HER2-overexpressing Tumors Research Articles

Abstract 1530: Anti-HER2 CAR monocytes demonstrate targeted anti-tumor activity and enable a single day cell manufacturing process

Abstract Recent advances in cell therapy have led to significant efficacy in hematologic malignancies, but solid tumors remain an intractable challenge. We have previously developed a CAR macrophage (CAR-M) adoptive cell therapy platform and demonstrated potent anti-tumor activity in pre-clinical models. CAR-M overcome several of the barriers to efficacy in the solid tumor setting - trafficking, immunosuppression, lymphocyte exclusion, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. In order to streamline the manufacturing process, improve cell yields, and potentially improve tumor infiltration, we sought to evaluate the feasibility of directly engineering CD14+ monocytes to express CAR. Using the chimeric adenoviral vector Ad5f35, we engineered primary human CAR-monocytes to target tumors overexpressing HER2. CAR expression and viability both exceeded 90%. Anti-HER2 CAR-monocytes produced pro-inflammatory cytokines in response to antigen, specifically phagocytosed HER2 overexpressing target cells, and eradicated HER2-overexpressing tumor cells in a time and dose-dependent manner. CAR-monocytes efficiently differentiated into CAR-expressing macrophages in response to various maturation factors and cytokines within 3-5 days. Adenoviral based gene modification resulted in the upregulation of several pro-inflammatory markers on CAR-monocytes and pre-conditioned these cells to differentiate into M1 macrophages in the absence of exogenous M1 polarization factors. The M1 phenotype was maintained when CAR-monocytes were challenged with immunosuppressive cytokines in vitro. These cells demonstrated potent effector function after differentiation into macrophages, regardless of exposure to GM-CSF, M-CSF, or immunosuppressive factors. Anti-HER2 CAR monocytes carried broadly expressed myeloid chemokine receptors and responded to a panel of chemotactic factors. In order to optimize the cell manufacturing process associated with CAR-M, we established a closed-system, ultra-rapid CAR monocyte process. This process enabled same day manufacturing of the final CAR monocyte cell product, significantly reducing the cost of goods associated with autologous cell therapy as well as “vein to vein” time. This study demonstrated the successful development of CAR-monocytes with direct anti-tumor activity and capacity to differentiate into M1 polarized CAR-M. In addition, we established an ultra-rapid same-day CAR monocyte manufacturing process. The CAR-monocyte platform represents an advance in the field of adoptive cell therapy. Citation Format: Linara Gabitova, Brett Menchel, Rashid Gabbasov, Stefano Pierini, Andrew Best, Kayleigh Ross, Yumi Ohtani, Daniel Blumenthal, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Anti-HER2 CAR monocytes demonstrate targeted anti-tumor activity and enable a single day cell manufacturing process [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 1530.

Nature-Inspired Nanoparticles as Paclitaxel Targeted Carrier for the Treatment of HER2-Positive Breast Cancer.

Simple SummaryGreat advances have been made in the treatment of an aggressive subtype of breast cancer, known as HER2-positive subtype. However, the antitumor therapy most widely employed in the clinical fight against it has severe side effects, and the apparition of treatment resistances is frequent. To overcome these drawbacks, nano-sized particles had already been developed as targeted vehicles for the drug and the antibody that are usually administered as first-line treatment for this subtype of breast cancer. These nanoparticles showed better results than the drug that they transported. Nevertheless, to further reduce the drug effective dose and its toxicity to normal tissues, other nanoparticles, more advantageous than the previous ones, were developed in the current work. Compared to the previous nanoparticles, those prepared here proved to be more efficacious, and their potential administration may constitute an excellent approach to improve the outcomes of patients suffering from HER2-positive breast cancer. Despite the advances made in the fight against HER2-positive breast cancer, the need for less toxic therapies and strategies that avoid the apparition of resistances is indisputable. For this reason, a targeted nanovehicle for paclitaxel and trastuzumab, used in the first-line treatment of this subtype of breast cancer, had already been developed in a previous study. It yielded good results in vitro but, with the aim of further reducing paclitaxel effective dose and its side effects, a novel drug delivery system was prepared in this work. Thus, polydopamine nanoparticles, which are gaining popularity in cancer nanomedicine, were novelty loaded with paclitaxel and trastuzumab. The effectiveness and selectivity of the nanoparticles obtained were validated in vitro with different HER2-overexpressing tumor and stromal cell lines. These nanoparticles showed more remarkable antitumor activity than the nanosystem previously designed and, in addition, to affect stromal cell viability rate less than the parent drug. Moreover, loaded polydopamine nanoparticles, which notably increased the number of apoptotic HER2-positive breast cancer cells after treatment, also maintained an efficient antineoplastic effect when validated in tumor spheroids. Thereby, these bioinspired nanoparticles charged with both trastuzumab and paclitaxel may represent an excellent approach to improve current HER2-positive breast cancer therapies.

172P Men with breast cancer: Biology and stage at diagnosis in Eastern Europe - Balkan Collaborative Project

Breast cancer (BC) in men is treated as per the recommendations for menopausal women with BC. Despite the favorable tumor biology in men, survival outcomes are worse as compared to women. We aimed to study the tumor biology and stage at diagnosis of men with BC in Bulgaria, Romania and Serbia. This is a retrospective study of men with invasive BC, diagnosed at Medical University Pleven, Bulgaria (2002-2020), Institute of Oncology Cluj-Napoca, Romania (2000-2020) and Institute of Radiology and Oncology in Belgrade, Serbia (2000-2018). A total of 318 men with BC were included. Mean age and stage distribution at diagnosis are shown in the table. The mean age at diagnosis was significantly lower in Romania, compared to Serbia. About 80% of patients from each country had invasive ductal cancer. The rate of invasive lobular cancer ranged from 0.6% in Romania to 8.9% in Serbia. The receptor status subtype was unknown in almost half of the patients from each country. Among patients with known receptor status, over 93% were positive for at least one of the hormonal receptors. None of the patients had a HER2-overexpressing tumor subtype.Table: 172PNumber of patients included per country, age and stage at diagnosisCountry (period)Bulgaria (2002-2020)Romania (2000-2020)Serbia (2000-2018)n%n%n%Total48100169100101100Mean age at diagnosis64.861.464.8Stage*4810010910097100I918.865.51414.4II1939.62825.74849.5III1531.35752.33435.1IV510.41816.511.0*Percentage of patients in each stage is calculated as percentage from patients with known stage at diagnosis. Open table in a new tab *Percentage of patients in each stage is calculated as percentage from patients with known stage at diagnosis. As a rare disease, BC in men is a difficult and uncommon diagnosis. This is why, not only in Eastern Europe, it is diagnosed at a later stage, compared to women. In our study more than 80% of patients have regional lymph node metastases at diagnosis. Similarly to other reports, the vast majority of our patients had invasive ductal histology and hormone receptors expressing tumors. Raising the awareness of male BC, closer follow up and optimized registration could improve the outcomes of this disease.

Expression of Estrogen Receptor- and Progesterone Receptor-Regulating MicroRNAs in Breast Cancer.

In ~70% of breast cancer (BC) cases, estrogen and progesterone receptors (ER and PR) are overexpressed, which can change during tumor progression. Expression changes of these receptors during cancer initiation and progression can be caused by alterations in microRNA (miR, miRNA) expression. To assess the association of BC progression with aberrant expression of miRNAs that target ER and PR mRNAs, we quantified miR-19b, -222, -22, -378a, and -181a in BC samples (n = 174) by real-time PCR. Underexpression of miR-222 and miR-378a in stage T2–T4 BC was characteristic for HER2-overexpressing tumors. In addition, the expression of miR-181a and miR-378a was higher in these tumors than in tumors with a HER2 IHC score of 0 or 1+. In tumors with a Ki-67 index ≥ 14%, all tested miRNAs were underexpressed in BC with a high Allred PR score (6–8). In ER-and-PR–negative tumors, miR-22, miR-222, miR-181a, and miR-378a underexpression was associated with Ki-67 index > 35% (median value). MiR-19b and miR-22 underexpression could be a marker of lymph node metastasis in ER- and/or PR-positive tumors with HER2 IHC score 0. Thus, the association of miR-19b, miR-22, miR-222, miR-378a, and miR-181a levels with BC characteristics is influenced by the status of tumor ER, PR, HER2, and Ki-67.

ES30.04 Monoclonal Antibodies Targeting the ErbB Receptors

Blockade of a critical oncogenic target is envisioned as a promising strategy for treating cancer. The blockade of ErbB receptors with anti-ErbB monoclonal antibody represents one such strategy that has been successful in the treatment of HER2-overexpressing multiple solid tumors including NSCLC. Approximately 6% of EGFR mutated NSCLC have EGFR exon 20 insertion (E20Ins) mutations. EGFR E20Ins is the third most common type of EGFR mutation Multiple subtypes and structural differences may limit development of novel targeted therapies. Amivantamab is a fully human EGFR-MET bispecific antibody targeting activating and resistance EGFR mutations and MET mutations and amplifications. Amivantamab demonstrated clinical activity across diverse EGFR mutant NSCLC. In CHRYSALIS phase 1study, amivantamab showed robust efficacy with tolerable safety profile in post-platinum EGFR E20Ins NSCLC. HER 2 mutation occurs in about 2% of lung adenocarcinoma. HER2 mutation is more frequently found in never-smoker, women, Asian, and adenocarcinoma. Contrast to HER2 mutation, HER2 overexpression and amplification is not a definite biomarker to HER2 directed therapy. Anti-HER2 monoclonal antibody as monotherapy or in combination has shown conflicting results in HER2 overexpressed or amplified NSCLC. Compared to EGFR and HER2, HER3 has been somwhat ignored as a therapeutic target most likely due to lack of intrinsc kinase activity. However, upon ligand binding and subsequent dimerization with HER2, HER3 potently activates PI3K pathway and MAPK pathway. HER3 Monoclonal antibody as a monotherapu may not sufficient to fully inhibit tumor cells and combination with other ErbB therapy, chemotherapy may enhance therapeutic effects. Recently, monoclonal antibody targeting HER3 exhibited clinical activity in NRG1 fusion+ NSCLC. In conclusion, rapid pace of targeted drug development transforms lives of NSCLC patients with these rare actionable mutations. ErbB receptor, monoclonal antibody, rare mutation

The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

Abstract PS6-44: The significance of body mass index and absolute lymphocyte count as a prognostic factor for disease-free survival in Korean breast cancer patients

Abstract Introduction: The purpose of our study is to identify the association between body mass index (BMI) and absolute lymphocyte count (ALC), and to determine the effect of these two factors on the prognosis of breast cancer patients.Methods: We retrospectively identified 16,375 healthy female and 1,226 primary invasive breast cancer patients from Gangnam Severance Hospital. BMI and complete blood count (CBC) information at the time of diagnosed was collected. A multivariate Cox proportional hazard model was used to variable associated with disease-free survival (DFS).Results: BMI and ALC has positive correlation in breast cancer patients as well as health female (both P < .001). However, the effect of BMI and ALC on prognosis was the opposite. Overweight and obese had worse DFS (HR, 2.00; 95% CIs, 1.36-2.96; P < .001) than underweight or normal weight, but patients with high ALC had better DFS than those with low ALC (HR, 0.42; 95% CIs, 0.28-0.63; P < .001). When the risk stratification group was divided according to BMI /ALC, the high-risk group who had high BMI and low ALC had worse DFS than low-risk group (HR, 2.61; 95% CIs, 1.51-4.51; P = .001). In subgroup analysis, Her2-overexpressing and early stage tumor was more affected by BMI/ALC.Conclusions: The impact of BMI and ALC on prognosis in breast cancer was the opposite although BMI and ALC had positive correlation. Patients with high BMI and low ALC had worse DFS than others. Weight management is essential to breast cancer patients, and additional study about correlation between BMI and ALC affecting prognosis is needed in the future. Citation Format: Sung Min Ko. The significance of body mass index and absolute lymphocyte count as a prognostic factor for disease-free survival in Korean breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-44.

Abstract PS10-13: Impact of trastuzumab on Ipsilateral breast tumor recurrence after breast conserving surgery

Abstract Background: Trastuzumab is well known to be effective to control locoregional recurrence and distant metastasis of human epidermal growth factor receptor 2 (HER2)-overexpressing breast tumor. However, few studies have reported the effect of ipsilateral breast tumor recurrence (IBTR) in spite of higher incidence of IBTR for HER2 overexpressing subtype than other subtypes. The purpose of this study is to investigate the difference in the incidence of IBTR of HER2-overexpressing breast tumor according to adjuvant trastuzumab. Methods: We retrospectively reviewed 996 patients who had done surgery for HER2-overexpressing breast cancer between January 2000 and December 2017 in our institution. Patients with tumors smaller than 0.5cm without axillar node metastasis were excluded. As regarding IBTR as recurrence “in” the ipsilateral breast, only patients who had done breast conserving surgery were included. Results: There were 735 patients who had finished adjuvant trastuzumab as first planned and 555 patients with hormone receptor positive. Median follow-up period for all patients was 70.7 months (range 12.7-239.6 months). The 10-year IBTR-free survival rate showed a significant benefit for the group treatment with trastuzumab than the group without trastuzumab (97.0% versus 91.9%; p=0.007). In a multivariate analysis, presence of lymphovascular invasion (Hazard ratio [HR], 2.53; 95% Confidence interval [CI], 1.19 - 5.41), closed or involved resection margin (HR, 2.62; 95% CI, 1.20 - 5.74), positive hormone receptor (HR, 3.70; 95% CI, 1.69 - 8.08), positive axillar lymph node (HR, 5.21; 95% CI, 1.75 - 15.57), and omitted or uncompleted adjuvant trastuzumab (HR, 2.72; 95% CI, 1.11 - 6.67) were independent predictors of IBTR. However, subgroup analysis of the patients with hormone receptor negative tumor showed no benefit of adjuvant trastuzumab (98.1% versus 96.6%, p=0.669) while it controlled IBTR for hormone receptor positive tumor (95.7% versus 86.2%; p=0.002). When additionally analzyed, trastuzumab showed benefit for 10-year locoregional recurrence-free survival (95.5% versus 89.7%, p=0.012) and distant metastasis-free survival (93.5% versus 77.8%, p<0.001). Conclusions: Trastuzumab has a clinical benefit in not only locoregional recurrence but also IBTR among HER2-overexpression breast cancer, especially with negative hormone receptor. Citation Format: Jong Ho Cheun, Han-Byoel Lee, Hyeong-Gon Moon, Dong-Young Noh, Wonshink Han. Impact of trastuzumab on Ipsilateral breast tumor recurrence after breast conserving surgery [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-13.

Abstract PS9-11: Health-related quality of life for margetuximab + chemotherapy vs. trastuzumab + chemotherapy in the phase 3 SOPHIA trial of patients with pretreated HER2+ metastatic breast cancer

Abstract Background: Margetuximab (M), an investigational Fc-engineered, immune-activating, HER2-targeted monoclonal antibody, shares specificity with trastuzumab (T) and is being studied in HER2 overexpressing tumors including MBC. The randomized phase 3 SOPHIA trial (NCT02492711) in pretreated HER2+ MBC demonstrated PFS superiority by blinded analysis of M + chemotherapy over T + chemotherapy. M demonstrated a tolerable safety profile. Here, patient-reported health-related quality of life (HRQOL) is reported. Methods: In SOPHIA, 536 patients with HER2+ MBC and ≥ 2 prior HER2+ directed therapies, including pertuzumab, were randomized 1:1 to M (15mg/kg IV q3w) or T (6 [8 for loading dose] mg/kg IV q 3wk). Each antibody was given with physician’s choice chemotherapy (standard dose capecitabine, eribulin, gemcitabine, or vinorelbine). HRQOL was measured at baseline and on D1 of each odd cycle using the Network-Functional Assessment of Cancer Therapy-Breast Cancer Symptom Index (NFBSI) - 16 and EuroQol 5-Dimension 5 level (EQ-5D-5L) questionnaires. Descriptive statistics were performed on NFBSI-16 total score (possible score range: 0 [most symptomatic] - 64 [least symptomatic]), EQ-5D-5L overall score (possible score range: 0 [most symptomatic] - 100 [least symptomatic]), and subscales of each measures. Lower scores reflect a higher impact on HRQOL domains. Changes from baseline in NFBSI-16 total score and in EQ-5D-5L utility score were assessed using mixed model repeated measures analysis (MMRM) with treatment group, stratification factors, time, and treatment group by time interaction as covariates. Each analysis used an unstructured covariance matrix, and least square mean estimates were calculated at each time point. A Cox proportional hazard model was used to assess time to symptom progression, defined as a ≥ 5-point decrease from baseline in NFBSI-16, using the same covariates as in MMRM analyses.Results: HRQOL questionnaire completion rates were comparable between M and T treatment groups. Mean NFBSI-16 total scores and EQ-5D-5L overall scores were similar between treatment arms at baseline and at end of treatment. During cycles 11-17, a greater proportion of patients in the M group reported being bothered by side effects and fatigue “quite a bit” or “very much” on the NFBSI-16. However, adverse event assessments at corresponding time points were largely attributed to concomitant chemotherapy. Also, a smaller proportion of patients on T completed these measures at these times. Overall, least mean square estimates (95% CIs) of change from baseline NFBSI-16 total scores were -1.99 (-3.395, -0.594) in the M group, and -2.13 (-3.794, -0.469) in the T group. A slightly higher proportion of patients in the M group had ≥ 5-point decrease from baseline NFBSI-16 total score compared to those in the T group (M: 105 [39.5%]; T: 100 [37%]). However, this difference was neither prespecified nor statistically significant (Cox model HR: 0.88, 95% CI: 0.672, 1.164; p=0.382). Overall least square mean estimates (95% CIs) of change from baseline in EQ-5D-5L total scores were -0.93 (-4.493, 2.637) in the M group, and -3.93 (-8.253, 0.387) in the T group, suggesting that patients on M had less signs of deterioration compared to the T group; statistical significance testing was not prespecified. Conclusions: Overall, HRQOL domains, including symptoms and functioning, were maintained. Changes from baseline were similar between the 2 treatment groups. Treatment-specific symptoms were consistent with side effects associated with chemotherapy, not antibody study therapy. Findings support similar, acceptable safety profiles demonstrated previously for margetuximab and trastuzumab. Citation Format: Fatima Cardoso, Javier Cortes, William Gradishar, Seock-Ah Im, Mark D. Pegram, Hope S. Rugo, Gail S. Wright, Michelino De Laurentiis, Christelle Levy, Jean-Marc Ferrero, Janine Mansi, Raul Oyola, Francesco Ricci, Erik H. Jakobsen, Beatriz Uziely, Daniel Egle, Aristoteles Giagounidis, Kirstin Williams, Shengyan Hong, Edwin Rock, Giuseppe Curigliano. Health-related quality of life for margetuximab + chemotherapy vs. trastuzumab + chemotherapy in the phase 3 SOPHIA trial of patients with pretreated HER2+ metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS9-11.

Drug Conjugates Based on a Monovalent Affibody Targeting Vector Can Efficiently Eradicate HER2 Positive Human Tumors in an Experimental Mouse Model.

Simple SummaryDrug conjugates, consisting of a tumor targeting part coupled to a highly toxic molecule, are promising for treatment of many different types of cancer. However, for many patients it is not curative, and investigation of alternative or complimentary types of drug conjugates is motivated. Here, we have devised and studied a novel cancer cell-directed drug conjugate ZHER2:2891-ABD-E3-mcDM1. We found that it could induce efficient shrinkage and, in some cases, complete regression of human tumors implanted in mice, and thus holds promise to become a therapeutic agent for clinical use in the future.The human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in a variety of cancers and therapies targeting HER2 are routinely used in the clinic. Recently, small engineered scaffold proteins, such as affibody molecules, have shown promise as carriers of cytotoxic drugs, and these drug conjugates may become complements or alternatives to the current HER2-targeting therapies. Here, we investigated if a monovalent HER2-binding affibody molecule, ZHER2:2891, fused with a plasma half-life extending albumin binding domain (ABD), may be used as carrier of the cytotoxic maytansine derivate mcDM1. We found that the resulting drug conjugate, ZHER2:2891-ABD-E3-mcDM1, had strong affinity for its cognate molecular targets: HER2 and serum albumin. ZHER2:2891-ABD-E3-mcDM1 displayed potent cytotoxic activity towards cells with high HER2 expression, with IC50 values ranging from 0.6 to 33 nM. In vivo, an unspecific increase in uptake in the liver, imparted by the hydrophobic mcDM1, was counteracted by incorporation of hydrophilic and negatively charged glutamate residues near the site of mcDM1 conjugation. A dose-escalation experiment showed that increasing doses up to 15.1 mg/kg gave a proportional increase in uptake in xenografted HER2-overexpressing SKOV3 tumors, after which the tumors became saturated. Experimental therapy with four once-weekly injection of 10.3 or 15.1 mg/kg led to efficient regression of tumors in all animals and complete regression in some. Weight loss was detected for some animals in the group receiving the highest dose, suggesting that it was close to the maximum tolerated dose. In conclusion, the monovalent HER2-targeting affibody drug conjugate presented herein have potent anti-tumor activity in vivo.

Development of HER2-Specific Aptamer-Drug Conjugate for Breast Cancer Therapy.

In this study, HER2 RNA aptamers were conjugated to mertansine (DM1) and the anti-cancer effectiveness of the conjugate was evaluated in HER2-overexpressing breast cancer models. The conjugate of HER2 aptamer and anticancer drug DM1 (aptamer-drug conjugate, ApDC) was prepared and analyzed using HPLC and mass spectrometry. The cell-binding affinity and cytotoxicity of the conjugate were determined using confocal microscopy and WST-1 assay. The in vivo anti-tumoral efficacy of ApDC was also evaluated in mice carrying BT-474 breast tumors overexpressing HER2. The synthesized HER2-specific RNA aptamers were able to specifically and efficiently bind to HER-positive BT-474 breast cancer cells, but not to HER2-negative MDA-MB-231 breast cancer cells. Also, the HER2-specific ApDC showed strong toxicity to the target cells, BT-474, but not to MDA-MB-231 cells. According to the in vivo analyses drawn from the mouse xenografts of BT-747 tumor, the ApDC was able to more effectively inhibit the tumor growth. Compared to the control group, the mice treated with the ApDC showed a significant reduction of tumor growth. Besides, any significant body weight losses or hepatic toxicities were monitored in the ApDC-treated mice. This research suggests the HER2 aptamer-DM1 conjugate as a target-specific anti-cancer modality and provides experimental evidence supporting its enhanced effectiveness for HER2-overexpressing target tumors. This type of aptamer-conjugated anticancer drug would be utilized as a platform structure for the development of versatile targeted high-performance anticancer drugs by adopting the easy deformability and high affinity of aptamers.

Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv.

e23sFv is a HER2-targeted single-chain variable fragment (scFV) that was characterized as the targeting portion of a HER2-targeted tumour proapoptotic molecule in our previous study. In vitro antibody affinity maturation is a method to enhance antibody affinity either by complementarity-determining region (CDR) mutagenesis or by framework region (FR) engraftment. In the present study, the affinity of e23sFv was enhanced using two strategies. In one approach, site-directed mutations were introduced into the FRs of e23sFv (designated EMEY), and in the other approach e23sFv FRs were substituted with FRs from the most homologous screened antibodies (designated EX1 and EX2). Notably, EX1 derived from the FR engraftment strategy demonstrated a 4-fold higher affinity for HER2 compared with e23sFv and was internalized into HER2-overexpressing cells; however, EMEY and EX2 exhibited reduced affinity for HER2 and decreased internalization potential compared with EX1. The 3D structure of EX1 and the HER2-EX1 complex was acquired using molecular homology modelling and docking and the HER2 epitopes of EX1 and the molecular interaction energy of the EX1-HER2 complex were predicted. In the present study, it was demonstrated that scFv affinity improvement based on sequence alignment was feasible and effective. Moreover, the FR grafting strategy was indicated to be more effective and simple compared with site-directed mutagenesis to improve e23sFv affinity. In conclusion, it was indicated that the affinity-improved candidate EX1 may present a great potential for the diagnosis and treatment of HER2-overexpressing tumours.

Targeting and imaging of HER2 overexpression tumor with a new peptide-based 68Ga-PET radiotracer

Human epidermal growth factor receptor 2 (HER2) overexpression, as a predictive biomarker, is associated with more tumor aggressiveness and worse clinical outcomes in cancer, whereas it’s accurate identification has led to the choice of effective treatments in many patients. In this study, a peptide-based PET probe (68Ga-DOTA-(Ser)3-LTVSPWY) was developed for imaging HER2 expression in tumors. The DOTA-(Ser)3-LTVSPWY was labeled with 68Ga and then was evaluated in vitro with HER2-positive SKOV-3 cell line; moreover, the in vivo biodistribution and PET/CT imaging were performed in xenografted tumor-bearing nude mice. The 68Ga-DOTA-(Ser)3-LTVSPWY displayed the high radiochemical purity greater than 95% and good stability in normal saline and human serum. The cellular binding experiments showed that the cell uptake in HER2-positive ovarian cancer cells could be effectively blocked by non-labeled peptide. The Kd and Bmax values for radiolabeled peptide were obtained at 2.5 ± 0.6 nM and (3.4 ± 0.2) × 105 sites per cell, respectively. Biodistribution study demonstrated that tumor-to-blood and tumor-to-muscle ratios were about 1.73 ± 0.36 and 3.78 ± 0.17 at 120 min after the injection of the radiolabeled peptide, respectively. Tumor imaging by PET/CT exhibited high contrast tumor image at 60 min after injection in animal models. Consequently, the results were indicative of the specific accumulation of 68Ga-DOTA-(Ser)3-LTVSPWY peptide in HER2-positive tumors and the suitability of its application as a PET probe for the diagnosis of HER2-overexpression tumor.

NIMG-01. MRI VIRTUAL BIOPSY AND TREATMENT OF PRIMARY OR BRAIN METASTATIC TUMORS WITH TARGETED NANOBIOCONJUGATES

Abstract Differential diagnosis of brain magnetic resonance imaging (MRI) enhancement(s) remains a significant problem, which may be difficult to resolve without biopsy that can be often dangerous or even impossible. Such MRI enhancement(s) can result from metastasis of primary tumors such as lung or breast, radiation necrosis, infections or a new primary brain tumor (glioma, meningioma). Neurological symptoms are often the same on initial presentation. METHODS: We present a noninvasive tumor-specific imaging approach “MRI virtual biopsy” by engineering new nano imaging agents (NIA) on polymalic acid polymer (PMLA) scaffold containing linear-Gd-DOTA or star-Gd-DOTA moieties for differential diagnosis of brain tumors. The strategy is that after non-invasive MRI diagnosis recognizing the brain lesion on the basis of its molecular signatures, the primary cancer or brain metastasis (BM) is suppressed by tumor-specific molecular inhibitor(s), which is structurally similar to the used NIA. Anti-TfR antibody or Angiopep-2 (AP-2) peptide used to cross blood-brain barrier (BBB) by receptor-mediated transcytosis, and targeting antibody against EFGR- or HER2-overexpressing tumors were covalently attached to PMLA to recognize the tissues of interest. Delivery of contrast agents across BBB was studied by optical imaging. MRI signals in healthy brain and tumors were quantified using 9.4-Tesla MRI system. RESULTS: High specific signal values prevailed for 3 hours for NIA, in comparison with clinical Gd (MultiHance) that declined rapidly. In newly developed double tumor xenogeneic mouse models of brain metastasis this method allowed differential diagnosis of HER2- and EGFR-expressing brain tumors. After MRI diagnosis, breast and lung cancer brain metastases were successfully treated with similar tumor-targeted nanoconjugates carrying molecular inhibitors of EGFR or HER2 instead of imaging contrast agent. The treatment resulted in significant increase of animal survival and markedly reduced immunostaining for several cancer stem cell markers. Support: NIH grants: R01 CA188743, R01 CA206220, R01 CA209921

Heterogeneity of CEACAM5 in breast cancer.

CEACAM5 is overexpressed in many primary breast carcinomas. However, the exact role of CEACAM5 in breast cancer tumorigenesis remains unresolved. Here, we examined a repository of 110 cryopreserved primary breast carcinomas by immunohistochemistry to assess the distribution of CEACAM5 in tumor subtypes. The majority of estrogen receptor-positive and HER2-overexpressing tumors were CEACAM5-positive, whereas most of Triple-negative tumors were negative. Assessing sample sets of paired primary breast cancers and corresponding lymph node lesions from a total of 59 patients revealed a high correlation between primary tumor and lymph node with regard to CEACAM5-status. However, a notable subset of sample sets demonstrated intratumoral heterogeneity in the primary tumor, the metastatic lesion or both, suggesting that both CEACAM5-positive and –negative cells can play a role in tumor dissemination. When examining the consequence of expression of CEACAM5 in breast cancer cell lines in culture assays we found that CEACAM5-expressing cells were less invasive. In survival analysis, using cohort studies of breast cancer, expression of CEACAM5 predicted different clinical outcomes depending on molecular subtypes. Altogether, our analysis suggests that CEACAM5 plays a context-dependent role in breast cancer that warrants further investigation.

Incidence and Clinicopathological Features of Breast Cancer in the Northern Emirates: Experience from Sharjah Breast Care Center.

BackgroundBreast cancer is the most frequently reported cancer among women in the Middle East and North Africa (MENA) region. However, the available data about women breast cancer from the MENA and particularly from the Northern Emirates region of the United Arab Emirates (UAE) are scarce and inconsistent. Therefore, this study estimated the incidence, patient-specific factors including 25(OH)D levels, and clinicopathological features of breast cancer in women from the Northern Emirates.MethodsWe conducted this retrospective case–control study on 1,048 women who were referred to the Sharjah Breast Care Centre at University Hospital Sharjah between March 2016 and July 2018. Multivariate logistic regression was used for the statistical analysis of clinical data.ResultsOut of 1048 women with breast-related conditions referred to our canter, 94 (10%) were diagnosed with breast cancer (1 in 11), and approximately 1 in 5 of these women was younger than 40 years. After adjusting for age, body mass index and menopause status, women with serum 25-hydroxyvitamin D [25(OH)D] levels lower than 20 ng/mL were found to be at higher risk of breast cancer (odd ratio, 4.63; 95% CI, 2.61–8.23). The majority of breast cancer cases had invasive-ductal carcinoma with hormone-positive receptor molecular subtype (78 cases out of 94, 83%). HER2 overexpressing tumor (3+ by immunohistochemistry (IHC) or by fluorescence in situ hybridization (FISH)) was seen more in women younger than 40 years as compared to older women (7 cases out of 19 HER2 expressed tumors, p=0.007).ConclusionOur study cohort showed a mean age of diagnosis of breast cancer in women a decade earlier than in the developed countries. Furthermore, women with breast cancer tend to be serum 25(OH)D deficient at diagnosis and to have luminal A tumors.

Brain metastases from breast cancer

BackgroundBreast cancer (BC) is one of the commonest causes of brain metastases (BM): approximately 10–16 % of patients diagnosed with metastatic breast cancer will eventually develop BM during the course of their disease, however, certain subtypes have a higher risk of this event. The aim of this analysis was therefore to evaluate the prognosis and the pattern and imaging features of BM according to different BC subtypes. Patients and methodsWe retrospectively reviewed the case records of patients with breast cancer and evidence of brain metastases from the database of IPO Porto between 2014–2018. The data obtained were statistically analysed. ResultsWe analysed 147 patients with BM from BC. The triple-negative subtype had the shortest overall survival (OS) after BM, besides a short period of time between BC and BM. HER2 overexpressing tumors had the longest OS. The estrogen-receptor positive group had the greatest interval between initial BC diagnosis and diagnosis of BM. Larger lesions showed a heterogeneous contrast enhancement and were heterogeneous pn T2WI sequences; a hyposignal on T2*WI was also associated with larger lesions. Triple-negative BC tended to have more heterogeneous lesions on T1WI. We noticed that the hippocampus is rarely affected by metastatic lesions. ConclusionsBased on the BC subtype it is possible to make a prediction about the prognosis of the disease and some imaging features of the BM, but not about their pattern of distribution. These data support further research concerning prevention, early detection, and treatment of BM from BC.

Abstract 738: Utility of Assessing HER2 RNA expression for precision medicine

Abstract Genomic analysis of tumors is rapidly becoming routine clinical practice. Most of the next generation sequencing (NGS)-based oncology assay focused on DNA alteration data for single nucleotide variants (SNVs), insertions and deletions (indels) and copy number variants (CNVs) of genes which have targeted drug. Although RNA sequencing technology is increasingly being explored, its clinical utility still needs to be clarified. As there is a growing number of HER2-targeted therapies, in this study, we dissected the association between HER2 amplification, RNA overexpression, HER2 protein expression by immunohistochemistry (IHC) and reverse phase proteomic arrays (RPPA) in 618 breast tumors in the TCGA. We conducted unsupervised clustering analysis based on HER2 RNA-seq gene expression and RPPA protein data to identify HER2 overexpression samples. As expected there was a strong correlation with ERBB2 copy number with HER2 RNA expression (R2 = 0.56, P <0.001), and HER2 protein expression by IHC (R2 = 0.25, P < 0.001). RNA expression also correlated with HER2 IHC (R2 = 0.40, P < 0.001) and HER2 protein expression by RPPA (R2 = 0.68, P < 0.001). We performed unsupervised clustering analysis based on RPPA protein data and gene expression data of the HER2 gene, all the breast cancer samples were classified as two distinct clusters. Among the two subgroups, one subgroup had significantly higher expression and protein level of HER2 gene. The clustering results of most breast tumors were consistent with the IHC score and copy number data. The gene expression cutoff value to distinguish the HER2 overexpression subgroup from the other was about 204 FPKM. In addition, we identified two HER2 overexpression tumors which were classified as HER2 negative based on IHC. Studies are planned to test transcriptomic based actionability in patient derived models. Our results suggest that RNA-seq gene expression data may identify patients with HER2 overexpression that may be classified as HER2 negative based on IHC. Further studies are needed to determine the role of HER2 RNA overexpression as a predictive marker and to determine HER2 RNA expression across tumor types. Citation Format: Yifei Shen, Christian Cruz Pico, Vakul Mohanty, Amber Johnson, Jia Zeng, Kenna Mills Shaw, Ken Chen, Funda Meric-Bernstam. Utility of Assessing HER2 RNA expression for precision medicine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 738.

Abstract CT262: Multicenter, phase I/II trial of anastrozole, palbociclib, trastuzumab, and pertuzumab in HR-positive, HER2-positive metastatic breast cancer

Abstract Background: Overexpression or amplification of HER2 occurs in approximately 15-20% of patients and roughly half of these tumors are hormone receptor (HR) positive. The use of aromatase inhibitors in the metastatic setting is well established while significant improvement in overall survival has been established with the use of trastuzumab or pertuzumab in HER2-overexpressing tumors. This study will examine the combination of endocrine therapy, palbociclib, and dual HER2 therapy with pertuzumab and trastuzumab in women with metastatic HR+, HER2+ breast cancer. Trial Design: Multicenter, phase I/II trial of Anastrozole, Palbociclib, Trastuzumab and Pertuzumab in HR+, Her2+ metastatic breast cancer. Eligibility Criteria: Newly diagnosed stage IV HR+, HER2+ breast cancer patients. Specific Aims: Phase I: To determine the maximum dose tolerated of palbociclib. Phase II: To determine the clinical benefit rate (CBR) of treatment with anastrozole, palbociclib, trastuzumab, and pertuzumab in HR+, HER2+ metastatic breast cancer patients. Exploratory: Examine potential biomarkers of response to palbociclib including expression of cyclin D1, cyclin E1 and E2, CDK 2, CDK 4, CDK 6, retinoblastoma, phosphorylated retinoblastoma, and p16. RNA sequencing will be used to assess for other predictors of response in an unbiased manner to assess for correlation with inhibition of Ki-67 and phosphorylated retinoblastoma expression as well as evaluate for potential mechanisms of resistance. Methods: This study will evaluate the maximum tolerated dose (MTD) of the Anastrozole, Palbociclib, Trastuzumab and Pertuzumab. Once the MTD is reached, we will assess the clinical benefit rate using a Simon's II stage design among a maximum 30 patients. Accrual: Maximum of 36 subjects. Current Enrollment: Enrollment to phase I ended in October 2018 with one dose escalation to palbociclib 125mg, at which level one DLT was observed. Thus, the MTD was determined at palbociclib 125 mg and subsequent phase II patients will be treated at this dose level. Enrollment to phase II began in February 2019. 39% of planned patients have been enrolled as of January 2020. Clinical trial registry number: NCT03304080. Citation Format: Krystal Pauline Cascetta, Poulikos Poulikakos, Charles Shapiro, Julie Fasano, Aarti Bhardwaj, Hanna Irie, Anupama Goel, Paula Klein, Meng Ru, Amy Tiersten. Multicenter, phase I/II trial of anastrozole, palbociclib, trastuzumab, and pertuzumab in HR-positive, HER2-positive metastatic breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT262.

Abstract 2011: HER2 expression and M2-like tumor infiltrating macrophages associated with Cabazitaxel activity in gastric cancer

Abstract Gastric cancer (GC), responsible for ~700,000 deaths worldwide annually, is a dismal disease, with median survival for metastatic disease less than 1 year. We examined the efficacy and safety of cabazitaxel, a novel 3rd generation taxane, in the 2nd line treatment setting in advanced GC. Comprehensive correlative studies were performed to identify genetic aberrations or tumor microenvironment signatures associated with cabazitaxel activity. Patients with previously treated GC were eligible for this multicenter phase II study of single agent cabazitaxel (NCT01757171). The progression-free survival (PFS) rate at 3-mo using RECIST 1.1 was 28% (95%CI 17-42%) in taxane-naïve and 35% (95%CI 16-57%) in taxane treated cohorts. A fresh tissue biopsy of the tumor and matching adjacent non-tumor tissue was obtained from each of the 66 patients (87% of the study population) and was examined using whole exome sequencing (WES) and bulk RNAseq. We performed CIBERSORT deconvolution of the RNA expression data into its constituent immune cell types. Tumor samples were segregated into those with high or low macrophage M2 levels using the cohort specific median M2 abundance as the threshold. 26 tumor samples were examined for validation of the M2 signature by immunohistochemistry (IHC) using CD68 (pan-macrophage), CD163 (M2), and iNOS (M1) markers. GC WES showed numerous somatic alterations including missense mutations, chromosomal rearrangements, SNVs, small indels and CNAs with prevalent mutations in TP53 (26/47 cases), RHOA, and RTK/RAS signaling. Clinically actionable alterations included BRAF V600E, EGFR amplifications (10/47) and HER2 amplifications (8/47). One patient had a KRAS Q61H mutation predictive of resistance to a broad spectrum of RTK inhibitors. Other alterations included mutations in RTK signaling components, deletions of MTOR and STK11 suppressor gene and mutations in PI3K/mTOR pathway. We found that HER2 amplification was significantly more prevalent in responders, 50% HER2 positive among patients with PR/SD vs 10% in patients with PD (p=0.003). Patients with HER2 positive tumors had better PFS (p=0.04) and OS (45% 2-year survival vs 15%, p=0.002). Deconvolution analysis revealed an enrichment of an M2 macrophage signature in a cohort of patients having an improved PFS (45% vs 20% at 12 months, p=0.031). IHC analysis also showed M2 enrichment in 65% tissue samples examined (n=26). The M2-like macrophage signature was associated with improved outcome independent of HER2 amplification/ over-expression. In 8 out of 10 matched on-treatment biopsies, the M2-like signature significantly decreased post treatment. We have identified two novel biomarkers, HER2 overexpression and M2-high tumor macrophage signature, associated with improved outcomes in patients with GC treated with cabazitaxel. Additional correlative analyses and integration are underway. Citation Format: Sandipto Sarkar, Prashant K. Thakkar, Heinz Lenz, Peter Enzinger, Andrew H. Ko, Allyson J. Ocean, Yao Lu, Chao Zhang, Bhavneet Bhinder, Olga Plotnikova, Nikita Kotlov, Feliz Frenkel, Aleksander Bagaev, Olivier Elemento, Doron Betel, Paraskevi Giannakakou, Meredith E. Pittman, Manish A. Shah. HER2 expression and M2-like tumor infiltrating macrophages associated with Cabazitaxel activity in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2011.