Tyrosine Kinase Inhibitor Neratinib Research Articles

Abstract 1828: Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor neratinib in HER2 mutant cancers

Abstract Background: Tumor genomic profiling has identified patients with cancers harboring activating ERBB2 (HER2) mutations that are sensitive to HER2 targeted therapies. In the SUMMIT phase II ‘basket' trial, a subset of patients with ERBB2 mutant cancers have exhibited significant clinical benefit from treatment with the pan-HER irreversible tyrosine kinase inhibitor (TKI) neratinib. However, durable responses to neratinib are few, suggesting mechanisms of de novo and acquired drug resistance. Thus, we sought to identify druggable mechanisms of resistance to neratinib. Methods: We utilized 5637 bladder cancer (with HER2S310F) and OVCAR8 ovarian cancer (with HER2G776V) cells. Drug resistant cells were developed by exposing cells to increasing concentrations of neratinib over 6 months (5637, 600 nM; OVCAR8, 2 µM). Neratinib resistant H1781 lung cancer cells (with HER2G776>VC) and MCF7 breast cancer cells (with L755S or V777L) knock-in mutations are currently being developed. For immunoblot and drug sensitivity assays, neratinib resistant cells were maintained drug-free for 96 hours and then retreated with neratinib and other inhibitors. Candidate pathways/genes driving neratinib resistance were identified by performing RNA sequencing and whole exome sequencing in drug-resistant and -sensitive cells. Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis of both cells treated with neratinib showed effective suppression of HER2, EGFR and HER3 phosphorylation. However, they exhibited a striking increase in S6 kinase (S6K) activity and S6 phosphorylation compared to drug-sensitive parental cells, which was maintained in the presence of supra-pharmacological levels of neratinib (1 µM). S6 phosphorylation and viability of drug resistant cells was completely ablated by the combination of neratinib and the mTORC1 inhibitor everolimus, but not with the PI3Kα inhibitor alpelisib, the pan-PI3K inhibitor buparlisib, or the AKT inhibitor MK-2206, suggesting PI3K- and AKT-independent activation of mTORC1. Gene set enrichment analysis (GSEA) of RNA seq data from the drug-resistant cells revealed significant enrichment of K-Ras pathway components in addition to mTORC1 pathway. Consistent with these results, whole exome sequencing revealed activating alterations of the Ras pathway including a truncating mutation in RASA2 and a P200L mutation in PIK3CA Ras binding domain; thereby suggesting potential Ras mediated mTOR activation driving neratinib resistance. Studies are underway to confirm the contribution of Ras pathway in mTOR mediated neratinib resistance. Conclusions: These data suggest that hyperactivation of mTORC1 promotes acquired resistance to neratinib across histologically distinct ERBB2-mutant cancers. Citation Format: Dhivya R. Sudhan, Ariella B. Hanker, Angel Guerrero-Zotano, Luigi Formisano, Yan Guo, Qi Liu, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Richard Bryce, Alan Auerbach, Carlos L. Arteaga. Hyperactivation of mTORC1 drives acquired resistance to the pan HER tyrosine kinase inhibitor neratinib in HER2 mutant cancers [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 1828.

Abstract 5533: 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 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.

Neratinib Approved for HER2+ Breast Cancer.

The FDA approved the tyrosine kinase inhibitor neratinib for extended adjuvant treatment of early-stage HER2-positive breast cancer. The decision adds another treatment option to help prevent recurrence, but its relatively small potential benefit must be weighed against the risk of serious side effects.

Abstract P3-03-03: An acquired HER2 T798I gatekeeper mutation induces resistance to neratinib in a patient with HER2 mutant-driven breast cancer

Abstract ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 irreversible tyrosine kinase inhibitor (TKI) neratinib has shown clinical activity against breast cancer cells harboring HER2 activating mutations. Here, we report for the first time an acquired gatekeeper HER2T798I mutation in a patient with HER2-mutant breast cancer after an initial exceptional response to neratinib. A patient with ER+/PR+/HER2-negative invasive lobular breast cancer progressing on standard therapy was found to harbor a L869R kinase domain mutation in HER2. HER2L869R is homologous to the known activating mutation EGFRL861R/Q. MCF10A breast epithelial cells expressing HER2L869R displayed enhanced HER2-mediated signaling and were resistant to lapatinib and trastuzumab but sensitive to neratinib. The patient was enrolled in the phase II SUMMIT trial (NCT01953926) and treated with neratinib, achieving a partial response lasting 16 months before developing progression. Next gen sequencing of DNA from both a new skin metastasis and plasma cell-free DNA (cfDNA) identified HER2L869R (8.7% cfDNA), whereas a novel HER2T798I mutation was detected only in plasma at 1.3%. Deep sequencing of pre-therapy tumor tissue and plasma did not detect HER2T798I, suggesting that this mutation arose upon resistance. HER2T798I has not been reported in TCGA, COSMIC, or among plasma samples from 17,345 cancer patients subjected to digital DNA sequencing using the Guardant360 assay. HER2T798I is homologous to the EGFRT790M, KITT670I and BCR-ABLT315I gatekeeper mutations known to mediate resistance to erlotinib/gefitinib and imatinib. To examine if HER2T798I mediates resistance to neratinib, we employed biochemical and biological assays and molecular modeling of wild-type (WT) HER2 and HER2T798I. Structural modeling showed the increased bulk of the isoleucine at position 798 would result in a steric clash with neratinib, thus reducing drug binding. We stably expressed HER2WT, HER2T798I, HER2L869R and HER2L869R/T798I in MCF10A cells and NR6 mouse fibroblasts. Neratinib (10-100 nM) blocked HER2-mediated signaling in cells expressing HER2WT or HER2L869R but did not in cells expressing HER2T798I. The EGFR irreversible TKI osimertinib (100 nM), which isselective for mutant EGFR (including EGFRT790M) and approved for treatment of NSCLC expressing EGFRT790M, failed to inhibit HER2WT, HER2L869R or HER2T798I. In contrast, either the EGFR/HER2 irreversible TKI afatinib or AZ5104, a metabolite of osimertinib, strongly blocked signaling induced by HER2WT, HER2L869R or HER2T798I. Cells expressing HER2T798M displayed a significantly higher IC50 to neratinib than cells expressing HER2WT, whereas afatinib or AZ5014 were very active against all cells (IC50<10 nM). Conclusions: The acquisition of a T798I gatekeeper mutation in HER2 upon development of clinical resistance to neratinib in a breast cancer with an initial activating mutation in HER2 strongly suggests that HER2L869R is a driver mutation. We speculate that HER2T798I may arise as a secondary mutation following response to effective HER2 TKIs in other cancers with HER2 activating mutations. Certain irreversible EGFR inhibitors may be effective in patients with HER2-driven breast cancer resistant to neratinib. Citation Format: Hanker AB, Red Brewer M, Sheehan JH, Koch JP, Lanman R, Hyman DM, Cutler, Jr. RE, Lalani AS, Cross D, Lovly CM, Meiler J, Arteaga CL. An acquired HER2 T798I gatekeeper mutation induces resistance to neratinib in a patient with HER2 mutant-driven breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-03.

LC–MS/MS assay for the quantitation of the tyrosine kinase inhibitor neratinib in human plasma

Neratinib is an orally available tyrosine kinase inhibitor targeting HER2 (ERBB2) and EGFR (ERBB). It is being clinically evaluated for the treatment of breast and other solid tumors types as a single agent or in combination with other chemotherapies. In support of several phase I/II clinical trials investigating neratinib combinations, we developed and validated a novel LC–MS/MS assay for the quantification of neratinib in 100μL of human plasma with a stable isotopic internal standard. Analytes were extracted from plasma using protein precipitation and evaporation of the resulting supernatant followed by resuspension. Chromatographic separation was achieved using an Acquity UPLC BEH Shield RP18 column and a gradient methanol-water mobile phase containing 10% ammonium acetate. An ABI 4000 mass spectrometer and electrospray positive mode ionization were used for detection. The assay was linear from 2 to 1,000ng/mL and proved to be accurate (98.9–106.5%) and precise (<6.2%CV), and met the FDA guidance for bioanalytical method validation. This LC–MS/MS assay will be an essential tool to further define the pharmacokinetics of neratinib.

Effects of neratinib and combination with irradiation and chemotherapy in head and neck cancer cells.

Prognosis of patients with head and neck squamous cell carcinoma (HNSCC) is still poor. Novel therapeutic approaches are of great interest to improve the effects of radiochemotherapy. We evaluated the effects of tyrosine kinase inhibitor neratinib on HNSCC cell lines CAL27, SCC25 and FaDu as a single agent and in combination with irradiation and chemotherapy. Effects of neratinib were evaluated in HNSCC cell lines CAL27, SCC25 and FaDu. Effect on cell viability of neratinib and combination with cisplatin and irradiation was measured using CCK-8 assays and clonogenic assays. Western blot analysis was performed to distinguish the effect on epithelial growth factor receptor and HER2 expression. Apoptosis was evaluated by flow cytometry analysis. Growth inhibition was achieved in all cell lines, whereas combination of cisplatin and neratinib showed greater inhibition than each agent alone. Apoptosis was induced in all cell lines. Combination of neratinib with irradiation or cisplatin showed significantly increased apoptosis. In clonogenic assays, significant growth inhibition was observed in all investigated cell lines. Neratinib, as a single agent or in combination with chemo-irradiation, may be a promising treatment option for patients with head and neck cancer.

Adaptive Randomization of Neratinib in Early Breast Cancer

BackgroundThe heterogeneity of breast cancer makes identifying effective therapies challenging. The I-SPY 2 trial, a multicenter, adaptive phase 2 trial of neoadjuvant therapy for high-risk clinical stage II or III breast cancer, evaluated multiple new agents added to standard chemotherapy to assess the effects on rates of pathological complete response (i.e., absence of residual cancer in the breast or lymph nodes at the time of surgery).MethodsWe used adaptive randomization to compare standard neoadjuvant chemotherapy plus the tyrosine kinase inhibitor neratinib with control. Eligible women were categorized according to eight biomarker subtypes on the basis of human epidermal growth factor receptor 2 (HER2) status, hormone-receptor status, and risk according to a 70-gene profile. Neratinib was evaluated against control with regard to 10 biomarker signatures (prospectively defined combinations of subtypes). The primary end point was pathological complete response. Volume changes on serial magnetic resonance imaging were used to assess the likelihood of such a response in each patient. Adaptive assignment to experimental groups within each disease subtype was based on Bayesian probabilities of the superiority of the treatment over control. Enrollment in the experimental group was stopped when the 85% Bayesian predictive probability of success in a confirmatory phase 3 trial of neoadjuvant therapy reached a prespecified threshold for any biomarker signature (“graduation”). Enrollment was stopped for futility if the probability fell to below 10% for every biomarker signature.ResultsNeratinib reached the prespecified efficacy threshold with regard to the HER2-positive, hormone-receptor–negative signature. Among patients with HER2-positive, hormone-receptor–negative cancer, the mean estimated rate of pathological complete response was 56% (95% Bayesian probability interval [PI], 37 to 73%) among 115 patients in the neratinib group, as compared with 33% among 78 controls (95% PI, 11 to 54%). The final predictive probability of success in phase 3 testing was 79%.ConclusionsNeratinib added to standard therapy was highly likely to result in higher rates of pathological complete response than standard chemotherapy with trastuzumab among patients with HER2-positive, hormone-receptor–negative breast cancer. (Funded by QuantumLeap Healthcare Collaborative and others; I-SPY 2 TRIAL ClinicalTrials.gov number, NCT01042379.)

Gene-set analysis based on the pharmacological profiles of drugs to identify repurposing opportunities in schizophrenia.

Genome-wide association studies (GWAS) have identified thousands of novel genetic associations for complex genetic disorders, leading to the identification of potential pharmacological targets for novel drug development. In schizophrenia, 108 conservatively defined loci that meet genome-wide significance have been identified and hundreds of additional sub-threshold associations harbour information on the genetic aetiology of the disorder. In the present study, we used gene-set analysis based on the known binding targets of chemical compounds to identify the 'drug pathways' most strongly associated with schizophrenia-associated genes, with the aim of identifying potential drug repositioning opportunities and clues for novel treatment paradigms, especially in multi-target drug development. We compiled 9389 gene sets (2496 with unique gene content) and interrogated gene-based p-values from the PGC2-SCZ analysis. Although no single drug exceeded experiment wide significance (corrected p<0.05), highly ranked gene-sets reaching suggestive significance including the dopamine receptor antagonists metoclopramide and trifluoperazine and the tyrosine kinase inhibitor neratinib. This is a proof of principle analysis showing the potential utility of GWAS data of schizophrenia for the direct identification of candidate drugs and molecules that show polypharmacy.

Abstract 132: HER2 activating mutations are potential targets for colorectal cancer treatment

Abstract Background: Cancer genome sequencing is identifying new genetic alterations and new driver events in human cancers. The Cancer Genome Atlas (TCGA) colorectal cancer project found that 7% of colorectal cancer (CRC) patients have HER2 somatic mutations or HER2 gene amplification. HER2 gene amplification in CRC is known to produce resistance to the EGFR monoclonal antibodies, cetuximab and panitumumab. However, the impact of HER2 somatic mutations in CRC has not been studied and it is open question as to whether HER2 mutations are clinically important in CRC. Results: Introduction of the HER2 mutations, S310F, L755S, V777L, and V842I, into immortalized colon epithelial cells increased cell signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of HER2 V842I mutation into colorectal cancer cell lines produced resistance to the EGFR antibodies, cetuximab and panitumumab. HER2 mutations are potently inhibited by low nanomolar doses of the second generation, irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple WT (KRAS, NRAS, BRAF, and PIK3CA WT) colorectal cancer patient derived xenografts (PDX’s) identified 4 PDX's with HER2 mutations (4/48 = 8.3%). Treatment of these PDX's with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus a tyrosine kinase inhibitor produced durable shrinkage of the tumors in mice. After the final timepoint in each PDX experiment, the mice were sacrificed and the tumors excised. The tumor histology demonstrated that dual HER2 targeted therapy caused reduction in tumor cellularity and acquisition of more differentiated features. Conclusions: These data demonstrate that HER2 activating mutations are a new drug resistance mechanism to EGFR monoclonal antibodies. More importantly, these data suggest that HER2 activating mutations may themselves be a drug target for the treatment of colorectal cancer. These data form a strong pre-clinical rationale for clinical trials targeting HER2 activating mutations in metastatic CRC patients. The NSABP Foundation is in the design stage for such a clinical trial and will use its living patient biorepository (MPR-1 trial) that contains 1350 colorectal cancer patients to identify subjects for this trial. Citation Format: Shyam M. Kavuri, Naveen Jain, Francesco Galimi, Francesca Cottino, Adam C. Searleman, Wei Shen, Livio Trusolino, Samuel A. Jacobs, Andrea Bertotti, Ron Bose. HER2 activating mutations are potential targets for colorectal cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 132. doi:10.1158/1538-7445.AM2015-132

HER2 activating mutations are targets for colorectal cancer treatment.

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer.

In vitro and in vivo studies of the combination of IGF1R inhibitor figitumumab (CP-751,871) with HER2 inhibitors trastuzumab and neratinib.

The insulin-like growth factor I receptor (IGF1R) has been linked to resistance to HER2-directed therapy with trastuzumab (Herceptin). We examined the anti-tumor activity of figitumumab (CP-751,871), a human monoclonal antibody that blocks IGF1R ligand binding, alone and in combination with the therapeutic anti-HER2 antibody trastuzumab and the pan-HER family tyrosine kinase inhibitor neratinib, using in vitro and in vivo breast cancer model systems. In vitro assays of proliferation, apoptosis, and signaling, and in vivo anti-tumor experiments were conducted in HER2-overexpressing (BT474) and HER2-normal (MCF7) models. We find single-agent activity of the HER2-targeting drugs but not figitumumab in the BT474 model, while the reverse is true in the MCF7 model. However, in both models, combining figitumumab with HER2-targeting drugs shows synergistic anti-proliferative and apoptosis-inducing effects, and optimum inhibition of downstream signaling. In murine xenograft models, synergistic anti-tumor effects were observed in the HER2-normal MCF7 model for the combination of figitumumab with trastuzumab, and, in the HER2-overexpressing BT474 model, enhanced anti-tumor effects were observed for the combination of figitumumab with either trastuzumab or neratinib. Analysis of tumor extracts from the in vivo experiments showed evidence of the most optimal inhibition of downstream signaling for the drug combinations over the single-agent therapies. These results suggest promise for such combinations in treating patients with breast cancer, and that, unlike the case for single-agent therapy, the therapeutic effects of such combinations may be independent of expression levels of the individual receptors or the single-agent activity profile.

Neratinib shows efficacy in the treatment of HER2/neu amplified uterine serous carcinoma in vitro and in vivo

ObjectivesUterine serous carcinoma (USC) represents an aggressive variant of endometrial cancer and accounts for a large proportion of deaths annually. HER2/neu amplification is associated with USC in approximately 30–35% of cases. The objective of this study was to determine the sensitivity of a panel of primary USC cell lines to the small tyrosine kinase inhibitor neratinib, an ErbB1 and HER2 inhibitor, both in vitro and in vivo. MethodsHER2/neu amplification was determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 24 USC cell lines. Flow cytometry was used to determine the effects of neratinib on cell viability, cell cycle distribution and signaling in vitro. Mice harboring HER2/neu amplified xenografts were treated with neratinib to assess the efficacy of the drug in vivo. ResultsHER2/neu amplification was noted in 8/24 primary cell lines. Data regarding the efficacy of neratinib was determined using 4 HER2 amplified cell lines and 4 non-amplified cell lines with similar growth rates. Data revealed that cell lines with HER2/neu amplification were exquisitely more sensitive to neratinib compared to non-amplified cell lines (mean±SEM IC50: 0.011μM±0.0008 vs. 0.312μM±0.0456 p<0.0001). Neratinib caused arrest in the G0/G1 phase of the cell cycle and resulted in decreased autophosphorylation of HER2 and activation of S6. Neratinib treated mice harboring xenografts of HER2/neu amplified USC showed delayed tumor growth and improved overall survival compared to vehicle (p=0.0019). ConclusionsNeratinib may be a potential treatment option for patients harboring HER2/neu amplified USC. Clinical trials for this subset of endometrial cancer patients are warranted.

E15. HER2 assessment in breast cancer

Because of the important therapeutic implications, a crucial issue in breast cancer pathology is the correct identification of true HER2 positivity in tissue specimens. The relevance of such assessment on HER2-positive breast cancer patients will be further enhanced if trastuzumab is administered to patients in monotherapy, such as trastuzumab emtansine (i.e. trastuzumab-DM1, T-DM1), an antibody–drug conjugate consisting of trastuzumab linked to the cytotoxin mertansine (DM1). T-DM1 is designed to target and inhibit HER2 signaling and to deliver the chemotherapy directly inside HER2-positive cancer cells. As a consequence, it will be of utmost importance for pathologists to recognize HER2-positive carcinomas with precision, because patients will not be administered any other chemotherapeutic agents and response to treatment will rely strictly on the pathological assessment of HER2 [1]. Activation of HER2 may be driven not only by HER2 gene amplification; indeed, in terms of the biology of HER2-positive tumors, a “brand new” topic is the recent demonstration by next-generation sequencing studies of the presence of HER2 mutations in breast cancer [2]. Bose and colleagues [3] have functionally characterized 13 HER2 mutations, and seven of these mutations were activating mutations sensitive to the irreversible HER2/EGFR tyrosine kinase inhibitor neratinib [3]. Estrogen-receptor(ER-)positive breast carcinomas, either HER2-negative (score 0/1+) or showing equivocal HER2 expression (score 2+), seem to be putative candidates for HER2 mutations. Here we discuss some of the “hot topics” concerning HER2 assessment.

Design, Synthesis, and Biological Evaluation ofN-Aryl-salicylamide Derivatives as Potential Antitumor Agents

According to the scaffold hopping, a series of N-aryl-salicylamide derivatives, which have fragments of tyrosine kinase inhibitors lapatinib and neratinib were designed and synthesized. Their structures were identified by IR, H NMR, C NMR and MS techniques. The target compounds were tested for cytotoxic activity against four cancer cell lines, including A549, MCF-7, SGC-7901, Bel-7402, by methyl thiazolyl tetrazolium (MTT) in vitro. All the compounds demonstrated certain antitumor abilities, and some of them were better than gefitinib.

The effects of lapatinib and neratinib on HER2 protein levels in breast cancer cell lines.

637 Background: HER2, a member of the c-erbB receptor tyrosine kinase family, is over-expressed (HER2 gene amplification/IHC 3+, &gt;30% cells) in approx. 25% of breast cancers. Pre-clinical studies have shown that the HER2-targeted small molecule tyrosine kinase inhibitor (TKI) lapatinib (LAP) can increase HER2 levels in cell line models and can potentiate trastuzumab-mediated antibody dependent cell-mediated cytotoxicity. To assess the potential effects of the next generation TKI neratinib (NER) on expression of HER2 in breast cancer, this study compares the effects of LAP and NER on HER2 protein levels in the HER2-amplified SKBR3 and HER2-non-amplified T47D cell lines in vitro. Methods: SKBR3 and T47D were treated with LAP or NER (0.2, 1 and 2 µM) for 12, 24 and 48 hours.HER2 protein levels were determined by ELISA, immunoblotting and high content analysis (HCA). HER2 protein was examined using two antibodies targeting the extracellular domain (ECD) and the intracellular domain (ICD) of HER2. pHER2, EGFR/pEGFR, MAPK/pMAPK and AKT/pAKT levels were determined by immunoblotting. Proliferation studies utilized an acid phosphatase-based assay. Results: ELISA analysis confirmedsignificantly lower HER2 expression in T47D (44 +/- 17 pg/µg) compared to SKBR3 (748 +/- 296 pg/µg), p = 0.015. NER proved a more potent inhibitor of pHER2 and pEGFR as analyzed by immunoblotting and in proliferation studies (NER IC50 - SKBR3 0.03 +/- 0.01 nM, T47D 199 +/- 70 nM, LAP IC50 - SKBR3 20 +/- 1 nM, T47D 1.2 +/- 0.2 µM). LAP induced an increase in both ECD and ICD-containing HER2 protein levels in SKBR3 and T47D cells. No increase in HER-2 levels was observed with NER treatment, as determined by HCA and immunoblotting. EGFR protein levels increased in response to lapatinib in both cell lines. Conclusions: Our results suggest that LAP and NER have differing effects on HER2 protein levels in the models examined. NER provides greater inhibition of HER2 signaling activity but does not increase HER2 protein levels as was observed with LAP. Further pre-clinical assessment of combinations of LAP and NER with HER2 and EGFR monoclonal antibody therapies is warranted in HER2-amplified, HER2-non-amplified and EGFR-expressing breast cancer models.

Abstract 1737: Overcoming trastuzumab resistance with the irreversible Pan-HER inhibitor neratinib

Abstract Overexpression of HER2 is seen in 15-20% of breast cancer and is a predictor of poor prognosis. Trastuzumab, a monoclonal antibody to the ECD of the HER2 receptor, has been shown to increase survival in patients especially in combination with chemotherapy. However, a significant number of patients do not respond to trastuzumab treatment and resistance develops in those given trastuzumab as a monotherapy. The purpose of this study was to investigate whether the small molecule irreversible tyrosine kinase inhibitor neratinib (HKI-272) could be used to overcome resistance in trastuzumab resistant BT474 and SKBR3 cell lines and to look at the effects of neratinib on trastuzumab naive cell lines. The effects of acute trastuzumab, neratinib, and trastuzumab and neratinib combination treatments were investigated in SKBR3 and BT474 cells. It has already been established that trastuzumab does not abolish pHER2 expression and this has been implicated in trastuzumab resistance. Western blotting showed an increase in pHER2 and pERK with acute trastuzumab treatment. Conversely, acute neratinib treatment alone and in combination with trastuzumab reduced pERK and pHER2 expression. Furthermore, although acute treatment with trastuzumab decreased the expression of pHER3 and pAKT, the combination of neratinib with trastuzumab gave a much greater decrease in expression. Fluorescence microscopy was also performed on both the resistant and native SKBR3 cells after acute trastuzumab, neratinib and combination treatments. Trastuzumab treatment increased pHER2 membrane localisation in both native and resistant cells in a dose dependent manner, whilst neratinib treatment or its combination with trastuzumab reduced membrane pHER2. Cell viability experiments on BT474 and SKBR3 cells showed that neratinib treatment was much more effective at reducing cell viability than trastuzumab(p=0.0078, p=0.0499) and that combined trastuzumab and neratinib treatment was more effective at reducing cell viability than neratinib alone (p=0.0028, p=0.0148). In the trastuzumab resistant SKBR3 and BT474 cells, withdrawal of trastuzumab lead to an increase in cell viability. However, neratinib treatment was able to reverse trastuzumab resistance and significantly reduce cell viability (p=0.0022, p=0.0087). Furthermore, combined treatment with trastuzumab and neratinib was more effective at reducing cell viability than neratinib alone(p=0.0087, p=0.0260) suggesting that trastuzumab treatment should be continued even when resistance develops. These results showed that the combined treatment with neratinib and trastuzumab was more effective in the inhibition of pHER2, pHER3, pAkt and pERK, and led to a greater decrease in cell viability than trastuzumab treatment alone. We would therefore recommend neratinib to be used in combination with trastuzumab in both trastuzumab naive and resistant HER2 over-expressing breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1737. doi:10.1158/1538-7445.AM2011-1737

Safety and Efficacy of Neratinib (HKI-272) in Combination with Vinorelbine in ErbB2+ Metastatic Breast Cancer.

Abstract Background: The irreversible pan-ErbB tyrosine kinase inhibitor neratinib (HKI-272) potently blocks downstream signaling of the ErbB1, -2, and -4 receptors and has demonstrated antitumor activity in patients with ErbB2+ breast cancer (BC). Vinorelbine has shown synergistic antitumor activity in combination with the ErbB2-specific monoclonal antibody trastuzumab. The objective of this ongoing phase 1/2 study is to evaluate the safety and efficacy of neratinib in combination with vinorelbine in patients with solid tumors and ErbB2+ metastatic BC.Material and Methods: This open-label, 2-part, phase 1/2 study enrolled patients with solid tumors (part 1) to evaluate safety and determine the maximum tolerated dose (MTD) of neratinib administered as ascending multiple daily oral doses (160 mg, 240 mg) in combination with IV vinorelbine 25 mg/m2 (days 1 and 8 every 3 weeks). In part 2, the clinical activity, safety, and pharmacokinetics (PK) of the combination will be assessed in patients ≥18 years of age with ErbB2+ metastatic BC (Eastern Cooperative Oncology Group status of 0 to 2) who had received ≤2 prior treatments with a trastuzumab-containing regimen for ≥6 weeks, with or without prior lapatinib exposure. The primary endpoint is objective response rate. Tumor responses are assessed by modified RECIST criteria every 6 weeks.Results: In part 1 (n = 12), no dose-limiting toxicities were observed in the neratinib 240-mg–vinorelbine 25-mg/m2 cohort (full standard doses; defined as the MTD). Two patients withdrew from the study because of adverse events (1 case each of neuropathy and progression of non-small cell lung cancer). As of the 1 May 2009 data cutoff, 1 patient with gastric cancer had received treatment with neratinib 160 mg plus vinorelbine for 183 days and 1 patient with BC had received treatment with neratinib 240 mg plus vinorelbine for 259 days. In part 2, as of 1 May 2009, 14 patients with ErbB2+ metastatic BC were enrolled and treated at the MTD (including 11 in the no prior lapatinib exposure cohort and 3 in the prior lapatinib exposure cohort; 100% were female). The median duration of treatment with neratinib was 5.9 weeks (range, 0-26). The most common drug-related treatment-emergent adverse events (all grades, ≥10% of patients) included diarrhea (86%), vomiting (36%), nausea (29%), fatigue (29%), headache (14%), and neutropenia (14%). No cases of rash were observed. The most common adverse events leading to dose reductions were diarrhea (n = 2) and vomiting (n = 1). One patient withdrew from the study because of diarrhea. Six patients from part 2 are evaluable for efficacy, all in the no prior lapatinib cohort. Among these 6 patients, best overall responses (based on investigator-assessed radiographic and clinical data) included 1 partial response and 4 cases of stable disease &amp;lt;24 weeks.Discussion: Preliminary results of this study show that the combination of neratinib and vinorelbine is tolerable and demonstrates promising antitumor activity in patients with ErbB2+ metastatic BC. Updated efficacy and safety data are forthcoming, as well as PK results. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5095.