Jose Manuel Vagace

Central nervous system chemotoxicity during treatment of pediatric acute lymphoblastic leukemia/lymphoma

In the last decades, increasing success rates are being obtained in the chemotherapy of pediatric leukemia and lymphoma. However, the cornerstone of this treatment is still formed by a reduced number of drugs with a highly toxic profile. In particular, central nervous system complications remain a challenging clinical problem, requiring rapid detection and prompt treatment to limit permanent damage. Furthermore, clinicians are often challenged to discriminate between CNS involvement by the disease, toxicity of drugs or infections. This clinically oriented review will help recognize and handle the main neurologic adverse effects induced by chemotherapy in pediatric patients with lymphoblastic leukemia/lymphoma. Different clinical entities and putative drugs involved are discussed in each chapter, with clinical cases illustrating the most relevant and challenging events. In addition, specific clinical-radiological patterns of some of these neurologic events are detailed. Finally, the role of pharmacogenetics, with special focus on those polymorphisms that could help explain the occurrence of neurotoxicity, is also discussed.

Impact of genetic polymorphisms on chemotherapy toxicity in childhood acute lymphoblastic leukemia

The efficacy of chemotherapy in pediatric acute lymphoblastic leukemia (ALL) patients has significantly increased in the last 20 years; as a result, the focus of research is slowly shifting from trying to increase survival rates to reduce chemotherapy-related toxicity. At the present time, the cornerstone of therapy for ALL is still formed by a reduced number of drugs with a highly toxic profile. In recent years, a number of genetic polymorphisms have been identified that can play a significant role in modifying the pharmacokinetics and pharmacodynamics of these drugs. The best example is that of the TPMT gene, whose genotyping is being incorporated to clinical practice in order to individualize doses of mercaptopurine. However, there are additional genes that are relevant for the metabolism, activity, and/or transport of other chemotherapy drugs that are widely use in ALL, such as methotrexate, cyclophosphamide, vincristine, L-asparaginase, etoposide, cytarabine, or cytotoxic antibiotics. These genes can also be affected by genetic alterations that could therefore have clinical consequences. In this review we will discuss recent data on this field, with special focus on those polymorphisms that could be used in clinical practice to tailor chemotherapy for ALL in order to reduce the occurrence of serious adverse effects.

Assessment of Iron Status by Erythrocyte Ferritin in Uremic Patients With or Without Recombinant Human Erythropoietin Therapy

Erythrocyte ferritin may be a better estimator of iron bioavailability than the conventional markers of iron stores (serum ferritin and transferrin saturation). To investigate the accuracy of these conventional markers in uremic patients compared with erythrocyte ferritin, we studied 29 chronic hemodialysis patients on erythropoietin (EPO) therapy, 18 without EPO therapy, and 22 healthy control subjects. Apart from the red blood cell indices, serum ferritin, transferrin saturation, and erythrocyte ferritin, the analytical study included red blood cell protoporphyrin and plasma aluminum levels. The control group showed erythrocyte ferritin concentrations between 8.3 and 12.5 attograms/cell (95% confidence interval). In the EPO group, red blood cell protoporphyrin correlated negatively with erythrocyte ferritin, but not with serum ferritin or transferrin saturation. In the non-EPO group, serum ferritin, erythrocyte ferritin, and transferrin saturation did not correlate with red blood cell protoporphyrin. Even though erythrocyte ferritin correlated well with serum ferritin in the EPO group (r = 0.61, P = 0.0003), the sensitivity of normal serum ferritin levels (30 to 300 ng/mL) to discard a low erythrocyte ferritin concentration (erythrocyte ferritin less than 7 ag/cell) was 0.53, while the sensitivity of serum ferritin at levels less than 30 ng/mL to indicate an absolute iron deficiency expressed as a low erythrocyte ferritin concentration was 0.28. Only values of serum ferritin and transferrin saturation greater than 300 ng/mL and 35%, respectively, could rule out a relative iron deficiency expressed as a low erythrocyte ferritin and high red blood cell protoporphyrin concentration. Plasma aluminum levels did not correlate with red blood cell protoporphyrin or erythrocyte ferritin levels in either uremic group.(ABSTRACT TRUNCATED AT 250 WORDS)

Methotrexate-induced subacute neurotoxicity in a child with acute lymphoblastic leukemia carrying genetic polymorphisms related to folate homeostasis†

Subacute methotrexate neurotoxicity (MTX-NT) may occur days to weeks after systemic or intrathecal (IT) MTX administration and is often manifest by stroke-like symptoms. The pathogenesis of MTX-NT has mainly been associated with cerebral folate homeostasis, but the specific mechanism leading to the development of this complication is mostly unknown and is likely to be multifactorial. Most of studies aimed to determine putative genetic determinants of this syndrome have been focused on the methylenetetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism (SNP). However, there are other functional polymorphisms that have also been identified in enzymes and transporters related to MTX and folate homeostasis. In this context, we carried out an extensive genetic analysis through the screening of 21 SNPs in 11 relevant genes in a five-year-old girl with acute lymphoblastic leukemia (ALL) who developed MTX-NT. The analysis revealed the presence of numerous genetic variants that may have accounted for the neurotoxicity observed. We discuss the putative role of MTX pharmacogenetics in the pathogenesis of MTX-NT.

Diagnostic utility of HFE variants in Spanish patients: Association with HLA alleles and role in susceptibility to acute lymphoblastic leukemia

Two single nucleotide polymorphisms (SNPs) in the Human Hemochromatosis (HFE) gene, C282Y and H63D, are the major variants associated to altered iron status and it is well known that these mutations are in linkage disequilibrium with certain Human Leukocyte Antigen (HLA)-A alleles. In addition, the C282Y SNP has been previously suggested to confer susceptibility to acute lymphoblastic leukemia (ALL). We have aimed to assess the diagnosis utility of these polymorphisms in a population of Spanish subjects with suspicion of hereditary iron overload and to evaluate the effect of their associations with HLA-A alleles on the susceptibility to ALL. Both the 63DD [OR=4.31 (1.7-11.2)] and 282YY (p for trend=0.02) genotypes were more frequently found among subjects with suspicion of iron overload than among controls. 282YY carriers displayed significantly higher transferrin saturation index (TSI) values (p<0.001) as well as serum iron (p=0.01) and ferritin (p=0.01) levels. In addition, transferrin levels were lower in these subjects (p=0.01). Likewise, patients who were carriers of the compound heterozygous diplotype (282CY/63HD) showed significantly higher TSI and serum iron and ferritin concentrations. The H63D SNP did not significantly affect the analytical parameters measured. All 282YY carriers and 69.2% of compound heterozygotes showed an altered biochemical index. The frequencies of the HFE SNPs in ALL pediatric patients were lower than those found in controls, whereas the HLA-A*24 allele was significantly overrepresented in the patients group [OR=3.76 (1.9-7.3)]. No HFE-HLA-A associations were found to modulate the ALL risk. These results suggest that it may be useful to test for both HFE H63D and C282Y polymorphisms in patients with iron overload, as opposed to just genotyping for the C282Y SNP, which is customary in some healthcare centers. These HFE variants and their associations with HLA-A alleles were not observed to be relevant for the susceptibility to ALL in our population.

Diagnostic and therapeutic challenges of primary autoimmune haemolytic anaemia in children

Autoimmune haemolytic anaemias (AIHAs) are extracorpuscular haemolytic anaemias produced by antierythrocyte autoantibodies which cause a shortened red blood cell life span. There are several reasons why the diagnosis and treatment of AIHAs in children represent a bigger challenge than in adult patients, including the presence of particular AIHA types, the uncertainty of serological tests and the limited clinical experience. All these facts have added up to a poor understanding and management of some topics in childhood AIHA. We discuss some of these questions, for example, the occurrence of AIHA with negative direct antiglobulin (Coombs) test, the correct diagnosis and actual incidence of paroxysmal cold haemoglobinuria, the most appropriate second-line therapy of AIHA in childhood or the management of transfusion procedures in these patients. This review takes a practical point of view, providing with some ground rules on how to identify and deal with these paediatric patients.

Chemotherapy Toxicity in Patients with Acute Leukemia

During the treatment for acute leukemia (AL) a patient may experience a wide variety of complications that mainly have three possible origins, namely the disease itself (leukemic infiltration), peripheral blood cell depression (because of hemorrhagic or infectious processes) and toxicity induced by chemotherapy. The toxicity of chemotherapy is a common cause of morbidity and mortality in cancer patients, as well as a frequent source of sequelae at mid-long term. These adverse effects are often the consequence of direct toxicity in healthy tissue, as a result of the low specificity displayed by these drugs. Furthermore, and regardless of their specificity, these compounds may also exacerbate complications derived from the tumor growth, as it is the case of pancytopenia or the Tumor Lysis Syndrome. Chemotherapy toxicity becomes more frequent as the treatment is intensified, thus challenging the clinician with both diagnostic and therapeutic problems. In this chapter we will discuss the major clinical signs of toxicity produced by chemotherapy drugs in patients with AL. Hematological and gastrointestinal (mucositis, nausea and vomiting) adverse effects will not be included, as their description suits better in an Initial Management chapter. In the last section of this chapter we will discuss recent data on whether pharmacogenetics may help individualize the therapy for AL, thus avoiding serious toxicity.

Ethylenediaminetetraacetic acid-dependent pseudomacrocytosis

We investigated the case of a 14-year-old girl with an ethylenediaminetetraacetic acid (EDTA)-dependent haemagglutination detected by macrocytosis, which was only evident by an abnormal red blood cell (RBC) population in the histogram. Investigations included haemograms with different anticoagulants and experimental conditions. Immunohaematological studies were performed using a gel-based technology. At admission, the patient had a low RBC count and an increased mean corpuscular volume with normal haemoglobin. A double population appeared in the RBC histogram. However, the peripheral blood smear was normal and macrocytosis was absent when heparin or citrate was used instead of EDTA. Later studies revealed that the patients serum was able to induce macrocytosis of control RBC only in the presence of EDTA. An EDTA-dependent panagglutinin was then indentified that produced mixed field agglutination. These findings provide evidence of a haemagglutination induced by EDTA as a source of pseudomacrocytosis.

Hyperhaemolysis syndrome responsive to splenectomy in a patient with δβ-thalassaemia: a discussion on underlying mechanisms

Dear Sir, Hyperhaemolysis syndrome (HS) is a life-threatening complication of transfusion, which was first described in patients with sickle-cell disease by Petz et al. 15 years ago1. Since then, the syndrome has also been detected in several thalassaemia patients as well as in subjects with other types of anaemia. The term HS is applied when: (i) the post-transfusion haemoglobin (Hb) levels are lower than pre-transfusion values; (ii) there is severe intravascular haemolysis and (iii) there is a fall in the absolute reticulocyte count from baseline levels1. Subsequent transfusions of compatible red blood cells (RBC) may worsen the haemolysis; it is, therefore, recommended that transfusions are avoided and therapy is initiated with intravenous immunoglobulin G (IgG) and methylprednisolone. We present the case of a paediatric patient with thalassaemia intermedia who developed HS that was responsive to splenectomy. We discuss several putative mechanisms that can help to explain the role of macrophages in this syndrome, whose pathological mechanisms are yet to be fully understood. A 9-year old patient visited our hospital for the study of suspected thalassaemia (both of his parents had thalassaemia traits). The boy presented with growth retardation, malar hypertrophy, pallor and palpable splenomegaly below the left costal margin (6 cm). The X-ray study showed bone deformities (broad metacarpal bones and thickening of the skull). His Hb concentration was 7.8 g/dL and his reticulocyte count was 150,000/mm3. Leucocyte and platelet concentrations were normal. Molecular tests revealed that the patient was a homozygous carrier of δβ-thalassaemia. A series of RBC transfusions was scheduled. Phenotype matching was not performed for antigens other than ABO and D. Two weeks after the second transfusion, the boy was admitted to our hospital because of fever, headache and haemoglobinuria. On examination, the patient was jaundiced and his spleen had further enlarged to become palpable 10 cm below the costal margin. Hb values progressively decreased in spite of additional transfusions (Figure 1). Eight days after admission the patient was transferred to the Intensive Care Unit because of severe anaemia. Results of laboratory analyses (Hb 4.4 g/dL, reticulocytes 53,000/mm3, haemoglobinuria+++, bilirubin 10.2 mg/dL, lactate dehydrogenase 2,821 IU/dL) were indicative of severe intravascular haemolysis. The study of the peripheral blood smear was negative for autoagglutination, schistocytes and acanthocytes. Direct antiglobulin tests, both total and monospecific, screening for atypical anti-RBC antibodies, serological cross-matching and screening for anti-HLA antibodies were all negative. Likewise, cytometric analysis for paroxysmal nocturnal haemoglobinuria and the Donath-Laudsteiner and microbiological tests (bacterial cultures, polymerase chain reaction and serological tests for herpes viruses) were also negative. Figure 1 Evolution of haemoglobin values and reticulocyte counts in the patient. A bone marrow aspirate was obtained and showed marked erythroid hyperplasia. The patient was diagnosed with HS and therapy with intravenous IgG and corticoids was then initiated. As haemolysis continued, an emergency splenectomy was conducted. Histological analysis of the spleen revealed a remarkable macrophage hyperplasia with no evidence of haemophagocytosis. After the splenectomy, the patient stopped haemolysing and the reticulocyte count recovered drastically (Figure 1). At present, the patient is receiving treatment with hydroxyurea and his Hb values have stabilised between 10 and 11.0 g/dL. No further transfusions have been needed. Glucose-6-phosphate dehydrogenase and pyruvate kinase levels determined after the patient’s recovery were normal. New immunohaematological tests conducted 6 months and 1 year after the events described remained negative. The patient described herein was diagnosed with HS, as he complied with the major criteria for this syndrome. The fact that the patient’s immunological tests were all negative raises several questions that the histological findings obtained may help solve. First, what was the origin of the fall in the post-transfusion Hb values? The marked erythroid hyperplasia observed in the bone marrow aspirate in our patient, as well as the rapid recovery of the reticulocyte count, suggest that reticulocytopenia was not due to suppression of erythropoiesis2, but that it probably originated from peripheral consumption. Second, why did the patient’s RBC suffer haemolysis during the transfusion reaction? In our case, complementary analyses ruled out the coexistence of several diseases that are known to cause intravascular haemolysis. The occurrence of an autoimmune haemolytic anaemia was also unlikely in our patient because the direct Coombs’ test was negative and remained so during the follow-up. An alternative explanation could be the so-called innocent bystander haemolysis, according to which the RBC are not the primary target of the antibody but they are lysed as a result of a complement-mediated immune reaction2. Sickle RBC show increased susceptibility to reactive lysis due to a defect in the regulation of membrane attack complex formation and, interestingly enough, similar findings have also been described in thalassaemia patients. However, this innocent bystander hypothesis calls for the existence of an antibody capable of activating the complex and no anti-RBC alloantibodies that could have played that role were identified in our patient. Tests were also negative for anti-HLA antibodies, whose participation has also previously been suggested2. So, how can we explain the haemolysis without alloantibodies? There are two possibilities: (i) there is an anti-RBC antibody that we failed to detect or (ii) there is no such antibody and the haemolysis is produced by other mechanism. The former possibility should not be entirely ignored, as agglutination-based techniques may not be sensitive enough to detect all haemolytic antibodies. The latter hypothesis, which points to a pivotal role of macrophages in the RBC lysis, seems to be supported by our findings. The increased splenomegaly coinciding with the haemolysis, the rapid recovery of the reticulocyte count after the splenectomy and especially the histological analyses, which revealed a macrophage hyperplasia without evidence of haemophagocytosis, suggest that peripheral consumption was caused by direct haemolysis induced by macrophages. Three theories for the specific mechanism responsible for the RBC destruction by macrophages in the absence of detectable antibodies have been proposed, although none of them has yet been proven in HS. First, it has been suggested that the pathogenic alloantibody could disappear from the serum because the IgG would easily bind the FcR1 receptor in the macrophage. This IgG would later react with RBC carrying the putative antigen by projecting its Fab domain from the macrophage (armed macrophage), thus destroying the RBC3. Second, the suicidal erythrocyte death theory suggests that phosphatidylserine exposed on the RBC surface enhances macrophage phagocytosis, and contributes to non-specific fixation of activated fractions of complement (C5b-C9)4. In this regard, it is noteworthy that plasma from HS patients has been shown to be capable of inducing this process in both transfused RBC and the patient’s own RBC5. Finally, the third hypothesis focuses on the role of adhesion molecules. Activated macrophages in patients with sickle cell disease express vascular cell adhesion molecule (VCAM-1), which interacts with α4b1 integrin. This receptor is expressed in immature reticulocytes both in sickle cell disease and in thalassaemia. Hence, it is tempting to speculate that reticulocytes in our patient could have been lysed by this interaction. Furthermore, the haemolysis of transfused RBC could also be mediated by the interaction between intracellular adhesion molecule (ICAM-4), an erythroid-specific membrane component, and the integrin CD11c-CD18, which is also overexpressed in activated macrophages. This last hypothesis involving adhesion molecules is more consistent with our histological findings, given the absence of haemophagocytosis both in the marrow and in the spleen. In summary, we have presented a case of HS, which was responsive to splenectomy, in a patient with thalassaemia intermedia. This case provides information that may help to clarify the role of macrophages in the pathophysiological mechanisms underlying this syndrome.

Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3.

Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606‐8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606‐8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606‐8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606‐8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early‐onset of iron overload.