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• W2071714652 abstract "Translocation of the mixed-lineage leukaemia gene (MLL) on chromosome 11q23 is associated with 80% of infant acute lymphoblastic leukaemia (ALL) cases (Pui et al, 2003; Kosaka et al, 2004). MLL rearrangements typically result in the amino-terminal portion of MLL being fused to the carboxyl portion of one of more than 50 different partner proteins (Meyer et al, 2006). Despite intensified treatment, infant ALL patients with MLL translocations do extremely poorly, regardless of translocation partner (Pieters et al, 2007). A better understanding of this particularly aggressive disease is essential for improvements in its treatment and prognosis. Xenografts using cell lines or primary patient material in immunocompromised mice are established as biologically relevant systems to study ALL of infancy and childhood (e.g. Pocock et al, 1995; Uckun et al, 1998) and continuous xenografts in non-obese diabetic severe combine immunodeficient (NOD/SCID) mice promise to be a useful adjunct as they also retain the cytological, immunophenotypic and chemosensitivity profiles of the original patient ALL samples, while providing a renewable source of patient cells (Liem et al, 2004 and references therein). Here we report effective and rapid engraftment of leukaemic cells from an infant ALL patient using the NOD/SCID model. Detailed examination of the cells derived from serial xenograft samples and comparison with patient samples revealed a complex translocation involving MLL and loci on chromosomes 2, 13 and 19. A 2-month-old infant girl presented with rapid onset of pallor, cutaneous petechiae, fever and tachypnoea associated with massive splenomegaly below the umbilicus. Blood examination showed haemoglobin 37 g/l, platelet count 13 × 109/l and white blood cell count 564 × 109/l (100% lymphoblasts) and ALL was diagnosed. Immunophenotyping of peripheral blood (PB) revealed expression of CD19, CD34 and TdT but CD10 was negative. Karyotype of PB blasts revealed 49,XX,+X,+X,+6[5]/46,XX[16]. Following leucopheresis, induction therapy using vincristine, prednisone, daunomycin and l-asparaginase according to Arm B of the Children’s Cancer Group protocol CCG1901 (Steinherz et al, 1993) was commenced, with morphological complete remission (CR) attained on day 28. Consolidation I and II were followed by allogeneic bone marrow transplant (BMT). Bone marrow relapse occurred 1 year later and re-induction according to protocol CCG1882 (Nachman et al, 1998) achieved CR2 by day 35. Twelve months maintenance therapy was followed by a second BMT at 2.75 years of age using the same donor. The subject remains in CR2 126 months with developmental delay and growth failure requiring growth hormone, but with a Karnofsky score of 100. The course of disease in this patient prompted investigation of chromosomal translocations involving MLL. Due to limited availability of patient material, initial investigations utilized patient leukaemia cells passaged in NOD/SCID mice. The studies were approved by the Animal Care and Ethics Committee of the University of New South Wales and the Hospital Institutional Review Board and informed consent was obtained from the patient’s parents. Bone marrow cells isolated at diagnosis (4.5 × 106) were engrafted in NOD/SCID mice as described previously (Liem et al, 2004). Engraftment was rapid, with 3–5% human cells detected in peripheral blood within 14 d and distinct infiltration of multiple organs, including brain. This represented the most rapid engraftment observed of any ALL cells previously tested in this model (Liem et al, 2004). For secondary and tertiary passages, 1 × 106 cells prepared from bone marrow or spleen again showed rapid engraftment by day 18–25, demonstrating reproducible propagation of patient cells. Following tertiary engraftment, cells derived from spleens showing 99% human leukaemic cell infiltration were processed for chromosome harvesting and karyotyping. Analysis of interphase and metaphase preparations by fluorescent in-situ hybridization (FISH) using the MLL break-apart probe, together with microscopic examination of the G-banded karyotype, revealed two displaced 3′MLL signals at chromosomal positions 2q37 and 13q32, indicating complex rearrangement of MLL (Fig 1A and B). Subsequent FISH using specific subtelomeric probes confirmed transfer of 11q material to chromosome 13q32 and revealed transfer of 13q material to 2q37, 2q material to 19p13.3 and 19p material to 11q23 (Fig 1C and D). Identification of a complex MLL translocation in xenografted patient sample. (A) G-banded karyotype of xenografted patient cells. Cells were cultured overnight in the presence of 125 ng/ml colcemid (GIBCO-BRL Karyomax, Invitrogen) and 37.5 ng/ml BrdU (Sigma) before fixation and visualization under a Leica DMLB microscope equipped with ×100 oil immersion lens. Images were acquired using CytoVysion v3.7 software (Applied Imaging, Santa Clara, CA). (B–D) Representative FISH images showing complex MLL translocation, visualized using an Olympus BX51 fluorescence microscope. (B) Dual-color FISH analyses were performed using a MLL dual-color break-apart rearrangement probe consisting of a 350 kb portion centromeric of the MLL breakpoint cluster region (bcr) labelled in SpectrumGreen and an approximately 190 kb portion spanning and extending telomeric of the bcr labeled in SpectrumOrange (Vysis, Abbott Laboratories, IL). (C) The TelVysion 11q subtelomere probe produces orange signals on the normal chromosome 11 and der(13). The PTEL13QG (13q subtelomere) probe produces green signals on the normal chromosome 13 and der(2). (D) The TelVysion 2q subtelomere probe produces an orange signal on the normal chromosome 2 and another on der(19). The TelVysion 19p subtelomere probe produces green signals on the normal chromosome 19 and der(11). (E) Diagram indicating derivative chromosomes present in clones 1 and 2. In clone 1 of the patient specimen, a reciprocal translocation between chromosomes 11 and 13 with disruption of a 3′ portion of MLL would account for the two apparently intact MLL fusion signals and the displaced 3′MLL signal observed with the break-apart probe (not shown here; see text). It is likely that clone 2 arose from a 3-way exchange of material between chromosome 2, chromosome 19 and the derivative chromosome 11 of the t(11;13) translocation from clone 1. This would have resulted in a second disruption, within the MLL bcr, resulting in fusion between 5′MLL and the 19p13.3 locus. (F) Partial sequence of fusion transcript indicating the identified MLL-MLLT1 fusion protein junction sequence. Primers binding to mRNA sequences of MLL and MLLT1 were used in polymerase chain reaction (PCR) reactions to test for expression of fusion transcripts. Following nested PCR and sequencing, an in-frame fusion of MLL exon 10 to MTTL1 exon 2 was identified. These findings were consistent with subsequent FISH analysis of the primary patient sample at diagnosis, for which only cells at interphase were available for hybridization with the MLL probe. In a small proportion of cells (12/100, data not shown), two apparently intact MLL fusion signals and an additional displaced 3′MLL signal were observed and the karyotype for this clone is 46,XX,t(11;13)(q23;q32) (clone 1, see Fig 1E). Extensive examination by interphase FISH with the MLL break-apart probe failed to detect the presence of this clone in xenograft cells, suggesting that this clone was unable to engraft. However, the majority of primary diagnosis cells (88/100) demonstrated split MLL signals on chromosomes 2 and 13, identical to that observed in the xenograft. We propose that the clone observed in the majority of patient cells at diagnosis and in the xenograft (clone 2), involving translocation of four chromosomal segments, actually arose through the evolution of clone 1 via 3-way exchange of material between chromosome 2 at band q37, chromosome 19 at band p13.3 and the derivative chromosome 11 at band q23 of the t(11;13) (Fig 1E). The karyotype for the major patient clone (clone 2) and xenografted cells is therefore 46,XX,der(2)(2pter->2q37::11q23->q23::13q32->qter),der(11)(11pter->q23::19p13.3->pter),der(13)(13pter->13q32::11q23->qter),der(19)(2qter->q37::19p13.3->qter). Despite the complex karyotype, it could be deduced that the amino-terminal portion of MLL was probably fused to a gene product of the 19p13.3 locus. Indeed, using cDNA derived from patient xenograft material we detected an in-frame fusion between MLL and MLLT1 (Fig 1F). While MLLT1 at 19p13.3 is a relatively common fusion partner of MLL and the 2q37 breakpoint observed here has been reported before in MLL translocations, involvement of the 13q32 locus has not (Meyer et al, 2006). Hence the significance of the initial t(11;13) detected in patient clone 1 is unclear, although its failure to engraft in NOD/SCID mice may reflect inherent biological differences between the two leukaemic clones. This report highlights the merit of utilizing a NOD/SCID xenograft model together with direct examination of patient material to identify novel and complex translocations involving MLL. Such complex rearrangements are important to our understanding of infant ALL, yet may be missed in routine clinical investigations. The authors thank Laura Piras for co-ordination of patient bone marrow sample storage. This work was supported by the National Health and Medical Research Council, The Cancer Council New South Wales, The Leukaemia Foundation, the Anthony Rothe Memorial Trust and The Children’s Leukaemia and Cancer Research Foundation, WA." @default.
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• W2071714652 title "A xenograft model of infant leukaemia reveals a complex MLL translocation" @default.
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