IHC is a standard method used in the diagnosis of breast cancer and research into breast cancer pathology. These markers are used to determine different breast cancer types, eg, in situ or invasive carcinoma, distinguishing normal breast cell types, eg, luminal, basal, and myoepithelial, and also proliferation and disease progression.

Here, we look at some of the most common primary IHC markers for breast cancer and some cell-type-specific biomarkers. We also recommend specific IHC antibodies for each biomarker.

ALDH1A1

Aldehyde dehydrogenase 1 family member A1 (ALDH1A1) has been identified as a putative cancer stem cell (CSC) marker in breast cancer¹.

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SLFN11

Schlafen 11 (SLFN11) is emerging as an important regulator of cellular response to DNA damage. Preclinical and emerging clinical trial data suggest that SLFN11 is a predictive biomarker of response to a wide range of therapeutics that cause DNA damage, raising exciting possibilities for its clinical application².

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Estrogen receptor alpha

Alternative names ER-a

Determining the distribution of estrogen receptor alpha (ER-α) in breast cancer samples is an important initial step for the diagnosis and treatment evaluation of the disease. Approximately 70% of breast cancer samples will give a positive staining signal for ER-α, making it a crucial biomarker for breast cancer diagnosis. ER-α is a nuclear protein with a ligand-dependent transcription factor function. It is also most commonly detected in both luminal A and B subtypes of breast cancer.

Figure 1. Immunohistochemical staining of paraffin-embedded human breast carcinoma tissue with ab32063 at a dilution of 1/5000. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP Polymer). The sample is counterstained with hematoxylin. Antigen retrieval was heat-mediated using ab93684 (Tris/EDTA buffer, pH 9.0).

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Progesterone receptor

Alternative names PR

The progesterone receptor (PR) is another biomarker important for the initial diagnosis and evaluation of breast cancer. PR is known to be induced by ER-α and plays a key role in ER-α protein regulation. PGR as a biomarker is therefore commonly used as an indicator of ER-α function. PR is highly expressed in luminal A-type breast cancer tissue and is associated with a good prognosis^3-5.

IHC stain (marker localization): Nuclear

Figure 2. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast cancer tissue sections labeling progesterone receptor with ab101688 at 1/400 dilution. Heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 minutes. Rabbit-specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Nuclear staining on human breast cancer tissue, performed on a Leica Biosystems BOND^® RX instrument.

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ErbB2/HER2

Overexpression of ErbB2/HER2 can be found in 20–30% of breast cancer tumors and is commonly found in more aggressive types of the disease. ErbB2/HER2 is used as a diagnostic indicator for the FDA-approved monoclonal antibody therapy, Trastuzumab (Herceptin). During this therapy, ErbB2/HER2 acts as the binding target of Trastuzumab^6-7.

IHC stain (marker localization): cytoplasmic, nuclear, and strongly in the cell membrane

Figure 3. Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling ErbB 2 with ab214275 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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p53

Mutated versions of the tumor suppressor p53 can be found in 80% of triple-negative breast cancer (TNBC) cases, ie, tumors lacking expression of ER, PR, and ErbB2/HER2. This makes mutant p53 an excellent biomarker for TNBC, one of the more difficult breast cancers to treat, as it will not respond to endocrine or anti-HER2 treatments. Currently, there are mixed findings on the prognostic potential of p53 expression in breast cancer. Some studies have associated it with a positive prognostic outcome, and others with a negative outcome. p53 IHC stains are commonly used for diagnostic purposes^8,9.

Figure 4. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrium cancer tissue sections labeling p53 with purified ab32389 at 1/5000 dilution (0.09 µg/mL).

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BRCA1

Mutations in the BRCA genes are some of the most well-known breast cancer-associated protein mutations. BRCA1 is a tumor suppressor gene and one of the most commonly mutated genes in breast cancer. Patients with a BRCA1 mutation have a 40–80% increased risk of developing the disease, making it a crucial biomarker in the diagnosis and prognosis of many breast cancer types¹⁰.

IHC stain (marker localization): Nuclear signal

Figure 5. Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling BRCA1 with ab213929 at 1/400 dilution, followed by rabbit-specific IHC polymer detection kit HRP/DAB (ab209101).

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EGFR

EGFR is a transmembrane receptor, and its expression is frequently found in TNBC and inflammatory breast cancer (IBC). It is most commonly associated with a poor prognosis. High levels of EGFR expression are linked to an increased ability of breast cancer to undergo metastasis, making it a common biomarker for aggressive breast cancer types^11,12.

IHC stain (marker localization): Cytoplasm and strong nuclear

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CK7 (Cytokeratin 7) and CK20 (Cytokeratin 20)

CK7 and CK20 are cytokeratins expressed in breast epithelia. Different CK7 and CK20 IHC expression patterns are commonly used to distinguish many carcinoma types, including breast carcinomas. Most breast cancers are CK7 positive and CK20 negative, making the combination of these cytokeratins an excellent biomarker combination. Approximately 80% of breast adenocarcinomas are CK7 positive and CK20 negative

IHC stain (marker localization) for CK7 and CK20:  Cytoplasm

Figure 6. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections analysis of human breast carcinoma tissue labeling Cytokeratin 7 with unpurified ab68459.

Figure 7. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 20 antibody [EPR1622Y] - Cytoskeleton Marker (ab76126)

CK7 and CK20 are cytokeratins expressed in breast epithelia. Different CK7 and CK20 IHC expression patterns are commonly used to distinguish many carcinoma types, including breast carcinomas. Most breast cancers are CK7 positive and CK20 negative, making the combination of these cytokeratins an excellent biomarker combination. Approximately 80% of breast adenocarcinomas are CK7 positive and CK20 negative

IHC stain (marker localization) for CK7 and CK20:  Cytoplasm

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Cyclin D1

Cyclin D1 is one of the main regulatory proteins of the cell cycle, promoting the cell cycle progression from G1 to S phase. Mutations in cyclin D1 are amplified in many different types of cancer, including many breast cancers. 50% of breast cancers will contain an overexpression of cyclin D1. It has been used as a biomarker for poor prognosis for many breast cancer types, but some studies are beginning to suggest that it could also suggest a positive outcome if expressed in some luminal subtypes of breast cancer^13,14.

IHC stain (marker localization): Nuclear

Figure 8. Immunohistochemical staining of paraffin-embedded human endometrial adenocarcinoma with purified ab134175 at a dilution of 1/100. An HRP goat anti-rabbit (ab97051) was used as the secondary antibody at a dilution of 1/500, and the sample was counterstained with hematoxylin.

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Ki67

Ki67 is an important protein involved in cell division and is commonly used as a marker of cellular proliferation. It is also used in breast cancer diagnosis to determine the level of cell proliferation, which can be a prognostic marker and a good indication of how well certain breast cancers will respond to endocrine therapy¹⁵.

IHC stain (marker localization):  Nuclear

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Cyclooxygenase-2 (COX-2)

COX-2 is an enzyme responsible for producing prostanoids. Its expression in breast tissue correlates strongly with breast cancer development. Overexpression of COX-2 has been shown to drive breast cancer phenotypes, and blocking the action of this protein also indicates potential for breast cancer therapy. There is also a strong link between COX-2 overexpression in breast adipose tissue and cancer progression linked to obesity¹⁶.

IHC stain (marker localization):  Cytoplasm

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Caveolin-1

Caveolin-1 is the main component of caveolae, small invaginations found in the cell membrane. It also plays a key role in cell proliferation, invasion, and breast cancer metastasis. It is also used as a biomarker for breast cancer treatments and disease outcomes. High expression of caveolin-1 is a sign of poor prognosis and indicates more aggressive metastatic breast cancer¹⁷.

IHC stain (marker localization): Cell membrane

Figure 9. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab32577)

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FOXA1

The transcription factor FOXA1 has been shown to have a unique distribution within breast cancer cells compared to other cell types. FOXA1 binding is crucial for chromatin opening and the transcriptional activation of ER-α-responsive genes within breast cancer cells. Studies have identified thousands of breast cancer-associated single-nucleotide polymorphisms (SNPs) within the enhancer regions for FOXA1^18,19.

IHC stain (marker localization):  Nuclear signal

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GATA-binding protein 3 (GATA3)

GATA3 is a zinc finger transcription factor crucial for breast luminal epithelium differentiation. It is also a diagnostic biomarker for both primary and metastatic breast cancer and is commonly found in luminal A and B breast cancer tissue. GATA3 also has a strong association with HER2-positive and ER-α-positive hormone response in luminal breast cancers²⁰.

IHC stain (marker localization): Nuclear signal

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Cytokeratin 5 (CK5)

CK5 is a cytokine found to be expressed in the basal cell layer of the mammary duct. Tumors that arise from these cells are also known to be CK5 positive, so CK5 is an excellent biomarker for the diagnosis of basal-type breast cancers. The CK5-positive progenitor cells may also differentiate into glandular and myoepithelial cancer types.

IHC stain (marker localization): Cytoplasm and cell membrane

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Smooth muscle myosin heavy chain (SMMHC)

Alternative names SMMHC

SMMHC is explicitly expressed in the contractile myoepithelial cells of the breast. A loss of the myoepithelial layer is commonly associated with invasive breast cancers. Markers such as SMMHC can be used in IHC to detect this layer more reliably than a hematoxylin and an eosin stain alone²¹.

IHC stain (marker localization): Cytoplasm

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E-cadherin

E-cadherin is expressed at the cell junction of normal breast epithelial cells. A loss of E-cadherin is associated with a phenotypic switch by these cells to become invasive and migratory breast cancer cells. Loss of E-cadherin expression is commonly used as a biomarker for metastatic lobular breast carcinomas²².

IHC stain (marker localization):  Cell junctions

Figure 10. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

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Alpha smooth muscle actin (ACTA2)

Alternative names ACTA2

Smooth muscle actin is highly expressed in normal breast myoepithelial cells. It is commonly used as a marker of this normal breast cell type. Smooth muscle actin is commonly used for histology in conjunction with SMMHC and calponin to mark out the myoepithelial layer. A combination of markers such as this is much more reliable than using hematoxylin and eosin stains alone²³.

Image 11. IHC image of alpha-smooth muscle actin staining in a human breast ductal carcinoma formalin-fixed paraffin-embedded tissue section, performed on a Leica Bond™ system using the standard protocol F.

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Calponin

Calponins are proteins found within the contractile components of the myoepithelium. Similarly to SMMHC and ACTA2, calponin stains are used as a marker of the myoepithelial layer in the diagnosis of invasive breast cancers²⁴.

Figure 12. Formalin-fixed, paraffin-embedded human breast tissue stained for Calponin 1 using ab227661 at 1/100 dilution in immunohistochemical analysis.

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Delta-1-Catenin

Delta-1-Catenin is a member of the p120 family and acts as a binder of E-cadherin. Delta-1-Catenin can be used in histology to detect invasive lobular breast cancer, high tumor-node-metastasis stage, and lymph node metastasis. It is also strongly associated with HER2-positive breast cancers and can be used as an indicator of poor prognosis, as it is thought to promote a malignant phenotype²⁵.

Figure 13. Formalin-fixed, paraffin-embedded human breast carcinoma tissue stained for delta 1 Catenin/CAS using ab227638 at 1/100 dilution in immunohistochemical analysis.

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Cytokeratin 14 (CK14)

Alternative names: Cytokeratin 14

CK14 is a cytokine found in the basal cell layer of the mammary duct. It is most commonly found co-expressing with CK5 in this tissue type. Similarly to CK5, the histological detection of CK14 is an excellent biomarker for the diagnosis of basal-type breast cancers²⁶.

Figure 14: Immunohistochemical analysis of paraffin-embedded human squamous cell carcinoma of cervix tissue labeling Cytokeratin 14 with ab181595 at 1/2000 dilution, followed by prediluted HRP polymer for Rabbit/Mouse IgG.

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References

1. Liu, Y. et al. ALDH1A1 expression correlates with clinicopathologic features and poor prognosis of breast cancer patients: a systematic review and meta-analysis.  BMC Cancer   14, 444 (2014).
2. Zhang, B. et al. A wake-up call for cancer DNA damage: the role of Schlafen 11 (SLFN11) across multiple cancers.  Br. J. Cancer   125, 1333–1340 (2021).
3. Hicks, D. et al. Immunohistochemical performance of estrogen and progesterone receptor antibodies on the Dako Omnis staining platform: evaluation in multicenter studies.  Appl. Immunohistochem. Mol. Morphol.   25, 313–319 (2017).
4. Lim, E., Palmieri, C. & Tilley, W. D. Renewed interest in the progesterone receptor in breast cancer.  Br. J. Cancer   115, 909–911 (2016).
5. Mohammed, H. et al. Progesterone receptor modulates ERα action in breast cancer.  Nature   523, 313–317 (2015).
6. Mitri, Z., Constantine, T. & O’Regan, R. The HER2 receptor in breast cancer: pathophysiology, clinical use, and new advances in therapy.  Chemother. Res. Pract.   2012, 1–7 (2012).
7. Piccart-Gebhart, M. J. & Gelber, R. D. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.  N. Engl. J. Med.   353, (2005).
8. Duffy, M. J., Synnott, N. C. & Crown, J. Mutant p53 in breast cancer: potential as a therapeutic target and biomarker.  Breast Cancer Res. Treat.   170, 213–219 (2018).
9. Li, J. et al. Association of p53 expression with poor prognosis in patients with triple-negative breast invasive ductal carcinoma.  Medicine   98, (2019).
10. Fackenthal, J. D. & Olopade, O. I. Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations.  Nat. Rev. Cancer   7, 937–948 (2007).
11. Masuda, H. et al. Role of epidermal growth factor receptor in breast cancer.  Breast Cancer Res. Treat.   136, 331–345 (2012).
12. Ali, R. & Wendt, M. K. The paradoxical functions of EGFR during breast cancer progression.  Signal Transduct. Target. Ther.   2, 1–7 (2017).
13. Mohammadizadeh, F. et al. Role of cyclin D1 in breast carcinoma.  J. Res. Med. Sci.   18, 1021–1025 (2013).
14. Ortiz, A. B. et al. Prognostic significance of cyclin D1 protein expression and gene amplification in invasive breast carcinoma.  PLoS ONE   12, 1–13 (2017).
15. Niazi, M. K. K. et al. Relationship between the Ki67 index and its area-based approximation in breast cancer.  BMC Cancer   18, 1–9 (2018).
16. Harris, R. E., Casto, B. C. & Harris, Z. M. Cyclooxygenase-2 and the inflammogenesis of breast cancer.  World J. Clin. Oncol.   5, 677–692 (2014).
17. Qian, X. L. et al. Caveolin-1: a multifaceted driver of breast cancer progression and its application in clinical treatment.  OncoTargets Ther.   12, 1539–1552 (2019).
18. Cowper-Sallari, R. et al. Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression.  Nat. Genet.   44, 1191–1198 (2012).
19. Meyer, K. B. & Carroll, J. S. FOXA1 and breast cancer risk.  Nat. Genet.   44, 1176–1177 (2012).
20. Shaoxian, T. et al. Characterisation of GATA3 expression in invasive breast cancer: differences in histological subtypes and immunohistochemically defined molecular subtypes.  J. Clin. Pathol.   70, 926–934 (2017).
21. Zaha, D. C. Significance of immunohistochemistry in breast cancer.  World J. Clin. Oncol.   5, 382–392 (2014).
22. Singhai, R. et al. E-Cadherin as a diagnostic biomarker in breast cancer.  N. Am. J. Med. Sci.   3, 227–233 (2011).
23. Zhang, D., Zhang, J. Y. & Wang, E. H. Δ-Catenin promotes the malignant phenotype in breast cancer.  Tumor Biol.   36, 569–575 (2015).
24. Laakso, M. et al. Cytokeratin 5/14-positive breast cancer: true basal phenotype confined to BRCA1 tumors.  Mod. Pathol.   18, 1321–1328 (2005).