Ian D. Dubé

The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein

The recurrent translocation t(8;16)(p11 ;p13) is a cytogenetic hallmark for the M4/M5 subtype of acute myeloid leukaemia. Here we identify the breakpoint-associated genes. Positional cloning on chromosome 16 implicates the CREB-binding protein (CBP), a transcriptional adaptor/coactivator protein. At the chromosome 8 breakpoint we identify a novel gene, MOZ, which encodes a 2,004-amino-acid protein characterized by two C4HC3 zinc fingers and a single C2HC zinc finger in conjunction with a putative acetyltransferase signature. In-frame MOZ–CBP fusion transcripts combine the MOZ finger motifs and putative acetyltransferase domain with a largely intact CBP. We suggest that MOZ may represent a chromatin-associated acetyltransferase, and raise the possibility that a dominant MOZ–CBP fusion protein could mediate leukaemogenesis via aberrant chromatin acetylation.

The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9.

The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated primarily with acute myeloid leukaemia (FAB M2 and M4). We present here the molecular definition of this translocation. On chromosome 7 positional cloning revealed the consistent rearrangement of the HOXA9 gene, which encodes a class I homeodomain protein potentially involved in myeloid differentiation. On chromosome 11 the translocation targets the human homologue of NUPP98, a member of the GLFG nucleoporin family. Chimaeric messages spliced over the breakpoint fuse the GLFG repeat domains of NUP98 in-frame to the HOXA9 homeobox. The predicted NUP98–HOXA9 fusion protein may promote leukaemogenesis through inhibition of HOXA9-mediated terminal differentiation and/or aberrant nucleocytoplasmic transport.

Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkins lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38β-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.

Diagnosis of acute promyelocytic leukaemia by RT-PCR: detection of PML-RARA and RARA-PML fusion transcripts

Summary. Acute promyelocytic leukaemia (APL; AML M3) is identified by a unique t(15;17) translocation which fuses the PML gene to the retinoic acid receptor alpha gene (RARA). Reverse transcription coupled with the polymerase chain reaction (RT‐PCR) has been used to develop a diagnostic test for APL based on the PML‐RARA fusion message. Separate PCR assays were designed to amplify either PML‐RARA (15q+ derived) or RARA‐PML (17q‐ derived) chimaeric transcripts. PML‐RARA transcripts were detected in every case from a series of 18 APL patients with cytogenetically confirmed t(15;17) translocations, whereas RARA‐PML messages were detected in only 67% (12/18) of these patients. This suggests that it is the 15q + derivative which mediates leukaemogenesis. Furthermore the PCR approach (or Southern analysis) may be used to identify in which of the alternative PML introns the breakpoint occurs; 52% of cases (15/29 patients) utilize a 5′ PML intron and 48% the 3′ intron (14/29 cases). Neither the choice of PML intron nor the expression of the 17q‐derivative could be correlated with the microgranular variant of APL (M3V), overall survival rate, age, sex or presence of coagulopathy. Finally, the fusion message is undetectable in five remission samples. This indicates a possible use for RT‐PCR in monitoring remission patients for evidence of relapse.

Characterization and developmental expression of Tlx-1, the murine homolog of HOX11

HOX11, a human homeobox gene with putative oncogenic potential, was originally discovered at the chromosome 10 breakpoint in T-cell acute lymphoblastic leukemias bearing the chromosomal translocation t(10;14)(q24;q11). To provide insight into the possible roles of this gene in development, we isolated and characterized its murine homolog, Tlx-1, and examined its profile of expression. Tlx-1 transcripts are first detected at E8.5 in the surface ectoderm and central mesenchyme of the first branchial arch. This expression subsequently extends to the 2nd, 3rd, and 4th branchial arches, as well as the presumptive pharynx, as these structures develop. Between E12.5 and E15.5, the profile of Tlx-1 expression becomes more complex; expression is observed in the developing pancreas and salivary glands, as well as in several components of the nervous system, including the trigeminal, glossopharyngeal and vestibulocochlear ganglia, the spinal cord, and the curvature of the pons-medulla. In addition, expression is seen in the pinna and external auditory meatus of the outer ear, the tooth primordia, and specific cell populations of the mandible and tongue. These complex patterns of expression are consistent with multiple and varied roles for Tlx-1 in development and suggest that Tlx-1 marks, amongst other cell populations, structures derived from cranial neural crest cells and migratory paraxial mesoderm that arise at corresponding levels along the rostral-caudal axis of the developing embryo.

Deregulation of NPM and PLZF in a variant t(5;17) case of acute promyelocytic leukemia.

Greater than 95% of acute promyelocytic leukemia (APL) cases are associated with the expression of PML-RARα. This chimeric protein has been strongly implicated in APL pathogenesis because of its interactions with growth suppressors (PML), retinoid signaling molecules (RXRα), and nuclear hormone transcriptional co-repressors (N-CoR and SMRT). A small number of variant APL translocations have also been shown to involve rearrangements that fuse RARα to partner genes other than PML, namely PLZF, NPM, and NuMA. We describe the molecular characterization of a t(5;17)(q35;q21) variant translocation involving the NPM gene, identified in a pediatric case of APL. RT – PCR, cloning, and sequence studies identified NPM as the RARα partner on chromosome 5, and both NPM-RARα and RARα-NPM fusion mRNAs were expressed in this patient. We further explored the effects of the NPM-RARα chimeric protein on the subcellular localization of PML, RXRα, NPM, and PLZF using immunofluorescent confocal microscopy. While PML remained localized to its normal 10 – 20 nuclear bodies, NPM nucleolar localization was disrupted and PLZF expression was upregulated in a microspeckled pattern in patient leukemic bone marrow cells. We also observed nuclear co-localization of NPM, RXRα, and NPM-RARα in these cells. Our data support the hypothesis that while deregulation of both the retinoid signaling pathway and RARα partner proteins are molecular consequences of APL translocations, APL pathogenesis is not dependent on disruption of PML nuclear bodies.

Regional cancer cytogenetics: A report on 1,143 diagnostic cases

The results of studies from a regional cancer cytogenetics diagnostic service are reported. In a 10-year period, 1,143 marrow samples from patients with newly diagnosed leukemia and myelodysplastic syndrome were referred. Successful studies were completed on 992 cases (87%). Among all referred cases, the rates of detection of cytogenetically abnormal clones were 95% for chronic myelogenous leukemia (CML), 54% for acute lymphoblastic leukemia (ALL), 51% for acute myeloid leukemia (ANLL), and 43% for myelodysplastic syndrome (MDS). Of 169 cases of CML studied, 90.5% bore the standard Philadelphia chromosome (Ph), 3.55% had an unusual Ph, and 5.33% were Ph-negative. Among the 59 cases of cytogenetically abnormal MDS, common abnormalities observed were trisomy 8 and changes resulting in loss of material from the long arm of chromosomes 5 and 7, and 20q-. Of the 168 abnormal ANLL, there was a strikingly non-random pattern of aneuploidy, with monosomy 7 and trisomy 8 predominating. Common structural changes observed were changes resulting in loss of material from the long arm of chromosomes 5 and 7, trisomy 8, rearrangements of 11q23, t(15;17), t(8;21), rearrangements of 12q13 and 3q, inversion 16, trisomy 11, Ph, trisomy 21, t(6;9) and t(1;22). The differences between adult and pediatric findings were minor, with the exception of chromosome 5 abnormalities, which were common among adults with ANLL but rare in the pediatric cases. There were 273 ALLs with abnormal cytogenetic findings. There was preferential gain of chromosomes 21, X, 14, 6, 4, 18, 17, and 10 (in decreasing order of frequency) in leukemic clones. Of the 193 ALLs with structural changes, many fell into-well-defined categories with established correlations to FAB subtypes. Common changes in ALL were rearrangements of 9p, 12p, 6q, TCR loci, 11q23, Ig loci, and 8q24, and duplication of 1q, Ph, i(17q), t(1;19), i(9q) and dic(9;12). The detailed documentation of the cytogenetic findings in this relatively large, single-institution study will likely facilitate the further characterization of rare, primary cytogenetic changes associated with leukemias and MDS. From a managed health care perspective, regional cancer cytogenetic services may be cost-effective alternatives to single-institution laboratories.

In Utero Injection of α-L-Iduronidase-Carrying Retrovirus in Canine Mucopolysaccharidosis Type I: Infection of Multiple Tissues and Neonatal Gene Expression

Canine alpha-L-iduronidase (alpha-ID) deficiency is caused by a single base pair mutation in the alpha-ID gene, resulting in no enzyme activity in homozygous affected pups. The disease clinically resembles human mucopolysaccharidosis type I (MPSI). We used the canine MPSI model system to address the efficacy of a new retroviral vector, MND-MFG, containing the human alpha-ID cDNA (MND-MFG-alpha-ID) for direct in utero gene delivery to MPSI cells. In vitro, the MND-MFG-alpha-ID vector showed high-level, long-term expression of the transgene in both canine and human alpha-ID-deficient fibroblasts. The effectiveness of this vector for in utero gene transfer and expression in multiple tissues was assessed by injecting viral supernatants into MPSI fetuses and evaluating transduction efficiency and enzyme expression at various times after birth. Transduction of a spectrum of cell types and tissues was observed in all seven live-born pups and in one stillborn pup. Although enzyme activity was not detected in adult tissues from the seven surviving pups, significant alpha-ID enzyme activity was detected in both the liver and kidney of the deceased pup. Our combined gene delivery vector and in utero transfer approach, while encouraging in terms of overall gene transfer efficiency to multiple tissues and successful short-term gene expression, was unable to meet the important requirement of sustained in vivo gene expression.

Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.