Two new translocations involving the 11q23 region map outside the MLL locus in myeloid leukemias

BACKGROUND AND OBJECTIVES: In acute leukemias, chromosomal translocations involving the 11q23 band are frequently, but not invariably, associated with MLL gene rearrangement and their finding is associated with a poor prognosis. We observed two new translocations with a breakpoint in the 11q23 region at standard cytogenetic analysis: a previously undescribed t(3;11)(q21;q23) in a 70-year old woman with a fulminating form of AML-M1 and a new translocation t(6;11)(q15;q23) in a 61-year old man with an atypical chronic myelogenous leukemia. In these two patients, involvement of the MLL gene was analyzed by molecular cytogenetic techniques which also allowed a more precise mapping of the breakpoints. DESIGN AND METHODS: The MLL gene was analyzed by Southern blot and by fluorescent in situ hybridization (FISH) with a double-color MLL probe. A panel of 11q, 3q and 6q cosmid/YAC probes mapping around the breakpoints was used for breakpoint mapping. RESULTS: In both patients, FISH analysis and Southern blot showed that the MLL gene was not rearranged; in patient 1, MLL was retained on the 11q+ derivative, whereas in patient 2 it moved to the 6q- chromosome. In the t(3;11) we localized the chromosome 11 breakpoint at 11q23.3, in a region flanked by CP-939H3 and cos1p3, distal to the MLL locus; in the t(6;11) the break occurred at 11q21, in a region flanked by CP-819A5 and CP-829A6, proximal to the MLL locus. INTERPRETATION AND CONCLUSIONS: Our cases add two new translocations to the list of chromosomal anomalies involving the long arm of chromosome 11, and show that apparent translocation t(11q23) may involve loci and genes other than MLL. Characterizing the molecular heterogeneity of 11q23 translocations may identify some prognostic significance.     

急性白血病11q23/MLL基因易位重排及其临床意义

目的：研究11q23/MLL基因易位重排在急性白血病（AL）中的发生率、产生融合基因的常见类型及其临床意义.方法：用荧光原位杂交技术，MLL双色断裂分离重排探针检测50例AL患者（49例初治，1例难治）的11q23/MLL基因，用流式细胞仪检测免疫表型，对于11q23/MLL基因易位重排阳性的患者，用巢式RTPCR方法检测11q23/MLL基因易位重排形成的6种常见融合基因类型.结果：6例AL有11q23/MLL基因易位重排，发生率为12%，2例为AML-M5，4例为ALL且均为B-ALL.2例11q23/MLL基因易位重排阳性的AML M5患者融合基因均为MLL/AF9，其中1例为初治，发病时左侧小腿有白血病细胞浸润，本例患者化疗1疗程获CR;1例为难治性AL患者，于第3个疗程化疗后才达CR.4例11q23/MLL基因易位重排阳性的B-ALL患者中有2例于诊断后3周内死于全身衰竭和感染，化疗未获CR，其中1例患者的融合基因为MLL/ENL，1例未扩出融合基因产物;1例于诊断后第2天因DIC脑出血死亡，未进行化疗，其融合基因为MLL/AF9;1例发病时胸椎有白血病细胞浸润，1疗程化疗后获CR，其融合基因产物未扩出.结论：荧光原位杂交技术是检测AL11q23/MLL基因易位重排快速、灵敏的方法，巢式RT-PCR是检测11q23/MLL基因易位重排所产生的融合基因类型简便可行的方法;有11q23/MLL基因易位重排的AL患者临床症状凶险，预后差。     

A study on 289 consecutive Korean patients with acute leukaemias revealed fluorescence in situ hybridization detects the MLL translocation without cytogenetic evidence both initially and during follow-up

Translocations involving the MLL gene on the chromosome 11 (11q23) are frequently observed in acute leukaemia. The detection of this genetic change has a unique significance as a result of its implication of poor prognosis. To reveal the utility of fluorescence in situ hybridization (FISH) in detecting the MLL translocation, we analysed 289 consecutive Korean patients (children and adults) with acute leukaemias using both conventional cytogenetic analysis (CC) and FISH, placing an emphasis on the result discrepancies. Twenty-two of 289 patients (7.6%) had the 11q23/MLL translocation. In nine of 22 patients (41%), only FISH detected the translocation. In eight of these 22 patients, a total of 19 follow-up examinations were performed, of which FISH detected a significant level of leukaemic cells harbouring the MLL translocation in five patients (26%) without cytogenetic evidence. In addition to the MLL translocation, FISH detected submicroscopic amplification, partial deletion of the MLL gene and trisomy 11 in 12 patients without cytogenetic evidence. In summary, up to 41% of the MLL translocations at initial work-up and 26% during follow-up were detected by FISH without cytogenetic evidence. Thus, we recommend that MLL FISH should be performed in the diagnosis and monitoring of acute leukaemias in combination with CC.     

Absence of MLL gene rearrangement in de novo myelodysplastic syndromes (MDS)

The Mixed Lineage Leukemia (MLL) gene has been identified in 11q23 translocations. The aim of the present study is the investigation of the frequency of MLL gene rearrangements in cases of de novo myelodysplastic syndromes (MDS). Sixty-two patients with de novo MDS were included in the analysis. The detection of MLL gene rearrangements was performed by Southern blot. Clonal karyotypic abnormalities were found in 15/50 (30%) cases. 11q23 abnormalities were not detected. One case with RAEB and a complex karyotype presented a del (11)(q13); further analysis by FISH revealed loss of one copy of MLL gene in all metaphases. Southern blot revealed germline bands in all cases using Eco RI and in 61/62 cases with Bam HI. The case with RAEB and a del (11)(q13) revealed a rearranged band following only Bam HI digestion, but not Eco RI. Rearrangements of MLL gene within exons 5–9 were not identified in this series of adult de novo MDS, indicating that this molecular abnormality is not involved in the pathogenesis of this group of hemopoietic disorders.     

Variability of 11q23 rearrangements in hematopoietic cell lines identified with fluorescence in situ hybridization

We mapped and ordered 17 cosmid, phage, and plasmid clones to chromosome 11, bands q22-q24, using fluorescence in situ hybridization (FISH). We then analyzed four hematopoietic cell lines with 11q23 rearrangements, Karpas 45, SUP-T13, RC-K8, and Karpas 422, using these probes. The studies showed that the translocation breakpoints of the Karpas 45 and SUP-T13 cell lines, which were derived from T-cell malignancies, were located in the same breakpoint cluster region of the MLL gene as the RS4; 11 cell line and patients with the t(9;11), t(11;19), and t(6;11) described previously. We confirmed that the translocation breakpoint of the RC-K8 cell line was located telomeric to the MLL gene, and found that the derivative 11 chromosome of the Karpas 422 cell line, which had been thought to contain a t(4;11) (q21;q23), was in fact formed through a deletion and an inverted tandem repeat of part of 11q.     

t(14;22)(q32;q11) in non-Hodgkin lymphoma and myeloid leukaemia: molecular cytogenetic investigations

Two non-Hodgkin lymphomas (NHL), one chronic lymphocytic leukaemia/small lymphocytic lymphoma and one diffuse large B-cell lymphoma and three cases of myeloid leukaemia, two chronic (CML) and one acute (AML), showed, by G-banding analysis, apparently identical chromosomal translocations t(14;22)(q32;q11), in three of the cases as the sole abnormality. Fluorescence in situ hybridisation (FISH) analysis with locus-specific probes for ABL at 9q34 [bacterial artificial chromosomes (BACs) 835J22 and 1132H12], IGH at 14q32 [P1 artificial chromosome (PAC) 998D24] and IGL (PAC 1019H10) and BCR (BAC 74M14) at 22q11, as well as multicolour in situ hybridisation (M-FISH) analyses were performed. A three-way variant translocation of the classical t(9;22)(q34;q11), t(9;22;14)(q34;q11;q32), involving both BCR and ABL, was unravelled by the molecular cytogenetic investigations in the three myeloid leukaemia cases; a similar variant translocation has previously been reported in seven CML. The two cases of NHL (one NHL with a similar 14;22-translocation has been reported previously) had no involvement of BCR or ABL, but instead the IGH and IGL genes were shown to be juxtaposed by the t(14;22)(q32;q11). How such a rearrangement with recombination of IGH and IGL might elicit a pathogenetic effect is completely unknown.     

Therapy-Related Acute Myeloid Leukemia 6 Years after Clonal Detection of inv(11)(q21q23) and MLL Gene Rearrangement

Results of recent studies with animal models suggest that expression of MLL fusion proteins promotes acute leukemogenesis. However, the most potent MLL fusion proteins are not sufficient for the development of acute myeloid leukemia (AML). The clinical data on the pathogenesis of this type of leukemia are limited. We analyzed the case of a patient with therapy-related AML with MLL rearrangement. The patient initially developed AML with t(8;21). Although the patient achieved complete remission with chemotherapy, an abnormal karyotype, inv(11)(q21q23), was detected. After 6-year persistence of a clone with the inversion 11 karyotype in the bone marrow, secondary AML developed. Results of fluorescence in situ hybridization analysis combined with magnet-activated cell sorting analysis showed that MLL rearrangement was detected in CD34+ and CD13+ fractions but not in a CD3+ fraction of the bone marrow. There were 2 important clinical findings. One was that MLL rearrangement was not sufficient for the development of leukemia. The other was that MLL rearrangement targets specific lineages.     

Translocation t(9;22) (p23;q11) in atypical chronic myeloid leukemia (aCML) presenting osteolytic lesions

A 58-year-old man with a 4-month history of atypical chronic myeloid leukemia (aCML), treated with INF-alpha and hydroxyurea, presented with severe localized bone pain with involvement of upper limbs on July 17, 2000. Cytogenetic analysis of peripheral blood cells showed 46,XY,t(9;22)(p23;q11) and no BCR-ABL fusion gene was detected by fluorescence in situ hybridization (FISH). On October 30,2000, x-rays revealed extended destruction of the bilateral proximal upper limbs; pain in the femoral bones appeared in December, and the patient couldn't walk. Roentgenograms taken on January 4, 2001, showed diffuse lytic changes in bilateral femoral bones. On January 23, 2001, fixation of pending fractures in the bilateral femoral bones with an intramedullary rod had produced good results. The infiltration of immature myeloid cells was diagnosed by the histological findings of a bone specimen from the right femur. Because the serum levels of parathyroid hormone (PTH), PTH related protein, and calcitonin were normal, we considered that the bone destruction was caused by the invasion of immature myeloid cells. Four months later, the patient showed a marked increase in peripheral immature granulocytes. A bone marrow specimen showed blastic marrow, and he died of a brain hemorrhage. This report suggests that aCML might cause destructive bone lesions prior to the disease progression. To our knowledge, this is the first published case of aCML in which the chromosomal abnormality t(9;22)(p23;ql 1) was detected.     

11q23 Aberration is an additional chromosomal change in de novo acute leukemia after treatment with etoposide and mitoxantrone

We report on 2 patients with acute leukemia who had an 11q23 chromosomal aberration as an additional change after treatment with etoposide and mitoxantrone, agents that affect topoisomerase II (Topo II). One patient with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (L2) received chemotherapy, including 1,000 mg of etoposide and 75 mg of mitoxantrone. She relapsed 10 months later. Analysis at time of relapse showed a chromosomal aberration of del(11)(q23) as an additional cytogenetic change. The other patient was diagnosed with acute monoblastic leukemia (M5a) and received two autologous peripheral blood stem-cell transplantations. Her cumulative doses of etoposide and mitoxantrone were 6,000 mg and 42 mg, respectively. She also relapsed, and analysis at that time revealed del(11)(q23) as an additional chromosomal aberration. The mixed lineage leukemia/(myeloid-lymphoid leukemia (MLL) gene was not rearranged in either case, making these cases distinct from previously described therapy-related leukemias caused by Topo II Inhibitors. Based on these two cases, it may be that Topo II inhibitors can cause clonal evolution affecting chromosome band 11q23. © 1996 Wiley-Liss, Inc.     

A novel gene, AF1q, fused to MLL in t(1;11) (q21;q23), is specifically expressed in leukemic and immature hematopoietic cells

Translocations involving chromosomal band 11q23 are associated with leukemias. These translocations fuse the MLL, a gene with sequence homology to the Drosophila trithorax, to genes from a number of other chromosomal loci. We have characterized two t(1;11)(q21;q23) translocations that fuse the MLL gene to a novel gene, AF1q on chromosomal band 1q21, in two infants with acute myelomonocytic leukemia (AMMOL). In one of these patients, der(11) represents an inframe fusion of the N-terminal portion of MLL gene to the complete AF1q open reading frame, whereas der(1) does not give rise to an open reading frame. This observation suggests that the N-terminal portion of MLL gene is critical for leukemogenesis in translocations involving band 11q23. The predicted wild-type AF-1q product is a 9-kD protein with no similarity to any other protein in the data banks. The AF1q mRNA is highly expressed in the thymus but not in peripheral lymphoid tissues. In contrast to its restricted distribution in normal hematopoietic tissue, AF1q was expressed in all leukemic cell lines tested.     

The t(X;7)(q22;q34) in paediatric T-cell acute lymphoblastic leukaemia results in overexpression of the insulin receptor substrate 4 gene through illegitimate recombination with the T-cell receptor beta locus

The t(X;7)(q22;q34), a translocation not previously reported in a neoplastic disorder, was identified and molecularly characterised in a paediatric T-cell acute lymphoblastic leukaemia (T-ALL), subsequently shown also to harbour a deletion of 6q, a STIL/TAL1 fusion and an activating NOTCH1 mutation. The t(X;7) was further investigated using fluorescence in situ hybridisation (FISH), real-time quantitative polymerase chain reaction (RQ-PCR) and Western blot analyses. FISH revealed a breakpoint at the T-cell receptor beta locus at 7q34 and mapped the corresponding breakpoint to Xq22.3. The latter region contains only two known genes, namely insulin receptor substrate 4 ( IRS4 ) and collagen, type IV, alpha 5 ( COL4A5 ), the expressions of which were analysed by the use of RQ-PCR. COL4A5 was not differentially expressed in the t(X;7)-positive sample compared to five T-ALL controls. However, a marked, 1000-fold overexpression of IRS4 was identified. Western blot analysis with a monoclonal antibody against IRS4 showed overexpression also at the protein level. Considering that forced expression of several members of the IRS family has been shown to result in increased cell proliferation, for example in haematopoietic cells, we hypothesise that the IRS4 up-regulation in T-ALL is pathogenetically important as a mitogenic stimulus.