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

1. Univ. Sapienza, Rome, Italy.
GASTROLEARNING
7 Aprile, 2014
Domenico ALVARO, Univ.“Sapienza” Rome, Italy
Epidemiologia ed aspetti patogenetici
del colangiocarcinoma

2. EXTRA-
HEPATIC
Distal
INTRAHEPATIC
CHOLANGIOCARCINOMA (CCA): a heterogeneus cancer !
Hilar
UICC classification
WHO classification
Klatskin t.
second-order bile ducts

3. CHOLANGIOCARCINOMA
Epidemiology
Risk Factors
Pathogenesis

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. 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. IH-CCA as a percentage of all primitive liver cancers.
Italy

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. 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. 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. 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. 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. 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. CHOLANGIOCARCINOMA
Epidemiology
Risk Factors
Pathogenesis

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. 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. 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. 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. 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. 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. 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. 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. CHOLANGIOCARCINOMA
Epidemiology
Risk Factors
Pathogenesis

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. HISTOLOGICAL VARIATION OF CCA(Komuta et al. Hepatology 2012)
EH-CCA IH-CCA
100 % 44 % 28 % 28 %
Mixed-CCAMucin-CCA

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)

27. CSCs and Liver Cancers
•Cancerogenesis
•Prognosis
•Target of treatment

28. 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)

29. 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

30. 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

31. IH-MIXED
Primary Cultures of Mucin-CCA and Mixed-CCA
IH-MUCIN IH-MIXED
Total cell population
Cells immunosorted for CSC surface markers

32. CCA CSCs: TUMORIGENIC POTENTIAL.
IN VITRO: Spheroid formation
IN VIVO:
1. Subcutaneous xenographts
2. Intrahepatic xenograpths
-normal liver
-CCL4-cirrhotic liver

33. 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-

34. 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

35. 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

36. 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

37. 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.

38. 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

39. CSC and cholangiocarcinoma
• CSCs were abundantly represented in human
CCA suggesting …..
CCA as a disease of stem/progenitor cells

40. 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 !

41. 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+)

42. (Cardinale V….Nature Rev. 2012)
Mucin-CCA: typical sites of emergence
correspond to the highest density of PBG !

43. 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

44. 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

45. 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% )

47. PBGs ARE INVOLVED IN
CCA
PRE-NEOPLASTIC LESIONS !

48. Hepatology 2012, 55: 2040-41.
Cystic and Papillary Neoplasm Involving Peribiliary Glands: A
Biliary Counterpart of Branch-Type Intraductal Papillary Mucinous
Cystic Neoplasm?

49. 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)

50. PBGs ARE ACTIVATED
IN PATHOLOGIES AT RISK
FOR CCA !