News Release

Title
Identification of novel neuroblastoma biomarkers using urine samples

Key Points• Comprehensive analysis of metabolites in urine samples obtained from children withneuroblastoma using liquid chromatography–mass spectrometry revealed three metabolic
markers important for neuroblastoma diagnosis. These new biomarkers (3-methoxytyramine
sulfate [3-MTS], cystathionine [CTN], and cortisol [COR]) were used for effectively
distinguishing patients with neuroblastoma from the control subjects.
• The predictive value of the conventional biomarker, HVA/VMA, was assessed in 15 children
with neuroblastoma, 2 of whom were false-negative cases. In contrast, the predictive value of
3-MTS, CTN, and COR was positive in all 15 patients with neuroblastoma.
• Identification of metabolic pathways specific to other cancer types and a combination ofmetabolites with high contribution to each cancer type may also provide useful biomarkers forcancer diagnosis.

Summary
A research group led by Assistant Professor Kazuki Yokota and Professor Hiroo Uchidaat the Department of Pediatric Surgery, Nagoya University Graduate School of Medicine,
Designated Professor Akinari Hinoki at the Department of Rare and Intractable Cancer
Analysis Research, with the cooperation of Hitachi, Ltd. could distinguish between 15 patientswith neuroblastoma and 39 control subjects using three new biomarkers—3-methoxytyramine
sulphate (3-MTS), cystathionine (CTN), and cortisol (COR).
Neuroblastoma is the most common childhood-onset solid tumor, except for braintumors. It is related to a poor prognosis and is the second leading cause of death among all
childhood cancers, with a 5-year progression-free survival rate of 30–40% for high-risk
neuroblastoma. HVA/VMA is used to diagnose neuroblastoma; however, these biomarkers cansometimes lead to false-negative diagnosis.
In this study, 15 children with neuroblastoma were subjected to diagnosis using thepredictive value of the conventional HVA/VMA biomarker, and 2 of these cases were false
negative. In contrast, the predictive value of the three new biomarkers, 3-MTS, CTN, and COR,
was positive for all 15 patients with neuroblastoma. These results indicated that the threeurinary metabolites associated with different metabolic systems identified in the present
comprehensive analysis may be useful biomarkers for neuroblastoma diagnosis.
Furthermore, identification of metabolic pathways specific to other cancer types andthe combination of urinary metabolites with high contribution to each cancer type may also
provide valuable biomarkers for cancer diagnosis.
The results of this research were published in the international scientific journal
Scientific Reports on February 18, 2021 (at 19 time in Japan).

Research BackgroundChildhood cancer is a rare disease, accounting for less than 1% of all cancers; however, it isthe second leading cause of death among children. Therefore, developing a method for early
diagnosis is desirable. In general, the mainstream cancer tests include blood tests and imaging
tests (such as CT/PET), but these tests are invasive, involve pain and radiation exposure, andsometimes require sedation associated with the tests. Recent studies on liquid biopsy havefocused on blood samples; however, in case of children, we believe that less invasive methods,
such as urine testing, are preferable over blood testing. Because urine contains manymetabolites, studies using polyamines and microRNAs have been conducted in various fields,
including pediatric oncology.
For neuroblastoma diagnosis, we focused on urinary metabolites (other thanHVA/VMA) that are associated with tumor characteristics and HVA/VMA. HVA/VMA
biomarker is used to diagnose neuroblastoma; however, it can provide false-negative results insome cases. Therefore, we aimed at the comprehensive analysis of urinary metabolites using
liquid chromatography–mass spectrometry to identify important metabolic pathways inchildren with neuroblastoma and to use these representative metabolites as biomarkers for
accurate and early diagnosis of neuroblastoma.

Research Results
We conducted a comprehensive analysis using urine samples from children withneuroblastoma and identified 998 metabolites. After removing exogenous substances fromthese metabolites, we identified the tyrosine, methionine, steroid, and leucine metabolic
pathways of the metabolites that were significantly increased in the neuroblastoma and control

groups.
We then attempted to distinguish the neuroblastoma group from the control groupusing the Orthogonal Projections to Latent Structures Discriminant Analysis model.
Examination of various combinations of substances with different metabolic systems confirmedthat the combination of 3-methoxytyramine sulfate (3-MTS), cystathionine (CTN), and cortisol(COR), related to different metabolic systems, resulted in fewer false negatives and higher
explanatory variables (R2) and predictive ability (Q2). This combination was therefore selected
as the new urinary biomarker for neuroblastoma. On comparing the predictive values of theconventional tumor markers (HVA and VMA) with those of the new urinary biomarkers (3-MTS,CTN, and COR) for assessing the risk of cancer, it was observed that the former resulted in 2
false-negative cases, whereas the latter was positive for all 15 children with neuroblastoma.
Positive predictive values indicate a high risk of cancer while negative predictive values
indicate a low risk of cancer. The findings suggested that the levels of 3-MTS, CTN, and COR
may be elevated in children aged less than 1 year.

Table1 ： Nineteen of the highest-ranking metabolites according to their contributionsevaluated by the random forest method.

Rank
1234910111314

Metabolites
homovanillate(HVA)
3-methoxytyramine
sulfatevanillylmandelate(VMA)vanillactate
3-methoxy-4-hydrox
yphenylglycol
cystathionine
3,4-dihydroxyphenyl
acetate3,4-dihydroxyphenyl
acetate sulfate
dopamine
3-O-sulfate

Fold-change

Increase or(neuroblastoma/control)

Decrease

17

increase<0.01

12increase

<0.01

27

increase

<0.01

31

increase

<0.01

19

increase

<0.01

11

increase

<0.01

19.

increase

<0.01

9.3

increase

<0.01

7.8

increase<0.01

ｐ valueSuperSub

pathway

pathway

Amino

Tyrosine

Acid

Metabolism

Amino

Tyrosine

Acid

Metabolism

Amino

Tyrosine

Acid

Metabolism

Amino

Tyrosine

Acid

Metabolism

Amino

Tyrosine

Acid

Metabolism

Amino

Methionine

Acid

Metabolism

Amino

Tyrosine

Acid

Metabolism

Amino

Tyrosine

Acid

Metabolism

Amino

Tyrosine

Acid

Metabolism

17dehydroascorbate

0.29

decrease

0.02

18

3-methoxytyrosine

13increase<0.01

3.6increase

<0.01

0.56

decrease

<0.01

1923

alpha-hydroxyisoval
erate
N2,N5-diacetylornit
hine

24

urea

0.77

decrease<0.0125

cortisol

10increase<0.0126

3-methoxytyramine

10increase<0.0128

homocitrulline

0.33

decrease<0.0129

tiglyl carnitine (C5)0.77

decrease

0.018

30

xanthurenate

0.49

decrease<0.01

Cofactor

Ascorbates

Metabolism

Amino

Tyrosine

Acid

Metabolism

Amino

LeucineAcid

Metabolism

Amino

Arginine

Acid

Metabolism

Amino

Arginine

Acid

Metabolism

Lipid

Corticostero
ids

Amino

Tyrosine

Acid

Metabolism

Amino

Arginine

Acid

Metabolism

Amino

LeucineAcid

Metabolism

Amino

Tryptophan

Acid

Metabolism

Fig. 1

OPLS-DA results（3-MTS、CTN、COR） showed that the control group formed a very narrowly
scattered pattern and the tumour group formed a widely scattered pattern.

Fig. 2(a)(b)(a) Predictive value analysis of the combination of VMA and HVA .
(b) Predictive value analysis of the combinations of three metabolites （3-MTS、CTN、COR）.

Research Summary and Future Perspective
Advances in mass spectrometry and other medical devices have enabled themeasurement of metabolites even at low concentrations that could not be measured in the past.
In this study, to identify biomarkers other than HVA/VMA for neuroblastoma diagnosis, we
performed a comprehensive analysis of urinary metabolites, identified important metabolic
pathways and metabolites, and developed a method to analyze neuroblastoma risk based on
the predictive value of the biomarkers. In the future, we plan to validate the urinary
biomarkers of neuroblastoma identified in this study (3-MTS, CTN, and COR). In addition, thisapproach may be valuable in future studies to identify new biomarkers in cancer types forwhich no effective biomarkers have been discovered till date.

Publication
Kazuki Yokota1, Hiroo Uchida1*, Minoru Sakairi2,3, Mayumi Abe2, Yujiro Tanaka1, Takahisa
Tainaka1, Chiyoe Shirota1, Wataru Sumida1, Kazuo Oshima1, Satoshi Makita1, Hizuru Amano1,Akinari Hinoki1,3
1 Department of Pediatric Surgery, Nagoya University Graduate School of Medicine, Nagoya

466-8550, Japan.
2 Hitachi, Ltd., R & D Group, Centre for Exploratory Research, Tokyo 185-8601, Japan3

Department of Rare/ Intractable Cancer Analysis Research, Nagoya University Graduate

School of Medicine, Nagoya 466-8550, Japan
Identification of novel neuroblastoma biomarkers in urine samples, Scientific Reports
Published February 18, 2021
DOI: 10.1038/s41598-021-83619-w
Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Sci_Rep_210218.pdf