肝癌生物标记物及靶点发现中的转化医学研究            John M Luk 陸 滿 晴   BSc (HKU), MPh (HKU), PhD (Karolinska), Postdoc (Harvard)
Translational Medicine in HCC Study       Proteomics                                      Dis. Biol.WGS       Clinical    ...
Translational Medicine in HCC Study  1. Biomarkers discovery     (proteins, genes, miRNAs)    o Diagnostic    o Prognostic...
Development and Progression of Hepatocellular Carcinoma
HCC Statistics: Worldwide            • Worldwide, HCC is the 5th most common              cancer            • Over 700,000...
HCC is a Chinese malignancy                              6
New Cancer Cases (Incidence) and Deaths (Mortality) in 2002               CA Cancer J Clin 2005;55:74-108.         7
Treatment Options for HCC   Surgery/hepatic resection (20%)   Local ablation therapies (20%)   Trans-arterial chemoembo...
Clinical Outcomes of HCC patients (n=651)•   Mortality rate:                                                              ...
The Unmet Medical Needs of HCC in China   3rd leading cause of cancer deaths in China (also in HK and    Singapore)   ~3...
How to improve the clinical outcomes for HCC patients      To detect the cancer earlier       when the tumors can be trea...
Translational Medicine Workflow in HCCClinical        Molecular     Clinical             Cell               Animal        ...
Liver Transplant & HBP Surgical TeamHKU Surgery, Queen Mary Hospital, Hong Kong   Tissue Biobank Team
Liver cancer or Hepatocellular carcinoma (HCC)SmallLarge           HCC                                                    ...
BioBanking -Clinical Samples                                 Patients          Follow-ups        OPD Liver Clinic        O...
I: Biomarkers Discovery for HCC   Biomarkers for separating tumors from non-    malignant liver tissues   Biomarkers for...
Clinical samples for the biomarker study:  A)Serum     • HCC n =120     • Cirrhosis n=120     • Healthy n=120  B) Resected...
Experimental workflow for proteomics 2-DE/MS platform                                     2-DE Gel         Protein extract...
1. Biomarker set distinguishes HCC from normal                                             20
Hsp27: AFP and survivals   Grp78: tumor venous invasion
Mimic HCCphenotypesLiver Functions& Structures
Proteomic markers for small (2cm) HCC     Spot Number    Protein Name (by MALDI-ToF/ToF MS/MS     SSp1615       Vimentin_H...
Vimentin and Lamin B1 are highly expressed in small HCC                          LMB1               SSP3717     SSP1615   ...
Circulating VIM detects small HCC in serum                                                                             Gra...
Predictive performance of vimentin and AFP for the detection of HCC                                  Non-neoplasm vs small...
Next step: multicenter clinical validation• Original dataset from Hong Kong• Multiethnic group test in Singapore• Biomarke...
II. miRNA as a diagnostic markerso    Identify miRNA biomarkers in both tissues & serumo     miRNAs are relatively stable ...
Table I: . Clinical characteristics ofpatients included in this study o miRNA as a diagnostic     biomarker in HCC o Espec...
Study ApproachExploration                         miRNA profiling of HCC tumor                        and adjacent non-tum...
microRNAs biomarkers for AFP-low HCC                      AFP-low TU tissues (<400 ng/ml)Selection criteria:              ...
Candidate miRNA biomarkers for AFP-low HCC
miRNAs are readily detected in             culture medium of HCC cells• miR-301 and miR-224 had very low abundance in the ...
Changes of serum miRNAs before and after surgery                                                 miR-15b                  ...
Measurements of serum miRNAs in an independent cohort (n=116)
miR-15b and miR-130b as a classifier in detecting HCC cases      • Four miRNAs tested: miR-15b, miR-21, miR-130b, and miR-...
The classifier could detect AFP-low HCC cases           HCC serum samples    HepB and healthy controlsAFP, 400        100 ...
The classifier could detect early-stage HCC cases               Liu AM et al., BMJ 2012
Conclusion 1:o The miRNA biomarkers are of great potential in detecting HCC  of low AFP levelo Independent validation with...
40
Genome-wide HCC data overview             Cell growth/             Proliferation/               survival                  ...
Prognostic genes can be identified from tumor and             adjacent normal tissues                             AN has m...
100 genes in TU
100 genes in AN
HepaPRINT: predicting prognostic outcomes
HepaPRINT: cross-validation in NCI samples                                              Overall Survival                  ...
Summary: •   Liver cancer is an aggressive malignancy with poor     outcome. Early detection can save many lives and     i...
Collaborators 合作伙伴                     NUS               HKUMerck• Hongyue Dai        • Ken Sung        • ST Fan• Ron Chen...
Thank You            23 Dec 2010
John Luk Shanghai Bioforum 2012-05-11
John Luk Shanghai Bioforum 2012-05-11
Upcoming SlideShare
Loading in …5
×

John Luk Shanghai Bioforum 2012-05-11

982 views

Published on

John Luk, May 11, 2012. Shanghai Bioforum Translational Medicine, Session S4, Shanghai, China

Published in: Health & Medicine
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
982
On SlideShare
0
From Embeds
0
Number of Embeds
3
Actions
Shares
0
Downloads
8
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

John Luk Shanghai Bioforum 2012-05-11

  1. 1. 肝癌生物标记物及靶点发现中的转化医学研究 John M Luk 陸 滿 晴 BSc (HKU), MPh (HKU), PhD (Karolinska), Postdoc (Harvard)
  2. 2. Translational Medicine in HCC Study Proteomics Dis. Biol.WGS Clinical Gene Biomarker Expression Samples Targets Genotyping 2
  3. 3. Translational Medicine in HCC Study 1. Biomarkers discovery (proteins, genes, miRNAs) o Diagnostic o Prognostic o Tx response 2. Target identification and assessment 3. Understanding of Disease Biology 3
  4. 4. Development and Progression of Hepatocellular Carcinoma
  5. 5. HCC Statistics: Worldwide • Worldwide, HCC is the 5th most common cancer • Over 700,000 new cases are diagnosed globally each year • HCC is the 3rd most common cause of cancer mortality and the main cause of death in cirrhotic patientsEl-Serag H, Rudolph KL. Gastroenterology. 2007;132:2557-2576; Garcia M, et al. Global Cancer Facts & Figures 2007. Atlanta, GA: 5American Cancer Society, 2007.
  6. 6. HCC is a Chinese malignancy 6
  7. 7. New Cancer Cases (Incidence) and Deaths (Mortality) in 2002 CA Cancer J Clin 2005;55:74-108. 7
  8. 8. Treatment Options for HCC Surgery/hepatic resection (20%) Local ablation therapies (20%) Trans-arterial chemoembolization (TACE) (25%) Liver Transplantation (<5%) Systemic chemotherapies/ Palliative care (>30%) Molecular targeted therapy- Sorafenib 8
  9. 9. Clinical Outcomes of HCC patients (n=651)• Mortality rate: KM curve: use tstage, TU• 1-year mortality rate 202/651=31.03% 1 I & II, 103 samples• 2-year mortality rate 345/651=52.99% III & IV, 127 samples• 3-year mortality rate 426/651=65.44% 0.8• 4-year mortality rate 490/651=75.27% E v e n tle s s P ro b a b ility• 5-year mortality rate 548/651=84.18%• 6-year mortality rate 604/651=92.78% 0.6• 7-year mortality rate 633/651=97.24%• 8-year mortality rate 651/651=100% 0.4 0.2 Short: < 1-year survival (31%) Chi2 = 19.84 P = 8.42e-006(wt power = 0) Medium: 1-3 year survival (36%) 0 0 20 40 60 80 Long: > 3-year survival (35%) Time(Months) Clinical stages predict survival Ke H, Luk JM et al, BMC Cancer (2009) 9
  10. 10. The Unmet Medical Needs of HCC in China 3rd leading cause of cancer deaths in China (also in HK and Singapore) ~300,000 new incidences per year ~80% HCC patients are inoperable at presentation in clinic Recurrence rate ~80% and some in early stages Poor prognosis due to:  Late detection  High tumor recurrence rate  Refractory to chemotherapies (Dox ~10% PR) 10
  11. 11. How to improve the clinical outcomes for HCC patients  To detect the cancer earlier when the tumors can be treated by curative surgery and/or radiotherapies.  To stratify high-risk subgroup of patients that may be benefited from target inhibitors (e.g. Avastin, Sunitinib/Sutent, Sorafenib/Nexavar)  To develop new/experimental drugs that can kill chemo-resistant HCC cancer cells and show survival advantages. 11
  12. 12. Translational Medicine Workflow in HCCClinical Molecular Clinical Cell Animal Clinicalspecimens studies data lines models trialsTissues; PBMC DNA/RNA/ Patients info Hypothesis Translational RandomizedSera Protein correlation Fx testings & target trials & cross- analyses validation center validation Clinical pathological Histopathology data Proteomics Genomics cDNA microarray • CGH • ROMA • SNP-CNV • miRNA HCC 2-D Gel Gene expression profiling
  13. 13. Liver Transplant & HBP Surgical TeamHKU Surgery, Queen Mary Hospital, Hong Kong Tissue Biobank Team
  14. 14. Liver cancer or Hepatocellular carcinoma (HCC)SmallLarge HCC 14
  15. 15. BioBanking -Clinical Samples Patients Follow-ups OPD Liver Clinic OT Surgery FU: 0,3,6,9,12,18,24,@6-12m Liver Blood / Biopsy tissues TU: Tumor AN: Normal Proteomics Histopathology Genomics cDNA microarray • CGH • ROMA Clinical data and • SNP-CNV Patient databases • miRNA 2-D Gel 15 Gene expression profiling
  16. 16. I: Biomarkers Discovery for HCC Biomarkers for separating tumors from non- malignant liver tissues Biomarkers for small-size (<2cm) HCC tumors Biomarkers for early tumor recurrence Biomarkers for prognostic outcomes 16
  17. 17. Clinical samples for the biomarker study: A)Serum • HCC n =120 • Cirrhosis n=120 • Healthy n=120 B) Resected Tissues • HCC n =103 • Matched non-tumor n=103 • Normal liver n=16 C) Recurrence (1/4 -1 year), ER = 33 Recurrence free (>1 year), RF = 35 17
  18. 18. Experimental workflow for proteomics 2-DE/MS platform 2-DE Gel Protein extraction Proteome image analysis Molecular biology analysis Statistical analysis Sequence with LC/Tandem mass spectrometry Protein identity 19 Search database
  19. 19. 1. Biomarker set distinguishes HCC from normal 20
  20. 20. Hsp27: AFP and survivals Grp78: tumor venous invasion
  21. 21. Mimic HCCphenotypesLiver Functions& Structures
  22. 22. Proteomic markers for small (2cm) HCC Spot Number Protein Name (by MALDI-ToF/ToF MS/MS SSp1615 Vimentin_HUMAN SSp2603 Heat shock 90kDa protein_HUMAN SSp2618 Glucose-regulated protein_78 HUMAN SSp3211 Cathepsin D SSp3717 Lamin B1_HUMAN SSp4111 Alternative splicing factor ASF-2-HUMAN SSp5605 Chain H, Cys302ser mutant of human mitochondrial aldehyde dehydrogenase complexed with Nad+ and Mg2 SSp6305 Keratin 10_HUMAN SSp7605 Mitochondrial aldehyde dehydrogenase 2, precursor_HUMAN SSp8613 Transferrin_HUMAN SSp9405 Phosphoinositol 4-phosphate adaptor protein- 2_HUMAN SSp9612 Aldehyde dehydrogenase 4A1, precursor_HUMAN
  23. 23. Vimentin and Lamin B1 are highly expressed in small HCC LMB1 SSP3717 SSP1615 VIM 24 Sun S et al. J Proteome Res. 2010
  24. 24. Circulating VIM detects small HCC in serum Gradient titration curve 0.350 y = 0.0003x + 0.0173 0.300 R2 = 0.9977 Absorbance 415nm 0.250 0.200 0.150 0.100 0.050 0.000 0 200 400 600 800 1000 1200 Vim entin ng/m l Sun S et al. J Proteome Res. 2010
  25. 25. Predictive performance of vimentin and AFP for the detection of HCC Non-neoplasm vs small HCC Non-neoplasm vs all HCC Vimentin AFP Vimentin AFPStatistical parameters (≥245ng/ml) (≥400ng/ml) (≥245ng/ml) (≥400ng/ml)Sensitivity, SEN 40.91% 16.28% 36.36% 30.23%Specificity, SPE 87.50% 85.19% 87.50% 85.19%False positive rate, FPR 12.50% 14.81% 12.50% 14.81%False negative rate, FNR 59.09% 83.72% 63.64% 69.77%Accuracy, AC 68.51% 42.86% 57.89% 43.36%Youden index 0.284 0.015 0.239 0.154Positive likeihood ratio, LR+ 3.273 1.099 2.909 2.041Negative likeihood ratio, LR_ 0.675 0.983 0.727 0.819Positive Predictive Value, PPV+ 69% 64% 80% 87%Negative Predictive Value, PPV- 68% 39% 50% 28%
  26. 26. Next step: multicenter clinical validation• Original dataset from Hong Kong• Multiethnic group test in Singapore• Biomarker assay development (MRM, Alphascreen, ELISA, biosensor)• International biomarker network: USA, EU, Africa
  27. 27. II. miRNA as a diagnostic markerso Identify miRNA biomarkers in both tissues & serumo miRNAs are relatively stable in blood plasma and serumo Tumor-derived miRNAs were detected in blood in mouse xenograft model (Mitchell P. et al., PNAS, 2008)o Diseases, such as colorectal cancer, lung cancer, and diabetes, had specific serum-miRNA profiles (Chen X. et al., Cell Res., 2008)
  28. 28. Table I: . Clinical characteristics ofpatients included in this study o miRNA as a diagnostic biomarker in HCC o Especially in AFP normal patients o look for miRNAs highly up- regulated in AFP normal tumor
  29. 29. Study ApproachExploration miRNA profiling of HCC tumor and adjacent non-tumor tissues (n = 96) Selection of 6 miRNAs Measurement of miRNAs in Selection/Filtering culture supernatant of HCC cell lines panel Selection of 4 miRNAs Detection of miRNAs in logitudinal HCC serum samples before and after surgical removal of tumors (n = 15) miR-15b and miR-130b Validation Validation in an independent cohort: • Healthy controls (n = 30) • Chronic hepatitis B carriers (n = 29) • HCC patients (n = 57)
  30. 30. microRNAs biomarkers for AFP-low HCC AFP-low TU tissues (<400 ng/ml)Selection criteria: > 2-fold Adjacent non-tumor tissues Tumor (TU) Adjacent non-tumor (AN) miR-15b miR-224 miR-130b miR-301 miR-21 miR-183
  31. 31. Candidate miRNA biomarkers for AFP-low HCC
  32. 32. miRNAs are readily detected in culture medium of HCC cells• miR-301 and miR-224 had very low abundance in the culture medium
  33. 33. Changes of serum miRNAs before and after surgery miR-15b miR-21 1400 600000 1200 500000 Copies / ng of RNA Copies / ng of RNA 1000 400000 800 300000 600 200000 400 200 100000 p = 0.0637 p = 0.0648 0 0 pre-opera on post-opera on pre-opera on post-opera on miR-130b miR-183 300 1200 250 1000 Copies / ng of RNA Copies / ng of RNA 200 800 150 600 100 400 50 200 p = 0.0158 p = 0.0084 0 0 pre-opera on post-opera on pre-opera on post-opera on
  34. 34. Measurements of serum miRNAs in an independent cohort (n=116)
  35. 35. miR-15b and miR-130b as a classifier in detecting HCC cases • Four miRNAs tested: miR-15b, miR-21, miR-130b, and miR-183 • Logistic regression: miR-15b and miR-130b a b
  36. 36. The classifier could detect AFP-low HCC cases HCC serum samples HepB and healthy controlsAFP, 400 100 20
  37. 37. The classifier could detect early-stage HCC cases Liu AM et al., BMJ 2012
  38. 38. Conclusion 1:o The miRNA biomarkers are of great potential in detecting HCC of low AFP levelo Independent validation with separate cohort of HCC serum samples (n=116) showed superior detection sensitivity and specificity of miR-15b and miR-130b classifiers (ROC >0.98)
  39. 39. 40
  40. 40. Genome-wide HCC data overview Cell growth/ Proliferation/ survival Metabolic process ECM, wound healing,inflammation, vadcular,, Tumor pattern 42 1: healthy, 2: Cirrhotic; 3: AN; 4:TU
  41. 41. Prognostic genes can be identified from tumor and adjacent normal tissues AN has more prognostic genes C.Zhang 43
  42. 42. 100 genes in TU
  43. 43. 100 genes in AN
  44. 44. HepaPRINT: predicting prognostic outcomes
  45. 45. HepaPRINT: cross-validation in NCI samples Overall Survival Disease-free SurvivalKM curve: OS, both 2 metaTU and metaAN, 60 matched TU and AN NCI sample curve: DFS, both 2 metaTU and metaAN, 31 matched TU and AN NCI sa KM 1 1 High High 0.9 Low 0.9 Low 0.8 0.8 0.7 0.7 Survival Probability Survival Probability 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 Chi2 = 10.08 P = 0.0015 0.1 Chi2 = 4.19 P = 0.0408 0 0 0 10 20 30 40 50 60 70 0 20 40 60 80 100 Time(Months) Time(Months) N=60 N=31
  46. 46. Summary: • Liver cancer is an aggressive malignancy with poor outcome. Early detection can save many lives and improve patients quality of life. Molecular profiling has allowed us to identify candidate biomarkers and molecular targets for detection and intervention of HCC Gene signature is potential clinically useful biomarkers for HCC outcome prediction WGS allows us to better understand the disease biology of HBV-associated HCC 48
  47. 47. Collaborators 合作伙伴 NUS HKUMerck• Hongyue Dai • Ken Sung • ST Fan• Ron Chen • Tony Wong • RT Poon• Carolyn Busser • Nikki Lee • Charlie Lee• James Hardwick • Pramila • TJ Yao• Andrey Loboda• Ke Hao • Angela Liu• Chunsheng Zhang FHCRC/Sage EHPH (Shanghai)Pfizer • Stephen Friend • C Gao• Mao Mao • Lee Hartwell 复旦大学中山医院 北京医科大学人民医院 • 王建华教授 • 冷希圣教授 49
  48. 48. Thank You 23 Dec 2010

×