Immunotherapy in Triple-Negative Breast Cancer

Abstract

Currently, immunotherapy has led to a paradigmatic shift in the treatment of many cancer types, including triple-negative breast cancer. Immunotherapy increases the efficacy of the immune system in treating cancer, with a durable effect due to immunologic memory. The PD-1 inhibitor, pembrolizumab, combined with neoadjuvant chemotherapy, improved event-free survival and is a new standard of care for patients with high-risk, early stage triple-negative breast cancer (TNBC), regardless of tumor PD-L1 expression. For metastatic TNBC, pembrolizumab combined with chemotherapy is a new standard of care for first-line therapy for PD-L1⁺ metastatic TNBC, and it improves overall survival. The PD-L1 inhibitor, atezolizumab, combined with nab-paclitaxel, is also approved for first-line treatment of metastatic PD-L1⁺ TNBC. The aim of this review is to examine the existing evidence and ongoing studies on immunotherapy in patients with early stage and metastatic triple-negative breast cancer (TNBC), including new combination strategies with several drugs, such as chemotherapy, targeted therapy, or radiation and to discuss immune checkpoint inhibitor (ICI) applications and the possibility of emerging strategies in different TNBC stages.

Key Summary Points

Immunotherapy increases the efficacy of the immune system in treating cancer.

The PD-1 inhibitor, pembrolizumab, combined with neoadjuvant chemotherapy, followed by adjuvant pembrolizumab, improved event-free survival and is a new standard of care for patients with high-risk, early stage triple-negative breast cancer (TNBC), regardless of tumor PD-L1 expression.

For metastatic TNBC, pembrolizumab combined with chemotherapy is a new standard of care as the first-line therapy for PD-L1 + metastatic TNBC, with improvement in overall survival. The PD-L1 inhibitor, atezolizumab, combined with nab-paclitaxel, is also approved as the first-line treatment for metastatic PD-L1 + TNBC.

The aim of this review is to examine the existing evidence and the ongoing studies on immunotherapy in patients with early stage and metastatic triple-negative breast cancer (TNBC), including new combination strategies with several drugs.

Introduction

Triple-negative (TN) breast cancer (BC) is a challenging disease to treat owing to its aggressive behavior and lack of actionable targets, which leads to routine management with chemotherapy (CT). The use of immune checkpoint inhibitors (ICIs) in this particular histological type of BC represents a new therapeutic approach.

TNBC is characterized by a high tumor mutational burden (TMB) compared with that of other subtypes of BC, a feature that is linked to increased response to immune checkpoint inhibitors (ICIs) [1].

The immune checkpoints (ICs) are negative regulators of T cell immune function. The main ICs are programmed death receptor 1 (PD-1) and cytotoxic T cell antigen 4 (CTLA-4) [2].

PD-1, which is expressed by T lymphocytes, interacts with programmed death ligand 1 and 2 (PD-L1, PD-L2) present on tumor cells.

This interaction causes:

• -the inhibition of T cell proliferation

• -the inhibition of the production of interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α)

• -a reduction in survival [3].

CTLA-4 inhibits the link between T cells and antigen-presenting cells (APCs), which reduce the immune response against neoplastic cells [4]. Tumor cells take advantage of this mechanism to create an immunosuppressive microenvironment [4]. Inhibiting immune checkpoints facilitates the immune response against neoplastic cells, which is the goal of anticancer treatment. The aim of this review is to summarize the available immunotherapy strategies in both neoadjuvant and adjuvant settings for the treatment of both early stage and metastatic triple-negative breast cancer.

This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors.

Early Stage TNBC

Compared with other breast cancer subtypes, triple-negative breast cancers (TNBCs) are characterized by a worse prognosis, earlier metastasis, and poor OS (overall survival) after recurrence. Anthracycline- and taxane-based chemotherapy is still the mainstay of treatment in the early stages, although several escalation approaches have been evaluated to improve survival outcomes. The addition of platinum salts to standard neoadjuvant chemotherapy (NACT) remains controversial owing to the lack of a clear survival advantage [5], and the use of the adjuvant capecitabine represents a valid treatment option for patients with TNBC with residual disease after NACT [6].

In addition to BRCA1/2 mutation status and PD-L1 expression, biomarkers identifying patients most likely to respond to systemic treatment are lacking.

Chemotherapy can be given to treat high-risk and early stage TNBC in the neoadjuvant and/or adjuvant setting. Given that there are no differences in survival between the adjuvant and neoadjuvant settings, NACT is now considered the standard approach for treating patients with high-risk TNBC to reduce the tumor burden and evaluate chemotherapeutic efficacy prior to surgical resection [7]. NACT provides a number of distinct advantages, including (1) the possibility of reducing the number of mastectomies for primary breast tumors (e.g., segmental instead of total mastectomy); (2) a reduced scope of axillary node resection to reduce the number of complete axillary node resections (e.g., sentinel node biopsy versus complete axillary node dissection); (3) time to consult plastic surgeons and genetic counselors prior to surgery; and (4), most significantly, an opportunity to assess tumor response, prognosis, and the potential need for additional or adjuvant treatments. NACT is interactive, quantitative, evidence-driven, and a preferred option compared with adjuvant chemotherapies, which are largely ineffective without the primary tumor as a surrogate marker postsurgery [8].

Completed Studies

Compared with metastatic disease, early TNBC has a reduced immunosuppressive phenotype [9]. Therefore, there is a strong biological rationale for testing immunotherapeutic strategies in both neoadjuvant and adjuvant settings for this subtype. Many chemotherapy regimens have immunomodulatory effects and are able to promote the death of tumor cells and release antigens that recruit antigen-presenting cells (APCs). This suggests that the combination of chemotherapy and immune therapy may produce better antitumor effects.

Neoadjuvant Chemotherapy + ICI

Several studies have investigated the impact of the addition of immunotherapy agents to NACT in patients with TNBC. The results have been published from phase I, II, and III studies evaluating the addition of pembrolizumab, atezolizumab, or durvalumab to different chemotherapy combinations.

The phase Ib KEYNOTE-173 study was conducted to assess the safety and preliminary antitumor activity of neoadjuvant chemotherapy plus pembrolizumab in patients with high-risk, early stage, nonmetastatic TNBC. The rate of pathological complete response (pCR, i.e., ypT0/Tis ypN0) was 60% and was positively correlated with tumor PD-L1 expression and stromal tumor-infiltrating lymphocyte (TIL) levels. [10]

The I-SPY2 study is an open-label, multicenter, adaptively randomized phase II platform trial for high-risk, stage II/III BC in which multiple arms are evaluated in parallel. Standard NACT is the common control arm; investigational agents are added to this backbone. Patients were randomized to receive taxane- or anthracycline-based NACT with or without pembrolizumab, followed by definitive surgery. The primary endpoint was pCR. The secondary endpoints were residual cancer burden (RCB), 3-year event-free survival (EFS), and distant recurrence-free survival. The pCR rate for patients with TNBC in the pembrolizumab arm was 60%, whereas it was 22% in the control population.

A lower percentage of patients in the pembrolizumab arm than in the control arm had RCB-III at the time of surgery [11].

The KEYNOTE-522 trial was the first prospective randomized controlled phase III study to show a benefit from adding pembrolizumab to NACT in patients with early TNBC. The trial randomized 602 patients to receive neoadjuvant treatment plus pembrolizumab or placebo with paclitaxel and carboplatin, followed by pembrolizumab or placebo plus doxorubicin and cyclophosphamide (AC) or EC. After surgery, patients received adjuvant pembrolizumab or placebo for 1 year. The primary endpoints were the pCR rate and EFS—the percentage of patients who responded to treatment was significantly greater in the pembrolizumab arm (64.8% versus 51.2%, delta 13.6, p = 0.0005). The efficacy of pembrolizumab was shown to be consistent regardless of PD-L1 status. Moreover, at a median follow-up of 39 months, the addition of pembrolizumab to NACT improved the 3-year EFS from 76.8 to 84.5% [hazard ratio (HR) 0.63, p = 0.0003] [12].

The NeoTRIPaPDL1 trial investigated the benefit of adding atezolizumab to NACT in patients with early TNBC. In this phase III study, 280 patients with early TNBC were randomized to receive neoadjuvant treatment with carboplatin and nab-paclitaxel with or without atezolizumab, followed by surgery and then adjuvant chemotherapy. The primary endpoint was 5-year EFS. The pCR rate after treatment with atezolizumab (48.6%) did not reach statistical significance compared with that after treatment without atezolizumab [44.4%, odds ratio (OR) 1.18]; follow-up for EFS is ongoing [13].

The IMpassion031 study is a randomized phase III trial in which 333 patients received datezolizumab or placebo in combination with nab-paclitaxel (weekly for 12 weeks), followed by atezolizumab or placebo every 2 weeks for four cycles with a dense AC (anthracycline and cyclophosphamide). After surgery, patients in the atezolizumab arm continued to receive immunotherapy for 11 cycles. The coprimary endpoints were the pCR rate in both the entire population and the PD-L1⁺ population, and the secondary endpoints were EFS in both populations. The pCR rate significantly improved in the atezolizumab arm (58% versus 41%, p = 0.004), regardless of PD-L1 status. There was a favorable trend in survival in the experimental arm; however, the EFS outcomes were still immature [HR 0.76; 95% confidence interval (CI) 0.40–1.44] after a follow-up of 20.6 months [14].

The GeparNuevo phase II trial evaluated the addition of durvalumab to standard anthracycline and taxane-based neoadjuvant therapy in 174 patients with early TNBC. Patients received durvalumab or placebo monotherapy 2 weeks prior to the start of chemotherapy (window phase), followed by treatment with durvalumab/placebo plus nab-paclitaxel and then with durvalumab/placebo plus dose-dense (DD) epirubicin and cyclophosphamide (EC). The primary endpoint was the pCR rate (ypT0 ypN0). The study was amended, and the window phase was interrupted after 117 patients were recruited. Subsequently, all patients started durvalumab/placebo plus chemotherapy on the first day. The addition of durvalumab increased the pCR rate in all patients, but this increase was not statistically significant (53.4% versus 44.2%; OR 1.45, 95% CI 0.80–2.63; p = 0.224). Of note, among the 117 patients who participated in the “window phase,” receiving a single dose of their assigned study group prior to NACT, those who received durvalumab had a significantly greater pCR (61.0% versus 41.4%, OR 2.22, 95% CI 1.06–4.64; p = 0.035), suggesting a potential benefit for this kind of “immune priming” [15].

Another phase I/II trial evaluated the addition of durvalumab to weekly nab-paclitaxel and dose-dense doxorubicin/cyclophosphamide (ddAC) neoadjuvant therapy for patients with stage I–III TNBC. Seven patients were included in the phase I phase of the study, four at 3 mg/kg and three at 10 mg/kg. In total, 52 patients were enrolled in the phase II phase at a 10 mg/kg dose. In phase II, where patients received a dose of 10 mg/kg durvalumab (N = 55), the pCR rate was 44% (N = 24, 95% CI 30–57%). Among the 57 patients who received durvalumab at any dose and completed surgery, the pCR rate was 46%, including two pCRs among the four patients who received 3 mg/kg durvalumab in the phase I part of the study. The response was related to PD-L1 expression and stromal tumor-infiltrating lymphocytes (sTILs) [16]Table 1.

Table 1.

Main clinical trials studying immune checkpoint inhibitors in early TNBC

Trial designation	Phase	Study population	Study treatment	Outcomes	Study completion	Biomarker
KEYNOTE-173 60 patients	Phase Ib	Stage I, II, and III TNBC	Neoadjuvant (pembrolizumab + six different chemotherapy regimens) → doxorubicin plus cyclophosphamide	pCR: 60%	18 November 2019	–
I-SPY NCT01042379	Phase II	Stages II and III TNBC Stages II–III HER2−	Neoadjuvant treatment (P→AC) ± pembrolizumab	pCR 60% versus 22% in the pembrolizumab and control arms, respectively	Ongoing	–
KEYNOTE-522 (NCT03036488) 602 patients	Phase III	Stages II and III TNBC	Neoadjuvant (P + Cp) ± pembrolizumab →  AC or EC ± pembrolizumab	pCR: 64.8% in pembrolizumab arm versus 51.2% in placebo (p = 0.00055) At 3-year: EFS: 84.5% in pembrolizumab arm versus 76.8% placebo (HR = 0.63, p = 0.0003)	Ongoing	PD-L1
NeoTRIP [18] (NCT02620280) 280 patients	Phase III	Stages II and III (allowed N3 +) TNBC	Neoadjuvant (Nab-P + Cp) ± atezolizumab	pCR: 48.6% in atezolizumab arm versus 44.4% control (p = 0.48)	7 January 2024	PD-L1
IMPASSION 031 (NCT03197935) 333 patients	Phase III	Stages II and III TNBC	Neoadjuvant Nab-P ± atezolizumab →  ddAC ± atezolizumab	PCR 57.6% in atezolizumab arm versus 41.1% placebo (p = 0.0044)	28 September 2022	PD-L1
GeparNuevo NCT02685059 174 patients	Phase II	Stages II–III TNBC	Neoadjuvant Nab-P ± durvalumab →  ddEC	pCR rate 53.4% in durvalumab arm versus 44.2% placebo	March 2018	–
NCT02489448 68 patients	Phase	Stages I–III TNBC	Neoadjuvant Nab-P ± durvalumab →  ddAC	pCR rate of 44% (durvalumab 10 mg/kg)	26 October 2022	–
An arm of the I-SPY2 409 patients				pCR rate 47% durvalumab + olaparib versus 27% placebo	2 June 2019	–

ICIs + PARP Inhibitors

Preclinical studies suggest the potential for synergistic improvement using a combination of immune checkpoint inhibitors and PARP inhibitors [17].

The impaired nucleotide and base excision repair caused by PARP inhibition can increase mutation and neoantigen loads, which are associated with increased sensitivity to immune checkpoint therapy due to recruitment of tumor-infiltrating lymphocytes (TILs) to tumor microenvironment [18].

One arm of the I-SPY2 trial investigated the combination of durvalumab and olaparib with weekly paclitaxel neoadjuvant therapy [durvalumab/olaparib/paclitaxel (DOP)] compared with chemotherapy alone in HER2⁻, stage II–III breast cancers (TNBC, HR+/HER2⁻). In patients with TNBC, durvalumab and olaparib improved the pCR rate from 27 to 47%, and there was a reduction in residual cancer across the entire spectrum of RCB scores in all HER2⁻ subtypes [19].

Ongoing Studies

The introduction of immunotherapy marked a revolution in the treatment of early stage TNBC. The results of the abovementioned studies have shown that, by unleashing anticancer immune responses through ICIs, long-term benefits can be obtained for the treatment of this aggressive BC subtype. However, this represents a starting point, and additional efforts are required to precisely implement immunotherapy for the treatment of TNBC [20].

The ongoing trials on early stage TNBC are summarized in Table 2.

Table 2.

Ongoing trials with checkpoint inhibitors plus chemotherapy in early breast cancer

Trial identifier	Phase	Setting	Study treatment	Principal endpoint	Study completion	Biomarker
NCT02950259 16 patients	Phase I	ESBC of any receptor subtype TNBC T1c +	IRX-2 regimen prior to chemotherapy	1. Number of surgeries delayed owing to adverse events from the IRX-2 regimen 2. TILs	Ongoing	–
NCT03756298 284 patients	Phase II	Early TNBC	Atezolizumab plus capecitabine adjuvant therapy	5 years—DFS	Ongoing	–
Neo ACT NCT04877821 46 patients	Phase II	Early TNBC	Sintilimab plus anlotinib combined with chemotherapy as neoadjuvant therapy	pCR	Ongoing	–
BIS-Program NCT05180006 210 patients (147 in TNBC cohort and 63 in HER2+)	Phase II	Early TNBC or HER2+ breast cancer	Atezolizumab monotherapy or in combination with other biologic agents (ipatasertib/bevacizumab/trastuzumab/pertuzumab)	Increased levels of activated GzmB+ CD8+ T cells from baseline to post-treatment	Ongoing	Activated GzmB+ CD8+T cells
NCT04848454 30 patients	Phase II	Early stage HER2− breast cancer (TNBC and HER2− hormone receptor positive)	6 cycles of neoadjuvant therapy of camrelizumab combined with vinorelbine and cisplatin (NP) as second-line neoadjuvant therapy	pCR	Ongoing	tumor infiltrating lymphocytes, expression of PD-L1
NCT03281954 1550 patients	Phase III	Early TNBC	Neoadjuvant chemotherapy (paclitaxel + carboplatin + followed by AC) plus atezolizumab/placebo and adjuvant atezolizumab/placebo	pCR 5 year-EFS	Ongoing	–
A-BRAVE NCT02926196 474 patients	Phase III	Early TNBC	1 year of adjuvant avelumab versus observation	DFS	Ongoing	–
IMpassion030 NCT03498716 2300 patients	Phase III	Stage II–III TNBC	Atezolizumab in combination with adjuvant anthracycline/taxane-based chemotherapy	iDFS	Ongoing	PD-L1

TNBC triple-negative breast cancer, pCR pathologic complete response, EFS event-free survival, DFS disease-free survival, iDFS invasive disease-free survival, TILs tumor-infiltrating lymphocytes

The efficacy of ICIs as adjuvant therapy for early stage TNBC is being assessed in the ongoing IMpassion030 (NCT03498716) trial, which tested atezolizumab in combination with adjuvant chemotherapy. Several trials are also evaluating the use of ICIs, including their combination with capecitabine (NCT03756298), for patients with invasive residual disease following neoadjuvant treatment [21].

NeoSACT (NCT04877821) is an open-label phase II trial evaluating the efficacy and safety of sintilimab plus anlotinib combined with chemotherapy as neoadjuvant therapy in patients with early stage TNBC.

The BIS-Program (NCT05180006) will help to assess whether short-term immunotherapy with atezolizumab monotherapy or in combination with other biologic agents (ipatasertib/bevacizumab/trastuzumab/pertuzumab) is associated with increased levels of activated GzmB+ CD8+ T cells from baseline to post-treatment (using immunohistochemistry of biopsies and surgically removed tumors).

NCT04848454 is a single-arm phase II clinical trial designed to test the efficacy and safety of camrelizumab (a PD-1 inhibitor) combined with vinorelbine and cisplatin as a second-line therapy for patients with HER2⁻ breast cancer who did not achieve a significant effect after two cycles of anthracycline plus taxane NACT (neoadjuvant chemotherapy).

Another ongoing study, NCT02950259, aimed to assess the safety and tolerability of the IRX-2 regimen in patients with ESBC and to estimate the pathologic complete response rate to neoadjuvant anthracycline-based and nonplatinum-containing chemotherapy in patients with triple-negative breast cancer who received the IRX-2 regimen before chemotherapy.

Moreover, NCT03281954 aimed to determine whether the usual chemotherapy regimen given before surgery (paclitaxel and carboplatin followed by doxorubicin and cyclophosphamide or epirubicin and cyclophosphamide) for breast cancer plus atezolizumab was better than the usual chemotherapy plus a placebo.

A-Brave NCT02926196 is a 1-year phase III randomized trial studying anti-PD-L1 antibody avelumab versus placebo as adjuvant or post-neoadjuvant treatment for patients with high-risk triple-negative breast cancer.

High-risk disease is defined as:

• -invasive residual disease (breast and/or nodes) after neoadjuvant chemotherapy (stratum A)

• -> pN2/any pT, pN1/pT2, or pN0/pT3 after primary surgery (stratum B)

The co-primary endpoints are disease-free survival (DFS) in the total population and in stratum A.

The secondary end point is OS.

Avelumab reduces the risk of death by 34% in the overall population but does not improve the co-primary endpoint of DFS [22].

Finally, Impassion030 is a phase III randomized trial studying atezolizumab plus standard adjuvant chemotherapy versus chemotherapy alone in stage II–III TNBC.

The primary endpoint is DFS in the intention-to-treat population.

The final analysis (32 months of follow-up) showed no disease-free or overall survival benefit with the addition of atezolizumab to adjuvant chemotherapy.

The subgroup analysis additionally confirmed the absence of benefit in the patients with node-positive disease and with PD-L1-expressing tumor [23].

Metastatic TNBC

Approximately 25% of patients with localized TNBC relapse with distant metastasis. For patients with advanced or stage IV disease, the median overall survival (OS) is almost 12 months, with fewer than 20% of patients alive at 4 years. Only 5% of patients with TNBC present with metastatic disease de novo [24]. Unfortunately, the majority of patients relapse following treatment with curative intent. TNBC is most commonly associated with visceral metastases, including lung, liver, and brain metastases [25].

Systemic chemotherapy is the backbone therapy recommended for TNBC, even in metastatic forms [7]. Standard chemotherapy regimens for TNBC are usually based on a combination of anthracyclines, alkylators, and taxanes [26].

The survival rates for patients with chemoresistant, relapsed, and metastatic TNBC have not improved significantly over the past 30 years [8]. High-risk and locally advanced TNBC tumors have high inter- and intratumor heterogeneity, which becomes more pronounced in chemoresistant, relapsed, and metastatic settings. The ability of chemoresistant TNBC to save more patients with progressive and metastatic disease has consistently challenged our ability to design better-targeted therapies [27].

Completed Studies

Monotherapy

ICI monotherapy demonstrated antitumor activity in the treatment of advanced TNBC, but the response rates were different. In terms of ICI first-line therapy in advanced TNBC, pembrolizumab showed an objective response rate (ORR) of 18.5% or 21.4% in the PD-L1⁺ subgroup [28, 29].

Atezolizumab had a 24% ORR. [29] However, the use of ICIs as second- or higher-line therapy in patients with advanced TNBC has low ORRs, at only 6% or 5.3%, respectively [30, 31].

A higher ORR was observed for first-line PD-1/PD-L1 inhibitor therapy.

The PFS of patients with advanced TNBC receiving ICI monotherapy is generally 1.4–2.1 months. The OS is generally 9–18 months.

The KEYNOTE-119 [32] phase III, open-label, randomized study compared pembrolizumab monotherapy (n = 312) with single-agent chemotherapy (n = 310) in previously treated patients with metastatic triple-negative breast cancer (mTNBC; 1–2 prior systemic treatments). The patients were stratified as PD-L1⁺ [combined positive score (CPS) ≥ 1] or PD-L1⁻ (CPS < 1). The primary endpoint was OS in patients with a combined positive score ≥ 10, patients with a CPS ≥ 1, and all patients. The secondary endpoints were PFS, ORR, and safety. Pembrolizumab did not improve OS in patients with a CPS ≥ 10 or a CPS ≥ 1. In an exploratory analysis of patients with a CPS ≥ 20, the median OS was 14 versus 12.5 months for patients treated with chemotherapy (HR: 0.58, 95% CI 0.38–0.88), and no improvement in PFS was observed.

The JAVELIN Solid Tumor trial was designed to evaluate the efficacy and safety of avelumab in patients with advanced breast cancer. One trial revealed that the antitumor efficacy of avelumab was not related to the amount of PD-L1 expressed in tumor cells but was associated with the amount of PD-L1 expressed in ICs. In patients with PD-L1⁺ or PD-L1⁻ ICs (10% threshold), the ORR was 16.7% versus 1.6% in the overall group (p = 0.039) and 22.2% versus 2.6% in the TNBC subgroup, respectively [33].

Generally, ICI monotherapy was effective but did not significantly improve outcomes in patients with advanced TNBC. Therefore, most studies currently focus on combination therapy.

Combination Therapy

Two drugs, pembrolizumab and atezolizumab, have been studied in phase III trials in the metastatic TNBC population [34–36]. The first of these, Impassion 130, studied the role of atezolizumab in combination with nab-paclitaxel for patients with unresectable, locally advanced or metastatic TNBC who had not received prior therapy in the metastatic setting[34]. After 12.9 months of follow-up, the intention-to-treat population showed a modest improvement in median progression-free survival (PFS) of 7.2 months for the atezolizumab and nab-paclitaxel groups compared with 5.5 months for the placebo and nab-paclitaxel groups (HR 0.80, 95% CI 0.69–0.92). There was also a modest improvement in the median overall survival (OS) of 21.3 months in the atezolizumab and nab-paclitaxel groups compared with 17.6 months in the placebo and nab-paclitaxel groups (HR 0.84, 95% CI 0.69–1.02). Although this was not preplanned from the original statistical plan, both PFS and OS were significantly greater among patients with PD-L1⁺ tumors, defined as those with PD-L1 staining in ≥ 1% of tumor-infiltrating immune cells. Among the PD-L1⁺ group, the median PFS was 7.5 months with atezolizumab and nab-paclitaxel versus 5.0 months with the placebo and nab-paclitaxel (HR 0.62, 95% CI 0.49–0.78), and the median OS was 25 months versus 15.5 months (HR 0.62, 95% CI 0.45–0.86), which was maintained at the final OS analysis [32]. This led to fast-track approval by the US Food and Drug Administration (FDA) to use a combination of atezolizumab and nab-paclitaxel for the treatment of metastatic TNBC.

However, it was later withdrawn following the publication of Impassion 131, which analyzed the combination of atezolizumab and nab-paclitaxel compared with a placebo and nab-paclitaxel. Despite a similar design, this study failed to show an improvement in PFS or OS among the intention-to-treat population and the PD-L1⁺ population [35]. The reasons postulated to explain this lack of benefit mainly included the choice of chemotherapy, the effect on the tumor microenvironment, and the use of steroids.

The European Medicines Agency (EMA) approved the use of nab paclitaxel for the treatment of patients with metastatic TNBC with PD-L1 ≥ 1% and no prior chemotherapy for metastatic disease.

The KEYNOTE-355 phase III study (NCT02819518) randomized 847 patients with mTNBC 2:1 to receive a physician’s choice of chemotherapy (paclitaxel, nab-paclitaxel, or gemcitabine and carboplatin) with either pembrolizumab or placebo in the first-line setting. [37] Eligible patients either had de novo mTNBC or relapsed at least 6 months after treatment in the early stage setting (compared with 12 months for the IMpassion trials). Among the 38% of patients whose tumors were PD-L1⁺, defined as a CPS > 10, the addition of pembrolizumab to chemotherapy significantly improved both PFS and OS (PFS: 9.7 versus 5.6 months, HR 0.66; p = 0.0012; OS 23.0 versus 16.1 months, HR 0.73; p = 0.0093). Both the ORR (53.2% versus 39.8%) and duration of response (19.3 versus 7.3 months) improved with pembrolizumab in patients who were PD-L1⁺ (CPS > 10) with mTNBC [38, 39].

On the basis of these data, the combination of pembrolizumab with the physician’s choice of chemotherapy (weekly paclitaxel, nab-paclitaxel, or gemcitabine and carboplatin) received accelerated approval for patients with PD-L1⁺ (CPS > 10) mTNBC by the US FDA in November 2020 and final approval in July 2021.

In addition, ICIs plus targeted drugs have also demonstrated feasible clinical efficacy for patients with advanced TNBC. The KEYNOTE 162 phase I/II trial evaluated the efficacy and safety of pembrolizumab plus niraparib (a PARP inhibitor) in patients with advanced TNBC. The ORR of the overall population was 29%. In particular, the ORR of the BRCA mutation subgroup was quite high, at 67%. Patients who were PD-L1⁺ (> 1%) had a greater ORR than those who were PD-L1⁻ (33% and 15%, respectively) [40].

There are also studies underway evaluating the safety and efficacy of combining a PARP inhibitor with cytotoxic chemotherapy, other targeted therapies, and/or ICIs. In particular, the combination of PARP inhibitors with ICIs is a promising area of research.

Trial	Phase	Treatment	Type of drugs	Principal endpoint	Study completion	Biomarkers
Keynote 012	Phase Ib	Pembrolizumab	Anti PD-1	ORR: 18,5% mPFS: 1.9 mOs: 11.2	Completed	PD-L1
Keynote 086 coorte A	Phase II	Pembrolizumab	Anti PD-1	ORR: 5,3% mPFS:2.0 mOS:9.0	Completed	PD-L1
Keynote 086 coorte B	Phase II	Pembrolizumab	Anti PD-1	ORR: 21,4% mPFS:2.1 mOS:18.0	Completed	PD-L1+
NCT01375842	Phase I	Atezolizumab	Anti PD-L1	ORR: 24% (first-line) mPFS: 1.4 mOS: 17.6	Completed	–
NCT02555657 Keynote-119	Phase III	Pembrolizumab	Anti PD-1	mPFS: 2.1 versus 2.1 (HR: 1.14) mOS: 12.7 versus 10.7 (HR: 0.78)	Completed	CPS
Javelin NCT01772004	Phase 1b	Avelumab	Anti PD-1	ORR: 5.2% mPFS: 1.5 mOS: 9.2	Completed	–
Impassion-130 NCT02425891	Phase III	Atezolizumab + nab-paclitaxel (comparator: placebo + nab-paclitaxel)	Anti PD-L1 + chemotherapy	ITT mPFS 7.2 mesi versus 5.5 (HR 0.80, IC 95% 0.69–0.92) mOS 21.3 mesi versus 17.6 mesi (HR 0.84, IC 95% 0.69–1.02) PD-L1 > 1% mPFS 7.5 mesi versus 5.0 mesi (HR 0.62, IC 95% 0.49–0.78)	Completed	–

CPS Combined Positive Score

In the phase II MEDIOLA trial (NCT02734004), 34 patients with germline BRCA1 or BRCA2 mutations and HER2⁻ metastatic breast cancer (MBC) were treated with a combination of the PD-L1 inhibitor durvalumab and olaparib until disease progression [46, 47]. The primary efficacy endpoint of disease control at 12 weeks was achieved in 24 patients (80%), the median duration of response was 9.2 months, and the median PFS was 8.2 months.

Liu et al. initially report the efficacy of combining anti -PD-1 antibody and antiangigenetic agents for advanced TNBC.

Patients received continuous or intermittent camrelizumab and apatinib oral 250 mg.

In the continuous-dose apatinib group, the ORR was 43.3%, while no objective response was found in the intermittent-dose apatinib group. Therefore, combination treatment with camrelizumab and continuous-dose apatinib demonstrated favorable antitumor activity and good tolerance for advanced TNBC. In addition, in this trial, in the continuous-dosing group, TILs > 10% were associated with a higher ORR and favorable PFS. [41]

In the phase Ib/II ENHANCE1 trial (NCT02513472), 167 patients with mTNBC who had received 0–2 prior lines of therapy were treated with the combination of pembrolizumab and the microtubule inhibitor eribulin [42]. In this study, the ORR was 25.8% for patients with no prior systemic anticancer therapies (n = 66) and 21.8% for patients with 1–2 prior systemic anticancer therapies (n = 101).

In another phase II study (NCT03044730), 30 patients with MBC (16 with mTNBC and 14 with hormone receptor-positive (HR+)/HER2⁻ MBC) were treated with pembrolizumab and capecitabine, with a median PFS and OS of 4 and 15.4 months, respectively [43], similar to those of historical controls treated with capecitabine alone.

In the first stage of the phase II TONIC trial (NCT02499367), 67 patients with mTNBC were randomized to receive a short induction regimen (irradiation, cyclophosphamide, cisplatin, doxorubicin, or no induction) followed by nivolumab. The ORR was 20%, with higher responses in the doxorubicin (35% ORR) and cisplatin (23% ORR) cohorts [44].

Doxorubicin or cisplatin induction resulted in the upregulation of immune-related genes involved in PD-1/PD-L1 and T cell cytotoxicity pathways. These data suggest that short-term treatment with low-dose doxorubicin and cisplatin could induce a more favorable tumor microenvironment (TME) and increase the likelihood of a response to anti-PD-1 therapy in patients with TNBC, which is currently under investigation.

Patients with mTNBC were treated with pembrolizumab plus the PARP inhibitor niraparib [48]. In 15 evaluable patients with tumor BRCA mutations, the ORR was 47%, and the median PFS was 8.3 months; in 27 patients with wild-type BRCA mutations, the ORR was 11%, and the median PFS was 2.1 months.

Therefore, the authors concluded that the combination of niraparib plus pembrolizumab provided promising antitumor activity, with higher response rates in those with tumor BRCA mutations.

In addition, tyrosine chain inhibitors are being studied in combination with ICIs.

In a recent phase Ib study (NCT03800836), the triple combination of ipatasertib, atezolizumab, and paclitaxel or nab-paclitaxel was evaluated in patients with advanced TNBC and mTNBC. Preliminary efficacy data demonstrated an ORR of 73% in the first 26 patients (Table 3) [49].

Trial + phase	Treatment	Type of drugs	Outcome
Keynote 012 Phase Ib	Pembrolizumab	Anti PD-1	ORR: 18,5% mPFS: 1.9 mOR: 11.2
Keynote 086 coorte A Phase II	Pembrolizumab	Anti PD-1	ORR: 5,3% mPFS:2.0 mOS:9.0
Keynote 086 coorte B Phase II	Pembrolizumab	Anti PD-1	ORR: 21,4% mPFS: 2.1 mOS: 18.0
NCT01375842 Phase I	Atezolizumab	Anti PD-1	ORR: 24% (first-line) mPFS: 1.4 mOS: 17.6
NCT02555657 Keynote-119 Phase III	Pembrolizumab	Anti PD-1	mPFS: 2.1 versus 2.1 (HR: 1.14) mOS: 12.7 versus 10.7 (HR: 0.78)
Javelin NCT01772004 Phase Ib	Avelumab	Anti PD-1	ORR: 5.2% mPFS: 1.5 mOS: 9.2
Impassion-130 NCT02425891 Phase III	Atezolizumab + nab-paclitaxel (comparator: placebo + nab-paclitaxel)	Anti PD-1 + chemotherapy	ITT mPFS 7.2 mesi versus 5.5 (HR 0.80, IC 95% 0.69–0.92) mOS 21.3 mesi versus 17.6 mesi (HR 0.84, IC 95% 0.69–1.02) PD-L1 > 1% mPFS 7.5 mesi versus 5.0 mesi (HR 0.62, IC 95% 0.49–0.78)

Table 3.

Completed studies of immunotherapy for metastatic TNBC

Trial	Phase	Treatment	Type of drugs	Endpoint	Study completion	Biomarkers
Keynote-162	Phase II	Pembrolizumab + niraparib	Anti PD-1 + parp inhibitor	mORR: 67% mPFS: 8.1	Completed	–
NCT03394287	Phase II	Camre + apatinib d1-14 (comparator: camre + apatinib d1-7)	Anti PD-1 + antiangiogenic	mORR: 43.3% versus 0 mPFS: 3.7 versus 1.9	Completed	(PD-L1, PD-1, VEGF-A, eg) in tumor tissue and peripheral blood
ENHANCE 1 NCT02513472	Phase Ib/II	pembrolizumab + eribulin	Anti PD-1 + tubulin polymerization inhibitor	mORR: 23.4 (25.8 if no prior therapy, 21.8 if 1–2 prior therapies) mPFS: 4.1 mOS: 16.1	Completed	CPS
NCT03044730	Phase II	Pembrolizumab + capecitabine	Anti PD-1 + chemotherapy	mORR: 13 mPFS: 4 mOS: 15.4	Completed	PD-L1
TONIC NCT02499367	Phase II	Nivolumab ± XRT or chemotherapy (cyclo, cis, doxo)	Anti PD-1 + radiation or chemotherapy	mORR:20 mOS: 1.9	Completed	–
MEDIOLA NCT02734004	Phase II	Durvalumab + olaparib	Anti PD-L1 + parp inhibitor	mORR: all cohort: 63.3 TNBC: 58.8 mPFS: all cohort: 8.2 TNBC: 4.9 mOS: all cohort: 21.5 TNBC 20.5	Completed	–
KEYNOTE-162 (TOPACIO)	Phase I/II	Pembro + niraparib	Anti PD-1 + parp inhibitor	mORR: overall: 21 gBRCAm: 47 mPFS: gBRCAm: 8.3	Completed	BRCA
NCT03800836	Phase Ib	Ipatasertib (AKTi) atezolizumab + paclitaxel or nab-paclitaxel	Akt inhibitor + anti PD-L1 + chemotherapy	mORR. 73	Completed	–
Impassion-131 NCT03125902	Phase III	Atezolizumab + paclitaxel (comparator: placebo + paclitaxel)	Anti PD-1 + chemotherapy	mPFS: 5.7 versus 6.0 (HR: 0.82, p = 0.20) in PD-L1+ population mOS: 22.1 versus 28.3 (HR: 1.12) in PD-L1+ population	Completed	PD-L1
Keynote-355 NCT02819518	Phase III	Pembrolizumab + nab-paclitaxel/paclitaxel/gemcitabine-carboplatin (comparator: placebo + nab-pac/pac/gem-carbo)	Anti PD-1 + chemotherapy	mOR: 53.2 versus 39.8 mPFS: 9.7 versus 5.6 in PD-L1 CPS > 10 HR = 0.66, p = 0.0012 mOS:23.0 versus 16.1 in PD-L1 CPS > 10 HR = 0.73, p = 0.0093	Completed	CPS

Ongoing Studies

Many ongoing studies are investigating the role of immunotherapy and combinations of ICIs such as pembrolizumab or atezolizumab in combination with paclitaxel, gemcitabine, or carboplatin or with targeted therapies such as ipataserib (an AKT inhibitor) or olaparib (a PARP inhibitor). Phase III ongoing studies are summarized in Table 4.

Table 4.

Ongoing phase III studies of immunotherapy for metastatic TNBC

Trial identifier	Setting	Study treatment	Primary endpoints	Biomarkers
IMpassion132 NCT03371017 595 patients	First-line therapy in metastatic TNBC with early recurrence (12 months)	Chemotherapy (carboplatin, carboplatin + gemcitabine or capecitabine) plus atezolizumab/placebo	OS in PD-L1+ OS in ITT population	PD-L1
NCT04177108 242 patients	First-line in metastatic TNBC	Paclitaxel plus ipatasertib/pbo plus atezolizumab/pbo	PFS	PD-L1
KEYLYNK-009 NCT04191135 932 patients	First-line in metastatic TNBC after induction chemotherapy with carboplatin gemcitabine plus pembrolizumab	Pembrolizumab ± olaparib maintenance	PFS OS	CPS
KEYNOTE-119 NCT02555657 692 patients	Second- or third-line in metastatic TNBC	Pembrolizumab versus chemotherapy (capecitabine, eribulin, gemcitabine, or vinorelbine)	OS in CPS ≥ 1 OS in CPS < 1 OS ITT population	CPS
MOIO NCT05078047 646 patients	Lung cancer Metastatic renal cell carcinoma Head and neck cancer bladder cancer Triple-Negative breast cancer, Merkel cell carcinoma, hepatocellular carcinoma, melanoma	Reduced dose intensity of immunotherapy (IO), such as treatment with anti-PD-1, PD-L1, or CTLA-4 inhibitors	PFS	–
TROPION-Breast02 NCT05374512 600 patients	Locally recurrent inoperable or metastatic triple-negative breast cancer	Dato-DXd versus paclitaxel Nab-paclitaxel Carboplatin Capecitabine Eribulin mesylate	PFS OS	–

TNBC triple-negative breast cancer, PFS progression-free survival, IO immunotherapy, OS overall survival, CPS combined positive score, ITT intention-to-treat

The efficacy and safety of atezolizumab plus chemotherapy compared with those of placebo plus chemotherapy in patients with inoperable TNBC were evaluated with IMpassion132 (NCT03371017). [50].

NCT04177108 will be used to evaluate the efficacy and safety of ipatasertib in combination with atezolizumab and paclitaxel in locally advanced or metastatic triple-negative breast cancer (TNBC) previously untreated in this setting. [51].

KEYLYNK-009 (NCT04191135) compares the efficacy of olaparib plus pembrolizumab versus chemotherapy plus pembrolizumab after induction with first-line chemotherapy plus pembrolizumab in terms of PFS and OS.

In the KEYNOTE-119 (NCT02555657) trial, patients with mTNBC were randomized to receive pembrolizumab or single-agent chemotherapy, consisting of capecitabine, eribulin, gemcitabine, or vinorelbine, in accordance with local regulations and guidelines. The primary endpoint was OS.

In the MOIO trial (NCT05078047), patients received a reduced dose of IO or current standard treatment.

The primary endpoint is disease progression. If proven right, this study will have a positive medico-economic impact by reducing the costs associated with the treatment and toxicity and increasing the patients’ quality of life.

New approaches include antibody drug conjugates (ADC), which consist of a monoclonal antibody covalently linked to a cytotoxic drug by a chemical linker. The aim of this pharmacological structure is to achieve pharmacological activity mainly in the cells targeted by the antibody.

Interest in ADCs has changed dramatically over the past few decades. Historically, anticancer drug discovery has focused solely on small-molecule chemotherapeutic agents, such as folate analogues, such as methotrexate, or DNA-damaging agents, such as nitrogen mustards. These agents target rapidly dividing cancer cells, but at the same time, other healthy dividing cells in the body are affected, causing severe side effects and limiting the administered dose, thus narrowing the therapeutic window. To address this issue, one pathway explored by researchers was the development of ADCs, involving the use of antibodies to deliver highly cytotoxic agents directly to tumor cells without affecting other dividing cells in the body. Theoretically, this concept should provide a significant increase in the therapeutic window compared with that of chemotherapy and radiation [53].

In recent years, the emergence of several antibody–drug conjugates, such as trastuzumab deruxtecan or sacituzumab govitecan, have dramatically changed the paradigm of HER⁺ disease, TNBC, and HER2-low patients. Current studies have focused on their positioning within therapeutic batteries for different types of breast cancer [55].

The primary objective of the TROPION-Breast02 trial (NCT05374512) was to demonstrate the superiority of Dato-DXd over ICC by assessing PFS in participants with locally recurrent inoperable or metastatic TNBC who are not candidates for PD-1/PD-L1 inhibitor therapy.

Dato-DXd is an ADC comprising a humanized anti-TROP2 IgG1 monoclonal antibody (mAb) conjugated to a potent topoisomerase I inhibitor payload (DXd) via a stable tetrapeptide-based cleavable linker [56].

There is a large range of different ongoing studies that are analyzing combination strategies. These studies focus not only on chemotherapy but also on other therapies, such as cryoablation, radiotherapy (NCT05233696), oncolytic viruses, antibody combinations, or targeted therapy.

The first three studies in Table 5 evaluate ICI combinations, such as atezolizumab with paclitaxel and bevacizumab (NCT04408118), pembrolizumab with either of two chemotherapy regimens, paclitaxel or capecitabine (NCT02734290), and spartalizumab monotherapy (NCT04802876).

Table 5.

Ongoing phase I–II studies of metastatic TNBC

Trial identifier	Phase	Setting	Study treatment	Principal endpoints	Biomarkers
ATRACTIB NCT04408118 100 patients	Phase II	Metastatic breast cancer Advanced breast cancer Triple-negative breast cancer	Atezolizumab in combination with paclitaxel and bevacizumab	PFS
NCT02734290 29 patients	Phase I–II	Unresectable/metastatic triple-negative breast cancer	Pembrolizumab + paclitaxel or pembrolizumab +  Capecitabine	Treatment-associated adverse events Number of patients who complete chemotherapy without a dose delay of more than 21 days
ACROPOLI NCT04802876 184 patients	Phase II	Patients with metastasis with programmed death-1 (PD1)-high-expressing tumors included TNBC	Spartalizumab or tislelizumab	ORR	PD1
IRENE NCT04445844 25 patients	Phase II	Stage IV breast cancer (AJCC version 8) locally advanced breast carcinoma, metastatic triple-negative breast carcinoma	INCMGA00012 and pelareorep	ORR Incidence of adverse events	PD-L1 T CELL RECEPTOR
BRACELET-1 S NCT04215146 48 patients	Phase II	Breast cancer, metastatic	Pelareorep in combination with chemotherapy and avelumab versus paclitaxel alone versus pelareorep plus avelumab	ORR To describe the safety and tolerability of the combinations of pelareorep, paclitaxel and avelumab as graded by the NCI CTCAE version 5.0	–
STOMP NCT03004183 57 patients	Phase I	Metastatic non-small cell lung cancer, metastatic triple-negative breast cancer	ADV/HSV-tk and SBRT Before Pembrolizumab	ORR	–
NCT03742349 64 patients	Phase I	TNBC	Spartalizumab + LAG525 in combination with NIR178, capmatinib, MCS110, or canakinumab,	Incidence of adverse events (AEs) and serious adverse events (SAEs) as a measure of safety Severity of adverse events (AEs) and serious adverse events (SAEs) as a measure of safety Incidence of dose-limiting toxicities (DLTs) of treatment (escalation only) Frequency of dose interruptions Frequency of dose reductions Dose intensities
Morpheus-TBNC NCT03424005 580 patients	Phase I–II	Metastatic or inoperable locally advanced triple-negative breast cancer	Atezolizumab + nab-paclitaxel, atezolizumab + nab-paclitaxel + tocilizumab, atezolizumab + sacituzumab govitecan, capecitabine, atezolizumab + ipatasertib, atezolizumab + SGN-LIV1A, atezolizumab + selicrelumab + bevacizumab, atezolizumab + chemo (gemcitabine + carboplatin or eribulin), inavolisib + abemaciclib + fulvestrant, inavolisib + ribociclib + fulvestrant, inavolisib (6 mg) + trastuzumab deruxtecan, inavolisib (9 mg) + trastuzumab deruxtecan	1. Objective response rate (ORR) 2. Number of participants with adverse events	-
NCT03387085 9 patients	Phase I–II	Patients with TNBC who have progressed on or after standard-of-care therapy	Aldoxorubicin HCl, N-803,ETBX-011 ETBX-051 ETBX-061 GI-4000—GI-6207 GI-6301 haNK avelumab Bevacizumab capecitabine cisplatin cyclophosphamide 5-fluorouracil Leucovorin nab-paclitaxel	Incidence of treatment-emergent adverse events (AEs) and serious adverse events (SAEs) Objective response rate by RECIST	-
NADiR NCT04837209 32 patients	Phase II	Metastatic, PD-L1 negative or immunotherapy-refractory triple-negative breast cancer	Dostarlimab and niraparib plus radiation therapy (RT)	Overall response rate (ORR)-RECIST	PD-L1
ATRiBRAVE NCT05582538 37 patients	Phase II	Triple-negative breast cancer, metastatic	Ceralasertib followed by durvalumab plus nab-paclitaxel	PFS
NCT03742349 64 patients	Phase I	Advanced or metastatic TBNC	Spartalizumab + LAG525 in combination with NIR178, capmatinib, MCS110, or canakinumab	Incidence of adverse events (AEs) and serious adverse events (SAEs) as a measure of safety Severity of adverse events (AEs) and serious adverse events (SAEs) as a measure of safety Incidence of dose-limiting toxicities (DLTs) of treatment (escalation only) Frequency of dose interruptions Frequency of dose reductions Dose intensities	-
NCT04683679 patients	Phase II	TNBC, metastatic breast cancer	Pembrolizumab, with or without olaparib, to standard radiation therapy	ORR	PD-L1
NCT03362060 20 patients	Phase II	TNBC, metastatic breast cancer	Pembrolizumab andPVX-410	Immune response following treatment with PVX-410 in combination with pembrolizumab	-
NCT04609215 20 patients	Phase Ib	TNBC, metastatic breast cancer	ALECSAT, carboplatin and gemcitabine	AEs and SAEs	–
AZTEC NCT03464942 54 patients	Phase II	Advanced triple-negative breast cancer	SABR followed by atezolizumab	PFS	–
BELLA NCT04739670 31 patients	Phase II	Metastatic triple-negative breast cancer	Carboplatin, gemcitabine, bevacizumab and atezolizumab	PFS	PD-L1 STROMAL TIL
Catalyst NCT05383170 6 patients	Phase I–II	Advanced head and neck squamous cell carcinoma, advanced breast cancer, advanced melanoma	CyPep-1 and pembrolizumab	Incidence, frequency, and seriousness of TEAEs Incidence of DLTs ORR based on radiological assessment according to the RECIST	PD-L1
NCT03289962 272 patients	Phase I	Locally advanced or metastatic cancer, including TNBC	Autogene cevumeran and atezolizumab	Percentage of participants with dose-limiting toxicities (DLTs) MTD/recommended phase II dose (RP2D) of autogene cevumeran Percentage of participants with adverse events (AEs) Percentage of participants with immune-mediated adverse events (imAEs) Percentage of participants by number of treatment cycles received Dose intensity of autogene cevumeran Change from baseline in targeted vital signs Change from baseline in targeted clinical laboratory test results Change from baseline in ECGs
AGADIR NCT03915678 247 patients	Phase II	Advanced solid tumors: pancreatic cancer, virus-associated tumors, non-small cell lung cancer, melanoma, bladder cancer, triple-negative breast cancer	Atezolizumab + BDB001 + radiotherapy	Assessment of the antitumor activity of atezolizumab combined with BDB001 and radiotherapy in patients with pancreatic cancer, virus-associated tumors, NSLC, soft-tissue sarcoma, bladder cancer, and TNBC	–
ABILITY NCT05086692 100 patients	Phase I–II	Locally advanced or metastatic solid tumor or unresectable solid tumor, including TNBC	MDNA11 monotherapy alone or in combination with checkpoint inhibitor	Recommended phase II dose (RP2D) for MDNA11 Incidence of treatment-related adverse events (TRAEs) Incidence of treatment-emergent adverse events (TEAEs)	–
NCT03589339 145 patients	Phase I	Metastatic triple-negative breast carcinoma and other advanced or metastatic tumors	Intratumoral injection of NBTXR3 activated by radiotherapy in combination with anti-PD-1 therapy (nivolumab or pembrolizumab)	1. Determination of the recommended dose 2. Evaluation of the antitumor response of R3/RT/PD-1 3. Assessment of the safety and feasibility of R3/RT/PD-1 4. Evaluation of the body kinetic profile of intratumorally injected NBTXR3	-
NCT05431270 38 patients		Advanced solid tumor and metastatic cancer and refractory cancer, including TNBC	PT199 (an anti-CD73 mAb) alone and in combination with a PD-1 inhibitor	Maximum tolerated dose (MTD), if reached RP2D of PT199 as a single agent and/or in combination with a PD-1 inhibitor	CD73, PD-L1
JAVELIN Medley NCT02554812 409 patients	Phase I–II	Locally advanced or metastatic solid tumors, including triple-negative breast cancer (TNBC)	Avelumab (MSB0010718C) in combination with other cancer immunotherapies Utomilumab, PF-04518600 PD 0360324 CMP-001	Number of partIcipats with DLT Objective response—number of participants with objective response	-
NCT05274451 54 patients FASE 1	Phase I	Relapsed and/or refractory ROR1 + triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC)	LYL797	Evaluate incidence of dose-limiting toxicities (DLTs) Evaluate incidence of treatment-emergent adverse events (TEAEs) Evaluate severity of treatment-emergent adverse events (TEAEs) Determine recommended phase II dose (RP2D)	-
NCT03761914 26 patients	Phase I–II	Advanced or metastatic cancer Colorectal (third or fourth line), ovarian (second or third line), small-cell lung cancer (second line), breast cancer (triple-negative; second line), acute myelogenous leukemia	Galinpepimut-S and Pembrolizumab	Number and frequency of TRAEs, including UARs, and SAEs (safety parameters)—for all tumor types Events [NCI overall response rate (ORR) by RECIST (RECIST)] Complete response (CR) rate	–

TBNC triple-negative breast cancer, ORR objective response rate, AEs adverse events, SAEs serious adverse events, DLTs dose-limiting toxicities, BORR best overall response rate, TRAEs treatment-related adverse events, TEAEs treatment-emergent adverse events, MTD maximum tolerated dose, DLT dose-limiting toxicities

Table 5 synthesizes phase I and phase II ongoing trials evaluating new treatments beyond ICIs, such as cancer vaccines, oncolytic viruses, cell therapies, and cytokine modulation. We are moving toward more personalized medicine.

For instance, oncolytic viruses constitute an antitumor tool through two mechanisms, either through nonreplicative viruses that infect the tumor cell, causing lysis, destroying the tumor vasculature, and activating the immune response against tumor cells, or by acting as vectors that generate cytotoxic proteins, inserting therapeutic genes, or sensitizing cancer cells to chemo-/radiotherapy, among others [57].

A wide variety of viruses have been studied, but only talimogene laherparepvec (T-VEC) in melanoma has been approved by the FDA [56]. A large number of preclinical studies on breast cancer have been reported, and some of them have also been evaluated in early clinical trials. However, to date, oncolytic viruses have not been as clinically successful as other immunotherapeutic approaches [54, 58, 59].

Table 5 summarizes trials involving oncolytic viruses.

The IRENE study (NCT04445844) is a phase II study investigating the safety and efficacy of the combination of INCMGA00012 and pelareorep and determining how well they work in treating patients with mTNBC [60].

INCMGA00012 is a monoclonal antibody that works by binding to programmed cell death protein 1 (PD-1) and blocking this pathway, allowing the immune system to recognize and attack cancer cells. Pelareorep is a type of virus called a reovirus that occurs naturally and may break down cancer cells. The administration of INCMGA00012 and pelareorep may slow the growth and spread of cancer to other parts of the body.

BRACELET-1 S (NCT04215146) has the purpose of determining the possible anticancer effect of pelareorep in combination with chemotherapy (paclitaxel) and avelumab in treating a type of breast cancer called hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2⁻) breast cancer, which is either locally advanced or metastasized (cancer that has spread throughout the body). This study investigated whether pelareorep in combination with paclitaxel and avelumab is more effective than paclitaxel alone or in combination with paclitaxel. The safety of the combination treatments will also be evaluated.

STOMP (NCT03004183), a phase II trial, is evaluating the efficacy and safety of stereotactic body radiation therapy (SBRT) and in situ oncolytic virus therapy used as a window of opportunity before pembrolizumab treatment in patients with metastatic TNBC and metastatic non-small cell lung cancer (NSCLC). Oncolytic virus therapy consists of adenovirus-mediated expression of herpes simplex virus thymidine kinase (ADV/HSV-tk) plus valacyclovir therapy.

Conclusions

The IMpassion130 study was the first to demonstrate the efficacy of immunotherapy plus chemotherapy in patients with metastatic PD-L1⁺ TNBC [34].

Immunotherapy is now also used for early stage PD-L1⁺ TNBC. The anticipated use of immunotherapy in the neoadjuvant/adjuvant setting is changing the therapeutic algorithm for metastatic disease.

The main prognostic and predictive biomarkers are PLD1 expression level, mismatch repair deficiency (dMMR), high level of microsatellite instability (MSI), tumor mutational board (TMB), and tumor infiltration lymphocytes (TILs). Nevertheless, more predictive biomarkers are required to identify patients who can respond better to a specific combination of treatments (for example, ICIs and targeted therapy). Current trials are discovering new biomarkers to identify responding patients and overcoming primary or secondary resistance. For instance, STAT4 promotes cell differentiation from native CD4⁺ T cell to T helper cell (Th1) and the production of interferon gamma (IFN-γ) in tumor microenvironment (TME) [61–63]. The transcriptional synergy between STAT4 and STAT3 starts a positive feedback loop through IL-12R: STAT4/IL-12R/JAK2–STAT3–STAT4. This pathway causes an increasing expression of PD-L1 and can be used to predict anti-PD1 treatment response [64]. The gut microbiota is also a new biomarker. The different composition of gut microbiota was associated with the heterogeneity of therapeutic effects, response to immunotherapy, and adverse events [65]. Specific bacteria species in the gut microbiota are associated with higher therapeutic efficacy or toxicities of ICs. The human microbiota produces a lot of of neoantigens that stimulate the innate immune responses through interaction of pathogen‐associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) that are located on intestinal epithelial cells and innate immune cells. This causes the activation and translocation of dendritic cells localized in gut‐associated lymphoid tissue (GALT) to mesenteric lymph nodes, stimulating naïve CD4⁺ and CD8⁺ T cells [66]. Improving new biomarkers is possible using NGS panel and monitoring mutations through the use of noninvasive analysis such as liquid biopsy to analyze circulating tumor cell (CTC), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), exosomes, and proteins. ctDNA can also be obtained from various non-blood sources such as urine and saliva through noninvasive methods and procedures [67]. These alternative sources can have a distinct yet complementary role to that of blood ctDNA analysis and consider various technical aspects of non-blood ctDNA assay development. We also reflect on the settings in which non-blood ctDNA can offer distinct advantages over plasma ctDNA and explore some of the challenges associated with translating these alternative assays from academic to clinical use. This is also important for optimizing clinical response, safety, and tolerability. Imagine examination can predict pCR during neoadjuvant treatment and control the clinical response [68]. The use of magnetic resonance imaging (MRI), ultrasound (US), and positron emission tomography (PET)/computed tomography (CT) has limitations. Image interpretation is not a standardized method because of different assessments made by radiologists. Secondly, frequent use of PET or MRI is also expensive, and additionally, PET is radioactive. Computer-aided diagnosis (CAD) helps with the development of deep convolutional neural network detection (CNNs), a type of learning algorithm applied to analyze and learn visual features from a large number of images used as input [68]. Recently, several studies have been published using CNN to predict breast cancer treatment responses through PET/CT and MRI images [68–71]. More studies are also required to better understand the effectiveness of immunotherapy as maintenance therapy and biological disease features for de-escalation treatment.

Author Contributions

Conception and design—Rossana Berardi and Alessandro Parisi. Methodology—Rossana Berardi, Elisa Tiberi, and Alessandro Parisi. Acquisition and collection of data—Elisa Tiberi, Alessandro Parisi, Mirco Pistelli, and Agnese Savini. Federica Galassi, Chiara Reschini, Debora Quintavalle, Riccardo Napoleoni, Carlo Ferrari, and Rossana Berardi. Writing—original draft preparation—Elisa Tiberi, Alessandro Parisi, and Rossana Berardi. Writing—reviewing and editing—Rossana Berardi. Writing—final approval—Elisa Tiberi, Alessandro Parisi, Mirco Pistelli, Agnese Savini, Federica Galassi, Chiara Reschini, Debora Quintavalle, Riccardo Napoleoni, Carlo Ferrari, Rossana Berardi.

Funding

No funding or sponsorship was received for this study or the publication of this article.

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Declarations

Conflicts of Interest

Alessandro Parisi received consultant/advisory board fees from AstraZeneca and Amgen and travel support from Merck, Daiichi-Sankio, and Accord. Elisa Tiberi received consultant/advisory board fees from Bayer and travel support from MSD and Merk. Agnese Savini received consultant/advisory board fees from AstraZeneca, Lilly, GSK, Gilead, Seagen, Genetic oncology, Roche, and Istituto Gentili. Mirco Pistelli received consultant/advisory board fees from AstraZeneca, Lilly, Gilead, Novartis, Pfizer, and Daiichi Sankyo and travel support from Roch and Msd. Federica Galassi, Chiara Reschini, Debora Quintavalle, Riccardo Napoleoni, and Carlo Ferrari declare no conflicts of interest. Rossana Berardi is an Editor-in-Chief of Oncology and Therapy. Rossana Berardi was not involved in the selection of peer reviewers for the manuscript nor any of the subsequent editorial decisions.

Ethical Approval

This article is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors.