Different Levels Of HER2 Research Articles

Abstract PO2-14-05: COMPARISON OF HER2 mRNA PCR LEVELS AND IMMUNOHISTOCHEMISTRY (IHC) IN HORMONE RECEPTOR POSITIVE (HR+)/HER2 NEGATIVE (HER2-) EARLY BREAST CANCER

Abstract BACKGROUND IHC assays were developed to identify HER2-positive breast cancers deemed to be treated with trastuzumab-based therapies. However, these assays lack sensitivity to reliably identify tumors with low levels of HER2 protein on the cell surface. HR+/HER2-low may benefit from Trastuzumab Deruxtecan (TDxd) and therefore accurate quantification of HER2 expression levels becomes a critical issue. As a result, additional technologies are needed to achieve a quantitative and reproducible detection of HER2 in the low range expression compared with the standard IHC testing. METHODS We conducted a retrospective study of all incident stage I-II HR +/HER2- breast cancer patient whose genomic risk was assessed with the Oncotype 21 gene assay from 2009 to 2022 in Hospital Clínico San Carlos (Spain). Local HER2 expression (IHC w/wo cerbB2 FISH) was performed in all patients according to ASCO/CAP guidelines available during the different periods. HER2 mRNA expression levels by RT-PCR were obtained from OncotypeDx patient reports. The primary objective was to determine the distribution of HER2 mRNA levels across the different HER2 expression groups. Other objectives were to compare the clinicopathological characteristics, management and OncotypeDX Recurrence Score (RS) in the different HER2 expression groups. Qualitative variables are presented with their frequency distribution. Quantitative variables are summarized in their mean and standard deviation (SD). Welch’s F-test ANOVA was used to measure association between the two HER2 measuring methods. RESULTS 500 early breast cancer patients with HER2 IHC 0, 1+ and 2+ (FISH negative) and HER2 mRNA expression by RT-PCR tumors were included. Among them, 169 (34%) were IHC 0, 211(42%) were IHC 1+ and 120 (24%) were IHC 2+ (FISH negative). The distribution of clinicopathological characteristics was similar between different IHC HER2 expression groups, and the distribution of chemotherapy treatment was also similar among them (Table 1). All patients had undergone surgery, and most of them had received hormone therapy (487, 97%) and radiotherapy when indicated (375, 77%). The mean OncotypeDx® recurrence score across HER2 IHC 0, 1, 2 showed not significant difference between groups (16,85, 17,36 and 17,89 respectively). The mean OncotypeDx® HER2 gene score expression across the different levels of HER2 (0, 1+, 2+) determined by IHC were 9.07, 9.25 and 9.51, respectively. One-way ANOVA and Games-Howell post-hoc test revealed that differences between all the groups were significant. The ranges of HER2 gene score data for the 3 IHC groups were widely overlapped, with IQR of [8,5-9,5], [8,83-9,7] and [9,07-10] respectively. CONCLUSIONS Our data show statistically significant differences between HER2 mRNA expression levels and IHC groups. This suggests that RT-qPCR could complement the data obtained by IHC. However, the wide range and dispersion of mRNA expression within groups limits its clinical utility. Clinicopathological characteristics and management between different IHC HER2 expression groups. Citation Format: Julia Tejerina-Peces, Marta Amann-Arévalo, Pablo Ballestín, Beatriz González-Diez, Mateo Paz-Cabezas, Alejandro Pascual, Alicia de Luna, Vanesa García-Barberán, Alfonso López de Sá, Jose Angel García-Sáenz, Fernando Moreno. COMPARISON OF HER2 mRNA PCR LEVELS AND IMMUNOHISTOCHEMISTRY (IHC) IN HORMONE RECEPTOR POSITIVE (HR+)/HER2 NEGATIVE (HER2-) EARLY BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-05.

Abstract 2111: Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy

Abstract Background: T-DXd is an FDA-approved anti-HER2 Antibody Drug Conjugate (ADC), which has shown clinical efficacy in breast cancers with either overexpression or low-level expression of HER2. The benefit of T-DXd across the spectrum of HER2 expression has called into question the relevance of HER2 expression level as relevant parameter for T-DXd sensitivity. We sought to experimentally address the relevance of HER2 expression level upon the degree of T-DXd response. Methods: A series of isogenic models was generated that expressed different levels of surface HER2 using a doxycycline-inducible promoter. These were analyzed for impact of differential expression level upon T-DXd internalization and drug response. Human breast cancer samples were analyzed for HER2 expression levels and association with clinical response to T-DXd under an IRB approved protocol. Results: Induction of different levels of HER2 at specified doxycycline concentrations was verified in multiple models using immunoblotting and FACS. These different levels appeared to correspond to clinical breast cancer immunohistochemistry (IHC) scoring levels to 0, 1+, 2+, and 3+. The models were analyzed for T-DXd response and showed increasing levels of HER2 led to increase lysosomal uptake of T-DXd (p<0.0001). Moreover, elevation of HER2 levels led to lower concentrations of T-DXd needed to suppress tumor growth. Analyses of the effects of increasing levels of HER2 upon xenograft response to T-DXd are ongoing. Finally, retrospective analyses of a cohort of 50 breast cancer patients treated with T-DXd revealed that higher levels of HER2 corresponded to longer time on treatment (time to progression). Conclusion: We observe a consistent relationship between elevated expression of HER2 and breast cancer response to T-DXd. The findings highlight the relevance of HER2 expression level as a biomarker of T-DXd response and support the potential value of therapeutic strategies that can induce HER2 surface expression levels to enhance response. Citation Format: Wanyi Chen, Joshua Drago, Avantika Gupta, Bo Liu, Sarat Chandarlapaty. Impact of HER2 Expression on trastuzumab deruxtecan(T-DXd) efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2111.

Abstract 92: Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune cells and cancer cells. Immune effector T-cells are a promising cancer therapy due to their innate cytotoxicity. In particular, CAR T-cell therapy uses genetically engineered T-cells that express a chimeric antigen receptor that binds to a specific antigen on tumor cells. Assessing the efficacy and potency of CAR T-cell therapies in vitro and at high throughputs is vital for the preclinical development of these promising therapies. Here, we describe an in vitro potency assay that uses an automated imaging platform to quantify immune cell-mediated cytotoxicity of cancer target cells by immune effector cells. A GFP fluorescent and HER-2 expressing lung cancer cell line, A549, was seeded into a 96-well cell culture plate. Then, HER-2 CAR T-cells were added at 24 hours post target cell seeding at various E:T ratios (1:10, 1:5, 1:2, 1:1, and 5:1). Fluorescent images of the target cells were captured every 6 hours by the Omni Pro 12, an automated, high-throughput, live-cell analysis platform designed for continuous multi-well imaging inside an incubator. Automated image analysis was performed using the Confluency Module in the Axion Portal to view attachment and proliferation and quantify cytolysis of fluorescent A549 target cells. Percent cytolysis of the target cells was calculated by comparing the green fluorescent confluency of treated wells to no treatment control wells. A549-GFP cells showed a dose-dependent decrease in confluency over time that correlated with increasing amounts of CAR T-cells. At 92 hours post HER-2 CAR T-cell addition, the 5:1 ET group demonstrated approximately 91.8% +/- 2.0% cytolysis of A549-GFP cells, while the 1:10 group demonstrated approximately 69.2% +/- 3.0% cytolysis. Future work will evaluate differences in CAR T-cell potency when co-cultured with target cells that express different levels of HER-2. Overall, the Omni Pro 12 platform enables continuous quantification of the potency and kinetics of immune cell-mediated cytolysis. Citation Format: Denise Sullivan, Lieke Stemkens, Inge Thijssen, Svenja Meiler, Danielle Califano, Stacie Chvatal, Daniel Millard. Assessment of an in vitro potency assay for evaluation of immune cell-mediated cytotoxicity using the Omni Pro 12 automated imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 92.

Abstract 3133: A novel bispecific antibody-drug conjugate targeting HER2 and Trop-2 displays potent antitumor activity in a variety of tumor models with promising preclinical characteristics

Abstract Antibody-drug conjugates (ADCs) have exhibited considerable advancements in cancer therapy, though their efficacy is often confined to specific patient subsets or results in modest clinical responses due to low and heterogenous expression of drug targets in tumors. Targeting more than one tumor-associated antigens (TAAs) represents a new strategy to broaden treatment applicability and minimize on-target toxicity of ADC. Our analysis of co-expression of HER2 and Trop-2, two clinically validated ADC targets, in tumors of various histological malignancies manifests the great potential for an ADC simultaneously targeting these two antigens. We describe here a novel bispecific ADC (BIO-201) designed to co-target HER2 and Trop-2. This compound comprises a bispecific anti-HER2/Trop-2 antibody conjugated with a novel topoisomerase I inhibitor via a cleavable linker. BIO-201 demonstrates increased or comparable cell binding and internalization compared to parental antibodies. In in vitro cytotoxicity assays, BIO-201 effectively kills cancer cells with an IC50 in the sub nM range, irrespective of whether they co-express HER2 and Trop-2 or express only one antigen. Co-culture studies reveal that BIO-201 possesses a good bystander killing effect on drug target-negative tumor cells. In vivo studies further demonstrate the superior anti-tumor activity of BIO-201 in various tumor xenograft models, including those expressing different levels of HER2 and/or Trop-2, as well as models resistant to HER-2-targeting therapies. Moreover, BIO-201 exhibits favorable plasma stability, preclinical pharmacokinetics (PK), and toxicity profiles. In conclusion, these findings suggest that the anti-HER2/Trop-2 bispecific ADC, BIO-201, holds significant promise as an effective therapy for a diverse range of cancers, especially those expressing heterogenous levels of HER2 or Trop-2, and has the potential to benefit a broad patient population. Citation Format: Haihong (Helen) Zhong, Yan Xiong, Han Huang, Yuming Pan, Na Wang, Bin Sun, Shen Liu, Wei Yuan, Dingguo Liu, Jan Fang, Haifeng Bao. A novel bispecific antibody-drug conjugate targeting HER2 and Trop-2 displays potent antitumor activity in a variety of tumor models with promising preclinical characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3133.

Biomimetic nanochannels for molybdate transport: application to sensitive electrochemical immunoassay for HER2

A nanochannel-based electrochemical immunoassay was developed for the detection of human epidermal growth factor receptor 2 (HER2), with molybdate as the reporter to explore the interaction occurring into the nanochannels. The presence of target increased steric hindrance of the antibody-functionalized nanochannels, thereby hindering the transport of molybdate. And the reporter could be monitored by working electrode modified with hydroxyapatite nanoparticles, based on the formation of the redox-active molybdophosphate. As a result, peak current obtained at ca. - 0.28V in square wave voltammograms could be applied to quantitative determination of HER2. The electrochemical signal increased linearly with the logarithm of the concentration of HER2 in a broad dynamic range of 0.1pg∙mL-1 to 10ng∙mL-1 with a detection limit of 0.05pg∙mL-1. The reliability of this immunoassay was validated by a recovery range of 99.5% to 111.7% for the detection of three different levels of HER2 in human serum samples. Integrating with multiple bionanochannels, this immunoassay is expected to provide a versatile approach for quantitative detection of various biomarkers in related disease diagnosis and therapy.

Abstract 957: Zenocutuzumab: An antibody that can overcome HER3 mediated HRG signalling in tumor cells by docking on HER2

Abstract HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The HER2/HER3 complex is most active in the presence of heregulin (HRG) and upregulation of HRG is one of the key resistance mechanisms after TKI treatment. Zenocutuzumab is an HER2/HER3 bispecific antibody that specifically and potently blocks HRG-driven signaling of the heterodimeric complex. Zenocutuzumab has demonstrated clinical activity in patients with advanced cancer where the HRG-driven HER2/HER3 pathway is active [Ref].Zenocutuzumab was discovered through a large unbiased screen with HER2×HER3 Biclonics® (bAbs). Zenocutuzumab specifically blocks HRG-induced signaling of HER2:HER3 but not of HER2:HER4, EGFR:HER2 or EGFR:HER3 heterodimers. Zenocutuzumab demonstrates an in vitro potency superior to other high affinity anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of HRG, thereby overcoming one of the resistance mechanisms of current HER2 therapies. The potent activity of Zenocutuzumab is based in-part on its binding to domain I of HER2. Docking of Zenocutuzumab in this position increases the effective affinity of the anti-HER3 Fab arm. As a result, Zenocutuzumab displaces HRG binding to HER3, even at high ligand concentrations, keeping HER3 in an inactive state and blocking HRG-mediated HER2/HER3 signaling. In binding experiments on cells expressing different levels of HER2 and HER3, HER2:HER3 ratios point to the anti-HER2 Fab binding arm being the main driver of Zenocutuzumab avidity. In an in vivo orthotopic breast cancer xenograft model that used Micro-PET imaging, the tumor penetration of a variant of Zenocutuzumab is HER2-dependent despite the high subnanomolar affinity of the HER3 Fab arm for its receptor. Gamma-counter quantification of radioactivity confirmed that antibody levels in the tumors with the HER2xHER3 bAb were 2.5-fold higher than for the parental HER3 mAb. Zenocutuzumab reduced tumor growth in an in vivo orthotopic pancreatic cancer xenograft model that expresses HRG. The brain is one of the organs with high HRG concentrations to which many tumors metastasize. Mice bearing intracranial HER2-amplified breast cancer cells presented at the end of the treatment period 100% survival when treated with MCLA-128, in contrast to 38% survival when treated with HER2 antibody drug conjugate T-DM1.Conclusion: Zenocutuzumab uses a Dock & Block™ mechanism for potent and specific tumor targeting and simultaneous inhibition of HRG-driven proliferation of HER2-amplified tumors, even at supramaximal concentrations of HRG. Citation Format: Cecile Geuijen, David Maussang-Detaille, Tristan Gallenne, Linda Hendriks, Arjen Kramer, Jan Gerlach, Mark Throsby, John de Kruif. Zenocutuzumab: An antibody that can overcome HER3 mediated HRG signalling in tumor cells by docking on HER2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 957.

Novel trastuzumab-DM1 conjugate: Synthesis and bio-evaluation.

Antibody-drug conjugates arenow of considerable interest and are recommended for the treatment of cancers. Linkers are having a crucial role in potency and efficacy of these drugs. Herein, for the first time, we have used a water-soluble poly-ethylene glycol based linker (succinimidyl-[(N-maleimido propionamido)-diethyleneglycol] [SM(PEG)2]) for lysine amide coupling of DM1 drug to trastuzumab considering evaluation of the effect of using a hydrophilic linker on physicochemical and biological properties of the resulting conjugate in comparison to the conjugate containing succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, which has a relative hydrophobic nature. The physicochemical properties of synthesized conjugates were investigated in terms of drug to antibody ratio, size variants and free drug quantities. In vitro biological activity of trastuzumab-DM1 conjugates was assessed on breast cancer cell lines expressing different levels of HER2 using binding affinity, antiproliferative, apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Synthesized conjugate containing hydrophilic linker, showed higher drug to antibody ratio, no aggregated form and higher cellular toxicity in comparison to SMCC bearing conjugate. Binding affinity and ADCC potential of conjugates was not affected upon the usage of hydrophilic linker. In conclusion, application of SM(PEG)2 for coupling of DM1 to trastuzumab enhance desirable characteristics of the resulting conjugate.

MI130004, a Novel Antibody-Drug Conjugate Combining Trastuzumab with a Molecule of Marine Origin, Shows Outstanding In Vivo Activity against HER2-Expressing Tumors.

In the search for novel payloads to design new antibody-drug conjugates (ADC), marine compounds represent an interesting opportunity given their unique chemical features. PM050489 is a marine compound that binds β-tubulin at a new site and disrupts the microtubule network, hence leading to mitotic aberrations and cell death. PM050489 has been conjugated to trastuzumab via Cys residues through a noncleavable linker, and the resulting ADC, named MI130004, has been studied. Analysis of MI130004 delivered data consistent with the presence of two molecules of PM050489 per antibody molecule, likely bound to both sides of the intermolecular disulfide bond connecting the antibody light and heavy chains. The antitumor activity of MI130004 was analyzed in vitro and in vivo in different cell lines of diverse tumor origin (breast, ovary, and gastric cancer) expressing different levels of HER2. MI130004 showed very high in vitro potency and good selectivity for tumor cells that overexpressed HER2. At the cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the microtubule network, which ultimately led to mitotic failure, mirroring the effect of its payload. Treatment with MI130004 in mice carrying histologically diverse tumors expressing HER2 induced a long-lasting antitumor effect with statistically significant inhibition of tumor growth coupled with increases in median survival time compared with vehicle or trastuzumab. These results strongly suggest that MI130004 is endowed with remarkable anticancer activity and confirm the extraordinary potential of marine compounds for the design of new ADCs. Mol Cancer Ther; 17(4); 786-94. ©2018 AACR.

Abstract B41: Transcriptome and proteome analyses of MCF7 cells with different expression profile of SPARC (secreted protein acidic in rich in cysteine) in the presence and absence of docetaxel

Abstract The SPARC (secreted protein, acidic, cysteine-rich) gene encodes a 32kDa protein that belongs to a family of matricellular proteins, which interact with cell-surface receptors, growth factors, and the ECM (extracellular matrix) components. SPARC plays a role in tissue remodeling, cell migration, angiogenesis, embryonic development, tumorigenesis, and chemosensitivity. Docetaxel is an effective chemotherapeutic drug for the treatment of advanced breast cancer. However, a considerable proportion of breast cancer patients do not respond positively to docetaxel. In a previous study, we identified SPARC as differentially expressed in mammary epithelial cells expressing different levels of HER-2. In the present study, we evaluate the effects of SPARC overexpression on the sensitivity of breast cancer cells to docetaxel. MCF7 cells were transfected with the expression vector pCMV6-SPARC or pCMV6-Neo. Real-time PCR, Western blot, and immunofluorescence were used to characterize the clones overexpressing SPARC. Using cDNA microarray and label-free approach for quantitative proteomic analysis, we evaluated the transcriptome and proteome changes in the comparison of the MCF7 cell control and overexpressing SPARC before and after docetaxel treatment. Several differentially expressed genes were identified as potentially involved in SPARC-mediated chemosensitivity to docetaxel. Among the molecular interaction networks from the differentially expressed proteins, extracellular matrix and cytoskeleton remodeling were the top pathway map observed in the comparison between MCF7 cells overexpressing SPARC and the control cells before docetaxel treatment. GTP metabolism pathway was the top pathway map observed in the comparison between MCF7 cells overexpressing SPARC and the control cells after docetaxel treatment. Transcriptome and proteome integrated data resulted in forty commonly up- and downregulated genes, with the WNT pathway represented among them. Our findings suggest that SPARC expression can influence docetaxel sensitivity in MCF7 cells by modulating genes involved in diverse biologic process that might be related to drug sensitivity. (Supported by Fapesp and CNPq.) Citation Format: Ana Carolina Pavanelli, Luciana Pizzatti Barboza, Eliana Saul Furquim Werneck Abdelhay, Maria Aparecida Nagai. Transcriptome and proteome analyses of MCF7 cells with different expression profile of SPARC (secreted protein acidic in rich in cysteine) in the presence and absence of docetaxel [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B41.

The correlation between HER-2 expression and the CEUS and ARFI characteristics of breast cancer.

ObjectiveThe purpose of the present study was to explore the correlation between the contrast-enhanced ultrasound (CEUS) and acoustic radiation force impulse (ARFI) characteristics of breast cancers and the expression of human epidermal growth factor receptor 2 (HER-2).MethodsHER-2 expression levels in the tumor masses of 167 clearly diagnosed cases of breast cancer were measured and analyzed. The enhancement features and time intensity curve (TIC) of CEUS and virtual touch tissue imaging (VTI) and virtual touch tissue quantification (VTQ) technology of ARFI were employed to analyze the relationship between HER-2 expression and the CEUS and ARFI characteristics of breast cancer.Results(1) Statistically significant differences in the distribution of the contrast agent, perforator blood flow, the overranging phenomenon and perfusion defects between the study groups with different HER-2 expression levels (P < 0.05) were observed on CEUS. In addition, statistically significant differences in the TIC peak time (PT), slope of the ascending branch (K) and area under the curve (AUC) were found in the groups expressing different levels of HER-2 (P < 0.05). In contrast, the degree of contrast agent enhancement and TIC peak intensity (PI) were found to be independent of the expression status of HER-2, as they were not statistically significant (P > 0.05). (2) Statistically significant differences in the VTQ results between the groups with different HER-2 expression levels were found (P < 0.05). However, no statistically significant differences in VTI image characteristics were detected between the groups expressing different levels of HER-2 (P > 0.05).ConclusionA correlation was found between the CEUS and ARFI characteristics of breast cancer and HER-2 expression levels. This correlation was principally reflected in perfusion defects, perforator blood flow, PI, PT, K and VTQ.

Abstract 2652: In vitro and in vivo antitumor activity of SYD985, a novel HER2-targeting ADC: a comparison with T-DM1

Abstract SYD985 is a HER2-targeting ADC based on trastuzumab and Synthon's proprietary cleavable linker-duocarmycin payload. The preclinical profile, including mechanism of action studies, PK, efficacy, and safety studies are described in the accompanying abstract. We have further compared SYD985 to T-DM1 (Kadcyla) and have focused on the in vitro and in vivo efficacies of both of these HER2-targeting ADCs. SYD985 and T-DM1 were compared head-to-head in a panel of 10 cell lines expressing different levels of HER2, including levels characterized as HER2 0, 1+, 2+, or 3+. SYD985 was 3 to 5 times more potent in one HER2 2+ and two HER2 3+ cell lines, while similar potencies were obtained on two other HER2 3+ cell lines. In cell lines with lower HER2 membrane expression (HER2 1+), SYD985 was a factor 7 to 300-fold more potent. These data show that the difference in potencies between SYD985 and T-DM1 becomes more pronounced at lower HER2 expression levels. Subsequently, we next studied how the in vitro data correlate with in vivo anti-tumor activities. In a xenograft with a breast BT-474 cell line (IHC HER2 3+) both SYD985 and T-DM1 dose-dependently reduced tumor growth after single dose (SD) i.v. administration. SYD985 was approximately 5-fold more potent than T-DM1 (tumor stasis at 1 mg/kg vs 5 mg/kg respectively). Subsequently, both ADCs were evaluated (SD i.v.) in a series of patient-derived xenografts (PDX) using HER2 FISH negative tumors from breast cancer patients. We have tested both ADCs in two models classified as Triple Negative Breast Cancer (TNBC; ER negative/PR negative/HER2 negative) with IHC HER2 1+ staining, two TNBC models with IHC HER2 2+ staining, and two Hormone Positive tumor models with IHC HER2 2+ staining. One model (TNBC IHC HER2 2+) did not respond to either SYD985 or T-DM1. In the other five models, SYD985 was more active than T-DM1. Corroborating with the in vitro data, the improved anti-tumor activity of SYD985 versus T-DM1 is most prominent in the PDX models with lowest HER2 expression. In the two TNBC IHC HER2 1+ models, SYD985 induced CR at 1-3 mg/kg, whereas T-DM1 was inactive at all dosages tested, up to 30 mg/kg. We conclude that i) SYD985 is more potent than T-DM1, both in vitro and in vivo in all responsive models; ii) In these studies, the potency advantage of SYD985 over T-DM1 is enhanced even further in the tumor cell lines and PDX models with low HER2 expression (i.e. IHC HER2 1+); iii) The preclinical profile of SYD985 enables extending the target population of BC patients that may respond to this treatment modality to include IHC HER2 1+ and 2+ / FISH negative patients. Early phase clinical trials with SYD985 in patients with this tumor profile, besides refractory BC patients who are IHC HER2 3+ or FISH positive, appear justified and warranted. Citation Format: Willem H.A. Dokter, Miranda van der Lee, Patrick Groothuis, Tanja van Achterberg, Eline Loosveld, Daniëlle Jacobs, Monique van der Vleuten, Patrick H. Beusker, Leon Hooftman, Peter Goedings, Gijs Verheijden, Marco Timmers. In vitro and in vivo antitumor activity of SYD985, a novel HER2-targeting ADC: a comparison with T-DM1. [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 2652. doi:10.1158/1538-7445.AM2014-2652

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.

Affibody-DyLight Conjugates for In Vivo Assessment of HER2 Expression by Near-Infrared Optical Imaging

PurposeAmplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression in vivo would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for in vivo evaluation of the receptor status by near-infrared (NIR) optical imaging.Experimental DesignAffibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested in vitro. For in vivo validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by ex vivo analysis.ResultsAffibody-DyLight conjugates showed high affinity to HER2 (KD = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue.ConclusionsThe results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.

P3-03-04: Targeted Modulation of HER Receptor Signaling in Breast Cancer Using miR-21.

Abstract Introduction: MicroRNAs (miRNAs) are multifunctional nucleic acids that regulate the stability and translational efficiency of targeted messenger RNAs. Endogenously altered miRNA levels have been linked in breast cancer to acquired chemoresistance and changed radiosensitivity. Thus the modification of miRNA expression represents an attractive therapeutic strategy. We now show that the targeted inhibition of miR-21 can modify expression of the HER2 receptor in breast cancer. Materials and Methods: Using TaqMan-based technology the expression of five selected miRNAs (miR-7b, miR-10a, miR-17-5p, miR-125a, and miR-21) was investigated in formalin-fixed, paraffin-embedded breast carcinoma tissues expressing different levels of HER2 (n=60). Possible regulation of HER2 was explored in cell lines using lentivirus to stably downregulate or upregulate HER2 and miR-21 expressions. The effects were analyzed using qRT-PCR, Western blots, proliferation and migration assays. Results: In tumor tissues we identified overexpression of miR-10a and miR-21, and underexpression of miR-17-5p and miR-125a in most of the tumor tissue samples. Significant associations (p≤0.05) were found between HER receptor expression and the expression of miR-17-5p and miR-125a, and miR-21 showed correlation with expression of ER and PR. Furthermore, analysis of miR-21 downregulation in breast cancer cell lines showed inhibition of cellular proliferation and HER2 protein synthesis. Additionally, downregulation of HER2 expression decreased miR-21 expression and showed moderate change in cellular proliferation activity. Conclusions: Based on these results we conclude that there is tight interplay between HER2 and miR-21 expression in breast cancer cells. Because miRNAs modulate gene expression we suggest that modulation of miR-21 has the potential to impact tumor therapy by interference with specific signaling pathways. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-04.

Quantitative Analysis of HER2 Receptor Expression In Vivo by Near-Infrared Optical Imaging

Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancers is associated with poor prognosis and resistance to therapy. Current techniques for estimating this important characteristic use ex vivo assays that require tissue biopsies. We suggest a novel noninvasive method to characterize HER2 expression in vivo, using optical imaging, based on HER2-specific probes (albumin-binding domain-fused-(ZHER2:342)2-Cys Affibody molecules [Affibody AB, Solna, Sweden], labeled with Alexa Fluor 750 [Molecular Probes, Invitrogen, Carlsbad, CA]) that could be used concomitantly with HER2-targeted therapy. Subcutaneous tumor xenografts, expressing different levels of HER2, were imaged with a near-infrared fluorescence small-animal imaging system at several times postinjection of the probe. The compartmental ligand-receptor model was used to calculate HER2 expression from imaging data. Correlation between HER2 amplification/overexpression in tumor cells and parameters, directly estimated from the sequence of optical images, was observed (eg, experimental data for BT474 xenografts indicate that initial slope, characterizing the temporal dependence of the fluorescence intensity detected in the tumor, linearly depends on the HER2 expression, as measured ex vivo by an enzyme-linked immunosorbent assay for the same tumor). The results obtained from tumors expressing different levels of HER2 substantiate a similar relationship between the initial slope and HER2 amplification/overexpression. This work shows that optical imaging, combined with mathematical modeling, allows noninvasive monitoring of HER2 expression in vivo.

Quantitative Analysis of HER2 Receptors Expression In Vivo by Near-Infrared Optical Imaging.

Abstract Purpose: HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. However, up to now, quantitative estimates of this important characteristic have been limited to ex vivo ELISA essays of tissue biopsies and/or PET-based analysis. We aim at developing a novel approach in optical imaging, involving specific probes that do not interfere with the binding of the therapeutic agents, thus, excluding competition between therapy and imaging. Affibody-based molecular probes seem to be ideal for in vivo analysis of HER2 receptors using near-infrared optical imaging. To achieve quantification of HER2 receptors and overcome experimental uncertainties, we analyze post-injection temporal variations of the fluorescence intensity in the tumor area.Experimental design: Quantitative in vivo near-infrared imaging uses HER2-specific albumin-binding domain-fused-(ZHER2:342)2 Affibody molecules, labeled with AlexaFluor750 dye, as targeting agents. Subcutaneous tumor xenografts, expressing different levels of HER2 (BT474, MDA-MB361, MCF7, U251), are imaged at different times post-injection. Mathematical modeling allowed assessment of correlation between measured characteristics and HER2 expression levels from ex-vivo assays of the same tumors.Results: Affibody-Alexa Fluor conjugates may be used as a specific near-infrared probe for the non-invasive imaging of HER2-positive tumors, while analysis of the imaging parameters, directly estimated from the sequence of optical images, provides quantitative insight on the HER2 overexpression, as suggested by observed correlation between tumor cell amplification/overexpression of HER2, and these parameters.Conclusions: Method might potentially be employed in clinic for noninvasive monitoring of the HER2 expression variation in response to therapeutic intervention. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5002.

HER/ErbB receptor interactions and signaling patterns in human mammary epithelial cells

BackgroundKnowledge about signaling pathways is typically compiled based on data gathered using different cell lines. This approach implicitly assumes that the cell line dependence is not important. However, different cell lines do not always respond to a particular stimulus in the same way, and lack of coherent data collected from closely related cellular systems can be detrimental to the efforts to understand the regulation of biological processes. To address this issue, we created a clone library of human mammary epithelial (HME) cells that expresses different levels of HER2 and HER3 receptors in combination with endogenous EGFR/HER1. Using our clone library, we have quantified the receptor activation patterns and systematically tested the validity of the existing hypotheses about the interaction patterns between HER1-3 receptors.ResultsOur study identified HER2 as the dominant dimerization partner for both EGFR and HER3. Contrary to earlier suggestions, we find that lateral interactions with HER2 do not lead to strong transactivation between EGFR and HER3, i.e., EGFR activation and HER3 activation are only weakly linked in HME cells. We also find that observed weak transactivation is uni-directional where stimulation of EGFR leads to HER3 activation whereas HER3 stimulation does not activate the EGFR. Repeating our experiments at lower cell confluency established that cell confluency is not a major factor in the observed interaction patterns. We have also quantified the dependence of the kinetics of Erk and Akt activation on different HER receptors. We found that HER3 signaling makes the strongest contribution to Akt activation and that, stimulation of either EGFR or HER3 leads to significant Erk activation.ConclusionOur study shows that clone cell libraries can be a powerful resource in systems biology research by making it possible to differentiate between various hypotheses in a consistent cellular background. Using our constructed clone library we profiled the cell signaling patterns to establish the role of HER2 in the crosstalk between EGFR and HER3 receptors in HME cells. Our results for HME cells show that the weak linkage between EGFR and HER3 pathways can lead to distinct downstream cellular signaling patterns in response to the ligands of these two receptors.

PP60 Correlation of absolute lymphocyte count with clinical benefit and overall survival: results of compassionate-use trial of ipilimumab in advanced melanoma at Memorial Sloan-Kettering Cancer Center

Results: Results across the cohort indicate Her2 MRM results correlate precisely with IHC and FISH demonstrating Her2 quantitation in Liquid Tissue lysates obtained by microdissecting multiple cancerous regions across the same tissue section and processing them together to produce a single lysate. Results of this assay across different regions within the same tumor tissue indicate different levels of Her2 are detected in different cancerous and stromal regions within the same tissue section that correlate precisely with IHC analysis in these various regions. Conclusion: These results indicate the need for a sampling strategy for measuring quantitative levels of specific proteins directly in patient tissue that requires tissue microdissection with implications for the molecular analysis of tumor tissue for advanced diagnostic applications.

Quantifying the effects of co-expressing EGFR and HER2 on HER activation and trafficking

The human epidermal growth factor receptor (HER) system is an intricately regulated system that plays critical roles in development and tumorigenesis. Here, we apply integrated experimentation and modeling to analyze HER receptor activation in a panel of cell lines expressing endogenous levels of EGFR/HER1 and different levels of HER2. A mathematical model that includes the fundamental processes involved in receptor activation and trafficking was used to fit the experimental data, and values of the independent parameters for active receptor dimer formation affinities, trafficking rates and relative phosphorylation levels were estimated. Obtained parameter values quantitatively support the existing ideas on the effect of HER2 on EGFR dynamics, and enable us to predict the abundances of various phosphorylated receptor dimers in the cell lines.

Redirected Activity of Human Antitumor Chimeric Immune Receptors is Governed by Antigen and Receptor Expression Levels and Affinity of Interaction

Novel Ab-based immunotherapeutic strategies have exploited T-cell receptor-like chimeric immune receptors (CIR) expressed on the surface of transduced human peripheral blood mononuclear cell (PBMC) to redirect potent non-major histocompatibility complex-dependent cytotoxicity to tumor cells expressing a tumor-associated antigens. We transduced human PBMC with 2 fully human CIRs that trigger through the zeta-chain of CD3 and contain either one of two human scFv specific for the same epitope on the extracellular domain of HER2 but with distinctly different affinities (KD 1616 and 1 nM) for this antigen. Potent direct CIR-mediated killing and in vitro tumor growth inhibition mediated by transduced PBMC were observed against targets expressing different levels of HER2. High-affinity CIR showed stronger ability to bind Ag and retain binding than low-affinity CIR. When lytic potential of the 2 CIRs was evaluated, their efficiency was comparable under conditions of high CIR and Ag expression, whereas low-affinity CIR was more efficient than high-affinity CIR in conditions of limiting Ag and CIR expression levels. When tumor growth inhibition was evaluated, Ag and CIR levels, rather than CIR affinity appeared relevant. Ag-driven CIR activation resulted in the production of soluble factors mediating efficient bystander effect. By carefully defining CIR surface expression and increasing affinity for a specific target antigen, it may be possible to selectively exclude CIR-mediated activity against targets expressing low levels of antigen, as normal cells. On the contrary, low antigen-expressing tumor variants could be eliminated by decreasing CIR affinity. Tuning CIR expression and affinity might help in discriminating different biologic contexts.