HER2-positive Breast Research Articles

Prognostic and Predictive Roles of HER2 Status in Non-Breast and Non-Gastroesophageal Carcinomas.

The oncogene ERBB2, also known as HER2 or c-ERB2, is located on chromosome 17 (q12). It encodes a tyrosine kinase receptor, the human epidermal growth factor receptor 2 (HER2), involved in neoplastic proliferation, tumor angiogenesis, and invasiveness. Over the past years, the introduction of various anti-HER2 therapies has significantly improved outcomes for patients with HER2-positive breast and gastroesophageal carcinomas. More recently, the introduction of a new antibody-drug conjugate, that is trastuzumab deruxtecan, expanded the therapeutic options to low-HER2 breast and gastroesophageal tumors. HER2 protein overexpression is investigated using immunohistochemistry, gene amplification using fluorescence in situ hybridization, and gene mutation using next-generation sequencing. This review evaluated the predictive and prognostic role of HER2 status in various types of epithelial malignant cancers beyond breast and gastroesophageal cancers. We critically analyzed the key published studies, focusing on utilized scoring systems and assays used, and analyzed clinical parameters and therapeutic approaches. Although the evidence about prognostic and predictive roles of HER2 in carcinomas other than breast and gastroesophageal has been widely increasing over the last decade, it still remains investigational, revealing a tumor site-related prognostic and predictive value of the different types of HER2 alterations. However, standardized and validated scoring system assays have not been well-established for many organs.

Usefulness of the baseline risk assessment guidelines to predict cancer therapy-related cardiac dysfunction in early-stage HER2 positive breast cancer

Abstract Introduction Treatment of HER 2-positive breast cancer (HER2+BC) with anthracyclines and HER2-targeted therapy is associated with a high incidence of cancer therapy-related cardiac dysfunction (CTRCD). Baseline risk assessment is crucial to determine patients at high risk of developing CTRCD. Heart Failure Association-International Cardio-Oncology (HFA-ICOS) risk stratification has recently been developed but validation with the new definition of CTRCD is needed (1). Purpose To investigate whether the HFA-ICOS baseline risk stratification is useful to identify patients at risk for CTRCD in patients with early-stage HER2+BC. Methods 95 patients with early-stage HER2+BC were consecutively included in a specialized cardio-oncology unit. In this cohort, patients with previous exposure to antineoplastic treatment, patients with left ventricular ejection fraction (LVEF) < 50% and patients with moderate-severe valvular heart disease had been excluded. All patients underwent a baseline echocardiography study and every 3 months until the end of the chemotherapy. Ultrasensitive troponin I was determined before and immediately after each cycle of chemotherapy. Results In our cohort, 48.4% of patients received sequential treatment with anthracyclines and anti-HER2 therapy, while 51.6% received anti-HER2 therapy without anthracyclines. HFA-ICOS baseline risk assessment identified 40 (42,1%) patients with low risk, 27 (28,4%) with moderate risk and 28 (29,5%) with high risk of CTRCD. Table 1 showed the distribution of HFA-ICOS risk factors in our cohort. Only 1 patient with high risk and 3 patients with moderate risk received cardioprotective drugs. At a median follow-up of 13,6 months, 38 patients (40%) did not develop CTRCD, 53 (55,8%) mild-CTRCD, 3 (3,2%) moderate-CTRCD, 1 (1,1%) severe-CTRCD. 3 out of the 4 patients who presented with moderate or severe CTRCD were properly identified as moderate or high risk. However, 52,5% of patients with low risk of CTRCD developed some degree of CTRCD and 19 out of the 38 patients (69,4%) who did not present CTRCD were wrongly identified as moderate or high risk. Table 2 showed the relation between HFA-ICOS risk assessment and the development of CTRCD. Conclusions Treatment of early-stage HER2+BC was associated with a high incidence of CTRCD. In our cohort of early-stage HER2+BC, HFA-ICOS baseline risk assessment was useful in identifying patients at risk of developing moderate or severe CTRCD, but was not useful in identifying patients at low risk of CTRCD.HFA-ICOS risk factors and CTRCDHFA-ICOS risk assessment and CTRCD

Trastuzumab-Related Cardiotoxicity in Adjuvant Setting: A Real-World Scenario.

Rachana Chennamaneni Trastuzumab, a humanized monoclonal antibody, significantly improves outcomes in HER 2-neu positive breast cancer. The incidence of cardiotoxicity with trastuzumab is approximately 8 to 10%. This study was designed to analyze the incidence and risk factors associated with trastuzumab-related cardiotoxicity in real-world settings. This was a single institutional retrospective analysis of the incidence of trastuzumab-related cardiotoxicity in nonmetastatic HER 2-positive, invasive breast cancer from January 2013 to December 2018. Trastuzumab-related cardiotoxicity was defined as symptomatic heart failure or asymptomatic decline in left ventricular ejection fraction (LVEF) by more than or equal to 10% or LVEF less than 50%. Risk factors analyzed were higher body mass index (≥30 kg/m 2 ), history of diabetes, hypertension, cardiac disease, left-sided radiotherapy (RT), and prior exposure to anthracyclines. Out of the 246 patients diagnosed with early stage HER 2-positive breast cancer, 117 (47.5%) received trastuzumab and constituted the study population. Trastuzumab-related cardiotoxicity was seen in a total of 16 (13.6%) patients. Eleven (9.4%) patients had an asymptomatic decline, while symptomatic LV dysfunction was seen in five (4.2%) patients. The median baseline ejection fraction was 65% (range, 56-72). The median time to development of cardiotoxicity was 18.5 weeks (range, 3-52) and the median trastuzumab cycle for cardiotoxicity was 6 (range, 2-16). Ten (62.5%) patients were rechallenged with trastuzumab following which one patient developed an asymptomatic decline in ejection fraction and one patient developed symptomatic heart failure. Cardiac-related mortality was seen in one (0.85%) patient. Left-sided RT to chest ( p = 0.012) and presence of more than or equal to two risk factors ( p = 0.01) had significant impact on incidence of cardiotoxicity. Approximately 14% developed trastuzumab-related cardiotoxicity that was slightly higher compared with that seen in clinical trials. Left-sided RT to chest and presence of two or more risk factors had significant impact on development of cardiotoxicity.

Safety and efficacy of trastuzumab biosimilar plus irinotecan or gemcitabine in patients with previously treated HER2 (ERBB2)-positive non-breast/non-gastric solid tumors: a phase II basket trial with circulating tumor DNA analysis

Human epidermal growth factor receptor 2 (HER2) (ERBB2)-directed agents are standard treatments for patients with HER2-positive breast and gastric cancer. Herein, we report the results of an open-label, single-center, phase II basket trial to investigate the efficacy and safety of trastuzumab biosimilar (Samfenet®) plus treatment of physician's choice for patients with previously treated HER2-positive advanced solid tumors, along with biomarker analysis employing circulating tumor DNA (ctDNA) sequencing. Patients with HER2-positive unresectable or metastatic non-breast, non-gastric solid tumors who failed at least one prior treatment were included in this study conducted at Asan Medical Center, Seoul, Korea. Patients received trastuzumab combined with irinotecan or gemcitabine at the treating physicians' discretion. The primary endpoint was the objective response rate as per RECIST version 1.1. Plasma samples were collected at baseline and at the time of disease progression for ctDNA analysis. Twenty-three patients were screened from 31 December 2019 to 17 September 2021, and 20 were enrolled in this study. Their median age was 64 years (30-84 years), and 13 patients (65.0%) were male. The most common primary tumor was hepatobiliary cancer (seven patients, 35.0%), followed by colorectal cancer (six patients, 30.0%). Among 18 patients with an available response evaluation, the objective response rate was 11.1% (95% confidence interval 3.1% to 32.8%). ERBB2 amplification was detected from ctDNA analysis of baseline plasma samples in 85% of patients (n= 17), and the ERBB2 copy number from ctDNA analysis showed a significant correlation with the results from tissue sequencing. Among 16 patients with post-progression ctDNA analysis, 7 (43.8%) developed new alterations. None of the patients discontinued the study due to adverse events. Trastuzumab plus irinotecan or gemcitabine was safe and feasible for patients with previously treated HER2-positive advanced solid tumors with modest efficacy outcomes, and ctDNA analysis was useful for detecting HER2 amplification.

Abstract CT278: ERBB2 amplification detected in ctDNA as a surrogate for tumor tissue FISH analysis of HER2 status in a phase 1 study with zanidatamab for the treatment of locally advanced or metastatic HER2 expressing cancers

Abstract Background: HER2-targeted therapies have substantially improved outcomes for patients with HER2-positive breast and gastric cancers. Several other cancers exhibit HER2 expression and/or amplification of its gene (ERBB2), suggesting that HER2-targeted agents may have broader therapeutic utility. Zanidatamab is a humanized, novel bispecific antibody directed against two non-overlapping domains of HER2. The aim of this Phase 1 dose-escalation and expansion study (NCT02892123) was to evaluate the safety and efficacy of zanidatamab across a range of solid tumors. Parallel to drug development, there has been rapid advancements in NGS technologies including the Guardant360 assay that can specifically sequence ctDNA and detect amplifications of the ERBB2 gene, which can lead to overexpression of HER2. FISH, the current gold standard for HER2 amplification detection, is a tissue-based assay that assesses the raw ERBB2 copy number as well as ratio of ERBB2 to a centromeric protein of chromosome 17 where the ERBB2 gene resides. We evaluated concordance of the FISH and Guardant360 assays to detect ERBB2 amplification in plasma samples. Unlike gene copy number in tissue analysis, the observed plasma copy number (pCN) is also a function of the tumor burden and rate of tumor shedding of ctDNA into the bloodstream. Methods: HER2 status was determined from a fresh tumor biopsy or in archival FFPE tissue samples by IHC and FISH according to ASCO-CAP guidelines from the Phase 1 study with zanidatamab in multiple cancer types (cholangiocarcinoma [21], colorectal carcinoma [27], all other [87]). Plasma samples were collected prior to the first cycle of zanidatamab and on-treatment for testing with Guardant360, 74 gene ctDNA NGS-based assays. Results: A concordance of 82% was observed in ERBB2/HER2 amplifications between the Guardant360 and FISH assays. An exploratory adjustment method based on tumor DNA shedding was developed by Guardant using the maximum mutant allele fraction (maxVAF) as a surrogate for tumor content. Majority of patients experienced a decrease in HER2 pCN post treatment, with 9 PD patients having the least and 21 PR patients the largest changes in ctDNA fraction (maxVAF). Conclusion: These results indicate that ERBB2 amplification detected by the Guardant360 assay could be used as a surrogate for FISH analysis in lieu of invasive surgical procedures. Citation Format: Diana Shpektor, Daryanaz Dargahi, Antonios Samiotakis, Sara Wienke, Ali Livernois, Arielle Yablonovitch, Geethika Yalamanchili, Elaina Gartner, Funda Meric-Bernstam. ERBB2 amplification detected in ctDNA as a surrogate for tumor tissue FISH analysis of HER2 status in a phase 1 study with zanidatamab for the treatment of locally advanced or metastatic HER2 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT278.

Autoimmune hypothyroidism and trastuzumab therapy: a rare association.

We report a case of a woman with a diagnosis of breast cancer who unintentionally started gaining weight, feeling tired, and constipated 44 weeks after the initiation of trastuzumab. Hypothyroidism secondary to an autoimmune thyroiditis associated with trastuzumab was diagnosed, the first case described in Portugal and the fourth case described worldwide. Our intention regarding the publication of this case report is to alert the clinicians treating people with trastuzumab that they should ask the patients about symptoms of hypothyroidism and should screen the thyroid function of the patients before, during, and after the initiation of trastuzumab. Trastuzumab is a humanized MAB used in HER2-positive breast and gastric cancer. Trastuzumab-associated autoimmune thyroid disease (AITD) is rare (incidence rate in an RCT of 0.3%). Manifestations of autoimmune thyroiditis associated with trastuzumab resemble those of hypothyroidism in other clinical contexts, but the presence of goiter is highlighted as a reason for medical evaluation. Biochemically, it is characterized by an increased thyroid-stimulating hormone (TSH) with or without a low FT4/FT3, and sonographically with a pattern of thyroiditis. The treatment consists of levothyroxine, in a dose of 1.6-1.8 µg/kg/day, with re-evaluation of the thyroid function in 4-6 weeks. We report the first case of autoimmune thyroiditis secondary to trastuzumab in Portugal. It is important to evaluate the thyroid function before, during, and after the initiation of this therapeutic agent.

Abstract P1-05-20: COVID-19 Lockdown Resulting in Advanced Cancer and Worse Histology at Presentation in Hispanics

Abstract Background In breast cancer, prognosis is marked by histology and stage at diagnosis. Patients presenting with HER-2 positive or triple negative breast (TNBC) often have a worse prognosis. Early detection of breast cancer is mainly based on yearly screening mammograms, which were disrupted during the lockdown stages of the COVID-19 pandemic. In general, underserved populations, especially the Hispanic population often lack access to preventive care due to lack of funding causing delays in access to timely care. The COVID-19 pandemic caused many patients to miss their annual mammogram screening due to lockdown causing subsequent presentation of more advanced cancer. We, therefore, hypothesized that more patients were diagnosed with advanced cancer after lockdown and worse histology in the Hispanic population compared to the non-Hispanic population of San Antonio, Texas. Methods We identified 3 cohorts retrospectively using chart review: Pre-covid-19 era was defined between 2018 to March 2020. Lockdown is defined as a period between April 2020 to December 2020 followed by the post-vaccine era from January 1st 2021 to 2022. Pearson’s Chi-squared and logistic regression tests were used to determine the relationship between time, histology at diagnosis and ethnicity. Results More Hispanic patients were found to present with HER2+ disease (OR: 1.65. p-value .047) compared to non-Hispanic women. When looking at presentation of HER2+ disease in the pre-covid-19 era, there was a 15.11% increase in the presentation of HER2+ disease in the post-vaccine era. When looking at the presentation of TNBC disease in women, there was not a significant correlation seen in the lockdown or post-vaccine period in Hispanic compared non-Hispanic women. Other factors such as funding status were associated with TNBC at presentation independently of ethnicity. In the lockdown era, the number of newly diagnosed breast cancer patients reached an all-time low and during the post-vaccine era, the patient numbers are back to the pre-covid era. Conclusion Ethnicity in part played a role in the number of patients presenting with more aggressive histology such as TNBC and Her 2 positive breast cancer in the post-vaccine era. These findings may be secondary to fact that certain ethnic groups are more likely to miss preventive screenings and the covid 19 pandemic lockdown exacerbated this problem. Diagnosis of advance cancer can further deter patients from seeking care due to socioeconomic factors and possibly increase mortality in these populations. These findings suggest that there seems to be a correlation between race and presentation of more aggressive histology caused by the effects of the pandemic in cancer care affecting minorities. Citation Format: Juzar Hussain, Gabriel Roman Souza, Tamarah Aldawoodi, Lauren C. Jameson, Lauren Rahman, Nomso C. Agim, Jonathan Gelfond, Marcela Mazo-Canola. COVID-19 Lockdown Resulting in Advanced Cancer and Worse Histology at Presentation in Hispanics [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 P1-05-20.

Abstract P5-02-21: RAB5 is a generic biomarker for ADC efficacy

Abstract Antibody-drug conjugates (ADCs) have demonstrated impressive activity in recent clinical trials in breast cancer. Such targeted therapeutics strongly depends on the presence of target molecules on the tumor cells, and the presence of such target molecules may determine the response of ADCs. However, ADCs are also dependent on cellular uptake, and factors regulating endocytosis as well as intracellular trafficking may strongly influence ADC activity. We have recently demonstrated that the activity of the HER2 targeted ADC trastuzumab emtansine (T-DM1) is dependent on the expression of RAB5A, a protein regulating endocytosis (1). A significant correlation between Rab5A expression and T-DM1 efficacy was found in a panel of HER2 expressing breast- and ovarian cancer cell lines. This result was verified in the I-SPY2 clinical trial where patients with high RAB5A expression were more likely to achieve a pathological complete response following T-DM1 as a neoadjuvant. The result was further validated in patients treated with T-DM1 in the Kamilla study where patients with a high RAB5A had a longer progression free survival. All ADCs should in principle be dependent on endocytosis to exert their activity. This triggered the investigation of proteins regulating endocytosis as predictive biomarkers for ADCs in general. METHODS: HER2-positive breast and ovarian cancer cell lines were evaluated with respect to the sensitivity and efficacy of treatment with T-DM1, trastuzumab deruxtecan, sacituzumab govitecan and the targeted toxin MH3B1/rGel.. The expression levels of proteins involved in endocytosis and endocytic trafficking including RAB4A, RAB5A and RAB11A were investigated in addition to the molecular drug targets (HER2 and TROP2). Cellular drug sensitivity was correlated to the expression levels of the investigated proteins using both RNA and protein as readout. RESULTS: The early endosome marker RAB5A, was found to correlate positively to the activity of trastuzumab deruxtecan, sacituzumab govitecan and MH3B1/rGel in the HER2 positive cell line panel, confirming the importance of RAB5A expression for the activity of these drugs. A significant correlation was found between RAB5A and drug efficacy using both protein and RNA as a readout. CONCLUSION: The present results indicate RAB5A as a generic predictive biomarker for both ADCs and targeted toxins which both depend on cellular uptake for cytotoxic efficacy. The results supports using both protein and RNA as a readout for RAB5A expression and point towards the development of a RAB5A stratification procedure for ADC and targeted toxin treatment. 1. Engebraaten O, Yau C, Berg K, Borgen E, Garred O, Berstad MEB, Fremstedal ASV, DeMichele A, Veer LV, Esserman L, Weyergang A. RAB5A expression is a predictive biomarker for trastuzumab emtansine in breast cancer. Nature communications 2021;12(1):6427 doi 10.1038/s41467-021-26018-z. Citation Format: Olav Engebraaten, Astrid Medhus, Ane Longva, Anette Weyergang, Kristian Berg. RAB5 is a generic biomarker for ADC efficacy [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 P5-02-21.

Magnetic resonance differential analysis for different hormone receptor expression status in HER-2-positive breast cancer.

Currently, it is difficult to assess the expression status of hormone receptor (HR) in breast malignant tumors with human epidermal growth factor receptor 2 (HER-2)-positive in the early preoperative stage, and it is difficult to predict whether it is non-invasively. This study aims to explore the value of MRI on the different HR expression status (HR+/HR-) in HER-2 positive breast cancer. Thirty patients with HR+ HER-2-positive breast cancer (HR+ group) and 23 patients with HR-HER-2-positive breast cancer (HR- group) from the First Hospital of Hunan University of Traditional Chinese Medicine between January 7, 2015 and November 26, 2021 were selected as subjects, and all the patients were examined by MRI and all were confirmed by surgery or pathological biopsy puncture. The immunohistochemical staining results were used as the gold standard to analyze the basic clinical conditions, peri-lesion conditions and MRI sign characteristics in the 2 groups. There were all significant differences in terms of mass margins, internal reinforcement features, and apparent diffusion coefficient (ADC) values between the HR+ group and the HR- group (all P<0.05). The logistic multivariate regression model showed that: when the lesion presented as a mass-type breast cancer on MRI, the internal enhancement features of the lesion were an independent predictor for differentiation in the 2 types of breast cancer [odds ratio (OR)=5.95, 95% CI: 1.223 to 28.951, P<0.05], and the mass margin (OR=0.386, 95% CI: 0.137 to 1.082, P>0.05) and ADC value (OR=0.234, 95% CI: 0.001 to 105.293, P>0.05) were not the independent predictors in distinguishing the 2 types of breast cancer. Multiparametric MRI has good diagnostic value for HR expression status in HER-2-positive breast cancer. Combined logistic regression analysis to construct a predictive model may be helpful to the identical diagnosis.

Antitumor Activities in Mouse Xenograft Models of Canine Fibroblastic Tumor by Defucosylated Mouse-Dog Chimeric Anti-HER2 Monoclonal Antibody (H77Bf).

Human epidermal growth factor receptor 2 (HER2) is a cell surface type I transmembrane glycoprotein that is overexpressed on a variety of solid tumors and transduces the oncogenic signaling upon homo- and heterodimerization with HER families. Anti-HER2 monoclonal antibodies (mAbs) including trastuzumab and its antibody-drug conjugate have been shown to improve patients' survival in HER2-positive breast, gastric, and lung cancers. Canine tumors have advantages as naturally occurring tumor models, and share biological and histological characteristics with human tumors. In this study, we generated a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) derived from H2Mab-77 (mouse IgG1, kappa). H77Bf possesses the high binding affinity (a dissociation constant: 8.7 × 10-10 M) for a dog HER2 (dHER2)-expressing canine fibroblastic tumor cell line (A-72). H77Bf exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity for A-72 cells. Moreover, intraperitoneal administration of H77Bf significantly suppressed the development of A-72 tumor compared with the control dog IgG in a mouse xenograft model. These results indicate that H77Bf exerts antitumor activities against dHER2-expressing canine cancers, which could provide a valuable information for canine cancer treatment.

Defucosylated mouse‑dog chimeric anti‑HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti-HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2-positive breast and gastric cancer. An anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding-affinity [a dissociation constant (KD): 7.5×10−10 M, as determined by flow cytometric analysis] for dog HER2-overexpressed CHO-K1 (CHO/dHER2) cells. H77Bf highly exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 ×enograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding-affinity (KD: 7.2×10−10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2-positive canine cancers, and could be valuable treatment regimen for canine cancers.

Neoadjuvant therapy for HER2-positive and negative simultaneous bilateral primary breast cancer.

Neoadjuvant therapy for HER2-positive and negative simultaneous bilateral primary breast cancer.

Abstract 1788: Engineered destabilized AU rich elements on the 3UTR of HER2 degrades HER2, inhibits proliferation, and induces apoptosis in HER2 positive trastuzumab resistant breast cancer cells

Abstract Resistance to molecular targeted therapy is a major challenge facing breast cancer patients and unfavorably impacts clinical outcomes, leading to hundreds of thousands of deaths yearly. Resistance to cancer therapies arises from many factors. One of the key factors driving resistance is oncogenes which initiate pro-growth, pro-survival and metastatic programs that enable cancers escape various therapies. In HER2+ breast cancer, the oncogene HER2 is the master driver of this tumor. Trastuzumab effectively targets HER2. While this is generally successful, some patients who have HER2+ breast cancer develop resistance to trastuzumab and there are limited treatment options for them. Moreover, for low HER2+ expressing breast cancer, there are limited treatment options for them. And in African American women who have HER2+ breast cancer, there is disparity in their treatment outcomes. We have discovered that 3’UTR of HER2 is enriched with poly U stabilizing AU rich elements (ARE). We have developed a novel technology wherein we engineered the stabilizing HER2 3’UTR ARE motifs to destabilizing motif in HER2+ and HER2+ trastuzumab resistant breast cancer cells to control HER2 transcript. We achieved complete degradation of HER2 within 4-8 days in HER2+ wildtype breast cancer cells and 9-11 days in HER2+ trastuzumab resistant breast cancer cells. We had loss of cancer cell viability by more than 90%. We also show that control of HER2 transcript downregulated HER2-dependent kinases, transcription factors, and HER2 interactome. Mechanistically, the control of HER2 transcript was achieved by destabilized ARE sequence specificity which triggered the proteins PARN, XRN1 and CNOT1 to degrade HER2 transcript and this led to induction of active caspase 3/7, reduction of cell size, and severe distortion and disruption of the cancer cell membrane leading to cell death. Taken together, we have a developed a novel approach to control HER2 transcript expression both on spatial and temporal scale. This novel approach offers applications for targeting HER2+ trastuzumab resistant breast cancer as well as other cancers resistant to HER2 targeted therapy and driven by pervasive oncogenic signals. Citation Format: Chidiebere U. Awah, Fu Dong, Fayola Levine, Leonard Ash, Yana Glemaud, Olorunseun Ogunwobi. Engineered destabilized AU rich elements on the 3UTR of HER2 degrades HER2, inhibits proliferation, and induces apoptosis in HER2 positive trastuzumab resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1788.

Abstract 5627: Functional tertiary lymphoid structure (TLS) and outcome in HER2-positive (HER2+) breast cancer in NCCTG N9831 (Alliance)

Abstract Background: Previous studies showed conflicting results regarding association between stromal tumor infiltrating lymphocytes (sTIL) and outcome in HER2+ breast cancer. While sTIL were not associated with outcome in patients (pts) treated with trastuzumab in N9831, immune functions genes are linked to outcome in these pts. TLS is an organized form of ectopic lymphoid aggregates (LA) with secondary lymphoid organ structure. Several studies showed that TLS associates with improved outcome in multiple cancers. However, distinguishing TLS and simple LA is challenging, particularly when germinal center is absent. In this study, we evaluated integrated pathological quantification and genomic data to assess functional TLS. Methods: Pathological evaluation of LA in H&amp;E slides from pts treated in Arm A (chemotherapy alone) and Arm C (chemotherapy with concurrent trastuzumab) in N9831 was performed. NanoString was used to quantify mRNA. Wilcoxon rank sum test, Chi squared test, Kaplan-Meier method and Cox regression model were used to evaluate association between LA and baseline characteristics as well as outcomes. Results: LA was quantified in 1011 pts (526 Arm A, 485 Arm C). Greater number of LA was significantly associated with higher tumor grade, ER/PR negativity and increasing sTIL, but not age, tumor size or lymph node status. Increasing numbers of LA were associated with improved RFS in both arms combined (p 0.028). However, using multivariable Cox regression analysis in each treatment arm, LA (≥ 1 vs 0) was associated with improved RFS only in Arm A (HR 0.6, 95%CI 0.43-0.84, p 0.003) but not Arm C (HR 0.72, 95%CI 0.47-1.1, p 0.134). Further evaluation of expression of TLS-related immune genes including IFNG, ICOSLG, CXCL13, CXCR3, BCL6, IL21R, ICOS, PDCD1, CXCR5, CXCL9, TBX21, CD38, CXCL10, CLXCL11, IL21, CD200 was carried out among 252 pts in Arm C with LA ≥ 1. Among these 16 TLS-related immune genes, as a continuous variable, higher expression of BCL6 (HR 0.61, 95%CI 0.41-0.92, p 0.019) and IL21R (HR 0.78, 95%CI 0.62-0.98, p 0.03) were associated with improved RFS in Arm C pts with LA ≥ 1. However, these genes were not significantly associated with outcome in Arm C pts without LA with BCL6 HR1.07 (95%CI 0.67-1.71, p 0.776) and IL21R HR 0.96 (95%CI 0.71-1.29, p 0.775). Conclusion: Similar to sTIL, greater number of LA was associated with improved outcome in HER2+ pts treated with chemotherapy alone but not chemotherapy in combination with trastuzumab. Using histogenomic integration with the combination of pathological LA and TLS-related immune genes, we identified that pts with functional TLS with LA ≥ 1 and higher expression of BCL6 or IL21R had significantly improved outcome when treated with trastuzumab. Future studies are needed to further confirm these findings. Support: U10 CA180821, U24 CA196171, https://acknowledgments.alliancefound.org; Genentech; Clinicaltrials.gov Id: NCT00005970 Citation Format: Saranya Chumsri, Tracy R. Shachner, Zhuo Li, Nadine Norton, Alvaro Moreno-Aspitia, Gerardo Colon-Otero, Edith A. Perez, E. A. Thompson, Aziza Nassar, Keith L. Knutson. Functional tertiary lymphoid structure (TLS) and outcome in HER2-positive (HER2+) breast cancer in NCCTG N9831 (Alliance) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5627.

Abstract 5334: Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers

Abstract Background: Human epithelial growth factor receptor 2 (HER2) is a well-established therapeutic target in HER2-positive breast and gastric cancer. Recently, novel HER2-targeting agents are being developed after trastuzumab, and HER2-targeted therapies have been attempted in across solid tumor types, including biliary tract cancer. In these aspects, it is essential to understand and elucidate the resistant mechanism of the HER2-targeting strategies. Yes-associated protein (YAP), a transcription factor of the Hippo pathway, function as proto-oncoproteins by inducing target genes involved in cancer cell survival and proliferation (Zhao B, et al. Genes Develop 2008). Also, YAP is emerging as a resistance mechanism of cytotoxic and targeted drugs. In this study, we explore the role of YAP in overcoming resistance to trastuzumab in HER2-positive cancer cells. Methods: Four trastuzumab-resistant (HR) cell lines were established using HER2-postive gastric and biliary tract cancer cell lines (N87, SNU216, SNU2670, and SNU2773) (Jin MH, et al. Mol Cancer Ther. 2017). YAP siRNA and Verteporfin (YAP-TEAD inhibitor) were used to downregulate YAP. MTT assay, cell cycle analysis, western blot and migration assay were performed to analyze the antitumor effects through YAP downregulation. In order to elucidate tumor cell immune-modulation by YAP, human PBMC co-culture was used and immune markers, such as programmed death-ligand 1 (PD-L1), were analyzed in HR cell lines. Results: Increase of receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) expression was observed in the HR cells. Relatively high expression of pYAP, YAP, and transcriptional co-activator with PDZ-binding motif (TAZ) were observed in the HR cells (SNU216HR, SNU2670HR and SNU2773HR) compared to parental cells. Reducing overexpressed YAP in HR cells resulted in tumor cell growth inhibition. In cell cycle analysis, the increase of subG1 phase through YAP downregulation was observed, and cyclinD, B, A and BCL-XL were effectively decreased. Migration was also inhibited by the decrease in YAP expression. By verteporfin treatment, changes in immune markers including increase of CD80 and decrease of PD-L1 was found. In addition, p-STAT3 and IL6, Which regulate PD-L1, were also decreased through the reduction of YAP. In HR cells treated with siYAP, there was a tendency to increase CD8+ T cells upon PBMC co-culture. Conclusion: YAP is upregulated in trastuzumab-resistant (HR) cells and upregulated YAP induce PD-L1 expression. Inhibition of YAP shows anti-tumor effects and modulates immune status. Collectively, our results suggest that inhibition of YAP is one of promising strategies to overcome trastuzumab resistance in HER2-positive cancers. Citation Format: Ah Rong Nam, Jeesun Yoon, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Tae-Yong Kim, Do-Youn Oh. Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5334.

Abstract 343: New derivatives of 1,3-dioxoisoindoline: Potential breast cancer therapeutics that are S6K1 inhibitors

Abstract Currently, more than 3.8 million women in the United States are diagnosed with breast cancer which is the second leading cause of cancer-related death in women. The correlation of the ribosomal protein S6 kinase 1 (S6K1) activity and breast cancer is evident from the amplification of S6K1 localized chromosomal region 17q23 in 20% of primary breast cancers. S6K1 is a conserved serine/threonine protein kinase, is the principal kinase effector downstream of the PI3K/mTOR regulatory signaling pathway. Over-expression of S6K1 has been associated with cell transformation and elevated proliferation rates in tumors, poor prognosis and an increased risk of local recurrence. S6K1 has been found to play an important role in the progression of ER-positive (ER+) breast cancer, HER2 positive (HER2+) breast cancer and node-negative premenopausal breast cancer. Inhibition of this kinase can prove to be beneficial for the treatment of several types of breast cancer. Our group has identified new molecules that are derivatives of 6-amido-4-aminoisoindoline-1,3-dione core structure as S6K1 inhibitors with low micromolar inhibition potency (IC50 = 2-5 μM). These compounds also inhibited the growth of HER2+ (SKBR3 and HER2Δ16), ER+ and triple negative breast cancer cells (MDA-MB-231 and MDA-MB-468) with a range of EC50 values (2 - 2000 μM). The EC50 values of growth inhibition of these compounds varied by the type of breast cancer cells and were in direct correlation with the expression levels of S6K1 in those cell lines. These results clearly indicate that high efficacy S6K1 inhibitors can function as potential therapeutics for multiple types of breast cancer. Future work involves in-vivo studies to understand the efficacy and pharmacokinetics of the three compounds. Citation Format: Satyendra Kumar, Rajesh Komati, Shahensha Shaik, Melyssa Bratton, Linh Tran, Rion Sam, Elijah Johnson-Henderson, Breyanah Graham, Christopher WIlliams, Jayalakshmi Sridhar. New derivatives of 1,3-dioxoisoindoline: Potential breast cancer therapeutics that are S6K1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 343.

Abstract 1141: Pharmacokinetic and pharmacodynamic evaluation of human tumor xenograft models treated upon administration of trastuzumab deruxtecan

Abstract Introduction: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate comprised of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor approved for the treatment of HER2 positive metastatic breast and gastric cancer. T-DXd has demonstrated antitumor activity in both HER2+ and HER2-low patient populations. Methods: To establish exposure profiles of T-DXd and link with tumor biomarker changes, we administered a single IV dose of T-DXd at 10 mg/kg in human tumor xenograft models representing HER2-positive (NCI-N87; nude) and Her2-low (Capan-1; NOD-SCID) and collected tumor and plasma from 6 h to 336 h post dose. We measured tumor volume in addition to total ADC, total antibody, and free payload in the plasma and assessed biomarkers related to DNA damage in the tumor by western blot (WB) and immunohistochemistry (IHC). Results: In HER2+ NCI-N87 tumor-bearing mice, T-DXd plasma AUC was 342.6ug/ml*day and T1/2 was 3.5 days, while in the HER2-low Capan-1 tumor-bearing mice, T-DXd plasma AUC was 297.2ug/ml*day and T1/2 was 1.4 days. Plasma exposures of free payload (DXd) were less than 1 ng/mL. Both models responded to T-DXd, demonstrating regression over the 14 day study (T/C =-6.08%, NCI-N87 and -96.1%, Capan-1). In NCI-N87, we observed rapid and sustained increases in gamma H2AX (gH2AX), with a 3.5-fold increase in % positive staining by IHC (H-score p&amp;lt;0.001) with gH2AX foci as early as 24h post treatment, and sustained out to 96h. Western blot analysis and quantification of gH2AX revealed a 4.3-fold increase at 48h (p&amp;lt;0.0001), which was sustained out to 96h. Further, we observed significant increases in pRAD50 at 24h (3.6-fold increase by IHC, H-score p&amp;lt;0.001) which was sustained out to 96h. In Capan-1, we observed more diffuse staining of gH2AX and non-significant 1.4-fold increase in gH2AX at 24h. pRAD50 increases were delayed in the Capan-1 model with a 2.8-fold increase observed at 48h and sustained out to 168h post treatment. Conclusions: Plasma exposure of T-DXd in NCI-N87 tumor bearing mice was prolonged compared to Capan-1, possibly due to mouse strain differences. The increased systemic exposure resulted in more rapid and sustained DNA damage as measured by gH2AX and pRAD50 in the NCI-N87 tumor compared to Capan-1. This profile suggests exploration of combinations with DNA damage response inhibitors to inform design of dose and schedule of combination therapy may be warranted. Citation Format: Theresa Proia, Jelena Urosevic, Christina Vasalou, Rebecca Sargeant, Matthew Griffin, Jiaqi Yuan, Anton I. Rosenbaum, Jerome Mettetal. Pharmacokinetic and pharmacodynamic evaluation of human tumor xenograft models treated upon administration of trastuzumab deruxtecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1141.

Abstract 5290: BSI-001, a novel anti-HER2 antibody exhibiting potent synergistic efficacy with trastuzumab

Abstract Background: Trastuzumab and pertuzumab are anti-HER2 antibodies that bind to two different epitopes of HER2. Although pertuzumab itself did not exhibit substantial clinical benefit as monotherapy, it was approved to be used in combination with trastuzumab because of its ability to enhance the clinical efficacy of trastuzumab. Currently, the combination of trastuzumab and pertuzumab is being used as first-line treatment for HER2-positive metastatic breast cancer. However, while the combination of trastuzumab and pertuzumab has been effective, there is still opportunity for greater effectiveness by identifying anti-HER2 mAbs that will be better synergistic partners of trastuzumab, allowing for better efficacy and/or broader indications. Methods: A SynAb࣪ Platform was used to identify BSI-001, an anti-HER2 mAb, through trastuzumab-based synergistic functional screening. A competitive ELISA was used to perform epitope binning of the anti-HER2 antibodies. BSI-001 was further characterized in combination with trastuzumab for its bioactivity in vitro, including inhibition of cell proliferation and antigen-mediated internalization of HER2 positive cell lines. The synergistic effect of BSI-001 with trastuzumab was also evaluated in trastuzumab-resistant cell lines. Multiple animal models were then employed to investigate the synergistic antitumor activity of the combination in vivo. Results: BSI-001 is the best functional partner of trastuzumab identified through the SynAb࣪ platform. BSI-001 binds to a unique epitope, distinct from the binding epitopes of trastuzumab and pertuzumab. It exhibits superior bioactivity and efficacy in vitro and in vivo when combined with trastuzumab. The combination of BSI-001 and trastuzumab inhibits HER2-positive cancer cell growth more potently than that of trastuzumab and pertuzumab. The combination of BSI-001 with trastuzumab also shows inhibition of cell growth of the trastuzumab-resistant cell line BT-474 Clone 5, whereas the combination of pertuzumab and trastuzumab did not. In addition, BSI-001 is able to synergize with trastuzumab to promote antigen-antibody internalization. In animal models, BSI-001 demonstrates superior synergistic antitumor activity when combined with trastuzumab as compared to pertuzumab. Summary: The combination of BSI-001 and trastuzumab showed significantly better in vitro and in vivo activity than the combination of trastuzumab and pertuzumab. In addition, because of the activity of the BSI-001/trastuzumab combination in a trastuzumab-resistant cancer cell line, there is the potential for this combination to be used to treat patients with trastuzumab-resistant cancer or patients who experienced relapse following trastuzumab and pertuzumab treatment. Citation Format: Shukai Xia, Zeyu Peng, Xiaoyu Ding, Wanjian Gu, Yujie Zhong, Jinyu Liu, Hongyan Li, Xiaoyao Hao, Xiaodong F. Liu, Mark Z. Ma, Lan Luo, Chunni Zhang, Mingjiu Chen. BSI-001, a novel anti-HER2 antibody exhibiting potent synergistic efficacy with trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5290.

Abstract 3333: Pre-clinical investigation of neratinib plus dasatinib in breast cancer

Abstract Background: HER2-targeted therapies have revolutionized the prognosis of HER2-positive (HER2+) breast cancer (BC). However, the emergence of acquired resistance or de novo resistance are persistent clinical problems. The objective of this study was to examine neratinib (NER), an irreversible pan-HER inhibitor approved for early-stage and metastatic HER2+ BC, and dasatinib (DAS), a multi-kinase inhibitor used to treat certain leukemias, as a new treatment for HER2-targeted therapy resistant HER2+ BC and triple negative BC (TNBC). Methods: The anti-proliferative effect of NER plus DAS was screened in a panel of 20 breast cancer cell lines using a 5-day acid phosphatase assay. This panel included 12 HER2+ (3 trastuzumab (TRAS) sensitive (SKBR3, BT474, EFM192A), 2 acquired TRAS resistant (BT474-T, SKBR3-T), 3 innately TRAS resistant (HCC1954, HCC1569, JIMT1), and 4 acquired neratinib resistant cell lines (HCC1954-N, HCC1569-N, SKBR3-N, BT474-N), 6 triple negative (BT20, HCC1143, HCC1937, MDA-MB- 157/231/468), and 2 estrogen receptor positive (ER+) cell lines (MCF7, ZR-75-1). Drug synergy was examined using Combenefit and Calcusyn software. Combination Index (CI) values &amp;lt; 1 indicated synergy, &amp;gt; 1 antagonism. Neratinib IC50 values &amp;lt; 150nM were considered physiologically relevant. Prolonged exposure in vitro efficacy was tested in these cell lines by treating twice weekly with NER +/- DAS until resistance emerged. Cell confluence was then quantified using a crystal violet-based method. The safety and efficacy of NER (10 mg/kg) plus DAS (15 mg/kg) was investigated in vivo in HER2+ HCC1954 xenografts in BALB/c nude mice. Results: NER was highly effective against HER2+ BC cell lines regardless of TRAS sensitivity (IC50 values range 1-172 nM), showed moderate activity in TNBC (IC50 value range 31 nM - &amp;gt;1 μM), and did not achieve &amp;lt;1 μM activity in ER+ BC cell lines. NER resistant cell lines had IC50 values greater than 150 nM DAS alone displayed IC50 values from 8-135 nM in 4/6 TNBC cell lines, with all 6 TNBC cell lines sensitive to either the single agents or the combination at concentrations less than 100 nM. DAS enhanced the anti-proliferative activity of NER in the HCC1954, HCC1569, JIMT1 cell lines (CI values = 0.27-0.62). The NER/DAS combination overcame NER resistance in all acquired NER-resistant cell line models and no antagonism was observed in any cell line tested. Matrix assays confirmed synergy at low nanomolar concentrations of both drugs. NER plus DAS had a greater long term in vitro anti-proliferative effect over NER in HCC1954, HCC1569, and JIMT1 cells, preventing or delaying the development of NER resistance. NER plus DAS was well tolerated in mice for over 60 days. The combination was better than single agents and showed prolonged anti-tumor effect against HCC1954 xenografts in vivo (p=0.02). Conclusions: This study suggests that the addition of DAS to NER may have potential as a new treatment strategy for BC, particularly for HER2+ BC. Citation Format: Neil T. Conlon, Sandra Roche, Fiona O'Neill, Justine Meiller, John Crown, Denis M. Collins. Pre-clinical investigation of neratinib plus dasatinib in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3333.

T-DM1 efficacy in trastuzumab-pertuzumab pre-treated HER2 positive metastatic breast cancer patients: a meta-analysis

BackgroundCurrent guidelines consider T-DM1 the standard 2nd line therapy for HER2 positive metastatic breast cancer (MBC) patients following trastuzumab (T) + pertuzumab (P) and taxane 1st line treatment. Despite this, there are no prospective studies supporting this sequence.MethodsWe performed a meta-analysis using real world data to determine the efficacy of T-DM1 after 1st line TP in HER2 positive MBC patients. We used a random-effect model to find differences in the rate of 1-year progression free survival (PFS) between TP pre-treated population and the EMILIA phase III pivotal trial.ResultsSeven studies were eligible. The meta-analysis showed a combined 1-year PFS risk difference for T-DM1 efficacy after TP in 2nd or more lines of -0.122, with lower and upper limits of -0.253 and 0.010, respectively (p = 0.07), with low heterogeneity among studies (I2 0.01%, p = 0.836). Considering the four studies on T-DM1 in 2nd line setting, 1-year PFS risk was -0.034 (95% CI -0.207 – 0,139; p = 0.701) (I2 0.01%, p = 0.91).ConclusionOverall, the efficacy of T-DM1 after TP seems to be similar to that previously reported in the EMILIA trial. In the second line setting, data are not mature enough to confirm T-DM1 efficacy in TP pre-treated population.