Combining gene expression analysis of gastric cancer cell lines and tumor specimens to identify biomarkers for anti-HER therapies-the role of HAS2, SHB and HBEGF.

BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.

Targetable HER3 functions driving tumorigenic signaling in HER2-amplified cancers.

Effective inactivation of the HER2-HER3 tumor driver has remained elusive because of the challenging attributes of the pseudokinase HER3. We report a structure-function study of constitutive HER2-HER3 signaling to identify opportunities for targeting. The allosteric activation of the HER2 kinase domain (KD) by the HER3 KD is required for tumorigenic signaling and can potentially be targeted by allosteric inhibitors. ATP binding within the catalytically inactive HER3 KD provides structural rigidity that is important for signaling, but this is mimicked, not opposed, by small molecule ATP analogs, reported here in a bosutinib-bound crystal structure. Mutational disruption of ATP binding and molecular dynamics simulation of the apo KD of HER3 identify a conformational coupling of the ATP pocket with a hydrophobic AP-2 pocket, analogous to EGFR, that is critical for tumorigenic signaling and feasible for targeting. The value of these potential target sites is confirmed in tumor growth assays using gene replacement techniques.

Extensive conformational and physical plasticity protects HER2-HER3 tumorigenic signaling.

Surface-targeting biotherapeutic agents have been successful in treating HER2-amplified cancers through immunostimulation or chemodelivery but have failed to produce effective inhibitors of constitutive HER2-HER3 signaling. We report an extensive structure-function analysis of this tumor driver, revealing complete uncoupling of intracellular signaling and tumorigenic function from regulation or constraints from their extracellular domains (ECDs). The canonical HER3 ECD conformational changes and exposure of the dimerization interface are nonessential, and the entire ECDs of HER2 and HER3 are redundant for tumorigenic signaling. Restricting the proximation of partner ECDs with bulk and steric clash through extremely disruptive receptor engineering leaves tumorigenic signaling unperturbed. This is likely due to considerable conformational flexibilities across the span of these receptor molecules and substantial undulations in the plane of the plasma membrane, none of which had been foreseen as impediments to targeting strategies. The massive overexpression of HER2 functionally and physically uncouples intracellular signaling from extracellular constraints.

Association of Adjuvant Hormone Therapy Timing With Overall Survival Among Patients With Hormone Receptor-Positive Human Epidermal Growth Factor Receptor-2-Negative Early Breast Cancer Without Chemotherapy.

Importance: Studies have shown that delayed initiation of surgery and adjuvant chemotherapy is associated with lower rates of breast cancer survival. However, it remains unclear whether delayed initiation of adjuvant hormone therapy (AHT) is associated with survival. Objective: To assess the association of time to adjuvant hormone therapy (TTH) with breast cancer survival and evaluate the factors associated with AHT. Design, Setting, and Participants: This cohort study examined data from the National Cancer Database from 2004 through 2014 to assess the association of TTH (stratified as ≤150 and >150 days) with cancer survival. All patients included were diagnosed with stage I to stage III hormone receptor-positive, human epidermal growth factor receptor-2 (ERBB2; formerly HER2)-negative invasive breast cancer and underwent AHT without chemotherapy. Data were analyzed from April 2019 to May 2020. Exposures: AHT was administered at different time points following surgical procedures for breast cancer treatment. Main Outcomes and Measures: An inverse probability of treatment weighting (IPTW) model was constructed to evaluate overall survival by adjusting for treatment facility, patient demographics, tumor characteristics, and treatment; multivariable logistic regression was conducted to assess factors associated with delayed treatment. Results: A total of 144 103 patients (median [IQR] follow-up, 36.6 months [25.5-49.2 months]; mean [SD] age, 63.7 [11.6] years) were identified, which included 142 916 (99.2%) women, 11 574 (8.0%) Black patients, and 126 013 (87.4%) White patients. Of these, 134 873 patients (93.6%) had a TTH of 150 days or less and 9230 patients (6.4%) had a TTH longer than 150 days. The IPTW-based Cox model demonstrated that patients with delayed AHT (ie, a TTH past 150 days) were associated with decreased survival (hazard ratio [HR], 1.31; 95% CI, 1.26-1.35; P < .001) compared with those receiving the timely treatment (TTH ≤150 days). Several sensitivity analyses (including IPTW with stabilized weight [HR, 1.31; 95% CI, 1.19-1.45; P < .001], propensity score matching [HR, 1.41; 1.13-1.76; P = .002], and propensity score regression adjustment [HR, 1.29; 95% CI, 1.16-1.43; P < .001]) and exploratory subgroup analyses yielded similar trends. Factors associated with delayed AHT included Black racial identity (OR, 1.66; 95% CI, 1.55-1.77), nonprivate insurance (eg, no insurance: OR, 1.46; 95% CI, 1.26-1.70), living in large metropolitan or metropolitan areas (reference vs urban, less urban, or rural: OR, 0.82; 95% CI, 0.76-0.87), treatment in a community hospital (reference vs academic or research: OR, 0.91; 95% CI, 0.84-0.98), Charlson-Deyo Comorbidity Index score 2 or higher (OR, 1.17; 95% CI, 1.04-1.32), poor grade differentiation (OR, 1.42; 95% CI, 1.32-1.53), II and III pathological stage (stage III: OR, 3.13; 95% CI, 2.76-3.54), estrogen receptor-positive (ER+)/progesterone receptor-negative (PR-) or ER-/PR+ (OR, 1.22; 95% CI, 1.13-1.31), receiving breast conservation surgery (reference vs mastectomy: OR, 0.87; 95% CI, 0.79-0.94), and radiotherapy (reference vs no radiotherapy: OR, 0.56; 95% CI, 0.52-0.61). Conclusions and Relevance: The delay of the initiation of AHT past 150 days was associated with diminished survival in hormone receptor-positive, ERBB2-negative patients with breast cancer who did not receive chemotherapy. Efforts should be made to address factors associated with delayed treatment to improve survival.

Photoactivatable Fluorescent Tags for Dual-Modality Positron Emission Tomography Optical Imaging.

Fluorescent protein conjugates are vital tools in a wide range of scientific disciplines from basic biochemical research to applications in clinical pathology and intraoperative surgery. We report the synthesis and characterization of photoactivatable fluorophores (PhotoTags) based on the functionalization of coumarin, fluorescein, BODIPY, rhodamine B, and cyanine dyes with a photochemically active aryl azide group. Photochemical labeling experiments using human serum albumin produced fluorescent proteins in high yields under irradiation with ultraviolet light for <15 min. We also synthesized DFO-RhodB-PEG3-ArN3─a photoactivatable compound that can be radiolabeled with 89Zr for applications in optical imaging and positron emission tomography. One-pot 89Zr-radiolabeling and light-induced protein conjugation produced [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab. Proof-of-concept studies in vitro and in vivo confirmed that [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab is a potential dual-modality agent for detecting human epidermal growth factor receptor 2 (HER2/neu) expression. Overall, the PhotoTag technology represents a rapid, synthetically versatile, and user-friendly approach for generating novel protein conjugates.

Real-World Outcomes of Everolimus and Exemestane for the Treatment of Metastatic Hormone Receptor-Positive Breast Cancer in Patients Previously Treated With CDK4/6 Inhibitors.

PURPOSE: Everolimus with exemestane (EVE+EXE) was FDA-approved to treat metastatic hormone receptor-positive breast cancer (mHRBC) based on BOLERO-2. However, none of those patients received prior CDK4/6 inhibitors. The purpose of this study is to evaluate the efficacy of EVE+EXE in mHRBC after CDK4/6 inhibitors. METHODS: A retrospective review of patients ≥18 years old with mHRBC treated with EVE+EXE, for ≥30 days, at our institution from January 1, 2012, to April 1, 2020 was conducted. Primary objective was to compare progression free survival (PFS) for EVE+EXE between patients with and without prior exposure to CDK4/6 inhibitors. Secondary outcomes included overall survival and safety. RESULTS: 192 patients were included in the study (n = 79, prior CDK4/6 inhibitor use; n = 113, no prior CDK4/6 inhibitor use). Baseline patient characteristics were similar between groups. Greater number of prior therapies before EVE+EXE use increased risk of disease progression (P = .017). Patients with prior CDK4/6 inhibitor use had a lower median PFS of 3.8 months (95% CI: 3.4-4.7) vs. 5.4 months (95% CI: 3.9-6.2) for patients without prior CDK4/6 inhibitor use, with a HR for progression of 1.46 (95% CI: 1.08 to 1.97, P = .013). Overall survival between groups was not significantly different. CONCLUSION: Patients who received a prior CDK4/6 inhibitor had a lower median PFS benefit from EVE+EXE compared to those who did not, without differences in overall survival. Although PFS is expected to decrease with subsequent lines of therapy, it is reasonable to use EVE+EXE after CDK4/6 inhibitors in selected patients, recognizing that additional benefit is modest.

Human induced pluripotent stem cell-derived platelets loaded with lapatinib effectively target HER2+ breast cancer metastasis to the brain.

Prognosis of patients with HER2+ breast-to-brain-metastasis (BBM) is dismal even after current standard-of-care treatments, including surgical resection, whole-brain radiation, and systemic chemotherapy. Radiation and systemic chemotherapies can also induce cytotoxicity, leading to significant side effects. Studies indicate that donor-derived platelets can serve as immune-compatible drug carriers that interact with and deliver drugs to cancer cells with fewer side effects, making them a promising therapeutic option with enhanced antitumor activity. Moreover, human induced pluripotent stem cells (hiPSCs) provide a potentially renewable source of clinical-grade transfusable platelets that can be drug-loaded to complement the supply of donor-derived platelets. Here, we describe methods for ex vivo generation of megakaryocytes (MKs) and functional platelets from hiPSCs (hiPSC-platelets) in a scalable fashion. We then loaded hiPSC-platelets with lapatinib and infused them into BBM tumor-bearing NOD/SCID mouse models. Such treatment significantly increased intracellular lapatinib accumulation in BBMs in vivo, potentially via tumor cell-induced activation/aggregation. Lapatinib-loaded hiPSC-platelets exhibited normal morphology and function and released lapatinib pH-dependently. Importantly, lapatinib delivery to BBM cells via hiPSC-platelets inhibited tumor growth and prolonged survival of tumor-bearing mice. Overall, use of lapatinib-loaded hiPSC-platelets effectively reduced adverse effects of free lapatinib and enhanced its therapeutic efficacy, suggesting that they represent a novel means to deliver chemotherapeutic drugs as treatment for BBM.

Cytotoxicity, anti-angiogenic, anti-tumor and molecular docking studies on phytochemicals isolated from Polygonum hydropiper L.

BACKGROUND: According to the recent global cancer statistics, breast cancer is the leading cause of deaths among women with 2.3 million new cases globally. Likewise, cervical cancer is also among the leading causes of mortality among women. Polygonum hydropiper is traditionally known for its cytotoxic effects and several bioactive cytotoxic compounds were isolated from it. This study was aimed to isolate potential anticancer compounds from its most potent fractions and evaluate their anticancer potentials. METHODS: Based on our earlier studies, active fractions including chloroform and ethyl acetate were subjected to column chromatography for isolation of compounds. Chemical structures of isolated compounds were confirmed via 1H NMR, 13C NMR, mass spectrometry. Purified compounds were tested for cytotoxicity against breast cancer cells (MCF-7), cervical cancer cells (HeLA) and NIH/3T3 fibroblasts cells cultures using MTT assy. Anti-angiogenic potentials of isolated compounds were evaluated via chorioallantoic membrane assay. Anti-tumor studies were done using Agrobacterium tumefaciens induced potato tumor assay. Furthermore, to understand the binding modes of Isolated compounds, molecular docking was performed against EGFR, HER2 and VEGFR using MOE as docking software. RESULTS: Two bioactive compounds PH-1 (4-methyl-5-oxo-tetrahydrofuran-3-yl acetate) and PH-2 (methyl 4-hydroxy-3-methoxybenzoate) were purified from the active fractions. In cytotoxicity studies, PH-1 exhibited highest cytotoxicity against HeLA cells with 87.50% lethality at 1 mgmL-1 concentration and LD50 of 60 µgmL-1. Likewise, PH-2 showed 82.33% cytotoxicity against HeLA cells with LD50 of 160 µgmL-1. Similarly, PH-1 and PH-2 exhibited LD50 of 170 and 380 µgmL-1 respectively. Moreover, PH-1 and PH-2 were also very potent cytotoxic compounds against NIH/3T3 cells with 81.45 and 85.55% cytotoxicity at 1 mgL-1 concentration and LD50 of 140 and 58 µgL-1 respectively. Isolated compounds exhibited considerable anti-angiogenic potentials with IC50 of 340 and 500 µgL-1 respectively for PH-1 and PH-2. In anti-tumor assay, PH-1 and PH-2 exhibited 81.15 and 76.09% inhibitions with LD50 of 340 and 550 µgL-1 respectively. Both compounds selectively binds with EGFR and HER2 receptors with low binding energies. Both compounds exhibited stronger interactions with VEGFR through binding pocket residues Lys868, Val916 and Asp1046. CONCLUSIONS: Both compounds cause considerable cytotoxicity against cancer cells. The anti-angiogenic and anti-tumor results suggests additional tumor suppressive properties. Docking analysis suggests that these compound not only has the ability to bind to EGFR and HER2 but also equally binds to VEGFR and may act as potential anti-angiogenic agents.

CCA-1.1, a Novel Curcumin Analog, Exerts Cytotoxic anti- Migratory Activity toward TNBC and HER2-Enriched Breast Cancer Cells.

OBJECTIVE: Chemoprevention curcumin Analog-1.1 (CCA-1.1) demonstrates antineoplastic effect toward cancer cells. By using triple-negative breast cancer (TNBC), 4T1, and human epidermal growth factor receptor 2 (HER2)-enriched metastatic cells (MCF-7/HER2), we evaluate the cytotoxic and antimigration activities from CCA-1.1. METHODS: The cytotoxic activities from a single treatment of CCA-1.1 and in combination with doxorubicin were determined through MTT assay. We also calculated the selectivity index and combination index of CCA-1.1 from the cytotoxic data. Migrating cells were evaluated using wound healing assay, and the MMP2 and MMP9 secretion levels were determined through gelatin zymography. RESULTS: As hypothesized, CCA-1.1 performed cytotoxic activity during treatment in 4T1 and MCF-7/HER2 cancer cells with good selectivity (Selectivity Index >2). In addition, CCA-1.1 demonstrated a synergistic effect in combinatorial treatment with a low dose of doxorubicin. A single treatment of CCA-1.1 repressed cell migration in 4T1 and MCF-7/HER2 cells. Under gelatin zymography, CCA-1.1 subsided the activities of MMP-9, thereby revealing the potency of CCA-1.1 as an anti-migratory agent. Moreover, MMP-9 was also eminently expressed in TNBC and HER2-enriched breast cancer cells when compared with that in other subtypes. CONCLUSION: Our preliminary study collectively reinforces the potential effect of CCA-1.1 through the inhibition of highly aggressive cell migration, particularly in breast cancer.

Is there a correlation between HER2 gene amplification level and response to neoadjuvant treatment with trastuzumab and chemotherapy in HER2-positive breast cancer?

There are contradictory data regarding the correlation between HER2 amplification level determined by in situ hybridization and evolution after treatment with anti-HER2 therapies. The aim of this study was to correlate quantitative results of FISH (ratio HER2/CEP17 and number of HER2 signals/nucleus) with pathological response achieved after neoadjuvant treatment with trastuzumab and chemotherapy. For this purpose, we analysed 100 consecutive HER2-positive cases of breast carcinoma treated with neoadjuvant therapy. HER2 amplification determined by FISH was found in 92 of the 100 cases studied. pCR was obtained in 58% of the patients whose tumours presented amplification. In contrast, no pCR was obtained in the 8 patients with non-amplified tumours. A significant direct correlation between HER2 high amplification (HER2/CEP17 ratio > 5 or HER2 signals/nucleus > 10) and pCR was found. In conclusion, HER2 amplification levels are clinically relevant because they provide oncologists with valuable information on the possibilities of achieving pCR after neoadjuvant treatment.

Discovery and development of trastuzumab deruxtecan and safety management for patients with HER2-positive gastric cancer.

Approximately 12-15% of gastric cancers (GCs) are human epidermal growth factor receptor-2 (HER2)-positive (HER2 immunohistochemistry 3 + or 2 + /in situ hybridization + [ERBB2/CEP17 ≥ 2.0]). While the anti-HER2 monoclonal antibody trastuzumab, in combination with chemotherapy, is the standard treatment for HER2-positive GC, other HER2-targeted therapies have not demonstrated survival benefits in patients with GC, despite showing efficacy in patients with HER2-positive breast cancer. This indicates that there are unique challenges to the use of currently available HER2-targeted therapies for the treatment of HER2-positive GC. Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate consisting of an anti-HER2 human monoclonal IgG1 antibody with the same amino acid sequence as trastuzumab, an enzymatically cleavable peptide-based linker, and DXd, a novel topoisomerase I inhibitor, as its released payload. T-DXd has a high drug-antibody ratio (approximately 8) and a demonstrated bystander antitumor effect. It has demonstrated significant efficacy when compared with standard therapies and is approved as third- or later-line treatment for HER2-positive GC in Japan and second- or later-line treatment in the US. T-DXd treatment is associated with gastrointestinal and hematological adverse events, and a risk of interstitial lung disease (ILD), with the ILD risk being higher in Japan than in countries other than Japan. However, most adverse events, including ILD, can be managed with proactive monitoring and T-DXd dose modification, and initiation of adequate treatment. In this review, we summarize the discovery and development of T-DXd and provide guidance for T-DXd safety management, including ILD monitoring, for patients with HER2-positive GC.

Hopes and failures in front-line advanced HER2-positive gastric cancer therapy.

Human epidermal growth-factor receptor 2 (HER2) was an important therapeutic target in gastric cancer. Through the last decade, strategy with trastuzumab-based chemotherapy remains the first-line standard of treatment in advanced HER2-positive gastric cancer. Based on the Trastuzumab for Gastric Cancer trial, trastuzumab plus systemic chemotherapy of cisplatin and fluoropyrimidine as the backbone was established as the first-line therapy in advanced HER2-positive gastric cancer. Since then, studies have explored the optimization of the front-line strategy, including the dose of trastuzumab, chemotherapy regimen and maintenance therapy. A large number of clinical trials were conducted to explore the optimal front-line therapy regimens, such as lapatinib and pertuzumab. Safe and effective first-line regimens are still lacking. Recently, two phase II studies of combining immune checkpoint inhibitor in first-line treatment of advanced HER2-positive gastric cancer showed promising results. The progress of immunotherapy has gradually promoted the development of front-line treatment of advanced HER2-positive gastric cancer to potential chemotherapy-free strategies. Therefore, this article reviewed these significant clinical trials and focus on the front-line treatment strategies for HER2-positive gastric cancer.

Pathological complete response of adding targeted therapy to neoadjuvant chemotherapy for inflammatory breast cancer: A systematic review.

BACKGROUND: The current use of targeted therapy plus neoadjuvant chemotherapy for inflammatory breast cancer (IBC) is based on data extrapolated from studies in non-IBC. We conducted a systematic review to determine whether neoadjuvant chemotherapy plus targeted therapy results in a higher pathologic complete response (pCR) rate than neoadjuvant chemotherapy alone in patients with IBC. METHOD AND FINDINGS: This systematic review was registered in the PROSPERO register with registration number CRD42018089465. We searched MEDLINE & PubMed, EMBASE, and EBSCO from December 1998 through July 2020. All English-language clinical studies, both randomized and non-randomized, that evaluated neoadjuvant systemic treatment with or without targeted therapy before definitive surgery and reported the pCR results of IBC patients. First reviewer extracted data and assessed the risk of bias using the Risk of Bias In Non-randomized Studies of Interventions tool. Second reviewer confirmed the accuracy. Studies were divided into 3 groups according to systemic treatment: chemotherapy with targeted therapy, chemotherapy alone, and high-dose chemotherapy with hematopoietic stem cell support (HSCS). Of 995 screened studies, 23 with 1,269 IBC patients met the inclusion criteria. For each of the 3 groups of studies, we computed a weighted average of the pCR rates across all studies with confidence interval (CI). The weighted averages (95% CIs) were as follows: chemotherapy with targeted therapy, 31.6% (26.4%-37.3%), chemotherapy alone, 13.0% (10.3%-16.2%), and high-dose chemotherapy with HSCS, 23.0% (18.7%-27.7%). The high pCR by targeted therapy group came from anti-HER2 therapy, 54.4% (44.3%-64.0%). Key limitations of this study included no randomized clinical studies that included only IBC patients. CONCLUSION: Neoadjuvant chemotherapy plus targeted therapy is more effective than neoadjuvant chemotherapy alone for IBC patients. These findings support current IBC standard practice in particular the use of anti-HER2 targeted therapy.

Clinical therapeutic effects of trastuzumab in HER2-positive breast cancer patients: A protocol for systematic review and meta-analysis.

BACKGROUND: Despite the developments in diagnosis and treatment of HER2-positive metastatic breast cancer, there is a high likelihood in the development of resistance to trastuzumab. In general, HER2-positive patients with deteriorated health face negative clinical outcomes. The present study is conducted to systematically explore the medicinal properties of trastuzumab in HER2-positive breast cancer patients. METHODS: Randomized controlled trials investigating the clinical properties of including trastuzumab to treat HER2-positive breast cancer cases will be sourced by exploring these online-based databases: MEDLINE, BIOSIS, China National Knowledge Infrastructure (CNKI), Cochrane Library, EMBASE, Central Register of Controlled Trials, and WanFang. Two independent authors will screen the literature, gather data, and assess the quality of selected studies. The significance of the relationship between the medical properties of trastuzumab when incorporated to treat HER2-positive breast cancer cases will be evaluated according to the relative risk, mean differences or standardized mean differences, and 95% confidence interval. RESULTS: The outcomes from this review shall be issued in a journal that will be reviewed by peers. CONCLUSION: The conclusions presented in this review will serve as a reference for clinical practitioners and scholars to determine whether trastuzumab is an effective and safety intervention for treating HER2-positive breast cancer patients. ETHICS AND DISSEMINATION: Since this study is a systematic review of published studies, an ethical approval is not needed. SYSTEMATIC REVIEW REGISTRATION NUMBER: March 31, 2021.osf.io/wvqkf (https://osf.io/wvqkf/).

Crizotinib induced antitumor activity and synergized with chemotherapy and hormonal drugs in breast cancer cells via downregulating MET and estrogen receptor levels.

MET is a receptor tyrosine kinase known to drive neoplastic transformation and aggressive tumor phenotypes. Crizotinib is an oral multi-targeted tyrosine kinase inhibitor of MET, ALK, RON, and ROS1 kinases. In this study, the anticancer effects of crizotinib on breast cancer cells were investigated in vitro along with the molecular mechanisms associated with these effects. Besides, the antiproliferative effects of crizotinib in combination with chemotherapy, hormonal drugs, and targeted agents were examined. Results showed that crizotinib produced dose-dependent antiproliferative effects in BT-474 and SK-BR-3 breast cancer cells with IC50 values of 1.7 µM and 5.2 µM, respectively. Crizotinib inhibited colony formation of BT-474 cells at low micromolar concentrations (1-5 µM). Immunofluorescence and Western blotting indicated that crizotinib reduced total levels of MET and estrogen receptor (ERα) in BT-474 cells. Also, crizotinib reduced the levels of phosphorylated (active) MET and HER2 in BT-474 cells. The combined treatment of crizotinib with doxorubicin and paclitaxel resulted in synergistic growth inhibition of BT-474 cells with combination index values of 0.46 and 0.35, respectively. Synergy was also observed with the combination of crizotinib with the hormonal drugs 4-hydroxytamoxifen and fulvestrant in BT-474 cells. Alternatively, the combination of crizotinib with lapatinib produced antagonistic antiproliferative effects in both BT-474 and SK-BR-3 cells. Collectively, these findings demonstrate the anticancer effects of crizotinib in breast cancer cells and reveal ERα as a potential therapeutic target of the drug apart from its classical kinase inhibitory activity. Crizotinib could be an appealing option in combination with chemotherapy or hormonal drugs for the management of breast cancer.

Frontiers in HER2-positive breast cancer in 2020.

PURPOSE OF REVIEW: The field of HER2-positive breast cancer has seen tremendous advances in the last 2 years with largest number of new drugs in decades. The present review aims to summarize the cutting-edge research of the past 2 years and future directions. RECENT FINDINGS: This review will go over four new drugs, three of which have gained FDA approval within the past 18 months, in the treatment of HER2-positive breast cancer. We will go over early and mature clinical data on these therapeutics and ongoing clinical trials further exploring their role in the treatment of patients with advanced HER2-positive breast cancer and HER2 low breast cancer. Will also discuss ongoing trials using immunotherapy and CDK4/6 inhibitors in the advanced HER2-positive setting. SUMMARY: : The therapies described in this review have quickly become standard of care for patients with HER2-positive breast cancer. Furthermore, they have the potential to change the landscape of breast cancer therapy further to include even patients with HER2 low breast cancer.

Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo.

Human EGF Receptor 2 (HER2) is an important oncogene driving aggressive metastatic growth in up to 20% of breast cancer tumors. At the same time, it presents a target for passive immunotherapy such as trastuzumab (TZM). Although TZM has been widely used clinically since 1998, not all eligible patients benefit from this therapy due to primary and acquired drug resistance as well as potentially lack of drug exposure. Hence, it is critical to directly quantify TZM-HER2 binding dynamics, also known as cellular target engagement, in undisturbed tumor environments in live, intact tumor xenograft models. Herein, we report the direct measurement of TZM-HER2 binding in HER2-positive human breast cancer cells and tumor xenografts using fluorescence lifetime Forster Resonance Energy Transfer (FLI-FRET) via near-infrared (NIR) microscopy (FLIM-FRET) as well as macroscopy (MFLI-FRET) approaches. By sensing the reduction of fluorescence lifetime of donor-labeled TZM in the presence of acceptor-labeled TZM, we successfully quantified the fraction of HER2-bound and internalized TZM immunoconjugate both in cell culture and tumor xenografts in live animals. Ex vivo immunohistological analysis of tumors confirmed the binding and internalization of TZM-HER2 complex in breast cancer cells. Thus, FLI-FRET imaging presents a powerful analytical tool to monitor and quantify cellular target engagement and subsequent intracellular drug delivery in live HER2-positive tumor xenografts.

Pathological complete response to neoadjuvant trastuzumab and pertuzumab therapy is related to human epidermal growth factor receptor 2 (HER2) amplification level in HER2-amplified breast cancer.

The human epidermal growth factor receptor 2 (HER2) is amplified in approximately 20% of breast cancers, and HER2 receptor targeting therapy is associated with a significant improvement in disease-free and overall survival. In several clinical trials, the pathologic complete response (pCR) rate was significantly increased with combined pertuzumab and trastuzumab treatment in HER2-amplified breast cancer. Although the efficacy and safety of anti-HER2 dual blockade therapy has been reported, the markers that predict the response are still unclear. This study aimed to investigate the relationship between the level of HER2 amplification and the pCR in trastuzumab and pertuzumab neoadjuvant therapy.Twenty-two HER2-amplified early breast cancer patients who had received neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP) therapy were included in this study. HER2/CEP17 ratio and average HER2 copy number were measured by fluorescence in situ hybridization analysis. The relationship between level of HER2 amplification and tumor pCR status was investigated.The median age was 47.5 years (range, 36-62). 31.8% of the patients were hormone receptor (HR) positive and 68.2%% of the patients were HR negative. The pCR (ypN0/is ypN0) rate in the breast and axilla was 68.2%. The patients who experienced a pCR had a median HER2/CEP17 ratio of 7.08 (range, 3.16-10.40) and average HER2 copy number of 17.00 (range, 5.85-37.50). The patients who did not experience a pCR had a median ratio of 4.70 (range, 1.06-9.00) and median HER2 copy number of 12.00 (range, 5.85-20.95) (P = .030, P = .174), respectively.pCR was highly correlated with HER2/CEP17 ratio in neoadjuvant anti-HER2 dual blockade. This suggests that the HER2/CEP17 ratio can be used as a predictive marker for pCR in neoadjuvant trastuzumab and pertuzumab therapy.

Evaluation of Absolute Oral Bioavailability and Bioequivalence of Ribociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor, in Healthy Subjects.

Ribociclib, a selective and potent cyclin-dependent kinase 4/6 inhibitor, has demonstrated safety and efficacy in combination with endocrine therapy in hormone receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer. In 2 open-label crossover studies in healthy participants, the absolute bioavailability of a single oral dose of a ribociclib 600-mg tablet (n = 16) was compared with a single intravenous ribociclib infusion of 150 mg (n = 16), and the bioequivalence of a ribociclib 600-mg tablet (n = 31) and a ribociclib 600-mg capsule (n = 31) was evaluated. The pharmacokinetics of ribociclib and its major metabolite, LEQ803, were assessed in both studies. The oral bioavailability of the 600-mg ribociclib tablet was 65.8% (90% confidence interval [CI], 59.1-73.2%). The geometric mean systemic clearance of ribociclib was moderate (40.2 L/h; 27.4% intersubject variability [CV%]) compared with hepatic blood flow, and the geometric mean volume of distribution was high (979 L; 25.2 CV%). LEQ803-to-ribociclib metabolic ratios were 0.198 for the oral administration and 0.125 for intravenous infusion. Bioequivalence of the tablet and capsule formulations was demonstrated for ribociclib. The geometric mean ratios of maximum concentration and area under the curve from time 0 to last quantifiable concentration and to infinity were 1.01, 1.00, and 0.937, respectively, within the predefined bioequivalence range of 0.80 to 1.25. The median time to reach maximum concentration was 3 hours with both formulations. No serious adverse events were observed in either study.