The etiology of Langerhans cell histiocytosis (LCH), a disease characterized by uncontrolled proliferation of Langerhans cells, is unknown. Although some believe that LCH is reactive, others support a neoplastic origin. We tested the hypothesis that LCH is neoplastic by investigating potential consistent chromosomal aberrations in LCH cells. We used multiparameter DNA flow cytometry to analyze the DNA ploidy LCH cells in 20 cases, performed karyotype analysis in 31 cases, array-based comparative genomic hybridization (arrayCGH) and single nucleotide polymorphism (SNP) arrays with DNA from flow-sorted CD1a-positive and CD1a-negative cells in 19 cases. Ploidy analysis revealed diploid DNA content in all cases. The karyotype of all patients analyzed was normal, excluding the presence of balanced translocations. ArrayCGH and SNP arrays did not show genome abnormalities. Despite positive TP53 protein immunohistochemical staining, sequencing of exon 5 to 8 of p53 gene showed no alterations in 7 cases. This study strongly suggests that gross chromosomal abnormalities do not cause LCH. Although we cannot exclude cryptic point mutations in as yet unidentified genes, this study of 72 LCH cases shows that LCH may be the result of restricted oligoclonal stimulation rather than unlimited neoplastic proliferation. (c) 2008 Wiley-Liss, Inc.
Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have an inferior prognosis compared with non-DS ALL patients. We reviewed methotrexate (MTX)/mercaptopurine (6MP) maintenance therapy data for children with DS treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL92 or the NOPHO ALL2000 protocols between 1992 and 2007. The 5-year event-free survival probability (pEFS(5 yr)) for the 66 DS patients was inferior to the 2602 non-DS patients (0.50 ± 0.07 vs 0.77 ± 0.01 (P<0.001)). The 48 DS patients in first remission at the beginning of maintenance therapy had pEFS(10 yr) below that of the 522 non-DS control patients (pEFS(10 yr): 0.58 (95% confidence interval (CI) 0.43-0.77) vs 0.83 (95% CI 0.80-0.86), respectively (P<0.0001)). The DS patients received lower median doses of MTX (median: 11.8 vs 15.4 (P<0.0001)) and 6MP (median: 43.6 vs 59.4 (P<0.0001)). In Cox regression analysis, male gender, presence of DS and high median maintenance therapy white blood cell levels (mWBC) were associated with increased risk for relapse. DS-ALL patients with mWBC above or below 3.5 × 10(9)/l (protocol target) had pEFS(10 yr) of 0.31 and 0.72 (P=0.02), and the mWBC hazard ratio for DS-ALL patients was 2.0 (P<0.0005). We conclude that insufficient treatment intensity during maintenance therapy of DS-ALL patients may contribute to their poor prognosis.
The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≈80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≈50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, ≥5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.
Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34+CD38− phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34+CD38− BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34+CD38− progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.
Chronic granulomatous disease (CGD) is an inherited deficiency of the superoxide-generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in recurrent, severe bacterial and fungal infections. The X-linked form of this disorder (X-CGD) results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase flavocytochrome b558. In this study, we used a murine model of X-CGD to examine the long-term function of retroviral vectors for expression of gp91phox based on the murine stem cell virus (MSCV) backbone. NADPH oxidase activity was reconstituted in neutrophils and macrophages for up to 18 to 24 months posttransplantation of transduced X-CGD bone marrow into lethally irradiated syngeneic X-CGD mice. Southern blot analysis and secondary transplant data showed proviral integration in multilineage repopulating cells. Although relatively small amounts of recombinant gp91phox (approximately 5% to 10% of wild-type levels) were detected in neutrophils after retroviral-mediated gene transfer, superoxide-generating activity was approximately 20% to 25% of wild-type mouse neutrophils. Expression of gp91phox is normally restricted to mature phagocytes. No obvious toxicity was observed in other hematopoietic lineages in transplant recipients, and provirus-marked cells were capable of reconstituting secondary transplant recipients, who also exhibited NADPH oxidase–positive neutrophils. MSCV-based vectors for long-term expression of gp91phox may be useful for gene therapy of human CGD targeted at hematopoietic stem cells.
We have confirmed, in two additional cases, that the recurrent T-ALL-associated t(12;14) results in overexpression of cyclin D2. The t(12;14) is the first neoplasia-associated translocation shown to result in overexpression of cyclin D2. Furthermore, it is the first example of a T-cell neoplasm with a targeted deregulation of a member of a cyclin-encoding gene family.
The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.
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