Low HER2-expressing Breast Cancer Research Articles

Multimodal image-guided surgery of HER2-positive breast cancer using [111In]In-DTPA-trastuzumab-IRDye800CW in an orthotopic breast tumor model

BackgroundCombining modalities using dual-labeled antibodies may allow preoperative and intraoperative tumor localization and could be used in image-guided surgery to improve complete tumor resection. Trastuzumab is a monoclonal antibody against the human epidermal growth factor-2 (HER2) receptor and dual-labeled trastuzumab with both a fluorophore (IRDye800CW) and a radioactive label (111In) can be used for multimodal imaging of HER2-positive breast cancer. The aim of this study was to demonstrate the feasibility of HER2-targeted multimodal imaging using [111In]In-DTPA-trastuzumab-IRDye800CW in an orthotopic breast cancer model.MethodsTrastuzumab was conjugated with p-isothiocyanatobenzyl (ITC)-diethylenetriaminepentaacetic acid (DTPA) and IRDye800CW-NHS ester and subsequently labeled with 111In. In a dose escalation study, the biodistribution of 10, 30, and 100 μg [111In]In-DTPA-trastuzumab-IRDye800CW was determined 48 h after injection in BALB/c nude mice with orthotopic high HER2-expressing tumors. Also, a biodistribution study was performed in a low HER2-expressing breast cancer model. In addition, multimodal image-guided surgery was performed in each group. Autoradiography, fluorescence microscopy, and immunohistochemically stained slices of the tumors were compared for co-localization of tumor tissue, HER2 expression, fluorescence, and radiosignal.ResultsBased on the biodistribution data, a 30 μg dose of dual-labeled trastuzumab (tumor-to-blood ratio 13 ± 2) was chosen for all subsequent studies. [111In]In-DTPA-trastuzumab-IRDye800CW specifically accumulated in orthotopic HER2-positive BT474 tumors (101 ± 7 %IA/g), whereas uptake in orthotopic low HER2-expressing MCF7 tumor was significantly lower (1.2 ± 0.2 %IA/g, p = 0.007). BT474 tumors could clearly be visualized with both micro-SPECT/CT, fluorescence imaging and subsequently, image-guided resection was performed. Immunohistochemical analyses of BT474 tumors demonstrated correspondence in fluorescence, radiosignal, and high HER2 expression.ConclusionsDual-labeled trastuzumab showed specific accumulation in orthotopic HER2-positive BT474 breast tumors with micro-SPECT/CT and fluorescence imaging and enabled image-guided tumor resection. In the clinical setting, [111In]In-DTPA-trastuzumab-IRDye800CW could be valuable for preoperative detection of (metastatic) tumors by SPECT/CT imaging, and intraoperative localization by using a gamma probe and fluorescence image-guided surgery to improve radical resection of tumor tissue in patients with HER2-positive tumors.

Abstract 5360: The preclinical profile of the duocarmycin-based HER2-targeting ADC SYD985 predicts for clinical benefit in low HER2-expressing breast cancers

Abstract SYD985 is a HER2-targeting ADC based on trastuzumab and vc-seco-DUBA, Synthon's proprietary cleavable linker-duocarmycin payload. Vc-seco-DUBA was coupled to cysteines after partial reduction on the interchain disulfides of trastuzumab. SYD985 was obtained after purification by Hydrophobic Interaction Chromatography to yield a well-defined ADC consisting of predominantly DAR 2 and DAR 4 species. To evaluate the therapeutic potential of this new ADC, and to prepare clinical development, mechanistic in vitro studies and in vivo PDX studies were conducted to compare SYD985 head-to-head to T-DM1 (Kadcyla®), another trastuzumab-based ADC with a toxin of the maytansinoid class in combination with a non-cleavable linker. SYD985 and T-DM1 had similar binding-affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3 to 50-fold more potent than T-DM1. In contrast to T-DM1, SYD985 efficiently induced bystander killing in vitro of HER2 negative (HER2 0) cells when mixed with HER2 3+, 2+, or 1+ cells. SYD985 efficiently killed HER2 0 cells, even in the presence of only 20% of HER2 3+ cells. At pH conditions relevant for tumors, cathepsin B cleavage studies showed efficient release of the active toxin from SYD985 but not from T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2 expressing tumors in vivo, allowing the treatment of tumor tissues with low or heterogeneous membrane expression of HER2. In line with this, in vivo anti-tumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models whereas T-DM1 only showed significant anti-tumor activity in HER2 3+ breast cancer PDX models. We conclude that the properties of SYD985 may enable expansion of the target population to patients who have low HER2 expressing breast cancer, a patient population with unmet high medical need. Early phase clinical evaluation with SYD985 is ongoing. Citation Format: Willem Dokter, Miranda van der Lee, Patrick Groothuis, Ruud Ubink, Monique van der Vleuten, Tanja van Achterberg, Eline Loosveld, Desirée Damming, Myrthe Rouwette, David Egging, Diels van den Dobbelsteen, Patrick Beusker, peter goedings, Gijs Verheijden, Jacques Lemmens, Marco Timmers. The preclinical profile of the duocarmycin-based HER2-targeting ADC SYD985 predicts for clinical benefit in low HER2-expressing breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2015-5360

Abstract 2466: Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy

Abstract The mainstay of HER2+ breast cancer treatment has been monoclonal antibody therapy with Trastuzumab, a humanized antibody that targets HER-2. However, the efficacy of Trastuzumab monotherapy is only 10-15%. Therefore, strategies are being developed to enhance its therapeutic efficacy. Our previous study demonstrated that stimulation of NK cells with an anti-CD137 agonistic mAb enhanced Trastuzumab-mediated Antibody Dependent Cellular Cytotoxicity (ADCC). Anti-CD137 agonistic mAb enhanced anti-breast cancer activity of Trastuzumab in vivo in a xenotransplanted human breast cancer model (Kohrt et al. J Clin. Invest, 2012, 22:3). We have developed a treatment regimen consisting of sequential administration of Trastuzumab followed by CD137 antibody as three weekly injections. This regimen demonstrated more potent antitumor activity than administration of anti-CD137 mAb followed by Trastuzumab. Our combination therapy was superior to Traztuzumab alone in a primary HER2-overexpressing-breast tumor xenotransplant model but was ineffective in low HER-2 expressing breast cancer model. Next, we evaluated the therapeutic efficacy of FDA approved antibody drug conjugate Trastuzumab Emtansine (T-DM1) in combination with CD137 antibody. We used HER2 over-expressing HER18 xenograft model and an SU-258 primary breast cancer model in our studies. Following tumor inoculation, mice received either T-DM1 on day 3 (for HER18 xenograft), or day 30 (for SU-258) and anti-CD137 antibody on day 4(or HER18) or day 31(for SU-258) with each treatment repeated weekly for a total of three weeks. Sequential antibody strategy with T-DM1 and CD137 significantly improved survival compared to T-DM1 alone or with Trastuzumab and CD137 antibody. Our results demonstrate that T-DM1 retains its potency and demonstrates synergistic activity with anti-CD137 mAb therapy against HER2-overexpressing breast cancer models. Our results support a novel, sequential antibody approach against HER-2+breast cancer, by targeting first the tumor with an antibody drug conjugate and then the host immune system. Clinical investigations are now planned to determine the clinical outcome of our immunotherapy strategy. Citation Format: Suparna Dutt, Narendiran Rajasekaran, Aurelien Marabelle, Roch Houot, Mohith Sadaram, Jonathan Hebb, Idit Sagiv-Barfi, Sid Ambulkar, Amanda Rajapaksa, Cariad Chester, Erin Waller, Holbrook Kohrt. Sequential tumor and immune targeted immunotherapy: Anti-tumor activity of antibody drug conjugate Trastuzumab Emtansine (T-DM1) with CD137 stimulation in HER-2+ breast cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2466. doi:10.1158/1538-7445.AM2015-2466

Gemcitabine treatment enhances HER2 expression in low HER2-expressing breast cancer cells and enhances the antitumor effects of trastuzumab emtansine.

Trastuzumab emtansine (T-DM1), trastuzumab-conjugated with a cytotoxic agent, has shown promising antitumor effects in breast cancer. Since a good therapeutic response using T-DM1 treatment requires high human epidermal growth factor receptor 2 (HER2) expression, breast cancers with low or no HER2 expression have not been used for T-DM1 treatment. The aim of the present study was to show that treatment of low HER2-expressing breast cancer cells with gemcitabine (GEM) enhanced HER2 expression using RT-qPCR, immunoblot and flow cytometric analysis. The results showed that GEM treatment significantly enhanced HER2 expression in MDA-MB-231, MCF7 and BT-20 breast cancer cells, while paclitaxel (PTX) treatment induced lower or no enhancement in HER2 expression. The expression of HER2 mRNA was also enhanced in GEM-treated MCF7 cells. Treatment with an inhibitor for nuclear factor-(NF)-κB suppressed GEM-induced HER2 upregulation, indicating that NF-κB activation by GEM may be associated with HER2 upregulation. T-DM1 binding to HER2 on MCF-7 cells was enhanced by GEM pretreatment and the combined treatment of GEM and T-DM1 synergistically inhibited the proliferation of MCF7 cells. Thus, the combined treatment with GEM and T-DM1 may be a promising therapeutic modality for low HER2-expressing breast cancers, which was facilitated by the unique HER2-upregulating effect of GEM.

The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers.

SYD985 is a HER2-targeting antibody-drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3- to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3+, 2+, or 1+ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need.

Abstract 3332: HER2-dependent RNA polymerase II binding in human breast tumors defines a regulon including a stem cell network

Abstract HER2-positive breast cancer accounts for approximately 25% of all cases and has a poor overall prognosis in spite of gains in targeted therapy. Multiple modes of resistance have been identified. We identified genes regulated in HER2 positive disease as a means of understanding the mechanisms of aggression and resistance to therapy. Chip-on-chip with anti-RNA polymerase II (POL II) in breast cancer cell lines with little or no HER2 expression was compared to cells with very high HER2 expression. Compared to MCF-7 cells with little or no HER2 expression, cells engineered to express large amounts of HER2 exhibits new POL II binding to 606 identified genes. Many of these genes, 379 and 486, also exhibited POL II binding in two breast cancer cell lines with naturally occurring amplified HER2: MDA453 and BT474 respectively. These observations indicate that the expression of HER2 in MCF-7 cells causes a massive increase in the number of genes bound by POL II. We performed whole genome expression analysis on the three HER2+/- cells and compared these to expression in 812 primary tumors stratified for HER2 +/- expression. Genes differentially expressed dependent on HER2 status were overwhelmingly regulated in the same direction in MCF7HER2, MDA453 and BT474 cell lines and in breast cancer tissues; 273/459 (60%), 335/502 (67%) and 349/502 (70%) respectively (Kappa ∼ 0.4, p < 0.0001). The same comparisons were performed on randomly selected genes and kappa values were calculated for 1000 rounds. The random kappa values averaged ∼0.05 indicating minimal false discovery. Second we identified genes that had HER2-dependent POLII binding in cell lines without significant expression. Of 737 such genes “poised” for expression, 113 were differentially expressed in breast tumors in a HER2 dependent manner, including a similar number of up and down-regulated genes in each class of tumors. We carried out pathway analysis of 113 “poised” genes identified having HER2-dependent POL II binding and exhibiting significant differential expression between high and low HER2-expressing breast cancer tissues. A pathway analysis tool (MetaCore) identified enrichment in GO terms for cell adhesion, regulation of translational initiation, inflammation, immune response especially for Interleukins -5 and 13, cytoskeleton remodeling, membrane domain ectodomain proteolysis (the proteolytic cleavage of trans membrane proteins and release of their extracellular domain), and development. The largest groups of genes are associated with stem cell and progenitor cell control as indicated by networks centered on NANOG and OCT3/4. Thus, the role of stem cells proliferation in HER2 regulated breast cancer is highly suggested. The massive gene regulation changes that are tissue context-dependent is a fundamental new insight for understanding HER2 mechanisms in breast cancer. Citation Format: Farah Rahmatpanah, Xin Chen, Zhenyu Jia, Bozhao Men, Michael McClelland, Dan Mercola. HER2-dependent RNA polymerase II binding in human breast tumors defines a regulon including a stem cell network. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3332. doi:10.1158/1538-7445.AM2014-3332

Abstract LB-146: A phase II randomized placebo-controlled study of AZD8931, an inhibitor of EGFR, HER2, and HER3 signaling, plus paclitaxel (P) vs P alone in patients (pts) with low HER2-expressing advanced breast cancer (BC) (THYME).

Abstract Background: AZD8931 is an oral, equipotent inhibitor of EGFR, HER2, and HER3 signaling. Preclinical evidence indicates that AZD8931 may be particularly potent when HER signaling is ligand driven, which is thought to be the signaling mechanism that is more prevalent in low HER2-expressing BC. Methods: In this double-blind multicenter phase II study, women with low HER2- expressing locally advanced or metastatic BC (ineligible by HER2 status for either trastuzumab or lapatinib) were randomized 1:1 to AZD8931 40mg bid + P (90mg/m2; d1, 8 and 15, q4 weeks) or matched placebo + P (NCT00900627). The primary objective was prolonged progression-free survival (PFS; assessed by RECIST v1.1); secondary endpoints included objective response rate (ORR), overall survival (OS), and safety. Results: Between July 2010 and April 2011, 190 pts (median age, 55 years [range, 32-80]; 83% metastatic disease) were randomized to AZD8931 + P (n=94) or placebo + P (n=96). No significant difference in PFS was observed with AZD8931 + P vs P alone (HR=1.08, 95% CI [0.76, 1.52], P=0.679; median PFS, 8.7 vs 9.1 months); this lack of treatment effect was consistent across subgroups. A significant increase in ORR (59% vs 41%; OR=2.02, 95% CI [1.09, 3.75], P=0.026) and in mean % change in tumor size at week 8 vs baseline (difference, -9.9%, 95% CI [-18.4%, -1.4%], P=0.023; exploratory objective) was observed with AZD8931 + P vs P alone. At data cut-off (April 2012), OS data were immature (52 [27%] pts had died), with no difference between treatment arms (HR=1.15, 95% CI [0.67, 2.01], P=0.607). The hazard ratio for the time to deterioration of health-related quality of life (assessed by FACT-B TOI; exploratory objective) was 1.46 (95% CI [1.04, 2.05], P=0.028). The most common adverse events (AEs) were rashes and acne (grouped term; 91% [AZD8931 + P] vs 26% [P alone]) and diarrhea (83% vs 35%). Grade ≥3 AEs were reported for 65% and 41% of pts in the AZD8931 + P and the P alone arm, respectively, the most common being rashes and acne (grouped term; 19% vs 2%), diarrhea (12% vs 4%), neutropenia (7% vs 15%) and peripheral neuropathy (7% vs 2%). Discontinuation of AZD8931 or placebo treatment due to AEs was reported for 12 (13%) and 8 (9%) pts, respectively. Fatal AEs were reported in two pts in the AZD8931 + P arm (neither considered AZD8931 treatment-related) and three pts in the P alone arm. No new or unexpected safety concerns were reported with this treatment combination. Paclitaxel delivery was not compromised by combination with AZD8931. Conclusion: The THYME study did not meet its primary objective of prolonging PFS when AZD8931 was added to weekly P in pts with low HER2-expressing advanced BC. AZD8931 40mg bid can be administered in combination with weekly P with a manageable toxicity profile. Exploratory biomarker analyses are ongoing and will be presented. Citation Format: José Baselga, Roberto Hegg, Maria Vidal Losada, Tatiana Vidaurre, Ana Lluch, Katerina Petrakova, Helen Mann, Serban Ghiorghiu, Mary Stuart, Dónal Landers, Kenneth Thress, Teresa Klinowska, Javier Cortes. A phase II randomized placebo-controlled study of AZD8931, an inhibitor of EGFR, HER2, and HER3 signaling, plus paclitaxel (P) vs P alone in patients (pts) with low HER2-expressing advanced breast cancer (BC) (THYME). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-146. doi:10.1158/1538-7445.AM2013-LB-146

Abstract 912: Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease.

Abstract Effective therapies for women with low-HER2 expressing breast cancer remain a significant unmet clinical need. We have detected significant phosphorylated-HER and HER2:HER3 dimer expression in clinical breast cancer samples without HER2 amplification suggesting that HER signaling may play a role in these tumors. It is our hypothesis that ligand-driven signaling is the major route of HER family activation in the absence of HER2 amplification. AZD8931 is an orally bioavailable and highly selective small molecule inhibitor of EGFR, HER2 and HER3 signaling, which shows greatest potency when HER signaling is ligand-driven(1). In a range of low HER2 breast cancer cell lines, AZD8931 inhibited heregulin-driven proliferation (GI50 range 0.05 to 0.38μM) HER2:HER3 signaling, HER3:PI3K interaction and downstream signaling. In vivo, twice-daily oral dosing of AZD8931 showed significant monotherapy anti-tumor efficacy in a xenograft model of low HER2 breast cancer (MDA-MB-175VII) at well-tolerated doses (90% TGI at 12.5 mg/kg/bid). We further evaluated the activity of AZD8931 in combination with paclitaxel, a commonly used standard of care chemotherapy for patients with advanced breast cancer expressing low levels of HER2. Pre-clinically, the combination of AZD8931 with paclitaxel showed at least additive activity in vitro in a range of breast cell lines and additive efficacy in vivo in a BT474 (high HER2) xenograft model at well tolerated doses (AZD8931 12.5 mg/kg/bid TGI 63%; paclitaxel 7.5mg/kg/qw TGI 41%; combination TGI 95%). Combination work in the low HER2 xenograft model is ongoing. These data demonstrate that AZD8931 inhibits ligand-driven HER family receptor activation, receptor dimerization, PI3K interaction and downstream signaling leading to anti-tumor activity in vivo. These data also support the potential clinical utility of AZD8931 for the therapeutic treatment of low HER2 expressing breast cancers in combination with paclitaxel. 1 Hickinson et al. Clin.Cancer Res (2010) 16:1159-69. Citation Format: Gayle Marshall, Susan Ashton, Georgina Speake, Celina D'Cruz, Michael Grondine, Cath Trigwell, Graham Bigley, Garry Beran, Katy Lynaugh, Teresa C. Klinowska. Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 912. doi:10.1158/1538-7445.AM2013-912

QLT0267, a small molecule inhibitor targeting integrin-linked kinase (ILK), and docetaxel can combine to produce synergistic interactions linked to enhanced cytotoxicity, reductions in P-AKT levels, altered F-actin architecture and improved treatment outcomes in an orthotopic breast cancer model

IntroductionSubstantial preclinical evidence has indicated that inhibition of integrin linked-kinase (ILK) correlates with cytotoxic/cytostatic cellular effects, delayed tumor growth in animal models of cancer, and inhibition of angiogenesis. Widely anticipated to represent a very promising therapeutic target in several cancer indications, it is increasingly evident that optimal therapeutic benefits obtained using ILK targeting strategies will only be achieved in combination settings. The purpose of this study was to investigate the therapeutic potential of the ILK small molecule inhibitor, QLT0267 (267), alone or in combination with chemotherapies commonly used to treat breast cancer patients.MethodsA single end-point metabolic assay was used as an initial screen for 267 interactions with selected chemotherapeutic agents. These in vitro assays were completed with seven breast cancer cell lines including several which over-expressed human epidermal growth factor receptor 2 (Her2). One agent, docetaxel (Dt), consistently produced synergistic interactions when combined with 267. Dt/267 interactions were further characterized by measuring therapeutic endpoints linked to phosphorylated protein kinase B (P-AKT) suppression, inhibition of vascular endothelial growth factor (VEGF) secretion and changes in cytoarchitecture. In vivo efficacy studies were completed in mice bearing orthotopic xenografts where tumor growth was assessed by bioluminescence and calliper methods.ResultsThe combination of 267 and Dt resulted in increased cytotoxic activity, as determined using an assay of metabolic activity. Combinations of cisplatin, doxorubicin, vinorelbine, paclitaxel, and trastuzumab produced antagonistic interactions. Further endpoint analysis in cell lines with low Her2 levels revealed that the 267/Dt combinations resulted in: a three-fold decrease in concentration (dose) of 267 required to achieve 50% inhibition of P-AKT; and a dramatic disruption of normal filamentous-actin cellular architecture. In contrast to Her2-positive cell lines, three-fold higher concentrations of 267 were required to achieve 50% inhibition of P-AKT when the drug was used in combination with Dt. In vivo studies focusing on low Her2-expressing breast cancer cells (LCC6) implanted orthotopically demonstrated that treatment with 267/Dt engendered improved therapeutic effects when compared with mice treated with either agent alone.ConclusionsThe findings indicate that the 267/Dt drug combination confers increased (synergistic) therapeutic efficacy towards human breast cancer cells that express low levels of Her2.