HER2-expressing Breast Cancer Research Articles

Development of a Nanostructured Platform for Identifying HER2-Heterogeneity of Breast Cancer Cells by Surface-Enhanced Raman Scattering.

Biosensor technology has great potential for the detection of cancer through tumor-associated molecular biomarkers. In this work, we describe the immobilization of the recombinant humanized anti-HER2 monoclonal antibody (trastuzumab) on a silver nanostructured plate made by pulsed laser deposition (PLD), over a thin film of Au(111). Immobilization was performed via 4-mercapto benzoic acid self-assembled monolayers (4-MBA SAMs) that were activated with coupling reagents. A combination of immunofluorescence images and z-stack analysis by confocal laser scanning microscopy (CLSM) allowed us to detect HER2 presence and distribution in the cell membranes. Four different HER2-expressing breast cancer cell lines (SKBR3 +++, MCF-7 +/−, T47D +/−, MDA-MB-231 −) were incubated during 24 h on functionalized silver nanostructured plates (FSNP) and also on Au(111) thin films. The cells were fixed by means of an ethanol dehydration train, then characterized by atomic force microscopy (AFM) and surface-enhanced Raman scattering (SERS). SERS results showed the same tendency as CLSM findings (SKBR3 > MCF-7 > T47D > MDA-MB-231), especially when the Raman peak associated with phenylalanine amino acid (1002 cm−1) was monitored. Given the high selectivity and high sensitivity of SERS with a functionalized silver nanostructured plate (FSNP), we propose this method for identifying the presence of HER2 and consequently, of breast cancer cells.

Abstract 818: NG-HER2 ADC (PF-06804103) is superior to trastuzumab emtansine in a mouse 'avatar' head-to-head clinical trial

Abstract Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice and represent personalized murine ‘avatars' of those tumors. PDXs are valuable tumor models for drug development since they recapitulate the complexity of the human tumor microenvironment more extensively than cell line xenografts (CLXs). Unlike CLXs, PDXs are never passaged in vitro, and therefore they more faithfully recapitulate native tumor biology and response to therapeutics. Thus, PDXs may more accurately predict clinical activity of therapeutic compounds than traditional CLXs. We are developing a next generation (NG)-HER2 antibody-drug conjugate (ADC), PF-06804103, that employs a proprietary site-specific conjugation technology that chemically links the clinically-validated linker-payload “ValCit-Aur0101” to an anti-HER2 antibody. The site-specific conjugation enables enhanced exposures and reduced off-target toxicities as previously described. We utilized our PDX collection to evaluate the breadth-of-efficacy of PF-06804103 versus trastuzumab emtansine (T-DM1), an FDA-approved ADC for metastatic breast cancer patients with high HER2 expression. Efficient cleavage of the ‘ValCit' linker and payload release in the early endosome is not impacted by the rapid recycling properties of the HER2 receptor, unlike T-DM1 which requires catabolism in the within the lysosomal milieu to efficiently release its payload. To date, we have enrolled >20 HER2-expressing breast, gastric and non-small cell lung cancer PDXs, with varying low to high HER2 expression levels, in a head-to-head ‘mouse avatar clinical trial' comparing activity of a single cycle of T-DM1 (6 mg/kg) to PF-06804103 (3 mg/kg). Impressively, PF-06804103 had more durable complete responses and a higher objective response rate (ORR) than T-DM1 (84% vs. 4%), including in low-moderate HER2 expressers. PF-06804103 prolonged median overall survival (OS) of mice with HER2-expressing tumors compared to T-DM1 (100 vs 45 days). Biomarker analysis showed that tumors at all levels of HER2 expression were more likely to receive more benefit with PF-06804103 than T-DM1 (HR < 0.45). By leveraging our novel PDX ‘avatar' clinic, we were able to demonstrate that PF-06804103 displays superior in vivo breadth-of-efficacy compared to T-DM1. Citation Format: Matthew S. Sung, Christine Hopf, Erik Upeslacis, Jonathon Golas, Mark Kaplan, Kiran Khandke, Manoj Charati, Frank Kotch, Frank Loganzo, Ken Geles, Judy Lucas, Hans-Peter Gerber, Puja Sapra, Edward Rosfjord. NG-HER2 ADC (PF-06804103) is superior to trastuzumab emtansine in a mouse 'avatar' head-to-head clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 818.

Abstract 3914: ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers

Abstract Therapies targeting HER2 have transformed the treatment of patients with HER2-expressing breast and gastric cancers. Unfortunately, many patients recur following HER2-targeted treatments and new therapies are needed. Multiple antibody-drug conjugate (ADC) technologies are being explored in this setting, some of which utilize the anti-HER2 antibody trastuzumab. Here we present the preclinical characterization of a new anti-HER2 biparatopic ADC, ZW49, which is generated from the conjugation of a novel N-acyl sulfonamide auristatin payload to the inter-chain disulfide bond cysteines of the bispecific anti-HER2 IgG1 antibody ZW25, via a protease cleavable linker. A series of in vitro and in vivo experiments were performed to characterize ZW49 as a potential therapeutic candidate. In cellular binding assays, it was confirmed that the payload conjugation to ZW25 did not affect the antibody's binding to HER2-expressing cells. ZW49 displayed potent in vitro cytotoxicity in multiple cancer cell lines expressing HER2 and was efficacious in multiple patient-derived xenograft (PDX) models. In mice bearing the HBCx-13b HER2 3+ PDX, two doses of ZW49 administered two weeks apart generated tumor regressions. Furthermore, preliminary results from PDX models with lower levels of HER2 expression treated with ZW49 also generated regressions. In nonhuman primates ZW49 administered intravenously every two weeks for three doses was well tolerated. Based on these findings, we are proceeding with further development of ZW49 as a therapeutic candidate in HER2-expressing cancers. Citation Format: Kevin J. Hamblett, Phil W. Hammond, Stuart D. Barnscher, Vincent K. Fung, Rupert H. Davies, Grant R. Wickman, Andrea Hernandez, Tong Ding, Adam S. Galey, Geoffrey C. Winters, Jamie R. Rich, John S. Babcook. ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3914.

Preliminary Investigation of Focused Ultrasound-Facilitated Drug Delivery for the Treatment of Leptomeningeal Metastases

Leptomeningeal metastases (LM) are a serious complication of cancer in the central nervous system (CNS) and are diagnosed in approximately 5% of patients with solid tumors. Effective treatment using systemically administered therapeutics is hindered by the barriers of the CNS. Ultrasound can mediate delivery of drugs through these barriers. The goal of this study was to test the feasibility of using ultrasound-mediated drug delivery to improve the treatment of LM. LM was induced in the spinal cord of athymic rats by injecting HER2-expressing breast cancer cells into the subarachnoid space of the thoracic spine. Animals were divided into three groups: no treatment (n = 5), trastuzumab only (n = 6) or trastuzumab + focused ultrasound + microbubbles (FUS + MBs) (n = 7). Animals in groups 2 and 3 were treated weekly with intravenous trastuzumab +/− FUS + MBs for three weeks. Suppression in tumor growth was qualitatively observed by MRI in the group receiving ultrasound, and was confirmed by a significant difference in the tumor volume measured from the histology data (25 ± 17 mm3 vs 8 ± 5 mm3, p = 0.04 in the trastuzumab-only vs trastuzumab + FUS + MBs). This pilot study demonstrates the potential of ultrasound-mediated drug delivery as a novel treatment for LM. Future studies will extend this work to larger cohorts and the investigation of LM arising from other cancers.

Oncodriver inhibition and CD4+ Th1 cytokines cooperate through Stat1 activation to induce tumor senescence and apoptosis in HER2+ and triple negative breast cancer: implications for combining immune and targeted therapies.

In patients with HER2-expressing breast cancer many develop resistance to HER2 targeted therapies. We show that high and intermediate HER2-expressing cancer cell lines are driven toward apoptosis and tumor senescence when treated with either CD4+ Th1 cells, or Th1 cytokines TNF-α and IFN-γ, in a dose dependent manner. Depletion of HER2 activity by either siRNA or trastuzumab and pertuzumab, and subsequent treatment with either anti-HER2 Th1 cells or TNF-α and IFN-γ resulted in synergistic increased tumor senescence and apoptosis in cells both sensitive and cells resistant to trastuzumab which was inhibited by neutralizing anti-TNF-α and IFN-γ. Th1 cytokines induced minimal senescence or apoptosis in triple negative breast cancer cells (TNBC); however, inhibition of EGFR in combination with Th1 cytokines sensitized those cells causing both senescence and apoptosis. TNF-α and IFN-γ led to increased Stat1 phosphorylation through serine and tyrosine sites and a compensatory reduction in Stat3 activation. Single agent IFN-γ enhanced Stat1 phosphorylation on tyrosine 701 and similar effects were observed in combination with TNF-α and EGFR inhibition. These results demonstrate Th1 cytokines and anti-oncodriver blockade cooperate in causing tumor senescence and apoptosis in TNBC and HER2-expressing breast cancer, suggesting these combinations could be explored as non-cross-reactive therapy preventing recurrence in breast cancer.

Abstract PD3-07: Safety and efficacy results from a phase 1 study of DS-8201a in patients with HER2 expressing breast cancers

Abstract Background DS-8201a is a HER2 targeting antibody-drug conjugate (ADC) with high drug to antibody ratio (8:1), a novel linker and topoisomerase I inhibitor as the payload. In preclinical studies, DS-8201a showed efficacy against both trastuzumab emtansine (T-DM1) resistant HER2 positive breast cancer (BC) and against BC with low HER2 expression. The current phase 1 trial is comprised of dose escalation (Part 1) and dose expansion (Part 2) cohorts including patients (pts) with BC, gastric cancer and other HER2 expressing tumors. Only the results from BC cohorts are presented here. Methods Part 1 used a modified continual reassessment model to identify the recommended phase 2 dose (RP2D) of DS8201a in pts with BC or gastric cancer of any HER2 status. Two out of planned 4 expansion cohorts included BC: T-DM1-treated HER2 positive BC (Cohort 2a; IHC 3+ or IHC 2+/ISH +) and HER2 low BC (Cohort 2c, IHC 2+/ISH- or IHC 1+). Efficacy endpoints of objective response rate (ORR), disease control rate (DCR: CR + PR + SD), durability of response, progression free survival (PFS) and adverse events (AEs) were assessed. Results Overall, 146 subjects were included (24 Part 1; 122 Part 2). DS-8201a was administered up to 8.0 mg/kg in Part 1 without any DLTs, maximum tolerated dose was not reached. Based on available results, the dose levels of 5.4 and 6.4 mg/kg IV every 3 weeks were chosen for Part 2. In Part 2, a total of 56 BC pts were enrolled; 46 pts in Cohort 2a and 10 pts in Cohort 2c. The median age was 56 and number of prior regimens in the metastatic setting was 5 (range: 1-16). 37 BC pts had follow-up imaging scans and were evaluable for response at the time of data cutoff. Confirmed ORR was 41% (15/37) including 1 CR; DCR was 97% (36/37). The median duration of treatment was not reached. For Cohort 2a, where all pts had received prior T-DM1, treatment with DS-8201a achieved a higher ORR of 41% (11/27) and DCR of 100% (27/27) compared to the reported response these patients had to their prior T-DM1 treatment with ORR of 23% (5/22) and DCR of 73% (16/22). In the subset of 24 pts from cohort 2a who had received prior treatment with pertuzumab and T-DM1, the confirmed ORR was 44% (11/25). In Cohort 2c, confirmed ORR was 40% (4/10), DCR was 90% (9/10). Of the 46 BC pts in Part 2 who received at least one dose of DS-8201a, 4 pts discontinued treatment due to disease progression and 3 pts discontinued due to AE. The most common AEs of any grades were nausea, decreased appetite, vomiting, alopecia, and diarrhea. Only 2 pts experienced grade 4 AEs (thrombocytopenia and neutropenia) and 46 % (21/46) experienced grade 3 AEs (most commonly anemia, neutropenia, thrombocytopenia, leukopenia, lymphocytepenia, and vomiting). Conclusions DS-8201a was well tolerated and has significant activity in pts with T-DM1 and T-DM1/ pertuzumab pretreated HER2 positive BC and in pts with HER2 low BC, with durable disease control. Promising efficacy of this ADC in BC warrants further investigation. Citation Format: Modi S, Tsurutani J, Takahashi S, Iwata H, Park H, Redfern CH, Doi T, Li B, Iwasa T, Taira S, Hattori M, Ma CX, Fisher JM, Naito Y, Yonemori K, Kawasaki Y, Saito K, Jikoh T, Shahidi J, Lee CC, Yver A, Tamura K. Safety and efficacy results from a phase 1 study of DS-8201a in patients with HER2 expressing breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-07.

Abstract P5-20-06: Phase 1 dose escalation with ZW25, a HER2-targeted bispecific antibody, in patients (pts) with HER2-high breast cancer (BC)

Abstract Background: ZW25, a novel IgG1-like bispecific antibody, targets the same domains of HER2 as trastuzumab (T) and pertuzumab (P). In preclinical studies, ZW25 demonstrated increased tumor cell binding density and internalization relative to T and activity in T-resistant cell lines as well as models of HER2-low to high cancers. Initial dose escalation data demonstrated that once-weekly ZW25 was well tolerated at all doses evaluated and associated with single-agent anti-tumor activity in pts with heavily pre-treated (tx) HER2-expressing cancers. Methods: 3+3 dose escalation of ZW25 given weekly (QW; 5, 10 or 15 mg/kg) or biweekly (Q2W; 20 mg/kg) in 4-week cycles. Eligibility included HER2 IHC 1, 2 or 3+ or FISH+ BC, progression after T, P and T-DM1, and measurable or non-measureable disease per RECIST 1.1. Active brain metastases were excluded. Baseline brain MRI was performed in QW cohorts only if pts had prior history (hx) of CNS mets, and in all Q2W pts regardless of hx. Assessments included AEs, LVEF, immunogenicity, PK and tumor response every 2 cycles. Results: 8 pts with HER2-high BC were tx with ZW25 QW at 5 (n=2), 10 (n=2) or 15 mg/kg (n=4); 20 mg/kg Q2W is enrolling. 5/8 pts were HR+; 7 had measurable disease, 6 visceral disease, and 3 stable CNS disease. Median years since initial dx was 6 (range 5-16). Prior tx included T and T-DM1 (n=8); P (n= 6), and lapatinib (n=5). Median number of prior HER2-targeted regimens for metastatic disease was 6 (range 3-7) and non-hormonal HER2 regimens was 5 (range 3-7). ZW25 was well tolerated with no DLTs or decreases in cardiac function. Most common related AEs (all Gr 1 or 2) were diarrhea (n=4), infusion reaction (IR) (n=4) and vomiting (n=3). All IRs occurred only with 1st dose. There were no treatment-related SAEs. Related Gr 3 AEs (hypophosphatemia, fatigue and arthralgia) were reported in 1 pt (10 mg/kg). At data cut-off, pts had received 2-10 cycles of treatment, with 3 pts active. Best overall response was 2 PR (10 mg/kg), 3 SD (1 at 5 mg/kg, 2 at 15 mg/kg), and 3 PD (1 at 5 mg/kg, 2 at 15 mg/kg) for a disease control rate of 63%. Decreases in target lesions were seen in 6/7 patients with at least one tumor re-staging. One pt with SD (5 mg/kg; active on study) had an 8% decrease after C2, and 29% decrease after C8. One PR pt with prior hx of brain mets had a 33% decrease after C2 and 44% decrease after C4, although was found to have new leptomeningeal disease (LMD) at that time. Two additional pts with systemic SD (15 mg/kg; no prior hx of CNS mets) were also considered to have PD due to symptomatic brain mets. One of these pts remains on study after receiving stereotactic radiotherapy. Conclusions: ZW25 was associated with single-agent anti-tumor activity and systemic disease control in HER2-high BC pts after a median of 6 prior HER2-targeted regimens for metastatic disease. Systemic disease control was maintained despite PD due to brain mets or LMD. The presence of CNS disease in an unscreened population is consistent with the biology of late-stage HER2-high BC. The activity and tolerability of ZW25 support further evaluation as a single agent and in combination including with CNS-directed therapies in early and late lines of treatment for HER2-expressing BC. Citation Format: Hamilton E, Meric-Bernstam F, Infante J, Murthy R, Patnaik A, Piha-Paul SA, Tolcher A, Hausman D, Royer N, Beeram M. Phase 1 dose escalation with ZW25, a HER2-targeted bispecific antibody, in patients (pts) with HER2-high breast cancer (BC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-20-06.

Heterologous human/rat HER2-specific exosome-targeted T cell vaccine stimulates potent humoral and CTL responses leading to enhanced circumvention of HER2 tolerance in double transgenic HLA-A2/HER2 mice

DNA vaccines composed of heterologous human HER2 and rat neu sequences induce stronger antibody response and protective antitumor immunity than either HER2 or neu DNA vaccines in transgenic mice. We previously developed HER2-specific exosome-targeted T-cell vaccine HER2-TEXO capable of stimulating HER2-specific CD8+ T-cell responses, but only leading to partial protective immunity in double-transgenic HLA-A2/HER2 mice with self-immune tolerance to HER2. Here, we constructed an adenoviral vector AdVHuRt expressing HuRt fusion protein composed of NH2-HER21-407 (Hu) and COOH-neu408-690 (Rt) fragments, and developed a heterologous human/rat HER2-specific exosome-targeted T-cell vaccine HuRt-TEXO using polyclonal CD4+ T-cells uptaking exosomes released by AdVHuRt-transfected dendritic cells. We found that the HuRt-TEXO vaccine stimulates enhanced CD4+ T-cell responses leading to increased induction of HER2-specific antibody (∼70 µg/ml) compared to that (∼40 µg/ml) triggered by the homologous HER2-TEXO vaccine. By using PE-H-2Kd/HER223-71 tetramer, we determined that HuRt-TEXO stimulates stronger HER2-specific CD8+ T-cell responses eradicating 90% of HER2-specific target cells, while HER2-TEXO-induced CD8+ T-cell responses only eliminating 53% targets. Furthermore, HuRt-TEXO, but not HER2-TEXO vaccination, is capable of suppressing early stage-established HER2-expressing 4T1HER2 breast cancer in its lung metastasis or subcutaneous form in BALB/c mice, and of completely protecting transgenic HLA-A2/HER2 mice from growth of HLA-A2/HER2-expressing BL6-10A2/HER2 melanoma. HuRt-TEXO-stimulated HER2-specific CD8+ T-cells not only are cytolytic to trastuzumab-resistant HLA-A2/HER2-expressing BT474/A2 breast tumor cells in vitro but also eradicates pre-established BT474/A2 tumors in athymic nude mice. Therefore, our novel heterologous human/rat HER2-specific T-cell vaccine HuRt-TEXO, circumventing HER2 tolerance, may provide a new therapeutic alternative for patients with trastuzumab-resistant HER2+ breast tumor.

Site-Specifically Labeled Antibody-Drug Conjugate for Simultaneous Therapy and ImmunoPET.

The conjugation of antibodies with cytotoxic drugs can alter their in vivo pharmacokinetics. As a result, the careful assessment of the in vivo behavior, and specifically the tumor-targeting properties, of antibody-drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide-alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin's Fc domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal 89Zr ( t1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the 89Zr-trastuzumab-MMAE immunoconjugate were validated in vivo using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of ∼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days.

Abstract A011: HER2-positive PDX model of breast cancer brain metastasis obtained from patient prior to T-DM1 therapy

Abstract Background: Ado-trastuzumab emtansine (T-DM1), a recently approved antibody-drug conjugate (ADC), is approved for treatment of high HER2-expressing (3+), trastuzumab-resistant breast and gastric cancers. While this agent is effective, HER2-positive breast cancer patients have a high risk of brain relapse, seen in up to 37% of patients of whom half die as a result of failure to control the intracranial disease. The aim of this study was to develop a model system that reflects the breast cancer brain metastases seen in patients undergoing T-DM1 therapy. We collected brain metastasis tissue from an HER2-positive breast cancer patient prior to receiving T-DM1 therapy. Methods: The high HER2-expressing (3+) breast cancer brain metastasis model designated ST3338 was established subcutaneously in athymic nude mice using biopsy material from a brain metastasis obtained from a 42-year-old breast cancer patient. The patient was on T-DM1 therapy following sample collection and had received prior treatment with trastuzumab, bevacizumab, and lapatinib. Immunohisotochemistry of both clinical and PDX tissues was applied to assess the estrogen (ER), progesterone (PR), and HER2 receptor levels. Drug sensitivity studies were performed in the ST3338 model established subcutaneously in athymic nude mice to single-agent T-DM1 administered intravenously as a single dose at 10 mg/kg. The ST3338 was also established orthotopically by stereotactic intracranial inoculation of enzymatically digested ST3338 tumor tissue. Tumor development was monitored by T2-weighted MR imaging and the study endpoint was survival by humane endpoints. Results: Immunohistochemistry of PDX tissues was scored as ER (0), PR (0), and HER2 (3+), which corresponded to the clinical brain metastasis sample: ER (0), PR (0), and HER2 (3+). A single dose of T-DM1 at 10 mg/kg showed tumor regression compared with Day 0 (T/C = 0%) in the subcutaneous setting. The ST3338 model was successfully established orthotopically determined by longitudinal T2-weighted MR imaging, and efficacy data of T-DM1 in the orthotopic/intracranial setting are pending. Conclusion: We have established a model system for evaluating drug sensitivity in HER2-positive breast cancer brain metastases. Treatment response to T-DM1 was observed in the subcutaneous setting. With the combination of subcutaneous and intracranial PDX models of breast cancer brain metastases, new drugs can be tested in preclinical models that more closely mimic the microenvironment and the challenges of drug delivery across the blood-brain barrier in breast cancer patients. Citation Format: Carsten H. Nielsen, Michael J. Wick, Lizette Gamez, Camilla S. Knudsen, Mette M. Jense, Melissa Rundle, Kyriakos P. Papadopoulos, Andreas Kjaer. HER2-positive PDX model of breast cancer brain metastasis obtained from patient prior to T-DM1 therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A011.

Theranostic pretargeted radioimmunotherapy of internalizing solid tumor antigens in human tumor xenografts in mice: Curative treatment of HER2-positive breast carcinoma.

In recent reports, we have shown that optimized pretargeted radioimmunotherapy (PRIT) based on molecularly engineered antibody conjugates and 177Lu-DOTA chelate (DOTA-PRIT) can be used to cure mice bearing human solid tumor xenografts using antitumor antibodies to minimally internalizing membrane antigens, GPA33 (colon) and GD2 (neuroblastoma). However, many solid tumor membrane antigens are internalized after antibody binding and it is generally believed that internalizing tumor membrane antigens are not suitable targets for PRIT. In this study, we tested the hypothesis that DOTA-PRIT can be performed successfully to target HER2, an internalizing membrane antigen widely expressed in breast, ovarian, and gastroesophageal junction cancers.Methods: DOTA-PRIT was carried out in athymic nude mice bearing BT-474 xenografts, a HER2-expressing human breast cancer, using a three-step dosing regimen consisting of sequential intravenous administrations of: 1) a bispecific IgG-scFv (210 kD) format (BsAb) carrying the IgG sequence of the anti-HER2 antibody trastuzumab and the scFv “C825” with high-affinity, hapten-binding antibody for Bn-DOTA (metal) (BsAb: anti-HER2-C825), 2) a 500 kD dextran-based clearing agent, followed by 3) 177Lu-DOTA-Bn. At the time of treatment, athymic nude mice bearing established subcutaneous BT-474 tumors (medium- and smaller-sized tumors with tumor volumes of 209 ± 101 mm3 and ranging from palpable to 30 mm3, respectively), were studied along with controls. We studied single- and multi-dose regimens. For groups receiving fractionated treatment, we verified quantitative tumor targeting during each treatment cycle using non-invasive imaging with single-photon emission computed tomography/computed tomography (SPECT/CT).Results: We achieved high therapeutic indices (TI, the ratio of radiation-absorbed dose in tumor to radiation-absorbed dose to critical organs, such as bone marrow) for targeting in blood (TI = 28) and kidney (TI = 7), while delivering average radiation-absorbed doses of 39.9 cGy/MBq to tumor. Based on dosimetry estimates, we implemented a curative fractionated therapeutic regimen for medium-sized tumors that would deliver approximately 70 Gy to tumors, which required treatment with a total of 167 MBq 177Lu-DOTA-Bn/mouse (estimated absorbed tumor dose: 66 Gy). This regimen was well tolerated and achieved 100% complete responses (CRs; defined herein as tumor volume equal to or smaller than 4.2 mm3), including 62.5% histologic cure (5/8) and 37.5% microscopic residual disease (3/8) at 85 days (d). Treatment controls showed tumor progression to 207 ± 201% of pre-treatment volume at 85 d and no CRs. Finally, we show that treatment with this curative 177Lu regimen leads to a very low incidence of histopathologic abnormalities in critical organs such as bone marrow and kidney among survivors compared with non-treated controls.Conclusion: Contrary to popular belief, we demonstrate that DOTA-PRIT can be successfully adapted to an internalizing antigen-antibody system such as HER2, with sufficient TIs and absorbed tumor doses to achieve a high probability of cures of established human breast cancer xenografts while sparing critical organs of significant radiotoxicity.

The Tumor-Suppressor Protein OPCML Potentiates Anti-EGFR- and Anti-HER2-Targeted Therapy in HER2-Positive Ovarian and Breast Cancer.

Opioid-binding protein/cell adhesion molecule-like (OPCML) is a tumor-suppressor gene that is frequently inactivated in ovarian cancer and many other cancers by somatic methylation. We have previously shown that OPCML exerts its suppressor function by negatively regulating a spectrum of receptor tyrosine kinases (RTK), such as ErbB2/HER2, FGFR1, and EphA2, thus attenuating their related downstream signaling. The physical interaction of OPCML with this defined group of RTKs is a prerequisite for their downregulation. Overexpression/gene amplification of EGFR and HER2 is a frequent event in multiple cancers, including ovarian and breast cancers. Molecular therapeutics against EGFR/HER2 or EGFR only, such as lapatinib and erlotinib, respectively, were developed to target these receptors, but resistance often occurs in relapsing cancers. Here we show that, though OPCML interacts only with HER2 and not with EGFR, the interaction of OPCML with HER2 disrupts the formation of the HER2-EGFR heterodimer, and this translates into a better response to both lapatinib and erlotinib in HER2-expressing ovarian and breast cancer cell lines. Also, we show that high OPCML expression is associated with better response to lapatinib therapy in breast cancer patients and better survival in HER2-overexpressing ovarian cancer patients, suggesting that OPCML co-therapy could be a valuable sensitizing approach to RTK inhibitors. Mol Cancer Ther; 16(10); 2246-56. ©2017 AACR.

Myc mediates cancer stem-like cells and EMT changes in triple negative breast cancers cells.

Women with triple negative breast cancer (TNBC) have poor prognosis compared to other breast cancer subtypes. There were several reports indicating racial disparity in breast cancer outcomes between African American (AA) and European American (EA) women. For example, the mortality rates of AA breast cancer patients were three times higher than of EA patients, even though, the incidence is lower in AA women. Our in vitro studies indicate that cancer stem-like cells (CSCs) derived from AA TNBC cell lines have significantly higher self-renewal potential (mammosphere formation) than CSCs derived from EA cell lines. TNBC tumors express high levels of Myc compared to luminal A or HER2 expressing breast cancers. We studied the effects of c-Myc overexpression on CSCs and chemotherapy in AA, and EA derived TNBC cell line(s). Overexpression of c-Myc in AA derived MDA-MB-468 (Myc/MDA-468) cells resulted in a significant increase in CSCs and with minimal changes in epithelial-to-mesenchymal transition (EMT) compared to the control group. In contrast, overexpression of c-Myc in EA derived MDA-MB-231(Myc/MDA-231) cells led to increased epithelial-to-mesenchymal transition (EMT), with a minimal increase in CSCs compared to the control group. Myc/MDA-468 cells were resistant to standard chemotherapeutic treatments such as iniparib (PARP inhibitor) plus cisplatin, / iniparib, cisplatin, paclitaxel and docetaxel. However, Myc/MDA-231 cells, which showed EMT changes responded to iniparib with cisplatin, but were resistant to other drugs, such as iniparib, cisplatin, paclitaxel and docetaxel. Collectively, our results indicate that intrinsic differences in the tumor biology may contribute to the breast cancer disparities.

A peptidomimetic with a chiral switch is an inhibitor of epidermal growth factor receptor heterodimerization.

Among different types of EGFR dimers, EGFR-HER2 and HER2-HER3 are well known in different types of cancers. Targeting dimerization of EGFR will have a significant impact on cancer therapies. A symmetric peptidomimetic was designed to inhibit the protein-protein interaction of EGFR. The peptidomimetic (Cyclo(1,10)PpR (R) Anapa-FDDF-(R)-Anapa)R, compound 18) was shown to exhibit antiproliferative activity with an IC50 of 194 nM in HER2-expressing breast cancer cell lines and 18 nM in lung cancer cell lines. The peptidomimetic has a Pro-Pro sequence in the structure to stabilize the β-turn and a β-amino acid, amino napthyl propionic acid. To investigate the effect of the chirality of β-amino acid on the structure of the peptide and its antiproliferative activity, diastereoisomers of compound 18 were designed and synthesized. Structure-activity relationships of these compounds indicated that there is a chiral switch at β-amino acid in the designed compound. The peptidomimetic with R configuration at β-amino acid and with a L-Pro-D-Pro sequence was the most active compound (18). Using enzyme complement fragmentation assay and proximity ligation assay, we show that compound 18 inhibits HER2:HER3 and EGFR:HER2 dimerization. Surface plasmon resonance studies suggested that compound 18 binds to the HER2 extracellular domain and in particular to domain IV. The anticancer activity of compound 18 was evaluated using a xenograft model of breast cancer in mice; compound 18 suppressed the tumor growth in mice compared to control. Compound 18 was also shown to have a synergistic effect with erlotinib on EGFR mutated lung cancer cell lines.

Abstract 3101: Hyperthermia at the single-cell level for disseminated cancer disease with immuno-magnetic nanoparticles

Abstract Hyperthermia using magnetic nanoparticles and alternating magnetic field is promising for cancer therapy. In most cases, the usefulness of this magnetic nanoparticle-based hyperthermia has been shown by applying magnetic nanoparticles to tumor tissue such as a subcutaneous tumor with direct injection technique; however, if the magnetic nanoparticles can be cancer cell-specifically delivered at the cellular level, the magnetic field hyperthermia would have the potential to treat various sorts of disseminated cancer diseases. The aim of this study is to prove this concept. Maghemite nanoparticles (MNPs) were conjugated with the anti-HER2 antibody (Tmab) for cancer targeting, and the Tmab-MNPs were applied to the co-culture flask containing both HER2-expressing breast cancer AU565 cells and normal fibroblast FEF3 cells. Thirty minutes later from the Tmab-MNPs administration, the medium was removed and the cells were washed to remove excess Tmab-MNPs. Prussian blue iron staining and immunostaining for Tmab showed co-localization of the maghemite nanoparticles and Tmab only on the cancer cells, which suggested that MNPs conjugation with Tmab was successful and that cancer specific delivery of MNPs at the cellular level was achieved in vitro. Moreover, application of alternating current magnetic field at 280 kHz for 1 hour caused apoptotic-like cell death only to cancer cells without any cell damage to normal fibroblast FEF3 cells. Intracellular hyperthermia using cancer-specific antibody-modified magnetic nanoparticles have the potential to be applied to the treatment of disseminated cancer diseases. Citation Format: Tetsuya Kagawa, Hiroyuki Kishimoto, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Takeshi Nagasaka, Satoshi Nohara, Ichiro Kato, Adarsh Sandhu, Hiromichi Aono, Toshiyoshi Fujiwara. Hyperthermia at the single-cell level for disseminated cancer disease with immuno-magnetic nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2017-3101

CD47 as a Target for Immune-Radiation Therapy for HER2-Expressing Breast Cancer Cells

CD47 as a Target for Immune-Radiation Therapy for HER2-Expressing Breast Cancer Cells

Inhibition of 6-phosphofructo-2-kinase (PFKFB3) suppresses glucose metabolism and the growth of HER2+ breast cancer.

Human epidermal growth factor receptor-2 (HER2) has been implicated in the progression of multiple tumor types, including breast cancer, and many downstream effectors of HER2 signaling are primary regulators of cellular metabolism, including Ras and Akt. A key downstream metabolic target of Ras and Akt is the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 isozyme (PFKFB3), whose product, fructose-2,6-bisphosphate (F26BP), is a potent allosteric activator of a rate-limiting enzyme in glycolysis, 6-phosphofructo-1-kinase (PFK-1). We postulate that PFKFB3 may be regulated by HER2 and contribute to HER2-driven tumorigenicity. Immunohistochemistry and Kaplan-Meier analysis of HER2+ patient samples investigated the relevance of PFKFB3 in HER2+ breast cancer. In vitro genetic and pharmacological inhibition of PFKFB3 was utilized to determine effects on HER2+ breast cancer cells, while HER2 antagonist treatment assessed the mechanistic regulation on PFKFB3 expression and glucose metabolism. Administration of a PFKFB3 inhibitor in a HER2-driven transgenic breast cancer model evaluated this potential therapeutic approach in vivo. PFKFB3 is elevated in human HER2+ breast cancer and high PFKFB3 transcript correlated with poorer progression-free (PFS) and distant metastatic-free (DFMS) survival. Constitutive HER2 expression led to elevated PFKFB3 expression and increased glucose metabolism, while inhibition of PFKFB3 suppressed glucose uptake, F26BP, glycolysis, and selectively decreased the growth of HER2-expressing breast cancer cells. In addition, treatment with lapatinib, an FDA-approved HER2 inhibitor, decreased PFKFB3 expression and glucose metabolism in HER2+ cells. In vivo administration of a PFKFB3 antagonist significantly suppressed the growth of HER2-driven breast tumors and decreased 18F-2-deoxy-glucose uptake. Taken together, these data support the potential clinical utility of PFKFB3 inhibitors as chemotherapeutic agents against HER2+ breast cancer.

CD44 correlates with clinicopathological characteristics and is upregulated by EGFR in breast cancer.

Cluster of differentiation 44 (CD44), a well-known transmembrane glycoprotein, serves as a promoting factor in the carcinogenesis and progression of a variety of neoplasms. Previous studies have demonstrated that aberrant expression of CD44 was associated with the initiation, invasion, metastasis, and therapy-resistance of breast cancer, but whether there was any association between CD44 and pathological characteristics of breast cancer or epidermal growth factor receptor (EGFR) has not been clearly elucidated. In this study, we utilized public microarray data analysis and tissue microarray technologies to display that CD44 level was enhanced in breast cancer and was significantly correlated with histological grade and the status of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 (HER2) and EGFR. Furthermore, mRNA expression of CD44 in breast tumors was positively correlated with basal cytokeratin markers KRT5 and KRT17, but inversely associated with luminal marker FOXA1. Besides, Kaplan-Meier analysis showed that high CD44 mRNA level had adverse impact on the progression-free survival of patients with HER2-expressing or basal-like breast cancer. Functionally, inhibition of EGFR activity by erlotinib impaired the invasion and migration ability of breast cancer cell lines. Western blot assays demonstrated that erlotinib treatment decreased the expression of CD44, accompanied with the reduced protein levels of mesenchymal and cancer stem cell markers. Collectively, this study suggested that the expression of CD44 was upregulated by EGFR pathway and CD44 had a robust impact on the development of breast cancer.

Abstract 2593: Association of mammographic density measures and breast cancer ‘intrinsic’ molecular subtypes

Abstract Percent mammographic density (PMD) is a risk factor for estrogen receptor (ER)-positive and ER-negative invasive breast cancer (BC). Gene expression profiling has identified molecular signatures that classify invasive BC into distinct subtypes that vary in their clinical behavior, response to treatment and likely, etiology. Immunohistochemical (IHC) staining of tumor sections using antibody panels can be used to classify these ‘intrinsic’ molecular subtypes. We evaluated whether density measures [PMD, absolute dense area (DA) and non-dense area (NDA)], are associated equally with all ‘intrinsic’ molecular subtypes. Pooled analysis of six cohort or case-control studies included 3492 women with invasive BC and 10,148 without, who underwent screening mammography a median 4 years prior to diagnosis (for cases). PMD, DA, and NDA were assessed from digitized film-screen mammograms using a computer-assisted thresholding technique, and categorized as 0-10%, 11-25%, 26-50% and 51%+ (PMD) or into quartiles (DA and NDA). Receptor status was abstracted from pathology records and supplemented by IHC staining. We classified tumors as Luminal A (ER+ and/or PR+ and HER2- and grade 1 or 2), Luminal B (ER+ and/or PR+ and HER2+ or Luminal A and grade 3), HER2 expressing (HER2+/ER-/PR-) and triple negative (TN) (ER-/PR-/HER2-). For TN, we also differentiated basal-like tumors (positive for EGFR and/or CK 5/6) from unclassified (negative on both markers). We used polytomous logistic regression to calculate the odds ratio (OR) of each ‘intrinsic’ subtype of BC by categories of PMD, DA or NDA, adjusting for age, body mass index and study. We tested for statistical heterogeneity of associations by subtype. Of 3492 invasive BC cases, 2217 (63%) were classified as Luminal A, 747 (21%) as Luminal B, 159 (5%) as HER2 expressing, and 369 (11%) as TN. Of TN, 203 were evaluated for CK 5/6 and EGFR, with 167 (82%) classified as basal-like and 36 (18%) unclassified. PMD was associated with BC risk across all subtypes. For Luminal A, compared to women with 11-25% PMD (reference), women with 0-10% had a reduced risk of BC (OR = 0.63 [95% confidence interval: 0.55, 0.74]) while women with 26-50% had an OR = 1.5 [1.3, 1.7] and women with 51%+ had the highest risk, OR = 2.3 [2.0, 2.7]. Similar BC associations were seen across PMD categories when comparing the five subtypes (P-heterogeneity = 0.63). Similar trends were seen for DA and BC across the five subtypes (P-het = 0.25). NDA was inversely associated with BC across subtypes, and there was suggestion of a stronger inverse trend among HER2-expressing BC compared to other subtypes (P-het = 0.09). Our results suggest mammographic density measures are associated with all ‘intrinsic’ molecular subtypes. However, NDA may be more strongly inversely associated with HER2-expressing than other subtypes. Understanding the importance of density measures for BC subtypes has significance for subtype-specific risk models. Citation Format: Aaron D. Norman, Rulla M. Tamimi, Christopher G. Scott, Kimberly A. Bertrand, Matthew R. Jensen, Daniel W. Visscher, Fergus J. Couch, John Shepherd, Bo Fan, Yunn-Yi Chen, Lin Ma, Andrew Beck, Vernon S. Pankratz, Karla Kerlikowske, Celine M. Vachon. Association of mammographic density measures and breast cancer ‘intrinsic’ molecular subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2593.

Combination inhibition of PI3K and mTORC1 yields durable remissions in mice bearing orthotopic patient-derived xenografts of HER2-positive breast cancer brain metastases.

Brain metastases represent the greatest clinical challenge in treating HER2-positive breast cancer. We report the development of orthotopic patient-derived xenografts (PDXs) of HER2-expressing breast cancer brain metastases (BCBM), and their use for the identification of targeted combination therapies. Combined inhibition of PI3K and mTOR resulted in durable tumor regressions in three of five PDXs, and therapeutic response correlated with reduction of 4EBP1 phosphorylation. The two non-responding PDXs showed hypermutated genomes with enrichment of mutations in DNA repair genes, suggesting an association of genomic instability with therapeutic resistance. These findings suggest that a biomarker-driven clinical trial of PI3K inhibitor plus an mTOR inhibitor should be conducted for patients with HER2-positive BCBM.