Trastuzumab Targeting Research Articles

Effects of immunorelated gene polymorphisms on trastuzumab targeting breast cancer cell in vitro.

Aim: To investigate the associations between genetic polymorphisms in immunorelated genes and PBMC-induced cytotoxicity to breast cancer cell with the treatment of trastuzumab in vitro.Methods: Trastuzumab-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) from 148 healthy donors and 13 BC patients was analyzed by flow cytometry. 16 SNPs in 7 immunorelated genes were genotyped by Sequenom Mass Array Genotype Platform.Results: Cytotoxicity in the trastuzumab treated PBMCs were significantly higher than those of the basal group. A wide variability in trastuzumab-mediated cytotoxicity was observed, and PBMC from individuals with the CD247 rs16859030 T genotype generated increased cytotoxicity than those with the CC genotype.Conclusion: The CD247 rs16859030 polymorphism affects trastuzumab-mediated cytotoxicity in vitro.

Efficacy and Safety of BP02 (Trastuzumab Biosimilar) in HER2-Positive Metastatic Breast Cancer: A Multicenter Phase III Study.

Trastuzumab targets human epidermal growth factor receptor 2 (HER2) receptors and is indicated for treating HER2-positive metastatic breast cancer. BP02,a recombinant IgG1 kappa humanized monoclonal antibody, is being developed as atrastuzumab biosimilar. The objective of this study was to evaluate the equivalence of BP02 with reference trastuzumab (RT: Herceptin®-EU) in patients withHER2-positive metastatic breast cancer. This double-blinded, 1:1 randomized, parallel-group, active-controlled, phase III equivalence trial recruited women aged 18-75 years with histologically/cytologically confirmed HER2- positive, locally recurrent or metastatic breast cancer with systemic metastasis, from 59 sites in India. Patients were randomly allocated 1:1 stratified by estrogen receptor/progesterone receptor status to receive BP02/RT (8-mg/kg loading dose on day 1-cycle 1, 6 mg/kg on day 1-cycles 2-8, of each 3-week cycle) combined with docetaxel (75mg/m2 on day 1-cycles 1-8) [induction phase]. Participants with complete or partial response, or stable disease at the end of the induction phase continued the study drug until disease progression/treatment discontinuation [maintenance phase]. The primary efficacy endpoint was the objective response rate per Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Between 23 September, 2020 and 16 September, 2022, 690 patients were recruited (n = 345 each to BP02/RT). At the end of the induction phase (intent-to-treat population), a similar proportion of patients achieved an objective response rate with BP02 (n = 231 [67.0%], 95% confidence interval [CI] 62.0, 71.9) and RT (n = 238 [69.0%], 95% CI 64.1, 73.9). The 95% CI of risk difference (-2.03, 95% CI -9.15, 5.09) and 90% CI of risk ratio (0.97, 90% CI 0.89, 1.06) were within equivalence margins of ±13% and (0.80, 1.25), respectively. Treatment-emergent adverse events leading to treatment withdrawal were reported in 2.9% and 3.2% patients with BP02 and RT, respectively. BP02 showed an equivalent efficacy and similar safety profile to RT at the end of 24 weeks. CTRI Number: CTRI/2020/04/024456.

Abstract LB014: The role of genetic alterations in metastatic development insights from a large-scale genomic database

Abstract Metastasis remains a major challenge in cancer treatment, with somatic genetic alterations being a crucial feature in progression. This study harnesses the whole genome sequencing data in the Genomics England biobank (GEL), cataloging of somatic alterations in the context of cancer metastasis. Our research aims to uncover the genetic underpinnings that facilitate the spread of cancer from its primary site to secondary locations by analyzing clinicogenomic data from 3047 cancer patients with metastatic progression. In this study, we analyzed the complex relationship between somatic alterations and cancer progression to uncover significant interactions among these alterations in a broad distribution of common tumor types among well-established oncogenic and tumor suppressor genes. In our study, we employed a Bayesian network approach, enabling us to not only pinpoint crucial genetic contributors to metastasis but also to consider other clinical variables, including patient treatment, sex, age, tumor grade and cancer types. In our analysis, LILRB2 was found to directly increase metastatic risk in hematologic cancer, with prior evidence suggesting its potential as a therapeutic biomarker in colorectal cancer and adversely related to patient prognosis in lung cancer. Similarly, MYL1, initially linked to tumor metastasis and immune infiltration in head and neck squamous cell carcinoma, was also found to increase the risk of metastasis in renal cancer, while interacting with the transcription factor E2F4, marker of poor prognosis in hepatocellular carcinoma. Our study not only confirmed the numerous key somatic gene alterations significantly linked to metastatic behavior across various cancers, but also established their significant roles.Our work highlights the potential of leveraging extensive genomic databases such as GEL to enhance our understanding of cancer metastasis. GEL’s paired germline and somatic data allows us to not only identify mutations unique to cancer cells but also to uncover germline variants that are associated with an elevated risk of cancer and the presence of more aggressive tumor characteristics. By characterizing metastatic risk features, we can explore the opportunities to stratify high-risk patients in clinical trials, with similar effort has been applied to brain metastasis in lung adenocarcinoma. Although tumor heterogeneity poses a treatment challenge due to cell diversity, previous work has demonstrated value in targeting the metastatic potential. For instance, trastuzumab target HER2-positive breast cancers prone to metastasis, markedly improving patient outcomes. Similarly, vemurafenib effectively controls metastatic Melanoma progression by targeting specific mutations like BRAFV600E. Understanding the genetic basis of metastasis could help identify high risk patients and lead to personalized combination therapies addressing various tumor subpopulations. Future research based on these findings could lead to the development of personalized combination treatment strategies that improve patient outcomes. Citation Format: Songjun Xu, Danyang Yu, Shuwei Li, Christopher Moy, Julio Molineros. The role of genetic alterations in metastatic development insights from a large-scale genomic database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB014.

Emerging insights into mechanisms of trastuzumab resistance in HER2-positive cancers.

HER2 is an established therapeutic target in breast, gastric, and gastroesophageal junction carcinomas with HER2 overexpression or genomic alterations. The humanized monoclonal antibody trastuzumab targeting HER2 has substantially improved the clinical outcomes of HER2-positive patients, yet the inevitable intrinsic or acquired resistance to trastuzumab limits its clinical benefit, necessitating the elucidation of resistance mechanisms to develop alternate therapeutic strategies. This review presents an overview of trastuzumab resistance mechanisms involving signaling pathways, cellular metabolism, cell plasticity, and tumor microenvironment, particularly discussing the prospects of developing rational combinations to improve patient outcomes.

Design, Synthesis, and Bioevaluation of a Novel Hybrid Molecular Pyrrolobenzodiazepine-Anthracenecarboxyimide as a Payload for Antibody-Drug Conjugate.

A novel series of hybrid molecules combining pyrrolobenzodiazepine (PBD) and anthracenecarboxyimide pharmacophores were designed, synthesized, and tested for in vitro cytotoxicity against various cancer cell lines. The most potent compound from this series, 37b3, exhibited a subnanomolar level of cytotoxicity with an IC50 of 0.17-0.94 nM. 37b3 induced DNA damage and led to tumor cell cycle arrest and apoptosis. We employed 37b3 as a payload to conjugate with trastuzumab to obtain the antibody-drug conjugate (ADC) T-PBA. T-PBA maintained its mode of target and internalization ability of trastuzumab. We demonstrated that T-PBA could be degraded through the lysosomal pathway to release the payload 37b3 after internalization. T-PBA showed a powerful killing effect on Her2-positive cancer cells in vitro. Furthermore, T-PBA significantly inhibited tumor growth in gastric and ovarian cancer xenograft mouse models without overt toxicity. Collectively, these studies suggest that T-PBA represents a promising new ADC that deserves further investigation.

Differential Gene Analysis of Trastuzumab in Breast Cancer Based on Network Pharmacology and Medical Images.

The purpose of this study was to use network pharmacology, biomedical images and molecular docking technology in the treatment of breast cancer to investigate the feasible therapeutic targets and mechanisms of trastuzumab. In the first place, we applied pubchem swisstarget (http://www.swisstargetprediction.ch/), (https://pubchem.ncbi.nlm.nih.gov/) pharmmapper (http://lilab-ecust.cn/pharmmapper/), and the batman-tcm (http://bionet.ncpsb.org.cn/batman-tcm/) database to collect the trastuzumab targets. Then, in NCBI-GEO, breast cancer target genes were chosen (https://www.ncbi.nlm.nih.gov/geo/). The intersection regions of drug and disease target genes were used to draw a Venn diagram. Through Cytoscape 3.7.2 software, and the STRING database, we then formed a protein-protein interaction (PPI) network. Besides, we concluded KEGG pathway analysis and Geen Ontology analysis by using ClueGO in Cytospace. Finally, the top 5 target proteins in the PPI network to dock with trastuzumab were selected. After screening trastuzumab and breast cancer in databases separately, we got 521 target genes of the drug and 1,464 target genes of breast cancer. The number of overlapping genes was 54. PPI network core genes include GAPDH, MMP9, CCNA2, RRM2, CHEK1, etc. GO analysis indicated that trastuzumab treats breast cancer through abundant biological processes, especially positive regulation of phospholipase activity, linoleic acid metabolic process, and negative regulation of endothelial cell proliferation. The molecular function is NADP binding and the cellular component is tertiary granule lumen. The results of KEGG enrichment analysis exhibited four pathways related to the formation and cure of breast cancer, containing Drug metabolism, Glutathione metabolism, Pyrimidine metabolism and PPAR signaling pathway. Molecular docking showed that trastuzumab has good binding abilities with five core target proteins (GAPDH, MMP9, CCNA2, RRM2, CHEK1). This study, through network pharmacology and molecular docking, provides new pieces of evidence and ideas to understand how trastuzumab treats breast cancer at the gene level.

Abstract 574: Allogenic HER2-CAR T cells overcome intrinsic trastuzumab resistance in vitro and in vivo in a preclinical model of breast cancer

Abstract The advent of anti-HER2 therapy has prominently prolonged the time of disease progression and survival for metastatic breast cancer patients, where a decent proportion is suffering from a HER2+ tumor. Beside classical approaches via antibodies against HER2, the use of Chimeric Antigen Receptor T (CAR-T) cells also in a solid cancer context is getting more and more attention. In our study, we evaluated the efficacy of Trastuzumab versus HER2 targeting CAR-T cells in a panel of human cancer cell lines (SK-OV3, Hs578T and JIMT-1) with different HER2 expression levels in vitro in 2D as well as 3D and in vivo in immunocompromised mice. In vitro, the tumor growth and invasion of the CAR-T cells was measured via fluorescence-based live cell imaging. On the last experiment day, a metabolic read-out (CellTiter-Glo, CTG assay) was performed. In vivo, we measured tumor growth inhibition (TGI) via caliper measurement and tumor tissue and hematopoietic organs were analyzed by flow cytometry (FC), Immunohistochemistry (IHC) and multiplex cytokine analysis. The HER2 targeting CAR-T cells eradicated the 3D spheroids of the HER2+ SK-OV3 as well as JIMT-1 in a dose-dependent manner. The untransduced control T cells did not influence the tumor growth in vitro. Trastuzumab displayed efficacy in 2D and 3D in SKOV3. As expected, JIMT-1 cells were resistant to trastuzumab treatment despite their positive HER2 status. The HER2- cell line Hs578T served as a negative control and proved to be resistant to any treatment in this study. The HER2-targeting CAR-T cells were applied in vivo in two different doses to JIMT-1 tumor bearing NSG mice (n=8/group) and tumor volume was measured over time. Again, the CAR-T cells were able to induce a complete remission. In contrast, the tumors displayed progressive disease under therapy with the untransduced T cells or Trastuzumab. Interestingly, the CAR-T cells induced a tumor swelling between eight- and twelve-days post injection very similar to the situation in the patient. Four weeks post injection the human T cells could be detected in spleen, peripheral blood, liver and bone marrow of the JIMT- bearing mice. The implication of this finding for possible side effects of the HER2 CAR´s remains to be elucidated. A PK/PD study is currently ongoing for this purpose. Taken together the 3D live cell imaging platform proved to be a feasible tool for efficacy testing of biologics as well as cellular therapies. Our in house developed HER2 CAR-T cells proved to be specific and effective in eradicating the targeted cancer cells in vitro and in vivo. The mechanism behind the modulated sensitivity of the HER2+ JIMT-1 cells against HER2-targeted treatment will help to shed some light in possible resistance mechanism and hopefully have some translational value for patients suffering from this disease. Citation Format: Eva Oswald, Gemma Moiset, Kanstantsin Lashuk, Sophie Vermond, Monique Hazenoot, Julia Schüler. Allogenic HER2-CAR T cells overcome intrinsic trastuzumab resistance in vitro and in vivo in a preclinical model of 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 574.

Synergistic activity of neratinib in combination with olaparib in uterine serous carcinoma overexpressing HER2/neu

IntroductionUterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with a poor prognosis. Approximately 30% of USC overexpress HER2/neu, a recognized target for trastuzumab in advanced/recurrent HER2/neu-positive USC. We evaluated the efficacy of the pan-c-erb inhibitor neratinib and the poly (ADP-ribose)-polymerase (PARP) inhibitor olaparib as single agents and in combination against USC cell lines and xenografts. MethodsIn-vitro cell-viability assays with olaparib, neratinib, and olaparib/neratinib were assessed using flow-cytometry based assays against a panel of USC cell lines with high and low HER2/neu expression. Homologous recombination deficiency (HRD) signatures were evaluated as described by Alexandrov et al. (Nature;2020;578:94–101) while downstream signaling affected by neratinib/olaparib exposure was assessed with immunoblotting. Efficacy of single- versus dual-agent inhibition was evaluated in-vivo using two USC-xenografts with 3+ HER2/neu expression. ResultsNeratinib was more potent than olaparib in suppression of in-vitro growth of HER2/neu 3+ cell lines (ARK1: p = 0.0047; ARK2: p = 0.0428) while no difference was noted against HER2/neu 1+ tumors (ARK4). Importantly, the combination of olaparib with neratinib synergistically improved tumor suppression compared to either single-agent in vitro. USC cells exposed to olaparib upregulated HER2/neu expression, while neratinib treatment increased PARP activity (ARK1: p < 0.0001; ARK2: p < 0.0001). Single-agent neratinib transiently inhibited in vivo growth of USC xenografts harboring HER2/neu gene amplification (ARK1: p < 0.05; ARK2: p < 0.05). In contrast, the combination of the two inhibitors caused a stronger and durable growth inhibition in both USC xenografts (ARK1: p < 0.05; ARK2: p < 0.05). ConclusionThe combination of olaparib and neratinib is active and synergistic against primary HER2/neu + USC. This combination may represent a novel therapeutic option for USC patients with HER2/neu+, homologous recombination-proficient tumors resistant to chemotherapy.

Host genetics influence response to targeted immunotherapy and effector cell capacity to perform key immune functions

Abstract Targeted immunotherapies have revolutionized cancer treatment, markedly improving patient cancer outcomes. Despite this rise to prominence as first line treatments for various malignancies, only a subset of patients respond to targeted immunotherapy. Trastuzumab targets tumor associated antigen (TAA) epidermal growth factor 2 (HER2). Differential response to trastuzumab suggests host genetics may influence response to treatment. To model this interaction, we utilize a genetically heterogeneous Diversity Outbred (DO) mouse model in an F1 cross (DOxBALB/c)F1. These genetically diverse mice (n=126) were orthotopically implanted with TUBO (BALB/c-syngeneic tumor) and treated with αNeu (7.16.4) mAb therapy. Like the human population, we observe a divergent response to therapy. Via genetic linkage analysis, we identify loci that correlate to a robust response. We confirm these loci in vivo using recombinant inbred collaborative cross (CC) strains containing/lacking driver loci (CCxBALB/c)F1. These loci alone are not sufficient to fully account for durable response to therapy; however, here we identify strains that uniformly eliminate tumor and conversely, are consistently refractory to treatment. Utilizing tissues from these strains, we evaluate canonical mechanisms of mAb therapy and show that tumor doubling time correlates with bone marrow derived macrophage capacity to phagocytose opsonized tumor in vitro via confocal microscopy. To better understand which cell populations of tumor infiltrate are expressing transcripts from these loci, we perform transcriptomic analysis on putative responder/non-responder strains, allowing us to see key differences in tumor infiltrating immune cell populations. Supported by NIH R37 CA220482, DMC Foundation, P30 CA022453

Abstract P1-02-03: Tumor-infiltrating lymphocytes but not HER2 copy number or ratio show prognostic value in trastuzumab-treated HER2-positive breast cancer

Abstract Background: Around 15% of all breast cancers are HER2+, a subtype that prior to HER2-targeted therapy was associated with a poor prognosis. With advances in treatment the survival has dramatically improved, and previous studies suggest that the level of HER2 copies are predictive of the effect of anti-HER2 therapies. However, some patients progress despite HER2-targeted therapy and routine HER2 testing is hampered by inaccuracy to discriminate patients with therapy-resistant disease. Furthermore, tumor-infiltrating lymphocytes (TILs) in the tumor stroma shows prognostic value not only in triple negative but also in HER2+ breast cancer. However, the clinical utility in the adjuvant setting for HER2+ disease needs further validation. The aim of this study was to investigate the prognostic significance of HER2/CEP17 ratio, HER2 copy numbers and TILs among early breast cancer patients treated with HER2-targeted therapy. Materials and methods: A retrospective study cohort comprising a total of 584 patients with HER2+ early breast cancer, all treated with the monoclonal antibody trastuzumab targeting HER2 during 2006-2014 in the Stockholm Region, Sweden, was identified and HER2 re-testing (silver in situ hybridization and immunohistochemistry (IHC)) was performed on 474 whole tissue sections prior to neoadjuvant (18.1%) or adjuvant therapy. Stromal TILs were manually assessed on all hematoxylin-eosin stained slides. Registry-based information on patient- and tumor characteristics including treatment were completed from medical records with a median follow-up of 8.0 years. Results: Among the 474 analyzed tumors, 11.4% were HER2 IHC score 2+ and 86.7% were 3+. In addition, 61.6% were ER+ and the median TIL score was 15%. The median HER2 copy number was 9.8 signals/cell and only 12.9% had &amp;lt;6.0 average HER2 signals/cell and 2.5% had a HER2/CEP17 ratio &amp;lt;2.0. Survival analysis showed no significant association between HER2 IHC score (recurrence: p=0.58; breast cancer specific death: p=0.69), HER2 signals (p=0.77; p=0.73), HER2/CEP17 ratio (p=0.73; p=0.85), ER status (p=0.55; p=0.11), PR status (p=0.97; p=0.46), Ki67 (p=0.14; p=0.77) or tumor grade (p=0.71; p=0.16) with risk of recurrence or breast cancer specific death. The hazard ratio (HR) for recurrence did not significantly differ by HER2 signals/cell (HR 1.12, CI 0.53-2.37, p=0.77) or ER status (HR 1.18, CI 0.68-2.05, p=0.55). Tumors with low TILs were independently associated with recurrence (cutoff &amp;lt;40%: HR 4.5, CI 1.4-14.9, p=0.012) and breast cancer specific survival (cutoff &amp;lt;30%: HR 3.5, CI 1.2-10.1, p=0.02). Among the 56 recurrences (11.8%), 37.5% had ER- primary tumors and 82.4% of the 34 HER2-analyzed recurrent tumors remained HER2+. Conclusions: This study demonstrates that routine clinicopathological data, including HER2 status, is insufficient to predict breast cancer recurrence among patients treated with HER2-targeted therapy. Neither HER2 levels, HER2/CEP17 ratio or ER status correlated to risk of recurrence. Importantly, TILs showed robust prognostic potential in trastuzumab-treated patients. Our results emphasize the need for detailed molecular analyses to understand the biology behind resistance to HER2-targeted therapy and to further tailor therapy for HER2+ breast cancer patients. Citation Format: Stephanie Robertson, Caroline Rönnlund, Irma Fredriksson, Theodoros Foukakis, Johan Hartman. Tumor-infiltrating lymphocytes but not HER2 copy number or ratio show prognostic value in trastuzumab-treated HER2-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-02-03.

Combination of Near-Infrared Photoimmunotherapy Using Trastuzumab and Small Protein Mimetic for HER2-Positive Breast Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer therapy based on a monoclonal antibody conjugated to a photosensitizer (IR700Dye) that is activated by near-infrared light irradiation. We previously reported on the use of NIR-PIT with a small protein mimetic, the Affibody molecule (6–7 kDa), instead of a monoclonal antibody. In this study, we investigated a combination of NIR-PIT for HER2-positive breast cancer cells (SK-BR3, MDA-MB361, and JIMT1) with HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate. HER2 Affibody and trastuzumab target different epitopes of the HER2 protein and do not compete. In vitro, the combination of NIR-PIT using both HER2 Affibody-IR700Dye conjugate and trastuzumab-IR700Dye conjugate induced necrotic cell death of HER2-positive breast cancer cells without damage to HER2-negative breast cancer cells (MCF7). It was more efficient than NIR-PIT using either the HER2 Affibody-IR700Dye conjugate alone or the trastuzumab-IR700Dye conjugate alone. Additionally, this combination of NIR-PIT was significantly effective against HER2 low-expressing cancer cells, trastuzumab-resistant cells (JIMT1), and brain metastatic cells of breast cancer (MDA-MB361). Furthermore, in vivo imaging exhibited the strong fluorescence intensity of both HER2 Affibody-IR700Dye conjugates and trastuzumab-Alexa488 conjugates in HER2-positive tumor, indicating that both HER2 Affibody and trastuzumab specifically bind to HER2-positive tumors without competing with each other. In conclusion, the combination of NIR-PIT using both HER2 Affibody and trastuzumab expands the targeting scope of NIR-PIT for HER2-positive breast cancer.

Quantification of HER2 in COS7 cells using quantum weak measurement.

A Mach-Zehnder interferometer system based on weak measurement was set up to determinate the concentration variation of molecule by measuring the phase difference change between the two optical paths. The spectrum of the light was recorded to monitor the concentration of trastuzumab (Herceptin), which is a humanised monoclonal antibody, targeted to human epidermal growth factor receptor 2 (HER2). The trastuzumab targeting to HER2 was real-time detected and continuously monitored, the HER2 numbers of COS7 cells on a coverslip was determined at pico-molar level. Our weak measurement enabled method proposes an alternative approach for the concentration detection of molecules, providing a promising functional tool for the quantification of HER2 in cancer cells, possibly promoting fields such as the diagnosis and treatment of cancer.

Leveraging outbred mice to identify genetic predictors of durable response to anti-tumor antibody

Abstract Trastuzumab is one of many monoclonal antibody (mAb) therapies targeting tumor-associated antigens (TAA) that have markedly improved cancer outcomes. Trastuzumab targets epidermal growth factor 2 (HER2). While it has long been considered the standard-of-care for HER2+ breast cancer treatment, differential response between patients suggests that host genetic background may contribute to an effective response to therapy. We utilize genetically heterogeneous Diversity Outbred (DO) mice to identify genetic elements that influence host response to TAA-mAb therapy. In a cohort of (DOxBALB/c)F1 mice bearing established BALB/c-syngeneic Neu+ (a HER2 homolog) TUBO mammary tumors, we find a divergent response to αNeu mAb therapy (clone 7.16.4). Several phenotypes were collected to compare immunological function to projected tumor doubling time (TDT). These include activation of peripheral anti-tumor T cells and IgG, natural killer cell ADCC competence, bone marrow derived macrophage ADCP/trogocytosis efficacy, and tumor immune cell infiltrate proportions. Contrary to the prospective mechanism of action of trastuzumab, we show that neither capacity for ADCC or ADCP correlate with a robust response to therapy. Following genetic linkage analysis, we identify a locus on Chr 10 that associates with TDT. To verify this locus, we use recombinant inbred collaborative cross (CC) mouse strains (CCxBALB/c) F1 mice that contain or lack this driver genotype. We find pooled survivorship between prospective responder/non-responder strains 2 months post tumor implantation differs by ~8%. This locus alone can’t fully account for a durable response to mAb therapy highlighting the multigenic nature of host immune response.

Dicer increases the indication for trastuzumab treatment in gastric cancer patients via overexpression of human epidermal growth factor receptor 2

The low proportion of gastric cancer (GC) patients with high HER2 expression limits the clinical application of trastuzumab, a humanized epidermal growth factor receptor 2 (HER2) antibody targeting for GC treatment. We found that Dicer was positively correlated with HER2 expression in GC tissue by immunostaining as well as induce HER2 overexpression without increasing invasiveness of GC cell. In addition, both the growth of GC referring to cell proliferation, invasion, migration and apoptosis was inhibited by Dicer overexpression. Moreover, the HER2 overexpression induced by Dicer provided more effective and additive target for trastuzumab to amplify the inhibition effect for GC cells in vitro and in vivo. Furthermore, as assessed in a subsequent experiment, calcitriol induced HER2 overexpression and amplified the inhibition effect of trastuzumab in GC cells referring to proliferation. Our finding demonstrated the calcitriol might increase indication of trastuzumab by inducing HER2 overexpression in GC patients. Dicer would be a potential target that extend the clinical indications of HER2 antibody in patients with low or negative HER2, who were not fit for HER2 antibody treatment before.

Novel Immunotherapeutic Approach in Gastric Cancer

Abstract Gastric cancer (GC) is suitable for immunotherapy because 80% of it display microsatellite and chromosomal instability, some mutations and DNA hypermethylation. Therefore, GC is more immunogenic. The immunotherapy with monoclonal antibodies, adoptive cell therapy and checkpoint inhibition are discussed. The commonly used monoclonal antibodies are Trastuzumab targeting HER2 and Bevacizumab suppressing VEGF and tumor angiogenesis. Treatment with tumor-specific T cells is called adoptive cell therapy. There is experience with the application of tumor infiltrating lymphocytes (TILs), cytotoxic T lymphocytes (CTLs) and cytokine-induced killer cells (CIK). This review discusses the therapy with innate immune cells with anti-tumor activity such as dendritic cells and NK cells. The checkpoint inhibition was also reviewed. In conclusion, it could be stated that the immunotherapy of GC has the potential to provide a more favorable outcome to patients with GC, but it also have some limitations which need to be considered.

Trastuzumab Specific Epitope Evaluation as a Predictive and Prognostic Biomarker in Gastric Cancer Patients

While human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) antibodies bind to the intracellular domain, trastuzumab binds to the extracellular epitope of HER2 receptor: target of drug action. We aimed to evaluate clinical significance of the new IHC method assessing the target of trastuzumab in gastric cancer (GC) patients and compare with conventional methods. Sixty-nine trastuzumab-treated GC patients were enrolled from two different cohorts. Additionally, we enrolled 528 consecutive GC patients to evaluate prognostic implications of HER2 test methods. HER2 status was assessed by trastuzumab IHC, HER2 IHC (4B5), and HER2 silver in situ hybridization (SISH). HER2 IHC showed 3+ in 48/69 trastuzumab-treated patients (69.6%), however, trastuzumab IHC showed 3+ in 25 (36.2%). Patients with trastuzumab IHC ≥2+ had significantly better progression-free survival (PFS) and overall survival (OS) than their counterpart (p = 0.014). In univariate analysis, trastuzumab IHC ≥2+ and HER2 IHC 3+ were only significant predictive factors for OS in trastuzumab-treated patients. Of the 528 consecutive GCs, patients with trastuzumab IHC ≥2+ had shorter disease-free survival (DFS) and OS (p = 0.008 and 0.031, respectively), while conventional methods failed to reveal any significant survival differences. HER2 assessment by trastuzumab IHC was different from conventional HER2 test results. Trastuzumab IHC was suggested to be a significant predictive factor for trastuzumab responsiveness and prognostic factor for consecutive GCs.

Investigating the utility of breast cancer (BC) molecular signatures and immune signature profiling (ISP) as predictors of pathological complete response (pCR) to neoadjuvant chemotherapy + trastuzumab (NAC+T) in HER2+ BC.

e12109 Background: Trastuzumab targets HER2 directly as well as contributes to activation of the immune system against the tumor. We investigated the utility of BC molecular signatures and ISP as predictors of pCR to NAC+T in HER2+ BC pts. Methods: The analysis included 73 consecutive HER2+ BC pts with pathological samples at diagnosis, who received NAC+T. RNA was extracted from 73/22 biopsy/surgical specimens (FFPE). RNA was analyzed using NanoString PanCancer Immunoncology 360 (IO360) panel with PAM50 spike-in. Raw gene counts were log2-transformed and normalized (using housekeeping genes). The analysis assessed 47 gene signatures including immune related and PAM50 signatures. Signatures were computed after removing samples with low expression of the housekeeping genes and normalization. Signature scores range from ~0 to 10 and have an average value of 5 in tumor samples from The Cancer Genome Atlas. Results: Overall, 46% of the pts achieved pCR; of 13 pts who were IHC 2+ and FISH positive, one achieved pCR. Significant associations were found between response to NAC+T (pCR, no response) and molecular signatures/ISP from the initial biopsy (Table). For pts who did not experience pCR and who underwent surgery, paired sample analysis was performed assessing molecular signatures/ISP in the initial biopsy sample vs the surgical sample. This analysis demonstrated a significant increase post-treatment (compared to the initial biopsy sample) in Risk of Recurrence (ROR) score (p=.036), interferon (IFN) downstream signature (p=.038), and antigen‐processing machinery (APM) loss (p=.002). Conclusions: Combining molecular BC signatures and ISP on biopsy samples may have a predictive value in HER2+BC pts for whom NAC+T is considered. Luminal A pts and IHC 2+ (FISH positive) are unlikely to achieve pCR with NAC+T. [Table: see text]

Heparan sulfate proteoglycans as trastuzumab targets in anoikis-resistant endothelial cells.

Anoikis is a form of programmed cell death induced by loss of contact from neighboring cells or from their extracellular matrix (ECM). Many tumorigenic cells are anoikis resistant, facilitating cancer progression and metastasis. Trastuzumab is a monoclonal antibody used for the treatment of breast and gastric cell cancer, but its mechanism of action is not well elucidated and its target molecules not well defined. Heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) play important roles in tumor development and in response of cancer cells to drugs. This study investigates the effect of trastuzumab on the expression of HSPGs and sulfated glycosaminoglycans (SGAGs) in anoikis-resistant endothelial cells. After trastuzumab treatment, endothelial cells resistant to anoikis show an increase in adhesion to fibronectin followed by a decrease in invasion, proliferation, and angiogenic capacity. In addition, a significant increase in the number of cells in the S phase of the cell cycle was also observed. In relation to HSPGs and SGAGs expression, we observed a decrease in syndecan-4 and perlecan expression, as well as in the heparan sulfate biosynthesis in anoikis-resistant endothelial cells after exposure to trastuzumab. Our results suggest that trastuzumab interacts with GAGs and proteoglycans of the cell surface and ECM and through this interaction controls cellular events in anoikis-resistant endothelial cells.

Paclitaxel reversed trastuzumab resistance via regulating JUN in human gastric cancer cells identified by FAN analysis.

In this study, we aim to use bioinformatics approach to identify paclitaxel-targeted modulators potentially involved in the process of reversing the trastuzumab resistance. Materials &methods: We extracted data from GSE77346 to identify potential trastuzumab resistance-related genes, used bioinformatics analysis and functional/activity network approach to find genes involved in trastuzumab resistance reversal. We identified hub differentially expressed genes related to trastuzumab resistance, trastuzumab targeting and paclitaxel targeting, respectively. We then found C-Jun may be critical in trastuzumab resistance reversal. This process may involve transcriptional activation of DUSP1by JUN, which lead to regulation of DUSP1-related signaling pathways. The present study revealed paclitaxel may reverse the trastuzumab resistance by JUN, which possibly in turn regulated DUSP1 and DUSP1-related signaling pathways.

Differential Co-expression and Regulatory Network Analysis Uncover the Relapse Factor and Mechanism of T Cell Acute Leukemia

The pediatric T cell acute lymphoblastic leukemia (T-ALL) still remains a cancer with worst prognosis for high recurrence. Massive studies were conducted for the leukemia relapse based on diagnosis and relapse paired samples. However, the initially diagnostic samples may contain the relapse information and mechanism, which were rarely studied. In this study, we collected mRNA and microRNA (miRNA) data from initially diagnosed pediatric T-ALL samples with their relapse or remission status after treatment. Integrated differential co-expression and miRNA-transcription factor (TF)-gene regulatory network analyses were used to reveal the possible relapse mechanisms for pediatric T-ALL. We detected miR-1246/1248 and NOTCH2 served as key nodes in the relapse network, and they combined with TF WT1/SOX4/REL to form regulatory modules that influence the progress of T-ALL. A regulatory loop miR-429-MYCN-MFHAS1 was found potentially associated with the remission of T-ALL. Furthermore, we proved miR-1246/1248 combined with NOTCH2 could promote cell proliferation in the T-ALL cell line by experiments. Meanwhile, analysis based on the miRNA-drug relationships demonstrated that drugs 5-fluorouracil, ascorbate, and trastuzumab targeting miR-1246 could serve as potential supplements for the standard therapy. In conclusion, our findings revealed the potential molecular mechanisms of T-ALL relapse by the combination of co-expression and regulatory network, and they provide preliminary clues for precise treatment of T-ALL patients.