Genetic of malignant and benign liver tumors

1
Genetic of malignant and benign liver tumors

• Jessica Zucman-Rossi
• DR Inserm U674
• CEPH
• 27 rue juliette dodu
• zucman_at_cephb.fr

2
Search for new tumor suppressor genes
Malignant liver tumors HCC hepatocellular
carcinoma
Somatic genetic alterations
Benign liver tumors Hepatocellular
adenoma Focal nodular hyperplasia
Germline genetic predisposition
Genotype-phenotype correlations
Clinical application diagnosis/ prognosis
3
Hepatocarcinogenesis is a multi-step process
Risk Pre-neoplastic Malignant factors
steps transformation
HCV
HCC
Small cell dysplasia
Cirrhotic nodules
HBV
AFB1
Alcohol
hemochromatosis
Gene mutations Oncogene activation tumor
suppressor gene inactivation
Monoclonality /- Chromosome imbalances /- Gene
expression modif /- Telomerase
4
Genetic alterations 1-Specific of etiological
factors 2- Non-specific of risk factors
5
Main pathways altered in hepatocarcinogenesis
P53
Wnt/ß-catenin
IGF/insulin growth factor
Rb/retinoblastoma
Chromatin assembling
Metabolism
TGFß
HNF1
Ras
6
How genetic alterations are accumulated in
tumors?Is there a relation with clinical
parameters?
7
Association between genetic alterations and
clinical characteristics of HCC
Genetic alterations
Clinical characteristics
Mechanisms
AXIN1, LOH 16p,16q, 1p, 4q
HBV infected
P53 LOH 13q, 17p
Low differentiation
Chromosome instability
I
9p, 6q LOH
Poor prognosis
No specific LOH
Non HBV infected
Chromosome stability
II
ß-catenin 8p LOH
Large tumors
Laurent-Puig, Gastroenterology, 2001
137 HCC
8
Is there a relation between genetic alterations,
global transcriptome and phenotype in HCC?
9
Design of a transcriptome/ genotype/phenotype
study
120 HCC, hepatectomy, 3 french hospital, HCV
(30), HBV (25), Alcohol (35)
10
Classification of HCC(non-supervised)
A
B
11
Search for prognostic predictors in HCC
Affy results 20 000 genes Curative resection
(test set n50 HCC)
Supervised analyses Early tum death lt3y Early
relapse lt2y Vascular invasion
140 tested genes in QRT/PCR (test set n50 )
12
5-genes signature predictive of overall survival
in HCC 16-gene signature predictive of the HCC
classification (G1 to G6)
Inserm patent, 2006
13
conclusions

• HCC is a heterogeneous group of tumors
• Several paths to HCC are found, specific or
  non-specific for risk factors and related to
  different tumor phenotypes

• Consequences
• A universal marker for diagnosis and prognosis
  does not exist but we have to identify a group of
  markers taking into account all HCC subgroups of
  tumors.
• gt transcriptome and proteome global analyses

• Classifying tumors in homogeneous subgroup may be
  important to test for new therapeutics and to
  identify new genetic susceptibility

14
Natural history of hepatocarcinogenesis
Chronic hepatitis
HCV
HBV
AFB1
Alcohol
Small cell dysplasia Macroregenerative nodules
hemochromatosis
90
HCC
Cirrhosis
Risk Pre-neoplastic Malignant factors
steps transformation
15
Genetic alterations in HA

• Biallelic mutations of TCF1/HNF1a gene
  inactivating the hepatocyte nuclear factor 1??are
  found in 50 of hepatocellular adenomas, mainly
  somatic (90), rarely germline Bluteau
  et al Nature Genetics 2002

• HNF1a mutations occur specifically in adenomas
  since no mutations were identified either in
  typical or telangiectatic focal nodular
  hyperplasia and only less than 3 of HCCs are
  mutated Bioulac-Sage et al
  Gastroenterology 2005

• Hepatocyte Nuclear factor 1??(HNF1?) germline
  mutations predispose to familial adenomatosis
  associated to diabetes MODY3 Bacq et al,
  Gastroenterology, 2003 Reznik et al, J Clin
  Endoc Metab, 2004

16
Genetic alterations in HA (2)

• Activation of the b-catenin pathway was
  recently found in HA
• Chen Hepatology 2002 Takayasu. Hum Pathol
  2002 Torbenson Mod Path 2002

• Activating mutations of b-catenin are found in
  20 35 of HCC, suggesting that b-catenin is the
  most frequently mutated oncogene in HCC
• de la Coste et al. PNAS 1998, Miyoshi et al.
  Cancer Res 1998

17
Aims

• To relate the molecular findings to
  histopathological and clinical features in
  hepatocellular adenomas

• In order to refine the classification of these
  hepatocellular tumors into quite homogeneous
  subgroups, possibly with different evolutive
  potential.

18
Patients and methods

• A multicentric series of 96 liver tumors were
  studied (13 french University hospitals)

Criteria for inclusion tumors with a firm or
possible diagnosis of adenoma, surgically
resected, and with an adequate sampling of fixed
and frozen tissues (-80C) with good quality of
nucleic acids

• Molecular analysis
• HNF1? mutations
• ß-catenin mutations and over-expression of
  targeted genes glutamine synthetase (GS) and
  GPR49

Réseau national détude des tumeurs
hépatocellulaires développées sur foie sain
19
Each case was reviewed by a panel of liver
pathologists, using a consensual report including
22 pathological items, without knowing of
genotyping results.

?




Ad / HNF

?



Inclassable


Autres

Commentai
res
.

agrément de réponses

_____ / _____


Adénomatose


Adénomes multiples

Commentaires .

20
Results
Sequencingdefined 3 groups of HA
No tumors showed both HNF1? and b-catenin
mutations
Search for correlations with pathological and
clinical features
21
Results HNF1? mutated adenomas
HNF1? mutated tumors
1
Stop/frameshift
n44
AA substitutions
black somatic mutations red germline mutations

• HNF1? mutations were identified in 46 of the 96
  cases
• In all cases bi-allelic inactivating mutations
  in the tumor

22
HNF1? mutated HA

• HNF1? mutations defined an homogeneous group of
  tumors, closely associated with
• marked steatosis plt 0.0001
• no inflammation plt 0.0001
• no cytological abnormalities plt 0.00001
• mainly, a firm diagnosis of adenoma
• plt 0.001

23
Comparison of germline HNF1? adenomas Vs somatic
HNF1 mutated
24
Results ?-catenin activated adenomas
2
ß-catenin act.
n13

• 13 cases with ß-catenin mutations (11 cases)
  and/or activation of the targeted genes GS and
  GPR49

25
adenoma
b catenin
In this group of 13 ?-catenin activated tumors
over-representation of male patients (38)
p0.02 - cytological abnormalities (p0.0002),
pseudo-glandular formation (p0.002)
- less frequently steatotic
(p0.0001)
26
Results non-mutated adenomas
3
No HNF1 or b-cat mutation
n39
27
Molecular classification and prognosis of HA
germline
1
ß-catenin act.
2
n13
Inflammatory Non-mutated
3
Non-inflammatory Non-mutated
4
n22
Zucman-Rossi et al, Hepatology 2006
28
Conclusion

• The molecular and pathological classification of
  HA enabled the identification of strong
  genotype/phenotype correlations

• The most usual group concerns HNF1? mutated HA,
  severely steatotic

• ?-catenin mutated HA have a higher risk of
  malignant transformation consequently,
  patients of this subgroup have to be more
  carefully followed

• A genetic counseling and a search for germline
  HNF1? mutation may be recommended for patients
  with familial adenoma and/or multiple steatotic
  adenomas and/or with familial maturity onset
  diabetes (MODY)

29
Germline genetic predisposition to develop
HNF1a?mutated adenomas

• Mutation constitutionnelle dHNF1a
• Bluteau O., Jeannot E. et al., Nature Genet.,
  2002
• Bacq Y. et al., Gastroenterology, 2003
• Reznik Y. et al., J Clin Endocrinol Metab, 2004

30
Present and future projects

• clinical translation and validation diagnostic
  and prognostic classification of 400 malignant
  and benign liver tumors using a 48 gene predictor
  in quantitative RT-PCR
• Inca funded project 2006-2008

• quantitative RT-PCR platform of validation
  Ligue national contre le cancer program to
  validate microarray analysis in the carte
  didentité des tumeurs project
• LNCC funded project 2006-2007

• professional exposition risk and genetic
  polymorphisms to develop HCC without cirrhosis
• Under construction

• CGH array analysis of tumors using SNP
  affymetrix microaarays to identify new tumor
  suppressor genes in homogeneous subgroups of
  tumors
• Under construction

31

• Sequencing genes implicated in cancer
• More than 40 genes are currently sequenced in our
  lab
• Search for correlation with tumor response to
  targeted or conventional therapies
• Search for associated trancriptome signature

32
GENTHEP Génétique des tumeurs hépatiques
développées sur foie sain Inserm Network
Inserm U674, Paris Jessica Zucman-RossiSandra
RebouissouEmmanuelle JeannotSandrine
BoyaultLucille MellotteeAurélie HéraultKarine
Poussin
Bordeaux Créteil Villejuif Lyon Lille Tours Anger
Saint-Antoine Bicêtre Caen Nice
Clichy Strasbourg Grenoble
French pathology, hepatology and surgical
departments
Inserm E361,Bordeaux Paulette Bioulac-sage Charles
Balabaud