Facts and challenges of immunotherapy in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive but common cancer subtype in clinical practice. Immune activation has been observed in a subgroup of TNBC, suggesting that immunotherapy may be a potential therapeutic option. With the widespread use of monotherapy, specific immune checkpoint inhibitors (ICIs) such as avelumab, pembrolizumab, and atezolizumab have made significant contributions to improving outcomes in both early and advanced TNBC. In addition, the expressions of immune regulators such as cytotoxic T-lymphocyte-associated protein 4, programmed cell death 1 (PD-1), and programmed cell death-ligand 1 (PD-L1), which are influenced by tumor-infiltrating lymphocytes (TILs), are also critical factors in determining the effect of immunotherapy in TNBC. This review focuses on the updates on the biological underpinnings of TNBC and the associated treatment advances. We present the current landscape of well-known immune regulators and widely used ICIs for TNBC and highlight the future directions that are significant for further improving the efficacy and effect of targeted therapeutic strategies to immunotherapy in TNBC and more reliable prognostic predictions for tailored therapy in the future.


Introduction
Fifteen to twenty percentages of all human breast cancers (BCs) are triple-negative breast cancer (TNBC). TNBC is characterized by the absence of expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR). TNBC frequently exhibits aggressive characteristics, including early recurrence and metastasis [1] . With regard to overall survival (OS), if a patient is found to have stage 1 TNBC, the 5-year survival rate of the patient is nearly 94.7% due to good immune condition and nutrition absorption. The 5-year survival rate of patients with stage 2 TNBC, where the cancer continues to spread but is still confined within the breast or has only affected adjacent lymph nodes, is about 86.37%. In stage 3 TNBC, the cancer has expanded past the tumor's local vicinity and may have even infiltrated adjacent muscles Based on the expression of ER, PR, and HER2, BC can be classified into four intrinsic subtypes: luminal A, luminal B, HER2+, and TNBC. In most cases, these subtypes have specific immunological characteristics, with different expression levels of tumor-infiltrating lymphocytes (TILs) [8] . In recent years, researchers have found that BC is immunogenetic regardless of its subtype. Lymphocyte-predominant BCs that have stromal or intratumorally lymphocytes make up more than 50 -60% of the tumor tissue [9,10] . Given that immunotherapy has improved survival in other solid tumors, it may also be a viable option for TNBC treatment. Immune checkpoint inhibitors (ICIs), which inhibit immunosuppressive receptors such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) to increase the cytotoxicity and proliferation of tumorinfiltrating cells, are the most effective immunotherapy drugs. ICIs, such as pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab [11] , which are monoclonal antibodies against PD-1, programmed cell death-ligand 1 (PD-L1), and CTLA-4, have produced longlasting responses in a variety of tumor types [12][13][14][15] .
Compared with other subtypes, TNBC is more likely to react to immunotherapy due to a number of factors. First off, TNBC contains higher levels of TILs than other BC subtypes, which have been found to be associated with more significant responses to ICIs and a better prognosis for TNBC in its early stages [16] . Second, TNBC has significantly different levels of PD-L1 expression on both immune and cancer tissues [17] , making it a direct target for ICIs and correlating with how well those treatments work in treating other malignancies [4] . Third, a better anti-tumor immune response has been mounted by neoantigen-specific T-cells when TNBC has a notable frequency of non-synonymous gene mutations, which lead to tumorspecific neoantigens [16] . These neoantigen-specific T-cell responses can be amplified by ICIs [17,18] . This review offers a framework for comprehending the most recent clinical data relating to immune checkpoint blockade (ICB) and other new immunotherapy drugs for TNBC. Future directions for the development of immunotherapy in TNBC are also explored, along with the development of immunotherapy biomarkers (Tables 1 and 2).

Triple-negative breast cancer characteristics
TNBC accounts for 15 -25% of all BCs and is widely recognized as the worst BC among all the subtypes of BC, posing a huge threat to patients diagnosed with BC [1] . TNBC can be classified into four robust subtypes based on their different transcriptomic characteristics: basallike (BL), immunomodulatory (IM), mesenchymal stemlike (MSL), and luminal androgen receptor (LAR) [30] . A large number of studies have indicated that age, sex, and even race can be risk factors of TNBC. According to research, BRCA and basal TNBC, as well as apocrine and neuroendocrine TNBC, are more common in younger and older women compared to the same age groups in men. It has been documented that Hispanic and African American women are at a higher risk of TNBC and have a poorer prognosis than other populations. In a case study, there was a 2.5% increased risk of TNBC in 187 TNBC patients who had taken oral contraceptives for more than a year, the risk of TNBC was 4.2% for women under the age of 40, and it was discovered that the risk rose as the duration of oral contraceptive use increased [17] . In the United States, TNBC accounts for 12% of BC cases, with 8 -16% 5-year survival rate.

Ovarian cancer
No and a high frequency of homologous recombination DNA repair deficiency (HRD). BLIS also shows a high mutation rate in TP53, complex genomic profiles, and an HRD-associated signature but are associated with significantly lower TILs [32] . The most noticeable feature about these subtypes is that BLIA and BLIS subtypes are, respectively, associated with the best and the worst disease-free survival.
To make a more vivid comparison among the subtypes, the LAR subtype displays mutations similar to those detected in luminal B cancers, and its microenvironment is described as "cold, " with low TILs, in comparison with the "desert" microenvironment in the MES subtype and the "hot" microenvironment in the BLIA subtype [33] . However, it should be noted that in these gene expression classification systems, the vast majority of TNBCs analyzed were of high grade; hence, it remains unclear as to how the lowgrade forms described above would fit into this taxonomy or if these low-grade forms would constitute completely different entities at the transcriptomic level [34] .
The risk factors of TNBC are discussed below. The first is related to age, in which 80% of BC cases (including TNBCs) are older than 50 years old [6] . Due to different sex hormonal stimulation, female sex is considered a higher risk for TNBC compared to male sex. In addition to these two factors, race is also associated with TNBC, in which the incidence of TNBC remains high among Caucasian non-Hispanic women [35] . With regard to breast tissue density, as per clinical practice, breasts can be categorized into lowdensity breasts, fatty breasts, and high-density breasts [36] . In postmenopausal and premenopausal women, breast density affects the risk of cancer, that is, the higher the https://doi.org/10.36922/td.v1i2.196

Tumor Discovery
Fact & challenges of immunotherapy in TNBC density, the higher the risk of BC [37] . Breast tissue density screening could be a promising and quick approach for rational surveillance. According to several epidemiological studies, obesity is a potential risk factor for BC [38,39] . Hence, engaging in physical activity is considered the best way to prevent BC. Alcohol and alcoholic beverages can also increase the risk of malignancy [40] .

Triple-negative breast cancer microenvironment
The tumor microenvironment (TME) contains various cell types, including fibroblasts, TILs, and lymphatic vascular channels. The active interaction between tumor cells and the microenvironment affects the pathogenesis and development of tumor. Research has indicated that high levels of TILs, especially in the IM subtype, are associated with better prognosis and response to chemotherapy in both neoadjuvant and adjuvant contexts [1,4] . Later, research has revealed that variations in gene overexpression of IM and MSL subtypes are derived from the TME, including infiltrating immune cells and tumor-associated mesenchymal tissue, respectively [41,42] . Intriguingly, and in agreement with the aforementioned findings, these genes are not expressed in cell lines when tests are conducted in vitro, where the microenvironment is absent. It is evident that TME has a significant influence on the development of tumor as well as the response and resistance to treatment. Furthermore, the elevated expression of immune regulators such as CTLA4, PD-1, and PD-L1 in TNBC, brought on by lymphocyte infiltration of the tumor, is likely linked to a response to ICIs. Several studies have purported the possibility that TILs may be a marker for improved survival outcomes [43][44][45] . All the preceding evidence suggests that focusing on TME in TNBC and further exploring the biomarker landscape are promising efforts for better immunotherapy.

Specific biomarkers
Due to the underlying heterogeneity of TNBC, there is a need for efficient biomarkers that can guide doctors in determining the best course of action. Therapeutic trials have been conducted on several suggested biomarkers for TNBC, with limited clinical benefits so far. Breast cancer gene (BRCA1/BRCA2) mutations have been found to be predictive of the effectiveness of poly (ADPribose) polymerase (PARP) inhibitors, and changes to other homologous recombination-related genes appear promising in this context [46][47][48][49] . It is possible to use the expression of PD-L1 protein in either immune cells (ICs) or tumor cells, or both, as a biomarker to predict how well an immune checkpoint inhibitor would work [15] . TILs are also considered an important prognostic factor in TNBC. Up to 15 studies have shown that 11% (median; range, 5 -26%) of breast cancers are lymphocyte-predominant breast cancers (LPBCs), among which TNBC accounts for the highest incidence (20%; range, 4 -37%) [50] . Moreover, CD8 + T-cell infiltrates have been observed in 60% of TNBC cases [41] . Various tests that employ different antibodies and scoring methods are commercially available. There is an ongoing debate over the optimal assay for TNBC and whether the findings hold for all ICIs.
A humanized antibody, pembrolizumab, is used in cancer immunotherapy to treat melanoma, lung, head and neck, stomach, cervical, and breast cancers, as well as Hodgkin lymphoma. Pembrolizumab is slowly injected into a vein. The IgG4 isotype antibody blocks the defense mechanism of cancer cells, thus enabling the immune system to eliminate them. Pembrolizumab targets the lymphocyte PD-1 receptor and functions by concentrating on the PD-1/PD-L1 biological pathway, which is present in some cancer cells and immune cells in the body [6,[54][55][56][57][58][59][60][61] . The PD-1 antagonist pembrolizumab, in the KEYNOTE-012 trial, which studied the safety and antitumor efficacy of pembrolizumab monotherapy in patients with advanced PD-L1-positive solid tumors, was initially assessed in PD-LI-positive advanced TNBC patients. PD-L1 expression was prescreened in 111 patients with advanced TNBC, in which 58.6% of them tested positive for PD-L1. The median number of prior therapies for advanced illness https://doi.org/10.36922/td.v1i2.196
By preventing the interaction of PD-L1 with PD-1 and CD80 receptors (B7-1Rs), atezolizumab can be used to treat dysplastic carcinoma, hepatocellular carcinoma (HCC), non-small-cell lung cancer (NSCLC), small-cell lung cancer (SCLC), and TNBC [63,64] . Atezolizumab is a monoclonal antibody of the IgG1 isotype that has been fully humanized and engineered to target the protein PD-L1 [13,[65][66][67] . In the PCD4989g trial, atezolizumab was tested in patients with advanced malignancies [68] , including 116 patients with advanced TNBC, 115 of whom had an objective response assessed. After the enrollment of the initial 25 patients, the eligibility was changed to permit the enrollment of patients with any PD-Ll status. These patients displayed PD-LI in IC, occupying <5% of the tumor area. With 58% of patients having received at least two prior lines of therapy for an incurable illness, the enrolled patients were severely treated [68,69] .

Cytotoxic T-lymphocyte-associated protein 4 inhibitors
Ipilimumab is a monoclonal antibody that works to activate the immune system by targeting CTLA-4, a protein receptor that downregulates the immune system. It boosts the immune response against cancer cells and prevents the inhibitory interruption of cytotoxic T-lymphocytes (CTLs), which can recognize and destroy cancer cells [70] . In a study on early-stage BC, 12 of 18 women received a single dose of neoadjuvant ipilimumab alone or with additional cryoablation; the other six patients received cryoablation alone. T-cell density was found to be significantly correlated with TIL count by hematoxylin and eosin (H&E). It was shown that about 5/6 patients who received ipilimumab alone had increased T-cell density in contrast to the decrease in the cryoablation group [71,72] .
Tremelimumab blocks the binding of antigenpresenting cell ligands B7.1 and B7.2 to CTLA-4, resulting in the inhibition of B7-CTLA-4-mediated downregulation of T-cell activation. Tremelimumab has been evaluated in various types of tumors. Experiments with tremelimumab in combination with exemestane have been carried out. Among 26 patients who received tremelimumab (3 -10 mg/kg) and exemestane (25 mg/kg daily), five patients developed dose-limiting toxicity when the dose of tremelimumab with 25 mg/day exemestane was about 6 mg/kg Q90D, four of which were diarrhea. Up to 42% of patients had stable disease for at least 12 weeks. The [73] .

Neoadjuvant ipilimumab
These groundbreaking studies offer solid proof in favor of using PD-1/PD-L1 and CTLA-4 inhibitors in both early and advanced TNBC. The FDA has authorized the use of pembrolizumab in conjunction with chemotherapy for the treatment of PD-L1-positive advanced TNBC, and health authorities now recommend the combination of atezolizumab and nab-paclitaxel [55] . To further understand the immunobiology of both early and late TNBC, wellcontrolled translational studies may be conducted using the data sets and tissue samples from these trials [49,60] . By identifying TNBC as a tumor that can react to immunotherapy, these studies collectively pave the way for the testing of cutting-edge ideas that can successfully harness the immune system to improve clinical outcomes for patients with this arduous condition (Table 3).

Unclear mechanism of tumor-infiltrating lymphocytes
It is essential to consider whether the number of TILs expressed in the primary tumor and the metastatic sites can affect the prognosis of patients with TNBC. Another issue is whether the heterogeneity of TILs at the original sites, the occurrence of residual invasive disease (RD) following the completion of neoadjuvant chemotherapy (NAC), and the metastatic locations can influence the choice of follow-up therapy options in TNBC [2,54] . Despite the fact that the discovery of biomarkers has given individuals who are looking to advance their skills an effective tool, one of the main limitations of the use of TILs at the moment is their reliance on manual measurement, which is subject to potential human error [41] . Surprisingly, there are many opportunities to employ computational techniques that extract spatial-morphologic predictive elements, thus making it possible for computer-aided diagnostics.
We, now, need to figure out how to assess TILs in combination with other biomarkers to direct a more focused course of treatment, as they have been established as distinct biomarkers in the early TNBC. In addition, it is still worthwhile to advocate the current belief that TILs serve as the starting point for the expression of additional biomarkers [16,51] .
In terms of immunomodulatory mechanisms, whether negative immunomodulatory regulation, as part of a standard feedback loop, has a positive and persistent https://doi.org/10.36922/td.v1i2.196

Tumor Discovery
Fact & challenges of immunotherapy in TNBC Schmid et al. [69] Miles et al. [74] IMpassion130  [76] A: Anthracycline, CPS: Combined positive score, IC: Immune cell, ITT: Intent-to-treat, mTNBC: metastatic TNBC, NR: Not reported, ORR: Objective response rate, OS: Overall survival, PD-L1: Programmed death-ligand 1, PFS: Progression-free survival, T: Taxane, TFI: Treatment-free interval. *Chemotherapy of physician's choice could be capecitabine, eribulin, or gemcitabine. #Chemotherapy of physician's choice could be paclitaxel, nab-paclitaxel, or gemcitabine + carboplatinum effect on tumor immune response is worth discussing. Furthermore, more exploratory work needs to be done to determine whether the possible mechanism above potentially defines a more immunogenic tumor. At the same time, we should continue to focus on the heterogeneity of TILs, the subpopulation classification of T cells, and how their respective molecular pathways regulate immunity [44,73] .

Unpredictable personal benefits
For the purpose of selecting patients who are most likely to benefit from immunotherapy and the development of combination treatments to overcome drug resistance, tumor molecular profiling is significant. Through gene expression profiling analysis of TNBC tumor samples, abnormal cell cycle-regulating and DNA repair-related gene expression has been observed in the BL1 subtype [77] . Possible therapeutic drugs for the BL1 subtype include PARP inhibitors and genotoxic agents; BL1 patients are usually sensitive to cisplatin treatment. On the other hand, the BL2 subtype has abnormal activation of signaling pathways, such as the epidermal growth factor receptor (EGFR), mesenchymal epithelial transition factor (MET), nerve growth factor (NGF), Wnt/β-catenin, and insulinlike growth factor-1 receptor (IGF-1R) pathways, and the potential targeted therapeutic drugs include mammalian target of rapamycin (mTOR) inhibitors and growth factor inhibitors (lapatinib, gefitinib, and cetuximab) [64] . Meanwhile, the IM subtype has significantly enriched immune cell-associated genes and signal transduction pathways, such as the Th1/Th2, NK cell, B-cell receptor, dendritic cell (DC), T-cell receptor, interleukin (IL)-12, and IL-7 pathways [78] ; thus, the IM subtype is highly similar to medullary carcinoma of the breast. PD1, PDL1, CTLA-4, and other immune checkpoint inhibitors are recommended for the treatment of patients with breast cancer of the IM subtype. The M subtype, on the other hand, has highly activated cell migration-related signaling https://doi.org/10.36922/td.v1i2.196

Tumor Discovery
Fact & challenges of immunotherapy in TNBC pathways (regulated by actin), extracellular matrixreceptor interaction pathways, and differentiation pathways (Wnt pathway, anaplastic lymphoma kinase pathway, and transforming growth factor [TGF]-β signaling). The M subtype has sarcoma-like or squamous epithelial cell-like tissue characteristics and is prone to developing resistance to chemotherapy drugs [79] . Patients with the M subtype may be treated with mTOR inhibitors or drugs that target epithelial-mesenchymal transition [15] . Compared with the M subtype, the MSL subtype shows a lower expression of cell proliferation-related genes but a higher expression of stem cell-related genes, HOX genes, and mesenchymal stem cell-specific markers. Presumably, patients with the MSL subtype can be treated with PI3K inhibitors, Src antagonists, and angiogenesis inhibitors [80] . Compared with other TNBC subtypes, the LAR subtype has a significantly different gene expression profile [81] . This subtype does not express ERs, but has highly activated hormone-related signaling pathways (e.g., steroid synthesis, porphyrin metabolism, and androgen/estrogen metabolism). Notably, androgen receptors (ARs) are highly expressed in breast cancers of the LAR subtype, with messenger ribonucleic acid (mRNA) levels nine times higher than in other TNBC subtypes. Immunohistochemistry has also shown that several metabolic markers of AR and its associated activators (24- Besides, biomarkers that predict the clinical benefit of immunotherapy in TNBC are also required. PD-L1 expression on immune cells and mismatch-repair deficiency are the only two validated biomarkers that are currently available [82,83] . The majority of patients with mTNBC are PD-L1-negative by the presently authorized SP142 test [83] , despite the fact that mismatch repair failure is uncommon in breast cancer but more frequent in the earlystage illness [73] . The variability of PD-L1 expression over time and at metastatic sites [84] , the discrepancy between PD-L1 assays, particularly when staining immune cells, the observation that some PD-L1-negative patients respond to ICIs [85] , and the recent trials in early disease setting that show little to no correlation of PD-L1 expression with benefit specific to ICIs, such as the KEYNOTE522 and NeoTRIPaPDL1 trials, are additional factors that limit the utility of PD-L1 [85,86] . Moreover, the IMpassion130 trial has shown a significant, modest improvement in progression-free survival (PFS) but a marked difference in OS [87] . The subgroup of patients with PDL1 > 1% (185/451 patients) benefited from atezolizumab; a trend toward a higher ORR was observed in patients with PD-L1-positive versus PD-L1-negative ICs in the overall population [69] . With the various outcomes from clinical trials, the focus of further research is on identifying more prognostic biomarkers to demonstrate the benefit for each patient receiving immunotherapy (Table 3).

Conclusion
TNBC, compared with other BC subtypes, has a poor prognosis and is still a complicated cancer for immunotherapy to be developed thus far. However, to improve the prognosis, immunotherapy techniques must be used. Research efforts should focus on using the ICB we have in relation to the molecular biology of TNBC to discover mono antibody therapies and other more effective drug combinations. For this highly diverse subtype of BC, personalized medicine appears to be of great importance. When deciding on a treatment plan, tumor molecular profiling should be carried out at the time of diagnosis, after each tumor recurrence or progression, and as needed. The functions of a cluster of biomarkers may be crucial to predicting an individual's response to future TNBC immunotherapy. https://doi.org/10.36922/td.v1i2.196

Tumor Discovery
Fact & challenges of immunotherapy in TNBC Ethics approval and consent to participate Not applicable.

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Tumor Discovery
Fact & challenges of immunotherapy in TNBC