Clinical trials in patients with HER2-positive breast cancer have exhibited modest results with immunotherapy. The combination of anti-PD-1 mAb (pembrolizumab) and trastuzumab was assessed for the treatment of HER2-positive progressive metastatic breast cancer, wherein a partial response was achieved in 15% of the enrolled patients with PD-L1 positive tumors (14). Moreover, dendritic cell (DC) vaccines that were primed against the HER2 protein proved beneficial in mammary tumor regression through activation of anti-HER2 CD4⁺ Th1 response in an early phase clinical trial (15). Preclinical investigations in immunocompetent mice suggest that PD-1 and CTLA-4 inhibition leads to a considerable increase in the immune-associated effects of HER2-based targeted therapies which is accomplished via synergistic activation of CD8⁺ T cells (16, 17). The PANACEA trial revealed that 15% of trastuzumab-resistant HER2-positive breast cancer patients harboring PD-L1-positive tumors elicited a partial response when treated with pembrolizumab plus trastuzumab (14). In the “Proceedings of the 2018 San Antonio Breast Cancer Symposium”, Emens et al. discussed the randomized phase II KATE2 trial, which revealed that patients with PD-L1-positive, HER2-positive, pre-treated metastatic breast cancer exhibited improved PFS following treatment with T-DM1 combined with atezolizumab relative to T-DM1 alone. Of note, CAR T-cell therapy, a popular example of adoptive T cell therapy, have proven successful in patients with hematological cancers and is currently being explored in solid tumors. Researchers have successfully expanded T cells specific for HER2 ex vivo in mice models and these were found to elicit antitumor activity (18). Administration of HER2 CAR T cells with CD28 costimulatory domain in the mice central nervous system resulted in the regression of HER2-positive metastatic breast carcinoma in the CNS (19). Nonetheless, clinical data regarding the application of adoptive T cell therapy for treating HER2-positive breast malignancy are still lacking. Preclinical and clinical observations, though limited, solidify the rationale for the clinical development of ICI for the treatment of HER2-positive breast carcinoma, and emphasize the need for more detailed research into the development of immunotherapeutic modules, especially in combination with HER2-targeted therapies.

Reportedly, factors such as a heavier tumor mutation load, increased frequency of TILs and enhanced expression of PD-L1 may contribute to increased immunogenicity for TNBC, thus, TNBC patients are expected to benefit from ICIs (20). The efficacy of pembrolizumab monotherapy as a first-line of therapy for metastatic TNBC (mTNBC) patients was evaluated in the phase II KEYNOTE-086 study (21). The results from the study showed favorable anti-tumor activities with median PFS of 2.1 months whereas the median OS was improved to 18 months. Many investigations are additionally aimed at establishing pembrolizumab monotherapy as a second-line or later therapeutic strategy in pre-treated mTNBC patients, including the KEYNOTE-086 (21), the KEYNOTE-012 (22) and the TAPUR basket study (23). In the phase III KEYNOTE-119 trial, efficacy of pembrolizumab monotherapy was assessed in comparison to standard chemotherapy in second or third line of treatment for patients with mTNBC. However, there was no evident improvement on the prognosis of 622 TNBC patients, who had progressed on 1-2 cycles of either taxane or anthracycline (24). Such results indicate the immediate need for additional large-scale randomized controlled trials and the need for combination approaches, especially in earlier lines of treatment. Atezolizumab and durvalumab are two more anti-PD-L1 antibodies that are yielding promising results (22). A phase II trial, consisting of 199 patients with no disease progression after 6-8 cycles of chemotherapy, has evaluated the role of durvalumab for TNBC treatment. In the exploratory subgroup analysis of TNBC patients (n = 82), durvalumab dramatically increased the OS (25), suggesting the rationale for additional investigations into using durvalumab as a therapy for TNBC patients with advanced disease. Avelumab, another PD-L1 inhibitor, is currently being tested as second-line or posterior-line therapy at JAVELIN basket trial, which has shown some promising results (26). Findings from the phase II TONIC study have indicated that addition of cisplatin and doxorubicin may exert better tumor response to immunotherapy in TNBC patients (27). The detailed insight into the underlying molecular mechanisms is still not thoroughly understood and remains an imperative area of future research focus. Nonetheless these reports strongly support the improved and durable clinical efficacy of PD-1/PD-L1 inhibition as an effective treatment modality for patients with TNBC.

It is important to note that not all subtypes of breast carcinoma equally respond to the effects of immunotherapy. For instance, in subjects bearing the luminal subtype of breast cancer, initial attempts used a combination of ICI with chemotherapy as a novel form of anti-cancer therapy- but that yielded disappointing results. One such study aimed at investigating the tumor suppressive effects of pembrolizumab combined with eribulin among patients harboring ER/PR-positive, HER2-negative metastatic breast carcinoma (28). However, the combination therapy did not lead to any noticeable improvement in the clinical outcome or prognosis of the metastatic luminal A-subtype breast cancer patients- the most possible reason underlying this pertains to the heavily-pretreated nature of the subjects in the study. A phase Ib non-randomized, open-label, multi-cohort study tried to evaluate the clinical impact of pembrolizumab plus abemaciclib in the presence or absence of anastrozole (endocrine therapy) in metastatic breast cancer patients with ER-positive, HER2-negative subtype. Patients had no prior exposure to CDK4/6 inhibitor treatment (29). Unfortunately, the anti-tumor efficacy of the combination was mitigated by the appearance of toxicity in the liver and lungs following therapy. In contrast, administration of letrozole, palbociclib, and pembrolizumab as front-line therapy in HR-positive, HER2-negative metastatic breast carcinoma was associated with good tolerance and favorable clinical efficacy in a phase I/II trial (30). For luminal B- subtype, the neoadjuvant phase II GIADA trial subjected patients to sequential anthracycline-associated chemotherapy prior to the treatment with nivolumab and endocrine therapy (31). A 16.3% pCR rate was observed followed by the identification and characterization of immune-based gene signatures and immune sub-populations that were correlated with the achieved pCR. While previous studies have found no notable benefit from pembrolizumab in a metastatic setting of the luminal subtype, addition of pembrolizumab to a sequential cycle of chemotherapy in a neo-adjuvant setting was found to elevate the pCR rate from 13 to up to 30% amongst patients with luminal breast cancer (32).

Utomilumab is a receptor IgG2 mAb agonist against 4-1BB, a co-stimulatory receptor that is involved in immune cell proliferation once activated. For the treatment of patients with advanced HER2-positive breast cancer, a phase I dose-escalation trial is investigating the combination of utomilumab with either trastuzumab or T-DM1 (NCT03364348). The effects of utomilumab plus avelumab is also being studied in a phase II trial (AVIATOR, NCT03414658). The preclinical findings revealed that utomilumab, when combined with a mAb targeting the PD-1/PD-L1 axis, can aid in a strong immune response (33). Another component associated with the adaptive immune response is the toll-like receptor, and activation of TLR4 can stimulate antigen processing and cross-presentation in vivo ( 34). In pre-clinical models, oligodeoxynucleotides with CpG motifs that activate TLR9 have been shown to induce active immune cytotoxicity (35). Activation of TLR2 in HER2-positive breast cancer preclinical models augments trastuzumab-related cytotoxicity (36). Such results inspired research undertakings for testing the therapeutic efficacy of TLR agonists in combination with HER2-based vaccines (NCT02276300). Synergistic interactions between trastuzumab and docetaxel chemotherapy have yielded a 60% response rate compared to 11% in the case of monotherapy (37). Taking these observations forward, clinical trials inspecting the efficacy of combining atezolizumab with HER2 mAbs plus chemotherapy in patients who are receiving early first line therapy for HER2-positive breast cancer are in progress. The findings from such trials are likely to provide new avenues for the treatment modalities (NCT03125928, NCT03726879) (38). While approaches combining immunotherapy and HER2-targeted therapies are promising candidates for the treatment of HER2-positive breast cancer, they can also be useful for a wider patient population including tumors exhibiting a modest/low HER2 expression level which are not usually eligible for HER2 mAbs.

One-third of patients with TNBC experience distant recurrences, and eventually succumb to death within 5 years post- diagnosis. Therefore, TNBC has a dire need for superior treatment options and precision medicine. A phase III clinical study, IMpassion130, interrogated the impact of immunotherapy in 902 patients with advanced TNBC, and tested the efficacy and safety of the PD-L1 antibody atezolizumab in conjunction with chemotherapeutic drug nab-paclitaxel (39). The promising data showed a significant improvement in the mean OS from 15.5 to 25 months among PD-L1-positive patients (40). The findings led to the FDA approval of the combination of atezolizumab and nab-paclitaxel for therapy-naïve patients having PD-L1-positive advanced TNBC in 2019 (41). Follow up phase III trials IMpassion131 and Impassion 132 are delineating the clinical impacts of atezolizumab with paclitaxel or first-line chemotherapy (carboplatin, gemcitabine or capecitabine) in multiple settings of TNBC (42). The primary goal of the IMpassion131 trial was to test the efficacy of weekly administration of paclitaxel as the chemotherapy backbone plus atezolizumab in a group of patients constituting of similar inclusion criteria as IMpassion130. Unfortunately, the results from this trial were not in sync with the observations from Impassion130. Of note, in an abstract presented at the “2021 ASCO Annual Meeting”, it was shown that several differences were present in the tumor microenvironment of tumor samples from patients enrolled in the two trials. Additional reasons underlying the observed discrepancy in results may be attributed to the differences in the body mass index and gut microbiota composition among the enrolled patients (43). Another phase III trial, KEYNOTE-355, tested the effects of integrating pembrolizumab with chemotherapy (albumin-bound paclitaxel, paclitaxel or gemcitabine/carboplatin) for the treatment of locally recurrent, inoperable or mTNBC patients who have not undergone prior therapy. There was a considerable prolonged PFS among PD-L1 positive population in the pembrolizumab-based group (44), paving the way for the accelerated FDA approval of pembrolizumab in combination with chemotherapy for the treatment of patients with locally recurrent unresectable or metastatic PD-L1-positive TNBC. Final results of the KEYNOTE-355 trial reported that the combination considerably benefitted the patients with a mean OS of 23 and 16.1 months in the combination versus chemotherapy alone group, respectively. Intriguingly, 22% of the TNBC patients in the pembrolizumab arm boasted of a disease-free interval between 6 and 12 months. Results from a phase II randomized trial demonstrated that chemotherapy or radiotherapy promoted a more favorable tumor microenvironment in TNBC patients that boosted the response to PD-1 blockade through nivolumab. Patients subjected to the combination regimen that included immunotherapy experienced a clear improvement in their median DFS and OS, relative to the individuals treated with monotherapy alone (27). Of interest, Oleclumab, a mAb specific for CD73, is being studied in the phase Ib/II BEGONIA study, as a combination therapy with durvalumab, plus paclitaxel, as first-line treatment for mTNBC individuals (NCT03742102). In the ENHANCE 1 trial enrolling 104 patients with mTNBC, eribulin, a microtubule inhibitor, when administered with pembrolizumab, displayed attractive tumor-suppressive activity (45). An objective response was achieved in (i) 26% of the evaluable patients, (ii) 25% of the 48 patients who were not exposed to any prior chemotherapy and (iii) 27% of the 34 subjects who had previously received chemotherapy. Again, in agreement with observations from other trials, patients harboring PD-L1-positive breast cancer boasted a better response than those having PD-L1-negative tumors. The exciting findings from such trials have elicited investigations into various combination regimes among TNBC patients. Chemotherapy regimens continue to be the frontline treatment strategy for a majority of breast cancer patients; however, it is associated with adverse side effects affecting the quality of life as well as therapy resistance leading to suboptimal response. While immunotherapy regimens are still being investigated for their long-term impact on quality of life, the combination treatment strategies combining chemotherapy and immunotherapy are presenting improved responses than monotherapy alone.

Multiple studies are underway focusing on determining the efficacy of immunotherapy with anti-angiogenesis agents. The GINECO A-TaXel phase II trial in TNBC reported a significant activity and tolerable safety profile for the combination of paclitaxel, capecitabine and bevacizumab, a recombinant humanized mAb against VEGF-A (46). In 2019, a single-arm trial investigated the effects of Nivolumab, paclitaxel and bevacizumab as first-line therapy in patients with HER2-negative metastatic breast carcinoma, consisting of both hormone-positive and TNBC populations. The treatment group exhibited a PFS of 8.1 months among the TNBC individuals and 19.1 months in the hormone receptor-positive subgroup (47). In addition, in heavily pretreated patients with advanced TNBC, a novel humanized mAb targeting PD-L1, TQB2450, was tested in combination with anlotinib, an anti-angiogenic small molecule inhibitor- the combination arm displayed an acceptable safety profile with potent activity (48). The capacity of HDAC inhibitors in upregulating antigen presentation genes and boosting tumor cell recognition by activated ICs suggest that they may act in augmenting the efficacy of ICIs. The cocktail of romidepsin, cisplatin and nivolumab indicated encouraging signs of efficacy in 34 pre-treated mTNBC patients, necessitating additional research in larger populations (NCT02393794). In contrast, evidences from a phase II study in people with advanced TNBC suggest that another HDAC inhibitor, entinostat, failed to improve PFS in combination with atezolizumab (NCT02708680), supporting the dire need for further investigation of the combination. With advances in research related to ADC, a randomized phase II trial is affirming the impact of pembrolizumab plus sacituzumab govitecan, composed of a topoisomerase I inhibitor (SN-38) and an anti-Trop2 monoclonal antibody, in patients with PD-L1 negative mTNBC (NCT04468061). Overexpression of Trop-2 is predictive of a more aggressive TNBC (49). Sacituzumab govitecan was found to serve as a potent immunomodulator, promoting antibody-driven cytotoxicity, depletion of T_reg cells and upregulation of MHC class I and PD-L1 expression in mice models, and it may overcome resistance to current immunotherapeutic strategies in PD-L1-negative tumors, which forms the rationale of the clinical trial. A recent presentation at the “2020 AACR annual meeting” demonstrated the potential medical application of another ADC with similar immunomodulatory features, ladiratuzumab-vedotin, an anti-LIV-1 ADC, in conjunction with pembrolizumab as first-line therapy in TNBC patients in a phase Ib/II study- the combination proved tolerable and exhibited promising anticancer activity. ICI therapy impedes the tumor-promoting signals that enable immune evasion by cancer cells. Combining this method with agents that function by potently targeting various other hallmarks of cancer, such as angiogenesis, epigenetics modulation and DNA damage response, can potentially results in synergistic effects that will ultimately lead to more successful response in breast carcinoma patients.

Various kinase inhibitors have been tested to target specific oncogenic pathways in breast cancer. Currently, many clinical studies are exploring combination regimens involving kinase inhibitors and immunotherapy. The phase II COLET study evaluated the therapeutic efficacy of atezolizumab, MEK1/2 inhibitor cobimetinib, and nab-paclitaxel or paclitaxel in locally advanced or mTNBC, wherein PD-L1-positive patients accomplished a visibly higher tumor ORR and PFS than PD-L1-negative individuals (50). The therapeutic outcome of combining PD-1 monoclonal antibody camrelizumab with apatinib for treating advanced TNBC was tested in a phase II study (51). The results were intriguing as they revealed an ORR as high as 32.5% compared to 18.5%, which is the highest recorded ORR for anti-PD-L1 monotherapy in TNBC, paving the groundwork for an effective alternative combinational approach for TNBC treatment. Moreover, a prospective phase II trial (FUTURE-C-PLUS) is ongoing that seeks to assess the efficacy and safety index of the combination of camrelizumab plus chemotherapy (nab-paclitaxel) and famitinib (multityrosine kinase inhibitor against VEGFR-2, PDGFR and c-kit) in mTNBCs. A major part (81.3%) of the population achieved objective responses with a 60.2% of 9-month PFS rate (52). These promising results inspired the ongoing phase II randomized trial FUTURE-SUPER (NCT04395989). A phase Ib/II study is presently determining the effects of tislelizumab, an anti-PD-1 IgG4-variant mAb, in combination with fruquintinib, a highly selective, oral tyrosine kinase inhibitor of VEGFR, in mTNBC, including patients who have been pretreated with immunotherapy in addition to naïve patients (NCT04579757). Meanwhile, patients suffering from AR-positive metastatic TNBC, when subjected to pembrolizumab combined with the AR regulator GTx-024 in a phase II clinical trial, demonstrated an ORR of 25% at 16 weeks (53). In another study, an AKT inhibitor ipatasertib was subsequently combined with the atezolizumab and paclitaxel or nab-paclitaxel cocktail as another candidate for front-line treatment. Irrespective of the expression of PD-L1 or alteration status of PIK3CA/AKT1/PTEN, 19 out of 26 patients showed a response accompanied with a significantly elevated ORR of 73%, thus advocating a novel therapeutic regime for treating TNBC (54). In addition to their traditional role in targeted inhibition of key proteins involved in cell survival and growth, kinase inhibitors eradicate tumors via certain immune-modulatory effects. Immunotherapy, when used in conjunction with such precision therapy, can suppress the toxicities associated with monotherapy and impart improved targeted anti-tumor functions even in breast cancer patients, who do not respond well to immunotherapy alone.

A more recent undertaking, which is currently recruiting patients with locally advanced or metastatic HER2-negative mammary carcinoma with homologous DNA repair deficiency, aims to uncover the efficacy of atezolizumab when incorporated with the PARP inhibitor, olaparib (NCT02849496). The TOPACIO/KEYNOTE-162 trial is a single-arm phase II study in advanced TNBC population that found a considerable anti-tumor activity and manageable safety profile for the combination of pembrolizumab and a PARP inhibitor, niraparib (55). Importantly, this combination was especially beneficial for patients harboring tumors with BRCA mutation. Furthermore, niraparib synergistically potentiates the anticancer functions of the anti-PD-1 antibody, BioXCell RMP1-14, in TNBC models through activation of the interferon signaling (56). Furthermore, co-administration of anti-PD-L1 monoclonal antibody, durvalumab and olaparib in advanced breast carcinoma with genomic BRCA mutation exhibited better survival rates in a phase I/II MEDIOLA study (57), denoting the alluring prospect of integrating PARP inhibitors with immunosuppressants as an efficient anticancer module for TNBC patients. Moreover, the SGNLVA-002 study attempts to assess the effects of the novel combination of pembrolizumab with ladiratuzumab vedotin, an ADC with great potential, as a front-line treatment choice for locally advanced or mTNBC (NCT03310957). A phase II randomized controlled trial in 2019 unveiled that PD-L1-positive or TNBC patients demonstrated a pronounced benefit when subjected to treatment with durvalumab with the median OS of durvalumab-treated PD-L1 positive or TNBC patients being 21 months and 26 months, respectively (NCT02299999). The efficacy of durvalumab plus the PARP inhibitor, olaparib, as a maintenance strategy for patients with platinum-sensitive advanced TNBC is being determined in the DORA study (NCT03167619). Another category of agents that holds imminent interest in combination modules with ICIs are the CDK inhibitors. In preclinical models of TNBC, dinaciclib, an intravenous CDK inhibitor, potentiated the antitumor effects of ICI through heightened immune cell activation and tumor infiltration. Following this, a phase Ib, dose-escalation trial tested dinaciclib plus pembrolizumab in patients with advanced TNBC, which revealed manageable toxicities upon reduction and delayed administration of the specified dose while the dose expansion part is ongoing. Furthermore, the CDK4-6 inhibitor, palbociclib, is being interrogated in combination with avelumab in AR-positive TNBC (NCT04360941). DNA-damaging agents, such as PARP inhibitors, can potentiate immune response through enhanced tumor mutational burden and improved neoantigen release, thereby rendering the tumor more amenable to immunotherapy. CDK4/6 inhibition is known to impart transcriptional reprogramming of immune as well as tumor cells, resulting in higher immunogenicity of cancer cells and an immune-rich TME, which is, again, more susceptible to immune-based therapies. Therefore, combining these approaches with immunotherapy can lead to positive response rates in a number of breast cancer patients, including those who are originally less responsive to ICI therapy.

Recent studies are also investigating the utility of combining different ICI regimes. Multiple CTLA-4 inhibitors have shown the efficacy in combination therapy for solid tumors, including breast carcinoma. While ipilimumab was FDA approved for better survival among advanced metastatic melanoma patients, its anti-tumor effect was modest in TNBC (58). A single-arm clinical study in patients with metastatic breast cancer, including TNBC population, tested the efficacy of durvalumab in conjunction with tremelimumab but the trial did not meet a successful completion (59).Treatment with dual anti-PD-1 and anti-CTLA-4 plus cisplatin resulted in activation of DCs and CD8⁺CD4⁺ T cells along with suppression of FOXP3⁺ T_reg cells and the effect was more pronounced in BRCA-1 deficient tumors (60). LAG3 is an immune checkpoint that blocks the activation of its host cell to facilitate further suppression in the immune response. LAG525 is an antibody raised against LAG3, which was tested in the setting of mTNBC in a phase II trial, in conjunction with PDR001, an anti-PD1 antibody in the presence or absence of carboplatin (61). The triplet arm showed an ORR of 32.4%. ICOS is a member of the CD28 superfamily that interacts with ligands expressed on B cells and phagocytes, thus promoting downstream signaling to regulate T cell proliferation and release of cytokines. In a phase I/II open-label study involving patients with advanced solid tumors, KY1044, a human anti-ICOS antibody, was tested as monotherapy and in combination with atezolizumab. KY1044 was well-tolerated in both the strategies and one complete response and four partial responses were noted in the TNBC cohort (NCT03829501). Results from the phase II part of the study are underway. Another immune-regulatory protein that suppresses T cell activation and cytokine production, VISTA, is capable of inducing an immunosuppressive environment. HMBD-002 is an antibody against VISTA, which is currently being studied in a phase I study among patients with advanced TNBC (NCT05082610). A summary of the clinical studies involving a combinatorial approach of immunotherapy plus some form of traditional anticancer therapeutic module that have demonstrated safe and favorable disease outcome so far is presented in Table 1 .

The application of immunotherapy in the neo-adjuvant setting, prior to any operative or adjuvant therapy, is expected to induce more beneficial results for cancer patients. This is based on preclinical evidences in animals that show improved immune responses and better survival rates when immunotherapy was administered before tumor resection or while the primary tumor plus the local lymph nodes were intact (62). This superior response is partly attributed to fact that immunotherapy, in a neoadjuvant environment, primes a stronger anti-tumor immune response prior to the changes in the tumor microenvironment or increased tumor antigen heterogeneity. Considering the attractive anticancer impacts of immunotherapy in treating patients with early-stage disease in the adjuvant setting, research now seeks to utilize ICI blockade for treating such patients in the pre-operative or neoadjuvant setup. A 2019 randomized phase II study, enrolling 174 patients with operable TNBC, administered durvalumab in addition to anthracycline/taxane-based chemotherapy in a neoadjuvant setting- the durvalumab-treated arm demonstrated a superior pCR, particularly in the PD-L1-positive subgroup (63). In agreement, a phase Ib study involving 60 high-risk, early-stage TNBC patients displayed a pCR rate of 60% following a combination of pembrolizumab and chemotherapy as neoadjuvant therapy (64). Interestingly, the results from the study also reported a positive correlation between pCR and PD-L1 expression along with stromal TILs. In addition, the IMpassion 031 study recruiting 333 patients explored the outcome of atezolizumab in conjunction with chemotherapy as neoadjuvant therapy in early TNBC subjects (65). Their results suggested that the combination treatment led to a dramatic increase in pCR rate, implying the potential application of atezolizumab as an alternative therapy for patients with TNBC. Besides, an ongoing MIRINAE study is comparing the efficacy of atezolizumab plus capecitabine versus capecitabine alone among TNBC patients having residual tumors following neoadjuvant therapy (NCT03756298). On a similar note, a phase III study examining pembrolizumab plus chemotherapy as neoadjuvant treatment for early-stage TNBC revealed that pembrolizumab successfully increased the pCR rate. Also, data hinted that patients with a heavier tumor burden, advanced stage of the disease and with positive lymph nodes may especially benefit from pembrolizumab (66). Also, neoadjuvant chemotherapy plus pembrolizumab in 250 patients prior to surgery showed a significantly higher pCR rate among the TNBC population, a result that is consistent with the findings from the KEYNOTE-522 study (32). The I-SPY 2 trial (NCT01042379), involving early-stage TNBC patients, initially showed that pembrolizumab administered with neoadjuvant paclitaxel followed by chemotherapy (doxorubicin and cyclophosphamide) resulted in a notably enhanced pCR rate from 22% to 60% and, this was most probably due to the known immunostimulatory effects of anthracyclines. The efficacy of a treatment regimen constituted of pembrolizumab in combination with paclitaxel plus carboplatin followed by anthracycline plus cyclophosphamide as neoadjuvant therapy prior to surgery, and cycles of pembrolizumab administration as adjuvant therapy, was investigated in the KEYNOTE-522 trial (NCT03036488). The pCR rates escalated from 51.2% to 64.8% (NCT03036488). Extensive follow-up research and long-term immune-related adverse effects need to be thoroughly determined to strengthen the observations. Two ongoing key trials are addressing the effect of a year-long adjuvant anti-PD-1/PD-L1 therapy on the survival of TNBC patients- firstly, the SWOG S1418/BR006 trial (NCT02954874) involving pembrolizumab for patients with residual disease and, secondly, the A-brave trial (NCT02926196) examining avelumab for individuals with high-risk or residual disease. In accordance, two additional trials are inspecting the efficacy of atezolizumab in combination with both neoadjuvant and adjuvant therapy on patient survival outcomes- the placebo-controlled NSABP B-59 trial (NCT03281954) testing the efficacy of atezolizumab plus neoadjuvant chemotherapy prior to adjuvant atezolizumab for one year, and the IMpassion030 trial (NCT03498716) studying the standard adjuvant chemotherapy with or without atezolizumab before an annual regime of adjuvant atezolizumab.

Cancer stem cells, in contrast to other cancer cells, are slow-dividing with a repressed tendency to undergo apoptosis and more agile in terms of DNA repair. These features render the cancer stem cells exceptionally refractory to traditional methods of treatment, like irradiation or chemotherapy. Cancer stem cells are known to express ABC drug transporters, which may explain the underlying mechanism towards their resistant nature (67). Disease relapse and tumor metastasis commonly arise from cancer stem cells that are not affected by traditional anticancer therapy. Elimination of breast cancer stem cells (BCSCs) may be accomplished through immunotherapy, which is likely to improve treatment outcomes for breast cancer patients. Although numerous attempts have been made to target specific CSC markers using preclinical models employing various immunotherapeutic approaches, the biggest hurdle has been posed by the non-uniqueness of these markers since most of them are also expressed by normal stem cells. CSCs found in TNBC patients are highly heterogeneous and dynamic, demonstrating variable responses to chemotherapy. Again, HER2-positive BCSCs are characterized by CD44^high/CD24^low phenotype and ALDH1 expression and they support resistance to anti-HER2 therapy, including trastuzumab. Importantly, this population of cells are frequently detected in recurrent breast cancer and not in primary tumors (68). Immunotherapeutic interventions seek to target BCSCs by utilizing immune cells such as NK cells, CD8⁺ T cells and γδ T cells (69). Till date, many surface markers have been reported for BCSCs including CD90, CD49, CD44, CD24, ALDH and EpCAM (70). Elimination of CSCs was achieved in vitro in breast cancer cell lines with ALDH-specific CD8⁺ T cells, which resulted in significant amelioration of mammary tumor development and metastases with prolonged survival (71, 72). Clinical trials are presently investigating CAR-T cells targeting CD44v6 (NCT04430595) and EpCAM (NCT02915445) surface antigens as an effective anticancer module for advanced breast carcinoma. Other studies recorded that BCSC-DCs can effectively inhibit BCSC proliferation when administered into the circulation of BCSC tumor-harboring rodents, suggesting the therapeutic potential of BCSC antigen-primed DCs for elimination of BCSCs (73). These results were further strengthened in additional murine models, wherein BCSC-primed DCs had a positive effect on the survival time by 70% (74).

Vaccination strategies based on DCs encompass either antigen-defined vaccines or polyvalent vaccines (75). In a mouse model of spontaneous mammary tumorigenesis, a DC-based vaccine specifically targeting HER-2/neu led to the production of anti-neu antibodies along with T-cell mediated expression of interferon-γ, resulting in tumor regression (76). Encouraging results were also observed in patients with metastatic breast carcinoma, who were administered with lysate-pulsed DCs (NCT02063893). Nonetheless, immunotherapy approaches that target a single antigen often fail to eradicate the population of cells that contribute to tumor initiation or cancer metastasis; therefore, this significantly lowers the long-term success of this strategy. To circumvent this concern, emerging studies suggest targeting of multiple antigens for an effective response and one way to accomplish this is through polyvalent vaccines. In stage IV melanoma, a DC/irradiated tumor vaccine displayed complete tumor remission in 3 patients and a partial disease remission in an additional 3 out of 46 patients (77). In an interesting study, human heterokaryons were prepared that expressed both breast tumor-associated antigens and costimulatory molecules derived from DCs. These functionally active fusion cells could successfully induce autologous T cell proliferation and stimulate cytotoxic-T lymphocyte activity to fight against autologous breast cancer cells (78). Development of a polyvalent vaccine for BCSCs requires identification of as many antigens as possible that are unique to BCSCs. Determining the presence of mutations that facilitate the stem cell-like phenotype in BCSCs and the underlying mechanisms may unearth important avenues for immunotherapy. Moreover, chemokine receptors that promote migration of BCSCs can also be explored as future targets for immunotherapy. Overall, harnessing DC-based vaccines may be a viable option for targeted elimination of BCSCs (79). Immuno-targeting of BCSCs holds great clinical significance in an adjuvant setting as it can abrogate the BCSC population and can, therefore, improve the outcome of existing therapies.

TILs are vital indicators of tumor immunogenicity (80), hence, the presence of TILs serves as a prognostic marker in many malignancies, including breast cancer (81). TILs collectively constitute of the T lymphocytes (CD8⁺, CD4⁺ and T_reg cells), B lymphocytes and natural killer cells present within the tumor. These lymphocytes impart crucial functions in breast carcinogenesis and immune recognition. The basic mode of action of CD8⁺ T cells is the induction of direct cytotoxicity to the cancer cells whereas CD4⁺ T cells primarily promote release of inflammatory cytokines to evoke anticancer immunity (82). On the other hand, the CD4⁺ T_reg population promotes an immune-suppressive environment by restricting the activation and subsequent function of CD8⁺ T cells (83). TILs are considered responsible for superior disease outcomes among breast cancer patients and are associated with relapse-free survival (84). However, we still do not entirely understand the T cell subtypes in breast carcinoma. One subset of the CD8⁺ TILs is represented by the CD8⁺ tissue-resident memory (TRM) cells that express cytotoxic molecules and immune checkpoint factors (85). These cells were found to positively correlate with higher relapse-free survival in TNBC patients (86). The presence of TRMs also favor improved prognosis among early-stage TNBC patients, denoted by better survival and reduced rates of tumor recurrence. Again, the presence of CD39⁺PD-1⁺CD8⁺ T cells in the tumors is intimately connected with prolonged DFS of breast cancer patients (87). Importantly, the FOXP3⁺ T_reg cells contribute to more aggressive outcomes in breast cancer, characterized by an enhanced likelihood of relapse and poor survival (88). A study found that the intra-tumoral infiltration of CD8⁺ T cells led to a notable drop in the risk of death among 12,439 breast cancer patients. This was especially evident for TNBC and HER2+ tumors, who demonstrated a 28% reduction in mortality while ER+, HER2+ tumors had a 27% reduction in mortality (89). TIL therapy involves isolation of TILs from patients and expanding them in an ex vivo setup with considerable amounts of IL-2 and other necessary cytokines, followed by their re-infusion into the patient (90). Since TNBC patients express increased number of neoantigens relative to other subtypes, as revealed by whole genome sequencing of breast cancer tissues, TNBC patients may serve as possible candidates for TIL therapy (91). In accordance, preliminary data from an ongoing trial (NCT01174121) has reported tumor regression in a subset of patients in response to TIL therapy (92).

Despite the emerging studies, the effects of the intra-tumoral population of immune cells in dictating response of breast cancer patients to different modes of treatment, specifically immunotherapy, are not fully defined. Importantly, the proportion of the intra-tumoral immune infiltrates is an important factor that determines breast cancer patient response to therapy. In the SweBCG91RT trial, early-stage breast cancer patients possessing immune infiltrates with anti-tumor effects exhibited a lower risk of tumor recurrence (93). Limited benefits were observed in the test subjects following addition of radiotherapy. A high TIL count has been shown to promote sensitization of tumors to chemotherapy, resulting in a high pCR to pre-operative chemotherapy among primary breast cancer patients (94). Another study involving around 3,000 breast cancer patients found that increased TIL counts exerted a survival benefit with an improved response to neoadjuvant chemotherapy in TNBC and HER2-enriched mammary tumors (95). On the contrary, a high TIL count was associated with adverse prognosis in luminal breast cancer. Furthermore, DFS was sharply worse for TNBC patients with TIL^low tumors compared to patients with TIL^high tumors (96). TILs are, therefore, intimately involved in tumor prognosis, chemotherapeutic outcome and selection of immunotherapy or adoptive cell therapy in TNBC patients (97). Till date, most studies have focused on the prognostic relevance of TILs in breast cancer. However, attractive properties such as diversity of the receptors, tumor specificity, and lack of toxicity have pushed TILs as a promising candidate for therapy (98).