www.nature.com/bjc ARTICLE Clinical Study Trastuzumab and paclitaxel in patients with EGFR mutated NSCLC that express HER2 after progression on EGFR TKI treatment Adrianus J. de Langen1,2, M. Jebbink1, Sayed M. S. Hashemi2, Justine L. Kuiper2, J. de Bruin-Visser2, Kim Monkhorst3, Erik Thunnissen4 and Egbert F. Smit1,2 BACKGROUND: HER2 expression and amplification are observed in ~15% of tumour biopsies from patients with a sensitising EGFR mutation who develop EGFR TKI resistance. It is unknown whether HER2 targeting in this setting can result in tumour responses. METHODS: A single arm phase II study was performed to study the safety and efficacy of trastuzumab and paclitaxel treatment in patients with a sensitising EGFR mutation who show HER2 expression in a tumour biopsy (IHC ≥ 1) after progression on EGFR TKI treatment. Trastuzumab (first dose 4 mg/kg, thereafter 2 mg/kg) and paclitaxel (60 mg/m2) were dosed weekly until disease progression or unacceptable toxicity. The primary end-point was tumour response rate according to RECIST v1.1. RESULTS: Twenty-four patients were enrolled. Nine patients were exon 21 L858R positive and fifteen exon 19 del positive. Median HER2 IHC was 2+ (range 1–3). For 21 patients, gene copy number by in situ hybridisation could be calculated: 5 copies/nucleus (n = 9), 5–10 copies (n = 8), and >10 copies (n = 4). An objective response was observed in 11/24 (46%) patients. Highest response rates were seen for patients with 3+ HER2 IHC (12 patients, ORR 67%) or HER2 copy number ≥10 (4 patients, ORR 100%). Median tumour change in size was 42% decrease (range −100% to +53%). Median duration of response was 5.6 (95% confidence interval [CI], 3.8 to 7.3) months. Treatment toxicity was mild with four patients experiencing grade ≥3 toxicity, including fatigue, neuropathy, neutropaenia, urinary tract infection, and pneumonitis. CONCLUSIONS: Trastuzumab-paclitaxel induces objective tumour responses in 46% of EGFR TKI pretreated patients with an activating EGFR mutation and HER2 expression. The treatment was well tolerated. The relation between response rate and HER2 expression level and copy number suggests effective HER2 targeting by trastuzumab, although the combination with paclitaxel does not allow to determine the relative contribution of the individual drugs in terms of treatment efficacy. British Journal of Cancer (2018) 119:558–564; https://doi.org/10.1038/s41416-018-0194-7 INTRODUCTION Another mechanism of treatment resistance is bypass track Non-squamous NSCLC that is characterised by an EGFR exon 19 activation. This means that other transmembrane receptors or deletion or an exon 21 L858R point mutation, is highly sensitive to downstream kinases are altered, thereby bypassing inhibition of treatment with EGFR tyrosine kinase inhibitors (TKIs). In metastatic the EGFR growth signal. Tumor biopsies upon progression on patients, multiple studies have shown superior clinical outcome EGFR TKI treatment have shown overexpression or amplification of for treatment with first (erlotinib, gefitinib) or second (afatinib) HER2 and MET, and mutations in BRAF and PIK3CA.2,4 The third generation EGFR TKIs, as compared to platinum doublet mechanism involves morphological alterations: dedifferentiation chemotherapy.1 by epidermal-mesenchymal transition (EMT) or change to small- However, almost all patients develop treatment resistance, cell-lung carcinoma (SCLC), including RB1 loss.2,4 typically 10–12 months after treatment initiation. Multiple resistance HER2 amplification and overexpression have been shown to be mechanisms have been described, involving three mechanisms: oncogenic in certain types of breast cancer and play an important altered drug target, bypass track activation, and morphological role in the development and progression of disease. In order to alterations.2 ThemostfrequentdrugtargetalterationistheT790M activate downstream signalling, HER family members, including mutation that accounts for 50–60% of secondary resistance to first EGFR, HER2, HER3, and HER4 have to form dimers. The major and second generation EGFR TKI therapies.2 Based on phase III data, partner for HER2 is EGFR and on this basis HER2 can modulate osimertinib (a third generation EGFR TKI) is the preferred treatment EGFR-signalling.5 Several reports suggest that HER2 signalling for patients whose tumour is T790M positive after progression on a contributes to resistance to EGFR TKIs in EGFR-mutated patients. In firstorsecondgenerationEGFRTKI.3 vitro, it was demonstrated that acquired resistance to EGFR TKIs 1Department of Thoracic Oncology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands; 2Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands; 3Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands and 4Department of Pathology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands Correspondence: Adrianus J. de Langen ([email protected]) Received: 13 March 2018 Revised: 2 July 2018 Accepted: 4 July 2018 Published online: 31 July 2018 © Cancer Research UK 2018 Trastuzumab and paclitaxel in patients with … A. J. de Langen et al. 559 and monoclonal antibodies can be mediated by activation of according to instruction from the manufacturer. The mean HER2.6,7 This acquired resistance could be overcome by inhibition number of HER2 copies per nucleus was calculated after counting of HER2. Amplification of HER2 can be found in 12% of EGFR TKI- 40 tumour cell nuclei. The copy number variation (CNV) was resistant cell lines8 and two clinical studies detected HER2- distributed in three categories: 1–4, 5–10, and >10. The ratio with amplification in 13% of NSCLC-patients with acquired EGFR TKI Ch17 was not used because the Ch17 copy number could not be resistance.4,9 calculated for the complete set of samples. Trastuzumab is a humanised IgG1 monoclonal antibody directed All biopsies were centrally reviewed prior to study inclusion by against HER2. It binds with high affinity and specificity to the one of two experienced thoracic pathologists (E.T. or K.M.). extracellular domain of the HER2 protein, thereby suppressing HER2 signalling. As a result, it inhibits the growth of tumour cells Trial design and treatment and mediates antibody-dependent cellular cytotoxicity in cancer Patients were treated weekly with trastuzumab (first dose 4 mg/ cells exhibiting HER2 protein expression.10,11 Trastuzumab is an kg, thereafter 2 mg/kg) and paclitaxel (60 mg/m2) until disease approved treatment for breast-cancer and gastric-cancer patients progression, the development of unacceptable side effects, or a and effective in the subgroup of patients with HER2 overexpression request by either the patient or the physician to discontinue or amplification.12,13 treatment. All patients provided written informed consent before When trastuzumab is combined with a taxane (e.g., paclitaxel or entering the trial. docetaxel), improved efficacy has been observed compared to Dose modifications were allowed for trastuzumab and pacli- trastuzumab monotherapy in breast cancer.14,15 taxel. In case of cardiac dysfunction (LVEF decreases by ≥10 EF Although multiple studies in NSCLC have failed to show benefit points and to <50%), trastuzumab was interrupted for 3 weeks. If a of HER2 directed treatment, these studies were performed in repeat LVEF measurement showed no improvement, trastuzumab patients with HER2 expression, irrespective of the co-occurrence of was permanently discontinued. Administration of paclitaxel an activating EGFR mutation and tested the hypothesis of HER2 required neutrophil count to be ≥1.0 × 109/L and platelet count being an oncogenic driver in NSCLC.16–22 ≥100 × 109/L. In case of grade II peripheral neuropathy or any We hypothesised that HER2 overexpression can drive thera- grade III toxicity paclitaxel was interrupted for 7 days (one cycle). peutic resistance in EGFR mutated NSCLC patients treated with In case of absence of improvement after 7 days or in case of grade EGFR TKIs and that these patients may benefit from treatment IV neutropaenia or grade III mucositis at any time, the paclitaxel with trastuzumab-paclitaxel. dose was decreased by 25%. Paclitaxel was discontinued in case of 1234567890();,: grade ≥3 peripheral neuropathy at any time. METHODS Trial end points A single centre single arm phase II study was designed to study The primary end point was confirmed response rate according to the safety and efficacy of weekly trastuzumab-paclitaxel treatment RECIST v1.1, as determined by investigator assessment. Secondary in EGFR mutation positive NSCLC patients who showed tumour objectives included toxicity according to CTC AE 4.0, disease membrane HER2 expression in a tumour biopsy (IHC ≥ 1) after control rate (DCR), median progression-free survival (PFS), dura- having experienced progressive disease on an EGFR TKI. The trial tion of response (DOR) and overall survival (OS). Preplanned has been approved by the local ethics committee and registered exploratory subgroup analyses for treatment outcome were on clinicaltrials.gov under the number NCT02226757. performed for the different HER2 expression and gene copy number levels. Eligibility criteria Eligible patients had pathologically confirmed stage IV non- Assessments squamous NSCLC with an activating EGFR mutation and had Baseline tumour assessments were performed within 28 days prior progressed on EGFR TKI monotherapy. A biopsy obtained after to initiation of study treatment. Brain