Pediatric Neuroblastoma Diagnosis Using Urinary Catecholamine Metabolites
Overview
Urinary catecholamine metabolite analysis is a valuable tool in the diagnosis of neuroblastoma. There is currently no agreement with regard to the method of sampling and variable catecholamine metabolite combinations being utilized. In this study, we assessed the reliability of spot urine samples in assessing the catecholamine metabolites to diagnose neuroblastoma.
Spot urine or 24-hour urine samples were obtained from both participants who had neuroblastoma and those who didn’t have neuroblastoma at diagnosis. Ultra-performance liquid chromatography with electrospray tandem mass spectrometry (UPLC-MS/MS)in addition to high-performance liquid chromatography with fluorescence detection (HPLC-FD) was used to measure vanillylmandelic acid (VMA), homovanillic acid (HVA), norepinephrine, epinephrine, dopamine, metanephrine, 3-methoxytyramine and normetanephrine. 400 participants with neuroblastoma and 571 participants without neuroblastoma had their urine assessed for catecholamine metabolite levels. 234 out of the 400 participants with neuroblastoma gave a 24-hour urine sample, while 166 gave spot urine samples. All participants without neuroblastoma served as the control group, and they all gave spot urine samples.
The diagnostic sensitivity and excretion level of the catecholamine metabolites were the same in spot urine samples and 24-hour urine samples. No significant variation was noted in the catecholamine metabolite levels among the two chosen analysis methods.
Spot urine samples are recommended by the Catecholamine Working Group for diagnosis in the standard of care. Vanillylmandelic acid and homovanillic acid levels were noted to have a lesser diagnostic accuracy compared to the complete panel comprising 8 catecholamine metabolites.
Introduction
Neuroblastoma
Neuroblastoma is the commonest solid tumor located extracranially in the pediatric population. It has a median age of diagnosis at 18 months. Urinary catecholamine metabolite measurement is a valuable tool in the diagnosis of neuroblastoma. 95% of patients diagnosed with neuroblastoma are noted to have high levels of urinary catecholamines. Previously, the 24-hour urine sample was recommended to assess the urinary catecholamine levels for diagnosis of neuroblastoma, this is because of the circadian rhythm fluctuations that may happen in the excretion of catecholamine metabolites in the urine noted in healthy adults.
The 24-hour urine sample collected has proven difficult on several occasions. A catheter or urine collection bag may be needed for 24-hour urine collection as the majority of patients are children. The use of urine collection bags has a higher rate of failure to accurately and completely collect the 24-hour urine sample.
Another method of urine sample collection for the measurement of urinary catecholamine levels is the spot urine sample, which is a sample collected randomly with catecholamine metabolites excretion being expressed in relation to creatinine excretion.
Several studies carried out previously demonstrated that 24-hour urine and spot urine samples had similar diagnostic sensitivities of the most commonly assessed urine markers such as VMA and HVA. This further suggests that spot urine samples can be used in place of 24-hour urine samples in assessing VMA and HVA levels.
However, a debate is currently ongoing as to whether vanillylmandelic acid and homovanillic acid should be included in the diagnostic investigations for neuroblastoma, owing to the fact that their combined sensitivity for diagnosis is just 85% in both spot urine and 24-hour urine samples.
Diagnostic sensitivity is however increased to 95% when the panel of 8 urinary catecholamine metabolites also called the extended panel are assessed including VMA and HVA plus their precursors which include norepinephrine, epinephrine, dopamine, metanephrine, normetanephrine and 3-methoxytyramine. Additionally, several cohort studies have shown that high levels of 3-methoxytyramine at diagnosis were associated with a poorer prognosis, also in patients who had high-risk neuroblastoma.
The diagnostic specificity of the urinary catecholamine metabolite extended panel has not been assessed in the past, and thus it is unclear if spot urine sample measurements can be used to reliably diagnose neuroblastoma. The analysis of catecholamine metabolites may differ among several neuroblastoma centers due to variations in the time of sample collection, catecholamine metabolite panels and methods of measurement.
Several methods used in the measurement of urinary catecholamine metabolites include ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography coupled with fluorescence detection or electrochemical detection (HPLCFD/ED).
High-performance liquid chromatography coupled with fluorescence detection or electrochemical detection (HPLCFD/ED) is commonly used, but it can be time-consuming. Additionally, common drugs can interfere with the assessment of catecholamine metabolites.
The accuracy and precision of detecting urinary catecholamine have been enhanced with the introduction of mass spectrometry.
The aims of the study includes the following:
- To assess the sensitivity of the 8-panel catecholamine metabolites using 24-hour urine and spot urine samples in the diagnosis of neuroblastoma,
- To analyze the accuracy of the 8-panel catecholamine metabolites in spot urine and the classic VMA and HVA panel in spot urine for the diagnosis of neuroblastoma,
- To demonstrate the methods of sample collection, methods of detection and panels of catecholamine metabolites in neuroblastoma centers in association with the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN).
Methods
400 patients who had been diagnosed with neuroblastoma and treated at the Amsterdam University Medical Centre (Amsterdam UMC) between the years 1990 and 2014 and at Princess Maxima Centre between the years 2018 to 2021 were retrospectively studied.
Diagnosis of all patients was done histologically in accordance with the International Neuroblastoma Staging System (INSS).
296 patients with neuroblastoma were included in the Amsterdam University Medical Center cohort, while 104 patients with neuroblastoma were included in the Princess Maxima Center cohort. Additionally, 39 patients which included patients with neuroblastoma and patients with other pediatric malignancies or no cancer at all from the Amsterdam University Medical Center and Princess Maxima Center were categorized into a separate cohort, and used for methodological comparisons between ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography coupled with fluorescence detection or electrochemical detection (HPLCFD/ED).
Also, 571 patients without neuroblastoma were recruited from the Wilhelmina Children’s Hospital. These patients were screened for the presence of other metabolic diseases and used as a control to establish catecholamine metabolite concentration reference values which are age-dependent.
The spot urine or 24-hour urine samples were obtained at the time of diagnosis of neuroblastoma. The time of collection was reported standardly on the application form. 24-hour urine collection which was not completely collected or failed was reported as a sample collection period of less than or more than 24 hours. There was no requirement for diet restriction before the urine sample was obtained.
Spot urine samples and 24-hour urine samples were taken and assessed using high-performance liquid chromatography coupled with fluorescence detection or electrochemical detection (HPLCFD/ED) at the Amsterdam University Medical Center. Urine samples were protected against light, acidified via the use of hydrochloric acid and stored properly at an adequate temperature of 4◦C prior to its analysis.
Spot Urine Samples were taken and assessed using ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS) at the Princess Maxima Centre. Urine samples were protected against light and were stored at a temperature of -20◦C prior to analysis without any acidification.
Urine samples were examined within a period of one week after it was collected at all three centers.
A retrospective analysis of data obtained from neuroblastoma patients was carried out. In addition to the methodological comparisons of testing techniques, catecholamine metabolite measurements in banked samples from both patients with neuroblastoma and patients without neuroblastoma was assessed. Catecholamine metabolite levels were said to be high if it exceeds the upper limit or 95th percentile of the reference range which is age-specific for spot urine samples and 24-hour urine samples.
Statistical analysis
Patient characteristics and diagnostic sensitivities between groups were compared using the chi-square test. Catecholamine metabolite excretion was compared using the Mann-Whitney U test. The number of patients with raised catecholamine metabolite concentrations divided by the sum total of patients diagnosed with neuroblastoma is defined as the diagnostic sensitivity, while the number of controls with no increase in catecholamine metabolites divided by the sum total of controls is defined as the diagnostic specificity.
The Wald method was used to calculate the confidence intervals of the specificity and sensitivity. Differences between the AUCs were calculated using the DeLong et al. method.
The GraphPad Prism was used to carry out statistical analysis. P-value <0.05 was significant statistically.
Results
In a survey carried out in SIOPEN centers, 21 out of 35 responses were received. The survey reported that 7 out of 21 SIOPEN centers used 24-hour urine sample collection or a combination of 24-hour urine collection and spot urine collection in the diagnosis of neuroblastoma. Also, high-performance liquid chromatography coupled with fluorescence detection or electrochemical detection (HPLCFD/ED) was the most commonly utilized method of detection. Additionally, a minimum of 6 panels of catecholamine metabolites were employed in diagnosis, with the 8 panels and VMA/HVA panels being the most commonly used across SIOPEN centers.
63 (27%) out of 234 patients had an unsuccessful 24-hour urine sample collection. Inability or failure to completely collect 24-hour urine samples was seen more in females and patients who had stage 4 disease. Additionally, there was a 20% to 30% failure to completely collect 24-hour urine samples in patients across all age groups which shows that the difficulties associated with this sample collection method are not only restricted to children.
There were no significant differences in the catecholamine excretion levels and diagnostic sensitivities for the 8-panel catecholamine metabolites between 24-hour urine samples and spot urine samples.
The diagnostic sensitivities of ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography coupled with fluorescence detection or electrochemical detection (HPLCFD/ED) in catecholamine metabolites measurements were also similar.
The diagnostic sensitivity and accuracy of the 8-panel catecholamine metabolites assay were observed to be higher than the panel consisting of just HVA and VMA, while the diagnostic specificity of the VMA/HVA panel was higher than that of the 8-panel catecholamine metabolites. HVA and normetanephrine were the metabolites most commonly elevated in neuroblastoma irrespective of the method of sample collection and analysis.
Conclusion
In this study, we established that the diagnosis of neuroblastoma can be carried out in an easier and more timely manner using the spot urine sample collection method rather than the 24-hour urine collection method. Additionally, we demonstrated the accuracy of the 8-panel catecholamine metabolites in the diagnosis of neuroblastoma, compared to the panel consisting of just HVA and VMA. Although the reliability of spot urine sample collection in the diagnosis of neuroblastoma has not yet been investigated, the steady catecholamine metabolite excretion associated with neuroblastoma will likely lead to a similar sensitivity between 24-hour urine samples and spot urine samples.
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