Univ. Sapienza, Rome, Italy.GASTROLEARNING 7 Aprile, 2014

Domenico ALVARO, Univ.“Sapienza” Rome, Italy

Epidemiologia ed aspetti patogeneticidel colangiocarcinoma

EXTRA-HEPATI

C Distal

INTRAHEPATIC

CHOLANGIOCARCINOMA (CCA): a heterogeneus cancer !

Hilar

UICC classification

WHO classification

Klatskin t.second-order bile ducts

CHOLANGIOCARCINOMA

Epidemiology

Risk Factors

Pathogenesis

Worldwide incidence (cases/100,000) of CCA

Canadian0.35

USA1.67

Costa Rica0.3 Puerto Rico

0.35

Australian 0.43

New Zeland 0.4

Philippines 1.2

Vietnam 0.1

Korea Gwangiu 8.75 Daegu 7.25Busan 7.1

Finland 1.05Denmark

1.27

UK 2.17Switzerland 0.45

France1.3

Spain 0.5Italy 3.36

Poland 0.7

Israel0.3

ThailandNorth East 85 North 14.6Central 14.4

Non rare cancer > 6/100,000

Rare cancer < 6/100,000

Taiwan 4.7

Japan Osaka 3.4Hiroshima 3.05

Singapore 1.45

ChinaShanghai 7.55Qidong 7.45

Hong Kong 2.25Guangzhou 0.97

South 5.7

China

Incidence IH-CCA vs EH-CCAIH-CCA EH-CCA

China

Korea

Philippines

SingaporeTaiwan

Thailand

VietNamUK-Scotland

Hong KongShanghai

Khon KaenChiang MaiBangkokSongkhla

GwangiuBusanDaegu

1.1& 0.35&

4.1* 0.6*

7.45* 0.22&+ 0.25&+

6.15&°+ 1.4&°+

4.55^$ 4.2^$

3.95^$ 3.15^$

4.1^$ 3.15^$

ItalyDenmark

JapanFrance

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)

EastIH > EH

WestEH > IH

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

Italy

• 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 !

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

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

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

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

CCA epidemiology: key concepts !

Canadian0.35

USA1.67

Costa Rica0.3 Puerto Rico

0.35

Australian 0.43

New Zeland 0.4

Philippines 1.2

Vietnam 0.1

Korea Gwangiu 8.75 Daegu 7.25Busan 7.1

Finland 1.05Denmark

1.27

UK 2.17Switzerland 0.45

France1.3

Spain 0.5Italy 3.36

Poland 0.7

Israel0.3

ThailandNorth East 85 North 14.6Central 14.4

Non rare cancer > 6/100,000

Rare cancer < 6/100,000

Taiwan 4.7

Japan Osaka 3.4Hiroshima 3.05

Singapore 1.45

ChinaShanghai 7.55Qidong 7.45

Hong Kong 2.25Guangzhou 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 ?)

CHOLANGIOCARCINOMA

Epidemiology

Risk Factors

Pathogenesis

CCA: DEFINITE RISK FACTORS

PSC

O.ViverriniC. Sinensis

Choledochal cysts, Caroli’s

Thorotrast

Hepatolithiasis

HCV

EH-CCAIH-CCA

Abnormal pancreatico-biliary junction with BD dilatation

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

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 !

CCA: probable risk factors

Biliary-enteric drainage

Toxins: dioxins, asbestos

CholedocolithiasisCholangitis

LiverCirrhosis

HBV

Cholelithiasis/cholecystectomy

Diabetes, Alcohol Obesity, tobacco

IBD

Hepatic Schistosmiasis

EH-CCA

IH-CCA

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/yearHBsAg- = 0.09/100,000/year HR = 4.8

Hepatology 2011

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.

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 !

CCA risk factors: key concepts !

Canadian0.35

USA1.67

Costa Rica0.3 Puerto Rico

0.35

Australian 0.43

New Zeland 0.4

Philippines 1.2

Vietnam 0.1

Korea Gwangiu 8.75 Daegu 7.25Busan 7.1

Finland 1.05Denmark

1.27

UK 2.17Switzerland 0.45

France1.3

Spain 0.5Italy 3.36

Poland 0.7

Israel0.3

ThailandNorth East 85 North 14.6Central 14.4

Non rare cancer > 6/100,000

Rare cancer < 6/100,000

Taiwan 4.7

Japan Osaka 3.4Hiroshima 3.05

Singapore 1.45

ChinaShanghai 7.55Qidong 7.45

Hong Kong 2.25Guangzhou 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 !

CHOLANGIOCARCINOMA

Epidemiology

Risk Factors

Pathogenesis

Chronic inflammation and CCA

Chronic Inflammation

(flukes,PSC..)

Apoptosis Proliferation

iNOS

NONitrosylation DNA basis

and DNA repair proteins, caspase 9

Mutagenesis

IL6TNF

COX-2PgE2

HISTOLOGICAL VARIATION OF CCA(Komuta et al. Hepatology 2012)

EH-CCA IH-CCA

100 % 44 % 28 % 28 %

Mixed-CCAMucin-CCA

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)

CSCs and Liver Cancers CSCs and Liver Cancers

•Cancerogenesis•Prognosis•Target of treatment

To investigate Cancer Stem Cellsin 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)

RESULTSImmunophenotype of Mixed-IHCCA

*Mixed-IHCCAs diffusely positive for K7, EpCAM, CD13 and CD133. *LGR5 restricted to few tumor epithelial cells (arrows). * CD90 and SMA mostly expressed by tumor stromal cells (arrows).α

PAS CK7 EpCAM CD133

LGR5 CD13 CD90 αSMA

60%

diffuse staining

Few cells

Immunophenotype of Mucin-IHCCA

*Mucin-IHCCAs diffusely positive for K7, EpCAM, LGR5, CD133; *CD13 restricted to few tumor epithelial cells (arrow); *CD90 and SMA mostly expressed by tumor stromal cellsα (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

IH-MIXED

Primary Cultures of Mucin-CCA and Mixed-CCA

IH-MUCIN IH-MIXED

Total cell population

Cells immunosorted for CSC surface markers

CCA CSCs: TUMORIGENIC POTENTIAL.

IN VITRO: Spheroid formation

IN VIVO:

1. Subcutaneous xenographts

2. Intrahepatic xenograpths -normal liver

-CCL4-cirrhotic liver

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

CD13+

*

CD13-

CD13+

* CD13-

CD90- CD90-

IH-Mixed IH Mucin

CD90+

*

IH-Mixed IH Mucin

CD133+

* CD133-

CD133+

*

CD133-

25µm

25µm IH-Mixed IH Mucin

Nu

mb

er s

ph

eroi

ds

(20

00

cel

ls)

LGR5+

*

LGR5+

*

LGR5- LGR5-

EPCAM+

*

EPCAM-

IH-Mixed IH Mucin

CD133+

*

CD133-

CD133+

*

CD133-

25-m IH-Mixed IH-Mucin

LGR5+

*

LGR5+

*

LGR5-LGR5-

25µm

CD13+

*CD13-

CD13+

*CD13- CD90- CD90-

IH-Mixed IH Mucin

CD90+

*

Spheroids formation by CCA CSCs subpopulations.

p< 0.01

Epithelial CSCs Quiescent CSCsMesenchymal 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

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

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

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.

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-MIXEDIH-MUCIN

CSC and cholangiocarcinoma

• CSCs were abundantly represented in human CCA suggesting …..

CCA as a disease of stem/progenitor cells

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 !

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+)

(Cardinale V….Nature Rev. 2012)

Mucin-CCA: typical sites of emergence correspond to the highest density of PBG !

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

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

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% )

PBGs ARE INVOLVED IN CCA

PRE-NEOPLASTIC LESIONS !

Hepatology 2012, 55: 2040-41.

Cystic and Papillary Neoplasm Involving Peribiliary Glands: A Biliary Counterpart of Branch-Type Intraductal Papillary Mucinous Cystic Neoplasm?

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)

PBGs ARE ACTIVATED IN PATHOLOGIES AT RISK FOR CCA !