Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Liver cancer with concomitant TP53 and CTNNB1 mutations: a case report

  • Juliane Friemel1, 2,
  • Markus Rechsteiner1,
  • Marion Bawohl1,
  • Lukas Frick1, 4,
  • Beat Müllhaupt3,
  • Mickaël Lesurtel4 and
  • Achim Weber1Email author
BMC Clinical PathologyBMC series – open, inclusive and trusted201616:7

DOI: 10.1186/s12907-016-0029-5

Received: 14 December 2015

Accepted: 21 May 2016

Published: 1 June 2016

Abstract

Background

In the spectrum of molecular alterations found in hepatocellular carcinoma (HCC), somatic mutations in the WNT/β-catenin pathway and the p53/cell cycle control pathway are among the most frequent ones. It has been suggested that both mutations occur in a mutually exclusive manner and they are used as molecular classifiers in HCC classification proposals.

Case presentation

Here, we report the case of a treatment-naïve mixed hepatocellular/cholangiocellular carcinoma (HCC/CCC) with morphological and genetic intratumor heterogeneity. Within the predominant part of the tumor with hepatocellular differentiation, a p.D32V mutation in exon 3 of the CTNNB1 gene occurred concomitantly with a TP53 intron 7/exon 8 splice site mutation.

Conclusion

Intratumor heterogeneity challenges the concept of CTNNB1 and TP53 gene mutations being mutually exclusive molecular classifiers in HCC, which has implications for HCC classification approaches.

Keywords

Hepatocellular carcinoma (HCC) Intratumor heterogeneity CTNNB1 TP53 Next generation sequencing

Background

Hepatocellular carcinoma (HCC) is the fifths most common cancer in men and the second most common cause for cancer-related death worldwide [1]. HCC mostly develop on the background of chronic liver diseases including chronic viral hepatitis due to infection with hepatitis B virus (HBV), or hepatitis C virus (HCV), alcohol-induced liver injury, fatty liver disease or exposure to toxic factors such as aflatoxin. The spectrum of somatic mutations related to liver carcinogenesis has been identified [2]. With marked geographic variation, TP53 and CTNNB1 represent two of the most common driver mutations in the African-Asian countries (TP53) and in the western world (CTNNB1). Several molecular classifications of HCC distinguish HCC with alterations in the p53/cell cycle control pathway from HCCs with alterations in the WNT/β-catenin pathway, including activating mutations of the CTNNB1 oncogene, AXIN1 or APC [3, 4]. Mutations of TP53 and CTNNB1 are largely considered to occur in a mutually exclusive manner [5]. Phenotypical and genetic intratumor heterogeneity with variable mutational status (i.e. wild type among mutated tumor cell clones) of TP53 and CTNNB1 in different tumor regions within the same tumor is frequently found in HCC [6]. Here, we describe a de novo, hepatitis C-related combined cholangiocellular and hepatocellular carcinoma with marked intratumor heterogeneity on three levels: morphology, immunohistochemical marker profile and mutational status with 3/14 tumor regions of solely hepatocellular differentiation harboring concomitant mutations of CTNNB1 and TP53.

Case presentation

A liver tumor was detected in a 72 year old male patient with liver cirrhosis Child-Pugh Stage A, a history of type 2 diabetes and chronic hepatitis C virus infection (HCV, genotype 1B), initially diagnosed 13 years ago. Liver enzymes were slightly elevated with alanine aminotransferase 89 U/L (reference: 10–50 U/L) and aspartate aminotransferase 65 U/L (reference: <50 U/L). The 4 cm tumor was detected by routine sonography and removed by laparoscopic liver segment resection.

Morphological analysis, immunohistochemistry and multiregional, next generation sequencing (NGS) was applied on representative tumor sections as described [6]. Table 1 and Fig. 1 illustrate histopathological and molecular findings in 14 individual tumor areas, which were grouped into three tumor regions (A, B and C) according to their predominant morphological and molecular characteristics. In summary, a multinodular, combined hepatocellular/cholangiocellular carcinoma, tumor stage T1 grade 2–3, was diagnosed. Intratumoral heterogeneous expression of five liver cell markers (CK7, CK19, glutamine synthetase, p53, β-catenin) was detected including a double positivity for glutamine synthetase, nuclear β-catenin and p53 in tumor region A.
Table 1

Morphology, immunohistochemistry and mutational status of individual tumor areas

 

Area

A1

A2

A3

B

C1

C2

C3

C4

C5

C6

C7

C8

C9

C10

 

Size in mm2

7.8

5.54

31.3

65

73.2

16.5

34.5

82.2

20.2

18.9

17.4

28.5

1.94

17.5

Morphology

Solid

+

+

+

 

+

+

+

 

+

+

 

+

  

Glandular (cholangiocellular)

   

+

          

Trabecular

      

+

+

  

+

 

+

+

Clear cell change

 

+

  

+

        

+

Fatty change

          

+

   
 

CK19

   

>50 %

 

>50 %

        

IC

CK7

   

70 %

SC

50 %

20 %

SC

40 %

SC

 

SC

 

50 %

glutamine synthetase

+

+

+

           

β-catenin nuclear

+

+

+

           

p53

+

+

+

           

Mutation

CTNNB1 sanger seq

D32

D32

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

CTNNB1 deep seq

D32

D32

D32a

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

TP53 sanger seq

IVS8-1

IVS8-1

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

TP53 deep seq

IVS8-1

IVS8-1

IVS8-1a

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

wt

sanger seq sanger sequencing, deep seq deep sequencing (NGS), wt wild type, SC single cells positive

alow frequency (<10 %)

Fig. 1

Combined HCC/CCC in a 72 year old male patient with history of hepatitis C and diabetes revealing morphological, immunohistochemical and genetic intratumor heterogeneity. a Gross morphology and slide overview (H&E staining) with annotations of defined tumor areas. b Microscopic images (40x) of different tumor areas (H&E staining, CK7, glutamine synthetase and β-catenin immunohistochemistry). c p53 and β-catenin nuclear positivity in consecutive slides of tumor area A2. d Nucleotide exchanges in CTNNB1 and TP53 genes (TP53 depicted as reverse sequence). e Mutated allele frequencies of double mutated areas A1-3 with number of gene copies analyzed. Reference sequences: NM 1904.1 (CTNNB1) and NM 001126112.2 (TP53)

Next generation sequencing was performed with a minimum coverage of 1329 (SD ± 725) reads per amplicon of every single tumor area. Sequencing results yielded a p.D32V (c.363 A > T) mutation and a TP53 ivs8-1 (c.783-1 G > A) splice site mutation in tumor region A. Comparing mutated allele frequencies, CTNNB1 and TP53 gene copies showed a similar range of both frequencies in area A1-3 (Fig. 1). All mutated and wild type tumor areas additionally displayed a SNP of exon 7 (rs17880604). Collectively, morphological and immunohistochemical findings together with sequencing results demonstrated that a tumor subclone with hepatocellular differentiation had concomitant CTNNB1 and TP53 gene mutations.

To date, after a follow-up time of 12 months, the patient had a local recurrence of a liver tumor which was inoperable and therefore treated by transarterial chemoembolization (TACE).

Conclusion

In this case of a HCV infection -related liver cancer, a CTNNB1/TP53 double mutation was detected in a tumor region of hepatocellular differentiation, among TP53 and CTNNB1 wild type tumor areas. The analysis of mutated allele frequencies using next generation sequencing techniques corroborates that the double mutation is located in the same tumor cell population. To our knowledge, this is the first detailed description of a CTNNB1/TP53 double mutation in a single liver cancer lesion. TP53 and CTNNB1 both are molecular classifiers for hepatocellular carcinoma. For instance, in the transcriptome-analysis based classification proposal by Boyault et al. [4], six HCC subgroups are distinguished: two groups are characterized by TP53 and two independent groups by CTNNB1 alterations. A study by Laurent-Puig et al. [5] on genetic alterations in hepatocarcinogenesis describes TP53 and CTNNB1 mutations as mutually exclusive. In agreement, a study by Tornesello et al. [7] records mutations of the two driver genes as being mutually exclusive.

CTNNB1 mutations are reported to be associated with hepatitis C infections [8]. TP53 point mutations frequently are reported to occur specifically at codon 249 after aflatoxin exposition. The frequency and the causal link between TP53 and CTNNB1 mutations in HCC have not been systematically investigated. A study by Ötztürk et al. [9] on HCC cell lines provides evidence that inactivation of TP53 could cause aberrant nuclear β-catenin accumulation, suggesting a link between the two genes. In the presented case study, the CTNNB1 mutation affected the GSK-3β phosphorylation site [10] which argues for a β-catenin accumulation independent from the TP53 mutation. The detected TP53 mutation affects a splice site of exon 8. Although splice sites in TP53 are not typical mutation sites, there is evidence that TP53 splicing mutations lead to exon dropping indicating biological relevance [11]. Furthermore, the nuclear accumulation of the dysfunctional p53 protein in immunohistochemical analysis found in this case supports a functional significance of this splice site mutation. As has been reported, wild type p53 is rapidly degraded while mutations lead to nuclear accumulation of the p53 protein [12, 13].

In summary, the molecular, immunohistochemical and morphological diversity in the presented case indicates a high level of intratumor heterogeneity and challenges the concept that TP53 and CTNNB1 are mutually exclusive driver alterations in HCC. Distinct parts of the tumor reveal multinodularity, and differ with respect to their biomarker expression and mutational status, indicative of distinct tumor subpopulations [14]. This finding illustrates the challenge to molecularly characterize individual HCC. Routine pathological analysis is based on testing a small piece of a tumor, assuming that it represents the whole tumor. When analyzing distinct pathways of hepatocarcinogenesis such as the WNT/β-catenin pathway as a potential therapeutic target in HCC [15], it will be pivotal in the future to also take into account the level of intratumor heterogeneity.

Abbreviations

CCC, cholangiocellular carcinoma; CTNNB1, Catenin β 1; HCC, hepatocellular carcinoma; TACE, transarterial chemoembolization; TP53, tumor protein 53

Declarations

Acknowledgements

We gratefully thank the patient and family members for allowing us to report this case.

Funding

This work was supported by the following grants: A grant from the Theiler-Haag Stiftung, Zurich to AW and LF. Grants from the Krebsliga Schweiz (Oncosuisse) and from the “Kurt and Senta Herrmann Stiftung”, Vaduz, Lichtenstein to AW.

Availability of data and materials

Not applicable.

Authors’ contributions

Conception: AW. Methods: JF, LF, MR, MB. Patient care and surgery: ML, BM. Writing of the manuscript: JF and AW. Critical revision of the manuscript: LF, ML, BM, AW, JF, MB. All authors read and approved the final manuscript.

Authors’ information

AW is Professor for Experimental and Molecular Pathology at the University (UZH) and University-Hospital (USZ) Zurich.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Ethics approval and consent to participate

Ethical approval for the study was given by the local ethics commitee (StV 26–2005 and KEK-ZH-Nr. 2013–0382).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Surgical Pathology, University and University Hospital Zurich
(2)
Leibniz Institute for Prevention Research and Epidemiology (BIPS)
(3)
Clinics of Hepatology and Gastroenterology, University and University Hospital Zurich
(4)
Swiss Hepato-Pancreato-Biliary Center, Department of Digestive and Transplant Surgery, University Hospital of Zurich

References

  1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–86.View ArticlePubMedGoogle Scholar
  2. Nault JC, Mallet M, Pilati C, Calderaro J, Bioulac-Sage P, Laurent C, Laurent A, Cherqui D, Balabaud C, Zucman-Rossi J. High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions. Nat Commun. 2013;4:2218.View ArticlePubMedPubMed CentralGoogle Scholar
  3. Guichard C, Amaddeo G, Imbeaud S, Ladeiro Y, Pelletier L, Maad IB, Calderaro J, Bioulac-Sage P, Letexier M, Degos F, et al. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma. Nat Genet. 2012;44(6):694–8.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Boyault S, Rickman DS, de Reynies A, Balabaud C, Rebouissou S, Jeannot E, Herault A, Saric J, Belghiti J, Franco D, et al. Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology. 2007;45(1):42–52.View ArticlePubMedGoogle Scholar
  5. Laurent-Puig P, Legoix P, Bluteau O, Belghiti J, Franco D, Binot F, Monges G, Thomas G, Bioulac-Sage P, Zucman-Rossi J. Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology. 2001;120(7):1763–73.View ArticlePubMedGoogle Scholar
  6. Friemel J, Rechsteiner M, Frick L, Bohm F, Struckmann K, Egger M, Moch H, Heikenwalder M, Weber A. Intratumor heterogeneity in hepatocellular carcinoma. Clin Cancer Res. 2015;21(8):1951–61.View ArticlePubMedGoogle Scholar
  7. Tornesello ML, Buonaguro L, Tatangelo F, Botti G, Izzo F, Buonaguro FM. Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular carcinoma associated with hepatitis B and hepatitis C virus infections. Genomics. 2013;102(2):74–83.View ArticlePubMedGoogle Scholar
  8. Huang H, Fujii H, Sankila A, Mahler-Araujo BM, Matsuda M, Cathomas G, Ohgaki H. Beta-catenin mutations are frequent in human hepatocellular carcinomas associated with hepatitis C virus infection. Am J Pathol. 1999;155(6):1795–801.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Cagatay T, Ozturk M. P53 mutation as a source of aberrant beta-catenin accumulation in cancer cells. Oncogene. 2002;21(52):7971–80.View ArticlePubMedGoogle Scholar
  10. de La Coste A, Romagnolo B, Billuart P, Renard CA, Buendia MA, Soubrane O, Fabre M, Chelly J, Beldjord C, Kahn A, et al. Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proc Natl Acad Sci U S A. 1998;95(15):8847–51.View ArticlePubMed CentralGoogle Scholar
  11. Lai MY, Chang HC, Li HP, Ku CK, Chen PJ, Sheu JC, Huang GT, Lee PH, Chen DS. Splicing mutations of the p53 gene in human hepatocellular carcinoma. Cancer Res. 1993;53(7):1653–6.PubMedGoogle Scholar
  12. Hsu HC, Tseng HJ, Lai PL, Lee PH, Peng SY. Expression of p53 gene in 184 unifocal hepatocellular carcinomas: association with tumor growth and invasiveness. Cancer Res. 1993;53(19):4691–4.PubMedGoogle Scholar
  13. Chen GG, Merchant JL, Lai PB, Ho RL, Hu X, Okada M, Huang SF, Chui AK, Law DJ, Li YG, et al. Mutation of p53 in recurrent hepatocellular carcinoma and its association with the expression of ZBP-89. Am J Pathol. 2003;162(6):1823–9.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Kanai T, Hirohashi S, Upton MP, Noguchi M, Kishi K, Makuuchi M, Yamasaki S, Hasegawa H, Takayasu K, Moriyama N, et al. Pathology of small hepatocellular carcinoma. A proposal for a new gross classification. Cancer. 1987;60(4):810–9.View ArticlePubMedGoogle Scholar
  15. Park JY, Park WS, Nam SW, Kim SY, Lee SH, Yoo NJ, Lee JY, Park CK. Mutations of beta-catenin and AXIN I genes are a late event in human hepatocellular carcinogenesis. Liver Int. 2005;25(1):70–6.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2016

Advertisement