IHC markers are used in both clinical and research settings to provide insights into a variety of processes that are of interest to breast cancer and disease research at large. Breast cancer, the most common malignancy in women worldwide, is classified by using microscopic morphologic criteria along with standard clinical immunohistochemistry markers for HER2 and ER/PR in order to predict if a patient’s tumor will respond to Herceptin or hormone therapy. IHC is further useful for determining rates of cell differentiation, elucidating molecular pathways, and highlighting the proteomics of tumor growth and metastatic potential. For example, IHC markers help distinguish in-situ from invasive carcinomas (KRT14 and KRT5), subtype ductal from lobular carcinomas (E-cadherin, p120, FOXA1, GATA3), determine mammary origin of a metastatic carcinoma (CK7, CK20, Mammaglobin A), and highlight tumor proliferation and apoptosis (Ki-67, BCL2, TP53). Furthermore, the myoepithelial markers SMA (Smooth Muscle Actin), Calponin, p63 and SMMHC can be used to determine whether or not a cancer has invaded, since benign and early lesions have an intact myoepithelial layer surrounding breast glands.
Targeting pathways through IHC:
IHC markers can be used to identify the deregulation or pathogenic activation of various cell-cycle pathways in breast cancer. These include BCL2, an anti-apoptotic protein which promotes immortality in cells, and TP53, which normally promotes apoptosis but is defective and easier to detect with IHC when mutated. Markers can also be useful for studying angiogenesis within tumors, examining proteins with a potential impact on tumor growth and survival such as CD31, CD34 and VEGFA.
Advanced and metastatic tumors:
SIAH2 and EGFR overexpression are predictors of advanced stage and metastatic potential in breast cancers, and antibodies to these targets have been found to perform well in metastatic breast carcinomas (Van Reesema, 2016). Additional useful targets include GATA3, which sees very strong and diffuse staining in breast metastases (Sangoi, 2017), as well as GCDFP-15/PIP.
TNBC:
For triple negative (ER, PR, HER2-negative) breast cancers with elevated MYC expression, PIM1 inhibitors that target PIM1 kinase (a protein that regulates cell death and tumorigenesis) are being studied as potential therapeutic agents (Brasó-Maristany, 2016; Zhao, 2017). Another useful IHC marker for TNBC is the ΔNp63 isoform of transcription factor TP63, a protein involved in regulating cell adhesion that is regularly overexpressed in triple negative tumors (Nekulova, 2016).
The Genetics of Breast Cancer:
For the 10% of patients with hereditary breast cancer syndromes, mutations have been identified within predisposing genes including BRCA1, BRCA2, PTEN, TP53, CDH1, GATA3 and STK11 (Gaynor, 2013; Nik-Zainal, 2017; Balmaña, 2009), all highly penetrant genes. BRCA1 and BRCA2 are repair enzymes involved in double-stranded DNA break repair, and mutations in these genes lead to genomic instability and increased rates of variation and are associated with a high risk of developing breast, ovarian, and fallopian tube cancers (Genetics of Breast Cancer: A Topic in Evolution, Annals of Oncology 26:1291, 2015).
In 2-3% of cases, mutations are found in moderate penetrance genes such as CHEK2, BRIP1, ATM, and PALB2 (Ripperger, 2008). Although these variations are often not detectable by IHC and require sequencing, proteins to amplified or overexpressed oncogenes can be detected using antibodies. Oncogenes such as MYC and tumor suppressors that are knocked out during tumorigenesis can also be confirmed by IHC.
BRCAness:
A subset of patients with breast cancers that are considered to be sporadic have tumors with dysfunctional or absent BRCA1 or BRCA2 but lack inherited mutations in these genes. The progression of their disease is similar to those with germline variants in these proteins, but the mechanisms of selective inactivation and somatic mutation are less clear and have been described as the BRCAness phenotype. Recent evidence suggests that inherited polymorphisms and epigenetic silencing in genes that interact with the BRCA repair genes, specifically PALB2 and RAD51C, are correlated with the same mutational signature and may result in BRCAness phenotype tumors in some patients (Polak, 2017; Foo, 2017).
APOBEC Mutation Signatures in Breast Cancer:
In a large cohort of breast cancers, localized hyper-mutation signatures have been detected that are attributed to the potential dysfunction of repair genes including polymerases and also enzymes in the APOBEC family (Nik-Zainal, 2012). This process of mutation, named “kataegis” by Nik-Zainal et al, has been described as regional clusters of mutations that exist in great abundance and within very close proximity to each other. Importantly, the same type of mutation, C>T or G>A, occurs multiple times typically in specific trinucleotides in the same immediate region on the same chromosomal strand, and these repetitive, successive variations seem to indicate a directed and tracked process of mutagenesis (Morganella, 2016). Nik-Zainal et al found that among these signatures, some (labeled 2 and 13) were present in up to 50% of tumors examined across more than 500 breast samples (Nik-Zainal, 2016; Nik-Zainal, 2012). Incidentally, they have also been found in other cancers such as lung adenocarcinoma (Campbell, 2016). The APOBEC family of cytidine deaminases has been proposed as one potential source of such localized, pathogenic hypermutation due to the specific function of these proteins in somatic hypermutation for antibody production in concert with AICDA (AID) (McBride, 2008; Hwang, 2015). A study in yeast has found distinct similarities between APOBEC function and kataegis mutation clusters (Lada, 2012). Furthermore, Kanu et al found that APOBEC3B is activated in tumors with loss of the PTEN tumor suppressor and amplification of transcription factor ERBB2. These events lead to DNA replication stress and chromosomal instability and consequential upregulation of APOBEC3B. The high rate of APOBEC mutation signature in these tumors implies that its upregulation may act as an important potential source of driving mutagenesis (Kanu, 2016). This connection has also been seen in work examining APOBEC3A and APOBEC3B upregulation, inherited mutation, the frequent spontaneous deletion of the gene FHIT and risk for breast cancer (Middlebrooks, 2016; Periyasamy, 2015; Volinia, 2017; Burns, 2013).
MMR Defects and Familial Risk for Breast Cancer:
A study from 2018 showed an important link between known pathogenic mutations in well characterized Lynch Syndrome progenitor genes MSH6 and PMS2 and breast carcinogenesis. Roberts et al show that the same variants in these mismatch repair genes that are known to cause microsatellite instability and cancer in Lynch Syndrome patients are associated with a two to three fold increased risk for breast cancer and may constitute an inherited basis for developing the disease (Roberts, 2017).
