HER2-low and ultra-low breast cancer (BC) have been recently proposed as new subcategories of HER2 BC, re-considering the immunohistochemical negative score of 0, 1+ or score of 2+/in situ hybridization (ISH) negative phenotype. In the present review we have outlined the needed criteria to exactly distinguish HER2 low and ultra-low BC. Recent clinical trials have demonstrated significant clinical benefits of novel HER2 directing antibody-drug conjugates (ADCs) in treating these group sof tumors. In particular, Trastuzumab-deruxtecan (T-Dxd), a HER2-directing ADC has been recently approved by the U.S. Food and Drug Administration as the first targeted therapy to treat HER2-low BC. Furthermore, ongoing trials, such as the DESTINY-Breast06, are currently evaluating ADCs in patients with HER2-ultra low BC. Finally we hope that new guidelines may help to codify HER2 low and ultra-low BC, increasing our knowledge of tumor biology and improving a targetable new therapeutical treatment.

Human epidermal growth factor receptor 2 (HER2) belongs to the ErbB family, a group of four transmembrane glycoproteins with tyrosine kinase activity, all structurally related to the epidermal growth factor receptor (EGFR). These tyrosine kinases are involved in the transmission of cellular signals controlling normal cell growth and differentiation. If this transmission goes awry, it can lead to dysregulated growth of the cell. HER2 specifically can be implicated in the pathogenesis of at least eight malignancies. HER2 positivity quickly became a well characterized indicator of aggressiveness and poor prognosis, with high rates of disease progression and mortality. After realizing the implication of HER2, it first became investigated as a target for treatment in breast cancer, and later expanded to areas of research in other cancers types. To this day, the most therapeutic advancements of anti-HER2 therapy have been in breast cancer, however, there have been strong advancements made in the incorporation of anti HER2 therapy in other cancer types as well. This comprehensive review dissects HER2 to its core incorporating the most up to date information. The topics touched upon are discussed in detail and up to 200 published sources from the most highly recognized journals have been integrated. The importance of knowing about HER2 is exemplified by the groundbreaking advancements that have been made, and the change in treatment plans it has brought to the oncological world in the last twenty years. Since, its ground breaking discovery there has been significant breakthroughs in knowledge regarding the actual receptor, the receptors biology, its mechanism of action, advancements in tests to detect HER2 and significant strides on how to best incorporate targeted treatment. Due to the success of this field thus far, the review concludes by discussing the future of novel anti-HER2 therapy currently in development that everyone should be aware of.

Up to 25% of early human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC) patients will relapse despite the improvement achieved by the introduction of HER2-targeted therapy. Beyond trastuzumab, other agents approved for early HER2+ BC include the monoclonal antibody pertuzumab, the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1) and the reversible HER2 inhibitor lapatinib. New agents, such as trastuzumab-deruxtecan or tucatinib in combination with capecitabine and trastuzumab, have also shown an improvement in the metastatic setting. Other therapeutic strategies to overcome treatment resistance have been explored in HER2+ BC, mainly in HER2+ that also overexpress estrogen receptors (ER+). One strategy has been to target the cyclin-dependent kinases 4/6 (CDK4/6) as they are downstream of HER2 and many of the cellular pathways associated with resistance to HER2-targeted therapies play a key role in cell cycle and proliferation. Different trials have explored these strategies with encouraging results, but definitive results are needed. In addition, HER2+ BC is known to be more immunogenic than other BC subgroups, with high variability between tumors. Different immunotherapeutic agents have been investigated in this setting, with promising but controversial results obtained to date.

Immune checkpoint blockade (ICB)-based therapy is revolutionizing cancer treatment by fostering successful immune surveillance and effector cell responses against various types of cancers. However, patients with HER2+ cancers are yet to benefit from this therapeutic strategy. Precisely, several questions regarding the right combination of drugs, drug modality, and effective dose recommendations pertaining to the use of ICB-based therapy for HER2+ patients remain unanswered. In this study, we use a mathematical modeling-based approach to quantify the growth inhibition of HER2+ breast cancer (BC) cell colonies (ZR75) when treated with anti-HER2; trastuzumab (TZ) and anti-PD-1/PD-L1 (BMS-202) agents. Our data show that a combination therapy of TZ and BMS-202 can significantly reduce the viability of ZR75 cells and trigger several morphological changes. The combination decreased the cell’s invasiveness along with altering several key pathways, such as Akt/mTor and ErbB2 compared to monotherapy. In addition, BMS-202 causes dose-dependent growth inhibition of HER2+ BC cell colonies alone, while this effect is significantly improved when used in combination with TZ. Based on the in-vitro monoculture experiments conducted, we argue that BMS-202 can cause tumor growth suppression not only by mediating immune response but also by interfering with the growth signaling pathways of HER2+ BC. Nevertheless, further studies are imperative to substantiate this argument and to uncover the potential crosstalk between PD-1/PD-L1 inhibitors and HER2 growth signaling pathways in breast cancer.

(1) Background: This study aimed to investigate the effect of increased HER-2 expression on tumor-infiltrating lymphocytes (TILs) and determine its impact on the prognosis of colorectal cancer (CRC) patients; (2) Methods: HER-2, CD4, CD8, CD19, LY6G, CD56, CD68, CD11b, and EpCam expression in CRC tissues and adjacent paracancerous tissues were assessed using multiplex fluorescence immunohistochemical staining. The correlation between HER-2 expression and the number of TILs in CRC tissues was analyzed. Kaplan-Meier and Cox proportional hazards models were used to analyze survival outcomes; (3) Results: The expression of HER-2 in tumor tissues was higher than that in paracancerous tissues (1.31 ± 0.45 vs. 0.86 ± 0.20, P < 0.05). Additionally, there was an increase in the numbers of CD4+, CD8+, CD19+, and CD68+ cells in CRC tissues (14.11 ± 1.10 vs. 3.40 ± 0.18, P < 0.005; 0.16 ± 0.12 vs. 0.04 ± 0.04, P < 0.005; 0.71 ± 0.46 vs. 0.25 ± 0.13, P < 0.0005; 0.27 ± 0.24 vs. 0.03 ± 0.11, P < 0.05). The increase in HER-2 expression was positively correlated with an increase in CD4, CD8, and CD19 (p < 0.0001). In HER-2-positive CRC tissues, CD68 expression was increased (0.80 ± 0.55 vs. 0.25 ± 0.22, P < 0.05). In HER-2-upregulated CRC tissues, CD4, CD8, CD19, CD68, CD11b, Ly6G, and CD56 expressions were elevated (0.70 ± 0.37 vs. 0.32 ± 0.17, P = 0.03; 0.22 ± 0.13 vs. 0.09 ± 0.06, P = 0.03; 0.31 ± 0.19 vs. 0.12 ± 0.08, P = 0.02; 1.05 ± 0.62 vs. 0.43 ± 0.21, P < 0.01; 1.34 ± 0.81 vs. 0.53 ± 0.23, P < 0.01; 0.50 ± 0.31 vs. 0.19 ± 0.10, P < 0.01; 1.26 ± 0.74 vs. 0.52 ± 0.24, P < 0.01). Furthermore, increased HER-2 expression is an independent risk factor for recurrence-free survival (RFS) in patients (P < 0.01, HR = 3.421); (4) Conclusions: The increased expression of HER-2 and its relationship with immune cells will provide new insights for immunotherapy in CRC patients.

Background and objectives: Primary brain tumors include any tumors arising in the brain whose prognosis is poor due to their histologic characteristics. The aim of this research was to evaluate the frequency of HER2 tumor marker in primary malignant brain tumors. Materials and Methods: This descriptive study was conducted on the samples admitted to the pathology laboratory with diagnosis of primary brain tumor during 2008–2015. Results: From among 107 patients (61.7% males and the rest females) with mean age of 40.4 years, the highest frequency of tumor location was in supratentorial region of the brain (including lobes and ventricles) (63.85% cases). High-grade astrocytoma had the highest prevalence at diagnosis (43.9%), followed by low-grade astrocytoma (37.4%). As for HER2 score, 42.1% of patients were HER2-positive (scores 2 & 3). On the other hand, 5.6% of patients were HER2-negative (-), 40.2% were positive (+), and 54.2% were positive (++). The patients with high-grade astrocytoma had older age (P < 0.001), higher HER2 positivity (P = 0.024) and percentage (P < 0.001) compared to the patients with low-grade astrocytoma. Conclusions: HER2 expression is dependent on the type of brain tumors. High expression of HER2 in high-grade astrocytoma may be useful for therapeutic purposes. The future research is needed to confirm these results with a large number of patients in different areas.

Since the discovery of the human epidermal growth factor receptor 2 (HER2) as an oncogenic driver in a subset of breast cancers and the development of HER2 directed therapies, the prognosis of HER2 amplified breast cancers has increased meaningfully. Next to monoclonal anti-HER2 antibodies and tyrosine kinase inhibitors, the antibody-drug conjugate T-DM1 is a pillar of targeted treatment of advanced HER2-positive breast cancers. Currently, several HER2 directed antibody-drug conjugates are under clinical investigation for HER2 amplified but also HER2 expressing but not amplified breast tumors. In this article, we review the current preclinical and clinical evidence of the investigational drugs A166, ALT-P7, ARX788, DHES0815A, DS-8201a, RC48, SYD985, MEDI4276 and XMT-1522.

Background: We aimed to identify overexpressed genes or related pathways associated with good responses to anti-HER2 therapy and to suggest a model for predicting drug response in neoadjuvant therapy with trastuzumab in HER2-positive breast cancer patients. Methods: We recruited 64 women with breast cancer and categorized them into three groups according to anti-HER2 therapy response. RNA from twenty core needle biopsy paraffin-embedded tissues and four cultured cell was extracted, reverse transcribed, and subjected to microarray. The obtained data were analyzed using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and database for annotation, visualization, and integrated discovery. Results: In total, 6,656 genes differentially expressed between trastuzumab-susceptible and trastuzumab-resistant cell lines (3,224 upregulated and 3,432 downregulated). Expression changes in 34 genes in several pathways were found to be related to the response to trastuzumab-containing treatment, interfering with adhesion to other cells or tissues (focal adhesion) and regulating extracellular matrix interactions and phagosome action. Thus, decreased tumor invasiveness and enhanced drug effects might be the mechanisms explaining the better drug response in complete response group. Conclusions: This multigene assay-based study provides insights into breast cancer signal transduction and the possible prediction of treatment response to targeted therapies such as trastuzumab.

The binding activity of various trastuzumab biosimilars versus the branded trastuzumab towards the glycosylated extracellular domain of the HER2 target in the presence of pertuzumab was in-vestigated. We employed size exclusion chromatography with tetra-detection methodology to simultaneously determine absolute molecular weight, concentration, molecular size, and intrinsic viscosity. All trastuzumab molecules in solution exhibit analogous behavior in their binary action towards HER2 regardless of the order of addition of trastuzumab/pertuzumab. This analogous behavior of all trastuzumab molecules, including biosimilars, highlights the robustness and con-sistency of their binding activity towards HER2. Furthermore, the addition of HER2 to a mixture of trastuzumab and pertuzumab leads to increased formation of high-order HER2 complexes, up to concentrations of one order of magnitude higher than in the case of sequential addition. The observed increase suggests a potential synergistic effect between these antibodies, which could enhance their therapeutic efficacy in HER2-positive cancers. These findings underscore the im-portance of understanding the complex interplay between therapeutic antibodies and their target antigens, providing valuable insights for the development of more effective treatment strategies.

Breast cancer patients with high levels of HER2 (human epidermal growth factor receptor 2) expression had worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa). This was then altered to produce H2Mab-77-mG2a-f, an afucosylated mouse IgG2a. In the present work, we examined the reactivity of H2Mab-77-mG2a-f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressed breast cancer cell line, was identified by H2Mab-77-mG2a-f with a strong binding affinity [a dissociation constant (KD): 5.0 × 10-9 M]. H2Mab-77-mG2a-f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in western blot analysis. Furthermore, H2Mab-77-mG2a-f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2Mab-77-mG2a-f. Additionally, in the BT-474-bearing tumor xenograft model, H2Mab-77-mG2a-f substantially suppressed tumor development when compared to the control mouse IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H2Mab-77-mG2a-f. These findings point to the possibility of H2Mab-77-mG2a-f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesopha-geal and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probes for radionuclide imaging. In clinical studies, the DARPin [99mTc]Tc-(HE)3-G3, labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)3), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G3C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)3-linker (designated as G3-(G3S)3C) would further improve the contrast of imaging using 99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinity in the range 1.9-5 nM. Biodistribution of [99mTc]Tc-G3-G3C, [99mTc]Tc-G3-(G3S)3C, and [99mTc]Tc-G3-E3C in mice was compared with the biodistribution of [99mTc]Tc-(HE)3-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-(HE)3-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.

HER2/neu positive breast tumors predict a high mortality and comprise 25-30% of breast cancer. We have shown that Flavokawain A (FKA) preferentially reduces the viabilities of HER2 overexpressing breast cancer cell lines (i.e. SKBR3 and MCF7/HER2) versus those with less HER2 expression (i.e. MCF7 and MDA-MB-468). FKA at cytotoxic concentrations to breast cancer cell lines also has minimal effect on the growth of non-malignant breast epithelial MCF10 cells. FKA induces G2M arrest in cell cycle progression of HER2 overexpressing breast cancer cell lines through inhibition of Cdc2 and Cdc25C phosphorylation and down-regulation of Myt1 and Wee1expression leading to increased Cdc2 kinase activities. In addition, FKA induces apoptosis in SKBR3 cells by increasing the protein expression of Bim and BAX and decreasing expression of Bcl₂, Bcl_x/L , XIAP and survivin. FKA also down-regulates the protein expression of HER-2 and inhibits AKT phosphorylation. Herceptin plus FKA treatment leads to enhanced growth inhibitory effect on HER-2 overexpressing breast cancer cell lines through down-regulation of Myt1, Wee1, Skp2, Survivin and XIAP. Our results suggest the promise of FKA as a novel apoptosis inducer and G2 checkpoint abrogating agent in combination with Herceptin for treatment of HER2 overexpressing breast cancer.

The HER2 overexpression occurs in 10–20% of breast cancer patients. HER2+ tumors are characterized by increase in Ki67, early relapse and increased metastasis. There is little known about factors influencing early stages of HER2- tumorigenesis and diagnostic markers. Previously, it was shown that deletion of NEDD9 in mouse models of HER2 cancer interferes with tumor growth, but the role of NEDD9 upregulation is currently unexplored. We report that NEDD9 is overexpressed in a significant subset of HER2+ breast cancers and correlates with limited response to anti-HER2 therapy. To investigate the mechanisms through which NEDD9 influences HER2-dependent tumorigenesis, we generated MMTV-Cre-NEDD9 transgenic mice. The analysis of mammary glands shows extensive ductal epithelium hyperplasia, increased branching, and terminal end bud expansion. The addition of oncogene Erbb2 (neu) leads to development of early hyperplastic benign lesions earlier (~16 weeks) with a significantly shorter latency than the control mice. Similarly, NEDD9 upregulation in MCF10A-derived acini leads to hyperplasia like DCIS. This phenotype is associated with activation of ERK1/2 and AURKA kinases leading to an increased proliferation of luminal cells. These findings indicate that NEDD9 is setting permissive conditions for HER2-induced tumorigenesis, thus identifying this protein as a potential diagnostic marker for early detection.

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti‐EGFR and human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients’ survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti‐EGFR mAb (E134Bf) and a mouse-dog chimeric anti‐HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermore, E134Bf-H77scFv exerted antibody‐dependent cellular cytotoxicity and complement‐dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.

Background: Human epidermal growth factor receptor 2 (HER-2) overexpression can be found in 15-20% of breast cancers, and it strongly correlates with aggressive clinical behavior and adverse prognosis. The first-line treatment for HER-2 positive metastatic breast cancers is the combination of trastuzumab, pertuzumab, and taxane (PTH). ABP 980 is a biosimilar of the innovator trastuzumab and is characterized by highly comparable effectiveness. Methods: The group of 61 patients with HER-2 positive MBC received biosimilar ABP 980 plus pertuzumab and docetaxel from November, 18, 2018 to December, 24, 2019. The response to therapy, overall survival (OS), progression-free survival (PFS), metastases, and adverse effects among patients were determined and analyzed. Results: Initially, 42 women responded partially to the treatment and their median PFS was 27 months. Median PFS for the whole group was 18 months. Cardiotoxicity of treatment was noticed in all patients in the form of the reduction in left ventricular ejection fraction but only in 2 cases, it was the reason for withdrawing from therapy. Conclusion: Biosimilar ABP 980 is registered in the same indications as the innovator trastuzumab and their effectiveness, as well as side effects, are comparable. The costs of biosimilar make the therapy more accessible and thus more patients with MBC around the world can receive relevant treatment.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel cancer-specific anti-HER2 antibody, named H₂Mab-250/H₂CasMab-2 (IgG₁, kappa). H₂Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H₂Mab-250 did not react with non-transformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs including H₂Mab-119 (IgG₁, kappa) reacted with both cancer and normal epithelial cells. Furthermore, a core-fucose deleted IgG_2a-type H₂Mab-250 (H₂Mab-250-mG_2a-f) could trigger the antibody-dependent cellular cytotoxicity activity to BT-474, but not to HaCaT cells. Furthermore, H₂Mab-250-mG_2a-f exhibited an in vivo antitumor effect against BT-474 xenograft. Immunohistochemical analysis demonstrated that H₂Mab-250 possesses much higher reactivity to the HER2-positive breast cancer tissues compared to H₂Mab-119, and did not react with normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H₂Mab-250. H₂Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

Human epidermal growth factor receptor 2 (HER2) is a major prognostic and predictive marker overexpressed in 15- 20 % of breast cancers. The diagnostic reference standard for selecting patients for HER2-targeted therapy is by analysis of tumor biopsies for HER2 expression and amplification. Previously patients were either defined as HER2-positive or negative, however, with approval of novel treatment options, specifically the antibody-drug conjugate trastuzumab deruxtecan, many breast cancer patients with tumors expressing low levels of HER2 and lacking HER2 amplification have become eligible for HER2-targeted therapy. Such patients will need to be reliably identified by suitable diagnostic methods. Biopsy-based diagnostics are invasive, and repeat biopsies are not always feasible. They cannot visualize heterogeneity of HER2 expression, leading to a substantial number of misdiagnosed patients. An alternative and highly accurate diagnostic method is mo-lecular imaging with radiotracers. In case of HER2, various studies demonstrate the clinical utility and feasibility of such approaches. Affibody-based radiotracers are clinically validated molecules with favorable characteristics for imaging. In this article we summarize the HER2-targeted therapeutic landscape, describe the experience with imaging diagnostics for HER2, and review the currently available clinical data on HER2-Affibody-based molecular imaging as novel diagnostic tool in breast cancer and beyond.

PURPOSE: Breast cancer tumors frequently have intratumor heterogeneity. Tumors with high intratumor heterogeneity have caused therapeutic resistance and have human epidermal growth factor receptor 2 (HER2) heterogeneity in response to HER2-targeted therapies. This study aimed to investigate whether high HER2 heterogeneity levels were clinically related to poor prognosis for HER2-targeted adjuvant therapies resistance in primary breast cancers. METHODS: This study included patients with primary breast cancer (n = 251) treated with adjuvant HER2-targeted therapies. HER2 heterogeneity was manifested by the shape of HER2 fluorescence in situ hybridization amplification (FISH) distributed histograms with HER2 gene copy number within a tumor sample. Those heterogeneity classified each tumor into the biphasic grade graph (high heterogeneity [HH]) group and the monophasic grade graph (low heterogeneity [LH]) group. Both groups were evaluated for disease-free survival (DFS) and overall survival (OS) for a median of ten years of annual follow-up. RESULTS: Of 251 patients with HER2-positive breast cancer, 46 (18.3%) and 205 (81.7%) were classified into the HH and LH groups, respectively. The HH group had more distant metastases and a poorer prognosis than the LH group (DFS: P < 0.001 (HH:63% vs LH:91% at 10 years), and OS: P = 0.012 (HH:78% vs LH:95% at 10 years). CONCLUSION: High HER2 heterogeneity is a poor prognostic factor in patients with HER2-positive breast cancer. A novel approach of heterogeneity, which is manifested by the shape of HER2 FISH distributions, might be clinically useful in the prognosis prediction of patients after HER2 adjuvant therapy.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab-deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) by flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) of H2Mab-300 and H2Mab-304 for CHO/mHER2 was 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD of H2Mab-300 and H2Mab-304 for NMuMG was 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

The binding affinity of trastuzumab and pertuzumab to HER2 has been studied using both experimental and in-silico methods. The experiments were conducted using the antibodies in their complete IgG form, as used in clinical therapy, and the extracellular domain of the HER2 protein in solution. This approach provides a precise, reproducible, and reliable view of the interaction between them in physiological conditions. Dynamic light scattering and size exclusion chromatography coupled with tetra detection were utilized to characterize the protein complexes, measure their concentrations, and calculate the equilibrium free binding energy, ΔGbind. In addition, PRODIGY, a QSAR-like model with excellent predictive ability, was employed to obtain in-silico ΔGbind estimations. The results obtained indicate that pertuzumab exhibits a slightly higher binding affinity to HER2 than trastuzumab. The difference in binding affinity was explained based on the contribution of the different interfacial contact (IC) descriptors to the ΔGbind value estimated by the PRODIGY model. Furthermore, experiments revealed that the pertuzumab IgG antibody binds preferentially to two HER2 proteins, one per Fab fragment, while trastuzumab mainly forms a monovalent complex. This finding was interpreted based on a geometrical model that identified steric crowding in the trastuzumab-HER2 complex as compared to the pertuzumab-HER2 complex.

The antibody Trastuzumab (Tz) targeting ERBB2 has improved the prognosis of patients with breast cancer (BCa) that overexpress this receptor. Resistance to Tz negatively impacts prognosis. Several mechanisms have been reported to cause Tz resistance. The objective of this study was to identify common mechanisms in in vitro models of acquired BCa Tz resistance. In particular, we used three widely available ERBB2+ BCa cell lines adapted to grow in Tz. We performed several analyses to address possible changes in phenotype, proliferation, and ERBB2 membrane expres-sion common to the three Tz-R cell lines compared to wt, but none was found. High-resolution mass spectrometry analysis identified, instead, a common set of differentially expressed proteins (DEPs) in Tz-R vs. wt cells. Furthermore, bioinformatic tools revealed that all three Tz-R cell models shared a modulation of proteins involved in the metabolism of lipids, organophosphate biosynthetic process, and macromolecule methylation. Ultrastructural analysis confirmed altera-tion of lipid droplets in resistant cells. Our data strongly support the hypothesis that complex metabolic adaptation, including lipid metabolism, protein phosphorylation, and possibly chro-matin remodeling, may fuel Tz resistance. The identification of a common set of 10 DEPs in all three TZ-resistant cell lines may provide novel targets for therapeutic intervention.

In the present day and age, cancer is still a life-limiting factor whose one of the therapeutic strategies is immunotherapy with vaccines. This research aims to prepare and characterize nanoliposomal vaccine formulation attached to HER2/neu-derived peptide (AE36) with or without CpG-ODN, and to evaluate its immunological responses to the therapy using BALB/c mice with HER2 overexpressing breast cancer. Methods: AE36 was conjugated to the liposomes containing DOTAP, DOPE and Cholesterol. Such formulations are able to produce CD8+ and CD4+ responses and induce synthesis of cytokines detectable via Enzyme-linked immunosorbent assay kits, cytotoxicity testing and intracellular cytokine staining combined with flow cytometry. Therapeutic and prophylactic effectiveness were evaluated through the formulation in samples. The highest effectiveness was found in DDC-peptide + CpG-ODN in both prophylactic and therapeutic studies, which decreased the size of tumors significantly and increased time of survival. These nanoliposomes linked to AE36 could be regarded as an appropriate candidate for the treatment and also for prophylaxis of HER2+ breast cancer; however further studies are needed.

Breast cancer stands as the prevailing malignancy across all six Gulf Cooperation Council (GCC) nations. In this literature review, we highlighted the incidence and trend of BC in the GCC. Most of the studies reported a consistent increase in BC incidence over the past decades, which was particularly attributed to the adoption of a westernized lifestyle in the region and the implications of emerging risk factors and other environmental and societal factors, the increase in screening uptake, as well as the improvement in data collection and reporting in the GCC. The data about BC risk factors in GCC was limited. In this geographic region, breast cancer frequently manifests with distinctive characteristics, including an early onset, typically occurring before the age of 50, an advanced stage at presentation, and a higher pathological grade. Additionally, it often exhibits more aggressive features such as human epidermal growth factor receptor 2 (HER2) positivity or the presence of triple-negative (TN) attributes, particularly among younger patients. Despite the growing body of literature on breast cancer in the GCC, data pertaining to survival rates are, regrettably, meager. Reports on breast cancer survival rates emanating from the GCC region are largely confined to Saudi Arabia and the United Arab Emirates (UAE). In the UAE, predictive modeling reveals 2-year and 5-year survival rates of 97% and 89%, respectively, for the same period under scrutiny. These rates, when compared to Western counterparts such as Australia (89.5%) and Canada (88.2%), fall within the expected range. Conversely, Saudi Arabia reports a notably lower 5-year survival rate, standing at 72%. This disparity in survival rates underscores the need for further research directed towards elucidating risk factors and barriers that hinder early detection and screening. Additionally, there is a pressing need for expanded data reporting on survival outcomes within the GCC. In sum, a more comprehensive and nuanced understanding of breast cancer dynamics in this region is imperative to inform effective strategies for prevention, early detection, and improved patient outcomes.

Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratifcation of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies and thereby the treatment will be more personalized and efficient. Phase I clinical evaluation of two scaffold protein-based imaging probes, [99mTc]Tc-(HE)3-G3 and [99mTc]Tc-ADAPT6, demonstrated that their injections are safe, well-tolerated and cause low level of absorbed and equivalent doses. The goal of this preclinical study was to select the best probe for patients’ stratification and for response monitoring. Biodis-tribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 ex-pression and MDA-MB-468 xenografts with very low expression. Changes in accumulation of both probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 permitted high contrast imaging of HER2-expressing tumors and clear discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [99mTc]Tc-(HE)3-G3 is capable to sense the decrease of HER2 expression on re-sponse to trastuzumab therapy only 24 h after injection of loading dose. This indicates that this tracer would be better for monitoring early response to such treatment. Results of this study should be considered in planning of further clinical development of HER2 imaging probes.

Neoadjuvant systemic therapy has now become the the standard in early breast cancer management. Chemotherapy in combination with trastuzumab +/- pertuzumab targeted therapy can improve rates of pathologic complete response (pCR) in patients with HER2-positive breast cancer. Achieving a pCR is considered a good prognostic factor, in particular in patients with more aggressive breast cancer subtypes such as TNBC or HER2 positive cancers. Furthermore, most studies demonstrate that chemotherapy in combination with trastuzumab and pertuzumab is well tolerated. The retrospective analysis presented here concentrates on neoadjuvant therapy with the TCbH-P regimen, with a particular emphasis on patients over 60 years of age. We analysed the factors affecting the achievement of pCR and presented adverse effects of the applied therapies, which opened a discussion about optimizing the therapy of older patients with HER-2 positive breast cancer.

De-escalation is currently taking place both in surgical and systemic treatment of breast cancer. The introduction of trastuzumab, the first monoclonal antibody against the HER2 receptor over 20 years ago was a milestone in the treatment of HER2 positive breast cancer and marked the beginning of a new era in targeted tumor therapy. In the sense of de-escalation, omitting non-targeted cytotoxic chemotherapy altogether is often hailed as the ultimate goal of oncological research. Especially in case of small, node-negative, HER2 positive early breast cancer, it remains a challenge for clinicians to establish the safest and most efficient treatment plan while considering the significant potential for toxic side effects associated with chemotherapy and HER2 targeted therapy, and the generally excellent prognosis. In this context, several ongoing studies are currently assessing chemotherapy-free regimens as part of strategies aimed at de-escalating therapy in the field of HER2 positive early breast cancer. Despite promising early results from these studies, combination of anti-HER2 treatment with chemotherapy backbone remains standard of care.

To assess AR’s role in TNBC treatment, various existing and completed clinical trials targeting AR or co-targeting AR with other pertinent signaling molecules were analyzed. Cyclin-dependent kinase 4/6 (CDK4/6), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17 lyase), and the phosphatidylinositol 3kinase (PI3K)/protein kinase B (AKT) signaling pathway were some of the most prevalent biomarkers used in combination therapy with AR inhibitors in these trials. Studying how AR functions in tandem with these molecules can have increasing breakthroughs in the treatment options for TNBC. Previous studies have been largely unsuccessful in utilizing AR as the sole drug target for systemic targeted treatment in TNBC. However, there is a lack of other commonly used drug target biomarkers in the treatment of this disease, as well. Thus, analyzing the clinical benefit rate (CBR) within clinical trials that are using combination therapy can prove to be imperative to the progression of improving treatment options and prognoses.

Cancers overexpressing the ERBB2 oncogene are aggressive and associated with a poor prognosis. Trastuzumab is a ERBB2 specific recombinant antibody employed for the treatment of these diseases since it blocks ERBB2 signaling causing growth arrest and survival inhibition. While the effects of Trastuzumab on ERBB2 cancer cells are well known, those on the extracellular vesicles released from these cells are scarce. This study focused on ERBB2+ breast cancer cells and aimed to establish what type of EVs they release and whether Trastuzumab affects their morphology and molecular composition. To these aims, we performed immunoelectron microscopy, immunoblot, and high-resolution mass spectrometry analyses on EVs purified by differential centrifugation of culture supernatant. Here we show that EVs released from ERBB2+ breast cancer cells are polymorphic in size and appearance, and that ERBB2 is preferentially associated with large (120 nm) EVs. Moreover, we report that Tz induces the expression of a specific glycosylated 50 kDa isoform of the CD63 tetraspanin and modulates the expression of 51 EVs proteins, including TOP1. As these proteins are functionally associated with organelle organization, cytokinesis, and response to lipids, we suggest that Tz may influence these cellular processes in target cells at distant sites via modified EVs.

Esophagogastric cancers are among the most common and deadly cancers worldwide. This review traces their chronology from 3000 BCE to the present. The first several thousand years were devoted to palliation, before advances in operative technique and technology led to the first curative surgery in 1913. Systemic therapies were introduced in 1910, and radiotherapy shortly thereafter. Operative technique improved massively over the 20th century, with operative mortality rates reducing from over 50% in 1933 to less than 5% by 1981. In addition to important roles in palliation, endoscopy became a key non-surgical curative option for patients with limited-stage disease by the 1990s. The first nonrandomized studies on combination therapies (chemotherapy +/- radiation +/- surgery) were reported in the early 1980s, with survival benefit only for subsets of patients. Randomized trials over the next decades had similar overall results, with increasing nuance. Disparate conclusions led to regional variation in global practice. Starting with the first FDA approval in 2017, multiple immunotherapies now encompass more indications and earlier lines of therapy. As standards of care incorporate these effective yet expensive therapies, care must be given to disparities and methods for increasing access.

Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuzumab and pertuzumab, were clinically approved. We previously developed a highly sensitive and specific anti-HER2 mAb, H2Mab-139 (mouse IgG1, kappa). In this study, we produced a defucosylated IgG2a version of anti‑HER2 mAb (H2Mab-139-mG2a-f) to enhance ADCC-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10‑9 M and 7.7 × 10‑9 M against HER2‑overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and could be useful for an antibody treatment regimen for HER2-positive human cancers.

(1) Background: This case presents a rare and challenging clinical scenario involving a 50-year-old female patient diagnosed with Inflammatory Carcinoma, a particularly aggressive and rapidly growing form of breast cancer. The disease is characterized by heat, redness, swelling and noticeable changes in the breast's skin texture, resembling the skin of an orange (peau d'orange). This case is important because it contributes to the limited clinical literature on such aggressive forms of breast cancer.; (2) Methods: The multidisciplinary approach was employed. Diagnostic procedures such as imaging tests, biopsy, and histopathological examination were performed to confirm the diagnosis of Inflammatory Carcinoma and assess the extent of the disease. The presence of the HER2 protein on the tumor cells supported the selection of a combination therapy involving systemic chemotherapy and targeted biological therapy.; (3) Results: The patient responded positively to the combination therapy consisting of systemic chemotherapy and targeted biological therapy. The aggressive nature of Inflammatory Carcinoma, characterized by rapid growth and distinct skin changes, was effectively addressed. The presence of the HER2 protein on the tumor cells indicated the potential efficacy of the combination therapy in aggressive forms of breast cancer.; (4) Conclusions: This case highlights the critical importance of a multidisciplinary approach in managing complex cases of breast cancer, especially rare and aggressive subtypes like Inflammatory Carcinoma. The positive response of the patient to the combination therapy involving systemic chemotherapy and targeted biological therapy demonstrates the potential for favorable outcomes in aggressive disease presentations.

Background and Objectives: Gastric cancer (GC) is one of the most commonly diagnosed cancer and the fourth cause of cancer death worldwide. Personalised treatment improves GC outcomes. A molecular classification is needed to choose the appropriate therapy. A classification that uses on-slide biomarkers and formalin-fixed and paraffin-embedded (FFPE) tissue is preferable to comprehensive genomic analysis. In 2016, Setia et al. proposed an on-slide classification, however this is not in widespread use. We propose a modification of this classification that has six subgroups: GC associated with Epstein-Barr virus (GC EBV+), GC with mismatch repair deficiency (GC dMMR), GC with epithelial-mesenchymal transformation (GC EMT), GC with functional loss of p53 due to mutation (GC p53m), CG with intact p53 (GC p53wt) and GC not otherwise specified (GC NOS). This classification also has provision for biomarkers for current or emerging targeted therapies (Her2, PD-L1 and Claudin18.2). Here we assess the implementation and feasibility of this inclusive working classification. Materials and Methods: We constructed a tissue microarray library from a cohort of 79 resection cases from FFPE tissue archives. We used a restricted panel of on-slide markers (EBER, MMR, E-cadherin, beta-catenin and p53), defined their interpretation algorithms, and assigned each case to a specific molecular subtype. Results: GC EBV(+) cases were 6%, GC dMMR cases were 20%, GC EMT cases were 14%, GC p53m cases were 23%, GC p53wt cases were 29% and GC NOS cases were 8%. Conclusions: This working classification uses markers that are widely available in Histopathology and are easy to interpret. A diagnostic subgroup is obtained for 92% of the cases. The proportion of cases in each subgroup is in keeping with other published series. Widescale implementation appears feasible. A study using endoscopic biopsies is warranted.

With the rise of novel immunotherapies able to stimulate the antitumor immune response, increasing literature data concerning the immunogenicity of breast cancer have been published in recent years. Numerous clinical studies have been conducted in order to identify novel biomarkers that could reflect the immunogenicity of BC and predict response to immunotherapy. In this regard, TILs have emerged as an important immunological biomarker related to the antitumor immune re-sponse in BC. TILs are more frequently observed in triple-negative breast cancer and HER2+ sub-types where increased TIL levels have been linked to better response to neoadjuvant chemotherapy and with improved survival. PD-L1 is a type 1 transmembrane protein ligand expressed on T lymphocytes, B lymphocytes and antigen-presenting cells and is considered a key inhibitory checkpoint involved in cancer immune regulation. PD-L1 immunohistochemical expression in breast cancer is observed in about 10–30% of cases and is extremely variable based on tumor stage and molecular subtypes . In detail, TNBC shows the highest percentages of PD-L1 positivity, followed by HER2+ tumors. On the other hand, PD-L1 is rarely expressed (0-10% of cases) in hormone-receptor positive BC. The prognostic role of PD-L1 expression in BC is still controversial since different immunohistochemistry (IHC) clones, cut-off points and scoring systems have been utilized across published studies. In the present paper an extensive review on the current knowledge of immune landscape of BC is provided. In detail TILS and PD-L1 expression across different BC subtypes is discussed also providing a guide for their pathological assessment and reporting.

This paper presents novel feature descriptors and classification algorithms for automated scoring of HER2 in Whole Slide Images (WSI) of breast cancer histology slides. Since a large amount of processing is involved in analyzing WSI images, the primary design goal has been to keep the computational complexity to the minimum possible level and to use simple, yet robust feature descriptors that can provide accurate classification of the slides. We propose two types of feature descriptors that encode important information about staining patterns and the percentage of staining present in ImmunoHistoChemistry (IHC) stained slides. The first descriptor is called a characteristic curve which is a smooth non-increasing curve that represents the variation of percentage of staining with saturation levels. The second new descriptor introduced in this paper is an LBP feature curve which is also a non-increasing smooth curve that represents the local texture of the staining patterns. Both descriptors show excellent interclass variance and intraclass correlation, and are suitable for the design of automatic HER2 classification algorithms. This paper gives the detailed theoretical aspects of the feature descriptors and also provides experimental results and comparative analysis.

Gastric cancer is an aggressive disease with increasing global incidence in recent years. Human epidermal growth receptor 2 (HER2) is overexpressed in approximately 10-20% of gastric cancers. The implementation of targeted therapy against HER2 as part of the standard of care treatment in metastatic disease has improved the prognosis of this subset of patients. However, gastric cancer still has high mortality rates and urgently requires new treatment strategies. The combination of immunotherapy with HER2-targeted therapies has shown synergistic effects in preclinical models, being the rationale behind exploring this combination in clinical trials in locally advanced and metastatic settings. Additionally, the irruption of antibody-drug conjugates and other novel HER2-targeted agents has led to the development of numerous clinical trials showing promising results. This review presents the molecular mechanisms supporting the use of HER2-targeted drugs in combination with immunotherapy and provides an overview of the therapeutic scenario of HER2-positive disease. We focus on the role of immunotherapy but also summarize emerging therapies and combinations under clinical research that may change the standard treatment in HER-2 positive disease in the future.

We explored the outcomes of germline BRCA1/2 pathogenic/likely pathogenic variants (PVs/LPVs) in the endocrine-sensitive disease treated with first line standard of care cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. Three studies retrospectively showed a reduction in overall survival (OS) and progression free survival (PFS) in gBRCA1/2m patients compared to both germinal BRCA1/2 wild type (gBRCA1/2wt) and to the untested population. Regarding the efficacy of PI3K inhibitors, there are no subgroup or biomarker analyses in which germinal BRCA status was explored. However, the biological interactions between the PIK3CA/AKT/mTOR pathway and BRCA1/2 at a molecular level could help us to understand the activity of these drugs when used to treat BC in BRCA1/2 PVs/LPVs carriers. The efficacy of trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate (ADC) targeting HER2 for HER2-low and HER2-positive (HER2+) BC, has been increasingly described. Unfortunately, data on T-DXd in HER2+ or HER2-low metastatic BC harboring germinal BRCA1/2 PVs/LPVs is lacking. Including germinal BRCA1/2 status in the subgroup analysis of the registration trials of this ADC would be of great interest, especially in the phase III trial DESTINY-breast04. This trial enrolled patients with HER2-negative (HER2-) and both HR+ and HR- metastatic disease, which can now be categorized as HER2-low. The HER2-low subgroup includes tumors that were previously classified as triple negative, so it is highly likely that some women were germline BRCA1/2 PVs/LPVs carriers and this data was not reported. Germline BRCA1/2 status will be available for a higher number of individuals with BC in the near future and data on the prognostic and predictive role of these PVs/LPVs is needed in order to choose the best treatment options.

The HER2-targeted antibody-drug conjugates (ADCs) trastuzumab emtansine (T-DM1) and Trastuzumab deruxtecan (T-DXd) improved outcomes in breast cancer. Novel combinations and sequential approaches are under investigation to overcome resistance to T-DM1 and T-DXd. Furthermore, the landscape of HER2-targeted therapy is rapidly advancing with the development of ADCs designed to attack cancer cells with greater precision and reduced toxicity. Disitamab Vedotin (RC48) incorporates a HER2 antibody with unique binding epitope and a potent microtubule inhibitor as its cytotoxic moiety. ARX-788 comprises a non-cleavable linker and a novel maytansinoid derivative as the cytotoxic drug, engineered for increased stability and a higher drug-to-antibody ratio. SYD985, a duocarmycin-based ADC, is designed to be activated only in the acidic environment of the tumor, thus reducing off-target effects. BL-M0701 targets a different epitope on the HER2 protein, which might be beneficial in circumventing resistance that affects the binding of other ADCs and incorporate a camptothecin derivative as its payload. Zanidatamab zovodotin (ZW25) is an ADC that binds to two different HER2 epitopes simultaneously, enhancing the internalization and delivery of its cytotoxic payload. These and other ADCs in phase I trials hold the potential to improve outcomes for HER2-positive and HER2-Low breast cancer and possibly other solid tumors.