Itaru Kuroda

Aberrant induction of LMO2 by the E2A-HLF chimeric transcription factor and its implication in leukemogenesis of B-precursor ALL with t(17;19)

LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.

Fms-like Tyrosine Kinase 3 Ligand Stimulation Induces MLL-Rearranged Leukemia Cells into Quiescence Resistant to Antileukemic Agents

Fms-like tyrosine kinase 3 (FLT3) is highly expressed in acute lymphoblastic leukemia with the mixed-lineage leukemia (MLL) gene rearrangement refractory to chemotherapy. We examined the biological effect of FLT3-ligand (FL) on 18 B-precursor leukemic cell lines with variable karyotypic abnormalities, and found that nine of nine MLL-rearranged cell lines with wild-type FLT3, in contrast to other leukemic cell lines, are significantly inhibited in their proliferation in a dose-dependent manner by FL. This inhibition was due to induction of the G0-G1 arrest. A marked up-regulation of p27 by suppression of its protein degradation and an abrogation of constitutive signal transducers and activators of transcription 5 phosphorylation were revealed in arrested leukemia cells after FL stimulation. Importantly, FL treatment rendered not only cell lines but also primary leukemia cells with MLL rearrangement resistant to chemotherapeutic agents. MLL-rearranged leukemia cells adhering to the bone marrow stromal cell line, which expresses FL as the membrane-bound form, were induced to quiescent state resistant to chemotherapeutic agents, but their chemosensitivity was significantly restored in the presence of neutralizing anti-FL antibody. The FL/FLT3 interaction between leukemia cells and bone marrow stromal cells expressing FL at high levels should contribute, at least in part, to persistent minimal-residual disease of MLL-rearranged leukemia in bone marrow.

Successful tandem (autologous-cord blood) SCT in advanced neuroblastomas with highly amplified MYCN

Although autologous tandem hematopoietic SCT has improved the prognosis of patients with advanced high-risk neuroblastoma, the results remain unsatisfactory. In an attempt to induce the graft-versus-tumor effect, we performed autologous PBSCT followed by allogeneic cord blood transplantation in three consecutive advanced neuroblastoma cases with marked BM infiltration and high MYCN amplification. Severe acute complications did not occur in any patient and they have maintained disease-free survival for 37–60 months. This strategy appears to be feasible and effective for the treatment of extremely high-risk neuroblastoma cases.

Natural pregnancy and delivery after allogeneic bone marrow transplantation in a Fanconi anaemia patient

Mohamad Mohty Jean El-Cheikh Philippe A. Cassier Catherine Faucher Sabine Fürst Norbert Vey Anne-Marie Stoppa Daniele Sainty Christine Arnoulet Luc Xerri Jean-Albert Gastaut Didier Blaise Reda Bouabdallah Unité de Transplantation et de Thérapie Cellulaire (UTTC), Institut Paoli-Calmettes, Département d’Hématologie, Institut Paoli-Calmettes, Département de Biopathologie, Institut Paoli-Calmettes, Université de la Méditerranée, Faculté de Médecine, and INSERM, UMR 599, Marseille, France. E-mail: mohtym@marseille.fnclcc.fr

BCR-ABL regulates death receptor expression for TNF-related apoptosis-inducing ligand (TRAIL) in Philadelphia chromosome-positive leukemia

Allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for chronic myeloid leukemia and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia, and the graft-vs-leukemia (GVL) effect can eradicate residual leukemia after allo-SCT. Ph(+) leukemia cells frequently express death-inducing receptors (DR4 and DR5) for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the cytotoxic ligands expressed on cytotoxic T cells and natural killer cells mediating the GVL effect. Here we demonstrate that imatinib specifically downregulated DR4 and DR5 expression in cell lines and clinical samples of Ph(+) leukemia. Second-generation tyrosine kinase inhibitors (dasatinib and nilotinib) and short hairpin RNA against bcr-abl also downregulated DR4 and DR5 expression in Ph(+) leukemia cells, and transfection of bcr-abl into a Ph(−) leukemia cell line induced DR4 and DR5 expression, which was abrogated by imatinib treatment. Accordingly, Ph(+) leukemia cells that had been pretreated with imatinib showed resistance to the pro-apoptotic activity of recombinant human soluble TRAIL. These observations demonstrate that BCR-ABL is critically involved in the leukemia-specific expression of DR4 and DR5 and in the susceptibility of Ph(+) leukemia to TRAIL-mediated anti-leukemic activity, providing new insight into the mechanisms of the tumor-specific cytotoxic activities of TRAIL.

Endoplasmic reticulum stress inducers, but not imatinib, sensitize Philadelphia chromosome-positive leukemia cells to TRAIL-mediated apoptosis.

Philadelphia-chromosome (Ph1)-positive leukemia cells frequently express death receptors DR4/DR5 for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and show high TRAIL-sensitivity. It has been reported that imatinib damaged cardiomyocytes by triggering endoplasmic reticulum (ER) stress and that ER stress inducers intensified TRAIL-sensitivity of some cancer cells by upregulating DR4/DR5 expression. In fact, ER stress inducers enhanced TRAIL-sensitivity of Ph1-positive leukemia cells by upregulating DR4/DR5 expression. In contrast, imatinib did not induce ER stress responses and unexpectedly downregulated DR4/DR5 expression, indicating that sensitization of Ph1-positive leukemia cells to TRAIL-mediated cellular immunity by imatinib through upregulation of DR4/DR5 expression is unlikely.

Specific induction of CD33 expression by E2A–HLF: the first evidence for aberrant myeloid antigen expression in ALL by a fusion transcription factor

Specific induction of CD33 expression by E2A–HLF: the first evidence for aberrant myeloid antigen expression in ALL by a fusion transcription factor

A specific linkage between the incidence of TP53 mutations and type of chromosomal translocations in B-precursor acute lymphoblastic leukemia cell lines.

Chromosomal translocation plays an essential role in leukemogenesis of childhood B-precursor acute lymphoblastic leukemia (ALL) in combination with specific gene mutations. Although several reports suggest correlation of TP53 mutation with the type of chromosomal translocation in childhood B-precursor ALL, it has not been directly confirmed in a single cohort study due to the limited incidence of TP53 mutation in primary samples at diagnosis. Leukemia cell lines generally show higher incidence of gene mutations compared with primary samples. Thus, to clarify possible differences in cooperation of the p53 pathway for leukemogenesis with the type of chromosomal translocation, TP53 gene mutation was analyzed in 32 B-precursor ALL cell lines. TP53 mutation was observed in 40.6% (13 cell lines), and missense mutation of R248Q was the most commonly observed (5 cell lines). Of note, TP53 mutation was frequently observed among MLL-rearranged and t(1;19)-positive ALL cell lines, but was very rare among Philadelphia chromosome-positive and t(17;19)-positive ALL cell lines. Although the number of samples analyzed is limited, this is the first direct observation of the correlation between the incidence of TP53 mutation and types of translocation in B-precursor ALLs, suggesting a functionally different fusion gene product in the p53 pathway for leukemogenesis.

Little impact of donor/recipient major mismatch for neutrophil-specific antigen NA2 on neutrophil recovery after allogeneic SCT.

The Fcγ receptor IIIb (FcγRIIIb), a receptor for the Fcγ region of IgG, is specifically expressed on neutrophils. It has two allelic polymorphisms, NA1 and NA2, which are highly immunogenic and act as targets in alloimmune or autoimmune neutropenia. Thus, neutrophil antigens (NA) compatibility of donor/recipient pairs might be expected to affect the engraftment of neutrophils after allogeneic SCT (allo-SCT). Here, the impact of NA compatibility of 17 patients and their donors undergoing allo-SCT with a myeloablative regimen was determined. Leukocyte depletion filters were used for all transfusions before and post-SCT; most patients received G-CSF after transplant. Major mismatches for NA1 and NA2 were present in 1 and 7 patient/donor pairs, respectively. These eight patients receiving NA major-mismatched allo-SCT were compared with nine patients who received NA compatible allo-SCT. Engraftment of neutrophils and the incidence of post-engraftment neutropenia were found to be identical in the two groups. Despite the limitations in statistical power because of the small number of patients analyzed, these observations suggest that the major mismatching for NA2 antigen has little impact on the engraftment of neutrophils after myeloablative allo-SCT, at least in patients transfused using leukocyte depletion filters and receiving G-CSF after transplantation.

Revision of the cytological diagnosis of CNS relapse into aseptic meningitis in a patient with TEL-AML1+ acute lymphoblastic leukaemia by FISH analysis of mononuclear cells in cerebrospinal fluid.

vesicles. Based on our results, the name Paneth cell-like metaplasia should be discarded, given that there are no immunohistochemical similarities between these granules, and true metaplasia does not occur. In our series, no clinical suspicion of sperm path obstruction existed, indicating that the presence of these changes cannot be used as a diagnostic marker of obstruction. The extensive necrosis that accompanies testicular tumours produces a massive release of substances for which the endocytic machinery of epididymis epithelial cells is not prepared. This excessive endocytosis probably leads to the accumulation of an increased number of secondary lysosomes in the cytoplasm. In conclusion, granular changes in epididymal epithelial cells represent an accumulation of lysosomes. This accumulation is probably caused by increased endocytic activity secondary to excessive fluid, which might be due not only to obstruction of the spermatic path but also to massive fluid release by a testicular tumour.