HER2-amplified Breast Cancer Patients Research Articles

BSBM-15 SYSTEMIC AND LOCAL IMMUNE RESPONSES FOLLOWING STEREOTACTIC RADIOSURGERY TO BRAIN METASTASES FROM HER2-AMPLIFIED BREAST CANCER

Abstract BACKGROUND Brain metastases (BMs) afflict 30-50% of HER2-amplified breast cancer (HER2-BC) patients. Stereotactic radiosurgery (SRS) is a highly effective focal treatment for BMs. Extracranial radiotherapy can promote anti-tumor immune responses that synergize with immunotherapy, but whether this applies to the brain microenvironment remains unclear. Here, we examined blood and BM samples from HER2-BC patients in the TROG 16.02 translational sub-study for immunological effects of SRS (ACTRN12616001265460). METHODS Blood samples from 10 patients taken pre- and 7-14 days post-SRS to intact HER2-BC BMs or post-resection BM cavities were analyzed by mass and flow cytometry for immune cell and cytokine modulation. One patient received pre-operative SRS for a BM that recurred 6 months after resection, followed by re-resection of the BM 7 days post-SRS. Tumors from pre- and post-SRS timepoints in this patient were analyzed by bulk RNAseq and immunohistochemistry. RESULTS Monocytes, CD8+ T effector memory and regulatory T cells were enriched in blood post-SRS, while conventional dendritic cells, CD4+ T early and CD4+ TEMRA cells were diminished. Stem cell factor (SCF) concentration, a growth factor important for monocyte production, was elevated in plasma post-SRS. In the tumor, SRS upregulated gene signatures for monocyte/macrophage processes (including SCF gene transcript levels), antigen presentation, and T cell activation. Cell type deconvolution from RNAseq suggested an SRS-induced loss of metastatic tumor cells and enrichment of macrophages and CD4+ T cells. Immunohistochemistry corroborated the influx of CD68+ macrophages and CD3+ T cells into tumor and brain regions post-SRS, with spatial co-localization evident between the cell types. Furthermore, the pre-SRS immune-excluded phenotype in tumor regions was no longer observed post-SRS. CONCLUSION Systemic and local immunological changes in this homogenous patient cohort receiving SRS to HER2-BC BMs suggest a radiation-induced adaptive immune response intracranially, involving the monocyte-macrophage lineage as antigen presenting cells, that should be further explored.

Targeting HER2-AXL heterodimerization to overcome resistance to HER2 blockade in breast cancer.

Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer.

Abstract P6-18-26: Ado-trastuzumab for the treatment of metastatic HER2-amplified breast cancer patients previously treated with pertuzumab

Abstract BACKGROUND: Ado-trastuzumab (T-DM1) is an antibody-drug conjugate of trastuzumab and a cytotoxic microtubule-inhibitory agent, emtansine. T-DM1 is approved for the treatment of advanced HER2-amplified breast cancer that progressed following trastuzumab-based therapies based on improvement in progression-free survival (PFS) and overall survival (OS) compared to the therapy of physician choice. However, T-DM1 trials were conducted prior to the widespread adoption of docetaxel, trastuzumab, and pertuzumab as standard frontline therapy for advanced HER2-amplified breast cancer. As such, none of the patients enrolled on T-DM1 studies had been exposed to pertuzumab, and the clinical benefit of T-DM1 in patients previously treated with pertuzumab therapy is unknown. METHODS: We completed a retrospective review of patients at our institution over the age of 18 with metastatic HER2-amplified breast cancer treated with pertuzumab prior to T-DM1 between February 2013 and May 2018. Data was collected on patient and tumor characteristics, number and duration of therapies in the metastatic setting, and clinical outcomes. The primary endpoint of this study was PFS in patients given T-DM1 after earlier exposure to pertuzumab. Secondary endpoints included overall response rate (ORR), prolonged duration of T-DM1 therapy (> 6 months), and OS. Adverse events following T-DM1 were collected using CTCAE 4.03, with a focus on cardiac dysfunction and peripheral neuropathy. Patient features and outcomes were summarized with descriptive statistics and time-to-event measures were analyzed using the Kaplan-Meier method and log-rank test. RESULTS: Twenty patients met the inclusion criteria and are included in this study. The patient population consisted of 18 non-Hispanic white and 2 black women, with a median age of 58.5 (range 34-68) years. The number of prior systemic therapies (excluding pertuzumab) ranged from 0-8 with a median of 1 therapy. The duration of T-DM1 therapy (started, on average, 24 months after metastatic diagnosis) ranged from < 1 month to 3.5 years with a median of 6 months. T-DM1 therapy was overall very well tolerated, with all adverse events being grade ≤2. Of note, 2 patients had grade 2 neuropathy, and one patient had grade 1 cardiotoxicity, without any change in left ventricular ejection. Among 18 patients evaluable for response, ORR was 16.7% (95% CI: 3.6% to 41.4%), with 3 patients achieving a partial response. No complete responses were noted. 10/18 (55.6%) patients had prolonged duration of therapy with T-DM1. Median follow-up time after initiation of T-DM1 was 15 months and 6/20 (30%) patients died while under observation. At the time of data cut-off, 10/20 patients had disease progression on T-DM1. Median PFS was 16 months, with a 1-year PFS rate of 54.5% (95% CI: 36.4% to 81.7%). The 1-year OS rate was 75.0% (95% CI: 58.2% to 96.6%). Patients with liver metastases (n=8) had a significantly worse PFS (p=0.003). CONCLUSION: T-DM1 following pertuzumab is well tolerated and shows excellent efficacy in the treatment of HER2-positive metastatic breast cancer. Comparing T-DM1 following pertuzumab to T-DM1 in pertuzumab-naïve patients should be explored in this patient population. Citation Format: Al Rabadi LS, Kaempf A, Lim JY, Saraceni MM, Savin MA, Mitri ZI. Ado-trastuzumab for the treatment of metastatic HER2-amplified breast cancer patients previously treated with pertuzumab [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-18-26.

Abstract 4641: Improved chemotherapeutic delivery to brain metastases with targeted nanoparticles in preclinical breast cancer brain metastasis models

Abstract Brain metastases present an increasing challenge to long-term survival for HER2-amplified breast cancer patients. While HER2 targeted therapies effectively control systemic metastases, they are largely ineffective against brain metastases due to their inability to cross the blood-brain barrier. We have recently demonstrated that brain uptake of targeted nanoparticles can be increased by adding an acid-cleavable linkage between transferrin (Tf) and the nanoparticle core [1]. Our lab has also developed a method for assembling targeted nanoparticles by complexing diol-containing polymer-drug conjugates and boronic acid-containing targeting agents [2]. Here, we report the first efficacy results of targeted nanoparticles formed via this assembly strategy in a preclinical model of breast cancer brain metastasis. Female Rag2-/-;Il2rg-/- mice were intracranially injected with human HER2-amplified BT-474 breast cancer cells, and formation of brain tumors was monitored by MRI. Mice were randomized into four groups of six mice per group (targeted nanoparticle, non-targeted nanoparticle, free drug, and saline). Targeted and non-targeted nanoparticles were prepared by functionalizing nanoparticle cores consisting of a mucic acid polymer conjugate of camptothecin (CPT) with either Tf-PEG or methoxy-PEG, respectively, using boronic acid-diol chemistry. The different formulations were administered intravenously once per week for four weeks at a dose of 4 mg/kg (CPT basis), and tumor volume was measured weekly by MRI. Targeted nanoparticles significantly reduced tumor growth in mice bearing intracranially implanted tumors compared to saline, free drug, and non-targeted nanoparticle controls. However, free drug and non-targeted nanoparticles also reduced tumor growth compared to saline, albeit to a lesser extent. Taken together, the results are highly suggestive that there isn't a completely intact blood-brain or blood-tumor barrier, and furthermore that artificial transport pathways may be introduced following intracranial implantation. To overcome limitations of the intracranial injection model, female Rag2-/-;Il2rg-/- mice were intracardiacly or intravenously (tail vein) injected with human HER2-amplified BT-474 breast cancer cells, and formation of metastatic brain tumors was monitored by MRI. Unlike brain tumors from intracranially injected cells, brain tumors formed from intravenous cell injections were not affected by systemic doses of either lapatinib or trastuzumab. Current investigations involve collection of efficacy data for the different formulations in the intracardiac and intravenous models that may provide further insight into the blood-brain barrier integrity and nanoparticle delivery. [1] Clark, AJ, Davis, ME, PNAS, 112 (2015) 12486-12491. [2] Han, H, Davis, ME, Mol. Pharmaceutics, 10 (2013) 2558-2567. Citation Format: Emily A. Wyatt, Mark E. Davis. Improved chemotherapeutic delivery to brain metastases with targeted nanoparticles in preclinical breast cancer brain metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4641.

Recent Insights into the Development of Preclinical Trastuzumab- Resistant HER2+ Breast Cancer Models

Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy. In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer. Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer.

Abstract B169: Neratinib has clinical activity in HER2-amplified breast cancer patients with tumors that have acquired activating mutations in HER2

Abstract Overexpression/amplification of HER2/ERBB2 occurs in 20% of breast cancers. Thanks to specific anti-HER2 agents, the prognosis of HER2-positive breast cancer has improved considerably. However, acquired resistance inevitably emerges over time and tumors escape pharmacologic pressure. In this work, we propose that acquisition of activating somatic mutations in HER2 upon anti-HER2 therapy may be more frequent than commonly reported and can reduce sensitivity to these agents. Moreover, we tested whether neratinib, an irreversible pan-HER inhibitor, is effective in tumors bearing both amplification and mutations of ERBB2. By targeted exome sequencing, we found that samples from metastatic breast cancer (MBC) patients relapsing to multiple lines of anti-HER2 therapy presented the acquisition of HER2 mutations. These mutations spanned from the extracellular domain (L313I, R456C) to the kinase domain (L755S, D769Y) of the receptor. To investigate the role of these mutations in drug resistance, we conducted functional studies by stably transducing the L755S HER2 mutation (the most frequent HER2 mutation in breast cancer) in two ERBB2-amplified breast cancer cell lines intrinsically sensitive to HER2 inhibition. In both models, we found that expression of L755S mutant-HER2 was sufficient to limit sensitivity to trastuzumab, lapatinib, or the combination of both agents. Consistently, neither trastuzumab nor lapatinib was effective in inhibiting tumor growth of patient-derived xenografts established from a patient with ERBB2-amplified/mutant (D769Y) breast cancer. However, neratinib treatment demonstrated marked sensitivity in this tumor model, resulting in significant tumor growth inhibition. The antitumor activity of neratinib was also explored in breast cancer patients with coexisting ERBB2 amplification and mutation, either by compassionate use after failure of standard-of-care therapy or as part of a “basket” trial (NCT01953926) enrolling ERBB2-mutant patients. In both settings, we observed durable clinical response to neratinib. MBC case #HER2 co-mutationResponse to neratinibDurability of response (mo)1D769YSD62L313ISD93Y772_A775dupSD44L755SSD55V777LPR6 Our findings indicate that acquired HER2 mutations may reduce the effectiveness of therapeutic agents commonly used for the management of ERBB2-amplified MBC. Moreover, we propose neratinib as an effective treatment option for patients whose tumors harbor both ERBB2 amplifications and mutations. Citation Format: Emiliano Cocco, F. Javier Carmona, Helen H. Won, Michael F. Berger, David M. Hyman, Valentina Rossi, Carmen Chan, Alyssa Moriarty, Kyriakos P. Papadopoulos, Michael J. Wick, James Cownie, Ivana Sarotto, Richard E. Cutler, Francesca Avogadri-Connors, Peter Savas, Alshad S. Lalani, Valentina Boni, Sherene Loi, Jose Baselga, Filippo Montemurro, Maurizio Scaltriti. Neratinib has clinical activity in HER2-amplified breast cancer patients with tumors that have acquired activating mutations in HER2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B169.

Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis

BackgroundThe importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear.MethodsBecause Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays.ResultsWe found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility.ConclusionThese findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2.

ERRα mediates metabolic adaptations driving lapatinib resistance in breast cancer.

Despite the initial benefits of treating HER2-amplified breast cancer patients with the tyrosine kinase inhibitor lapatinib, resistance inevitably develops. Here we report that lapatinib induces the degradation of the nuclear receptor ERRα, a master regulator of cellular metabolism, and that the expression of ERRα is restored in lapatinib-resistant breast cancer cells through reactivation of mTOR signalling. Re-expression of ERRα in resistant cells triggers metabolic adaptations favouring mitochondrial energy metabolism through increased glutamine metabolism, as well as ROS detoxification required for cell survival under therapeutic stress conditions. An ERRα inverse agonist counteracts these metabolic adaptations and overcomes lapatinib resistance in a HER2-induced mammary tumour mouse model. This work reveals a molecular mechanism by which ERRα-induced metabolic reprogramming promotes survival of lapatinib-resistant cancer cells and demonstrates the potential of ERRα inhibition as an effective adjuvant therapy in poor outcome HER2-positive breast cancer.

Phospho-PRAS40Thr246 predicts trastuzumab response in patients with HER2-positive metastatic breast cancer.

Resistance to trastuzumab is frequently observed during the treatment of patients with human epidermal growth factor 2 (HER2)-positive metastatic breast cancers. The aim of the present study was to determine if the phosphorylated proline-rich Akt substrate of 40 kDa (phospho-PRAS40Thr246), a novel biomarker for phosphoinositol-3 kinase (PI3K) pathway activation, could predict the response of HER2-positive metastatic breast cancers to treatment with trastuzumab. Formalin-fixed, paraffin-embedded tumor tissue samples were retrospectively collected from 55 trastuzumab-treated patients. Next, the expression of phospho-PRAS40Thr246 and phosphatase and tensin homolog (PTEN) was assessed by immunohistochemistry. In total, five common phosphoinositol-3 kinase α catalytic subunit mutations, namely E542K, E545K, E545D, H1047R and H1047L, were identified by the amplification-refractory mutation system, using the allele-specific polymerase chain reaction. The activation of the PI3K pathway, as determined by low PTEN expression or the presence of oncogenic PIK3CA mutations, was observed in 49.1% (27 cases) of the 55 HER2-positive metastatic breast cancer tissues. In total, 40% of the tumors were defined as being phospho-PRAS40Thr246-positive. Furthermore, the results revealed that phospho-PRAS40Thr246 expression was associated with the PI3K pathway activation status and an increased risk of tumor progression in HER2-positive metastatic breast cancer patients who had received trastuzumab-based therapy. Therefore, phospho-PRAS40Thr246 expression levels may reflect the PI3K pathway activation status and act as a biomarker for HER2-amplified breast cancer patients who are unlikely to respond to trastuzumab-based therapy.

Abstract 4519: In vitro cell and in vivo animal studies of the demethylating agent in treating HER2-amplified breast cancer

Abstract PURPOSE: The recent evidence shows that HER2-amplified breast cancers exhibit more frequent CpG island methylation than other types of breast cancers, suggesting epigenetic alterations are involved in HER2-mediated tumorigenesis. In this study, we addressed the possibility of the use of the demethylating agent (5-aza-2’-deoxycytidine, decitabine) for treatment of HER2-overexpressing breast cancer. EXPERIMENTAL DESIGN: Two cell model systems were used in this study: murine primary Her2-overexpressing tumor cell line (A6O5, derived from the tumor naturally developed in HER2/neu transgenic mice) and human HER2-overexpressing luminal breast cancer cell line MCF7-HER2-18. Cells were cultured in regular cell culture dishes with the serum-containing medium (adhesive culture) or in ultra-low attachment dishes with the serum-free sphere culture medium (tumorsphere culture) for drug testing. Tumors developed from A6O5 cells injected into mammary glands of HER2/neu transgenic mice were used as the xenograft model. The early spontaneous mammary tumors naturally developed in HER2/neu transgenic mice (size < 5 mm) were used for drug treatment. RESULTS: The in vitro growth of A6O5 was more significantly inhibited by decitabine than that of non-tumorigenic murine mammary epithelial cells. Consistently, MCF7-HER2-18 cells were more sensitive to decitabine-induced growth suppression than its control vector-transfected cell line MCF7-Neo. These results suggest that HER2 overexpression renders cancer cells more sensitive to the effect of decitabine. Surprisingly, the tumorsphere formation of A6O5 and MCF7-HER2-18 was dramatically abrogated by decitabine, suggesting that self-renewal of cancer stem cells in these two cell lines was annihilated by decitabine. Importantly, decitabine inhibited the in vivo growth of A6O5 xenograft tumors. For proof-in-principle of this study, we treated the HER2/neu transgenic mice bearing early developing mammary tumors with decitabine. Indeed, decitabine effectively suppressed the in vivo tumor growth of the decitabine-treated group compared to that of the vehicle-treated group. CONCLUSIONS: This study for the first time explores the potential of the demethylating agent in the treatment of HER2-amplified breast cancers using in vitro cell and in vivo animal models. These findings shed light on the translational application of epigenetic modifiers with the HER2-signaling blockers to treatment of HER2-amplified breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4519. doi:10.1158/1538-7445.AM2011-4519

A Central Role for HER3 in HER2-Amplified Breast Cancer: Implications for Targeted Therapy

Epidermal growth factor receptor (EGFR) and HER3 each form heterodimers with HER2 and have independently been implicated as key coreceptors that drive HER2-amplified breast cancer. Some studies suggest a dominant role for EGFR, a notion of renewed interest given the development of dual HER2/EGFR small-molecule inhibitors. Other studies point to HER3 as the primary coreceptor. To clarify the relative contributions of EGFR and HER3 to HER2 signaling, we studied receptor knockdown via small interfering RNA technology across a panel of six HER2-overexpressing cell lines. Interestingly, HER3 was as critical as HER2 for maintaining cell proliferation in most cell lines, whereas EGFR was dispensable. Induction of HER3 knockdown in the HER2-overexpressing BT474M1 cell line was found to inhibit growth in three-dimensional culture and induce rapid tumor regression of in vivo xenografts. Furthermore, preferential phosphorylation of HER3, but not EGFR, was observed in HER2-amplified breast cancer tissues. Given these data suggesting HER3 as an important therapeutic target, we examined the activity of pertuzumab, a HER2 antibody that inhibits HER3 signaling by blocking ligand-induced HER2/HER3 heterodimerization. Pertuzumab inhibited ligand-dependent morphogenesis in three-dimensional culture and induced tumor regression in the heregulin-dependent MDA-MB-175 xenograft model. Importantly, these activities of pertuzumab were distinct from those of trastuzumab, a monoclonal antibody currently used for treatment of HER2-amplified breast cancer patients. Our data suggest that inhibition of HER3 may be more clinically relevant than inhibition of EGFR in HER2-amplified breast cancer and also suggest that adding pertuzumab to trastuzumab may augment therapeutic benefit by blocking HER2/HER3 signaling.