HER2 Protein Research Articles

Establishment of a murine model of breast cancer expressing human epidermal growth factor receptor 2 (4T1-HER2).

Although people with HER2-positive breast cancer benefit from approved HER2-targeted therapy, acquiring resistance to the therapies occurs. Animal models can play a part in gaining a deep understanding of such a process and addressing questions concerning developing and improving immunotherapy approaches. To develop such a model, we transfected murine 4T1 cells with the pCMV6-Neo-HER2 construct and evaluated HER2 expression and its effects on the established cell line behavior in vitro and in vivo. Data illustrated that human HER2 protein was expressed on isolated 4T1-HER2 clones in vitro and in vivo. Except for proliferation over 48 hours, such expression did not change 4T1-HER2 characteristics compared to 4T1 in vitro. Notwithstanding the reduction in proliferation, the rate of tumorigenicity was 90% in challenged mice and Herceptin therapy significantly decreased tumors' growth and metastasis compared to the control group. We describe a murine model for HER2-positive breast cancer not only helping shed light on the mechanisms by which the tumor evades antitumor immunity but also playing a key role in making breast cancer more sensitive to novel immunotherapy modalities.

Abstract 561: Development of competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies

Abstract The human epidermal growth factor receptor (HER) family of receptors has been implicated in several human cancers. Among them, HER2 is overexpressed in several cancers with or without gene amplification with prognostic and predictive implications. In 15-30% of breast cancers, HER2 overexpression and amplification are associated with shorter disease-free and overall survivals along with resistance to hormonal agents as well as increased risk of brain metastasis. Two types of HER2 targeting have been developed: anti-HER2 monoclonal antibodies and small molecule tyrosine kinase inhibitors. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular segment and blocks its signaling. It is the first therapeutic antibody targeting Her2 overexpression approved by the FDA. More recently, two antibody drug conjugates using trastuzumab have been approved: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu to deliver the topoisomerase inhibitor deruxtecan. We are reporting here the development of fully human internalizing anti-Her2 antibodies that compete or not with trastuzumab for binding to Her2. These antibodies have been developed by immunizing fully human (TC-Mab mouse) mice with recombinant Her2 protein. After production of hybridoma secreting fully human immunoglobulins, the screening process included inhibition of binding of trastuzumab to Her2 by enzyme linked immunoassay and by Octet epitope binning as well as internalization assay. Several internalizing antibodies able to compete or not with trastuzumab and with high affinity (Kd ranging from 10−9 M to 10−12 M) were selected. They were next investigated for their ability to deliver a cytotoxic payload in HER2 overexpressing cells breast cancer cells such as SKBR3, BT474 and AU565. Two antibodies: one competing with trastuzumab and the other one non-competing were further characterized. Data related to these antibodies’ biochemical characteristics as well as their ability to inhibit proliferation in vitro and in vivo in mouse xenografts studies will be presented here. In conclusion, the use of fully human mice in our laboratory provides a powerful and attractive approach to develop fully human monoclonal antibodies against cancer targets allowing to by-pass the need for humanization and affinity maturation of antibodies. Citation Format: Ginette Serrero, Binbin Yue, Jianping Dong, Chun Dong, Jun Hayashi. Development of competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies [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 561.

Abstract 3916: role of her3 v104l mutation on tumor growth and her3 stabilization

Abstract We aimed to determine if naturally occurring HER3 mutations could drive oncogenic activity of HER2+ HCC1569 cells in which endogenous HER3 was knocked out using CRISPR-Cas9 technology. A series of HER3 mutations found in breast cancer patients (F94L, V104L, G284R, D297Y, T355I, and E928G) were introduced via lentiviral transduction and stable cell lines were generated in HER3 knock out (KO) HCC1569 cells and were maintained in 0.5 mg/mL puromycin. Our data indicated that cells stably transduced with HER3V104L mutation had significantly higher cell growth with higher p-HER3 expression versus wild-type (wt) and empty-vector (EV) HER3. We found that HCC1569HER3KO cells with WT and V104L were sensitive to increasing concentration of neratinib (0.1-0.5 µM) as indicated by crystal violet assay. Next we examined if V104L mutation rendered resistance to another FDA- approved irreversible HER2 tyrosine kinase inhibitor, tucatinib. Our results showed that V104L cells were sensitive to higher concentration of tucatinib compared to neratinib. In other experiments, we used COS7 cells to examine the signaling pathways of HER3V104L mutation. Our western blot data demonstrated that transient transfection of COS7 cells with HER3V104L mutant significantly upregulated p-HER3 and p-HER2 expression versus WT HER3 in a NRG- dependent manner. In addition, we found that the V104L mutation stabilizes HER3 protein expression independent of ligand stimulation. Our western blot data showed that V104L induced HER3 stabilization is independent on HER3 binding partners HER1, HER2 and HER4. Our cycloheximide chase data indicated that V104L mutation stabilizes HER3 expression in COS7 cells as well as low HER3 expressing colon cancer cell line, SNUC5. Our western blot data indicates that V104L mutation activates ERK signaling in NRG dependent manner over AKT pathway in COS7 cells. In addition, we are using various PDXs with different HER3 mutations (V104M and E928D) to determine the role of these driver HER3 mutations in cancer and how this can be targeted in the clinic using HER targeted therapy. Citation Format: Rosalin Mishra, Mary Kate Kilroy, Wasim Feroz, Hima Patel, Samar Alanazi, Joan T. Garrett. role of her3 v104l mutation on tumor growth and her3 stabilization. [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 3916.

In silico Molecular Modelling and Docking Studies on Kinase Inhibitors as Potential Anti-Cancer Target in HER2-associated Breast Cancer

Abstract: Breast cancer is one of the most frequent invasive malignancies in women globally and the leading cause of mortality. The HER2 target is an important therapeutic option for treating breast cancer. In the present study, efforts have been made to virtually screen the natural kinase inhibitors through molecular docking. A total of 800 HER2 protein inhibitor compounds were selected to screen out the potential inhibitors of the HER2 protein. The docking study demonstrated that these HER2 protein inhibitors confirm the strong binding interaction with HER2 protein based on the docking score, indicating that kinase inhibitors can play a major role in preventing breast cancer. Among all the inhibitors, the flavanone compound named 6-C-(3-Hydroxyisopentyl) eriodictyol, IUPAC: 2-(3, 4- dihydroxyphenyl)-5, 7-dihydroxy-6-(3-hydroxy-3-methylbutyl)- 2, 3-dihydrochromen-4-one observed to have the maximum docking score value of (-8.717), indicating the highest binding affinity with HER2 protein which might serve as the promising compound for the development of a new class of drug to combat breast cancer.

Agonistic Effects of Deinoxanthin on Tamoxifen Antiproliferative Activity on HER2 Positive Breast Cancer: An In vitro Study on MDA-MB-453

A good understanding of the pathogenesis of breast cancer, in which in 2020 an estimated 684,996 women across the world died, plays a vital role in the development of treatment methods. In recent years, estrogen receptor (ER), progesterone receptor (PR), and Epidermal Growth Factor receptor-2 (HER2) expression levels have been substantial prognostic markers. The combinational therapy approach is the use of two or more active ingredients or methods. The most important benefit of this treatment method is to reduce the development of drug resistance since the probability of the carcinoma being resistant to more than one drug at the same time is low. Tamoxifen, an estrogen receptor competitive and nonsteroidal drug, has been used for nearly 20 years to treat patients with hormone receptor-positive breast cancer. Deinoxanthin is a xanthophyll derivative purified from the cell wall of a radiation-resistant bacterium, Deinococcus radiodurans. This xanthine derivative has been shown in some studies to have proapoptotic and antiproliferative effects on some types of cancer. Within the study's scope, it aimed to increase the effectiveness of Tamoxifen with deinoxanthin. Using the ACTB gene as a reference to investigate the synergistic effect of Tamoxifen and deinoxanthin on the MDA-MB-453 breast cancer cell line; Expression levels of BAX, CASP-3, BCL-2, and HER2 genes were examined by RT-qPCR method. The ELISA method determined the amounts of BAX, CASP-3, BCL-2, and HER2 proteins and compared them with RT-qPCR results. It was determined that the antiproliferative effect of Tamoxifen on the HER2 positive breast cancer cell line was increased with the combination of deinoxanthin.

Molecular imprinting of miR-559 on a peptide-immobilized poly L-DOPA/silica core-shell and in vitro investigating its effects on HER2-positive breast cancer cells.

In a significant percentage of breast cancers, increased expression of the HER2 receptor is seen and is associated with the spread and worsening of the disease. This research aims to investigate the effect of miR-559 (which targets HER2 mRNA) on SKBR3 breast cancer cells and the possibility of their effective delivery with polymeric nanoparticles and tumor-targeting peptides. L-DOPA monomers were polymerized on the surface of silica nanoparticles in the presence of miR-559 (as a molecular template for molecular imprinting) then an anti-HER2 peptide coupled to the surface of these polymeric nanocomposites (miR-NC-NL2), and the effects of this construct against a HER2-positive breast cancer cells (SKBR3 cells) investigated in vitro conditions. The results showed that miR-NC-NL2 is selective for HER2-positive cells and delivers the miR-559 to them in a targeted manner. miR-NC-NL2 decreased the proliferation of SKBR3 cells and reduced the expression and production of HER2 protein in these cells. Effective and targeted delivery of miR-559 to HER2-positive cancer cells by the miR-NC-NL2 promises the therapeutic potential of this nascent structure based on its inhibitory effect on cancer growth and progression. Of course, animal experiments require a better understanding of this structure's anti-tumor effects.

Analysis of HER2 protein expression and clinical prognosis based on CT texture analysis in the arterial phase of gastric cancer

ObjectiveTo assess the relationship of CT texture feature parameters in the arterial phase of gastric cancer with the expression of HER2 protein. MethodsA total of 39 patients with gastric cancer confirmed by pathology were included and further divided into the HER2 negative and positive groups. According to tumor invasion depth, they were assigned to the early and advanced groups. The maximum diameter of the tumor lesions on the CT axial plane was measured, and 3-year survival was followed up and recorded. ResultsThe maximum axial diameter of early gastric cancer was 4.84 ± 0.52 cm, and the 3-year follow-up survival time was 2.41 ± 0.56 years; the maximum diameter of advanced gastric cancer was 6.58 ± 0.42 cm, and the 3-year follow-up survival time was 1.21 ± 0.21 years with a significance between them (P < 0.05). The CT texture parameters in the arterial phase elucidated significant differences in the 10th percentile, 90th percentile, maximum, median, average, and minimum value between HER2 positive group and negative group (P < 0.05) without difference in texture features between the two groups (P > 0.05). ROC curve showed when the 10th percentile was 13.5, the 90th percentile was 70.2, the maximum value was 101.5, the median was 24.2, the average was 39.4, and the minimum of −7 was the best cut-off value. The specificity and sensitivity for distinguishing HER2-negative or positive were high, and the area under curve (AUC) was about 0.707–0.813, of which the 90th percentile was the most significant. ConclusionThe maximum diameter of the tumor in the CT axial plane is related to early and advanced gastric cancer and the prognosis. Based on CT arterial phase texture parameters, HER2 expression in gastric cancer can be judged to provide a basis for clinical diagnosis and treatment.

Olaparib (Lynparza)

&#x0D; CADTH recommends that Lynparza be reimbursed by public drug plans for the adjuvant treatment of adult patients with deleterious or suspected deleterious germline BRCA-mutated (gBRCAm), human epidermal growth factor receptor 2 (HER2)-negative high-risk early breast cancer who have been treated with neoadjuvant or adjuvant chemotherapy if certain conditions are met.&#x0D; Lynparza should only be covered in patients with a confirmed type of inherited (germline) abnormal BRCA gene, whose early-stage breast cancer tests negative for the HER2 protein, who are at high risk for breast cancer recurrence, and who have received chemotherapy before or after surgery.&#x0D; Lynparza should only be reimbursed if prescribed by clinicians with expertise and experience in treating breast cancer, and if the cost is reduced.&#x0D;

An ultrasensitive label-free fluorescent aptamer sensor based on pH-gated release coumarin for detect HER2

Evaluation of human epidermal growth factor receptor 2 (HER2) molecular markers is a very suitable option for early diagnosis of breast cancer. Metal-organic frameworks (MOFs) have large porosity and surface interactions such as π-π stacking, electrostatics, hydrogen bonding, and coordination. Here, we integrated the HER2 aptamer and fluorescent probe coumarin (COU) with zeolite imidazolic acid framework-8 (ZIF-8) to construct a label-free fluorescent aptamer sensor with pH-gated release of COU. In the presence of the target-HER2, the aptamer adsorbed on the surface of ZIF-8@COU specifically recognizes and falls off the HER2 protein, exposing a portion of the pore size of ZIF-8@COU while reducing the negative charge on the sensor surface, under alkaline hydrolysis conditions, a large number of COU fluorescent molecules can be produced and released in the detection system.The aptamer fluorescence sensor has good detection performance, sensitivity and low background interference, the detection linearity range of HER2 protein is 0.05–10 ng/mL, the detection limit is 0.0005 ng/mL, and it has good recovery rate for the serum detection of clinical breast cancer patients. Therefore, this sensor has high potential in detecting and monitoring HER2 levels for the care and clinical diagnosis of breast cancer patients.

Outcome comparison of pyrotinib with current standard of care in the second/third line setting in advanced non-small cell lung cancer patients with HER2 mutation.

Outcome comparison of pyrotinib with current standard of care in the second/third line setting in advanced non-small cell lung cancer patients with HER2 mutation.

Multinational Study of the Efficacy and Safety of Humanized Anti-HER2 Monoclonal Antibody in Women Who Have HER2-Overexpressing Metastatic Breast Cancer That Has Progressed After Chemotherapy for Metastatic Disease.

Overexpression of the HER2 protein occurs in 25% to 30% of human breast cancers and leads to a particularly aggressive form of the disease. Efficacy and safety of recombinant humanized anti-HER2 monoclonal antibody as a single agent was evaluated in women with HER2-overexpressing metastatic breast cancer that had progressed after chemotherapy for metastatic disease. Two hundred twenty-two women, with HER2-overexpressing metastatic breast cancer that had progressed after one or two chemotherapy regimens, were enrolled. Patients received a loading dose of 4 mg/kg intravenously, followed by a 2-mg/kg maintenance dose at weekly intervals. Study patients had advanced metastatic disease and had received extensive prior therapy. A blinded, independent response evaluation committee identified eight complete and 26 partial responses, for an objective response rate of 15% in the intent-to-treat population (95% confidence interval, 11% to 21%). The median duration of response was 9.1 months; the median duration of survival was 13 months. The most common adverse events, which occurred in approximately 40% of patients, were infusion-associated fever and/or chills that usually occurred only during the first infusion, and were of mild to moderate severity. These symptoms were treated successfully with acetaminophen and/or diphenhydramine. The most clinically significant adverse event was cardiac dysfunction, which occurred in 4.7% of patients. Only 1% of patients discontinued the study because of treatment-related adverse events. Recombinant humanized anti-HER2 monoclonal antibody, administered as a single agent, produces durable objective responses and is well tolerated by women with HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. Side effects that are commonly observed with chemotherapy, such as alopecia, mucositis, and neutropenia, are rarely seen.

Association between progression-free survival and overall survival in women receiving first-line treatment for metastatic breast cancer: evidence from the ESME real-world database

BackgroundOverall survival (OS) is the gold standard endpoint to assess treatment efficacy in cancer clinical trials. In metastatic breast cancer (mBC), progression-free survival (PFS) is commonly used as an intermediate endpoint. Evidence remains scarce regarding the degree of association between PFS and OS. Our study aimed to describe the individual-level association between real-world PFS (rwPFS) and OS according to first-line treatment in female patients with mBC managed in real-world setting for each BC subtype (defined by status for both hormone-receptor [HR] expression and HER2 protein expression/gene amplification).MethodsWe extracted data from the ESME mBC database (NCT03275311) which gathers deidentified data from consecutive patients managed in 18 French Comprehensive Cancer Centers. Adult women diagnosed with mBC between 2008 and 2017 were included. Endpoints (PFS, OS) were described using the Kaplan–Meier method. Individual-level associations between rwPFS and OS were estimated using the Spearman’s correlation coefficient. Analyses were conducted by tumor subtype.Results20,033 women were eligible. Median age was 60.0 years. Median follow-up duration was 62.3 months. Median rwPFS ranged from 6.0 months (95% CI 5.8–6.2) for HR-/HER2 − subtype to 13.3 months (36% CI 12.7–14.3) for HR + /HER2 + subtype. Correlation coefficients were highly variable across subtypes and first-line (L1) treatments. Among patients with HR − /HER2 − mBC, correlation coefficients ranged from 0.73 to 0.81, suggesting a strong rwPFS/OS association. For HR + /HER2 + mBC patients, the individual-level associations were weak to strong with coefficients ranging from 0.33 to 0.43 for monotherapy and from 0.67 to 0.78 for combined therapies.ConclusionsOur study provides comprehensive information on individual-level association between rwPFS and OS for L1 treatments in mBC women managed in real-life practice. Our results could be used as a basis for future research dedicated to surrogate endpoint candidates.

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells.

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.

Synthesis, characterization, molecular docking and in vitro anti-cancer activity studies of new and highly selective 1,2,3-triazole substituted 4-hydroxybenzohyrdazide derivatives

In this study, 16 new hybrid compounds 6(a-h) and 7(a-h) were synthesized starting from methylparaben. These new compounds consist of eight 1,2,3-triazole-arylidenehydrazide derivatives and eight 1,2,3-triazole-thiazolidin-4-one derivatives. All compounds were tested against MCF-7 breast cancer cells and MCF10A breast healthy tissue to determine their anti-cancer activity. Molecular docking studies were also carried out against receptor proteins (HER2, EGFR, and VEGFR1) that are related to the growth factor of cancer cells to understand the inhibition mechanism. According to the results of in silico and in vitro activity studies, compounds 6g and 7g were found as the highest selective and active against breast cancer cells.The IC50 values of compounds 6g and 7g against MCF7 cells were found as 8.48 and 4.38 µM, respectively. In addition, the IC50 values of compounds 6g and 7g against MCF10A cells were found as 114.80 and 170.60 µM. When comparing the selectivity of the 6g and 7g to the reference drug doxorubicin, 6g and 7g had higher selectivity (13.5 and 39 respectively) compared to doxorubicin (4). The compound 7g also showed activity against cancer cells at lower doses and was found to be safe against healthy cells at high doses. Molecular docking results showed that both 6g and 7g had higher binding scores for all 3 target enzymes and especially for HER2 protein related to breast cancer with binding scores of -8.059 kcal and -9,008 kcal respectively, while doxorubicin had a binding score of -7.854 kcal. According to the results of the in vitro, and in silico study, compound 7g which was synthesized in this study is a promising, potent, and selective anti-cancer drug candidate for the treatment of breast cancer. All the tested compounds were very well characterized by spectroscopic methods and they have at least 95% HPLC purity.

Abstract P5-06-03: Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms

Abstract Background 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 biopsy is otherwise infeasible. In addition, the development of quantitative, reproducible, and more sensitive HER2 assays is expected to enable the identification of patients with HER2-low MBC that may benefit from novel HER2-targeted therapies. Here we report a comprehensive liquid biopsy platform including immunofluorescent HER2 and ER quantitative protein expression in CTCs (ctcIF) coupled with the determination of ERBB2 amplification and the number of Large-scale State Transitions (LST) by single-cell CTC genomics (ctcDNA), and ctDNA alterations in plasma. Methods Blood samples were collected for cell-based and cell-free analysis from 62 patients with documented MBC and from 24 blood donors (HD) with no known cancer history. After isolation, nucleated cells were plated, and slides and plasma were bio-banked. CTCs were identified using Epic Sciences digital imaging and machine learning algorithms, and ctcIF enables quantitation of HER2 and ER protein expression. ctcDNA was analyzed by low-pass whole-genome sequencing (WGS), allowing detection of ERBB2 amplification (ERBB2amp) and quantification of large-scale state transitions (LST+) in individual CTCs. cfDNA from plasma was analyzed using a validated NGS panel (56 genes of interest) to detect ctDNA alterations. Results Within this cohort of 62 MBC patients, the presence of CTCs, ctcDNA (LST+) and ctDNA alterations were detected in 87%, 70%, and 59%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2amp in MBC patients (11%, and 2%, respectively). A variant allele frequency (VAF) of &amp;gt; 40%, which is required for detecting a two-fold ERBB2 amplification by ctDNA, was not present among 86% and 0% of MBC detected by the ctcDNA and ctDNA platforms, respectively, suggesting that the ctcDNA platform can identify ERBB2amp among patients with a low ctDNA fraction. HER2+ or ER+ expression by ctcIF were detected in 37% and 58% of MBC patients, respectively. At the cellular level, across 62 patients, CTC with detectable ERBB2amp by ctcDNA had a higher median expression of HER2 protein by ctcIF compared to CTC with ERBB2nonamp (1772 MFI vs 122.5 MFI, respectively; p&amp;lt; 0.001). Similarly, at the patient level, among patients with circulating ERBB2amp, HER2 protein was detected by ctcIF in 100% of MBC patients (p&amp;lt; 0.001), suggesting a very high positive correlation between the presence of ctcDNA genomic ERBB2amp and ctcIF HER2 protein expression. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that among MBC, 11% were ERBB2amp, 26% were HER2 expressing (HER2+ and ERBB2nonamp), and 60% were HER2neg (HER2- and ERBB2nonamp). Combination models of the three individual platforms (ctcIF, ctcDNA, and ctDNA) were able to provide potentially clinically actionable biomarker data (LST+ CTC, ERBB2amp CTC, HER2+ CTC, ER+ CTC and 1A+ SNVs) to 79% of MBC patients while retaining 100% specificity. Conclusions Here we reported a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in late MBC patients. The comprehensive liquid biopsy platform’s combined utility can aid biomarker profiling of MBC among often biologically heterogeneous tumor sites of metastatic disease and those inaccessible by conventional tissue biopsy. Citation Format: Giuseppe Di Caro, Ernest T. Lam, Megan M. Slade, Anna Lundberg, Martin Blankfard, Nilesh Dharajiya, Alisa Tubbs, Rick Wenstrup, Lee Schwartzberg. Analyzing the results of liquid biopsy in the identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: comparison and combination of cell and cell-free platforms [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-03.

Abstract P4-06-08: HER2-protein expression is a predictive marker for treatment response in patients with HER2-positive breast cancer who received neoadjuvant chemotherapy with dual HER2-blockade

Abstract Background In HER2-positive breast cancer, pathologic complete response (pCR) has been known to be a surrogate marker for favorable prognosis after neoadjuvant chemotherapy. We aimed to identify the clinico-pathologic factors related to pCR in patients with HER2-positive breast cancer treated with neoadjuvant dual HER2-targeted therapy. Methods Two-hundred ninty-five patients with HER2-positive breast cancer who received neoadjuvant chemotherapy with docetaxel, carboplatin, trastuzumab, and pertuzumab (TCHP) were included in this retrospective analysis. We assessed the association between age, clinical T, N stage, pathologic factors such as histologic grade, hormone receptor (HR), tumor-infiltrating lymphocytes, the membranous expression of HER2 protein evaluated by immunohistochemistry (IHC) and pCR. The fluorescent in situ hybridization (FISH) was performed in a tumor with HER2 score of 2+. Results Of the 295 patients, 195 (66.1%) achieved pCR (ypT0/is and ypN0). Besides, 240 (81.4%) patients were HER2 score of 3+, and 55 (18.6%) patients were HER2 score of 2+. The pCR was frequently observed in patients with HER2 score of 3+ (64 of 176 [73.3%]) than in those with HER2 score of 2+ (19 of 55 [34.5%]), regardless of HR status. After adjusting other clinicopathologic factors, the high HER2 protein expression was only an independent factor for pCR (adjusted OR 3.85, 95% CI, 1.66-8.91, p=0.002). Conclusions Our results suggest that high expression of HER2 protein assessed by IHC is important in predicting neoadjuvant TCHP response in HER2-positive breast cancer. Citation Format: Soong June Bae, Ji Soo Jang, Yoonwon Kook, Seung Ho Baek, Jee Hung Kim, Sung Gwe Ahn, Joon Jeong. HER2-protein expression is a predictive marker for treatment response in patients with HER2-positive breast cancer who received neoadjuvant chemotherapy with dual HER2-blockade [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-06-08.

Abstract P3-08-05: Correlation of Trop2 expression with in vivo sensitivity to sacituzumab govitecan in a panel of breast XPDX models

Abstract Background: Sacituzumab govitecan (SG) is an antibody-drug conjugate targeting Trop2 with an SN-38 payload recently approved for pretreated patients with locally advanced or metastatic triple-negative breast cancer (TNBC). The XenoSTART Patient-Derived Xenograft (XPDX) breast cancer platform includes over 180 models spanning all subtypes characterized with immunohistochemistry (IHC) including ER, PR, and HER2 protein levels, genomic and transcriptomic sequencing, and in vivo drug sensitivity. To better understand potential benefit of SG in breast cancers other than TNBC and further annotate our platform, Trop2 protein levels were determined in all breast models by IHC. We evaluated tumor growth inhibition by SG in 125 of our XPDX breast models and compared protein expression with agent activity. Methods: 180 breast XPDX models were evaluated for Trop2 expression (AF650, R&amp;D Systems) and 125 were evaluated in vivo against SG; responses were grouped by ER and Trop2 status (+/-). Models were grown subcutaneously in female athymic nude mice and ER+ models supplemented with estradiol. Models were also characterized for PR, HER2, and AR protein expression by IHC and profiled using WES and RNAseq. For in vivo studies, SG was administered by intravenous injection biweekly for two cycles at 1 mg, flat; endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C&amp;lt; 0%) versus Day 0 tumor volume was also reported. Results: 180 breast models were examined by IHC with 75/180 (42%) classified as ER+ and 105/180 (58%) ER-. In ER+ models 38/75 (51%) were Trop2+ and 37/75 (49%) Trop2-, and in ER- models 41/105 (39%) were Trop2+ and 64/105 (61%) Trop2-. In vivo, 20% of ER+/Trop2+ models reported sensitivity to SG, most notably models from patients with acquired resistance to CDK4/6 inhibitors, including STM001 and ST4316B. Interestingly, &amp;gt;70% of ER+/HER2+/Trop2+ models were insensitive to SG, including ST225 and ST340. Of 41 ER-/Trop2+ models, approximately 40% reported some response to SG with 50% of these sensitive to therapy, including ST5954 established from a patient who began treatment with SG following sample collection and is currently in remission. &amp;gt;75% of Trop2- models were insensitive to SG regardless of ER status. Conclusion: We screened 180 models in our XPDX breast cancer platform for Trop2 expression and compared expression with in vivo SG efficacy in 125 models. Analysis is underway to correlate receptor and molecular profiles with SG sensitivity in breast models and we are expanding expression and in vivo testing to additional indications. Citation Format: Alyssa Simonson, Johnnie Flores, Ebony Anderson, Crystal Moreno, George Plasko, Kyriakos P. Papadopoulos, Amita Patnaik, Drew Rasco, Gladys Rodriguez, Amy Lang, Muralidhar Beeram, Luis Rodriguez, Ronald Drengler, Steven Abbate, Hanni Salih, Lon Smith, Maryam Elmi, Brittany DeBerry, Arthur Rosenthal, Tatiana Hernandez, Nehal Lakhani, Manish Sharma, Michael Wick. Correlation of Trop2 expression with in vivo sensitivity to sacituzumab govitecan in a panel of breast XPDX models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-05.

HER2 Gene Protein Assay: A Robust Tool for Evaluating HER2 Status and Intratumoral Heterogeneity in Endometrial Cancers.

Human epidermal growth factor receptor 2 (HER2) status in endometrial cancer is usually determined by immunohistochemistry and/or in situ hybridization. We employed a novel HER2 gene protein assay (GPA) to simultaneously assesses HER2 gene amplification and protein expression in high-grade endometrial cancers. We performed GPA in 180 endometrial cancers, including 106 serous carcinomas, 34 carcinosarcomas, and 40 mixed epithelial carcinomas. HER2 status was determined using the 2018 HER2 guidelines for breast carcinoma, and HER2 intratumoral heterogeneity (ITH) was examined. Clinicopathologic characteristics were collected and correlated with HER2 status. HER2 positivity was noted in 32% of serous carcinomas, significantly higher than in carcinosarcomas (5.9%) and mixed carcinomas (12.5%). HER2 ITH was detected in 32% of serous carcinomas, significantly greater than in carcinosarcomas (8.8%) and mixed carcinomas (10%). Patients with carcinosarcoma had a significantly lower overall survival than patients with serous or mixed epithelial carcinoma, but HER2 status caused no difference in survival in patients with serous carcinoma. HER2 GPA can be used to accurately determine HER2 status in endometrial cancers and is a highly valuable tool for identifying HER2 heterogeneity.

Cellular 1H MR Relaxation Times in Healthy and Cancer Three-Dimensional (3D) Breast Cell Culture.

Noninvasive measurements of 1H Magnetic Resonance Imaging (MR) relaxation times in a three-dimensional (3D) cell culture construct are presented. Trastuzumab was used as a pharmacological component delivered to the cells in vitro. The purpose of this study was to evaluate the Trastuzumab delivery by relaxation times in 3D cell cultures. The bioreactor has been designed and used for 3D cell cultures. Four bioreactors were prepared, two with normal cells and two with breast cancer cells. The relaxation times of HTB-125 and CRL 2314 cell cultures were determined. An immunohistochemistry (IHC) test was performed before MRI measurements to confirm the amount of HER2 protein in the CRL-2314 cancer cells. The results showed that the relaxation time of CRL2314 cells is lower than normal HTB-125 cells in both cases, before and after treatment. An analysis of the results showed that 3D culture studies have potential in evaluating treatment efficacy using relaxation times measurements with a field of 1.5 Tesla. The use 1H MRI relaxation times allows for the visualization of cell viability in response to treatment.

Abstract P2-19-01: Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a significant role in the pathogenesis of several human cancers. Among them, HER2 is overexpressed in several cancers. In particular, overexpression with or without gene amplification occurs in 15-30% of breast cancers and this has both prognostic and predictive implications. HER2 overexpression and amplification is associated with shorter disease-free and overall survivals and with resistance to certain hormonal agents as well as increased risk of metastasis to the brain. Two types of treatment targeting HER2 biological activity have been developed: anti-Her2 monoclonal antibodies and small molecule tyrosine kinase inhibitors such as lapatinib and neratinib targeting HER2 and EGFR and Afatinib targeting all HER family members. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular segment and blocks its signaling. It is the first therapeutic antibody targeting Her2 overexpression approved by the FDA. It is administered in combination with standard of care chemotherapy. More recently, since it was shown that Trastuzumab was an internalizing antibody, two antibody drug conjugates using trastuzumab have been approved and used in the standard of care: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu). Enhertu is another antibody drug conjugate using Trastuzumab to deliver a topoisomerase inhibitor deruxtecan. Our laboratory has been focused in developing fully human internalizing anti-Her2 antibodies that compete or not with trastuzumab for binding to Her2. These antibodies have been developed by immunizing fully human mice with recombinant Her2 protein. Human Ab producing Tc mice (TC-mAb mice) stably maintain a mouse-derived engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. After production of hybridoma secreting fully human immunoglobulins, the screening process included inhibition of binding of trastuzumab to Her2 by enzyme linked immunoassay and by Octet epitope binning as well as internalization assay. Several internalizing antibodies with Kd ranging from 10-9 M to 10-12 M were selected. They were stratified by their ability to compete or not with trastuzumab. They were further characterized for their ability to deliver a cytotoxic payload in Her2 overexpressing cells as well as for their efficacy to inhibit proliferation, signaling and migration of Her2 overexpressing breast cancer cells. Data related to these antibodies will be presented here. In conclusion, the use of fully human TC mice in our laboratory represents an attractive approach to develop fully human monoclonal antibodies to high value cancer targets that can by-pass the need for humanization and affinity maturation of antibodies. Citation Format: Ginette Serrero, Jianping Dong, Binbin Yue, Udaya Yerramalla, Jun Hayashi. Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-01.