HER2 Protein Expression Levels Research Articles

Development and Validation of a HER2-Low Focused Immunohistochemical Scoring System With High-Interobserver Concordance: The Australian HER2-Low Breast Cancer Concordance Study

The DESTINY Breast-04 trial revealed survival advantages of trastuzumab deruxtecan for women with metastatic HER2-low breast cancer (1+ or 2+ immunohistochemistry [IHC], without amplification). Although this trial applied the 2018 Americal Society of Clinial Oncology (ASCO)/College of American Pathologists (CAP) HER2 IHC scoring criteria, the subjectivity and imprecision in IHC scoring have raised concerns that patients’ treatment may be misaligned. Our group of 9 experienced breast pathologists collated a deidentified set of 60 breast cancer core biopsies from 3 laboratories, evaluated with the Ventana 4B5 HER2 assay and mostly scored locally as HER2 0 or 1+. Based on ASCO/CAP 2018 criteria and our extensive experience of reporting HER2 IHC, we specified scoring conventions for cancers with low levels of HER2 protein expression, articulating specific scoring pitfalls. Each pathologist then reviewed digitized whole slide images of the IHC slides and scored the HER2 expression for each case. At a subsequent consensus workshop, we reviewed the cases jointly to establish consensus scores for each case and determine the percentage of HER2 expressing tumor cells. Consensus was reached on all cases, with 40 classified as 1+ and 3 as 2+ (not amplified), totaling 43 (71.7%) HER2-low cancers. The remaining cases were HER2 0. In 93.3% of cases (56/60), the consensus score matched with the majority opinion of pathologists’ independent scores. Seven (41.2%) of the 17 cases reported locally as HER2 0 were classified as HER2 low. Conversely, among 32 cases with local scores of 1+, 7 (21.8%) were reclassified as ultralow or null. Individual pathologists’ accuracy in matching the consensus scores ranged from 73.3% to 91.67% (mean, 80.74%). Among HER2-low cancers those in which <20% of the tumor cells expressed HER2 had the lowest concordance levels. Observers Cohen’s κ coefficients for concordance were excellent for 4, good in 1, and moderate in the 4 observers. This reference set of cases with expert consensus HER2 scores will be invaluable for peer training and development of our national external quality assurance program for HER2-low cancers. For assessing breast cancers at the low end of HER2 protein expression, our targeted scoring criteria and explicit instruction on pitfalls improved pathologists’ accuracy and concordance.

Abstract PO5-04-04: Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer

Abstract Introduction Blood-based liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional tissue biopsy is not feasible. Recently, the novel anti-HER2 antibody-drug conjugate, Trastuzumab Deruxtecan (T-DXd), has shown effectiveness against MBC with low HER2 expression detected by tissue biopsy characterized as HER2-low (immunohistochemically 1+ or 2+ and ERBB2 amplification-negative via in situ hybridization) and is being explored in an ultra-low population. Here we report on the application of a liquid biopsy platform designed to sensitively detect low levels of HER2 protein expression in CTCs from MBC patients. Methods Blood samples from 704 metastatic breast cancer patients as well as control cells lines were collected for cell-based and cell-free DNA analysis. After plasma isolation, nucleated cells deposited on glass slides went through immunofluorescent staining and imaging. CTCs were identified using Epic Sciences’ digital imaging and machine learning algorithms, and a subset of CTCs were isolated and sequenced for genomic quantification of large scale-state transitions (LSTs) as well as copy number variants (CNVs). The HER2 status was based upon on ASCO-CAP guidelines and the previous tissue biopsy results from the ordering healthcare provider, as documented on the DefineMBC™ test requisition form. Results Within a DefineMBC early-access program (EAP), 93% of patients with MBC had detectable CTCs. Among ERBB2-non-amp patients tested with the DefineMBC assay, 25% of patients with MBC had findings that were reported as consistent with HER2-low in the CAP-CLIA setting. To explore the lower boundaries of HER2 protein expression, we created and validated an MCF-7 HER2-negative ERBB2 knock-out cell line and utilized its 95% confidence level threshold (271 MFI) as a negative fluorescence cutoff for very low HER2 expression. Based upon publicly available CCLE datasets, MDA-MB-453, MCF7, and MCF7 ERBB2 KO breast cancer cell lines were selected for their high and low HER2 RNA expression levels and were corroborated via observed HER2 immunofluorescence median levels of 15740, 381, and 188 MFI respectively. In an exploratory analysis, among ERBB2-non-amp patients, we retrospectively applied the 271 MFI HER2 cutoff to the EAP cohort and identified 38% of patients with very low HER2 expression. We examined the impact of the HER2 ultra-low cutoff in patients not eligible for T-DXd treatment. Among patients reported as IHC=0 or with unknown IHC status from the most recent tissue biopsy, 44% and 42%, respectively, may qualify for treatment with T-DXd. Conclusion Using our higher sensitivity assay that combines CTC immunofluorescence and single-cell genomic analysis, we examined the lower boundary of HER2 protein expression. Feasibility data support further characterization of a very low HER2 expression phenotype, for the eventual inclusion into our comprehensive cancer profiling solution. To date, studies with biomarker expression have been limited to tissue biopsy, which may not always yield contemporaneous sampling in the metastatic setting. These results offer a liquid biopsy test that might identify patients potentially responsive to HER2-directed therapies. Citation Format: Giuseppe Di Caro, David Bourdon, Andrew Kunihiro, Alessandra Cunsolo, Ernest Lam, Megan Slade, Martin Blankfard, Lee Schwartzberg. Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer [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 PO5-04-04.

Abstract PO3-13-11: Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies

Abstract Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² &amp;gt; 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [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 PO3-13-11.

Abstract 3704: Whole genome and targeted proteome analysis of single cancer cells

Abstract The simultaneous genome and proteome analysis of very rare single cells would enable addressing important biological and diagnostic questions particularly in cancer. Here, we introduce a novel genome and proteome (G&amp;P) analytic workflow, a method allowing comprehensive analysis of single-cell DNA and targeted profiling of protein expression in single cells. As a proof of concept, we demonstrate the capability of the method by quantifying copy number of the ERBB2 gene alongside with protein expression levels of HER2 and phospho-HER2 in single cells. The G&amp;P workflow utilizes antibodies conjugated with DNA oligonucleotides designed to enable their amplification alongside the single-cell genome facilitating parallel genome and proteome profiling by either quantitative PCR (qPCR) or sequencing. We utilized antibodies targeting cytokeratin 8/18/19, HER2 and phospho-HER (Tyr1248). Performance of each DNA-conjugated antibody was assessed by immunofluorescence staining in a variety of breast cancer cell lines (MDA-MB231, MDA-MB453, BT474 and MCF7) and sample types. Stained cells were isolated by manual micromanipulation and subjected to whole genome amplification (WGA). Protein expression levels, derived from quantification of the amplified DNA tags, were measured by qPCR. Shallow whole genome sequencing was employed to facilitate genome-wide profiling of copy number variations (CNVs), including the ERBB2 locus. We successfully developed and tested a G&amp;P workflow allowing parallel genome-wide CNV profiling and quantification of three target proteins. G&amp;P measurement yielded concordant results to immunofluorescence quantification for all tested markers. Furthermore, the workflow allowed direct correlation of copy numbers of the ERBB2 gene with HER2 expression in single cancer cells. Notably, the method enabled measurements of cellular and molecular responses at the single-cell level to lapatinib treatment, i.e. monitoring of the HER2 pathway activation level. Our novel multiplex G&amp;P technique is capable of generating multiparametric measurements linking DNA copy numbers and expression levels of selected genes of interest. The method is optimized for analysis of single cells from rare cell populations and is readily translatable to study circulating tumor cells (CTCs) and disseminated cancer cells (DCCs), enabling for the first time pathway analysis via phosphoprotein analyses of these cells. Citation Format: Atieh Alyasin, Kathrin Weidele, Catherine Botteron, Christoph A. Klein, Zbigniew T. Czyż. Whole genome and targeted proteome analysis of single cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3704.

HER2-low metastatic breast cancer: molecular insights and therapeutic strategies

The identification of HER2-low metastatic breast cancer as a novel subgroup with therapeutic implications underscores the intricacies in breast cancer classification. This subset, comprising about 60% of breast cancer cases, presents a challenge due to its heterogeneous nature, characterized by varying HER2 protein expression levels. This heterogeneity complicates diagnosis and treatment decisions. The advent of trastuzumab deruxtecan (T-DXd), a second-generation antibody-drug conjugate (ADC), instills renewed hope for HER2-low breast cancer patients, having demonstrated effectiveness in clinical trials. The article also explores the evolution of HER2 testing guidelines, notably the 2023 ASCO/CAP guidelines that acknowledge the potential benefits of HER2-targeted therapies for this subgroup. In summary, this article emphasizes the significance of collaborative efforts between Pathologists and Oncologists in the era of precision medicine. It also highlights the potential for innovative, tailored therapies for HER2-low breast cancer, promising enhanced treatment outcomes and a broader range of therapeutic options.

Exosomal Linc00969 induces trastuzumab resistance in breast cancer by increasing HER-2 protein expression and mRNA stability by binding to HUR

BackgroundBreast cancer (BC) is the most common malignant disease in female patients worldwide. In HER-2+ BC patients, trastuzumab therapy is associated with a better prognosis. However, many HER-2+ BC patients experience recurrence or metastasis because of trastuzumab resistance. The mechanisms underlying trastuzumab resistance remain unclear. Recently, substantial evidence has suggested that exosomes are associated with drug resistance, and lncRNAs have attracted increasing attention due to their potential role in the regulation of trastuzumab resistance.MethodsWe collected the exosomes from the plasma of BC patients with and without trastuzumab resistance, sequenced the whole transcriptomes, identified differentially expressed lncRNAs, and identified lncRNA Linc00969, which was overexpressed in trastuzumab-resistant patients. Then, we established trastuzumab-resistant BC cell lines and explored the role of exosomal Linc00969 in trastuzumab resistance in vitro and in vivo by silencing or overexpressing Linc00969 and performing a series of functional analyses. Furthermore, to explore the mechanism by which exosomal Linc00969 contributes to trastuzumab resistance, we measured changes in HER-2, HUR and autophagy-related protein expression levels after regulating Linc00969 expression. In addition, we investigated the interaction between Linc00969 and HUR via pull-down and RIP assays and the effect of HUR on HER-2 expression and trastuzumab resistance after blocking HUR.ResultsWe first found that exosomal lncRNA Linc00969 was overexpressed in trastuzumab-resistant BC patients and that exosome-mediated Linc00969 transfer could disseminate trastuzumab resistance in BC. Then, we found that silencing Linc00969 could reduce trastuzumab resistance and that overexpressing Linc00969 could enhance trastuzumab resistance. Furthermore, our results showed that Linc00969 could upregulate HER-2 expression at the protein level and maintain the stability of HER-2 mRNA by binding to HUR. Additionally, we found that exosomal Linc00969 could regulate trastuzumab resistance by inducing autophagy.ConclusionsIn this study, we first identified that exosomal lncRNA Linc00969 could induce trastuzumab resistance by increasing HER-2 protein expression and mRNA stability by binding to HUR, and Linc00969 might also be involved in trastuzumab resistance by inducing autophagy. Our results elucidate a novel mechanism underlying trastuzumab resistance, and Linc00969 might be a new target for improving the treatment of HER-2+ BC patients.

Differences between the efficacy of HER2(2+)/FISH-positive and HER2(3+) in breast cancer during dual-target neoadjuvant therapy.

This study investigated the differences in efficacy between IHC(2+)/FISH-positive and IHC(3+) in HER2-positive breast cancer (BC) during neoadjuvant chemotherapy (NAC) combined with trastuzumab and pertuzumab. The research also aimed to provide insight into treatment strategies for clinical HER2(2+)/FISH-positive and HER2(3+) BC. A retrospective analysis was performed on the clinical and pathological data of patients with confirmed diagnoses of invasive BC treated via combined NAC and dual-target therapy who underwent surgery at the Breast Surgery Center of Sichuan Cancer Hospital between June 2019 and June 2022. The correlation between the clinicopathological characteristics and pathological complete response (pCR) was analyzed via the χ2 test, while logistic regression was performed using the SAS 9.4 statistical analysis software. This study examined 224 patients with an overall pCR rate of approximately 59.82%, which included 36 IHC(2+)/FISH-positive and 188 IHC(3+) cases with approximate pCR rates of 41.67% and 63.30%, respectively. Univariate and multifactorial analysis of the clinical and pathological data determined that age, menstrual status, family history, Ki67 expression, number of treatment cycles, and treatment regimen did not influence pCR. No statistical differences were evident between the univariate and multivariate models. However, the clinical stage, hormone receptor, and HER2 expression status significantly impacted pCR, with considerable consistent differences between the univariate and multifactor analyses. HER2 IHC(3+) BC displays a higher pCR rate than HER2 IHC(2+)/FISH-positive BC (p≤0.05), with a positive correlation between the HER2 protein expression levels and the response to anti-HER2 therapy.

Low interobserver agreement among subspecialised breast pathologists in evaluating HER2-low breast cancer

AimsMetastatic HER2-low breast cancer (HLBC) can be treated by trastuzumab deruxtecan. Assessment of low levels of HER2 protein expression suffers from poor interobserver reproducibility. The aim of the study was...

Bioinformatics combined with clinical data to analyze clinical characteristics and prognosis in patients with HER2 low expression breast cancer.

Human epidermal growth factor receptor 2 (HER2) is a landmark protein in determining the targeted treatment of breast cancer (BC). However, the latest research shows that different intensity of HER2 protein expression levels in BC leads to different clinical characteristics, treatment, and prognosis, especially in HER2 low expression patients. Therefore, this study intends to analyze and compare the clinicopathologic features and prognosis of BC patients with low and zero HER2 expression from The Cancer Genome Atlas (TCGA) database and the data collected by our center. First, the BC dataset was downloaded from TCGA database, including 345 eligible and with complete clinical information BC patients, to compare the difference between HER2 low expression groups and HER2 zero expression groups and their correlation with estrogen receptor (ER) and progesterone receptor (PR) expression. Then, the clinicopathological data and follow-up of 405 patients with HER2 low expression and HER2 zero expression diagnosed with BC admitted to the Affiliated Hospital of Youjiang Medical University for Nationalities (YJMU) from January 2017 to December 2021 were collected to verify the consistency of the results of the two data sets. Both the clinical samples and the TCGA data showed that the ER and PR rates were higher in the HER2 low expression group compared with the HER2 zero expression group. There were no significant differences in tumor size, lymph node metastasis, distant metastasis, and disease-free survival (DFS). In addition, the data analysis of 405 clinical samples also showed that the HER2 low expression group had a lower 3-year recurrence or metastasis rate compared with the HER2 zero expression group. Compared with HER2 zero expression, HER2 low patients express more ER and PR, and have less short-term recurrence and metastasis, but there is no obvious difference in DFS between the two groups.

Synergistic Cytotoxic and Antimigratory Effect of Brazilein and Doxorubicin on HER2-Overexpressing Cells.

The present research aims to report cytotoxic and antimigratory activities of the oxidized form of brazilin, i.e., brazilein, and the effects of the combination of brazilein-doxorubicin on MCF-7/HER2 cells. The MTT assay was conducted to test the cytotoxic activity, while flow cytometry with PI and PI-annexin V staining were respectively performed for cell cycle and apoptosis analyses. Migration and invasion analyses were assessed via Boyden chamber assay, while HER2, Rac1, p120, MMP2, and MMP9 protein levels were determined by immunoblotting and gelatin zymography. Molecular docking of ligands with HER2, Src, PI3Kα, PI3KΔ, and PI3Kγ proteins was evaluated using MOE 2010. The MTT assay showed that the IC50 value of brazilein against MCF-7/HER2 cells was 51 ± 2.1 µM. Moreover, brazilein and its combination with doxorubicin-induced G2/M accumulation and apoptosis. Combination of brazilein-doxorubicin inhibited cell migration and tended to decrease HER2, Rac1, p120, MMP2, and MMP9 protein expression levels. Based on our molecular docking study, the docking score of brazilein with PI3Kγ is comparable to that of the native ligand. Taken together, a combination of brazilein-doxorubicin exhibited synergistic cytotoxic and antimigratory effects on MCF-7/HER2 cells.<br />.

Enhanced antitumor efficacy by combining afatinib with MDV3100 in castration-resistant prostate cancer.

Background: Patients with prostate cancer often develop resistance to androgen deprivation therapy, a condition called castration-resistant prostate cancer (CRPC). Enzalutamide (MDV3100) can prolong the survival of patients with CRPC after chemotherapy, but ∼50% of patients eventually relapse and develop resistance to MDV3100. Thus, it is necessary to explore new treatment methods to improve the therapeutic effect of MDV3100. Tyrosine kinases play an essential role in the pathogenesis of CRPC. Methods: MTT assay was used to detect the inhibitory effects of MDV3100 and tyrosine kinase inhibitor on prostate cancer cells. CompuSyn version 1.0 was used to calculate the combination index (CI) values using the Chou-Talalay method. Clone formation and EdU assay were used to detect the effect of afatinib combined with MDV3100 on the proliferation of 22Rv1 cells. RT-qPCR and Western blot were used to explore the mechanism of drug combination. The aim of the present study was to determine the effects of several tyrosine kinase inhibitors (TKIs) when used in combination with MDV3100 in vitro. Results: The results demonstrated that TKIs combined with MDV3100 exerted a synergistic effect on a variety of PCa cells. Afatinib combined with MDV3100 could suppress the proliferation and migration of 22RV1 cells, as well as cause their cell cycle arrest and apoptosis. Mechanistically, afatinib effectively reduced the protein expression levels of HER2 and HER3 and inhibited EGFR phosphorylation, thereby enhancing the effect of MDV3100 and suppressing CRPC. Conclusions: These findings suggested that afatinib treatment improved the effect of MDV3100 on 22RV1 cells, highlighting this drug as a potential therapeutic strategy for patients with CRPC.

Survival analysis in breast cancer using proteomic data from four independent datasets

Breast cancer clinical treatment selection is based on the immunohistochemical determination of four protein biomarkers: ESR1, PGR, HER2, and MKI67. Our aim was to correlate immunohistochemical results to proteome-level technologies in measuring the expression of these markers. We also aimed to integrate available proteome-level breast cancer datasets to identify and validate new prognostic biomarker candidates. We searched studies involving breast cancer patient cohorts with published survival and proteomic information. Immunohistochemistry and proteomic technologies were compared using the Mann–Whitney test. Receiver operating characteristics (ROC) curves were generated to validate discriminative power. Cox regression and Kaplan–Meier survival analysis were calculated to assess prognostic power. False Discovery Rate was computed to correct for multiple hypothesis testing. We established a database integrating protein expression data and survival information from four independent cohorts for 1229 breast cancer patients. In all four studies combined, a total of 7342 unique proteins were identified, and 1417 of these were identified in at least three datasets. ESR1, PGR, and HER2 protein expression levels determined by RPPA or LC–MS/MS methods showed a significant correlation with the levels determined by immunohistochemistry (p < 0.0001). PGR and ESR1 levels showed a moderate correlation (correlation coefficient = 0.17, p = 0.0399). An additional panel of candidate proteins, including apoptosis-related proteins (BCL2,), adhesion markers (CDH1, CLDN3, CLDN7) and basal markers (cytokeratins), were validated as prognostic biomarkers. Finally, we expanded our previously established web tool designed to validate survival-associated biomarkers by including the proteomic datasets analyzed in this study (https://kmplot.com/). In summary, large proteomic studies now provide sufficient data enabling the validation and ranking of potential protein biomarkers.

Investigation of The Synergistic Effects of Trastuzumab And Gambogic Acid in Her-2 Positive Breast Cancer Cell Line

HER2 positive breast cancer is one of the biggest health problems in the world, causing millions of deaths every year. Drug combination modeling studies are extensively evaluated in treating many diseases. Pharmacological studies over the last half-century have shown that gambogic acid has potent anti-tumor activity against many types of cancer, including breast cancer. In this study, we examined the synergistic anticancer effect of gambogic acid and trastuzumab in HER2 positive breast cancer cell line (MDA-MB-453). In-vitro synergistic and antiproliferative effects of trastuzumab plus gambogic acid studies were determined with XTT method and the combination index (CI) values of the trastuzumab and gambogic acid combination were calculated by CompuSyn software. To determine molecular mechanisms of the trastuzumab and gambogic acid combination, we analyzed HER2, caspase-9 and Bax gene and protein expression levels quantitative reverse transcription-PCR (qRT-PCR) and ELISA techniques. The combination of 50 µg/ml trastuzumab and 5 µM gambogic acid showed the best synergistic effect at 24 h incubation in MDA-MB-453 cells according to the in-vitro cell proliferation, RT-qPCR and ELISA test. Gambogic acid effects on HER2 positive breast cancer cell line shows its potential as natural compound to inhibit breast cancer cell proliferation in combination with trastuzumab.

Quinazoline Based HSP90 Inhibitors: Synthesis, Modeling Study and ADME Calculations Towards Breast Cancer Targeting

A new 2-thioquinazolinones series was designed and synthesized as HSP90 inhibitors based on the structure of hit compound VII obtained by virtual screening approach. Their in vitro anti-proliferative activity was evaluated against three human cancer cell lines rich in HSP90 namely; colorectal carcinoma (HCT-116), and cervical carcinoma (Hela), breast carcinoma (MCF-7). Compounds 5a, 5d, 5e and 9h showed a significant broad spectrum anti-proliferative activity against all tested cell lines. They were characterized by potent effect against breast cancer in particular with IC50 of 11.73, 8.56, 7.35 and 9.48 μM, respectively against Doxorubicin (IC50 4.17 μM). HSP90 ATPase activity inhibition assay were conducted where compound 5d exhibited the best IC50 with 1.58 μM compared to Tanespimycin (IC50 = 2.17 μM). Compounds 5a and 9h showed higher IC50 values of 3.21 and 3.41 μM, respectively. The effects of 5a, 5d and 9h on Her2 (a client proteins of HSP90) and HSP70 were evaluated in MCF-7 cells. All tested compounds were found to reduce Her2 protein expression levels and induce Hsp70 protein expression levels significantly, emphasizing that antibreast cancer effect is a consequence of HSP90 chaperone inhibition. Cell cycle analysis of MCF-7 cells treated with 5d showed cell cycle arrest at G2/M phase 38.89% and pro-apoptotic activity as indicated by annexin V-FITC staining by 22.42%. Molecular docking studies suggested mode of interaction to HSP90 via hydrogen bonding. ADME properties prediction of the active compounds suggested that they could be used as orally absorbed anticancer drug candidates.

Targeted next generation sequencing to expand HER2 status detection: Implication for newer HER2-directed agents.

1047 Background: Establishment of ERBB2 ( HER2) amplification status in breast carcinoma (BC) and gastric carcinoma (GC) is essential for treatment selection, but no anti- HER2 therapies have been approved for tumors with low level of HER2 expression. The clinical trial ( NCT02564900 ) was initiated to evaluate the safety and efficacy of trastuzumab deruxtecan (DS-8201a) in BC patients with lower HER2 expression by current methods. This study aims to validate NGS-based detection for HER2 low expression. Methods: 275 BC and 425 GC were collected and subjected to NGS for genomic alteration detection. The testing was carried out by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory, Shanghai, China. Protein expression was analyzed by using IHC. FISH was carried out on 108 samples, including 63 BC and 45 GC. To set up NGS cutoff, FISH was performed on additional 34 samples. Sensitivity, specificity and accuracy were evaluated based on FISH as a gold-standard reference. Results: In BC, the expression level of HER2 protein detected by IHC was overall IHC 0 in 28.7%, 1+ in 18.9%, 2+ in 27.3% and 3+ in 25.1%, respectively, while in GC, the expression level was 60.7%, 18.6%, 14.8% and 5.9%, respectively. Log2ratio was used to assess ERBB2 amplification status detected by NGS. According to the FISH results of 34 other samples with high sensitivity (98%) and specificity (100%), the threshold was determined as 0.5. 8 and 10 samples of IHC 1+/2+ met the cutoff in BC and GC, respectively. In 63 BC, there were 17 positive and 46 negative by FISH. According to the threshold, the sensitivity and specificity of NGS detection was 94.1% and 97.8%, respectively. The proportion of samples with IHC 2+ that couldn't determine NGS ERBB2 status was 49.2%. However, except for false positive and false negative, the NGS results were concordant with FISH. In 45 GC, there were 5 positive and 40 negative by FISH. The specificity and sensitivity was 97.5% and 40%, respectively. In 4 samples with IHC 2+, 2 of them were discordant with the results of NGS and FISH. All IHC 1+ didn't meet the cutoff of NGS and was FISH negative in 108 samples. The accuracy of NGS in ERBB2 detection was 96.8% and 91.1% for BC and GC, respectively. Conclusions: Our data indicated that the NGS-based detection of ERBB2 amplification had high sensitivity, specificity and accuracy. In samples with IHC 1+/2+, the results of NGS detection were high concordant with FISH detection.

Evolving concepts in HER2 evaluation in breast cancer: Heterogeneity, HER2-low carcinomas and beyond.

The human epidermal growth factor receptor 2 (HER2) is a well-known negative prognostic factor in breast cancer and a target of the monoclonal antibody trastuzumab as well as of other anti-HER2 compounds. Pioneering works on HER2-positive breast cancer in the 90s' launched a new era in clinical research and oncology practice that has reshaped the natural history of this disease. In diagnostic pathology the HER2 status is routinely assessed by using a combination of immunohistochemistry (IHC, to evaluate HER2 protein expression levels) and in situ hybridization (ISH, to assess HER2 gene status). For this purpose, international recommendations have been developed by a consensus of experts in the field, which have changed over the years according to new experimental and clinical data. In this review article we will document the changes that have contributed to a better evaluation of the HER2 status in clinical practice, furthermore we will discuss HER2 heterogeneity defined by IHC and ISH as well as by transcriptomic analysis and we will critically describe the complexity of HER2 equivocal results. Finally, we will introduce the clinical impact of HER2 mutations and we will define the upcoming category of HER2-low breast cancer with respect to emerging clinical data on the efficacy of specific anti-HER2 agents in subgroups of breast carcinomas lacking the classical oncogene addition dictated by HER2 amplification.

Plasma HER2ECD a promising test for patient prognosis and prediction of response in HER2 positive breast cancer: results of a randomized study - SAKK 22/99

BackgroundThe HER2 extracellular domain shed in blood (HER2ECD) is reported to rise and fall in parallel with HER2+ breast cancer behavior. In this study, we evaluated the clinical relevance of plasma HER2ECD values in patients with metastatic breast cancer treated in the SAKK22/99 trial comparing trastuzumab monotherapy followed by trastuzumab-chemotherapy combination at progression versus upfront combination therapy.MethodsQuantitative assessment of plasma HER2ECD was performed in 133 patients at baseline; after 2–24 h; at 3 weeks; at first response evaluation (8–9 weeks); and at tumor progression. Associations with tumor characteristics, disease course and trial treatment were evaluated.ResultsBaseline HER2ECD levels were stable within 24 h after the first trastuzumab injection. These plasma values correlated positively with the HER2 gene ratio (rs = 0.39, P < 0.001) and HER2 protein expression levels (rs = 0.36, P < 0.001) but not with ER/PR status of the primary tumor. HER2ECD baseline levels were positively associated with the presence of visceral disease (P = 0.05) and poor patients’ outcome (Cox-regression: P = 0.009). Patients with high baseline levels (> 35 ng/ml) had the worst overall survival (P = 0.03) if treated with upfront combination therapy. Conversely, patients with low HER2ECD baseline values (< 15 ng/ml) had longer time to progression on combined trastuzumab-chemotherapy when first treated with trastuzumab monotherapy (P = 0.02). Monitoring HER2ECD levels during the course of the trial revealed significant time (P = 0.001) and time-treatment arm interactions (P = 0.0007). Under upfront trastuzumab alone, the HER2ECD levels remained stable until just before disease progression. In patients responding to combination treatment HER2ECD levels decreased to > 20%.ConclusionsPlasma HER2ECD levels in patients with metastatic breast cancer reflect HER2 disease status. This robust biomarker might help identifying patients without visceral disease profiting from a sequential treatment’s modality. Monitoring HER2ECD levels during trastuzumab monotherapy could help defining the optimal time to introduce chemotherapy.Trial registrationRegistration Number by ClinicalTrials.gov: NCT00004935, Trial number: SAKK22/99. Registered on 27 January 2003.

Changes in oncogenic protein levels in peri-implant oral malignancy: a case report

BackgroundOral squamous cell carcinoma (OSCC) constitutes a group of tumors that exhibit heterogeneous biology, histopathology, and clinical behaviors.Case presentationA 73-year-old male had a whitish leukoplakia-like lesion around inflamed peri-implant area (#42, #43, and #44), and this lesion had transformed to OSCC within 3 years. He underwent mass resection, selective neck dissection, and reconstructive surgery. To detect any carcinogenesis progression, we examined the removed tumor tissue as well as the patient’s preoperative and postoperative sera to identify causative oncogenic proteins using immunoprecipitation high-performance liquid chromatography (IP-HPLC).ConclusionsThe protein expression levels of p53, E-cadherin, β-catenin, MMP-10, HER2, NRAS, Met, HER2, and ERb were significantly lower in the serum collected on postoperative day 10 than in the preoperative serum, and if these proteins are consistently not elevated in the serum 3 months after surgery compared with the preoperative serum, these proteins can be potential oncogenic proteins. However, we also found that the serum extracted 3 months after the operation had elevated levels of oncogenic proteins compared with that of the preoperative and 10-day postoperative serum indicating the possibility of tumor recurrence. At postoperative follow-up period, ipsilateral neck metastasis and second primary lesion were found and additional surgery was performed to the patient. IP-HPLC using the patient’s serum shows the possibility of oncogenic protein detection. However, follow-up IP-HPLC data is needed to find out patient-specific prognostic factors.

IP‐HPLC serum analysis of potential oncogenic proteins in peri‐implant oral malignancy

Background Oral squamous cell carcinoma (OSCC) constitutes a group of tumors that exhibit heterogeneous biology, histopathology, and clinical behaviors. Aim/Hypothesis Changes of oncogenic protein expressions in patient diagnosed as peri-implant oral malignancy (PIOM) were observed through immunoprecipitation high performance liquid chromatography (IP-HPLC). Material and Methods A 73-year-old male had a whitish leukoplakia like lesion around inflamed peri-implant area (#42, #43, and #44), and this lesion had transformed to OSCC within three years. He underwent mass resection, selective neck dissection, and reconstructive surgery. To detect any carcinogenesis progression, we examined the removed tumor tissue as well as the patient's preoperative and postoperative sera to identify causative oncogenic proteins using immunoprecipitation high performance liquid chromatography (IP-HPLC). Results The protein expression levels of p53, E-cadherin, β-catenin, MMP-10, HER2, NRAS, Met, HER2, and ERb were significantly lower in the serum collected on postoperative day 10 than in the preoperative serum, and if these proteins are consistently not elevated in the serum three months after surgery compared with the preoperative serum these proteins can be potential oncogenic proteins. However, we also found that the serum extracted three months after the operation had elevated levels of oncogenic proteins compared with that of the preoperative and 10-day postoperative serum indicating the possibility of tumor recurrence. At postoperative follow-up period, ipsilateral neck metastasis and second primary lesion were found, the patient was performed additional surgery. Conclusion and Clinical Implications IP-HPLC using patient`s serum show the possibility of oncogenic protein detection. However, follow-up IP-HPLC data are needed to find out patient-specific prognostic factors.

Abstract 29: Activity of trastuzumab emtansine (T-DM1) in 3D cell culture

Abstract Introduction: Cell spheroids/aggregates generated from three dimensional (3D) cell culture methods are similar to real tumors in terms of tissue morphology, biology, and gene expression. We performed a unique 3D cell culture drug efficacy study with Trastuzumab emtansine (T-DM1) across a number of breast cancer cell lines that were previously investigated in 2D cell culture (Lewis Phillips, et al, 2008). We obtained significantly different results for some cell lines grown as 3D spheroids/aggregates when compared to those grown as 2D cultures. Methodology: We performed 3D cell culture and produced FFPE blocks (using novel methods) from 3D spheroids/aggregates to determine HER2 (IHC) protein expression levels for five cell lines: SK-BR-3; BT-474; MDA-MB-361; MDA-MB-175 and MCF-7. We also performed HER2 gene-protein assay (HER2 GPA) to determine HER2 gene amplification along with IHC protein expression status in four cell lines: SK-BR-3; BT-474; MDA-MB-361 and MDA-MB-175. Drug (T-DM1) activity testing using CellTiterTM-Glo 3D cell viability assay was performed on 3D cell spheroids/aggregates for comparison with 2D cells. Images were obtained of T-DM1 internalization in BT-474 cells and spheroids using pHrodo™ iFL Human IgG Labeling Reagent. Results: In 3D spheroids/aggregates, HER2 IHC staining and GPA assay showed for SK-BR-3 and BT-474 HER2 3+ expression and HER2/CEP17 of ≥ 2; MDA-MB-361 cells with HER2 2+ expression and HER2/CEP17 of ≥ 2.0; MDA-MB-175 cells with HER2 1+ expression and HER2/CEP17 &amp;lt; 2.0 and MCF-7 cells with HER2 0+(IHC staining only without HER2 GPA data). Some of the 3D spheroids/aggregates in MDA-MB-361 cells showed heterogeneous expression of HER2 protein. The IC50 values of 3D spheroids/aggregates for some cell lines were significantly higher than were demonstrated for cell lines grown in 2D cell cultures. The fold changes between 3D spheroids and 2D cells (72h T-DM1 treatment time) are: 4.2 for SK-BR-3; ≳ 10 for BT-474 and 22 for MDA-MB-361. Additionally, the fluorescent images showed that a longer incubation time was required for the T-DM1 drug (3 µg/ml) to be internalized effectively into BT-474 3D spheroids; for example, about 120h for 3D spheroids in comparison to about 36h in 2D cells. Interestingly, the 3D spheroids incubated for 120h with T-DM1 (470 µm) are smaller in size than 3D spheroids in the control group (600 µm) incubated for 120h without T-DM1 treatment. Conclusions: Drug efficacy studies performed on 3D cultured spheroids/aggregates are expected to be very important and biologically relevant for determining drug activity in tumor tissue. Our drug efficacy study using 3D cell culture demonstrated greater concentrations of T-DM1 and longer incubation times were required than for cells grown as 2D in some cell lines, likely due to less efficient internalization. Citation Format: Jean Z. Boyer, Gail Lewis Phillips, Hiro Nitta, Karl Garsha, Eric May, Brittany Admire, Robert Kraft, Megan Peccarelli, Andre Zamorano, Scott Gill, Eslie Dennis, Liz Vela, Penny Towne. Activity of trastuzumab emtansine (T-DM1) in 3D cell culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 29.