NMR based Metabolome to characterize and compare hepatocellular carcinoma with chronic liver disease and normal liver

Mr. Rakesh.N M.Phil trainee CERTC, Trivandrum

Metabonomics
Metabonomics is formally defined as, “The quantitative measurement of dynamic multi-parametric metabolic response of living systems to physiological stimuli or genetic modifications”. It summarizes the entire pool of metabolites in a bio-fluid, thereby promising a powerful diagnostic tool in future.
Metabolome or Metabolic profile
Ref: Nicholson et al., 1999

Metabonomics in health so far
• Diagnosis and classification of diseases (tumor types) • Time course disease progression • Learn pathological mechanisms • identify new biomarkers • Responses to treatment (efficiency, toxicity) • Drug design (decrease development time) • Generating databases (HMDB, tumor metabolome database)

Evolution of Metabonomics ?
Post-genomic Era of Biology
Genome Transcriptomics

Metabonomics
Genomics Gene expression Metabolism Proteins

Environmental stressors

Proteomics

gas chromatography. LC.Technologies commonly used in Metabolic profiling • NMR: Nuclear magnetic resonance spectroscopy • MS: Mass spectroscopy (coupled with GC or LC) • FTIR: Fourier transformed infra red spectra • CE: capillary electrophoresis GC.liquid chromatography .

Overview NMR-based metabonomic approach Tissue or biofluid sample NMR spectroscopy Measure the metabolite profile Multivariate analysis Treat profile as statistical ‘object’ for classification purposes Explore profile to gain mechanistic insight into the stress response 1H • Minimal sample preparation • Rapid analysis • Unbiased detector • Molecular structure .

4 3 2.46 hypotaurine carnitine arginine • extremely congested spectra (raw data) with hundreds of overlapping peaks 1.58 2.6 2.50 Chemical shift (ppm) 2.8 2.2 2 1.54 2.Sample spectrum with metabolic profiling Intensity 1-D 1H NMR spectra dimethylglycine ? acetate 2.6 .62 zoom Intensity alanine 2.8 Chemical shift (ppm) 1.4 2.

via the identification of metabolic profile.We hypothesize that NMR-based metabonomics can provide evidence for the diagnosis of diseases. Generate a hypothesis Record limited dataset specific to that hypothesis Determine if hypothesis is true or false (next phase) Hypothesis-driven research .

(2007) .E. called hepatocellular carcinoma 90% of all primary liver cancer is HCC 20 to 50% of patients presenting with hepatocellular carcinoma had previously undiagnosed cirrhosis.CANCER OF THE LIVER hepatocellular carcinoma Chronic liver cell injuries and regeneration stimulates a pathway of increased liver cell activation resulting in malignant transformation of hepatocytes.H. Ref:Blum.

000 deaths from HCC all over the world. • In the year 2000.9%. Ref: Mohandas. • The prevalence of HCC in an autopsy study among Indians were low.2% to 1. and varies from 0. it was projected that there will be 430.Global burden of HCC • Fifth most common malignancy in men & eighth in women worldwide • Age and male sex have a positive correlation • A rise in incidence due to cohort effect of hep B and C infections during 1980’s.K (2004) .

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1 9.1 2.4 6.3 0.000 people.9 3.77 in males and 1.7 1.5 9.7 2.2 22.7 0.28 in females per 100.9 1.1 1.8 4.0 1.1 1.5 9.5 3.7 36.2 1.1 2.1 1.Age standardised incidence rate of HCC in different cities of India compared with rest of the world Place Japan (Osaka) Hong Kong China (Shanghai) Singapore Chinese Singapore Indians US SEER (Blacks) US SEER (Whites) Mumbai Trivandrum Bangalore Chennai Delhi Bhopal Karunagapally Barshi Men 46. .8 5.0 3.8 Women 11.2 2.6 2.5 2.5 1.2 28.7 The mean age adjusted incidence of HCC in 6 Indian populations is 2.

hepatitis. corn. or too much iron in the liver (hemochromatosis) • Aflatoxins (from fungus that can contaminate peanuts. wheat.Etiology of Liver cancer • Hepatitis B (HBV) and hepatitis C (HCV) • Cirrhosis due to alcohol. soybeans. groundnuts. and rice) • Tobacco use .

Imaging • Ultrasound of the liver.Diagnosis of hepatocellular carcinoma ? recommendations from EASL (European association for studies on liver) Serology • liver function tests. • Blood tests for Hepatitis B and C. • Angiogram of the liver. • Biopsy. • AFP (alpha fetoprotein) blood test. • Laparoscopy. • CT scan or MRI scan of liver. .

According to EASL recommendations • The Gold standard in the diagnosis of liver cancer depends on the size of nodules. imaging techniques + appropriate serology can confidently establish the diagnosis without confirmation from a positive biopsy. . 2001. • nodule size ≤ 2 cm . (imaging techniques do not have sufficient accuracy to distinguish HCC from other conditions & AFP levels usually remain within normal or slightly elevated) • nodules > 2 cm. Ref: Bruix J et al.biopsy is recommended.

Rationale for Metabolic profiling in HCC .

The secondary objective was to find optimal cutoff’s. patients with cirrhotic liver (CLD) and apparently normal people from a basket of metabolome library.Objective of the study To identify & characterise the distribution of significant metabolites in serum of patients with HCC. which differentiates between these conditions .

.Methodology This study is a venture to adopt epidemiological principles into basic science research and is a sole attempt to highlight the use of Nuclear magnetic resonance (NMR) spectroscopy and Metabonomics principle in the diagnosis of hepatocellular carcinoma.

.Research question Is there a different pattern of metabolite levels distributed in human serum samples analysed using 1H NMR based metabolome. which can distinguish patients with hepatocellular carcinoma from cirrhotic liver or people with apparently normal liver admitted to a tertiary care centre.

and (1-) at 95%. .Study design: Descriptive study Sample size: Keeping sensitivity of new test as 95 %. precision of population parameter as 10 %. the sample size was 18 positive cases.

Study setting • Gastroenterology department. . Medical College. (tertiary care centre) • NIST (National institute of science and technology) Pappanamcode. with results of USG and appropriate serology tests (Inclusion criteria) Exclusion criteria • Patients not giving consent. Trivandrum. Study subjects Cases consecutively admitted to Gastroenterology department in Medical College. • Patients without or not willing for further tests. Trivandrum. for the confirmation of diagnosis as HCC or no HCC.

Reference Test  USG along with appropriate serology and expert opinions. (relatively imperfect gold standard when compared to histopathology)  approval from human ethical committee were obtained.  Data collection using Performa after informed consent  Performa was completed by patient’s attending physician according to medical files. .

chronic liver disease and normal liver. • The patients were grouped as Hepatocellular carcinoma. • CHILD Pugh score were recorded (as a summary index in progression of liver cirrhosis). • Tests for AFP and USG were measured on all patients.Stepwise protocol in sample collection • Blood samples were collected and routine biochemical parameters were measured. CT and MRI were used wherever required. . • The serum samples were sent for NMR spectroscopy.

(250 metabolites considered.Methodology for in vitro NMR spectroscopy • Sample collection. dilution with D2O. 137 metabolites were selected) . serum separation. • Metabolic profiles generated . storage. loading into NMR tubes. • Water suppression experiment. • Preprocessing of spectrum in CHENOMX NMR SUITE. • Spectrum was obtained from Bruker 400x NMR spectroscopy.

(considered as 137 independent tests) 2x2 tables for each metabolite at different levels ROC curve plotted & optimal cut off to discriminate HCC and no HCC Sn & 1-Sp was calculated at different levels AUC was computed. .test's ability to discriminate between HCC and no HCC.Data mining using ROC analysis 137 Metabolites.I (95%) Z test) Area under the ROC curves . (P-value and C.

.Metabolites with significant AUC & their Optimal cutoff’s Measures of accuracy (Sn. if any one test is positive is considered as test positive) Combinations in the metabolome which can improve accuracy of diagnosis of HCC will be summarized .e. Sp. LR+ & LR-) Combinations Metabolome Parallel combinations of metabolites in metabolome (i.

Results Baseline characters of total study subjects .

Baseline characters of hepatocellular carcinoma group .

Experimental spectrum after processing .

92) AST (IU/L) ALT (IU/L) ALP (IU/L) Bilirubin (mg/dl) CHILD-Pugh AST (aspartate transferase).636 (1. 95% C.94-74. ALP(alkaline phospatase) .02-225.35 (20.03) 2.49-70.900 (0.04 (136.60) 22.26 (183. ALT (alkaline transferase).56) 0.950-3.25 (55.067-6.083) 5.98) 80.43) 58. 95% C.10) 4.93) 165.36) 23.72-25.18-91.19) 284.I) 180 (134.74) 104.34-25.90-9.21 (45.07 (80.069) 11 (10. 95% C.26-11.Biochemical parameters of the total samples Characteristics HCC group CLD group Normal liver n=20 n=28 n= 20 (Mean.71-127.I) (Mean.98) 65.75 (7.43-385.068 (2.68 (70.60 (5.04-194.I) (Mean.35 (19.322) 8.28-5.717-1.

805 0. 2-phosphoglycerate 13.865 0.589 0.747 0.729 0.911 0.003 0.001 0.583 0.894 0.827 0.835 0. acetoacetate 18.621 0. citrate 24.000 0. 2-hydroxyglutarate 7.634 0.001 0.000 0.872 0.004 0.893 0.000 0. 3-dimethylurate 3. 2-oxovalerate 12.001 0.644 0.851 0.775 0.922 0.754 0.940 0.781 0.628 0.930 0. 2-oxoisocaproate 11.763 0.894 0.660 0. butanone 23.741 0. ascorbate 21.760 0.871 0.728 0.734 0.001 0. 2-ethylacrylate 6.890 0.704 0.850 0.880 0. 2-methylglutarate 8.925 0. acetone 19. 2-aminoadipate 5.699 0.639 0.000 0. cystathionine 25.779 0.927 0.668 0.000 0.001 0.593 0.650 0.835 0.881 0.000 0.622 0. 6-anhydro-ß-dglucose 4.809 0. dimethylamine Area under curve 0.000 0.Metabolome with significant Area under the ROC curve Sl Metabolites No 1.936 0. 2-oxobutyrate 9.000 0. 5-aminolevulinate 17.821 0.803 0.750 0.700 0. 2-oxocaproate 10.000 0.000 0.001 0.906 0.755 0.865 0.720 0.000 0.001 0.695 0.879 0. 3-dihydroxyacetone 2. alanine 20.807 p 95% Confidence value Interval 0.659 0.005 0.776 0.911 .878 0.765 0.664 0.756 0. 3-methylglutarate 15.931 0.000 0.724 0. 3-hydroxy-3methylglutarate 14.661 0. aspartate 22.000 0.720 0.924 0.828 0.000 0.879 0.000 0.731 0.796 0. 4-hydroxybutyrate 16.

707 0.678 0. 37.557 0.885 0. 31.739 0.860 0.623 0.611 0.792 0.802 0.009 0.003 0.886 0.818 0.847 .753 0.854 0. 44.873 0.846 0.949 0.878 0.892 0.000 0.916 0.001 0.634 0.843 0.559 0.007 0.732 0. 32.849 0.811 0.561 0. 48.768 0.848 0.004 0. 40.648 0.000 0.936 0. 30.767 0.000 0. 46. 27.001 0.001 0.808 0. 34.008 0.755 0.818 0.930 0.705 0.000 0. 45.621 0.006 0.717 0.000 0.748 0.002 0.604 0. 38.705 0.857 0. 36.000 0.573 0.714 0.719 0.710 0.000 0.921 0.743 0.756 0.836 0.005 0.589 0.691 0.000 0.842 0.573 0. 47.853 0.001 0. 29. 43. 33.704 0.923 0.870 0.701 0.002 0.009 0. 41.643 0. 28. ethylene glycol fructose galactarate galactitol galactonate glucarate glucose glutamate glutarate glutaric acid monomethyl ester glycine glycolate glycylproline guanidoacetate homoserine isobutyrate isocitrate levulinate malate mannitol methionine methylamine methylguanidine methylsuccinate n-dimethylglycine 0.010 0.567 0. 39.765 0.700 0.595 0. 35.714 0.912 0. 49.001 0.905 0.914 0.703 0. 42. 50.820 0.866 0.722 0.26.000 0.615 0.

731 0.785 0.930 0.911 0.713 0.000 0. 61. 55.000 0.810 0.656 0.862 0.654 0. 66.783 0.000 0.573 0.000 0.859 0.702 0.911 0.000 0. 73.830 0. 60.000 0.000 0.659 0. 64. 59.000 0.850 0.736 0.000 0.000 0.724 0.910 0. 68.855 0.798 0.784 0.580 0. n-carbamoylaspartate n-carbamoyl-ß-alanine o-phosphoserine pimelate proline propionate propylene glycol pyruvate sarcosine serine ß-alanine s-sulfocysteine suberate succinate succinylacetone sucrose tartrate threonate trans-4-hydroxy-l-proline trimethylamine trimethylamine n-oxide valine xylose 0.733 0. 71.003 0.804 0.000 0.710 0.661 0.918 0.004 0.720 0. 53.731 0.875 0.901 0. 54. 56. 65.816 0.898 0.699 0.51.794 0.873 0. 52.000 0.606 0. 63.900 0.777 0.590 0.675 0.804 0. 62.899 0.895 0.776 0.003 0.002 0.005 0.821 0. 67.683 0.671 0.000 0.720 0. 69.785 0.898 0.612 0.000 0.571 0. 57.893 0.777 0. 58.671 0.000 0.721 0.919 0.688 0.921 0.578 0.786 0.867 . 72.698 0.898 0.004 0. 70.000 0.007 0.670 0.913 0.

75 680.3998 60.086 00.6427 30.21 00. 3.00 900.28 20.00 680.42 580.91 20.086 00.49 30.6246 20.077 00.7143 0.00 900.92 +LR 30.32 30.22 00.33 540.20 00.95 -LR 00.7171 20. 5.00 900.00 720.14 30.00 900.16 20.00 950. 6-anhydro-ß-dglucose 24. 13.00 900. 7.3481 130.00 850. O-phosphoserine 12.23 00.00 950.72 10. ascorbate 23.20 20. 4.127 40.19 00. 15.72 20.33 580.00 850.1926 40. 6.00 950.7027 120.54 20.28 20.06 30.00 Specificity 700.00 850.75 560.22 00.17 00.15 00.4457 60.8257 00. s-sulfocysteine 22.40 30.21 00.17 790.88 20.72 20.33 560.00 850.8658 120.5898 60.92 720.26 20. 14.14 20.00 720.00 850.92 700.170 10.00 900. 9. metabolites acetoacetate methylamine 2-oxovalerate dimethylamine 2-oxocaproate galactarate 2-hydroxyglutarate acetone glucarate Cut off’s (mM/L) 30.75 640.13 00. 2.88 20.94 50.25 850.42 750.8376 50.17 750.0864 100. glutamate 20.00 900.0808 10.58 580.17 00.9835 50.13 00.8223 240.092 00.83 640.Metabolome and their measures of accuracy Sl no 1.08 750.00 850.15 00.3513 120.75 680. succinate 25.83 680.27 20.00 850.7368 50.00 800.5652 10. 8.15 00.071 00.00 950.8376 40.27 00.22 00.27 00. 3-hydroxy-3methylglutarate 19.68 20. threonate 11. alanine .18 00.75 680.27 10.00 850.00 850.25 770.71 30.60 20. sarcosine propionate butanone citrate 16.7186 Sensitivit y 950.00 900.58 600.219 200.00 800. glutarate 21. tartrate 17.21 00. trimethylamine noxide 18.00 800.07 40.

83 700. levulinate 37.37 10.00 750.6971 110.00 750.40 00.40 00.44 29.58 00. glycine 28.5508 40.57 00.00 750. 4-hydroxybutyrate 60.342 carbamoylaspartate 50.42 830.30 10.35 20. proline 47.28 20.83 700.75 00.67 540.1551 120.00 750.6589 10.92 720.77 00.9003 30.3824 90.4469 20.58 00.57 00.36 20.89 00.83 700.35 20.83 680.41 20.37 30.00 750.00 700.00 800.35 20.00 750.00 700. xylose 43. glycolate 42.00 00. ß-alanine 39.29 20.77 00. propylene glycol 38.00 700. 3-dimethylurate 48.00 700.00 720.83 700.24 00.34 20.74 00.00 750.9556 80.9707 90.00 750. succinylacetone 41.2389 50.92 700.83 680.00 700.00 700.83 700.74 00. 2-phosphoglycerate 330.2818 20. glycylproline 44. valine 35.00 750.34 20.57 00. 3-methylglutarate 50.00 750.40 00.83 700.77 00.33 20.4628 80. n150.25 00.36 00.35 20.00 750.00 750. cystathionine 46. trans-4-hydroxy-lproline 34.4765 110.3909 30.38 20.75 20.1598 120.20 00.0304 200. pimelate 33. 2-aminoadipate 36.00 800.41 20.42 20.92 720.00 700.3615 .75 660.8817 00. pyruvate 32. 3-dihydroxyacetone 45.00 800.40 20.17 750.56 00.41 40.92 700.92 720.00 800.3623 40. malate 100.36 30. isobutyrate 31. suberate 40.00 700.75 660.00 720.80 00.28 20.35 20.67 600.188 49.17 750.35 20.57 00.3447 800.00 750.6442 140.57 00.83 680.34 20.33 790.57 00.8691 20.939 60. 2-methylglutarate 27.26.

8712 10.50 10.44 20.67 620.4011 30.44 20.67 660.83 660.00 700.75 680.4669 40.52 .9397 20.00 700.40 00.00 650. galactitol 68.00 600.00 700. guanidoacetate 63.64 00.75 660.3727 100.0926 00.60 00. homoserine 59.44 20.1396 30.24 00. 5-aminolevulinate 57. trimethylamine 61.49 10.50 10.48 20.51 20.50 10. galactonate 58.95 00.33 750.48 20. n-carbamoyl-ßalanine 10.98 00.5 10.45 10.47 20.23 00.08 20. methylsuccinate 67.0561 100.53 10.56 10.00 700.42 580.00 700.62 00. glutaric acid monomethyl ester 53.00 650.42 600.8915 60.10 00.23 00.4794 30.00 700.92 700.42 830. n-dimethylglycine 65.58 30.00 650.33 770. serine 54.45 20. 2-oxoisocaproate 69. methylguanidine 62. aspartate 56.77 00.6588 20.77 00.50 600.75 680.46 10.42 600.67 640.2698 160.00 700.00 700.51. methionine 64.23 110.1585 700.9777 00.68 00.60 00. ethylene glycol 52.00 650.49 20.24 00.00 680.73 00. isocitrate 66.24 00.7126 60.00 650.77 00.00 650.83 700.00 700. mannitol 60.00 650.8293 190.58 600.10 00. 2-ethylacrylate 55.00 600.00 720.505 70.

67 660.Combinations of metabolite to improve the diagnostic measures Sl No 1 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Metabolite Combinations Galactorate and Cystathionine Succinate and Cystathionine Pyruvate and Cystathionine 2-Hydroxyglutarate and Galactarate Butanone and pyruvate 3 hydroxy-3-methylglutarate and Succinate Cystathionine and Succinylacetone Serine and 2-Ethylacrylate Guanidoacetate and 1.40 20.13 00.0 10.17 0 0 00.07 00.90 LR00.10 .20 00.6-Anhydro-ß-D-glucose and Ethylene glycol 2-methylglutarate and guanidoacetate Methylamine and Propionate 2-Oxocaproate and Methylamine Acetone and 2 ethylacrylate Sensi tivity 90 85 80 95 85 85 75 100 95 95 90 85 100 100 90 85 95 95 85 100 100 95 Specificity LR+ 75 75 75 700.07 00.55 20.58 620.58 640.58 640.85 20.6-Anhydro-ßD-glucose 2 hydroxyglutarate and pimelate Pimelate and Cystathionine Glycylproline and Methylamine Methylamine and Galactarate Galactarate and Trimethylamine Noxide Alanine and Pimelate Acetone and cystathionine 1.08 00.53 20.82 20.06 00.23 0 0 00.83 680.23 00.91 20.50 600.05 20.20 30.67 660.35 0 00.60 30.75 680.91 20.67 640.83 700.15 00.57 30.21 00.40 20.27 00.20 30.83 700.15 20.08 00.0 20.40 30.54 20.82 20.11 00.42 50 50 50 30.21 00.6-Anhydro-ß-Dglucose 2-Methylglutarate and 1.26 20.70 20.83 700.42 600.75 660.58 640.

• To improve the specificity. as well as ultrasound can be made. • Parallel combinations of metabolites can improve sensitivity.al. (Miller.  Different cut off  Serial combinations (Consider the test as positive.Discussion of Results compared with AFP • An approximate comparison of sensitivity and specificity of metabolites with AFP.et. (Arrieta.O et.2005) • Overall sensitivity and specificity of NMR based metabolome in the diagnosis of HCC have to be established.C.J.al. 2007) • Improved sensitivity of many metabolites than the techniques in current practise. if both metabolites are positive) .

4 970.2 100 410.4 39 Sp 76 Sn 13 21 Sp 97 93 45 100 AFP 200 (ng/mL) Sn Sp AFP 400 (ng/mL) Sn Sp .6 310.1 990.6 950.3 32 100 0–64 100 65 41 60 87 94 900.Accuracy of AFP at different cut off’s (Literature search) Authors AFP 20 (ng/mL) AFP 100 (ng/mL) Sn Oka et al 1994 Pateron et al 1994 Peng et al 1999 Tong et al 2001 Trevisani et al 2001 Gebo et al 2002* Nguyen et al 2002 Gupta et al 2003* Farinati et al 2006 Arrieta et al 2007 60 63 41-65 54 600.2 980.3 100 80 80-94 18 200.8 22 99 170.9 470.2 99 360.

• More precise estimates can be obtained if large molecular sized proteins are removed.Recommendations • The Metabolome requires further validation with perfect gold standard. • Many metabolites like 2 butanone. is unique in hepatocellular carcinoma which is also reported in another recent study. • Higher frequency NMR spectroscopy (>500 MHz). can give greater separation. • NMR based metabonomics are cheaper (400Rs/sample) and faster (~ 5 min). • The cut off’s suggested in this study is arbitrary (screening tool) and have to be changed. if using as a confirmatory test. • Known concentration of internal standards to improve the reliability. .

• Optimal cut off’s to discriminate between hepatocellular carcinoma were determined. • Many metabolites in the serum with high sensitivity and reasonable specificity which after validation can be used in routine clinical practise.Conclusion • A Metabolome was identified for hepatocellular carcinoma. .

for their helps. teachers and fellow trainees. who directly & indirectly gave me their strength and wisdom to complete this thesis. stimulating suggestions and encouragements .Acknowledgement Express my gratitude towards one and every body. I am deeply indebted to my supervisors.

THANKING YOU .

USG.Diagnostic measures for AFP. & other imaging .

Comparison of Spectral images .

CHILD Pugh Score MELD Score (Model for end stage liver disease) .

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(considering each one as independent test) • Sensitivity & 1. specificity and likelihood ratios were computed for all metabolites individually at this cut off levels • Metabolites were combined parallel. if any one test is positive is considered as test positive) improves the Sensitivity at minimal cost of Specificity. • Area under the curve for each metabolite was calculated. (i.70 was picked up for further analysis. which differentiates into HCC and no HCC were identified. • A suitable cut off for each metabolite.e. • Most significant AUC. . • Sensitivity..Specificity was computed at different levels. above 0.Data mining using ROC analysis • ROC analysis were done for each metabolites (137 metabolites).

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