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Introduction: NER is a pan-HER tyrosine kinase inhibitor (TKI) approved for the treatment of HER2+ BC in the adjuvant setting following trastuzumab and in combination with capecitabine for advanced disease. Resistance to small molecule TKIs like NER can develop in the clinic. Pre-clinical studies have highlighted that retinoic acid can inhibit BC growth and modulate HER2 signalling pathways. The RAR family of nuclear transcription factors consists of RARα, RARβ and RARγ. The synthetic retinoic acid fenretinide (FN) acts as a pan-RAR agonist, while AGN194310 (AG) acts as a pan-RAR antagonist. In order to investigate the anti-proliferative potential of co-targeting RAR and HER2 pathways in sensitive and resistant BC cell line models, we examined the effect of FN and AG in combination with NER in two HER2+, estrogen receptor-negative, trastuzumab-resistant cell lines HCC1569 and HCC1954, and their NER-resistant (NR) sub-lines HCC1569-NR and HCC1954-NR. Methods: HCC1569 and HCC1954 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. NR cell lines were generated by continuous exposure to 150nM NER for 6 months. 10 mM stock solutions of FN (H7779-Sigma), AG (SML2665-Sigma) and NER (supplied by Puma Biotechnology, Inc) were made in DMSO. Proliferation was measured as percentage growth versus DMSO control using an acid phosphatase based assay after 5 days drug exposure. The half-maximal inhibitory concentration (IC50) was calculated for each drug using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50) using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research found that the NR cell lines were >10-fold resistant to NER (HCC1569-NR IC50 0.44 ± 0.1 μM, HCC1954-NR IC50 0.198 ± 0.019 μM) compared to the parental HCC1569 (IC50 0.018 ± 0.015 μM) and HCC1954 (IC50 0.017 ± 0.001 μM) cell lines. Pan-RAR agonism by FN had a potent anti-proliferative effect in the HCC1569 (FN IC50 0.22 ± 0.02 μM) and the HCC1569-NR cell lines (FN IC50 0.28 ± 0.13 μM), with the HCC1954 and HCC1954-NR cell lines proving less sensitive (IC50 6.47 ± 1.3 μM and 1.9 ± 0.2 μM, respectively). When combined with NER, FN produced a strong antagonistic effect in the HCC1569 cell line (CI value: 15.63 ± 9.5) and a strong synergistic effect in the HCC1954 cell line (CI value: 0.42 ± 0.06). For the NR cell line models, the NER/FN combination proved synergistic (HCC1569-NR, CI value: 0.84 ± 0.46) or additive (HCC1954-NR, CI value: 0.97 ± 0.15). Next, we wanted to assess the impact of antagonising rather than activating RAR activity in our cell line models. All four cell lines were less sensitive to antagonist AG (IC50 >8μM for all cell lines) compared to FN. The addition of AG to NER resulted in responses diametrically opposed to the FN/NER combination. The AG/NER combination produced a strong synergistic effect in the HCC1569 cell line (CI value: 0.52 ± 0.17), an antagonistic effect in the HCC1954 cell line (CI value: 2.1 ± 0.4) and an antagonistic effect in both NR cell lines (HCC1954-NR, CI value: 2.69 ± 0.6 and HCC1569-NR, CI value: 1.58 ± 0.12). Conclusions: This pre-clinical study suggests involvement of the RAR signalling pathway in response to NER and the development of NR. Results also suggest pan-RAR agonism, rather than pan-RAR antagonism, as a potential therapeutic strategy to overcome NR. Further investigation is warranted to determine how targeting the RAR signalling pathway may assist in the treatment of HER2+ BC. Citation Format: Debbie O’Reilly, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-09.

Overall sequencing CONSORT diagram. Flow diagram of study patients and analyzed biospecimens processed through and selected for central sequencing using MSK-IMPACT and Guardant360. cfDNA, cell-free DNA; FFPE, formalin-fixed paraffin-embedded; MSK-IMPACT, Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets; QC, quality control.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib.Significance:HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer.This article is highlighted in the In This Issue feature, p. 161

Supplementary data

Purpose:We wanted to determine the prognosis and the phenotypic characteristics of hormone receptor–positive advanced breast cancer tumors harboring an ERBB2 mutation in the absence of a HER2 amplification.Experimental Design:We retrospectively collected information from the American Association of Cancer Research-Genomics Evidence Neoplasia Information Exchange registry database from patients with hormone receptor–positive, HER2-negative, ERBB2-mutated advanced breast cancer. Phenotypic and co-mutational features, as well as response to treatment and outcome were compared with matched control cases ERBB2 wild type.Results:A total of 45 ERBB2-mutant cases were identified for 90 matched controls. The presence of an ERBB2 mutation was not associated with worse outcome determined by overall survival (OS) from first metastatic relapse. No significant differences were observed in phenotypic characteristics apart from higher lobular infiltrating subtype in the ERBB2-mutated group. ERBB2 mutation did not seem to have an impact in response to treatment or time-to-progression (TTP) to endocrine therapy compared with ERBB2 wild type. In the co-mutational analyses, CDH1 mutation was more frequent in the ERBB2-mutated group (FDR < 1). Although not significant, fewer co-occurring ESR1 mutations and more KRAS mutations were identified in the ERBB2-mutated group.Conclusions:ERBB2-activating mutation was not associated with a worse OS from time of first metastatic relapse, or differences in TTP on treatment as compared with a series of matched controls. Although not significant, differences in coexisting mutations (CDH1, ESR1, and KRAS) were noted between the ERBB2-mutated and the control group.

Supplementary Figure from Natural History and Characteristics of ERBB2-mutated Hormone Receptor–positive Metastatic Breast Cancer: A Multi-institutional Retrospective Case–control Study from AACR Project GENIE

Supplementary Table from Natural History and Characteristics of ERBB2-mutated Hormone Receptor–positive Metastatic Breast Cancer: A Multi-institutional Retrospective Case–control Study from AACR Project GENIE

Background: HER2 mutations are oncogenic drivers in a subset of metastatic breast cancers (MBC). Neratinib (N) is an oral, irreversible pan-HER tyrosine kinase inhibitor with preclinical and clinical activity against HER2 mutations. Genomic analyses from paired biopsies following N ± fulvestrant (F) suggest that resistance to N may occur via amplification of the mutant allele or by acquisition of secondary HER2 mutations. Addition of trastuzumab (T) to N+F showed encouraging clinical activity with durable responses in the SUMMIT trial in hormone receptor-positive (HR+), HER2-mutant MBC, including patients (pts) who had previously received cyclin-dependent kinase 4 & 6 inhibitors (CDK4/6i) [Jhaveri et al. SABCS 2020]. On the basis of these findings, and in order to better understand the contribution of N to the activity of the N+F+T combination, SUMMIT has recently been expanded to include a randomized Simon 2-stage comparison of N+F+T vs. F+T vs. F in pts with HR+, HER2-mutated, HER2-negative MBC who were exposed to CDK4/6i. Enrollment for stage 1 is now complete (N+F+T, n=7; F+T, n=7; F, n=7), and results will be forthcoming once the data are mature. Here we report updated findings from the breast cancer cohorts of the SUMMIT trial for which data are currently available. Methods: The phase 2 SUMMIT trial (NCT01953926) enrolled pts with HR+, HER2-negative MBC whose tumors harbored activating HER2 mutation(s) identified by genomic sequencing. Prior to starting the randomized portion of the trial, these patients were enrolled in a non-randomized cohort and received N+F+T (oral N 240 mg/d, i.m. F 500 mg d1&15 of cycle 1 then q4w, i.v. T 8 mg/kg initially then 6 mg/kg q3w). Following initiation of the randomized portion of the trial, these pts received N+F+T, F+T or F (1:1:1 ratio; dose schedules as above). Pts with HER2-mutant triple-negative breast cancer (TNBC) were enrolled in a non-randomized cohort and received N+T (dose schedules as above). Loperamide prophylaxis was mandatory during the first 2 treatment cycles. There was no restriction on the number of prior lines of systemic therapy for MBC. Efficacy endpoints: investigator-assessed objective response rate and clinical benefit rate (RECIST v1.1 or other defined criteria); duration of response; best overall response. Results: Prior to enrolling the randomized cohort, 24 pts with HR+, HER2-mutated MBC who had previously received CDK4/6i were enrolled in the non-randomized cohort and received N+F+T, and 17 pts with HER2-mutant TNBC were enrolled and received N+T, as of 18-Jun-2021. Data for randomized pts are not yet mature. HER2 allelic variants across both cohorts (pts may have >1 mutation): kinase domain hotspots (n=26); exon-20 insertion (n=9); extracellular domain hotspot (n=4); exon-19 deletion (n=1); transmembrane domain missense (n=1); kinase domain non-hotspot (n=2). Efficacy findings are reported in the Table. Diarrhea was the most commonly reported adverse event: N+F+T (non-randomized cohort), 96%; N+T (TNBC cohort), 94%. No grade 4 diarrhea was reported. Conclusions: N+F+T is a promising combination for pts with HR+, HER2-mutated MBC with prior exposure to CDK4/6 inhibitors. N+T also showed encouraging activity in HER2-mutated TNBC. The first results from the randomized comparison of N+F+T vs. F+T vs. F in pts with HR+, HER2-mutated MBC (Simon stage 1 analysis) will be presented at the meeting. Table: Efficacy findingsHR+, HER2-mutated, HER2-non-amplified MBCHER2-mutant TNBCN+F+T (n=24)N+T (n=17)Confirmed objective response,a n (%)11 (46)5 (29)CR0 (0)1 (6)PR11 (46)4 (24)ORR, % (95% CI)46 (26–67)29 (10–56)Best overall response, n (%)13 (54)7 (41)CR0 (0)1 (6)PR13 (54)6 (35)Best overall response rate, % (95% CI)54 (33–74)41 (18–67)Medianb DOR, months (95% CI)14.4 (6.4–NR)NRClinical benefit, n (%)14 (58)6 (35)CR or PR11 (46)5 (29)SD ≥24 weeks3 (13)1 (6)CBR,b % (95% CI)58 (37–78)35 (14–62)aORR defined as either a CR or PR confirmed no less than 4 weeks after the response criteria are met; bCBR defined as confirmed CR or PR or SD for ≥24 weeks. Note: Tumor response is based on investigator tumor assessments per RECIST v1.1 for HR+, HER2-mutated cohort, and RECIST v1.1 or modified PERCIST for HER2-mutated TNBC cohort. CBR, clinical benefit rate; CI, confidence interval; CR, complete response; DOR, duration of response; F, fulvestrant; HR+, hormone receptor-positive; MBC, metastatic breast cancer; N, neratinib; NR, not reached; ORR, objective response rate; PERCIST, Positron Emission Tomography Response Criteria in Solid Tumors; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; T, trastuzumab, TNBC, triple-negative breast cancer. Citation Format: Komal Jhaveri, Haeseong Park, James Waisman, Jonathan W Goldman, Angel Guerrero-Zotano, Valentina Boni, Barbara Haley, Ingrid A Mayer, Adam Brufsky, Eddy S Yang, José A García-Sáenz, François-Clement Bidard, John Crown, Bo Zhang, Aimee Frazier, Irmina Diala, Lisa D Eli, Brian Barnett, Hans Wildiers. Neratinib + fulvestrant + trastuzumab for hormone receptor-positive, HER2-mutant metastatic breast cancer and neratinib + trastuzumab for triple-negative disease: Latest updates from the SUMMIT trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS4-10.

Background: HER2-positive (+) breast cancer (BC) represents a significant clinical burden due to an aggressive phenotype and propensity for brain metastasis. Up to 70 % of HER2+ BCs are also oestrogen receptor (ER)-positive, with receptor crosstalk often attributed to anti-HER2 therapeutic resistance. Neratinib is a pan-HER tyrosine kinase inhibitor, currently approved for the treatment of HER2+ BC. Dual-blockade with anti-HER2 and endocrine therapies (ETs) has shown positive outcomes in clinical trials, without the added toll of traditional chemotherapy. Fulvestrant is the only currently approved selective ER degrader (SERD), but it is hindered by poor bioavailability, leading to several orally available SERDs being developed. CDK4/6 inhibitors have been approved for ER+ BC, and trials are ongoing to see if their addition also benefits ER+/HER2+ BC patients. The aim of this study is to outline the benefit of ETs in HER2+ BC and to investigate neratinib/ET combinations with potential for the treatment of HER2+/ER+ BC. Methods: BC patient overall (OS) and recurrence free (RFS) survival and clinical data for a number of independent breast cancer studies was sourced from the METABRIC study via cBioportal and from KMplotter. Patients were stratified according to HER2 status, ER status and treatment with ET. GraphPad Prism 8 was then used to produce Kaplan-Meier graphs and to calculate difference in patient survival according to treatment with ET. Fulvestrant (SERD, #S1191), AZD9496 (novel, orally bioavailable SERD, #S8372) and ribociclib (CDK4/6 inhibitor, #S7440) were obtained from Selleckchem. Neratinib was obtained from Puma Biotechnology, Inc. Estradiol (Sigma, #E2758) treatment was carried out at 1nM. To assess drug effect, the HER2+/ER+ BC cell line BT-474 was treated for 5 days before growth inhibition was measured using an acid phosphatase-based proliferation assay. Compounds were combined at fixed ratios. Drug IC50 and combination index (CI) values were calculated using Calcusyn. Results: Analysis of the METABRIC study shows HER2+ BC patients who receive ET, numerically experience a longer median survival, with particular separation seen in early OS (ET = 113.8 months vs no ET= 85.50 months, HR= 0.8797, 95% CI: 0.6402 - 1.209, p=0.0987). In addition, analysis of KMPlotter data shows HER2+ patients who receive ET may also experience greater 5 year RFS (HR= 0.7639, 95% CI: 0.5452 to 1.070, p= 0.0539). Despite this potential benefit, the METABRIC dataset showed within a cohort of HER2+ patients, 10.5% were ER+ but did not receive ET. An in vitro cell line model of HER2+/ER+ BC was highly sensitive to neratinib (N) at nanomolar concentrations (IC50 = 2.0549± 0.7 nM). In contrast, fulvestrant (F), AZD9496 (A) and ribociclib (R) were less effective as single agents and an IC50 was not achieved (% growth @ 5µM: F = 54.7 ± 8.5%, A = 61.3 ± 7.8% and R = 95.6 ± 8.7%). The combination of N with F was compared to N with A. Both combinations demonstrated synergy, defined as CI < 1 @ ED50 (N+F CI value = 0.21385 ± 0.15, N+A CI value = 0.25448 ± 0.26). The combination of N + R did not improve the efficacy of N alone ( % growth at max, N = 6.8 ± 1.7% vs N +R = 10.8 ± 2.8%, p>0.9999). Further, triplet combinations with R did not significantly affect cell growth for either the N + F or the N + A combinations (% growth @ max conc., N + F= 5.8 ± 1.6%, N+F+R = 7.2 ± 1.2 %, N+A= 8.2 ± 2%, N+A+R= 9.8 ± 1.7%, p>0.9999). Estradiol supplementation at time of drug treatment significantly increased the growth inhibitory effect of A (p = 0.0489 @ 5µM). Conclusions: Using publicly available data, our results highlight the positive impact ET has on HER2+ breast cancer survival outcome. In vitro data supports the combination of novel SERDs and neratinib as a therapeutic strategy that warrants further investigation in HER2+/ER+ breast cancer. Citation Format: Amira F Mahdi, Neil T Conlon, Lisa D Eli, Irmina Diala, John Crown, Denis Collins. Investigation of neratinib and endocrine therapy combinations in HER2 positive breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-39.

Introduction: Treatment strategies for HER2+ breast cancer (BC) involves first line treatment with the monoclonal antibody trastuzumab, however resistance to treatment can occur. The pan-HER tyrosine kinase inhibitor neratinib is an approved adjuvant treatment for early stage HER2+ BC after one year of treatment with trastuzumab. Recent studies have highlighted the potential benefits of using retinoic acid as an adjuvant treatment for BC. The retinoic acid receptor (RAR) family of nuclear transcription factors consists of RARα, RARβ and RARγ. Interestingly ~30% of HER2+ BC have a co-amplification of the RARA gene, which codes for RARα. We examined the effect of RARα agonists (Fenretinide & AM580) and antagonist (AGN194310) in combination with neratinib in two HER2+ cell lines: SKBR3, has co-amplification of ERBB2 and RARA, and HCC1569 has amplified ERBB2 but are not RARA-amplified. Methods: SKBR3 and HCC1569 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. 10mM stock concentrations of fenretinide (H7779-Sigma), AM580 (A8843-Sigma), AGN194310 (SML2665-Sigma) and neratinib (Puma Biotechnology, Inc) were prepared in DMSO. For proliferation assays, cells were seeded in 96 well plates at a density of 3 X 104 cells per well for 24h. Cells were treated with a drug alone (2X concentration) or combination of drugs (4X concentration) in 100μL of medium. Proliferation was measured using an acid phosphatase-based assay after 5 days as percentage growth versus DMSO control. The half maximal inhibitory concentration (IC50) was calculated for each drug, using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50), using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research has found that the synthetic retinoic acid fenretinide, a pan-RAR (α, β and γ) activator, was more potent in the HCC1569 cells compared to the SKBR3 cell line with an IC50 of 0.21 ± 0.03μM compared to 4.55 ± 0.87 μM, respectively. However, when combined with neratinib there was a strong antagonistic effect observed in the HCC1569 cell line (CI value: 15.63 ± 9.5). Conversely, fenretinide enhanced the effect of neratinib in the SKBR3 cell line (CI value: 0.83 ± 0.4). The RARα-specific activator AM580 had an IC50 >10μM for both cell lines. When combined with neratinib there was an additive effect observed in the RARα negative HCC1569 cells (CI value: 0.97 ± 0.4), and a synergistic effect observed in the RARα upregulated SKBR3 cell line (CI value: 0.78 ± 0.08). Next we wanted to determine if inhibiting RAR activity would have any effect on the proliferation of these cell lines. The IC50 for the pan-RAR (α, β and γ) antagonist AGN194310 was >10μM for each cell line. However, when combined with neratinib treatment there was a strong synergistic effect observed in the HCC1569 cell line (CI value: 0.52 ± 0.17) and the SKBR3 cell line (CI value: 0.66 ± 0.19). Conclusions: This study suggests a differential effect of RAR agonists when combined with neratinib in RARα amplified versus non-amplified HER2+ BC. Targeting RAR signalling, particularly with a RAR antagonist, in combination with the pan-HER inhibitor warrants further investigation in HER2+ BC. Citation Format: Debbie O'Reilly, Nicola Gaynor, Neil Conlon, Irmina Diala, Lisa D Eli, John Crown, Denis M Collins. Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-28.

Purpose: Neratinib plus capecitabine (N+C) demonstrated significant progression-free survival (PFS) benefit in NALA (NCT01808573), a randomized phase III trial comparing N+C with lapatinib + capecitabine (L+C) in 621 patients with HER2-positive (HER2+) metastatic breast cancer (MBC) who had received ≥2 prior HER2-directed regimens in the metastatic setting. We evaluated correlations between exploratory biomarkers and PFS. Patients and Methods: Somatic mutations were evaluated by next-generation sequencing on primary or metastatic samples. HER2 protein expression was evaluated by central IHC, H-score, and VeraTag/HERmark. p95 expression (truncated HER2) was measured by VeraTag. HRs were estimated using unstratified Cox proportional hazards models. Results: Four hundred and twenty samples had successful sequencing: 34.0% had PIK3CA mutations and 5.5% had HER2 (ERBB2) mutations. In the combined patient populations, PIK3CA mutations trended toward shorter PFS [wild-type vs. mutant, HR = 0.81; 95% confidence interval (CI), 0.64–1.03], whereas HER2 mutations trended toward longer PFS [HR = 1.69 (95% CI, 0.97–3.29)]. Higher HER2 protein expression was associated with longer PFS [IHC 3+ vs. 2+, HR = 0.67 (0.54–0.82); H-score ≥240 versus Conclusions: PIK3CA mutations were associated with shorter PFS whereas higher HER2 expression was associated with longer PFS. Higher HER2 protein expression was also associated with a greater benefit for N+C compared with L+C.

Background: Cutaneous melanoma showed the highest HER4 genomic alteration frequency (17.34%) across 33 different cancer types (cBioPortal). HER4 is a tyrosine kinase receptor belonging to the human epidermal growth factor receptor (HER/ErbB) family. The role of HER4 in cancer has not been fully studied, potentially due to its complex biology (four isoforms, different stability/tissue-specific expression patterns, and oncogenic/tumor suppressor roles). Using computational analysis, we identified two potentially oncogenic HER4 mutations in melanoma (HER4 R106C and R711C). We then developed CRISPR/Cas9-modified selection-free melanoma cell lines for interrogating the role of these mutations as biomarkers of response to the oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER). Methods: Two melanoma cell lines (WM115, SKMEL24) were genome-edited by using a selection-free CRISPR/Cas9 approach. Single guide RNAs (sgRNAs) were designed by using the CRISPRon (v1.0) web tool, and Homology Directed Repair (HDR) single-stranded OligoDeoxyriboNucleotides (ssODNs), containing the HER4 mutations, were designed with the SnapGene Software (v6.1.1). The RNP complex (Cas9 and sgRNAs) and the HDR_ssODNs, for each HER4 mutation, were delivered into the melanoma cells through electroporation. Upon validation of the CRISPR/Cas9-efficiency via PCR and Sanger Sequencing, the CRISPR/Cas9-modified cells were single-cell sorted via FACS, by using two singlets gating (FSC-A/FSC-H and SSC-A/SSC-H) and a live/dead (propidium iodide) gating, to perform single-cell cloning. Colonies were then amplified and screened via PCR and Sanger sequencing to isolate pure selection-free HER4 CRISPR/Cas9 knock-ins clones of melanoma cells. Results: The WM115 and SKMEL24 melanoma cell lines were found to be wild-type for the HER4 receptor in the Depmap Portal (mutation 22Q2, 2022 data release 2). R106C and R711C CRISPR/Cas9 knock-ins were created in the melanoma cell lines three days after the electroporation of the CRISPR/Cas9 complex. The efficiency of the HER4 R106C CRISPR/Cas9 knock-in was 22% and 7% in the SKMEL24 and WM115 melanoma cells, respectively, with a respective total indel percentage of 76% (SKMEL24) and 55% (WM115). The HER4 R711C CRISPR/Cas9 knock-in efficiency was 15% in the SKMEL24 (with 26% total indels) and 1% in the WM115 (with 33% total indels). Single-cell colonies of the HER4 CRISPR/Cas9-mediated knock-ins (HER4 R106C and R711C), were formed at four days (SKMEL24) and at one week (WM115) after single-cell sorting. Conclusions: CRISPR/Cas9 technology can be utilised to introduce single-point mutations in HER4 to melanoma cell lines. In vitro functional characterisation of the isolated HER4 CRISPR/Cas9-edited melanoma cell lines will be performed in order to understand the activating potential of HER4 R106C and R711C and their sensitivity to NER. Citation Format: Marta Valenti, Yonglun Luo, Neil T. Conlon, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Development of selection-free HER4-mutated CRISPR/Cas9 knock-ins in cutaneous melanoma cell lines. [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 3962.

Background: Neratinib is a potent, irreversible pan-HER inhibitor that inhibits the ErbB family members EGFR, HER2, and HER4 and downstream signal transduction of these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks druggable levels of receptors for estrogen, progesterone and HER2 and therefore challenging to treat. There is evidence that some cases of TNBC have activated signaling pathways mediated by ErbB family members that may contribute to aggressive behavior. The purpose of this preclinical study was to identify and validate kinases whose targeting may enhance the antitumor activity of neratinib in TNBC cell lines. Methods: In vitro proliferation assays were used to evaluate the efficacy of neratinib in TNBC cell lines. Baseline and post-neratinib-treatment expression of EGFR and phosphorylated EGFR (phospho-EGFR) were assessed via Western blot analysis in 18 TNBC cell lines. Reverse-phase protein array (RPPA) was used to profile and validate the signaling networks induced by neratinib. To identify potential targets or pathways that may synergize with neratinib treatment, we performed high-throughput RNA interference (HT RNAi) screening using a 709-kinome library. CellTiter-Blue, sulforhodamine B, and soft-agar assays were performed to evaluate the antiproliferative effect of neratinib alone and with target inhibitor. Mammary fat pad xenograft models were used to evaluate the efficacy of neratinib alone or with inhibitor in vivo. Results: In vitro proliferation assays showed that the half-maximal inhibitory concentration (IC50) of neratinib in tested TNBC cell lines ranged from 0.16 µM to 1.25 µM. RPPA and Western blot analyses revealed that the efficacy of neratinib correlated with phospho-EGFR expression levels across the TNBC cell lines tested (R2 = 0.3245). Among the tested TNBC cell lines, SUM149 cells (PIK3CA wild-type) were selected for high throughput RNAi screening because this cell line has high EGFR expression and is moderately sensitive to neratinib (IC50 = 0.35 µM). We identified the 40 most relevant kinase targets by the sensitivity index analysis, and further pathway analysis identified PI3K/AKT/mTOR (drug: everolimus) and MAPK (drug: trametinib) as major canonical pathways whose targeting enhanced the cytotoxic effect of neratinib. Everolimus (mTOR inhibitor) produced a strong antiproliferative effect when combined with neratinib in most tested TNBC cell lines (12 of 15 cell lines; combination index [CI] values, 0.1-0.5) and was more effective in PIK3CA-mutated compared to wildtype cell lines. Trametinib (MEK inhibitor) showed a moderate antiproliferative effect (effective in 10 of 15 cell lines; CI values, 0.2-0.9). Synergistic antitumor effects of neratinib combined with everolimus or with trametinib were also observed in anchorage-independent growth conditions (P < 0.05). In vivo experiments demonstrated that neratinib plus everolimus and neratinib plus trametinib combinations inhibited tumor growth in the SUM149 xenograft model for than single drug (neratinib, 42.3% growth inhibition; everolimus, 29.7%; trametinib, 47.1%; neratinib plus everolimus, 69.7%; neratinib plus trametinib, 77.7%; P < 0.0001). Conclusion: Combining neratinib with everolimus or with trametinib enhanced the antitumor effects of these drugs in TNBC regardless of PIK3CA mutation status, and clinical investigations evaluating these combination regimens for the treatment of TNBC are warranted. Citation Format: Jangsoon Lee, Troy Pearson, Huey Liu, Jon A. Fuson, Toshiaki Iwase, Irmina Diala, Alshad S. Lalani, Lisa D. Eli, Debu Tripathy, Bora Lim, Naoto T. Ueno. Identification of novel molecules that enhance neratinib efficacy in triple-negative breast cancer by high-throughput RNA interference [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-07.

Introduction: Human epidermal growth factor 2 (HER2/ERBB2) is frequently amplified or mutated across various cancer types. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. All three TKIs bind and inhibit the kinase domain of HER2 but differ both in the mechanism of binding and in specificity for other HER family members. Direct comparisons to differentiate the pre-clinical efficacy of the three TKIs have been limited to small-scale studies and novel biomarkers of response to further define appropriate patient populations are required. Methods: In this study, the anti-proliferative effects of the three TKIs were compared using a 115-cancer cell line panel, including 12 breast cancer cell lines and 22 cell lines harbouring point mutations or amplifications of EGFR, HER2, or HER3. Hierarchical clustering analysis was carried out to compare the IC50 “fingerprint” of the three TKIs to 168 other anti-cancer agents. Novel markers of TKI sensitivity and resistance were identified through cross-analysis of each drug response profile with mutation, copy number variation, and gene expression data. Results: All three TKIs were effective against HER2-positive breast cancer models; neratinib showed the most potent activity, followed by tucatinib and lapatinib respectively (Table 1). Neratinib displayed the greatest anti-proliferative activity in HER2-mutant and EGFR-mutant cell lines. Clustering analysis revealed that the anti-proliferative profile of tucatinib was most similar to trastuzumab, while neratinib and lapatinib were most like other HER family inhibitors. Mutation and gene expression analysis identified potential markers of response for each TKI. High expression of four genes (HER2, VTCN1, CDK12, and RAC1) correlated with response to all three TKIs. DNA damage repair genes were significantly associated with resistance to the HER2-targeted TKIs. BRCA2 mutation was correlated with neratinib and tucatinib response, and high gene expression of ATM, BRCA2, and BRCA1 were all associated with neratinib resistance. Conclusions: Neratinib was the most effective HER2-targeted TKI against HER2-amplified, -mutant, and EGFR-mutant cell lines. This analysis revealed possible mechanisms that may be exploited using combinatorial strategies involving CDK inhibitors, immunotherapies, and targeting DNA repair pathways. Table: IC50 values for neratinib, lapatinib, and tucatinib in the HER2+ breast cancer cell linesIC50 values (nM)Cell linesNeratinibLapatinibTucatinibAU-56520294125BT-4745926229HCC195413814262122MDA-MB-453306228445928SKBR3715222 Citation Format: Neil T Conlon, Jeffrey J Kooijman, Suzanne JC van Gerwen, Winfried R Mulder, Guido JR Zaman, Irmina Diala, Lisa D Eli, Alshad S Lalani, John Crown, Denis Collins. Comparative analysis of anti-proliferative effects and gene profiling of lapatinib, neratinib, and tucatinib [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-06.

Background: HER2 mutations are oncogenic in hormone receptor positive (HR+) metastatic breast cancer (MBC), and may confer resistance to prior endocrine therapy but retain sensitivity to neratinib. Neratinib is an oral, irreversible, pan-HER tyrosine kinase inhibitor with clinical activity either as a single agent or in combination with fulvestrant in HER2-mutated, HER2-non-amplified MBC. Genomic analyses suggest that acquired resistance to neratinib can occur via additional HER2 alterations, which may alter HER2-pathway signaling. We investigated whether dual HER2-targeted therapy could improve clinical benefit in a cohort of patients with HER2-mutant, HR+ MBC treated with neratinib + trastuzumab + fulvestrant (N+T+F) from SUMMIT - a phase 2 basket trial (NCT01953926). Methods: Patients with HR+ MBC with known or suspected pathogenic HER2 mutation(s) identified by genomic sequencing were eligible to receive N+T+F (oral neratinib 240 mg/day, i.v. trastuzumab 8 mg/kg initially followed by 6 mg/kg every 3 weeks, and i.m. fulvestrant 500 mg on days 1&15 of month 1, then on day 1 every 4 weeks). Loperamide prophylaxis was mandatory during the first 2 treatment cycles. There was no restriction on the number of prior lines of systemic treatment for MBC. Efficacy endpoints: confirmed objective response rate and clinical benefit rate (RECIST v1.1); duration of response; progression-free survival. Results: As of 22-May-2020, 46 patients were enrolled in the N+T+F cohort and received at least 1 dose of study medication (safety population). 14 unique HER2 allelic variants were identified: 8 kinase domain missense; 1 extracellular domain missense; 2 transmembrane domain missense; 2 exon-20 insertion; 1 exon-19 deletion. The most common HER2 mutant variant was L755S (n=15, 33%) Median number of prior systemic regimens for metastatic disease was 4 (range 0-10); 34 (74%) patients had received prior fulvestrant, and 31 (67%) patients had received prior cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor therapy. 16 (35%) patients had ductal histology, 29 (63%) had lobular carcinoma, and 1 (2%) had mixed ductal and lobular carcinoma. At this time, 30/46 patients had RECIST measurable disease and are efficacy evaluable (ongoing patients who did not have the opportunity for their first post-baseline tumor assessment were excluded); clinical activity - see Table. Diarrhea was the most commonly reported adverse event (80% any grade) with 15 (33%) patients reporting grade 3 diarrhea (no grade 4 diarrhea). 10 patients (22%) had a neratinib dose reduction due to diarrhea but no patients discontinued treatment due to diarrhea. Conclusions: The combination of N+T+F demonstrated encouraging clinical activity in heavily pre-treated HER2-mutant, HR+, HER2-non-amplified MBC, including patients who had previously received either fulvestrant and/or CDK4/6 inhibitor-based therapies. While the rate of grade 3 diarrhea was higher than that observed with single-agent neratinib in SUMMIT, this was manageable through loperamide prophylaxis, and no patients discontinued study treatment due to diarrhea. SUMMIT has recently been amended to evaluate N+T+F, T+F and F (1:1:1 randomization) and continues to enroll patients. RECIST measurable and efficacy evaluable patients (n=30)Confirmed objective response,a n (%)12 (40)CR0PR12ORR, % (95% CI)40 (23-59)Best overall response, n (%)18 (60)CR0PR18Best overall response rate, % (95% CI)60 (41-77)Medianb DOR, months (95% CI)8.4 (4.1-NE)Clinical benefit,c n (%)14 (47)CR or PR12SD ≥24 weeks2CBR, % (95% CI)47 (28-66)Medianb PFS, months (95% CI)8.3 (4.2-12.5)aORR is defined as either a CR or a PR that is confirmed no less than 4 weeks after the criteria for response are initially met; bKaplan-Meier analysis; cCBR is defined as confirmed CR or PR or SD for ≥24 weeks; CR, complete response; CBR, clinical benefit rate; DOR, duration of response; NE, not estimable; ORR, objective response rate; PFS, progression-free survival; PR, partial response; SD, stable disease. Citation Format: Komal Jhaveri, Cristina Saura, Angel Guerrero-Zotano, Iben Spanggaard, François-Clement Bidard, Jonathan W Goldman, José A García-Sáenz, Andrés Cervantes, Valentina Boni, John Crown, Adam Brufsky, Sherene Loi, Barbara Haley, Ingrid A Mayer, Stephen Chia, Janice Lu, James Waisman, Noa Efrat Ben-Baruch, Mark E Burkard, Jennifer M Suga, Lucía González-Cortijo, Bruno Perrucci, Feng Xu, Sofia Wong, Jie Zhang, Lisa D Eli, Alshad S Lalani, Hans Wildiers. Latest findings from the breast cancer cohort in SUMMIT - a phase 2 ‘basket’ trial of neratinib + trastuzumab + fulvestrant for HER2-mutant, hormone receptor-positive, metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-05.

BackgroundHuman epidermal growth factor 2 (HER2/ERBB2) is frequently amplified/mutated in cancer. The tyrosine kinase inhibitors (TKIs) lapatinib, neratinib, and tucatinib are FDA-approved for the treatment of HER2-positive breast cancer. Direct comparisons of the preclinical efficacy of the TKIs have been limited to small-scale studies. Novel biomarkers are required to define beneficial patient populations.MethodsIn this study, the anti-proliferative effects of the three TKIs were directly compared using a 115 cancer cell line panel. Novel TKI response/resistance markers were identified through cross-analysis of drug response profiles with mutation, gene copy number and expression data.ResultsAll three TKIs were effective against HER2-amplified breast cancer models; neratinib showing the most potent activity, followed by tucatinib then lapatinib. Neratinib displayed the greatest activity inHER2-mutant andEGFR-mutant cells. High expression ofHER2,VTCN1,CDK12, andRAC1correlated with response to all three TKIs. DNA damage repair genes were associated with TKI resistance.BRCA2mutations were correlated with neratinib and tucatinib response, and high expression ofATM,BRCA2, andBRCA1were associated with neratinib resistance.ConclusionsNeratinib was the most effective HER2-targeted TKI againstHER2-amplified, -mutant, andEGFR-mutant cell lines. This analysis revealed novel resistance mechanisms that may be exploited using combinatorial strategies.

Background: In the SUMMIT trial, original cohorts of patients with HR+, HER2-negative (locally assessed), HER2-mutant MBC received N alone or N+F, with promising clinical response rates but abbreviated duration. Clinical progression coincided with emergence of additional HER2 mutations and/or amplification of the mutant allele [Smyth et al. Cancer Discov 2020;10:198–213]. Addition of T to the combination was postulated to prolong response; the combination of N+F+T in heavily pretreated patients with HR+, HER2-mutant MBC who had received CDK4/6 inhibitors (n=51) yielded a confirmed overall response rate (ORR) of 35.3%, median duration of response (DOR) of 14.3 months, clinical benefit rate (CBR) of 47.1%, and median progression-free survival (PFS) of 8.2 months [Jhaveri et al. J Clin Oncol 2022;40:1028]. Seven of these 51 patients were part of a randomized (1:1:1) cohort who received N+F+T, F+T, or F alone. Patients randomized to F+T or F could crossover to N+F+T upon progression. No patients responded to F or F+T; however, one of four patients who crossed over to N+F+T upon progression on F+T responded to the triplet, as did two of six who crossed over upon progression on F. We undertook ctDNA sequencing in patients who responded to N+F+T upfront and after crossover. Methods: Patients with HR+, HER2-negative MBC with activating HER2 mutation(s) and prior CDK4/6i received N+F+T (oral N 240 mg/d with loperamide prophylaxis, im F 500 mg d1, d15, and d29 of cycle 1 then q4w, iv T 8 mg/kg initially then 6 mg/kg q3w) or F+T or F alone. Efficacy endpoints included investigator-assessed ORR and CBR (RECIST v1.1), DOR, and best overall response. ctDNA was collected at baseline, every third cycle during treatment, and at the end of treatment and analyzed by next-generation sequencing. Samples were analyzed using the TEMPUS xF assay. Results: Sequencing results from ctDNA analysis are pending at the time of this abstract submission. Baseline HER2 mutations and co-alterations will be reported and compared with those found in tissue samples. Genomic spectrum and variant allele frequencies in samples taken at baseline, on treatment, and at the end of treatment from patients who experienced complete or partial response to N+F+T and then progressed (n=25) will be sequenced and mechanisms of acquired resistance will be posited. ctDNA genomic profiles of serial time points from patients randomized to F or F+T before and after crossover to N+F+T (n=10) will also be evaluated. Conclusions: Similarities and differences between the mechanisms of acquired resistance to N+F+T, and those previously reported to be associated with progression on N or N+F, will be discussed. Citation Format: Cynthia Ma, James Waisman, Adam M. Brufsky, Eddy S. Yang, Hans Wildiers, John P. Crown, Sarina A. Piha-Paul, Jennifer M. Suga, José Ángel García-Sáenz, Valentina Gambardella, Angel Guerrero, Salomon Stemmer, Ron Bose, Tonya Novara-Demgen, Daniel DiPrimeo, Lisa D. Eli, Komal Jhaveri. Genomic analysis of circulating tumor DNA (ctDNA) from patients with HR+, HER2-mutant metastatic breast cancer (MBC) enrolled in SUMMIT: mechanisms of acquired resistance to neratinib + fulvestrant + trastuzumab (N+F+T) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-01.

Background: HER2 mutations define a subset of metastatic breast cancers (MBCs) with a unique mechanism of oncogenic addiction to HER2 signaling. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been shown to have encouraging clinical activity when combined with fulvestrant in HER2-mutant, hormone receptor-positive (HR+) MBC [Smyth et al. SABCS 2018]. Genomic analyses suggest that acquired resistance to neratinib may occur by the acquisition of additional HER2 alterations, which may amplify HER2 pathway signaling [Won et al. AACR 2019]. We therefore explored whether dual HER2-targeted therapy may improve clinical benefit in this setting. Here we describe initial results from a cohort of patients with HER2-mutant, HR+ MBC treated with neratinib + trastuzumab + fulvestrant (N+T+F) from the phase 2 SUMMIT ‘basket’ trial (NCT01953926). Methods: Patients with HR+ MBC and known oncogenic driver HER2 mutations identified by genomic sequencing were eligible to receive combination treatment with oral neratinib 240 mg daily, intravenous trastuzumab 8 mg/kg initially followed by 6 mg/kg every 3 weeks, and intramuscular fulvestrant 500 mg on days 1 and 15 of month 1, then on day 1 every 4 weeks (N+T+F). Loperamide prophylaxis was mandatory during cycle 1. There was no restriction on the number of prior lines of systemic treatment for MBC. Efficacy endpoints included: confirmed objective response rate and clinical benefit rate - all defined according to RECIST v1.1 - as well as duration of response and progression-free survival. Genomic profiling from fresh/archival tumor tissues and/or plasma cfDNA was performed retrospectively by next-generation sequencing (MSK-IMPACT). Results: As of 01-May-2019, 19 patients were enrolled into the N+T+F cohort and received study treatment (safety population). 20 HER2 mutations were identified in the 19 patients: 14 kinase domain missense mutations, 3 extracellular domain missense mutations, and 3 exon-20 insertion mutations. Median number of prior systemic regimens for metastatic disease was 4 (range 0-10) and histologies were evenly split between lobular and ductal carcinomas. While the majority of patients remain on study treatment (n=15), only 13 of the 19 enrolled patients are efficacy evaluable at this time (having had ≥1 post-baseline tumor assessment). Clinical activity is summarized in the Table. Diarrhea was the most commonly reported adverse event (84.2% any grade) with 5 patients reporting Grade 3 diarrhea (there were no Grade 4 diarrhea events). Three patients (15.8%) reduced neratinib dose due to diarrhea but no patient discontinued treatment due to diarrhea. Conclusions: The combination of N+T+F resulted in an encouraging response rate and was a well-tolerated regimen in predominantly heavily pretreated HER2-mutant HR+ breast cancers. Based on a pre-planned interim analysis, the cohort has been expanded to enroll a total of 50 patients. Updated efficacy and safety data will be presented. Neratinib + trastuzumab + fulvestrant(n=13)Confirmed objective response rate, % (95% CI)39 (13.9-68.4)Complete response0Partial response5 (39)Duration of responses range, months4.2*-10.4*Median progression-free survivala,b, months (95% CI)NA (1.9-NA)*Response ongoing; aKaplan-Meier analysis; bincludes all patients enrolled (n=19); NA, not applicable. Citation Format: Hans Wildiers, Valentina Boni, Cristina Saura, Mafalda Oliveira, Komal Jhaveri, Helen Won, François-Clément Bidard, Adam M Brufsky, Mark E Burkard, Andrés Cervantes, Carlos Fernández-Martos, Barbara Haley, Sherene Loi, Iben Spanggaard, Stefano Panni, Janice Lu, Melanie E Dujka, Feng Xu, Sonia Macia, Lisa D Eli, Alshad S Lalani, Sarina Piha-Paul, Funda Meric-Bernstam, David B Solit, David M Hyman. Neratinib + trastuzumab + fulvestrant for HER2-mutant, hormone receptor-positive, metastatic breast cancer: Updated results from the phase 2 SUMMIT ‘basket’ trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-08.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib. Significance: HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer. This article is highlighted in the In This Issue feature, p. 161

Human epidermal growth factor receptor (EGFR) 2 (HER2) is overexpressed/amplified in about 25% of all breast cancers, and EGFR is overexpressed in up to 76% and amplified in up to 24% of triple-negative breast cancers (TNBC). Here, we aimed to identify inhibitors that may enhance the anti-tumor activity of neratinib for HER2+ breast cancer and TNBC. By conducting a non-biased high-throughput RNA interference screening, we identified PI3K/AKT/mTOR and MAPK as two potential inhibitory synergistic canonical pathways. We confirmed that everolimus (mTOR inhibitor) and trametinib (MEK inhibitor) enhances combinatorial anti-proliferative effects with neratinib under anchorage-independent growth conditions (p &lt, 0.05). Compared to single agent neratinib, the combination therapies significantly enhanced tumor growth inhibition in both SUM190 HER2+ breast cancer (neratinib plus everolimus, 77%, neratinib plus trametinib, 77%, p &lt, 0.0001) and SUM149 TNBC (neratinib plus everolimus, 71%, neratinib plus trametinib, 81%, 0.0001) xenograft models. Compared to single-agent neratinib, everolimus, or trametinib, both everolimus plus neratinib and trametinib plus neratinib significantly suppressed proliferation marker Ki67 and enhanced antitumor efficacy by activating the apoptosis pathway shown by increased Bim and cleaved-PARP expression. Taken together, our data justify new neratinib-based combinations for both HER2+ breast cancer and TNBC.

Background: Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that inhibits PI3K/Akt and MAPK signaling pathways after HER2 receptor activation. The ExteNET study showed that neratinib significantly improved 5-year invasive disease-free survival (iDFS) in women who completed trastuzumab-based adjuvant therapy for early breast cancer (EBC). We assessed the prognostic and predictive significance of PIK3CA alterations in patients in ExteNET. Methods: Participants were women aged ≥ 18 years (≥ 20 years in Japan) with stage 1–3c (modified to stage 2–3c in February 2010) operable breast cancer, who had completed (neo)adjuvant chemotherapy plus trastuzumab ≤ 2 years before randomization, with no evidence of disease recurrence or metastatic disease at study entry. Patients were randomized to oral neratinib 240 mg/day or placebo for 1 year. Formalin-fixed, paraffin-embedded primary tumor specimens underwent polymerase chain reaction (PCR) PIK3CA testing for two hotspot mutations in exon 9, one hot-spot mutation in exon 20, and fluorescence in situ hybridization (FISH) analysis for PIK3CA amplification. The primary endpoint (iDFS) was tested with log-rank test and hazard ratios (HRs) estimated using Cox proportional-hazards models. Results: Among the intent-to-treat population (n = 2840), tumor specimens were available for PCR testing (991 patients) and PIK3CA FISH (702 patients). Overall, 262 samples were PIK3CA altered: 201 were mutated (77%), 52 (20%) were amplified, and 9 (3%) were mutated and amplified. iDFS was non-significantly worse in placebo-treated patients with altered vs wild-type PIK3CA (HR 1.34; 95% CI 0.72–2.50; P = 0.357). Neratinib’s effect over placebo was significant in patients with PIK3CA-altered tumors (HR 0.41; 95% CI 0.17–0.90, P = 0.028) but not PIK3CA wild-type tumors (HR 0.72; 95% CI 0.36–1.41; P = 0.34). The interaction test was non-significant (P = 0.309). Conclusions: Although there was a greater absolute risk reduction associated with neratinib treatment of patients with PIK3CA-altered tumors in ExteNET, current data do not support PIK3CA alteration as a predictive biomarker of response to neratinib in HER2-positive EBC. Trial registration: ClinicalTrials.gov , NCT00878709 . Trial registered April 9, 2009.

Background: HER4 is a member of the human epidermal growth factor receptor (HER/ErbB) family of receptor tyrosine kinases. HER4 is activated through ligand binding, transmitting intracellular signals by homodimerizing, or by heterodimerizing with other family members (EGFR, HER2, HER3). The role of HER4 in cancer or normal tissue has not been studied as comprehensively as EGFR and HER2, potentially due to a more complex biology that includes four different isoforms and different stability/tissue-specific expression patterns, and oncogenic or oncosuppressor roles. The oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER) is clinically active against HER2-mutant and HER2-amplified cancers. Using publicly available datasets including cBioPortal and COSMIC and predicted structural and functional impacts, this study aimed to identify HER4 mutations with the greatest oncogenic potential that may have value as potential biomarkers of response to NER. Methods: The frequency of HER4 mutations across 33 cancer types was determined by using the clinical database cBioPortal and TCGA PanCancer Atlas datasets (n=10967). A shortlist of potentially oncogenic HER4 mutations was created based on the occurrence in cBioPortal and COSMIC databases, annotation as putative driver and cancer hotspot, structural and functional impacts according to OncoKB, Mutation Assessor, SIFT and Polyphen-2 software, and HER4 protein folding studies. Analysis was carried out using the R-studio statistical environment. Results: HER4 amplification is rare in cancer, with the majority of genetic changes reported as point mutations; particularly missense (31.93%), synonymous (6.43%) and nonsense (3%) substitutions. Melanoma and esophagogastric (EG) cancers showed the highest HER4 alteration frequency, 17.34% and 13.42%, respectively, across different cancer types in the cBioPortal database. Melanoma and EG cancers showed the second and third highest absolute count of HER4 genomic alterations (n=77 and n=69, respectively). Based on the criteria outlined in methods, a priority list of HER4 mutations with greatest oncogenic potential was curated for melanoma (HER4 R106C, R306C, R711C, G936E, F1003I), stomach (HER4 C217R, S774N, K1002R, Y1150C) and esophageal (HER4 R488W, S449Y) adenocarcinomas. Mutations at these sites, located across ligand-binding, dimerization and intracellular signaling domains, have the potential to impact HER4 activity and thus possibly act as biomarkers of response to an irreversible pan-HER TKI like NER. Conclusions: These data suggest that melanoma and EG cancers are the cancer types with the highest rate of HER4 mutations. In vitro functional characterization of the identified HER4 mutations is now required to further explore their value as biomarkers of response to NER. Citation Format: Marta Valenti, Neil T. Conlon, Lisa D. Eli, Irmina Diala, John Crown, Denis M. Collins. Identification of HER4 mutations with potential as biomarkers of response to neratinib [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 3988.

Purpose: We wanted to determine the prognosis and the phenotypic characteristics of hormone receptor–positive advanced breast cancer tumors harboring an ERBB2 mutation in the absence of a HER2 amplification. Experimental Design: We retrospectively collected information from the American Association of Cancer Research-Genomics Evidence Neoplasia Information Exchange registry database from patients with hormone receptor–positive, HER2-negative, ERBB2-mutated advanced breast cancer. Phenotypic and co-mutational features, as well as response to treatment and outcome were compared with matched control cases ERBB2 wild type. Results: A total of 45 ERBB2-mutant cases were identified for 90 matched controls. The presence of an ERBB2 mutation was not associated with worse outcome determined by overall survival (OS) from first metastatic relapse. No significant differences were observed in phenotypic characteristics apart from higher lobular infiltrating subtype in the ERBB2-mutated group. ERBB2 mutation did not seem to have an impact in response to treatment or time-to-progression (TTP) to endocrine therapy compared with ERBB2 wild type. In the co-mutational analyses, CDH1 mutation was more frequent in the ERBB2-mutated group (FDR < 1). Although not significant, fewer co-occurring ESR1 mutations and more KRAS mutations were identified in the ERBB2-mutated group. Conclusions: ERBB2-activating mutation was not associated with a worse OS from time of first metastatic relapse, or differences in TTP on treatment as compared with a series of matched controls. Although not significant, differences in coexisting mutations (CDH1, ESR1, and KRAS) were noted between the ERBB2-mutated and the control group.

Càncer de coll uterí; Assaig clínic; Mutant HER2 Cáncer de cuello uterino; Ensayo clínico; Mutante HER2 Cervical cancer; Clinical trial; HER2 mutant Objective: Somatic HER2 mutations occur in ~5% of cervical cancers and are considered oncogenic and associated with poor prognosis. Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, is active in multiple HER2-mutant cancers. SUMMIT is a phase II basket trial investigating the efficacy and safety of neratinib in solid tumors. Methods: Patients with HER2-mutant, persistent, metastatic/recurrent cervical cancer with disease progression after platinum-based treatment for advanced/recurrent disease received oral neratinib 240 mg/day with mandatory loperamide prophylaxis during cycle 1. The primary endpoint was confirmed objective response rate (ORR). Secondary endpoints included: response duration (DOR); clinical benefit rate (CBR); progression-free survival (PFS); overall survival (OS); safety. Results: Sixteen eligible patients were enrolled; 10 (62.5%) had endocervical adenocarcinoma. The most common HER2 mutation was S310F (63% of patients). Three of 12 RECIST-measurable patients had confirmed partial responses (ORR 25%; 95%CI 5.5-57.2%); 3 had stable disease ≥16 weeks (CBR 50%; 95%CI 21.1-78.9%). DOR for responders were 5.6, 5.9, and 12.3 months. Median PFS was 7.0 months (95%CI 0.7-18.3 months); median OS was 16.8 months (95%CI 4.1-NE months). Diarrhea (75%), nausea (44%), and decreased appetite (38%) were the most common adverse events. One patient (6%) reported grade 3 diarrhea. There were no grade 4 events, and no diarrhea-related treatment discontinuations. Conclusions: Neratinib monotherapy showed evidence of activity in heavily pretreated patients with HER2-mutant cervical cancer, with no new safety signals. Given the few effective options for cervical cancer after platinum-based therapy failure, neratinib warrants further investigation in this molecularly defined patient population. This work was supported by Puma Biotechnology Inc . 10880 Wilshire Blvd, Suite 2150, Los Angeles, CA 90024, USA.

Background: We recently reported that acquired resistance to the dual HER1/2 TKI lapatinib (Lap) was mediated by HER2 L755S, while resistance to the pan-HER TKI neratinib (Nrb) was associated with co-acquisition of an additional pathogenic PIK3CA mutation. Though the role of HER2 mutations is gaining attention in HER2-positive (+) BC, less is known about their role and clinical implications in next generation TKI resistance, particularly when co-occurring with PIK3CA mutations in HER2+ BC. Investigating optimal treatment combinations and the development of new clinically relevant 3D models are warranted.Materials and Methods: HER2+ BT474 parental (P) cells and models with acquired resistance to Lap (LapR) and Nrb (NrbR) (SABCS20-PD3-09) were used. Xenografts established in mice using P, LapR, and NrbR cells and 3D organoids derived from these xenografts using the Hans Clevers (HC, PMID 29224780) or Mark Burkard (MB, PMID 31175091) method were characterized by qRT-PCR and western blot. Drug efficacy was assessed by growth changes in 2D and 3D models using the IncuCyte system or by microscopy-based analysis. Results: We previously showed that Lap and Nrb resistance confers cross-resistance to tucatinib (Tuca) and trastuzumab, and that targeting the HER and downstream PI3K pathway, especially using small molecule agents that are key for treatment of CNS lesions, is effective only in combination with Nrb or poziotinib (Pozio), but not Tuca. Our new studies revealed that the MEK inhibitor (i) AZD6244 (selumetinib; Sel), mTORi everolimus (Eve), and selective estrogen receptor degrader fulvestrant (Ful) were not effective as single agents in inhibiting the growth of either LapR or NrbR models. Whilst the LapR cells were highly sensitive to the irreversible HER1/2 TKI afatinib (Afa) and the irreversible dual/pan-HER TKI pyrotinib (Pyro) as single agents, the NrbR models were cross-resistant to both TKIs, highlighting the importance of the co-occurring PIK3CA mutation in resistance. Interestingly, Afa and Pyro were only partly effective when combined with Eve+Ful, Sel+Eve, or Sel+the PIK3CAi alpelisib in inhibiting NrbR growth. Consistent with our previously reported findings for Nrb and Pozio, Pyro was highly effective with TDM1. As opposed to the P xenografts, the LapR and NrbR tumors grew in the presence of the respective TKI, confirming their resistant phenotype in vivo. P and resistant xenograft-derived organoids (XDOs) were successfully established using the HC but not MB method, but the HC-derived XDOs were subsequently grown in MB condition and used for molecular and functional studies. Preliminary characterization showed that the LapR tumors and XDOs harbor HER2 L755S, whereas the NrbR tumors and XDOs also have a concomitant PIK3CA E542V mutation, findings that are in line with our 2D results, suggesting that the xenografts and XDOs retain and recapitulate the molecular profile of their 2D or tumor counterparts. Early drug efficacy studies indicate that, akin to the 2D models, the LapR XDOs are highly sensitive to Nrb, whereas both the LapR and NrbR XDOs exhibit cross-resistance to Tuca but remain sensitive to Pozio.Conclusions: Our data suggest that the potency of next generation irreversible HER TKIs in HER2+ BC may be challenged by the emergence of mutations in HER2, together with other co-occurring downstream mutations, such as PIK3CA. Our findings present a clear roadmap for the development of combinatorial therapies that should be individualized for patients with HER2+ BC. Our newly developed XDO strategy may offer a new platform to confirm and prioritize optimal drug combinations to overcome this resistance and may facilitate the near future development of patient-derived organoids for precision medicine of resistant HER2+ BC. Citation Format: Sreyashree Bose, Ragini Mistry, Chia Chia Liu, Sarmistha Nanda, Lanfang Qin, Pier Selenica, Andrea Gazzo, Yingjie Zhu, Michael A. Mancini, Fabio Stossi, Irmina Diala, Lisa D. Eli, Britta Weigelt, Jorge S. Reis-Filho, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Jamunarani Veeraraghavan. Resistance to next generation tyrosine kinase inhibitors (TKIs) in HER2-positive breast cancer (BC): Role of HER and PIK3CA mutations and development of new treatment strategies and study models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-01.

Background: Neratinib is a pan-HER tyrosine kinase inhibitor that blocks the PI3K/Akt and MAPK signaling pathways downstream from HER2. The international, randomized, placebo-controlled phase III ExteNET trial showed that a 1-year course of neratinib after trastuzumab-based adjuvant therapy significantly improved 2-year invasive disease-free survival (iDFS) in early-stage HER2+ breast cancer (HR 0.67; 95% CI 0.50–0.91; p=0.0091) [Chan et al. Lancet Oncol 2016]. Furthermore, the effects of neratinib on iDFS were shown to be durable at 5 years' follow-up (HR 0.73; 95% CI 0.57–0.92; p=0.008) [Martin et al. ESMO 2017]. PIK3CA alterations are common in HER2+ breast cancers, and in general are associated with a worse prognosis. We sought to assess the prognostic and predictive significance of PIK3CA alterations in an exploratory substudy of the ExteNET trial. Methods: ExteNET is an international, multi-center, randomized, double-blind, placebo-controlled phase III trial (Clinicaltrials.gov: NCT00878709). Patients received oral neratinib 240 mg/day or placebo for 1 year. Of the intent-to-treat (ITT) population (n=2840), primary formalin-fixed paraffin-embedded (FFPE) tumor specimens were available from 991 patients for PIK3CA mutation testing by RT-PCR for two hot-spot mutations in exon 9 (E542K, E545K/D) and one hot-spot mutation in exon 20 (H1047R). 702 FFPE tumor slides underwent FISH analysis for PIK3CA amplification with a ratio of ≥2.2 considered as amplified. Primary endpoint: iDFS. iDFS events were tested by 2-sided log-rank tests, and HR (95% CI) were estimated using Cox proportional-hazards models. Data cut-off: March 2017. Results: Baseline demographics and disease characteristics between treatment arms of the correlative cohort (n=1201) were balanced. Overall, 21.2% (n=210) of primary tumors harbored one of the specified PIK3CA mutations, and 8.7% (n=61) were PIK3CA FISH-amplified. Patients with PIK3CA-altered tumors (i.e. PIK3CA mutations or FISH-amplified) had fewer iDFS events with neratinib compared with placebo (HR 0.41; 95% CI 0.17-0.90, p=0.028). The interaction test was not significant (p=0.1842). Results of the various correlative analyses within treatment arms are shown in the table. NeratinibPlacebo iDFS iDFS 2-sidedPopulationnevents, nnevents, nHR (95% CI)P valueaITT142011614201630.73 (0.57–0.92)b0.008bCorrelative cohort59345608700.67 (0.45–0.96)0.0317PIK3CA-mutation positive1047106170.43 (0.17–1.01)0.056PIK3CA-mutation negative38527396420.66 (0.40-1.06)0.089PIK3CA-amplified3312840.20 (0.01-1.33)0.106PIK3CA-non-amplified31629325360.85 (0.52-1.39)0.521PIK3CA-altered1308132200.41 (0.17-0.90)0.028a. Log-rank test; b. Stratified analysis Conclusions: One year of neratinib treatment after trastuzumab-based adjuvant therapy significantly improves iDFS after 5 years in patients with early-stage HER2+ breast cancer. From this modest-sized exploratory cohort, it appears that PIK3CA may be a biomarker for differential sensitivity to neratinib after 1 year of trastuzumab in the adjuvant setting.These exploratory results should be validated in a larger subset. Citation Format: Chia SKL, Martin M, Holmes FA, Ejlertsen B, Delaloge S, Moy B, Iwata H, von Minckwitz G, Mansi J, Barrios CH, Gnant M, Tomašević Z, Denduluri N, Šeparović R, Kim S-B, Hugger Jakobsen E, Harvey V, Robert N, Smith II J, Harker G, Lalani AS, Zhang B, Eli LD, Buyse M, Chan A. PIK3CA alterations and benefit with neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: Correlative analyses of the phase III ExteNET trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-12.

Somatic mutations of ERBB2 and ERBB3 (which encode HER2 and HER3, respectively) are found in a wide range of cancers. Preclinical modelling suggests that a subset of these mutations lead to constitutive HER2 activation, but most remain biologically uncharacterized. Here we define the biological and therapeutic importance of known oncogenic HER2 and HER3 mutations and variants of unknown biological importance by conducting a multi-histology, genomically selected, 'basket' trial using the pan-HER kinase inhibitor neratinib (SUMMIT; clinicaltrials.gov identifier NCT01953926). Efficacy in HER2-mutant cancers varied as a function of both tumour type and mutant allele to a degree not predicted by preclinical models, with the greatest activity seen in breast, cervical and biliary cancers and with tumours that contain kinase domain missense mutations. This study demonstrates how a molecularly driven clinical trial can be used to refine our biological understanding of both characterized and new genomic alterations with potential broad applicability for advancing the paradigm of genome-driven oncology.

Background: We previously showed preclinical and clinical activity of combined inhibition of EGFR and PARP with lapatinib and veliparib in triple negative breast cancer (Nowsheen et al. PLoS One 2012;7(10):e46614; Reasor et al. J Clin Onc 2018; 36(15_suppl):1095), due to an induced homologous recombination repair defect with EGFR inhibition sensitizing cells to PARP inhibition. In this study, we tested the in vitro and in vivo activity of the irreversible pan-HER tyrosine kinase inhibitor, neratinib, with the PARP inhibitor olaparib or niraparib. Methods: Human triple negative breast cancer (TNBC) cells MDA-MB231 (BRCA wt), MDA-MB453 (BRCA wt), and MDA-MB436 (BRCA1 mutant) and Mouse TNBC 4T1 cells (BRCA wt) were used. Cell proliferation and colony formation assays were performed to assess the in vitro activity of various concentrations of neratinib and olaparib. Markers of DNA damage were measured via gamma-H2AX western blot analysis. In vivo effects of combination treatments on tumor metastasis were tested using intracardiac injection of tumor cells labelled with luciferase and mice were monitored for survival. Results: Combining neratinib with olaparib or niraparib significantly decreased proliferation compared to either agent alone in MDA-MB231, MDA-MB453, MDA-MB436 and 4T1 cells. Similar results were observed using colony formation assays. Cytotoxicity was associated with increased gamma-H2AX levels, indicating persistent DNA damage. Mice treated with 20mg/kg/day of neratinib and 100mg/kg/day of olaparib after MDA-MB231-Luc intracardiac injection exhibited reduced tumor metastases compared to either agent alone, resulting in better survival (p=0.0088). Conclusions: Combining neratinib with PARP inhibition induces synthetic lethality resulting in increased anti-tumor activity in TNBC in cell lines and in xenografts. Future clinical trials testing these combinations are warranted. Citation Format: Chuan Xing, Zhuo Zhang, Deborah Della Manna, Irmina Diala, Lisa Eli, Alshad S. Lalani, Eddy Shih-Hsin Yang. Neratinib induces synthetic lethality with PARP inhibitors in triple negative breast cancer cells in vitro and in vivo [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 1181.

Hundreds of somatic ERBB4 mutations have been described in cancer tissues with very limited information about their functional significance. However, analyses of individual mutants have indicated that activating ERBB4 mutations, such as ERBB4 K935I, do exist. Understanding the functional consequences of ERBB4 mutations is needed in order to assess the relevance of targeting ERBB4 in human cancers with matched therapies such as neratinib. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that binds to and potently inhibits ERBB4 kinases activity in-vitro, and is currently approved clinically for the treatment of early stage and metastatic HER2+ breast cancers. We set up to address the transforming potential of individual ERBB4 variants by selecting 18 mutations (Table 1) from cBioPortal data (www.cbioportal.org) using the following criteria: 1) the mutations were recurrent, 2) they were analogous to activating mutations described for other oncogenic ERBB family members and/or 3) their position at receptor dimerization interfaces suggested functional relevance. Retroviral pBABE-puro-gateway vector was used to achieve functional, but still near-physiological expression levels of ERBB4 in different cell backgrounds. A screen for optimal cellular background to assess the transforming potential of ERBB4 variants indicated that 1) IL-3 independent growth of mouse lymphoid Ba/F3 cells, 2) focus formation analysis of mouse NIH-3T3 fibroblasts, and 3) ligand-induced proliferation of human mammary epithelial MCF-10A provided read-outs with robust differences when the effects of the positive control mutant ERBB4 K935I was compared to wild-type ERBB4. These models are now being used to address the transforming potential of the 18 mutations (Table 1). Table 1.Somatic ERBB4 mutations chosen for functional analyses.R106CE452KR711CL798RG870RK1223TS303FR524CG741EV840IG907ES1289AR393WR544WS774GR847HR992CR1304W Citation Format: Peppi Suominen, Deepankar Chakroborty, Kari J. Kurppa, Irmina Diala, Lisa D. Eli, Alshad S. Lalani, Klaus Elenius. Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib [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 2420.

9068 Background: The phase 2 SUMMIT basket trial (NCT01953926) demonstrated efficacy of neratinib in patients with EGFR exon 18-mutant NSCLC [Boni et al. WCLC 2020]. Neratinib also has documented activity in HER2+ metastatic breast cancer with CNS metastases [Saura et al. SABCS 2020 & J Clin Oncol 2020]. Here we report neratinib efficacy in a subgroup of patients with EGFR exon 18-mutant NSCLC and CNS involvement from SUMMIT. Methods: Patients with EGFR exon 18-mutant NSCLC were treated with single-agent neratinib (240 mg po daily). Prior EGFR tyrosine kinase inhibitors (TKIs), chemotherapy, and checkpoint inhibitors (IO) were allowed. Patients with stable, asymptomatic CNS metastasis were enrolled. Study endpoints: objective response rate (ORR) at week 8 (±1 week); ORR (RECIST 1.1 confirmed); duration of response (DOR); clinical benefit rate (CBR); progression-free survival (PFS); safety; biomarkers. Results: Baseline characteristics of 11 patients with EGFR exon 18-mutant NSCLC: median age 67 (range 56–83) years; ECOG PS 0/1 (45%/55%). Prior lines of therapies: 2 (range 1–3): EGFR TKIs (91%); chemotherapy (55%); IO (27%). 3/11 patients had baseline CNS metastasis and received radiation 8–22 months prior to study enrollment. Best CNS response in these 3 patients was stable disease with overall individual PFS of 1.9 (censored), 6.9 and 9.1 months and OS of 2.6 (censored), 17.7 (censored), and 17.9 months. Efficacy is summarized in Table. Efficacy summary: TKI-pretreated EGFR exon 18-mutant NSCLC cohort receiving neratinib monotherapy. Conclusions: Activity of single-agent neratinib was observed in prior TKI-exposed patients with EGFR exon 18-mutant NSCLC. Despite the small sample size of only 3 patients with baseline CNS metastases, findings suggest a potential role for neratinib as a systemic treatment option for patients with NSCLC and difficult-to-treat uncommon mutations with CNS involvement. The SUMMIT trial continues to enroll patients with EGFR exon 18-mutant NSCLC. Clinical trial information: NCT01953926. [Table: see text]

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Pre-existent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively these data define HER2 mutations as a therapeutic target in breast cancer and suggest that co-existence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib.

310 Background: FLC, a rare liver cancer of young adults, has no effective systemic therapies. Surgical resection is used extensively with non-curative intent. FLC is associated with a DNAJB1- PRKACA chimeric transcript that produces a fusion protein with retained kinase activity and increased expression of several oncogenic signaling pathways including, but not limited to, HER2 ( ERBB2). Methods: N (240 mg oral daily) was studied in FLC pts in the SUMMIT study (NCT01953926); and later under compassionate use for N-based combinations (combo): P (2 mg/kg q3w), E (7.5 mg daily), and V (240 mg q2w) in doublet or triplet regimens. Eligible pts: ≥12y; histologically confirmed FLC; adequate organ function; any number of prior therapies. Primary endpoint: objective response rate (ORR; RECIST v1.1). Secondary endpoints: duration of response; clinical benefit rate (CBR); safety (CTCAE v4.0); somatic and germline sequencing (MSK IMPACT). Results: As of 03-Sep-2020, 15 pretreated pts received N in SUMMIT (confirmed ORR 0%; CBR 13%). Efficacy data for 5 pts from SUMMIT and 2 more pts receiving combo under compassionate use (4 male, 3 female, median age 26 years, median 0 [range 0–4] prior systemic therapies) are in shown in the table. The most common adverse events (AE) with single-agent N (n = 5) were diarrhea (grade 1 80%; grade 2 20%) and nausea (grade 1 60%); other AEs were grade ≤1 in ≤20% of pts. Conclusions: N monotherapy had limited benefit as a single agent in FLC pts. Several case studies evaluating N-based combo with checkpoint inhibitors administered under compassionate use demonstrated that NP led to 1 PR, and the triplet of NPE to prolonged SD. These are case-limited observations but are critical and worth evaluating further in upcoming clinical trials given the continued lack of a standard of care therapy for pts with FLC. Clinical trial information: NCT01953926. [Table: see text]

Purpose: Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer (MBC) is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. We and others previously described acquired activating hotspot HER2 (ERBB2) mutations in ~5% of ER+ MBC that conferred resistance to multiple ER-targeting therapies, including the selective estrogen receptor degrader fulvestrant. These tumors could be re-sensitized to fulvestrant in vitro through addition of the irreversible pan-HER tyrosine kinase inhibitor neratinib, suggesting a possible clinical combination strategy for patients. Although some HER2 mutations are relatively more frequent in tumors, there is a “long tail” of rare HER2 mutations that have not been characterized but remain clinically important for patients whose tumors harbor them. Therefore, there is biological and clinical value in prospectively characterizing all possible missense mutations in HER2. Methodology: Since only activating HER2 mutations conferred resistance to fulvestrant (and not passenger or inactivating mutations), resistance to fulvestrant in ER+ breast cancer cells can be used as a surrogate kinase assay for HER2 activity. Therefore, we are performing a saturation mutagenesis screen of HER2, using fulvestrant resistance as a readout for activating mutants. Screen optimization included: a) testing selected mutants cloned into a custom vector to identify appropriate positive and negative controls for screen QC, b) custom screen design (transduction under ER inhibition, and an empirically-determined ratio of growth in fulvestrant vs DMSO) to enable recovery of mutants of varying growth phenotypes. We also designed PCR conditions to enable efficient amplification of HER2 (the largest ORF ever tested by saturation mutagenesis) from genomic DNA, and custom-designed and built a comprehensive HER2 library that includes built-in controls such as stop codons. The saturation mutagenesis screen is currently underway, and will generate putative activating HER2 mutations (i.e., not growth-inhibited in fulvestrant versus complete media), that will be validated in a “minipool” screen. Validated hits will be further tested for sensitivity to the combination of fulvestrant+neratinib or other kinase inhibitors. Summary and conclusions: We have designed a saturation mutagenesis screen to recover a spectrum of activating HER2 mutations, the largest such library generated to date, and using resistance to the ER inhibitor fulvestrant as a novel surrogate kinase assay. This screen will generate a comprehensive reference table of HER2 mutant phenotypes in terms of response and resistance to ER and HER2 targeting agents. These findings will have translational applicability, and may suggest promising precision medicine approaches for clinical management of patients harboring somatic HER2 mutations. Citation Format: Utthara Nayar, Federica Piccioni, Xiaoping Yang, David Root, J T. Neal, Lisa D. Eli, Irmina Diala, Alshad S. Lalani, Nikhil Wagle. Phenotypic characterization of a comprehensive set of HER2 missense mutants in ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1914.

The 'Competing interests' statement of this Article has been updated; please see the accompanying Amendment. The original Article has not been corrected online.

Background: Somatic mutations in ERBB2 occur across various tumor types at relatively low frequencies and can lead to constitutive kinase signaling and oncogenic transformation. SUMMIT is an ongoing basket trial of neratinib, an irreversible pan-HER tyrosine kinase inhibitor, in pts with ERBB2 mutant cancers (NCT01953926). Methods: Pts with advanced solid tumors and locally documented ERBB2 mutations received neratinib 240 mg daily (N=8) +/- fulvestrant at the labeled dose for pts with ER+ breast cancer (N=6) as part of the global Phase II SUMMIT basket study. Pre- and post-treatment tumor and cfDNA were subjected to NGS using MSK-IMPACT for tissue (410 genes) and MSK-ACCESS for plasma (129 genes) to identify SNVs, indels, and copy number alterations. Results: In total, 14 pts had successful sequencing of both paired tissue and cfDNA sample (10 breast, 2 gallbaldder, 1 bladder, and 1 unknown primary). All pts achieved clinical benefit on neratinib including 2 CRs, 7 PRs, and 5 SDs. In 1 gallbladder pt, paired tissue sequencing identified loss of the pretreatment clonal ERBB2 mutation but retention of a truncal TP53 mutation, suggesting emergence of a distantly related ERBB2 wildtype clone. In the remaining 13 patients, the pretreatment ERBB2 mutation was retained in tissue at progression. 64% (9/14) pts had at ≥1 acquired alteration in tissue including 3 pts who acquired secondary alterations in ERBB2, both clonal and subclonal ERBB2 mutations as well new focal amplifications. Consistent with this finding, plasma cfDNA sequencing revealed 57% (8/14) of pts acquired ≥1 secondary ERBB2 mutation with the majority occurring at known activating hotspots. Analysis of mutant allele frequencies of these emergent ERBB2 alterations, in comparison to other variants, suggested the majority were subclonal with evidence of multiple independent subclones arising in the same patient. Two pts developed known ERBB2 gatekeeper mutations (T798I and L785F). Conclusion: In pts with ERBB2-mutant solid tumors with clinical benefit on neratinib, a potential on-target resistance mechanism was identified in 71% (10/14, including 7 with acquired gain-of-function ERBB2 mutations, 2 with ERBB2 both gain-of-function and gatekeeper mutations, and 1 with outgrowth of an ERBB2 wildtype clone). Collectively, these data provide additional evidence that ERBB2 mutations lead to oncogene addiction in solid tumors. We also demonstrate that tumor and cfDNA sequencing provides complementary information that can be integrated to more fully elucidate potential resistance mechanisms. Citation Format: Helen H. Won, S. Duygu Selcuklu, Sarina A. Piha-Paul, Cristina Saura, Jordi Rodon, Ingrid A. Mayer, Sherene Loi, Geoffrey I. Shapiro, Janice Lu, Adam Brufsky, Catherine Zimel, Myra Melcer, Maurizio Scaltriti, Lisa D. Eli, Richard E. Cutler Jr., Alshad S. Lalani, Richard P. Bryce, Carlos Arteaga, Funda Meric-Bernstam, Michael F. Berger, David B. Solit, Alison Schram, David M. Hyman. Paired tumor and cfDNA in patients with HER2-mutant solid tumors treated with neratinib reveals convergence of multiple on-target resistance mechanisms: Results from the SUMMIT "Basket" Trial [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 929.

An estimated 15-20% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2/ERBB2/neu). Two small-molecule tyrosine kinase inhibitors (TKIs), lapatinib and neratinib, have been approved for the treatment of HER2-positive (HER2+) breast cancer. Lapatinib, a reversible epidermal growth factor receptor (EGFR/ERBB1/HER1) and HER2 TKI, is used for the treatment of advanced HER2+ breast cancer in combination with capecitabine, in combination with trastuzumab in patients with hormone receptor-negative metastatic breast cancer, and in combination with an aromatase inhibitor for the first-line treatment of HER2+ breast cancer. Neratinib, a next-generation, irreversible pan-HER TKI, is used in the US for extended adjuvant treatment of adult patients with early-stage HER2+ breast cancer following 1 year of trastuzumab. In Europe, neratinib is used in the extended adjuvant treatment of adult patients with early-stage hormone receptor-positive HER2+ breast cancer who are less than 1 year from the completion of prior adjuvant trastuzumab-based therapy. Preclinical studies have shown that these agents have distinct properties that may impact their clinical activity. This review describes the preclinical characterization of lapatinib and neratinib, with a focus on the differences between these two agents that may have implications for patient management. Published version

Background: Mutations in ERBB2, encoding the HER2 protein, occur in up to 4% of breast cancers. A subset of ERBB2 mutations have been shown to have oncogenic potential through constitutive activation of the HER2 kinase. SUMMIT (NCT01953926) is a phase II ‘basket' study investigating the use of the irreversible pan-HER tyrosine kinase inhibitor neratinib, in combination with the estrogen degrader fulvestrant, for the treatment of patients with advanced hormone-positive breast cancers harboring ERBB2 mutations. We collected and sequenced serial samples of circulating cell-free DNA (cfDNA) from participating patients in order to identify biomarkers of response and resistance. Methods: Patients were treated with neratinib 240 mg daily and fulvestrant at the labelled 500 mg dose. Plasma was collected and cfDNA was extracted from patients at baseline and on Day 1 of each cycle (occurring every 4 weeks). Next generation sequencing (NGS) was performed on cfDNA samples collected from 12 patients using the Guardant360 test (Guardant Health, CA), which identifies select point mutations, small insertions and deletions, copy number changes, and structural rearrangements in 73 genes. Paired samples were sequenced from 9 of the 12 patients, including baseline and progression samples from 7 patients. cfDNA NGS results were compared to pretreatment tissue NGS results obtained 0-6 years prior (median: 6 months). Results: 90% (9/10) baseline cfDNA NGS detected the locally reported ERBB2 mutation identified by tissue NGS. In one patient with only a sample obtained at progression, the ERBB2 mutation was identified. No ERBB2 mutation was identified in a singular on-treatment sample obtained from a patient with continued response to therapy. Eight ERBB2 mutations were identified in the baseline cfDNA sample of one patient with a hypermutated tumor, 6 of which were not identified by tissue NGS. Emergence of at least one additional ERBB2 mutation was seen in 3/7 patients with paired baseline and progression samples, including patients with 2 and 5 acquired ERBB2 mutations. The ERBB2 mutant allele fraction (MAF) increased compared to baseline in 4/7 of patients at the time of progression and declined in 2/2 patients with continued response to therapy. cfDNA NGS detected mutations, unidentified by tissue NGS in 9/10 baseline samples and in 11/12 patients, including 5 ESR1 mutations in 3 patients. Conclusion: cfDNA NGS was sensitive for the detection of ERBB2 mutations. MAF dynamics typically tracked response to, or progression on therapy, at the time of cfDNA collection. The emergence of additional ERBB2 mutations was common at the time of progression; however, no T798 gatekeeper mutation was observed. Many subclonal mutations were identified on cfDNA NGS that were not observed on tissue NGS. Citation Format: Alison M. Schram, S. Duygu Selcuklu, Rebecca J. Nagy, Lillian M. Smyth, Lisa D. Eli, Richard E. Cutler, Alshad S. Lalani, David M. Hyman, Richard B. Lanman. Cell-free DNA sequencing in ERBB2-mutant breast cancer patients treated with neratinib and fulvestrant: Exploratory analysis from the Phase 2 SUMMIT ‘basket' trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5533.

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

e12554 Background: ERBB2 mutations in the absence of gene amplification are rare, with an incidence of 2-4%. Neratinib is a HER2/EGFR tyrosine kinase inhibitor being evaluated for use in ERBB2 mutated breast cancer. Neratinib has been found to have clinical activity on heavily pre-treated ERBB2 mutant breast cancer patients. We are evaluating the response and genomic profiles of 3 postmenopausal patients with metastatic ERBB2 mutant/non-amplified breast cancer receiving neratinib and fulvestrant NCT01953926, NCT01670877. Methods: Samples were collected at different points during treatment and CTCs were identified. Other representative cells were tracked but not classified as CTCs. CD45-CK+ cells with cytoplasmic and/or nuclear apoptosis were defined as CTC-Apoptotic. CD45- cells expressing little to no CK but otherwise meeting morphological criteria for CTCs are CTC-LowCK. CD45-CK+ cells with small nuclear size are CTC-SmallCK. Single CTCs will be analyzed using whole genome copy number variation to determine chromosomal alterations. Results: The patients had an average of 4.7 lines of therapy for metastatic disease before neratinib. Two had stable disease and one had progression. The patient who progressed had a rise in the number of CTCs from 37 to 52 cells/ml and drop in apoptotic cells from 5 to 0 cells/ml. Conclusions: The high CTC count of the patient who progressed may suggest more aggressive disease. The drop in the apoptotic cell count may correlate with a failure to respond to therapy. Samples have been sent for copy number variation profiling. The goal is to identify any genomic amplifications or deletions associated with clinical response and progression after targeted therapy. We hope to demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the use of fluid biopsies to monitor disease progression. [Table: see text]

Background: HER3 is widely accepted as a potent heterodimerization partner for HER2 tyrosine kinase. HER3 activation may promote intrinsic and/or acquired resistance to HER2-targeted therapies. Therapeutic strategies that target HER3 directly or in combination with HER2 are being evaluated both preclinically and clinically. Historically, such strategies have focused exclusively on tumors that exhibit HER2 gene-amplification and/or over-expression (HER2 high/positive). Recent studies suggest distinct or additional utility in non-amplified, low HER2 tumor settings (HER2 low/negative). Attempts to identify appropriate HER2-positive patient populations that are likely to benefit from HER3-targeted therapies have revealed an important need for more quantitative, sensitive methods of measuring HER2 and HER3 protein expression other than traditional IHC, and also for quantitative, directed measurements of HER3 activation. Methods: Dual-antibody proximity-binding assays (VeraTag) were used to measure HER2 total (HERmark®), HER3 total, HER2-HER3 heterodimer, phospho-HER3 and HER3-PI3 kinase (p85) protein expression across a broad dynamic range in a group of HER2-(low) and HER2+ (high) FFPE breast tumors (HER2 status determined by IHC and verified by HERmark). Dual-antibody proximity-binding assays developed for use in high HER2 FFPE tumor specimens (PloS ONE: 28 Jan 2011 10.1371/journal.pone.0016443) were optimized for use in low HER2 samples. Results: HER2 expression spanned nearly a 2-log10 dynamic range in the HER2 breast tumor set. HER3 expression spanned a 1.5-log10 dynamic range in these tumors and trended with HER2 expression levels. HER2-HER3 heterodimerization correlated more closely with HER2 expression than HER3 expression in both low and high HER2 breast tumors, suggesting that HER2 may be the primary driver of heterodimerization. Correlations between phospho-HER3, HER3-PI3K, and HER2-HER3 heterodimer measurements were observed, with each analyte spanning a ∼1-log10 dynamic range for the corresponding measurement. HER3 signaling patterns in low and high HER2 breast tumors were compared to assess the level of HER3 activation relative to HER2 expression levels. Conclusions: We have developed multiple assays for quantitative measurements of HER3 activation in FFPE breast tumors samples that express low and high levels of the HER2 receptor. These assays are presently being investigated preclinically to define mechanism of drug action, and in clinical trials to predict response to targeted therapies. Citation Format: Jerry Wallweber, Ahmed Chenna, Roy Ravanera, Weidong Huang, David Stathas, Gayle Marshall, Chris Womack, Alison Pritchard, Ken Thress, Carl Barrett, Christos Petropoulos, Lisa DeFazio Eli, John Winslow. Profiling HER3/ErbB3 activation in formalin-fixed, paraffin-embedded (FFPE) breast tumor samples that express high and low HER2/ErbB2 levels using proximity-based immunoassays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2013-3029