Abstract
Breast cancer (BC) remains the most prevalent malignancy among women worldwide, with incidence and mortality rates varying across regions due to disparities in screening, diagnosis, and treatment accessibility. Molecular classification has redefined BC management, categorizing tumors into luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and triple-negative breast cancer (TNBC), each with distinct prognostic and therapeutic implications. Advances in tumor microenvironment (TME) research highlight its critical role in cancer progression, immune evasion, and treatment resistance, underscoring the importance of tumor-infiltrating lymphocytes, natural killer cells, and macrophages in shaping BC outcomes. Traditional therapies, including chemotherapy, endocrine therapy, and targeted treatments such as cyclin-dependent kinase 4 and 6 (CDK4/6) and phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K) inhibitors, continue to evolve to overcome resistance mechanisms. Immunotherapy, particularly immune checkpoint inhibitors targeting programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), has demonstrated clinical benefits, especially in TNBC. Additionally, novel strategies, including chimeric antigen receptor T-cell therapy and cancer vaccines, are emerging as promising therapeutic avenues. This review provides a comprehensive analysis of BC epidemiology, molecular subtypes, TME interactions, and cutting-edge therapeutic strategies, emphasizing the need for personalized, immune-based approaches to enhance treatment efficacy and patient outcomes.
- Breast cancer
- human epidermal growth factor receptor 2 (HER2)
- luminal A
- luminal B
- triple-negative breast cancer (TNBC)
- review
Introduction
In 2022, worldwide statistics recorded approximately 2.3 million new cases of breast cancer (BC) among women, making it the most commonly detected cancer in 17 regions and the leading cause of death in 14 (1). GLOBOCAN 2022 reported that high-income countries have the highest incidence rates of female BC, accounting for 25.4% compared to low-income countries (24.5%), this might be due to early detection and advanced screening programs which increase the number of newly diagnosed BC cases in high-income countries (1). However, the mortality rates are significantly higher in low-income countries (19.4%) compared to high-income countries (14.7%), owing to delays in diagnosis and limited access to high-quality treatments (1). The Eastern Mediterranean Region, classified by the World Health Organization, includes 21 member states in the Middle East, North Africa, and Central Asia. The Middle East comprises 15 countries with an estimated population of over 411 million (2). The incidence and mortality rates of female BC in the Middle East are significantly increasing, accounting for about 31.2% of all new cancer cases. In 2022, BC was the most common cancer among females in the region, with around 140,021 cases and 49,178 deaths (1). These rising numbers highlight the critical need for heightened cancer awareness, improved screening programs, access to advanced diagnostic modalities, and effective personalized treatments.
Molecular Classification of Breast Cancer
BC is a highly heterogeneous disease that is classified into four main molecular subtypes varying in prognosis and clinical presentation, these are Luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and triple-negative breast cancer (TNBC). The molecular classification of BC is based on the expression of hormone receptors (HR) estrogen receptor (ER) and progesterone receptor (PR), HER2, and the cell proliferation marker (Ki-67) using immunohistochemistry (IHC) and complementary DNA microarray-based gene-expression profiling (3-5). The survival rate of patients with BC can vary depending on the molecular subtype and treatment strategy. However, The National Cancer Institute reported that women diagnosed with luminal A subtype have the best survival rate at 94.4%, followed by those with luminal B subtype with 90.7%. Lower survival rates were seen in women having HER2 subtype at 84.8%, followed by TNBC subtype with the worst survival rate at 77.1% (6).
Luminal A subtype. Luminal A is the most prevalent BC subtype accounting for around 40% of BC and is associated with early-stage presentation. It is defined as HR+, HER2−, and Ki-67low based on the overexpression of HR (ER with/without PR), the absence of HER2 expression, and the low expression of the cell proliferation marker Ki-67 (7). On the clinical level, luminal A tumors are well-differentiated and have controlled cell growth, low histological grade, and the best prognosis. It is also characterized by fewer episodes of relapse and higher survival rates (Figure 1) (3).
Molecular subtypes of breast cancer including hormone expression, prognosis, and therapeutic approaches. BRCA1/2: BRCA DNA repair-associated 1/2 gene; ER: estrogen receptor; HER2: human epidermal growth factor receptor 2; FISH: fluorescence in situ hybridization; IHC: immunohistochemistry; Ki-67: cell proliferation marker; PR: progesterone receptor.
Luminal B subtype. Luminal B is less common and represents 10-20% of BC and is associated with early-stage presentation. These tumors are defined as HR+/HER2+ and HR+/HER2−, Ki-67high (8). Unlike luminal A tumors, cancer cells in this subtype are either HER2+ or HER2− and are highly proliferative as indicated by high levels of Ki-67 marker (9). Clinically, they grow and divide faster, resulting in worse prognosis compared to luminal A tumors. The expression of Ki-67 can be used to distinguish between luminal cancer as well as assessing the aggressiveness of tumors (Figure 1) (9, 10).
HER2-enriched subtype. HER2+ cancer makes up 10-15% of BC. These tumors are characterized by overexpression/amplification of HER2 with the absence of both ER and PR, and thus defined as HR−, HER2+ (11). Notably, a new advanced classification of HER2+ BC has evolved to better identify cases: HER2low, HER2ultralow, and HER2null subtypes (12). They are characterized by expressing low levels of HER2 protein compared to classic HER2+ BC. If the IHC result is 0 with no membranous staining, the cancer is considered HER2null and will not respond to HER2-targeted treatments. If the IHC result is 0 with membranous staining in 0-10% of tumor cells, it is classified as HER2ultralow, and the cancer may respond to antibody–drug conjugates. Moreover, tumors with IHC results of 1+ or 2+ with a negative HER2 fluorescent in situ hybridization are considered HER2low and may also respond to antibody–drug conjugates. Finally, those with IHC 2+ and positive HER2 fluorescent in situ hybridization or IHC 3+ are HER2+ and are typically treated with HER2-targeted drugs (13, 14). HER2+ BC are fast-growing tumors that exhibit worse prognosis (Figure 1). However, the use of HER2-targeted therapy was shown to improve prognosis (3).
Triple-negative breast cancer. The TNBC subtype is defined as HR−/HER2− and is negative for all three hormone receptors, making it unresponsive to hormonal and targeted therapies. TNBC is the most aggressive subtype and is associated with the worst prognosis, accounting for 15-20% of all cases (Figure 1). In addition, women with TNBC are at risk of relapsing within the first 3 years of diagnosis, which decreases after 5 years. It was reported that some subtypes of TNBC might be associated with genetic mutations [tumor-suppressor genes BRCA DNA repair-associated 1 (BRCA1) and BRCA2] (15). Compared to the luminal and HER2-enriched breast subtypes, TNBC tends to metastasize to the lungs and brain more likely than to the bones. Histologically, TNBC cells are poorly differentiated and exhibit heterogeneous histopathological features (8).
New advancements in gene-expression profiling have been employed to further classify TNBC into six distinct subtypes, each with a unique behavior: basal-like immune-suppressed (BLIS), basal-like immune-activated (BLIA), luminal androgen receptor (LAR), immunomodulatory, mesenchymal, and mesenchymal stem-like. A subtype expressing the basal marker (CK5/6) is defined as a basal-like subtype (16). Cancer in the basal-like subtypes often exhibit chromosomal rearrangements and BRCA mutations. The BLIS subtype makes up 17.9% of all TNBC. It is particularly characterized by enriched cell-cycle response genes, activated DNA-repair mechanisms such as ataxia telangiectasia and RAD3-related protein, and a lack of immune cell activation, making it sensitive to DNA damage-inducing agents. BLIA, on the other hand, are less common constituting 11.1% of TNBC, and they express high levels of growth factor receptors (epidermal growth factor receptor, insulin-like growth factor 1 receptor, and myoepithelial markers) and have poor responses to chemotherapy (8, 17, 18). The LAR subtype represents 9.2% of all TNBC cases and is distinguished by the overexpression of androgen receptor which justifies its high response to androgen receptor antagonists. In comparison to other TNBC subtypes, the LAR subtype has a gene-expression pattern similar to that of luminal cancer. Additionally, the LAR subtype was found to be associated with mutations in the phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K) pathway, making PI3K inhibitors as well as inhibitors of androgen receptor effective treatments (19). Immunomodulatory tumors are greatly enriched with immune cells and cytokine signaling pathways along with high levels of tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment (TME) which makes immune checkpoint inhibitors (ICIs) a promising therapy (18). In fact, the immunomodulatory subtype accounts for 21.5% of TNBC (8). Lastly, both mesenchymal and mesenchymal stem-like tumors are characterized by epithelial–mesenchymal transition, and cell motility signaling pathways and are associated with PI3KCA-activating mutations. The mesenchymal subtype is considered to be more common, accounting for 20.8% compared with the mesenchymal stem-like subtype, which accounts for just 6.5% of all TNBC (Figure 1) (8). It has been shown that dasatinib (ABL/SRC inhibitor) and PI3K/mechanistic target of rapamycin kinase (mTOR) inhibitors are effective against these cancer types (17, 20).
The Tumor Microenvironment
The TME of BC is a highly complex network of cellular and non-cellular components that regulates cancer progression, metastasis, immune escape, and response to anticancer treatments. The TME consists of different components: cancer cells, stromal cells (fibroblasts and endothelial cells), tumor-infiltrating immune cells [T- and B-lymphocytes, natural killer (NK) cells, macrophages and dendritic cells], and the extracellular matrix. Understanding this complex dynamic environment and the interplay between immune cells and TME is crucial for enhancing the effectiveness of therapies (21). This section will focus on the interplay between immune cells and tumor cells within the TME and their implications in tumor suppression and prognosis.
The abundance and presence of different intratumoral immune cells vary between BC subtypes, resulting in heterogeneity which can affect the spread of the tumors and treatment resistance (22, 23). In line with this, a study by Denkert et al. reported an increase in immune cell infiltration in patients with different BC subtypes and treated with neoadjuvant chemotherapy (24). Nevertheless, it was positively associated with better survival and prognosis in TNBC and luminal HER2+ subtypes and negatively associated with increased TIL infiltration in luminal HER2− subtypes. This study illustrates how the composition of the tumor-immune microenvironment influences the clinical outcomes of BC.
Among TILs, CD4+ and CD8+ T-cells have been considered central players in shaping and maintaining immunity in BC. A study by Shohdy et al. found that CD4+ T cells were positively associated with better survival in patients with BC (25). In addition, Gu-Trantien et al. reported that follicular helper T-cells had better prognostic effects and response to chemotherapy in patients with BC (26). On the other hand, Huang et al. found that CD4+ Th17 and Treg cells exhibited negative prognostic effects on BC survival (27). Other studies have also focused on the role of CD8+ T-cells as key players in tumor immunity. Oshi et al. demonstrated that high levels of infiltrating CD8+ T-cells with increased frequency of tumor CD4+ memory T-cells, results in improved survival rates in patients with TNBC (28). Likewise, another report suggested the positive role of tissue resident CD8+ T-cells within the TME in improving survival in BC (29). Further in vivo study by Dobrzanski et al. emphasized on the role of type 1 CD8+ T-cells in suppressing tumor growth and promoting and activating the function of endogenous CD4+ and CD8+ T-cells in the TME (30). Previous research has highlighted the impact of T-cells on the TME, clinical presentation of BC, and the development of targeted therapies. NK cells have also been extensively studied in cancer due to their ability in recognizing aberrant surface markers such as epidermal growth factor receptor on the surface of cancer cells and attacking cancer cells downregulating major histocompatibility complex class I molecules (31, 32). NK cells recognize their target cells using different inhibitory and activating receptors that are critical players in immunosurveillance. They also produce several cytokines and chemokines such as interleukins 2, 12, 21, 15 and 18; tumor necrosis factor-α; interferon-γ; CXC chemokine receptor 3; and granulocyte-macrophage colony-stimulating factor, which attack and eliminate target cells such as cancerous cells thereby promoting antitumor immunity (33-35). Despite the capabilities of NK cells to recognize and eliminate tumor cells, it was found that TME components can suppress and impair the cytotoxic effects of NK cells. The review by Ran et al. discussed the mechanism by which an immunosuppressive TME inhibits the effector functions of NK cells (32). Generally, the TME components downregulate the expression of NK cell-activating receptors, including natural killer cell protein 80 (NKp80/KLRF1), natural killer cell protein 30 (NKp30/NCR3), DNAX accessory molecule-1 (DNAM-1/CD226), and Fc gamma receptor III (CD16/FcγRIII), reducing NK cell responsiveness. In addition, the release of transforming growth factor-β, interleukin 10, indoleamine 2,3-dioxygenase, and prostaglandin E2 by tumor cells, fibroblasts, and regulatory T-cells can also hinder the effector functions of NK cells against tumor cells (32).
Endothelial cells constitute an essential part of the TME as they facilitate cancer angiogenesis which in turn allow cancer to spread to secondary sites. It was demonstrated that BC cells transfected with angiogenic stimulatory peptides had rapid growth and gained metastatic potential (36). It was also reported that the presence of fibroblasts within TME plays an important role in the development of tumors by releasing structural proteins such as elastin and collagen type I-V, which contribute to the formation of basal membranes (37), induce inflammation (38), and stimulate angiogenesis of tumors (39). Furthermore, fibroblasts contribute to BC development by releasing stromal cell-derived factor 1(SDF-1/CXCL12) which promote proliferation, migration, immune evasion, and survival of BC cells (40). In BC, tumor-associated macrophages are one type of TIL found in abundance within the TME and were shown to play opposite roles in BC. Tumor-associated macrophages differentiate into M1 and M2 phenotypes in response to tumor-derived cytokines. M1 macrophages exhibit pro-inflammatory and antitumor activity while M2 macrophages mediate an anti-inflammatory activity and act as pro-tumoral macrophages that support tumor growth (41). Furthermore, there is evidence that tumor-associated macrophages promote the metastasis of breast tumors and stimulate their proliferation by producing interleukin 10 (IL-10) and epidermal growth factor (EGF) (42, 43).
The latest research findings on the interplay between tumor cells and the TME components strengthen our understanding of their significant role in cancer immunity. Earlier findings emphasize the need for improving BC immunity through the development and advancement of ICIs.
Current Therapies of Breast Cancer
Several BC treatment approaches have been developed to improve survival rates and prognosis, in addition to prevent relapses and metastases. Besides local treatment options (such as radiotherapy and surgery), systemic monotherapies including chemotherapy, hormone therapy, targeted therapy, or immunotherapy have been significantly effective in treating BC patients. However, the overall mortality rate remains relatively high because of metastasis and the fact that some cancer subtypes, such as TNBC, are resistant to treatment. In light of this, new targeted and effective therapies are essential for treating the disease in combination with existing treatments (44). Previous studies and clinical trials have shown that monotherapies are more effective, tend to be less toxic, and provide a better risk-benefit profile compared with chemotherapy and radiation therapy. Before discussing immunotherapies and the latest advances in this topic, we will give a quick review of the most widely used monotherapies in the treatment of different BC subtypes.
Chemotherapy. A combination of chemotherapy agents has been developed for treating different BC subtypes. Anthracyclines (doxorubicin and epirubicin) and taxanes (paclitaxel and docetaxel) are effective chemotherapeutic regimens considered the backbone of chemotherapy. Paclitaxel and docetaxel work by inhibiting cell division, and they have been significantly effective in the treatment of early and advanced stages of BC (45, 46). Lagunes and Pezo reviewed the use of current chemotherapy regimens in metastatic TNBC and highlighted the importance of taking BRCA1/2 mutations into consideration when making treatment decisions (47). They emphasized that anthracyclines are the first treatment option for patients with metastatic TNBC who have not been on this treatment before. In comparison, taxanes are more effective in patients with metastatic TNBC who have been on anthracycline agents or have developed resistance to them. Considering BRCA1/2 mutations in metastatic TNBC, platinum-based targeted therapy is considered more beneficial compared to other agents (47). Furthermore, platinum agents, including cisplatin and carboplatin, have shown promising results, particularly in TNBC due to their role in inhibiting DNA synthesis and suppressing the division of cancer cells. Platinum-based therapy has been considered an alternative treatment strategy in patients with metastatic cancer showing resistance to anthracyclines or taxanes (48).
Anthracyclines such as doxorubicin and epirubicin exert antitumor activity by inhibiting the proliferation of cancer cells, resulting in apoptosis. They use different mechanisms to damage DNA in cancer cells, such as topoisomerase-II inhibition, and free radical formation (49). Despite their similar anticancer properties, doxorubicin was found to be associated with adverse effects such as cardiotoxicity in patients with BC (50).
Hormone/endocrine therapy. Approximately 70% of all BC cases are HR+ (ER+ and/or PR+), and, therefore, hormone or endocrine therapy targeting estrogen, and its signaling pathways are considered the cornerstone treatment for HR+ BC. These endocrine therapies are divided into three classes, namely aromatase inhibitors (AIs), selective estrogen receptor degraders (SERDs), and selective estrogen receptor modulators (SERMs) (44). SERDs are effective estrogen antagonists with no estrogenic effects. Fulvestrant is the most promising SERD that was approved by the U.S. Food and Drug Administration (FDA) for the treatment of ER+ BC. It primarily works by attaching and damaging ERs in cancer cells, which disrupts the ER activation function-1 (AF-1) and (AF-2) signaling pathways, resulting in ER degradation and inhibition of cancer cell proliferation (51, 52). SERMs such as tamoxifen, toremifene and raloxifene are widely used in ER+ BC treatment. Basically, all SERMs work at the receptor level by competing with estrogen for ERs, which blocks and inactivates estrogen–ER interaction. This stops estrogen supply and eventually inhibits breast tumors from growing and spreading to secondary tissues (53, 54). Furthermore, early clinical trials have demonstrated that tamoxifen is particularly effective in managing early and advanced stages of ER+ BC in premenopausal and postmenopausal women as already reviewed by Moyer and Brown (55). AIs (e.g., letrozole, anastrozole, and exemestane) function by blocking the enzyme aromatase, thereby preventing the biosynthesis of estrogen. This blockade results in a significant reduction in estrogen level, subsequently suppressing cancer cell growth and proliferation (56). In addition, a study reported that AIs can significantly lower circulating estrogen levels in postmenopausal women with BC, resulting in improved clinical outcomes and disease-free survival rates (57). Lower estrogen levels can have an indirect impact on the TME. Specifically, reduced estrogen production can cause changes in gene expression related to cell proliferation and survival, ultimately leading to apoptosis of cancer cells (58).
Previous studies have shown that resistance to endocrine therapies is rising and becoming more challenging in BC management. These resistance mechanisms include genetic mutations in estrogen receptor 1 (ESR1) and PI3KCA genes, as well as loss of ESR1. Activating alternative signaling pathways such as PI3K/AKT/mTOR, RAS/mitogen-activated protein kinase (MAPK), and CDK4/6-retinoblastoma protein (RB)-E2F (CDK4/6-RB-E2F) pathways have significantly contributed to endocrine therapy resistance. Thus, newer generations of novel anti-estrogen therapies have been developed to reduce resistance, side-effects, and improve efficacy. Additionally, combining endocrine therapy with targeted therapy was another treatment approach to overcome resistance challenges in BC management (59).
New approaches for endocrine therapy in ER+ breast cancer: The next generation of oral SERDs including camizestrant, giredestrant, accelerant, and imlunestrant are being investigated extensively for the treatment of early and metastatic BC (60, 61). Several clinical trials have shown show promising results for these new SERDs in overcoming resistance to previous endocrine therapies and were effective in improving progression-free survival (PFS) in patients with ER+ BC having ESR1 mutations (62). Lasofoxifene, a next-generation SERM, has also been considered a potential treatment for metastatic BC. Administration of lasofoxifene to in vivo ESR1-mutant models suppressed cancer progression at primary and distant sites (63). In addition, the ELAINE I clinical trial revealed that lasofoxifene improved the PFS in patients with BC having ESR1 mutations (64). Elacestrant is an oral SERM/SERD hybrid that was recently approved by the FDA for the treatment of advanced stages of ER+ and HER2− BC. It has shown superior effect associated with improved PFS compared with standard endocrine therapies (65).
Other mechanisms to overcome resistance to endocrine therapy are also being studied, notably proteolysis targeting chimerics (PROTACs), complete estrogen receptor antagonists (CERANs), selective estrogen receptor covalent antagonists (SERCAs), selective human ER partial agonists (ShERPA), and selective estrogen receptor covalent antagonists (ShERPA) (59, 66). PROTACs regulate the interaction between the target protein (e.g., ER) and E3 ubiquitin ligase, resulting in ubiquitination and proteasomal degradation of ER through the ubiquitin–proteasome system (67). On the other hand, CERANs causes degradation of ER and total blockade in its transcription pathways through the recruitment of nuclear receptor co-repressors which subsequently block the activation domains F1 and AF2 of ER signaling (68). SERCAs form a covalent bond at cysteine residues on ER, causing its inactivation and permanent inhibition of ER signaling. ShERPAs are another class of drugs that partially activate the ER to reduce cancer cell proliferation and maintain normal estrogen activity (59). According to previous research, ShERPAs is a good option for patients with BC showing resistance to traditional SERMs such as tamoxifen (69).
Targeted therapy. As BC treatment resistance continues to increase, oncology research has, in turn developed new therapies that selectively target oncogenic proteins and mutations to disrupt signaling pathways and inhibit cancer progression. Among these therapeutic agents, CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) target the cell-cycle machinery by inhibiting kinases 4 and 6, and preventing phosphorylation of retinoblastoma protein, resulting in cell-cycle arrest (70). Interestingly, CDK4/6 inhibitors have been shown to improve the PFS in patients with HER2+ and HER2− BC and were found to be most effective when combined with SERDs or AIs (71, 72). Likewise, PI3K inhibitors (alpelisib, pilaralisib)/AKT inhibitors (capivasertib)/mTOR inhibitors (everolimus), and dual PI3K/mTOR inhibitors (voxtalisib) act on specific components of PI3K/AKT/mTOR signaling pathways that regulate cell proliferation and tumorigenesis (73). Moreover, vascular endothelial growth factor (VEGF) inhibitors such as bevacizumab function by inhibiting the interaction between VEGF-A and VEGF receptors, leading to angiogenesis inhibition (74). Another targeted therapeutic agent includes inhibitors of poly (ADP-ribose) polymerase (olaparib, talazoparib, niraparib) induce DNA damage and apoptosis of cancer cells. In particular, olaparib has been shown to be effective in patients with BC harboring BRCA1/2 mutations (75).
Immunotherapy
BC is generally not considered immunogenic. However, the presence of TILs in TNBC and HER2+ BC subtypes, and their correlation with pathological complete response to therapeutic agents, highlights the critical role of the immune system in BC treatment (76, 77).
Immunotherapy has been proven to be a groundbreaking approach in the treatment of BC, particularly TNBC, since this type of cancer does not respond to endocrine therapy or HER2−-targeted therapy due to lack of ER, PR, and HER2 expression (44). Unlike other therapeutic approaches, immunotherapies are used to activate the patient’s own immune cells to attack cancer cells and inhibit their growth (44). Ongoing clinical trials (NCT01491737, NCT02734004, NCT02614794, NCT03036488, NCT02819518, NCT02425 891) evaluating the efficacy of various immunotherapies alone or in combination with other therapies in BC are showing promising results. However, the efficiency of immunotherapies relies greatly on several factors related to the TME, genetic and molecular characteristics, as well as previous treatments (44). For instance, a study by Sato et al. identified a gene signature consisting of 18 differentially expressed genes as an effective biomarker to identify patients with TNBC who will benefit from immunotherapy (78). Immunotherapies are classified based on their mechanisms of action into cytokine-based therapy, immune-checkpoint inhibitors, adoptive T-cell therapy and anticancer vaccines.
Several cytokines such as interleukins, interferons, and tumor necrosis factors have been utilized in cancer therapy in a large number of clinical trials [reviewed in (79)]. Cytokines have been shown to play an important role in different steps of cancer immunity, including cancer antigen presentation, activation and proliferation of T-cells, infiltration of T-cells into the TME, and apoptosis of cancer cells (79, 80). However, clinical trials revealed that the efficacy of cytokines in cancer therapy is limited due to their short half-life in the blood which require frequent doses and long-term therapeutic effect. Additionally, previous studies reported that administration of high doses of cytokines is frequently associated with adverse effects in cancer patients such as flu-like symptoms, cardiac abnormalities, musculoskeletal pain, gastrointestinal problems, and other adverse effects related to the immune system such as anaphylaxis and anemia (79).
Immune-checkpoint inhibitors (ICIs). Immune checkpoint proteins are expressed on immune cells and have immune-suppressive characteristics, leading to pro-tumorigenic effects. Different immune checkpoint molecules have been identified as therapeutic targets in cancer treatment. The most developed and used checkpoint proteins in various cancer treatment include programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) (81). Other emerging checkpoint proteins, including lymphocyte-activation gene 3 (LAG3), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), T-cell immunoglobulin and ITIM domain (TIGIT), and B7-H3, are under clinical investigation for the treatment of BC (82). ICIs work by preventing checkpoint proteins from binding with their respective ligands/receptors, thus maintaining T-cells in an active state and enabling them to effectively target and destroy tumor cells (83).
PD1/PD-L1 inhibitors. PD-L1 is expressed on both tumor and immune cells. Upon binding to PD1, activation of T-lymphocytes is inhibited, causing exhaustion of T-cells and consequently favoring the growth of malignant cells. Blocking or disrupting the interaction between PD1 and PD-L1 may result in reactivating T-cells, causing them to recognize and attack cancer cells, thus halting the growth of tumors (Figure 2A) (83). Tumor immunotherapy with PD1/PD-L1 inhibitors has shown clinical activity across multiple solid tumor types; however, not all types of BC have the same levels of PD1/PD-L1 expression, as it varies according to the tumor stage and molecular subtype (84). Based on IHC, TNBC has higher levels of PD-L1 expression when compared to HER2+ BC (84). In addition, it has been reported that patients with TNBC exhibit higher levels of PD-L1 expression, increased TILs, and a high tumor mutation burden, which are associated with better treatment response and prognosis (85, 86). In line with these studies, Alkhayyal et al. reported a higher percentage of TILs and PD-L1 expression in the TME of patients with TNBC compared to patients with other molecular subtypes. Additionally, there was a strong association between PD-L1 expression and the presence of TILs in the BC microenvironment, highlighting a possible role of PD-L1 in regulating the infiltration of lymphocytes within the BC microenvironment (87).
Overview of immunotherapeutic approaches in breast cancer management. (A) Restoring T-cell function and suppressing breast cancer tissue via programmed cell death protein 1 (PD1)/programmed cell death ligand 1 (PDL-1) inhibitors. (B) Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) inhibitors are effective in preventing T-cell deactivation and promoting cancer cell death. (C) Genetic modification of T-cells to express chimeric antigen receptors (CARs) increases the effectiveness of T-cells in targeting antigen-specific cancer cells in patients with breast cancer. (D) Development of peptide-based and DNA/mRNA-based vaccines stimulate the immune system to recognize and target tumor-specific antigens in patients with breast cancer. APC: Antigen-presenting cell; MHC: major histocompatibility complex; TCR: T-cell receptor.
PD1/PD-L1 inhibitors combined with chemotherapy have significant success when compared to ICIs as monotherapy. According to the IMpassion130 trial, patients with PD-L1+ TNBC treated with PD-L1 inhibitor (atezolizumab) plus nab-paclitaxel had a 7-month median improvement in PFS (88). Consequently, the FDA has approved this combination therapy as a first-line treatment for metastatic PD-L1+ TNBC (89). Other PD1/PD-L1 inhibitors, including avelumab, pembrolizumab, nivolumab, and durvalumab are also being extensively evaluated to be used in BC treatment (82). Previous study has demonstrated that avelumab has the potential to be used in BC treatment, where it showed modest antitumor activity in patients with metastatic BC, with a relatively low objective response rate of 3% in the metastatic BC group and 5.2% in the TNBC group (90). It is worth mentioning that patients with PD-L1+ tumors within the TNBC group had a higher objective response rate of 22.2% compared with those with PD-L1+ tumors in the overall population (16.7%). In the same trial, tumor shrinkage was also assessed and found to be higher in the TNBC group (45.7%) compared to those with metastatic BC (27.9%).
In addition, it was found that avelumab enhanced the cytotoxic properties of NK cells against TNBC cells (91). Avelumab is still being evaluated in phase III clinical trial as adjuvant or post-neoadjuvant treatment for patients with TNBC (NCT02926196).
A systematic review reported that patients with metastatic TNBC treated with pembrolizumab had a PFS of 2.66 and overall survival (OS) of 12.26 months (92). Several clinical trials are being conducted with pembrolizumab in combination with other therapeutic agents for patients with TNBC (NCT05159778, NCT02755272, NCT02513472, NCT06246968, and NCT04895358). The use of nivolumab in patients with TNBC is also being investigated, but in combination with other therapies. According to phase IB/II study, combining nivolumab with eribulin (a chemotherapy that inhibits formation of the mitotic spindle in cancer cells, leading to cell-cycle arrest and apoptosis) resulted in median PFS of 5.6 and 3.0 months in the HR+HER2− and TNBC patients, respectively (93). Another study showed that combination of nivolumab and capecitabine (an oral chemotherapeutic agent that disrupts DNA synthesis and suppresses tumor growth) significantly improved the immunoscore, indicating better immune response in patients with TNBC (94).
Another study examined the predictive power of several biomarkers in patients with metastatic TNBC treated with immunotherapy. These markers included the Immunotherapy Against Gastric Cancer (IMAGiC) model, PD-L1 combined positive score (CPS), intra-tumoral tumor-infiltrating lymphocytes (iTILs), and stromal TILs (sTILs), where the combination of IMAGiC and iTILs as a biomarker was found to guide clinical decisions in the immunotherapy of metastatic TNBC (95).
Durvalumab is also showing promise in BC treatment, particularly in HER2− BC when paired with other therapies. The combination of durvalumab with eribulin improved the clinical outcome for patients with previously treated HER2− BC with an objective response rate of 55%, median PFS of 6.2 months, and median OS of 15.0 months (96). The SAFIR02-Immuno phase II trial explored the efficacy of durvalumab in patients with HR+/HER2− and TNBC subtypes. As compared to maintenance chemotherapy, durvalumab led to lower PFS in patients with HER2− metastatic BC but improved OS in patients with TNBC and PD-L1+ subgroups (97). These findings suggest that PD-L1 inhibitors exhibit clinical benefits in specific BC subtypes particularly when combined with other therapeutic agents.
CTLA4 inhibitors. CTLA4 protein inhibits T-cell activation and differentiation by competitively binding to B7 ligand with the co-stimulatory receptor CD28, thereby inducing T-cell anergy. CTLA4 inhibitors function by blocking the ability of B7 to bind to CTLA4. By blocking this pathway, T-cells remain activated, allowing them to attack and eliminate cancerous cells (Figure 2B) (83). The two main CTLA4 inhibitors are tremelimumab and ipilimumab, which have been approved by the FDA for the treatment of other malignancies, such as unrespectable hepatocellular carcinoma and metastatic melanoma (98-100). For BC, CTLA4 inhibitors are generally evaluated in combination with other ICIs or endocrine therapies (82, 83). A preclinical study evaluated the synergistic effect of anti-CTLA4 and anti-PD1 in mouse models of TNBC and colon cancer. This study found that the combination of both therapies was more effective in inhibiting tumor progression in TNBC when compared to monotherapy (101). A pilot study was conducted on 19 patients with BC to assess the effect of combination therapy of cryoablation and ipilimumab on the immune response. In this study, the combination treatment resulted in increased proportion of CD4+ and CD8+ T-cells, indicating improved cytotoxic effect of immune cells against tumor cells (102). Other research was done to evaluate the effect of tremelimumab combined with exemestane on patients with advanced stage HR+ BC. It was found that patients treated with the combined treatment had higher percentages of circulating activated CD4+ and CD8+ T-cells, indicating immune activation. However, the combined treatment did not result in partial or complete objective response (103). These studies have demonstrated that CTLA4 inhibitors can function as immune activators. More studies are needed to thoroughly evaluate the efficacy of CTLA4 inhibition against BC when combined with other therapies.
Other ICIs. LAG3 is expressed on activated T-cells, NK cells, and other immune cells and interacts with major histocompatibility complex class II molecules. In addition, LAG3 is expressed on TILs within the TME of BC and other solid tumors. A study has shown that LAG3 expression was detected in 11% of patients with BC and demonstrated a strong association with advanced tumor stages, increased tumor size, and aggressive BC subtype such as HER2, and basal-like subtype (104). In patients with TNBC, LAG3 expression was significantly correlated with PD1 and PD-L1 expression, suggesting their use as potential targets in a combinatorial immunotherapy (105). Infiltration of LAG3+ TILs along with high expression of PD1/PD-L1 suggests the potential of LAG3 and PD1/PD-L1 checkpoint blockade as a combination treatment option for BC (104). TIM3 is indicated as a good checkpoint to target in the treatment of BC. Significant correlation was found between poor prognosis and higher TIM3 expression in BC tissues compared to normal tissues. TIM3 plays a significant role in BC progression, proliferation, and metastasis (106). TIM3 has been shown to effectively modulate the immune response in patients with TNBC by interacting with ligands such as galectin-9, resulting in enhanced anticancer activity (107). It has been discovered that TIM3 expression is linked to resistance to PD1 inhibitors. Using bispecific antibodies inhibiting both checkpoint proteins has been examined in mouse models resistant to anti-PD1 and resulted in better treatment outcomes (108). These findings emphasize the potential of combining TIM3 inhibitors with PD1/PD-L1 inhibitors to improve clinical outcomes and enhance the efficacy of BC immunotherapies, particularly in resistant cases. On the other hand, TIGIT and B7-H3 are highly expressed in BC tissues and correlate with poor prognosis and tumor aggressiveness. Both TIGIT and B7-H3 have been evaluated as potential targets for immunotherapy due to their significant roles in tumor metastasis and immune evasion (109). Given their role in inhibiting T-cells and limiting cytotoxic effects of immune cells, combining therapies targeting both checkpoint proteins is of great importance to restore cytotoxicity of T-cells and to enhance efficacy of immunotherapies in BC (110). While targeting these pathways shows promising results, challenges still remain including the need for further clinical validation and understanding of the TME complexities.
Adoptive T-cell therapy. In order to enhance the efficacy of adjuvant systemic therapy in solid tumors, adding activated T-lymphocyte-targeted treatment to standard adjuvant systemic therapies showed promising effects in terms of patients’ survival (111). Adoptive T-cell therapy using chimeric antigen receptor T-cells (CAR-T) has emerged as an advanced treatment approach for challenging BC such as TNBC (Figure 2C). CAR-T therapy has demonstrated successful and promising results in hematological malignancies (112, 113). However, its use in other cancer types including BC remains challenging due to the complex immunosuppressive TME, antigen escape, and difficulty in identifying BC neoantigens that might be used as targets (114, 115). A clinical trial has shown that the adoptive transfer of bone marrow T-cells can significantly induce tumor antigen-reactive type-1 T-cells in the blood circulation and result in longer OS in responding patients compared to patients with bone metastases (116). However, further work is needed to assess the therapeutic potential of this therapy in solid cancer, including BC. Several ongoing clinical trials aimed at assessing the safety, tolerability, feasibility, and efficacy of different CAR-T targets including mesenchymal-epithelial transition factor (MET), mucin 1 (MUC1), Tn antigen-modified mucin 1 (TnMUC1), natural killer group 2 member D (NKG2D) ligand, and receptor tyrosine kinase-like orphan receptor 1 (ROR1) for TNBC treatment. In addition, other clinical trials are investigating different CAR-T targeting mesothelin and MUC1 antigen in different BC subtypes such as metastatic HER2− subtype, aiming at expanding the applicability of CAR-T therapy and overcoming the challenges of this therapy in BC subtypes including aggressive subtypes as TNBC (117). Recently, a phase I trial (NCT01837602) assessed the feasibility of intravenous infusions of RNA-electroporated cMET-directed CAR-T in three patients with metastatic melanoma and four with metastatic TNBC. IHC of tumor tissues revealed increased levels of CD8 and CD3 T-cell infiltration and a decrease in proliferation markers (pS6 and Ki-67), indicating immune activation within the TME (118). MUC1 and TnMUC1-CAR-T-cells have also shown promising cytotoxic effects against TNBC in vivo and in vitro (119). This study reported a significant reduction in TNBC tumor growth and increased release of granzyme B, interferon-γ and other Th1 type cytokines and chemokines resulting in enhanced cytotoxicity. Co-stimulated NKG2D CAR-T-cells were shown to elicit antitumor activity against various TNBC cell lines as well as suppressing tumor growth in vivo (120). ROR1-CAR-T has been considered an effective therapeutic approach in 3D models of lung and breast cancer (121). However, studies have reported lower efficacy in TNBC due to the immunosuppressive TME, particularly through transforming growth factor-β-mediated suppression of CAR-T-cell function. Notably, the therapeutic outcomes of ROR1-CAR-T-cells in TNBC can be enhanced significantly via blocking transforming growth factor β receptor signaling (122).
In preclinical models of TNBC, NK cell immunotherapy showed anticancer effects (123). One study reported that intertumoral injection of allogenic NK cells was effective in inducing cell apoptosis and enhancing tumor infiltration resulting in tumor regression. Moreover, combining intertumoral injection of NK cells with systemic chemotherapy resulted in superior efficacy in suppressing tumor growth and enhancing apoptosis (124).
Anticancer vaccines. A variety of anticancer vaccines are being developed for BC treatment, particularly, TNBC and HER2+ subtypes (125). Peptide-based vaccinations have been widely investigated in clinical trials due to their low toxicity, efficiency in inducing immune responses, and fast production. Citrullinated-enolase 1 peptide vaccine is a peptide-based vaccine that exhibited anticancer effects and inhibited tumor growth in preclinical models of TNBC (126). The HER2 peptide nelipepimut-S-based vaccine has shown efficacy in patients with HER2+ BC by eliciting immune responses against tumor tissues and lowering the risk of recurrence (Figure 2D) (127). Additionally, use of DNA/mRNA-based vaccines may become a leading treatment strategy for BC because of their unique ability to stimulate cellular, humoral and innate immunity (Figure 2D). DNA-based vaccination involves delivery of potential antigens that are overexpressed on cancer cells but not on normal cells such as MUC1, HER2 fragments, and neoantigens (125). A newly developed vaccine containing three components, SZU251 (toll-like receptor 7 agonist), MUC1 and aluminum adjuvants, has been found effective in inducing a humoral and cellular immune response indicated by high antibody titer and elevated infiltration of CD4+ and cytotoxic CD8+ T-cells against tumors expressing MUC1 in mouse models of BC (128). Another DNA-based vaccine target MAM-A has also been reported to be safe and effective in eliciting effector immune response in patients with TNBC. Clinical trials have reported that MAM-A DNA vaccine significantly increased CD8+ T-cells and interferon-γ producing T-cells, and CD4+ T-cells with high expression of inducible T-cell co-stimulator, which contributes to activation and function of T-cells, leading to lysis of BC cells expressing MAM-A in patients with BC (129-131). Despite the promising results of various vaccines in preclinical studies and clinical trials, the FDA has not yet approved any BC vaccine for clinical use. Currently, the main goal is to improve vaccine efficacy through various approaches, including nanotechnology and combination therapies, to prevent cancer recurrence and immunosuppression, particularly in aggressive subtypes with poor prognosis such as TNBC. For instance, it was demonstrated that MUC1 mRNA vaccines delivered by nanoparticles in combination with CTLA4 inhibitors significantly suppressed tumor growth in mouse models with TNBC compared to either treatment alone (132). This combined therapy also elicited a robust cytotoxic T-lymphocyte immune response against cancer cells, emphasizing the crucial role of combining vaccines with other therapeutic agents in treating challenging BC subtypes.
Conclusion
This review demonstrates that the molecular classification of BC has provided a comprehensive understanding of the disease’s heterogeneity and the diversity between its subtypes, directing significant advances in treatment strategy. Indeed, molecular subtyping of BC has become essential in determining a personalized treatment approach, particularly in TNBC and HER2+ subtypes. Additionally, the combination of immunotherapy with different traditional treatments has shown efficacy and safety in modulating immune system responses against breast tumors. While adoptive T-cell therapy and cancer vaccines are both promising components of BC treatment, immunotherapy remains a promising treatment option due to the major role of TILs in regulating cancer progression and immune escape. Research is still ongoing to evaluate the efficacy of combined therapies as well as the new emerging therapeutic agents in different subtypes of BC. Our current focus should be on identifying predictive biomarkers to improve therapeutic effectiveness, reduce adverse reactions, and overcome resistance to immunotherapeutic agents and other treatment modalities. Continued research and clinical trials are essential to address these challenges and further improve the outcomes for patients with BC.
Footnotes
Authors’ Contributions
D.K. conducted the primary literature search and wrote the first draft of the manuscript. N.M.E., N.A., I.M.T. and R.B. revised and edited the drafts of the manuscript. All Authors read and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest related to this work.
Funding
This work was conducted without any external funding.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine-learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received May 7, 2025.
- Revision received June 3, 2025.
- Accepted June 9, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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