High-Throughput Functional Evaluation of Variants of Unknown

Published OnlineFirst July 2, 2018; DOI: 10.1158/1078-0432.CCR-18-0991

Personalized Medicine and Imaging Clinical Cancer Research High-Throughput Functional Evaluation of Variants of Unknown Signiﬁcance in ERBB2 Masaaki Nagano1,2, Shinji Kohsaka3, Toshihide Ueno1, Shinya Kojima1, Kanju Saka4, Hirotaro Iwase4, Masahito Kawazu3, and Hiroyuki Mano1,5

Abstract

Purpose: The advent of next-generation sequencing tech- prevalence of concurrent ERBB2 mutation with gene amplifi- nologies has enabled the identification of several activating cation and found that approximately 30% of ERBB2-amplified mutations of Erb-B2 receptor tyrosine kinase 2 (ERBB2) urothelial carcinomas simultaneously carried ERBB2 muta- among various cancers. However, the significance of infre- tions, altering their sensitivity to trastuzumab, an mAb against quent mutations has not been fully investigated. Herein, we ERBB2. Furthermore, the MANO method was applied to comprehensively assessed the functional significance of the evaluate the functional significance of 17 compound muta- ERBB2 mutations in a high-throughput manner. tions within ERBB2 reported in the COSMIC database, reveal- Experimental Design: We evaluated the transforming activ- ing that compound mutations involving L755S were sensitive ities and drug sensitivities of 55 nonsynonymous ERBB2 to osimertinib but insensitive to afatinib and neratinib. mutations using the mixed-all-nominated-in-one (MANO) Conclusions: Several ERBB2 mutations showed varying method. sensitivities to ERBB2-targeted inhibitors. Our comprehensive Results: G776V, G778_S779insG, and L841V were newly assessment of ERBB2 mutations offers a fundamental database revealed to be activating mutations. Although afatinib, ner- to help customize therapy for ERBB2-driven cancers. atinib, and osimertinib were shown to be effective against We identified several ERBB2 mutations as activating muta- most of the ERBB2 mutations, only osimertinib demonstrated tions related to tumorigenesis. In addition, our comprehensive good efficacy against L755P and L755S mutations, the most evaluation revealed that several ERBB2 mutations showed common mutations in breast cancer. In contrast, afatinib and varying sensitivities to ERBB2-targeted inhibitors, and thus, neratinib were predicted to be more effective than other the functional significance of each variant should be inter- inhibitors for the A775_776insYVMA mutation, the most preted precisely to design the best treatment for each patient. frequent ERBB2 mutation in lung cancer. We surveyed the Clin Cancer Res; 1–11. 2018 AACR.

Introduction ERBB2-positive gastric and breast cancers (8, 9). ERBB2 mutations were first reported in 2% to 4% of lung adenocarcinomas (10), Erb-B2 receptor tyrosine kinase 2 (ERBB2) is a member of the and cancer genome resequencing with next-generation sequenc- ErbB tyrosine receptor family, which also includes EGFR, ERBB3, ing technologies has identified many ERBB2 mutations across a and ERBB4. ERBB2 dimerizes with itself or other ErbB members to variety of cancers, such as breast, lung, gastric, colorectal, liver, activate two major downstream signaling pathways: PI3K–AKT ovarian, and urothelial cancers (11–16). and MEK–ERK (1). ERBB2 gene amplification occurs in a wide Some ERBB2 mutations are known to be activating mutations variety of human cancers (2–4). ERBB2-targeted therapies, such as that induce oncogenic transformation (11, 17), but many other trastuzumab, lapatinib, and pertuzumab, have improved out- mutations are still variants of unknown significance (VUS; refs. 16, comes in patients with ERBB2 amplification-positive cancers 18). In fact, although over 300 ERBB2 nonsynonymous mutations (5–7), and these drugs have been approved by the FDA against are reported in the COSMIC database (http://cancer.sanger.ac.uk/ cosmic/), only 74 of these are annotated in the OncoKB database (http://oncokb.org/). Furthermore, only a few studies have exam- 1Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 2Department of General Thoracic Surgery, Graduate ined the sensitivity of these mutations to drugs (11, 18, 19). Phase School of Medicine, The University of Tokyo, Tokyo, Japan. 3Department of I and II clinical trials have shown that irreversible ErbB receptor Medical Genomics, Graduate School of Medicine, The University of Tokyo, Tokyo, family inhibitors, such as afatinib, neratinib, and dacomitinib, are Japan. 4Department of Forensic Medicine, Graduate School of Medicine, The effective for tumors expressing ERBB2 mutations (20–22). Recent- 5 University of Tokyo, Tokyo, Japan. National Cancer Center Research Institute, ly, the efficacy of neratinib monotherapy was tested across 21 Tokyo, Japan. cancer types in the SUMMIT trial (23), and the greatest clinical Note: Supplementary data for this article are available at Clinical Cancer activity was observed in breast cancer (objective response rate at Research Online (http://clincancerres.aacrjournals.org/). week 8, 32%). Moreover, a recent study has demonstrated osi- Corresponding Authors: Shinji Kohsaka, Department of Medical Genomics, The mertinib, a third-generation tyrosine kinase inhibitor (TKI) University of Tokyo, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, against EGFR T790M, as a potential ERBB2-targeting agent Tokyo 113-0033, Japan. Phone: 813-5841-0633; Fax: 813-5841-0634. E-mail: (24). However, there has been no large clinical trial of afatinib [email protected]; and Hiroyuki Mano, [email protected] or osimertinib conducted for ERBB2 mutation–positive cancers. doi: 10.1158/1078-0432.CCR-18-0991 Moreover, recent studies have also reported the emergence of 2018 American Association for Cancer Research. ERBB2 K753E or L755S mutation after trastuzumab treatment or

www.aacrjournals.org OF1

Nagano et al.

Virus production and infection Translational Relevance The recombinant plasmids were transduced together with The development of next-generation sequencing technolo- packaging plasmids (Takara Bio) into HEK293T cells to achieve gies has identified many Erb-B2 receptor tyrosine kinase 2 recombinant retroviral particles. The 3T3 cells were infected in (ERBB2) mutations across a wide range of cancers. Several 12-well plates with ecotropic recombinant retroviruses using ERBB2 mutations are known to be activating mutations, but 4-mg/mL Polybrene (Sigma-Aldrich) for 24 hours. Ba/F3 cells many other mutations are still variants of unknown signifi- were plated in retronectin-coated (Takara Bio) 12-well plates cance. Moreover, only a few studies have examined the sen- and infected with the retroviruses in RPMI1640 medium con- sitivity of the ERBB2 mutations to drugs. In this study, we taining 20 U/mL IL3. comprehensively assessed the transforming activities and drug sensitivities of 55 nonsynonymous ERBB2 mutations in a Focus formation assay high-throughput manner using the mixed-all-nominated-in- For the focus formation assay, 3T3 cells expressing various one (MANO) method. We identified several ERBB2 mutations ERBB2 mutants were cultured in DMEM-F12 supplemented with as activating mutations related to tumorigenesis and revealed 5% bovine calf serum for 2 weeks. The cells were then stained with that several ERBB2 mutations showed varying sensitivities to Giemsa solution. ERBB2-targeted inhibitors. Thus, the functional significance of each variant should be interpreted precisely to design the best The MANO method treatment for each patient, and the MANO method might be Schematic representation of the MANO method is shown in beneficial for the determination of the best treatment for Supplementary Fig. S1. This method uses a retroviral vector that cancers harboring ERBB2 mutations. enables the stable integration of individual genes into the genome of assay cells (such as mouse 3T3 fibroblasts or the IL3-dependent, murine pro–B-cell line Ba/F3) along with 6-basepair (bp) barcode sequences. Individually transduced assay cells are subsequently pooled and cultured in a competitive manner to evaluate their of ERBB2 T798I mutation after neratinib treatment, suggesting transforming potential or drug sensitivity, either in vitro or that acquired secondary mutations may be a mechanism of in vivo. At the end of expansion period, genomic DNA was resistance to ERBB2-targeted inhibitors (25, 26). obtained from cell lysates using the QIAamp DNA Mini Kit Herein, we searched for nonsynonymous ERBB2 mutations (Qiagen), followed by amplification by PCR using primers that are recurrently reported in the COSMIC database (v78) and 50-TGGAAAGGACCTTACACAGTCCTG-30 and 50-GACTCGTT- comprehensively evaluated their transforming activity and GAAGGGTAGACTAGTC-30. The obtained products were puri- drug sensitivity in a high-throughput manner using the mixed- fied using AMPure beads (Beckman Coulter), and the sequenc- all-nominated-in-one (MANO) method, which was recently ing libraries were prepared using the NEB NextUltra DNA developed in our laboratory (27). This method is beneficial in Library Prep Kit (NEB) according to the manufacturer's instruc- that it enables the assessment of not only the oncogenic potential tions. The library quality was evaluated using a Qubit 2.0 but also the drug sensitivity of hundreds of gene mutations within fluorometer (Thermo Fisher Scientific) and the Agilent 2200 a short period of time in a competitive manner with positive and TapeStation system. The library was sequenced on an Illumina negative controls. MiSeq using the Reagent Kit V2 (300 cycles), and 150-bp paired-end reads were created. The barcode sequence Materials and Methods 50-CTAGACTGCCXXXXXXGGATCACTCT-30 (where X denotes Cell lines any nucleotide) was included in the sequencing results, and the Human embryonic kidney (HEK) 293T cells and 3T3 cells were number of each barcode in each mutant was quantified. purchased from the ATCC and cultured in DMEM-F12 supplemented with 10% FBS, 2 mmol/L glutamine, and 1% penicillin/ Functional annotation of ERBB2 mutations using the MANO streptomycin (all from Thermo Fisher Scientific). Ba/F3 cells were method maintained in RPMI1640 medium (Thermo Fisher Scientific) 3T3 cells expressing various ERBB2 mutants were mixed 2 days supplemented with 10% FBS, 2 mmol/L glutamine, 1% penicil- after mutant infection of the cells. The mixed cell population was lin/streptomycin, and mouse IL3 (20 U/mL; Sigma). maintained in DMEM-F12 with 10% FBS for 12 days. Ba/F3 cells harboring different ERBB2 mutants were mixed in a similar Establishment of retroviral vector with random barcodes manner and cultured in RPMI1640 medium without IL3. Cell The pcx5 vector was created by inserting random 6-bp DNA pellets were stored every 3 days, and the experiment was carried barcode sequences upstream of the start codon of the genes of out in triplicate in both cell lines. We determined the time when interest into the pcx4 vector (28). The full-length wild-type cDNAs cells were mixed as day 0, and cell mixtures obtained on day 0 of human EGFR and ERBB2 were cloned into the pcx5 vector. were used as the reference control for scaling each cell clone signal: Plasmids encoding EGFR and ERRB2 mutations were developed the signal from each cell pellet collected every 3 days was eval- using the QuikChange II Site-Directed Mutagenesis Kit (Agilent uated as 100 (average read number across replicates)/(average Technologies) with mutation-specific primers. The plasmids were read number of the mixed cell population on day 0). The fold sequenced with the BigDye Terminator version 3.1 Cycle Sequenc- change in the ratio of ERBB2-mutant cell number on day 12 ing Kit (Applied Biosystems) and analyzed with a 3730 ABI relative to day 0 was compared with that of wild-type ERBB2 capillary electrophoresis system, with confirmation by Sanger cell number to perform paired t test. ERBB2 mutants whose sequencing that the plasmids harboring different ERBB2 mutants fold changes increased significantly (P < 0.05) were regarded as carried different 6-bp barcodes. activating mutants.

OF2 Clin Cancer Res; 2018 Clinical Cancer Research

Evaluation of Drug Sensitivity of ERBB2 Mutations

Inhibitor assays using the MANO method or osimertinib at high doses (20 mg/kg body weight; n ¼ 5 mice Ba/F3 cells expressing each EGFR or ERBB2 mutant were for each group). All drugs were dissolved in 2% DMSO, cultured in RPMI1640 medium without IL3. The transformed 30% polyethylene glycol 300, and sterile ultrapure water. The Ba/F3 cells, which showed IL3-independent growth, were average tumor volume in each group was calculated using the mixed in equal amounts and incubated for 72 hours with formula 1/2 (large diameter) (small diameter)2. the indicated concentrations of each inhibitor: trastuzumab (1 ng/mL to 100 mg/mL), lapatinib (0.1 nmol/L–10 mmol/L), Pharmacokinetic studies sapitinib(0.1nmol/L–10 mmol/L), afatinib (0.01 nmol/L– Serial blood samples were collected at 0.5, 1, 2, 4, 6, and 24 1 mmol/L), neratinib (0.01 nmol/L–1 mmol/L), and osimerti- hours after intraperitoneal injection of single doses (n ¼ 3 mice for nib (0.1 nmol/L–10 mmol/L). The experiment was conducted each group). Each sample was spiked with crizotinib (LC Labo- in triplicate. We calculated the number of each barcode using ratories) as an internal standard and processed by acetonitrile the MANO method. Considering the different doubling times protein precipitation. The samples were then analyzed using a of the transduced cells, DMSO-treated cell mixtures were used validated high-performance liquid chromatography tandem mass as the reference control for scaling each cell clone signal. The spectrometry method. Drug-to-internal standard peak area ratios relative growth inhibition of each cell clone was calculated as for the standards were used to create a calibration curve. Plasma 100 (average read number across triplicates)/(average read concentrations of the samples were quantified by comparing the number of the DMSO control). Six ERBB2-targeted drugs used ratios for each sample with those in the relevant calibration curve. in the inhibitor assay were purchased commercially: trastuzu- The lower limit of quantification was 5 and 10 ng/mL for afatinib mab (Chugai Pharmaceutical Co.), lapatinib (LC Laborato- and osimertinib, respectively. ries), sapitinib (Selleckchem), afatinib (LC Laboratories), neratinib (LC Laboratories), and osimertinib (LC Laboratories). Statistical analysis Statistical significance was evaluated using Student t test for Alamar Blue cell viability assay comparisons between two mutants in vitro (each ERBB2 mutant The transformed Ba/F3 cells expressing each EGFR or ERBB2 vs. wild-type ERBB2). Statistical comparisons between two groups mutant were cultivated in 96-well plates (with 100 mLof in vivo (DMSO/vehicle vs. each ERBB2-targeted drug) were con- culture medium/well) in RPMI1640 medium without IL3, and ducted using one-way ANOVA with a Dunnett multiple compar- each ERBB2 inhibitor was added at different concentrations: isons test. For all comparisons, P < 0.05 was considered statisti- trastuzumab (1 ng/mL–100 mg/mL), afatinib (0.01 nmol/L– cally significant. 1 mmol/L), neratinib (0.01 nmol/L–1 mmol/L), and osimertinib (0.1 nmol/L–10 mmol/L). Next, 10 mL of Alamar Blue (Thermo Fisher Scientific) was added to the plates 72 hours Results after exposure to these inhibitors, and the fluorescence was Spectrum of ERBB2 mutations identified in the COSMIC measured (excitation 530 nm, emission 590 nm) at the indicated database times (29). Wells without cells were used as negative controls, and In the COSMIC database, a total of 55 nonsynonymous muta- survival data were graphically analyzed using GraphPad Prism tions in ERBB2 are reported as recurrent mutations (Supple- software version 7.0 for Windows (GraphPad Software Inc.). mentary Table S1). Comparing similarities and differences of ERBB2 mutations reported recurrently in the COSMIC, TCGA, The In Vivo MANO method MSK-IMPACT, or Cancer Hotspots database (30, 31), the COS- Individually transduced 3T3 cell clones were mixed in equal MIC database stored the largest number of mutations among all numbers, and 3.0 106 cells of the mixture (i.e., 0.5 105 cells databases (Supplementary Fig. S2; Supplementary Table S2A). from each of the 60 cell clones) were subcutaneously injected into Because we prioritized recurrently reported mutations to evaluate 6-week-old female nude mice according to the animal use pro- the significance, we determined to use the COSMIC database tocol reviewed and approved by the University of Tokyo Animal rather than the OncoKB database for this study. The 55 recurrent Care and Use Committee. All drugs were dissolved in 2% DMSO, mutations in the COSMIC database are found across various 30% polyethylene glycol 300 (Sigma Aldrich), and sterile ultra- cancer subtypes, and such mutations are most frequently reported pure water. Tumor size was evaluated every 2 days by caliper in lung cancer, followed by breast and bladder urothelial carci- measurements, and the average tumor volume was calculated noma (Fig. 1A). We then examined the location of these muta- using the formula 1/2 (large diameter) (small diameter)2. The tions in the ERBB2 protein structure (Fig. 1B). The 55 mutations tumors were resected and mechanically homogenized using a included 16 mutations in the ectodomain, two mutations in the gentleMACS Dissociator (Miltenyi Biotec) according to the juxtamembrane domain, 29 mutations in the intracellular tyro- "homogenization of tissue for total RNA isolation" protocol set sine kinase domain, and eight mutations in the C-terminal domain. forth by the manufacturer. The relative abundance of each cell Note that E770_A771insAYVM and A775_G776insYVMA muta- clone was calculated using the MANO method. tions lead to the identical amino acid changes causing YVMA duplication. The most frequent ERBB2 mutation in lung cancer Xenograft tumor assays was A775_G776insYVMA, while L755S mutations constituted For xenograft generation, 1.0 106 3T3 cells expressing the approximately 40% of ERBB2 mutations in breast cancer. ERBB2 L755P were subcutaneously injected into 6-week-old We next analyzed the datasets of TCGA and MSK-IMPACT (30) female nude mice. The mice were treated every 2 days by an stored in cBioPortal (http://www.cbioportal.org) to determine intraperitoneal injection of either vehicle control, afatinib at low the combined status of ERBB2 mutations with gene amplification doses (8 mg/kg body weight), afatinib at high doses (40 mg/kg in each type of cancer (Supplementary Tables S2B–S2D). Muta- body weight), osimertinib at low doses (8 mg/kg body weight), tions and amplifications of ERBB2 were observed at a similar

www.aacrjournals.org Clin Cancer Res; 2018 OF3

Nagano et al.

Figure 1. Spectrum and functional annotation of ERBB2 mutants. A, Patterns of ERBB2 mutations in different cancers. The bar chart illustrates the number of tumor samples with ERBB2 mutations in the COSMIC database according to cancer types. Mutation type are coded in green (missense mutation), purple (frameshift), orange (complex insertion/substitution), and red (insertion). B, Distributions of ERBB2 mutations detected in the COSMIC database. C, The fold change in the relative proportion of Ba/F3 (x-axis) and 3T3 (y-axis) cells harboring each ERBB2 mutant on day 12, which were normalized to that on day 0, is plotted. Blue circles highlight the ERBB2 mutations that were evaluated as activating mutations in both cell lines. D, Venn diagram revealing the numbers of oncogenic ERBB2 mutations observed in 3T3 cells (green) and Ba/F3 cells (pink).

frequency in the two datasets (Supplementary Fig. S3). The mutants in both cell lines, in which G776V, G778_S779insG, frequency of ERBB2 mutations was highest in bladder urothe- and L841V were newly shown activating mutants. In parallel with lial carcinoma followed by esophageal and cervical cancers, the MANO method, we also assessed the transforming potential whereasthatofamplification was highest in esophageal cancer of ERBB2 mutants using the focus formation assay and obtained followed by stomach and breast cancers. It is notable that a similar results to those obtained with the MANO method (Sup- certain number of tumors carried both ERBB2 mutation and plementary Fig. S6). amplification (Supplementary Table S2E). In particular, approximately 30% of bladder urothelial carcinomas carried Evaluation of the sensitivity of ERBB2 mutants to ERBB2- simultaneous ERBB2 amplification and mutation. targeted drugs in vitro To evaluate the drug sensitivity of ERBB2 mutants, we treated Functional annotation of somatic ERBB2 mutations the mixture of Ba/F3 cells expressing 57 different types of muta- To assess the transforming activity of ERBB2 mutations, we tions with six different targeted drugs (small compounds and an used the MANO method to calculate the temporal changes in the antibody) at various concentrations. We excluded Ba/F3 cells relative proportions of 3T3 and Ba/F3 cells harboring each ERBB2 harboring GFP, A775G, or E914K from this analysis because these mutant in the entire cell culture population from day 0 to 12 cells did not show IL3-independent growth. As shown in Fig. 2A (Supplementary Fig. S4; Supplementary Table S3). The fold and Supplementary Table S4, wild-type ERBB2 was sensitive to all changes in the read counts on day 12 were then normalized to six drugs. Whereas EGFR L858R was sensitive to afatinib, nerati- those on day 0 (Fig. 1C; Supplementary Fig. S5). As shown nib, and osimertinib, EGFR T790M_L858R was sensitive only to in Fig. 1D, 18 ERBB2 mutants were evaluated as activating osimertinib, and EGFR T790M_C797S was resistant to all six

OF4 Clin Cancer Res; 2018 Clinical Cancer Research

Evaluation of Drug Sensitivity of ERBB2 Mutations

Figure 2. The sensitivities of ERBB2 mutations to ERBB2-targeted drugs. A, Ba/F3 cells expressing wild-type ERBB2, 53 ERBB2, and three EGFR mutants (indicated at the top) were treated with DMSO or ERBB2-targeted drugs (trastuzumab, lapatinib, sapitinib, afatinib, neratinib, and osimertinib) at the indicated concentrations. Wild-type ERBB2 and three EGFR mutants (L858R, T790M_L858R, and T790M_C797S) were grouped together on the right. The relative viability of the treated cells with each drug compared with the corresponding DMSO-treated cells was measured, and the results are illustrated according to the color-coded scale indicated at the top right. WT, wild type; TM, transmembrane; AS, autophosphorylation site. B, Ba/F3 cells expressing wild-type ERBB2, L755P, A775_776insYVMA, G776>LC, V777L, T798M, T798M_C805S, EGFR_L858R, EGFR_T790M, or EGFR_T790M_C797S mutant were treated with the indicated concentrations of afatinib, neratinib, or osimertinib for 72 hours. Viable cells were measured using the Alamar Blue cell viability assay and plotted relative to the untreated controls. The results are shown as the mean values of ﬁve independent experiments. Error bars, SEM.

drugs. These results confirmed the validity of the MANO E770_A771insAYVM and A775_776insYVMA than it was against method. Most of the ectodomain and the C-terminal domain other mutations. ERBB2 mutants conferred sensitivity to trastuzumab, whereas The Alamar Blue cell viability assay was performed to precisely mostofthetyrosinekinasedomainmutationswereresistantto determine the sensitivity of the ERBB2 mutants to afatinib, the antibody. Furthermore, L755P/S mutants were evaluated neratinib, and osimertinib (Fig. 2B). EGFR L858R was sensitive as resistant to lapatinib, which was compatible with the find- to all three drugs, and well-known TKI-resistant mutations ings of previous studies (32, 33). E770_A771insAYVM, (ERBB2 T798I_C805S, and EGFR T790M_C797S) were shown A775_776insYVMA, G776>LC, G776>VC, S779_P780insVGS, to be resistant to all the drugs, confirming the validity of our test. and V842I mutations were also resistant to lapatinib. Several The IC50 values of afatinib and neratinib against ERBB2 L755P ERBB2 mutants were insensitive to sapitinib (IC50 > 100 nmol/L) were 8.94 and 7.03 nmol/L, respectively, and were higher than in this study. Afatinib and neratinib revealed similar sensitivities those against TKI-sensitive mutations such as V777. Osimertinib, to most of the mutations, and they were partially insensitive to the in contrast, demonstrated a high IC50 value against ERBB2 L755P mutation. Considering the IC50 values of Ba/F3 cells A775_776insYVMA (170 nmol/L) compared with the other expressing EGFR T790M_L858R (5.8 nmol/L), wild-type EGFR ERBB2 mutants, including L755P. Then, the IC50 values of these (752 nmol/L), and wild-type ERBB2 (10.4 nmol/L) in previous drugs for EGFR mutations were tested because afatinib and studies (24, 34), osimertinib was effective against most of the osimertinib are commonly used drugs for EGFR mutation– ERBB2 mutants including the L755P mutation. However, this positive lung cancer (Supplementary Fig. S7). As reported previ- drug was less effective against exon 20 insertions such as ously (27, 35), the IC50 values of afatinib and neratinib against

www.aacrjournals.org Clin Cancer Res; 2018 OF5

Nagano et al.

Figure 3. Inhibition of tumor growth in vivo by ERBB2-targeted drugs. A, 3T3 cells expressing either wild-type ERBB2, ERBB2 mutants, or EGFR mutants were subcutaneously injected into 6-week-old female nude mice. EGFR mutants (L858R, T790M_L858R, and T790M_C797S) were included as controls. The mice were treated every 2 days with an intraperitoneal injection of each ERBB2-targeted drug (n ¼ 10 mice for each drug). Tumor volumes on day 12 in the groups treated with osimertinib, neratinib, or afatinib were statistically smaller than those in the control group (one-way ANOVA with a Dunnett multiple comparisons test). , P < 0.05; , P < 0.01. Error bars, SD. B, The tumors were resected 13 days after injection of the 3T3 cell mixtures. The relative proportion of each cell clone in the mixed cell population was determined using the MANO method. For each sample, the barcode number for each cell line was calculated and converted to the relative contribution of each cell line to the tumor. The graph shows the relative growth of the top 13 mutant cell clones in the mixture, which accounted for more than 1% of the total (one-way ANOVA with a Dunnett multiple comparisons test). , signiﬁcant difference between the treated group and the control group for each mutant. Error bars, SEM. C, A total of 1.0 106 3T3 cells expressing ERBB2 L755P were subcutaneously injected into nude mice. The mice were treated as in A (n ¼ 5 mice for each group). , P < 0.01. ns, not signiﬁcant. Error bars, SD, one-way ANOVA with a Dunnett multiple comparisons test.

ERBB2 exon 20 insertions were lower than those against EGFR have weak efficacy against many ERBB2 mutants and were not exon 20 insertions. In contrast, the IC50 value of osimertinib considered promising treatment for cancers harboring those against ERBB2 A775_776insYVMA was similar to that against mutations. Tumor volumes decreased significantly by day 12 in EGFR V769_D770insASV and higher than those against other the group treated with any inhibitor but lapatinib compared with EGFR exon 20 insertions. those in the control group (Fig. 3A). The tumors were excised from the mice on day 12, and the relative percentage of each cell clone Evaluation of the sensitivity of ERBB2 mutants to ERBB2- was quantitated using the MANO method. The fold changes in targeted drugs in vivo read numbers in the control group on day 12 compared with day 0 We next measured the effectiveness of each ERBB2 inhibitor are shown in Supplementary Fig. S8 and Supplementary Table S5. in vivo. 3T3 cells expressing 60 different genes, including GFP, In the vehicle-treated group, 13 mutant cell clones accounted wild-type or mutant forms of ERBB2, or EGFR mutants (listed in for more than 1% of the total reads and were thus considered Supplementary Table S1), were pooled and injected en bloc into evaluable. We then compared the relative proportions treated nude mice. The mice were treated every 2 days with an intraper- with each inhibitor of these 13 mutant cell clones to those of the itoneal injection of either vehicle control, lapatinib (100 mg/kg vehicle control group. As shown in Fig. 3B and Supplementary body weight), afatinib (40 mg/kg body weight), neratinib Table S6, lapatinib was effective against ERBB2 V777L and L841V. (40 mg/kg body weight), or osimertinib (20 mg/kg body weight) The relative proportion of cell clones expressing ERBB2 mutants for 12 days (n ¼ 10 mice for each group). We did not examine decreased significantly in the group treated with afatinib and trastuzumab or sapitinib in vivo for further analysis because in vitro neratinib, whereas that of the cell clones expressing the TKI- experiments using the MANO method showed that the two drugs resistant EGFR mutants (T790M_L858R and T790M_C797S)

OF6 Clin Cancer Res; 2018 Clinical Cancer Research

Evaluation of Drug Sensitivity of ERBB2 Mutations

increased. In contrast, the proportion of cell clones harboring relatively higher than those of the clinical standard dose of E770_A771insAYVM and A775_776insYVMA was increased sig- afatinib (Supplementary Fig. S9; refs. 38, 39). nificantly in the group treated with osimertinib. Although cell clones carrying the L755P mutation demon- Detection and analysis of ERBB2 compound mutations strated a significant decrease when they were treated with EGFR compound mutations, defined as multiple mutations in afatinib and neratinib in vivo, these mutations were shown to the EGFR gene, constitute 14% to 30% of all EGFR mutations be less sensitive to the two drugs in vitro. Therefore, we further (27, 40, 41). We expected that similar compound mutations explored the in vivo antitumor efficacy of afatinib and osimer- might also exist in ERBB2 and indeed found 15 types of ERBB2 tinib against the xenograft of L755P. As shown in Fig. 3C, a low compound mutations in the COSMIC database (Supplementary dose of afatinib (8 mg/kg) could not inhibit tumor growth, Table S7A). S310F is most frequently found in the compound whereas a similar dose of osimertinib (8 mg/kg) demonstrated mutation form (Fig. 4A), with six compound mutations in 33 asignificant inhibitory effect on tumor growth to an extent samples analyzed by whole-exome or RNA sequencing. ERBB2 similar to high-dose afatinib (40 mg/kg) and high-dose osi- compound mutations were identified in many cancer types mertinib (20 mg/kg). The pharmacokinetics of these two drugs (Supplementary Table S7B), the most common of which was were monitored to ensure the validity of the drug concentration breast cancer (six cases), followed by bladder urothelial carcino- used in the in vivo assays. The geometric mean plasma concen- ma (five cases). tration–time profile after single doses of low-dose osimertinib We then evaluated the transforming potential of these com- (Cmax ¼ 230 nmol/L) was similar to that of the clinical standard pound mutations using the focus formation assay (Supplemen- dose of osimertinib (36, 37). In contrast, the plasma concen- tary Fig. S10) and the MANO method (Supplementary Fig. S11). trations of low-dose afatinib (Cmax ¼ 343 nmol/L) were The transforming potential of such compound mutations tends to

Figure 4. Analysis of ERBB2 compound mutations. A, The number of tumor samples with ERBB2 compound mutations in the COSMIC database. Blue and orange bars show the compound and simple mutations, respectively. B, Ba/F3 cells expressing wild-type ERBB2, 18 ERBB2 simple mutations, 17 ERBB2 compound mutations, and three EGFR mutations (indicated at the top) were treated with DMSO or ERBB2-targeted drugs (trastuzumab, lapatinib, sapitinib, afatinib, neratinib, or osimertinib) at the indicated concentrations. Wild-type ERBB2 and three EGFR mutants (L858R, T790M_L858R, and T790M_C797S) were grouped together on the right. The relative viability of the cells treated with each drug compared with the corresponding DMSO-treated cells was measured, and the results are illustrated according to the color-coded scale indicated at the top left. WT, wild type. C, Ba/F3 cells expressing wild-type ERBB2 or different ERBB2 mutants were treated with the indicated concentrations of trastuzumab, afatinib, neratinib, or osimertinib for 72 hours. EGFR mutants (L858R, T790M, and T790M_C797S) were also included as controls. Viable cells were measured using the Alamar Blue cell viability assay and plotted relative to the untreated controls. The results are presented as the mean values of ﬁve independent experiments. Error bars, SEM.

www.aacrjournals.org Clin Cancer Res; 2018 OF7

Nagano et al.

Figure 5. Assessment of drug sensitivity for ERBB2 mutants. The drug sensitivities of the indicated ERBB2 mutants were categorized as sensitive (green), partially sensitive

(yellow), and resistant (red) based on the IC50 of each drug against each mutant. WT, wild type; TM, transmembrane; AS, autophosphorylation site.

be stronger than that of simple mutations, and 13 of the 17 neratinib showed good efficacy for most of the ERBB2 mutations, ERBB2 compound mutations were evaluated to confer trans- but they were less effective against several mutations, including forming activities in both 3T3 and Ba/F3 cells (Supplementary L755P/S. Osimertinib, however, seems to be a good candidate Fig. S12A and S12B). We then assessed the drug sensitivity of the drug against L755 mutations, whereas it showed lower efficacy ERBB2 compound mutations using the MANO method (Fig. 4B) against E770_A771insAYVM and A775_776insYVMA. and the Alamar Blue cell viability assay (Fig. 4C). We excluded Ba/F3 cells harboring GFP, G135E, or D277H from the analysis because these cells did not show IL3-independent growth. Inter- Discussion estingly, three compound mutations involving L755S were The current findings revealed that most of the ERBB2 mutations shown to be sensitive to osimertinib (IC50 < 50 nmol/L) but with transforming potential in both 3T3 and Ba/F3 cells were less sensitive to afatinib or neratinib (IC50 > 5 nmol/L). Inter- located in the tyrosine kinase domain (16/18 mutations, 89%). estingly, the sensitivity of the compound mutations to trastuzu- Our data (Fig. 5) and those of previous preclinical studies (42, 43) mab was generally between those of the two single mutations further suggest that trastuzumab and lapatinib may not be pref- (Supplementary Fig. S13). erable for the treatment of cancers with several ERBB2-activating mutations. This suggestion is also supported by the MyPathway Drug sensitivity profile of ERBB2 mutations basket trial, showing that only four of 36 patients (11%) with A summary of the estimated drug sensitivities is shown in Fig. 5. cancers harboring ERBB2 mutations had objective responses to On the basis of this evaluation, trastuzumab and lapatinib were treatment with trastuzumab plus pertuzumab (44). Moreover, effective against most of the ERBB2 ectodomain mutations, but assessments of both mutation and amplification of ERBB2 are several mutations in the tyrosine kinase domain were shown to likely to be of great clinical relevance, especially in bladder confer resistance against the two drugs. Although there was no urothelial carcinoma, because concurrent mutations/amplifica- definitive resistant mutation to sapitinib, many of the ERBB2 tion may reduce the efficacy of trastuzumab and lapatinib (45). In mutations were partially insensitive to sapitinib. Afatinib and fact, among 41 patients with both ERBB2 amplification and

OF8 Clin Cancer Res; 2018 Clinical Cancer Research

Evaluation of Drug Sensitivity of ERBB2 Mutations

mutation in the MSK-IMPACT dataset, 19 (46.3%) and 12 To our knowledge, this report is the first to comprehensively (29.2%) patients had ERBB2 mutations that were resistant to evaluate ERBB2 compound mutations, although a few ERBB2 trastuzumab and lapatinib, respectively. compound mutations have been reported previously (50). Our Although afatinib, neratinib, and osimertinib were found to study showed that 6.1% (MSK-IMPACT dataset) and 8.1% be effective against most of the ERBB2 mutants analyzed in this (TCGA dataset) of ERBB2 mutant–positive patients had com- study, several ERBB2 mutants, such as L755 mutations and pound mutations. Our study further discovered that each exon 20 insertions, showed different sensitivities to the three compound mutation had a varying degree of sensitivity to each drugs. Whereas some studies have suggested that afatinib and ERBB2-targeted drug. For instance, the simple mutation of neratinib are effective against L755S (11, 18, 46), our in vitro S310F was sensitive to trastuzumab and lapatinib, whereas the study showed that the IC50 values of the two drugs against compound mutation of S310F and L755S was resistant to both L755P/S mutations were relatively higher than those against drugs. The transforming potential of ERBB2 compound muta- other ERBB2 mutants. Moreover, a low-dose treatment with tions was generally stronger than that of simple mutations, afatinib (8 mg/kg) could not inhibit the in vivo growth of the suggesting that the compound mutations contributed an tumors with ERBB2(L755P). In the SUMMIT trial, the median advantage to tumor growth. best percentage change from baseline in breast cancers harbor- Potential limitations of this study include the following ing the L755S mutation was lower than those in breast cancers points. First, retroviral transduction of ERBB2 mutations into with other ERBB2 mutations, including S310, V777,andexon cell lines results in elevated ERBB2 protein overexpression 20 insertions. The MutHER trial also showed that the clinical compared with endogenous ERRB2 expression. The evaluation benefitofneratinibforpatientswithERBB2(L755S)waspoor of transforming potential is complicated because overexpres- (one partial response, one stable disease <24 weeks, and four sion of wild-type ERBB2 itself confers some transforming progressive disease; ref. 47). In contrast, osimertinib demon- activity. Therefore, we compared the transforming potential of strated better efficacy against the L755 mutations in this study. ERBB2 mutants with that of wild-type ERBB2 in this study. We found that the osimertinib IC50 values against L755 muta- Second, our preclinical data are not yet completely supported tions were lower than those against wild-type EGFR and similar by clinical data. Like the SUMMIT trial, the sensitivities of to those against TKI-sensitizing ERBB2 mutations, such as different types of ERBB2 mutations to ERBB2-targeted inhibi- S310F and V777L. Our in vivo study also showed that low-dose tors, including osimertinib, should be evaluated in open bas- osimertinib (8 mg/kg) could inhibit the growth of tumors ket–type clinical trials. Third, we were unable to determine with the L755P mutation, compared with low-dose afatinib whether ERBB2 compound mutations occur in cis or trans (8 mg/kg) treatment. Osimertinib, therefore, may be a candi- because the two mutations are located in different exons far date drug for cancers carrying the L755 mutation. Importantly, away from each other in the genome, making the assessment in addition to the most frequent de novo mutation in breast technically difficult. We previously reported that all EGFR cancers, the L755S mutation has recently been reported to be an compound mutations are present in the cis allele (27), and acquired mutation that is resistant to trastuzumab treatment thus, ERBB2 compound mutations may also exist in a similar (25). However, considering that osimertinib as well as afatinib manner. Fourth, because the drugs used in vivo were adminis- could not completely inhibit the progression of tumors expres- tered intraperitoneally but not orally, plasma concentrations sing the L755P mutant in vivo,whetherosimertinibwouldbea achieved in mice were higher than those in humans as shown in promising treatment for patients with cancers harboring the Supplementary Fig. S9 (36, 38). Therefore, we should be careful L755 mutant is still controversial and should be assessed in of applying the results to humans. Finally, our study did not further clinical trials. take into account other gene mutations such as PIK3CA and It should be noted, however, that osimertinib is likely ineffec- ERBB3 mutations that possibly cooccur in cancers harboring tive against E770_A771insAYVM and A775_776insYVMA ERBB2 mutations and can affect drug sensitivity (23, 47). (IC50 >100 nmol/L). In fact, one patient was shown to acquire In conclusion, a comprehensive evaluation of ERBB2 muta- the ERBB2(A775_776insYVMA) mutation after receiving osimer- tions was successfully performed using the MANO method. In tinib for EGFR mutant–positive lung cancer in the AURA study particular, osimertinib was first evaluated for the treatment of (48). In contrast, neratinib may be effective against these muta- ERBB2-positive cancer and might be effective against most of tions because the IC50 was less than 2 nmol/L, whereas the IC50 the ERBB2 mutants including the L755 mutants, which are the against EGFR exon 20 insertions was 16 to 96 nmol/L based on most common in breast cancer and acquired resistant muta- our evaluation. However, the efficacy of neratinib against ERBB2 tions after trastuzumab or lapatinib treatment. Therefore, the exon 20 insertions should be interpreted with caution considering efficacy of osimertinib treatment against these ERBB2 mutants that the IC50 values against wild-type ERBB2 and EGFR were both should be evaluated in future clinical trials. Given that recent reduced (0.3 and 0.7 nmol/L). According to the SUMMIT study, studies have reported several acquired ERBB2 mutations after neratinib was effective against ERBB2 exon 20 insertions in treatment with ERBB2-targeted drugs, it has become increas- breast cancers but not in lung cancers. The same is true for afatinib ingly important to evaluate the significance of ERBB2 muta- treatment. Although afatinib showed efficacy against tumors tions, including minor mutations, amplifications with muta- driven by the ERBB2(A775_776insYVMA) mutation in some tions, or compound mutations. Therefore, the MANO method preclinical studies (17, 35), the clinical benefit of afatinib may become a beneficial approach for the functional evalua- for patients with lung cancers harboring the ERBB2 tion of ERBB2 mutants, enabling determination of the best (A775_776insYVMA) mutation remains unclear (49). These treatment for cancers harboring ERBB2 mutations. However, results may suggest that neratinib or afatinib treatment for cancers our preclinical data obtained in this study are not validated by harboring ERBB2 exon 20 insertions may be dependent on the clinical data, and thus must be confirmed in further investiga- tumor origin and should be evaluated in larger cohort studies. tions of large-scale clinical studies.

www.aacrjournals.org Clin Cancer Res; 2018 OF9

Nagano et al.

Disclosure of Potential Conflicts of Interest Acknowledgments No potential conflicts of interest were disclosed. The authors thank A. Maruyama for technical assistance. This study was financially supported in part through grants from the Leading Advanced Projects Authors' Contributions for Medical Innovation (LEAP) under grant number JP17am0001001, the Practical Research for Innovative Cancer Control under grant number Conception and design: M. Nagano, S. Kohsaka, H. Mano JP17ck0106252, and the Project for Cancer Research And Therapeutic Evolution Development of methodology: M. Nagano, S. Kohsaka (P-CREATE) under grant number JP17cm0106502 from the Japan Agency for Acquisition of data (provided animals, acquired and managed patients, Medical Research and Development, AMED. provided facilities, etc.): M. Nagano, S. Kohsaka, K. Saka Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Nagano, S. Kohsaka, T. Ueno, S. Kojima The costs of publication of this article were defrayed in part by the payment of advertisement Writing, review, and/or revision of the manuscript: M. Nagano, S. Kohsaka, page charges. This article must therefore be hereby marked in H. Iwase, M. Kawazu, H. Mano accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Nagano, S. Kohsaka, M. Kawazu Received March 29, 2018; revised June 4, 2018; accepted June 25, 2018; Study supervision: S. Kohsaka, H. Mano published first July 11, 2018.

References 1. Tamaskovic R, Schwill M, Nagy-Davidescu G, Jost C, Schaefer DC, Verdur- in genes involved in sister chromatid cohesion and segregation. Nat Genet men WP, et al. Intermolecular biparatopic trapping of ErbB2 prevents 2013;45:1459–63. compensatory activation of PI3K/AKT via RAS-p110 crosstalk. Nat Com- 17. Greulich H, Kaplan B, Mertins P, Chen TH, Tanaka KE, Yun CH, et al. mun 2016;7:11672. Functional analysis of receptor tyrosine kinase mutations in lung cancer 2. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. identifies oncogenic extracellular domain mutations of ERBB2. Proc Natl Studies of the HER-2/neu proto-oncogene in human breast and ovarian Acad Sci U S A 2012;109:14476–81. cancer. Science 1989;244:707–12. 18. Kavuri SM, Jain N, Galimi F, Cottino F, Leto SM, Migliardi G, et al. HER2 3. McKenzie SJ, DeSombre KA, Bast BS, Hollis DR, Whitaker RS, Berchuck A, activating mutations are targets for colorectal cancer treatment. Cancer et al. Serum levels of HER-2 neu (C-erbB-2) correlate with overexpression Discov 2015;5:832–41. of p185neu in human ovarian cancer. Cancer 1993;71:3942–6. 19. Breslin S, Lowry MC, O'Driscoll L. Neratinib resistance and cross-resistance 4. Press MF, Pike MC, Hung G, Zhou JY, Ma Y, George J, et al. Ampli- to other HER2-targeted drugs due to increased activity of metabolism ficationandoverexpressionofHER-2/neuincarcinomasofthe enzyme cytochrome P4503A4. Br J Cancer 2017;116:620–5. salivary gland: correlation with poor prognosis. Cancer Res 1994;54: 20. De Greve J, Teugels E, Geers C, Decoster L, Galdermans D, De Mey J, et al. 5675–82. Clinical activity of afatinib (BIBW 2992) in patients with lung adenocar- 5. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. cinoma with mutations in the kinase domain of HER2/neu. Lung Cancer Use of chemotherapy plus a monoclonal antibody against HER2 for 2012;76:123–7. metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344: 21. Gandhi L, Bahleda R, Tolaney SM, Kwak EL, Cleary JM, Pandya SS, et al. 783–92. Phase I study of neratinib in combination with temsirolimus in patients 6. Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, et al. with human epidermal growth factor receptor 2-dependent and other solid Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N tumors. J Clin Oncol 2014;32:68–75. Engl J Med 2006;355:2733–43. 22. Kris MG, Camidge DR, Giaccone G, Hida T, Li BT, O'Connell J, et al. 7. Baselga J, Cortes J, Kim SB, Im SA, Hegg R, Im YH, et al. Pertuzumab plus Targeting HER2 aberrations as actionable drivers in lung cancers: phase II trastuzumab plus docetaxel for metastatic breast cancer. N Engl J Med trial of the pan-HER tyrosine kinase inhibitor dacomitinib in patients with 2012;366:109–19. HER2-mutant or amplified tumors. Ann Oncol 2015;26:1421–7. 8. Goldhirsch A, Gelber RD, Piccart-Gebhart MJ, de Azambuja E, Procter M, 23. Hyman DM, Piha-Paul SA, Won H, Rodon J, Saura C, Shapiro GI, et al. HER Suter TM, et al. 2 years versus 1 year of adjuvant trastuzumab for HER2- kinase inhibition in patients with HER2- and HER3-mutant cancers. positive breast cancer (HERA): an open-label, randomised controlled trial. Nature 2018;554:189–94. Lancet North Am Ed 2013;382:1021–8. 24. Liu S, Li S, Hai J, Wang X, Chen T, Quinn MM, et al. Targeting HER2 9. Cameron D, Casey M, Oliva C, Newstat B, Imwalle B, Geyer CE. Lapatinib aberrations in non-small cell lung cancer with osimertinib. Clin Cancer Res plus capecitabine in women with HER-2-positive advanced breast cancer: 2018;24:2594–604. final survival analysis of a phase III randomized trial. Oncologist 2010; 25. Zuo WJ, Jiang YZ, Wang YJ, Xu XE, Hu X, Liu GY, et al. Dual characteristics of 15:924–34. novel HER2 kinase domain mutations in response to HER2-targeted 10. Stephens P, Hunter C, Bignell G, Edkins S, Davies H, Teague J, et al. Lung therapies in human breast cancer. Clin Cancer Res 2016;22:4859–69. cancer: intragenic ERBB2 kinase mutations in tumours. Nature 2004; 26. Hanker AB, Brewer MR, Sheehan JH, Koch JP, Sliwoski GR, Nagy R, et al. An 431:525–6. acquired HER2T798I gatekeeper mutation induces resistance to neratinib 11. Bose R, Kavuri SM, Searleman AC, Shen W, Shen D, Koboldt DC, et al. in a patient with HER2 mutant-driven breast cancer. Cancer Discov Activating HER2 mutations in HER2 gene amplification negative breast 2017;7:575–85. cancer. Cancer Discov 2013;3:224–37. 27. Kohsaka S, Nagano M, Ueno T, Suehara Y, Hayashi T, Shimada N, et al. A 12. Mazieres J, Peters S, Lepage B, Cortot AB, Barlesi F, Beau-Faller M, et al. Lung method of high-throughput functional evaluation of EGFR gene variants of cancer that harbors an HER2 mutation: epidemiologic characteristics and unknown significance in cancer. Sci Transl Med 2017;9:pii:eaan6566. therapeutic perspectives. J Clin Oncol 2013;31:1997–2003. 28. Akagi T, Sasai K, Hanafusa H. Refractory nature of normal human diploid 13. Lee JW, Soung YH, Seo SH, Kim SY, Park CH, Wang YP, et al. Somatic fibroblasts with respect to oncogene-mediated transformation. Proc Natl mutations of ERBB2 kinase domain in gastric, colorectal, and breast Acad Sci U S A 2003;100:13567–72. carcinomas. Clin Cancer Res 2006;12:57–61. 29. Page B, Page M, Noel C. A new fluorometric assay for cytotoxicity measure- 14. Bekaii-Saab T, Williams N, Plass C, Calero MV, Eng C. A novel mutation in ments in-vitro. Int J Oncol 1993;3:473–6. the tyrosine kinase domain of ERBB2 in hepatocellular carcinoma. BMC 30. Zehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, et al. Mutational cancer 2006;6:278. landscape of metastatic cancer revealed from prospective clinical sequenc- 15. The Cancer Genome Atlas Research Network. Integrated genomic analyses ing of 10,000 patients. Nat Med 2017;23:703–13. of ovarian carcinoma. Nature 2011;474:609–15. 31. Chang MT, Bhattarai TS, Schram AM, Bielski CM, Donoghue MTA, Jonsson 16. Guo G, Sun X, Chen C, Wu S, Huang P, Li Z, et al. Whole-genome and P, et al. Accelerating discovery of functional mutant alleles in cancer. whole-exome sequencing of bladder cancer identifies frequent alterations Cancer Discov 2018;8:174–83.

OF10 Clin Cancer Res; 2018 Clinical Cancer Research

Evaluation of Drug Sensitivity of ERBB2 Mutations

32. Kancha RK, von Bubnoff N, Bartosch N, Peschel C, Engh RA, Duyster J. 41. Kobayashi S, Canepa HM, Bailey AS, Nakayama S, Yamaguchi N, Goldstein Differential sensitivity of ERBB2 kinase domain mutations towards lapa- MA, et al. Compound EGFR mutations and response to EGFR tyrosine tinib. PLoS One 2011;6:e26760. kinase inhibitors. J Thorac Oncol 2013;8:45–51. 33. Trowe T, Boukouvala S, Calkins K, Cutler RE Jr, Fong R, Funke R, et al. EXEL- 42. Boulbes DR, Arold ST, Chauhan GB, Blachno KV, Deng N, Chang WC, et al. 7647 inhibits mutant forms of ErbB2 associated with lapatinib resistance HER family kinase domain mutations promote tumor progression and can and neoplastic transformation. Clin Cancer Res 2008;14:2465–75. predict response to treatment in human breast cancer. Mol Oncol 34. Uchibori K, Inase N, Araki M, Kamada M, Sato S, Okuno Y, et al. Brigatinib 2015;9:586–600. combined with anti-EGFR antibody overcomes osimertinib resistance in 43. Mishra R, Hanker AB, Garrett JT. Genomic alterations of ERBB receptors in EGFR-mutated non-small-cell lung cancer. Nat Commun 2017;8:14768. cancer: clinical implications. Oncotarget 2017;8:114371–92. 35. Kosaka T, Tanizaki J, Paranal RM, Endoh H, Lydon C, Capelletti M, et al. 44. Hainsworth JD, Meric-Bernstam F, Swanton C, Hurwitz H, Spigel DR, Response heterogeneity of EGFR and HER2 exon 20 insertions to covalent Sweeney C, et al. Targeted therapy for advanced solid tumors on the basis of EGFR and HER2 inhibitors. Cancer Res 2017;77:2712–21. molecular profiles: results from MyPathway, an open-label, phase IIa 36. Planchard D, Brown KH, Kim DW, Kim SW, Ohe Y, Felip E, et al. multiple basket study. J Clin Oncol 2018;36:536–42. Osimertinib Western and Asian clinical pharmacokinetics in patients and 45. Zabransky DJ, Yankaskas CL, Cochran RL, Wong HY, Croessmann S, Chu healthy volunteers: implications for formulation, dose, and dosing fre- D, et al. HER2 missense mutations have distinct effects on oncogenic quency in pivotal clinical studies. Cancer Chemother Pharmacol 2016;77: signaling and migration. Proc Natl Acad Sci U S A 2015;112:E6205–14. 767–76. 46. Xu X, De Angelis C, Burke KA, Nardone A, Hu H, Qin L, et al. HER2 37. Yates JW, Ashton S, Cross D, Mellor MJ, Powell SJ, Ballard P. Irreversible reactivation through acquisition of the HER2 L755S mutation as a mech- inhibition of EGFR: modeling the combined pharmacokinetic-pharmaco- anism of acquired resistance to HER2-targeted therapy in HER2(þ) breast dynamic relationship of osimertinib and its active metabolite AZ5104. Mol cancer. Clin Cancer Res 2017;23:5123–34. Cancer Ther 2016;15:2378–87. 47. Ma CX, Bose R, Gao F, Freedman RA, Telli ML, Kimmick G, et al. Neratinib 38. Wind S, Schnell D, Ebner T, Freiwald M, Stopfer P. Clinical pharmacoki- efficacy and circulating tumor DNA detection of HER2 mutations in HER2 netics and pharmacodynamics of afatinib. Clin Pharmacokinet 2017;56: nonamplified metastatic breast cancer. Clin Cancer Res 2017;23:5687–95. 235–50. 48. Ramalingam SS, Yang JC, Lee CK, Kurata T, Kim DW, John T, et al. 39. Sparidans RW, van Hoppe S, Rood JJ, Schinkel AH, Schellens JH, Beijnen Osimertinib as first-line treatment of EGFR mutation-positive advanced JH. Liquid chromatography-tandem mass spectrometric assay for the non-small-cell lung cancer. J Clin Oncol 2018;36:841–9. tyrosine kinase inhibitor afatinib in mouse plasma using salting-out 49. Smit EF, Peters S, Dziadziuszko R, Dafni U, Wolf J, Wasag˛ B, et al. A single- liquid-liquid extraction. J Chromatogr B Analyt Technol Biomed Life Sci arm phase II trial of afatinib in pretreated patients with advanced NSCLC 2016;1012–3:118–23. harboring a HER2 mutation: the ETOP NICHE trial. J Clin Oncol 2017;35 40. Kim EY, Cho EN, Park HS, Hong JY, Lim S, Youn JP, et al. Compound EGFR (15_suppl):9070. mutation is frequently detected with co-mutations of actionable genes and 50. de Martino M, Zhuang D, Klatte T, Rieken M, Roupret M, Xylinas E, et al. associated with poor clinical outcome in lung adenocarcinoma. Cancer Impact of ERBB2 mutations on in vitro sensitivity of bladder cancer to Biol Ther 2016;17:237–45. lapatinib. Cancer Biol Ther 2014;15:1239–47.

www.aacrjournals.org Clin Cancer Res; 2018 OF11

High-Throughput Functional Evaluation of Variants of Unknown Significance in ERBB2

Masaaki Nagano, Shinji Kohsaka, Toshihide Ueno, et al.

Clin Cancer Res Published OnlineFirst July 2, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-0991

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2018/11/01/1078-0432.CCR-18-0991.DC2

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2018/08/29/1078-0432.CCR-18-0991. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.