Giuliana Cangemi

Improved Survival of Children With Neuroblastoma Between 1979 and 2005: A Report of the Italian Neuroblastoma Registry

PURPOSE To describe treatment, clinical course, and survival of a cohort of Italian patients with neuroblastoma. PATIENTS AND METHODS The study includes data from 2,216 children (age 0 to 14 years) diagnosed between 1979 and 2005. Overall survival (OS) was analyzed by clinical and biologic features at presentation and periods of diagnosis: 1979 to 1984, 1985 to 1991, 1992 to 1998, and 1999 to 2005. The relative risk of second malignant neoplasm (SMN) was assessed by the standardized incidence ratio (SIR), with the Italian population selected as referent. RESULTS Yearly patient accrual increased over time from 58 to 102. Patients age 0 to 17 months represented 45.6% of the total population, and their incidence increased over time from 36.5% to 48.5%. The incidence of stage 1 patients increased over time from 5.8% to 23.2%. A total of 898 patients (40.5%) developed disease progression or relapse, 19 patients developed SMN, and two patients developed myelodysplasia. The cumulative risk of SMN at 20 years was 7.1%, for an SIR of 8.4 (95% CI, 5.1 to 13.2). A total of 858 patients (39%) died (779 of disease, 71 of toxicity, six of SMN, and two of tumor-unrelated surgical complications). Ten-year OS was 55.3% (95% CI, 53.0% to 57.6%) and increased over time from 34.9% to 65.0%; it was significantly better for females and patients age 0 to 17 months at diagnosis, with extra-abdominal primary, and stage 1 and 2 disease. OS improved significantly over time in stage 1 and 3 patients. In patients with stage 4 disease, the improvement occurred between the first and second time cohorts (6.7% v 23.5%), but not afterward. CONCLUSION The outcome of children with neuroblastoma has progressively improved. Long-term survivors bear a significant risk of SMN.

Diagnostic potential of hepcidin testing in pediatrics

Hepcidin, a peptide hormone released by hepatocytes into circulation is the main regulator of dietary iron absorption and cellular iron release. Although commercial tests are available, assay harmonization for hepcidin has not been yet reached, making reference intervals and consequent clinical decisions still elusive for each assay and specific population. The aim of this study is to set up hepcidin measurement in pediatric age and to investigate its potential usefulness in the diagnosis and management of iron disorders in children.

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically

Natural killer (NK) cells have the capability of lysing targets that have down‐regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV‐infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti‐p70, anti‐p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV‐infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV‐infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer‐cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV‐infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA‐A and HLA‐B and did not modify the expression of HLA‐C, but also that NK cell clones expressing the “activating” form of the anti HLA‐C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV‐infected autologous target cells. J. Med. Virol. 62:354–363, 2000.

Urinary homovanillic and vanillylmandelic acid in the diagnosis of neuroblastoma: Report from the Italian cooperative group for neuroblastoma

BACKGROUND Urinary homovanillic and vanillylmandelic acid (HVA and VMA) are well known biomarkers for the management of neuroblastoma (NB). Very few and contradictory publications on their diagnostic performance are present in the literature. The aim of this study is to review the results of HVA/Cr and VMA/Cr obtained by the reference laboratory of the Italian Cooperative Group for NB within a 7-year period using HPLC-EC. PROCEDURE Updated reference intervals based on age as a continuous variable were calculated by using a multivariate statistical analysis. The diagnostic performance of the two biomarkers has been established by calculating their specificity and sensitivity and by receiver operating characteristics (ROC) curves for different ages and stages of disease. RESULTS Accurate age-related reference intervals were obtained from 648 HVA/Cr and 671 VMA/Cr results derived from patients in which the diagnosis of neuroendocrine tumors was excluded. Sensitivity, specificity and ROC curves were obtained from 169 HVA/Cr and 179 VMA/Cr results from confirmed NB patients. The best diagnostic performance was obtained in stage 4S tumors and in children <18months. CONCLUSIONS This is the first report, to our knowledge, that analyzes in depth the diagnostic performance of HVA/Cr and VMA/Cr for NB in different stages and age subgroups. In addition, the present work provides cut-off points able to discriminate between NB patients and negative subjects suspected to have NB and could be of help in taking medical decisions.

Reference values for urinary neutrophil gelatinase-associated lipocalin (NGAL) in pediatric age measured with a fully automated chemiluminescent platform

Abstract Background: Neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as the most promising biomarker of acute kidney injury. However, there are no reliable data on analytical sensitivity and reference limits of urinary NGAL (uNGAL) assay in pediatric age. The aim of the present study is to evaluate the analytical sensitivity and the reference range of uNGAL measured in urine specimens of pediatric age with the fully automated platform ARCHITECT® i1000. Methods: A total of 333 urine samples were collected from 25 healthy newborns (16 males and 9 females; age 1–4 days) and 308 children (150 males and 147 females; mean age 80.7 months, range 0.63–248 months) and assayed for uNGAL by two different Italian centers (Department of Laboratory Medicine of the Fondazione Toscana G. Monasterio of Pisa and Massa and the Clinical Pathology Laboratory Unit of Istituto Giannina Gaslini of Genova). Results: The calculated limits of blank (LOB) and detection (LOD) values were 0.5 ng/mL and 0.95 ng/mL, respectively. The distribution of uNGAL values approximated a log-normal distribution (median 5.2 ng/mL, interquartile range 2.5–12.8 ng/mL, 99th percentile 117.6 ng/mL). uNGAL values of the 25 neonates were significantly higher than those of 308 children (neonates: mean 44.2 ng/mL, median 30.3 ng/mL, range 5.2–137.4 ng/mL; children: mean 10.2 ng/mL, median 4.6 ng/mL, range 0.2–146.7 ng/mL; p<0.0001). Conclusions: uNGAL assay shows a good analytical sensitivity and imprecision, which allows the measurement of uNGAL values around the cut-off value (i.e., 117.6 ng/mL) with an imprecision <5 CV%. The distribution of uNGAL values in pediatric age approximates a log-normal distribution, with values which are higher in neonates compared to children.

Prognostic value of ferritin, neuron-specific enolase, lactate dehydrogenase, and urinary and plasmatic catecholamine metabolites in children with neuroblastoma.

Different plasma and urinary parameters have been tested as valuable prognostic markers for children with neuroblastoma (NB), but conclusive results from multivariate analyses are still lacking. Samples collected at diagnosis from 505 patients diagnosed in Italy between June 1994 and November 2010 were analyzed at the Italian reference laboratory according to standard methodologies. Patient clinical data were retrieved from the Italian NB Registry. For statistical analysis, patients were grouped according to stage, age, MYCN status, and outcome. Cumulative survival was calculated by the Kaplan–Meier procedure using the first quartile of the marker distribution as a cut-off value to stratify the patients. Multivariate analysis was performed by the Cox regression model by considering only the significant variables. When the entire cohort of patients was considered, none of the different parameters had an independent prognostic value. However, in patients with localized disease without MYCN amplification the significant positive associations between urinary and plasmatic vanillylmandelic acid (VMA)/homovanillic acid (HVA) ratio and a better prognosis remained significant (P < 0.05 and P < 0.01, respectively), as well as, the positive association between high lactate dehydrogenase (LDH) values and a worse prognosis (P < 0.001). Moreover, in stage 4 patients without MYCN amplification, neuron-specific enolase levels above 200 ng/mL and LDH levels above 2500 IU/mL maintained their significant association with a worse outcome (P = 0.01 and P = 0.0001, respectively). In conclusion, LDH had an independent prognostic value in patients of all stages without MYCN amplification. Moreover, the urinary and plasmatic VMA/HVA ratio was an independent predictor of prognosis in patients with localized disease without MYCN amplification. Since LDH and catecholamine metabolites are measured in all patients at diagnosis, these findings may be helpful for an easy, cost-effective, patient risk stratification.

Comparison of antibody-conjugated magnetic immunoassay and liquid chromatography-tandem mass spectrometry for the measurement of cyclosporine and tacrolimus in whole blood

The aim of this work is to compare the results of a commercially available liquid chromatography tandem mass spectrometry (LC-MS/MS) method in a clinical pathology laboratory for routine Therapeutic Drug Monitoring (TDM) of cyclosporine (CsA) and tacrolimus (Tacr) in pediatric patients with those obtained with the current antibody-conjugated magnetic immunoassay (ACMIA). Whole blood levels of CsA (n= 135) and Tacr (n=100) were sequentially analyzed by using ACMIA and LC-MS/MS on pediatric transplanted patients. The differences were analyzed by using the Passing Bablok regression analysis and the Bland and Altman test. The LC-MS/MS method showed excellent reproducibility and lower limits of quantification compared to the ACMIA. A linear relationship between ACMIA and LC-MS/MS was obtained for both CsA (r= 0.9449; P<0.0001, 95% CI 0.9234 to 0.9605) and Tacr (r=0.9275; P<0.0001, 95% CI 0.8941 to 0.9506). No significant inter-method biases were observed. The analytical performances of the LC-MS/MS method make it suitable for the accurate measurement of CsA and Tacr in pediatric transplanted patients. However ACMIA results are also accurate and reliable. For this reason the choice of the method to be used in a routine clinical pathology laboratory can be made on the bases of non-analytical considerations such as costs, organization, availability of skilled personnel.

A validated HPLC method for the monitoring of thiopurine metabolites in whole blood in paediatric patients with inflammatory bowel disease.

Therapeutic drug monitoring (TDM) of major metabolites of thiopurine drugs is a widely used tool for assessing treatment efficacy and toxicity in patients with inflammatory bowel disease (IBD). We report the laboratory and clinical validation of a simple and reliable high performance liquid chromatography (HPLC) method for the measurement of 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) on paediatric patients with IBD. The aim of this paper is to develop and validate a method for the measurement of 6-TGN and 6-MMP applicable to routine practice and to evaluate the usefulness of the TDM of thiopurine drugs in children with IBD attending our Gastroenterology Unit. The HPLC method was validated following international guidelines starting from red blood cells (RBC) and whole blood (WB). A comparison between RBC and WB was assessed. The usefulness of TDM was then evaluated using the new method from WB in 47 paediatric patients with IBD treated with thiopurine drugs. WB and RBC resulted in interchangeable matrices. The majority of patients had the metabolite levels inside the therapeutic ranges. A moderate correlation was found between 6-MMP concentration and the dose of thiopurines. A higher percentage of non responders was found among patients with lower levels of 6-TGN. Toxicity was found in eight patients and was evaluated in respect to the metabolite concentration. The described HPLC method is applicable to routine practice and it is suitable for its use in multicentric studies. Our results of TDM on paediatric IBD patients can contribute to clarify its role in their therapeutic management.

Quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma by liquid chromatography/tandem mass spectrometry

Therapeutic drug monitoring is a cornerstone of antibacterial therapy, especially in an era of increasing antibacterial resistance in individualizing antimicrobial therapy. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was used for the simultaneous measurement of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in 50 μl plasma samples over a wide range. The overall turnaround time for the assay was 20 minutes. Intra-assay precision and accuracy for quality control samples ranged within 1·8–8·5 and 91·4–106·7%, respectively. Inter-assay precision and accuracy ranged within 1·3–14·4 and 95·8–104·6%, respectively. The lower limit of quantification was below 1·5 μg/ml for all the five antibiotics. No ion suppression due to matrix effects was found. A simple and rapid LC–MS/MS method which provides high specificity, precision and accuracy for the simultaneous quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma has been developed and validated. The present method is suitable for therapeutic drug monitoring in paediatrics.

Plasma Levels of Soluble HLA-E and HLA-F at Diagnosis May Predict Overall Survival of Neuroblastoma Patients

The purpose of this study was to identify the plasma/serum biomarkers that are able to predict overall survival (OS) of neuroblastoma (NB) patients. Concentration of soluble (s) biomarkers was evaluated in plasma (sHLA-E, sHLA-F, chromogranin, and B7H3) or serum (calprotectin) samples from NB patients or healthy children. The levels of biomarkers that were significantly higher in NB patients were then analyzed considering localized or metastatic subsets. Finally, biomarkers that were significantly different in these two subsets were correlated with patients outcome. With the exception of B7H3, levels of all molecules were significantly higher in NB patients than those in controls. However, only chromogranin, sHLA-E, and sHLA-F levels were different between patients with metastatic and localized tumors. sHLA-E and -F levels correlated with each other but not chromogranin. Chromogranin levels correlated with different event-free survival (EFS), whereas sHLA-E and -F levels also correlated with different OS. Association with OS was also detected considering only patients with metastatic disease. In conclusion, low levels of sHLA-E and -F significantly associated with worse EFS/OS in the whole cohort of NB patients and in patients with metastatic NB. Thus, these molecules deserve to be tested in prospective studies to evaluate their predictive power for high-risk NB patients.