HER2-expressing Breast Cancer Research Articles

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.

A phase 1 study of a heterologous prime-boost vaccination involving a truncated HER2 sequence in patients with HER2-expressing breast cancer

A phase 1 clinical trial was conducted to assess the safety, tolerability, and preliminary efficacy of a heterologous prime-boost strategy involving plasmid DNA (pHM-GM-CSF, expressing truncated human epidermal growth factor receptor 2 (HER2) and granulocyte macrophage colony-stimulation factor (GM-CSF) as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in patients with stage III–IV metastatic breast cancer expressing HER2. Nine eligible subjects were divided into three cohorts based on the dosages (2, 4, and 8 mg/patient/visit) of pHM-GM-CSF used as the primer, which was intramuscularly injected three times at weeks 0, 2, and 4. It was followed by a single injection of Ad-HM (3 × 109 virus particles), used as a booster, at week 6. During the 6-month follow-up period, adverse events (AEs), pharmacokinetics and pharmacodynamics, and HER2-specific cellular and humoral immune responses were evaluated. Seven cases of minor grade 1 toxicities in four of nine subjects and no serious drug-related AEs were reported. HER2-specific cell-mediated or humoral immunity was produced in all (100%) or three subjects (33%), respectively. One subject showed a partial response, and seven subjects had stable diseases. However, there were no differences in clinical tumor response and HER2-specific immune responses among the cohorts. These results showed that intramuscular injections of pHM-GM-CSF and Ad-HM were well tolerated and safe.

Abstract 3783: Adipocytes inhibit trastuzumab-mediated ADCC via induction of GDF15

Abstract Background : Trastuzumab is a monoclonal antibody that has been approved in the treatment of HER2-expressing breast cancer. Despite its efficacy, resistance often occurs. Little is known about the role of the tumor microenvironment in resistance to trastuzumab. Recent studies have shown rising role of adipose tissue in promoting cancer progression and inducing resistance to chemotherapy. Given the abundance of adipose tissue in breast and its proximity to cancer cells, we investigated the impact of adipocytes on antibody-dependent cellular cytotoxicity (ADCC), one of the main mechanisms of action of trastuzumab. Methods: We set up a co-culture system in the presence of undifferentiated or differentiated human multi-potent adipose-derived stem (hMADS) cells, HER2-expressing human breast cancer cell lines (BT474, MDA-453, SKBR3, MDA-MB-361), and human natural killer NK-92 expressing the Fc receptor CD16. Trastuzumab-mediated cytotoxicity was assessed by calcein-release assay. Morphological changes on cancer cells and NK-92-CD16 cells were investigated by microscopy and flow cytometry. Results: We found that adipocytes, as well as pre-adipocytes, inhibited trastuzumab-mediated ADCC by approximately 30%. Notably, this inhibition was enhanced in hypoxic conditions with 1% O2 (up to 60% inhibition). The inhibition of ADCC by adipose cells was not due to a titration or degradation of the antibody, but via factors that are secreted by adipose cells into the conditioned media. Analysis of cell phenotype did not reveal any modification of NK cell receptors (CD16, NKG2D, NKG2A, NKp30, NKp44) or NK cell activation markers (CD25, CD69, CD107a), nor of HER2 levels in the presence of adipocytes. Pre-incubation of NK cells with the conditioned medium from adipocytes did not alter NK cytotoxicity. Conversely, pre-incubation of cancer cells with adipocyte-conditioned medium reduced sensitivity of cancer cells to ADCC. Using transcriptomic approach, we found GDF15 to be rapidly up-regulated in cancer cells exposed to adipocyte-conditioned medium. Down-regulation of GDF15 by siRNA reversed the adipocyte-induced inhibition of ADCC. Conclusion: Adipocytes inhibit antitumor activity of trastuzumab in HER2-expressing breast cancer cells via secretion of factors that reduce cancer cell sensitivity to ADCC. Our findings underline the importance of adipose tissue in the resistance to trastuzumab, and suggest that development of approaches targeting GDF15 may sensitize cancer cells to trastuzumab-based therapy. Citation Format: Minh Ngoc Duong, Aurore Cleret, Eva-Laure Matera, Kamel Chettab, Doriane Poloni, Sandrine Valsesia-Wittmann, Béatrice Clémenceau, Charles Dumontet. Adipocytes inhibit trastuzumab-mediated ADCC via induction of GDF15. [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 3783. doi:10.1158/1538-7445.AM2014-3783

Abstract CT414: Efficient induction of cellular and humoral immune responses by heterologous prime-boost therapeutic vaccination, involving plasmid DNA and adenoviral vector, in subjects with HER2-expressing breast cancer: Results from a phase Ib study

Abstract A phase I clinical trial was conducted to assess safety, tolerability and preliminary efficacy of a heterologous prime-boost strategy, involving a plasmid DNA (pHM-GM-CSF, expressing truncated HER2 and GM-CSF as a bicistronic message) and an adenoviral vector (Ad-HM, containing the same modified HER2 sequence only), in subjects with stage III-IV metastatic breast cancer expressing HER2 (1+∼3+ by immunohistochemistry). Nine eligible subjects were divided into three cohorts based on three different DNA doses (2, 4 and 8 mg) of pHM-GM-CSF, which were intramuscularly injected three times on weeks 0, 2, and 4 (i.e. 6, 12, and 24mg in total), followed by a single injection of Ad-HM (3x109 viral particle) on week 6. Relevant parameters were measured during the 6-month follow-up period. Seven minor grade 1 toxicities were found in four of nine patients, while no drug-related serious adverse event was reported. HER2-specific cell-mediated or humoral immunities were produced in all (100 %) or three patients (33 %), respectively. One patient showed a partial response, and seven subjects had stable diseases. No clear correlation was observed between DNA dosages and induction of immunogenicity and clinical tumor response. Our data showed that intramuscular injection of pHM-GM-CSF 24mg and Ad-HM 3x109 VP were well tolerated and safe. To our knowledge, this is the first clinical study demonstrating an efficient induction of HER2-specific cellular and humoral responses in patients with metastatic breast cancer expressing HER2. Citation Format: SUNGBAE KIM, KyungHae Jung, JinHee Ahn, Jeongeun Kim. Efficient induction of cellular and humoral immune responses by heterologous prime-boost therapeutic vaccination, involving plasmid DNA and adenoviral vector, in subjects with HER2-expressing breast cancer: Results from a phase Ib study. [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 CT414. doi:10.1158/1538-7445.AM2014-CT414

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

188Re-HYNIC-trastuzumab enhances the effect of apoptosis induced by trastuzumab in HER2-overexpressing breast cancer cells.

The development of radioimmunotherapy has provided an impressive alternative approach in improving trastuzumab therapy. However, the mechanisms of trastuzumab and radiation treatment combined to increase therapeutic efficacy are poorly understood. Here, we try to examine the efficacy of cytotoxicity and apoptosis induction for (188)Re-HYNIC-trastuzumab in cancer cell lines with various levels of Her2. Fluorescence flow cytometry was used to detect the alterations of apoptosis induction after (188)Re-HYNIC-trastuzumab treatment in two breast cancer cell lines with different levels of HER2 (BT-474 and MCF-7) and a colorectal carcinoma cell line (HT-29) for control. Our results indicated that (188)Re-HYNIC-trastuzumab led to cell death of breast cancer cells specifically in HER2 level-dependent and radioactivity dose-dependent fashions. In BT-474 cells, 370kBq/ml of (188)Re-HYNIC-trastuzumab enhanced the cytotoxicity to a level nearly 100-fold that of trastuzumab-alone treatment. The results also revealed that the mitochondria-dependent pathway attenuated irradiation-induced apoptosis in HER2-expressing breast cancer cells after (188)Re-HYNIC-trastuzumab treatment. In contrast, only after 48h of (188)Re-HYNIC-trastuzumab treatment, BT-474 cells exhibited typical apoptotic changes, including exposure of phospholipid phosphatidylserine on the cell surface, or fragmented DNA formation, in a radioactivity dose-dependent manner. Briefly, our study demonstrates that (188)Re-labeled HYNIC-trastuzumab not only enhances cell death in a radioactivity dose-dependent fashion, but may also prolong the effects of apoptosis involved with the mitochondria-dependent pathway in HER2-overexpressing breast cancer cells. It is possible that the (188)Re-HYNIC-trastuzumab treatment induced a second round of apoptosis to prolong the effects of cell kill in these cancer cells. These data revealed that (188)Re-HYNIC-trastuzumab has the potential for use as a therapeutic radiopharmaceutical agent in HER2-overexpressing breast cancer cell treatment.

Abstract P3-03-08: A comparison of PI3K inhibition by eribulin, other microtubule targeting agents and a DNA-damaging chemotherapeutic in triple negative and HER2 expressing breast cancer cell lines

Abstract Background. Eribulin is a microtubule-targeting agent with significant benefits in treating refractory metastatic breast cancer. Tumors from patients with Triple negative breast cancer (TNBC) have high levels of Akt expression and consistently show activation of the PI3K-mTOR pathway. Our objective was to compare Eribulin's ability to inhibit PI3K pathway activity and cell growth with two other microtubule targeting agents, Paclitaxel and Vinblastine, as well as a conventional DNA damaging chemotherapeutic Cisplatin. Methods. MDA468 and BT549 TNBC cell lines and SKBR3 HER2 overexpressing breast cancer cell lines were used for this study. Western blot analysis was used to evaluate the expression of phosphorylated Akt-Ser473 (pAkt) and S6K1 (pS6K1) at different time points from 2 to 24 hours of treatment with Eribulin, Paclitaxel, Vinblastine, or Cisplatin. MTT assays were used to assess growth inhibition after 72 hours of treatment. Results. Western blot analysis for MDA468 cells treated with Eribulin in varying concentrations confirm partial inhibition of pAkt expression as early as 4 hours at 100 pM concentration. Complete inhibition is reached at 50 nM. Partial inhibition of pS6K1 can be seen as early as 4 hours at 500 nM. Western analysis for MDA468 cells treated with Vinblastine in varying concentrations confirms inhibition of pAkt and pS6K1 beginning at 50 nM at 24 hours. Western analysis for MDA468 cells treated with Paclitaxel in varying concentrations showed increases in pAkt expression in a dose responsive fashion with significant increase in pAkt beginning at 5 nM concentration as well as increase in pS6K1 at 24 hours. Cisplatin markedly increases pAkt at 24 hours in a dose responsive fashion and decreases pS6K1 at 500 nM to 1000 nM concentration range in BT549. The IC50's for Eribulin ranged from 0.06 nM to 0.3 nM at 72 hours by MTT assay. The IC50's for Vinblastine ranged from 0.5 nM to 0.9 nM. Paclitaxel has reported IC50's in the 2 nM to 75 nM range in these cell lines, and Cisplatin has IC50's ranging from to ∼500 nM to ∼2000 nM at 72 hours in these cell lines. Conclusion. Our study shows that for microtubule targeting agents such as Eribulin and Vinblastine that block polymerization of tubulin into microtubules, both pAkt and pS6K1 expression is suppressed. Growth inhibition is also confirmed, and is seen at doses when pAkt and pS6K1 are not suppressed. Eribulin inhibits pAkt and pS6K1 at lower concentrations than Vinblastine. With Paclitaxel, a microtubule-targeting agent that enhances polymerization of tubulin to microtubules, and Cisplatin, a conventional DNA damaging chemotherapeutic we observe an increase in pAkt expression, with variable effects on pS6K1. Enhancement of Akt activity is a likely survival response by cancer cells to chemotherapy, yet in the case of microtubule polymerization blockade as seen with Eribulin and Vinblastine, Akt activity is suppressed, along with downstream mTOR activity. The potential role of microtubule polymerization blockade in inhibition of the PI3K pathway needs further study. It may be a factor in the novel finding of pAkt and pS6K1 inhibition by Eribulin. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-08.

Abstract P3-03-07: Combination of eribulin and PI3K inhibitors in triple negative and HER2 expressing breast cancer cell lines results in synergistic growth inhibition and enhanced inhibition of the PI3K pathway

Abstract Background. Patients with triple negative breast cancer (TNBC) have high levels of pAkt expression and activation of the PI3K-mTOR pathway. Eribulin is a microtubule-targeting agent with benefits in treating taxane and anthracycline refractory breast cancer. Our objective was to evaluate its efficacy in inhibiting PI3K pathway activity and cell growth both alone and in combination with PI3K/MTOR inhibitors BEZ 235 and BKM 120. Methods. TNBC cell lines MDA468, BT549, HS587T, MDA231, and HER2 expressing breast cancer cell line SKBR3 were used for this study. Both MDA468 and BT549 have PI3K- related mutations. The tetrazolium salt, 3-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assays were used to assess growth inhibition after 72 hour treatment with eribulin, BEZ 235 and BKM 120 both alone and in combination. Combination indices (CI) generated by Chou-Talalay plots were used to quantify synergy. Western blots were used to evaluate the expression of phosphorylated Akt (pAkt), S6K1 (pS6K1) and S6 (pS6) from 30 min to 24 hours of treatment at different doses. Results. Eribulin has IC50 ranging from 60 pM to 300 pM, BEZ 235 has IC50 ranging from 50 nM to 80 nM, and BKM has IC50 ranging from 500 nM or higher. Standard dilutions of eribulin in combination with BEZ 235 resulted in synergistic growth inhibition (CI<1) in both MDA468 and BT549 cells at all doses tested, but required higher concentrations (500 to 2000 nM) for BKM 120. Western blot analysis for all cell lines treated with eribulin showed pAkt inhibition by eribulin alone with doses as low as 1 nM and as early as 4 hours. PI3K inhibitor alone confirmed inhibition of pAkt, pS6K1 and pS6 at early time points with feedback increase in pAkt at 24h. While both BEZ 235 and BKM 120 treatment increased pAkt in a dose dependent fashion at 24 hours, combination treatment with eribulin showed a dose dependent decrease in pAkt. Conclusion. Our study shows significant synergistic growth inhibition with the combination of eribulin and PI3K inhibitors. This may be related to inhibition by eribulin of the feedback increase in pAkt seen with PI3K inhibitors alone at later time points. These findings point to a potential role for combination therapy of both eribulin and PI3K inhibitors in treating refractory metastatic disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-07.

Abstract C104: Preclinical sensitivity of low and high-HER2 expressing START-PDX breast models to T-DM1 and effects of HER3 expression on drug antitumor activity.

Abstract Background: Ado-trastuzumab emtansine (T-DM1), a recently approved antibody-drug conjugate (ADC), is comprised of the monoclonal antibody trastuzumab linked to the cytotoxic agent mertansine. Trastuzumab targets and inhibits growth of HER2/neu-receptor expressing tumors and mertansine enters and kills the cells by binding to tubulin. Trastuzumab alone is sufficient to effectively treat high (3+) HER2-expressing breast cancers; however, whether T-DM1 is effective in lower HER2-expressing cancers is unclear. To better understand how HER-2 expression correlates with activity of trastuzumab or T-DM1 we characterized HER2 expression in our START-PDX breast panel and screened trastuzumab and T-DM1 efficacy in each model. Study endpoints included tumor volume and time from treatment initiation. Reported results included tumor growth inhibition or delay and regression. Methods: Methods: START-PDX breast models were established in immune-deficient mice from primary or metastatic patient tissue and once established were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each model, DNA was extracted and subjected to exon sequencing of 207 known oncogenes; growth factor receptor and ligand densities were interrogated using immunohistochemistry and quantitative RNA in situ hybridization. Drug sensitivity studies were performed evaluating sensitivity of models to single agent trastuzumab and T-DM1, administered for at least four weeks. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: HER2 or HER3 and comparative heregulin expression occurred in 67% of models. EGFR expression was reported in 50% of models Efficacy studies revealed T-DM1 but not trastuzumab was active both in several high- and low-HER2 expressing models. Interestingly, models reporting higher T-DM1 sensitivity had no detectable HER3 expression. For example ST996 and ST910 showed similar HER2 and EGFR expression but ST996 (HER3 3+) was insensitive to T-DM1 whereas ST910 (HER3 0+) reported significant tumor growth inhibition compared with control. Conclusion: We have established a panel of breast PDX models and characterized mutation status, receptor and ligand density and drug sensitivity. We found T-DM1 active towards a number of breast PDX models even with low HER2 expression. HER3 co-expression may serve as a negative predictor for T-DM1 activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C104. Citation Format: Michael J. Wick, Jennifer Brown, Teresa L. Vaught, Justin Meade, Anthony W. Tolcher, Drew Rasco, Amita Patnaik, Amy Lang, Murali Beeram, Kyriakos P. Papadopoulos. Preclinical sensitivity of low and high-HER2 expressing START-PDX breast models to T-DM1 and effects of HER3 expression on drug antitumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C104.

Effect of eribulin on cell growth and PI3K pathway activity with and without RAD001 in triple-negative and HER2-expressing breast cancer.

173 Background: Patients with triple-negative breast cancer have high levels of Akt expression and activation of the PI3K-mTOR pathway. Eribulin is a microtubule-targeting agent with benefits in treating refractory triple negative disease. Our objective was to evaluate its efficacy in inhibiting PI3K pathway activity and cell growth both alone and in combination with the mTOR inhibitor RAD001. Methods: MDA468, BT549 and SKBR3 breast cancer cell lines were used for this study. MTT assays were used to assess growth inhibition after 72 hour treatment with eribulin alone and in combination with RAD001. Combination indices (CI) generated by Chou-Talalay plots were used to quantify synergy. Western blots were used to evaluate the expression of phosphorylated Akt-Ser473 (pAkt) and S6K1 after 24 hours of treatment with both agents. Results: Both MDA468 and SKBR3 cells treated with eribulin in varying concentrations showed inhibition of pAkt expression. Standard dilutions of eribulin in combination with log dilutions of RAD001 resulted in marked synergistic growth inhibition (CI<<1) in both MDA468 and BT549 cells. Western blot analysis for MDA468 cells treated with the combination erubulin and RAD001 showed a dose related suppression of pAkt along with complete inhibition of pS6K1, while RAD001 alone increased pAkt. Conclusions: Our study shows dose related inhibition of Akt activation as well as inhibition of cell growth in triple negative breast cancer and HER2 cell lines treated with eribulin alone or combined with RAD001. We also show reversal of the pAkt feedback response seen with mTOR inactivation, and a significant synergistic growth inhibition with combination treatment. These findings point to a potential role for eribulin and RAD001 in the treatment of refractory triple-negative breast cancer. [Table: see text]

Targeting cancer cell-specific RNA interference by siRNA delivery using a complex carrier of affibody-displaying bio-nanocapsules and liposomes

BackgroundSmall interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required.ResultsIn this study, we used a carrier that combined ZHER2-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA.ConclusionsThese findings show that, in the field of nucleic acid medicine, ZHER2-BNC/LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.

Bond oracle HER2 IHC assay for identifying low to intermediate HER2-expressing breast cancer.

e11509 Background: HER2 targeted therapies are effective treatment for breast tumors that overexpress the HER2 protein to a high level (IHC score of 3+) or have amplification of the HER2 gene (HER2/CEP17 >2). However, the majority of breast tumors exhibit low to intermediate expression of HER2 (IHC 1+ and 2+), for which there are limited treatment options. A new cancer vaccine candidate, E75 or nelipepimut-S, offers prevention of reoccurrence in these patients by generating a specific immune response to a HER2 peptide. To date, no companion diagnostic test is validated to differentiate HER2 at these levels of expression. The BOND Oracle HER2 IHC assay (Leica Biosystems) is being validated to reliably identify IHC HER2 1+ and 2+ cases by correlating to independent analytical measures of HER2 expression. Methods: Quantitative PCR was used to determine HER2 DNA copy number and mRNA expression in eight breast cancer cell lines spanning HER2 IHC scores of 0 to 3+ and 45 formalin fixed, paraffin embedded (FFPE) invasive breast tumor samples. HER2 receptor load was determined for the eight cell lines by flow cytometry. Data was correlated to HER2 expression levels as determined by manual interpretation using the BOND Oracle HER2 IHC assay. Results: Previous testing by Leica Biosystems (Table) demonstrates the relationship between HER2 receptor load, HER2 copy number, and HER2 IHC status on four assay control breast cancer cell lines. Preliminary results also show that HER2 mRNA expression correlates with IHC staining with the BOND Oracle HER2 IHC Assay in FFPE breast cancer tissue samples. Conclusions: Improving the discrimination of HER2 protein expression (IHC 1+, 2+) using the Bond Oracle HER2 IHC assay in breast cancer tumors will identify patients for new treatments in development such as E75. [Table: see text]

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

Abstract P5-16-05: The combination of trastuzumab and HER2-directed peptide vaccines is safe in HER2-expressing breast cancer patients

Abstract Background: Cardiotoxicity is the most concerning toxicity associated with the commonly used HER2-directed immunotherapy, trastuzumab (Tz). In general, a significant decline of left ventricular ejection fraction (EF) in asymptomatic patients is accepted as a decrease of at least 10% or an absolute value of below 50%. We are currently conducting multiple trials of HER2-directed peptide vaccines, often given either concurrently or in close temporal proximity to Tz. This has raised the issue that combining therapies could increase the risk of cardio-toxicity. Here, we present safety data from multiple trials in which the combination of these HER2-directed therapies was administered. Methods: Phase I and II trials were conducted in disease-free breast cancer patients after completion of chemotherapy when indicated. Patients (pts) who were determined by treating oncologists to qualify for Tz received this therapy per standard-of-care. These pts were enrolled onto HER2-directed peptide vaccine trials per each trial's inclusion criteria, with vaccinated (VG) pts receiving peptide + GM-CSF and control (CG) pts receiving GM-CSF alone. All patients were monitored for local and systemic toxicity to peptide inoculations (graded by the NCI's Common Terminology Criteria for Adverse Events). In addition, patients who received Tz had EF tracked through either echocardiogram or MUGA according to local standard of practice. Our database was queried for patients who received Tz and peptide, and had documented measures of EF pre-vaccine (Pre), during vaccine (D) and post-vaccine (Post). These pts were then placed in two groups based on the timing of Tz and vaccine therapy: concurrent(C) group and sequential(S) group. Mean EF at each time point was compared using a t-test. Results: Overall, the peptide vaccines were well tolerated (max local tox: 1% Grade 0, 65% Gr 1, 33% Gr 2, 1% Gr 3; max systemic tox: 19% Gr 0, 63% Gr 1, 18% Gr 2, 0% Gr 3). These toxicities are likely secondary to the GM-CSF immunoadjuvant as control pts receiving GM-CSF alone have similar toxicity profiles (max local tox: 0% Gr 0, 76% Gr 1, 23% Gr 2, 1% Gr 3; max systemic tox: 20% Gr 0, 65% Gr 1, 15% Gr 2, 0% Gr 3). There have been no serious or non-serious cardiac-related adverse events in our trials. In total, 71 pts treated with Tz and enrolled in a vaccine trial had EF measurements available for analysis; 54 in the S group (35 VG, 19 CG) and 17 in the C group (10 VG, 7 CG). Overall, neither VG nor CG pts had significant changes in EF (VG Pre: 65±0.8%, D: 63±0.9%, Post: 64±0.3%; CG Pre: 63±1.2%, D: 64±1.8%, Post: 63±1.1%). Separating VG pts into C and S pts, there were again no significant changes in EF, (C Pre: 65±1.0%, D: 63±1.0%, Post: 63±1.4%; S Pre: 65±1.7%, D: 61±1.3%, Post: 65±1.8%). Conclusions: HER2-directed peptide vaccines are safe and well tolerated. Initial data indicate that the combination of Tz and HER2-directed peptide vaccines, whether concurrent or sequential, does not cause significant cardiac toxicities as measured by changes in the EF during and after therapy. We will continue to track this safety data to confirm early findings as we pursue additional combination trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-16-05.

Granting specificity for breast cancer cells using a hepatitis B core particle with a HER2-targeted affibody molecule

Capsid-like particles consisting of a hepatitis B core (HBc) protein have been studied for their potential as carriers for drug delivery systems (DDS). The hollow HBc particle, which is formed by the self-assembly of core proteins comprising 183 aa residues, has the ability to bind to various cells non-specifically via the action of an arginine-rich domain. In this study, we developed an engineered HBc particle that specifically recognizes and targets human epidermal growth factor receptor-related 2 (HER2)-expressing breast cancer cells. To despoil the non-specific binding property of an HBc particle, we genetically deleted the C-terminal 150-183 aa part of the core protein that encodes the arginine-rich domain (ΔHBc). Then, we genetically inserted a Z(HER2) affibody molecule into the 78-81 aa position of the core protein to confer the ability of target-cell-specific recognition. The constructed Z(HER2)-displaying HBc (Z(HER2)-ΔHBc) particle specifically recognized HER2-expressing SKBR3 and MCF-7 breast cancer cells. In addition, the Z(HER2)-ΔHBc particle exhibited different binding amounts in accordance with the HER2 expression levels of cancer cells. These results show that the display of other types of affibody molecules on HBc particles would be an expandable strategy for targeting several kinds of cancer cells that would help enable a pinpoint DDS.

Response to trastuzumab by HER2 expressing breast tumour xenografts is accompanied by decreased Hexokinase II, glut1 and [18F]-FDG incorporation and changes in 31P-NMR-detectable phosphomonoesters

Trastuzumab, effective in about 15 % of women with breast cancer, downregulates signalling through the Akt/PI3K and MAPK pathways. These pathways modulate glucose and phospholipid metabolism which can be monitored by [(18)F]FDG-PET and (31)P-NMR spectroscopy, respectively. Here, the relationship between response of HER-2 overexpressing tumours and changes in [(18)F]-FDG incorporation and (31)P-NMR-detectable phosphomonoesters were examined. Xenografts derived from HER2-overexpressing MDA-MB-453 human breast tumour cells were grown in SCID mice, treated with trastuzumab for 15 days, then [(18)F]-FDG uptake determined and (31)P-NMR carried out on chemical extracts of the tumours. Western blots were carried out to determine protein expression of Hexokinase II and glut1. [(18)F]-FDG incorporation, Hexokinase II and glut1 protein expression and the concentration of phosphocholine and phosphoethanolamine in chemical extracts subjected to (31)P-NMR were significantly decreased in the xenografts in the trastuzumab-treated mice compared with xenografts from the PBS-injected group. Changes in FDG incorporation and (31)P-NMR spectral changes can accompany response of HER2-expressing breast cancer xenografts to trastuzumab. This is the first study to show parallel changes in [(18)F]FDG- and (31)P-NMR-detectable metabolites accompany response to targeted anticancer treatment.

Complex carriers of affibody-displaying bio-nanocapsules and composition-varied liposomes for HER2-expressing breast cancer cell-specific protein delivery

A bio-nanocapsule (BNC), a hollow particle composed of hepatitis B virus (HBV) surface antigen (HBsAg), and liposome (LP) conjugation method (BNC/LP) has been recently developed by . The BNC/LP complex carrier could successfully deliver fluorescence-labeled beads (100 nm) into liver cells. In this study, we report the promising delivery of proteins incorporated in the complex carriers, which were prepared by the BNC/LP conjugation method with specificity-altered BNC and composition-varied LPs. The specificity-altered BNC, ZHER2-BNC was developed by replacing the hepatocyte recognition site of BNC with ZHER2 binding to HER2 receptor specifically. Using green fluorescent protein (GFP; 27 kDa) and cellular cytotoxic protein (exotoxin A; 66 kDa) for the delivery, we herein present the impact of different charges attributed to the composition of the LP on specific cell targeting and cellular uptake of the complex carriers. In addition, we demonstrate that the mixture prepared by mixing LPs with helper lipid possessing endosomal escaping ability boosts the functional expression of the cellular cytotoxic exotoxin A activity specifically. Finally, we further show the blending ratio of the LP mixture and ZHER2-BNC is a critical factor in determining the highly-efficient expression of the cytotoxic activity of exotoxin A.

TOPOGRAPHY MEDIATED REGULATION OF HER-2 EXPRESSION IN BREAST CANCER CELLS

This article demonstrates that the surface micro-topography regulates the biology of breast cancer cells, including the expression of HER-2 gene and protein. The breast tumor microenvironment is made up of heterogenous mixture of pores, ridges and collagen fibers with well defined topographical features. Although, significant progress has been achieved towards elucidating the biochemical and molecular mechanisms that underlie breast cancer progression, quantitative characterization of the associated mechanical/topographical properties and their role in breast tumor progression remains largely unexplored. Therefore, the aim of this study is to investigate the effect of topography on the adhesion and biology of breast cancer cells in in vitro cultures. Polydimethylsiloxane (PDMS) surfaces containing different topographies were coated with polyelectrolyte multilayers (PEMs) to improve cell adhesion and maintain cell culture. HER-2 expressing breast cancer cells, BT-474 and SKBr3, were cultured on these PDMS surfaces. We demonstrate that micro-topography affects the cell adhesion and distribution depending on the topography on the PDMS surfaces. We also report for the first time that surface topography down-regulates the HER-2 gene transcription and protein expression in breast cancer cells when cultured on PDMS surfaces with micro-topographies compared to the tissue culture polystyrene surface (TCPS) control. Results from this study indicate that micro-topography modulates morphology of cells, their distribution and expression of HER-2 gene and protein in breast cancer cells. This study provides a novel platform for studying the role of native topography in the progression of breast cancer and has immense potential for understanding the breast cancer biology.

Fractionated Therapy of HER2-Expressing Breast and Ovarian Cancer Xenografts in Mice with Targeted Alpha Emitting 227Th-DOTA-p-benzyl-trastuzumab

BackgroundThe aim of this study was to investigate therapeutic efficacy and normal tissue toxicity of single dosage and fractionated targeted alpha therapy (TAT) in mice with HER2-expressing breast and ovarian cancer xenografts using the low dose rate radioimmunoconjugate 227Th-DOTA-p-benzyl-trastuzumab.Methodology/Principal FindingsNude mice carrying HER2-overexpressing subcutaneous SKOV-3 or SKBR-3 xenografts were treated with 1000 kBq/kg 227Th-trastuzumab as single injection or four injections of 250 kBq/kg with intervals of 4–5 days, 2 weeks, or 4 weeks. Control animals were treated with normal saline or unlabeled trastuzumab. In SKOV-3 xenografts tumor growth to 10-fold size was delayed (p<0.01) and survival with tumor diameter less than 16 mm was prolonged (p<0.05) in all TAT groups compared to the control groups. No statistically significant differences were seen among the treated groups. In SKBR-3 xenografts tumor growth to 10-fold size was delayed in the single injection and 4–5 days interval groups (p<0.001) and all except the 4 weeks interval TAT group showed improved survival to the control groups (p<0.05). Toxicity was assessed by blood cell counts, clinical chemistry measurements and body weight. Transient reduction in white blood cells was seen for the single injection and 4–5 days interval groups (p<0.05). No significant changes were seen in red blood cells, platelets or clinical chemistry parameters. Survival without life threatening loss of body weight was significantly prolonged in 4 weeks interval group compared to single injection group (p<0.05) for SKOV-3 animals and in 2 weeks interval group compared with the 4–5 days interval groups (p<0.05) for SKBR-3 animals.Conclusions/SignificanceThe same concentration of radioactivity split into several fractions may improve toxicity of 227Th-radioimmunotherapy while the therapeutic effect is maintained. Thus, it might be possible to increase the cumulative absorbed radiation dose to tumor with acceptable toxicity by fractionation of the dosage.