with a 12-base-long spacer on the normal chromosome 11, close to the breakpoint, we speculate that the t(11;14) chromosome translocation in CLL may be sequence specific and may involve the recombination system for immunoglobulin gene segment (V-D-J) joining.

Abnormalities of chromosome 1, including trisomy for all or a portion of the long arm, have been frequently reported in many cancers. Anomalies of chromosome 19 are far less common, although a t(1;19)(q23;p13) translocation has been reported in association with pre-B-cell leukemia. We have observed a t(1;19)(q12;p13) translocation in three cases of advanced melanoma, with the translocation chromosome representing an extra dose of 1q in each instance. The breakpoint on 1q was within the centromeric heterochromatin, proximal to the site in pre-B-cell leukemia, but the breakpoint on 19p appeared identical. The gene for human insulin receptor has recently been mapped to this region of chromosome 19 (p13.2-13.3). This gene shares structural and sequence homologies with the epidermal growth factor receptor (erb-B oncogene) and members of the src family of oncogenes, suggesting that alterations in the insulin receptor, resulting from chromosomal translocation, could lead to a role in tumorigenesis. The present findings may permit this possibility to be examined in a neoplasm of neuroectodermal origin.

A number of clinically significant human diseases are associated with rearrangements of chromosome #22. These include the t(9;22) of chronic myelogenous leukemia (CML), the t(8;22) of Burkitt's lymphoma (BL), the t(11;22)(q23;q11) constitutional rearrangement and the t(11;22) of Ewing's sarcoma (ES) and neuroepithelioma (NE). All of these translocations have breakpoints in 22q11. Using a molecular cytogenetic approach and various cloned portions of the lambda light chain gene as probe, we have assigned a linear order within 22q11 to these breakpoints. Using chromosomal in situ hybridization we have determined that the 22q11 breakpoint in BL2, a t(8;22) Burkitt's lymphoma is proximal to the breakpoint of the t(9;22) of CML. We have demonstrated that the 22q11 breakpoint of PA682, another t(8;22) BL cell line, interrupts the lambda light chain locus. Using a combination of variable and constant region probes and PA682 cells, we have shown that the lambda light chain locus is oriented such that V lambda is proximal to C lambda in 22q11. Our results for the constitutional t(11;22) indicate that the 22q11 breakpoint is distal to that of BL, proximal both to that of CML and ES and, in addition, it interrupts the C lambda gene cluster. Our studies of the 22q11 breakpoint of the t(11;22) of ES and NE suggest that they are the most distal of the breakpoints we studied on chromosome #22.

The T-cell receptor (TCR) alpha/delta chain locus on chromosome 14q11 is nonrandomly involved in translocations and inversions in human T-cell neoplasms. We have analyzed three acute T-lymphoblastic leukemia samples carrying a t(10;14)(q24;q11) chromosome translocation by means of somatic cell hybrids and molecular cloning. In all cases studied the translocation splits the TCR delta chain locus. Somatic cell hybrids containing the human 10q+ chromosome resulting from the translocation retain the human terminal deoxynucleotidyltransferase gene mapped at 10q23-q24 and the diversity and joining, D delta 2-J delta 1, regions of the TCR delta chain, but not the V alpha region (variable region of the TCR alpha chain), demonstrating that the split occurred within the V alpha-D delta 2 region. Molecular cloning of the breakpoint junctions revealed that the TCR delta chain sequences involved are made from the D delta 2 segment. The chromosome breakpoints are clustered within a region of approximately 263 base pairs of chromosome 10. The results suggest that the translocation of the TCR delta chain locus to a locus on 10q, which we have designated TCL3, results in deregulation of this putative oncogene, leading to acute T-cell leukemia.

A 75-year-old man with chronic myelomonocytic leukemia was found to have a constitutional t(6;16)(q13;q22) translocation, as did his healthy daughter. Chromosomal in situ hybridization studies of the daughter's lymphocytes did not indicate translocation-mediated interruption of the metallothionein gene cluster, at 16q22, although this locus has been reported to be involved in the eosinophilic variant of acute myelomonocytic leukemia. Lymphocytes from the daughter and from the patient's brother (who had a normal karyotype), had no increased fragility at 16q22. The findings do not provide evidence for an association between the familial chromosome abnormality and this patient's leukemia.

We have produced cell hybrids between mouse myeloma cells, which do not produce immunoglobulin chains, and Burkitt lymphoma cells (Daudi), which express surface IgM. Daudi Cells carry a reciprocal chromosome translocation between chromosomes 8 and 14, described as (8;14)(q24;q32). The hybrids were studied for the expression of human immunoglobulin chains and human isozyme markers, for the presence of human chromosomes, and for the presence of the human genes for heavy chain variable regions (VH) and mu and gamma chain constant (C) regions. The results indicate that the expressed mu chain gene is on normal chromosome 14 in Daudi cells. We have also determined that the chromosome 14 involved in the translocation (14q+) carries the gene for C mu and C gamma 1-4 and probably several genes for the variable region (V). Certain hybrids had lost both the chromosomes 14 but had retained the abnormal chromosome 8 (8q-) that carries the terminal end of the long arm of chromosome 14. These hybrids were studied for the presence of human VH, C mu,, and C gamma DNA sequences, and the results indicated that the hybrid cells with the 8q- chromosome contained VH genes that not C genes. Therefore, we conclude that, in the Daudi Burkitt lymphoma, the break in chromosome 14 occurred within the chromosome segment containing V region genes. As a result of the translocation some of these VH genes became associated with chromosome 8. It is possible that the expression of malignancy in Burkitt lymphoma is caused by immunoglobulin V region gene translocation resulting in activation of a gene on the long arm of human chromosome 8.

We have analyzed the oncogene rearrangements involving BCL2 and MYC in the leukemia cells of a patient with an aggressive prolymphocytic leukemia that had an abnormal karyotype including a t(14;18) translocation and a chromosome 17q+. Molecular analysis showed that BCL2 was rearranged in the major breakpoint cluster region and had joined into the immunoglobulin heavy chain gene as in follicular lymphoma. Cloning and sequence analysis of the rearranged MYC gene revealed that MYC was truncated at the Pvu II site at the end of the first exon of MYC and had joined into the regulatory elements of a gene that we called BCL3 (B-cell leukemia/lymphoma 3). The BCL3 locus was mapped to chromosome 17 band q22. We found BCL3 transcribed as a message of 1.7 kilobases in many hematopoietic cell lines representing all hematopoietic lineages. In the patient's leukemia cells, the truncated MYC gene was highly expressed under the influence of BCL3 regulatory elements, leading to an aggressive B-cell leukemia that presumably had been derived from an indolent lymphoma carrying a rearranged BCL2 gene.

Mouse lymphoma cells were hybridized with two human acute T-cell leukemias with a t(11;14) (p13;q11) translocation and the segregated hybrids were examined for the presence of the DNA segments coding for the constant (C) and the variable (V) regions of the alpha chain (C alpha and V alpha) of the T-cell receptor. The C alpha segment was translocated to the involved chromosome 11 (11p+) while the V alpha segment remained on the involved chromosome 14 (14q-). The data indicate that the locus for the alpha chain of the T-cell receptor is split by the chromosomal breakpoint between the V alpha and the C alpha gene segments, and that the V alpha segments are proximal to the C alpha segment within chromosome band 14q11.2.

In situ chromosomal hybridization of a probe for part of the lambda light chain constant region (C lambda) has demonstrated that the 22q11 breakpoints of chronic myelogenous leukemia (CML) t(9;22) and Burkitt lymphoma t(8;22) are not identical. For CML, the breakpoint is distal to the IGLC genes, whereas for Burkitt lymphoma it is proximal. The study provides direct evidence for regional assignment of the IGLC gene cluster to 22q11.

We have performed gene rearrangement studies on the leukemic blasts of a patient with acute pre-B-cell leukemia. The patient had a 5 year history of follicular lymphoma, which developed into acute pre-B-cell leukemia. The leukemic blasts revealed a karyotype with two translocations, t(8; 14) and t(14; 18), characteristic for Burkitt's lymphoma and follicular lymphoma. The cells are TdT positive, do not possess surface immunoglobulin, and they show immunoglobulin gene rearrangement. The mu heavy chain and kappa light chain constant (C mu and C kappa) loci are deleted, while the gamma and lambda light chain constant (C gamma and C lambda) region genes are rearranged. Both alleles of the heavy chain joining segment (JH) are rearranged on chromosome 14q+, one of them with the bcl-2 oncogene from chromosome 18. The breakpoint of the t(14; 18) translocation occurs in the major breakpoint cluster region in the 3' untranslated region of bcl-2. On chromosome 8 a c-myc rearrangement was mapped immediately 5' to the c-myc first exon in a region involved in sporadic Burkitt lymphoma. The data are consistent with our previous hypothesis on the evolution of B-cell malignancies: a rare pre-B cell develops a t(14; 18) translocation during immunoglobulin VDJ joining that results in an expansion of a follicular lymphoma clone carrying an activated bcl-2 gene. Within the clone of pre-B cells a second translocation, t(8; 14), occurs during heavy chain isotype switching that results in the deregulation of the c-myc involved in the translocation.

From an acute B-cell leukemia cell line, a DNA probe was obtained that was specific for chromosome 18 and flanked the heavy chain joining region of the immunoglobulin heavy chain locus on chromosome 14. This probe detected rearrangement of the homologous DNA segment in the leukemic cells and in follicular lymphoma cells with the t(14:18) chromosome translocation but not in other neoplastic or normal B or T cells. The probe appears to identify bcl-2, a gene locus on chromosome 18 (band q21) that is unrelated to known oncogenes and may be important in the pathogenesis of B-cell neoplasms with this translocation.

The chromosomal breakpoint of chronic lymphocytic leukemia (CLL) cells of the B-cell type carrying the translocated long arms of chromosomes 11 and 14 [t(11;14) (q13;q32)] was cloned. The breakpoint was found to be within the joining segment of the human heavy chain locus on the translocated long arm of chromosome 14. A probe that is specific for chromosome 11 and that maps immediately 5' to the breakpoint on the 14q+ chromosome was isolated. The probe detected a rearrangement of the homologous genomic DNA segment in the parental CLL cells and also in DNA from a diffuse large cell lymphoma with the t(11;14) translocation. This rearranged DNA segment was not present in Burkitt lymphoma cells with the t(8;14) translocation or in nonneoplastic human lymphoblastoid cells. The probe can thus be used to identify and characterize a gene located on band q13 of chromosome 11 that appears to be involved in the malignant transformation of human B cells carrying the t(11;14) translocation. This gene, named bcl -1, appears to be unrelated to any of the known retrovirus oncogenes described to date.

We analyzed a t(1;14)(p32;q11) chromosomal translocation in a human lymphohemopoietic stem cell line derived from a patient with acute T-lymphoblastic leukemia. The chromosomal joining on the 1p+ chromosome occurred at the T-cell receptor delta diversity (D delta 2) segment, and the reciprocal chromosomal joining on the 14q- chromosome occurred at the T-cell delta diversity segment D delta 1. The involvement of delta diversity segments at the translocation junctions suggests that the translocation occurred during an attempt at D delta 1-D delta 2 joining in a stem cell. The segment of chromosome 1 at band p32, adjacent to the chromosomal breakpoint, encodes a transcriptional unit designated TCL5 (T-cell leukemia/lymphoma 5). The differential expression of the TCL5 RNA transcripts in this lymphohemopoietic stem cell line relative to several other T- and B-cell lines suggests that TCL5 gene expression is an integral event in the pathogenesis of the T-cell leukemia. Rearrangement of the TCL5 locus in a human melanoma cell line carrying a del(1p32) further implies that the TCL5 gene may play a role in malignant transformation.

We have studied somatic cell hybrids between mouse myeloma and JI Burkitt lymphoma cells carrying a t(2;8) chromosome translocation for the expression of human kappa chains. and for the presence and rearrangements of the human c-myc oncogene and kappa chain genes. Our results indicate that the c-myc oncogene is unrearranged and remains on the 8q+ chromosome of JI cells. Two rearranged C kappa genes were detected: the expressed allele on normal chromosome 2 and the excluded kappa allele that was translocated from chromosome 2 to the involved chromosome 8 (8q+). The distribution of V kappa and C kappa genes in hybrid clones retaining different human chromosomes indicated that C kappa is distal to V kappa on 2p and that the breakpoint in this Burkitt lymphoma is within the region carrying V kappa genes. High levels of transcripts of the c-myc gene were found when it resided on the 8q+ chromosome but not on the normal chromosome 8, demonstrating that translocation of a kappa locus to region distal to the c-myc oncogene enhances c-myc transcription.

The majority of chromosomal rearrangements observed in Burkitt lymphomas involve a translocation between 8q and 14q, while the remaining minority carry variant translocations between chromosome 8 and either 2 or 22. We have studied the JI Burkitt lymphoma cell line carrying the variant 2;8 chromosome translocation using a combination of high-resolution and molecular cytogenetic techniques. We have determined that the chromosome 2 breakpoint of the 2;8 translocation in these cells is in the distal portion of 2p11.2. In situ hybridization of a DNA probe for kappa light chain variable (V kappa) region demonstrated that this 2p11.2 breakpoint is within the V kappa region. There was significant hybridization of the probe to both the 2p- and 8q+ chromosomes, with 23% of all grains considered to be specific for V kappa located over the middle one-third of the long arm of the 8q+ chromosome. Thus, there is translocation of the entire kappa constant (C kappa) region and a portion of the region carrying V kappa genes from 2p to a region 3' of the c-myc oncogene on the involved chromosome 8, resulting in transcriptional activation of the c-myc that is quite distant from the 5' end of the C kappa gene. These results provide direct evidence for translocation-related rearrangement of the kappa immunoglobulin gene cluster in this Burkitt lymphoma and for the assignment of the V kappa locus to 2p11.2.

Chromosome studies were done on 7 patients with chronic T cell leukemia. Their lymphocytes responded in culture to one or more T cell mitogens: PHA, Con A, or the calcium ionophore A23187. Clones of cytogenetically-abnormal cells were present in all seven patients, but on occasion the frequency of such cells varied greatly in cultures stimulated with different mitogens. There was no consistent chromosome change, but alterations of chromosome 2 were noted in four individuals and of chromosome 14 in three. In two patients, there was a translocation to the long arm of chromosome 14, producing a 14q+, with the break point in the terminal portion, an abnormality previously observed in B cell lymphomas. One of these patients also showed evidence of clonal evolution in sequential cytogenetic studies, but more data are needed to determine whether such investigations are of prognostic value with respect to the clinical course of the disease.

Two human T-cell leukemias carrying a t(8;14)(q24;q11) chromosome translocation were studied for rearrangements and expression of the c-myc oncogene. For one leukemia, rearrangement was detected in a region immediately distal (3') to the c-myc locus; no rearrangements of c-myc were observed in the second case (DeF). However, studies with hybrids between human and mouse leukemic T cells indicated that in the leukemic cells of DeF, the breakpoint in chromosome 14 occurred between genes for the variable (V alpha) and the constant (C alpha) regions for the alpha chain of the T-cell receptor. The C alpha locus had translocated to a region more than 38 kilobases 3' to the involved c-myc oncogene. Since human c-myc transcripts were expressed only in hybrids carrying the 8q+ chromosome but not in hybrids containing the normal chromosome 8, it is concluded that the translocation of the C alpha locus 3' to the c-myc oncogene can result in its transcriptional deregulation.

A woman with thrombocytopenia that progressed to aplastic anemia, and her brother, who had persistent thrombocytopenia, both had a constitutional t(13;14) translocation. Six other family members, in three generations, had the same translocation, but no hematologic disorder. There was evidence suggestive of increased chromosomal fragility in lymphocyte cultures from two members of the kindred, but not in the two patients. The findings support a postulated association between the t(13;14) and hematologic disorders, but whether the mechanism involves an inherited defect in chromosomal stability is unproved.

Human leukemic T cells carrying a t(10;14)(q24;q11) chromosome translocation were fused with mouse leukemic T cells, and the hybrids were examined for genetic markers of human chromosomes 10 and 14. Hybrids containing the human 10q+ chromosome had the human genes for terminal deoxynucleotidyltransferase that has been mapped at 10q23-q25 and for C alpha [the constant region of TCRA (the alpha-chain locus of the T-cell antigen receptor gene)], but not for V alpha (the variable region of TCRA). Hybrids containing the human 14q- chromosome retained the V alpha genes. Thus the 14q11 breakpoint in the t(10;14) chromosome translocation directly involves TCRA, splitting the locus in a region between the V alpha and the C alpha genes. These results suggest that the translocation of the C alpha locus to a putative cellular protooncogene located proximal to the breakpoint at 10q24, for which we propose the name TCL3, results in its deregulation, leading to T-cell leukemia. Since hybrids with the 10q+ chromosome also retained the human terminal deoxynucleotidyltransferase gene, it is further concluded that the terminal deoxynucleotidyltransferase locus is proximal to the TCL3 gene, at band 10q23-q24.

We hybridized neoplastic cells from a patient with chromic lymphocytic leukemia of the B-cell type, which carried a reciprocal chromosomal translocation between chromosomes 11 (q13) and 14 (q32) with mouse plasmacytoma cells. The hybrid cells were studied for the presence, rearrangement, and expression of the human immunoglobulin mu chain locus. The results indicate that the expressed mu chain gene is located on the normal chromosome 14, whereas the 14q+ translocation chromosome carries the excluded immunoglobulin constant (C) region mu chain allele (C mu) but does not contain variable (V) region heavy chain genes (VH). Since we found that the heavy chain joining region DNA (JH) of the excluded mu chain gene is on the 14q+ chromosome, we can conclude that the chromosomal break observed in the leukemic cells occurred in a chromosomal region within or 5' of the JH region. With these results, it is logical to postulate that a gene, for which we suggest the name bcl-1, is located on band q13 of chromosome 11 and is activated by its translocation into close proximity with the rearranged heavy chain locus on chromosome 14q+, contributing to the neoplastic transformation of the B cells with the t(11;14) chromosomal translocation.

We have detected and cloned two rearrangements in the T-cell receptor alpha locus from a clone of somatic cell hybrids carrying a t(14;14)(q11;q32) chromosomal translocation derived from an ataxia telangiectasia patient with T-cell chronic lymphocytic leukemia. The T-cell clone carrying the t(14;14) chromosomal translocation was known to be present for greater than 10 years before the onset of overt leukemia. One molecular rearrangement of the T-cell receptor alpha locus corresponded to a functional variable-joining region (V-J) joining, whereas the other derived from the breakpoint of the t(14;14)(q11;q32) translocation. Chromosomal in situ hybridization of the probe derived from the t(14;14) breakpoint localized the breakpoint region to 14q32.1, apparently the same region that is involved in another ataxia telangiectasia characteristic chromosome translocation, t(7;14)(q35;q32). The 14q32.1 breakpoint is at least 10,000 kilobase pairs (kbp) centromeric to the immunoglobulin heavy chain locus. Sequence analysis of the breakpoint indicates the involvement of a J alpha sequence during the translocation. Comigration of high-molecular weight DNA fragments involved with t(7;14) and t(14;14) translocations suggests the presence of a cluster of breakpoints in the 14q32.1 region, the site of a putative oncogene, TCL1.

Among 46 patients with chronic lymphocytic leukemia (CLL) (40 B cell, 6 T cell) and 40 patients with cutaneous T cell lymphoma (CTCL), a chromosomally abnormal neoplastic clone was identified in 43 cases. A translocation involving 14q32 was present in nine cases (five B-CLL, two T-CLL, two CTCL). The donor chromosomal site was 11q13 in four patients and 1q12, 4q25-27, 17q21-22, 18q21, and 22q11 in one case each. The next most frequent abnormalities were rearrangements involving 6q21-23 (four cases), and trisomy 12 (four cases, all B-CLL). In one CTCL patient, the t(11;14) translocation was present in one of three apparently unrelated T cell clones. Recent studies indicate that the selective advantage conferred by the 14q+ chromosome in B cell neoplasms appears to result from an oncogene being brought adjacent to a rearranged and transcriptionally active immunoglobulin heavy chain locus. The present findings suggest that similar mechanisms may operate in certain T cell neoplasms, although the activating gene is not necessarily the same.

Primary cultures and established cell lines derived from human T-cell leukemias were analyzed for genomic rearrangements in the region 3' of the MYC locus. A T-cell leukemia line, HUT 78, whose 3' MYC region is rearranged, carries a chromosome t(2;8) juxtaposition; i.e., a locus derived from chromosome region 2q34 is attached to the 3' end of one MYC allele. The t(2;8) rearrangement in the HUT 78 cell line results in expression of a fused transcript encompassing the MYC gene and a locus designated TCL4 (T-cell leukemia/lymphoma 4), which normally resides on chromosome 2. The steady-state level of MYC-TCL4 fusion transcripts in HUT 78 cells is significantly higher than the MYC RNA level found in several other B- and T-cell lines. The production of fused MYC-TCL4 transcripts in a leukemic cell line raises the possibility that other B- and T-cell leukemias may express MYC fusion transcripts as an integral step in their pathogenesis.