Immune checkpoint inhibitors: Key trials and an emerging role in breast cancer.

This review focuses on immune checkpoint inhibitors - immunomodulatory agents that aim to relieve tumour-mediated immune-cell suppression. Immune checkpoint proteins can be expressed on the tumour-cell or immune-cell populations. Immune checkpoint proteins dampen the immune response by inactivating immune cells capable of tumour destruction. Blockade of immune checkpoints has shown impressive results in a range of solid cancers, particularly melanoma and non-small cell lung cancer. The potential benefit of this class of drugs is widespread across most cancer types and an unprecedented number of clinical studies are underway to examine the benefit of these agents. The aims of this review are to: provide an overview of the key early immune checkpoint inhibitor trials involving drugs targeting programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) in multiple disease types; provide an overview of emerging therapies aimed at these targets; and provide a detailed exploration of the status of immune checkpoint inhibitors in breast cancer.

Alterations in immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients receiving HER2-targeted neo-adjuvant therapy.

BACKGROUND: The phase II neo-adjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy in combination with trastuzumab, lapatinib or both in patients with HER2+ breast cancer. We studied circulating immune cells looking for alterations in phenotype, genotype and cytotoxic capacity (direct and antibody-dependent cell-mediated cytotoxicity (ADCC)) in the context of treatment response. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from pre- (n = 41) and post- (n = 25) neo-adjuvant treatment blood samples. Direct/trastuzumab-ADCC cytotoxicity of patient-derived PBMCs against K562/SKBR3 cell lines was determined ex vivo. Pembrolizumab was interrogated in 21 pre-treatment PBMC ADCC assays. Thirty-nine pre-treatment and 21 post-treatment PBMC samples were immunophenotyped. Fc receptor genotype, tumour infiltrating lymphocyte (TIL) levels and oestrogen receptor (ER) status were quantified. RESULTS: Treatment attenuated the cytotoxicity/ADCC of PBMCs. CD3+/CD4+/CD8+ T cells increased following therapy, while CD56+ NK cells/CD14+ monocytes/CD19+ B cells decreased with significant post-treatment immune cell changes confined to patients with residual disease. Pembrolizumab-augmented ex vivo PBMC ADCC activity was associated with residual disease, but not pathological complete response. Pembrolizumab-responsive PBMCs were associated with lower baseline TIL levels and ER+ tumours. CONCLUSIONS: PBMCs display altered phenotype and function following completion of neo-adjuvant treatment. Anti-PD-1-responsive PBMCs in ex vivo ADCC assays may be a biomarker of treatment response.

Comparative analysis of drug response and gene profiling of HER2-targeted tyrosine kinase inhibitors.

BACKGROUND: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations. METHODS: In this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data. RESULTS: All three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity in HER2-mutant and EGFR-mutant cells. High expression of HER2, VTCN1, CDK12, and RAC1 correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance. BRCA2 mutations were correlated with neratinib and tucatinib response, and high expression of ATM, BRCA2, and BRCA1 were associated with neratinib resistance. CONCLUSIONS: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.

Targeting c-Met in triple negative breast cancer: preclinical studies using the c-Met inhibitor, Cpd A.

Introduction Triple negative breast cancer (TNBC) represents a heterogeneous subtype of breast cancer that carries a poorer prognosis. There remains a need to identify novel drivers of TNBC, which may represent targets to treat the disease. c-Met overexpression is linked with decreased survival and is associated with the basal subtype of breast cancer. Cpd A, a kinase inhibitor selective/specific for Met kinase has demonstrated preclinical anti-cancer efficacy in TNBC. We aimed to assess the anti-cancer efficacy of Cpd A when combined with Src kinase, ErbB-family or hepatocyte growth factor (HGF) inhibitors in TNBC cell lines. Methods We determined the anti-proliferative effects of Cpd A, rilotumumab, neratinib and saracatinib tested alone and in combination in a panel of TNBC cells by acid phosphatase assays. We performed reverse phase protein array analysis of c-Met and IGF1Rß expression and phosphorylation of c-Met (Y1234/1235) in TNBC cells and correlated their expression/phosphorylation with Cpd A sensitivity. We examined the impact of Cpd A, neratinib and saracatinib tested alone and in combination on invasive potential and colony formation.Results TNBC cells are not inherently sensitive to Cpd A, and neither c-Met expression nor phosphorylation are biomarkers of sensitivity to Cpd A. Cpd A enhanced the anti-proliferative effects of neratinib in vitro; however, this effect was limited to cell lines with innate sensitivity to Cpd A. Cpd A had limited anti-invasive effects but it reduced colony formation in the TNBC cell line panel.Conclusions Despite Cpd A having a potential role in reducing cancer cell metastasis, identification of strong predictive biomarkers of c-Met sensitivity would be essential to the development of a c-Met targeted treatment for an appropriately selected cohort of TNBC patients.

HER-targeted tyrosine kinase inhibitors enhance response to trastuzumab and pertuzumab in HER2-positive breast cancer.

Despite trastuzumab and pertuzumab improving outcome for patients with HER2-positive metastatic breast cancer, the disease remains fatal for the majority of patients. This study evaluated the anti-proliferative effects of adding anti-HER2 tyrosine kinase inhibitors (TKIs) to trastuzumab and pertuzumab in HER2-positive breast cancer cells. Afatinib was tested alone and in combination with trastuzumab in HER2-positive breast cancer cell lines. TKIs (lapatinib, neratinib, afatinib) combined with trastuzumab and/or pertuzumab were tested in 3 cell lines, with/without amphiregulin and heregulin-1ß. Seven of 11 HER2-positive cell lines tested were sensitive to afatinib (IC50 < 80 nM). Afatinib plus trastuzumab produced synergistic growth inhibition in eight cell lines. In trastuzumab-sensitive SKBR3 cells, the TKIs enhanced response to trastuzumab. Pertuzumab alone did not inhibit growth and did not enhance trastuzumab-induced growth inhibition or antibody-dependent cellular cytotoxicity. Pertuzumab enhanced response to trastuzumab when combined with lapatinib but not neratinib or afatinib. In two trastuzumab-resistant cell lines, the TKIs inhibited growth but adding trastuzumab and/or pertuzumab did not improve response compared to TKIs alone. Amphiregulin plus heregulin-1ß stimulated proliferation of SKBR3 and MDA-MB-453 cells. In the presence of the growth factors, neither antibody inhibited growth and the TKIs showed significantly reduced activity. The triple combination of trastuzumab, pertuzumab and a TKI showed the strongest anti-proliferative activity in all three cell lines, in the presence of exogenous growth factors. In summary, addition of anti-HER2 TKIs to combined anti-HER2 monoclonal antibody therapy results in enhanced anticancer activity. These data contribute to the rationale for studying maximum HER2 blockade in the clinic.

The HSP90 inhibitor NVP-AUY922 inhibits growth of HER2 positive and trastuzumab-resistant breast cancer cells.

As HER2 is a client protein of the molecular chaperone Hsp90, targeting Hsp90 may be beneficial in HER2-positive breast cancer. In this study, the activity of the Hsp90 inhibitor NVP-AUY922 was assessed in HER2 overexpressing breast cancer cell lines, including two cell line models of acquired trastuzumab-resistance. The seven HER2-positive breast cancer cell lines tested showed significant sensitivity to NVP-AUY922 in vitro, with IC50 values between 6 and 17 nM. Combining NVP-AUY922 with chemotherapy did not improve response. NVP-AUY922 in combination with trastuzumab, significantly enhanced growth inhibition in three of the seven cell lines tested. In conclusion, our data shows that NVP-AUY922 displays potent anti-cancer activity in HER2-positive and trastuzumab-resistant breast cancer cells, and supports further testing of NVP-AUY922 in patients with HER2-positive breast cancer.

Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

BACKGROUND: Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcÆ´R+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. METHODS: HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. RESULTS: HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. CONCLUSIONS: In the presence of effector cells with high cytotoxic capacity, TKIs have the ability to augment the passive immunotherapeutic potential of trastuzumab in SKBR3, a model of HER2+ breast cancer. ADCC levels detected by LDH release assays are extremely low in MCF-7; the flow cytometry-based CFSE/7-AAD method is more sensitive and consistent for the determination of ADCC in HER2-low models.

Evaluation of ABT-751, a novel anti-mitotic agent able to overcome multi-drug resistance, in melanoma cells.

PURPOSE: Drug efflux transporter associated multi-drug resistance (MDR) is a potential limitation in the use of taxane chemotherapies for the treatment of metastatic melanoma. ABT-751 is an orally bioavailable microtubule-binding agent capable of overcoming MDR and proposed as an alternative to taxane-based therapies. METHODS: This study compares ABT-751 to taxanes in vitro, utilizing seven melanoma cell line models, publicly available gene expression and drug sensitivity databases, a lung cancer cell line model of MDR drug efflux transporter overexpression (DLKP-A), and drug efflux transporter ATPase assays. RESULTS: Melanoma cell lines exhibit a low but variable protein and RNA expression of drug efflux transporters P-gp, BCRP, and MDR3. Expression of P-gp and MDR3 correlates with sensitivity to taxanes, but not to ABT-751. The anti-proliferative IC50 profile of ABT-751 was higher than the taxanes docetaxel and paclitaxel in the melanoma cell line panel, but fell within clinically achievable parameters. ABT-751 IC50 was not impacted by P-gp-overexpression in DKLP-A cells, which display strong resistance to the P-gp substrate taxanes compared to DLKP parental controls. The addition of ABT-751 to paclitaxel treatment significantly decreased cell proliferation, suggesting some reversal of MDR. ATPase activity assays suggest that ABT-751 is a potential BCRP substrate, with the ability to inhibit P-gp ATPase activity. CONCLUSION: Our study confirms that ABT-751 is active against melanoma cell lines and models of MDR at physiologically relevant concentrations, it inhibits P-gp ATPase activity, and it may be a BCRP and/or MDR3 substrate. ABT-751 warrants further investigation alone or in tandem with other drug efflux transporter inhibitors for hard-to-treat MDR melanoma.

Preclinical evaluation of Insulin-like growth factor receptor 1 (IGF1R) and Insulin Receptor (IR) as a therapeutic targets in triple negative breast cancer.

Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, has fewer successful therapeutic therapies than other types of breast cancer. Insulin-like growth factor receptor 1 (IGF1R) and the Insulin receptor (IR) are associated with poor outcomes in TNBC. Targeting IGF1R has failed clinically. We aimed to test if inhibiting both IR/IGF1R was a rationale therapeutic approach to treat TNBC. We showed that despite IGF1R and IR being expressed in TNBC, their expression is not associated with a negative survival outcome. Furthermore, targeting both IR/IGF1R with inhibitors in multiple TNBC cell lines did not inhibit cell growth. Linsitinib, a small molecule inhibitor of both IGF1R and IR, did not block tumour formation and had no effect on tumour growth in vivo. Cumulatively these data suggest that while IGF1R and IR are expressed in TNBC, they are not good therapeutic targets. A potential reason for the limited anti-cancer impact when IR/IGF1R was targeted may be because multiple signalling pathways are altered in TNBC. Therefore, targeting individual signalling pathways may not be sufficient to inhibit cancer growth.

Modulation of P-gp expression by lapatinib.

Chemotherapy drug resistance is a major obstacle in the treatment of cancer. It can result from an increase in levels of cellular drug efflux pumps, such as P-glycoprotein (P-gp). Lapatinib, a growth factor receptor tyrosine kinase inhibitor, is currently in clinical trials for treatment of breast cancer. We examined the impact of co-incubation of chemotherapy drugs in combination with lapatinib in P-gp over-expressing drug resistant cells. Unexpectedly, lapatinib treatment, at clinically relevant concentrations, increased levels of the P-gp drug transporter in a dose- and time-responsive manner. Conversely, exposure to the epidermal growth factor (EGF), an endogenous growth factor receptor ligand, resulted in a decrease in P-gp expression. Despite the lapatinib-induced alteration in P-gp expression, use of accumulation, efflux and toxicity assays demonstrated that the induced alteration in P-gp expression by lapatinib had little direct impact on drug resistance.

Research Strategies for Low-Survival Cancers.

While substantial progress has been made to improve the diagnosis, prognosis, and survivorship of patients with cancer, certain cancer types, along with metastatic and refractory disease, remain clinical challenges. To improve patient outcomes, ultimately, the cancer research community must meet and overcome these challenges, leading to improved approaches to treat the most difficult cancers. Here, we discuss research progress aimed at gaining a better understanding of the molecular and cellular changes in tumor cells and the surrounding stroma, presented at the 56th Irish Association for Cancer Research (IACR) Annual Conference. With a focus on poor prognosis cancers, such as esophageal and chemo-resistant colorectal cancers, we highlight how detailed molecular knowledge of tumor and stromal biology can provide windows of opportunity for biomarker discovery and therapeutic targets. Even with previously characterized targets, such as phosphoinositide 3-kinase (PI3K), one of the most altered proteins in all human cancers, new insights into how this protein may be more effectively inhibited through novel combination therapies is presented.

Effects of HER Family-targeting Tyrosine Kinase Inhibitors on Antibody-dependent Cell-mediated Cytotoxicity in HER2-expressing Breast Cancer.

PURPOSE: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. EXPERIMENTAL DESIGN: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array-determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry-based protocols. RESULTS: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell-mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. CONCLUSIONS: TKIs differentially alter tumor cell phenotype which can impact NK cell-mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Novel Strategies for Cancer Treatment: Highlights from the 55th IACR Annual Conference.

While conventional cancer treatments, such as surgery, radiotherapy and chemotherapy, have been combined for decades in an effort to treat cancer patients, the emergence of novel fields of cancer research have led to a renewed interest in combining conventional treatments with more innovative approaches. The realisation that cancer progression is not exclusively due to changes in the cancer epithelial cells, but also involves changes in the tumour microenvironment, has opened new avenues for combination treatments. Here we discuss the use of combination therapies presented at the 55th Irish Association for Cancer Research (IACR) Annual Conference, highlighting examples of novel therapeutic strategies which, combined with conventional therapies, may greatly enhance not only the overall outcome for patients, but also the quality of life for cancer survivors. Among the novel treatment strategies, immune metabolism, epigenetic therapies and physical exercise are presented. In addition, novel technologies in the field of precision medicine, which will be useful to discover new therapeutics and to stratify patients for combination treatments, are also discussed.

Acquired Resistance to Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) combine the tumor selectivity of antibodies with the potency of cytotoxic small molecules thereby constituting antibody-mediated chemotherapy. As this inherently limits the adverse effects of the chemotherapeutic, such approaches are heavily pursued by pharma and biotech companies and have resulted in four FDA (Food and Drug Administration)-approved ADCs. However, as with other cancer therapies, durable responses are limited by the fact that under cell stress exerted by these drugs, tumors can acquire mechanisms of escape. Resistance can develop against the antibody component of ADCs by down-regulation/mutation of the targeted cell surface antigen or against payload toxicity by up-regulation of drug efflux transporters. Unique resistance mechanisms specific for the mode of action of ADCs have also emerged, like altered internalization or cell surface recycling of the targeted tumor antigen, changes in the intracellular routing or processing of ADCs, and impaired release of the toxic payload into the cytosol. These evasive changes are tailored to the specific nature and interplay of the three ADC constituents: the antibody, the linker, and the payload. Hence, they do not necessarily endow broad resistance to ADC therapy. This review summarizes preclinical and clinical findings that shed light on the mechanisms of acquired resistance to ADC therapies.

Preclinical Characteristics of the Irreversible Pan-HER Kinase Inhibitor Neratinib Compared with Lapatinib: Implications for the Treatment of HER2-Positive and HER2-Mutated Breast Cancer.

An estimated 15-20% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2/ERBB2/neu). Two small-molecule tyrosine kinase inhibitors (TKIs), lapatinib and neratinib, have been approved for the treatment of HER2-positive (HER2+) breast cancer. Lapatinib, a reversible epidermal growth factor receptor (EGFR/ERBB1/HER1) and HER2 TKI, is used for the treatment of advanced HER2+ breast cancer in combination with capecitabine, in combination with trastuzumab in patients with hormone receptor-negative metastatic breast cancer, and in combination with an aromatase inhibitor for the first-line treatment of HER2+ breast cancer. Neratinib, a next-generation, irreversible pan-HER TKI, is used in the US for extended adjuvant treatment of adult patients with early-stage HER2+ breast cancer following 1 year of trastuzumab. In Europe, neratinib is used in the extended adjuvant treatment of adult patients with early-stage hormone receptor-positive HER2+ breast cancer who are less than 1 year from the completion of prior adjuvant trastuzumab-based therapy. Preclinical studies have shown that these agents have distinct properties that may impact their clinical activity. This review describes the preclinical characterization of lapatinib and neratinib, with a focus on the differences between these two agents that may have implications for patient management.

Innovative Technologies Changing Cancer Treatment.

Conventional therapies for cancer such as chemotherapy and radiotherapy remain a mainstay in treatment, but in many cases a targeted approach is lacking, and patients can be vulnerable to drug resistance. In recent years, novel concepts have been emerging to improve the traditional therapeutic options in cancers with poor survival outcomes. New therapeutic strategies involving areas like energy metabolism and extracellular vesicles along with advances in immunotherapy and nanotechnology are driving the next generation of cancer treatments. The development of fields such as theranostics in nanomedicine is also opening new doors for targeted drug delivery and nano-imaging. Here we discuss the use of innovative technologies presented at the Irish Association for Cancer Research (IACR) Annual Meeting, highlighting examples of where new approaches may lead to promising new treatment options for a range of cancer types.