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It has been proposed that genomic instability is essential to account for the multiplicity of mutations often seen in malignancies. Using the X-linked PIG-A gene as a sentinel gene for spontaneous inactivating somatic mutations, we previously showed that healthy individuals harbor granulocytes with the PIG-A mutant (paroxysmal nocturnal hemoglobinuria) phenotype at a median frequency (f) of approximately 12 x 10(-6). Herein, we used a similar approach to determine f in blast cells derived from 19 individuals with acute lymphoblastic leukemia (ALL) and in immortalized Epstein-Barr virus-transformed B-cell cultures (human B-lymphoblastoid cell lines) from 19 healthy donors. The B-lymphoblastoid cell lines exhibited a unimodal distribution, with a median f value of 11 x 10(-6). In contrast, analysis of the f values for the ALL samples revealed at least two distinct populations: one population, representing approximately half of the samples (n = 10), had a median f value of 13 x 10(-6), and the remaining samples (n = 9) had a median f value of 566 x 10(-6). We conclude that in ALL, there are two distinct phenotypes with respect to hypermutability, which we hypothesize will correlate with the number of pathogenic mutations required to produce the leukemia.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease in children. The most significant clinical features of PNH include: bone marrow failure, intravascular hemolysis, and thrombosis. To further characterize the clinical presentation and outcome to treatment we performed a retrospective analysis of pediatric patients with PNH. PROCEDURE: We reviewed the medical records of 12 consecutive pediatric patients with PNH diagnosed at our institution from 1992 to 2010. RESULTS: Presenting clinical symptoms included: bone marrow failure (N = 10); gross hemoglobinuria with isolated red cell anemia (N = 1); and jaundice, hepatitis, and isolated thrombocytopenia (N = 1). Immunosuppressive therapy was the initial treatment for 8 patients. Five patients had myelodysplastic features without developing excessive blasts or leukemic transformation. Thrombosis occurred in 6 patients. Five patients underwent hematopoietic stem cell transplant (HSCT) of whom 3 patients are alive and disease-free. Three patients received anti-complement therapy with eculizumab. Two patients died following complications related to thrombosis and 2 patients are transfusion independent with stable disease. CONCLUSION: This report highlights a high rate of bone marrow failure along with a low rate of hemoglobinuria at presentation, a high rate of thrombosis, and for some patients the spontaneous resolution of myelodysplastic features. Delay in diagnosis is common and we recommend appropriate PNH testing in all patients with AA, MDS, unexplained Coombs-negative hemolysis, or thrombosis. While HSCT remains the only curative option the high prevalence of hemolysis and thrombosis should warrant the consideration of early treatment with anti-complement therapy. Pediatr Blood Cancer 2012;59:525-529. (c) 2011 Wiley Periodicals, Inc.

Background Thrombosis is the major risk factor for death in patients with paroxysmal nocturnal hemoglobinuria. Previous case reports indicate that venous thrombosis in patients with paroxysmal nocturnal hemoglobinuria is amenable to thrombolysis. DESIGN AND METHODS: We reviewed the outcome of thrombolytic therapy for patients with paroxysmal nocturnal hemoglobinuria who had thromboses refractory to anticoagulation at our institutions. RESULTS: In this study of 41 patients who had at least one thrombotic event, we confirmed a very high incidence of recurrence despite anticoagulation. Nine patients with thrombosis were regarded as eligible for administration of intravenous tissue plasminogen activator, which was effective in reversing thrombi in all of 15 occasions in which it was given. Serious hemorrhagic complications developed in three cases. At last follow-up visit, of the nine patients treated, three had died, and six were in very good to excellent condition in terms of clinical outcome and radiological findings. The only patient in whom thrombolysis may have contributed to a fatal outcome also had complications of "heparin induced thrombocytopenia with thrombosis", which we diagnosed in three additional patients. In our review of the literature, nine out of 15 patients treated with thrombolysis have had a good outcome. Conclusions Although it is associated with a significant but manageable risk of bleeding, systemic thrombolysis is a highly effective treatment for reversing venous thromboses in patients with paroxysmal nocturnal hemoglobinuria.

It has been hypothesized that the early acquisition of genomic instability is essential to account for the high number of mutations commonly identified in human cancers. Previous reports have demonstrated that MYC overexpression results in gene amplification. Given the importance of MYC in Burkitt's lymphoma, we investigated the effects of MYC on spontaneous inactivating somatic mutations, using an assay that we have developed, based on the PIG-A gene. PIG-A is X-linked, thus a single mutation can disrupt its function. PIG-A encodes an enzyme required for the biosynthesis of glycosylphosphatidylinositol (GPI), which is required for the expression of a set of membrane proteins on the cell surface (e.g., CD48, CD52, CD55, and CD59). Expansion of PIG-A mutant stem cells occurs in PNH, and it is known from this disorder that a very broad spectrum of mutations can inactive PIG-A. Using PIG-A, we have previously determined the mutation rate (mu ) in EBV transformed B-lymphoblastoid cell lines (BLCLs) from normal human donors, and we have demonstrated genomic instability in cell lines derived from hematologic malignancies. Here we have measured mu in P493, a cell line derived from normal human B cells, which overexpress MYC (normalized levels 17 x higher than BLCLs). P493 can also be induced to express lower levels of MYC, resulting in a lower growth rate, using tetracycline and estradiol. To measure mu, pre-existing mutants are first eliminated from the culture by staining the cells with an antibody specific for CD59 and sorting to collect the upper 50th percentile of the population. This purified GPI(+) population is then returned to culture for 3 weeks, with careful cell counts taken to estimate the number of cell divisions (d) occurring in vitro. After expansion, the frequency (f) of mutants arising in vitro is measured by first incubating with a mixture of mouse antibodies specific for CD48, CD52, CD55, and CD59, followed by a rabbit anti-mouse PE conjugate, followed by a FITC conjugated antibody specific for a transmembrane protein (CD45 or HLA-DR). Live cells are identified based on exclusion of propidium iodide and based on FITC fluorescence; GPI(-) cells are defined as having <4% of the mean PE fluorescence as the GPI(+) population. f is calculated as # GPI(-) cells/total gated live cells. The mutation rate is then calculated as mu = f/d. In initial experiments, 6 separate cultures of P493 were analyzed, demonstrating a median mu of 15 x 10^-7 mutations per cell division (range 11 - 33 x 10^-7). For comparison, the positive control, a hypermutable mantle cell lymphoma cell line (HBL2), demonstrated a mu of 674 x 10^-7, and five BLCLs derived from normal donors demonstrated a median mu value of 37 x 10^-7 (range 11 to 57 x 10^-7). In parallel experiments, P493 was also grown in the presence of tetracycline and estradiol to reduce MYC: in 5 separate experiments, P493 growing in these conditions demonstrated a median mu of 6 x 10^-7. However, in 2 subsequent experiments, we observed a higher mu under "lower MYC" conditions: 81 x 10^-7 (SEM +/-15 x 10^-7) vs 13 x 10^-7 (+/-2.2 x 10^-7) and 65 x 10^-7 (+/-6.2 x 10^-7) vs 21 x 10^-7(+/-3.3 x 10^-7). We then isolated 6 clones of P493: for 5 clones, under "high MYC" conditions, mu was similar to the previous experiments (26 x 10^-7, SEM +/-3.4 x 10^-7), and for 3 of these clones mu went up in the "lower MYC" conditions by 5 to 9 fold. For 2 of these clones, mu was similar in the "high MYC" and "lower MYC" conditions. One of the 6 clones, surprisingly, did not adopt a lower growth rate in the presence of tetracycline and estradiol and demonstrated a reversed pattern: mu was 70 x 10^-7 under "high MYC" conditions and 16 x 10^-7 under "lower MYC" conditions. We next analyzed 7 cell lines derived from Burkitt's neoplasms which also overexpressed MYC (mean normalized levels ~ 3 x higher compared with normal BLCLs). Here we found a bimodal pattern, as we have previously described for primary acute lymphoblastic leukemia samples: for 4 of the cell lines mu was < 25 x 10^-7, whereas 3 samples exhibited mu values comparable to the hypermutable positive control: 229 x 10^-7, 562 x 10^-7, and 1786 x 10^-7 respectively. We conclude that overexpression of MYC is not sufficient to produce hypermutability in human B cells, based on this assay for gene-inactivating mutations. Hypermutability-as detected here-is a common feature of cell lines derived from Burkitt's neoplasms but does not seem to be necessary for its development