Questioning How to Define the "Ultra-High-Risk" Subgroup of Neuroblastoma Patients.

Neuroblastic tumours exhibit heterogeneity, which results in different therapeutic outcomes. Neuroblastoma is categorized into three major risk groups (low, intermediate, high risk). Recent identification of new genes raised the possibility of new biomarkers to identify sub-risk groups. In this retrospective cross-sectional study, we aimed to assess new biomarkers defining the ultra-high-risk subgroup within the high-risk group that differ in clinical situation with very bad prognosis. Twenty-five low- and 29 high-risk groups of patients were analysed for their expression of ALK, ATRX, HIF1a, HIF2a (EPAS), H2AFX, and ETV5 genes at the RNA level. Immunohistochemistry was performed to confirm the protein expression level of ALK. The risk group of patients was determined according to the International Neuroblastoma Risk Group Stratification System. Spearman correlation analysis and Mann-Whitney-U nonparametric test were used to assess the importance of expression levels among the groups. P < 0.05 was considered as significant. Sensitivity of the results was checked by ROC curve analysis. All analysed genes were found to be highly expressed in the high-risk group compared to the low-risk group, except for ETV5. When the ultra-high-risk and highrisk groups were compared, ALK was found to be highly expressed in the ultra-high-risk group. Our results show that ALK may be a candidate gene whose mRNA expression levels can distinguish the ultrahigh- risk subgroup of patients in the high-risk group of patients with non-familial neuroblastoma.

Carbon monoxide attenuates bacteria-induced Endothelin-1 expression in second trimester placental explants.

INTRODUCTION: The pro-inflammatory mediator and potent vasoconstrictor Endothelin-1 (ET-1) is known to be expressed in the placenta. We have recently demonstrated that very low, non-toxic doses of carbon monoxide (CO), prevented infection-induced preterm birth in mice. However the effect(s) of CO on human gestational tissues is yet to be fully explored. We hypothesize that CO will have a protective role against inflammation-induced E. coli by down-regulating the ET axis in placental explants. METHODS: Twenty placentas from elective termination of pregnancy in the second trimester were analyzed with or without exposure to heat killed E. coli over the course of 30 h. Placental ET-1, along with its biologically inactive precursor Big ET-1, and Endothelin Converting Enzyme-1 (ECE-1, responsible for the cleavage of Big ET-1 to ET-1), were analyzed by ELISA. Gene expression for ET-1 (EDN1), ECE-1 and the ETA receptor (EDNRA) were analyzed using qPCR. Localization of ET-1 expression was also demonstrated using immunohistochemistry. RESULTS: E. coli significantly increased ET-1 transcription and secretion of BIG ET-1 and ET-1 in a time dependant manner which was ameliorated when exposed to CO at later time points. In the presence of CO, mRNA levels of ECE-1 were significantly reduced at 3 and 24 h, while EDNRA was significantly reduced at 6 and 18 h. CONCLUSIONS: Up-regulation of ET-1 production in human placenta in the setting of infection can be attenuated by low doses of CO. Our results further explore the anti-inflammatory and regulatory mechanism(s) of CO on the ET axis components at the maternal fetal interface.

Protective effect of acetyl-l-carnitine against cisplatin ototoxicity: role of apoptosis-related genes and pro-inflammatory cytokines.

OBJECTIVES: Cisplatin is an anti-neoplastic agent treatment with which causes many side effects including ototoxicity. The aim of this study was to investigate whether acetyl-L-carnitine would have protective effects on cisplatin-induced ototoxicity in vitro, and if present, to reveal roles of apoptotic gene expressions and pro-inflammatory cytokines. MATERIALS AND METHODS: House Ear Institute-Organ of Corti 1 cell line was used for this study. Apoptotic genes were evaluated with an apoptosis PCR array and pro-inflammatory cytokine levels were measured using ELISA. RESULTS: Apoptotic cell death reduced by around 22% with acetyl-L-carnitine-cisplatin treatment compared to cisplatin alone. Genes displaying increase in expression of apoptosis, related to cisplatin treatment, were Casp8, Bcl10, Bcl2, Bcl2l1, Bcl2l2, Bid, Naip1, Bnip3l, Card6, Pak7, Cd40, Trp 53inp1, Cideb and Cd70. The acetyl-L-carnitine-cisplatin combination caused reduced expression of genes Casp8, Fas, Casp1, Tnfrsf11b, Tnfrsf10b induced by cisplatin. Acetyl-L-carnitine-cisplatin also caused reduced levels of IL-6, IL-1ß and TNF-α, pro-inflammatory cytokines, induced by cisplatin. CONCLUSION: Protective mechanisms of aceytl-L-carnitine against cisplatin induced apoptosis, mainly due to activation of anti-apoptotic Bcl family members' genes, and in an Akt-related gene expression dependent manner. This is the first study to indicate that acetyl-L-carnitine can be an effective agent against cisplatin ototoxicity in auditory cells, with induction of anti-apoptotic gene expression and attenuating levels of pro-inflammatory cytokines.

High-dose oral methylprednisolone therapy in childhood hemangiomas.

The authors report their experience with high-dose oral methylprednisolone therapy (HDMP) in 15 infants with complicated hemangiomas. The starting dose for methylprednisolone was 30 mg/kg/day for 5 days, then the dose was tapered gradually every 5 days to 20, 10, 5, 2.5, and finally to 1 mg/kg/day. Therapy was then stopped and the patients were followed. An initial response was evident in 12 patients. Nine out of 12 responders showed regrowth signs. After regrowth, 4 cases received prednisolone at doses between 1 to 5 mg/kg/day and 3 patients received a second course with HDMP as additional corticosteroid therapy. Overall, 9 out of 15 cases were responders; very good and good responses were obtained in 5, partial response in 4, and therapy failure in 5 cases. One child was not available for evaluation of response. A very rapid initial response was observed in subglottic and periocular hemangiomas. Side effects were not serious and resolved after discontinuation of treatment. Although the number of patients is small in this study, overall response rate with HDMP regimen seems not to be superior to the regimens that use lower doses (5 mg/kg/day), but it provides a high initial response rate and the duration of therapy is short. Therefore, it may be useful for treating hemangiomas that fail to respond with low doses, especially in centers with limited resources where other treatment modalities cannot be used at the moment.

Bleomycin-induced hyperpigmentation and hypersensitivity reactions to etoposide and vinblastine in a child with endodermal sinus tumor.

We report a pediatric case who developed bleomycin-induced hyperpigmentation and hypersensitivity reactions to both etoposide and vinblastine while receiving chemotherapy for germ cell tumor. Skin hyperpigmentation related to chemotherapeutic agents has been reported only rarely in pediatric patients. This patient developed a characteristic skin hyperpigmentation which was "flagellate" in appearance. Two features of the hyperpigmentation were noteworthy: development at a low cumulative dose of bleomycin and persistence after cessation of chemotherapy. Additive effect of cisplatinum-induced hyperpigmentation was suggested. Although hypersensitivity reactions to etoposide have been previously reported, hypersensitivity reactions to vinblastine are almost unknown. To our knowledge, this is the first report of hypersensitivity reaction to vinblastine in a child in English literature.

A rare tumor of craniofacial bones in children: a pediatric chondroblastic osteosarcoma case with diagnostic and therapeutic problems.

Osteosarcoma of the cranial facial region is uncommon and only rarely involves the ethmoid or sphenoid bones. The authors report on an unusual case of a 17-year-old male presenting with chondroblastic osteosarcoma of the maxillary, ethmoid, and sphenoid sinuses who remains well and disease-free at 46 months. He was treated with anterior craniofacial resection followed by postoperative radiotherapy to the sight of the primary tumor. He did not receive chemotherapy because of emerging hepatitis-B infection and vasculitis. The literature on extragnathic craniofacial osteosarcomas is reviewed with particular emphasis on treatment options of this rare tumor.

A case with extraosseous Ewing's sarcoma: a late effect related to bone marrow transplantation for thalassemia or a component of a familial cancer syndrome?

Allogeneic bone marrow transplantation has proved to be a radical form of cure in patients with beta-thalassemia major who have a human leukocyte antigen identical donor. Although malignant neoplasms are serious late complications of bone marrow transplantation, very few reports describing the development of malignant tumors after allografting for thalassemia appeared in the literature. A case is presented here of extraosseous Ewing's sarcoma that developed 8 years after allogeneic bone marrow transplantation performed for beta-thalassemia major. The phenotypic features of the patient's family fulfill the criteria for Li-Fraumeni syndrome. The patient was treated with chemotherapy and radiotherapy and died with recurrent disease. To the authors' knowledge, this is the first case of extraosseous Ewing's sarcoma after bone marrow transplantation for thalassemia. The possible contribution of transplantation procedure and the genetic factors as well as the primary genetic hemoglobinopathy to the development of this malignant tumor are discussed.

Tropisetron in the prevention of chemotherapy-induced acute emesis in pediatric patients.

We evaluated the antiemetic efficacy of tropisetron for control of acute emesis during grade 3 or 4 emetogenic chemotherapy in children. Tropisetron was administered as a single intravenous dose of 0.2 mg/kg on the first day and intravenously or orally with the same dose on subsequent days. A total of 125 courses of highly emetogenic chemotherapy was administered to 22 children with a median age of 14 years (range: 3-18 years). All 22 patients received tropisetron for at least two courses. Overall complete response on day 1 was observed in 80 out of 125 courses (64%). The response rates were consistent over multiple courses; a complete and major response rate on the first day of Course I (n: 22 courses) and Course II (n: 22 courses) was observed in 73 and 77 percent of cases, respectively. When the results were analyzed according to the daily schedules, overall complete response for grade 4, grade 3 and grade 1-2 emetogenic treatment days was 59, 85 and 75 percent, respectively. In this study, cost effectiveness for tropisetron was also determined; the cost per successfully controlled course was 162 USD. No side effects of tropisetron other than mild diarrhea and dry-mouth were documented in this study. In conclusion, the results of this study confirmed that tropisetron is a safe, well tolerated and effective antiemetic drug for the prevention of acute emesis in children and adolescents during highly emetogenic chemotherapy.

Platelet activation during the early neonatal period.

The first week of life is a time when hereditary or more frequently acquired factors lead to some important differences in the hemostatic mechanism of the newborn. It has been well known that ill neonates are prone to both hemorrhage and thrombosis. The aim of this study was to answer the question of whether there is a difference in platelet activation in healthy neonates during the first days of life that may contribute to both hemorrhage and thrombosis in the presence of additional pathologic insults. Platelet activation was determined with flow cytometry using monoclonal antibodies in 63 healthy children (29 neonates, 17 infants, and 17 older children). There was no significant difference in platelet activation among these three age groups (p > 0.05). In addition, platelet activation did not show any significant relationship to age, sex, mode of delivery, or blood bilirubin concentration (p > 0.05). It has been previously reported that platelet activation occurs at the time of birth. We could not find any evidence that healthy newborns during the first 3 days of life exhibit increased platelet activation. Further studies on platelet activation in ill neonates will help to clarify whether platelet activation plays a role in the pathogenesis of thrombotic and/or hemorrhagic disorders.

Posterior fossa pseudotumour due to viral encephalitis in a child.

A 14-year-old boy had a 1-month history of diplopia (due to a VI nerve palsy), motor ataxia and dizziness. Brain MRI showed a 1.5-cm mass posterior to the pons. Histopathological examination of a biopsy specimen showed the lesion to be of viral origin. After 3 months, the ataxia and dizziness had resolved and the MRI findings returned to normal. By 5 months the abducens paralysis had also resolved. Viral encephalitis should be considered in the differential diagnosis of posterior fossa tumours.

Platelet activation in congenital heart diseases.

The research presented here investigated platelet activation in cyanotic and acyanotic congenital heart diseases (CHD). Children with cyanotic CHD are prone to both thrombosis and hemorrhage. However, patients with acyanotic CHD may also have a mild bleeding disorder. The platelet activation in CHD was investigated in support of a hypothesis that platelet activation may play a role in the hemostatic abnormalities reported in these patients. Platelet activation was determined by using flow cytometry with anti-CD62 monoclonal antibody (mAb), which has been shown to be a specific marker of platelet activation. Thirteen children with cyanotic CHD, 33 children with acyanotic CHD and 17 healthy children serving as controls were studied. Platelet activation was significantly higher in the cyanotic group and also in the acyanotic group compared with the healthy children (P = 0.0000 and P = 0.019, respectively). In the cyanotic group, platelet activation showed a direct correlation with arterial O2 saturation (SaO2) (P = 0.014). There was no correlation between platelet activation and erythrocyte related parameters in either group. Platelet activation occurs in CHD, particularly in patients with cyanotic CHD (even in patients with no evidence of clinical thrombosis) and it may play a role in the pathogenesis of thrombotic disorders seen in these patients.

Hyperglycemia and acute parotitis related to L-asparaginase therapy.

In a boy with non-Hodgkin's lymphoma (NHL), two different complications developed concurrently associated with L-asparaginase (L-ASP) therapy. A non-ketotic hyperglycemic state was observed simultaneously with bilateral acute parotitis after the patient was subjected to L-ASP. The hyperglycemia with normal insulin levels and the absence of plasma and urine ketones was controlled with insulin therapy and no residual impairment of glucose tolerance was demonstrated later. Bilateral acute parotitis, which is a rare complication associated with L-ASP, resolved spontaneously within a week after cessation of L-ASP. The rarely observed toxic effects of L-ASP, such as parotitis, should be recognized as promptly as the better-known complications, e.g., hyperglycemia, to avoid the continuation of this antineoplastic agent.

Atypical hemolytic uremic syndrome.

The pathogenesis of atypical uremic syndrome (HUS), which is rarely encountered in childhood, is poorly understood and its mortality and morbidity rates are high. A wide variety of therapeutic approaches has been attempted and the literature contains numerous conflicting reports about the results of these approaches. In a case diagnosed as recurrent atypical HUS, pulse methyl prednisolone, fresh frozen plasma infusions and plasma exchange transfusion were used at different stages of the disease with satisfactory response.

Relationship between serum unconjugated bilirubin levels and the autofluorescence of white blood cells in neonatal jaundice.

In this study, using flow cytometry, we investigated the autofluorescence emitted by lymphocytes, monocytes and neutrophils exposed to different unconjugated bilirubin concentrations and investigated the relationship between these parameters. Different unconjugated bilirubin concentrations were prepared from a newborn serum with an unconjugated bilirubin concentration of 800 m mumol/l. The same concentrations of unconjugated bilirubin have been prepared from pure bilirubin. 10 microliters of cord blood were incubated at room temperature for 15 min with 90-microliter solutions with different bilirubin concentrations prepared from both serum and pure bilirubin. After incubation, cells were washed three times with PBS, erythrocytes were lysed by lysing buffer and run through flow cytometry immediately. Autofluorescence was measured by recording mean fluorescence channels for lymphocytes, monocytes and neutrophils. There was a good correlation between serum concentrations of unconjugated bilirubin that cells were exposed to and the autofluorescence intensity of neutrophils (r = 0.904, p < 0.005), monocytes (r = 0.759, p < 0.05) and lymphocytes (r = 0.766, p < 0.01). Results obtained with pure bilirubin were also similar. Autofluorescence emitted by lymphocytes was lower than that of monocytes (p < 0.01) or neutrophils (p < 0.0005).

Assessment of clinical impact and predisposing factors for neonatal thrombocytopenia.

Thrombocytopenia is a common hemostatic abnormality in the newborn infant. The early diagnosis of thrombocytopenia and the underlying primary pathology process play an important role in reducing the risk of severe complications and mortality. We performed a 2-year prospective study of 643 neonates admitted to our neonatology unit to determine the frequency, predisposing factors, and clinical impact of thrombocytopenia. Thrombocytopenia developed in 18.2% of the preterm neonates and 0.8% of the term neonates. Prematurity, sepsis, hypoxia, intrauterine growth retardation, and disseminated intravascular coagulation were identified as predisposing factors for thrombocytopenia. The incidence of complications and mortality were higher in thrombocytopenic infants. Especially the prognosis was worse in cases who had mucosal hemorrhage, without a relation with the degree of thrombocytopenia. The thrombocytopenia occurred by day 2 in 43% of the infants, and resolved by day 8 in 61%. The platelet count nadir occurred by day 2. Since thrombocytopenic infants are at greater risk for bleeding, and the thrombocytopenia itself may have contributed to the high mortality, predisposing factors such as prematurity, infections, hypoxia must be eliminated by providing better care, giving adequate hygiene of both mother and the baby during the prenatal, natal, and neonatal period.

Nutrition, immunity and infections: T lymphocyte subpopulations in protein--energy malnutrition.

Protein energy malnutrition (PEM) is one of the most frequent causes of secondary immune deficiency states. Alterations either in cellular or humoral immune mechanisms increase the susceptibility to infections in the malnourished organism. Infections aggravate the interrelationship of malnutrition to immune deficiency and infections, resulting in future adverse effects of malnutrition on humoral and cellular immune systems, IgG, IgM, IgA, C3, and T lymphocyte subpopulations were identified in 29 patients with PEM and 15 healthy infants serving as the control group, ranging between 3 and 24 months of age. Patients with PEM demonstrated elevated levels of IgG, IgM and IgA when compared to the control group (P < 0.01, P < 0.01, P < 0.01), C3 levels were significantly lower than the values of the control group (P < 0.01).