The RCK gene associated with t(11;14) translocation is distinct from the MLL/ALL-1 gene with t(4;11) and t(11;19) translocations.

We previously demonstrated that the 11q23 breakpoint region, designated the RCK locus, of the RC-K8 B-lymphoma cell line with t(11;14)(q23;q32) is centromeric to PBGD, while breakpoints of infantile leukemia cell lines with t(11;19)(q23;p13) are detectable by pulsed-field gel electrophoresis with the CD3D probe. In the present study, using a probe within 1.0 kilobase of the t(11;14) breakpoint, we isolated a partial complementary DNA clone for the putative RCK gene, which detects a 7.5-kilobase mRNA. Sequence analysis predicted a novel protein of 472 amino acids which demonstrated sequence homology to a translation initiation factor/helicase family. We also isolated a phage clone from the CD3D/G yeast artificial chromosome clone (yB22B2) which detects 11- and 12-kilobase mRNAs, most likely for the MLL/ALL-1 gene associated t(4;11)(q21;q23) and t(11;19)(q23;p13) translocations. By pulsed-field gel electrophoresis after NotI digestion, this recombinant clone is on a 96-kilobase fragment, while RCK and PBGD probes are on a more telomeric 690-kilobase NotI fragment. These results, altogether, suggested that two different genes, RCK and MLL/ALL-1, are associated with 11q23 translocation of hematopoietic tumors.     

Molecular cloning of the chromosomal breakpoint of a B-cell lymphoma with the t(11;14)(q23;q32) translocation

The breakpoint of t(11;14)(q23;q32) chromosome translocation in a B-cell lymphoma line, RC-K8, was cloned. Immunoglobulin heavy chain (IGH) constant gene, C gamma 2 at the 5' end, was involved in this translocation, and the DNA segment juxtaposed to the C gamma 2 was proved to be derived from chromosome 11 by somatic cell hybrid study. The normal counterpart of chromosome 11 was also isolated. With a DNA probe near the breakpoint of chromosome 11, Southern blot analysis of RC-K8 and 10 other cases with translocation involving the 11q23 region was conducted, but no rearrangement bands have been observed thus far except for RC-K8.     

The oncogene ETS1 is distant from the chromosome 11 breakpoint in leukaemic cells with the t(11;19)(q23;p13)

Field inversion gel electrophoresis of DNA from a leukemic clone characterized by the t(11;19)(q23;p13) allowed us to exclude any genomic rearrangement within more than 900 kb of DNA encompassing ETS1 on chromosome 11. Although ETS1 was moved to the derivative chromosome 19 as a result of the t(11;19), we conclude that this oncogene is not close to the chromosome 11 breakpoint and is unlikely to be involved in this leukaemia.     

Molecular Cloning of 19p13 Breakpoint Region in Infantile Leukemia with t(11;19)(q23;p13) Translocation

We studied the breakpoint regions involved in t(11;19)(q23;p13) translocation associated with infantile leukemias. Southern blot analysis with the partial cDNA clone for the MLL gene at 11q23 which we had isolated previously detected gene rearrangements in all three cell lines and three leukemia samples from the patients with t(11;19) translocation, indicating that these breakpoints were clustered within the 8.5 kb Bam HI germline fragment detected by the probe. To study the breakpoint region, a genomic library of one of the cell lines, KOCL-33, was made. We have isolated the der(19) allele containing the breakpoint as well as the germline alleles at 19p13 and 11q23. Using the genomic probes on chromosome 19 near the breakpoint, Southern blot analysis was performed. The breakpoints at 19p13 of the two other cell lines and the three leukemia samples were not located within 36 kilobases of the KOCL-33 breakpoint, although pulsed-field gel electrophoresis showed that the breakpoints of all three cell lines were on the same Nru I fragment of 230 kilobases. These results showed that the breakpoints at 19p13 were not clustered like those at 11q23 in t(11;19) translocation.     

Molecular characterization of a t(11;14)(q23;q32) chromosome translocation in a B-cell lymphoma

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).     

A t(8;14)(q24;q11) translocation in a T-cell leukemia (L1-ALL) with c-myc and TcR-alpha chain locus rearrangements

Cell lines established from T-cell leukemias have recently been reported to exhibit a chromosome translocation t(8;14) involving proto-oncogene c-myc and the gene of the T-cell receptor alpha-chain(TcR-alpha). In this work, we have studied a case of T-cell leukemia presenting a t(8;14)(q24;q11) translocation that was found in fresh leukemic cells taken during relapse, but was absent in cells collected at diagnosis. Hybridization analysis showed that the breakpoint on chromosome 8 was located 3' to the c-myc exon 3. A TcR-alpha-specific original probe (D14S7, Mathieu-Mahul et al., 1985) revealed two differently rearranged patterns in DNA from leukemic cells obtained at diagnosis and during relapse. In contrast, the rearranged TcR-beta-gene DNA pattern did not change during the course of the disease, indicating that leukemic cells were clonally related. These data indicate that the chromosome breakpoint in 14q11 is situated in the TcR-alpha locus. These results suggest that translocations t(8;14) involving TcR-alpha and c-myc genes in T-cell malignancies are analogous to variant t(2;8) and t(8;22) translocations observed in Burkitt lymphoma. They also establish that the same types of molecular rearrangements due to a t(8;14)(q24;q11) translocation, at first described in T-cell lines established in culture, also exist in vivo and may play a role in the evolution of the leukemic process.     

Cloning of the t(11;14)(q13;q32) translocation breakpoints from two human leukemia cell lines

The t(11;14)(q13;q32) translocation has been associated with several subtypes of human leukemia and lymphoma. It has been proposed that this translocation activates a proto-oncogene designated BCL1. In an effort to better understand the mechanism by which this translocation leads to malignancy, we have studied this translocation in two human cell lines. MO1094 and MO2058 were derived from patients with prolymphocytic variants of chronic lymphocytic leukemia. Southern blotting of the MO2058 cell line documented that the translocation linked the Jh region in the immunoglobulin heavy chain gene to the previously described BCL1 major translocation cluster (MTC). Using the polymerase chain reaction, we cloned this translocation and showed that the chromosome 11 breakpoint was within 7 bp of two other samples reported previously. Southern blotting of the MO1094 cell line suggested that the translocation in this cell line might link Jh sequences to a new region in the BCL1 locus on chromosome 11. Therefore, the MO1094 breakpoint was cloned from a genomic library. Comparison with normal cloned DNA from the BCL1 locus showed that the chromosome 11 breakpoint occurred 24 kb telomeric of the MTC. This work reinforces the concept that translocation breakpoints in the BCL1 locus are scattered over at least 63 kb.     

Molecular analysis of a t(9;14)(p11;q32) translocation occurring in a case of human alpha heavy chain disease

We performed the cloning and sequencing of the der(14) breakpoint of a new chromosomal translocation involving the 14q32 immunoglobulin locus. This t(9;14)(p11;q32) translocation was found in a case of malignant lymphoma occurring in human alpha heavy chain disease. A rearranged alpha 1 gene fragment was cloned and shown to contain chromosome 9 information by Southern blotting on sorted chromosomes and by in situ hybridization. Sequence analysis of the junction point region established that the breakage occurred 3' to the heavy chain joining region. In contrast to the data obtained in other translocations affecting 14q32 immunoglobulin locus, the recombination did not involve the immunoglobulin heavy chain locus specific recombination signals on chromosome 14, or homologous sequences on chromosome 9. In the present case, the existence of two almost perfect inverted repeats flanking the junction point suggests that the translocation originated from a local pairing of the two chromosomes 9 and 14. Chromosome 9 fragments sequenced in the vicinity of the breakpoint did not share significant homology with sequences listed in GenBank and EMBL data bases.     

The T-ALL specific t(11;14)(p13;q11) translocation breakpoint cluster region is located near to the Wilms' tumour predisposition locus

A breakpoint cluster region (T-ALLbcr) has been previously described on 11p13 for T-ALL carrying t(11;14)(p13;q11). One further T-ALL breakpoint is described bringing to 5 out of 6 such translocations which are found to break within a maximum of 6.7 kb on chromosome 11p13. Studies of somatic cell hybrids derived from t(11;14)(p13;q11) T-ALL placed the T-ALLbcr between the genes for catalase (CAT) and the beta-subunit of follicle stimulating hormone (FSHB). This suggested a link between the T-ALLbcr and the Wilms' tumour predisposition locus (WT) since constitutional 11p13 deletions predispose to Wilms' tumour. Utilising somatic cell hybrids from patients with Wilms' tumours and aniridia, we show that while the T-ALLbcr maps distal to the catalase gene at 11p13, it maps outside the shortest region of overlap of a series of 11p13 deletions associated with Wilms'-Aniridia. The data suggest the order of genes at 11p13 to be: centromere-CAT-T-ALLbcr-WT-aniridia-FSHB-telomere. Therefore, the T-ALLbcr must lie very close to but may be distinct from the Wilms' predisposition locus at 11p13.     

Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(11;14)(q13;q32) translocation.

The proto-oncogene PRAD1 (parathyroid adenoma 1) on chromosome 11q13 was found to be overexpressed in all five B-cell lines with t(11;14)(q13;q32) translocation tested. One B-cell lymphoma and four myeloma cell lines with this translocation demonstrated more than 10-fold overexpression as determined by Northern blot analysis, when compared with normal lymphoid tissues such as thymus, spleen and lymph node. Hematopoietic cell lines without the translocation were also examined, but none of these demonstrated the overexpression, confirming that overexpression of the PRAD1 gene is associated with t(11;14) translocation. A truncated form of mRNA was seen in one of five cell lines with the translocation, SP-49. Hybridization with different regions of the PRAD1 cDNA revealed that the truncated form of mRNA retained the coding region but had lost the 3' untranslated region. Southern blot analysis demonstrated a gene rearrangement in this SP-49 cell line. To study the genetic alteration responsible for the truncated form of mRNA in this cell line, the rearranged allele as well as the germline allele were cloned. The restriction map revealed that the rearranged portion was at the 3' end of the PRAD1 gene, eliminating the mRNA-destabilizing signal AUUUA. Human-rodent hybrid cell analysis demonstrated that the region introduced 3' of PRAD1 was derived from chromosome 11, suggesting that the PRAD1 gene region is deleted at the 3' end. Over-expression of the PRAD1 gene in association with t(11;14)(q13;q32) translocation suggested that in these cases the regulation of PRAD1 was altered by the juxtaposed gene, most likely the immunoglobulin heavy-chain gene from chromosome 14.     

Acute myeloid leukemia with t(5;11): two case reports.

A case of acute monocytic leukemia (AMoL) with t(5;11)(q31;q23) and a case of acute myelomonocytic leukemia (AMMoL) with t(5;11)(q35;q13.1) are reported. The translocation between the long arm of chromosome 11q and that of chromosome 5q with leukemia have been rarely reported. Though breakpoint of both cases were subtlety different, they had morphologically monocytic character and showed hyperleukocytosis and chemoresistance.     

Chromosomal localization of the human G-CSF gene to 17q11 proximal to the breakpoint of the t(15;17) in acute promyelocytic leukemia

The G-CSF gene encodes a hematopoietic colony-stimulating factor (CSF) that promotes growth, differentiation, and survival of neutrophilic granulocytes. By analysis of somatic cell hybrids and in situ chromosomal hybridization, we localized this gene to human chromosome 17, at bands q11 to q12, the region of the breakpoint on chromosome 17 in the 15;17 translocation [t(15;17)] characteristic of acute promyelocytic leukemia. To determine the position of the G-CSF gene in relation to the breakpoint junctions and to evaluate the possible role of G-CSF in the pathogenesis of promyelocytic leukemia, we applied the techniques of in situ chromosomal hybridization and Southern blot analysis to leukemia cells from eight acute promyelocytic leukemia patients who had a t(15;17). Our results indicate that the G-CSF gene is proximal to the breakpoint of the t(15;17) and that this gene remains on the rearranged chromosome 17. Southern blot analysis using conventional and pulsed-field gel electrophoresis did not reveal rearranged restriction fragments, indicating that no rearrangements had occurred within the G-CSF gene or in surrounding sequences.     

MLL-AF1q fusion resulting from t(1;11) in acute leukemia.

The MLL gene, located on chromosome band 11q23 is fused to different partner genes as a result of various chromosomal translocations in hematopoietic malignancies. A t(1;11) (q21;q23) resulting in a MLL-AF1q fusion gene has previously been reported. Cytogenetic studies on six cases are reported, including one three-way translocation. FISH analysis using a YAC encompassing the MLL gene and a YAC encompassing the AF1q locus showed splitting in three cases and two patients, respectively. PCR analysis of two cases confirmed that AF1q is specifically associated with t(1;11)(q21;q23). The MLL-AF1q fusion mRNA was similar to that previously described in one case and involved MLL exon 7 in the other. This study confirms the specific involvement of AF1q in t(1;11) (q21;q23)-positive acute leukemia with monocytic involvement.     

Deregulation of the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) gene in a B-cell chronic lymphocytic leukemia with a t(12;14)(q23;q32)

The t(12;14)(q23;q32) breakpoints in a case of B-cell chronic lymphocytic leukemia (B-CLL) were mapped by fluorescence in situ hybridization (FISH) and Southern blot analysis and cloned using an IGH switch-gamma probe. The translocation affected a productively rearranged IGH allele and the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) locus at 12q23, with a reciprocal break in intron 2 of the CHST11 gene. CHST11 belongs to the HNK1 family of Golgi-associated sulfotransferases, a group of glycosaminoglycan-modifying enzymes, and is expressed mainly in the hematopoietic lineage. Northern Blot analysis of tumor RNA using CHST11-specific probes showed expression of two CHST11 forms of abnormal size. 5'- and 3'-Rapid Amplification of cDNA Ends (RACE) revealed IGH/CHST11 as well as CHST11/IGH fusion RNAs expressed from the der(14) and der(12) chromosomes. Both fusion species contained open reading frames making possible the translation of two truncated forms of CHST11 protein. The biological consequence of t(12;14)(q23;q32) in this case presumably is a disturbance of the cellular distribution of CHST11 leading to deregulation of a chondroitin-sulfate-dependent pathway specific to the hematopoietic lineage.     

A novel recurrent translocation t(11;14)(p11;q32) in splenic marginal zone B cell lymphoma.

A novel recurrent translocation t(11;14)(p11;q32) was found in three patients with splenic marginal zone B cell lymphoma (MZBCL). Fluorescence in situ hybridization (FISH) studies with IgH probes revealed in all cases involvement of the IgH locus, with breakpoint downstream of the IGVH sequences. Partner genes at 11p11 were not identified. The translocation defined the stem line in two patients, who carried additional cytogenetic aberrations, including a 17p deletion, present in both cases. In one patient a 7q- chromosome was the primary cytogenetic defect, the t(11;14) having been found in four out of 11 abnormal metaphase cells at the time of transformation into high-grade MZBCL. Hematological features in all cases included splenomegaly with peripheral blood (PB) involvement by a monoclonal B cell population consisting of lymphocytes with villous projections and several blast-like cells. The immunophenotype was CD19+; CD22bright+; CD23-, CD10-, CD5-, surface Igbright+. A bone biopsy in one patient revealed an interstitial infiltration with an intrasinusoidal pattern of growth. Histological studies on spleen specimens in two patients showed an expanded marginal zone, with small lymphocytes and several blast-like cells. One patient had a therapy-demanding disease, with partial, short-term responses to cytotoxic treatment; one patient transformed into a high-grade MZBCL involving the gut, the PB and the bone marrow 2 years after diagnosis; one patient was unresponsive to cytotoxic treatment and underwent splenectomy. The t(11;14)(p11;q32) may define a subset of splenic MZBCL with a high-grade component and a relatively aggressive clinical behavior.     

Myxoid liposarcoma with t(12;16) (q13;p11) contains site-specific differences in methylation patterns surrounding a zinc-finger gene mapped to the breakpoint region on chromosome 12

The q13 to q15 region of human chromosome 12 is frequently and consistently rearranged in malignant and benign adipose tissue tumors as well as benign tumors of smooth muscle and salivary glands. A reciprocal translocation, (12;16) (q13;p11), is characteristic of the myxoid subtype of liposarcoma, whereas translocations within 12q13-14 are frequently observed in benign lipomas. We are using pulsed-field gel electrophoresis to study the 12q13-q14 region in order to detect and clone the respective translocation breakpoints in these tumors. The locus GLI, which encodes a zinc-finger protein, has been mapped to the same region as the myxoid liposarcoma breakpoint. Pulsed-field analysis of myxoid liposarcoma and lipoma DNA has allowed us to construct a 600-kilobase physical map surrounding the GLI locus, which shows that breakpoints in both types of tumor are outside this region. However, myxoid liposarcoma DNA samples contained altered restriction fragments detectable with GLI probes that were highly specific and reproducible from case to case. These altered fragments are due to highly specific and reproducible methylation differences that are unique to myxoid liposarcoma DNA. These methylation changes may prove to be useful clinically as a diagnostic tool to differentiate subtypes of liposarcoma.