Urinary Metabolite alterations in Wilms Tumor.
Dawn L. MacLellan, MD FRCSC1, Tobias K. Karakach, PhD2, Rodrigo L.P. Romao, MD1, Conrad V. Fernandez, MD1.
1Dalhousie University, Halifax, NS, Canada, 2National Research Council of Canada, Halifax, NS, Canada.
Identification of urinary molecular signatures linked to histology and risk of progression and relapse would be valuable for the management of Wilms tumor(WT). Biopsy of renal masses carries risk and may not be representative of the tumor. Detection of urinary biomarkers may allow non-invasive testing of the underlying histology of renal masses as well as risk recurrence, and thus appropriate risk stratification for therapy. In addition, it may help understand tumor-related biological pathways.
Ninety-one frozen urine samples from males with WT studied through the National Wilms Tumor Study Group trials and the AREN03B2 biology and classification study were obtained from the Children's Oncology Group tissue bank. Voided urine specimens of 10 healthy male children were collected from a pediatric urology clinic. Diagnoses included phimosis, hydrocele, testicular pain, and enuresis. Sample distribution with regards to stage, histology and relapse were as follows: Stage 1 (n=20), Stage 2 (n=27), Stage 3 (n=28), and Stage 4 (n=16); favorable histology (n=76), diffuse anaplasia (n=15); relapse (n=38).
One-dimensional Proton Nuclear Magnetic Resonance (1D 1H NMR) spectra of the urine samples were acquired and analyzed using advanced multivariate statistical methods, principal component analysis (PCA) and partial least squares with discriminant analysis (PLS-DA).
Urinary metabolomics allows metabolite signatures of control and WT samples to be distinctly separated (see Figure 1). Levels of creatine, creatinine, acetate and citrate are shown to be lower in children with WT, while the levels of amino acids, sugars (glucose, sucrose, lactose, mannitol ), dimethylamine, 2-oxoglutarate, alanine, and branched chain amino acids (including : leucine, isoleucine, iso-valerate, 2-hydroxybutyrate, and 2-oxoisovalerate) are elevated relative to control subjects. Metabolite profiles for different stages of WT showed distinct and separable profiles as did metabolite profiles of anaplasia and favorable histology samples. Levels of acetate, alanine and 3-hydroyisovalerate are higher in anaplasia samples, whereas favorable histology urine samples had higher levels of creatine, glucose, amino acids, dimethylamine/succinate, glutamate/glutamine, and 2-oxoglutarate when compared to anaplastic samples. Within the favorable histology group, there was separation of relapse and relapse free samples with some overlap. Relapse samples had higher levels of 2-oxoglutarate and alanine; whereas relapse free samples had higher levels of creatine, 3-hydroxyisovalerate, and glutamate/glutamine.
Patients with favorable histology WT have a distinct urinary metabolite profile compared to normal controls; these signatures vary between different stages of WT and might be able to distinguish patients prone to relapse. Tumors with diffuse anaplasia exhibit a distinctive metabolite signature compared to those depicting favorable histology. Validation in a larger sample size is required.
Fig. 1: PLS-DA scores plot indicating dissimilarity in the NMR spectral profiles of the normal pediatric urine from their counterparts with Wilms tumor at different stages and histologies
Figure 2. : PLS-DA scores plot indicating dissimilarity in the NMR spectral profiles of diffuse anaplasia (DIFINA, yellow) vs. Favourable histology samples (blue). Sample stage is denoted in roman numerals.
Figure 3. PLS-DA scores plot indicating dissimilarity in the NMR spectral profiles of urine specimens with favourable histology with (blue/"yes" ) and without (yellow/"no") relapse.
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