Rab27a: A key to melanosome transport in human melanocytes.

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.

Cost-effectiveness of cefepime + netilmicin or ceftazidime + amikacin or meropenem monotherapy in febrile neutropenic children with malignancy in Turkey.

Infection remains the major cause of morbidity and mortality in immunocompromised children with malignancy. In addition, the economic impact of antibiotic treatment should always be evaluated, especially in developing countries. In our center between January 1998 and January 1999, 73 children with hematological malignancies [acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML)]; 9 children with solid tumors (rhabdomyosarcoma, neuroblastoma) had 87 febrile neutropenic episodes (related to chemotherapy). These children were randomized prospectively into three treatment groups. The first group (n: 28) received cefepime plus netilmicin, while the second group (n: 29) was treated with ceftazidime plus amikacin and the third (n: 30) with meropenem as monotherapy. The aim of the study was to compare the success rates and cost of fourth generation cephalosporin plus aminoglycoside and monotherapy of meropenem with ceftazidime plus amikacin, which is the standard therapy for febrile neutropenia. Microbiologically documented infections were 29.9%, clinically documented infections were 9.2% and 60.9% of the febrile neutropenic episodes were considered to be FUO. Gram-positive microorganisms were the most commonly isolated agents from blood cultures [MRSA (Methicillin Resistant Staphylococcus aureus) in 6 patients and MSSA (Methicillin Sensitive Staphylococcus aureus) in 4 patients]. The success rates were 78.5%, 79.3% and 73.3 % for the 1st, 2nd and 3rd groups respectively. In 4 patients (4.5%) fever responded only to amphotericin-B therapy. There was no statistically significant difference between the three treatment regimens with respect to efficacy, safety and tolerance (chi2 test, p>0.05), but while the third and fourth generation cephalosporins + aminoglycosides were comparable for cost, the monotherapy regimen was the most expensive. The main determining factors for the choice of treatment of febrile neutropenic children, especially in a developing country, are cost, presence of indwelling catheter and the bacterial flora of the unit, as well as efficacy.

Results of rhabdomyosarcoma treatment in a developing country.

Fifty-one children (median age: 4.5 years; 4 months-16 years) diagnosed with rhabdomyosarcoma were treated in our center between 1980-1999. The primary sites were head and neck in 31.4%, the genito-urinary system in 21.6%, and extremities in 9.8% of the patients. The histopathologic subtypes were embryonal in 80.4%, alveolar in 9.8%, and undifferentiated in 9.8%. The majority of the patients were considered group III (47%) and group IV (25.5%) according the criteria of the Intergroup Rhabdomyosarcoma Study (IRS). Primary total tumour resection was performed in only 27.5% of the patients. The patients were treated with assigned regimens of IRS II and IRS III protocols. Radiotherapy was applied to 92.1% of the patients. Thirty-four patients (66.7%) were lost to follow up, and of the remaining 17 patients, 7 patients (41.2%) died, relapse occurred in 9 patients (52.9%) and 10 patients (58.8%) are alive. The percentage of cases lost to follow up during the first 10 years and the following 9 years of the study were 77.4% and 50%, respectively. In compliance with cancer treatment remains a major problem in developing countries.

A case with acute leukemia presenting with cardiac tamponade.

Pericardial effusions and cardiac tamponade are rare and severe complications of leukemia. They often develop during the radiation therapy, chemotherapy, or infections in the course of leukemia. However, some cases present with pericardial effusion and tamponade. We report a three-year-old girl who was admitted with cardiac tamponade and needed urgent pericardiocentesis. Clinical evaluation and laboratory results revealed myeloid markered-T cell acute lymphoblastic leukemia (ALL) and pericardial invasion. She is the youngest patient with cardiac tamponade who was diagnosed acute lymphoblastic leukemia in the English-language literature.

Fludarabine, cytarabine, G-CSF and idarubicin (FLAG-IDA) for the treatment of relapsed or poor risk childhood acute leukemia.

The prognosis of relapsed acute leukemia or chronic leukemia in acute blast crisis is poor and new chemotherapeutic regimens could be useful for these patients. Six relapsed acute lymphoblastic leukemia (ALL), nine relapsed acute myeloblastic leukemia (AML), one chronic myelomonocytic leukemia (CMML) and one chronic myeloid leukemia (CML) in acute blast crisis between three to 18 years (median 10 years) received fludarabine, cytarabine, G-CSF and idarubicin (FLAG-IDA) chemotherapy (CT). Five of the AML relapses were after bone marrow transplantation (BMT) and four were recurrent relapses. At the end of the second course only three patients (2 AML, 1 ALL) were in complete remission (CR). Of the three patients in CR, one patient with AML had her first donor lymphocyte transfusion (DLT) on the 7th day of the second FLAG-IDA course and she is disease-free on the 30th month of the second remission. The remaining two patients were transplanted from unrelated donors in a BMT center abroad on the 5th and 8th month of the last remission and both died with BMT-related complications. Out of 25 courses, seven resulted in fatal infections. The regimen was ineffective in B-cell ALL as in acute blastic crisis of CMML and CML. We could not evaluate the remission-inducing effect accurately in most of the patients due to induction failure. FLAG-IDA appears to be a myelotoxic therapy for relapsed or poor risk leukemia in a developing country. It is not cost-effective; dose modifications or a regimen without IDA may be tried if there is an available marrow donor.

The effect of bone marrow transplantation on systemic and oral health in Fanconi's aplastic anemia.

Fanconi's anemia (FA) is an autosomal-recessive disorder characterized by a progressive pancytopenia, diverse congenital abnormalities and increased predisposition to malignancy. Sixteen children with FA, aged between 4 to 16 were divided into two groups according to treatments. Nine children had bone marrow transplantation and seven children were treated with steroid and/or anapolan. The changes in dental caries, caries-associated microflora, salivary status and periodontal health were investigated in children with FA. Data were analyzed by one-way ANOVA. A statistically significant difference was found in hematological findings between children who have received bone marrow transplantation (BMT+) and the others, who have not received (BMT-). There was no significant difference in dental caries experience, salivary flow rate, buffering capacity, mutans streptococci and Lactobacilli levels between the study groups. A statistically significant difference was found in gingival index, plaque index, bleeding on probing, probing depth scores between the patients with FA in BMT(+) and BMT(-) groups (p<.05). In conclusion, besides systemic control, additional preventive measures during their whole life to maintain oral health is necessary in these children.

Acute lymphoblastic leukemia: dental health of children in maintenance therapy.

The dental health of 41 children aged 4-16 years who were in maintenance therapy from acute lymphoblastic leukemia (ALL) was examined in relation to the period of time in maintenance. There was no significant difference in dental experience and salivary flow rate between the control group and patients with leukemia. Performed treatment index (PTI) and required treatment index (RTI) scores reflected that children, who were in maintenance therapy had insufficient dental care and needed more dental treatments. A statistically significant difference in salivary pH was found between the children, who were in maintenance therapy for 12-24 months and less than 12 months and also the control group; but the pH scores of all groups were observed in normal limits.

Could Parvovirus B19 Induce a Rejection After Bone Marrow Transplantation in a Patient with Diamond-Blackfan Anemia?

A four year-old-girl with Diamond-Blackfan anemia (DBA) that was resistant to corticosteroid treatment and transfusion dependent underwent (bone marrow transplantation) BMT from HLA identical sibling. The patient was conditioned with busulfan and cyclophosphamide and achieved complete marrow engraftment and mixed chimerism in DNA analysis. For the following 13 months she was not transfusion dependent and had a 100% Karnofsky score. But on the 14th month she had anemia ollowing fever, rash and enteritis. Parvovirus B19 IgM seropositivity confirmed Parvovirus infection. Although intravenous immunoglobulin was administered, bone marrow morphology and DNA analysis revealed rejection. Although mixed chimerism detected shortly after the BMT procedure might raise the possibility of an ongoing slow graft rejection during the relatively stable remission period, we think that parvovirus B19 had also contributed rejection.

Prognostic significance of wilms tumor 1 gene in childhood acute Lymphoblastic Leukemia.

Wilms tumor 1 (WT1) gene is a tumor supressor gene, expressed in malignant and normal hematopoietic progenitor cells. Prognostic significance of this gene in childhood acute lymphoid leukemia (ALL) is not clear. We evaluated the presence of WT1 expression in bone marrow samples of 28 children with de novo ALL at diagnosis by two step RT-PCR. Expression of WT1 gene was detected in 78.5% of patients. There was no correlation between WT1 gene expression and age, sex, FAB type, leukocyte count, and presence of t(4;11) and t(9;22). All patients were treated with modified BFM 86 protocol. There was no difference in the complete remission (CR) rate between WT1 positive and negative patients. Event free survival (EFS) and overall survival (OS) of WT1 positive and negative patients were also not significant. We conclude that expression of WT1 gene is not associated with specific characteristics of ALL blast cells and is not a prognostic factor for CR, remission duration and overall survival.

Detection of BCR/ABL transcripts by reverse transcriptase polimerase chain reaction in pediatric acute Lymphoblastic Leukemia: incidence and clinical eatures.

BCR/ABL expression, which is the molecular equivalent of the Philadelphia chromosome, is an independent poor risk factor in acute lymphoblastic leukemia (ALL). We used a two-step (nested) reverse transcriptase polimerase chain reaction (RT-PCR) assay to examine BCR/ABL expression in the diagnostic bone marrow specimen of children with ALL, prospectively. Among 75 de novo ALL patients, 4 (%5.3) were found to be BCR/ABL- ositive, whereas 4 of 17 relapsed patients (23.5%) were positive. This preliminary study in Turkish children showed an incidence similar to reports from Europe and the U.S.A. More intensive chemotherapies and allogeneic bone marrow transplantations (BMT) uring the first remission were planned if a donor was available. Out of 8 BCR/ABL-positive patients, complete remission (CR) was achieved in 7 patients and partial remission (PR) was achieved in 1 patient. Three patients underwent allogeneic BMT during the first CR and 1 under went autologous BMT during the first PR. The Kaplan-Meier estimate of vent-free survival (EFS) of BCR/ABL negative de novo ALL patients was 78.36% at 3 years, whereas the EFS of positive patients was 31.25% at 26 ± 6.4 months. Molecular screening for the Philadelphia chromosome should become a part of the routine diagnostic panel in ALL patients in order to predict which patients have a poor prognosis and need tailored therapy.

Differential diagnosis of Fanconi anemia by nitrogen mustard and diepoxybutane.

Fanconi anemia (FA) is an autosomal recessive inherited disorder which is associated with a variety of congenital anomalies. These include morphometric abnormalities involving mainly the head and face, skeletal malformations particularly of the radial ray, growth retardation, abnormal skin pigmentation, deafness, and renal, ocular, genital, and cardiac defects. The cardinal clinical feature is a severe progressive pancytopenia. The overall aim of our study was to compare two different alkylating agents that would permit rapid and unequivocal detection of FA. A total of 271 patients underwent nitrogen mustard (NTM) and diepoxybutane (DEB) tests in our laboratory; baseline chromosomal breakage was studied for all of them. After the results of the chromosomal breakage studies, 72 patients were diagnosed as affected and 136 patients as unaffected by FA. We also studied 63 family members of FA patients. According to our study, NTM seems more specific to identify chromosomal breakages in FA parents than DEB.

Is AML1/ETO gene expression a good prognostic factor in pediatric acute myeloblastic leukemia?

To assess the clinical significance of AML1/ETO gene detected by nested reverse transcriptase polymerase chain reaction, the outcome of 7 patients with acute myeloblastic leukemia between 3 and 14 years of age were presented. All patients had complete remission (CR) at the end of induction (AML-MRC 10 protocol) and 4 underwent unpurged autologous, 2 allogeneic (from matched siblings) non-T-cell-depleted bone marrow transplantations (BMT) in first CR. One patient died due to allogeneic BMT-related complications, and 4 patients relapsed at 13, 17, 18, and 26 months. Only one patient achieved second CR. All relapsed patients died between 18 and 36 months with resistant disease (n = 3) or infection during salvage chemotherapy (n = 1). Two patients who had autologous BMT are alive and disease free at 44 and 50 months. Although statistical significance could not be shown, event-free survival and overall survival rates of AML1/ETO-positive patients (28.57 and 28.57%, respectively) at 3.5 years were even lower than those of AML1/ETO-negative patients. The results confirm some previous reports that AML1/ETO gene in children and adolescents is not a favorable prognostic factor.

Two genes are responsible for Griscelli syndrome at the same 15q21 locus.

Griscelli syndrome is a rare autosomal recessive disease characterized by pigment dilution, variable cellular immunodeficiency, and an acute phase of uncontrolled T lymphocyte and macrophage activation. We previously mapped the disease locus to 15q21 and showed that a MyoVa gene (HGMW-approved symbol MYO5A) defect leads to Griscelli syndrome. We report a second MyoVa mutation in a new patient, confirming this first finding. However, in four other Griscelli syndrome patients analyzed, the MYOVA protein is expressed, and no mutation can be detected in the MyoVa gene coding sequence, even in the alternatively spliced region for which exon-intron boundaries were characterized. Linkage analysis performed in 15 Griscelli families thus far studied confirms the first localization. However, fine haplotype analysis in three families strongly suggests the existence of a second gene at the same locus for Griscelli syndrome less than 7.3 cM distant from the MyoVa gene.

Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.