HER2 Variants Research Articles

Gene Editing for Unraveling the Regulatory Role of a HER2-Associated Enhancer with lncRNA GAS5 and Related Genes in Breast Cancer Cells

Background: The HER2/neu gene belongs to the epidermal growth factor receptor (EGFR) family and encodes a 185 kDa protein with tyrosine kinase activity. HER2 amplification or overexpression has been associated with tumorigenesis and cancer progression, notably in breast cancer (BC). The long non-coding RNA GAS5 has been recognized as a tumor suppressor in BC, promoting apoptosis and inhibiting cell proliferation. Moreover, GAS5 regulates key signaling pathways in breast cancer, including PI3K/AKT/mTOR and PTEN signaling. The PI3K/AKT pathway is critical in various cancer processes, such as proliferation, apoptosis, invasion, and drug resistance. Breast cancer stem cells (BCSCs) and stemness genes also contribute to tumor growth and therapy resistance. Therefore, targeting key regulatory regions in the HER2 gene could offer potential therapeutic strategies. Objectives: In this study, we aimed to investigate the regulatory role of a putative enhancer located in the HER2 gene in the 17q12 region (GH17J039694; we referred to it as Her2-Enhancer1 in our previous study) on HER2 variants and genes that are connected to it through different pathways. Methods: We analyzed the effects of enhancer knockout on HER2 variants, GAS5, PTEN, and stemness genes in HER2-positive and HER2-negative breast cancer cells using the CRISPR/Cas9 system and real-time PCR. Results: Our results showed significantly reduced HER2 variant expression in both cell lines after genetic editing. Furthermore, the expression of some other target genes was affected, including GAS5, P53, and PTEN. Interestingly, we observed altered expression of stemness genes like NANOG, SOX2, and OCT4, indicating possible implications for cancer stem cell properties. Conclusions: Our study highlights the potential regulatory role of the studied region as an enhancer in the HER2 gene and sheds light on its influence on various cancer-related pathways, providing valuable insights into breast cancer pathogenesis and potential therapeutic targets.

Targeting Cardiomyocytes with Adeno Associated Viruses to Protect Hearts from Trastuzumab-induced Cardiotoxicity

Amplification of HER2 occurs in 20% of breast cancers. HER2 (known as ErbB2 in mouse) belongs to the Epidermal Growth Factor Receptor (EGFR) family that is responsible for cell growth and survival. While HER2 does not bind ligands, it forms heterodimers with HER1 (also known as EGFR) in the presence of its ligand EGF, and HER4 following stimulation with Neuregulin. In HER2-positive breast cancer where HER2 expression is high, HER2 homodimerizes leading to excessive activation of downstream signalling and cell proliferation. Current HER2-positive breast cancer treatment involves chemotherapy with drugs called anthracyclines, such as Doxorubicin, and an antibody targeting HER2 called Trastuzumab. This combination is effective in killing HER2-positive breast tumours, however, Trastuzumab alone or in combination with anthracyclines leads to severe cardiotoxicity and heart failure in 2.6-11% of cases, making it a dose-limiting side effect. We hypothesise that cardiac toxicity can be mitigated by targeting cardiomyocytes exclusively to express a mutated (but functional) version of HER2 that is not recognised by trastuzumab. We designed 3 HER2 variants wherein the trastuzumab binding domain was mutated to disrupt binding. These HER2 mutants were transfected into HEK293T cells, and we confirmed that all 3 successfully localised to the cell membrane, and did not bind trastuzumab. We have used bioluminescence resonance energy transfer to examine the capacity of all 3 mutants to activate recruitment of Grb2 to the HER2 as part of HER1/HER2 and HER4/HER2 heterodimers; Western blotting also confirming mutants were capable of stimulating ERK and Akt phosphorylation. Future studies will evaluate the effcacy of using AAVs to instruct cardiomyocytes to express these HER2 mutants in a murine model of breast cancer. We predict that this new therapy would not only mitigate cardiac toxicity, but would also permit higher doses of trastuzumab to be used to treat breast cancer. This work is supported by Australia's National Health and Medical Research Council and the Australian Research Council. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prevalence and treatment of human epidermal growth factor receptor 2-altered non-small cell lung cancer: a retrospective analysis and systematic literature review.

Human epidermal growth factor receptor 2 (HER2) is known for its oncogenic activities in diverse cancers, including non-small cell lung cancer (NSCLC). However, the prevalence of HER2 alterations in Malaysian NSCLC patients remains unreported. This study examined the prevalence and characteristics of HER2 mutations and amplification in a Malaysian cohort. Additionally, a systematic review was conducted to evaluate the global prevalence of HER2 alterations in NSCLC, as well as the efficacy of HER2-targeted therapies observed in clinical trials. NSCLC tumour samples received from October 2019 to December 2022 for next-generation sequencing diagnostics were included in the retrospective analysis. In this patient cohort, HER2 alteration was present in 5.8% of patients; 3.9% had HER2 mutations, 1.5% had HER2 amplifications and 0.4% were both HER2-mutated and amplified. HER2 exon 20 insertions were the most common HER2 variants, detected in 47/59 (79.7%) of HER2-mutated patients. Among cases with HER2 exon 20 insertions, the Y772_A775dup variant was found in 34 patient samples. HER2-mutated patients were significantly younger than non-HER2-mutants (61 versus 64 years old; p = 0.046) and were inclined to be female and never-smokers, albeit not statistically significant. Patients with HER2 amplification were more likely to have progressed post-tyrosine kinase inhibitor therapy (p = 0.015). The systematic review highlighted a global variation in the prevalence of HER2 alterations in NSCLC, ranging from 0.3% to 9.1% for mutations and 0.2% to 19% for amplification. Finally, phase II clinical trials involving HER2-altered NSCLC patients demonstrated promising treatment outcomes with trastuzumab deruxtecan, trastuzumab emtansine, pyrotinib, pyrotinib + apatinib and trastuzumab + pertuzumab + docetaxel. In conclusion, the prevalence of HER2 alteration among Malaysian NSCLC patients falls within the global range. A systematic review of clinical trials revealed promising treatment outcomes and Malaysian NSCLC patients with HER2 alterations are anticipated to similarly benefit from HER2-targeted therapies.

Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells

HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer sequences can deeply affect the expression and transcriptional regulation of downstream genes, which can cause some physiological and pathological changes, including tumor progression. A therapeutic approach that directly targets the genomic sequence alterations is of high importance, with low side effects on healthy cells. Here, we employed the CRISPR/Cas9 method to genetically knockout an expressed putative enhancer (GH17J039694; we coined it as Her2-Enhancer1) located within the HER2 gene, 17q12: 39,694,339–39,697,219 (UCSC-hg38). We then investigated the potential regulatory effect of Her2-Enhancer1 on HER2 and HER2-interacting genes. To evaluate the cis and trans effects of Her2-Enhancer1, genetic manipulation of this region was performed in HER2-positive and -negative breast cancer cells. Our bioinformatics and real-time PCR data revealed that this putative enhancer region is indeed expressed, and acts as an expressed enhancer. Further functional analysis on edited and unedited cells revealed a significant alteration in the expression of HER2 variants, as well as some other target genes of HER2. Moreover, the apoptosis rate was considerably elevated within the edited cells. As we expected, Western blot analysis confirmed a reduction in protein levels of HER2, GRB7, the gene interacting with HER2, and P-AKT in the PI3K/AKT pathway. Altogether, our findings revealed an enhancer regulatory role for Her2-Enhancer1 on HER2 and HER2-interacting genes; and that this region has a potential for targeted therapy of HER2-positive cancers.

Clinical characteristics and treatment outcomes of HER2 mutation and HER2 fusion in 22 patients with advanced breast cancer.

The clinical characteristics and efficacy of human epidermal growth factor receptor-2 (HER-2)-directed agents against HER2 mutations and HER2 fusions in breast cancer are obscure due to their low frequency. We conducted a retrospective study in patients with advanced breast cancer harboring HER2 mutations and/or HER2 fusions between January 1, 2017 and January 1, 2021. Among a total of 22 patients, 17 HER2 mutations were detected, including L755S, S310F, R100=, V777L, R897W, T862A, 440-17C > G, H878Y, V842I, 73 + 9G > C, T278fs, E1069K, L755P, 226-11C > T, 574 + 12C>T, L114V and P128L. The majority of patients had ductal carcinoma, which mostly coexisted with HER2 amplification/overexpression. The median progression-free survival (PFS) of the 22 patients was 6.9 months (95% CI: 4.7, 9.1) in the first-line setting. The median PFS of patients who received first-line trastuzumab-based regimens was significantly longer than that of patients who received a first-line tyrosine kinase inhibitor (TKI) (10.8 months [95% CI: 2.9, 18.7] vs. 1.9 months [95% CI: 0.8, 3.0], p < 0.005). A total of 14 patients were treated with anti-HER2 antibody-drug conjugate (ADC), among whom the median treatment line of first-time of administration of anti-HER2 ADC was 4.5 (range, 1-10). Anti-HER2 ADC reached an objective response rate (ORR) of 42.9%, a disease control rate (DCR) of 85.7% and a median PFS of 7.3 months (95% CI: 4.4-10.1) from the first-time of administration. Our data demonstrated the clinical benefit of anti-HER2 treatment in Chinese breast cancer patients harboring HER2 mutation and/or HER2 fusion. The value of immunotherapy and treatment selection among individual HER2 variants needs further study.

HER2Δ16 Engages ENPP1 to Promote an Immune-Cold Microenvironment in Breast Cancer.

The tumor-immune microenvironment (TIME) is a critical determinant of therapeutic response. However, the mechanisms regulating its modulation are not fully understood. HER2Δ16, an oncogenic splice variant of the HER2, has been implicated in breast cancer and other tumor types as a driver of tumorigenesis and metastasis. Nevertheless, the underlying mechanisms of HER2Δ16-mediated oncogenicity remain poorly understood. Here, we show that HER2∆16 expression is not exclusive to the clinically HER2+ subtype and associates with a poor clinical outcome in breast cancer. To understand how HER2 variants modulated the tumor microenvironment, we generated transgenic mouse models expressing either proto-oncogenic HER2 or HER2Δ16 in the mammary epithelium. We found that HER2∆16 tumors were immune cold, characterized by low immune infiltrate and an altered cytokine profile. Using an epithelial cell surface proteomic approach, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as a functional regulator of the immune cold microenvironment. We generated a knock-in model of HER2Δ16 under the endogenous promoter to understand the role of Enpp1 in aggressive HER2+ breast cancer. Knockdown of Enpp1 in HER2Δ16-derived tumor cells resulted in decreased tumor growth, which correlated with increased T-cell infiltration. These findings suggest that HER2Δ16-dependent Enpp1 activation associates with aggressive HER2+ breast cancer through its immune modulatory function. Our study provides a better understanding of the mechanisms underlying HER2Δ16-mediated oncogenicity and highlights ENPP1 as a potential therapeutic target in aggressive HER2+ breast cancer.

Abstract 3877: Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer

Abstract The estrogen receptor-positive (ER+) subtype accounts for 70% of breast cancer, and endocrine therapies that target ER are effective in the early-stage clinical setting. However, endocrine resistance is ubiquitous in late-stage metastatic breast cancer (MBC). The chief mechanism is the acquisition of ER mutations (ERmuts) in 25% of endocrine-resistant tumors; ERmuts are strongly resistant to aromatase inhibitor (AI) endocrine therapy. We previously identified four novel acquired activating mutations in human epidermal growth factor receptor 2 (HER2) by whole exome sequencing (WES) of paired ER+ MBC in a clinical cohort. These HER2 mutations (HER2muts) were enriched in multiple resistant tumors, and like ERmuts, were resistant to AIs. In contrast to ERmuts, these HER2muts also conferred complete resistance to all other classes of endocrine therapy, as well as cross-resistance to combination therapy with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors; the latter is standard-of-care in the refractory setting in ER+ MBC. At the same time, these HER2muts had variable responses to HER2-targeting therapies used in HER2+ breast cancer. Thus, HER2muts represent only the second validated mechanism of clinical endocrine resistance in ER+ MBC, accounting for ~8% of cases, and that is therapeutically intractable. Systematic phenotypic characterization of HER2muts in ER+ MBC is a prerequisite for effective targeting of this class of resistance. We used cBioPortal to identify &amp;gt;20 novel HER2 variants of unknown significance (VUS) that were frequent pan-cancer, in ER+ breast tumors, or annotated in a domain-restricted HER2 saturation mutagenesis screen, and that had not been previously characterized in ER+ MBC. We also identified a novel HER2 VUS by WES in an ER+/HER2+ tumor with resistance to HER2 therapy, from a clinical cohort of breast cancer patients. We phenotypically characterized all VUS through clinically comprehensive targeted therapy viability assays, and downstream activation assays. In sum, we identified seven novel activating HER2muts, spanning domains, with clinically important phenotypes in ER+ MBC. We found these HER2muts also conferred complete resistance to all classes of endocrine therapies, and cross-resistance to CDK4/6 inhibition. While novel HER2muts had variable responses to HER2-targeting therapies, they could be endocrine re-sensitized by combination with an irreversible tyrosine kinase inhibitor. Lastly, we report that constitutive HER2 homodimerization via reduction-sensitive disulfide bridges that occur as a consequence of mutations in the extracellular and juxtamembrane domains is a more common mechanism of HER2 hyperactivation than previously appreciated. This finding has unique functional consequences, and provides important structure-function insight into HER2 biology, beyond its immediate clinical ramifications in ER+ MBC. Citation Format: Nelson Vicente Guevara, Zirui Zhu, Esha Jain, Jennifer Kavran, Selime Arslan, Nikhil Wagle, Utthara Nayar. Systematic functional and phenotypic characterization of HER2 missense mutants and their impact in ER+ metastatic breast cancer. [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 3877.

Multiple genetic variants predict the progression-free survival of bevacizumab plus chemotherapy in advanced ovarian cancer: A retrospective study.

Bevacizumab (BV) plus chemotherapy is broadly used in advanced ovarian cancer (OC). However, the efficacy of BV-based regimens for advanced OC patients is not satisfactory. Therefore, it is urgent to explore the predictive genetic biomarkers for BV.Tumor tissues from advanced OC patients receiving BV-based regimens were analyzed with a 150-gene targeted panel for next generation sequencing. The associations between gene alterations or clinicopathology features and progression-free survival (PFS) were analyzed by Kaplan–Meier curves or Cox regression. The association of the genetic alteration in potential predictive genes and expressions of 11 vascular endothelial growth factor-related genes were analyzed in The Cancer Genome Atlas cohort using 292 OC cases.Sixty two Chinese advanced OC patients treated with BV-based therapy were included. The median PFS of was 6.9 months, and objective response rate was 14.5%. In multivariate Cox regression analysis, the status of endothelial growth factor receptor (EGFR) (hazard ratio = 6.39, 95% confidence interval [CI] 2.25–18.13, P < .001) and human epidermal growth factor receptor 2 (HER2) (hazard ratio = 3.58, 95% CI 1.27–10.08, P = .016) were significantly correlated with PFS. MYC Proto-Oncogene amplification seemed to have a positive trend (hazard ratio = 0.21, 95% CI 0.05–1.02, P = .052). Moreover, EGFR and HER2 alterations were not prognostic factors of overall survival for OC in The Cancer Genome Atlas OC cohort. The vascular endothelial growth factor-related signature analysis indicated vascular endothelial factor A expression was upregulated with EGFR alterations (P = .034) which may be involved in BV resistance, and HER2 alterations were associated with hypoxia inducible factor 1 subunit alpha overexpression significantly (P = .029).EGFR or HER2 alterations are negative predictors of PFS for OC patient treated with BV plus chemotherapy. Therefore, the clinicians may consider to use alternative regimens such as anti-EGFR or anti-HER2 targeted therapy instead of BV-based regimens on these patients when standard care fail.

HER2 splice variants in breast cancer: investigating their impact on diagnosis and treatment outcomes.

Overexpression of the HER2 receptor occurs in approximately 20% of breast cancer patients. HER2 positivity is associated with poor prognosis and aggressive tumour phenotypes, which led to rapid progress in HER2 targeted therapeutics and diagnostic testing. Whilst these advances have greatly increased patients’ chances of survival, resistance to HER2 targeted therapies, be that intrinsic or acquired, remains a problem.Different forms of the HER2 protein exist within tumours in tandem and can display altered biological activities. Interest in HER2 variants in breast cancer increased when links between resistance to anti-HER2 therapies and a particular variant, Δ16-HER2, were identified. Moreover, the P100 variant potentially reduces the efficacy of the anti-HER2 therapy trastuzumab. Another variant, Herstatin, exhibits ‘auto-inhibitory’ behaviour. More recently, new HER2 variants have been identified and are currently being assessed for their pro- and anti-cancer properties.It is important when directing the care of patients to consider HER2 variants collectively. This review considers HER2 variants in the context of the tumour environment where multiple variants are co-expressed at altered ratios. This study also provides an up to date account of the landscape of HER2 variants and links this to patterns of resistance against HER2 therapies and treatment plans.

Clinical outcomes of patients with HER2-mutant advanced lung cancer: chemotherapies versus HER2-directed therapies.

Background:Lung cancer is now the leading cause of cancer mortality worldwide for both men and women. In non-small cell lung cancer (NSCLC), matching a specifically targeted drug to the identified driver mutation in each patient resulted in dramatically improved therapeutic efficacy, often in conjunction with decreased toxicity. Mutations in HER2 have been identified as an oncogenic driver gene for NSCLC. This retrospective study was conducted to better understand the clinical outcomes of advanced lung cancer patients harboring HER2 mutations treated with chemotherapies and HER2-targeted agents, as well as the optimal clinical choice.Methods:Patients who were diagnosed with advanced lung cancer (stage IIIB/IV) and had undergone molecular testing at Zhongshan Hospital, Fudan University, Shanghai, China from April 2016 to December 2018 were reviewed. For patients that had HER2 mutant advanced lung cancer, we analyzed their clinical and molecular features and clinical outcomes, including overall survival (OS), progression-free survival (PFS), disease control rate (DCR) and objective response rate (ORR).Results:We identified 44 patients harboring HER2 mutations. Their median age was 56 years, with the majority being women (n = 24), never smokers (n = 32), and having the adenocarcinoma genotype (n = 42). Amongst the HER2 mutations present, a 12 base pair in-frame insertion in exon 20 with p.771insAYVM was the most common subtype in patients with known detail variants of HER2 mutation (9/27). The median OS from the date of advanced disease diagnosis was 9.9 months with 24 deaths, and a median follow-up of 12.7 months for survivors. For patients with a known HER2 exon 20 insertion mutation, OS tended to be superior (though not statistically) in the first-line HER2-TKI group to that in the group receiving chemotherapy (10.8 versus 9.8 months, p = 0.40). However, patients that received first-line chemotherapy had a median PFS of 5.9 months, numerically longer than that of the HER2-TKI group (4.6 months, p = 0.63). Patients who received HER2-targeted therapy as first-line therapy had an improved OS (10.8 versus 10.1 months, p = 0.30) and PFS (4.6 versus 2.8 months, p = 0.36) relative to those who received HER2-targeted therapy as subsequent-line therapy, although they did not meet the threshold for statistical significance. Furthermore, patients with AYVM mutation were associated with poor clinical outcomes.Conclusion:Pemetrexed-based chemotherapy remains an important component of care for patients with HER2-mutant NSCLC. HER2-TKI given as an initial therapy may bring more clinical benefits than when given as a subsequent-line therapy. Refining the patient population based on patterns of HER2 variants may help improve the efficacy of anti-HER2 treatment in lung cancer. Developing highly effective and tolerable HER2-targeted agents is urgently needed for this population.

Mutation Variants and Co-Mutations as Genomic Modifiers of Response to Afatinib in HER2-Mutant Lung Adenocarcinoma.

Human epidermal growth factor receptor 2 (HER2)-mutant lung cancer remains an orphan of specific targeted therapy. The variable responses to anti-HER2 therapies in these patients prompt us to examine impact of HER2 variants and co-mutations on responses to anti-HER2 treatments in lung cancer. Patients with stage IV/recurrent HER2-mutant lung cancers identified through next-generation sequencings were recruited from seven hospitals. The study comprised a cohort A to establish the patterns of HER2 variants and co-mutations in lung cancer and a cohort B to assess associations between HER2 variants, co-mutations, and clinical outcomes. The study included 118 patients (cohort A, n = 86; cohort B, n = 32). Thirty-one HER2 variants and 35 co-mutations were detected. Predominant variants were A775_G776insYVMA (49/118, 42%), G778_P780dup (11/118, 9%), and G776delinsVC (9/118, 8%). TP53 was the most common co-mutation (61/118, 52%). In cohort B, objective response rates with afatinib were 0% (0/14, 95% confidence interval [CI], 0%-26.8%), 40% (4/10, 14.7%-72.6%), and 13% (1/8, 0.7%-53.3%) in group 1 (A775_G776insYVMA, n = 14), group 2 (G778_P780dup, G776delinsVC, n = 10), and group 3 (missense mutation, n = 8), respectively (p = .018). Median progression-free survival in group 1 (1.2 months; 95% CI, 0-2.4) was shorter than those in group 2 (7.6 months, 4.9-10.4; hazard ratio [HR], 0.009; 95% CI, 0.001-0.079; p < .001) and group 3 (3.6 months, 2.6-4.5; HR, 0.184; 95% CI, 0.062-0.552; p = .003). TP53 co-mutations (6.317; 95% CI, 2.180-18.302; p = .001) and PI3K/AKT/mTOR pathway activations (19.422; 95% CI, 4.098-92.039; p < .001) conferred additional resistance to afatinib. G778_P780dup and G776delinsVC derived the greatest benefits from afatinib among HER2 variants. Co-mutation patterns were additional response modifiers. Refining patient population based on patterns of HER2 variants and co-mutations may help improve the efficacy of anti-HER2 treatment in lung cancer. Human epidermal growth factor receptor 2 (HER2)-mutant lung cancers are a group of heterogenous diseases with up to 31 different variants and 35 concomitant genomic aberrations. Different HER2 variants exhibit divergent sensitivities to anti-HER2 treatments. Certain variants, G778_P780dup and G776delinsVC, derive sustained clinical benefits from afatinib, whereas the predominant variant, A775_G776insYVMA, is resistant to most anti-HER2 treatments. TP53 is the most common co-mutation in HER2-mutant lung cancers. Co-mutations in TP53 and the PI3K/AKT/mTOR pathway confer additional resistance to anti-HER2 treatments in lung cancer. The present data suggest that different HER2 mutations in lung cancer, like its sibling epidermal growth factor receptor, should be analyzed independently in future studies.

Abstract 347: Identification of poziotinib alone or in combination with TDM1 as a pan-HER2 inhibitor

Abstract Clinical studies of HER2 targeting agents have shown mutational variant &amp; cancer-specific differences in patient outcomes. Furthermore, the drug sensitivity profiles of HER2 mutational variants have not been fully characterized. To this end, we expressed the 16 most frequently detected HER2 mutations across exons 19-21 into Ba/F3 cells &amp; determined activating potential &amp; drug sensitivity to 13 HER1/2 targeting agents including afatinib, neratinib, dacomitinib, tarloxotinib-TKI, poziotinib, pyrotinib, TDM-1, &amp; trastuzumab. Third generation EGFR TKIs, osimertinib, ibrutinib, &amp; nazartinib were not effective at inhibiting cell viability in cells expressing exon 20 mutations; however, 3rd generation TKIs demonstrated activity against cells expressing D769 exon 19 variants &amp; exon 21 variants. By comparison, covalent, quinazoline-based TKIs, afatinib, neratinib, dacomitinib, tarloxotinib-TKI, &amp; poziotinib, differentially inhibited HER2 mutants across all three exons. Across all HER2 mutation variants &amp; TKIs tested, poziotinib had the lowest average IC50 (1.73nM) &amp; was significantly more effective in reducing cell viability than neratinib &amp; tarloxotinib-TKI (p&amp;lt;0.001 &amp; p=0.018). In addition, while poziotinib was more efficacious than either neratinib or tarloxotinib-TKI against HER2 exon 19 &amp; 20 mutations, there was no significant difference in average IC50 for exon 21 mutants, suggesting that mutation location impacts drug binding. Furthermore, within exon 19, L755S &amp; L755P variants had significant differences in drug sensitivity across all TKIs tested, &amp; L755P &amp; exon 20 insertion mutations were refractory to most inhibitors, but remained sensitive to poziotinib. We next tested the anti-tumor activity of HER2 targeting agents in human breast (MCF10A), colorectal (CW-2), &amp; kidney (TUHR14TKB) cell lines with HER2 mutations in exons 19-20 &amp; observed that poziotinib was the most potent inhibitor of the 13 TKIs tested. Furthermore, in a CW-2 xenograft model, poziotinib treatment resulted in a 58% reduction in tumor volume compared to the control group (p=0.011). In comparison, neratinib &amp; afatinib treatment did not reduce tumor growth compared to control animals. Lastly, in a HER2 mutant NSCLC PDX model (HER2 Y772dupYVMA), the combination of low dose poziotinib (2.5mpk) &amp; a single dose of TDM1 (10mpk) resulted in complete tumor regression in 9/9 mice, compared to 2/9 mice receiving TDM1 alone or 0/9 mice receiving low dose poziotinib (p&amp;lt;0.0001). In conclusion, HER2 exon 20 insertions &amp; L755P mutations are resistant to the majority of HER2 TKIs, but remain sensitive to poziotinib. Combination of low dose poziotinib &amp; TDM1 caused complete tumor regression in a HER2 exon 20 mutant PDX model. These data suggest that poziotinib alone &amp; in combination with TDM1 has activity against the most common HER2 variants across diverse malignancies &amp; that clinical studies testing these agents in HER2 mutant cancers are warranted. Citation Format: Jacqulyne P. Robichaux, Monique B. Nilsson, Fahao Zhang, Limei Hu, Junqin He, Marlese Pisegna, Alissa Poteete, John V. Heymach. Identification of poziotinib alone or in combination with TDM1 as a pan-HER2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 347.

Her2Ile655Val polymorphism and its association with breast cancer risk: an updated meta-analysis of case-control studies

Breast cancer (BC) is one of the most common types of cancer in women worldwide. Several factors including genetic and environmental have been linked with susceptibility to development of BC. Her2 is a transmembrane protein with tyrosine kinase activity, overexpressed in several cancers including BC. Various studies in different populations have shown association of Her2 variants with susceptibility to BC, however these results were inconsistent, inconclusive and controversial. To obtain a common conclusive finding, we performed meta-analysis of 35 case-control studies reported earlier including 19, 220 cases and 22, 306 controls. We observed significant association of Her2Ile655Val polymorphism with susceptibility to development of breast cancer (Overall allele Val vs Ile: OR = 1.130, 95% CI = 1.051–1.216, p = 0.001; Ile-Val vs Ile-Ile: OR = 1.100, 95% CI = 1.016–1.192, p = 0.019; Val-Val+Ile-Val vs Ile-Ile: OR = 1.127, 95% CI = 1.038–1.223, p = 0.004). Subgroup analysis indicated a significant association with susceptibility to breast cancer in African and Asian populations. However, such association was not observed in other ethnic groups. Our findings suggested that Her2Ile655Val polymorphism is associated with breast cancer risk in overall, Asian and African populations, and can be used as diagnostic marker for BC.

Outcomes of Pemetrexed-based chemotherapies in HER2-mutant lung cancers

BackgroundHER2 mutation has been found to be an oncogenic driver gene in non-small cell lung cancers(NSCLC) and HER2-directed therapies have shown promising results in this unique population, while little is known about its association with outcomes of chemotherapy. The aim of this study was to investigate the efficacy of first line chemotherapy in patients with advanced HER2-mutant lung adenocarcinomas.MethodsPatients with advanced NSCLC(N = 1714) initially underwent testing for EGFR, KRAS, BRAF mutations and ALK, ROS1 rearrangements, and negative cases were then assessed for HER2 mutations using the method of amplification refractory mutation system(ARMS). The efficacy of first line pemetrexed-based chemotherapy was investigated in patients with HER2-mutant and those with EGFR-mutant, ALK/ROS1-rearranged and KRAS-mutant advanced adenocarcinomas.ResultsHER2 mutations were detected in 29 of 572(5.1%) specimens from a selected population of EGFR/KRAS/BRAF/ALK/ROS1 negative patients. All of them are adenocarcinomas. Among patients with HER2-mutant lung cancers, 25 received pemetrexed-based first line chemotherapy. The objective response rate(ORR) was 36.0%. Their median progression free survival(PFS) was 5.1 months, which was similar with that of KRAS-mutant group (n = 40,5.0 months, p = 0.971), numerically shorter than that of EGFR-mutant group(n = 74, 6.5 months, p = 0.247) and statistically significantly shorter than that of ALK/ROS1-rearranged group (n = 39,9.2 months, p = 0.004). Furthermore, HER2 variants subgroup analysis showed that PFS was inferior in A775_G776insYVMA group compared with other variants (4.2 vs 7.2 months, p = 0.085).ConclusionsPatients with advanced HER2-mutant lung adenocarcinomas showed an inferior outcome of first line pemetrexed-based chemotherapy compared to those with ALK/ROS1 rearrangements, which strengthen the need for effective HER2-targeted drugs in clinical practice.

Rigidity of the extracellular part of HER2: Evidence from engineering subdomain interfaces and shared-helix DARPin-DARPin fusions.

The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated "open" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a "tethered" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the "open" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the "open" conformation.

Abstract CT001: Neratinib in HER2 or HER3 mutant solid tumors: SUMMIT, a global, multi-histology, open-label, phase 2 "basket" study

Abstract Background: Somatic HER2 (ERBB2) and HER3 (ERBB3) mutations can lead to constitutive HER2 activation in the absence of gene amplification. They occur in a variety of solid tumors with a prevalence that does not exceed 5-10% in any tumor type. Activating HER2 mutations occur in the HER2 receptor extracellular and transmembrane domains, and at multiple hotspots in the kinase domain, with no single mutation predominating. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, inhibits growth of HER2- and HER3-mutant tumors in preclinical models. We explored the clinical activity of neratinib in pts with HER2/HER3 mutant cancers in SUMMIT, a global (9 countries), multicenter, multi-histology, phase II ‘basket’ trial. Methods: Pts with advanced solid tumors and locally documented HER2/HER3 mutations received neratinib 240 mg daily with high-dose loperamide prophylaxis during cycle 1. Primary endpoint: objective response rate at week 8 (ORR8) per RECIST 1.1; pts without RECIST-measurable disease were assessed separately using FDG-PET. Retrospective confirmation of HER2/HER3 mutations, as well as a broader cancer gene profiling, was performed using both tumor and plasma-derived cfDNA. For each tumor type, using Simon’s optimal 2-stage design, a true ORR8 ≤10% was considered unacceptable (null hypothesis) whereas a true ORR8 ≥30% (alternative hypothesis) merited further study. Interim data cut: 16-Dec-2016. Clinicaltrials.gov: NCT01953926. Results: 141 (125 HER2, 16 HER3) pts have received neratinib including 125 efficacy-evaluable pts (110 HER2, 15 HER3). 21 unique cancer types were enrolled (most common: breast, lung, bladder, colorectal cancer). 30 HER2 and 12 HER3 mutations were observed, the most frequent HER2 variants involving S310, L755, A755_G776insYVMA and V777. In the HER2-mutant cohort, clinical responses were observed in tumors with S310, L755, V777, P780_Y781insGSP, and A775_G776insYVMA. When stratified by tumor type, responses were observed in pts with breast, cervical, biliary, salivary and non-small-cell lung cancers, which led to indication-specific cohort expansions. No activity was observed in the HER3-mutant cohort. Tumor and/or cfDNA from 116 pts has been sequenced centrally. Of these, the locally reported HER2/3 mutation was confirmed in 108 (93%), a discordant HER2/3 mutation detected in 5 (4%), and not detected in 3 (3%). Co-mutation patterns in different tumor types and their association with neratinib sensitivity/resistance are under review. Conclusions: SUMMIT provides the largest body of clinical data to date on the use of a pan-HER inhibitor in pts with solid tumors with somatic HER2/HER3 mutations. Responses in HER2 mutants varied by type of tumor and the specific HER2 mutation, suggesting: 1) mutant HER2 is a driver oncogene in some cancers; and 2) not all mutations generate the same level of HER2 hyperactivity and/or oncogene dependence. Citation Format: David M. Hyman, Sarina A. Piha-Paul, Jordi Rodon, Cristina Saura, Geoffrey I. Shapiro, David I. Quinn, Victor Moreno, Ingrid A. Mayer, Carlos Arteaga, Valentina Boni, Emiliano Calvo, Sherene Loi, Albert C. Lockhart, Lillian M. Smyth, Joseph Erinjeri, Maurizio Scaltriti, Gary Ulaner, Jean Torrisi, Juber Patel, Jiabin Tang, Fanli Meng, Duygu Selcuklu, Helen Won, Nancy Bouvier, Michael F. Berger, Richard E. Cutler, Feng Xu, Anna Butturini, Lisa D. Eli, Grace Mann, Alshad S. Lalani, Richard P. Bryce, Funda Meric-Bernstam, José Baselga, David B. Solit. Neratinib in HER2 or HER3 mutant solid tumors: SUMMIT, a global, multi-histology, open-label, phase 2 "basket" study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT001. doi:10.1158/1538-7445.AM2017-CT001

In vitro functional analysis of HER2 variants in lung cancers to evaluate their oncogenic activity and predict clinical response to HER2 targeted therapies.

e23150 Background: HER2 variants are implicated as oncogenic drivers in a variety of cancers. However, not all HER2 variants found on next-generation sequencing (NGS) have been studied, raising the question if these variants of unknown significance (VUS) are oncogenic. This study explores the use of an in-vitro functional assay on HER2 and other variants found on NGS in patients (pts) with lung cancer, and its predictive value for response to HER2 targeted therapy. Methods: Pts with lung cancer at Memorial Sloan Kettering identified to have a HER2 variant by NGS on MSK IMPACT are eligible. NGS data on HER2 variants and co-mutations were analyzed. Functional status was determined by the NovellusDx FACT platform. Mutations were synthesized and transfected into HeLa cells with a fluorescently tagged signaling pathway reporter (MAPK, NFKB, JAK-STAT). Fluorescent microscopy quantified the translocation of reporters to the nucleus, inferring gain of function. Afatinib was added and its effect on pathway activation analyzed. Functional information was correlated with clinical outcomes. Results: As of 2/1/17, 13 pts were included, 9 unique HER2 variants analyzed. Known activating HER2 mutations analyzed (exon 20 insertions (A775_G776insYVMA, G778_P780insGSP, G776delinsVC), point mutations (V659E, L755P, S301F)) showed in vitro gain of function. HER2 VUS analyzed included D277Y showed no gain of function; exon 16 splice variant c.1889 showed gain of function. Afatinib was added to 4 cell lines with gain of function variants (G778_P780insGSP, V659E, L755P, exon 16 splice variant c.1889) all had inhibited pathway activation suggesting in vitro sensitivity to afatinib. Clinically, no response was seen in 2 pts treated with afatinib but more pts are planned to be treated. Objective responses to ado-trastuzumab emtansine (NCT02675829) were seen in 5 pts, all of which had HER2 variants with gain of function. Conclusions: In vitro functional analysis of HER2 variants based on NGS reports is feasible, and has the potential to identify novel actionable oncogenic drivers from VUS. Its predictive value for clinical response to targeted therapy requires further study.

Abstract 2644: AP32788, a potent, selective inhibitor of EGFR and HER2 oncogenic mutants, including exon 20 insertions, in preclinical models

Abstract In non-small cell lung cancer (NSCLC), multiple classes of activating mutations have been identified in EGFR and HER2 that vary widely in their sensitivity to available tyrosine kinase inhibitors (TKIs). Erlotinib, gefitinib, and afatinib are approved for use in patients with the most common forms of EGFR activating mutations (ie, exon 19 deletions or L858R substitutions). However, no TKIs are approved for patients with EGFR activated by any other mutation, including exon 20 insertions or other uncommon substitutions, or for patients with any class of HER2 activating mutation (including exon 20 insertions). As inhibition of wild-type (WT) EGFR is associated with dose-limiting toxicities, a TKI that inhibits oncogenic EGFR and HER2 variants more potently than WT EGFR is more likely to be able to be dosed to efficacious levels. AP32788 is a potent inhibitor of all oncogenic forms of EGFR and HER2, including exon 20 insertions, with selectivity over WT EGFR. Activity of AP32788 and other TKIs was assessed by measuring viability of Ba/F3 cell lines engineered to express 20 mutant variants of EGFR (n = 14) or HER2 (n = 6): 4 EGFR variants containing a common activating mutation with or without a T790M resistance mutation, 8 EGFR/HER2 variants containing an exon 20 activating insertion (eg, EGFR ASV, HER2 YVMA), and 8 EGFR/HER2 variants containing other uncommon activating mutations (eg, EGFR G719A, HER2 G776V). Inhibition of WT EGFR was assessed by measuring effects on EGFR phosphorylation in cells (A431) that over-express WT EGFR. Consistent with their clinical activity, erlotinib and gefitinib generally only inhibited the 2 EGFR variants with common activating mutations more potently than WT EGFR (IC50s 71 and 56 nM, respectively), and afatinib generally only inhibited EGFR with common activating mutations or uncommon substitutions more potently than WT EGFR (IC50 4 nM). In contrast, AP32788 inhibited all 14 mutant variants of EGFR (IC50s 2.4-22 nM), and all 6 mutant variants of HER2 (IC50s 2.4-26 nM), more potently than it inhibited WT EGFR (IC50 35 nM), including all 8 variants with exon 20 activating insertions. In mice implanted with a patient-derived tumor containing an EGFR exon 20 activating insertion, or with engineered Ba/F3 cells containing a HER2 exon 20 activating insertion, once daily oral dosing of AP32788 induced regression of tumors at doses that were well tolerated (30-100 mg/kg). In vivo efficacy was associated with inhibition of EGFR signaling in the tumor. AP32788 potently inhibited all activated forms of EGFR and HER2 tested, including exon 20 insertions, more potently than WT EGFR, suggesting it may have the selectivity necessary to achieve efficacious levels of exposure in patients. A phase 1/2 clinical trial of AP32788 in NSCLC patients is planned. Citation Format: Francois Gonzalvez, Xiaotian Zhu, Wei-Sheng Huang, Theresa E. Baker, Yaoyu Ning, Scott D. Wardwell, Sara Nadworny, Sen Zhang, Biplab Das, Yongjin Gong, Matthew T. Greenfield, Hyun G. Jang, Anna Kohlmann, Feng Li, Paul M. Taslimi, Meera Tugnait, Yongjin Xu, Emily Y. Ye, Willmen W. Youngsaye, Stephan G. Zech, Yun Zhang, Tianjun Zhou, Narayana I. Narasimhan, David C. Dalgarno, William C. Shakespeare, Victor M. Rivera. AP32788, a potent, selective inhibitor of EGFR and HER2 oncogenic mutants, including exon 20 insertions, in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2644.

Analysis of different HER-2 mutations in breast cancer progression and drug resistance.

Studies over the last two decades have identified that amplified human epidermal growth factor receptor (HER-2; c-erbB-2, neu) and its overexpression have been frequently implicated in the carcinogenesis and prognosis in a variety of solid tumours, especially breast cancer. Lots of painstaking efforts were invested on the HER-2 targeted agents, and significantly improved outcome and prolonged the survival of patients. However, some patients classified as 'HER-2-positive' would be still resistant to the anti-HER-2 therapy. Various mechanisms of drug resistance have been illustrated and the alteration of HER-2 was considered as a crucial mechanism. However, systematic researches in regard to the HER-2 mutations and variants are still inadequate. Notably, the alterations of HER-2 play an important role in drug resistance, but also have a potential association with the cancer risk. In this review, we summarize the possible mutations and focus on HER-2 variants' role in breast cancer tumourigenesis. Additionally, the alteration of HER-2, as a potential mechanism of resistance to trastuzumab, is discussed here. We hope that HER-2 related activating mutations could potentially offer more therapeutic opportunities to a broader range of patients than previously classified as HER-2 overexpressed.

Polymorphic Ala-allele carriers at residue 1170 of HER2 associated with Parkinson's disease

ObjectiveHER2, a receptor tyrosine kinase, was originally identified based on its role in cancer research. The protein has subsequently received attention for its role in nerve injury and neurodevelopment. We investigated the polymorphic association of HER2 variants at amino acid residues 655 and 1170 with Parkinson's disease (PD), a neurodegenerative disorder. Design and methodsPolymerase chain reaction (PCR) was used to amplify DNA samples from PD patients and control subjects. The resulting PCR fragments, which spanned HER2 residues 655 and 1170, were analyzed by restriction fragment length polymorphism and/or direct nucleotide sequencing. ResultsThe genetic distribution at residue 655 in PD patients did not differ from that in controls. However, homozygosity for genes encoding Pro at residue 1170 (Pro/Pro) occurred at a significantly lower rate among PD subjects. In other words, Ala-allele carries higher frequency in PD, especially among female PD subjects. ConclusionDifferent signals or potency of the kinase activities resulting from the Ala1170Pro allele of HER2 may be associated with vulnerability to stress on dopaminergic neurons in PD.