Primary HER2 Research Articles

1297P A phase II study of pyrotinib combined with apatinib in first-line treatment of advanced non-small cell lung cancer patients with primary HER-2 mutations/amplification

1297P A phase II study of pyrotinib combined with apatinib in first-line treatment of advanced non-small cell lung cancer patients with primary HER-2 mutations/amplification

Prognostic and Predictive Significance of Primary Tumor Localization and HER2 Expression in the Treatment of Patients with KRAS Wild-Type Metastatic Colorectal Cancer: Single-Centre Experience from Serbia.

The treatment of patients with metastatic colorectal cancer (mCRC) is complex and is impacted by the location of the primary tumor (LPT). Our study aims to emphasize the importance of LPT as a prognostic and predictive marker as well as to examine the significance of HER2 overexpression in patients with mCRC, particularly in relation to the response to Epidermal Growth Factor Receptor Antibody treatment (anti-EGFR therapy). In this study, 181 patients with Kirsten RAS (KRAS) wild-type mCRC who received anti-EGFR therapy were included. Among them, 101 had left colon cancer (LCC) and 80 had right colon cancer (RCC). Results demonstrated that patients with KRAS wild-type LCC had better median overall survival (OS) (43 vs. 33 months, p = 0.005) and progression-free survival (PFS) (6 vs. 3 months, p < 0.001) compared to those with RCC. Multivariate analysis identified mucinous adenocarcinoma (p < 0.001), RCC location (p = 0.022), perineural invasion (p = 0.034), and tumors at the resection margin (p = 0.001) as independent predictors of OS, while mucinous adenocarcinoma (p = 0.001) and RCC location (p = 0.004) independently correlated with significantly shorter PFS. In addition, human epidermal growth factor receptor 2 (HER2) positive expression was significantly associated with worse PFS compared to HER2 negative results (p < 0.001). In conclusion, LPT is an important marker for predicting outcomes in the treatment of wild-type mCRC using anti-EGFR therapy, since patients with RCC have a statistically significantly shorter PFS and OS. Further investigation is needed to understand the role of HER2 overexpression in wild-type mCRC, as these patients also exhibit shorter survival.

A population-based study on trajectories of HER2 status during neoadjuvant chemotherapy for early breast cancer and metastatic progression

BackgroundThis study aimed to investigate the distribution and changes of HER2 status in untreated tumours, in residual disease and in metastasis, and their long-term prognostic implications.MethodsThis is a population-based cohort study of patients treated with neoadjuvant chemotherapy for breast cancer during 2007–2020 in the Stockholm–Gotland region which comprises 25% of the entire Swedish population. Information was extracted from the National Breast Cancer Registry and electronic patient charts to minimize data missingness and misclassification.ResultsIn total, 2494 patients received neoadjuvant chemotherapy, of which 2309 had available pretreatment HER2 status. Discordance rates were 29.9% between primary and residual disease (kappa = 0.534), 31.2% between primary tumour and metastasis (kappa = 0.512) and 33.3% between residual disease to metastasis (kappa = 0.483). Adjusted survival curves differed between primary HER2 0 and HER2-low disease (p < 0.001), with the former exhibiting an early peak in risk for death which eventually declined below the risk of HER2-low. Across all disease settings, increasing the number of biopsies increased the likelihood of detecting HER2-low status.ConclusionHER2 status changes during neoadjuvant chemotherapy and metastatic progression, and the long-term behaviours of HER2 0 and HER2-low disease differ, underscoring the need for obtaining tissue biopsies and for extended follow-up in breast cancer studies.

Abstract PO5-24-07: HER2 EXPRESSION HETEROGENEITY PATTERN IN INVASIVE BREAST CARCINOMAS: FREQUENCY, DISTRIBUTION AND RELATION TO MORPHOLOGICAL VARIABLES

Abstract Human epidermal growth factor receptor 2 (HER2), a critical contributor to breast cancer development, is found to be amplified and overexpressed in approximately 15-20% of cases, making it a significant factor in carcinogenesis. This scenario was targeted by anti-HER2 drugs approved by several regulatory agencies in the past 15 years. Recently, novel anti-HER2 antibody-drug conjugates (ADCs) were approved to metastatic breast cancer with some HER2 expression albeit not due to amplification of the gene. Despite being classified as HER2 negative, most of these tumors exhibit detectable amounts of HER2 protein, reinforcing the challenging task of HER2 expression evaluation. In this scenario, it is very important to evaluate the spectrum of HER2 expression within a tumor due to its heterogeneity. We designed a retrospective linear study to reanalyze all HER2 immunohistochemical core biopsy slides of patients with primary invasive breast carcinomas (IBC) diagnosed and treated at São Paulo Federal University Hospital between 2019 and 2023. Pathological variables (histological subtype, histological grading, and hormonal receptors expression) were collected in pathology reports. Only patients with available slides and blocks were enrolled. HER2 slides were evaluated by three observers in consensus to access ASCO-CAP 2018 guidelines HER2 expression; presence or absence of HER2 expression heterogeneity; pattern of heterogeneity; frequency of the primary and secondary HER2 score in heterogeneous cases; and frequency of HER2 score in cases with homogeneous expression. Statistical analyses were performed with Chi-Square and Mann–Whitney U test using SSPS (26.0) program. 353 cases were included in this study. 104 (29,5%) were under 50 years and 249 (70,5%) over 50 years. 54,1% cases were left sided and 45,9% right sided. IBC of no special type comprehended 324 cases (91,8%) and invasive lobular carcinomas (ILC) were represented by 26 cases (7,4%); less than 1% of the cases were rare subtypes. Overall, HER2 results was negative in 296 (83,9%) cases and positive in 42 (11,9%) cases; HER2 2+ accounted by 15 cases (4,2%). Out of the negative cases, 235 (79.4%) were classified as 0+ and 61 (20.6%) were classified as 1+. Homogenous HER2 expression was observed in 287 cases (81,3%) with predominance of 0+ expression (80,8% - 232 cases), followed by 1+ (9,4% - 27 cases). ITH was present in 66 cases (18,7%), in which the subtyping was represented by 39 scattered (59,1%), 25 clustered (37,9%) and 2 mosaic (3%). The most prevalent score in this group was 1+ (51,5% - 34 cases), followed by 3+ (25,8% - 17 cases); the secondary score was predominantly categorized as 0+ (58,3%), followed by 1+ (20%) and 2+ (20%); only one case showed as 3+ expression. In the ILC group, 24 cases (92,3%) exhibited homogeneous HER2 expression, with predominant 0+ score (75% - 18 cases), while 2 cases (7,7%) showed heterogeneous expression. Out of the cases with heterogeneous HER2 expression, one exhibited a clustered heterogeneity pattern, with a primary HER2 score of 1+ and a secondary score of 0+. The other case displayed a scattered heterogeneity pattern, with a primary HER2 score of 3+ and a secondary score of 2+. There was a statistically significant association between HER2 expression heterogeneity and histologic grade (p = 0.005), and a marginal association with estrogen (p = 0.060) and progesterone (p = 0.060) expression. Tumor size wasn’t associated with heterogeneity (p = 0,071). We conclude that heterogeneity is prevalent in HER2 expression, especially in IBC, and should be addressed in pathology reports, especially in the ADCs scenario. Citation Format: Angela Flavia Waitzberg, Lucas de Figueiredo Barbosa, Adilson Monteiro dos Santos Filho, Ana Luiza da Cruz, Lisandra Gonzalez Porta Nova, Karla Calaça Kabbach Prigenzi. HER2 EXPRESSION HETEROGENEITY PATTERN IN INVASIVE BREAST CARCINOMAS: FREQUENCY, DISTRIBUTION AND RELATION TO MORPHOLOGICAL VARIABLES [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-07.

Abstract PS11-07: High prevalence of HER2-Low and AR expression in breast cancer brain metastases provide novel therapeutic options

Abstract Background: Although survival rates for patients with advanced breast cancer (BC) have improved overall, rates of BC brain metastases (BCBM) have increased over the past two decades, occurring in up to 15% to 30% of patients. BCBM greatly reduce quality of life and overall survival (OS) and are now the leading cause of BC patient mortality. Patients with primary HER2+ and triple negative BC (TNBC) are at highest risk for BM. Locoregional therapy with surgery and radiation are currently standard of care (SOC), but these treatments have significant morbidity and BCBM progressing despite these interventions are hard to treat. Systemic therapies to reduce cognitive impairment and increase survival are desperately needed, particularly in HER2-negative patients. Currently, immune checkpoint inhibitors (ICIs) and small molecules such as PARP inhibitors are among the limited systemic options that have BCBM activity in HER2-negative BC. However, the benefit of these therapies is limited to a select group of patients. For example, androgen receptor (AR) is expressed in 60-80% of all BC and, in our recently completed clinical trial (NCT03206203), patients with AR+ metastatic TNBC treated with carboplatin +/- atezolizumab received no clinical benefit from the addition of atezolizumab. Alternatives to ICIs are needed for patients with AR+ TNBC. HER2-low (H2L) BC is a newly defined subset of HER2-negative BC with a HER2 immunohistochemical (IHC) score of 1+ ( &amp;gt;10%) or 2+/in situ hybridization (ISH) negative phenotype. Recent clinical trials have demonstrated significant clinical benefit from novel HER2 antibody-drug conjugates (ADCs) in treating H2L BC. Trastuzumab-deruxtecan (T-Dxd), a HER2-directing ADC (topoisomerase I inhibitor conjugated to a humanized HER2-targeted antibody) was recently FDA approved as the first targeted therapy to treat H2L BC and can penetrate the blood–brain barrier. DESTINY-Breast04 demonstrated notable PFS and OS benefit of T-DXd compared to SOC in patients with H2L metastatic BC and included 61 patients with BCBM (results anticipated shortly). The overall rates of AR and H2L expression in patients with BCBM is unknown. The goal of this study is to investigate the prevalence of AR and HER2-low expression in a large cohort of BCBM treated at our institution. Methods: We retrospectively assessed rates of H2L and AR positivity (AR+) among patients with BCBM whose treatment included surgery at our institution between 2005-2023. Results were correlated with clinical receptor status (HR+/HER2-, HER2+/HR+, HER2+/HR- and TNBC) and clinical characteristics. Results of ER, PR, and HER2 testing performed for clinical purposes on the brain metastases and primary tumors were obtained from pathology reports. We retrieved formalin-fixed paraffin-embedded tissue blocks of the brain metastases and performed IHC for AR (SP107) and prognostic markers for cases with missing data. Results: We identified 101 cases of BCBM. Based on clinical prognostic markers, BCBM subtypes were HR+/HER2- (22%), HR+/HER2+ (14%), HER2+/HR- (21%) and TNBC (43%). Forty-five percent of the BCBM (n=45) were AR+ ( &amp;gt;10%) and 24% (n=24) demonstrated &amp;gt; 50% AR+. Among tumors with &amp;gt;50% AR+, 42% (n=10) are H2L (7 HR+[30%] and 3 TNBC [13%]). Forty-two percent of BCBM were H2L. Among the HER2-negative BCBM (n= 66), 64% (n=42) were H2L, including 23 of 44 TNBC (52%) and 19 of 22 HR+ (86)%. Fifteen percent of BMBC changed clinical subtype from the primary, including conversion to TNBC and H2L. Conclusion: AR+ positive (24% &amp;gt; 50% expression) and HER2-Low expressing (42%) tumors represent a significant proportion of our BCBM cohort providing exciting new options for treatment. Trials re-evaluating AR antagonists in combination with SOC utilizing high AR+ thresholds may benefit patients with BCBM. At least 10% of BCBM are both H2L and demonstrate &amp;gt;50% AR positivity providing the rationale for the combination of ADCs such as T-DXd and AR-targeted therapies for BCBM. Citation Format: Guadalupe Garcia, Paula Gonzalez-Ericsson, Brian Lehmann, Bret Mobley, Jennifer Pietenpol, Laura Kennedy, Ben Park, Melinda Sanders. High prevalence of HER2-Low and AR expression in breast cancer brain metastases provide novel therapeutic options [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-07.

Abstract PS11-06: Analysis of HER2 expression changes from breast primary to brain metastases including HER2 Low and impact on overall survival

Abstract Background: Previous studies have shown that breast cancer receptor subtype switching between matched primary and metastatic brain metastases (BrM) is common. HER2 expression at low levels, termed “HER2 Low”, has emerged as a new therapeutic biomarker for highly active antibody drug conjugates with potential intracranial activity. Trastuzumab deruxtecan is FDA approved for the treatment of HER2 Low and HER2 positive breast cancer with emerging evidence of CNS responses and clinical benefit in patients with BrM. This study aimed to investigate the relationship between HER2 expression including HER2-Low between matched breast cancer primary tumors and brain metastases. We also sought to evaluate the impact of low HER2 expression on overall survival in breast cancer brain metastases. Methods: We identified pts with MBC seen at least once at a single, NCI-designated center and had a diagnosis of BrM between 2003 and 2023. Of 1252 MBC pts with a diagnosis of BrM, 265 pts underwent resection/biopsy for BrM with available ER/PR/HER2 status. Only pts with available matched primary and BrM tissue were analyzed. HER2 expression was defined as: HER2+ (3+ or 2+/ISH amplified), HER2-Low (1+, 2+/ISH neg) or HER2-0 [by ASCO-CAP guidelines]. Estrogen receptor (ER) status was defined as ER≥1%. Multivariate overall survival (OS) analyses by Cox proportional hazard models were determined from time of BrM resection to death or last follow-up between HER2+, HER2-Low and HER2-0 patients controlling for ER and age. Results: 197 matched breast cancer primary and BrMs with available ER, PR, and HER2 testing were evaluated. Median age at BrM resection was 51 yrs. ER was positive in 48% (N=95) of BrMs. Of 265 resected BrMs, HER2 expression was found in 72%: 49.8% HER2+ (N=132), 22.2% HER2-Low (N=59), and 27.9% HER2-0 (N=74). HER2 expression by 197 primary tumors was: 57% HER2+ (N=112), 24% HER2-Low (N=48), 19% HER2 0 (N=37). Amongst 112 HER2+ primary tumors, 97% (N=109) had concordant HER2+ BrMs, 3 (2.6%) switched to HER2-Low, with 100% retaining some HER2 expression. Of 37 HER2-0 primaries, 35% (13/37) gained HER2-Low expression and 5.4% (2/37) gained HER2+ expression in the BrM. Amongst 48 HER2-Low primary tumors, half (52%) changed expression to either HER2+ (21%) or HER2-0 (31.2%) in the brain. 66 tumors had extracranial tissue biopsied prior to BrM resection available for HER2 analysis. Of 15 HER2-0 extracranial tumors, 47% gained HER2-Low and 13% gained HER2+ at time of BrMs. After adjusting for age and ER status at the time of BrMs, patients with HER2+ BrM had a statistically significant lower risk of death at time of follow up than those with HER2-Low BrM (HR=0.41, P=0.0006). The risk of death between patients with HER2-0 BrM did not differ from HER2-Low BrM after adjustment for ER and age (HR=0.96, p =0.9). Median survival from BrM resection was 12.4m, 16.1m, 47.6m for HER2-Low, HER2-0 and HER2+ respectively (p = .0004). Conclusions: HER2 expression amongst primary, extracranial and brain metastases is dynamic with frequent change. Over 40% of HER2-0 primary or extracranial tumors gain HER2 expression in the brain. These changes have therapeutic implications given HER2 targeting antibody drug conjugates with improved OS and emerging evidence of CNS activity, highlighting the need for better diagnostics, including those that do not require invasive tissue sampling, to determine HER2 status in the brain. Patients with HER2-Low and HER2-0 BrMs have inferior survival and are a clinical unmet need. Citation Format: Alyssa Pereslete, Melissa Hughes, Alyssa Patterson, Janet Files, Kyleen Nguyen, Lauren Buckley, Ashka Patel, Abigail Moore, Eric Winer, Tianyu Li, Sara Tolaney, Nancy Lin, Sarah Sammons. Analysis of HER2 expression changes from breast primary to brain metastases including HER2 Low and impact on overall survival [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-06.

Abstract IA21: Mechanisms of Resistance to HER2-Targeted Therapies in HER2-positive Breast Cancer

Abstract Recent advancements in HER2-targeted therapies for HER2+ BC have revolutionized patient outcome. Yet, intrinsic and acquired resistance, especially in the advanced setting remains a clinical challenge. In the era of precision oncology, better understanding of these mechanisms of resistance is key to identifying new strategies to overcome resistance and for developing personalized treatment approaches. Our recent studies suggest that a subset of HER2+ BCs, associated with high and homogeneous levels of HER2 gene amplification, protein, and activity, are addicted to HER2 and may therefore benefit from chemotherapy-sparing HER2-targeted regimens. However, even in such tumors, constitutive activation of the PI3K/AKT pathway due to deregulations in the downstream PI3K pathway can lead to resistance. In all HER2+ BCs, failure to achieve a comprehensive blockade of the HER receptor layer is one of the major mechanisms of resistance due to the functional redundancy of the HER receptors and compensatory signaling within the pathway. But, in some cases, effective inhibition of HER receptors might be challenged by molecular masking of the receptors (e.g., MUC4) or by epi/genetic or post-translational alterations in the receptors itself (e.g., HER2 L755S, p95HER2). Indeed, we recently reported that acquisition of HER2 L755S, either alone or together with PIK3CA mutation, and hyperactive EGFR signaling due to acquired EGFR amplification or excess HER ligands are associated with resistance to HER2-targeted therapy, especially tyrosine kinase inhibitors. Likewise, clinically, HER mutations have been reported to be further enriched in the metastatic vs. primary HER2+ tumors. When HER2 does remain sustainably inhibited by HER2-targeted agents, resistance may arise due to the emergence of alternative signaling pathways transmitting compensatory proliferative and survival signals, such as ER, receptor tyrosine kinases, or downstream/intracellular signaling. Further, the emergence of metabolic pathways as alternative pathways of resistance have also been documented. We recently reported the activation of the cholesterol biosynthetic mevalonate (MVA) pathway as an escape mechanism that provides alternative signals through the YAP/TAZ-mTORC1-survivin axis to evade HER2 blockade. This resistance could be overcome using MVA pathway inhibitors (e.g., statins). Additionally, activation of the cell cycle regulatory complex cyclin D1/CDK4 has been shown to be associated with HER2-targeted therapy resistance, which could be exploited as a potential therapeutic vulnerability by using CDK4/6 inhibitors. Finally, response to HER2-targeted therapy could potentially be also modulated by the tumor microenvironment itself, including components of the host immune system and extracellular matrix. Together, a better understanding of the underlying mechanisms of resistance in the context of tumor and host biology, and treatment setup is fundamental to develop new treatment strategies and to guide patient stratification for personalized treatments and improved patient outcomes. Citation Format: Jamunarani Veeraraghavan, Fu-Tien Liao, Lanfang Qin, Tia Gordon, Alekya Raghavan, Caroline Sabotta, Rachel Kaplan, Carolina Gutierrez, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. Mechanisms of Resistance to HER2-Targeted Therapies in HER2-positive Breast Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA21.

Identification and characterization of a potent and selective HUNK inhibitor for treatment of HER2+ breast cancer

Human epidermal growth factor receptor 2 (HER2)-targeted agents have proven to be effective, however, the development of resistance to these agents has become an obstacle in treating HER2+ breast cancer. Evidence implicates HUNK as an anti-cancer target for primary and resistant HER2+ breast cancers. In this study, a selective inhibitor of HUNK is characterized alongside a phosphorylation event in a downstream substrate of HUNK as a marker for HUNK activity in HER2+ breast cancer. Rubicon has been established as a substrate of HUNK that is phosphorylated at serine (S) 92. Findings indicate that HUNK-mediated phosphorylation of Rubicon at S92 promotes both autophagy and tumorigenesis in HER2/neu+ breast cancer. HUNK inhibition prevents Rubicon S92 phosphorylation in HER2/neu+ breast cancer models and inhibits tumorigenesis. This study characterizes a downstream phosphorylation event as a measure of HUNK activity and identifies a selective HUNK inhibitor that has meaningful efficacy toward HER2+ breast cancer.

Evolution and prognostic significance of HER-2 conversion from primary to residual disease in HER-2 negative patients with breast cancer after neoadjuvant chemotherapy.

This study aimed to analyze HER-2 zero or HER-2 low conversion in HER-2 negative patients after neoadjuvant chemotherapy (NAC) and evaluate its prognostic significance. HER-2 negative patients with breast cancer with residual disease after NAC and paired pre- and post-therapeutic HER-2 testing results were analyzed retrospectively. HER-2 low, defined as immunohistochemistry (IHC) scores of 1+ or 2+/in situ hybridization (ISH), were not amplified. HER-2 zero is defined as an IHC score of 0. A total of 571 patients were enrolled, including primary HER-2 zero (n=201, 35.2%) and HER-2 low (n=370, 64.8%). The overall HER-2 change rate was 32.4%. Multivariable logistic regression showed that patients with hormone receptor-positive status before NAC was significantly associated with the conversion of HER-2 zero to low (OR=3.436, P < 0.0001). The median follow-up time was 50.0 months. In patients who are primary HER-2 zero, HER-2 zero to low was significantly associated with better disease-free survival (DFS) than constant HER-2 zero (HR=0.49, P=0.01) after adjustment (4-year DFS 80.1% vs 55.7%, Log-rank P=0.033). Subgroup analysis revealed that among patients who are primary HER-2 zero with hormone receptor-positive, HER-2 zero to low had a significantly better DFS than constant HER-2 zero (Log-rank P=0.037). In contrast, patients with hormone receptor-negative status did not. In conclusion, almost one-third of patients who are HER-2 negative underwent HER-2 zero or HER-2 low conversion after NAC. HER-2 zero to low conversion was associated with better DFS in patients who are HER-2 zero. These results provide a valuable reference for the potential application of anti-HER-2 ADC in an adjuvant setting for patients with residual disease after NAC.

Integrative whole-genome and transcriptome analysis of HER2-amplified metastatic breast cancer

BackgroundIn breast cancer, the advent of anti-HER2 therapies has made HER2+ tumors a highly relevant subgroup. However, the exact characteristics which prohibit clinical response to anti-HER2 therapies and drive disease progression are not yet fully known. Integrative whole-genome and transcriptomic sequencing data from both primary and metastatic HER2-positive breast cancer will enhance our understanding of underlying biological processes.MethodsHere, we used WGS and RNA sequencing data of 700 metastatic breast tumors, of which 68 being HER2+, to search for specific genomic features of HER2+ disease and therapy resistance. Furthermore, we integrated results with transcriptomic data to associate tumors exhibiting a HER2+-specific gene expression profile with ERBB2 mutation status, prior therapy and relevant gene expression signatures.ResultsOverall genomic profiles of primary and metastatic HER2+ breast cancers were similar, and no specific acquired genomics traits connected to prior anti-HER2 treatment were observed. However, specific genomic features were predictive of progression-free survival on post-biopsy anti-HER2 treatment. Furthermore, a HER2-driven expression profile grouped HER2-amplified tumors with ERBB2-mutated cases and cases without HER2 alterations. The latter were reported as ER positive in primary disease, but the metastatic biopsy showed low ESR1 expression and upregulation of the MAPK pathway, suggesting transformation to ER independence.ConclusionsIn summary, although the quantity of variants increased throughout HER2-positive breast cancer progression, the genomic composition remained largely consistent, thus yielding no new major processes beside those already operational in primary disease. Our results suggest that integrated genomic and transcriptomic analyses may be key in establishing therapeutic options.

Liquid biopsy based HER2 amplification status in gastric cancer patients indicates clinical response

Gastric carcinomas are among the most common cancers in Germany, with approximately 18,000 new cases per year.About 10 years ago, based on results of the Trastuzumab for gastric cancer (ToGA) trial, the addition of the monoclonal antibody trastuzumab to a platinum-fluoropyrimidine chemotherapy backbone became the standard-of-care 1st-line therapy for human epidermal growth factor receptor 2 (HER2)-positive gastric cancers. Only patients with primary HER2 gene amplification benefit from this therapy. Thus, accurate HER2 gene amplification detection is predictive and critical for therapy selection. As a gold standard the HER2 status is currently determined in tumor tissue specimens using immune histochemistry and fluorescent in situ hybridisation. However, HER2 amplification is detectable in only about 20 % of gastric carcinomas. The recent approval of an antibody-drug conjugate Trastuzumab deruxtecan (T-DXd) and the establishment of a new subgroup of HER2-low tumors due to the bystander effect associated with T-DXd increases the relevance of precise HER2 diagnostics.Aim of this analysis was to determine the HER2 amplification status from circulating DNA fragments in blood using a HER2 Copy Number Variation assay to establish a minimal invasive approach.For the present study, a digital droplet PCR-based method was validated relative to established tissue-based methods. Furthermore and most importantly, the changes of HER2 status during therapy were investigated in seven patients indicating that the changes of HER2 status and number of HER2 copies detected in blood can reflect on therapy efficiency and uncover treatment resistance.

Changes of HER2 low expression status in primary and recurrent/metastatic breast cancer

Objective: To investigate the evolution and clinical significance of HER2 low expression status in HER2 negative patients in primary and recurrent/metastatic breast cancers. Methods: The data and archived sections of 259 breast cancer patients with recurrence/metastasis and HER2-negative primary foci were collected from January 2015 to January 2022 at the Fourth Hospital of Hebei Medical University, and the HER2 status of primary and recurrence/metastasis foci was determined by immunohistochemistry (IHC), among which IHC 2+patients were subject to fluorescence in situ hybridization (FISH). The HER2 status was classified as HER2-0 group; patients with IHC 1+, IHC 2+and no FISH amplification were classified as HER2 low expression group; and patients with IHC 3+, IHC 2+and FISH amplified were classified as HER2-positive group. The changes of HER2 status in patients with HER2 low expression in primary versus recurrent/metastatic breast cancer foci were compared, and their clinicopathologic characteristics and prognosis were analyzed. Results: The overall concordance rate between primary and recurrent/metastatic HER2 status in breast cancer was 60.6% (157/259, κ=0.178). A total of 102 patients (102/259, 39.4%) had inconsistent primary and recurrent/metastatic HER2 status; 37 patients (37/259, 14.3%) had HER2-0 at the primary foci and HER2-low expression at the recurrent/metastatic; and 56 patients (56/259, 21.6%) had HER2-low expression in the primary foci and HER2-0 in the recurrent/metastatic. The recurrent/metastatic foci became low-expressing compared with the recurrent/metastatic foci which remained HER2-0 patients, with longer overall survival time, higher ER and PR positivity, lower Ki-67 positivity index, and lower tumor histological grade; all with statistically significant differences (all P<0.05). In the primary HER2-low group, patients with recurrent/metastatic foci became HER2-0 while those with recurrent/metastatic foci remained low expression; there were no statistically significant differences in clinicopathological features and overall survival time (all P>0.05). Conclusions: Unstable HER2 status in patients with HER2-0 and low expression in primary versus recurrent/metastatic breast cancer foci, and HER2-0 in the primary foci but low HER2 expression status in recurrence/metastasis is associated with favourable prognosis, and testing HER2 status in recurrence/metastasis can provide more treatment options for such patients.

Abstract 3417: Nanocage delivered engineered destabilized HER2 3UTR ARE reduced growth of primary EGFR T790M HER2 overexpressing osimertinib and trastuzumab resistant non small cell lung cancer and inhibited liver and lung metastasis in vivo

Abstract Non-small cell lung cancer (NSCLC) caused more deaths in 2017 than breast cancer, prostate cancer and brain cancers combined. This is primarily due to its metastatic abilities to organs such brain, liver, bones, and adrenal glands leading to death of the patient. And there are no clinically approved drugs that can target metastasis. NSCLC with EGFR T790M overexpressing HER2 shows resistance to osimertinib and trastuzumab starting 10-18 months after therapy. For these patients, prospects are grim. To address the challenge of HER2+ drug resistant NSCLC, we have developed an innovative approach based on genome engineering of the 3’ untranslated region (3’UTR) of the HER2 gene to destabilize and degrade HER2 transcript, protein expression, HER2 dependent kinases and interactome. We report the development of two very effective HER2 destabilizing constructs which when transfected into cells lead to the specific loss of HER2 transcript, protein, downstream kinases, and loss of cell viability. In vivo, we administered the constructs complexed with a nanocage to mice bearing NSCLC. We achieved very significant control of primary tumors p=0.0001 and inhibited liver and lung metastasis and prevented hepatomegaly in mice bearing tumors that received our engineered constructs. Mechanistically, the destabilized mRNA creates premature termination codon which triggers the upregulation of nonsense mediated decay proteins UPF3B and then there is subsequent uncapping of the mRNA and the exonucleases XRN1 and CNOT1 degrades the transcript specifically and the proteins are not made available. The exogenous destabilized constructs are continuously transcriptionally active dominant negative which outcompetes the native endogenous HER2 and co-opts its mRNA for degradation. The loss of HER2 leads to loss of WNK1 and YES1 leading to activation of caspase 3,9 mediated cell death. The migration and invasion of the tumor is impaired. The constructs safely integrated into non-coding regions of the genome and caused no genome rearrangements, and we found no blood, renal, liver or electrolytes toxicities in the animals that received them. Taken together, we have developed a novel therapeutic agent that inhibits the growth of primary NSCLC and metastasis to the liver and lung. Citation Format: Chidiebere U. Awah, Joo Sun Mun, Baris Boylu, Alooka Paragodaarachchi, Chika Ochu, Junfei Zhang, Hiroshi Matsui, Olorunseun O. Ogunwobi. Nanocage delivered engineered destabilized HER2 3UTR ARE reduced growth of primary EGFR T790M HER2 overexpressing osimertinib and trastuzumab resistant non small cell lung cancer and inhibited liver and lung metastasis in vivo [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 3417.

Abstract 6799: A porous silicon particle based particulate anti-cancer vaccine for immunotherapy

Abstract Background: Anti-cancer vaccine is a widely-studied strategy that activates innate and adaptive host immunity via its adjuvant components and tumor antigens against cancer. Unfortunately, its response rate in clinical trials was not promising. On the vaccine part, sufficient stimulation of strong and long-lasting local and systemic immunity and efficient promotion of intratumoral infiltration of cytotoxic T lymphocytes are among the critical issues determining the therapeutic efficacy of cancer vaccines in addition to the innate features of tumor. Our project aims to develop a porous silicon particle based particulate vaccine against cancer, and investigate the therapeutic mechanism and combinational effects with current first-line strategies. Method: We first screened the combinations of ligands of Toll-like receptors (TLRs) expressing on antigen presenting cells (APCs) and selected the most powerful combo for immune stimulation. We then loaded the identified adjuvants in addition to antigen peptides to produce vaccine particles. Stimulation and licensing of APCs were tested in vitro, and its therapeutic efficacies were evaluated on multiple orthotopic and systemic metastasis cancer murine models of breast and colon cancers. Furthermore, we investigated the vaccine’s synergistic effects and impacts on tumor microenviroment (TME) with an immunogenic chemotherapeutic oxaliplatin on a CT26 colon cancer model. Result: According to the adjuvant screening assays, we identified a capable combination of CpG1826 (CpG) and 2’3’-cGAMP (cGAMP), which extensively stimulated the activation, maturation and type-I interferon secretion of APCs. A µGCVax formulation composed by these two adjuvants and antigen peptides was then applied on primary and metastatic HER2+ breast cancer TUBO bearing mice. µGCVax potently promoted the activation and lymph node migration of CD8+ and CD103+ dendritic cells in vivo. Distal metastatic tumor nodules in all different tissues vanished after two vaccinations. Functional cure of cancer and extension of animal survival were achieved. Moreover, oxaliplatin treatment on colon cancer caused immunogenic cell death and generated an immune favorable TME in a subcutaneous CT26 colon cancer model. The combination of µGCVax with the oxaliplatin effectively blocked the progression of large tumor mass. We finally revealed that the reprogramming of TME and the promotion of efficacy were mostly achieved from diminishing myeloid-derived suppressor cells (MDSCs) in tumor. Conclusion: µGCVax is an effective and board-spectrum formulation for cancer immunotherapy. It effectively induces APC activation and licensing, and stimulates the innate and adaptive immunity against multiple cancers. The combination with oxaliplatin further promotes the therapeutic efficacy of µGCVax by decreasing tumor MDSCs. Citation Format: Junhua Mai, Yongbin Liu, Dongfang Yu, Haifa Shen. A porous silicon particle based particulate anti-cancer vaccine for immunotherapy. [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 6799.

Abstract P4-07-28: Prognostic Impact of Delaying Multimodality Treatment in HER2 Positive Breast Cancer

Abstract Objective: Multimodality treatment in breast cancer is a key to the improved survival outcomes. The effects of delays in multimodality treatment in HER2 amplified breast cancer were studied. Patients and Methods: Of the patients with primary HER2 amplified breast cancer who were treated between 2009 and 2018 in a single institution, 1,075 patients met the inclusion criteria. The patterns of the multimodality treatments and the prognostic effects of treatment delays were studied. Kaplan-Meier method was used to estimate the relapse free survival (RFS) and overall survival (OS). Hazard ratio (HR), their 95% confidence interval (CI) and p value were computed using the Cox proportional-hazards model adjusting for 11 covariates including age, ethnicity, clinical T stage, clinical N stage, ER, PR, Ki67, LVI, histologic grade, surgery type (mastectomy versus lumpectomy), neoadjuvant versus adjuvant chemotherapy. The Harrell’s C statistics were used to determine the Cox model accuracy. Adjusted multivariate analyses of recurrence/death and death were computed using Cox proportional-hazards model. Delays in treatment were defined as starting treatment beyond 60 days after diagnosis or after the completion of the leading treatment. Results: Of the patients included, 49% received neoadjuvant chemotherapy/HER2 target treatment and surgery, 43% had adjuvant treatments and surgery and 8% had surgery only without chemotherapy. Timely commencement of treatment in the 3 groups were 85.7%, 72.1% and 78.4% respectively. The 5-year RFS was 88.7% and OS was 98.2%. Patients who received neoadjuvant and adjuvant chemotherapy were combined, and six treatment groups with delays in various treatments were compared: no delay in chemotherapy/target treatment and surgery (1); surgery delay (2); chemotherapy delay (3); delays in both treatments (4) and surgery only groups with (5) or without delays (6). Concordance statistics showed that the covariate adjusted Cox proportional-hazards model had a better accuracy than the unadjusted. Compared to those without delays, patients with both chemotherapy and surgery delayed had worse recurrence/death (adjusted HR = 4.11, 95% CI: 1.39-12.5, p= 0.0161). Delays in either surgery or chemotherapy also increased recurrence/death, although to a lesser magnitude. The adjusted death in surgery delay, chemotherapy delay and delays in both had HR 2.91; 2.22; and 2.29 respectively when compared to the group without delays in either treatment although not significant at p &amp;lt; 0.05. Adjusted recurrence/death and death were similar between neoadjuvant and adjuvant groups (HR 0.99 &amp; HR 0.85 respectively). The group that received surgery only without chemotherapy was associated with worse recurrence/death (adjusted HR=1.78, 95% CI: 0.19 – 16.9) Conclusion: Delays in any treatment adversely impacted recurrence and death and chemotherapy/target treatment is a critical component in treating patients with operable HER2 positive breast cancer. Citation Format: Helena Chang, Jeffrey Gornbein, Sin Yee Lim. Prognostic Impact of Delaying Multimodality Treatment in HER2 Positive Breast Cancer [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 P4-07-28.

The Poly (ADP-ribose) polymerase inhibitor olaparib and pan-ErbB inhibitor neratinib are highly synergistic in HER2 overexpressing epithelial ovarian carcinoma in vitro and in vivo

IntroductionOvarian cancer (OC) is associated with the highest gynecologic cancer mortality. The development of novel, effective combinations of targeted therapeutics remains an unmet medical need. We evaluated the preclinical efficacy of the Poly (ADP-ribose) polymerase (PARP) inhibitor (olaparib) and the pan-ErbB inhibitor (neratinib) as single agents and in combination in ovarian cancer cell lines and xenografts with variable HER2 expression. MethodsIn vitro cell viability with olaparib, neratinib, and their combination was assessed using flow-cytometry based assays against a panel of OC primary cell lines with variable HER2 expression. Immunoblotting experiments were performed to elucidate the mechanism of activity and synergism. The in vivo antitumor activity of the olaparib/neratinib combination versus single agents was tested in HER2 positive xenograft OC models. ResultsHER2 + OC cell lines demonstrated higher sensitivity to olaparib and neratinib when compared to HER2 negative tumors (i.e., IC50: 2.06 ± 0.33 μM vs. 39.28 ± 30.51 μM, p = 0.0035 for olaparib and 19.42 ± 2.63 nM vs. 235.0 ± 165.0 nM, p = 0.0035 for neratinib). The combination of olaparib with neratinib was more potent when compared to single-agent olaparib or neratinib both in vitro and in vivo, and demonstrated synergy in all primary HER2 + OC models. Western blot experiments showed neratinib decreased pHER2/neu while increased Poly(ADP-ribose) (PAR) enzymatic activity; olaparib increased pHER2/Neu expression and blocked PAR activatio. Olaparib/neratinib in combination decreased both pHER2/Neu as well as PAR activation. ConclusionThe combination of olaparib and neratinib is synergistic and endowed with remarkable preclinical activity against HER2+ ovarian cancers. This combination may represent a novel therapeutic option for ovarian cancer patients with HER2+, homologous recombination-proficient tumors resistant to chemotherapy.

Whole Genome Sequencing of Single-Circulating Tumor Cell Ameliorates Unraveling Breast Cancer Heterogeneity.

Because tumor tissues are most frequently heterogeneous and hard to characterize, the resulting therapeutic strategy could be misled. The most active and invasive tumor cells are circulating tumor cells (CTCs). In this study, we investigated the feasibility of individualized treatment of breast cancer patients based on whole genome sequencing (WGS) of single cell CTC. Twenty-four CTCs were identified in three breast cancer patients. For each patient, one polyploid CTC was captured, and on which the WGS was performed. WGS was considered due to its sequencing robustness compared to conventional sequencing approaches. Based on the histopathological Her-2 status in tumor tissue and the HER2 gene status in WGS results of CTC, we adjusted treatment strategies and monitored disease progression. Patients ID1 and ID2 are found to be Her-2 positive in primary tumors and HER2 gene amplification in the DNA of CTCs. In-patient ID3, histopathological examination of the primary tumor and liver metastases revealed Her-2 negative, but the WGS analysis of CTC showed HER2 gene amplification. After adjusting treatment by adding Her-2 inhibitors according to the results of CTC sequencing, liver metastases and pleural effusion were significantly reduced two months later, CTC number and ctDNA burden were decreased, and 18-months progression-free survival (PFS) was recorded. In addition, some potential therapeutic targets and mutations in drug-resistant genes were detected. The results of CTC sequencing effectively guided the treatment of a patient with HER2 gene amplification in CTC but with Her-2 negative on tumor tissue. Therefore, CTC sequencing could help resolve the heterogeneity of tumors and provide precision medicine for patients.

BIOM-05. THE HER2 FLIP: HER2 AMPLIFICATION OF TUMOR CELLS IN THE CEREBROSPINAL FLUID (CSF-TCS) OF PATIENTS WITH SOLID TUMOR LEPTOMENINGEAL METASTASIS

Abstract INTRODUCTION Patients with leptomeningeal metastasis (LM) have limited treatment options and a poor outcome. However, they may benefit from targeted therapy. LM patients with HER2-positive primary breast cancer treated with IT trastuzumab demonstrated clinical benefit (Malani, 2020), and improved PFS (Figura, 2019). HER2 amplification in the primary and metastatic tumors can be divergent. Biocept’s CNSideTM is a CLIA validated test that can detect CSF tumor cells (CSF-TCs) and interrogate those cells with FISH and NGS. We analyzed HER2 amplification on CSF-TCs in LM patients with breast cancer, non-small cell lung cancer (NSCLC) and upper GI cancer. METHODS CSF was collected from patients with suspected or confirmed LM with breast cancer (N =134 patients), NSCLC (Nf28 patients) or upper GI cancer (Nf2 patients). CSF TCs were tested for HER2 amplification by FISH using CNSide. RESULTS HER2 amplification in CSF-TCs was detected in 46% (76/164) of all patients. Of the breast cancer patients, 37% (49/134) flipped HER2 status in the LM tumor, 38% (41/108) switched from a HER2 negative or equivocal primary tumor to HER2 positive CSF-TCs, and 27% (7/26) from a HER2 positive primary tumor to HER2 negative CSF-TCs. For the NSCLC patients, 50% (14/28) showed HER2 amplification on the CSF-TCs. For the upper GI cancer patients, HER2 amplification in CSF-TCs was detected in both patients, one patient had an equivocal HER2 primary tumor and HER2 positive CSF-TCs. CONCLUSION HER2 amplification was detected in a substantial fraction of CSF-TCs from patients with LM from breast, upper GI, and NSCL cancers. This finding may have important therapeutic (justifying the use of intra-CSF trastuzumab), prognostic (HER2 positivity in NSCLC is associated with poorer prognosis and a higher frequency of CNS metastases) and pathophysiologic implications (a potential role of HER2 amplification in the genesis of CNS metastases). Additional investigations are underway.

Prognostic nomogram for female patients suffering from non-metastatic Her2 positive breast cancer: A SEER-based study.

This paper aimed at constructing and validating a novel prognostic nomogram, so that physicians forecast the overall survival (OS) rates of female patients suffering from non-metastatic human epidermal growth element receptor-2 (HER2) positive breast.Information of primary female her2 positive breast cancer patients without metastasis was obtained from the Surveillance, Epidemiology, and End Results (SEER) database with given inclusion and exclusion standards. Independent variables were obtained greatly by performing univariable and multivariate analyses. Based on those independent predictors, a novel prognostic nomogram was constructed for predicting the survival of those with 3- and 5-year OS. Then, concordance index (C-index), receiver operating characteristic curve (ROC), and calibration plot were adopted for the assessment of the predictive power of the nomogram.A total of 36,083 eligible patients were classified into a training cohort (n = 25,259) and a verification cohort (n = 10,824) randomly. According to the identification of multivariate analysis, survival was predicted by age at diagnosis, marital status, race, site, T stage, N stage, progesterone receptor (PR) status, estrogen receptor (ER) status, surgery, radiation, and chemotherapy independently. A nomogram was established by applying the training cohort. The nomogram displayed excellent discrimination and performance as indicated by the C-index (0.764, 95% confidence interval: 0.756–0.772), and the 3- and 5-year area under the curve of ROC (AUC) values (0.760 and 0.692 respectively). The calibration plots for forecasting the 3- and 5-year OS were in great agreement.The OS for female her2 positive breast cancer patients without metastasis was predicted by constructing a nomogram on basis of the SEER database. A precise survival prediction could be offered for each patient.

Discordance of positive immunohistochemical results among different primary HER2 antibodies used in breast cancer: A comparison between Histofine® HER2 Kit (MONO) SV2-61γ antibody and Ventana I-VIEW PATHWAY™ HER2 4B5 antibody

IntroductionImmunohistochemical (IHC) testing for human epidermal growth factor receptor 2 (HER2) expression status renders variable scores among different primary antibodies. Materials and methodsWe assessed HER2 expression status by IHC score using two types of kits, i.e., Roche Ventana I-VIEW PATHWAY™ HER2 using the 4B5 antibody that recognizes the intracellular domain (ICD) and Nichirei Biosciences Histofine® HER2 Kit (MONO) using the SV2-61γ antibody that recognizes the extracellular domain (ECD). In addition, we determined the presence or absence of the ECD in samples by Western blotting (WB) and examined its relationship with the IHC score. ResultsOf 101 samples, 44 and 9 samples received a score of 2+/3+ by IHC with 4B5 and SV2-61γ, respectively. A verification by WB in samples with a discrepancy of IHC scores found that samples with the score of 2+/3+ with SV2-61γ showed a significantly higher protein intensity of p185HER2 with the ECD, whereas samples with a score of 2+/3+ with 4B5 but as 0/1+ with SV2-61γ showed a significantly higher protein intensity of p95HER2 without the ECD. ConclusionThe nature of primary antibodies used for IHC and the presence or absence of the ECD in samples may contribute to different positive rates between 4B5 and SV2-61γ.