HER2 Receptor In Breast Cancer Research Articles

Abstract PO3-15-07: False-negative results of hyperexpression of HER2 receptor in breast cancer at a public tertiary hospital in Brazil

Abstract Introduction: Breast cancer (BC) is the second leading cause of cancer death in women worldwide. One of the major advances in BC management was its molecular classification, especially regarding the Human Epidermal growth factor Receptor 2 (HER2). HER2 positive tumors are at greater risk of visceral metastasis and are associated with worse survival rates. Thus, the use of specific drugs to target this pathway is essential and false-negative have a high impact on patient care. HER2 status is usually accessed using immunohistochemistry (IHC), but false-negatives results occur. When IHC results are inconclusive, in-situ hybridization (ISH) testing is necessary. Objective: to determine if there is variability between the positivity rates between laboratories that performed the ISH, according to the IHQ, and to evaluate if there are false-negative results and possible prognostic impact. Methods: retrospective, observational study in a public hospital in the city of Curitiba (Brazil), followed by cross-sectional analysis of histological samples. All cases of BC that underwent HER2 assessment by IHC and ISH between January 2008 and December 2018 were included. Participants classified as HER2 negative by IHC and patients whose medical records were not available were excluded. A new analysis of all IHC slides and of cases with negative ISH was performed. Results: We identified 205 people with an average age of 53.52 (± 11.89) years. Most cases were classified as clinical stage I and II (68.3%), and the most prevalent histological features were Invasive Ductal Carcinoma (82.4%) and luminal molecular subtype (49.8%). Initial ISH testing was performed by four laboratories. There was a significant difference in HER2 positivity rates between these laboratories, even after individual reanalysis of all IHC slides. Of the 114 cases with negative ISH, it was possible to obtain histological material in 82 of them to perform a new standardized ISH. The false-negative rate for HER2 in this 82 patients was 41.46%, with 10 cases out of 24 for positive IHC (3+) and 22 out of 58 for inconclusive IHC (2+). In this paper, false-negative results did not impact in survival, most likely to differences between groups. Conclusion: our work showed different rates of positivity for HER2 among laboratories that performed confirmatory test with ISH. The performance of a second standardized ISH proved that the difference between the laboratories was due to false-negative results. There was no difference in recurrence and cancer-specific survival in this false-negative sample. Keywords: breast cancer. Biomarkers. Health technology assessment. HER2 receptor. DIfferences between laboratories reagarding HER2 false negatives –insert figrue image– Citation Format: Bruno Batista, Sergio Ioshii, Sergio Padilha, Jacqueline Nabhen, Tayza Ostroski, Caroline Batista. False-negative results of hyperexpression of HER2 receptor in breast cancer at a public tertiary hospital in Brazil [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-07.

Peptide-drug conjugate designated for targeted delivery to HER2-expressing cancer cells.

Targeted therapy of the highest globally incident breast cancer shall resolve the issue of off-target toxicity concurring with augmented killing of specific diseased cells. Thus, the goal of this study was to prepare a peptide-drug conjugate targeting elevated expression of HER2 receptors in breast cancer. Towards this, the rL-A9 peptide was conjugated with the chemotherapeutic drug doxorubicin (DOX) through a N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker. The synthesized peptide-drug conjugate, rL-A9-DOX, was characterized by mass spectrometry. Molecular docking studies, based on binding energy data, suggested a stronger interaction of rL-A9-DOX with the HER2 receptor in comparison to the unconjugated peptide, rL-A9. The cytotoxic effect of the rL-A9-DOX conjugate was observed to be higher in HER2-positive SKOV3 cells compared to HER2-negative MDA-MB-231 cells, indicating selective cell killing. Cellular internalization of the rL-A9-DOX conjugate was evident from the flow cytometry analysis, where a noticeable shift in mean fluorescent intensity (MFI) was observed for the conjugate compared to the control group. This data was further validated by confocal microscopy, where the fluorescent signal ascertained nuclear accumulation of rL-A9-DOX. The present studies highlight the promising potential of rL-A9-DOX for targeted delivery of the drug into a defined group of cancer cells.

HER2-Positive Metastatic Breast Cancer: Available Treatments and Current Developments

For several years, the overexpression of the HER2 receptor in breast cancer has been correlated with a poor prognosis and an increased risk of developing brain metastases. Currently, the combination of anti-HER2 double blockade and taxane and trastuzumab emtansine (T-DM1) are considered the standard treatments for metastatic breast cancer overexpressing these receptors in the first and second line. Very recently, the development of a new antidrug conjugate, trastuzumab-deruxtecan, has improved the overall survival of patients, even in second-line treatment. However, trastuzumab-deruxtecan has become a new standard. Despite the benefits of these antidrug conjugates, this benefit in patients with brain metastases remains unclear. Tucatinib is a new tyrosine kinase inhibitor that has given hope for the treatment of these patients. The objective of this article was to review data on the established drugs and novel agents for HER2-positive MBC and to discuss how to incorporate anti-HER2 therapies in first and later-line settings.

In silico study of baicalin as an inhibitor of HER-2 receptor in breast cancer

Breast cancer is the second cause of female death in the world. Lapatinib targeting HER-2 is the common inhibitor for breast cancer therapy. This study reports the potential of baicalin as an inhibitor of HER-2 through in silico molecular docking. The study was conducted by structure optimization of baicalin and lapatinib, preparation of HER-2 (PDB ID: 3PP0) target protein, validation of the molecular docking method, and docking of baicalin and lapatinib. The results showed that baicalin had an affinity for HER-2 with a binding energy of -6.0 kcal/mol, while the binding energy of the 03Q native ligand and lapatinib to HER-2 was -4.79 kcal/mol and -3.98 kcal/mol, respectively. This finding indicated that baicalin is a potential breast anticancer through the inhibition of the HER-2 protein.

In Silico Studies of Green Tea Catechins Against HER-2 Receptor in Breast Cancer

In Silico Studies of Green Tea Catechins Against HER-2 Receptor in Breast Cancer

Design and development of PEGylated liposomal formulation of HER2 blocker Lapatinib for enhanced anticancer activity and diminished cardiotoxicity

Breast cancer is most frequently diagnosed cancer and fifth leading cause of death in women. About 20–30% of all breast cancers overexpress HER2/neu receptors. Lapatinib is a dual tyrosin kinase inhibitor of EGFR and HER2. It exhibits its anticancer effect via blocking intracellular domain of HER2 receptor in breast cancer. Lapatinib belongs to class II of BSC classification due to its poor solubility restricting its clinical application. Due to presence of HER2 receptor on cardiomyocytes, it is associated with generation of cardiotoxicity. The present study was aimed to design a PEGylated liposomal formulation of Lapatinib and evaluate its anticancer potential. Lapatinib liposomes were prepared using lipid layer hydration method and its characterization was done by determining its particle size, zeta potential, entrapment efficiency and in vitro release profiling. The anti-tumor activity of PEGylated liposomal formulation was evaluated in xenografted tumor induced by MDA-MB-453 breast cancer cells in chick embryos. The anti-tumor effect of lapatinib was enhanced by its PEGylated liposomal preparation as it led to the reduction in tumor size to a greater extent compared to the embryos treated with free lapatinib. Flowcytometric analysis and immunoflurescence study using cleaved PARP antibody demonstrated the enhanced apoptotic potential of PEGylated liposomes of lapatinib. SGOT levels, marker for cardiotoxicity and hepatotoxicity, significantly decreased in serum of embryos treated with PEGylated liposomes of lapatinib compared to free drug treated embryos. Hence, the PEGylated liposomal formulation of lapatinib can be used as a therapeutic strategy against HER2 positive breast cancer either alone or in combination with conventional anticancer agents and hormonal therapies.

The Core Needle and Surgical Biopsy Concordance to Detect Estrogen, Progesterone, and Her-2 Receptors in Breast Cancer: A Comparative Study

Evaluation of estrogen receptor (ER), progesterone receptor (PR), and (human epidermal growth factor receptor-2) Her-2 on core needle biopsies (CNBs) is increasingly in use to diagnosis early breast cancer, but its concordance with surgical excision (SE) is not well documented. The study included 100 formalin fixed, paraffin-embedded specimens of invasive breast carcinoma archived in Pathology Department of Cancer Institute, Tehran, Iran, from 2011 to 2014. Immunohistochemistry was applied to detect ER, PR, and Her-2. The current study findings indicated a significant correlation of 90% between CNB and SE specimens for ER expression. The correlation between CNB and SE specimens was estimated as 81% and 97.3% for PR and Her-2, respectively. CNB can be performed confidently to determine ER and Her-2. For PR, results obtained from CNB should be considered.

Correlation of high levels of HER2 measured by multiplex mass spectrometry with increased overall survival in patients treated with anti-HER2-based therapy.

649 Background: There is evidence that increased absolute levels of the HER2 receptor in breast cancer positively correlates with a clinical benefit from anti-HER2 therapies. The current method of HER2 evaluation, immunohistochemistry (IHC), does not allow for absolute HER2 quantification and is prone to false-positives. Thus, a significant portion of patients classified as HER2-positive actually express low levels of the receptor. We quantitated absolute levels of HER2 using a mass spectrometry-based assay and correlated expression levels with the anti-HER2 therapy benefit. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor sections were microdissected and proteins were solubilized and digested by trypsin in Liquid Tissue buffer. After digestion, internal standards were added and absolute quantitation for multiple proteins was performed using selected reaction monitoring (SRM) mass spectrometry. Results: 26 HER2-positive (IHC 3+) breast cancer samples from patients treated with anti-HER2 therapy wer...

The HER2 Receptor in Breast Cancer: Pathophysiology, Clinical Use, and New Advances in Therapy

Human epidermal growth factor receptor 2 (HER2) is overexpressed in around 20–30% of breast cancer tumors. It is associated with a more aggressive disease, higher recurrence rate, and increased mortality. Trastuzumab is a HER2 receptor blocker that has become the standard of care for the treatment of HER2 positive breast cancer. The effectiveness of Trastuzumab has been well validated in research as well as in clinical practice. The addition of Trastuzumab to standard of care chemotherapy in clinical trials has been shown to improve outcomes for early stage as well as metastatic HER2 positive breast cancer. The most clinically significant side effect of Trastuzumab is the risk of cardiac myocyte injury, leading to the development of congestive heart failure. The emergence of patterns of resistance to Trastuzumab has led to the discovery of new monoclonal antibodies and other targeted agents aimed at overcoming Trastuzumab resistance and improving survival in patients diagnosed with HER2 positive breast cancers.

Abstract 5543: Hetmap: Evaluating tumor heterogeneity in immunohistochemistry-stained breast cancer tissue

Abstract Immunohistochemisty assays examining the HER2 receptor in breast cancer is the most widely adopted example of a companion diagnostic approach, which seeks to dictate therapeutic strategy based on a molecular description of a patient's disease. There are well-established guidelines for selecting patients for anti HER2 adjuvant therapies in breast cancer treatment, yet the current HER2 companion diagnostic approach is qualitative, does not sufficiently account for intratumor heterogeneity, and does not utilize any additional information about tumor cells that score beyond a specific threshold level. A major contributing factor for the failure of both treatment and diagnostic paradigm is thought to be intratumor heterogeneity. This lack of information in the scoring paradigm may contribute to inappropriate patient selection and explain why the disease in many trastuzumab treated patients progresses or becomes recurrent. Thus, a quantitative clinical measurement of heterogeneity in immunohistochemistry staining would be useful in better predicting patient therapeutic response. To answer this, we created a heterogeneity scoring approach (HetMap) that allows the visualization of an individual patient's IHC heterogeneity in the context of a cell population. We combined HER2 semi-quantitative analysis with the use of ecology diversity statistics to evaluate cell-level heterogeneity (consistency of protein expression within neighboring cells in a tumor nest) and tumor-level heterogeneity (differences of protein expression across a tumor as represented by a tissue section). We evaluated the approach on HER2 immunohistochemistry stained breast cancer samples, using 200 specimens across two different CLIA laboratories, with three pathologists at each laboratory each outlining regions of tumor for scoring by automatic cell-based image analysis. HetMap was evaluated using three different scoring schemes: HER2 scoring according to ASCO/CAP guidelines, H-Score and a new continuous HER2 score (HER2cont). Cell-level heterogeneity, reported either as an average or the maximum area of heterogeneity across a slide, had low levels of dependency on the pathologist choice of region. Tumor-level heterogeneity measurements had more dependence on the pathologist choice of regions. Significantly, discordant pathologist assessments of IHC scores in the +2, or equivocal score, range occurred most in tumors with high heterogeneity, leading to potentially major clinical impact. Thus, HetMap is a measure of heterogeneity, by which pathologists, oncologists, and drug development organizations can view cell-level and tumor-level heterogeneity for a patient for a given marker in the context of an entire patient cohort. Including such measures of heterogeneity in diagnostic approaches can help establish better thresholds for patient selection, thereby improving patient response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5543. doi:1538-7445.AM2012-5543

P3-03-04: Targeted Modulation of HER Receptor Signaling in Breast Cancer Using miR-21.

Abstract Introduction: MicroRNAs (miRNAs) are multifunctional nucleic acids that regulate the stability and translational efficiency of targeted messenger RNAs. Endogenously altered miRNA levels have been linked in breast cancer to acquired chemoresistance and changed radiosensitivity. Thus the modification of miRNA expression represents an attractive therapeutic strategy. We now show that the targeted inhibition of miR-21 can modify expression of the HER2 receptor in breast cancer. Materials and Methods: Using TaqMan-based technology the expression of five selected miRNAs (miR-7b, miR-10a, miR-17-5p, miR-125a, and miR-21) was investigated in formalin-fixed, paraffin-embedded breast carcinoma tissues expressing different levels of HER2 (n=60). Possible regulation of HER2 was explored in cell lines using lentivirus to stably downregulate or upregulate HER2 and miR-21 expressions. The effects were analyzed using qRT-PCR, Western blots, proliferation and migration assays. Results: In tumor tissues we identified overexpression of miR-10a and miR-21, and underexpression of miR-17-5p and miR-125a in most of the tumor tissue samples. Significant associations (p≤0.05) were found between HER receptor expression and the expression of miR-17-5p and miR-125a, and miR-21 showed correlation with expression of ER and PR. Furthermore, analysis of miR-21 downregulation in breast cancer cell lines showed inhibition of cellular proliferation and HER2 protein synthesis. Additionally, downregulation of HER2 expression decreased miR-21 expression and showed moderate change in cellular proliferation activity. Conclusions: Based on these results we conclude that there is tight interplay between HER2 and miR-21 expression in breast cancer cells. Because miRNAs modulate gene expression we suggest that modulation of miR-21 has the potential to impact tumor therapy by interference with specific signaling pathways. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-04.

Molecular targeting of HER2 for diagnosis and therapy of breast cancer.

Abstract Abstract #6004 Background: Expression of HER2 receptors in breast cancer is correlated with poor prognosis and may be different in distant metastases as compared to the primary tumor. We are developing methods to assess global expression of HER2 in vivo and to deliver therapeutic agents specifically to HER2-positve cells.
 Materials and Methods: As the targeting agent we use an Affibody molecule (http://www.affibody.com). These very stable and highly soluble proteins are relatively small (8.3 kDa) and bind to HER2 receptors with high affinity (22 pM). For imaging with PET, SPECT, or optical methods, an appropriate group containing the imaging beacon can be attached by a selective chemical reaction to a unique C-terminal cysteine residue of Affibody. For therapeutic purposes, they can be conjugated to multifunctional nanoparticles containing both imaging and therapeutic agents. We have conjugated affibody molecules with thermo-sensitive liposomes or gold nanoparticles (subsequently activated with neutrons) and characterized their biodistribution using optical imaging and SPECT, respectively. We used PET imaging with 18F-ZHER2-Affibody to monitor the down-regulation of HER2 following four doses (50 mg/kg) of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin, 17-DMAG, an inhibitor of Hsp90 known to decrease HER2 expression. Animals were scanned before and after treatment. Immediately after the last scan, the mice were euthanized and tumors were frozen for ex-vivo analysis of receptor expression. For optical imaging, we used AlexaFluor dyes conjugated with affibody molecules containing an albumin binding domain that extended their circulation time. We have attached nanoparticles (liposomes and gold) to Affibody molecules using the same type of maleimide chemistry.
 Results: Our results showed that Affibody molecules do not affect the targeted cells and that their binding does not interfere with either the binding or the effectiveness of trastuzumab. 18F-ZHER2-Affibody was eliminated quickly from blood and normal tissues, providing high tumor/blood and tumor/muscle ratios by 1h post injection. The signal obtained from PET and optical imaging correlated well with the number of receptors expressed in the studied tumors as assessed by western blot, ELISA, and IHC. Following 17-DMAG treatment, the level of HER2 expression, estimated by PET imaging, in BT474 and Mcf-7/clone18 tumors decreased 70% and 30%. This change was confirmed by the biodistribution studies, ELISA and western blot.
 Discussion: This strategy, involving assessment of target presence and distribution in an individual patient followed by optimized, target-specific drug delivery, may significantly improve efficacy of breast cancer treatment while reducing side effects. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6004.

Non-linear relationship between HER-2 expression and response to trastuzumab in breast cancer cells

13083 Background: Currently, eligibility for treatment with the mAb trastuzumab (T) in breast cancer patients is assessed by semiquantitative immunohistochemical (IHC) analysis of HER-2 expression, where 3+ score represents overexpression, and/or by Fluorescence In Situ Hybridization (FISH), which detects HER-2 gene amplification. However, both methods do not measure small but potentially significant differences in protein expression. Methods: We modulated HER-2 expression by RNA interference (siRNA) in the human HER-2-overexpressing breast cancer cell line SKBR3. Protein expression was measured by Western Blot analysis. Cells expressing different levels of HER-2 protein were then treated with T, to assess the mAb effect on cell proliferation and apoptosis. Results: Compared with the parental cell line, SKBR3 cells transfected with 10, 20, and 30 nM HER2 specific siRNAs, showed a reduction of HER-2 protein levels of 40%, 55% and 70%, respectively. Despite up to a 70% decrease in HER-2 expression, transfected cells still scored 3+ at IHC (Dako HercepTest®). Interestingly, this reduction rendered cancer cells more sensitive to T treatment. In fact, we found that sensitivity to T follows a bell shaped curve, depending on different amounts of HER-2. When T was administered at the standard dose of 10 μg/mL, cells showing a 40% reduction of the HER-2 resulted more sensitive to the growth inhibitory effect of T, compared with parental SKBR3 cells and with cells showing lower levels of receptor. Furthermore, these cells were sensitive to T concentrations lower than standard (6 vs 10 μg/mL), which were markedly less effective in cells expressing higher or lower levels of HER-2 receptor. Conclusions: The observation that cells expressing significantly different amounts of HER-2 protein are all scored as “overexpressors” by conventional IHC suggests that quantitative analysis of HER-2 receptor in breast cancer might be more appropriate for the selection of candidates to T treatment. Moreover, we showed that the inhibitory effect of T depends in a non-linear manner on the quantity of HER-2 protein and that the level of HER-2 expression can influence the sensitivity to T, which requires an optimal drug/receptor ratio. No significant financial relationships to disclose.

Expression of HER2 and its association with AP-2 in breast cancer

The aim of the study was to investigate the relationship between the expression of human epidermal growth factor receptor 2 (HER2) and activator protein 2 (AP-2), as well as the prognostic significance of HER2 in breast cancer. HER2 and AP-2 expressions were immunohistochemically (IHC) analysed in a large prospective, consecutive series of 425 breast cancer patients diagnosed and treated between 1990 and 1995 at the Kuopio University Hospital, Kuopio, Finland. In a subset of patients ( n=71), the gene amplification status was examined by using a chromogenic in situ hybridisation (CISH) analysis. Expression of HER2 was studied in relation to AP-2, clinicopathological parameters and patients' survival. Pathological membranous overexpression of the HER2 receptor was seen in 13% of the carcinomas, which was related both to gene amplification (78% of the cases) and high nuclear expression of AP-2 (67%, P=0.007). HER2-positivity was most often seen in carcinomas having both high nuclear and high cytoplasmic AP-2 expression ( P<0.001). In the univariate survival analyses HER2-positivity predicted a shorter recurrence-free survival (RFS) ( P<0.0001) and a shorter breast cancer-related survival (BCRS) ( P=0.0063) in the whole patient group, as well as in the subgroup of node-negative patients. In the node-positive patients, HER2-positivity predicted only a shorter RFS. Combined expression of HER2 and nuclear AP-2 resulted in the separation of four groups with different prognoses, the best prognosis being for patients in the HER2-/AP-2+ group and the worse prognosis for those in the HER2+/AP-2- group. In the multivariate survival analyses, HER2-positivity independently predicted a shorter RFS in the whole patient group ( P=0.0067), as well as in the subgroup of node-positive patients ( P=0.0209). In conclusion, pathological membranous overexpression of the HER2 receptor in breast cancer is related both to gene amplification and a high AP-2 expression. Combining HER2 and AP-2 expressions may provide valuable information on the prognosis of breast cancer patients.