An immunohistochemical approach to detect oncogenic CTNNB1 mutations in primary neoplastic tissues

Abstract

The Wnt/β-catenin signaling pathway is dysregulated in different types of neoplasms including colorectal cancer (CRC). Aberrant activation of this signaling pathway is a key early event in the development of colorectal neoplasms, and is mainly caused by loss of function mutations in Adenomatous Polyposis Coli (APC), and less frequently by β-catenin stabilization mutations via missense or interstitial genomic deletions in CTNNB1. In this study, we have defined an immunohistochemical algorithm to dissect Wnt pathway alterations in formalin-fixed and paraffin-embedded neoplastic tissues. Basically, consecutive sections of tumor specimens were stained by immunohistochemistry with two different monoclonal antibodies against β-catenin: one (anti-active β-catenin antibody) recognizes hypo-phosphorylated β-catenin and the other recognizes the total pool of β-catenin. We validated the strategy in the HCT116 CRC cell line which has an in-frame deletion of β-catenin serine 45, and then studied human tumor microarrays containing colon adenomas, CRCs, solid pseudopapillary neoplasms of the pancreas as well as the whole tissue sections of CRCs, desmoid fibromatosis, and pilomatrixoma of the skin. In some tumors, we found strong β-catenin cytoplasmic and/or nuclear staining with the total β-catenin antibody but no staining with the anti-active β-catenin antibody. This was inferred to be an altered/mutant β-catenin staining pattern. All six colon adenomas of the 126 total adenomas studied for the altered/mutant β-catenin staining pattern had presumptively pathogenic point mutations or deletions in CTNNB1. Four of 10 CRCs with the alterated/mutant β-catenin staining pattern studied in depth, from 181 total CRCs from tissue microarray, had pathogenic CTNNB1 mutations. The frequencies of CTNNB1 alterations in non-colonic tumors with altered/mutant β-catenin staining ranged between 46 and 100%. Our results demonstrate that the immunohistochemical approach described here can detect oncogenic forms of β-catenin in primary tissue samples and can also highlight other tumors with presumptive novel defects activating the Wnt/β-catenin pathway.

Analysis of the Clinicopathologic Characteristics of Lung Adenocarcinoma With CTNNB1 Mutation

“…With the advent of genomics, molecular or genetic variants affecting disease risk can be identified (Field, 2008). Mutations in the gene encoding b-catenin (CTNNB1) have been detected in numerous human malignancies, including lung cancer (Woenckhaus et al, 2008), malignant mesothelioma (Shigemitsu et al, 2001), desmoid tumors (Colombo et al, 2013), colon cancer (Akyol et al, 2019), and others. Woenckhaus et al (2008) identified a number of differentially expressed genes in smoke-exposed bronchial epithelium and nonsmall cell lung cancers (NSCLCs), they found in adenocarcinomas, the cytoplasmic expression of beta-catenin was associated with shorter survival (p = 0.012).…”

“…Shigemitsu et al (2001) found CTNNB1 is infrequently mutated in lung cancer. Akyol et al (2019) defined an immunohistochemical algorithm to dissect Wnt pathway alterations in formalin-fixed and paraffin-embedded neoplastic tissues and found all six colon adenomas of the 126 total adenomas studied for the altered/ mutant b-catenin staining pattern had presumptively pathogenic point mutations or deletions in CTNNB1. The N-terminus of b-catenin, with contains conserved phosphorylated threonine/ serine amino acid residues, is the most frequent location of cancer-related CTNNB1 mutations (Dar et al, 2017).…”

“…The N-terminus of b-catenin, with contains conserved phosphorylated threonine/ serine amino acid residues, is the most frequent location of cancer-related CTNNB1 mutations (Dar et al, 2017). The level of free b-catenin in the cytoplasmic pool is regulated by ubiquitination and proteasomal degradation (Akyol et al, 2019). b-catenin is a member of the Wnt signaling cascade and is associated with cadherin-mediated cell-cell adhesion systems (Woenckhaus et al, 2008).…”

Clinicopathological and molecular analyses of linearly expanded epithelial cells with β‐catenin alterations, “β‐catenin signature”, in the normal fallopian tube

“…15,16 Hence, accumulation of clinicopathological evidences regarding the mutant cells and their localisation in normal epithelium is essential. However, it is currently difficult to detect mutant cells histopathologically in situ, because mutations leading to the expression of aberrant proteins that can be detected immunohistochemically are limited for genes such as TP53, 17 CTNNB1, 18,19 ARID1A, 20,21 SMARCB1, 22 SMARCA4, 23 IDH1, 24 and BRAF. 25 Clonal expansion of epithelial cells harbouring oncogenic mutations in the female genital tract was documented prior to the NGS era.…”

Response to: An immunohistochemical approach to detect oncogenic CTNNB1 mutations in primary neoplastic tissues

“…In a recent issue of Laboratory Investigation, Akyol et al describe an immunohistochemical approach to detect oncogenic CTNNB1 mutations in primary neoplastic tissues [1]. The β-catenin signaling pathway is one of the most commonly deregulated pathways among various types of cancers [2].…”