Sebastiano Barco

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.

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.

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.

A validated LC–MS/MS method for the quantification of piperacillin/tazobactam on dried blood spot

Background Therapeutic drug monitoring (TDM) of antibiotics in children is of fundamental importance for effective patient management. The use of dried blood spot (DBS) offers a number of advantages over conventional blood collection. The aim of this study is to develop and validate a method to measure piperacillin/tazobactam in DBS. Results The analysis was performed by using LC-MS/MS operating in multiple reaction monitoring mode. The method has been validated by applying EMA guidelines and its suitability for TDM was evaluated by using samples from low birth weight neonates. Conclusion This paper describes a fast and cost-effective micromethod for the simultaneous determination of piperacillin/tazobactam levels on dried blood spot that is suitable for TDM in children.

Plasma total adiponectin levels in pediatrics: reference intervals calculated as a continuous variable of age.

OBJECTIVE Plasma total Adiponectin (tAN) is a novel biomarker with interesting potentialities in pediatrics. Age-related intervals are needed for the correct interpretation of results. In this study, we calculated the reference values for tAN using a large number of results derived from pediatric patients. DESIGN AND METHODS tAN was determined by ELISA in more than 4000 samples and the results collected in the Laboratory Information System were used for the calculation of reference intervals. RESULTS tAN reference values for the different age intervals were obtained and age related tAN reference intervals were calculated. Some differences were observed in males and females. In obese children, no correlation between age and tAN could be observed. On the contrary, significant relations were observed in the total group of patients and in healthy control subjects. For this reason, the parameters of the equations to calculate tAN reference values using age as a continuous variable were defined. CONCLUSION The use of continuous tAN reference intervals, calculated on age, should be considered a useful laboratory tool, at least in the pediatric population, for those biomarkers whose reference values have been shown to change from birth to the late adolescence.

Volumetric adsorptive microsampling-liquid chromatography tandem mass spectrometry assay for the simultaneous quantification of four antibiotics in human blood: Method development, validation and comparison with dried blood spot

HighlightsA new VAMS‐LC–MS/MS method for quantification of antibiotics from 10 &mgr;L blood is shown.The new method has been validated following international guidelines.The absence of HCT on the method performance has been verified in comparison with a DBS method.The method has been applied on samples derived from pediatric patients under therapy. Summary In this paper we show the development and validation of a volumetric absorptive microsampling (VAMS™)‐LC–MS/MS method for the simultaneous quantification of four antibiotics: piperacillin‐tazobactam, meropenem, linezolid and ceftazidime in 10 &mgr;L human blood. The novel VAMS‐LC–MS/MS method has been compared with a dried blood spot (DBS)‐based method in terms of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics were extracted from VAMS and DBS by protein precipitation with methanol after a re‐hydration step at 37 °C for 10 min. LC–MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS‐LC–MS/MS method is selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics.

Interchangeability between 24-hour collection and single spot urines for vanillylmandelic and homovanillic acid levels in the diagnosis of neuroblastoma.

The determination of the two urinary catecholamine metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) is of crucial importance for the diagnosis and follow‐up of neuroblastoma (NB). The standard practice for their measurement requires the use of 24‐hour collections that are time consuming and difficult to obtain. In this article, we directly demonstrate that 24‐hour collections and single spot urines are interchangeable for the determination of HVA and VMA expressed as ratio on creatinine concentration. This study can be useful for a faster management of NB at onset. Pediatr Blood Cancer 2013;60:E170–E172.

CD4+CD25hiCD127− Treg and CD4+CD45R0+CD49b+LAG3+ Tr1 cells in bone marrow and peripheral blood samples from children with neuroblastoma

ABSTRACT Metastatic spread in the bone marrow (BM) at diagnosis is the worst prognostic factor for neuroblastoma (NB) patients. Here, we analyzed the presence of two immunosuppressive cell subsets, CD4+CD25hiCD127− regulatory T (Treg) cells and CD4+CD45R0+CD49b+LAG3+ type 1 regulatory (Tr1) cells, in BM and peripheral blood (PB) samples from NB patients and controls. Frequency of both regulatory cell subsets was lower in BM and PB samples from NB patients than in respective healthy controls. No correlation was found between the frequency of Treg and Tr1 cells and prognostic factors at diagnosis, such as age and stage. Only MYCN amplification correlated to a higher number of Treg in BM and of Tr1 in PB. These findings suggested an altered trafficking of regulatory T cells in NB, but delineated a limited role of these subsets in BM microenvironment and/or periphery in NB. These observations should be considered designing immunotherapeutic approaches for metastatic NB.