a method of high-throughput functional evaluation of egfr ...(mano) method, to evaluate the...

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1Department of Medical Genomics, Graduate School of Medicine, The University ofTokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2Department of CellularSignaling, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo,Bunkyo-ku, Tokyo 113-0033, Japan. 3Department of Thoracic Surgery, GraduateSchool of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 4Department of Orthopedic Surgery, Graduate School of Medicine,Juntendo University, Bunkyo-ku, Tokyo 113-8431, Japan. 5Department of Human Pa-thology, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Tokyo 113-8431, Japan. 6Department of Respiratory Medicine, Graduate School of Medicine,JuntendoUniversity, Bunkyo-ku, Tokyo113-8431, Japan. 7DepartmentofGeneral ThoracicSurgery, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Tokyo 113-8431,Japan. 8National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan.*Corresponding author. Email: [email protected] (S.K.); [email protected] (H.M.)†These authors contributed equally to this work.

Kohsaka et al., Sci. Transl. Med. 9, eaan6566 (2017) 15 November 2017

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A method of high-throughput functional evaluation ofEGFR gene variants of unknown significance in cancerShinji Kohsaka,1*† Masaaki Nagano,2,3† Toshihide Ueno,2 Yoshiyuki Suehara,4 Takuo Hayashi,5

Naoko Shimada,6 Kazuhisa Takahashi,6 Kenji Suzuki,7 Kazuya Takamochi,7

Fumiyuki Takahashi,6 Hiroyuki Mano2,8*

Numerous variants of unknown significance (VUS) have been identified through large-scale cancer genome projects,although their functional relevance remains uninvestigated. We developed a mixed-all-nominated-mutants-in-one(MANO) method to evaluate the transforming potential and drug sensitivity of oncogene VUS in a high-throughputmanner and applied this method to 101 nonsynonymous epidermal growth factor receptor (EGFR) mutants. Wediscovered a number of mutations conferring resistance to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib-and erlotinib-insensitive missense mutations within exon 19 and other gefitinib-resistant mutations, such as L833V,A839T, V851I, A871T, and G873E. L858R-positive tumors (12.8%) harbored compound mutations primarily in the cisallele, which decreased the gefitinib sensitivity of these tumors. The MANOmethod further revealed that some EGFRmutants that are highly resistant to all types of TKIs are sensitive to cetuximab. Thus, these data support the impor-tance of examining the clinical relevance of uncommonmutations within EGFR and of evaluating the functions of suchmutations in combination. Thismethodmaybecome a foundation for the in vitro and in vivo assessment of variants ofcancer-related genes and help customize cancer therapy for individual patients.

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INTRODUCTIONSince transforming mutations in epidermal growth factor receptor(EGFR) were first identified in non–small cell lung carcinoma (NSCLC)(1–3), advancements in the diagnostics for suchmutations and the evo-lution of targeted therapeutics against EGFR have greatly improved themanagement and outcome of patients with this lethal disease (4). How-ever, extensive next-generation sequencer–driven analyses of theNSCLC genome have revealed a large number of variants of unknownsignificance (VUS) in EGFR and other regions in the cancer genomethat await further investigation (5).

The approval of gefitinib, erlotinib, or afatinib as first-line treatmentsfor advanced lung cancers requires the presence of classical/sensitizingEGFRmutations, such as exon 19deletions or theL858Rpointmutation(6, 7). Furthermore, thoracic oncologists may generally agree with theclinical usage of EGFR tyrosine kinase inhibitors (TKIs) for uncommonsensitizing mutations, including exon 18 insertions/deletions (indels),E709 mutations (8, 9), G719 mutations (10, 11), exon 19 insertions(12), the insertion of A763_Y764insFQEA (13), the S768I mutation(14), and the L861Q mutation, although preclinical and clinical trialdata suggest that uncommon EGFR mutations are frequently less sen-sitive to first-generation EGFR TKIs (11, 15). The main mechanismunderlying resistance to gefitinib/erlotinib is the acquisition of theT790M mutation in EGFR (16, 17), which can be overcome by the

third-generationEGFRTKI osimertinib (18), but such efficacy is furtherbypassed by C797 compound mutations (19). Another class of EGFRTKI–insensitive mutations includes exon 20 insertions (20, 21).

In addition to these genomic alterations, the cancer genome containsa large number of nonsynonymous mutations with unknown clinicalsignificance. In the COSMIC (Catalogue of Somatic Mutations inCancer) database of somatic mutations (v78; http://cancer.sanger.ac.uk/cosmic/); for example, a total of 770 nonsynonymous mutationshave been reported for EGFR. Similarly, 442 of such mutations havebeen deposited for the ALK gene. However, the clinical relevance re-mains unknown for the vast majority of such alterations. Thus, we de-signed an approach, termed the mixed-all-nominated-mutants-in-one(MANO) method, to evaluate the transforming ability and drug sensi-tivity of hundreds of such VUS.

RESULTSEstablishment of a high-throughput functional assayThe MANO method uses a retroviral vector that enables the stable in-tegration of individual genes into the genome of assay cells [such asmouse 3T3 fibroblasts or the interleukin-3 (IL-3)–dependent, murinepro-B cell line Ba/F3] along with 6–base pair (bp) bar code sequences(Fig. 1). Individually transduced assay cells are subsequently pooled andcultured in a competitive manner to evaluate their transformingpotential or drug sensitivity in vitro or in vivo. At the end of the expan-sion period, genomic DNA (gDNA) is extracted from the assay cells topolymerase chain reaction (PCR)–amplify the bar code sequences,which are subsequently subjected to deep sequencing with the IlluminaMiSeq platform to quantify their relative abundance.

To determine the feasibility and the sensitivity of theMANOmethod,we transduced the cDNAofNSCLC-relatedoncogenes, includingEML4-ALK, KIF5B-RET, KRAS(G12V), CD74-ROS1, EGFR(E746_A750del),or EGFR(L858R) (22–25), with individual bar codes into Ba/F3 cells. Atotal of 20,000 each of the transduced cells with mutations other thanL858R was mixed together with a different number (100 to 20,000 cells)of the EGFR (L858R) mutant cells. gDNA was subsequently isolated

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http://cancer.sanger.ac.uk/cosmic/

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from the pools and subjected to bar code sequencing. As shown in Fig. 2A,the number of bar code reads corresponding to the former five cells wasconstant in all mixtures, whereas that corresponding to Ba/F3 cellsexpressing EGFR(L858R) varied proportionally to the initial inputnumber (r = 0.99). Thus, the MANO method is highly sensitive,enabling the quantitative detection of as little as 0.1% of the initial input.

We next tested whether the MANO method could evaluate thetransforming potential of cDNAs. First, we used the standard 3T3focus-formation assay for measuring the transforming activity of 14EGFRmutants and their wild-type counterparts (Fig. 2B and fig. S1). Othertransforming cDNAs, such as BRAF(V600E), MET exon 14 skipping,ERBB2(V777L) (26–28), and those shown in Fig. 2B, were similarlyinvestigated.

Subsequently, the MANO method was conducted using 3T3 cellsand the same cDNA samples as in Fig. 2B (Fig. 2C). Culturing 3T3 cells


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under different medium conditions resulted in similar alterations ofeach gene proportion, although rapid alterations in the relative cellnumber were observed when the cells were cultured under low concen-trations of fetal bovine serum (FBS) (fig. S2A). We used the MANOmethod to investigate the correlation of the growth of 3T3 cells betweenculture with 10% FBS and that with other serum concentrations andobserved a good correlation among the different settings (fig. S2B). Inaddition, apositive correlationwasobservedbetween the focus-formationassay and the MANO method (r = 0.63; Fig. 2D and fig. S3A).

The MANO method was further conducted using 3T3 cells in anude mouse tumorigenicity assay (Fig. 2C). Whereas cells expressinggreen fluorescent protein (GFP) or the wild-type forms of EGFR, ERBB2,or MET were depleted by day 11, the cells expressing EGFR(L858R) orEGFR(E746_A750del) gradually increased during the sameperiod. Thisobservationwas consistentwith the results obtained in the in vitro assay(Fig. 2D), supporting the feasibility of this method for assessingtransforming activity in vivo. We also transduced the same 25 genesinto Ba/F3 cells and monitored the relative proportion of cellsexpressing each gene. In the presence of IL-3, the proportion of all cellswas relatively constant until day 20 (fig. S3B), whereas several variantswere depleted at about day 6 in the absence of IL-3 (Fig. 2C). The cellswhose ratio of relative occupancy at day 6 compared to day 0 was>0.01 showed IL-3–independent cell growth (fig. S3C).

We next treated a pool of 16 Ba/F3 cells expressing active EGFRmu-tants (n=11) or other oncoproteins (n=5)with various TKIs. Consider-ing the different doubling times of the transduced cells, we comparedeach TKI-treated Ba/F3 cell to vehicle-treated controls to calculate therelative growth inhibition of each cell clone (Fig. 3A and table S1).Whereas treatment with the cytotoxic compound puromycin induceduniform cell death across the cell clones, treatment with EGFRTKIs (ge-fitinib, erlotinib, afatinib, osimertinib, and rociletinib) resulted in thedose-dependent death of cells for five TKI-sensitive EGFR mutants(L858R, E746_A750 del, G719S, E861Q, and S768I) in the pool (Fig.3A). As expected, Ba/F3 cells expressing EGFR(T790M) were resistantto first- and second-generation EGFR TKIs (gefitinib, erlotinib, and afa-tinib) but sensitive to third-generation EGFR TKIs (osimertinib and ro-ciletinib). By contrast, cells expressing EGFR (T790M_C797S) showedresistance to these third-generation TKIs. Similarly, crizotinib, a TKIfor ALK and ROS1 (29, 30), suppressed the growth of cells expressingEML4-ALK or CD74-ROS1, and another inhibitor for ALK and RET,alectinib (31, 32), inhibited the growth of the cells expressing EML4-ALK or KIF5B-RET (Fig. 3A). To independently evaluate the sensitivityof the EGFR mutants to each EGFR TKI, the number of viable cellswas also determined using the alamarBlue cell viability assay, amethod for quantifying cell viability based on mitochondrial enzymeactivity (fig. S4) (33). The relative fold changes of read counts (repre-senting cell number) in theMANOmethodwere consistentwith thoseof the alamarBlue cell viability assay (r = 0.89; Fig. 3B).

Furthermore, 25 independent 3T3 clones were pooled and sub-cutaneously xenografted into mice that were further treated for 14 dayswith vehicle, erlotinib, or afatinib. Erlotinib treatment induced amarkedreduction in the relative abundance of six of six TKI-sensitizing EGFRmutants (L858R, E746_A750del, L861Q, G719C, G719S, and S768I),whereas the number of cells carrying both TKI-resistant EGFRmutants(T790M and T790M_C797S) and nine of nine clones harboring otheroncogenes increased during the same period (Fig. 3C). Similar resultswere obtainedwith afatinib treatment (fig. S5). These experiments dem-onstrated the feasibility of theMANOmethod to assess drug sensitivityin vivo.

Fig. 1. Schematic representation of the MANOmethod.Mouse 3T3 or Ba/F3 cellswere infected with recombinant retrovirus expressing oncoproteins withcorresponding 6-bp bar codes. Equal numbers of the stably transduced cells weremixed and culturedwith different types ofmedium and/or treatedwith TKIs or vehicle.gDNA was harvested from themixture of the remaining viable cells at the appropriateperiods for each assay. The bar code sequences were PCR-amplified and subjected todeep sequencing onMiSeq sequencers to quantitate their relative abundance (a directreflection of the cell number). To evaluate the transforming potential of each oncoprotein,the read number for each bar code was normalized to that of day 0. To evaluate theinhibition profile of test compounds across transduced clones in the mixture, the readnumber for each bar code was normalized to that of the vehicle-treated control. WT,wild-type.

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Functional annotation of VUS within EGFRWe subsequently used the MANO method to evaluate 101 EGFR mu-tants reported 4 to 7386 times in the COSMIC database (tables S2 andS3), including 13mutations in the extracellular (EC) domain (primarilypresent in glioblastoma), 86 mutations in the intracellular TK domain,and 2 mutations in the C-terminal domain.


We performed paired t tests to examine the transforming potentialamong the 101 EGFR mutations under several serum concentrations.Compared with wild-type EGFR, 27, 28, 25, 22, and 34 EGFR mutantswere predicted to confer higher transforming potential in 3T3 cells assayedunder 10, 5, 2, and 1%FBS or 5% bovine serum albumin, respectively. Asdepicted in fig. S6A, 55.9% of the total mutations induced oncogenicity

Fig. 2. TheMANOmethod in vitro and in vivo. (A) Mouse 3T3 cells were infectedwith a retrovirus expressing the oncoprotein shown at the top. The indicated number of cellswasmixed together in thewells of a six-well tissue culture plate, and gDNAwas prepared from themixture the next day. The 6-bp bar codeswere PCR-amplified and subjected todeep sequencing. The relative read numbers of bar codes in themixtures were compared to those of the initial input of cells infected with a retrovirus for EGFR(L858R) (n = 4).(B) Mouse 3T3 cells were infected with a retrovirus expressing an oncogene or the corresponding wild-type gene (indicated on the right) and subjected to a focus-formationassay. On day 14, the cells were stainedwith Giemsa solution. A retrovirus expressing GFPwas used as a negative control. (C) Temporal changes in the proportion of 3T3 or Ba/F3cells expressing each oncoprotein, the corresponding wild-type protein, or GFP are shownwith different colors as indicated in the lower right panel. Mouse 3T3 cells were eithercultured inDMEMcontaining10%FBS (upper left) or subcutaneously injected intonu/numice (upper right), and the relativeproportionof cell cloneswas assessed at the indicatedtimes using theMANOmethod. The read number for each cell clonewas normalized to that of day 0. Ba/F3 cell clones expressing oncoproteinswere cultured in vitro in RPMI 1640without IL-3 and similarly analyzed using theMANOmethod (lower left). The yellow arrowhead indicates the complete depletion of several cell clones at day 6. (D) The correlationbetween the focus-formation assay and the MANO method. The Giemsa-stained area (%) in the 3T3 focus-formation assay in (B) was compared to the fold change of the readnumber on day 8 relative to day 0 in 3T3 cells cultured in vitro using the MANO method in (C). The Pearson’s correlation coefficient (r) was calculated.

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across all serum conditions, implying that oncogenic potentialmay par-tially depend on assay conditions.

We subsequently investigated the correlation of transforming abilityof the 101 EGFR mutants evaluated by the focus-formation assay andtheMANOmethod. About 70% of themutations were predicted as on-cogenic using both methods (fig. S6B). Furthermore, Ba/F3 cells trans-


duced with the mutant cDNAs were individually examined for IL-3–independent growth. Every EGFR mutant with cytokine abrogationpotential was also predicted as an oncogenicmutation using theMANOmethod (fig. S6C).

Sixty-two and 57 mutations within EGFR were predicted to confertransforming potential in 3T3 and Ba/F3 cells, respectively (Fig. 4A).

Fig. 3. TKI sensitivity assessed by theMANOmethod. (A) Ba/F3 cells expressing each of the 16 genes shown on the rightweremixed and cultured in the presence of differentconcentrations of gefitinib, erlotinib, afatinib, osimertinib, rociletinib, crizotinib, alectinib, or puromycin. Bar code read numbers of the compound-treated cells were normalized tothose of the dimethyl sulfoxide (DMSO)–treatedmixture, and the relative viability (%) of each cell clone onday 5 is color-coded according to the indicated scheme. (B) Comparisonof cell viabilitymeasuredwith alamarBlue cell viability assay and theMANOmethod for Ba/F3 cells with 10 EGFRmutants in (A). Each data point was normalized to vehicle-treatedcells. Pearson’s correlation coefficient (r) was calculated as 0.89 (P < 0.0001). The low ratio area is magnified in the right panel. (C) Changes in the relative cell populations in thetumors ofmice treatedwith TKIs. TheMANOmethodwas used to quantify the bar code read numbers of tumors in 10 erlotinib-treated and 10 vehicle-treatedmice. The bar codenumber for each cell line was normalized to the total bar code numbers of the tumor on day 18, and the calculated number was subsequently used to determine the percentagecontribution of each cell line to the tumors. Themean relative cell population (%)within the tumors is shown formice treatedwith either vehicle (green circle) or erlotinib (orangecircle). The blue and pink arrows represent decrease and increase in the relative cell populations, respectively, in the tumors treatedwith erlotinib compared to those treatedwithvehicle. Error bars denote SD.

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We further examined whether the remaining 37 mutations that did notprovide transforming potential in either cell line may have some func-tional role in TKI resistance. These mutants were individually trans-duced into Ba/F3 cells to investigate their TKI sensitivities. TheA839T mutation showed a marked resistance to any generation ofEGFR TKIs (Fig. 4B and fig. S7).

We also evaluated the TKI sensitivity of other EGFR mutants usingthe MANO method. As shown in Fig. 4C and table S4, the L858R andE746_A750del mutants were sensitive to any TKI, whereas the T790M,C797S, T790M_C797S_Cis, and T854A substitutions and exon 20 in-sertions showed resistance to some TKIs, suggesting that the MANOmethod well recapitulates the sensitivity data obtained in previousstudies (34). We revealed that compared with L858R or the exon19 deletion, the EC domain mutations (at exons 2 to 15), E709 muta-tions (exon 18), exon 19missensemutations, V769L (exon 20), and exon21mutations (L833V,V851I, A871T, andG873E)were all insensitive toTKIs (Fig. 4C). Although the exon 20 insertions showed decreased sen-sitivity to osimertinib and rociletinib in general, the N771_P772insN


change conferred higher sensitivity, and D770_N771insSVD conferredhigher resistance than the other mutants (Fig. 4C).

Compound mutations in EGFRTo investigate whether infrequent mutations within EGFR play an im-portant role in the tolerance against TKIs in clinical settings, we se-quenced EGFR exons among 11 rebiopsy specimens from cases thatrecurred after gefitinib treatmentwithout acquiring theT790Mmutation.Three (27%) of 11 cases harbored compound mutations correspondingto L858R plus E709G or E709A (table S5). Because the allelic frequenciesofL858R andE709G/Awere almost identical, these compoundmutationsare likely present in the same subclone in tumors (table S5).

Thus, we further performedEGFR exon sequencing among 195 spec-imens of EGFR(L858R)-positive NSCLC and identified 39 casesharboring compound mutations (19.5%), including another 9 cases(4.6%) with compound mutations of L858R and E709A/G/K (Fig. 5Aand table S6). We further identified 24 compound mutations in 195additional cases formerly identified as positive for the EGFR exon

Fig. 4. Functional annotation of VUS in EGFR. (A) Venn diagram revealing the numbers of oncogenic EGFRmutants seen in 3T3 cells using a focus-formation assay (3T3 FF),3T3 cells by the MANO method (3T3 MANO), Ba/F3 cells using the MANO method (Ba/F3 MANO), or BA/F3 cells individually examined for IL-3–independent growth (Ba/F3individual). The method for the evaluation of the transforming activity in each assay is described in detail in the legend for fig. S6. (B) Ba/F3 cells expressing the L858R, A839T, orT790M_C797S_Cismutants of EGFRwere treatedwith the indicated concentrations of gefitinib, erlotinib, afatinib, or osimertinib for 72 hours. Viable cells (%)weremeasuredusingthe alamarBlue cell viability assay, and the results are shown as the means of five independent experiments. (C) Ba/F3 cells expressing 86 EGFR mutants (indicated at top) weretreated with either DMSO or EGFR TKIs (gefitinib, erlotinib, afatinib, osimertinib, or rociletinib) at 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 mM, and the relativeviability of TKI-treated cells relative to the corresponding DMSO-treated cells is color-coded according to the scheme indicated at the top left. Missense, deletion, and insertionmutations are shown in green, brown, and red, respectively.

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19 deletion,G719mutations, or L861Q. Notably, more than 90% of theG719 mutations existed as compound mutations in the examined co-hort. Furthermore, in the COSMIC database, more than 75% of theE709 mutations existed as compound mutations, primarily withL858R (35%) orG719mutations (32%) (table S7). To investigate whetherthe compoundmutations exist in cis or trans allele, we performed gDNA-or cDNA-based amplicon sequencing (table S6) or Droplet Digital PCR(fig. S8) and observed that all compound mutations were present in cisalleles in all cases analyzed. Thus, the transforming potential of com-pound mutations may be stronger than that of minor mutations alone,as determined using theMANOmethod (Fig. 5B) and the 3T3 focus-formation assay (figs. S9 and S10).

We further evaluated the TKI sensitivity of EGFR compound mu-tants using the MANO method. As shown in Fig. 5C and table S8, theTKI sensitivity of compound mutations was between those of the two


single mutations. The IC50 of gefitinib in Ba/F3 cells expressing EGFRwith L858R_E709 mutations was 20 to 80 times higher than that ofBa/F3 cells expressingEGFRwithL858Raloneaccording to the alamarBluecell viability assay (Fig. 5D). Clinically, none of the tumors with com-pound mutations of L858R and E709A/G responded to EGFR TKIs(table S5). These findings suggest a mechanism of TKI resistancemediated by the combination of E709A/G and L858R. Although thecompound mutants were less sensitive to gefitinib and erlotinib, theirsensitivity to afatinib was not remarkably different (IC50 values of lessthan 0.1 nM).

Cetuximab sensitivity of EGFR mutations assessed using theMANO methodWe further used the MANO method to evaluate the sensitivities ofEGFR mutants to cetuximab, a monoclonal antibody to EGFR. All of

Fig. 5. Compound mutations in EGFR. (A) The frequency and patterns of EGFR compound mutations. Three hundred ninety cases formerly identified as positive for EGFRmutations (L858R, exon 19 deletion, G719 mutations, and L861Q) were examined for EGFR target sequence to investigate the frequency and the patterns of EGFR compoundmutations. (B) The fold change in the ratio of 3T3 cell numberwith each EGFR compoundmutation in cis (x axis) or trans (y axis) on day 12 comparedwith that of the correspondingEGFR single mutation is plotted. (C) Ba/F3 cells expressing 106 EGFR mutants (indicated at top) were treated with either DMSO or EGFR TKIs (gefitinib, erlotinib, afatinib, orosimertinib) at 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 mM, and the viability of TKI-treated cells relative to the corresponding DMSO-treated cells is color-codedaccording to the scheme indicated at the top left. (D) Ba/F3 cells expressing single or compound EGFR mutations (indicated at the right) were treated with the indicated con-centrations of gefitinib, erlotinib, afatinib, or osimertinib for 72 hours without IL-3. The percentage of viable cells relative to that of parental Ba/F3 cells similarly treated in thepresence of IL-3 was measured using the alamarBlue cell viability assay (n = 5). The mean median inhibitory concentration (IC50) values of the EGFR mutants for each TKI werecalculated from five independent experiments.

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the missense mutations in the EC domain except for S492R (L62R,R108K, A289D/T/V, H304Y, P596L, and G598V), E709 mutations,K714R, L718Q, V769mutations, V774M, C797S, and L833V, were sen-sitive to cetuximab (Fig. 6A and table S9). An additional L858R muta-tion in the cis allele conferred varying degrees of resistance to cetuximab,according to the alamarBlue cell viability assay (Fig. 6B). The growth of3T3 cellswithEGFRmutations (R108K, L718Q, andR108K_L858R_Cis)was completely inhibited in vivo after cetuximab treatment, andno tumorgrowth was observed for more than 1 month, whereas the growth ofEGFR(L718Q_L858R_Cis) cells was initially inhibited but reboundedwithin 3 weeks (Fig. 6C).

The estimated drug sensitivities are summarized in Fig. 7, and therecommended drug for each mutation is suggested in table S10. On thebasis of this evaluation, among theL858R-compoundmutations (n= 39),25, 7, and 6 mutations showed sensitivities distinct from L858R alone


toward gefitinib, erlotinib, and afatinib, respectively (table S6). Thus,in total, 12.8, 3.6, and 3.1% of the L858Rmutations showed altered drugsensitivity for gefitinib, erlotinib, and afatinib, respectively. In theexamined cohort, clinical data were available for seven patients withEGFR compoundmutations. Among these, four cases with EGFR com-poundmutations predicted as sensitive to EGFRTKIs obtained a partialresponse, whereas the other three caseswithmutations predicted as par-tially sensitive exhibited stable disease after EGFR TKI treatment.

EGFR mutants resistant to all EGFR TKIs tested (S768I,D770_N771insSVD, T790M_C797S_Cis, L718Q_L858R_Cis, andT790M_G719A_Cis) were further examined to detect sensitivitiesto other TKIs. As shown in fig. S11, the alamarBlue cell viability assaywith dacomitinib (an irreversible pan-ErbB inhibitor) (35), nazartinib(an irreversible mutant-selective EGFR inhibitor) (36), or neratinib(a highly selective HER2 and EGFR inhibitor) (37) revealed that

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Fig. 6. Cetuximab sensitivity of EGFR mutations assessed by the MANOmethod. (A) Ba/F3 cells expressing 158 EGFR mutants (indicated at top) were treated with eitherDMSO or cetuximab at 0.001, 0.01, 0.1, 1, 10, or 100 mg/ml, and the viability of cetuximab-treated cells relative to that of the corresponding DMSO-treated cells is color-codedaccording to the scheme indicated at the top right. (B) Ba/F3 cells expressing the indicated mutants of EGFR were treated with the indicated concentrations of cetuximabfor 72 hours. Viable cells (%) were measured using the alamarBlue cell viability assay, and the results are shown as the means of five independent experiments. (C) Evaluation ofcetuximab sensitivity in vivo. Mouse 3T3 cells (1 × 106) transfected with the indicated expression constructs were injected into the subcutaneous tissue of mice (n = 5 per group).Tumor volumes were calculated as described in Materials and Methods. Error bars, SD; *P < 0.01, the tumor volume of the cells expressing R108K_L858R_Cis (left) orL718Q_L858R_Cis (right) treated with vehicle is compared with that of the corresponding cells treated with cetuximab at day 20. †P < 0.01, the tumor volume of the cellsexpressing R108K (left) or L718Q (right) treated with vehicle is compared with that of the corresponding cells treated with cetuximab at day 20 (left) or day 38 (right).

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DISCUSSIONRecently, a number of high-throughput genetic perturbation assayshave been developed to investigate the functional significance ofmutantgenes. Kim et al. (38), for example, assembled a collection of 1163cDNAs (including 474 mutants reported in fewer than three tumors)for the in vivo assessment of transforming potential. Berger et al. (39)developed an expression-based variant impact phenotyping methodthat measures the ability to induce gene expression changes across aset of landmark transcripts, enabling the classification of VUS intogain-of-function, loss-of-function, or change-of-function mutations.In contrast with these technologies, the approach presented hereinapplies a high-throughput functional analysis using cytokine-dependentcells (Ba/F3) to investigate the susceptibility of the given variants to ther-apeutic drugs. The MANO method thus enables an assessment of notonly the oncogenic potential but also the drug sensitivity of hundreds ofVUS of genes of interest within a short period of time.

Using the MANO method, the EC domain mutants of EGFR weredemonstrated to be relatively insensitive to gefitinib and erlotinib com-paredwith L858Ror exon 19 deletions, providing a possible explanationfor the poor activity of erlotinib against gliomas. Because EC domainmutants exhibited high sensitivity to afatinib and osimertinib, individualswith gliomas harboring these mutations may be eligible for afatinib orosimertinib treatment. Similarly, E709 mutations may be suitable fortreatment with afatinib, whereas G719 mutations showed varying drugsensitivities to EGFR TKIs, depending on the precise amino acid substi-


tution: G719C is sensitive to any type of EGFR TKI, whereas G719A issensitive to only afatinib. Consistent with the results of a previous study(13), the MANOmethod revealed that all exon 20 insertions, except forA763_Y764insFQEA, are resistant to gefitinib, erlotinib, and afatinib.

Our report also shows that the activatingmissensemutations withinexon 19 are generally insensitive to gefitinib and erlotinib but sensitive toafatinib and osimertinib. Notably, in the COSMIC database, there are ninelung cancer patients with EGFR exon 19 missense mutations who weretreated with gefitinib, and none of these individuals responded to TKIs.

Considering that the plasma concentration of osimertinib at 24 hoursafter 80-mg oral administration is about 200 nM (40), we set the sensi-tivity threshold of osimertinib to an IC90 of 100 nM. According to thisthreshold, we observed distinct sensitivities to osimertinib among variousEGFR exon 20 insertions. Compared to T790M, A763_Y764insFQEAwas more sensitive, whereas other exon 20 insertions, includingD770_N771insSVDandV769_D770insASV (the twomost common ex-on 20 insertions), exhibited resistance. These results suggest that thesensitivity of each exon 20 insertion should be carefully interpreted.Similarly, a different mutation within exon 21 showed divergentsensitivities to EGFR TKIs. Our findings demonstrate that L833V,A839T, V851I, A871T, and G873E are resistant to gefitinib.

These data reveal that compound mutations of EGFR (L858R andE709A/G) are a relatively frequent mechanism for gefitinib resistanceamong tumors without the T790Mmutation. Provided that mutationsfor E709A/G and L858R exist in cis and that only specific combinationsemerge in TKI-resistant tumors, such double changes likely affect theprotein structure of EGFR. Considering that 12.8% of L858R-positivetumors potentially have compoundmutations that limit gefitinib effica-cy, not only the hotspot mutation but also sequencing analysis of the

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Fig. 7. Assessment of drug sensitivity for EGFRmutants. Thedrug sensitivities of the indicated EGFRmutantswere evaluatedusing theMANOmethod inBa/F3 cells. The drugsensitivity was categorized as sensitive, partially sensitive, or resistant based on the IC90 of each mutant with each drug.

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entire EGFR gene/cDNA is clinically important to select suitable TKIsfor each patient. Because the transforming potential of compoundmutations is stronger than that of minor mutations alone, compoundmutations likely result from multistep mutagenesis within one gene.All compound mutations observed in our study were present in cis,although the underlying biological mechanism remains elusive.

TheMANOmethod also showed the utility of cetuximab for tumorswith several minor mutants of EGFR, including L718Q, which is resist-ant to all types of EGFR TKIs (41), providing a treatment option forpatients with thesemutations. Mutants sensitive to cetuximab likely de-pend on the EGF ligand signal and subsequent receptor dimerizationfor transforming activities.

In the DNA-mutation Inventory to Refine and Enhance CancerTreatment (DIRECT) database, 42 EGFR mutations were associatedwith disease progression after gefitinib or erlotinib therapy (42), and10 of these mutations were also evaluated in the present study. Eightsuch mutations (80%) were predicted as partially sensitive or resistantto gefitinib or erlotinib using the MANOmethod, confirming that ourpreclinical data are consistent with the findings of other studies.

The limitation of our study is the lack of sufficient clinical data forvalidating the prediction by the MANOmethod. Especially, the resultsregarding cetuximab remain to be verified by clinical evidence becauselittle has been done to examine the sensitivity of EGFR minor mutantsto cetuximab in human. Although suitable TKIs for a given EGFRmu-tant can be readily predicted using theMANOmethod (and such com-binations have been retrospectively verified in a small cohort), theclinical application of the output of theMANOmethod should be care-fully translated. For example, patients with different types of minorEGFR mutations predicted as sensitive using the MANO method maybe enrolled in open basket–type clinical trials, because the incidenceof eachmutation is too rare to conduct clinical trials for each subtype.

The annotation of VUS in terms of drug sensitivity and transformingpotential is urgently needed in the catalog of the cancer genome atlas.Thus, we propose that the MANO method could accelerate the evalua-tion of the VUS of TKs, enabling the determination of the best drug foreach mutation.

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MATERIALS AND METHODSStudy design and patient specimensTo evaluate the tumorigenicity and drug sensitivity of EGFR mutants,EGFR mutants reported more than four times in COSMIC databasewere selected, and their cDNAs were constructed by site-directed mu-tagenesis from wild-type EGFR cDNA in a retroviral vector. 3T3 cellsand Ba/F3 cells overexpressing EGFR mutants were subjected to theMANO method. All in vitro experiments for the assessment of EGFRmutants by the MANO method (Figs. 4, A and C; 5C; 6A; and fig. S6)were performed in triplicate. To evaluate the tumorigenicity and drugsensitivity of oncogenic mutants, an in vivo MANO method was con-ducted in 10 mice per each group. Mice were sacrificed to obtain DNAfrom the tumors at the indicated day in each experiment (Figs. 2C and3C and fig. S5). To investigate the involvement of infrequent mutationswithin EGFR in the tolerance against TKIs in clinical settings, we per-formed target amplicon sequencing of EGFR of 52 primary biopsy andrebiopsy specimens from21 cases that recurred after gefitinib treatment,without acquiring theT790Mmutation (obtained from theDepartmentof Respiratory Medicine, Juntendo University, Graduate School ofMedicine, Tokyo, Japan). We excluded the data of 10 cases, becausewe could not detect the EGFR mutations that were reported in the


primary biopsy specimens of those cases. The samples included endo-bronchial biopsies, computed tomography–guided core needle biopsies,and small surgical biopsies. To determine the prevalence of EGFR com-pound mutations, 420 surgically resected NSCLC specimens positive forEGFR(L858R), EGFR exon 19 deletion, G719mutations, or L861Q wereobtained from the Department of General Thoracic Surgery, JuntendoUniversity, Graduate School of Medicine, Tokyo, Japan. We excludedthedataof30cases, becausewecouldnotdetect theEGFRmutationswhichthe specimens of those cases were reported to have. To investigate whetherthe compound mutations exist in the cis or trans allele, we performedgDNA- or cDNA-based amplicon sequencing in 12 and 27 specimens,respectively. If both mutations were on the same exon and closer than200 nucleotides, then gDNA-based amplicons were used for sequencing,andotherwise, cDNA-based ampliconswere used for sequencing becausegDNA amplicons covering two different EGFR exons are too big for thelibrary preparation. We could not evaluate the allele status of the other23 specimens harboring compound mutations because we could notobtain reliable cDNA libraries and therefore stopped the sequencing anal-ysis. Tumor tissue specimens were collected and analyzed under aprotocol approved by the institutional review boards of The Universityof Tokyo (no. G3546) and Juntendo University (no. 2014176). Informedconsent was obtained from all patients involved in the present study.

Cell linesHuman embryonic kidney–293T (HEK293T) cells and mouse 3T3fibroblasts were obtained from the American Type Culture Collec-tion and maintained in Dulbecco’s modified Eagle’s medium–F12(DMEM-F12) supplemented with 10% FBS (both from ThermoFisher Scientific). Ba/F3 cells were cultured in RPMI 1640 (ThermoFisher Scientific) supplemented with 10% FBS and mouse IL-3 (20 U/ml;Sigma-Aldrich).

Construction of retroviral vector with random bar codesThe pcx5bleo vector was developed by modifying the pcx4bleo vector(43) toharbor the6-bpDNAbar code sequenceupstreamof the start codonof the genes of interest. Plasmids encoding wild-type human EGFR cDNAwere isolated by PCR and ligated into pcx5bleo. The cDNAs encodingthe EGFRmutants were generated using the QuikChange II Site-DirectedMutagenesis kit (Agilent Technologies) and ligated into pcx5bleo.

Preparation of retrovirus and transduction of cell linesThe recombinant plasmids were introduced together with packagingplasmids (TakaraBio) intoHEK293Tcells toobtain recombinant retroviralparticles. For the focus-formation assay, 3T3 cells were infected with eco-tropic recombinant retroviruses using polybrene (4 mg/ml) (Sigma-Aldrich)for 24 hours and further cultured in DMEM-F12 supplemented with 5%calf serum for up to 2 weeks. Cell transformation was assessed througheither phase-contrast microscopy or staining with Giemsa solution.

MANO detectiongDNA from the cell lysates was PCR-amplified using the primers 5′-TGGAAAGGACCTTACACAGTCCTG-3′ and 5′-GACTCGTT-GAAGGGTAGACTAGTC-3′. The PCR products were purified usingAMPure beads (Beckman Coulter). The sequencing libraries were gen-erated using the NEBNext Ultra DNA Library Prep Kit (New EnglandBiolabs) according to the manufacturer’s instructions, and index barcodes were added. The library quality was assessed using a Qubit 2.0fluorometer (Thermo Fisher Scientific) and theAgilent 2200 TapeStationsystem. The library was sequenced on an Illumina MiSeq using Reagent

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Kit V2 (300 cycle) with the 150-bp paired-end option. These sequencereads included the bar code sequence 5′-CTAGACTGCCXXXXXXG-GATCACTCT-3′ (where X denotes any nucleotide) and theircomplementary sequences. We detected the amount of each cDNA bycounting these bar code sequences. DMSO-treated cell mixtures wereused as the reference control for scaling of each cell clone signal. Thus,the signal from each treated cell linewas calculated as 100 × (median readnumber across replicates)/(median read number of the DMSO control).

In vivo MANO methodIndividually transduced cell clones were mixed in equal numbers, and2.5 × 106 cells of this mixture (1 × 105 cells from each of 25 cell clones)were subcutaneously injected into 20 6-week-old female nude miceaccording to the animal use protocol reviewed and approved by theUniversity of Tokyo Animal Care and Use Committee. The mice weretreated once daily for 16 days by gavage with the EGFR TKI erlotinib(50 mg/kg body weight), afatinib (20 mg/kg body weight), or vehiclecontrol (1% sodium carboxymethyl cellulose), beginning 5 days afterinjection of the cell lines. The tumors were resected and homogenizedwith gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) toobtain gDNA from each component of the tumor uniformly. The barcode number for each cell line was normalized to the total bar codenumbers of the tumor, and the calculated number was subsequentlyused to determine the percentage contribution of each cell line to thetumors, treated either with EGFR inhibitor (n = 10) or with vehiclealone (n = 10).

AlamarBlue cell viability assayAfter incubating the cells in 96-well plates (with 100 ml of culture me-dium per well), 10 ml of alamarBlue (Thermo Fisher Scientific) wasadded, and the fluorescence was measured by a microplate reader(2030 ARVO X3, PerkinElmer) (excitation, 530 nm; emission, 590 nm)at the indicated times. Wells without cells were assayed as negativecontrols. Adjustment for fluorescence gain for every well was performedagainst the well with the maximum fluorescence intensity.

EGFR sequencing and allele quantificationgDNAwas prepared from frozen or formalin-fixed paraffin-embeddedsamples using theDNeasy kit fromQiagen.EGFR exons were PCR-am-plified using the following 13 primer sets: set 1: 5′-ACGAGTAA-CAAGCTCACGCA-3′, 5′-ATTCTGCCCAGGCCTTTCTC-3′; set 2:5′-TTGCCCTCAACACAGTGGAG-3′, 5′-TTATGAACCCC-CAGCCTTGG-3′; set 3: 5′-CTGCGACATCCCTGGGAAAT-3′, 5′-CATCTTACCAGGCAGTCGCT-3′; set 4: 5′-ACTTACCT-CACTTGCCCAGC-3′, 5′-GACAAGGATGCCTGACCAGT-3′; set5: 5′-AAGCCAAAGGAGGATGGAGC-3′, 5′-AGGCCCTTCG-CACTTCTTAC-3′; set 6: 5′-TTCTCTTGCAGTCGTCAGCC-3′, 5′-GGACCCATTAGAACCAACTCCA-3′; set 7: 5′-TGTGCCCACTA-CATTGACGG-3′, 5′-TTGCCGGAAAACTTGGGAGA-3′; set 8: 5′-TCCACCTCATTCCAGGCCTA-3′ , 5 ′-ACTGCTAATGG-CCCGTTCTC-3′; set 9: 5′-AGCCTCTTACACCCAGTGGA-3′, 5′-ACAGCTTGCAAGGACTCTGG-3′; set 10: 5′-GGCACCATCTCA-CAATTGCC-3′, 5′-AAAAGGTGGGCCTGAGGTTC-3′; set 11: 5′-TCATGCGTCTTCACCTGGAA-3′, 5′-AGGTACTGGGAGCCAA-TATTGT-3′; set 12: 5′-CACAGCAGGGTCTTCTCTGT-3′, 5′-GGT-GTCAGGAAAATGCTGGC-3′; and set 13: 5′-GGCTCTGTGCA-GAATCCTGT-3′, 5′-CAGGCTAATTTGGTGGCTGC-3′. Sequen-cing libraries were generated from the PCR products and sequencedon the MiSeq platform. The nucleotides in the sequence reads with a Q


value of <20 were masked, and we further extracted unique reads thatwere subsequentlymapped to the reference human genome (hg38) usingBWA (http://bio-bwa.sourceforge.net/), Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml), and NovoAlign (www.novocraft.com/products/novoalign/).Mutationswere called by an in-house pipelinebased on the following detection rules: (i) Mutations are detected at aposition of total read depth of≥1000, (ii) themutation allelic frequencyfor the tumor is≥0.01, and (iii) the mutations were supported by bothstrands of the genome.

Investigation of compound mutation allelic statusRNA was isolated from the frozen samples using the RNeasy kit fromQiagen, and 1 ml of total RNA (1 mg) and 4 ml of SuperScript IV VILOMaster Mix (Invitrogen) were mixed with 15 ml of nuclease-free water,followed by incubation at 25°C for 10 min, 50°C for 10 min, and 85°Cfor 5 min. The resulting cDNA was amplified by reverse transcription(RT)–PCR using PrimeSTAR HS DNA polymerase (Takara Bio) andspecific primer sets to obtain a fragment containing both compoundmutations. The following primer sets were used: set 1: 5′-GTCT-TGAAGGCTGTCCAACGAATG-3′ and 5′-TCCAATGCCATC-CACTTGATAGGC-3′ for detecting E709 or L718Q and L858R; set2 : 5 ′-TCTGGATCCCAGAAGGTGAGAAAG-3 ′ and 5 ′-TCCAATGCCATCCACTTGATAGGC-3′ for detecting I744M orS768I and L858R; set 3: 5′-ATCTGCCTCACCTCCACCGTG-3′ and5′-TCCAATGCCATCCACTTGATAGGC-3′ for detecting T790Mand L858R; set 4: 5′-GTCTTGAAGGCTGTCCAACGAATG-3′ and5′-AGGTGAGGCAGATGCCCAGCA-3′ for detecting G719 andS768I; set 5: 5′-GTCTTGAAGGCTGTCCAACGAATG-3′ and 5′-CTTTGCGATCTGCACACACCAGTTG-3′ for detecting G719 andT790M; and set 6: 5′-TCTGGATCCCAGAAGGTGAGAAAG-3′ and5′-CTTTGCGATCTGCACACACCAGTTG-3′ for detectingE746_A750del and T790M. The PCR amplification was conductedfor 40 cycles at 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min. ThePCR products were purified using AMPure beads and subjected tolibrary construction with NEBNext Ultra DNA Library Prep Kit. Thelibrary was sequenced on an Illumina MiSeq using Reagent Kit V2(300 cycles) with 150-bp paired-end option.

Droplet digital PCROne microliter of total RNA (=1 mg) and 1 ml of 2 mM gene-specificreverse primer (5′-GTCCTGGTAGTGTGGGTCTC-3′) were mixedwith 1 ml of 10 mM deoxynucleotide triphosphate mix and 10 ml ofnuclease-free water and heated at 65°C for 5min, followed by cooling onice for 1 min. Subsequently, 1 ml of dithiothreitol (100 mM), 4 ml of 5×SuperScript IV buffer, 1 ml of RNaseOUT recombinant RNase inhibitor,and 1 ml of SuperScript IV reverse transcriptase (Invitrogen) were added.The combined reactionmixture was incubated at 23°C for 10min, 50°Cfor 10 min, and 80°C for 10 min. The resulting cDNAs were subse-quently amplified by RT-PCR using the same procedure as describedabove. Digital PCR was performed using the QX100 Droplet DigitalPCR system (Bio-Rad Laboratories) with EGFR-E709K primers (5′-CCAACCAAGCTCTCT-3′ and 5′-GCCCAGCACTTTGAT-3′) andthe EGFR-E709K BHQ1 probe (HEX-GAGGATCTTGAAGAAAACT-GA) at a final concentration of 1 mM primers and 250 nM probe, andEGFR-L858R primers (5′-GTATTCTTTCTCTTCCGCA-3′ and 5′-CAGCATGTCAAGATCACA-3′) and EGFR-L858R BHQ1 probe(FAM-TTGGGCGGGCCAAAC)at a final concentrationof 1mMprimersand 250 nMprobe. The 20-ml aqueous-volume digital PCR contained finalconcentrations of 1× Droplet Digital PCR supermix for probes (Bio-Rad

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Laboratories), each primer and probe (EGFR-L858R and EGFR-E709K),and templates.We used 600-pg to 60-ng cDNA, 100 to 10,000 copies ofpcx5-plasmid, or 100 to 10,000 copies of PCR products as templates persample. The reaction mixture was loaded onto a plastic cartridge with70 ml of droplet generation oil and placed in the droplet generator. Thedroplets generated from each sample were transferred to a 96-well PCRplate, and the PCR amplification was conducted using a T100 ThermalCycler with the following conditions: 95°C for 10 min followed by45 cycles at 94°C for 30 s and 50°C for 90 s, a 10-min incubation at 98°C,and a final hold at 4°C. After amplification, the digital PCR data werecollected and analyzed using a Bio-Rad QX100 droplet reader andQuantaSoft v1.3.2.0 software. Crosshair gating was used to splitthe data into four quadrants in a procedure analogous to that appliedin flow cytometry analysis (44). About 15,000 droplets were analyzedper well.

Xenograft tumor assaysFor xenograft generation, 1.0 × 106 cells were subcutaneously injectedinto 6-week-old female nude mice. The mice were treated twice a weekwith an intraperitoneal injection of either cetuximab (10 mg/kg bodyweight) (Merck Serono) or vehicle control, beginning 5 days after theinjection of the cell lines. The average tumor volume in each group wasexpressed in cubic millimeters and calculated using the formula p/6 ×(large diameter) × (small diameter)2. Tumor injections and volumemeasurements were performed blinded to the constructs expressed bythe cells used for injection. All procedures in mice were performedaccording to the protocols reviewed and approved by the Universityof Tokyo Animal Care and Use Committee.

Statistical analysisData are means ± SD or means only, as stated in the figure legends. Dif-ferences between two experimental groups were determined by two-tailed Student’s t test. P < 0.05 was considered statistically significant.

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SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/416/eaan6566/DC1Fig. S1. Quantification of focus-formation assay.Fig. S2. Temporal changes in the proportion of 3T3 cells expressing 25 different genes.Fig. S3. Transforming activity evaluation using the MANO method in vitro.Fig. S4. AlamarBlue cell viability assay in Ba/F3 cells expressing EGFR mutants.Fig. S5. In vivo evaluation of sensitivity to afatinib using the MANO method.Fig. S6. In vitro transforming activity of 101 EGFR mutants evaluated by the MANO method.Fig. S7. Immunoblot analysis of Ba/F3 cells expressing EGFR mutants.Fig. S8. The droplet digital PCR assay for the detection of EGFR E709A and L858R.Fig. S9. Focus-formation assay of 3T3 cells with EGFR L858R compound mutations.Fig. S10. Focus-formation assay of 3T3 cells expressing EGFR G719 compound mutations orexon 19 deletion compound mutations.Fig. S11. The sensitivity of EGFR mutants to dacomitinib, nazartinib, and neratinib.Table S1. The raw data of Fig. 3A (provided as an Excel file).Table S2. One hundred one recurrent EGFR mutations in COSMIC database analyzed with theMANO method (provided as an Excel file).Table S3. Barcode sequences used in the MANO method for EGFR compound mutations(provided as an Excel file).Table S4. The raw data of Fig. 4C (provided as an Excel file).Table S5. Recurrent NSCLC cases after gefitinib treatment without the EGFR T790Mmutation.Table S6. Clinical and molecular characteristics of surgically resected lung adenocarcinomawith EGFR compound mutations (provided as an Excel file).Table S7. The frequency and pattern of E709 compound mutations.Table S8. The raw data of Fig. 5C (provided as an Excel file).Table S9. The raw data of Fig. 6A (provided as an Excel file).Table S10. Candidate drug for each EGFR mutation (provided as an Excel file).


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Acknowledgments: We thank A. Maruyama and H. Tomita for technical assistance. We alsothank T. Akagi and K. Sasai for providing the pcx4bleo plasmid. Funding: This study wasfinancially supported in part through grants from the Leading Advanced Projects for MedicalInnovation (LEAP) and the Practical Research for Innovative Cancer Control from the JapanAgency for Medical Research and Development, AMED. Author contributions: S.K. and H.M.conceived the project and wrote the paper. S.K. designed and performed the experiments andanalyses. M.N. performed the experiments. T.U. performed the bioinformatic analyses. Y.S.,T.H., N.S., K. Takahashi, K.S., K. Takamochi, and F.T. provided clinical specimens and performedthe analyses. Competing interests: The authors declare that they haveno competing interests.

Submitted 17 May 2017Accepted 15 October 2017Published 15 November 201710.1126/scitranslmed.aan6566

Citation: S. Kohsaka, M. Nagano, T. Ueno, Y. Suehara, T. Hayashi, N. Shimada, K. Takahashi,K. Suzuki, K. Takamochi, F. Takahashi, H. Mano, A method of high-throughput functionalevaluation of EGFR gene variants of unknown significance in cancer. Sci. Transl. Med. 9,eaan6566 (2017).

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significance in cancer gene variants of unknownEGFRA method of high-throughput functional evaluation of

Takahashi, Kenji Suzuki, Kazuya Takamochi, Fumiyuki Takahashi and Hiroyuki ManoShinji Kohsaka, Masaaki Nagano, Toshihide Ueno, Yoshiyuki Suehara, Takuo Hayashi, Naoko Shimada, Kazuhisa

DOI: 10.1126/scitranslmed.aan6566, eaan6566.9Sci Transl Med

treatment response for each mutation pattern found in patients.epidermal growth factor mutations alone and in combinations, which should help predict tumor behavior and

. developed a high-throughput method for assessing the effects ofet alpotentially pathogenic mutations, Kohsaka genomics studies, but their effects on the tumor phenotype were often uncertain. To identify and classify thesefor driving cancer progression and drug resistance. A variety of mutations in this gene have been reported in

Mutations in the epidermal growth factor are present in a variety of tumor types and are often responsibleNo longer unknown

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