HER2-positive Tumor Cells Research Articles

Evaluation of Potency and Specificity of Cryptophycin-Loaded Antibody-Drug Conjugates.

An enhanced variant of the antimitotic toxin cryptophycin was conjugated to the anti-Her2 monoclonal antibody (mAb) Trastuzumab upon Michael addition. Either antibodies with freed hinge-region cysteines or THIOMAB formats with engineered cysteines in the mAbs light chain were added to a maleimide derivative of cryptophycin. These Antibody-Drug Conjugates (ADCs) showed retained binding to Her2 positive tumor cells and highly efficient cell killing in double-digit pM range on high Her2-expressing SK-BR-3 cells. Two ADCs (DAR6, DAR3) showed superior cell killing of the cell lines JIMT-1 and RT112 with medium receptor expression level in comparison with a DAR6 MMAE ADC serving as reference. The observed cell cytotoxicity is target-dependent since no impact on cell viability was observed for low Her2-expressing MDA-MB468 cells. Particularly the DAR3 ADC in THIOMAB format exhibiting desirable biophysical properties and high potency emerged as a promising candidate for further in vivo investigations.

HER2-CD3-Fc Bispecific Antibody-Encoding mRNA Delivered by Lipid Nanoparticles Suppresses HER2-Positive Tumor Growth.

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor and tumor-associated antigen abnormally expressed in various types of cancer, including breast, ovarian, and gastric cancer. HER2 overexpression is highly correlated with increased tumor aggressiveness, poorer prognosis, and shorter overall survival. Consequently, multiple HER2-targeted therapies have been developed and approved; however, only a subset of patients benefit from these treatments, and relapses are common. More potent and durable HER2-targeted therapies are desperately needed for patients with HER2-positive cancers. In this study, we developed a lipid nanoparticle (LNP)-based therapy formulated with mRNA encoding a novel HER2-CD3-Fc bispecific antibody (bsAb) for HER2-positive cancers. The LNPs efficiently transfected various types of cells, such as HEK293S, SKOV-3, and A1847, leading to robust and sustained secretion of the HER2-CD3-Fc bsAb with high binding affinity to both HER2 and CD3. The bsAb induced potent T-cell-directed cytotoxicity, along with secretion of IFN-λ, TNF-α, and granzyme B, against various types of HER2-positive tumor cells in vitro, including A549, NCI-H460, SKOV-3, A1847, SKBR3, and MDA-MB-231. The bsAb-mediated antitumor effect is highly specific and strictly dependent on its binding to HER2, as evidenced by the gained resistance of A549 and A1847 her2 knockout cells and the acquired sensitivity of mouse 4T1 cells overexpressing the human HER2 extracellular domain (ECD) or epitope-containing subdomain IV to the bsAb-induced T cell cytotoxicity. The bsAb also relies on its binding to CD3 for T-cell recruitment, as ablation of CD3 binding abolished the bsAb's ability to elicit antitumor activity. Importantly, intratumoral injection of the HER2-CD3-Fc mRNA-LNPs triggers a strong antitumor response and completely blocks HER2-positive tumor growth in a mouse xenograft model of human ovarian cancer. These results indicate that the novel HER2-CD3-Fc mRNA-LNP-based therapy has the potential to effectively treat HER2-positive cancer.

A high hydrophobic moment arginine-rich peptide screened by a machine learning algorithm enhanced ADC antitumor activity.

Cell-penetrating peptides (CPPs) with better biomolecule delivery properties will expand their clinical applications. Using the MLCPP2.0 machine algorithm, we screened multiple candidate sequences with potential cellular uptake ability from the nuclear localization signal/nuclear export signal database and verified them through cell-penetrating fluorescent tracing experiments. A peptide (NCR) derived from the Rev protein of the caprine arthritis-encephalitis virus exhibited efficient cell-penetrating activity, delivering over four times more EGFP than the classical CPP TAT, allowing it to accumulate in lysosomes. Structural and property analysis revealed that a high hydrophobic moment and an appropriate hydrophobic region contribute to the high delivery activity of NCR. Trastuzumab emtansine (T-DM1), a HER2-targeted antibody-drug conjugate, could improve its anti-tumor activity by enhancing targeted delivery efficiency and increasing lysosomal drug delivery. This study designed a new NCR vector to non-covalently bind T-DM1 by fusing domain Z, which can specifically bind to the Fc region of immunoglobulin G and effectively deliver T-DM1 to lysosomes. MTT results showed that the domain Z-NCR vector significantly enhanced the cytotoxicity of T-DM1 against HER2-positive tumor cells while maintaining drug specificity. Our results make a useful attempt to explore the potential application of CPP as a lysosome-targeted delivery tool.

Phase 1 study of ZV0203, a first in class pertuzumab ADC, in patients with HER2 positive advanced solid tumors.

e15004 Background: ZV0203, a first in class (FIC) pertuzumab antibody-drug conjugate (ADC), targets HER2 positive tumor cells with tubulin inhibitor DUO5 as its payload via a stable and protease-cleavable valine-citrulline linker. In preclinical studies, ZV0203 demonstrated superior antitumor activity compared to Kadcyla in multiple tumor cell line xenograft models with no noticeable toxicity. GLP toxicity studies indicated that ZV0203 was well tolerated with an estimated therapeutic index of 35. Methods: This was an open-label, multicenter, phase 1, dose-escalation study in patients (pts) with HER2+ advanced solid tumors. Primary objectives were safety, tolerability and RP2D. Secondary objectives included PK, immunogenicity and preliminary clinical efficacy. ZV0203 was administered intravenously Q3W on a 21-day cycle. An accelerated titration design was used for the 0.3 and 0.6 mg/kg doses, followed by a 3+3 design for 1.2, 1.8, 2.7 and 3.6 mg/kg dose levels. Results: As of January 18, 2024, 15 pts with solid tumors (breast [11], colorectal [2], gastric [1], lung [1]) were enrolled across doses of 0.3-3.6 mg/kg with a median age of 52 years old (range 35-65), of which 9 pts received prior HER2-targeted therapy (trastuzumab ± pertuzumab) and 14 pts completed 21-day DLT assessment (0.3 mg/kg and 0.6 mg/kg [1 each], 1.2 mg/kg, 1.8 mg/kg, 2.7 mg/kg and 3.6mg/kg [3 each]) with no DLT incidence. Treatment-related adverse events (TRAE) occurred in 14 (93.3%) pts. The most frequent TRAEs were corneal epitheliopathy [9], elevated liver enzymes [6], dry eyes [6], neutropenia [4], anemia [4], leukopenia [3], and proteinuria [3], and most were grade 1-2 in severity. 5 pts experienced grade 3 TRAEs: lymphocytopenia (1.8mg/kg [1]) , corneal epitheliopathy (3.6 mg/kg [1]) and blurred vision (2.7 mg/kg [2], 3.6 mg/kg [1]), which were manageable during treatment. No unexpected safety signals were reported. TRAEs led to 13.3% (2/15) treatment discontinuation. Among 14 pts evaluated, 5 pts achieved a best tumor response of PR; 4 pts had SD; 1 pt had Non-CR/Non-PD per RECIST v1.1. Thus, disease control rate was 71.4% (10/14). PK results demonstrated that the PK profile of total antibody was similar to that of ZV0203 ADC, whose systematic exposure increased in a dose-dependent manner and remained stable after repeated administration. Concentration of DUO5 remained scarce, suggesting good stability of ZV0203. Conclusions: ZV0203 was well tolerated and showed clinically encouraging anti-tumor activity in heavily pretreated pts with HER2 positive cancer. Clinical trial information: NCT05423977 .

Innovative tumor interstitial fluid-triggered carbon dot-docetaxel nanoassemblies for targeted drug delivery and imaging of HER2-positive breast cancer

In this study, we have developed an innovative pH-triggered nanomedicine delivery system, targeting HER2-positive breast cancer cells for effective low-cost, imaging-guided drug delivery and precise therapy. The key feature of this system lies in its unique tumor interstitial fluid microenvironment-responsive drug release behavior which achieved tumor site-specific drug delivery. Our in vitro experiments demonstrated that the carbon dot-integrated material achieves more efficient DTX release (96.13 % at 72 h) in the tumor interstitial fluid microenvironment (pH 6.5), thereby boosting drug concentration at the tumor site and enhancing therapeutic efficacy. Further cell experiments confirmed the system's significant inhibitory effect on HER2-positive tumor cells SKBR3 in a pH 6.5 environment, and apoptosis assays indicating a notable increase in early cell apoptosis (from 8.39 % to 24.61 % compared with pH 7.4). Furthermore, the integration of HER2 aptamer within the carbon dot-based system enables targeted recognition and binding to tumor cells, ensuring more precise delivery of DTX while minimizing potential side effects. Crucially, the carbon dots in this system emit superior red fluorescence (the QY = 47.64 % excited at 535 nm compared with Rodamine 6G), enabling real-time visualization of the drug delivery process. This feature provides valuable feedback on treatment effectiveness, facilitating necessary adjustments. The small size (1.88 ± 0.48 nm) of carbon dots significantly improved their ability to penetrate biological barriers, while their low toxicity (no significant cell toxicity under 350 μg/mL) contributed to the formulation’s outstanding biocompatibility. Overall, this carbon dot-enhanced drug delivery system offers immense potential for enhancing drug efficacy, minimizing side effects, and providing real-time treatment monitoring, thus proposing a innovate strategy for breast cancer therapy.

Nanobody targeted photodynamic therapy induces cell death of HER2-positive tumor cells

SignificanceHuman epidermal growth factor receptor 2 (HER2) protein promotes the growth of cancer cells. Photodynamic therapy (PDT) using nanobodies (NBs) targeting HER2 is a promising treatment strategy for patients with HER2-positive cancers. ApproachIn this study, we evaluated the affinity of pheophorbide A (Pa)-conjugated HER2-targeted NBs (Pa-NB) conjugates for HER2-positive cells and the cytotoxicity induced by PDT using Pa-NB conjugates in vitro. MCF-7 cells with low HER2 expression and SKBR-3 with high HER2 expression were used. ResultsPa-NB conjugates had e high affinity for SKBR-3 cells and selectively induced cell death in them after laser irradiation. Pa-NB conjugates selectively accumulated in vitro. Upon laser irradiation, the Pa-NB conjugates selectively induced significant cytotoxicity in HER2-positive cells. ConclusionsPDT using Pa-NB conjugates, has been suggested as an attractive new therapeutic modality for HER2-positive cancers. Further studies are needed to assess the selectivity and antitumor effects of the Pa-NB conjugates in vivo.

Development of a novel HER2-CAR monocyte cell therapy with controllable proliferation and enhanced anti-tumor efficacy.

One of the significant challenges for cell therapies, such as chimeric antigen receptor (CAR)-T cell therapy, is the poor infiltration of immune cells into tumor tissues. CAR-monocytes/macrophages (CAR-M) are promising therapies because of their enrichment in the tumor microenvironment. Thus, we constructed a novel CAR-M to facilitate the infiltration of T cells and other immune cells. The suicide gene inducible caspase-9 (iCasp9) and anti-erb-b2 receptor tyrosine kinase 2 (HER2) CAR elements were transfected into THP1 (an immortalized human monocyte cell line) by lentivirus. The suicide efficiency and specific anti-tumor efficacy were assessed using flow cytometry, inCucyte, and tumor-bearing BALB/c-nude mouse models. The activation of related signaling pathways in CAR-THP1 activation was explored by transcriptome sequencing. Finally, the synergistic therapeutic efficacy of CAR-THP1 combined with RAK cell treatment was demonstrated in tumor-bearing NOD.CB17-Prkdcscid Il2rgtm1/Bcgen mouse models. We developed a novel CAR-THP1 which incorporated iCasp9, CD3ζ and CD147 intracellular segments, based on the first-generation HER2-CAR backbone. By constructing and comparing a series of CARs with different permutations, CAR-CD3ζ-CD147-iCasp9-THP1 was selected as the optimal combination. CAR-CD3ζ-CD147-iCasp9-THP1 initiated suicide quickly and efficiently under the control of iCasp9 gene, which enabled us to achieve controlled proliferation of CAR-THP1. CAR-THP1 also exhibited robust specific anti-tumor efficacy independently of T cells in vitro and in vivo. Through transcriptional sequencing, we found that CAR-THP1 tended to differentiate into the M1 phenotype and bridged innate and adaptive immunity. A combination of CAR-THP1 and Retronectin actived killer cells (RAKs) showed better therapeutic efficiency, as the metalloproteinases (MMPs) secreted by CAR-THP1 facilitated the degradation of the dense tumor matrix. This further assisted intratumoral infiltration of T cells and augmented the anti-tumor immune response. CAR-THP1 might be effective against HER2-positive tumor cells and has great potential for combination therapy with other immune cells.

Shearing of surface mucin saps tumor cell strength

Aberrant expression of transmembrane mucins promotes tumor progression and interferes with immunological and medicinal elimination of cancer cells. In a recent article, Pedram et al. directed an attenuated bacterial mucin-specific protease to HER2-positive tumor cells and observed decreased tumor growth rates and extended survival of mice bearing HER2-positive tumors.

Anti-PD-1/Her2 Bispecific Antibody IBI315 Enhances the Treatment Effect of Her2-Positive Gastric Cancer through Gasdermin B-Cleavage Induced Pyroptosis.

The majority of patients with human epidermal growth factor receptor 2 (Her2)-positive gastric cancer develop refractory to Her2-targeted therapy, whereupregulation of immune checkpoints plays an essential role. Herein, a recombinant fully human IgG1 bispecific antibody IBI315 targeting both PD-1 and Her2 is developed and its antitumor efficacy as well as the underlying mechanism is investigated. IBI315 crosslinksthe physical interaction between Her2-positive tumor cells and PD-1-positive T cells, resulting in significantly enhanced antitumor effects compared to each parent antibody or their combination, both in vitro and in vivo mouse tumor models reconstituted with human immune cells using patient-derived xenografts and organoids. Moreover, IBI315 treatment also induces the recruitment and activation of immune cells in tumors. Mechanistically, IBI315 triggers gasdermin B (GSDMB)-mediated pyroptosis in tumor cells, leading to the activation and recruiments of T cells. The activated T cells secret IFNγ, enhancing GSDMB expression and establishing a positive feedback loop of T cell activation and tumor cell killing. Notably, GSDMB is found to be elevated in Her2-positive gastric cancer cells, providing a rationale for IBI315's efficacy. IBI315 is supported here as a promising bispecific antibody-based immunotherapy approach for Her2-positive gastric cancer in preclinical studies, broadening the therapeutic landscape of this patient population.

Glutamine metabolic microenvironment drives M2 macrophage polarization to mediate trastuzumab resistance in HER2-positive gastric cancer.

Trastuzumab is a first-line targeted therapy for human epidermal growth factor receptor-2 (HER2)-positive gastric cancer. However, the inevitable occurrence of acquired trastuzumab resistance limits the drug benefit, and there is currently no effective reversal measure. Existing researches on the mechanism of trastuzumab resistance mainly focused on tumor cells themselves, while the understanding of the mechanisms of environment-mediated drug resistance is relatively lacking. This study aimed to further explore the mechanisms of trastuzumab resistance to identify strategies to promote survival in these patients. Trastuzumab-sensitive and trastuzumab-resistant HER2-positive tumor tissues and cells were collected for transcriptome sequencing. Bioinformatics were used to analyze cell subtypes, metabolic pathways, and molecular signaling pathways. Changes in microenvironmental indicators (such as macrophage, angiogenesis, and metabolism) were verified by immunofluorescence (IF) and immunohistochemical (IHC) analyses. Finally, a multi-scale agent-based model (ABM) was constructed. The effects of combination treatment were further validated in nude mice to verify these effects predicted by the ABM. Based on transcriptome sequencing, molecular biology, and in vivo experiments, we found that the level of glutamine metabolism in trastuzumab-resistant HER2-positive cells was increased, and glutaminase 1 (GLS1) was significantly overexpressed. Meanwhile, tumor-derived GLS1 microvesicles drove M2 macrophage polarization. Furthermore, angiogenesis promoted trastuzumab resistance. IHC showed high glutamine metabolism, M2 macrophage polarization, and angiogenesis in trastuzumab-resistant HER2-positive tumor tissues from patients and nude mice. Mechanistically, the cell division cycle 42 (CDC42) promoted GLS1 expression in tumor cells by activating nuclear factor kappa-B (NF-κB) p65 and drove GLS1 microvesicle secretion through IQ motif-containing GTPase-activating protein 1 (IQGAP1). Based on the ABM and in vivo experiments, we confirmed that the combination of anti-glutamine metabolism, anti-angiogenesis, and pro-M1 polarization therapy had the best effect in reversing trastuzumab resistance in HER2-positive gastric cancer. This study revealed that tumor cells secrete GLS1 microvesicles via CDC42 to promote glutamine metabolism, M2 macrophage polarization, and pro-angiogenic function of macrophages, leading to acquired trastuzumab resistance in HER2-positive gastric cancer. A combination of anti-glutamine metabolism, anti-angiogenesis, and pro-M1 polarization therapy may provide a new insight into reversing trastuzumab resistance.

T cells expressing a HER2-specific chimeric antigen receptor as treatment for breast cancer.

Human endothelial growth factor receptor-2 (HER2) is a leucine kinase receptor that is closely related to cell growth and differentiation. It is very weakly expressed in a few epithelial cells in normal tissue. Abnormal expression of HER2 usually leads to sustained activation of downstream signaling pathways, enabling epithelial cell growth, proliferation, and differentiation; this disturbs normal physiological processes and causes tumor formation. Overexpression of HER2 is related to the occurrence and development of breast cancer. HER2 has become a well-established immunotherapy target for breast cancer. We chose to construct a second-generation CAR targeting HER 2 to test whether it kills breast cancer. We constructed a second-generation CAR molecule targeting HER2, and we generated cells expressing this second-generation CAR through lentivirus infection of T lymphocytes. LDH assay and flow cytometry were perform to detect the effect of cells and animal models. The result indicated that the CARHER2 T cells could selectively kill cells with high Her2 expression. The PBMC-activated/CARHer2 cells had stronger in vivo tumor suppressive activity than PBMC-activated cells, and administration of PBMC-activated/CARHer2 cells significantly improved the survival of tumor-bearing mice, and induced the production of more Th1 cytokines in tumor-bearing NSG mice. We prove that the generated T cells carrying the second-generation CARHer2 molecule could effectively guide immune effector cells to identify and kill HER2-positive tumor cells and inhibit tumors in model mice.

Cbl induced ubiquitination of HER2 mediate immune escape from HER2-targeted CAR-T.

Breast cancer (BC) with high HER2 expression has higher recurrence rate and worse prognosis, and its immunotherapy is promising.Based on the high expression of HER2, develop Chimeric Antigen Receptor T-cell (CAR-T) and PDL-1 immunotherapy, and study the molecular pathways of related immune cells and recurrence.HER2-CAR-T cells were constructed using retroviruses, and their specific recognition and immune effects on HER2+ BC cells were verified by in vivo and in vitro experiments. PDL-1 was used as adjuvant immunotherapy, knocking down PDL-1 in tumor cells or dendritic cells, or depleted macrophages to study immune pathways. The negative regulation of HER2 by cbl was determined by IP, ubiquitination experiments, and segmented plasmids, elucidating the molecular mechanism of HER2+ BC recurrence after immunotherapy.HER2-CAR-T specifically recognizes HER2-positive tumor cells and inhibits tumor growth in vivo and in vitro, and anti-PDL1 treatment enhances the therapeutic effect of HER2-CAR-T on tumors. HER2-CART therapy eradicated solid tumors after PDL1 knockdown in dendritic cells. Immunotherapy of relapsed tumors lost HER2 expression by upregulating cbl.HER2-CAR-T shows specific recognition of HER2+ cells and can mediate immune response therapy with the cooperation of PDL-1.

Disruption of the sialic acid/Siglec-9 axis improves antibody-mediated neutrophil cytotoxicity towards tumor cells.

Upregulation of surface expressed sialoglycans on tumor cells is one of the mechanisms which promote tumor growth and progression. Specifically, the interactions of sialic acids with sialic acid-binding immunoglobulin-like lectins (Siglecs) on lymphoid or myeloid cells transmit inhibitory signals and lead to suppression of anti-tumor responses. Here, we show that neutrophils express among others Siglec-9, and that EGFR and HER2 positive breast tumor cells express ligands for Siglec-9. Treatment of tumor cells with neuraminidases or a sialyl transferase inhibitor significantly reduced binding of a soluble recombinant Siglec-9-Fc fusion protein, while EGFR and HER2 expression remained unchanged. Importantly, the cytotoxic activity of neutrophils driven by therapeutic EGFR or HER2 antibodies in vitro was increased by blocking the sialic acid/Siglec interaction, either by reducing tumor cell sialylation or by a Siglec-9 blocking antibody containing an effector silenced Fc domain. In vivo a short-term xenograft mouse model confirmed the improved therapeutic efficacy of EGFR antibodies against sialic acid depleted, by a sialyltransferase inhibitor, tumor cells compared to untreated cells. Our studies demonstrate that sialic acid/Siglec interactions between tumor cells and myeloid cells can impair antibody dependent tumor cell killing, and that Siglec-9 on polymorphonuclear cells (PMN) is critically involved. Considering that PMN are often a highly abundant cell population in the tumor microenvironment, Siglec-9 constitutes a promising target for myeloid checkpoint blockade to improve antibody-based tumor immunotherapy.

M1 polarization enhances the antitumor activity of chimeric antigen receptor macrophages in solid tumors

BackgroundChimeric antigen receptor macrophage (CAR-M) therapy is a novel cancer immunotherapy approach that integrates CAR structure and macrophage functions. CAR-M therapy has shown unique and impressive antitumor effects in immunotherapy for solid tumors. However, the polarization state of macrophages can affect the antitumor effect of CAR-M. We hypothesized that the antitumor activity of CAR-Ms may be further improved after inducing M1-type polarization.MethodsIn this report, we constructed a novel HER2-targeting CAR-M, which was composed of humanized anti-HER2 scFv, CD28 hinge region and FcγRI transmembrane domain and intracellular domain. Phagocytosis, tumor-killing capacities, and cytokine release of CAR-Ms were detected with or without M1-polarization pretreatment. Several syngeneic tumor models were used to monitor the in vivo antitumor activity of M1-polarized CAR-Ms.ResultsAfter polarization with LPS combined with interferon-γ in vitro, we found that the phagocytic and tumor-killing capacities of CAR-Ms against target cells were significantly enhanced. The expression of costimulatory molecules and proinflammatory cytokines was also significantly increased after polarization. By establishing several syngeneic tumor models in vivo, we also demonstrated that infusing polarized M1-type CAR-Ms could effectively suppress tumor progression and prolong the survival of tumor-bearing mice with enhanced cytotoxicity.ConclusionsWe demonstrated that our novel CAR-M can effectively eliminate HER2-positive tumor cells both in vitro and in vivo, and M1 polarization significantly enhanced the antitumor ability of CAR-M, resulting in a stronger therapeutic effect in solid cancer immunotherapy.

ICG-Dimeric Her2-Specific Affibody Conjugates for Tumor Imaging and Photothermal Therapy for Her2-Positive Tumors.

Human epidermal growth factor receptor 2 (Her2) is abundantly expressed in various solid tumors. The Her2-specific Affibody (ZHer2:2891) has been clinically tested in patients with Her2-positive breast cancer and is regarded as an ideal drug carrier for tumor diagnosis and targeted treatment. Indocyanine green (ICG) can be used as a photosensitizer for photothermal therapy (PTT), in addition to fluorescent dyes for tumor imaging. In this study, a dimeric Her2-specific Affibody (ZHer2) based on ZHer2:2891 was prepared using the E. coli expression system and then coupled to ICG through an N-hydroxysuccinimide (NHS) ester reactive group to construct a novel bifunctional protein drug (named ICG-ZHer2) for tumor diagnosis and PTT. In vitro, ICG-ZHer2-mediated PTT selectively and efficiently killed Her2-positive BT-474 and SKOV-3 tumor cells rather than Her2-negative HeLa tumor cells. In vivo, ICG-ZHer2 specifically accumulated in Her2-positive SKOV-3 tumor grafts rather than Her2-negative HeLa tumor grafts; high-contrast tumor optical images were obtained. However, Her2-negative HeLa tumor grafts were not detected. More importantly, ICG-ZHer2-mediated PTT exhibited a significantly enhanced antitumor effect in mice bearing SKOV-3 tumor grafts owing to the good photothermal properties of ICG-ZHer2. Of note, ICG-ZHer2 did not exhibit acute toxicity in mice during short-term treatment. Overall, our findings indicate that ICG-ZHer2 is a promising bifunctional drug for Her2-positive tumor diagnosis and PTT.

1705P HER2 intratumoral genetic and non-genetic heterogeneity in metastatic colorectal cancer

HER2-positive metastatic colorectal cancer (mCRC) is a distinct subtype for which HER2-targeted therapy is established. HER2 intratumoral heterogeneity (ITH), containing two types of heterogeneity–genetic heterogeneity (GH, a mixture of HER2 positive and negative tumor cells) and non-genetic heterogeneity (NGH, a mixture of HER2 positive tumor cells and amplified HER2 gene tumor cells without HER2 protein expression)– is a predictor of response to HER2-targeted treatment in breast cancer (BC). Here, using samples with mCRC, we evaluated the diagnostic accuracy of the Gene-Protein Assay (GPA), an assay assessing HER2 ITH by a combined HER2 immunohistochemistry (IHC) and bright-field HER2 in situ hybridization (ISH) on the same tissue section, and the frequency of HER2 ITH and heterogeneous patterns.

Herceptin-conjugated magnetic polystyrene-Agsbox nanoparticles as a theranostic agent for breast cancer

Breast cancer is a malignant tumor, which has derived from cells of the breast. Further, a relatively rapid metastasis, and resistance development against all the conventional drug combinations are major clinical issues in breast cancer patients as well as limitations like toxicity, genetic mutation, and metastasis make difficult the use of conventional therapy methods such as chemotherapy, radiotherapy, and local surgery. Therefore, considering the urgent needs, and high death rate in breast cancer cases, the development of new diagnosis and treatment regimens which diagnosed at the early stage and protected normal tissues required for clinical applications. Recently, the combination of tumor diagnosis and treatment within a single platform is a novel perspective, and magnetic nanoparticles are potential candidate owing to their low toxic effect, biocompatibility, biological degradability, superior magnetic properties, and targeting ability to overcome the problems of conventional diagnosis and therapy techniques. Considering these restrictions and requirements, the goal of this research was to investigate the potential of an innovative theranostic agent, which is soybean oil-based polystyrene (PS)-g-soybean oil graft copolymer containing AgNPs (PS-Agsbox) for treatment and MRI-based diagnosis of cancer. Herein, we designed targeted magnetic PS-Agsbox nanoparticles carrying thymoquinone (TQ) that is known for its anticancer potential against breast cancer, and herceptin (HER), which is to bind to the HER2 receptor protein on the surface of HER2-positive tumor cells, and acts by blocking the effects of it. We have successfully demonstrated selective binding, effective uptake of HER-conjugated magnetic PS-Agsbox nanoparticles into MDA-MB-231 (human breast carcinoma cells, a HER2-underexpressing cell line) and SKBR-3 (human breast cancer cells, a HER2-overexpressing breast cancer cell line) cell lines while no effect against L929 (mouse fibroblast cell line). Moreover, the magnetic resonance (MRI) properties of HER-conjugated magnetic PS-Agsbox nanoparticles were also confirmed.

Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray\u2122\u2013HER2-FISH workflow

BackgroundIn breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology.Methods50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany).ResultsApplying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive.ConclusionsThe DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

Molecular Classification of Gastric Cancer With Emphasis on PDL-1 Expression: The First Report From Iran.

Background:Gastric cancer is one of the lethal cancers and there is no effective treatment for these patients and still, 5-year survival rate is about 25% to 30%. Finding reliable biomarkers for early-stage diagnosis, targeted therapy, and survival prediction is a priority in this cancer.Objectives:In this study we were trying to know about the molecular classification of gastric cancers in a group of patients from the South of Iran.Patients and Methods:In a cross sectional study, 50 specimens of gastric cancer were selected that have enough tissue to be stained by immunohistochemistry (IHC). IHC was performed for Her-2, mismatch repair genes (MLH-1, MSH-2, MSH-6, and PMS-2), and PDL-1. Frequency of positive makers was compared with survival and outcome.Results and Conclusion:In our study, deficient MMR (dMMR) was detected in 4 patients (8.0%). PD-L1 expression in tumor cells (TC) was observed in 1 of 4 cases (25%) with PMS2 loss. However, PD-L1 in TCs and TILs (tumor infiltrating lymphocytes) was negative in 1 case with MLH1 loss and in 3 of 4 cases with PMS2 loss, which was not statistically significant. All of our 50 cases were positive for MSH2 and MSH6, 24% of which showed TCs with PDL-1 expression and 32% of them in TIL. HER2 was positive in 2 (2/50, 4.0%) cases, among which all of the cases were positive for PD-L1 expression in TCs and TILs, respectively. However, in HER2-negative group, 26.2% (11/42) and 28.6% (12/42) of tumors were positive for PD-L1 in TCs and TILs, respectively. The expression rate of PD-L1 in HER2 negative TCs was significantly higher than that in HER2 positive TCs (P = .033). Immunohistochemistry for Her-2 was equivocal in 6 cases (12.0%) none of which expressed PD-L1 in tumor cells. In our study minimum and maximum survival times from detection of gastric cancer were 1 and 87 months, respectively. The mean ± SD and median ± SD of overall survival time were 30.69 ± 4.88 and 18 ± 1.45 months, respectively. One and 3-year survival rates of 40% and 24%, respectively. PD-L1 expression was not associated with survival, but its expression was associated with intestinal type Lauren classification and negative HER-2. PD-L1 positivity in tumor cells or tumor infiltrating lymphocytes was not an independent prognostic factor in gastric cancer.

Immunotoxin IHP25-BT with low immunogenicity and off-target toxicity inhibits the growth and metastasis of trastuzumab-resistant tumor cells

Human epidermal growth factor receptor 2 (HER2) is overexpressed in some breast and gastric cancer patients. As the first HER2-targeteed therpeutic antibody, trastuzumab could significantly improve the prognosis of HER2-positive cancer patients. However, even responding patients inevitably get worse due to acquired resistance to trastuzumab after a period of treatment. Many HER2-targeted antibody drugs used wild-type tumor cells to conduct their corresponding preclinical experiments in vitro and in vivo. However, it is impossible to determine whether these newly developed drugs have antitumor effective to trastuzumab-resistant tumor cells. In the study, two trastuzumab-resistant HER2-positive tumor cell populations NCI-N87-TR and BT474-TR were generated. Then, we examined the anti-tumor effects of newly constructed immunotoxins with low immunogenicity and off-target toxicity based on the trastuzumab-resistant tumor cells both in vitro and in vivo. Results demonstrated that the immunotoxin IHP25-BT could not only effectively inhibit tumor growth but also inhibit liver metastasis of tumor cells in a mouse xenograft model. Furthermore, tumor tissue transcriptome sequencing was performed to clarify the potential mechanisms of inhibiting tumor cell distant metastasis by immunotoxin. In conclusion, this work describes a series of attractive therapeutic immunotoxins, the low immunogenicity and off-target toxicity making them promising for trastuzumab-resistant cancer therapy.