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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

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- gas chromatography, LC- 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

Sample spectrum with metabolic profiling


Intensity

1-D 1H NMR spectra

dimethylglycine ?

acetate
2.62

zoom
Intensity

alanine

2.58 2.54 2.50 Chemical shift (ppm)

2.46

hypotaurine carnitine

arginine

extremely congested spectra (raw data) with hundreds of overlapping peaks


1.4

2.8

2.6

2.4 2.2 2 1.8 Chemical shift (ppm)

1.6

We hypothesize that NMR-based metabonomics can provide evidence for the diagnosis of diseases, via the identification of metabolic profile.

Generate a hypothesis

Record limited dataset specific to that hypothesis

Determine if hypothesis is true or false (next phase)

Hypothesis-driven research

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, called hepatocellular carcinoma
90% of all primary liver cancer is HCC 20 to 50% of patients presenting with hepatocellular carcinoma had previously undiagnosed cirrhosis.
Ref:Blum,H.E.(2007)

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 1980s.
In the year 2000, it was projected that there will be 430,000 deaths from HCC all over the world. The prevalence of HCC in an autopsy study among Indians were low, and varies from 0.2% to 1.9%.
Ref: Mohandas,K (2004)

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
46.7 36.2 28.2 22.1 9.4 6.5 3.0 3.9 3.1 2.7 2.5 2.2 2.1 2.7 1.8

Women
11.5 9.5 9.8 5.8 4.6 2.0 1.2 1.9 1.1 1.3 0.5 1.1 1.1 1.7 0.7

The mean age adjusted incidence of HCC in 6 Indian populations is 2.77 in males and 1.28 in females per 100,000 people.

Etiology of Liver cancer


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

Diagnosis of hepatocellular carcinoma ?


recommendations from EASL (European association for studies on liver)

Serology
liver function tests. AFP (alpha fetoprotein) blood test.

Blood tests for Hepatitis B and C.


Imaging Ultrasound of the liver. CT scan or MRI scan of liver. Biopsy. Angiogram of the liver. Laparoscopy.

According to EASL recommendations


The Gold standard in the diagnosis of liver cancer depends on the size of nodules.

nodule size 2 cm - biopsy is recommended.


(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, imaging techniques + appropriate serology can confidently establish the diagnosis without confirmation from a positive biopsy.
Ref: Bruix J et al, 2001.

Rationale for Metabolic profiling in HCC

Objective of the study


To identify & characterise the distribution of significant metabolites in serum of patients with HCC, patients with cirrhotic liver (CLD) and apparently normal people from a basket of metabolome library.
The secondary objective was to find optimal cutoffs, 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.

Study design: Descriptive study

Sample size:

Keeping sensitivity of new test as 95 %, precision of population parameter as 10 %, and (1-) at 95%, the sample size was 18 positive cases.

Study setting
Gastroenterology department, Medical College, Trivandrum. (tertiary care centre)
NIST (National institute of science and technology) Pappanamcode, Trivandrum.

Study subjects
Cases consecutively admitted to Gastroenterology department in Medical College, with results of USG and appropriate serology tests (Inclusion criteria)

Exclusion criteria
Patients not giving consent. Patients without or not willing for further tests, 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 patients attending physician according to medical files.

Stepwise protocol in sample collection


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

The serum samples were sent for NMR spectroscopy.

Methodology for in vitro NMR spectroscopy


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

Data mining using ROC analysis


137 Metabolites, (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, (P-value and C.I (95%) Z test)

Area under the ROC curves - test's ability to discriminate between HCC and no HCC.

Metabolites with significant AUC & their Optimal cutoffs

Measures of accuracy (Sn, Sp, LR+ & LR-)

Combinations

Metabolome

Parallel combinations of metabolites in metabolome


(i.e., 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

Results
Baseline characters of total study subjects

Baseline characters of hepatocellular carcinoma group

Experimental spectrum after processing

Biochemical parameters of the total samples


Characteristics HCC group CLD group Normal liver n=20 n=28 n= 20 (Mean, 95% C.I) (Mean, 95% C.I) (Mean, 95% C.I) 180 (134.02-225.98) 80.68 (70.18-91.19) 284.26 (183.43-385.10) 4.068 (2.067-6.069) 11 (10.26-11.74) 104.07 (80.71-127.43) 58.21 (45.49-70.93) 165.04 (136.04-194.03) 2.636 (1.950-3.322) 8.75 (7.90-9.60) 22.35 (19.34-25.36) 23.35 (20.72-25.98) 65.25 (55.94-74.56) 0.900 (0.717-1.083) 5.60 (5.28-5.92)

AST (IU/L) ALT (IU/L) ALP (IU/L) Bilirubin (mg/dl) CHILD-Pugh

AST (aspartate transferase), ALT (alkaline transferase), ALP(alkaline phospatase)

Metabolome with significant Area under the ROC curve


Sl Metabolites No 1. 3-dihydroxyacetone 2. 3-dimethylurate 3. 6-anhydro--dglucose 4. 2-aminoadipate 5. 2-ethylacrylate 6. 2-hydroxyglutarate 7. 2-methylglutarate 8. 2-oxobutyrate 9. 2-oxocaproate 10. 2-oxoisocaproate 11. 2-oxovalerate 12. 2-phosphoglycerate 13. 3-hydroxy-3methylglutarate 14. 3-methylglutarate 15. 4-hydroxybutyrate 16. 5-aminolevulinate 17. acetoacetate 18. acetone 19. alanine 20. ascorbate 21. aspartate 22. butanone 23. citrate 24. cystathionine 25. dimethylamine Area under curve 0.835 0.755 0.796 0.809 0.729 0.835 0.775 0.756 0.776 0.724 0.805 0.763 0.828 0.754 0.720 0.765 0.850 0.803 0.779 0.750 0.747 0.827 0.821 0.781 0.807 p 95% Confidence value Interval 0.000 0.741 0.930 0.001 0.639 0.871 0.000 0.661 0.931 0.000 0.003 0.000 0.000 0.001 0.000 0.004 0.000 0.001 0.000 0.001 0.005 0.001 0.000 0.000 0.000 0.001 0.001 0.000 0.000 0.000 0.000 0.695 0.593 0.734 0.660 0.634 0.659 0.583 0.699 0.644 0.731 0.628 0.589 0.650 0.760 0.700 0.664 0.621 0.622 0.728 0.720 0.668 0.704 0.924 0.865 0.936 0.890 0.878 0.893 0.865 0.911 0.881 0.925 0.880 0.851 0.879 0.940 0.906 0.894 0.879 0.872 0.927 0.922 0.894 0.911

26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

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

0.748 0.753 0.857 0.719 0.743 0.802 0.722 0.818 0.820 0.756 0.808 0.792 0.767 0.714 0.700 0.704 0.732 0.818 0.811 0.710 0.768 0.846 0.701 0.739 0.703

0.001 0.001 0.000 0.005 0.002 0.000 0.004 0.000 0.000 0.001 0.000 0.000 0.001 0.006 0.010 0.008 0.003 0.000 0.000 0.007 0.001 0.000 0.009 0.002 0.009

0.623 0.621 0.765 0.589 0.615 0.691 0.595 0.705 0.717 0.634 0.705 0.678 0.648 0.573 0.557 0.573 0.604 0.714 0.707 0.567 0.643 0.755 0.561 0.611 0.559

0.873 0.885 0.949 0.848 0.870 0.914 0.849 0.930 0.923 0.878 0.912 0.905 0.886 0.854 0.843 0.836 0.860 0.921 0.916 0.853 0.892 0.936 0.842 0.866 0.847

51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73.

n-carbamoylaspartate n-carbamoyl--alanine o-phosphoserine pimelate proline propionate propylene glycol pyruvate sarcosine serine -alanine s-sulfocysteine suberate succinate succinylacetone sucrose tartrate threonate trans-4-hydroxy-l-proline trimethylamine trimethylamine n-oxide valine xylose

0.731 0.720 0.798 0.783 0.733 0.804 0.794 0.804 0.785 0.776 0.786 0.830 0.810 0.816 0.777 0.724 0.821 0.785 0.784 0.710 0.777 0.720 0.736

0.003 0.004 0.000 0.000 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.004 0.000 0.000 0.000 0.007 0.000 0.005 0.002

0.590 0.578 0.683 0.656 0.612 0.698 0.688 0.699 0.670 0.654 0.675 0.731 0.702 0.713 0.661 0.573 0.721 0.671 0.671 0.571 0.659 0.580 0.606

0.873 0.862 0.913 0.911 0.855 0.911 0.899 0.910 0.901 0.898 0.898 0.930 0.919 0.918 0.893 0.875 0.921 0.900 0.898 0.850 0.895 0.859 0.867

Metabolome and their measures of accuracy


Sl no 1. 2. 3. 4. 5. 6. 7. 8. 9. metabolites acetoacetate methylamine 2-oxovalerate dimethylamine 2-oxocaproate galactarate 2-hydroxyglutarate acetone glucarate Cut offs (mM/L) 30.0808 10.6246 20.8257 00.6427 30.1926 40.8223 240.7171 20.3513 120.3481 130.219 200.170 10.0864 100.7368 50.5898 60.9835 50.8376 50.8376 40.8658 120.4457 60.7143 0.3998 60.127 40.5652 10.7027 120.7186 Sensitivit y 950.00 950.00 950.00 950.00 950.00 900.00 900.00 900.00 900.00 900.00 900.00 900.00 900.00 850.00 850.00 850.00 850.00 850.00 850.00 850.00 850.00 850.00 800.00 800.00 800.00 Specificity 700.83 640.58 580.33 580.33 540.17 790.17 750.00 680.75 680.75 640.58 600.42 580.33 560.25 770.08 750.00 720.92 720.92 700.83 680.75 680.75 680.75 560.25 850.42 750.00 720.92 +LR 30.26 20.68 20.28 20.28 20.07 40.32 30.60 20.88 20.88 20.54 20.27 20.16 20.06 30.71 30.40 30.14 30.14 20.91 20.72 20.72 20.72 10.94 50.49 30.20 20.95 -LR 00.071 00.077 00.086 00.086 00.092 00.13 00.13 00.15 00.15 00.15 00.17 00.17 00.18 00.19 00.20 00.21 00.21 00.21 00.22 00.22 00.22 00.27 00.23 00.27 00.27

10. threonate 11. O-phosphoserine 12. 13. 14. 15. sarcosine propionate butanone citrate

16. tartrate 17. trimethylamine noxide 18. 3-hydroxy-3methylglutarate 19. glutamate 20. glutarate 21. s-sulfocysteine 22. ascorbate 23. 6-anhydro--dglucose 24. succinate 25. alanine

26. 2-methylglutarate 27. glycine 28. malate

100.3909 30.6442 140.3447

800.00 800.00 800.00 800.00 800.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 750.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00

700.83 700.83 680.75 660.67 540.17 750.00 720.92 720.92 720.92 700.83 700.83 700.83 700.83 700.83 700.83 680.75 660.67 600.42 830.33 790.17 750.00 720.92 720.92 700.83 680.75

20.74 00.28 20.74 00.28 20.56 00.29 20.40 00.30 10.75 00.37 30.00 00.33 20.77 00.34 20.77 00.34 20.77 00.34 20.57 00.35 20.57 00.35 20.57 00.35 20.57 00.35 20.57 00.35 20.57 00.35 20.40 00.36 20.25 00.37 10.89 00.41 40.20 00.36 30.36 00.38 20.80 00.40 20.58 00.41 20.58 00.41 20.40 00.42 20.24 00.44

29. 2-phosphoglycerate 330.9003 30. isobutyrate 31. pyruvate 32. pimelate 33. trans-4-hydroxy-lproline 34. valine 35. 2-aminoadipate 36. levulinate 37. propylene glycol 38. -alanine 39. suberate 40. succinylacetone 41. glycolate 42. xylose 43. glycylproline 44. 3-dihydroxyacetone 45. cystathionine 46. proline 47. 3-dimethylurate 48. 3-methylglutarate 50.2818 20.939 60.1551 120.8817 00.9707 90.8691 20.9556 80.2389 50.3824 90.5508 40.3623 40.4765 110.6971 110.6589 10.4628 80.0304 200.4469 20.1598 120.188

49. n150.342 carbamoylaspartate 50. 4-hydroxybutyrate 60.3615

51. ethylene glycol 52. glutaric acid monomethyl ester 53. serine 54. 2-ethylacrylate 55. aspartate 56. 5-aminolevulinate 57. galactonate 58. homoserine 59. mannitol 60. trimethylamine 61. methylguanidine 62. guanidoacetate 63. methionine 64. n-dimethylglycine 65. isocitrate 66. methylsuccinate 67. galactitol 68. 2-oxoisocaproate 69. n-carbamoyl-alanine

10.9397 20.2698 160.8712 10.3727 100.4794 30.7126 60.0561 100.8915 60.0926 00.5 10.4669 40.1396 30.9777 00.8293 190.23 110.6588 20.4011 30.505 70.1585

700.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00 700.00 650.00 650.00 650.00 650.00 650.00 650.00 650.00 600.00 600.00

680.75 680.75 680.75 660.67 660.67 640.58 600.42 600.42 600.42 580.33 750.00 720.92 700.83 700.83 660.67 620.50 600.42 830.33 770.08

20.24 00.44 20.24 00.44 20.24 00.44 20.10 00.45 20.10 00.45 10.98 00.46 10.77 00.50 10.77 00.50 10.77 00.50 10.68 00.51 20.60 00.47 20.40 00.48 20.23 00.49 20.23 00.49 10.95 00.53 10.73 00.56 10.64 00.58 30.60 00.48 20.62 00.52

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 1,6-Anhydro--Dglucose 2-Methylglutarate and 1,6-Anhydro-D-glucose 2 hydroxyglutarate and pimelate Pimelate and Cystathionine Glycylproline and Methylamine Methylamine and Galactarate Galactarate and Trimethylamine Noxide Alanine and Pimelate Acetone and cystathionine 1,6-Anhydro--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 75 700.83 700.83 700.83 700.83 680.75 680.75 660.67 660.67 660.67 640.58 640.58 640.58 640.58 620.50 600.42 600.42 50 50 50 30.60 30.40 30.20 30.26 20.91 20.91 20.57 30.20 30.05 20.85 20.70 20.55 20.82 20.82 20.54 20.40 20.53 20.40 20.15 20.0 20.0 10.90 LR00.13 00.20 00.27 00.07 00.21 00.21 00.35 0 00.07 00.08 00.15 00.23 0 0 00.11 00.23 00.06 00.08 00.17 0 0 00.10

Discussion of Results compared with AFP


An approximate comparison of sensitivity and specificity of metabolites with AFP, as well as ultrasound can be made.
(Arrieta.O et.al, 2007)

Improved sensitivity of many metabolites than the techniques in current practise. (Miller.J.C.et.al,2005)

Overall sensitivity and specificity of NMR based metabolome in the diagnosis of HCC have to be established.

Parallel combinations of metabolites can improve sensitivity. To improve the specificity, Different cut off Serial combinations (Consider the test as positive, if both metabolites are positive)

Accuracy of AFP at different cut offs (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 54 600.6 950.9 470.2 99 360.3 100 80 80-94 18 200.2 100 410.4 970.3 32 100 064 100 65 41 60 87 94 900.6 310.2 980.8 22 99 170.1 990.4 39 Sp 76 Sn 13 21 Sp 97 93 45 100 AFP 200 (ng/mL) Sn Sp AFP 400 (ng/mL) Sn Sp

Recommendations
The Metabolome requires further validation with perfect gold standard. The cut offs suggested in this study is arbitrary (screening tool) and have to be changed, if using as a confirmatory test. Many metabolites like 2 butanone, is unique in hepatocellular carcinoma which is also reported in another recent study.

Known concentration of internal standards to improve the reliability. More precise estimates can be obtained if large molecular sized proteins are removed. Higher frequency NMR spectroscopy (>500 MHz), can give greater separation. NMR based metabonomics are cheaper (400Rs/sample) and faster (~ 5 min).

Conclusion
A Metabolome was identified for hepatocellular carcinoma.
Optimal cut offs 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.

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

THANKING YOU

Diagnostic measures for AFP, USG, & other imaging

Comparison of Spectral images

CHILD Pugh Score

MELD Score (Model for end stage liver disease)

Data mining using ROC analysis


ROC analysis were done for each metabolites (137 metabolites), (considering each one as independent test) Sensitivity & 1- Specificity was computed at different levels.

Area under the curve for each metabolite was calculated.


Most significant AUC, above 0.70 was picked up for further analysis. A suitable cut off for each metabolite, which differentiates into HCC and no HCC were identified. Sensitivity, specificity and likelihood ratios were computed for all metabolites individually at this cut off levels Metabolites were combined parallel, (i.e., if any one test is positive is considered as test positive) improves the Sensitivity at minimal cost of Specificity.

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