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Il colangiocarcinoma: Epidemiologia, Fattori di rischio e patogenesi - Gastrolearning®

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Gastrolearning II modulo/7a lezione
Il colangiocarcinoma: Epidemiologia, Fattori di rischio e patogenesi
Prof. D. Alvaro - Università di Roma La Sapienza

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Il colangiocarcinoma: Epidemiologia, Fattori di rischio e patogenesi - Gastrolearning®

  1. 1. Univ. Sapienza, Rome, Italy. GASTROLEARNING 7 Aprile, 2014 Domenico ALVARO, Univ.“Sapienza” Rome, Italy Epidemiologia ed aspetti patogenetici del colangiocarcinoma
  2. 2. EXTRA- HEPATIC Distal INTRAHEPATIC CHOLANGIOCARCINOMA (CCA): a heterogeneus cancer ! Hilar UICC classification WHO classification Klatskin t. second-order bile ducts
  3. 3. CHOLANGIOCARCINOMA Epidemiology Risk Factors Pathogenesis
  4. 4. Worldwide incidence (cases/100,000) of CCA Canadian 0.35 USA 1.67 Costa Rica 0.3 Puerto Rico 0.35 Australian 0.43 New Zeland 0.4 Philippines 1.2 Vietnam 0.1 Korea Gwangiu 8.75 Daegu 7.25 Busan 7.1 Finland 1.05 Denmark 1.27 UK 2.17 Switzerland 0.45 France1.3 Spain 0.5 Italy 3.36 Poland 0.7 Israel 0.3 Thailand North East 85 North 14.6 Central 14.4 Non rare cancer > 6/100,000 Rare cancer < 6/100,000 Taiwan 4.7 Japan Osaka 3.4 Hiroshima 3.05 Singapore 1.45 China Shanghai 7.55 Qidong 7.45 Hong Kong 2.25 Guangzhou 0.97 South 5.7 China
  5. 5. Incidence IH-CCA vs EH-CCA IH-CCA EH-CCA China Korea Philippines Singapore Taiwan Thailand VietNam UK-Scotland Hong Kong Shanghai Khon Kaen Chiang Mai Bangkok Songkhla Gwangiu Busan Daegu 1.1& 0.35& 4.1* 0.6* 7.45* 0.2 2&+ 0.25&+ 6.15& + 1.4& + 4.55^$ 4.2^$ 3.95^$ 3.15^$ 4.1^$ 3.15^$ Italy Denmark Japan France 51.45& 0.25& 6.1& 0.3& 1.95& 0.2& 1.05&* 0.15&* 0.1&* 0 Qidong 1.05& 0.4& 1.1* 0.1* 0.88* 1.55* 0.62# 0.65# 0.2^ 1.1^ Hiroshima Osaka 1.25& 1.8& 1.3& 2.1& (# = ICD – 01) (* = ICD – 03) (^ = ICD – 10) (& = ICD – 02) ( = ICD-V9) (+ = ICD-V10) USA 0.58* 0.88* ($ = ICD-0) East IH > EH West EH > IH
  6. 6. IH-CCA as a percentage of all primitive liver cancers. Italy
  7. 7. • Lack of uniform classification ! (morphology only, morphology + topography) •Histological heterogeneity,lack specific markers ! • Anatomical origin difficult to establish(advanced diagnosis) ! •Hilar EH-CCA often classified as IH-CCA ! IH- and EH-CCA epidemiology ! Biases and criticisms ! USA SEER-9 registries, Welzel TM et al. (J Natl Cancer Inst 2006) ICD-01/02: overreporting of IH-CCA by 13% underreporting of EH-CCA by 15 % IH-CCA EH-CCA Morphology Morphology+ topography Cancer registries IH-CCA = 20-30 % EH-CCA = 40-50 % NOS = 20-40% IH- and EH-CCA epidemiology ! Misclassification of hilar CCA !
  8. 8. Temporal trends in IH- and EH-CCA incidence/mortality in 1980-2000. Temporal trends in IH-CCA and EH-CCA mortality in men from 1979 to 1997 (Khan SA. J. Hepatology 2002) In different countries, in the 1980-2000 decades incidence/mortality… ↑ for IH-CCA =↓ for EH-CCA More recent data on incidence …. EH-CCA IH-CCA
  9. 9. Germany Italy EH-CCA Temporal trends in IH-/EH-CCA incidence in Korea. IH-CCA IH-CCA EH-CCA Von Hahn et el. Scand J Gastro 2011. Alvaro D. et al. Dig Liver Dis. 2010. Shin HR et al. J Korean Med Sci 2010 Korea 0.5 1.5 2 2.5 3 3.5 4 1 IH-CCA EH-CCA x 100,000 1999 2000 2001 2002 2003 2004 2005
  10. 10. Germany Italy EH-CCA Temporal trends in IH- and EH-CCA incidence in Italy, Germany, France, England-Wales, USA. IH-CCA IH-CCA EH-CCA Von Hahn et el. Scand J Gastro 2011. Alvaro D. et al. Dig Liver Dis. 2010. x 1,000,000 IH-CCA EH-CCA England-Wales France Khan SA et al. J. Hepatology In Press IH-CCA EH-CCA Khan SA et al. J. Hepatology In Press USA 0.1 0.2 0.3 0.4 0,5 0.6 0.7 0,. 0.9 1 1.1 x 100,000 IH-CCA EH-CCA Lepage C. et al. J. Hepatology 2011 IH-CCA EH-CCA Khan SA et al. J. Hepatology In Press USA
  11. 11. Temporal trends in IH- and EH-CCA incidence in Denmark. Denmark 0.2 0.6 0.8 1.0 1.2 0.4 Jepsen P. et al. J Natl Cancer Inst 2007 x 100,000 IH-CCA EH-CCA
  12. 12. CCA epidemiology: key concepts ! Canadian 0.35 USA 1.67 Costa Rica 0.3 Puerto Rico 0.35 Australian 0.43 New Zeland 0.4 Philippines 1.2 Vietnam 0.1 Korea Gwangiu 8.75 Daegu 7.25 Busan 7.1 Finland 1.05 Denmark 1.27 UK 2.17 Switzerland 0.45 France1.3 Spain 0.5 Italy 3.36 Poland 0.7 Israel 0.3 Thailand North East 85 North 14.6 Central 14.4 Non rare cancer > 6/100,000 Rare cancer < 6/100,000 Taiwan 4.7 Japan Osaka 3.4 Hiroshima 3.05 Singapore 1.45 China Shanghai 7.55 Qidong 7.45 Hong Kong 2.25 Guangzhou 0.97 South 5.7 China EH-CCA > IH-CCA IH-CCA > EH-CCA * EH-CCA incidence stable/decreasing… last 2-3 decades ! * IH-CCA incidence increased in ‟80-2000‟, now stable ! (trend similar to HCC ?)
  13. 13. CHOLANGIOCARCINOMA Epidemiology Risk Factors Pathogenesis
  14. 14. CCA: DEFINITE RISK FACTORS PSC O.Viverrini C. Sinensis Choledochal cysts, Caroli’s Thorotrast Hepatolithiasis HCV EH-CCAIH-CCA Abnormal pancreatico-biliary junction with BD dilatation
  15. 15. CCA > HCC = 8:1 with O. Viverrini CCA < HCC = 1:8 without O. Viverrini O. Viverrini, C. Sinensis and CCA: Meta-analysis of published literature. Shin HR et al. Cancer Sci 2010 Sripa B. et al. Curr Opin Gastro. 2008. More than 35 million people worldwide infected ! 20-30 years recurrent pyogenic cholangitis →IH-> EH-CCA x100,000 Liver Flukes: control of foodborn infection, mass anthelmintic therapy
  16. 16. HCV and CCA: Meta-analysis of published literature. Shin HR et al. Cancer Sci 2010 HCV and CCA: 8/11 studies only IH-CCA 3/11 studies IH-CCA EH-CCA O.R. El-Serag 2009 2.55 1.50 (NS) (USA, cohort) Shahib 2007 7.9 2.8 (NS) (USA, case-control) Welzel 4.4 1.5 (NS) (USA, case-control) HCV definite risk factor only for IH-CCA More advanced is the liver disease more significant the association !
  17. 17. CCA: probable risk factors Biliary-enteric drainage Toxins: dioxins, asbestos Choledocolithiasis Cholangitis Liver Cirrhosis HBV Cholelithiasis/cholecystectomy Diabetes, Alcohol Obesity, tobacco IBD Hepatic Schistosmiasis EH-CCA IH-CCA
  18. 18. HBV and CCA: Meta-analysis of published literature. Shin HR et al. Cancer Sci 2010 7/10 studies only IH-CCA examined (3/7 NS) 2/3 studies EH-CCA not associated with HBV ! Therefore, HBV probable risk factor only for IH-CCA ! In general, higher HBV prevalence higher the significance of the association with CCA ! Incidence rate of IH-CCA HBsAg+ = 0.43/100,000/year HBsAg- = 0.09/100,000/year HR = 4.8 Hepatology 2011
  19. 19. IBD enhances the risk of CCA in PSC pts ? Boberg KM. 2002: 394 PSC, CCA associated with IBD(p<0.001) IBD as potential risk factor for CCA PSC: CCA incides at 30-50 yrs. Lifetime Risk= 5-15%; 0.3-1.5%/year. Burak K. 2004: 167 PSC, CCA not associated with IBD However…. PSC was not controlled for in the analysis of IBD ! Therefore, …unclear if IBD is an independent risk factor for CCA nor if it confers additional risk in PSC pts.
  20. 20. CCA risk factors IH-CCA EH-CCA n= 116 n= 102 Positive hepatitis virus markers 35 (30.2%) 19 (18.6%) p= 0.048 No putative risk factor in 60% CCA !
  21. 21. CCA risk factors: key concepts ! Canadian 0.35 USA 1.67 Costa Rica 0.3 Puerto Rico 0.35 Australian 0.43 New Zeland 0.4 Philippines 1.2 Vietnam 0.1 Korea Gwangiu 8.75 Daegu 7.25 Busan 7.1 Finland 1.05 Denmark 1.27 UK 2.17 Switzerland 0.45 France1.3 Spain 0.5 Italy 3.36 Poland 0.7 Israel 0.3 Thailand North East 85 North 14.6 Central 14.4 Non rare cancer > 6/100,000 Rare cancer < 6/100,000 Taiwan 4.7 Japan Osaka 3.4 Hiroshima 3.05 Singapore 1.45 China Shanghai 7.55 Qidong 7.45 Hong Kong 2.25 Guangzhou 0.97 South 5.7 China EH-CCA > IH-CCA IH-CCA > EH-CCA *HCV, HBV (treated) *PSC *LiverFlukes *HBV, HCV (untreated) *Hepatolithiasis * Increased incidence of IH-CCA in „80-2000 decades linked with the burden of HCV infection !? * > 60% CCA, no putative risk factor !
  22. 22. CHOLANGIOCARCINOMA Epidemiology Risk Factors Pathogenesis
  23. 23. Chronic inflammation and CCA Chronic Inflammation (flukes,PSC..) Apoptosis  Proliferation iNOS NO Nitrosylation DNA basis and DNA repair proteins, caspase 9 Mutagenesis IL6 TNF COX-2 PgE2
  24. 24. HISTOLOGICAL VARIATION OF CCA(Komuta et al. Hepatology 2012) EH-CCA IH-CCA 100 % 44 % 28 % 28 % Mixed-CCAMucin-CCA
  25. 25. Mucin-producing CCA From PBGs or mucin-producing cells “mixed” IH-CCA, cholangiolo-CCA, From HPC or non mucin-producing cuboidal cells in C. Hering and bile ductules CLASSIFICATION OF PRIMITIVE LIVER CANCERs: based on cells of origin (Alvaro D. Hepatology 2012)
  26. 26. CSCs and Liver Cancers •Cancerogenesis •Prognosis •Target of treatment
  27. 27. To investigate Cancer Stem Cells in human CCA and its subtypes, in primary cultures of human CCA and in established CCA cell lines. CSC markers: CD44 (hyaluronan receptor) “Mesenchymal”: CD90 (Thy-1) “Quiescent”: CD13(amino peptidase N) “Epithelial”: CD133 (prominin-1) EpCAM (pan-epithelial differentiation antigen) LGR5(receptor for Wnt-agonistic R-spondins)
  28. 28. RESULTS Immunophenotype of Mixed-IHCCA *Mixed-IHCCAs diffusely positive for K7, EpCAM, CD13 and CD133. *LGR5 restricted to few tumor epithelial cells (arrows). * CD90 and αSMA mostly expressed by tumor stromal cells (arrows). PAS CK7 EpCAM CD133 LGR5 CD13 CD90 αSMA 60% diffuse staining Few cells
  29. 29. Immunophenotype of Mucin-IHCCA *Mucin-IHCCAs diffusely positive for K7, EpCAM, LGR5, CD133; *CD13 restricted to few tumor epithelial cells (arrow); *CD90 and αSMA mostly expressed by tumor stromal cells(arrows) No difference between IH- and EH- mucin-CCAs. LGR5 = Mucin-CCA > Mixed-IHCCA (p<0.05) CD13 = Mixed-CCA > Mucin-CCA (p<0.05) PAS CK7 EpCAM CD133 LGR5 CD13 CD90 αSMA 60% diffuse staining Few cells
  30. 30. IH-MIXED Primary Cultures of Mucin-CCA and Mixed-CCA IH-MUCIN IH-MIXED Total cell population Cells immunosorted for CSC surface markers
  31. 31. CCA CSCs: TUMORIGENIC POTENTIAL. IN VITRO: Spheroid formation IN VIVO: 1. Subcutaneous xenographts 2. Intrahepatic xenograpths -normal liver -CCL4-cirrhotic liver
  32. 32. Spheroid formation by CCA CSCs subpopulations. CD13+ CD13- merge CD13- 50µm 25µm CD13+ merge CD90+ CD90- CD90+ merge CD90- merge 50µm 25µm CD133+ CD133- CD133- mergeCD133+ merge 50µm 25µm EPCAM-EPCAM+ EPCAM- mergeEPCAM+ merge 50µm LGR5+ LGR5- LGR5+ merge LGR5- merge 25µm 50µm 0 10 20 30 Numberspheroids (2000cells) IH-MucinIH-Mixed CD13+ * CD13- CD13+ * CD13- 0 10 20 30 Numberspheroids (2000cells) CD90+ * CD90- CD90- IH-Mixed IH Mucin CD90+ * 0 20 40 60 80 100 Numberspheroids (2000cells) IH-Mixed IH Mucin CD133+ * CD133- CD133+ * CD133- 25µm 0 20 40 60 80 IH-Mixed IH Mucin EPCAM+ * Numberspheroids (2000cells) EPCAM- 25µm 0 20 40 60 80 IH-Mixed IH Mucin Numberspheroids (2000cells) LGR5+ * LGR5+ * LGR5- LGR5- EPCAM+ * EPCAM-
  33. 33. 0 10 20 30 40 50 60 70 80 90 Numberspheroids (2000cells) IH-Mixed IH Mucin CD133+ * CD133- CD133+ * CD133- 25-m IH-Mixed IH-Mucin LGR5+ * LGR5+ * LGR5- LGR5- 25µm IH-Mixed IH Mucin EPCAM+ * EPCAM- EPCAM+ * EPCAM- IH-MucinIH-Mixed CD13+ * CD13- CD13+ * CD13- CD90+ * CD90- CD90- IH-Mixed IH Mucin CD90+ * Spheroids formation by CCA CSCs subpopulations. p< 0.01 Epithelial CSCs Quiescent CSCs Mesenchymal CSCs •Cells expressing epithelial markers, CD133, EpCam, Lgr5 formed the highest number of spheroids; •CD13+ from Mixed-CCA > Mucin-CCA •CD90+ or CD133+ cells from Mucin-CCA > Mixed-CCA
  34. 34. In vivo tumorigenicity: subcutaneous tumor xenographts. 1/5 CD90+ CD90- CD90+ CD90- CD13+ CD13- CD13+ CD13- p<0.05 p<0.05 1/5 1/5 1/5
  35. 35. Subcutaneous tumor xenographts, Mucin- vs Mixed-CCA. H&E H&EH&E CD133+ CD13+ CD90+ PCNA αSMAH&E PAS K19 Xenographts from CD133+/mucin-CCA Xenographts from CD90+/mixed-CCA Subcutaneous Xenographts from mucin-CCA Xenographts from mixed-CCA CD133 CD13 CD90 CD133 CD13 CD90 PAS + + - - - - K19 + + - + + + Ductular-like structures NO NO NO YES YES YES
  36. 36. In vivo tumorigenicity: intrahepatic tumor xenographts. Cancers only reproduced by iniecting CSCs in the cirrhotic (CCL4) but not normal livers and only with cells immunosorted from mucin-CCA. CD133+ Original Human Mucin-CCA Tumor xenographt; CD133+ immunosorted from mucin-CCA and injected in the cirrhotic liver. Tumor xenographt; CD90+ immunosorted from mucin-CCA and injected in the cirrhotic liver.
  37. 37. Effect of the PI3-kinase/AKT inhibitors, NVP-BEZ235 and MK2206 (AKT inhibitor), on cell proliferation in primary cultures of Mucin- or Mixed-CCAs. NVP-BEZ235 (PI3-kinase inhibitor) and, to a lower extent MK2206 (AKT inhibitor) are highly active against Mucin- and Mixed-CCAs. IH-MIXED IH-MUCIN 0 20 40 60 80 100 120 0 1 5 10 50 100 1000 Cellviability(%) NVP-BEZ235 (nM) NVP-BEZ235: Ic50 = 1.26 ± 0.88 nM NVP-BEZ235: Ic50 = 1.1 ± 0.14 nM 0 20 40 60 80 100 120 0 0.1 0.5 1 5 10 50 Cellviability(%) MK2206 (µM) MK2206: Ic50 = 4.54 ± 2.3 µM MK2206: Ic50 = 5.6 ± 0.53 µM
  38. 38. CSC and cholangiocarcinoma • CSCs were abundantly represented in human CCA suggesting ….. CCA as a disease of stem/progenitor cells
  39. 39. Therefore, cholangiocarcinoma may be a disease of stem/progenitor cells, which can be detected by the increasing expression of albumin in combination with stem/progeni- tor markers. N. 34 periphereal IH-CCA associated with O. Viverrini. Coexpression of albumin and K-19 found in the majority of CCA cells ! Since K19/Albumin coexpression normally found in hepatic progenitor cells before differentiation into cholangiocytes......CCA develops from the intermediate cell type according to the maturation arrest theory !
  40. 40. Mucin-CCA… a PBG cancer ??? Cardinale V,…. Alvaro D. Hepatology 2011, J. Anatomy 2012, Nature Rev . 2012. Gland base (near fibromuscular layer) Undifferentiated phenotype (10%) (EpCAM+ ⁄ - /Lgr5+/CD133+) Transit-amplifying progenitors ( 25%) (EpCAM + ⁄ - /PDX1 + ⁄ - ⁄SOX17 +/- ⁄ Lgr5-) Gland body (middle of the duct wall) Neck of PBGs (close contact with surface epithelium) Mature cells (cholangiocytes, goblet cells, pancreatic cells, hepatocytes) Intermediate phenotype (EpCAM+⁄PAS+, EpCAM+ ⁄ SR+, EpCAM+insulin+, EpCAM+ ⁄ albumin+/K19+)
  41. 41. (Cardinale V….Nature Rev. 2012) Mucin-CCA: typical sites of emergence correspond to the highest density of PBG !
  42. 42. Mucin-producing cholangiocarcinoma might derive from Biliary Tree Stem/progenitor Cells located in PBGs. Cardinale V….Carpino G, Gaudio E, D. Alvaro. Hepatology 2012. Mucin-CCAM
  43. 43. Mucin-CCA, Pancreatic cancer and Colorectal cancer. Similar cancers originating from similar glands ? Cardinale V,…. Alvaro D. Hepatology 2011, J. Anatomy 2012, Nature Rev . 2012. Peribiliary Glands Stem cells: CD133, EpCAM, Lgr5 Mucin-CCA CSCs: CD133, EpCAM, Lgr5 Pancreatic duct glands Pancreatic ductal adenoK CD133, EpCAM, Lgr5 Colon crypts CD133, EpCAM, Lgr5 CD133, EpCAM, Lgr5 Human ColoRectal Cancer CD133, EpCAM, Lgr5
  44. 44. KRAS mutations are frequent in Mucin-CCAs (Komuta M et al. Hepatol. 2012) EH-CCA IH-CCA 28 % 28 %100 % 44 % Kras mutation reflects the different cholangiocytes pheno- and genotype in intrahepatic cholangiocarcinoma. KRAS mutations only in muc-CCAs (41.4% )
  45. 45. PBGs ARE INVOLVED IN CCA PRE-NEOPLASTIC LESIONS !
  46. 46. Hepatology 2012, 55: 2040-41. Cystic and Papillary Neoplasm Involving Peribiliary Glands: A Biliary Counterpart of Branch-Type Intraductal Papillary Mucinous Cystic Neoplasm?
  47. 47. CCA develops through a multi-step process involving separate precursor pathways. Similarities between CCA and pancreatic cancer. Intraductal Papillary Biliary Neoplasm (IPBN) Mucin-CCAPeribiliary Glands Pancreatic duct glands Pancreatic duct adenoKIntraductal Papillary Muc. Neoplasm (IPMN)
  48. 48. PBGs ARE ACTIVATED IN PATHOLOGIES AT RISK FOR CCA !

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