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Immunotherapy in breast cancer: An overview of modern checkpoint blockade strategies and vaccines
Author’s Accepted Manuscript Immunotherapy in breast cancer: An overview of modern checkpoint blockade strategies and vaccines Katherine Sanchez, David Page, Heather L. McArthur www.elsevier.com/locate/cpcancerv
PII: DOI: Reference:
S0147-0272(16)30075-7 http://dx.doi.org/10.1016/j.currproblcancer.2016.09.009 YMCN312
To appear in: Current Problems in Cancer Received date: 21 September 2016 Accepted date: 21 September 2016 Cite this article as: Katherine Sanchez, David Page and Heather L. McArthur, Immunotherapy in breast cancer: An overview of modern checkpoint blockade strategies and vaccines, Current Problems in Cancer, http://dx.doi.org/10.1016/j.currproblcancer.2016.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Immunotherapy in breast cancer: An overview of modern checkpoint blockade strategies and vaccines Authors: Katherine Sanchez, MD1, David Page, MD1, and Heather L. McArthur, MD, MPH2 1Providence
Cancer Center / Earle A. Chiles Research Institute, Portland, OR
2Cedars-Sinai
Medical Center, Los Angeles, CA
Disclosures: Dr. McArthur has participated in advisory boards for Celgene, Merck, OBI, Peregrine, Spectrum Pharmaceuticals and Syndax Pharmaceuticals and has research supported by Bristol-Myers Squibb, MedImmune, LLC/AstraZenica, Eli Lilly, and Merck Pharmaceuticals. Dr. Page has participated in advisory boards for Celgene and Peregrine, and has research supported by Medimmune and Merck Pharmaceuticals. Research support: No research funds were used in the development of this manuscript Key words (from MeSH database): Breast neoplasms; immunotherapy; vaccines; CTLA-4 Antigen; programmed cell death 1 receptor; lymphocytes, tumor-infiltrating
Abstract Immune therapy has recently emerged as a standard-of-care strategy for the treatment of melanoma, lung cancer, bladder cancer, among other malignancies. However, the role of immune therapy in the treatment of breast cancer is still being determined. Two current strategies for harnessing the immune system to treat cancer include drugs that modulate key T cell inhibitory checkpoints and vaccines. Specifically, modern immune therapy strategies can facilitate T-cell mediated tumor regression by priming the immune system against specific tumor associated antigens, by modulating immunoregulatory signals, or both. In breast cancer, preliminary data from preclinical and early clinical studies are promising. In fact, clinical data with checkpoint blockade as monotherapy has been reported in multiple breast cancer subtypes to date, with durable responses observed in a significant proportion of women with chemotherapy resistant disease. However, because the number of genetic mutations and thus, the number of neoantigens available for immune response are modest in most breast cancers when compared with other cancers, most breast cancers may not be inherently sensitive to immune modulation and therefore may require strategies that enhance tumor associated antigen presentation if immune modulation strategies are to be effective. To that end, studies that combine checkpoint blockade with other strategies including established systemic therapies (including hormone therapy and chemotherapy), radiation therapy, and localized therapy including tumor freezing (cryoablation) are underway in breast cancer. Studies that combine checkpoint blockade with vaccines are also planned. Herein, we provide a brief summary of key components of the immune response against cancer, a rationale for the use of immune therapy in breast cancer, data from early clinical trials of checkpoint blockade and vaccine strategies in breast cancer, and future directions in the field. Introduction Immunotherapy, or the use of the body’s immune system to fight cancer, is a rapidly evolving field. Cancer-specific immune responses are initiated when the immune system effectively recognizes abnormally expressed proteins from cancer cells, called tumorassociated antigens (TAAs). Immune responses are tightly regulated by a balance of stimulatory and inhibitory mechanisms. Modern immune therapy strategies in cancer aim to prime the immune system toward specific TAAs, to modulate immune-regulatory signals
towards a favorable inflammatory state, or both. To achieve these aims, investigators are exploring several therapeutic strategies including immune checkpoint antibodies which block inhibitory signals to exploit existing immunity, antibodies to other immune targets, tumor vaccines which prime the immune system against TAAs, intratumoral oncolytic viruses, and chimeric antigen receptor (CAR) T cell therapies. To date, immune checkpoint blockade antibodies and vaccines have been most extensively studied in breast cancer. Emerging data suggest, however, that efficacy can be maximized by combining multiple immunotherapy strategies, for example, immune checkpoint blockade antibodies plus cancer vaccines. Herein we describe the rationale for immune checkpoint antibodies and vaccines in the treatment of breast cancer, summarize the relevant literature, and highlight future approaches. An Overview of Immunotherapy Principles T-lymphocyte activation The adaptive immune response against cancer depends on antigen-specific T cell activation. In this process, T cells survey tissues for TAAs that are presented in peptide complexes bound to a cellular surface protein called major histocompatibility complex-1 (MHC-1). The T cell receptor (TCR) binds the antigen-MHC complex and, in turn, induces activation of T cells. Although MHC-1 molecules are present on many cell types, including normal and tumor cells, specialized cells called antigen presenting cells (APCs) have costimulatory molecules and are highly effective in presenting peptides and activating T cells. Classical examples of APCs include dendritic cells (DCs), macrophages, and Langerhans cells. This interaction can occur in the tumor, in secondary lymphoid organs, or in peripheral tissues.1 APCs engulf apoptotic cells and extracellular peptides . These peptides are then digested and expressed on the APC surface chaperoned by MHC-1 molecules. Activation requires two signals: the first originates from the binding of the MHCpeptide complex to the TCR, and the second originates from the binding of co-stimulatory molecules CD80/86 (B7-1/2) on APCs to CD28 on T cells. Once activated, downstream TCR signaling is regulated by inhibitory signaling via cellular surface proteins, including programmed death (PD)-1 (which binds to PD-ligands 1 or 2) and cytotoxic T-lymphocyte antigen 4 (CTLA-4, which competes for binding with B7-1/2). These regulatory signals are
physiologically important to prevent uncontrolled inflammation and autoimmune disease. However, the same regulatory signals may also impair anti-tumor immune responses. There are multiple type of T-cells, classified by their function and expression of surface proteins. Two subtypes are CD8+ and CD4+ T cells. CD8+ T-cells, also called cytotoxic T-cells, are activated by type 1 MHC molecules expressed on tumor cells, and are capable of directly killing tumor cells. CD4+ T-cells are called helper T-cells, and are activated upon binding to antigen-loaded type 2 MHC molecules on APCs. These helper Tcells can further differentiate into T-helper type 1 (Th1), type 2 (Th2), type 17 (Th17), or regulatory T cells (T reg) based on the cytokine milieu. Once activated, Th1 cells facilitate an anti-tumor response by releasing cytokines, which directly activate and recruit other immune cells. Th1 cells secrete interferon-gamma (IFN) and support the anti-tumor response. Th2 cells secrete IL-4 and IL-13, any may hamper the anti-tumor response. The role of Th17 cells in this process has not been clearly elucidated. Tregs have been implicated in suppression of tumor-specific immune responses and thus, tumor immune escape.2 Strategies that preferentially activate Th1 and CD8+ anti-tumor immune responses, and suppress Th1 and Treg responses, are under development. Cancer vaccines Similar to vaccines used for infectious diseases, cancer vaccines prime the adaptive immune system by providing exogenous antigens such as TAAs. These TAAs are placed into vaccine vectors that facilitate TAA-specific T-cell activation, which in turn would facilitate immune-mediated destruction of TAA-expressing tumor cells. The best studied vaccine target in breast cancer is the human epidermal growth factor receptor 2 (HER2) protein. The HER2 protein is a tyrosine kinase receptor that is present in normal tissue, but overexpressed in a subset of breast cancers. There are numerous HER2- directed vaccines in different stages of development and clinical trials. Nelipipimut-S (NeuVax, Galenda Biopharma) is the most extensively studied HER2-cancer vaccine and is currently being studied in a phase III clinical trial evaluating clinical efficacy in early stage breast cancer (NCT01479244). The relevant TAA, E75, is a peptide from the extracellular domain of HER2.3 Cytotoxic-T cells are activated against the human leukocyte antigen (HLA)presented HER2 epitope, causing immune activation against breast cancers that express
this TAA. It is hoped that the induction of HER2-specific immune response will translate into long term immunity from recurrence and thus, cure.
Immune checkpoint directed antibody therapy Immune checkpoint antibodies facilitate T-cell activation by either binding and agonizing co-stimulatory signals, or binding and antagonizing co-inhibitory signals. Drugs that bind and antagonize a potent regulator of T-cell co-inhibition such as CTLA-4, for example, likely suppress immune activation via a variety of mechanisms. Specifically, downstream CTLA-4 signaling may directly inhibit T-cell activation; however, CTLA-4 also competitively binds to the B7 ligand on APCs, thereby impairing co-stimulatory signaling mediated by CD28 binding to B7.4 Finally, CTLA-4 is expressed on Treg cells, and may promote suppressive Treg activity. Therefore, drugs that bind to CTLA-4 may function by inhibiting downstream signaling, by preventing competitive binding of B7 or by depleting CTLA-4-expressing Tregs in the microenvironment via antibody-dependent cell-mediated cytotoxicity (ADCC). The first clinical signal for CTLA-4 blockade as an effective anti-cancer strategy was demonstrated in advanced melanoma. For example, in one of the early pivotal phase III clinical trials, patients with advanced melanoma were treated with ipilimumab (Yervoy, Bristol Myers Squibb) +/- gp100 vaccine, versus gp100 vaccine alone. Compared to gp100 alone, median overall survival was increased from 6.4 to 10.1 months, and 24-month survival nearly doubled.5 This was the first report of a survival benefit with systemic therapy in advanced melanoma and consequently, ipilimumab became the first immune checkpoint antibody to be approved by the United States (US) Food and Drug Administration (FDA) for the treatment of cancer. Given the success with this strategy in advanced melanoma, many studies of CTLA-4 blockade have been undertaken with ipilimumab or tremelimumab (MedImmune/AstraZeneca), in a variety of tumor types including breast cancer.6 PD-1 is another inhibitory receptor expressed on T-cells that binds to PD-L1/L2 on tumor cells and APCs and mediates T-cell suppression. Nivolumab (Opdivo, Bristol Myers Squibb), pembrolizumab (Keytruda, Merck), and atezolizumab (Tecentriq, Genentech) are all US FDA-approved checkpoint antibodies that target PD-1 or its ligand, PD-L1.7 T-cells
that are chronically stimulated by antigens express PD-1 as part of co-inhibitory pathway that ultimately leads to T-cell exhaustion. Furthermore, interferon gamma (IFNγ) secreted by T cells can promote up-regulation of PD-L1 on both immune cells and tumor cells, which may bind PD-1 and further promote T-cell exhaustion. PD-1/L1 antibodies are thought to function by blocking PD-1-mediated T-cell suppressive signals, thereby re-invigorating Tcells that are specific to tumor TAAs. PD-1/L1 blocking antibodies have been US FDAapproved for the treatment of metastatic melanoma,8,9 non-small cell lung cancer,10,11 renal cell carcinoma, bladder cancer, and Hodgkin lymphoma. Several other anti PD-1/L1 directed antibodies are in development, including durvalumab (anti-PD-L1, Medimmune) and avelumab (anti-PD-L1, EMD Serono). Rationale for Immunotherapy in Breast Cancer Tumor Infiltrating Lymphocytes In some cancers, immune cells infiltrate the tumor or surrounding stroma. The majority of these immune cells are lymphocytes and are therefore called tumor infiltrating lymphocytes (TILs). The presence of TILs suggests endogenous immune recognition of TAAs in some tumors, which in turn, has been associated with improved prognosis, and may also portend inherent responsiveness to immunotherapy. Notably, an effort to standardize TIL reporting in breast cancer was undertaken by an International Working Group and it was ultimately recommended that the percentage of tumor stroma infiltrated by TILs be reported.12 Tumors that are densely infiltrated by TILs may be particularly sensitive to PD1/L1 directed therapies, which function by re-invigorating previously activated T-cells. For example, as first described in metastatic melanoma, tumors with higher baseline TILs were more likely to respond to anti-PD-1, compared to patients with few or no TILs.13 A similar observation has been demonstrated in early stage breast cancer (ESBC) wherein the presence of TILs has been shown to be a potent favorable prognostic and predictive marker, especially in triple negative breast cancer and HER2-positive breast cancer.14-19 Contrastingly, one study of invasive lobular carcinomas reported that the presence of TILs were associated with an adverse prognosis.20 Although this retrospective study did not meet statistical significance and was subject to confounding by other variables, it contributes to a growing body of literature indicating that different breast cancer subtypes
may have different patterns of immune cell recruitment both at baseline and in response to conventional chemotherapies. There is also a growing body of literature demonstrating the impact of TILs in predicting responses to specific systemic therapy strategies in breast cancer. For example, a pooled analysis of TILs as a biomarker in 991 women with triple negative breast cancer (TNBC) participating in five adjuvant anthracycline-based chemotherapy trials was recently reported. In this study, increased quantity of stromal TILs was associated with improved recurrence-free and overall survival,14 independent of conventional prognostic factors including tumor size and lymph node involvement. A recent review summarized data from 12,439 breast cancer patients participating in multiple studies and correlated TILs with response to chemotherapy. Similar results were reported, them majority of studies showed that TILs were predictive of pathological complete response, disease free survival, and overall survival.21 In other subsets of breast cancer, including HER2-positive breast cancer, TILs have also been shown to have prognostic implications;22 however, the predictive impact of TILs for response to trastuzumab has been conflicting. For example, in the FinHER study of chemotherapy with or without nine weeks of adjuvant trastuzumab15, TIL count predicted response to HER2 directed therapy. However, in North Central Cancer Treatment Group (NCCTG) N9831, a North American study of chemotherapy with or without a year of adjuvant trastuzumab, TIL count was not predictive of response to trastuzumab.19 Theoretically, immunotherapy agents should facilitate tumoral TIL recruitment; however, there have been limited studies evaluating TILs in breast cancers treated with immune checkpoint therapy or cancer vaccines to date. PD-L1 expression in breast cancer The model of personalized cancer therapy generally involves evaluating a tumor for the presence or absence of specific biomarkers, and then tailoring treatment recommendations accordingly. Examples in breast cancer include measurement of HER2 positivity (by immunohistochemistry [IHC] or in situ hybridization [ISH]) to guide treatment recommendations about HER2-targeted therapies, or measurement of hormone receptors to guide treatment recommendations about anti-estrogen agents. This same model has been applied to immune therapy with tumoral or stromal membranous PD-L1 expression status by IHC, informing decisions about anti-PD-1/L1 therapy in some settings. For example, in the first clinical trial of the anti-PD1 agent, nivolumab, responses in
melanoma were not observed in patients with tumors that were classified as negative for PD-L1 expression.23 Notably, however, only 36% of patients with PD-L1 positive tumors had an objective response to therapy, indicating that PD-L1 positivity does not absolutely predict for responses, an observation which may be explained, at least in part, by the dynamic nature of PD-L1 expression (i.e. PD-L1 expression is upregulated in tumor cells, for example, in response to IFN secretion by activated T cells). The observation of PD-L1 expression enriching for responses to PD-1/PD-L1 directed therapy has since been recapitulated in subsequent trials of both lung cancer and melanoma, with fewer responses reported in tumors classified as PD-L1 negative. On the basis of these findings, attempts have been made to classify breast cancers as PD-L1 positive or negative. However, it should be noted that determination and reporting of PD-L1 expression has not been standardized, and consequently, comparison of predictive impact of PD-L1 expression across clinical trials have not been feasible. Specifically, to date, there has been no standardization of assays, percentage cutoff for establishing positivity, or scoring methodology. For example, some investigators have classified positivity as PD-L1 expression in tumor cells only, whereas others have measured expression in TILs only. Some studies measure “hotspots” (areas of maximal expression), whereas others measure average expression. The use of different techniques to measure PD-L1 expression further confounds cross-study comparisons. One clinical trial of anti-PD-L1 therapy in metastatic breast cancer attempted to classify tumors with a proprietary IHC PD-L1 assay, similar to method used in other tumor types. They reported 58% PD-L1 positivity in TNBC and 19% in hormone positive/HER2-negative tumors.24,25 Another proposed method of reporting PD-L1 positivity is using PD-L1 RNA expression data from the Cancer Genome Atlas (TCGA) and PD-L1 IHC expression in microarrays: in one related study, PD-L1 expression was much higher in TNBC compared to hormone receptor positive breast cancer, although only 19% of TNBC specimens expressed PD-L1.26 In another trial, in which eligibility was not restricted to patients with tumoral PDL1 expression, PD-L1 expression was not predictive of response to PD-L1 directed therapy.27 However, the presence of PD-L1 “hotspots,” in areas of aggregated TIL’s, did appear to predict for response. Given that PD-L1 expression appears to enrich for responses to PD-1 or PD-L1 directed therapy in other settings, most related breast cancer trials require PD-L1 expression or positivity for eligibility. However, standardized methods for PD-L1 evaluation and reporting are needed. Furthermore, because significant clinical
responses have also been observed in PD-L1 “negative” tumors, considerable efforts are underway to identify additional biomarkers of response. Immunotherapy in Breast Cancer Cancer vaccines for breast cancer Cancer vaccines facilitate immune recognition of tumors by providing an exogenous source or TAAs, sometimes delivered with substances to enhance response, called immune adjuvants. Because cancer vaccines promote specific responses to one or several antigens, they are generally well tolerated. No breast cancer vaccines are approved by the FDA currently, however multiple vaccine strategies are being investigated.28,29 There are currently three classes of cancer vaccines in development, including monovalent peptide vaccines, polyvalent peptide vaccines, and cellular vaccines. Monovalent vaccines Monovalent vaccines facilitate immune responses against a single TAA. These vaccine targets are focused on antigens that are expressed by a large proportion of tumor cells, and with higher expression in tumor cells compared to normal tissue. Single peptide vaccines are the simplest class of cancer vaccine. These vaccines can be made at low cost compared to other classes, and are easily generated with high purity. Multiple targets have been proposed including HER2, mucin 1 (MUC1), carcinoembryonic antigen (CEA), p53, survivin, and many others.29 The most well studied TAAs peptide vaccines in breast cancer are against HER2 and MUC1. Monovalent vaccines depends on robust immunological responses against a single antigen, or further propagation of the immune response to other antigens in a process called epitope spreading. One potential challenge of monotherapy is the outgrowth of resistant tumor cells that have down-regulated the peptide target. This observation was noted clinically in a HER2 pre-operative vaccine trial whereby residual tumor exhibited a loss of HER2 expression.30 Another important observation in vaccine development is that the amount of Th1 immune activation relative to Th2 immune activation is integral for the success of tumor eradication. Th1 responses are effective in facilitating anti-tumor immunity via secretion of IFN, IL-2, and IL-12.31 One strategy used in vaccine development is to select specific peptides that are more likely to potentiate Th1 responses.32
HER2-directed vaccines The HER2-protein may be an effective antigen target because it is overexpressed frequently in breast cancer. In the clinical setting, HER2 overexpression is reported in all breast cancers cases during pathology review. Multiple studies have shown that the HER2protein is immunogenic and can induce CD4+ and CD8+ lymphocyte antigen-specific immune responses.33,34 The most studied HER2-protein vaccine is E75, or nelipepimut-S (NeuVax). This peptide vaccine expresses an HLA-class 1 restricted peptide from the extracellular domain of the HER2/neu protein. The E75 antigen has been shown to be immunogenic and stimulate a CD8+ cytotoxic response specific to HER2.35 The E75 vaccine has also been evaluated in combination with GM-CSF in the adjuvant setting. The vaccine was intended for treatment of tumors with any degree of overexpression of HER2, improved outcomes (disease free survival and immune responses) were observed moreso in patients with low or intermediate HER2 expression. This observation suggests that patients who have overexpression may have preexisting immune-tolerance to HER2, which may impair immunogenic response following vaccination. The GP2 HER2 peptide is another HER2 antigen target in development. This peptide is an HLA-A2-restricted peptide from the transmembrane domain of the HER2/neu protein. Although this antigen has been shown to have a weaker affinity for HLA-A2 than E75, preclinical studies have shown it to be an effective immunogenic target.36 Current phase II/III clinical trials with HER2 vaccines and GM-CSF are currently underway using both the E75 antigen (NCT01479244) and the GP2 antigen (NCT00524277). Carbohydrate antigens The MUC1 glycoprotein is a member of the mucin family, it is another well-suited target for breast cancer vaccines because it highly expressed. Similar to HER2, this glycoprotein is also implicated in tumor growth and metastatic potential. The most well studied MUC1 vaccine is a carbohydrate Sialyl-Tn epitope of MUC1, named TheratopeTM. The vaccine is a synthetic O-linked disaccharide liked to keyhole limpet hemcyanin (STnKLH). Although this epitope of MUC1 was shown to be an effective immunomodulator in preclinical studies and in a phase II trial,37 there was no improvement in time to progression in a phase III trial in metastatic breast cancer patients.38
Cellular vaccines The third class of vaccine involves the transfer of whole cellular or modified cellular components from tumor or immune cells modified ex vivo from either patient-derived or donor-derived cells. The overall concept is to prime the immune system to multiple TAA to provoke a more robust response. The DRribbles vaccine (UbiVac) is created by treating patient-derived or donor-derived tumors utilizing the autophagy process to create shortlived proteins (SLiPs) and defective ribosomal products (DRiPs), DRibbles is most studied in lung cancer, and as been shown to stimulate both cytotoxic and helper T-cell cells against neoantigens or TAAs specific to the tumor.39 A phase II clinical trial is planned in breast cancer. GVAX (Aduro BioTech) is another cellular vaccine derived from irradiated autologous tumor cells that are genetically engineered to express GM-CSF. The GVAX vaccine has been studied in multiple tumor types.40 Another strategy is to engineer immune cells ex vivo against TAAs. Sipuleucil-T is a cellular vaccine to treat prostate cancer. Autologous peripheral blood is leukopheresed and treated with GM-CSF linked to prostatic acid phosphatase. In a phase III clinical trial, it improved survival in prostate cancer, and is FDA approved for this indication.41 Dendritic cell (DC) vaccines are also being investigated in breast cancer. Patient-derived DCs were harvested prior to lumpectomy in DCIS trials. DCs were treated with cytokines and synthetic HER2-peptides and then injected intranodally. Vaccination was associated with tumor shrinkage, a reduction of HER-2/neu expression, and specific T-cell activation against HER2,30 with an 88% reduction of HER2 expression with patients with residual disease. These results indicate that the vaccine caused either immune mediated destruction of HER2-positive cells or downregulation of HER2 expression. Immune Checkpoint Therapy Checkpoint Blockade Monotherapy Strategies Anti PD-1/L1 agents have shown promise as monotherapy in multiple tumor types, and are currently being evaluated in breast cancer. Most studies use PD-L1 “positivity” as a criterion for eligibility, although as previously discussed, determination of PD-L1 status has not been standardized to date. Some studies define PD-L1 positivity as expression in at least 1% of tumor cells while others use a cutoff of expression in at least 5% of stromal TILs. The
first report of PD-1/PD-L1 blockade in breast cancer came from a phase 1b study of pembrolizumab, an anti-PD-1 directed antibody, in 27 patients with pretreated metastatic TNBC. Women with PD-L1 positive disease, defined as at least 1% expression in tumor or stroma were eligible. Pembrolizumab in that setting was associated with an overall response rate (ORR) of 18.5%, and progression-free survival of 23% at 6 months.24 Soon thereafter the results from a phase 1 expansion cohort of 21 women with metastatic TNBC treated with atezolizumab, a PD-L1 directed antibody were reported. In that study, an ORR of 19% and progression-free survival of 27% at 6 months were reported.42,43 Thus, the first two studies of PD-1/PD-L1 directed therapy in similar populations of women with chemotherapy-resistant, PD-L1 positive TNBC conferred similar benefits. Although most PD-1/PD-L1 breast cancer studies have focused on TNBC populations (as a consequence of the higher prevalence of PD-L1 “positivity” in this subtype), these strategies have also been explored in women with other subtypes of breast cancer. For example, 25 women with hormone receptor positive, HER2-neu negative breast cancer participated in Keynote-028, a phase Ib multicohort trial of pembrolizumab monotherapy for PD-L1-positive advanced tumors with an ORR of 12% and a clinical benefit rate of 20% reported.25 Of note, 11 of the 25 women who participated in this study received at least 5 lines of prior treatment and all women received prior palliative chemotherapy. In the JAVELIN study, 168 women with breast cancer of any subtype who were unselected for PD-L1 expression were treated with avelumab, a PD-L1 directed antibody.27 An ORR of 8.6% and 2.8% were reported in women with TNBC and hormone receptor positive breast cancer, respectively, and responses did not correlate with various PD-L1 cut-offs for positivity. However, an ORR of 33% was observed in patients for whom immune cell clusters or “hotspots” were identified. It is unclear whether the overall responses in the JAVELIN study were more modest than those reported in other studies because of the lack of selection for PD-L1 positivity; however, the fact that responses have been observed with monotherapy in chemotherapy-resistant TNBC and hormone receptor positive disease indicates that immune therapy benefits some subgroups of breast cancer patients and warrants further study. Studies exploiting other checkpoint blockade mechanisms, such as CTLA-4-mediated blockade with tremelimumab monotherapy are also underway in advanced solid tumors including breast cancer (i.e. NCT02527434).
Dual Checkpoint Blockade Therapy Strategies In melanoma, the combination of ipilimumab (anti-CTLA-4) and nivolumab (antiPD-1) resulted in improved progression free survival compared to monotherapy, however the effect on overall survival is uncertain.44 One important drawback of combination therapy was an increase in reported adverse events. Multiple clinical trials are evaluating combination immune checkpoint therapy in other tumor types, including breast cancer. A phase I/II multi-cohort clinical trial of ipilimumab plus nivolumab in advanced solid tumors has been completed (NCT02499367) but the results for the metastatic TNBC cohort have not yet been reported. As previously outlined, a pilot study of pre-operative ipilimumab, nivolumab and cryoablation in the treatment of early stage breast cancer is underway (NCT02833233), as are multiple other studies combining checkpoint therapies with or without other agents in breast cancer (i.e. entinostat, nivolumab and ipilimumab in NCT02453620; tremelimumab and durvalumab in NCT02639026). Strategies That Combine Checkpoint Blockade with Other Systemic Therapies Hormone therapy combinations Although hormone therapy is a cornerstone of palliative therapy for women with advanced hormone receptor positive breast cancer, hormone sensitive tumors typically become resistant to anti-estrogen treatment over time. Consequently, rational combinations of hormone therapies with other agents, including checkpoint blockade strategies, are being investigated. For example, in a phase 1 study the CTLA-4 directed antibody, tremelimumab (MedImmune) was combined with the aromatase inhibitor exemestane (Aromasin, Pfizer) in 26 women with hormone receptor positive metastatic breast cancer, with tremelimumab administered at different doses and schedules (3-10 mg/kg every 28 or 90 days).45 Although no objective responses were reported with the combination, disease stability was observed in 42%, including 4 of the 5 women who had previously progressed on exemestane. Furthermore, potentially favorable changes in immune correlates were observed in most patients treated with tremelimumab, as evidenced by a systemic increase in CD4+ and CD8+ T cells expressing inducible costimulator (ICOS), a proposed marker of T cell activation, and changes in the ratios of specific T cell subtypes. These data, in combination with data from anti-PD-1/L1
monotherapy studies in hormone receptor positive breast cancer, supports further exploration of hormone therapy-immune therapy combinations in this setting. To that end, a large phase II trial of pembrolizumab plus anti-estrogen therapy is currently underway (NCT02648477). Cytotoxic chemotherapy combinations Chemotherapy is not only directly cytotoxic, but may also induce an immune response by releasing cytokines and TAAs.46,47 This immune response could, in turn, be augmented when combined with immune checkpoint therapy, a hypothesis that is supported by a growing body of pre-clinical data. Clinical trials in lung cancer and melanoma also suggest potential benefits with combination chemotherapy and immunotherapy.48,4950 A phase III trial of ipilimumab plus etoposide and platinum in advanced small lung cell cancer recently reported no difference in overall survival or disease free progression.51 The first clinical trial report of chemotherapy with checkpoint blockade in breast cancer was a phase 1b clinical trial of atezolizumab plus nanoparticle albumin-bound paclitaxel (Abraxane, Celgene) in women with advanced TNBC. The confirmed ORR was 46% in the first-line setting and 38% overall, with no significant correlation observed between responses and PD-L1 expression.52 Furthermore, after a median follow-up of 6 months, 6 of the 12 responders remain on therapy. Durability of responses has been reported in other checkpoint blockade studies in breast and other cancers, and is a hallmark of this strategy. Several trials of chemotherapy-immune therapy are underway in different phases (NCT02309117, NCT02331251). Prospectively collected data from randomized studies comparing chemotherapy with or without checkpoint blockade are ultimately needed. Other systemic therapy combinations Combination strategies may be further improved when combined with targeted agents. Specifically, agents that mediate downstream signaling and thereby prevent or overcome hormone therapy resistance have not only been successfully combined with hormone therapies but have also been shown to modulate PD-L1 expression. For example, the AKT inhibitor, rapamycin, has been shown to inhibit the PI3K signaling pathway and modulate PD-L1 expression.26 Thus, immune therapy in combination with drugs that target the PI3K
pathway may further enhance antitumor responses. Other targeted therapy combinations are underway including a trial of checkpoint blockade with the anti-angiogenesis drug, bevacizumab (Avastin, Genentech; NCT02484404). Cancer Vaccines There have been no trials of cancer vaccines with checkpoint blockade therapy in breast cancer to date. However, this strategy has been studied in a phase II clinical trial in melanoma. This study combined a dendritic cell vaccine, TriMixDC-MEL, and ipilimumab with encouraging results (38% ORR).53 Trials of vaccines with checkpoint blockade in breast cancer are planned. Strategies That Combine Checkpoint Blockade with Local Therapies Radiation and ablation can not only cause physical damage to tumors, but can also induce potentially beneficial inflammatory effects.54 Three types of ablative treatments have been used for the treatment of cancer: cyroablation (freezing), radiofrequency ablation (RFA) with exposure to medium frequency current, or thermal ablation (heating). Through physical disruption, these local therapies can induce release of TAAs and, in turn, TAA-specific T-cell responses which may be further augmented when combined with immune modulation.55 In mice, radioablation or cryoablation in combination with checkpoint blockade therapy has been synergistic.56,57 In a pilot study, women with early stage breast cancer for whom mastectomy was planned were treated with a single dose of pre-operative tumor cryoablation and/or ipilimumab. The combination of cryoablation and ipilimumab was safe and did not incur any delays in the standard-of-care surgical planning.58 Moreover, immune activation was noted in the tumor and the peripheral blood with the combination. For example, an increase in serum T cell populations by Ki67 and ICOS expression was observed, suggesting increased T cell activation and proliferation, respectively. Furthermore, expansion of intratumoral T-cell clones were detected by T-cell receptor signaling, indicating that TAA-specific T cell populations were being generated in response to the intervention.59 A pilot study of pre-operative ipilimumab, nivolumab and cryoablation in breast cancer is underway (NCT02833233) and will directly inform a planned randomized study of peri-operative cryoablation with checkpoint blockade, versus standard care, in early stage breast cancer.
Radiation, a treatment modality that is commonly used with palliative and curative intent for the treatment of many tumors, is immunogenic and therefore, may synergize with checkpoint blockade therapy. In one pre-clinical trial, for example, radiation was combined with CTLA-4 and PD-L1 directed therapy with synergistic effect.60 Furthermore, radiation in combination with checkpoint blockade therapy has not only been well tolerated in both prostate cancer and melanoma, but has also been effective in inducing tumor responses at sites distant to the radiation field in some cases. This so-called abscopal effect, whereby tumor regression is observed beyond the radiation site, suggests that a tumor-specific immune response has successfully been induced.61-63 In a mouse model of TNBC, radiation plus anti-CTLA-4 blockade showed both a decrease in tumor size and improved survival.64 There are currently several trials evaluating combinations of radiation plus immune checkpoint inhibitors in breast cancer including a trial of tremelimumab (anti-CTLA-4) plus brain radiation with or without trastuzumab (NCT02563925) in women with breast cancer brain metastases, and a trial of pembrolizumab with radiation in women with metastatic triple negative breast cancer (NCT02730130). Conclusion Immunotherapy is a rapidly evolving area of research in the treatment of cancer with encouraging results in multiple tumor types including breast cancer. However, because breast cancer is a heterogeneous disease and because most breast cancers demonstrate limited innate immunogenicity, strategies that combine checkpoint blockade with other drugs or treatment modalities - including vaccines - are likely needed as are biomarkers that predict responses to specific strategies. Ultimately, it is hoped that these combination strategies will improve responses for women with breast cancer and ultimately translate into long-term tumor-specific immunity and increased cure rates.
References 1. Page DB, Bourla AB, Daniyan A, et al. Tumor Immunology and Cancer Immunotherapy: Summary of the 2014 SITC Primer. Journal for immunotherapy of cancer 2015;3. 2. O'Shea JJ, Paul WE. Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 2010;327:1098-102. 3. Mittendorf EA, Clifton GT, Holmes JP, et al. Clinical trial results of the HER-2/neu (E75) vaccine to prevent breast cancer recurrence in high-risk patients: from US Military Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer 2012;118:2594-602. 4. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science 1996;271:1734-6. 5. Hodi FS, O'Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711-23. 6. Tarhini AA. Tremelimumab: a review of development to date in solid tumors. Immunotherapy 2013;5:215-29. 7. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol 2012;24:207-12. 8. Robert C, Long GV, Brady B, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med 2015;372:320-30. 9. Robert C, Schachter J, Long GV, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med 2015;372:2521-32. 10. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 2015;372:2018-28. 11. Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015;373:1627-39. 12. Salgado R, Denkert C, Demaria S, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol 2015;26:259-71. 13. Tumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014;515:568-71. 14. Loi S, Drubay D, Adams S, et al. Pooled individual patient data analysis of tumor infiltrating lymphocytes (TILs) in primary triple negative breast cancer (TNBC) treated with anthracycline-based chemotherapy. San Antonio Breast Cancer Symposium; 2015 12/18/2015; San Antonio, TX. 15. Loi S, Michiels S, Salgado R, et al. Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial. Ann Oncol 2014;25:1544-50. 16. Adams S, Gray RJ, Demaria S, et al. Prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancers from two phase III randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199. J Clin Oncol 2014;32:2959-66. 17. Denkert C, Loibl S, Noske A, et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010;28:10513. 18. Salgado R, Denkert C, Campbell C, et al. Tumor-Infiltrating Lymphocytes and Associations With Pathological Complete Response and Event-Free Survival in HER2-Positive Early-Stage Breast Cancer Treated With Lapatinib and Trastuzumab: A Secondary Analysis of the NeoALTTO Trial. JAMA Oncol 2015;1:448-54.
19. Perez EA, Ballman KV, Tenner KS, et al. Association of Stromal Tumor-Infiltrating Lymphocytes With Recurrence-Free Survival in the N9831 Adjuvant Trial in Patients With EarlyStage HER2-Positive Breast Cancer. JAMA Oncol 2016;2:56-64. 20. Desmedt C, Salgado R, Buisseret L, et al. Characterization of lymphocytic infiltration in invasive lobular breast cancer. San Antonio Breast Cancer Symposium; 2015 December 8-12, 2015; San Antonio, Texas. 21. Dushyanthen S, Beavis PA, Savas P, et al. Relevance of tumor-infiltrating lymphocytes in breast cancer. BMC medicine 2015;13:202. 22. Dieci MV, Goubar A, Mathieu M-C, et al. Prognostic and predictive value of tumor infiltrating lymphocytes (TIL) in two phase III randomized adjuvant breast cancer (BC) trials. ASCO Meeting Abstracts 2014;32:11087. 23. Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of antiPD-1 antibody in cancer. N Engl J Med 2012;366:2443-54. 24. Nanda R, Chow LQ, Dees EC, et al. Pembrolizumab in Patients With Advanced TripleNegative Breast Cancer: Phase Ib KEYNOTE-012 Study. J Clin Oncol 2016. 25. Rugo H, DeLord JP, Im S-A, et al. Preliminary efficacy and safety of pembrolizumab (MK3475) in patients with PD-L1-positive, estrogen receptor positive (ER+)/HER2-negative advanced breast cancer enrolled in KEYNOTE-028. San Antonio Breast Cancer Symposium; 2015 December 8-12, 2015; San Antonio, TX. 26. Mittendorf EA, Philips AV, Meric-Bernstam F, et al. PD-L1 expression in triple-negative breast cancer. Cancer Immunol Res 2014;2:361-70. 27. Dirix LY, Takacs I, Nikolinakos P, et al. Avelumab (MSB00107118C), an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast canceR: A phase Ib JAVELIN solid tumor trial. San Antonio Breast Cancer Symposium; 2015 December 8-12, 2015; San Antonio, TX. 28. Page DB, Naidoo J, McArthur HL. Emerging immunotherapy strategies in breast cancer. Immunotherapy 2014;6:195-209. 29. Harao M, Mittendorf EA, Radvanyi LG. Peptide-based vaccination and induction of CD8+ T-cell responses against tumor antigens in breast cancer. BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy 2015;29:15-30. 30. Sharma A, Koldovsky U, Xu S, et al. HER-2 pulsed dendritic cell vaccine can eliminate HER-2 expression and impact ductal carcinoma in situ. Cancer 2012;118:4354-62. 31. Castellino F, Germain RN. Cooperation between CD4+ and CD8+ T cells: when, where, and how. Annu Rev Immunol 2006;24:519-40. 32. Cecil DL, Holt GE, Park KH, et al. Elimination of IL-10-inducing T-helper epitopes from an IGFBP-2 vaccine ensures potent antitumor activity. Cancer research 2014;74:2710-8. 33. Baxevanis CN, Sotiropoulou PA, Sotiriadou NN, Papamichail M. Immunobiology of HER2/neu oncoprotein and its potential application in cancer immunotherapy. Cancer Immunol Immunother 2004;53:166-75. 34. Ward RL, Hawkins NJ, Coomber D, Disis ML. Antibody immunity to the HER-2/neu oncogenic protein in patients with colorectal cancer. Human immunology 1999;60:510-5. 35. Benavides LC, Gates JD, Carmichael MG, et al. The impact of HER2/neu expression level on response to the E75 vaccine: from U.S. Military Cancer Institute Clinical Trials Group Study I01 and I-02. Clin Cancer Res 2009;15:2895-904. 36. Carmichael MG, Benavides LC, Holmes JP, et al. Results of the first phase 1 clinical trial of the HER-2/neu peptide (GP2) vaccine in disease-free breast cancer patients: United States Military Cancer Institute Clinical Trials Group Study I-04. Cancer 2010;116:292-301.
37. Rochman S. New peptide vaccine for HER2-expressing breast tumors. J Natl Cancer Inst 2015;107. 38. Julien S, Picco G, Sewell R, et al. Sialyl-Tn vaccine induces antibody-mediated tumour protection in a relevant murine model. Br J Cancer 2009;100:1746-54. 39. Hilton TL, Hulett TW, Dubay C, et al. DPV-001 an autophagosome-enriched cancer vaccine in phase II clinical trials contains 25 putative cancer antigens, DAMPS, HSPS and agonists for TLR 2, 3, 4, 7 and 9. SITC 28th Annual Meeting; 2013 November 8-10, 2013; National Harbor, MD. 40. Karan D, Van Veldhuizen P. Combination immunotherapy with prostate GVAX and ipilimumab: safety and toxicity. Immunotherapy 2012;4:577-80. 41. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-T immunotherapy for castrationresistant prostate cancer. N Engl J Med 2010;363:411-22. 42. Emens LA. Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer (TNBC) AACR Annual Meeting; 2015 April 20, 2015; Philidelphia, PA. 43. Emens LA, Kok M, Ojalvo LS. Targeting the programmed cell death-1 pathway in breast and ovarian cancer. Current opinion in obstetrics & gynecology 2016;28:142-7. 44. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med 2015;373:23-34. 45. Vonderheide RH, LoRusso PM, Khalil M, et al. Tremelimumab in combination with exemestane in patients with advanced breast cancer and treatment-associated modulation of inducible costimulator expression on patient T cells. Clin Cancer Res 2010;16:3485-94. 46. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of cancer chemotherapy. Nat Rev Immunol 2008;8:59-73. 47. Sistigu A, Yamazaki T, Vacchelli E, et al. Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy. Nat Med 2014. 48. Reck M, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line therapy in extensive-disease-small-cell lung cancer: results from a randomized, double-blind, multicenter phase 2 trial. Ann Oncol 2013;24:75-83. 49. Lynch TJ, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line treatment in stage IIIB/IV non-small-cell lung cancer: results from a randomized, double-blind, multicenter phase II study. J Clin Oncol 2012;30:2046-54. 50. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 2011;364:2517-26. 51. Reck M, Luft A, Szczesna A, et al. Phase III Randomized Trial of Ipilimumab Plus Etoposide and Platinum Versus Placebo Plus Etoposide and Platinum in Extensive-Stage SmallCell Lung Cancer. J Clin Oncol 2016. 52. Adams S, Diamond J, Hamilton E, et al. Phase Ib trial of atezolizumab in combination with nab-paclitaxel in patients with metastatic triple-negative breast cancer (mTNBC). 2016 ASCO Annual Meeting; 2016 June 3-7, 2016; Chicago, IL. 53. Wilgenhof S, Corthals J, Heirman C, et al. Phase II Study of Autologous MonocyteDerived mRNA Electroporated Dendritic Cells (TriMixDC-MEL) Plus Ipilimumab in Patients With Pretreated Advanced Melanoma. J Clin Oncol 2016. 54. Chu KF, Dupuy DE. Thermal ablation of tumours: biological mechanisms and advances in therapy. Nature reviews Cancer 2014;14:199-208. 55. !!! INVALID CITATION !!! 56. Shi L, Chen L, Wu C, et al. PD-1 Blockade Boosts Radiofrequency Ablation-Elicited Adaptive Immune Responses against Tumor. Clin Cancer Res 2016;22:1173-84.
57. Waitz R, Solomon SB, Petre EN, et al. Potent induction of tumor immunity by combining tumor cryoablation with anti-CTLA-4 therapy. Cancer research 2012;72:430-9. 58. McArthur HL, Diab A, Page DB, et al. A pilot study of preoperative single-dose ipilimumab and/or cryoablation in women with early-stage breast cancer with comprehensive immune profiling. Clin Cancer Res 2016. 59. Page D, Yuan J, Redmond D, Wen HY, Durack JC, Emerson RO. T-cell Receptor DNA Deep Sequencing as a biomarker of tumor infiltrating lymphocytes and immunotherapy-associated clonal lymphocyte expansion in breast cancer. Cancer Immunol Res 2016. 60. Twyman-Saint Victor C, Rech AJ, Maity A, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature 2015;520:373-7. 61. Slovin SF, Higano CS, Hamid O, et al. Ipilimumab alone or in combination with radiotherapy in metastatic castration-resistant prostate cancer: results from an open-label, multicenter phase I/II study. Ann Oncol 2013. 62. Barker CA, Postow MA, Khan SA, et al. Concurrent Radiotherapy and Ipilimumab Immunotherapy for Patients with Melanoma. Cancer Immunol Res 2013;1:92-8. 63. Postow MA, Callahan MK, Barker CA, et al. Immunologic correlates of the abscopal effect in a patient with melanoma. N Engl J Med 2012;366:925-31. 64. Demaria S, Kawashima N, Yang AM, et al. Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. Clin Cancer Res 2005;11:728-34.
PII: DOI: Reference:
S0147-0272(16)30075-7 http://dx.doi.org/10.1016/j.currproblcancer.2016.09.009 YMCN312
To appear in: Current Problems in Cancer Received date: 21 September 2016 Accepted date: 21 September 2016 Cite this article as: Katherine Sanchez, David Page and Heather L. McArthur, Immunotherapy in breast cancer: An overview of modern checkpoint blockade strategies and vaccines, Current Problems in Cancer, http://dx.doi.org/10.1016/j.currproblcancer.2016.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title: Immunotherapy in breast cancer: An overview of modern checkpoint blockade strategies and vaccines Authors: Katherine Sanchez, MD1, David Page, MD1, and Heather L. McArthur, MD, MPH2 1Providence
Cancer Center / Earle A. Chiles Research Institute, Portland, OR
2Cedars-Sinai
Medical Center, Los Angeles, CA
Disclosures: Dr. McArthur has participated in advisory boards for Celgene, Merck, OBI, Peregrine, Spectrum Pharmaceuticals and Syndax Pharmaceuticals and has research supported by Bristol-Myers Squibb, MedImmune, LLC/AstraZenica, Eli Lilly, and Merck Pharmaceuticals. Dr. Page has participated in advisory boards for Celgene and Peregrine, and has research supported by Medimmune and Merck Pharmaceuticals. Research support: No research funds were used in the development of this manuscript Key words (from MeSH database): Breast neoplasms; immunotherapy; vaccines; CTLA-4 Antigen; programmed cell death 1 receptor; lymphocytes, tumor-infiltrating
Abstract Immune therapy has recently emerged as a standard-of-care strategy for the treatment of melanoma, lung cancer, bladder cancer, among other malignancies. However, the role of immune therapy in the treatment of breast cancer is still being determined. Two current strategies for harnessing the immune system to treat cancer include drugs that modulate key T cell inhibitory checkpoints and vaccines. Specifically, modern immune therapy strategies can facilitate T-cell mediated tumor regression by priming the immune system against specific tumor associated antigens, by modulating immunoregulatory signals, or both. In breast cancer, preliminary data from preclinical and early clinical studies are promising. In fact, clinical data with checkpoint blockade as monotherapy has been reported in multiple breast cancer subtypes to date, with durable responses observed in a significant proportion of women with chemotherapy resistant disease. However, because the number of genetic mutations and thus, the number of neoantigens available for immune response are modest in most breast cancers when compared with other cancers, most breast cancers may not be inherently sensitive to immune modulation and therefore may require strategies that enhance tumor associated antigen presentation if immune modulation strategies are to be effective. To that end, studies that combine checkpoint blockade with other strategies including established systemic therapies (including hormone therapy and chemotherapy), radiation therapy, and localized therapy including tumor freezing (cryoablation) are underway in breast cancer. Studies that combine checkpoint blockade with vaccines are also planned. Herein, we provide a brief summary of key components of the immune response against cancer, a rationale for the use of immune therapy in breast cancer, data from early clinical trials of checkpoint blockade and vaccine strategies in breast cancer, and future directions in the field. Introduction Immunotherapy, or the use of the body’s immune system to fight cancer, is a rapidly evolving field. Cancer-specific immune responses are initiated when the immune system effectively recognizes abnormally expressed proteins from cancer cells, called tumorassociated antigens (TAAs). Immune responses are tightly regulated by a balance of stimulatory and inhibitory mechanisms. Modern immune therapy strategies in cancer aim to prime the immune system toward specific TAAs, to modulate immune-regulatory signals
towards a favorable inflammatory state, or both. To achieve these aims, investigators are exploring several therapeutic strategies including immune checkpoint antibodies which block inhibitory signals to exploit existing immunity, antibodies to other immune targets, tumor vaccines which prime the immune system against TAAs, intratumoral oncolytic viruses, and chimeric antigen receptor (CAR) T cell therapies. To date, immune checkpoint blockade antibodies and vaccines have been most extensively studied in breast cancer. Emerging data suggest, however, that efficacy can be maximized by combining multiple immunotherapy strategies, for example, immune checkpoint blockade antibodies plus cancer vaccines. Herein we describe the rationale for immune checkpoint antibodies and vaccines in the treatment of breast cancer, summarize the relevant literature, and highlight future approaches. An Overview of Immunotherapy Principles T-lymphocyte activation The adaptive immune response against cancer depends on antigen-specific T cell activation. In this process, T cells survey tissues for TAAs that are presented in peptide complexes bound to a cellular surface protein called major histocompatibility complex-1 (MHC-1). The T cell receptor (TCR) binds the antigen-MHC complex and, in turn, induces activation of T cells. Although MHC-1 molecules are present on many cell types, including normal and tumor cells, specialized cells called antigen presenting cells (APCs) have costimulatory molecules and are highly effective in presenting peptides and activating T cells. Classical examples of APCs include dendritic cells (DCs), macrophages, and Langerhans cells. This interaction can occur in the tumor, in secondary lymphoid organs, or in peripheral tissues.1 APCs engulf apoptotic cells and extracellular peptides . These peptides are then digested and expressed on the APC surface chaperoned by MHC-1 molecules. Activation requires two signals: the first originates from the binding of the MHCpeptide complex to the TCR, and the second originates from the binding of co-stimulatory molecules CD80/86 (B7-1/2) on APCs to CD28 on T cells. Once activated, downstream TCR signaling is regulated by inhibitory signaling via cellular surface proteins, including programmed death (PD)-1 (which binds to PD-ligands 1 or 2) and cytotoxic T-lymphocyte antigen 4 (CTLA-4, which competes for binding with B7-1/2). These regulatory signals are
physiologically important to prevent uncontrolled inflammation and autoimmune disease. However, the same regulatory signals may also impair anti-tumor immune responses. There are multiple type of T-cells, classified by their function and expression of surface proteins. Two subtypes are CD8+ and CD4+ T cells. CD8+ T-cells, also called cytotoxic T-cells, are activated by type 1 MHC molecules expressed on tumor cells, and are capable of directly killing tumor cells. CD4+ T-cells are called helper T-cells, and are activated upon binding to antigen-loaded type 2 MHC molecules on APCs. These helper Tcells can further differentiate into T-helper type 1 (Th1), type 2 (Th2), type 17 (Th17), or regulatory T cells (T reg) based on the cytokine milieu. Once activated, Th1 cells facilitate an anti-tumor response by releasing cytokines, which directly activate and recruit other immune cells. Th1 cells secrete interferon-gamma (IFN) and support the anti-tumor response. Th2 cells secrete IL-4 and IL-13, any may hamper the anti-tumor response. The role of Th17 cells in this process has not been clearly elucidated. Tregs have been implicated in suppression of tumor-specific immune responses and thus, tumor immune escape.2 Strategies that preferentially activate Th1 and CD8+ anti-tumor immune responses, and suppress Th1 and Treg responses, are under development. Cancer vaccines Similar to vaccines used for infectious diseases, cancer vaccines prime the adaptive immune system by providing exogenous antigens such as TAAs. These TAAs are placed into vaccine vectors that facilitate TAA-specific T-cell activation, which in turn would facilitate immune-mediated destruction of TAA-expressing tumor cells. The best studied vaccine target in breast cancer is the human epidermal growth factor receptor 2 (HER2) protein. The HER2 protein is a tyrosine kinase receptor that is present in normal tissue, but overexpressed in a subset of breast cancers. There are numerous HER2- directed vaccines in different stages of development and clinical trials. Nelipipimut-S (NeuVax, Galenda Biopharma) is the most extensively studied HER2-cancer vaccine and is currently being studied in a phase III clinical trial evaluating clinical efficacy in early stage breast cancer (NCT01479244). The relevant TAA, E75, is a peptide from the extracellular domain of HER2.3 Cytotoxic-T cells are activated against the human leukocyte antigen (HLA)presented HER2 epitope, causing immune activation against breast cancers that express
this TAA. It is hoped that the induction of HER2-specific immune response will translate into long term immunity from recurrence and thus, cure.
Immune checkpoint directed antibody therapy Immune checkpoint antibodies facilitate T-cell activation by either binding and agonizing co-stimulatory signals, or binding and antagonizing co-inhibitory signals. Drugs that bind and antagonize a potent regulator of T-cell co-inhibition such as CTLA-4, for example, likely suppress immune activation via a variety of mechanisms. Specifically, downstream CTLA-4 signaling may directly inhibit T-cell activation; however, CTLA-4 also competitively binds to the B7 ligand on APCs, thereby impairing co-stimulatory signaling mediated by CD28 binding to B7.4 Finally, CTLA-4 is expressed on Treg cells, and may promote suppressive Treg activity. Therefore, drugs that bind to CTLA-4 may function by inhibiting downstream signaling, by preventing competitive binding of B7 or by depleting CTLA-4-expressing Tregs in the microenvironment via antibody-dependent cell-mediated cytotoxicity (ADCC). The first clinical signal for CTLA-4 blockade as an effective anti-cancer strategy was demonstrated in advanced melanoma. For example, in one of the early pivotal phase III clinical trials, patients with advanced melanoma were treated with ipilimumab (Yervoy, Bristol Myers Squibb) +/- gp100 vaccine, versus gp100 vaccine alone. Compared to gp100 alone, median overall survival was increased from 6.4 to 10.1 months, and 24-month survival nearly doubled.5 This was the first report of a survival benefit with systemic therapy in advanced melanoma and consequently, ipilimumab became the first immune checkpoint antibody to be approved by the United States (US) Food and Drug Administration (FDA) for the treatment of cancer. Given the success with this strategy in advanced melanoma, many studies of CTLA-4 blockade have been undertaken with ipilimumab or tremelimumab (MedImmune/AstraZeneca), in a variety of tumor types including breast cancer.6 PD-1 is another inhibitory receptor expressed on T-cells that binds to PD-L1/L2 on tumor cells and APCs and mediates T-cell suppression. Nivolumab (Opdivo, Bristol Myers Squibb), pembrolizumab (Keytruda, Merck), and atezolizumab (Tecentriq, Genentech) are all US FDA-approved checkpoint antibodies that target PD-1 or its ligand, PD-L1.7 T-cells
that are chronically stimulated by antigens express PD-1 as part of co-inhibitory pathway that ultimately leads to T-cell exhaustion. Furthermore, interferon gamma (IFNγ) secreted by T cells can promote up-regulation of PD-L1 on both immune cells and tumor cells, which may bind PD-1 and further promote T-cell exhaustion. PD-1/L1 antibodies are thought to function by blocking PD-1-mediated T-cell suppressive signals, thereby re-invigorating Tcells that are specific to tumor TAAs. PD-1/L1 blocking antibodies have been US FDAapproved for the treatment of metastatic melanoma,8,9 non-small cell lung cancer,10,11 renal cell carcinoma, bladder cancer, and Hodgkin lymphoma. Several other anti PD-1/L1 directed antibodies are in development, including durvalumab (anti-PD-L1, Medimmune) and avelumab (anti-PD-L1, EMD Serono). Rationale for Immunotherapy in Breast Cancer Tumor Infiltrating Lymphocytes In some cancers, immune cells infiltrate the tumor or surrounding stroma. The majority of these immune cells are lymphocytes and are therefore called tumor infiltrating lymphocytes (TILs). The presence of TILs suggests endogenous immune recognition of TAAs in some tumors, which in turn, has been associated with improved prognosis, and may also portend inherent responsiveness to immunotherapy. Notably, an effort to standardize TIL reporting in breast cancer was undertaken by an International Working Group and it was ultimately recommended that the percentage of tumor stroma infiltrated by TILs be reported.12 Tumors that are densely infiltrated by TILs may be particularly sensitive to PD1/L1 directed therapies, which function by re-invigorating previously activated T-cells. For example, as first described in metastatic melanoma, tumors with higher baseline TILs were more likely to respond to anti-PD-1, compared to patients with few or no TILs.13 A similar observation has been demonstrated in early stage breast cancer (ESBC) wherein the presence of TILs has been shown to be a potent favorable prognostic and predictive marker, especially in triple negative breast cancer and HER2-positive breast cancer.14-19 Contrastingly, one study of invasive lobular carcinomas reported that the presence of TILs were associated with an adverse prognosis.20 Although this retrospective study did not meet statistical significance and was subject to confounding by other variables, it contributes to a growing body of literature indicating that different breast cancer subtypes
may have different patterns of immune cell recruitment both at baseline and in response to conventional chemotherapies. There is also a growing body of literature demonstrating the impact of TILs in predicting responses to specific systemic therapy strategies in breast cancer. For example, a pooled analysis of TILs as a biomarker in 991 women with triple negative breast cancer (TNBC) participating in five adjuvant anthracycline-based chemotherapy trials was recently reported. In this study, increased quantity of stromal TILs was associated with improved recurrence-free and overall survival,14 independent of conventional prognostic factors including tumor size and lymph node involvement. A recent review summarized data from 12,439 breast cancer patients participating in multiple studies and correlated TILs with response to chemotherapy. Similar results were reported, them majority of studies showed that TILs were predictive of pathological complete response, disease free survival, and overall survival.21 In other subsets of breast cancer, including HER2-positive breast cancer, TILs have also been shown to have prognostic implications;22 however, the predictive impact of TILs for response to trastuzumab has been conflicting. For example, in the FinHER study of chemotherapy with or without nine weeks of adjuvant trastuzumab15, TIL count predicted response to HER2 directed therapy. However, in North Central Cancer Treatment Group (NCCTG) N9831, a North American study of chemotherapy with or without a year of adjuvant trastuzumab, TIL count was not predictive of response to trastuzumab.19 Theoretically, immunotherapy agents should facilitate tumoral TIL recruitment; however, there have been limited studies evaluating TILs in breast cancers treated with immune checkpoint therapy or cancer vaccines to date. PD-L1 expression in breast cancer The model of personalized cancer therapy generally involves evaluating a tumor for the presence or absence of specific biomarkers, and then tailoring treatment recommendations accordingly. Examples in breast cancer include measurement of HER2 positivity (by immunohistochemistry [IHC] or in situ hybridization [ISH]) to guide treatment recommendations about HER2-targeted therapies, or measurement of hormone receptors to guide treatment recommendations about anti-estrogen agents. This same model has been applied to immune therapy with tumoral or stromal membranous PD-L1 expression status by IHC, informing decisions about anti-PD-1/L1 therapy in some settings. For example, in the first clinical trial of the anti-PD1 agent, nivolumab, responses in
melanoma were not observed in patients with tumors that were classified as negative for PD-L1 expression.23 Notably, however, only 36% of patients with PD-L1 positive tumors had an objective response to therapy, indicating that PD-L1 positivity does not absolutely predict for responses, an observation which may be explained, at least in part, by the dynamic nature of PD-L1 expression (i.e. PD-L1 expression is upregulated in tumor cells, for example, in response to IFN secretion by activated T cells). The observation of PD-L1 expression enriching for responses to PD-1/PD-L1 directed therapy has since been recapitulated in subsequent trials of both lung cancer and melanoma, with fewer responses reported in tumors classified as PD-L1 negative. On the basis of these findings, attempts have been made to classify breast cancers as PD-L1 positive or negative. However, it should be noted that determination and reporting of PD-L1 expression has not been standardized, and consequently, comparison of predictive impact of PD-L1 expression across clinical trials have not been feasible. Specifically, to date, there has been no standardization of assays, percentage cutoff for establishing positivity, or scoring methodology. For example, some investigators have classified positivity as PD-L1 expression in tumor cells only, whereas others have measured expression in TILs only. Some studies measure “hotspots” (areas of maximal expression), whereas others measure average expression. The use of different techniques to measure PD-L1 expression further confounds cross-study comparisons. One clinical trial of anti-PD-L1 therapy in metastatic breast cancer attempted to classify tumors with a proprietary IHC PD-L1 assay, similar to method used in other tumor types. They reported 58% PD-L1 positivity in TNBC and 19% in hormone positive/HER2-negative tumors.24,25 Another proposed method of reporting PD-L1 positivity is using PD-L1 RNA expression data from the Cancer Genome Atlas (TCGA) and PD-L1 IHC expression in microarrays: in one related study, PD-L1 expression was much higher in TNBC compared to hormone receptor positive breast cancer, although only 19% of TNBC specimens expressed PD-L1.26 In another trial, in which eligibility was not restricted to patients with tumoral PDL1 expression, PD-L1 expression was not predictive of response to PD-L1 directed therapy.27 However, the presence of PD-L1 “hotspots,” in areas of aggregated TIL’s, did appear to predict for response. Given that PD-L1 expression appears to enrich for responses to PD-1 or PD-L1 directed therapy in other settings, most related breast cancer trials require PD-L1 expression or positivity for eligibility. However, standardized methods for PD-L1 evaluation and reporting are needed. Furthermore, because significant clinical
responses have also been observed in PD-L1 “negative” tumors, considerable efforts are underway to identify additional biomarkers of response. Immunotherapy in Breast Cancer Cancer vaccines for breast cancer Cancer vaccines facilitate immune recognition of tumors by providing an exogenous source or TAAs, sometimes delivered with substances to enhance response, called immune adjuvants. Because cancer vaccines promote specific responses to one or several antigens, they are generally well tolerated. No breast cancer vaccines are approved by the FDA currently, however multiple vaccine strategies are being investigated.28,29 There are currently three classes of cancer vaccines in development, including monovalent peptide vaccines, polyvalent peptide vaccines, and cellular vaccines. Monovalent vaccines Monovalent vaccines facilitate immune responses against a single TAA. These vaccine targets are focused on antigens that are expressed by a large proportion of tumor cells, and with higher expression in tumor cells compared to normal tissue. Single peptide vaccines are the simplest class of cancer vaccine. These vaccines can be made at low cost compared to other classes, and are easily generated with high purity. Multiple targets have been proposed including HER2, mucin 1 (MUC1), carcinoembryonic antigen (CEA), p53, survivin, and many others.29 The most well studied TAAs peptide vaccines in breast cancer are against HER2 and MUC1. Monovalent vaccines depends on robust immunological responses against a single antigen, or further propagation of the immune response to other antigens in a process called epitope spreading. One potential challenge of monotherapy is the outgrowth of resistant tumor cells that have down-regulated the peptide target. This observation was noted clinically in a HER2 pre-operative vaccine trial whereby residual tumor exhibited a loss of HER2 expression.30 Another important observation in vaccine development is that the amount of Th1 immune activation relative to Th2 immune activation is integral for the success of tumor eradication. Th1 responses are effective in facilitating anti-tumor immunity via secretion of IFN, IL-2, and IL-12.31 One strategy used in vaccine development is to select specific peptides that are more likely to potentiate Th1 responses.32
HER2-directed vaccines The HER2-protein may be an effective antigen target because it is overexpressed frequently in breast cancer. In the clinical setting, HER2 overexpression is reported in all breast cancers cases during pathology review. Multiple studies have shown that the HER2protein is immunogenic and can induce CD4+ and CD8+ lymphocyte antigen-specific immune responses.33,34 The most studied HER2-protein vaccine is E75, or nelipepimut-S (NeuVax). This peptide vaccine expresses an HLA-class 1 restricted peptide from the extracellular domain of the HER2/neu protein. The E75 antigen has been shown to be immunogenic and stimulate a CD8+ cytotoxic response specific to HER2.35 The E75 vaccine has also been evaluated in combination with GM-CSF in the adjuvant setting. The vaccine was intended for treatment of tumors with any degree of overexpression of HER2, improved outcomes (disease free survival and immune responses) were observed moreso in patients with low or intermediate HER2 expression. This observation suggests that patients who have overexpression may have preexisting immune-tolerance to HER2, which may impair immunogenic response following vaccination. The GP2 HER2 peptide is another HER2 antigen target in development. This peptide is an HLA-A2-restricted peptide from the transmembrane domain of the HER2/neu protein. Although this antigen has been shown to have a weaker affinity for HLA-A2 than E75, preclinical studies have shown it to be an effective immunogenic target.36 Current phase II/III clinical trials with HER2 vaccines and GM-CSF are currently underway using both the E75 antigen (NCT01479244) and the GP2 antigen (NCT00524277). Carbohydrate antigens The MUC1 glycoprotein is a member of the mucin family, it is another well-suited target for breast cancer vaccines because it highly expressed. Similar to HER2, this glycoprotein is also implicated in tumor growth and metastatic potential. The most well studied MUC1 vaccine is a carbohydrate Sialyl-Tn epitope of MUC1, named TheratopeTM. The vaccine is a synthetic O-linked disaccharide liked to keyhole limpet hemcyanin (STnKLH). Although this epitope of MUC1 was shown to be an effective immunomodulator in preclinical studies and in a phase II trial,37 there was no improvement in time to progression in a phase III trial in metastatic breast cancer patients.38
Cellular vaccines The third class of vaccine involves the transfer of whole cellular or modified cellular components from tumor or immune cells modified ex vivo from either patient-derived or donor-derived cells. The overall concept is to prime the immune system to multiple TAA to provoke a more robust response. The DRribbles vaccine (UbiVac) is created by treating patient-derived or donor-derived tumors utilizing the autophagy process to create shortlived proteins (SLiPs) and defective ribosomal products (DRiPs), DRibbles is most studied in lung cancer, and as been shown to stimulate both cytotoxic and helper T-cell cells against neoantigens or TAAs specific to the tumor.39 A phase II clinical trial is planned in breast cancer. GVAX (Aduro BioTech) is another cellular vaccine derived from irradiated autologous tumor cells that are genetically engineered to express GM-CSF. The GVAX vaccine has been studied in multiple tumor types.40 Another strategy is to engineer immune cells ex vivo against TAAs. Sipuleucil-T is a cellular vaccine to treat prostate cancer. Autologous peripheral blood is leukopheresed and treated with GM-CSF linked to prostatic acid phosphatase. In a phase III clinical trial, it improved survival in prostate cancer, and is FDA approved for this indication.41 Dendritic cell (DC) vaccines are also being investigated in breast cancer. Patient-derived DCs were harvested prior to lumpectomy in DCIS trials. DCs were treated with cytokines and synthetic HER2-peptides and then injected intranodally. Vaccination was associated with tumor shrinkage, a reduction of HER-2/neu expression, and specific T-cell activation against HER2,30 with an 88% reduction of HER2 expression with patients with residual disease. These results indicate that the vaccine caused either immune mediated destruction of HER2-positive cells or downregulation of HER2 expression. Immune Checkpoint Therapy Checkpoint Blockade Monotherapy Strategies Anti PD-1/L1 agents have shown promise as monotherapy in multiple tumor types, and are currently being evaluated in breast cancer. Most studies use PD-L1 “positivity” as a criterion for eligibility, although as previously discussed, determination of PD-L1 status has not been standardized to date. Some studies define PD-L1 positivity as expression in at least 1% of tumor cells while others use a cutoff of expression in at least 5% of stromal TILs. The
first report of PD-1/PD-L1 blockade in breast cancer came from a phase 1b study of pembrolizumab, an anti-PD-1 directed antibody, in 27 patients with pretreated metastatic TNBC. Women with PD-L1 positive disease, defined as at least 1% expression in tumor or stroma were eligible. Pembrolizumab in that setting was associated with an overall response rate (ORR) of 18.5%, and progression-free survival of 23% at 6 months.24 Soon thereafter the results from a phase 1 expansion cohort of 21 women with metastatic TNBC treated with atezolizumab, a PD-L1 directed antibody were reported. In that study, an ORR of 19% and progression-free survival of 27% at 6 months were reported.42,43 Thus, the first two studies of PD-1/PD-L1 directed therapy in similar populations of women with chemotherapy-resistant, PD-L1 positive TNBC conferred similar benefits. Although most PD-1/PD-L1 breast cancer studies have focused on TNBC populations (as a consequence of the higher prevalence of PD-L1 “positivity” in this subtype), these strategies have also been explored in women with other subtypes of breast cancer. For example, 25 women with hormone receptor positive, HER2-neu negative breast cancer participated in Keynote-028, a phase Ib multicohort trial of pembrolizumab monotherapy for PD-L1-positive advanced tumors with an ORR of 12% and a clinical benefit rate of 20% reported.25 Of note, 11 of the 25 women who participated in this study received at least 5 lines of prior treatment and all women received prior palliative chemotherapy. In the JAVELIN study, 168 women with breast cancer of any subtype who were unselected for PD-L1 expression were treated with avelumab, a PD-L1 directed antibody.27 An ORR of 8.6% and 2.8% were reported in women with TNBC and hormone receptor positive breast cancer, respectively, and responses did not correlate with various PD-L1 cut-offs for positivity. However, an ORR of 33% was observed in patients for whom immune cell clusters or “hotspots” were identified. It is unclear whether the overall responses in the JAVELIN study were more modest than those reported in other studies because of the lack of selection for PD-L1 positivity; however, the fact that responses have been observed with monotherapy in chemotherapy-resistant TNBC and hormone receptor positive disease indicates that immune therapy benefits some subgroups of breast cancer patients and warrants further study. Studies exploiting other checkpoint blockade mechanisms, such as CTLA-4-mediated blockade with tremelimumab monotherapy are also underway in advanced solid tumors including breast cancer (i.e. NCT02527434).
Dual Checkpoint Blockade Therapy Strategies In melanoma, the combination of ipilimumab (anti-CTLA-4) and nivolumab (antiPD-1) resulted in improved progression free survival compared to monotherapy, however the effect on overall survival is uncertain.44 One important drawback of combination therapy was an increase in reported adverse events. Multiple clinical trials are evaluating combination immune checkpoint therapy in other tumor types, including breast cancer. A phase I/II multi-cohort clinical trial of ipilimumab plus nivolumab in advanced solid tumors has been completed (NCT02499367) but the results for the metastatic TNBC cohort have not yet been reported. As previously outlined, a pilot study of pre-operative ipilimumab, nivolumab and cryoablation in the treatment of early stage breast cancer is underway (NCT02833233), as are multiple other studies combining checkpoint therapies with or without other agents in breast cancer (i.e. entinostat, nivolumab and ipilimumab in NCT02453620; tremelimumab and durvalumab in NCT02639026). Strategies That Combine Checkpoint Blockade with Other Systemic Therapies Hormone therapy combinations Although hormone therapy is a cornerstone of palliative therapy for women with advanced hormone receptor positive breast cancer, hormone sensitive tumors typically become resistant to anti-estrogen treatment over time. Consequently, rational combinations of hormone therapies with other agents, including checkpoint blockade strategies, are being investigated. For example, in a phase 1 study the CTLA-4 directed antibody, tremelimumab (MedImmune) was combined with the aromatase inhibitor exemestane (Aromasin, Pfizer) in 26 women with hormone receptor positive metastatic breast cancer, with tremelimumab administered at different doses and schedules (3-10 mg/kg every 28 or 90 days).45 Although no objective responses were reported with the combination, disease stability was observed in 42%, including 4 of the 5 women who had previously progressed on exemestane. Furthermore, potentially favorable changes in immune correlates were observed in most patients treated with tremelimumab, as evidenced by a systemic increase in CD4+ and CD8+ T cells expressing inducible costimulator (ICOS), a proposed marker of T cell activation, and changes in the ratios of specific T cell subtypes. These data, in combination with data from anti-PD-1/L1
monotherapy studies in hormone receptor positive breast cancer, supports further exploration of hormone therapy-immune therapy combinations in this setting. To that end, a large phase II trial of pembrolizumab plus anti-estrogen therapy is currently underway (NCT02648477). Cytotoxic chemotherapy combinations Chemotherapy is not only directly cytotoxic, but may also induce an immune response by releasing cytokines and TAAs.46,47 This immune response could, in turn, be augmented when combined with immune checkpoint therapy, a hypothesis that is supported by a growing body of pre-clinical data. Clinical trials in lung cancer and melanoma also suggest potential benefits with combination chemotherapy and immunotherapy.48,4950 A phase III trial of ipilimumab plus etoposide and platinum in advanced small lung cell cancer recently reported no difference in overall survival or disease free progression.51 The first clinical trial report of chemotherapy with checkpoint blockade in breast cancer was a phase 1b clinical trial of atezolizumab plus nanoparticle albumin-bound paclitaxel (Abraxane, Celgene) in women with advanced TNBC. The confirmed ORR was 46% in the first-line setting and 38% overall, with no significant correlation observed between responses and PD-L1 expression.52 Furthermore, after a median follow-up of 6 months, 6 of the 12 responders remain on therapy. Durability of responses has been reported in other checkpoint blockade studies in breast and other cancers, and is a hallmark of this strategy. Several trials of chemotherapy-immune therapy are underway in different phases (NCT02309117, NCT02331251). Prospectively collected data from randomized studies comparing chemotherapy with or without checkpoint blockade are ultimately needed. Other systemic therapy combinations Combination strategies may be further improved when combined with targeted agents. Specifically, agents that mediate downstream signaling and thereby prevent or overcome hormone therapy resistance have not only been successfully combined with hormone therapies but have also been shown to modulate PD-L1 expression. For example, the AKT inhibitor, rapamycin, has been shown to inhibit the PI3K signaling pathway and modulate PD-L1 expression.26 Thus, immune therapy in combination with drugs that target the PI3K
pathway may further enhance antitumor responses. Other targeted therapy combinations are underway including a trial of checkpoint blockade with the anti-angiogenesis drug, bevacizumab (Avastin, Genentech; NCT02484404). Cancer Vaccines There have been no trials of cancer vaccines with checkpoint blockade therapy in breast cancer to date. However, this strategy has been studied in a phase II clinical trial in melanoma. This study combined a dendritic cell vaccine, TriMixDC-MEL, and ipilimumab with encouraging results (38% ORR).53 Trials of vaccines with checkpoint blockade in breast cancer are planned. Strategies That Combine Checkpoint Blockade with Local Therapies Radiation and ablation can not only cause physical damage to tumors, but can also induce potentially beneficial inflammatory effects.54 Three types of ablative treatments have been used for the treatment of cancer: cyroablation (freezing), radiofrequency ablation (RFA) with exposure to medium frequency current, or thermal ablation (heating). Through physical disruption, these local therapies can induce release of TAAs and, in turn, TAA-specific T-cell responses which may be further augmented when combined with immune modulation.55 In mice, radioablation or cryoablation in combination with checkpoint blockade therapy has been synergistic.56,57 In a pilot study, women with early stage breast cancer for whom mastectomy was planned were treated with a single dose of pre-operative tumor cryoablation and/or ipilimumab. The combination of cryoablation and ipilimumab was safe and did not incur any delays in the standard-of-care surgical planning.58 Moreover, immune activation was noted in the tumor and the peripheral blood with the combination. For example, an increase in serum T cell populations by Ki67 and ICOS expression was observed, suggesting increased T cell activation and proliferation, respectively. Furthermore, expansion of intratumoral T-cell clones were detected by T-cell receptor signaling, indicating that TAA-specific T cell populations were being generated in response to the intervention.59 A pilot study of pre-operative ipilimumab, nivolumab and cryoablation in breast cancer is underway (NCT02833233) and will directly inform a planned randomized study of peri-operative cryoablation with checkpoint blockade, versus standard care, in early stage breast cancer.
Radiation, a treatment modality that is commonly used with palliative and curative intent for the treatment of many tumors, is immunogenic and therefore, may synergize with checkpoint blockade therapy. In one pre-clinical trial, for example, radiation was combined with CTLA-4 and PD-L1 directed therapy with synergistic effect.60 Furthermore, radiation in combination with checkpoint blockade therapy has not only been well tolerated in both prostate cancer and melanoma, but has also been effective in inducing tumor responses at sites distant to the radiation field in some cases. This so-called abscopal effect, whereby tumor regression is observed beyond the radiation site, suggests that a tumor-specific immune response has successfully been induced.61-63 In a mouse model of TNBC, radiation plus anti-CTLA-4 blockade showed both a decrease in tumor size and improved survival.64 There are currently several trials evaluating combinations of radiation plus immune checkpoint inhibitors in breast cancer including a trial of tremelimumab (anti-CTLA-4) plus brain radiation with or without trastuzumab (NCT02563925) in women with breast cancer brain metastases, and a trial of pembrolizumab with radiation in women with metastatic triple negative breast cancer (NCT02730130). Conclusion Immunotherapy is a rapidly evolving area of research in the treatment of cancer with encouraging results in multiple tumor types including breast cancer. However, because breast cancer is a heterogeneous disease and because most breast cancers demonstrate limited innate immunogenicity, strategies that combine checkpoint blockade with other drugs or treatment modalities - including vaccines - are likely needed as are biomarkers that predict responses to specific strategies. Ultimately, it is hoped that these combination strategies will improve responses for women with breast cancer and ultimately translate into long-term tumor-specific immunity and increased cure rates.
References 1. Page DB, Bourla AB, Daniyan A, et al. Tumor Immunology and Cancer Immunotherapy: Summary of the 2014 SITC Primer. Journal for immunotherapy of cancer 2015;3. 2. O'Shea JJ, Paul WE. Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 2010;327:1098-102. 3. Mittendorf EA, Clifton GT, Holmes JP, et al. Clinical trial results of the HER-2/neu (E75) vaccine to prevent breast cancer recurrence in high-risk patients: from US Military Cancer Institute Clinical Trials Group Study I-01 and I-02. Cancer 2012;118:2594-602. 4. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science 1996;271:1734-6. 5. Hodi FS, O'Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711-23. 6. Tarhini AA. Tremelimumab: a review of development to date in solid tumors. Immunotherapy 2013;5:215-29. 7. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor immunity. Curr Opin Immunol 2012;24:207-12. 8. Robert C, Long GV, Brady B, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med 2015;372:320-30. 9. Robert C, Schachter J, Long GV, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med 2015;372:2521-32. 10. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 2015;372:2018-28. 11. Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015;373:1627-39. 12. Salgado R, Denkert C, Demaria S, et al. The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014. Ann Oncol 2015;26:259-71. 13. Tumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014;515:568-71. 14. Loi S, Drubay D, Adams S, et al. Pooled individual patient data analysis of tumor infiltrating lymphocytes (TILs) in primary triple negative breast cancer (TNBC) treated with anthracycline-based chemotherapy. San Antonio Breast Cancer Symposium; 2015 12/18/2015; San Antonio, TX. 15. Loi S, Michiels S, Salgado R, et al. Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial. Ann Oncol 2014;25:1544-50. 16. Adams S, Gray RJ, Demaria S, et al. Prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancers from two phase III randomized adjuvant breast cancer trials: ECOG 2197 and ECOG 1199. J Clin Oncol 2014;32:2959-66. 17. Denkert C, Loibl S, Noske A, et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 2010;28:10513. 18. Salgado R, Denkert C, Campbell C, et al. Tumor-Infiltrating Lymphocytes and Associations With Pathological Complete Response and Event-Free Survival in HER2-Positive Early-Stage Breast Cancer Treated With Lapatinib and Trastuzumab: A Secondary Analysis of the NeoALTTO Trial. JAMA Oncol 2015;1:448-54.
19. Perez EA, Ballman KV, Tenner KS, et al. Association of Stromal Tumor-Infiltrating Lymphocytes With Recurrence-Free Survival in the N9831 Adjuvant Trial in Patients With EarlyStage HER2-Positive Breast Cancer. JAMA Oncol 2016;2:56-64. 20. Desmedt C, Salgado R, Buisseret L, et al. Characterization of lymphocytic infiltration in invasive lobular breast cancer. San Antonio Breast Cancer Symposium; 2015 December 8-12, 2015; San Antonio, Texas. 21. Dushyanthen S, Beavis PA, Savas P, et al. Relevance of tumor-infiltrating lymphocytes in breast cancer. BMC medicine 2015;13:202. 22. Dieci MV, Goubar A, Mathieu M-C, et al. Prognostic and predictive value of tumor infiltrating lymphocytes (TIL) in two phase III randomized adjuvant breast cancer (BC) trials. ASCO Meeting Abstracts 2014;32:11087. 23. Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of antiPD-1 antibody in cancer. N Engl J Med 2012;366:2443-54. 24. Nanda R, Chow LQ, Dees EC, et al. Pembrolizumab in Patients With Advanced TripleNegative Breast Cancer: Phase Ib KEYNOTE-012 Study. J Clin Oncol 2016. 25. Rugo H, DeLord JP, Im S-A, et al. Preliminary efficacy and safety of pembrolizumab (MK3475) in patients with PD-L1-positive, estrogen receptor positive (ER+)/HER2-negative advanced breast cancer enrolled in KEYNOTE-028. San Antonio Breast Cancer Symposium; 2015 December 8-12, 2015; San Antonio, TX. 26. Mittendorf EA, Philips AV, Meric-Bernstam F, et al. PD-L1 expression in triple-negative breast cancer. Cancer Immunol Res 2014;2:361-70. 27. Dirix LY, Takacs I, Nikolinakos P, et al. Avelumab (MSB00107118C), an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast canceR: A phase Ib JAVELIN solid tumor trial. San Antonio Breast Cancer Symposium; 2015 December 8-12, 2015; San Antonio, TX. 28. Page DB, Naidoo J, McArthur HL. Emerging immunotherapy strategies in breast cancer. Immunotherapy 2014;6:195-209. 29. Harao M, Mittendorf EA, Radvanyi LG. Peptide-based vaccination and induction of CD8+ T-cell responses against tumor antigens in breast cancer. BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy 2015;29:15-30. 30. Sharma A, Koldovsky U, Xu S, et al. HER-2 pulsed dendritic cell vaccine can eliminate HER-2 expression and impact ductal carcinoma in situ. Cancer 2012;118:4354-62. 31. Castellino F, Germain RN. Cooperation between CD4+ and CD8+ T cells: when, where, and how. Annu Rev Immunol 2006;24:519-40. 32. Cecil DL, Holt GE, Park KH, et al. Elimination of IL-10-inducing T-helper epitopes from an IGFBP-2 vaccine ensures potent antitumor activity. Cancer research 2014;74:2710-8. 33. Baxevanis CN, Sotiropoulou PA, Sotiriadou NN, Papamichail M. Immunobiology of HER2/neu oncoprotein and its potential application in cancer immunotherapy. Cancer Immunol Immunother 2004;53:166-75. 34. Ward RL, Hawkins NJ, Coomber D, Disis ML. Antibody immunity to the HER-2/neu oncogenic protein in patients with colorectal cancer. Human immunology 1999;60:510-5. 35. Benavides LC, Gates JD, Carmichael MG, et al. The impact of HER2/neu expression level on response to the E75 vaccine: from U.S. Military Cancer Institute Clinical Trials Group Study I01 and I-02. Clin Cancer Res 2009;15:2895-904. 36. Carmichael MG, Benavides LC, Holmes JP, et al. Results of the first phase 1 clinical trial of the HER-2/neu peptide (GP2) vaccine in disease-free breast cancer patients: United States Military Cancer Institute Clinical Trials Group Study I-04. Cancer 2010;116:292-301.
37. Rochman S. New peptide vaccine for HER2-expressing breast tumors. J Natl Cancer Inst 2015;107. 38. Julien S, Picco G, Sewell R, et al. Sialyl-Tn vaccine induces antibody-mediated tumour protection in a relevant murine model. Br J Cancer 2009;100:1746-54. 39. Hilton TL, Hulett TW, Dubay C, et al. DPV-001 an autophagosome-enriched cancer vaccine in phase II clinical trials contains 25 putative cancer antigens, DAMPS, HSPS and agonists for TLR 2, 3, 4, 7 and 9. SITC 28th Annual Meeting; 2013 November 8-10, 2013; National Harbor, MD. 40. Karan D, Van Veldhuizen P. Combination immunotherapy with prostate GVAX and ipilimumab: safety and toxicity. Immunotherapy 2012;4:577-80. 41. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-T immunotherapy for castrationresistant prostate cancer. N Engl J Med 2010;363:411-22. 42. Emens LA. Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer (TNBC) AACR Annual Meeting; 2015 April 20, 2015; Philidelphia, PA. 43. Emens LA, Kok M, Ojalvo LS. Targeting the programmed cell death-1 pathway in breast and ovarian cancer. Current opinion in obstetrics & gynecology 2016;28:142-7. 44. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med 2015;373:23-34. 45. Vonderheide RH, LoRusso PM, Khalil M, et al. Tremelimumab in combination with exemestane in patients with advanced breast cancer and treatment-associated modulation of inducible costimulator expression on patient T cells. Clin Cancer Res 2010;16:3485-94. 46. Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of cancer chemotherapy. Nat Rev Immunol 2008;8:59-73. 47. Sistigu A, Yamazaki T, Vacchelli E, et al. Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy. Nat Med 2014. 48. Reck M, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line therapy in extensive-disease-small-cell lung cancer: results from a randomized, double-blind, multicenter phase 2 trial. Ann Oncol 2013;24:75-83. 49. Lynch TJ, Bondarenko I, Luft A, et al. Ipilimumab in combination with paclitaxel and carboplatin as first-line treatment in stage IIIB/IV non-small-cell lung cancer: results from a randomized, double-blind, multicenter phase II study. J Clin Oncol 2012;30:2046-54. 50. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 2011;364:2517-26. 51. Reck M, Luft A, Szczesna A, et al. Phase III Randomized Trial of Ipilimumab Plus Etoposide and Platinum Versus Placebo Plus Etoposide and Platinum in Extensive-Stage SmallCell Lung Cancer. J Clin Oncol 2016. 52. Adams S, Diamond J, Hamilton E, et al. Phase Ib trial of atezolizumab in combination with nab-paclitaxel in patients with metastatic triple-negative breast cancer (mTNBC). 2016 ASCO Annual Meeting; 2016 June 3-7, 2016; Chicago, IL. 53. Wilgenhof S, Corthals J, Heirman C, et al. Phase II Study of Autologous MonocyteDerived mRNA Electroporated Dendritic Cells (TriMixDC-MEL) Plus Ipilimumab in Patients With Pretreated Advanced Melanoma. J Clin Oncol 2016. 54. Chu KF, Dupuy DE. Thermal ablation of tumours: biological mechanisms and advances in therapy. Nature reviews Cancer 2014;14:199-208. 55. !!! INVALID CITATION !!! 56. Shi L, Chen L, Wu C, et al. PD-1 Blockade Boosts Radiofrequency Ablation-Elicited Adaptive Immune Responses against Tumor. Clin Cancer Res 2016;22:1173-84.
57. Waitz R, Solomon SB, Petre EN, et al. Potent induction of tumor immunity by combining tumor cryoablation with anti-CTLA-4 therapy. Cancer research 2012;72:430-9. 58. McArthur HL, Diab A, Page DB, et al. A pilot study of preoperative single-dose ipilimumab and/or cryoablation in women with early-stage breast cancer with comprehensive immune profiling. Clin Cancer Res 2016. 59. Page D, Yuan J, Redmond D, Wen HY, Durack JC, Emerson RO. T-cell Receptor DNA Deep Sequencing as a biomarker of tumor infiltrating lymphocytes and immunotherapy-associated clonal lymphocyte expansion in breast cancer. Cancer Immunol Res 2016. 60. Twyman-Saint Victor C, Rech AJ, Maity A, et al. Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer. Nature 2015;520:373-7. 61. Slovin SF, Higano CS, Hamid O, et al. Ipilimumab alone or in combination with radiotherapy in metastatic castration-resistant prostate cancer: results from an open-label, multicenter phase I/II study. Ann Oncol 2013. 62. Barker CA, Postow MA, Khan SA, et al. Concurrent Radiotherapy and Ipilimumab Immunotherapy for Patients with Melanoma. Cancer Immunol Res 2013;1:92-8. 63. Postow MA, Callahan MK, Barker CA, et al. Immunologic correlates of the abscopal effect in a patient with melanoma. N Engl J Med 2012;366:925-31. 64. Demaria S, Kawashima N, Yang AM, et al. Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. Clin Cancer Res 2005;11:728-34.