Combining PD-1/PD-L1 inhibitors with platinum-based chemotherapy (PBC) may increase tumor immunogenicity [36]. PBC causes DNA damage and induces cell death, thereby attracting antigen presenting cells (APC) [37]. PBC also increases TMB, and tumor-specific neoantigens are presented by MHC-1 and cause cytotoxic T cell activation [38]. While MHC-1 is often downregulated in cancer, in vitro experiments have shown that PBC induces MHC-1 on tumor cells [36,39,40]. IL-12 is essential for antigen presentation; in vivo knockout experiments showed that PBC increases dendritic cell (DC) maturation and leads to an increased ability of DCs to present antigens in an IL-12 dependent manner, resulting in the hypothesis that PBC sensitizes tumors for immune recognition [41]. Experiments in a murine model revealed that T cell costimulatory molecules such as CD80/CD86 are increased in tumor infiltrating immune cells after cisplatin treatment, suggesting that CD80/CD86 expression can be modulated by cisplatin treatment [42]. In vitro experiments showed that PBC induces PD-L1, making PD-L1 an interesting target to inhibit after PBC [39,43,44,45]. PBC may also decrease PD-L2 expression via modulation of the transcriptional regulator STAT6 [46]. As PD-L2 competes with PD-L1 to bind PD-1, decreased expression of PD-L2 after PBC results in enhanced affinity of PD-L1 to PD-1, and increases the relevance of PD-L1 for ICI [47]. The beneficial effects of the addition of PBC to PD-1/PD-L1 blockade is summarized in Figure 2A.

 

The favorable effects of combined ICI and PBC have been shown in numerous studies [48]. In an MB49 BC subcutaneous model, anti-PD-L1 combined with PBC was more effective than either therapy alone [49]. In an MBT-2 subcutaneous model, anti-PD-L1 combined with PBC was more effective than PBC alone, but counterintuitively, anti-PD-L1 monotherapy was most effective, suggesting that combined treatment results are model-dependent [49]. Immune profiling of baseline tumors by flow cytometry did not demonstrate differences of tumor immune cell populations, and both MB49 and MBT-2 tumors were highly enriched with myeloid-derived suppressor cells (MDSCs). However, expression of the TIGIT checkpoint proteins on immune cells were decreased after PBC treatment in the MB49 vs. MBT-2 model, and the authors suggest that TIGIT may play a role in the diverse anti-tumor responses between models.

 

Multiple clinical trials evaluate a combination of PBC with PD-1/PD-L1 ICI in MIBC (Table 1) [13]. Neoadjuvant pembrolizumab plus gemcitabine/cisplatin showed a partial response rate to NMIBC at RC of 61% (n = 36) (NCT02365766) [50]. In BLASST-1, a multicenter Phase II trial, PBC combination therapy with nivolumab demonstrated a very high pCR rate of 65.8% (n = 27) [51]. Gemcitabine combined with pembrolizumab for cisplatin-ineligible patients yielded results comparable to pembrolizumab alone in NCT02365766, a Phase 1b/II study: 45.2% achieved pCR, whereas 51.6% reached <pT1N0 [52]. Interim analysis of SAKK06/17 showed pCR in 30% (n = 9) after treatment with gemcitabine and durvalumab pre- and post-operatively, whereas 20% of patients reached non-muscle invasive disease (pT1/pTis) [53]. Preclinical studies and interim study reports are encouraging and suggest a synergistic effect of chemotherapy with PD-L1 blockade. However, pCR rates are a surrogate marker for actual clinical outcome, thus long-term results analyzing disease progression are necessary. More information on treatment sequence and timing of PBC with ICI is also needed before these alternative treatment paradigms can be implemented.

 

Ionizing radiation induces double-stranded DNA breaks and produces reactive oxygen species (ROS) leading to cell cycle arrest, apoptosis and cell death [54,55]. Radiation elicits immunogenic effects within the tumor microenvironment (TME) by upregulating and activating the complement pathway and stimulation of IFN-γ release, both implicated in tumor eradication [56,57]. Radiotherapy enhances the adaptive immune system by upregulation of MHC-I, increasing cytotoxic T cell activity. Combining radiotherapy with immune-modulation may inhibit cancer via APC cross-presentation and improvement of T cell priming [58,59,60,61]. By inducing genotoxic stress, radiation prompts NK-mediated innate immune responses [62]. Interestingly, radiation also elicits chemotactic signals that attract MDSCs, instigating T-cell suppression [63,64]. Immunosuppressive effects can also be induced via activation of TGF-β, leading to upregulation of PD-L1 on tumor cells [65]. Combined radiotherapy with PD-L1 inhibition led to synergistic anti-tumor effects in vivo, thus providing the rationale for combined regimens with anti-PD-1/PD-L1 drugs (mechanism depicted in Figure 2A) [66,67,68,69]. Anti-PD-L1 therapy started one day prior and continued until two weeks after local irradiation (12Gy) in an HT1197/MB49 mouse model led to significant tumor growth delay compared to monotherapy [70].

 

Radiation may have immune-mediated distant effects, termed the abscopal effect, which refers to regression of non-irradiated lesions located elsewhere than the primary radiated site [62]. The abscopal effect is more often detected in mice treated with a combination of radiotherapy and PD-1/PD-L1 than radiation alone [65,71,72]. The underlying mechanism remains unclear, but it is hypothesized that anti-tumor immune responses are amplified. Taken together, radiation therapy stimulates tumor immunogenicity and suggests higher PD-1/PD-L1 susceptibility, hence combined regimes are appealing for investigation in patients with localized BC who prefer a bladder-sparing treatment [59,60,73]. Issues that need to be resolved include determining the optimal fractioning of radiotherapy, timing of ICI and an appropriate balance with regard to side effects associated with each modality [74,75]. Currently, multiple trials investigating radiotherapy and PD-1/PD-L1 blockade in BC are underway but none have been published so far (Table 1) [11].

For optimal cytotoxic T cell-mediated tumor response, co-stimulation by CD80/CD86 binding to CD28 is required. CD28-CD80/CD86 interaction leads to increased T cell proliferation, differentiation and survival [76]. CTLA-4, a CD28 homolog, is another checkpoint-inhibitor expressed by T cells. CTLA-4 binds competitively with CD80/CD86 on APC’s, resulting in downregulation of T cell proliferation and response. Hence, CTLA-4 blockade can facilitate T cell recognition and increases eradication of malignant cells, thus strengthening the immunological response (illustrated in Figure 2B) [77,78].

In vivo mouse models studying melanoma, ovarian and colon carcinoma showed higher tumor rejection rates and increased tumor induced lymphocyte activity and proliferation after combined therapy with CTLA-4 and PD-1 compared to monotherapy [79,80]. Combining CTLA-4 and PD-L1 inhibitors yields increased CD8+ and CD4+ counts, reduced CD4+ Tregs, increased expression of pro-inflammatory cytokines and a polarization shift from M2 to M1 macrophages [81]. In another study, MB49 BC cells were injected subcutaneously in C57Bl/6 female mice to investigate the response to anti-PD-1 vs. anti-CTLA-4 alone or combined treatment and found that the combination showed increased circulating CD8+ T cells and superior overall survival compared to each monotherapy. In a control group, BCG treatment had no effect on tumor growth [82]. These results were confirmed in a second study that investigated a combination of anti-PD-1/anti-CTLA-4 in an orthotopic mouse model [83].

 

The NABUCCO-trial recently demonstrated a 46% pCR rate after treatment with ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) prior to RC in cisplatin-ineligible stage III BC patients [84]. Of 24 patients, 14 (58%) reached either non-muscle invasive disease or pCR. Biomarker analysis revealed that tumors from pCR had higher frequencies of DDR alterations compared to non-pCR tumors (p = 0.03), and trended towards a higher TMB (p = 0.056) [84]. An increased TGF-β expression signature was associated with non-response, while response was independent of baseline CD8+ presence or T-effector signatures. However, upregulation of B cell-related genes at baseline correlated with non-response. Non-responding tumors exhibited more stromal B cells than responding tumors (p = 0.043) whereas the density of B cells in the tumor compartment was higher (p = 0.07) [84]. Overall, NABUCCO indicates that combination regimens with anti-CTLA-4 blockade should be investigated further, as the pCR results are promising. Other studies investigating CTLA-4 plus PD-1/PD-L1 combinations are illustrated in Table 1 and Table 2. No combination regimes have reported data in HR-NMIBC, but considering toxicity profiles, ICI combinations might also prove useful in BCG-unresponsive patients. The extent to which auto-immune symptoms will develop and whether they are manageable will likely determine if combination therapies are feasible and safe [85].

 

Poly (ADP-ribose) polymerase (PARP) is involved in DDR. PARP inhibitors are synthetically lethal against tumors with BRCA1/2 mutations, as these cancer cells are the result of genomic instability [86]. Secondly, PARP inhibitor induced genomic instability leads to more somatic mutations and a higher TMB, which in turn may increase the neoantigen load and immunogenicity, which can be exploited using ICI (Figure 2C) [87,88]. In mice, PARP increased expression of PD-L1, while dual blockade of both PARP and PD-L1 may lead to synergistic effects and increased tumor cell death [89]. In a Phase II study in MIBC, combined inhibition of PARP with olaparib and anti-PD-L1 resulted in a systemic immune response demonstrated by release of chemokines, TNF-α, angiogenic factors, tumor infiltrating lymphocytes (TIL) and increased IFN-y expression [90]. Preliminary results showed a pCR of 44.5% (n = 28) after treatment with anti-PDL1 and olaparib [91].

 

TGF-β is overexpressed in many malignancies and has long been implicated as a stimulator of cancer progression by regulation of apoptosis, cell proliferation and initiation of epithelial mesenchymal transition (EMT) [92]. An RNA-based TGF-β signature was associated with non-response to anti-PD-1 therapy in locally advanced BC and anti-PD-L1 therapy in metastatic BC [84,93]. Murine models showed that blocking of TGF-β reduces TGF-β signaling in stromal cells, facilitated T-cell penetration and provoked strong anti-tumor immunity and tumor regression [93]. Interestingly, a bi-functional fusion protein (Bintrafusp-α) targeting anti-PD-L1 and TGF-β has recently been developed, and it is expected to be tested in localized BC [94]. Vascular endothelial growth factor (VEGF) impairs hematopoietic differentiation and like TGF- β causes an immunosuppressive microenvironment in BC, thereby facilitating evasion of the immune response [95]. Anti-VEGF therapy may eliminate immunosuppressive effects, decreases tumor vasculature, and increases perfusion and influx of T cells [96]. Several trials tested anti-VEGF with concurrent chemotherapy, but this did not result in improved clinical outcome for metastatic BC patients [97]. Thus far, no trials have reported on ICI combinations with anti-VEGF in localized BC yet, but in other cancer entities synergistic effects have been identified [98]. The collagen discoidin domain receptor (DDR2), a kinase, is recognized as a metastases promoter [88]. DDR2 depletion increases sensitivity to anti-PD-1 treatment in vivo in BC and multiple other murine models of cancer [99]. Treatment with the anti-tyrosine kinase inhibitor dasatinib (anti-DDR2) and anti-PD-1 resulted in increased CD8+ tumor lymphocytes compared to monotherapy and a reduced tumor load [99]. Thus, a combination treatment of ICI with dasatinib appears to be worth exploring in localized BC. In summary, the synergistic mechanisms of anti-TGF-β, anti-VEGF and anti-DDR2 with PD-1/PD-L1 blockade are depicted in Figure 2D.

 

Finally, Toll-like receptor (TLR) agonists are being investigated in localized BC. Treatment with BCG in vitro triggers TLRs and prevents deprivation of MHC-II expression, thereby promoting immunogenicity [100,101]. Treatment with the TLR-4 agonist tasquinimod had weak anti-tumorigenic effects in AY-27 rats and MBT-2 mice models [102]. Combination treatment was tested only in mice, but anti-PD-L1 modulated M1/M2 tumor-infiltrating myeloid cells and increased activity and proliferation of CD8+ T-cells were observed, resulting in tumor regression [102]. To summarize, targeted therapies seem promising when combined with anti-PD-1/PD-L1 treatment, though clinical studies are now needed to assess whether these combination strategies truly enhance clinical efficacy of ICI. An overview of currently ongoing combination trials using targeted treatment combinations with PD-1/PD-L1 blockade are included in Table 2.