Urinary nuclear magnetic resonance spectroscopy of a Bangladeshi cohort with hepatitis-B hepatocellular carcinoma: A biomarker corroboration study.
OBJECTIVE
To establish if a distinct urinary metabolic profile could be identified in Bangladeshi hepatitis-B hepatocellular carcinoma (HCC) patients compared to cirrhosis patients and controls.


METHODS
Urine samples from 42 Bangladeshi patients with HCC (39 patients with hepatitis-B HCC), 47 with cirrhosis on a background of hepatitis B, 46 with chronic hepatitis B, and seven ethnically-matched healthy controls were analyzed using nuclear magnetic resonance (NMR) spectroscopy. A full dietary and medication history was recorded for each subject. The urinary NMR data were analyzed using principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) techniques. Differences in relative signal levels of the most discriminatory metabolites identified by PCA and OPLS-DA were compared between subject groups using an independent samples Kruskal-Wallis one-way analysis of variance (ANOVA) test with all pairwise multiple comparisons. Within the patient subgroups, the Mann-Whitney U test was used to compare metabolite levels depending on hepatitis B e-antigen (HBeAg) status and treatment with anti-viral therapy. A Benjamini-Hochberg adjustment was applied to acquire the level of significance for multiple testing, with a declared level of statistical significance of P < 0.05.


RESULTS
There were significant differences in age (P < 0.001), weight (P < 0.001), and body mass index (P < 0.001) across the four clinical subgroups. Serum alanine aminotransferase (ALT) was significantly higher in the HCC group compared to controls (P < 0.001); serum α-fetoprotein was generally markedly elevated in HCC compared to controls; and serum creatinine levels were significantly reduced in the HCC group compared to the cirrhosis group (P = 0.004). A three-factor PCA scores plot showed clustering of the urinary NMR spectra from the four subgroups. Metabolites that contributed to the discrimination between the subgroups included acetate, creatine, creatinine, dimethyamine (DMA), formate, glycine, hippurate, and trimethylamine-N-oxide (TMAO). A comparison of relative metabolite levels confirmed that carnitine was significantly increased in HCC; and creatinine, hippurate, and TMAO were significantly reduced in HCC compared to the other subgroups. HBeAg negative patients showed a significant increase in creatinine (P = 0.001) compared to HBeAg positive patients in the chronic hepatitis B subgroup, whilst HBeAg negative patients showed a significant decrease in DMA (P = 0.004) in the cirrhosis subgroup compared to HBeAg positive patients. There were no differences in metabolite levels in HCC patients who did or did not receive antiviral treatment.


CONCLUSIONS
Urinary NMR changes in Bangladeshi HCC were identified, corroborating previous findings from Egypt and West Africa. These findings could form the basis for the development of a cost-effective HCC dipstick screening test.
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
I Jane
IJ
Cox
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Abil E
AE
Aliev
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Mary Me
MM
Crossey
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Mahvish
M
Dawood
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Mamun
M
Al-Mahtab
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Sheikh M
SM
Akbar
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Salimur
S
Rahman
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Antonio
A
Riva
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Roger
R
Williams
I Jane Cox, Antonio Riva, Roger Williams, Institute of Hepatology, London, Foundation for Liver Research, London WC1E 6HX, United Kingdom.
Simon D
SD
Taylor-Robinson
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000704:Analysis of Variance; D000998:Antiviral Agents / Q000627:therapeutic use; D001459:Bangladesh; D014408:Biomarkers, Tumor / Q000097:blood / Q000652:urine*; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000473:pathology / Q000652:urine* / Q000821:virology; D005260:Female; D006509:Hepatitis B / Q000097:blood / Q000150:complications* / Q000175:diagnosis / Q000188:drug therapy; D006513:Hepatitis B e Antigens / Q000097:blood; D006801:Humans; D016018:Least-Squares Analysis; D008113:Liver Neoplasms / Q000097:blood / Q000473:pathology / Q000652:urine* / Q000821:virology; D008297:Male; D055432:Metabolomics / Q000379:methods*; D008875:Middle Aged; D011237:Predictive Value of Tests; D025341:Principal Component Analysis; D066244:Proton Magnetic Resonance Spectroscopy; D012189:Retrospective Studies; D016482:Urinalysis; D055815:Young Adult
D000998:Antiviral Agents; D014408:Biomarkers, Tumor; D006513:Hepatitis B e Antigens
Bangladesh; Hepatitis B; Hepatocellular carcinoma; Nuclear magnetic resonance spectroscopy; Urinary metabolic profiling