HER2 Protein Expression Research Articles

Abstract PR-04: Association of HER2/GRB7 co-expression with Indigenous American ancestry and breast cancer clinical-pathological features

Abstract Introduction: Breast cancer is the most prevalent cancer in women worldwide. It is a heterogeneous disease that has been classified into intrinsic subtypes based on gene expression analyses. Previous studies have reported a higher proportion of HER2-positive (HER2+) tumors in Hispanics/Latinas, compared to Non-Hispanic White women, characterized by their aggressive phenotype. A gene expression analysis of breast tumor tissues from Colombian women reported that patients with a higher Indigenous American (IA) ancestry expressed higher mRNA levels of ERBB2, and of other genes in the HER2-amplicon region (17q12) such as GRB7. Although the expression of HER2 and GRB7 is highly correlated, it has been reported that these proteins might also be expressed independently. It is still unclear whether IA ancestry is associated with the co-expression of HER2/GRB7, or only with one of these two proteins. Aim of the study: To explore differences in HER2 and GRB7 protein expression according to genetic ancestry and the presentation of clinical-pathological features in Colombian women with breast cancer. Methods: A total of 517 Colombian breast cancer patients from different health institutions were included. For genetic ancestry estimation, 106 ancestry informative markers were genotyped from non-tumoral DNA. The protein expression of HER2 and GRB7 was evaluated in paraffin blocks with tumoral tissue by immunohistochemistry by a single pathologist. We tested the association between genetic ancestry and protein expression of HER2, GRB7, and the co-expression of both using logistic and multinomial logistic regression models. Results: Higher IA ancestry fractions were observed in patients with HER2+ compared to HER2- tumors (46% vs 41%, respectively, p = 0.013). We found higher odds of having HER2+ (OR=1.65, 95% CI, 1.05-2.63, p=0.0293) and GRB7+ tumors (OR=1.92, 95% CI, 1.03-3.56, p=0.0376) for every 25% increase in the IA ancestry proportion. The association between IA ancestry and HER2/GRB7 co-expression was influenced by estrogen receptor (ER) status. Patients with a higher IA ancestry (>42%) and with ER+ subtypes presented higher odds of having HER2+/GRB7- breast tumors (OR=3.59, 95% CI, 1.11-11.65, p=0.0327), whereas ER- patients with high IA ancestry tended to have HER2+/GRB7+ tumors (OR=2.27, 95% CI, 0.69 - 7.45, p=0.1730). The co-expression of these proteins was found associated with advanced clinical stages (p=0.018), less differentiated tumors (p=0.005), and a higher proliferation index (p<0.001), compared to HER2+/GRB7- and HER2-/GRB7- tumors. Conclusions: Our results confirm the previously reported association between IA ancestry and HER2 expression in breast tumors and suggest that HER2/GRB7 co-expression in patients with high AI ancestry may change according to the ER status. This event also correlates with unfavorable clinical-pathological features. Citation Format: Laura Rey Vargas, Lina Maria Bejarano, Juan Carlos Mejia Henao, Luz Fernanda Sua Villegas, Jhon Faustino Bastidas Andrade, Carlos Andrés Ossa Gómez, Laura Fejerman, Maria Carolina Sanabria Salas, Silvia Juliana Serrano Gomez. Association of HER2/GRB7 co-expression with Indigenous American ancestry and breast cancer clinical-pathological features [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR-04.

CERBB-2/Her-2 Neu Overexpression and Prognostic Significance in Uterine Carcinosarcoma.

There is not enough data in the literature regarding Her-2 overexpression in uterine carcinosarcomas or its association with the prognosis. The aim of this study was to determine the Her-2 overexpression rate in uterine carcinosarcoma and to evaluate its relationship with the prognosis. Her-2 protein and gene status were evaluated by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively, in hysterectomy specimens from 51 patients with uterine carcinosarcoma. Her-2 protein expression in the epithelial component was negative in 42 patients (score 0 in 33 cases, score (+1) in 9 cases), score (+2) in 7 patients and score (+3) in 2 patients. None of the patients had Her-2 protein expression within the sarcomatous component of the tumors. Her-2 gene was not amplified in epithelial or mesenchymal tumor areas according to the FISH method. There was no difference between the Her-2 overexpression negative and positive groups in terms of disease-free survival (DFS) and overall survival (OS). Her-2 overexpression was significantly higher in tumors of patients diagnosed at 65 years or older (p=0.046). In our study, no relationship could be shown between Her-2 overexpression and prognosis in uterine carcinosarcoma. More comprehensive studies are needed to illustrate the relationship between Her-2 overexpression and carcinosarcoma prognosis.

Interaksi Senyawa Taxifolin dari Buah Kasturi (Mangifera casturi) sebagai Antikanker Payudara: Evaluasi Docking Molekular

Taxifolin also called dihydroquercetin is a flavonoid. Taxifolin is included in the flavonol subclass and polyphenol class. Taxifolin has been found in the leaves, bark and flesh of Mangifera casturi. Taxifolin is thought to have potential as an antibreast cancer through its activity as an anti-neoplastic agent. The occurrence of breast cancer begins with overexpression of the Human Epidermal Growth Factor Receptor 2 (HER-2) protein which can induce dimerization and autophosphorylation of the tyrosine kinase enzyme, resulting in migration and metastasis of breast cancer cells. This study aims to determine the interaction of taxifolin with HER-2 protein by molecular docking. Molecular docking using the SwissDock web server. Parameters of molecular docking include Gibbs free energy and interaction of taxifolin ligands and HER-2 residues. The results of docking molecular expressed by free energy Gibbs of taxifolin compounds with HER-2 protein is -7,99 kcal / mol, while the free energy Gibbs of the native ligand bond with HER-2 protein is -10.76 kcal / mol. The residues on HER-2 that interacts with taxifolin are GLY727, GLY804, LEU726, THR798, ASP863, ALA730, SER728, ARG849, GLY729, CYS805, THR862, VAL734, LEU852, LYS753, ALA751, ASP808, ARG849, ASN850. The value of Gibbs free energy and the interaction of residues indicate that taxifolin compounds have potential as anticancer breasts. Taxifolin as an inhibitor of HER-2 protein expression.

HER2/p53 ratios among canine mammary tumours and respective derived cell culture

In this study, the expression of p53 and human epidermal growth factor receptor 2 (HER2) proteins were investigated in different canine mammary tumors (CMT) and cell cultures derived from them, for the purpose of determining neoplastic behavior in vitro. For this purpose, immunohistochemistry and immunocytochemistry were performed and results were compared according to histological diagnose. CMT were collected and divided into two halves, one for histological and immunohistochemistry analyses and another for cell cultures. Cell markers p53 and HER2 proteins were detected by immunohistochemistry in the original tumors and immunocytochemistry in CMT-derived cell lines. A total of CMT (n = 17) were classified and divided into benign mixed tumor (BMT), complex carcinoma (CC), simple carcinoma (SC), and squamous cell carcinoma (SCC). Low expression of p53 was verified in BMT and CC, averagely in SC and undetectable in SCC. In vitro, there was an increase in p53 expression by cultured cells. However, HER2 protein expression was not significant in BMT, CC and SCC. In contrast, MCT-derived cells were considered HER2-positive in all MCT types. In conclusion, the in vitro model is essential for the study of neoplastic behavior and understanding of the carcinogenic mechanisms that occur in vivo.

80P HER2 expression in primary bladder tumors and paired regional lymph nodes: Relationship to cell cycle profile

The HER2 receptor plays an important role in the aberrant proliferation of cancer cells with modification of the cell cycle profile. Phosphorylation of HER2 (pHER2) which represents the functional and active form of HER2 is a precondition for downstream signalling network involved in cell growth, invasion and metastasis. The aim of our study was to investigate the HER2 protein expression in both primary tumors and paired regional lymph nodes in relation to cell cycle profile in bladder cancer.

Therapeutic Mechanism of Lapatinib Combined with Sulforaphane on Gastric Cancer

Background Lapatinib is a small-molecule tyrosine kinase inhibitor that plays important roles in cell proliferation and survival. Administration of lapatinib with capecitabine is an effective treatment for HER2-positive metastatic BC. However, the effects of lapatinib on gastric cancer (GC) remain to be clear. In this study, we aimed to investigate the therapeutic effects of lapatinib combined with sulforaphane on GC and its underlying mechanisms. Methods SGC-7901 and lapatinib-resistant SGC-7901 cells were treated with lapatinib (0.2 μM), sulforaphane (5 μM), or their combinations. Cell viability, invasion, cycle, and apoptosis of SGC-7901 and lapatinib-resistant SGC-7901 cells were evaluated by thiazolyl blue tetrazolium bromide (MTT), Boyden chamber assay, and flow cytometer. The protein expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were detected by Western blotting. Results We observed that lapatinib combined with sulforaphane significantly decreased cell viability and inhibited cell migration of drug-sensitive and drug-resistant cells. Lapatinib sulforaphane also remarkably induced cell apoptosis with G0/G1 arrest. In addition, Western blotting revealed that the expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were downregulated by lapatinib-sulforaphane treatment. Conclusion Combination of lapatinib and sulforaphane might be a novel and promising therapeutic treatment for lapatinib-sensitive or lapatinib-resistant GC patients.

DHES0815A, a novel antibody-drug conjugate targeting HER2/neu, is highly active against uterine serous carcinomas in vitro and in vivo

ObjectiveUterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2 overexpressing tumors at a distinct epitope from that bound by trastuzumab and pertuzumab after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A against primary USC cell lines and xenografts. MethodsTwelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for C-erbB2 gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability and bystander killing in USC cell lines after exposure to DHES0815A, the non-targeted ADC, and the unconjugated antibody (i.e. MHES0488A) were evaluated using flow cytometry-based-assays. In vivo activity of DHES0815A was tested against HER2/neu overexpressing USC xenografts. ResultsHigh HER2/neu protein expression was seen in 25% (3/12) of the primary USC cell lines. USC cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (p < 0.001). DHES0815A did not induce significant bystander killing of HER2/neu negative tumors when admixed with HER2/neu positive tumors. DHES0815A caused growth-inhibition and increased survival in USC HER2/neu overexpressing xenografts when compared to controls (p < 0.01). ConclusionsDHES0815A is both highly selective and toxic to USC tumors overexpressing HER2/neu both in vitro and in vivo. HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy.

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.

Differential expression of pyruvate dehydrogenase E1A and its inactive phosphorylated form among breast cancer subtypes

AimsPyruvate dehydrogenase E1A (PDH-E1A) is one of the key regulators of metabolic pathways that determines pyruvate entry into the citric acid cycle or glycolysis. When PDH-E1A is phosphorylated (P-PDH-E1A), it loses its activity, shifting the metabolism towards glycolysis. Breast cancer (BC) is a highly heterogeneous disease by which different breast cancer subtypes acquire distinct metabolic profiles. Assessing PDH-E1A and P-PDH-E1A expressions among BC subtypes might reveal their association with the distinct molecular profiles of BCs. MethodsThe expressions of PDH-E1A and P-PDH-E1A were investigated in BC cell lines and 115 BC tissues using Western blot and immunohistochemistry, respectively. Besides, PDHE1A mRNA expression was assessed in 1084 BCE patients' transcriptomics data retrieved from Cancer Genome Atlas database. Statistical analyses were performed to assess the correlation of PDH-E1A and P-PDH-E1A expressions with patients' clinicopathological characteristics. Kaplan-Meier method was used to evaluate their prognostic value. Key findingsMultivariate analysis revealed a significant association between PDH-E1A/P-PDH-E1A expressions and the molecular subtype, histological type, and tumor size of breast cancer tissues. The hormonal receptors (ER and PR), HER-2, and Ki67 protein expressions were significantly associated with PDH-E1A and P-PDH-E1A protein expressions. Similar findings were observed when PDHA1 mRNA expression was assessed. The increased protein expression of PDH-E1A could be an independent prognostic factor for unfavorable overall survival (OS). In contrast, high PDHA1 mRNA expression had better OS. SignificanceThis study revealed the differential expression of PDH-E1A and P-PDH-E1A among breast cancer subtypes and suggested PDH-E1A expression as a prognostic factor for BC patients' OS.

In vitro and in vivo activity of DHES0815A, an antibody-drug conjugate targeting HER2/neu in uterine serous carcinoma

<h3>Objectives:</h3> Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2-amplified USC tumors at a distinct epitope from that bound by trastuzumab, pertuzumab, and T-DM1 (trastuzumab emtansine) after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A as a single agent against primary USC cell lines and xenografts. <h3>Methods:</h3> Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for <i>C-erbB2</i> gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability in USC primary cell lines after exposure to DHES0815A, the non-targeted ADC, the unconjugated antibody MHES0488A, and T-DM1 were evaluated using flow cytometry-based-assays. <i>in vivo</i> activity of these respective agents was tested against HER2/neu overexpressing USC (ARK2) xenografts. <h3>Results:</h3> High HER2/neu protein expression by IHC and <i>C-erbB2</i> gene amplification by FISH was seen in 25% (3/12) of the primary USC cell lines. Primary tumor cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (<i>p<0.05</i>). DHES0815A caused growth-inhibition and increased survival in HER2/neu overexpressing xenografts when compared to controls (<i>p<0.05</i>). The animals also tolerated DHES0815A treatment with no significant change in their body weight [Figure 1]. <h3>Conclusions:</h3> DHES0815A as a single agent is both highly selective and toxic to USC tumors overexpressing HER2/neu both <i>in vitro</i> and <i>in vivo.</i> HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel and less toxic treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy.

MTT assay based inhibition analysis of A2780 cells proliferation using Mollugo nudicaulis Lam. extract with CXCR4 and HER2 expression.

It is of interest to document the inhibition of A2780 cell proliferation using Mollugo nudicaulis Lam.(M.nudicaulis) extract by MTT assay and by monitoring the CXCR4 and HER2 expression through RT-PCR analysis. Results shown that the n-hexane extract of M.nudicaulis have anticancer activity IC50 values of 32.46±0.92 µg/mL on A2780 cell lines. It is further found that the CXCR4 and HER2 mRNA and protein expression were significantly reduced in M.nudicaulis treated A2780 cell lines. Thus, the n-hexane extract of M.nudicaulis is a natural source of bioactive compounds as potential anticancer agents.

HER2 Protein Overexpression and Gene Amplification in Tubo-Ovarian High-grade Serous Carcinomas.

Most tubo-ovarian high-grade serous carcinomas (TO-HGSC) are diagnosed in advanced stages. Although the majority of patients achieve initial remission with cytoreductive surgery and chemotherapy, mortality rate remains high due to recurrent/progressive disease. The addition of trastuzumab to carboplatin-paclitaxel improved progression-free survival of patients with human epidermal growth factor receptor 2 (HER2)-positive uterine serous carcinoma. After this encouraging result of transtuzumab in HER2-positive uterine serous carcinoma, we aimed to determine the frequency of HER2 overexpression/amplification in TO-HGSC and reveal the utility of 2018 ASCO/CAP HER2 testing guideline in breast cancer for TO-HGSC. For 100 cases, HER2 protein expression was assessed by immunohistochemistry and scored from 0 to 3+ according to 2018 ASCO/CAP HER2 testing guideline. HER2 gene amplification was assessed by florescence in situ hybridization for all the 2+ and 3+ cases as well as 5 of the 0/1+ cases. Among 100 cases, immunohistochemistry scores were 0/1+ in 81 cases, 2+ in 18 cases and 3+ in 1 case. By florescence in situ hybridization, the only 3+ case and 1 of the 2+ cases were HER2-amplified and all 5 of the 0/1+ cases were HER2 nonamplified. Subclonal HER2 overexpression/amplification was identified in 1 of the neoadjuvant cases comprising <10% of the entire tumor. In summary, HER2 overexpression/amplification was found in 2% of TO-HGSC. The 2018 ASCO/CAP HER2 testing guideline in breast cancer can be utilized for TO-HGSC. Future studies are needed to explore HER2-targeted therapies in TO-HGSC and expand the patient population who may benefit from HER2-targeted therapies such as patients with activating mutations in HER2 gene without overexpression/amplification.

Abstract 2718: Digital spatial profiling in HER2 positive breast cancer: The road to precision medicine

Abstract Background: Breast cancer (BC) has been considered immunologically quiescent, this could be explained by tumor and tumor immune microenvironment (TIME) characteristics. We aimed to characterize the TIME of human epidermal growth factor 2 positive (HER2+) BC using GeoMx Digital spatial profiling (DSP) in primary and metastatic samples. Methods: DSP was performed on 15 primary and metastatic FFPE samples from 8 patients; including 2 brain metastases. Visualization markers for Pan-Cytokeratin (PanCK, tumor), CD45 (immune), CD3 (T cells) were used to guide selection of immune cold, warm and hot regions of interest (ROI). Each ROI segmented tumor (PanCK+) or stroma (PanCK-) based on PanCK tumor + or - areas of illumination (AOI). For each AOI 71 protein targets were tested. Gene expression profiling for PAM50 subtypes and Tumor Inflammation Score (TIS) was done using NanoString's nCounter PanCancer IO360 assay with PAM50 spike. TIS was determined by threshold of 6.5. Results: Primary HER2+ samples had the highest amount of tumor infiltrating lymphocytes (CD3) and overall immune cells (CD45) compared to all metastatic disease in tumor and stroma (Table 1). Brain metastases had lowest TIS. Stroma of primary tumors had higher protein expression of immune activation and checkpoint markers CD27, ICOS, 41BB and TIM3 compared to brain and soft tissue metastases. HER2 protein expression most correlated with metastases. Metastatic tumor had higher proliferation by Ki67. Patient 2 had archival primary tumor (n=2) and soft tissue metastasis (ST) (n=1). TIS was obtained from immune hot, warm and cold ROI of each sample. Primary tumor stroma had highest expression of STING and Granzyme B compared to ST, suggesting that the tumor underwent immune escape. Conclusions: Primary tumors had a higher number of immune cells than metastatic sites. These findings suggest that immunotherapy in early stage BC could be more effective than in the advanced BC. This project will be part of the GeoMx Breast Cancer Consortium. Table 1.Summary of patient, tissue, diagnosis, ER/PR/HER2 status, PAM50 subtype and TIS statusPatientTissueDiagnosisER(%)/PR(%)/HER2(+)PAM50 SubtypeTIS Status1BreastIDC, DCIS2/0/3Luminal AHigh1BreastTumor emboliN/ALuminal AHigh1LungMetastatic0/0/3HER2 enrichedHigh2BreastICD, DCIS0/0/3HER2 enrichedLow2BreastResidual IDC0/0/N/AHER2 enrichedHigh2Soft tissueLocal recurrence0/0/3HER2 enrichedLow3BrainMetastatic15/0/3HER2 enrichedLow4BreastIDC0/0/3Basal likeHigh4BreastLocal recurrenceN/ALuminal AHigh5BrainMetastatic0/0/3Basal likeLow6BreastIDC, DCIS80/70/2*Luminal AHigh7BreastIDC, DCIS0/0/3HER2 enrichedHigh7BreastLocal recurrenceN/ABasal likeHigh8BreastIDC, DCIS10/15/1Luminal ALow8Soft tissueLocal recurrence0/0/3Basal likeLowAbbreviations: ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor 2; TIS: tumor inflammation score; IDC: invasive ductal carcinoma; DCIS: ductal carcinoma in situ*HER2/CEP17 ratio: 2.13 Citation Format: Ilana Schlam, Sarah E. Church, Krysta Chaldekas, Brent T. Harris, Briana M. Hudson, Andrew M. White, Sandra M. Swain. Digital spatial profiling in HER2 positive breast cancer: The road to precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2718.

Abstract 1373: Primary or iPSC-derived cell-based cytotoxicity assays to assess potential safety risks of engineered T cell therapies in vitro

Abstract Engineered T cell therapies, such as Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) T cells, but also bi-specific antibody therapies, play a major role in the field of immuno-oncology and offer great promise in becoming a new wave of highly specific therapies against solid tumors. Solid tumors generally lack the expression of tumor specific target antigens, posing a significant safety challenge. Selected solid tumor target antigens are often expressed at low levels in healthy tissues throughout the human body, risking the activation of the engineered T cells against these tissues. This can potentially result in life-threatening side effects, as such, assessing the safety of engineered T cell therapies is a critical step during early development and before filing for Investigational New Drug (IND) status. We have generated an in vitro safety profile for CAR-T cells targeting the ‘Human Epidermal growth factor Receptor 2' (HER2). The gene encoding this receptor is found to be amplified and/or overexpressed in 20-30% of invasive breast carcinomas and ovarian cancers. To determine which tissues are most at risk for unwanted reactivity by the CAR-T cells, in silico analysis for expression of the HER2 gene was performed. Subsequently, HER2 protein expression in various tissues was validated by staining cells with a HER2 antibody and determining the percentage of positive cells by flow cytometry. Primary tissues and iPSC-derived cell with high and low HER2 protein expression were selected, characterized and utilized for in vitro co-culture assays to evaluate on-target off-tumor and/or off-target cytotoxicity of the HER2 targeting CAR-T cells. Readouts for the in vitro cytotoxicity assays included measuring target cell viability by flow cytometry and/or HCA and T cell activation by cytokine release. Our study generated high quality data that provided insight into the safety of the HER2 targeting CAR-T cells. Moreover, we were able to demonstrate the value of using iPSC-derived cells in de-risking selected tissues against unwanted reactivity of engineered T cell therapies. Our strategy to generate a safety profile for T cell therapies and bi-specific antibodies is robust can be applied during both early stage development and late stage testing of the therapeutic product. Engineered T cell therapies have the capacity to fill tremendous unmet medical needs and are moving into the clinic at a high rate. This expedited timetable, although good news for patients with late stage disease, results in less time to conduct the rigorous safety tests required before FDA approval. The use of primary tissues and iPSC derived cells to test the safety of these therapies in vitro has been and will be essential in getting these novel drugs on the market. Citation Format: Sanne L. Holt, Sophie Vermond, Monique Hazenoot, Rene McLaughlin, Marco Guadagnoli, Marijn Vlaming. Primary or iPSC-derived cell-based cytotoxicity assays to assess potential safety risks of engineered T cell therapies in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1373.

Abstract 1078: Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features

Abstract Trastuzumab and trastuzumab emtansine (T-DM1) targeted therapies are the main choice for the treatment of HER2-overexpressing breast cancer patients. However, de novo or acquired resistance is still the major obstacle in clinical practice. It has been shown that several pathways, including HER2, can lead to HIF-α stabilization in breast cancer cells. Thus, the purpose of our study was to analyse the effect of hypoxia in acquired resistance to anti-HER2 therapies. We used HER2-overexpressing BT-474 and non-overexpressing MCF-7 human breast cancer cell lines. As an acute hypoxia model, we added CoCl2 (100 µM) to cell culture medium. The hypoxic status of the cells was evaluated by a Western blot analysis, showing a peak of HIF-1α expression after 6 hours of CoCl2 exposure. This result correlated with VEGF induction, as measured by RT-qPCR (p&amp;lt;0.05). Interestingly, under hypoxia, BT-474 cells treated with increasing concentrations of trastuzumab and T-DM1 for 72 hours presented a significantly higher viability compared with non-hypoxic cells (p&amp;lt;0.05). However, although the drugs decreased membrane HER2 protein expression (p&amp;lt;0.01), HER2 levels did not change under hypoxic conditions, as shown by flow cytometry and Western blot assays. Moreover, drug effects on MCF-7 cells did not change under hypoxic conditions. To further analyse this cytoprotective effect of hypoxia, proliferation-associated markers such as PCNA expression and AKT activation were evaluated by Western blot, while apoptosis frequency was measured by flow cytometry in BT-474 cells. PCNA and pAKT expression remained unaltered, while T-DM1-mediated apoptosis was significantly reduced under hypoxia (p&amp;lt;0.05). Then, using different open gene expression datasets, we identified a group of four genes (COL12A1, DKK1, CEACAM5 and ANTXR1) differentially expressed in T-DM1 resistant HER2-overexpressing breast cancer BT-474 and KPL-4 cell lines (p&amp;lt;0.05), which were coexpressed with hypoxia-related genes (HIF1A, EPAS1 and VEGFA) in HER2-overexpressing breast cancer tumours and cell lines from TCGA (p&amp;lt;0.01). Further in silico analyses revealed a direct association between those genes and the fibronectin gene (FN1, p&amp;lt;0.05). Remarkably, they were involved in TGF-β and angiogenesis pathways, as shown by a functional enrichment analysis (p&amp;lt;0.01). In addition, we found that BT-474 cells under hypoxia presented a higher mammosphere formation efficiency than non-hypoxic cells (p&amp;lt;0.05), showing a tendency towards a cancer stem cell phenotype. It is known that hypoxia represents a common feature of metastatic tumours and has a role in regulating stem cell behaviour. According to this, our results highlight a link between anti-HER2 therapy resistance and epithelial-to-mesenchymal transition characteristics, where hypoxia plays a key role which deserves further study. Citation Format: Virginia Judith Wolos, Ivana Jaqueline Tapia, Marianela Abrigo, Ezequiel Lacunza, Elisa Dora Bal de Kier Joffé, Gabriel León Fiszman. Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1078.

Abstract 911: In vitro and in vivo activity of DHES0815A, an antibody-drug conjugate targeting HER2/neu in uterine serous carcinoma

Abstract Objective: Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2 overexpressing tumors at a distinct epitope from that bound by trastuzumab and pertuzumab after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A against primary USC cell lines and xenografts. Methods: Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for C-erbB2 gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability and bystander killing in USC cell lines after exposure to DHES0815A, the non-targeted ADC, and the unconjugated antibody (i.e. MHES0488A) were evaluated using flow cytometry-based-assays. In vivo activity of DHES0815A was tested against HER2/neu overexpressing USC xenografts. Results: High HER2/neu protein expression was seen in 25% (3/12) of the primary USC cell lines. USC cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (p&amp;lt;0.001). DHES0815A did not induce significant bystander killing of HER2/neu negative tumors when admixed with HER2/neu positive tumors. DHES0815A caused growth-inhibition and increased survival in USC HER2/neu overexpressing xenografts when compared to controls (p&amp;lt;0.01). Conclusions: DHES0815A is both highly selective and toxic to USC tumors overexpressing HER2/neu both in vitro and in vivo. HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy. Citation Format: Joan Tymon R. Tymon-Rosario, Elena Bonazzoli, Stefania Bellone, Arancha Manzano, Paola Manara, Luca Zammataro, Silvia Pelligra, Adele Guglielmi, Barbara Gnutti, Burak Zeybek, Justin Harold, Dennis Mauricio, Elena Ratner, Peter Schwartz, Alessandro D. Santin. In vitro and in vivo activity of DHES0815A, an antibody-drug conjugate targeting HER2/neu in uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 911.

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.

Association of HER2 expression with pathologic features and prognosis in stage II and III colon cancer.

3524 Background: We examined the frequency, tumor characteristics, and prognostic impact of HER2 protein expression in stage II and III colon cancer after curative resection. Methods: Paraffin-embedded tumors from consecutive primary stage II and III colon cancer patients were analyzed for HER2 protein expression by immunohistochemistry between April, 2013 and May, 2020. HER2 determination of immunohistochemistry scores (0/1+/2+/3+) was according to HERACLES diagnostic criteria. Results: A total of 2088 stage II and III colon cancer patients were included (53.8% stage II, 46.3% stage III). HER2 scored positive (3+) was detected in 48(2.3%) tumors, and was correlated with younger age (P &lt; 0.001), well/moderate differentiation (P = 0.026), proficient mismatch repair (pMMR) (P = 0.045) and KRAS wild-type (P &lt; 0.001). HER2 scored positive (3+) was not significantly associated with disease-free survival (DFS) compared with HER2 scored negative (0/1+), neither in stage III patients (multivariable HR, 0.86; 95CI, 0.38 to 1.94; P = 0.717), nor in stage II patients (multivariable HR, 1.68; 95CI, 0.74 to 3.84; P = 0.218). In a separate analysis involving stage II patients without any high-risk factor (n = 741), those with HER2 scored positive (3+) tumors (n = 16) showed significantly reduced DFS (multivariable HR, 2.91; 95CI, 1.04 to 8.81; P = 0.041) compared with patients with HER2 scored negative (0/1+) tumors, independent of sex, age and MMR status. Conclusions: HER2 scored positive (3+) was independently associated with poor DFS in stage II colon cancer patients without high-risk factors. HER2 expression determination may help to judge the prognosis of those patients and guide adjuvant chemotherapy.

Breast cancer and molecular testing in Brazil.

e13602 Background: Breast cancer is a common malignancy among women worldwide. Regardless of the economic status of a country, breast cancer poses a burden in prevention, diagnosis and treatment.Accurate pathologic and molecular diagnosis of high quality in breast cancer is a challenge in low- and middle-income countries (LMIC). The two most important aspects needed are the hormonal and epidermal growth factor receptor 2 (HER2) status as they are used as prognostic factor and as predictive biomarker for treatment. The most commonly used tests for assessing HER2 status are immunohistochemistry (IHC) assays for HER2 protein expression and fluorescence in situ hybridization (FISH) for HER2 gene amplification.In this study, we evaluated the hormonal and HER2 status of women with breast cancer and the need for FISH in HER2+++. Methods: We retrospectively reviewed the database of the Hospital Evangelico de Cachoeiro de Itapemirim, a regional cancer center from the south of Espirito Santo State – Brazil.The period covered in the study was from 01/08/2019 to 01/08/2020. We analyzed all new cases of invasive breast cancer diagnosed by IHC for the estrogen (ER), progesterone (PR) and HER2 receptors. All the HER 2++ and HER2 +++ were tested by FISH. We had during this period 152 newly diagnosed cases, 11 patients were excluded due to missing data. Results: The 141 cases were separated into four categories by hormonal status: 109 ER+PR+ (77,3%), 9 ER+PR− (6,4%), 2 ER−PR+ (1,4%) and 21 ER−PR− (14,9%). When these results were associated to HER 2 status we found 11 (7,8 %) triple negative breast cancer (TNBC) , 26 (18,43%) HER2 ++ and 23 (16,31%) HER 2+++. The HER2++ were tested by FISH and 8 (30,7%,) were found positive, 10 (38,6%) negative and 8(30,7%)indeterminate. Among the HER 2 +++ tested by FISH we found 15 (65,3 %) positive and 8 (34,7 %) indeterminate. Conclusions: Our study demonstrated a lower incidence of TNBC and higher incidence of hormone-receptor positive cases as compared with other institutions. Different from other countries, all HER 2++ and HER2+++ in Brazil need to go to molecular testing. Altough trastuzumab is available in the public Brazilian Health System (SUS) for HER 2 positive women, diagnosis with IHC alone is not permitted and there is a need for molecular testing confirmation. Therefore, the most troublesome data in this study was that 34,7% of HER 2+++ had indeterminate FISH results, in these cases SUS does not offer traztuzumab. Newer guidelines allow the use of IHC only in HER2 +++ . We consider important for SUS to review the recommendation for need of molecular testing for all HER2 +++ so this will avoid subtreating women in Brazil

Evaluation of the Expression of HER2 and c-KIT Proteins as Prognostic Markers in Superficial Bladder Urothelial Carcinoma.

BackgroundThe roles of c-KIT and HER2 protein expression in bladder cancer are still debated, and the prognostic value of these proteins as markers of tumor progression is inconclusive.ObjectiveTo assess the impact of HER2 and c-KIT protein expressions in the progression of non-muscle-invasive bladder cancer.MethodsAll patients undergoing transurethral resection of bladder tumors for non-muscle-invasive urothelial carcinoma, with standard regimen of BCG, between January 2017 and November 2019, were evaluated pathologically and immunohistochemically for HER1 and c-KIT proteins in urothelial carcinoma cells. Follow-up cystoscopy was performed for 100 patients every 3 months for the first 2-years and any recurred tumors were excised and examined pathologically, as well as stained for HER2 and c-KIT protein expression.ResultsHER2 and c-KIT positive expressions were detected in 49% and 38% of cases, respectively. After a mean follow-up of 26.4±7.2 months, the overall recurrence and progression rates were significantly correlated with overexpression of HER2 and c-KIT. In high-grade non-invasive muscle neoplasms, tumor cells showed weak expression for both HER2 and c-KIT proteins, but with progression to muscle-invasion, tumor cells strongly expressed HER2 and lost expression to c-KIT. In the multivariate model, overexpression of HER2 rather than c-KIT protein significantly predicted increased progression.ConclusionRecurrence and progression of non-muscle-invasive bladder cancer correlate with overexpression of HER2 and c-KIT proteins in tumor cells.