HER2 Gene Expression Research Articles

Abstract 968: Evaluation of Her2 RNA expression as a potential predictive biomarker for anti-Her2 therapy

Abstract Trastuzumab deruxtecan (Enhertu) is effective in "HER2 Low" breast cancer, defined by 1+ or 2+ expression by immunohistochemistry (IHC). Interest has now turned toward defining a sub-population of IHC 0+ tumors that may have HER2 expression below the limit of IHC detection/quantification and may thus also be responsive. We previously validated a high dynamic range HER2 RNA expression assay run as part of our comprehensive genomic profiling test, StrataNGS. Herein, we evaluated the HER2 RNA expression data together with copy number and clinical outcome data from the Strata Clinical Molecular Database (SCMD) in advanced breast cancer (n = 3,063) and other advanced solid tumors (n = 26,715). As expected, HER2 gene expression was significantly higher in tumors with DNA amplification (>=6 copies; median: 13.9 vs. 10.0 in log2 units; p < 1e-100). Despite similar copy number levels in amplified breast vs. other cancers (median: 21.8 vs. 19.8 copies), HER2 expression levels were ~2-fold higher (median: 14.5 vs. 13.5; p = 1.3e-10). Similarly, HER2 expression levels were higher in non-amplified breast vs. other cancers (median: 10.7 vs. 9.9; p<1e-100), suggesting that DNA amplification and cell lineage affect HER2 expression. Using our previously validated HER2 threshold, among 75 eligible SCMD breast cancer patients treated with 1st or 2nd line systemic trastuzumab or pertuzumab containing therapy, HER2 RNA High patients (n=46, 59%) had significantly longer time to next therapy (TTNT) compared to HER2 RNA Not High patients (median TTNT 26.9 vs. 5.6 months, adjusted hazard ratio 0.31, p=0.005 when adjusted for 1st vs. 2nd line, pertuzumab inclusion, and inclusion of chemotherapy or hormonal therapy). In patients with available IHC data (n = 388), HER2 RNA expression trended with IHC across the 0-3+ range, however, while 3+ tumors had distinctly high RNA expression (median: 14.4), 0-2+ tumors had lower expression with overlapping distributions (median: 10.5, 10.9, 11.5, respectively), suggesting that 0-2+ tumors do not represent distinct biological groups, but rather a continuum of low expression. We defined a HER2 RNA Low threshold (>10.6), corresponding to the top 75% of IHC 1-2+ breast cancers. Importantly, at this threshold, nearly half (44.1%) of 0+ breast cancers were also classified as HER2 RNA Low. Additionally, 25.8% of all non-breast solid tumors were classified as HER2 RNA Low. Given that HER2 RNA High predicted benefit from 1st generation anti-HER2 therapies, future studies should consider HER2 RNA Low as an alternative biomarker to Her2 IHC Low, with the opportunity to further expand trastuzumab deruxtecan use into the IHC 0+ breast cancer population and potentially to additional solid tumors. Citation Format: Laura E. Lamb, Nickolay A. Khazanov, Daniel H. Hovelson, Kat Kwiatkowski, D. Bryan Johnson, Daniel R. Rhodes, Scott A. Tomlins. Evaluation of Her2 RNA expression as a potential predictive biomarker for anti-Her2 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 968.

Baicalein suppresses HER2-mediated malignant transformation of HER2-overexpressing ovarian cancer cells by downregulating HER2 gene expression.

The upregulation of the HER2 oncogene is associated with a variety of human cancers and is associated with poor prognosis. Baicalein is reported to have anti-tumor activity, but the molecular mechanism of this effect in HER2-positive cancer cells has not been studied. In this study, our data showed that baicalein can inhibit the proliferation and transformation potential of ovarian cancer cells overexpressing HER2. Baicalein treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level. Baicalein acted on ovarian cancer cells overexpressing HER2 to downregulate the PI3K/Akt signaling pathway downstream of HER2 and inhibit the expression or activity of downstream targets, such as VEGF and cyclin D1 and MMP2. Oral administration of baicalein supplemented with a pharmaceutical excipient significantly inhibited the growth of HER2-overexpressing ovarian SKOV-3 cancer xenografts in mice. These results suggest that downregulation of HER2 gene expression by baicalein at the transcriptional level contributes to inhibit the in vitro and in vivo proliferation and HER2-mediated malignant transformation of HER2-overexpressing ovarian cancer cells.

Biomarker Data from the Phase III KATHERINE Study of Adjuvant T-DM1 versus Trastuzumab for Residual Invasive Disease after Neoadjuvant Therapy for HER2-Positive Breast Cancer.

In KATHERINE, adjuvant T-DM1 reduced risk of disease recurrence or death by 50% compared with trastuzumab in patients with residual invasive breast cancer after neoadjuvant therapy (NAT) comprised of HER2-targeted therapy and chemotherapy. This analysis aimed to identify biomarkers of response and differences in biomarker expression before and after NAT. Exploratory analyses investigated the relationship between invasive disease-free survival (IDFS) and HER2 protein expression/gene amplification, PIK3CA hotspot mutations, and gene expression of HER2, PD-L1, CD8, predefined immune signatures, and Prediction Analysis of Microarray 50 intrinsic molecular subtypes, classified by Absolute Intrinsic Molecular Subtyping. HER2 expression on paired pre- and post-NAT samples was examined. T-DM1 appeared to improve IDFS versus trastuzumab across most biomarker subgroups, except the HER2 focal expression subgroup. High versus low HER2 gene expression in residual disease was associated with worse outcomes with trastuzumab [HR, 2.02; 95% confidence interval (CI), 1.32-3.11], but IDFS with T-DM1 was independent of HER2 expression level (HR, 1.01; 95% CI, 0.56-1.83). Low PD-L1 gene expression in residual disease was associated with worse outcomes with trastuzumab (HR, 0.66; 95% CI, 0.44-1.00), but not T-DM1 (HR, 1.05; 95% CI, 0.59-1.87). PIK3CA mutations were not prognostic. Increased variability in HER2 expression was observed in post-NAT versus paired pre-NAT samples. T-DM1 appears to overcome HER2 resistance. T-DM1 benefit does not appear dependent on immune activation, but these results do not rule out an influence of the tumor immune microenvironment on the degree of response.

Expression of epidermal growth factor receptor and human epidermal growth factor receptor 2 in urothelial bladder carcinoma in an Egyptian cohort: Clinical implication and prognostic significance.

Bladder cancer (BC) has a particular importance in Egyptian patients due to aggressive behavior and absence of prognostic markers. To evaluate the expression of gene and protein expression of HER2 and epidermal growth factor (EGFR) in Egyptian patients with BC and ultimately to investigate their clinical implication and prognostic significance. The study was carried out on 46 patients with urothelial bladder BC. Tissue were obtained from transurethral resection (N = 22) and radical cystectomy (N = 24) specimens. The original hematoxylin and eosin slides were re-evaluated and the formalin fixed, paraffin-embedded (FFPE) tissues which had sufficient tumor tissue (>75%) and minimal or absent tumor necrosis were selected for immunohistochemistry (IHC) and RNA extraction. Furthermore, five control biopsies were obtained from patients with cystitis. Follow-up data were retrieved from the medical records which included the treatment regimen, disease recurrence and/or progression, and survival. EGFR and HER2 protein were overexpressed in 35% and 46% of patients respectively. EGFR was correlated with the tumor size, grade and pathological stage, with a similar trend for HER2. The recurrence rate was higher in patients with expression of any of the markers. Gene expression was significantly higher (10.6-folds) for EGFR and (21-folds) for HER2 in patients with BC in comparison to control patients. Survival analysis showed lower median disease-free survival in association with HER2 protein overexpression. Our data highlighted the prognostic significance of EGFR and HER in BC and proposed their possible use as predictive markers and potential therapeutic targets.

Effects of Alkanna bracteosa extract on the expression level of HSP90α and HER2 genes in human gastric cancer cell line

Gastric cancer was classified as the third most deadly cancer among all other cancer types. The HSP90 and HER2 genes play essential roles in the stability and function of high-expression proteins that cause malignancy. The aim of this research was to investigate the influence of the alcoholic Alkanna bracteosa extract on the expression of HSP90α and HER2 genes in AGS cell line. Therefore, the methanolic extraction was isolated from aerial parts of the plant and AGS and HuGu cell lines were analyzed using 102.4–0.05 mg ml−1 dose concentrations in serial dilution; to measure the cell toxicity by MTT assay. Furthermore, real-time PCR analysis measured the expression level of HSP90α and HER2 genes using the IC50 dose concentrations. Quantification of apoptosis was analyzed by Annexin/PI kit in flow cytometry and DNA fragmentation tests. The results of MTT assay represented the IC50 dose concentration of 0.8 and 3.2 mg ml−1 for AGS and HuGu respectively. The rate of HER2 gene expression was significantly decreased in AGS cells treated with 0.8 mg ml−1 dose concentration compared to control. The exposure of AGS treated cells with 0.8 mg ml−1 dose concentration after 24 h represented 24.3% apoptosis and 13.3% necrosis. The agarose gel represented the DNA fragmentation pattern of apoptosis. This study demonstrated the significant differences between the cell viability rate, gene expression level, and apoptosis of the Alkanna bracteosa extract on AGS cells. These results demonstrated the first report of which the Alkanna braceteosa would be an effective candidate for possible treatment of Gastric cancer.

Expression and Clinical Significance of HER2 Gene and DNMT1 in Non-Small-Cell Lung Cancer.

Objective To explore the expression and clinical significance of HER2 and DNMT1 in non-small-cell lung cancer. Methods The patients with non-small-cell lung cancer treated in the First Affiliated Hospital of Jiamusi University between 2018 and 2020 were enrolled in this study. The serum DNMT1 concentration and the expression of HER2 protein in lung cancer and adjacent tissues of the two groups were analyzed. Results The DNMT1 protein concentration was significantly correlated with gender, age, and smoking history of patients. HER2-positive expression was significantly related to tumor type, tumor size, tumor differentiation degree, and lymph node metastasis. However, HER2 levels were not related to the gender and smoking history of patients. Conclusion High expression of DNMT1 protein in serum may increase the risk of non-small-cell lung cancer and may play an important role in the early development of lung cancer. HER2-positive expression may promote the development of advanced and metastatic non-small-cell lung cancer.

Long-term prognostic significance of HER2-low and HER2-zero in node-negative breast cancer

BackgroundRecently, novel antibody––drug conjugates (ADCs) showed clinical activity in a subset of advanced human epidermal growth factor receptor 2 (HER2)-negative patients. We investigated the prognostic significance of HER2-low and HER2-zero tumours. Patients and methodsThe retrospective cohort study included 410 consecutive node-negative breast cancer patients without adjuvant systemic therapy treated between 1985 and 2000 (median follow-up: 16.73 [IQR 8.58–23.45] years). 351 (85.6%) were HER-2 negative and subdivided into HER2-zero (immunohistochemistry [IHC] score 0) and HER2-low (IHC score 1+ or 2+/in situ hybridisation [ISH]-negative). HER2 gene expression was available in 170 (48.4%) patients. Differences in HER2 status for immunohistochemistry, gene expression and clinico-pathologic parameters were assessed using Fisher's exact test, Pearson's correlation and Mann–Whitney test. Prognosis was investigated using the Kaplan–Meier method and Cox regression analyses. ResultsOf the 351 HER2-negative patients, 198 (56.4%) had HER2-low tumours and 153 (43.6%) were HER2-zero. Significant differences between HER2-zero and HER2-low tumours were found in histologic grading (P = 0.001), Ki-67 (P = 0.013) and HER2 gene expression (P = 0.002). HER2-low patients had significantly longer disease-free survival (DFS) (15-year rate: 67.5% [95% CI 61.0–74.7] vs. 47.3% [95% CI 39.9–56.1], P < 0.001) and overall survival (OS) (15-year rate: 75.4% [95% CI 69.4–81.9] vs. 66.8% [95% CI 59.5–74.9], P = 0.009). The OS difference was observed in hormone receptor (HR)-positive (P = 0.039) but not HR-negative (P = 0.086) tumours. The results of multivariable analyses confirmed the independent prognostic significance of HER2 status (DFS: HR, 0.546; 95% CI, 0.402–0.743; P < 0.001; OS: HR, 0.653; 95% CI, 0.458–0.932; P = 0.019). ConclusionHER2-low patients had a better survival than HER2-zero patients.

HER2-ResNet: A HER2 classification method based on deep residual network.

BACKGROUND: HER2 gene expression is one of the main reference indicators for breast cancer detection and treatment, and it is also an important target for tumor targeted therapy drug selection. Therefore, the correct detection and evaluation of HER2 gene expression has important value for clinical treatment of breast cancer.OBJECTIVE: The study goal is to better classify HER2 images.METHODS: For general convolution neural network, with the increase of network layers, over fitting phenomenon is often very serious, which requires setting the value of random descent ratio, and parameter adjustment is often time-consuming and laborious, so this paper uses residual network, with the increase of network layer, the accuracy will not be reduced.RESULTS: In this paper, a HER2 image classification algorithm based on improved residual network is proposed. Experimental results show that the proposed HER2 network has high accuracy in breast cancer assessment.Conclusion: Taking HER2 images in Stanford University database as experimental data, the accuracy of HER2 image automatic classification is improved through experiments. This method will help to reduce the detection intensity and improve the accuracy of HER2 image classification.

C-MET positivity and its relationship with histopathological findings in gastric carcinomas exhibiting HER2 gene expression.

Co-expressions of receptor tyrosine kinases such as c-MET and HER2 were reported in many studies. The concomitant expression is associated with more aggressive clinical course. In this study, it was intended to investigate the correlation of the positivity of c-MET and HER2 with histopathologic findings and their impacts on prognosis. After the decision of the ethics committee, a total of 64 cases, whose HER 2 status was studied by dual silver in situ hybridization/immunohistochemistry method, were included in the study. Immunohistochemical staining for c-MET was performed to all cases and the evaluation was performed similarly to the criteria for HER2 evaluation, but cytoplasmic staining was also considered significant. The data were analyzed using SPSS 20 for Windows. c-MET positivity which is considered by the score of 2+ and 3+ was found only in 34.4% of HER2 positive cases while it was 59.3% in HER2 negative cases (P = 0.045). The sole histopathological feature associated with c-MET positivity was distal gastric localization (P = 0.016). Even though higher rates of c-MET positivity in HER2 positive cases were stated in the literature, contrary results were obtained in this study. Comparing the HER2+/c-MET + co-expression group with the other groups, no difference was found about age, sex, macroscopic and microscopic characteristics. The presence of c-MET positivity in cases with HER2 expression suggests that c-MET expression might be associated with the resistance to Trastuzumab.

ELISA and Fourier-transform infrared spectroscopy

ELISA and FTIR assay techniques were used to identify HER2 gene expression in the blood serum of female dogs and to characterise the biochemical composition. ELISA tests assess the stage of primary tumour development and evolution, while FTIR allows for a complete characterisation of biomolecules associated with the tumoral process. Blood serum samples from 30 female dogs were analysed. Concentrations of the HER2/neu protein were detected using ELISA kits specific for canine and human detection. Infrared spectroscopy (IR) was conducted in absorbance mode at a frequency range of 400–4000 cm-1 and a resolution of 4 cm-1 over 50 scans. The ELISA cut-off for HER2 protein concentration in blood serum was determined using the receiver operating characteristic (ROC) curve and by estimating the area under the curve (AUC) at a 95% confidence interval (CI=95%). The ROC curves in the canine and human ELISA tests were 0.75 and 0.45, respectively. The representative IR spectra for HER2 gene expression corresponded to lipids (1161 cm-1, 1452 cm-1, 2851 cm-1). This study contributes to the knowledge of HER2 through the identification of biochemical features associated with the changes in the HER2/neu+ and HER2/neu- states.

Diagnostic Potential of miR-30a and miR-200c in Invasive Breast Ductal Carcinoma

Background: Breast cancer is one of the most common cancers worldwide and is responsible for the death of many people. It is mainly found in women; however, it is rarely observed in men. Nowadays, extensive research has been conducted on the detection, diagnosis, and prognosis of biomarkers of this type of cancer, as well as other types. MicroRNAs play a major role in regulating the onset and progression of breast cancer. To date, many significant increases and decreases in microRNAs levels have been reported during various cancers, and also tumor inhibitory roles have been reported for these small non-coding RNA molecules. Methods: In this study, miR-30a and miR-200c were studied to find new expression patterns in breast cancer patients. For this purpose, a quantitative polymerase chain reaction was used to analyze the relationships between expression levels of miR-30a and miR-200c and several clinical and pathological features of the disease, such as tumor stage, HER2 gene expression, and lymphatic metastasis. Results: Our results showed that the expression of miR-200c and miR-30a in tumor samples was significantly lower than in normal samples (P &lt; 0.01). Also, at higher stages of the disease, the expression of both miRNAs was remarkably reduced (P &lt; 0.01). We also found that the expression of two miRNAs in patients with HER2 gene expression and metastasis in lymphatic regions was significantly lower than in HER2-negative and non-metastatic patients (P &lt; 0.05 and P &lt; 0.01, respectively). Conclusions: We identified the relationship between miR-200c and miR-30a and breast cancer. These findings indicate that these microRNAs can be considered biomarkers in breast cancer.

COMPREHENSIVE REVIEW ON ETIOPATHOGENESIS, TREATMENT AND EMERGING THERAPIES OF BREAST CANCER

Breast cancer (BC) appears to the most predominant form of cancer for women and the second largest factor in malignancy-associated mortality in women globally. The major predicts indicators for BC comprise lower menarche, late pregnancy, later menopause, obesity, alcohol intake, immobility, and another factor that influence the development of BC. Even though, it’s rising prevalence, BC death has decreased due to screening, early diagnosis, and access to adjuvant therapies for their treatment. Chemotherapy, surgery, targeted therapy, hormone therapy, and radiation therapy are all treatments for BC, but due to adverse side effects and several drug resistances, they are becoming largely unsuccessful. BC is a complex disorder for several different forms and classification has depended on the cell morphology and immunohistochemical evaluation focused on the deregulation gene expression of various molecular biomarkers, that is, BRCA1, BRCA2, mTOR, PI3K, RB, MDM2, TQK53, and HER2, microRNA is responsible for BC occurrences. Several in vitro, in vivo, and clinical studies are underneath assessment and many other types of research, clinical studies are promising better effectiveness against cancerous cell proliferation and related pathways involved in tumor growth and angiogenesis. Hence, in the present review, we summarized and compiled the complete information of BC about its molecular pathogenesis, biomarkers, staging system, emerging therapies, and current advancement of BC.

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

BackgroundCell spheroids and aggregates generated from three-dimensional (3D) cell culture methods are similar to in vivo tumors in terms of tissue morphology, biology, and gene expression, unlike cells grown in 2D cell cultures. Breast cancer heterogeneity is one of the main drug resistant mechanisms and needs to be overcome in order to increase the efficacy of drug activity in its treatments.MethodsWe performed a unique 3D cell culture and drug efficacy study with trastuzumab emtansine (Kadcyla®, T-DM1) across five breast cancer cell lines (BT-474, SK-BR-3, MDA-MB-361, MDA-MB-175, and MCF-7) that were previously investigated in 2D cell culture. We performed HER2 IHC staining, cell viability experiments, Gene-protein-assay (GPA), and T-DM1 internalization studies.ResultsWe obtained significantly different results including higher IC50 for some of the cell lines. Our GPA showed some significant heterogeneous HER2 gene and protein expression in 3D cultured spheroids or aggregates. The fluorescent images also showed that a longer incubation time is needed for T-DM1 to be internalized effectively into 3D cultured spheroids or aggregates.ConclusionOur study demonstrated that the difference of T-DM1 drug activity in 3D spheroids or aggregates might be due to tumor heterogeneity and less efficient internalization of T-DM1 that is not seen using 2D cell culture models. Drug studies using 3D cell culture are expected to provide biologically relevant models for determining drug activity in tumor tissue in future drug response and resistance research.

HER2 Expression, Test Deviations, and Their Impact on Survival in Metastatic Gastric Cancer: Results From the Prospective Multicenter VARIANZ Study.

PURPOSETrastuzumab is the only approved targeted drug for first-line treatment of human epidermal growth factor receptor 2–positive (HER2+) metastatic gastric cancer (mGC). However, not all patients respond and most eventually progress. The multicenter VARIANZ study aimed to investigate the background of response and resistance to trastuzumab in mGC.METHODSPatients receiving medical treatment for mGC were prospectively recruited in 35 German sites and followed for up to 48 months. HER2 status was assessed centrally by immunohistochemistry and chromogenic in situ hybridization. In addition, HER2 gene expression was assessed using qPCR.RESULTSFive hundred forty-eight patients were enrolled, and 77 had HER2+ mGC by central assessment (14.1%). A high deviation rate of 22.7% between central and local test results was seen. Patients who received trastuzumab for centrally confirmed HER2+ mGC (central HER2+/local HER2+) lived significantly longer as compared with patients who received trastuzumab for local HER2+ but central HER2− mGC (20.5 months, n = 60 v 10.9 months, n = 65; hazard ratio, 0.42; 95% CI, 8.2 to 14.4; P < .001). In the centrally confirmed cohort, significantly more tumor cells stained HER2+ than in the unconfirmed cohort, and the HER2 amplification ratio was significantly higher. A minimum of 40% HER2+ tumor cells and a HER2 amplification ratio of ≥ 3.0 were calculated as optimized thresholds for predicting benefit from trastuzumab.CONCLUSIONSignificant discrepancies in HER2 assessment of mGC were found in tumor specimens with intermediate HER2 expression. Borderline HER2 positivity and heterogeneity of HER2 expression should be considered as resistance factors for HER2-targeting treatment of mGC. HER2 thresholds should be reconsidered. Detailed reports with quantification of HER2 expression and amplification levels may improve selection of patients for HER2-directed treatment.

Cell specific tumor suppressor effect of Hsa-miR-1226-3p through downregulation of HER2, PIK3R2, and AKT1 genes.

PI3K/AKT signaling has a crucial role in breast cancer incidence and finding the miRNAs that regulate this pathway enhances our understanding of breast cancer regulation. Firstly, our bioinformatics analysis suggested miR-1226-3p as a bona fide regulator of HER2, PIK3R2, and AKT1 putative target genes. Secondly, RT-qPCR, ELISA test and western blotting showed that overexpression of miR-1226 was followed by reduced expression of HER2, PIK3R2, and AKT1 putative targets genes in SKBR3 cells. Third, dual luciferase assay verified direct interaction of miR-1226-3p with 3'UTR sequences of these target genes. Then, overexpression of miR-1226 in SKBR3 cells brought about increased population of sub-G1 and decreased populations of G1 cells, measured by flow cytometry. This was consistent to the reduction of p-AKT protein and increased BAX protein levels, detected by western analysis and consistent to decreased CCND1 genes expression, detected by RT-qPCR. The reduced survival and increased apoptosis rate of these cells was also verified through MTT, Annexin V-FITC and Live-Dead cell staining assays. Our results suggest that miR-1226-3p is a tumor suppressor in SKBR3 cells. However, following the overexpression of miR-1226 in MDA-MB-231 cells, Bax/Bcl2 ratio and CCND1 genes expression levels were not significantly changed, sub-G1 and G1 cell cycle population were reduced while, S and G2/M cell populations were increased, consistent to the results acquired from the apoptosis and staining assays. Finally, TCGA data analysis and RT-qPCR against 20 pairs of Normal/Tumor breast tissues indicated that miR-1226-3p has been downregulated in breast cancer. Overall, the present study gathered shreds of evidence that suggest miR-1226-3p as a tumor suppressor that exerts its inhibitory effect on SKBR3 cells through targeting of HER2, PIK3R2, and AKT1 genes and downregulates PI3K/AKT pathway.

Concordance of the 21-gene assay between core needle biopsy and resection specimens in early breast cancer patients

BackgroundAdjuvant therapy decisions may be partly based on the results of a multigene quantitative reverse transcription-polymerase chain reaction (RT-PCR)-based assay: the 21-gene recurrence score (RS) test of resection specimens. When necessary, core needle biopsy (CNB) may be considered as a surrogate. Here, we evaluated the concordance in gene expression according to results from RT-PCR-based RS testing between paired CNBs and resection specimens.MethodsCNBs and resection specimens from 50 breast cancer (BC) patients were tested to calculate RSs. First, we examined the concordance of the ER, PR and HER-2 status of tissue samples indicated by immunohistochemical (IHC) and RT-PCR analyses. Then, we compared the IHC findings of ER, PR, HER-2 and Ki-67 staining across paired samples. Ultimately, the RS and single-gene results for ER, PR, HER-2 and Ki-67 were explored between paired samples.ResultsThe concordance between IHC and RT-PCR was 100%, 80.0% and 100% for ER, PR and HER-2, respectively, in both resection specimens and CNBs. The concordance for IHC ER, PR, HER-2 and Ki-67 status was 100%, 94.0%, 52.0% and 82.0%, respectively, between paired samples. RS results from paired samples showed a strong correlation. The overall concordance in RS group classification between samples was 74%, 72% and 78% based on traditional cutoffs, TAILORx cutoffs and ASCO guidelines, respectively. ER, PR, HER-2 and Ki-67 were modestly- to- strongly correlated between paired samples according to the RT-PCR results.ConclusionA modest- to- strong correlation of ER, PR, HER-2 and Ki-67 gene expression and RS between CNBs and resection specimens was observed in the present study. The 21-gene RS test could be reliably performed on CNBs. ER, PR and HER-2 status showed remarkable concordance between the IHC and RT-PCR analyses. The concordance between paired samples was high for the IHC ER, PR and Ki-67 results and low for HER-2.

Calcium based siRNA coating: a novel approach for knockdown of HER2 gene in MCF-7 cells using gold nanoparticles

Surface functionalization of nanoparticles (NPs) for therapeutic siRNA delivery into cancer cells has gained interest. The present study was designed for surface functionalization of gold nanoparticles (AuNPs) for efficient siRNA delivery and knockdown in cancer cells. In order to achieve this objective, AuNPs were coated with HER2-siRNA in the presence of 11-mercaptoundecanoic acid (11-MUA), calcium chloride (CaCl2) and polyethyleneimine (PEI) in alternate charge bearing successive layers. MCF-7 cells were cultured and transfected with fabricated assembly of AuNPs. Cytotoxicity analysis revealed that the half inhibitory concentration (IC50) for the formulation was 45.35 nM . Total RNA was isolated from transfected cells, reverse transcribed into complementary DNA (cDNA) and real-time polymerase chain reaction (RT-PCR) was performed. The RT-PCR based delta-delta Ct analysis in treated cells revealed a significant 18.94 times decrease (p&lt;0.001) in the expression of HER2 gene standardized with ACTB housekeeping gene as compared to untreated cells, which makes this formulation a potent approach for siRNA delivery and gene knockout.

Abstract 738: Utility of Assessing HER2 RNA expression for precision medicine

Abstract Genomic analysis of tumors is rapidly becoming routine clinical practice. Most of the next generation sequencing (NGS)-based oncology assay focused on DNA alteration data for single nucleotide variants (SNVs), insertions and deletions (indels) and copy number variants (CNVs) of genes which have targeted drug. Although RNA sequencing technology is increasingly being explored, its clinical utility still needs to be clarified. As there is a growing number of HER2-targeted therapies, in this study, we dissected the association between HER2 amplification, RNA overexpression, HER2 protein expression by immunohistochemistry (IHC) and reverse phase proteomic arrays (RPPA) in 618 breast tumors in the TCGA. We conducted unsupervised clustering analysis based on HER2 RNA-seq gene expression and RPPA protein data to identify HER2 overexpression samples. As expected there was a strong correlation with ERBB2 copy number with HER2 RNA expression (R2 = 0.56, P &amp;lt;0.001), and HER2 protein expression by IHC (R2 = 0.25, P &amp;lt; 0.001). RNA expression also correlated with HER2 IHC (R2 = 0.40, P &amp;lt; 0.001) and HER2 protein expression by RPPA (R2 = 0.68, P &amp;lt; 0.001). We performed unsupervised clustering analysis based on RPPA protein data and gene expression data of the HER2 gene, all the breast cancer samples were classified as two distinct clusters. Among the two subgroups, one subgroup had significantly higher expression and protein level of HER2 gene. The clustering results of most breast tumors were consistent with the IHC score and copy number data. The gene expression cutoff value to distinguish the HER2 overexpression subgroup from the other was about 204 FPKM. In addition, we identified two HER2 overexpression tumors which were classified as HER2 negative based on IHC. Studies are planned to test transcriptomic based actionability in patient derived models. Our results suggest that RNA-seq gene expression data may identify patients with HER2 overexpression that may be classified as HER2 negative based on IHC. Further studies are needed to determine the role of HER2 RNA overexpression as a predictive marker and to determine HER2 RNA expression across tumor types. Citation Format: Yifei Shen, Christian Cruz Pico, Vakul Mohanty, Amber Johnson, Jia Zeng, Kenna Mills Shaw, Ken Chen, Funda Meric-Bernstam. Utility of Assessing HER2 RNA expression for precision medicine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 738.

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.

Predictive and prognostic value of HER2 gene expression and HER2 amplification in patients with metastatic colorectal cancer (mCRC) enrolled in CALGB/SWOG 80405 (Alliance).

4086 Background: The randomized phase III CALGB/SWOG 80405 trial found no difference in overall survival (OS) in first-line mCRC pts treated with either bevacizumab (Bev) or cetuximab (Cet) combined with the same chemotherapy. We investigated the potential prognostic and predictive value of HER2 amplification and HER2 gene expression using NGS and Nanostring data. Methods: Primary tumor DNA from 559 patients (pts) was profiled for HER2 amplification by NGS (Foundation One). Tumor tissue from 925 pts was tested for Nanostring gene expression using an 800 gene panel. OS and progression free survival (PFS) were the endpoints as time-to-event variables. Cox proportional hazard models with gene expression fitted with linear spline (one internal knot at median) were used, adjusting for pts baseline characteristics, treatment assignment, and molecular features (microsatellite instability, BRAF, all RAS). Results: Of 505 tumors with both NGS and Nanostring data, 16 harbored HER2 amplification (copy number variation &gt; 6), limited to microsatellite stable tumors and significantly associated with HER2 expression ( P &lt; 0.001) and wild-type RAS ( P = 0.036). HER2 amplification was neither prognostic nor predictive for OS or PFS. Conversely, HER2 expression higher than median was associated with longer PFS ( P = 0.018) but not OS ( P = 0.13). Among pts with HER2 not amplified, higher HER2 expression was associated with better OS (hazard ratio [HR], 0.83; 95%CI, 0.72-0.97; P = 0.016) and PFS (HR, 0.85; 95%CI, 0.74-0.98; P = 0.027) when the expression was less than median. Additionally, in pts with no HER2 amplification and HER2 expression lower than median, treatment with Cet was associated with worse PFS compared to Bev (HR, 1.46; 95%CI, 1.12-1.90; P = 0.005). This effect was not observed with expression higher than median regardless of HER2 amplification status. Conclusions: To our knowledge, this is the largest analysis of HER2 amplification and gene expression in mCRC pts treated with standard therapy. Our results provide novel insight on the predictive and prognostic value of HER2 gene expression in pts treated with Cet- and Bev-based regimens. Upon validation, these findings could inform pts selection and the design of more effective treatment options for pts with low HER2 expression. Clinical trial information: NCT00265850.