Wild-type HER2 Research Articles

Population Pharmacokinetic Analysis of Tucatinib in Healthy Participants and Patients with Breast Cancer or Colorectal Cancer.

Tucatinib is a highly selective, oral, reversible, human epidermal growth factor receptor 2 (HER2)-specific tyrosine kinase inhibitor. Tucatinib is approved at a 300-mg twice-daily dose in adults in combination with trastuzumab and capecitabine for advanced HER2-postitive (HER2+) unresectable or metastatic breast cancer and in combination with trastuzumab for RAS wild-type HER2+ unresectable or metastatic colorectal cancer. This study sought to characterize the pharmacokinetics (PK) and assess sources of PK variability of tucatinib in healthy volunteers and in patients with HER2+ metastatic breast or colorectal cancers. A population pharmacokinetic model was developed based on data from four healthy participant studies and three studies in patients with either HER2+ metastatic breast cancer or metastatic colorectal cancer using a nonlinear mixed-effects modeling approach. Clinically relevant covariates were evaluated to assess their impact on exposure, and overall model performance was evaluated by prediction-corrected visual predictive checks. A two-compartment pharmacokinetic model with linear elimination and first-order absorption preceded by a lag time adequately described tucatinib pharmacokinetic profiles in 151 healthy participants and 132 patients. Tumor type was identified as a significant covariate affecting tucatinib bioavailability and clearance, resulting in a 1.2-fold and 2.1-fold increase in tucatinib steady-state exposure (area under the concentration-time curve) in HER2+ metastatic colorectal cancer and HER2+ metastatic breast cancer, respectively, compared with healthy participants. No other covariates, including mild renal or hepatic impairment, had an impact on tucatinib pharmacokinetics. The impact of statistically significant covariates identified was not considered clinically meaningful. No tucatinib dose adjustments are required based on the covariates tested in the final population pharmacokinetic model. NCT03723395, NCT03914755, NCT03826602, NCT03043313, NCT01983501, NCT02025192.

IAM1363-01: A phase 1/1b study of a selective and brain-penetrant HER2 inhibitor for HER2-driven solid tumors.

TPS3186 Background: HER2 activation promotes carcinogenesis, prompting the development of HER2-directed therapies in cancers with HER2 alterations. There is a critical need for novel agents to treat HER2-driven cancers, as current inhibitors display significant toxicity and lack efficacy against certain mutations. IAM1363 (formerly IAM-H1 and ENT-H1) is an irreversible and brain-penetrant small molecule inhibitor targeting both wildtype HER2 and oncogenic HER2 mutants, including the recalcitrant exon 20 mutations. IAM1363, discovered through Iambic Therapeutics' AI-driven discovery platform, represents the only example of a Type II HER2 inhibitor. It was designed to avoid off-target toxicities from EGFR inhibition thus expanding the therapeutic index over existing HER2 inhibitors. In preclinical studies, IAM1363 shows preferential tumor enrichment, a unique feature observed across tumor types and anatomic sites. The promising target specificity and pharmacokinetic profile of IAM1363 offers the potential to bridge gaps left by current standard of care HER2 targeted treatments. Methods: IAM1363-01 is a first-in-human, multicenter, open-label, Phase 1/1b dose escalation and expansion trial evaluating IAM1363 as monotherapy and in combination with trastuzumab. The study will enroll patients with relapsed/refractory malignancies that have documented HER2 gene alterations (amplification or tyrosine kinase domain mutations) or protein overexpression. Prior treatment with HER2 directed therapies is allowed after a washout period. Part 1, dose escalation, will enroll patients with any tumor type. Part 2 will optimize IAM1363 dosing as monotherapy and in combination with trastuzumab. Part 3 consists of a Simon 2-Stage evaluation of IAM1363 as monotherapy in patients with colorectal, non-small cell lung, and bladder cancer with or without brain metastases and in combination with trastuzumab in breast cancer patients with brain metastases. Key objectives are to establish the safety and tolerability of IAM1363, characterize PK/PD, and determine the recommended Phase 2 dose. Exploratory analysis will correlate HER2 expression level/mutation status with tumor response and tolerability. IAM1363 will be given orally once daily at a starting dose of 120 mg/day in up to 4 planned dose levels. Dose escalation employs an accelerated titration design that transitions to a standard 3 + 3 design based on protocol-specific criteria. Up to 287 patients will be enrolled at approximately 20 sites and treated until disease progression or unacceptable toxicity. The trial is designed to establish the safety profile of IAM1363 as a single agent and in combination with trastuzumab and to provide initial proof of antitumor activity in malignancies with HER2 overexpression, gene amplification, or tyrosine kinase mutations, including in patients with brain metastases. Clinical trial information: NCT06253871 .

MR-based radiomics predictive modelling of EGFR mutation and HER2 overexpression in metastatic brain adenocarcinoma: a two-centre study

ObjectivesMagnetic resonance (MR)-based radiomics features of brain metastases are utilised to predict epidermal growth factor receptor (EGFR) mutation and human epidermal growth factor receptor 2 (HER2) overexpression in adenocarcinoma, with the aim to identify the most predictive MR sequence.MethodsA retrospective inclusion of 268 individuals with brain metastases from adenocarcinoma across two institutions was conducted. Utilising T1-weighted imaging (T1 contrast-enhanced [T1-CE]) and T2 fluid-attenuated inversion recovery (T2-FLAIR) sequences, 1,409 radiomics features were extracted. These sequences were randomly divided into training and test sets at a 7:3 ratio. The selection of relevant features was done using the least absolute shrinkage selection operator, and the training cohort’s support vector classifier model was employed to generate the predictive model. The performance of the radiomics features was evaluated using a separate test set.ResultsFor contrast-enhanced T1-CE cohorts, the radiomics features based on 19 selected characteristics exhibited excellent discrimination. No significant differences in age, sex, and time to metastasis were observed between the groups with EGFR mutations or HER2 + and those with wild-type EGFR or HER2 (p > 0.05). Radiomics feature analysis for T1-CE revealed an area under the curve (AUC) of 0.98, classification accuracy of 0.93, sensitivity of 0.92, and specificity of 0.93 in the training cohort. In the test set, the AUC was 0.82. The 19 radiomics features for the T2-FLAIR sequence showed AUCs of 0.86 in the training set and 0.70 in the test set.ConclusionsThis study developed a T1-CE signature that could serve as a non-invasive adjunctive tool to determine the presence of EGFR mutations and HER2 + status in adenocarcinoma, aiding in the direction of treatment plans.Clinical relevance statementWe propose radiomics features based on T1-CE brain MR sequences that are both evidence-based and non-invasive. These can be employed to guide clinical treatment planning in patients with brain metastases from adenocarcinoma.

Abstract LB044: Selective therapeutic antibodies against oncogenic mutations of the HER2 extracellular region

Abstract Oncogenic mutations of HER2 have been identified in HER2-low tumors. Such mutations in the extracellular region could serve as tumor-specific antigens that are accessible to antibody therapeutics. Although multiple antibody therapeutics have been successfully developed against cancers that overexpress wild-type HER2, none is specifically applicable to treating tumors driven by HER2 mutants expressed at a low level. Here, we developed antibodies exquisitely selective to HER2 S310F and S310Y, two most common oncogenic mutations in the HER2 extracellular region. Cryo-EM structures of the homodimer of HER2 S310F and an antibody bound to HER2 S310F revealed that these antibodies recognize the mutations in a manner that mimic the dimerization arm of HER and inhibit HER2 dimerization. These antibodies as T-cell engagers selectively killed a HER2 S310F-driven cancer cell line in vitro and in a mouse xenograft model. These results validate HER2 extracellular region mutations as actionable therapeutic targets and offer promising candidates toward clinical development. More generally, they suggest that point mutations in the extracellular region of cell-surface receptors as actionable targets for anti-cancer therapy. Citation Format: Injin Bang, Takamitsu Hattori, Nadia Leloup, Alexis Corrado, Atekana Nyamaa, Akiko Koide, Ken Geles, Elizabeth Buck, Shohei Koide. Selective therapeutic antibodies against oncogenic mutations of the HER2 extracellular region [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB044.

Abstract 1979: Preclinical characterization of NVL-330, a selective and brain penetrant HER2 tyrosine kinase inhibitor with broad activity on HER2 oncogenic alterations

Abstract Background: Oncogenic mutations and gene amplifications in the HER2 receptor tyrosine kinase are detected in approximately 2 - 4% and 1 - 5%, respectively, of non-small cell lung cancers (NSCLC) in the US. Exon 20 insertion mutations (exon20ins) are the predominant HER2 mutations in NSCLC, and ~50% of HER2-mutant metastatic NSCLC patients develop brain metastases. The antibody drug conjugate trastuzumab deruxtecan (T-DXd) has received FDA accelerated approval for HER2-mutant NSCLC, but currently there are no approved tyrosine kinase inhibitors (TKIs) for this indication. NVL-330 is a novel, brain-penetrant, HER2-selective TKI, designed to address the combined medical need of treating HER2-mutant tumors, avoiding treatment related adverse events due to off-target inhibition of wild-type EGFR, and treating brain metastases. Here we characterized the preclinical pharmacological profile of NVL-330 in comparison to other investigational HER2-targeting TKIs and T-DXd. Methods: TKIs were profiled in cellular phospho-HER2, phospho-EGFR, and viability assays. In vivo antitumor studies were performed in nude mice with HER2-altered subcutaneous or intracranial tumors. T-DXd concentration was measured in an ELISA assay. The unbound brain-to-plasma partitioning ratio (Κp,uu) was determined at one hour after oral 10 mg/kg dosing in Wistar Han rats. Results: NVL-330 broadly inhibited HER2 oncogenic alterations, including amplified wild-type HER2, HER2 exon20ins and non-exon20ins mutants, with potency and selectivity over wild-type EGFR comparable to investigational HER2-selective TKI zongertinib. By contrast, NVL-330 had a higher Kp,uu than zongertinib in rats. In a patient-derived xenograft model harboring HER2 exon20ins, treatment with NVL-330 suppressed phospho-HER2 and downstream signaling, increased total HER2 and caused dose-dependent tumor regression at well tolerated doses. In the HER2amp NCI-N87 subcutaneous xenograft model, oral administration of 30 mg/kg NVL-330 twice per day induced tumor regression, similar to that achieved by intravenous administration of 10 mg/kg T-DXd once every three weeks, a human-relevant dose in mice. By comparison, in the intracranial setting with the same tumor model and dosing regimens, NVL-330 induced tumor regression, whereas T-DXd provided tumor stasis. Pharmacokinetic analysis in these intracranial tumor bearing mice indicated that NVL-330 had significantly higher brain penetrance than T-DXd. Conclusions: NVL-330 was broadly and selectively active against HER2 oncogenic alterations in vitro and in vivo, and demonstrated brain penetrance in rodents exceeding that of HER2 investigational TKI zongertinib and T-DXd. This preclinical profile supports the potential for NVL-330 to address a medical need for patients with HER2-driven cancers, including those with brain metastases. Citation Format: Yuting Sun, Kristin L. Andrews, Anupong Tangpeerachaikul, Tuan M. Nguyen, Baudouin Gerard, Nancy E. Kohl, Joshua C. Horan, Henry E. Pelish. Preclinical characterization of NVL-330, a selective and brain penetrant HER2 tyrosine kinase inhibitor with broad activity on HER2 oncogenic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1979.

HER2 phosphorylation induced by TGF-β promotes mammary morphogenesis and breast cancer progression.

Transforming growth factor β (TGF-β) and HER2 signaling collaborate to promote breast cancer progression. However, their molecular interplay is largely unclear. TGF-β can activate mitogen-activated protein kinase (MAPK) and AKT, but the underlying mechanism is not fully understood. In this study, we report that TGF-β enhances HER2 activation, leading to the activation of MAPK and AKT. This process depends on the TGF-β type I receptor TβRI kinase activity. TβRI phosphorylates HER2 at Ser779, promoting Y1248 phosphorylation and HER2 activation. Mice with HER2 S779A mutation display impaired mammary morphogenesis, reduced ductal elongation, and branching. Furthermore, wild-type HER2, but not S779A mutant, promotes TGF-β-induced epithelial-mesenchymal transition, cell migration, and lung metastasis of breast cells. Increased HER2 S779 phosphorylation is observed in human breast cancers and positively correlated with the activation of HER2, MAPK, and AKT. Our findings demonstrate the crucial role of TGF-β-induced S779 phosphorylation in HER2 activation, mammary gland development, and the pro-oncogenic function of TGF-β in breast cancer progression.

Abstract A065: Lineage tracing oncogenic isoforms of HER2 in a mouse model of pancreatic cancer

Abstract Among the numerous clinical challenges of pancreatic ductal adenocarcinoma (PDAC) is the eventual development of therapeutic resistance. Although previous studies report that only about 3% of PDAC cases are HER2 gene amplified, as many as 82% of PDAC tumors may have HER2 overexpressed. Anti-HER2 therapy with trastuzumab has yielded tremendous therapeutic success in breast cancer and multiple gastrointestinal malignancies, however minimal benefit is found in PDAC. Experimental mouse models have demonstrated initiation and acceleration of pancreatic tumorigenesis through overexpression of activated mutant HER2, yet the role of HER2 alone as a driver of PDAC and treatment resistance is still unknown. Multiple HER2 isoforms exist that are generated either via alternative splicing and loss of exon 16 (d16 HER2) or through N-terminal truncations (p95 HER2). These isoforms are implicated in differential responses to anti-HER2 therapy in the breast. Previously we described a HER2+ cancer rainbow mouse model in which human wild-type HER2 (WT HER2), d16 HER2, and p95 HER2 were expressed in the same mouse and lineage traced through co-expression of fluorescent protein reporters. Studies in the mammary gland revealed that each isoform drives distinct tumor phenotypes. Herein, we hypothesize that these isoforms drive HER2 positive PDAC. HER2 Crainbow mice were crossed with mice expressing PDX1-Cre recombinase to initiate recombination and expression of each fluorescently labelled HER2 isoform during pancreas development. PDX1-Cre/HER2 Crainbow (PHBOW) mice were followed for up to one year and sacrificed to lineage trace each isoform and register HER2 lineages with histopathology. Within our pilot cohort, early-stage pre-malignant pancreatic intraepithelial neoplasia (PanIN-1) was observed in 75% of PDAC HER2bow mice as early as five weeks. By one year, 50% demonstrated high grade PanIN (i.e. PanIN-3) and features of early invasion. Although WT HER2 was expressed abundantly throughout the pancreas, lineage tracing revealed WT HER2 was not found in pre-malignant lesions. Conversely, the pre-malignant PanIN lesions and early invasive histopathologic features traced to d16 HER2 expressing cells, while p95 HER2 traced with areas of acinar-to-ductal metaplasia, lesions known to be early initiators of pancreatic dysplasia. Our data show how the oncogenic HER2 isoforms, and not WT HER2, are able to drive PanIN lesions and, at lower frequency, invasive PDAC after one year. Future work seeks to complete HER2 Crainbow lineage tracing in PHBOW mice, test HER2 targeted therapy in PHBOW mice, and quantify HER2 isoforms in HER2+ human PDAC cases. Citation Format: Elishama N. Kanu, Joelle Sills, Rebecca Shelley, Joshua D. Ginzel, Joseph Fernandes, Ashley A. Fletcher, Peter J. Allen, Daniel P. Nussbaum, Joshua C. Snyder. Lineage tracing oncogenic isoforms of HER2 in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A065.

Abstract A038: Selective therapeutic antibodies against oncogenic mutations of HER2 ectodomain

Abstract Dysregulation of HER2 is a well-established mechanism of oncogenesis. Small molecule drugs targeting its kinase domain, and antibody-based drugs such as trastuzumab and pertuzumab targeting its ectodomain have shown efficacy against HER2-overexpression tumors. Separately from HER2 overexpression, oncogenic mutations in the ectodomain in HER2 have been identified in several tumors. As disease drivers that are readily accessible to biologics, these mutants are potentially attractive but still unvalidated therapeutic targets. Current anti-HER2 therapeutic antibodies are indiscriminatory of the mutational state of HER2 and their cross-reactivity to wild-type HER2 can cause toxicity. Here, we have developed antibodies exquisitely selective to HER2 S310F and S310Y, two most common oncogenic mutations in the HER2 ectodomain. These antibodies bound to both HER2 S310F/Y with >100-fold selectivity over the wild-type. Cryo-EM structures of the homodimer of HER2 S310F and an antibody bound to HER2 S310F revealed that these antibodies mimic the dimerization arm of HER2 and inhibit dimerization. The developed antibodies as T cell engagers selectively killed a HER2 S310F-driven cancer cell line in vitro. These results establish the feasibility of targeted therapy against S310F/Y-driven cancers and offer promising candidates toward clinical development. Citation Format: Injin Bang, Takamitsu Hattori, Nadia Leloup, Alexis Corrado, Atekana Nyamaa, Akiko Koide, Ken Geles, Elizabeth Buck, Shohei Koide. Selective therapeutic antibodies against oncogenic mutations of HER2 ectodomain [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A038.

The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer.

HER2 is amplified or overexpressed in around 20% of breast cancers (BC). HER2-targeted therapies have significantly improved the prognosis of patients with HER2+ BC, however, de novo and acquired resistance to anti-HER2 treatment is common. Activating mutations in the PIK3CA gene are reported in ∼30% of HER2+ BC and are associated with resistance to anti-HER2 therapies and a poor prognosis. Here, we investigated the in vitro and in vivo antitumor efficacy of the alpha-specific PI3K inhibitor alpelisib alone or in combination with anti-HER2 therapy using a panel of HER2+ BC cell lines. We also generated models of acquired resistance to alpelisib to investigate the mechanisms underlying resistance to alpha-specific PI3K inhibition. PIK3CA mutant (HCC1954, KPL4 and JMT1) and wild-type (BT474 and SKBR3) HER2+ BC cell lines were used. The HCC1954 and KPL4 cells were chronically exposed to increasing concentrations of alpelisib or to alpelisib + trastuzumab in order to generate derivatives with acquired resistance to alpelisib (AR) and to alpelisib + trastuzumab (ATR). The transcriptomic profiles of HCC1954, KPL4 and their AR and ATR derivatives were determined by RNA sequencing. Cell growth was assessed by MTT assay. Changes in the protein levels of key PI3K pathway components were assessed by Western blotting. Gene expression, cellular and patients' data from the Cancer Dependency Map (DepMap) and KMPlot datasets were interrogated. HER2+ BC cell lines harboring activating mutations in PIK3CA were less sensitive to single or dual anti-HER2 blockade compared to PIK3CA wild-type cells. Alpelisib treatment resulted in dose-dependent inhibition of the growth of cells with or without PIK3CA mutations and enhanced the antitumor efficacy of anti-HER2 therapies in vitro. In addition, alpelisib greatly delayed tumor growth of HCC1954 xenografts in vivo. Functional annotation of the significantly differentially expressed genes suggested the common activation of biological processes associated with oxidation reduction, cell proliferation, immune response and RNA synthesis in alpelisib-resistant models compared with native cells. Eight commonly upregulated genes (log2 fold-change >1, False Discovery Rate [FDR] <0.05) in models with acquired resistance to alpelisib or alpelisib + trastuzumab were identified. Among these, AKR1C1 was associated with alpelisib-resistance in vitro and with a poor prognosis in patients with HER2+ BC. Our findings support the use of an alpha-selective PI3K inhibitor to overcome the therapeutic limitations associated with single or dual HER2 blockade in PIK3CA-mutant HER2+ breast cancer. Future studies are warranted to confirm the potential role of candidate genes/pathways in resistance to alpelisib.

HER2 testing in the MOUNTAINEER trial: Analysis of treatment response based on central HER2 assessment using IHC/ISH and NGS.

3528 Background: Tucatinib (TUC) is a highly selective HER2-directed TKI approved by the FDA in combination w/trastuzumab (Tras) for treatment (tx) of pts w/ RAS wild-type HER2+ unresectable or metastatic colorectal cancer (mCRC) that has progressed following tx w/ fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy. There are currently no established best practices for HER2 testing/interpretation in mCRC. Here we present results of central HER2 testing across multiple platforms and response to tx for MOUNTAINEER pts treated w/ TUC + Tras based on central HER2 status. Methods: MOUNTAINEER (NCT03043313) enrolled pts w/ local HER2+ mCRC using ≥1 method: IHC, ISH, and/or NGS testing; retrospective central assessment of HER2 status was performed on multiple platforms. Pts in cohorts A+B were treated w/ TUC + Tras; pts in cohort C were treated w/ TUC monotherapy. Confirmed objective response rates (cORRs) using RECIST v1.1 per BICR, DOR, and PFS were calculated for pts treated w/ TUC + Tras based on each central testing method. Results: 114 pts were enrolled in cohorts A (n = 45), B (n = 39), and C (n = 30) w/ HER2+ tumors per ≥1 local testing method. Of samples submitted for central testing for Cohorts A and B, 70 per IHC/FISH, 50 per tissue NGS, and 71 per blood NGS had evaluable results. In all cohorts, there was 81.0% (95% CI, 68.6-90.1) agreement between blood and tissue NGS, 92.6% (95% CI, 83.7-97.6) between IHC/FISH and tissue NGS, and 79.5% (95% CI, 69.2-87.6) between IHC/FISH and blood NGS. In cohorts A and B, pts w/ HER2+ tumors by central IHC/ISH had a mDOR of 16.4 months (95% CI: 10.6-25.5) and mPFS of 10.1 months (95% CI: 4.2-15.2). cORR was 41.1% to 47.7% for the 3 assays. Detailed HER2 results are presented in the Table. Conclusions: Percent agreement of HER2 status was highest w/ tissue-based platforms. Detection of HER2 amplification by ctNDA NGS is useful; however, pts w/o HER2 amplification should be confirmed w/ a tissue-based assay. HER2 status by all 3 platforms predicted tx response to TUC + Tras. cORR in IHC2+/ISH+ was numerically lower but remained clinically relevant. These data support use of the above methods to identify HER2+ mCRC patients that may benefit from TUC + Tras. Clinical trial information: NCT03043313 . [Table: see text]

Abstract 4019: Preclinical activity of ELVN-002: A potent, selective, and irreversible HER2 and pan-HER2 mutant small molecule inhibitor for the treatment of HER2 driven malignancies

Abstract Mutation or amplification of the human epidermal growth factor receptor 2 (HER2) gene has been identified in numerous solid tumor types, including non-small cell lung cancer (NSCLC), breast, and colorectal cancer. These genetic alterations are believed to confer elevated HER2 activity resulting in oncogenic transformation. In NSCLC, an estimated 3% of patients harbor activating mutations in the HER2 gene. Enhertu (T-DXd), a HER2-specific antibody-drug conjugate was recently approved as an important therapy for this patient population. However, for patients who are not candidates for T-DXd or have discontinued treatment due to an adverse event or disease progression, there exist few therapeutic options. Accordingly, HER2 inhibitors have the potential to provide meaningful therapeutic benefit to these patients. Due to significant structural homology between EGFR and HER2, most investigational small molecule HER2 kinase inhibitors are dual EGFR/HER2 inhibitors and are substantially dose-limited by EGFR-related toxicities in patients. These toxicities likely limit their clinical activity. Currently, tucatinib is the only HER2-selective small molecule inhibitor approved for metastatic breast cancer in combination with trastuzumab and capecitabine. However, it lacks sufficient potency against several of the key clinically relevant mutations, including HER2 YVMA, the most common exon 20 insertion mutation, prevalent in HER2 mutant NSCLC. ELVN-002 is a potent, irreversible inhibitor of HER2 with a &amp;gt;100-fold selectivity over EGFR. ELVN-002 potently inhibits the phosphorylation of HER2 in cell lines endogenously expressing HER2 or in those engineered to express specific clinically relevant HER2 mutants. This activity readily translates into marked anti-proliferative activity against cell lines dependent upon HER2 for their growth and survival. In addition, ELVN-002 is highly selective for HER2 and HER2 mutants versus wild-type EGFR as demonstrated via assessment of both EGFR-driven proliferation and EGFR phosphorylation in cells. Furthermore, ELVN-002 is highly active in mouse models of HER2-driven cancers, including subcutaneous models driven by wild type or mutant HER2. ELVN-002 is also active in an NCI-N87 wild-type HER2 intracranial model. In all models tested, ELVN-002 treatment is well tolerated and resulted in tumor regressions at exposures believed to be clinically achievable. Due to its selectivity and breadth of activity versus the clinically relevant HER2 mutants, ELVN-002 has the potential to be an effective treatment for HER2 mutant NSCLC as well as other HER2 mutant amplified solid tumors, including metastatic HER2 positive breast cancer. Additionally, ELVN-002 could provide a meaningful therapeutic option for patients with CNS metastases. ELVN-002 has been selected for clinical development. Citation Format: Monette Aujay, Amanda J. Broad, Stefan D. Gross, Li Ren, Joseph P. Lyssikatos, Samuel Kintz, Qi Wang, Helen Collins. Preclinical activity of ELVN-002: A potent, selective, and irreversible HER2 and pan-HER2 mutant small molecule inhibitor for the treatment of HER2 driven malignancies. [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 4019.

A randomized phase 2 study of trastuzumab and pertuzumab (TP) compared to cetuximab and irinotecan (CETIRI) in advanced/metastatic colorectal cancer (mCRC) with HER2 amplification: SWOG S1613.

140 Background: HER2 ( ERBB2) over-expression and amplification (HER2+) is seen in a small but distinct subset (2-3%) of mCRC and is enriched in RAS/BRAF wild type (WT) tumors. This subset is characterized by a limited response to anti-epidermal growth factor receptor monoclonal antibody-based (anti-EGFR) therapy and a promising response to dual-HER2 inhibition. Methods: In this multicenter, open label, randomized, phase 2 trial, we enrolled 54 patients with RAS/BRAF WT HER2+ mCRC who had had disease progression after 1 or 2 previous therapies. HER2 status was confirmed centrally with immunohistochemistry (IHC) and in-situ hybridization (ISH). HER2+ was defined as IHC 3+ or 2+ and ISH amplified (dual-probe HER2/CEP17 ratio &gt; 2.0). Patients were then randomly assigned in a 1:1 ratio to receive either TP (trastuzumab [loading 8 mg/kg then 6 mg/kg] + pertuzumab [loading 840 mg then 420 mg] every 3 weeks) or CETIRI (cetuximab 500 mg/m2 + irinotecan 180 mg/m2 every 2 weeks). Crossover was allowed for patients on CETIRI arm to TP (cTP) after progression. Restaging (per RECIST v1.1) was performed at 6 and 12 weeks and then every 8 weeks until progression. The primary endpoint was progression-free survival (PFS). Key secondary endpoints were overall response rate (ORR), overall survival (OS) and safety. Results: A total of 54 (out of planned 62 due to low accrual) patients were randomized to TP (26) and CETIRI (28) between 10/2017 and 12/2021. By 8/18/2022, 20 patients had crossed over to cTP arm. One CETIRI patient was not analyzable. The results for key endpoints by protocol defined stratification factors, prior irinotecan (Piri) (yes or no) and HER2/CEP17 ratio (HCR) (&gt;5 or ≤5), are summarized as of data cut-off of 9/6/2022. PFS did not vary significantly by treatment: medians 4.4 (95%CI: 1.9 – 7.6) months in TP group and 3.7 (95%CI: 1.6 – 6.7) months in CETIRI group (p = 0.35). Grade ≥3 adverse events occurred in 23%, 46% and 40% of patients in TP, CETIRI and cTP groups. Conclusions: Dual-HER2 inhibition with TP appears to be a safe and effective treatment option for patients with RAS/BRAF WT HER2+ mCRC with a promising response rate of 31%. Higher level of HER2 amplification may provide a greater degree of clinical benefit from TP compared to CETIRI. Future correlative efforts will explore biomarkers of response/resistance with this strategy. Clinical trial information: NCT03365882 . [Table: see text]

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer

BackgroundPrecision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism.MethodsWe generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient’s cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug’s efficacies against HER2 E401G.ResultsWe confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective.ConclusionsAfatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.

HER2 induces cell scattering and invasion through ∆Np63α and E-cadherin.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer constitutes approximately 30% of human breast cancers and is associated with poor outcomes. ∆Np63 is considered a metastasis inhibitor involved with cancer progression. This study aimed to clarify the roles and mechanisms of HER2 and ∆Np63 on scattering and invasion of MCF10A cells. Wild-type or mutant HER2 was cloned and transfected into MCF10A cells. Cell counting and transwell assays were applied to unveil the impact of HER2 upregulation and mutation on proliferation, cell scattering, and invasion. Western blotting and cell accounting were used to investigate the roles of ∆Np63 and p27. In vivo lung colonization assay was used to reveal the influences of HER2 and ∆Np63a on tumor metastasis. The results indicated HER2 remarkably enhanced cell proliferation, invasion, and scattering. Overexpression of either ΔNp63 or E-cadherin led to attenuated HER2-mediated regulation of cell migration, invasion, and scattering. Furthermore, we confirmed that HER2 enhanced cell proliferation but not migration through p27 and independent ∆Np63a. The results revealed that ∆Np63α contributed to the inhibition of HER2-induced metastasis. Collectively, our findings may further our understanding of the regulation of tumor progression and metastasis.

Abstract 5376: Pleclinical investigation of T-DXd internalization and payload release in HER2 mutant non-small cell lung cancer cells

Abstract Introduction: T-DXd (trastuzumab deruxtecan) is an antibody-drug conjugate (ADC) targeting human epidermal growth factor receptor 2 (HER2) composed of a humanized anti-HER2 monoclonal antibody conjugated to a DNA topoisomerase I inhibitor, DXd through a linker. Internalization of HER2-T-DXd followed by DXd release is a crucial process for T-DXd to exert its antitumor activity. T-DXd induced robust and durable responses in HER2 mutant metastatic non-small cell lung cancer (NSCLC) patients in DESTINY-Lung01 study, including those with low HER2 expression levels (Li BT, et al. NEJM 2021). One hypothesis of this T-DXd sensitivity of HER2 mutant NSCLC cells involves the potential of enhanced receptor and ADC internalization in these cells (Li BT, et al. Cancer Discov 2020). To examine this possibility further, we investigated the effect of HER2 mutations on T-DXd internalization and DXd release in transgenic cells expressing various HER2 mutants, as well as on T-DXd sensitivity in vivo. Methods: We established transgenic NCI-H322 cells expressing wild-type (WT) HER2 or various HER2 mutants and evaluated T-DXd internalization in these cells. Antitumor activity of T-DXd was examined in xenograft mouse models of transgenic NCI-H322 cells and compared between WT and each mutant. DXd concentration in tumor was also determined. In addition, in vitro internalization activity and antitumor activity of T-DXd were evaluated using NCI-H1781, the NSCLC cell line harboring an endogenous HER2 mutation (G776delinsVC). Results: Transgenic NSCLC cells expressing various HER2 mutants showed higher T-DXd internalization than WT HER2 expressing cells. There also was a negative correlation between the surface HER2 expression level and T-DXd internalization among various mutants and WT expressing cells. In xenograft models with transgenic cells, some HER2 mutant models showed higher response to T-DXd than WT HER2 model, with greater than 2-fold increase in G660D, YVMA, G776delinsVC, V777_G778insGSP, and V842I. Tumor DXd concentration was positively correlated with the antitumor activity. In an endogenous HER2 mutant NSCLC cell line (NCI-H1781), T-DXd also showed high internalization activity and potent antitumor activity. Conclusion: In vitro study with transgenic NSCLC cells suggested that T-DXd internalization in HER2 mutant tumors was more efficient than in WT HER2 tumor. Some of those HER2 mutant cell lines showed increased T-DXd sensitivity and higher payload release in the tumor in vivo, suggesting the enhanced internalization played a role in the mechanism of T-DXd sensitivity in these HER2 mutant transgenic NSCLC cells. Some mutants may be more or less clinically relevant. However, the relationship between the in vitro internalization efficiency and in vivo antitumor activity in each mutant was not consistent among different mutants, thus these results need to be interpreted cautiously. Citation Format: Atsushi Yamasaki, Misa Ikuta, Manabu Abe, Tsuneo Deguchi, Yasuki Kamai, Kenji Nakamaru, Toshinori Agatsuma. Pleclinical investigation of T-DXd internalization and payload release in HER2 mutant non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5376.

Abstract 2691: Expression pattern and prognostic significance of HER family members and EGFRvIII in pancreatic cancer

Abstract Pancreatic cancer is one of the most aggressive, heterogeneous, and fatal types of human cancers. There is urgent need for discovery of biomarkers of prognostic significance, predictive value and as well as therapeutic targets in patients with pancreatic cancer. In this study, we examined the relative expression and prognostic significance of all HER family members and the type III EGFR mutant (EGFRvIII) in patients with pancreatic ductal adenocarcinoma (PDC). The expression levels of wild-type EGFR (wtEGFR), HER2, HER3, HER4 and EGFRvIII were determined in tumor specimens from 43 patients by immunohistochemistry using antibodies DAK-H1-WT, Neu 3B5, SP71, HFR1 and NBP2-50599 respectively. The staining was scored based on the percentage of positive tumor cells, the intensity (i.e. strong 3+, moderate 2+, weak 1+) and the cellular location of immunostaining (i.e. membranous, cytoplasmic or nuclear). The association between the expression level of the biomarkers and patient overall survival was evaluated using Chi-squared, Kaplan-Meier survival curves and log rank test. At cut-off value of above 5%, we found weak to moderate expression of wtEGFR, HER2, HER3, HER4 and EGFRvIII in 4.7%, 14%, 14%, 20.9% and 2.3% of the cases examined respectively. The cellular location of wtEGFR and HER2 was found to be predominantly cytoplasmic, HER-3 membranous and cytoplasmic whereas HER4 expression was found to be mainly nuclear. There was no statistically significant association between the expression level of these markers and patient overall survival. The expression levels of the above biomarkers were also evaluated in tissue microarrays (TMAs) from 40 cases of pancreatic ductal adenocarcinoma. Interestingly, at the same cut-off value, 62.5%, 75%, 0%, 55% and 5% of the TMAs were positive for wtEGFR, HER2, HER3, HER4 and EGFRvIII respectively. Overall, we found that the expression pattern of HER family members was heterogenous, and with EGFRvIII expression being very low in both whole tumor sections and TMAs from patients with pancreatic cancer. Taken together, our results provide support for more detailed studies of co-expression of all HER family members in a larger group of pancreatic cancer patients. Such studies should help to unravel their potential as targets for therapeutic interventions as well as their prognostic significance and predictive value for response to therapy. Citation Format: Tanzeel Khan, Said A. Khelwatty, Izhar Bagwan, Alan Seddon, Angus Dalgleish, Satvinder Mudan, Helmout Modjtahedi. Expression pattern and prognostic significance of HER family members and EGFRvIII in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2691.

Abstract 5340: GATA3-VMP1 axis induces trastuzumab resistance via promoting autophagy in HER2-positive breast cancer cells

Abstract Trastuzumab is the most important agent for HER-2 positive breast cancer both in neoadjuvant/adjuvant and metastatic settings. However, primary or acquired resistance to trastuzumab often compromises the efficacy and impairs patients’ survival. Vacuole Membrane Protein-1(VMP1), a transmembrane protein located in the endoplasmic reticulum membrane, has been reported to induce chemotherapy resistance by promoting autophagy. Our preliminary study found that the expression of GATA Binding Protein 3(GATA3) and VMP1 are positively correlated in tissue samples from HER-2 positive breast cancer patients. GATA3 and VMP1 were significantly increased in both protein and mRNA levels in trastuzumab-resistant breast cancer cell lines as compared to the sensitive counterparts. Furthermore, the expression autophagy signature protein P62 significantly decreased in trastuzumab-resistant cell lines, while the expression of autophagy activation protein ATG5 increased, indicating a close relationship between autophagy and trastuzumab resistance. In tissue samples from patients treated with neoadjuvant regiments containing trastuzumab, the expression of VMP1 significantly increased in the non-pCR (non-pathological complete response) group than the pCR group. Overexpression of VMP1 in wild-type breast cancer cells decreased their sensitivity to trastuzumab. On the contrary, when VMP1 was knocked-down in trastuzumab-resistance cells, the sensitivity of cell lines to trastuzumab was increased. Moreover, overexpressed GATA3 in wild-type HER-2 positive cell lines, the expression of VMP1 increased as well as the expression of ATG5, the signature protein of autophagy, suggesting that the transcription factor GATA3 may play a role in the transcriptional activation of VMP1. The promoter sequence was predicted, and it was found that GATA3 regulatory core sequence "TGATA" existed in front of the transcription start site from -327bp to -322bp, indicating that GATA3 may transcriptionally activate the expression of VMP1 and promote the occurrence of autophagy. TCGA (The Cancer Genome Atlas) correlation analysis showed that, both VMP1 and GATA3 are positively correlated with classical autophagy related factors ATG2B, ATG7 and BCL1, indicating VMP1 and GATA3 may be involved in the positive regulation of autophagy in HER-2 positive breast cancer. Collectively, our results suggest that GATA3 transcriptionally activates VMP1 to promote autophagy, resulting in the induction of trastuzumab resistance in HER-2 positive breast cancer. Further mechanistic study on autophagy in contribution to trastuzumab resistance is warranted. Citation Format: hao-yu Lin, Yu-cui Gu, Zhen-hao Wang, Kai-yuan Huang, He-xing Sun, De Zeng, Yuan-ke Liang. GATA3-VMP1 axis induces trastuzumab resistance via promoting autophagy in HER2-positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5340.

Activity and mechanism of acquired resistance to tarloxotinib in HER2 mutant lung cancer: an in vitro study.

BackgroundHER2 (ERBB2) activating mutations are present in 2–3% of lung adenocarcinomas; however, no targeted therapy is approved for HER2-altered lung cancers. A novel pan-HER inhibitor, tarloxotinib, is designed to release the active form (tarloxotinib-E) under hypoxic conditions in tumor tissues after being administered as a prodrug. Following the evaluation of the in vitro activity of tarloxotinib-E in HER2-mutant cells, we explored the mechanisms of resistance to tarloxotinib-E in these cells.MethodsGrowth inhibitory assays were performed with tarloxotinib-E and its prodrug using Ba/F3 cells expressing one of six HER2 mutations or wild-type (WT) HER2, in addition to H1781 cells with HER2 exon 20 insertions. Resistant clones were established from N-ethyl-N-nitrosourea (ENU)-treated HER2-mutant Ba/F3 cells and H1781 cells by chronic exposure to tarloxotinib-E.ResultsTarloxotinib-E showed potent activity against HER2-mutant Ba/F3 cells and H1781 cells. Furthermore, the half maximal inhibitory concentration (IC50) of tarloxotinib (inactive form) for WT HER2 was 180 times higher than that of tarloxotinib-E, indicating a wide therapeutic window of tarloxotinib. We established 30 resistant clones with secondary mutations of HER2 by ENU mutagenesis, all of which harbored C805S in exon 20. In the analysis of H1781 cells that acquired resistance to tarloxotinib-E, we found that increased HER3 expression was the molecular mechanism of tarloxotinib-E resistance.ConclusionsTarloxotinib-E exhibited potent activity against cell line models with HER2 mutations. We identified a secondary C805S HER2 mutation and HER3 overexpression as the mechanisms of acquired resistance to tarloxotinib-E.

Abstract 514: Specific anti-HER2 host immunity conferred by HER2-targeted antibody drug conjugate therapy and checkpoint blockade

Abstract Using HER2-targeted antibody drug conjugates (ADC), we tested the therapeutic efficacy, direct impact on cancer stem cells (CSCs), and potential added benefit when combined with PD-L1 blockade in immunocompetent mouse tumor models. Two murine mammary tumor models were used in this study: D2F2/E2, a cell line resulting from stable transfection of parental D2F2 murine mammary tumor cells with wild-type human HER2, and 4T1-HER2, derived from parental 4T1 by stable transduction with wild-type human HER2. We demonstrate that HER2-targeted ADC treatment induced both cellular and humoral anti-HER2 adaptive immune responses which effectively targeted both bulk tumor and cancer stem cells. We recently investigated induction of host immunity using a tumor re-challenge strategy in tumor free animals subjected to ADC or ADC + anti-PD-L1 therapy. D2F2/E2 tumor-bearing mice were treated with ADC or ADC + anti-PD-L1 to generate tumor free mice. These tumor free animals were re-challenged with the relevant tumor D2F2/E2 or irrelevant tumor WT 4T1 respectively. While all the tumor free animals re-challenged with WT 4T1 showed aggressive tumor growth, ADC-treated tumor free animals re-challenged with D2F2/E2 demonstrated significantly (p&amp;lt;0.0001) slower tumor growth compared with the age-matched naive mice, and this protection was significantly (p=0.0078) enhanced in ADC + anti-PD-L1-treated tumor free animals, resulting in completely rejection of the re-challenge and thus documenting immunological memory formation. B cell activation was obvious by the significant upregulation of Ki67 and GL7 CD86 on both CD45+CD19+ tumor-infiltration lymphocytes (TILs) and splenocytes. B cell differentiation was evident by the upregulation of GL7 on both TILs and splenocytes, as well as the expression of CD138 on splenotytes. We found that IgG1 was the predominant isotype in treated mice vs. IgG2a, IgG2b, IgG3, and ADC + anti-PD-L1 dual treatment drastically increased the amount of IgG1 in serum. In addition, ADC+ anti-PD-L1 dual treatment significantly increased the frequency of CD8+IFNγ+ cells in TILs. Both ADC alone (p=0.0004) and ADC+anti-PD-L1 dual treatment (p=0.01) significantly decreased CD4+FoxP3+ regulatory T cells. These results provide evidence that HER2-targeted ADC + anti-PD-L1 immunotherapy-induced B cell activation and differentiation along with the modulation of T cell subsets could confer host anti-HER2 immunity. Citation Format: You Qin, Hannah E. Dobson, Frank I. Comer, Alfred E. Chang, Max S. Wicha, Qiao Li. Specific anti-HER2 host immunity conferred by HER2-targeted antibody drug conjugate therapy and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 514.

Mutational analysis of rare insertions and deletions in exon 18 and 19 of HER2 in Chinese patients with different cancer types.

3069 Background: HER2 belongs to the same family with EGFR and is known as an important cancer driver gene. Kinase domain insertions and deletions (KD indels) are frequent driver mutations in both EGFR and HER2. The most common HER2 KD indels are the exon 20 insertions while others are rarely reported. Our study aimed to investigate the role of less common HER2 KD indels in solid tumors. Methods: This study was performed in 63,267 Chinese patients including 53,591 patients with lung cancer, 5,888 patients with colorectal cancer, 3,774 patients with breast cancer and 14 patients with renal pelvis cancer. Tissue or plasma samples from the patients were subjected to capture-based targeted sequencing covering HER2 and other cancer related genes. The sequencing data of each patient were retrospectively collected and analyzed. The HER2 exon 18/19 indels identified in our study were compared with data from COSMIC and MSKCC. In vitro analysis in Ba/F3 cell lines was performed to assess drug response of different HER2 exon 18/19 indels. Results: We identified recurrent HER2 KD indels in exon 18 and 19, with a frequency of 0.04% (25/63,267). The data from COSMIC and MSKCC reported the prevalence of HER2 exon 18/19 indels ranging from 0.04% to 0.23% among breast, cervical, and pancreatic cancers. In our study, HER2 exon 18/19 indels were identified in 20 patients with lung cancer (0.037%), two with colorectal cancer (0.034%), two with breast cancer (0.053%) and one with renal pelvis cancer (7.143%). Only two patients (8%) harbored concurrent actionable driver mutations including EGFR mutation and MET amplification. Meanwhile, high level of normalized allelic frequency of HER2 exon 18/19 indels was presented in most patients (22/25, 88%). In lung cancer, the presence of EGFR driver mutation was less common in patients with HER2 exon 18/19 indels than wild type HER2 (5% vs. 47.4%, p &lt; 0.01). The rates of concurrent driver mutations in lung cancer were comparable between HER2 exon 18/19 indels and the two established HER2 drivers, exon 20 insertions and S310 mutations. The in vitro proliferation assay demonstrated that E698_P699insLL mutation in HER2 exon 18 and L755_E757delinsPQ mutation in HER2 exon 19 both conferred a survival advantage to Ba/F3 cells. Dose-response curves showed inhibitory effects on cell viability of several HER2 tyrosine kinase inhibitors including neratinib, lapatinib, poziotinib and afatinib. In particular, lapatinib, poziotinib and afatinib demonstrated comparable or stronger inhibitory ability toward the two HER2 mutants than wild type HER2 in terms of IC50. Conclusions: Our study revealed a novel class of HER2 KD indels in exon 18/19 that may act as driver mutations in several cancer types. The drug response observed in vitro indicated the potential to use anti- HER2 targeted therapies for HER2 exon 18/19 indels. Further studies on this rare type of HER2 mutation are warranted.