Professor John Scott, folate and neural tube defects.

John Scott (1940-2013) was born in Dublin where he was to spend the rest of his career, both as an undergraduate and subsequently Professor of Biochemistry and Nutrition at Trinity College. His research with the talented group of scientists and clinicians that he led has had a substantial impact on our understanding of folate metabolism, mechanisms of its catabolism and deficiency. His research established the leading theory of folate involvement with vitamin B12 in the pathogenesis of vitamin B12 neuropathy. He helped to establish the normal daily intake of folate and the increased requirements needed either in food or as a supplement before and during pregnancy to prevent neural tube defects. He also suggested a dietary supplement of vitamin B12 before and during pregnancy to reduce the risk of neural tube defects. It would be an appropriate epitaph if fortification of food with folic acid became mandatory in the UK and Ireland, as it is in over 70 other countries.

How I treat transfusional iron overload.

Patients with ß-thalassemia major (TM) and other refractory anemias requiring regular blood transfusions accumulate iron that damages the liver, endocrine system, and most importantly the heart. The prognosis in TM has improved remarkably over the past 10 years. This improvement has resulted from the development of magnetic resonance imaging (MRI) techniques, especially T2*, to accurately measure cardiac and liver iron, and from the availability of 3 iron-chelating drugs. In this article we describe the use of MRI to determine which adult and pediatric patients need to begin iron chelation therapy and to monitor their progress. We summarize the properties of each of the 3 drugs, deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX), including their efficacy, patient acceptability, and side effects. We describe when to initiate or intensify therapy, switch to another drug, or use combined therapy. We also discuss the management of refractory anemias other than TM that may require multiple blood transfusions, including sickle cell anemia and myelodysplasia. The development of a potential fourth chelator FBS 0701 and the combined use of oral chelators may further improve the quality of life and survival in patients with TM and other transfusion-dependent patients.

X-linked sideroblastic anemia due to carboxyl-terminal ALAS2 mutations that cause loss of binding to the ß-subunit of succinyl-CoA synthetase (SUCLA2).

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the ß subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.

The JAK3-selective inhibitor PF-956980 reverses the resistance to cytotoxic agents induced by interleukin-4 treatment of chronic lymphocytic leukemia cells: potential for reversal of cytoprotection by the microenvironment.

Extensive evidence suggests that the malignant cells of chronic lymphocytic leukemia (CLL) patients are in close contact with activated T lymphocytes, which secrete a range of cytoprotective cytokines including interleukin-4 (IL-4). IL-4 induced the rapid phosphorylation and activation of the signal transducer and activator of transcription 6 transcription factor in CLL cells in vitro. Longer incubation with IL-4 resulted in up-regulation of the antiapoptotic proteins, Mcl-1 and Bcl-X(L). All of these events were blocked by the JAK3-selective inhibitor, PF-956980. A dye reduction cytotoxicity assay showed that IL-4 induced resistance to the cytotoxic drugs fludarabine and chlorambucil and to the novel p53-elevating agent nutlin 3. IL-4-induced drug resistance was reversed by PF-956980. These conclusions were confirmed by independent assays for apoptosis induction (annexin V binding, cleavage of poly[ADP-ribose] polymerase, and morphologic analysis). Coculture with bone marrow stromal cells in the presence of supernatants derived from activated T-lymphocyte cultures also protected CLL cells from apoptosis induction by chlorambucil. Protection by these combined signals was reversed by PF-956980. The data here provide a preclinical rationale for the possible therapeutic use of PF-956980 in conjunction with conventional cytotoxic drugs to achieve more extensive killing of CLL cells by overcoming antiapoptotic signaling by the microenvironment.

2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.

We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

Minimal residual disease is a significant predictor of treatment failure in non T-lineage adult acute lymphoblastic leukaemia: final results of the international trial UKALL XII/ECOG2993.

The predictive value of molecular minimal residual disease (MRD) monitoring using polymerase chain reaction amplification of clone-specific immunoglobulin or T-cell Receptor rearrangements was analysed in 161 patients with non T-lineage Philadelphia-negative acute lymphoblastic leukaemia (ALL) participating in the UK arm of the international ALL trial UKALL XII/Eastern Cooperative Oncology Group (ECOG) 2993. MRD positivity (> or =10(-4)) in patients treated with chemotherapy alone was associated with significantly shorter relapse-free survival (RFS) at several time-points during the first year of therapy. MRD status best discriminated outcome after phase 2 induction, when the relative risk of relapse was 8.95 (2.85-28.09)-fold higher in MRD-positive (> or =10(-4)) patients and the 5-year RFS 15% [95% confidence interval (CI) 0-40%] compared to 71% (56-85%) in MRD-negative (<10(-4)) patients (P = 0.0002) When MRD was detected prior to autologous stem cell transplantation (SCT), a significantly higher rate of treatment failure was observed [5-year RFS 25% (CI 0-55%) vs. 77% (95% CI 54-100%) in MRD-negative/<10(-4), P = 0.01] whereas in recipients of allogeneic-SCT in first complete remission, MRD positivity pre-transplant did not adversely affect outcome. These data provide a rationale for introducing MRD-based risk stratification in future studies for the delineation of those at significant risk of treatment failure in whom intensification of therapy should be evaluated.

p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism.

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.

Minimal residual disease tests provide an independent predictor of clinical outcome in adult acute lymphoblastic leukemia.

PURPOSE: Investigation of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) using molecular markers has proven superior to other standard criteria (age, sex, and WBC) in distinguishing patients at high, intermediate, and low risk of relapse. The aim of our study was to determine whether MRD investigation is valuable in predicting outcome in Philadelphia-negative adult patients with ALL. PATIENTS AND METHODS: MRD was assessed in 85 adult patients with B-lineage ALL by semiquantitative immunoglobulin H gene analysis on bone marrow samples collected during four time bands in the first 24 months of treatment. Fifty patients received chemotherapy only and 35 patients received allogeneic (n = 19) or autologous (n = 16) bone marrow transplantation (BMT) in first clinical remission. The relationship between MRD status and clinical outcome was investigated and compared with age, sex, immunophenotype, and presenting WBC count. RESULTS: Fisher's exact test established a statistically significant concordance between MRD results and clinical outcome at all times. Disease-free survival (DFS) rates for MRD-positive and -negative patients and log-rank testing established that MRD positivity was associated with increased relapse rates at all times (P <.05) but was most significant at 3 to 5 months after induction and beyond. MRD status after allogeneic BMT rather than before was found to be an important predictor of outcome in 19 adult patients with ALL tested. In patients receiving autologous BMT (n = 16), the MRD status before BMT was more significant (P =.005). CONCLUSION: The association of MRD test results and DFS was independent of and greater than other standard predictors of outcome and is therefore important in determining treatment for individual patients.

Role of autoimmune gastritis, Helicobacter pylori and celiac disease in refractory or unexplained iron deficiency anemia.

BACKGROUND AND OBJECTIVES: Conventional endoscopic and radiographic methods fail to identify a probable source of gastrointestinal blood loss in about one third of males and post-menopausal females and in most women of reproductive age with iron deficiency anemia (IDA). Such patients, as well as subjects refractory to oral iron treatment, are often referred for hematologic evaluation. DESIGN AND METHODS: Patient clinic, screened for non-bleeding gastrointestinal conditions including celiac disease (antiendomysial antibodies), autoimmune atrophic gastritis (hypergastrinemia with strongly positive antiparietal cell antibodies) and H. pylori infection (IgG antibodies confirmed by urease breath test). RESULTS: The mean age of all subjects was 39+/-18 years, and 119 of 150 were females. We identified 8 new cases of adult celiac disease (5%). Forty IDA patients (27%) had autoimmune atrophic gastritis of whom 22 had low serum vitamin B12 levels. H. pylori infection was the only finding in 29 patients (19%), but was a common co-existing finding in 77 (51%) of the entire group. Refractoriness to oral iron treatment was found in 100% of patients with celiac disease, 71% with autoimmune atrophic gastritis, 68% with H. pylori infection, but only 11% of subjects with no detected underlying abnormality. H. pylori eradication in previously refractory IDA patients in combination with continued oral iron therapy resulted in a significant increase in hemoglobin from 9.4+/-1.5 (mean +/- 1SD) before, to 13.5+/-1.2 g/ dL (p<0.001 by paired t test) within 3 to 6 months. INTERPRETATION AND CONCLUSIONS: The recognition that autoimmune atrophic gastritis and H. pylori infection may have a significant role in the development of unexplained or refractory IDA in a high proportion of patients should have a strong impact on our daily practice of diagnosing and managing IDA.

Molecular analysis of minimal residual disease in adult acute lymphoblastic leukaemia.

Despite intensive chemotherapy and stem cell transplantation (SCT) programmes, overall survival in adult acute lymphoblastic leukaemia (ALL) remains poor compared to that in childhood ALL. Despite clinical and morphological remission being achieved by over 80% of patients, 5-year survival is limited to 40% of patients, clearly indicating that morphology is insufficient in predicting future outcome. Molecular assessment of residual disease in bone marrow using immunoglobulin genes as markers of clonality has recently been evaluated in a large adult ALL study in our institution. Analysis of disease-free survival (DFS) rates for minimal residual disease-(MRD-) positive and -negative patients established that MRD positivity was associated with increased relapse rates at all times, being most significant at 3-5 months post-induction and beyond. Pre-autologous SCT tests are predictive of outcome, but for allogeneic SCT outcome is related to results of the tests after the procedure rather than before. The association of MRD test results and DFS was independent of, and greater than, other standard predictors of outcome and is therefore important in determining treatment for individual patients.

V(H) gene usage differs in germline and mutated B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: Given the prognostic relevance that the identification of mutated and germline subgroups of chronic lymphocytic leukemia (CLL) has recently acquired we set out to analyze in depth individual VH gene usage rearrangements in patients with mutated and germline CLL. DESIGN AND METHODS: Using sequence analysis of FR1/JH polymerase chain reaction products, the VH immunoglobulin gene configuration was analyzed in 159 rearranged IgH alleles from 154 CLL patients. Having previously identified a spatial relationship between VH gene usage and JH proximity in patients with acute lymphocytic leukemia (ALL), we performed linear and Poisson regression analysis on patients with germline and mutated CLL against VH rearrangements from normal peripheral blood. RESULTS: Sequence analysis showed that 102 patients (64%) had mutated sequences (>2% DNA base pair changes) while 57 (36%) had germline sequences. The germline CLL group showed JH proximal overusage similar to that reported in ALL patients, while the mutated CLL group showed a pattern comparable to that of the control group (peripheral blood rearranged VH sequences). The CDR3 region was statistically longer in the patients with germline CLL than in those with mutated CLL. INTERPRETATION AND CONCLUSIONS: This study highlights differences in the VDJ profile in mutated and germline CLL, consistent with the suggestion that CLL comprises two subgroups. The interpretation of these differences is that the B-cell of CLL, particularly in the germline group, may derive from a pool that has been unable to follow or complete the normal pathway of B-cell differentiation.

Iron deficiency and overload.

In the past seven years numerous genes that influence iron homeostasis have been discovered. Dr. Beutler provides a brief overview of these genes, genes that encode HFE, DMT-1, ferroportin, transferrin receptor 2, hephaestin, and hepcidin to lay the groundwork for a discussion of the various clinical forms of iron storage disease and how they differ from one another. In Section I, Dr. Beutler also discusses the types of hemochromatosis that exist as acquired and as hereditary forms. Acquired hemochromatosis occurs in patients with marrow failure, particularly when there is active ineffective erythropoiesis. Hereditary hemochromatosis is most commonly due to mutations in the HLA-linked HFE gene, and hemochromatosis clinically indistinguishable from HFE hemochromatosis is the consequence of mutations in three transferrin receptor-2 gene. A more severe, juvenile form of iron storage disease results from mutations of the gene encoding hepcidin or of a not-yet-identified gene on chromosome 1q. Autosomal dominant iron storage disease is a consequence of ferroportin mutations, and a polymorphism in the ferroportin gene appears to be involved in the African iron overload syndrome. Evidence regarding the biochemical and clinical penetrance of hemochromatosis due to mutations of the HFE gene is rapidly accumulating. These studies, emanating from several centers in Europe and the United States, all agree that the penetrance of hemochromatosis is much lower than had previously been thought. Probably only 1% of homozygotes develop clinical findings. The implications of these new findings for the management of hemochromatosis will be discussed. In Section II, Dr. Victor Hoffbrand discusses the management of iron storage disease by chelation therapy, treatment that is usually reserved for patients with secondary hemochromatosis such as occurs in the thalassemias and in patients with transfusion requirements due to myelodysplasia and other marrow failure states. Tissue iron can be estimated by determining serum ferritin levels, measuring liver iron, and by measuring cardiac iron using the MRI-T2* technique. The standard form of chelation therapy is the slow intravenous or subcutaneous infusion of desferoxamine. An orally active bidentate iron chelator, deferiprone, is now licensed in 25 countries for treatment of patients with thalassemia major. Possibly because of the ability of this compound to cross membranes, it appears to have superior cardioprotective properties. Agranulocytosis is the most serious complication of deferiprone therapy and occurs in about 1% of treated patients. Deferiprone and desferoxamine can be given together or on alternating schedules. A new orally active chelating agent ICL 670 seems promising in early clinical studies. In Section III, Dr. James Cook discusses the most common disorder of iron homeostasis, iron deficiency. He will compare some of the standard methods for identifying iron deficiency, the hemoglobin level, transferrin saturation, and mean corpuscular hemoglobin and compare these with some of the newer methods that have been introduced, specifically the percentage of hypochromic erythrocytes and reticulocyte hemoglobin content. The measurement of storage iron is achieved by measuring serum ferritin levels. The soluble transferrin receptor is a truncated form of the cellular transferrin receptor and the possible value of this measurement in the diagnosis of iron deficiency will be discussed. Until recently iron dextran was the only parental iron preparation available in the US. Sodium ferric gluconate, which has been used extensively in Europe for many years, is now available in the United States. It seems to have a distinct advantage over iron dextran in that anaphylactic reactions are much less common with the latter preparation.

Mycophenolate mofetil for the treatment of refractory auto-immune haemolytic anaemia and auto-immune thrombocytopenia purpura.

The treatment of both auto-immune haemolytic anaemia (AIHA) and auto-immune thromobocytopenic purpura (AITP) remains unsatisfactory in those refractory to first-line management. Mycophenolate mofetil (MMF) is an immunosuppressive agent originally used to prevent acute rejection of solid organ transplants and used more recently in the management of auto-immune conditions. We report its use in four patients with AIHA and six patients with AITP. All four patients with AIHA and five of the six patients with AITP showed a complete or good partial response to treatment with MMF, confirming a possible role in the treatment of these conditions.