HER2 Density Research Articles

Multiparametric profiling of HER2-enriched extracellular vesicles in breast cancer using Single Extracellular VEsicle Nanoscopy

BackgroundPatients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy – such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids.ResultsUsing Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls.ConclusionsSEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.Graphical abstract

HER2-low and tumor infiltrating lymphocytes in triple-negative breast cancer: Are they connected?

Most patients with triple-negative breast cancer (TNBC) are not candidates for targeted therapy, leaving chemotherapy as the primary treatment option. Recently, immunotherapy has demonstrated promising results in TNBC, due to its immunogenicity. In addition, a novel antibody–drug conjugate, namely, trastuzumab-deruxtecan, has shown effectiveness in TNBC patients with low-HER2 expression (HER2-low). These novel treatment options raise the question about the potential association between the density of stromal tumor-infiltrating lymphocytes (sTILs) and the level of HER2 expression. We aimed to evaluate the association between the level of HER2 expression (HER2-low versus HER2-0) and density of sTILs in TNBC patients, and how they impact the response to neoadjuvant chemotherapy (NAC). This was a retrospective multicenter study including all TNBC patients diagnosed between 2018 and 2022. Central pathology review included sTILs percentages and level of HER2 expression. Tumors were reclassified as either HER2-0 (HER2 IHC 0) or HER2-low (IHC 1 + or 2 + with negative reflex test). Various clinicopathologic characteristics, including sTILs density, and response to NAC were compared between HER2-0 and HER2-low cases. In total, 753 TNBC patients were included in this study, of which 292 patients received NAC. Interobserver agreement between the original pathology report and central review was moderate (77% had the same IHC status after reclassification in either HER2-0 or HER2-low; k = 0.45). HER2-low TNBC represented about one third (36%) of the tumors. No significant difference in sTILs density or complete pathologic response rate was found between HER2-0 and HER2-low cases (p = 0.476 and p = 0.339, respectively). The density of sTILs (≥ 10% sTILs vs. < 10%) was independently associated with achieving a pCR (p = 0.011). In conclusion, no significant association was found between HER2-low status and density of sTILs nor response to NAC. Nonetheless, sTILs could be an independent biomarker for predicting NAC response in TNBC patients.

Lentiviral vector-based xenograft tumors as candidate reference materials for detection of HER2-low breast cancer.

The human epidermal growth factor receptor 2 (HER2) is an important biomarker that plays a pivotal role in therapeutic decision-making for patients with breast cancer (BC). Patients with HER2-low BC can benefit from new HER2 targeted therapy. For ensuring the accurate and reproducible detection of HER2-low cancer, reliable reference materials are required for monitoring the sensitivity and specificity of detection assays. Herein, a lentiviral vector was used to transduce the HER2 gene into MDA-MB-231 cells that exhibited low HER2 density, and the cells were characterized by droplet digital PCR to accurately determine the copy number variation. Then, the formalin-fixed paraffin-embedded (FFPE) samples from xenografts were prepared and evaluated for suitability as candidate reference materials by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The FFPE reference materials were selected on the basis of IHC score of 2+ and negative FISH result to meet the requirement for HER2-low BC detection. Furthermore, the FFPE reference materials exhibited typical histological structures that resembled the clinical BC specimens. These novel FFPE reference materials displayed the high stability and homogeneity, and they were produced in high quantity. In summary, we generated high-quality reference materials for internal quality control and proficiency testing in HER2-low detection.

Abstract 896: An antifungal, Itraconazole, induces cell death by targeting HER2 signaling and stem-like properties in trastuzumab-resistant HER2-positive breast cancer

Abstract Trastuzumab resistance in HER2-positive breast cancer leads to a poorer prognosis and shorter overall survival. Identifying existing drugs for another purpose of use may be considered as a part of new approaches to overcome resistance in cancer biology. Itraconazole is a FDA-approved drug, which is one of anti-fungal drugs. This drug has been reported to exploit as a cancer therapeutic agent in several cancer types. We sought to investigate the anti-cancer effects of itraconazole on cell proliferation, apoptosis, autophagy and breast cancer stem cell-like properties in terms of challenging trastuzumab resistance in HER2-positive breast cancer. The effect of itraconazole on trastuzumab-resistant cell line, JIMT-1, in vitro was examined in aspects of cell viability, apoptosis, autophagy, and impact on cancer stem cells. As in vivo experimental model, trastuzumab-resistant JIMT-1 cells were implanted to generate xenografts to study anti-tumor efficacy of itraconazole. Treatment of itraconazole significantly suppressed the growth of JIMT-1 cells with marked induction of apoptosis. Itraconazole downregulated p185HER2 and truncated-p95HER2, and their phosphorylation levels in JIMT-1 cells. In addition, the outcome of the decrease in the levels of Beclin-1 and p62 and the increase of LC3 I/II after the exposure to itraconazole supported that itraconazole induced autophagy as well. Importantly, itraconazole not only killed proliferating tumor cells but also effectively eradicated cancer stem-like populations. To elucidate eradication of cancer stem-like population by itraconazole, ALDH1 activity assay and FACS analysis of CD44+/CD24- stem-like phenotype in JIMT-1 cells were carried out. As a result, stem-cell like populations were impaired as evidenced by a significant decrease in ALDH1 activity and CD44+/CD24- stem-like populations. Through in vivo mouse model, the physiological relevance of in vitro observations was confirmed. Evident inhibition of tumor burden and growth in trastuzumab-resistant xenografts was shown in consequence of itraconazole administration, accompanying by substantial downregulation of HER2, ALDH1 and microvessel density as well as dramatic decrease of p62 in vivo. No injury to liver or kidneys by itraconazol was found based on no statistically significant difference in serum levels of ALT, AST and BUN between vehicle- and itraconazole-administrated groups. We have demonstrated that itraconazole, which is FDA-approved drug as an anti-fungal drug, exerts anti-tumor activity in trastuzumab-resistant HER2-positive breast cancer by targeting cancer stem-like properties, suppression of the HER2 signaling as well as induction of autophagy. These findings support the notion that itraconazole might be a new strategic approach as a treatment for trastuzumab-resistant HER2-positive breast cancers. Citation Format: Jung Min Park, Soeun Park, Minsu Park, Seongjae Kim, Juyeon Seo, Dongmi Ko, Kee Dal Nam, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. An antifungal, Itraconazole, induces cell death by targeting HER2 signaling and stem-like properties in trastuzumab-resistant HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 896.

Molecular Assessment of HER2 to Identify Signatures Associated with Therapy Response in HER2-Positive Breast Cancer.

Simple SummaryThe HER2 status of breast cancers is accurately determined by measuring HER2 protein overexpression and gene amplification. However, these clinical diagnostic tests cannot predict the response to therapy. Single molecule imaging approaches can quantify molecular features of HER2, such as receptor nano-organization, with exquisite spatial resolution and sensitivity. The aim of our study was to assess how the molecular features of HER2 varied with the therapy response. According to our results in cultured cell lines and six patient specimens, the therapy response was associated with high detected HER2 densities and clustering. This advanced imaging approach can thus provide key data to complement the current diagnostic standards.Trastuzumab, the prototype HER2-directed therapy, has markedly improved survival for women with HER2-positive breast cancers. However, only 40–60% of women with HER2-positive breast cancers achieve a complete pathological response to chemotherapy combined with HER2-directed therapy. The current diagnostic assays have poor positive-predictive accuracy in identifying therapy-responsive breast cancers. Here, we deployed quantitative single molecule localization microscopy to assess the molecular features of HER2 in a therapy-responsive setting. Using fluorescently labeled trastuzumab as a probe, we first compared the molecular features of HER2 in trastuzumab-sensitive (BT-474 and SK-BR-3) and trastuzumab-resistant (BT-474R and JIMT-1) cultured cell lines. Trastuzumab-sensitive cells had significantly higher detected HER2 densities and clustering. We then evaluated HER2 in pre-treatment core biopsies from women with breast cancer undergoing neoadjuvant therapy. A complete pathological response was associated with a high detected HER2 density and significant HER2 clustering. These results established the nano-organization of HER2 as a potential signature of therapy-responsive disease.

Caveolin-1 temporal modulation enhances antibody drug efficacy in heterogeneous gastric cancer

Resistance mechanisms and heterogeneity in HER2-positive gastric cancers (GC) limit Trastuzumab benefit in 32% of patients, and other targeted therapies have failed in clinical trials. Using patient samples, patient-derived xenografts (PDXs), partially humanized biological models, and HER2-targeted imaging technologies we demonstrate the role of caveolin-1 (CAV1) as a complementary biomarker in GC selection for Trastuzumab therapy. In retrospective analyses of samples from patients enrolled on Trastuzumab trials, the CAV1-high profile associates with low membrane HER2 density and low patient survival. We show a negative correlation between CAV1 tumoral protein levels – a major protein of cholesterol-rich membrane domains – and Trastuzumab-drug conjugate TDM1 tumor uptake. Finally, CAV1 depletion using knockdown or pharmacologic approaches (statins) increases antibody drug efficacy in tumors with incomplete HER2 membranous reactivity. In support of these findings, background statin use in patients associates with enhanced antibody efficacy. Together, this work provides preclinical justification and clinical evidence that require prospective investigation of antibody drugs combined with statins to delay drug resistance in tumors.

Abstract P2-13-19: Statin modulation of antibody drug conjugate activity in breast cancer models and patients

Abstract Background: Membrane HER2 expression levels strongly influence the activity of anti-HER2 therapies directed at the HER2 extracellular domain (1,2). Tumoral cell-surface caveolin-1 (CAV-1) regulates receptor tyrosine kinase membrane trafficking mechanisms (1,3). In xenograft models, CAV-1 can be modulated with cholesterol-depleting drugs, such as statins. We hypothesized that preclinical and clinical use of statins might influence the expression of CAV-1 and thereby affect the efficacy of anti-HER2 antibody-drug conjugates (ADCs) in breast cancer. Methods: Preclinically, statins were given alone and in combination with HER2 antibody drug conjugates such as T-DM1 to assess HER2 levels, drug uptake, and antitumor efficacy in HER2-positive cancer models. Mice received an intravenous injection of T-DM1, oral doses of lovastatin, or a combination of T-DM1 and lovastatin, for 5 weeks. Clinically, we performed retrospective analyses of HER2-positive MBC patients at MSKCC who received T-DM1 and consented to molecular profiling and clinical data abstraction. Progression-free survival (PFS) on T-DM1 was estimated using Kaplan Meyer methods and compared using the log rank test. Univariable and multivariable Cox proportional hazards models were constructed using relevant clinical covariates. Results: Tumor models with high levels of CAV-1 exhibit low HER2 density on the cell surface and show low T-DM1 targeted immunoPET binding when compared with CAV-1-low tumors. Mechanistic studies showed that CAV-1 depletion in HER2-positive BC cells temporally stabilizes HER2 on the surface and delays T-DM1 recycling. In preclinical BC models, CAV-1 depletion induced by synthetic oligonucleotides or statins enhances T-DM1 binding and efficacy in tumors with incomplete HER2 membranous reactivity. For clinical analyses, a total of 164 patients who received T-DM1 were included in the final analysis. Twenty-one (12.8%) of these patients were recorded to be taking statins concurrently with T-DM1, as part of their routine clinical care. Compared with patients with no recorded statin use, patients receiving statins were older on average (median age 58 vs. 50 years, p = 0.007), but did not differ in their race, histologic breast cancer type, ER status, or T-DM1 treatment line. Median PFS on T-DM1 in the overall cohort was 5.5 months. Median PFS in patients who received statins was 14 months vs. 5.4 months in patients who had no recorded statin use (p = 0.1). In univariable Cox analysis, the association between statin use and PFS did not reach statistical significance (p=0.119). In a multivariable analysis including age, ER status, treatment line, and breast cancer histology, no variable reached statistical significance. However, statin use had the greatest observed degree of association with PFS (p = 0.17). Conclusions: Statins modulate surface HER2 levels and T-DM1 efficacy preclinically via CAV-1. Although only a numerical difference in outcomes was observed in the MSKCC cohort, the effect appeared meaningful and therefore assessment in larger patient cohorts is now underway. Based on the results of these forthcoming analyses, prospective trials may be justified that integrate these well-tolerated and low-cost agents into HER2 ADC treatment regimens.

Isoforms of IDH in breast carcinoma: IDH2 as a potent prognostic factor associated with proliferation in estrogen-receptor positive cases.

Isocitrate dehydrogenase (IDH) is an important enzyme that oxidatively decarboxylates isocitrate to α-ketoglutarate, and three isoforms (IDH1-3) have been identified. Overexpression and/or downregulation of IDH isoforms was reported in several human malignancies, suggesting importance of IDH in oncogenesis. However, significance of IDH isoforms remains largely unclear in the breast carcinoma. We immunolocalized IDH1, IDH2 and IDH3α in 226 breast carcinomas and evaluated their clinical significance. Subsequently, we examined effects of IDH2 on proliferation in breast carcinoma cells. Immunoreactivity of IDH1-3α was detected in 53%, 38% and 41% of breast carcinomas, and the non-neoplastic epithelium was IDH1-positive, IDH2-negative and IDH3α-positive. IDH1 immunoreactivity was inversely associated with pathological T factor (pT) and Ki-67 in the breast carcinoma, while IDH3α immunoreactivity was not significantly associated with clinicopathological factors. IDH2 status was positively correlated with stage, pT, histological grade, HER2, Ki-67 and microvessel density. Moreover, IDH2 status was significantly associated with worse prognosis of the patients, and it turned out an independent prognostic factor for estrogen-receptor (ER) positive patients. These findings were more evident in the IDH1-negative / IDH2-positive/IDH3α-negative subgroup which is the opposite immunohistochemical IDH phenotype of normal mammary epithelium. In vitro studies demonstrated that RNA interference of IDH2 significantly decreased proliferation activity of T47D and SKBR-3 cells. These results suggest that IDH2 is associated with an aggressive phenotype of breast carcinoma through increasing cell proliferation, different from IDH1 and IDH3α, and immunohistochemical IDH2 status is a potent prognostic factor especially in ER-positive breast cancer patients.

T cell circuits that sense antigen density with an ultrasensitive threshold.

Overexpressed tumor-associated antigens [for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)] are attractive targets for therapeutic T cells, but toxic "off-tumor" cross-reaction with normal tissues that express low levels of target antigen can occur with chimeric antigen receptor (CAR)-T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive-feedback circuit that allows human cytotoxic T cells to discriminate targets on the basis of a sigmoidal antigen-density threshold. In this circuit, a low-affinity synthetic Notch receptor for HER2 controls the expression of a high-affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells-it increases both CAR expression and activation-leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100 times as much HER2, both in vitro and in vivo.

Molecular Assessment of HER2 in Breast Cancer

A standard method to diagnose disease is classical light microscopy. However, its limit of resolution is becoming more constraining as the paradigm for diagnosis shifts towards precision medicine. Super-resolution microscopy approaches can shed light on disease at the level of single molecules. As the capabilities of these approaches (which are well-established for in vitro studies) are brought to bear on clinically relevant questions, corresponding methodologies must be developed to provide rigorous assessment of patient specimens. Here, Quantitative Single Molecule Localization Microscopy (qSMLM) was advanced as a research tool to probe the molecular features of HER2-positive breast cancer. We first optimized qSMLM in cultured cell lines and then developed the approach for tissue specimens. When an anti-HER2 therapy (antibodies trastuzumab and pertuzumab) was used as a fluorescent reporter, qSMLM was able to quantify how many HER2 receptors harbor an accessible drug binding site. In cultured cells, qSMLM detected unique populations of “drug accessible” HER2 receptors. We used qSMLM to compare trastuzumab-sensitive cells to -resistant cells: resistant cells exhibited lower HER2 density and a lower degree of HER2 clustering. We developed corresponding methods to robustly assess specimens taken from women with breast cancer. Our tissue-optimized methodology rigorously probed specimens within one working day. Compared to women who achieved a complete pathological response after neoadjuvant chemotherapy and trastuzumab, poor responders had lower detected HER2 density and lower HER2 clustering. This approach, which provided a preliminary signature of therapy response (six patients), may lead to new research tools to assess breast cancer.

Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2

All breast cancers are assessed for levels of human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) and immunohistochemistry are currently used to determine if a patient is eligible for anti-HER2 therapy. Limitations of both tests include variability and relatively long processing times. Additionally, neither test determines whether HER2 contains the extracellular domain. While truncated in some tumors, this domain is required for binding of the therapeutic antibody trastuzumab. Here, trastuzumab was used to directly detect HER2 with quantitative single molecule localization microscopy (qSMLM). In proof of concept studies, our new method rapidly quantified both HER2 density and features of nano-organization. In cultured cells, the method was sensitive to subtle variations in HER2 expression. To assess patient samples, we combined qSMLM with tissue touch preparation (touch prep-qSMLM) and examined large areas of intact membranes. For cell lines and patient samples, HER2 copy numbers from FISH showed a significant positive correlation with detected densities from qSMLM and trended with HER2 cluster occupancy.

Bispecific CD3/HER2 Targeting FynomAb Induces Redirected T Cell-Mediated Cytolysis with High Potency and Enhanced Tumor Selectivity

CD3 bispecific therapies retargeting T cells to tumors have recently demonstrated striking activity in patients. Several CD3 bispecific antibodies directed against various tumor targets are currently being investigated in the clinic across different tumors. One limitation of these therapies is the risk of target-related toxicity due to low-level expression of tumor antigen in normal tissue. In this study we have engineered a bispecific CD3/HER2 FynomAb, COVA420, which redirects T cells with high potency and selectivity to tumor cells with high HER2 expression in vitro and in vivo. COVA420 activity depends on high HER2 density as no activity was observed on cells with lower HER2 levels as found in human normal tissue. These results suggest that COVA420 may spare normal tissue expressing low levels of HER2 while still having uncompromised efficacy on tumor cells with high HER2 expression. This concept may be applied to other cancer antigens that otherwise cannot be targeted by T cell redirecting approaches, and may therefore expand the applicability of CD3 bispecific FynomAbs to a larger number of solid tumors.

Trastuzumab Labeled to High Specific Activity with (111)In by Site-Specific Conjugation to a Metal-Chelating Polymer Exhibits Amplified Auger Electron-Mediated Cytotoxicity on HER2-Positive Breast Cancer Cells.

Our objective was to evaluate the cytotoxicity toward HER2-positive human breast cancer (BC) cells of trastuzumab modified site-specifically with a metal-chelating polymer (MCP) that presents multiple DTPA chelators for complexing (111)In. (111)In emits subcellular range Auger electrons that induce multiple lethal DNA double-strand breaks (DSBs) in cells. MCPs were synthesized with a polyglutamide backbone with 24 or 29 pendant DTPA groups, with or without nuclear translocation sequence (NLS) peptide modification and a terminal hydrazide group for reaction with aldehydes generated by sodium periodate (NaIO4)-oxidation of glycans on the Fc-domain of trastuzumab. Trastuzumab was site-specifically modified with two DTPA and labeled with (111)In for comparison (trastuzumab-NH-Bn-DTPA-(111)In). The maximum specific activity (SA) for labeling trastuzumab-Hy-MCP with (111)In was 90-fold greater than for trastuzumab-NH-Bn-DTPA-(111)In [8.9 MBq/μg (1.5 × 10(6) MBq/μmol) vs 0.1 MBq/μg (1.2 × 10(4) MBq/μmol)]. Trastuzumab-Hy-MCP-(111)In was bound, internalized, and imported into the nucleus of SK-BR-3 cells. NLS peptide modification of MCPs did not increase nuclear importation. A greater density of DNA DSBs was found for BC cells exposed to high SA (5.5 MBq/μg) than low SA (0.37 MBq/μg) radioimmunoconjugates. At 20 nmol/L, high SA trastuzumab-Hy-MCP-(111)In was 6-fold more effective at reducing the clonogenic survival (CS) of HER2 overexpressed and HER2 gene-amplified SK-BR-3 cells (1.3 × 10(6) receptors/cell) than low SA MCP-radioimmunoconjugates (CS = 1.8 ± 1.3 vs 10.9 ± 0.7%; P = 0.001). Low SA trastuzumab-NH-Bn-DTPA-(111)In (20 nmol/L) reduced the CS of SK-BR-3 cells to 15.8 ± 2.1%. The CS of ZR-75-1 cells with intermediate HER2 density (4 × 10(5) receptors/cell) but without HER2 gene amplification was reduced to 20.5 ± 4.3% by high SA trastuzumab-Hy-MCP-(111)In (20 nmol/L). The CS of HER2-overexpressed (5 × 10(5) HER2/cell) but trastuzumab-resistant TrR1 cells was decreased to 17.1 ± 1.6% by high SA trastuzumab-Hy-MCP-(111)In. Unlabeled trastuzumab (20 nmol/L) was 18-fold less potent than high SA trastuzumab-Hy-MCP-(111)In at reducing the CS of SK-BR-3 cells (CS = 37.0 ± 5.3%) and 3-fold less effective against Zr-75-1 cells (CS = 53.1 ± 9.8%). Unlabeled trastuzumab had no effect on the survival of TrR1 cells. We conclude that increasing the SA for labeling with (111)In by site-specific conjugation of MCPs to trastuzumab greatly amplified the cytotoxic potency against HER2-overexpressed and gene-amplified BC cells and extended its cytotoxicity to cells with intermediate HER2 expression but without gene amplification and to cells that are HER2 overexpressed but trastuzumab-resistant.

Abstract 3330: HER2 overexpression induces membrane deformation that increases cell motility

Abstract Approximately 20% of breast cancers (BC) are characterized by the gene amplification and overexpression of HER2, a member of the ErbB/HER receptor family. While targeted therapies against HER2 effectively delay disease progression in this BC subtype, details of how overexpressed HER2s drive these tumors to malignancy are still unclear. To better understand this process, we investigated various cellular responses to HER2 overexpression in individual live cells. We developed novel single receptor diffusion analyses that determine the activation status of HER2 by diffusivity, to estimate average HER2 activity per single cell. Surprisingly, we found HER2 overexpression induces membrane deformation, which depends only on the HER2 density, but not the receptor activation status. Moreover, this membrane deformation lowers the available surface area for formation of focal adhesion sites, resulting in reduced cell adhesion and increased cell motility. These findings suggest there are signaling-independent roles of HER2 overexpression in disease progression of HER2 positive BCs. Citation Format: Inhee Chung, Mike Reichelt, Donald Dowbenko, Ira Mellman, Mark Sliwkowski. HER2 overexpression induces membrane deformation that increases cell motility. [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 3330. doi:10.1158/1538-7445.AM2014-3330

HER2 Overexpression Induces Membrane Deformation that Increases Cell Motility

Approximately 20% of breast cancers (BC) are characterized by the gene amplification and overexpression of HER2, a member of the ErbB/HER receptor family. While targeted therapies against HER2 effectively delay disease progression in this BC subtype, details of how overexpressed HER2s drive these tumors to malignancy are still unclear. To better understand this process, we investigated cellular responses to HER2 overexpression in individual live cells. We developed novel single receptor diffusion analyses to probe the spatial distribution of HER2s and determine their activation status by their diffusivity. Surprisingly, we found that HER2 overexpression induced deformation of basal cell membranes, which depended only on the HER2 density, regardless of the receptor signaling activity. Moreover, this membrane deformation lowered the focal adhesion coverage on the cell surface, which reduced cell adhesion and increased cell motility. These findings suggest that there is a signaling-independent role of HER2 overexpression in disease progression of HER2 positive BCs.

Abstract P4-12-14: Metal chelating polymer-conjugated trastuzumab labeled to high specific activity with indium-111 is cytotoxic to trastuzumab-sensitive and resistant breast cancer cells through the emission of auger electrons

Abstract Purpose: Auger electrons are very low energy electrons emitted by Indium-111(111In) that cause lethal DNA damage when emitted in close to the cell nucleus. Our laboratory reported previously that trastuzumab modified with nuclear localizing sequence (NLS) peptides and labeled with 111In(111In-NLS-DTPA-trastuzumab) was internalized and transported to the nucleus of HER2-overexpressing breast cancer (BC) cells, reducing their clonogenic survival (CS) to ≤10%. However, a limitation was that at the specific activity (SA) achieved (0.24 MBq/μg) for 111In-DTPA-trastuzumab without or with NLS, only 1 in 50 molecules was radiolabeled. This reduced the potency for cells with low or intermediate HER2 density. Our purpose was to dramatically increase the SA of 111In-labeled trastuzumab by conjugation to a metal chelating polymer (MCP) that displays multiple DTPA chelators for 111In and compare its potency to 111In-NLS-trastuzumab on BC cells with a wide range of HER2 density including cells resistant to trastuzumab. Methods: An MCP with a polyglutamide backbone, 29 DTPA groups, and a hydrazide reactive end-group was site-specifically linked to periodate-oxidized carbohydrates on the Fc domain of trastuzumab. HER2 immunoreactivity, internalization and nuclear importation were evaluated by cell binding and fractionation studies using HER2-overexpressing SK-Br-3 human BC cells. The SA achievable was assessed by radiolabeling 1, 5, 50 and 100 μg of the immunoconjugates with 9.25 MBq of 111In. The effect of high SA (5.5 MBq/μg) 111In-MCP-trastuzumab to on the CS of human BC cells with a wide range of HER2 density (5×105-1.3×106 HER2 receptors/cell) that were sensitive or resistant to trastuzumab was compared to low SA (0.37 MBq/μg) 111In-labeled trastuzumab conjugates. Studies are also in progress to assess DNA double-strand breaks caused by the Auger electrons emitted by 111In using the γH2AX assay. Results: MCP-trastuzumab bound specifically to HER2 with high affinity (KD = 10.7±1.5nM) and was internalized and imported into the nucleus of HER2-positive BC cells. The smallest mass that could be labeled (labeling efficiency&amp;gt;80%) with 111In for MCP-trastuzumab was 1 μg (SA = 8.9 MBq/μg) compared to 50 μg for DTPA-trastuzumab (SA = 0.08 MBq/μg). High SA 111In-MCP-trastuzumab reduced the CS of SK-Br-3 cells with high HER2 density to 1.8±1.3% and the CS of TrR1 trastuzumab-resistant BC cells with intermediate HER2 density to 17.1±1.6%. These represent a 4-5 and 5-6 fold greater potency compared to low SA 111In-trastuzumab conjugates on cells with high or intermediate HER2 expression, respectively. Conclusion: 111In-MCP-trastuzumab was labeled to 37-fold higher SA than 111In-DTPA-trastuzumab. High SA increased its cytotoxicity potency for HER2-positive BC cells that were sensitive or resistant to trastuzumab. Auger electron radioimmunotherapy is a promising strategy for HER2-positive BC which may extend to trastuzumab-resistant tumours. Supported by a grant from the Canadian Breast Cancer Research Alliance with funds from the Canadian Cancer Society. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-14.

Human epidermal growth factor receptor 2-positive breast cancer: which cytotoxic agent best complements trastuzumab&amp;#39;s efficacy in vitro?

IntroductionDespite trastuzumab having enhanced selectivity for human epidermal growth factor receptor 2 (HER-2) overexpressing breast cancer cells, treatment is hampered by interindividual variation and tumors with high mitogenic potential. The lack of significant clinical benefit in certain patient cohorts suggests that HER-2 expression is ineffective as a sole prognostic indicator of response to therapy. Therefore, optimizing the clinical role of trastuzumab in drug combinations remains critical for clinical success.AimTo investigate the effects of trastuzumab in combination with either doxorubicin or geldanamycin on in vitro cell viability, cell cycling, apoptosis and relative HER-2 expression in HER-2-positive (SK-BR-3) and estrogen receptor-positive (MCF-7) breast adenocarcinoma models.ResultsHER-2-rich SK-BR-3 cells demonstrated a greater sensitivity to the effects of doxorubicin than MCF-7 cells. Concurrent trastuzumab exposure resulted in a further reduction in cell viability. This decreased cell viability induced by doxorubicin was associated with activation of executioner caspases as well as with alterations in cell-cycle kinetics, primarily promoting S-phase accumulation. Doxorubicin had no effect on surface HER-2 density expression. Geldanamycin reduced cell viability significantly greater in SK-BR-3 than MCF-7 cells, and was associated with G2 cell-cycle accumulation. The addition of trastuzumab did not augment these effects. Geldanamycin promoted substantial reductions in relative surface HER-2 density in SK-BR-3 cells.ConclusionThe in vitro data supported the rationale for using doxorubicin in trastuzumab-based therapies. Therefore, despite the incidence of cardiotoxicity, doxorubicin could retain a fundamental role in treating HER-2-positive breast cancer. While geldanamycin is a potent cytotoxic agent, its concurrent use with trastuzumab requires further research into the transient or permanent nature of alterations in HER-2 status in cell progeny.

Trastuzumab Labeled to High Specific Activity with 111In by Conjugation to G4 PAMAM Dendrimers Derivatized with Multiple DTPA Chelators Exhibits Increased Cytotoxic Potency on HER2-Positive Breast Cancer Cells

To conjugate trastuzumab with/without NLS peptides to G4 PAMAM dendrimers derivatized with DTPA and determine the specific radioactivity (SA) for (111)In labeling, HER2 immunoreactivity and cytotoxicity on breast cancer (BC) cells. G4 dendrimers were reacted with DTPA then conjugated through a thiol to maleimide-derivatized trastuzumab. The SA achievable was determined by incubating 2 to 20 μg with 60 MBq of (111)In. HER2 immunoreactivity, internalization and nuclear importation were measured. The effect of (111)In-DTPA-G4-trastuzumab (5.9 MBq/μg) on the clonogenic survival (CS) of SK-Br-3 or MDA-MB-231 cells with high or low HER2 density, respectively was compared to (111)In-DTPA-NLS-trastuzumab (0.5 MBq/μg). DNA double-strand breaks (DSBs) were measured. DTPA-G4-trastuzumab was labeled with (111)In to a SA (23.6 MBq/μg) which was 100-fold higher than (111)In-DTPA-NLS-trastuzumab. (111)In-DTPA-G4-trastuzumab and (111)In-DTPA-G4-NLS-trastuzumab retained HER2 immunoreactivity and were internalized and imported into the nucleus of BC cells. G4-radioimmunoconjugates were 2-4 fold and 9-fold more cytotoxic to SK-Br-3 and MDA-MB-231 cells, respectively than (111)In-DTPA-NLS-trastuzumab which was associated with an increase in DNA DSBs. Conjugation of trastuzumab to G4 PAMAM dendrimers modified with 30 DTPA permitted high SA (111)In labeling which increased their cytotoxic potency for BC cells with high or low HER2 density.

111In-Bn-DTPA-nimotuzumab with/without modification with nuclear translocation sequence (NLS) peptides: an Auger electron-emitting radioimmunotherapeutic agent for EGFR-positive and trastuzumab (Herceptin)-resistant breast cancer

Increased expression of epidermal growth factor receptors (EGFR) in breast cancer (BC) is often associated with trastuzumab (Herceptin)-resistant forms of the disease and represents an attractive target for novel therapies. Nimotuzumab is a humanized IgG(1) monoclonal antibody that is in clinical trials for treatment of EGFR-overexpressing malignancies. We show here that nimotuzumab derivatized with benzylisothiocyanate diethylenetriaminepentaacetic acid for labelling with the subcellular range Auger electron-emitter, (111)In and modified with nuclear translocation sequence (NLS) peptides ((111)In-NLS-Bn-DTPA-nimotuzumab) was bound, internalized and transported to the nucleus of EGFR-positive BC cells. Emission of Auger electrons in close proximity to the nucleus caused multiple DNA double-strand breaks which diminished the clonogenic survival (CS) of MDA-MB-468 cells that have high EGFR density (2.4 × 10(6) receptors/cell) to less than 3 %. (111)In-Bn-DTPA-nimotuzumab without NLS peptide modification was sevenfold less effective for killing MDA-MB-468 cells. (111)In-Bn-DTPA-nimotuzumab with/without NLS peptide modification were equivalently cytotoxic to MDA-MB-231 and TrR1 BC cells that have moderate EGFR density (5.4 × 10(5) or 4.2 × 10(5) receptors/cell, respectively) reducing their CS by twofold. MDA-MB-231 cells have intrinsic trastuzumab resistance due to low HER2 density, whereas TrR1 cells have acquired resistance despite HER2 overexpression. Biodistribution and microSPECT/CT imaging revealed that (111)In-NLS-Bn-DTPA-nimotuzumab exhibited more rapid elimination from the blood and lower tumour uptake than (111)In-Bn-DTPA-nimotuzumab. Tumour uptake of the radioimmunoconjugates in mice with MDA-MB-468 xenografts was high (8-16 % injected dose/g) and was blocked by administration of an excess of unlabelled nimotuzumab, demonstrating EGFR specificity. We conclude that (111)In-Bn-DTPA-nimotuzumab with/without NLS peptide modification are promising Auger electron-emitting radioimmunotherapeutic agents for EGFR-positive BC, but (111)In-Bn-DTPA-nimotuzumab may be preferred due to its higher tumour uptake in vivo.

Tumor Targeting Using Affibody Molecules: Interplay of Affinity, Target Expression Level, and Binding Site Composition

Radionuclide imaging of cancer-associated molecular alterations may contribute to patient stratification for targeting therapy. Scaffold high-affinity proteins, such as Affibody molecules, are a new, promising class of probes for in vivo imaging. The effects of human epidermal growth factor receptor 2 (HER2) affinity and binding site composition of HER2-binding Affibody molecules, and of the HER2 density on the tumor targeting, were studied in vivo. The tumor uptake and tumor-to-organ ratios of Affibody molecules with moderate (dissociation constant [K(D)] = 10(-9) M) or high (K(D) = 10(-10) M) affinity were compared between tumor xenografts with a high (SKOV-3) and low (LS174T) HER2 expression level in BALB/C nu/nu mice. Two Affibody molecules with similar affinity (K(D) = 10(-10) M) but having alternative amino acids in the binding site were compared. In SKOV-3 xenografts, uptake was independent of affinity at 4 h after injection, but high-affinity binders provided 2-fold-higher tumor radioactivity retention at 24 h. In LS174T xenografts, uptake of high-affinity probes was already severalfold higher at 4 h after injection, and the difference was increased at 24 h. The clearance rate and tumor-to-organ ratios were influenced by the amino acid composition of the binding surface of the tracer protein. The optimal affinity of HER2-binding Affibody molecules depends on the expression of a molecular target. At a high expression level (>10(6) receptors per cell), an affinity in the low-nanomolar range is sufficient. At moderate expression, subnanomolar affinity is desirable. The binding site composition can influence the imaging contrast. This information may be useful for development of imaging agents based on scaffold affinity proteins.