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ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 3  |  Page : 677-680

Immunohistochemical expression of beta-catenin in hepatoblastoma and its clinical significance


1 Department of Pathology, Sri Ramachandra Medical College, Chennai, Tamil Nadu, India
2 MBBS Student, Sri Ramachandra Medical College, Chennai, Tamil Nadu, India
3 Department of Pathology, Sree Balaji Medical College, Chennai, Tamil Nadu, India
4 Department of Paediatric Oncology, Sri Ramachandra Medical College, Chennai, Tamil Nadu, India

Date of Submission08-Sep-2021
Date of Acceptance22-Sep-2021
Date of Web Publication25-Jul-2022

Correspondence Address:
Lawrence D'Cruze
Department of Pathology, Sri Ramachandra Medical College, Porur, Chennai - 600 116, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.jcrt_1575_21

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 > Abstract 


Background: Primary hepatic malignancies account for 0.5-2% of all solid tumours in childhood. Hepatoblastoma, a rare embryonic tumour in the general population, represents the most frequent primary hepatic malignancy in the paediatric age group, with an incidence of one new case per million under 15 years of age, median age at diagnosis being 1 year. Aberrant activation of the Wnt/beta-catenin signalling pathway is likely to result in tumorigenesis of hepatoblastoma. The nuclear and intra-cytoplasmic accumulations of beta-catenin correlate with the likely prognosis of the disease. Nuclear expression of beta catenin is associated with a shorter survival, higher stage, and seen in embryonal/undifferentiated types.
Aim: To study the expression of beta-catenin in hepatoblastoma by immunohistochemistry and correlate it with the tumour histology and survival outcome.
Materials & Methods: This is a retrospective study of 11 children over a period of 5 years with the diagnosis of hepatoblastoma. These children underwent partial hepatectomy or liver transplantation at the Department of Paediatric Surgery. The clinical, histological and survival data were collected. Immunohistochemical analysis with beta-catenin was done and analysed.
Results: Mean birth weight of the children was 2.75kg.63.6% had an epithelial type of histology.Beta catenin expression by IHC was studied in 11 cases and found to be positive in 4 cases. Nuclear positivity was noted in 2/4 cases of embryonal type and Cytoplasmic and membranous positivity was seen in the other 2/4 cases. Normal liver showed a membranous pattern of positivity in one case. Negative staining was seen in 6 out of 11 cases.
Conclusion: Beta catenin is considered to be an useful tool for assessing the prognosis of patients with hepatoblastoma and its expression is associated with a poor survival outcome. There are no validated biomarkers for prognosis so far. However, larger studies incorporating molecular profiling is warranted to establish prognostic factors for planning effective treatment strategies.

Keywords: Beta-catenin, immunohistochemistry, liver tumor, pediatrics


How to cite this article:
Archana B, D'Cruze L, Nazneen S, Thanka J, Scott JX. Immunohistochemical expression of beta-catenin in hepatoblastoma and its clinical significance. J Can Res Ther 2022;18:677-80

How to cite this URL:
Archana B, D'Cruze L, Nazneen S, Thanka J, Scott JX. Immunohistochemical expression of beta-catenin in hepatoblastoma and its clinical significance. J Can Res Ther [serial online] 2022 [cited 2022 Aug 10];18:677-80. Available from: https://www.cancerjournal.net/text.asp?2022/18/3/677/351810




 > Introduction Top


Primary hepatic malignancies account for 0.5%–2% of all solid tumors in childhood. Hepatoblastoma is the most common pediatric primary hepatic malignancy with an incidence of 1.5 new cases per 1 million children under 15 years of age.[1] The median age at diagnosis was 1 year. It is known to occur more frequently in males. It is also seen in association with risk factors such as prematurity, low birth weight, maternal alcohol consumption, maternal smoking, oral contraceptive use, and methods of assisted reproduction. Hepatoblastomas are also seen to be associated with syndromes such as Beckwith–Wiedemann syndrome, familial adenomatous polyposis, Li-Fraumeni syndrome, trisomy 18, and other metabolic disorders.[2] Histology is very important and has distinct clinical associations. Histology has been incorporated as a risk stratification parameter in the Children's Oncology Group (COG) protocols. Histological diversity of these lesions has proven to be a challenge to the pathologist in diagnosing and subclassifying the distinct variants of hepatoblastoma. Among the variants of hepatoblastoma, well-differentiated mitotically inactive fetal-type hepatoblastomas are associated with a better outcome compared to mitotically active type, embryonal, and small cell types.[3] The standard treatment includes surgery with or without chemoradiation.[4] In inoperable cases, liver transplantation may be considered and has offered potential cure.

Aberrant activation of the Wnt/beta-catenin signaling pathway is likely to result in the tumorigenesis of hepatoblastomas and is considered a hallmark of hepatoblastoma.[5],[6] The nuclear and cytoplasmic accumulations of beta-catenin correlate with the likely prognosis of the disease. Nuclear expression of beta-catenin is associated with a shorter survival and higher stage and predominantly seen in the embryonal/small cell undifferentiated variants. However, in our study, the staining pattern was not statistically significant. Thus, differences in beta-catenin staining in hepatoblastoma could impact tumor behavior, alter treatment, and require further rigorous investigations incorporating larger sample size and further molecular profiling.

Aims

To study the expression of beta-catenin in hepatoblastoma by immunohistochemistry and correlate it with the tumour histology and survival outcome.


 > Materials and Methods Top


The retrospective study was carried out on 11 children diagnosed with hepatoblastoma over a period of 5 years. Ethical approval was obtained by the institutional ethics committee before the commencement of the study. Guardians of the patients gave consent for the study after verbal counseling. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 2000. Inclusion criteria: All cases with biopsy/resection-proven hepatoblastoma irrespective of the prior therapy given. Exclusion criteria: Infections, other benign and malignant tumors of the liver. Clinical and demographic data were collected and documented. Laboratory parameters such as complete blood counts, liver function test, and serum alpha-fetoprotein levels were recorded. Radiological data on the pretreatment extent of tumor (PRETEXT) staging done by computed tomography scan for hepatoblastomas were collected. Histopathology was studied and the results were analyzed. Formalin-fixed/paraffin-embedded tissue blocks were sectioned at 5 μm and lightly stained with hematoxylin–eosin. Each block contained representative tumor areas together with nonneoplastic adjacent liver parenchyma. Immunohistochemistry for beta-catenin was done. It was performed by the automated method. The prepared section in barrier slides was placed in Xmatrx where the slides were deparaffinized with xylene and ethanol. Antigen retrieval was done, endogenous peroxidases was blocked with 0.3% hydrogen peroxide, and nonspecific binding was blocked with casein containing 0.9% sodium azide. Slides were incubated overnight at 4°C with primary antibody (beta-catenin 1:100 dilution) along with chromogen diaminobenzidine (DAB). Slides were counterstained with hematoxylin. All samples were run with a positive and negative control. Staining pattern was recorded based on the positivity or negativity and the location of staining (nuclear, cytoplasmic, or membranous) independently by two observers. Data on the survival outcome were collected and the results were statistically analyzed.


 > Results Top


The mean age of the study group with 11 participants was 16 months. The M: F ratio was 3.6:1. The mean birth weight of these children was 2.75 kg. Thrombocytosis was seen in 7/11 (63.6%) cases. Deranged LFT was noted in 4 (36%. 4) children. Serum alpha-fetoprotein levels were elevated in 9 (81%) children; however, in those with very low AFP levels, the prognosis was poor [Table 1]. The majority belonged to Stage I (36.4%) disease. Histologically, out of the 11 children, 7 (63.6%) had epithelial histology and 4 (36.4%) had mixed epithelial and mesenchymal histology [Figure 1]. Among biopsies with epithelial histology, pure fetal type was seen in 3 (27%), fetal mitotically active in 1 (9%), mixed epithelial pattern with a combination of fetal and macrotrabecular in 2 (18%), and fetal and embryonal pattern in 2 (18%). Mixed epithelial–mesenchymal variants included a combination of different epithelial types (fetal, embryonal, and cholangioblastic) with a mesenchymal component (spindle cells and osteoid formation).
Table 1: Clinicopathological profile and beta-catenin staining in hepatoblastoma

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Figure 1: (a) Mitotically active fetal hepatoblastoma (H and E) ×20. (b) Sheets and trabecular arrangement of tumor cells suggesting a fetal type of hepatoblastoma (H and E) ×40. (c) Embryonal type (H and E) ×400

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Beta-catenin expression by IHC was studied in 11 (eleven) cases. Positive pattern of expression was noted in 4 cases. Beta-catenin focal nuclear expression was noted in 2/8 cases which were predominantly of embryonal type. Cytoplasmic expression was noted in 2/8 cases of fetal type. Normal liver in one case showed a membranous pattern of positivity [Figure 2] (Graph 5). Negative staining was seen in the remaining 6/11 cases.
Figure 2: (a) Immunohistochemistry for beta-catenin in mixed hepatoblastoma showing membranous positivity, ×400. (b) Immunohistochemistry for beta-catenin showing cytoplasmic and membranous positivity in the fetal type with cholangioblastic differentiation, ×200. (c) Immunohistochemistry for beta-catenin in embryonal type showing nuclear positivity, ×400

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Among the 11 children, survival data could be collected for 9: 3 died and 6 responded well to treatment. One child has been planned for transplantation [Table 1]. Statistically, by Chi-square test, the percentage of those who survived/died was similar in those who were positive or negative for beta-catenin (P = 0.956). No significance was noted in the survival outcome with respect to the histological favorability (P = 0.845). Beta-catenin in our study showed nuclear expression in embryonal tumors, which represents tumors with poor prognosis in the literature. However, due to low sample size because of rarity of these lesions, significance could not be established. More studies involving bigger sample size and molecular confirmation are needed to establish its role as a potential therapeutic target.


 > Discussion Top


Hepatoblastomas are rare pediatric liver tumors and very limited studies from India have been published. Only eight centers from India have published their data.[7] The diagnosis of these tumors is challenging due to a lack of current consensus classification system.[3] The PRETEXT staging is used for risk stratification; this system includes annotation factors that determine the prognosis.[8] Thrombocytosis is a regular feature noticed. Low serum alpha-fetoprotein levels are associated with an adverse outcome like that noticed by us. Histopathological subtypes also play a vital role and hence histology is incorporated in the COG protocols for planning treatment.[7] The small cell undifferentiated type is associated with an adverse prognosis. Complete surgical resection with or without neoadjuvant chemotherapy is the main modality of treatment, and in surgically unresectable nonmetastatic cases, liver transplantation is considered.[9] Activation of beta-catenin is a hallmark noticed in hepatoblastomas and plays a critical role in its tumorigenesis. In a study done by Purcell et al. aberrant beta catenin expression was noted in 87% of tumor samples studied.[5] By IHC, tumor cells demonstrated strong expression of beta-catenin positivity in the small cell undifferentiated, embryonal, and macrotrabecular types, which carries a poor prognosis. This confirms that activation of beta-catenin is associated with an aggressive tumor growth and carries an adverse outcome. Fetal types showed a cytoplasmic and membranous staining pattern. Normal liver demonstrated a membranous pattern of staining in the hepatocytes and a membranous/cytoplasmic pattern in bile ducts. IHC staining needs to be further correlated with the molecular pattern of expression. So far, no validated molecular biomarkers are established for treatment and prognosis in hepatoblastoma.[10] Recently, a study done by Sumazin et al. revealed three risk stratification molecular profiles in hepatoblastoma involving HNF1A, NFE2L2, SALL4, HMGA2, and LIN28B genes.[10] Studies have demonstrated an aberrant Wnt/beta-catenin signaling with a high mutation involving the CTNNB1 gene in hepatoblastomas.[6] It is, however, unclear whether this signaling pathway is important in hepatoblastoma genesis or prognosis.[11] The beta-catenin signaling pathway occurs in association with other pathways such as YAP1, mTOR1, nF-kB, TNF, and PARP1.[12] It alone is insufficient for tumorigenesis. Different mutations such as deletions, small deletions, and point mutations in beta-catenin impact tumor behavior differently. Bell et al. has reported a study which showed that 60-70% of hepatoblastomas have a high frequency of interstitial deletions and missense mutations in CTNNB1 gene which encodes for beta–catenin, leading to overaccumulation of cytoplasmic and nuclear beta-catenin.[6] Deletion mutations of exon 3 have more often been associated with fetal type histology, compared to small deletions and point mutations that are associated with embryonal and small cell undifferentiated types. Molecular analysis revealed somatic mutations involving beta-catenin exon 3 extending into exon 4. Like our case, literature review indicates that beta-catenin overexpression is noticed at the protein level in the epithelial as well as in the mesenchymal subtypes.[13] Targeted inhibition of Wnt/beta-catenin signaling using siRNA, miRNA, and pharmacological agents has potential to attenuate the progression of hepatoblastomas, thus providing the needed cure in children affected by them.


 > Conclusion Top


Hence, the role of beta-catenin/Wnt signaling pathway with a better understanding of hepatoblastoma pathology remains elusive and new treatments are needed for advanced-stage tumors to improve clinical management and develop therapeutic strategies. More researches on molecular biomarkers are warranted to develop consensus classifications and develop therapeutic protocols to improve patient outcome.

Financial support and sponsorship

This study was supported by Chancellor's Summer Research Project.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Herzog CE, Andrassy RJ, Eftekhari F. Childhood cancers: Hepatoblastoma. Oncologist 2000;5:445-53.  Back to cited text no. 1
    
2.
Chen H, Guan Q, Guo H, Miao L, Zhuo Z. The Genetic Changes of Hepatoblastoma. Frontiers in oncology 2021;11:690641.  Back to cited text no. 2
    
3.
López-Terrada D, Alaggio R, de Dávila MT, Czauderna P, Hiyama E, Katzenstein H, et al. Towards an international pediatric liver tumor consensus classification: Proceedings of the Los Angeles COG liver tumors symposium. Mod Pathol 2014;27:472-91.  Back to cited text no. 3
    
4.
Schnater JM, Aronson DC, Plaschkes J, Perilongo G, Brown J, Otte JB, et al. Surgical view of the treatment of patients with hepatoblastoma. Cancer 2002;94:1111-20.  Back to cited text no. 4
    
5.
Purcell R, Childs M, Maibach R, Miles C, Turner C, Zimmermann A, et al. HGF/c-Met related activation of β-catenin in hepatoblastoma. Journal of experimental & clinical cancer research 2011;30:1-0.  Back to cited text no. 5
    
6.
Bell D, Ranganathan S, Tao J, Monga SP. Novel advances in understanding of molecular pathogenesis of hepatoblastoma: a Wnt/β-catenin perspective. Gene expression 2017;17:141.  Back to cited text no. 6
    
7.
Archana B, Thanka J, Sneha LM, Xavier Scott JJ, Arunan M, Agarwal P. Clinicopathological profile of hepatoblastoma: An experience from a tertiary care center in India. Indian J Pathol Microbiol 2019;62:556-60.  Back to cited text no. 7
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8.
Roebuck DJ, Aronson D, Clapuyt P, Czauderna P, de Ville de Goyet J, Gauthier F, et al. 2005 PRETEXT: A revised staging system for primary malignant liver tumours of childhood developed by the SIOPEL group. Pediatr Radiol 2007;37:123-32.  Back to cited text no. 8
    
9.
Shanmugam N, Scott JX, Kumar V, Vij M, Ramachandran P, Narasimhan G, et al. Multidisciplinary management of hepatoblastoma in children: Experience from a developing country. Pediatr Blood Cancer 2017;64:e26249.  Back to cited text no. 9
    
10.
Sumazin P, Chen Y, Treviño LR, Sarabia SF, Hampton OA, Patel K, et al. Genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups. Hepatology 2017;65:104-21.  Back to cited text no. 10
    
11.
Cairo S, Armengol C, De Reyniès A, Wei Y, Thomas E, Renard CA, et al. Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer. Cancer Cell 2008;14:471-84.  Back to cited text no. 11
    
12.
Sha YL, Liu S, Yan WW, Dong B. Wnt/β-catenin signaling as a useful therapeutic target in hepatoblastoma. Biosci Rep 2019;39:BSR20192466.  Back to cited text no. 12
    
13.
Wei Y, Fabre M, Branchereau S, Gauthier F, Perilongo G, Buendia MA. Activation of β-catenin in epithelial and mesenchymal hepatoblastomas. Oncogene 2000;19:498-504.  Back to cited text no. 13
    


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