Personalized Medicine and ImagingSee related commentary by Hirsch et al., p. 4422
Biomarker Analyses from a Placebo-Controlled Phase IIStudy Evaluating Erlotinib � Onartuzumab in AdvancedNon–Small Cell Lung Cancer: MET Expression Levels ArePredictive of Patient Benefit
Hartmut Koeppen1, Wei Yu1, Jiping Zha6, Ajay Pandita3, Elicia Penuel1, Linda Rangell1, Rajiv Raja1,Sankar Mohan3, Rajesh Patel1, Rupal Desai1, Ling Fu1, An Do1, Vaishali Parab1, Xiaoling Xia1, Tom Januario1,Sharianne G. Louie1, Ellen Filvaroff1, David S. Shames1, Ignacio Wistuba5, Marina Lipkind1, Jenny Huang1,Mirella Lazarov4, Vanitha Ramakrishnan1, Lukas Amler1, See-Chun Phan1, Premal Patel1, Amy Peterson2, andRobert L. Yauch1
AbstractPurpose: In a recent phase II study of onartuzumab (MetMAb), patients whose non–small cell lung
cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a
significant benefit with onartuzumab plus erlotinib (OþE) versus erlotinib. We describe development and
validation of a standardizedMET IHC assay and, retrospectively, evaluatemultiple biomarkers as predictors
by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA.
Results: A positive correlation between IHC,Western blotting, andMETmRNA expression was observed
in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-
based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best
differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement
with OþE in patients with high MET copy number (mean �5 copies/cell by FISH); however, benefit was
maintained in "MET IHC-positive"/MET FISH-negative patients (HR, 0.37; P ¼ 0.01). MET, EGFR,
amphiregulin, epiregulin, orHGFmRNA expression did not predict a significant benefit with onartuzumab;
a nonsignificant OS improvement was observed in patients with high tumorMETmRNA levels (HR, 0.59;
P ¼ 0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of
0.519 (P ¼ 0.09) in favor of onartuzumab treatment.
Conclusions: MET IHC remains the most robust predictor of OS and progression-free survival benefit
from OþE relative to all examined exploratory markers. Clin Cancer Res; 20(17); 4488–98. �2014 AACR.
IntroductionMET, a receptor tyrosine kinase (RTK) that binds
hepatocyte growth factor (HGF) is frequently overex-
pressed in a variety of human malignancies. MET acti-vation has been implicated in tumorigenesis, and METsignaling can be dysregulated through a variety of geneticor epigenetic mechanisms in cancer (1, 2). In non–smallcell lung cancer (NSCLC), tumor MET receptor proteinexpression, HGF protein expression, and high MET genecopy number are indicative of poor prognosis (3–6).Although focal amplification of the MET gene is rare inprimary lung tumors (�1%–7%; ref. 4), it is associatedwith oncogenic addiction, and with sensitivity, in pre-clinical models, to small-molecule inhibitors (SMI) tar-geting MET (7, 8). No activating mutations have beenidentified in the kinase domain of MET in NSCLC;however, somatic variants causing exon 14 skipping, canresult in an alternatively spliced MET receptor lacking thejuxtamembrane domain that sustains enhanced ligand-dependent MET signaling (9). Finally, genetic poly-morphisms have been linked to enhanced MET signaling
Authors' Affiliations: 1Genentech Inc., SouthSan Francisco; 2Medivation,Inc., San Francisco; 3OncoMDx, Inc., Palo Alto; 4Gilead Sciences, Inc.,Foster City, California; 5Department of Translational Pathology, The Uni-versity of TexasMDAndersonCancerCenter, Houston, Texas; and 6CrownBioscience Inc., Jiangsu Province, China
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
H. Koeppen, W. Yu, and J. Zha contributed equally to this article.
Corresponding Author: Robert L. Yauch, Department of Molecular Diag-nostics and Cancer Cell Biology, Genentech, Inc., 1 DNA Way, South SanFrancisco, CA 94080. Phone: 1-650-467-7037; Fax: 1-650-742-5179;E-mail: [email protected]
(R970C, T990I; ref. 10), as well as to lower HGF-bindingaffinity (N375S; ref. 11).A growing bodyof evidence has emerged to support a link
between the MET and EGF receptor (EGFR) signaling path-ways. These RTKs are often coexpressed in tumors, andevidence exists for functional transactivation that mayamplify downstream signals (12). For example, activationof EGFR may occur through MET amplification or HGF-mediated induction of EGFR ligands (13). MET activationhas been associated with resistance to EGFR inhibitors bothpreclinically and clinically (14–16). Collectively, these find-ings support the rationale for dual inhibition of MET andEGFR signaling.Onartuzumab (MetMAb) is a recombinant, humanized,
monovalent monoclonal antibody targeting MET (17). Aphase II study (OAM4558g) evaluated onartuzumab pluserlotinib (OþE) versus placebo plus erlotinib (pþE) inpatients with second-/third-line NSCLC therapy (18).Patient tumor samples were evaluated for MET expressionby immunohistochemistry (IHC) and were classified asMET-positive or MET-negative, after randomization, butbefore unblinding the treatment assignment. There wasneither a progression-free survival (PFS; HR, 1.09; P ¼0.69), nor overall survival (OS) benefit (HR, 0.80; P ¼0.34), in the intent-to-treat (ITT) population. However, thecombination of OþE in MET-positive disease resulted inimproved PFS and OS (HR, 0.53; P ¼ 0.04; HR, 0.37; P ¼0.002, respectively; ref. 18).In this article, we describe the development and vali-
dation of the specific IHC assay and the correspondingscoring system that was used to assess MET proteinexpression in the OAM4558g clinical trial. In addition,we carry out retrospective analyses to further assess thediagnostic cutoff point and evaluate additional biomar-
kers related to the MET and/or EGFR pathways, as pre-dictors of benefit from OþE.
Materials and MethodsPatients
Patients ages �18 years with measurable and previouslytreated (up to twoprior regimens) stage IIIB/IVNSCLCwereeligible. Submission of tumor tissue (archival permitted), aseither a tissue block or unstained serial slides, was required.Written informed consent was obtained before any study-specific screening procedures. A total of 137 patients wererandomized; 68 to pþE and 69 to OþE. Demographic andbaseline characteristics were generally well-balanced acrossthe treatment arms in the ITT and MET diagnostic sub-groups,with fewexceptions noted. A similar pattern in thesecharacteristics was also observed across the treatment armsin the subgroups of FISH evaluable patients and quantita-tive reverse transcription PCR (qRT-PCR) evaluable patients(data not shown). Prioritization of specific biomarker anal-yses and number of tissues examined for the given analysiswas as follows: MET IHC (n ¼ 128), EGFR/KRAS mutationanalysis (n¼ 112),MET/EGFR FISH (n¼ 96),MET exon14(n¼ 87) andN375S genotyping (n¼ 113) and finally, qRT-PCR (n ¼ 67). Plasma HGF levels were evaluated from 96patients. The overlap in biomarker analyses is shown inSupplementary Table S1.
Study designThis was a phase II double-blinded, multicenter, inter-
national randomized clinical trial (OAM4558g). Patientswere randomly assigned in a 1:1 ratio to receive eitheronartuzumab (15 mg/kg) or placebo (both administeredby intravenous infusion every 3 weeks), plus erlotinib (oral150 mg daily). The coprimary endpoints were PFS in theITT- and MET-positive populations. Approximately, 120patients would be needed to estimate the PFS benefit inboth these populations. Further details of the study designhave been published previously (18).
MET expression by IHCMET expression levels were evaluated using CONFIRM
anti-totalMET (SP44) rabbitmonoclonal primary antibody(Ventana Medical Systems, Inc.; cat no. 790-4430), and acomposite scoring system was devised to determine thestatus of MET. The IHC cutoff point for positivity wasprospectively defined in OAM4558g. The scoring ofOAM4558g specimens was independently reviewed by asecond pathologist, with an 88.3% concordance in callingMET-positive between pathologists. We did not observe adifference in outcome based upon the interpretation ofpathologist #2 of the data. Further details are included inthe Supplementary Methods section.
Western blot analysisCell lines used for Western blotting were obtained from
the American Type Culture Collection (ATCC), NationalCancer Institute Division of Cancer Treatment and Diag-nosis Tumor Repository (Bethesda, MD); or Japan Health
Translational RelevanceA hallmark of the successful clinical development of
targeted therapies is the identification of the patientsubgroups most likely to benefit from these agents.Recent clinical studies evaluating molecules targetingtheMET signaling pathway, including onartuzumab andrilotumumab, have demonstrated benefit in patientswhose tumors express high levels of MET protein. Wedescribe the development, performance, and scoringcriteria of a MET IHC test that differentiated patientoutcomes for onartuzumab plus erlotinib versus erloti-nib alone in a recent phase II study in non–small celllung cancer. In a retrospective analysis of this study,multiple biomarkers related to MET and/or EGFR sig-naling were additionally examined. It was determinedthat the IHC test to detect tumor MET levels representedthe best predictor of benefit. These results have guidedthe phase III clinical development strategy for onartu-zumab and provided further support for development ofthe MET IHC test as a potential companion diagnostic.
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Sciences Foundation (Tokyo, Japan) and were authenticat-ed. For authentication, short tandem repeat (STR) profileswere determined for each line using the Promega PowerPlex16 System. This was performed once and compared withexternal STR profiles of cell lines (when available) to deter-mine cell line ancestry. Sixteen loci (fifteen STR loci andAmelogenin for gender identification) were analyzed,including D3S1358, TH01, D21S11, D18S51, Penta E,D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, AMEL, vWA, D8S1179, and TPOX. NSCLC cell lineswere lysed in T-PER tissue extraction reagent (ThermoScientific) supplemented with protease and phosphataseinhibitors. Proteins resolved by SDS–PAGE were electro-phoretically transferred to polyvinylidene difluoride mem-brane and Western blots were carried out using standardtechniques. The primary antibodies used in this study were:MET clone SP44 (Ventana Medical Systems), MET cloneL41G3 (Cell Signaling Technology), MET clone 5D5 (HB-11895; ATCC), and actin (cat. no. sc-8432; Santa CruzBiotechnology). A polyclonal antibody raised againstMST1R was a kind gift from Dr Amitabha Chaudhuri(Genentech, Inc.).
Molecular assessmentsMET and EGFR gene copy numbers were evaluated by
FISH; a CEP7 centromere probe (Abbott Molecular) wasused as a control. High-levelMET amplificationwas definedas tight gene clusters of�15 copies in�10% of tumor cells,or a MET: CEP7 ratio of �2. A cutoff of �5 copies of MET/cell was predefined as the criterion for FISH-positive status(FISHþ), based on prior prognostic data supporting thiscutoff in NSCLC (4). Tumors were considered EGFR FISHþ
based on a scoring system used in multiple clinical studies(19). Further details are included in the SupplementaryMethods section.
DNA and RNA were isolated from macrodissected tissueto enrich for tumor content. EGFR and KRAS mutationswere evaluated using the DxS Genotyping Kit.MET exon 14variants were evaluated by Surveyor nuclease digestion anddetection by WAVE analysis (Transgenomics, Inc.). A poly-morphism at position N375S of MET was evaluated bypyrosequencing on the Pyromark Q24 (11). Expression ofMET, HGF, EGFR, amphiregulin (AREG), and epiregulin(EREG)mRNA transcripts was evaluated by qRT-PCRon theBiomark platform (Fluidigm). The primer/probes used forprofiling are shown in Supplementary Table S2 and furtherdetails are included in the Supplementary Methods section.
HGF quantification by ELISAPlasma HGF levels were measured by ELISA, as described
previously (20).
Statistical analysisPFS was defined as the time from randomization to the
first occurrence of disease progression or death on studyfrom any cause, or to the last tumor assessment date ifpatients were progression free. OS was determined fromrandomization to the date of death, or the last known
patient contact. For each treatment arm, the median PFSand OS were estimated from Kaplan–Meier curves. The PFSand OS treatment comparisons were based on a log-ranktest at the 0.05 level of significance (two-sided). Estimatedtreatment effects for OþE relative to pþE were expressed asHRs with 95% confidence intervals (CI), derived from anunstratified Cox model. Survival analyses were performedin biomarker subgroups for PFS and OS.
ResultsDevelopment of the SP44 IHC test
To identify antibodies that would be suitable for detect-ing MET protein expression levels in formalin-fixed paraf-fin-embedded tissue specimens, we initially tested 16 com-mercially or internally available antibodies for their spec-ificity and staining intensity on control tissues and cell lines(data not shown). The CONFIRM anti-total cMET (SP44)rabbit monoclonal antibody (mAb), generated against anintracellular epitope ofMET,was chosen for further analysisbased upon its initial reactivity pattern in NSCLC cell linesengineered to knockdown or ectopically express the METgene (Supplementary Fig. S1). Furthermore, SP44 did notcross-react with the Ron receptor (MST1R), the closesthomolog of MET (Supplementary Fig. S1C). IHC stainingof NSCLC tissues (Fig. 1A) and cell lines (SupplementaryFig. S2A) revealed a range of staining intensities character-ized as no staining (negative), weak, moderate, or strong,with a pattern that was primarily membranous with avariably strong cytoplasmic component. In NSCLC celllines, the SP44 staining intensities associated with thedegree of MET protein expression, as determined by West-ern blot analysis using both SP44, as well as an alternativeanti-MET antibody (L41G3; Fig. 1B). SP44 staining inten-sities also corresponded with the levels of MET detected onthe surface of NSCLC cell lines using flow cytometry whenprobed with the bivalent form of the parent mAb to onar-tuzumab (5D5) and no IHC staining was observed withSP44 in cell lines in which 5D5 did not bind (Supplemen-tary Fig. S2B and S2C). Finally, a statistically significantassociation was observed between the intensity of IHCstaining with SP44 and MET mRNA levels across a largerpanel of NSCLC cell lines (Supplementary Fig. S3) furthersupporting the specificity of this reagent.
In NSCLC tissues, heterogeneity in the intensity wasfrequently observed within individual NSCLC tumors(Supplementary Fig. S4). To account for this heterogeneity,a comprehensive clinical scoring system was developedthat evaluated bothmembranous and cytoplasmic stainingintensity and the percentage of cells staining at a givenintensity level (Supplementary Table S3). A proportionalcutoff of�50%was selected to ensure that amajority of thecells within a given specimen expressed MET at either aweak (clinical score¼ 1þ),moderate (clinical score¼ 2þ),or strong (clinical score ¼ 3þ) intensity level. Specimenswith no or equivocal staining in tumor cells or <50% oftumor cells staining at any given intensity were considerednegative (clinical score ¼ 0). Using these IHC scoringcriteria, we continued to observe a statistically significant
Koeppen et al.
Clin Cancer Res; 20(17) September 1, 2014 Clinical Cancer Research4490
(Jonckheere–Terpstra P < 0.0001) relationship with METmRNA expression in tumor specimens (Fig. 1C). NSCLCtumors expressing moderate or strong levels of MET in�50% of cells (clinical IHC score 2þ or 3þ) were classifiedas MET-positive (MET IHC diagnostic positive). To under-
stand how intratumoral heterogeneity may affect thisclinical diagnostic score, we evaluated MET expression in10 patients for whom more than one paraffin block hadbeen submitted for analysis (Supplementary Table S4).Although variation was observed in the proportion of cellsstaining at a given intensity, there was complete concor-dance in the final IHC score for the multiple tissue speci-mens in these patients. Finally, using these scoring criteria,we determined that 58%(�6%)of nonsquamous and 26%(�11%) of squamous cell tumor specimens were MET-positive across three separate series of NSCLC tumor speci-mens (Table 1).
Evaluation of alternative scoring systemsWe also evaluated the H-score method of semiquantita-
tively assessingMET expression as an alternative to take intoaccount the intensity and heterogeneity of MET staining(21). Both membranous and cytoplasmic staining werecombined into a single intensity score. We observed a rangeof H-scores from 0 to 300, with a median H-score of 160in the set of tissue samples collected from patients inOAM4558g. Most specimens within each clinical scorecategory (0, 1þ, 2þ, and 3þ) were distributed around theH-scores of 0, 100, 200, and 300, respectively (Supplemen-tary Figs. S5 and S6), as would be expected if staining wascompletely homogeneous. When heterogeneity in stainingintensity was present (n ¼ 64 specimens exhibiting tumorcell populations with multiple staining intensity levels),positivity was primarily distributed between two differentstaining intensity levels (n ¼ 48). Only 13% of specimens(n ¼ 16) exhibited a significant degree of heterogeneity inwhich staining intensities were noted across three differentlevels. Therewere four specimensbelow themedianH-scorethat were considered MET-positive when applying the clin-ical scoring algorithm; conversely, there was one specimenat themedianH-score that wasMET-negative by the clinicalscoring system.
Defining the best cutoff for the MET IHC assayThe IHC cutoff point for positivity was prospectively
defined in OAM4558g. An analysis evaluating the appro-priateness of this cutoff point revealed that alternativeproportional cutoff points to define MET positivity of�10% or �90% of tumor cells staining moderately orstrongly did not result in a better differentiation of patientoutcomes (18). To evaluate alternative cutoff points inmore detail, we examined PFS (Fig. 2A) and OS (Fig. 2B)based upon individual staining intensities using a 10%,50%, or 90% proportional cutoff. Consistent with priorresults, the best differentiation of patient outcomes wasobserved at the 50% cutoff and the differentiation was notgreatly altered by applying different cutoffs to differentstaining intensities. More importantly, PFS was also ana-lyzed in two subsets of patients whose IHC scores bor-dered the diagnostic cutoff: group 1, patients categorizedas MET-positive using a 10% cutoff but as MET-negativeusing 50% cutoff (patients whose tumors exhibitedmoderate to strong staining in 10%–49% of tumor
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Figure 1. Characterization of SP44. A, representative images depictingthe range of SP44 staining intensities (negative, weak, moderate, andstrong staining) in malignant NSCLC tissues. B, relationship of SP44immunohistochemical staining intensity and MET protein levels byWestern blotting in NSCLC cell lines using two anti-MET antibodies;four representative cell lines and staining intensity are shown. C,correlation of IHC score with MET mRNA levels in NSCLC tissuesdetermined by qRT-PCR.
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cells; Fig. 2C); and group 2, patients categorized as MET-positive using 50% cutoff, but as MET-negative using a90% cutoff (patients whose tumors exhibited moderate tostrong staining in 50%–89% of tumor cells; Fig. 2D). Ingroup 1 (n ¼ 21), patients treated with OþE had shorterPFS than patients treated with pþE (HR, 3.065; P ¼ 0.04),suggesting that using a 10% cutoff may incorporatepatients who may not benefit from the study drug. Out-comes in group 2 patients were not statistically differentbetween treatment arms; however, there was a trendtoward benefit in those who received OþE (n ¼ 19; HR,0.47; P ¼ 0.23). Similar findings were observed for OS(data not shown).
Evaluation of alternative predictive markers ofonartuzumab þ erlotinib activity
Pathway mutations. EGFR and KRAS genotyping datawere obtained from112 patients, mutations were identifiedin 13 (12%) and 26 (23%) samples, respectively, and weremutually exclusive. Nine of 13 EGFRmut cases and 13 of the26 KRASmut cases were MET-positive. EGFRmutations wereidentified in 6 of the 7 patients with objective responses(Supplementary Tables S5 and S6); there were no responsesin patients with KRASmut tumors. Exon 14, encoding thejuxtamembrane domain of MET, was genotyped in 87patients. A splice site deletion was detected in only oneindividual and alternative splicing was confirmed at themRNA level (data not shown). Two additional patients(both on pþE) had tumors harboring exon 14 polymorph-
isms R970C and T990I. Polymorphisms in MET N375Swere found in 12 of 113 (11%) tumors tested; because of animbalance between treatment subgroups, no further anal-ysis was carried out.
MET/EGFR copy number. MET and EGFR copy numberwere evaluated by FISH for 96 patients. The median METcopy number was 3.44 copies per cell (range, 1.6–25.0),with 19 (20%) evaluable tumors being MET FISHþ,including eight (8%) harboring high-level MET amplifi-cation. The relationship between MET copy number andMET IHC clinical score is shown in Supplementary Fig.S7, with a statistically significant association of FISHpositivity with IHC 3þ cases observed (c2, P < 0.0001).No high-level MET gene amplifications were identified inpatients who had objective responses to therapy (Sup-plementary Table S6); however, a high-level MET ampli-fication was identified in an EGFRmut patient who did notrespond to OþE.
The estimated HR for OS for patients who were METFISHþ was 0.60 (P ¼ 0.35) in favor of OþE (Fig. 3A). Noimprovement in OS was observed using lower cutoffs forMET copy number (HR, 0.89; P ¼ 0.79 for �4 copies andHR, 0.75; P ¼ 0.37 for �3 copies; Supplementary Fig. S8Aand S8B). To evaluate the impact of EGFR mutations onpatient outcomes, we examined outcomes in the knownEGFRwt population: OS HR for patients with MET FISHþ
tumors receivingOþEwas 0.30 (P¼0.06); PFSHRwas 0.58(P ¼ 0.38; Fig. 3B). As observed in the ITT population, noimprovement in OS was seen in the EGFRwt populations
Table 1. Prevalence of MET expression by SP44 IHC assay in NSCLC tumor specimens
Frequency (%)
Study Group Subgroup Total (n) Dx-negative Dx-positive 0 1 2 3
Figure 2. Evaluation of patientoutcomes with OþE treatmentbaseduponalternative IHCcutoffs.A, the forest plot for PFS fromOAM4558g according to differentIHC intensities and differentproportional cutoffs (10%, 50%,and 90%); B, the forest plot for OSin OAM4558g according todifferent IHC intensities anddifferent proportional cutoffs (10%,50%, and 90%). Kaplan–Meierestimates for PFS in patients withIHC scores bordering thediagnostic cutoff; C, patientsdefined as MET-positive using a10% cutoff and MET-negativeusing a 50% cutoff; D, patientsdefined as MET-positive using a50% cutoff and MET-negativeusing a 90% cutoff.
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further defined by�4 (HR, 0.76; P¼ 0.55) or�3 (HR, 0.75;P ¼ 0.40) copies ofMET/cell (Supplementary Fig. S8C andS8D, respectively).
Todeterminewhether theMET FISHþpatients influencedthe benefit observed in the MET-positive subgroup, OS andPFS analyses were performed in both the MET IHCþ/FISH�
subgroups of the ITT and EGFRwt populations. AnOSHR of0.37 (P ¼ 0.01) and a PFS HR of 0.24 (P ¼ 0.003) wereobserved in theMET IHCþ/FISH� ITT population (Fig. 3C).An OS HR of 0.51 (P ¼ 0.13) and a PFS HR of 0.44 (P ¼0.10) was observed in the MET IHCþ/FISH�and EGFRwt
patients (Fig. 3D).Because of chromosome 7 polyploidy, gains inMET gene
copy number have been shown to associate with high EGFRcopy number gains (4). To ensure an unbiased analysis,EGFR and MET copy number variations were evaluatedusing identical criteria, as previously adopted for use inEGFR FISH testing (19). A significant association betweenEGFR and MET copy number was observed (Fisher exact,P¼ 2.2� 10�5), with 24% (n¼ 23) of evaluable cases beingpositive for gains in bothEGFR andMET copynumber (datanot shown). However, no clinical benefit was observed in
this subpopulation (Supplementary Fig. S8E): OS HR, 1.36(P ¼ 0.54); PFS HR, 1.0 (P ¼ 1.0).
MET/EGFR pathway transcript expression. Expression ofmRNA transcripts for MET, HGF, and EGFR, and the EGFRligands, AREG and EREG, was evaluated by qRT-PCR intumor biopsies from 67 patients. In this biomarker sub-population, anonsignificant improvement inOS (HR, 0.49;P¼ 0.11) and PFS (HR, 0.57; P¼ 0.22)was observed in IHCMET–positive patients. In patients with high tumor METmRNA levels (median�3.16), anOSHRof 0.59 (P¼ 0.23),and a PFS HR of 0.76 (P ¼ 0.51) was observed (Fig. 4;Supplementary Table S7). No similar improvements inoutcome were seen in any other mRNA subgroups forpatients receiving OþE.
Plasma HGF. Pretreatment plasma protein levels ofHGF were evaluated in 96 patients. The median level ofHGFwas 480.2 pg/mL (range, 203–11,375pg/mL), with nosignificant difference in distribution between MET IHCdiagnostic subgroups (Supplementary Fig. S9). No correla-tion of plasma HGF with tumor HGF mRNA levels wasobserved (data not shown). In this subpopulation ofpatients with evaluable plasma HGF data, both OS and PFS
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Figure 3. Evaluation of patient outcomes with OþE based upon MET gene copy number. Kaplan–Meier estimates of OS according to MET FISH positivity(�5 copies of MET/cell) in the ITT (A; n ¼ 19) and EGFRwt (B; n ¼ 15) populations. Kaplan–Meier estimates of OS in patients with MET-positive and METFISH-negative (<5 copies of MET/cell) tumors in the ITT (C) and EGFRwt (D) populations.
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Clin Cancer Res; 20(17) September 1, 2014 Clinical Cancer Research4494
were consistent with what was observed in the generalpopulation: HR, 0.39 (P ¼ 0.006) in OS and HR, 0.52(P¼ 0.06) in PFS, respectively, in MET-positive patients. Inpatients who had baseline HGF protein below the median,the OS HR was 0.52 (P ¼ 0.09) and PFS HR was 0.56 (P ¼0.10) favoringOþE (Fig. 5A). In patients with baselineHGFprotein �median, the OS HR was 1.23 (P ¼ 0.56; Fig. 5B).In addition, a Cox proportional hazard regression modelrevealed no statistically significant association between OSandHGF level (HR, 0.81; 95%CI, 0.52–1.25; P¼ 0.339) orbetween OS and HGF level and treatment (HR, 1.68; 95%CI, 0.91–3.10; P ¼ 0.096).
DiscussionIn the OAM4558g study, the addition of onartuzumab to
erlotinib resulted in improvements in both PFS and OS inpatients whose tumor specimens were determined to beMET-positive by IHC (18). This benefit was not seen in theoverall population, and patients withMET-negative tumorswho received OþE had a worse outcome versus thosetreated with erlotinib alone. Therefore, the data indicatethat the MET IHC assay may be a reliable diagnostic test foridentifying NSCLC patients most likely to benefit fromOþE; and as such, this test is currently being developed asa companion diagnostic for onartuzumab in combinationwith erlotinib (clinicaltrials.gov identifier: NCT01456325;ref. 22). In this report, we describe the development andperformance of this IHC test and compare its performancerelative to alternative biomarkers.
Our investigation has identified that the SP44mAb bindsMET with high specificity and generates a range of stainingintensities that correlate well with MET mRNA levels andMET protein levels determined by alternative means. Aswith many histologic tests, we observed intratumoral het-erogeneity in MET protein expression levels in NSCLCtissues. To account for such heterogeneity, we examinedvarious scoring systems that incorporate both stainingintensity and the percentage of malignant cell positivity.We reasoned that a cutoff that captures the MET expressionsignal in the majority of the malignant cell population in aspecimen (i.e., �50%) would likely produce more repro-ducible findings. When using a cutoff of 50% of malignantcells in a biopsy sample with moderate and/or strongstaining intensity, we observed that our call for positivitydid not changewhen evaluating intratumoral heterogeneityin a small set of cases for which sections representing morethan one area of the same resection specimenwere availablefor analysis (Supplementary Table S4). More importantly,we demonstrated that this 50% cutoff was the criterion thatbest differentiated patient outcomes in the phase IIOAM4558g trial. A less stringent cutoff of 10%ofmalignantcells staining atmoderate and/or strong intensity resulted inthe inclusion of a subset of patients who did not benefitfromOþE therapy in this trial, as shownbyworse outcomes(PFSHR, 3.065;P¼ 0.04). Conversely, implementation of amore stringent cutoff of 90% of malignant cells staining atmoderate and/or strong intensity would result in the loss ofa subset of patients who trended toward improved
p+E O+E
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All patients 67 31 7.4 36 8.7 0.949 (0.493–1.827) 0.876
Baseline risk Total Median Median factor (median) n n (mo) n (mo) HR (95% CI) P O+E Better p+E Better
MET (3.16)
Low 33 19 15.3 14 11.7 1.481 (0.535–4.100) 0.447
High 34 12 6.7 22 8.7 0.591 (0.250–1.395) 0.225
AREG (7.11)
Low 33 18 6.9 15 6.5 1.314 (0.532–3.245) 0.552
High 34 13 8.3 21 0.767 (0.295–1.992) 0.584
EREG (0.26)
Low 33 18 9.2 15 8.1 1.336 (0.495–3.602) 0.566
High 34 13 6.9 21 10.2 0.669 (0.275–1.627) 0.372
EGFR (7.75)
Low 33 18 7.1 15 11.7 0.880 (0.332–2.332) 0.797
High 34 13 8.3 21 7.1 1.103 (0.441–2.757) 0.834
HGF (0.25)
Low 33 15 6.9 18 6.5 1.083 (0.459–2.555) 0.856
High 34 16 9.2 18 11.7 0.876 (0.316–2.431) 0.799
Figure4. Evaluationof patient outcomeswithOþEbasedupon tumormRNAexpression. The forest plot showingassociationofMET,AREG,EREG,EGFR, andHGF mRNA expression as determined by quantitative PCR with OS. High, mRNA levels � median; low, mRNA levels < median.
Biomarkers for Onartuzumab þ Erlotinib Therapy in NSCLC
www.aacrjournals.org Clin Cancer Res; 20(17) September 1, 2014 4495
outcomes with OþE, although this did not achieve statis-tical significance (PFS HR, 0.464; P ¼ 0.23). Additionalevaluations of outcome based on different staining inten-sities supported these proposed criteria. Overall, these datastrongly suggest that the diagnostic cutoff that was prospec-tively defined represents a suitable scoring system for futureclinical studies evaluating OþE in NSCLC. Other scoringcriteria, such as H-score, were considered. However, explor-atory analysis did not show that H-score could alter theinterpretation of the outcomes fromprevious analyses (datanot shown).
To determine whether MET IHC represented the bestpredictor of patient benefit in this study, additional bio-markers related to theMET and/or EGFR signaling pathwayswere measured. EGFR-activating mutations represent thestrongest predictor of response to EGFR-targeted therapiesinNSCLC.As expected in aprimarilyCaucasianpopulation,approximately 12% of patients harbored EGFR mutationsin this study. Of the 7 patients who reported an objectiveresponse, all but one had an EGFRmut tumor. One EGFRwt
patient had an objective response on OþE; however, it wasnot possible to associate this response to any other bio-marker examined, including EGFR or MET gene amplifica-tion,METmutation or high tumor/serumHGF levels. Most
importantly, EGFR mutations did not drive the benefitobserved in the MET-positive population, as themagnitudeof the treatment benefit was maintained in MET-positive,EGFRwt patients (OS HR, 0.46; P ¼ 0.03; ref. 18).
KRAS is a downstream effector of both EGFR and METsignaling and mutations in this gene provide a criticalpredictive marker for efficacy of EGFR-targeted antibodiesin colorectal cancer. Although KRASmutations predict for alack of radiologic response to EGFR-targeted SMIs inNSCLC, any similar impact on PFS and OS remains unre-solved (23, 24). In this study, the incidence of KRASmuta-tions wasmutually exclusive from EGFRmutations, and didnot significantly affect outcomes in patients receiving OþEin the ITT, MET-positive or MET-negative populations.However, given limitation in sample size, it remains to bedetermined whether this subgroup would indeed benefitfrom a MET inhibitor.
The role of high-levelMET gene amplification has gainedconsiderable attention, as it predicts for enhanced sensitivityto MET-targeted SMIs in preclinical models (7, 8), hasrecently been linked to anecdotal responses onMET-targetedagents in the clinic (25), and, importantly, represents amechanism of acquired resistance to EGFR SMIs in patientswithEGFRmutNSCLC(14–16). In addition, general increasesinMET copynumberhavebeen reported inmany, but not allstudies, to represent a negative prognostic factor in patientswith NSCLC and may be relevant in the context of EGFR-targeted combination therapy, given that EGFR and METcopy number gains can be associated because of chromo-some7 ploidy. In this study, the frequency of high-levelMETgene amplification and highMET copy number was 8% and20%, respectively,whichwas in accordance (albeit at thehighend) with previous studies. The association of MET FISHpositivity with the MET IHC 3þ subgroup suggests that highMET gene copy number leads to high protein expression inthis subgroup. Although the MET IHC 2þ score is alsorepresented within the MET-positive subgroup, there wasno corresponding association with high copy number gains.Nonsignificant improvements in PFS and OS were observedin the MET FISHþ ITT and EGFRwt populations. However,this benefit was not maintained when lower copy numbercutoffs were evaluated. This cutoff for MET FISH positivity(�5 copies ofMET/cell) has been linked with shortened OSand advanced-stage disease (4) and, hence, may identify apoor prognostic population who could benefit from OþE.However, a statistically significant benefit was also observedin theMET-positive/FISH-negative population. These resultswould suggest that MET IHC status, rather than MET FISHstatus, performs better for predicting benefit, and that FISHstatus alone wouldmiss a large population whomay benefitfrom OþE. No improvement in outcome was observed inpatients harboring gains in bothMET and EGFR copy num-ber, further suggesting that thebenefit to thisMETandEGFR-targeted combination therapy was not driven by the cases ofgeneral chromosome 7 ploidy.
Tumor mRNA expression of key MET and EGFR pathwaygenes was additionally evaluated as alternative predictivebiomarkers. This not only included expression of MET/
1.0A
p+E (n = 27; patients with an event = 18)
O+E (n = 21; patients with an event = 11)
0.8
0.6
0.4
Sur
viva
l pro
babi
lity
0.2
0.00 2 4 6
Time (mo)8 10 12 16
1.0B
p+E (n = 22; patients with an event = 15)
O+E (n = 26; patients with an event = 16)
0.8
0.6
0.4
Sur
viva
l pro
babi
lity
0.2
0.00 2 4 6
Time (mo)8 10 12 16
14
14
Plasma HGF low
Plasma HGF high
6.9 10.4
HR 0.52 (95% CI, 0.24–1.12); P = 0.09
7.7 7.9
HR 1.23 (95% CI, 0.60–2.52); P = 0.56
Figure 5. Evaluation of patient outcomes with OþE based upon plasmaHGF levels. Kaplan–Meier estimates of OS in patients with low(<median) baseline HGF levels (A) and with high (�median) baseline HGFlevels (B).
Koeppen et al.
Clin Cancer Res; 20(17) September 1, 2014 Clinical Cancer Research4496
HGF, but expression levels of two EGFR ligands that can betranscriptionally regulated by MET activation, AREG andEREG. MET was the only mRNA biomarker examined thatshowed a trend toward improvement in OS and PFS, albeitthis did not achieve statistical significance. This could likelybe attributed to the small sample size, as only approximate-ly 1/2 of the patients (n ¼ 67) had material remainingfollowing the prioritization of other biomarker assessmentsfor transcriptional analysis. Regardless, evaluation of METmRNA levels as an alternative predictive biomarker maywarrant further investigation in future studies.High serum or plasma HGF levels have been associated
with poor prognosis in several cancers, including NSCLC(26–28). In a study of patients with NSCLC treated withEGFR SMIs, high-serum HGF was associated with worseoutcome (tumor response, PFS, and OS; ref. 26). Despiteour expectations that the addition of onartuzumab toerlotinib could improve outcomes versus erlotinib alonein patients with high-plasma HGF at baseline, we did notobserve a statistically significant association. In fact, anonsignificant trend toward benefit was observed inpatients with low baseline HGF. The study cited fromKasahara and colleagues evaluated serum HGF, whereasour study evaluated plasma HGF. However, this wouldunlikely explain the difference, as strong correlationsbetween plasma and serum HGF levels have been observed(data not shown). High-plasma HGF also failed to show aprognostic link in the pþE arm in our study (OS HR, 0.75;P ¼ 0.41), suggesting that the prognostic significance ofcirculating HGF may be influenced by differences in thepopulation tested. It remains to be elucidated how HGFlevels in circulation relate to intratumoral levels of HGF.Although elevated circulating HGF levels have beenobserved in subsets of patients with cancer, elevated circu-lating HGF levels have additionally been described in othersettings, including certain viral or bacterial infections, graftversus host disease, and surgical procedures (29–31).In conclusion, MET IHCwas a reliable and accurate assay
for identifying patients with NSCLC most likely to benefitfrom OþE, and outperformed the other examined explor-atorymarkers. The retrospective, exploratory nature of theseanalyses, combined with small sample size, limit the scopeof definitive conclusions to be drawn from these alternativebiomarkers. Further investigations of these biomarkersusing larger sample sizes are necessary to determinewhetherany would be useful in conjunction withMET IHC to betterdefine subpopulations most likely to benefit from OþE. Inaddition, it will be imperative to determine whether suchbiomarkers will be useful in determining benefit to otheronartuzumab-based therapies and/or other antagonists ofthe MET signaling pathway. The anti-HGF antibody, rilo-tumumab, in combination with standard-of-care chemo-therapy exhibited a benefit in both PFS and OS comparedwith chemotherapy alone (OS HR, 0.29; P ¼ 0.012) inpatients with gastric or gastroesophageal cancer withMETHIGH tumors by IHC (32). Interestingly, similar to theOAM4558g NSCLC trial with onartuzumab, patients withMETLOW tumors receiving HGF-targeted therapy seemed to
do worse compared with patients receiving chemotherapyonly (HR, 1.84; P value not reported).However, tumorMETexpression did not predict for benefit on rilotumumab in aphase II study in castration-resistant prostate cancer (33).Hence, the role of MET IHC in predicting benefit to onar-tuzumab and/or other MET-targeting therapies will likelyneed to be carefully evaluated within a given indication.Currently, the test reported here is being developed as acompanion diagnostic for onartuzumab and is being usedto screen and enroll onlyMET-positive patients in the phaseIII study comparing onartuzumab in combination witherlotinib versus placebo plus erlotinib (clinicaltrials.govidentifier: NCT01456325; ref. 22).
Disclosure of Potential Conflicts of InterestH. Koeppen has ownership interest in Roche. L. Fu is an employee of
Roche. T. Januario, M. Lazarov, and L.C. Amler are employees of Genentech.I.I. Wistuba reports receiving a commercial research grant from Genentech/Roche and is a consultant/advisory boardmember forGenentech/Roche andVentana. S.C. Phan is an employee of and has ownership interest (includingpatents) inGenentech.Nopotential conflicts of interestwere disclosedby theother authors.
Authors' ContributionsConception and design:W. Yu, J. Zha, A. Pandita, E. Filvaroff, M. Lazarov,L. Amler, A. Peterson, R.L. YauchDevelopment of methodology: W. Yu, J. Zha, A. Pandita, L. Rangell,S. Mohan, R. Patel, L. Fu, S.G. Louie, E. Filvaroff, J. Huang, R.L. YauchAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): H. Koeppen, J. Zha, A. Pandita, L. Rangell,S. Mohan, R. Patel, L. Fu, A. Do, V. Parab, X. Xia, T. Januario, E. Filvaroff,D.S. Shames, I. Wistuba, A. Peterson, R.L. YauchAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): H. Koeppen, W. Yu, J. Zha, A. Pandita,E. Penuel, S.Mohan, V. Parab, T. Januario, E. Filvaroff,D.S. Shames, J.Huang,M. Lazarov, V. Ramakrishnan, L. Amler, S.-C. Phan, P. Patel, A. Peterson,R.L. YauchWriting, review, and/or revision of the manuscript: H. Koeppen,W. Yu, J. Zha, A. Pandita, L. Fu, V. Parab, T. Januario, I. Wistuba,J. Huang, M. Lazarov, V. Ramakrishnan, L. Amler, S.-C. Phan, P. Patel,A. Peterson, R.L. YauchAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): W. Yu, A. Pandita, L. Rangell,R. Raja, E. Filvaroff, M. Lipkind, J. Huang, V. Ramakrishnan, A. PetersonStudy supervision: J. Zha, A. Pandita, E. Filvaroff, L. Amler, P. Patel,A. Peterson, R.L. YauchDeveloped biomarker assays and methodologies, performed theassays and data analysis: R. RajaMutation analysis and interpretation of data: R. Desai
AcknowledgmentsThe authors thank F. Hoffmann-La Roche Ltd. for third-party writing
assistance for this article. The authors thank the OAM4558g investigators onthis study, including Drs. D.R. Spigel, T.J. Ervin, R. Ramlau, D.B. Daniel, J.H.Goldschmidt Jr, G.R. Blumenschein Jr, M.J. Krzakowski, G. Robinet,B. Godbert, F. Barlesi, R. Govindan, T. Patel, S.V. Orlov, andM.S. Wertheim;as well as the patients enrolled on OAM4558g and their families forsupporting this work.
Grant SupportThis work was supported by Genentech, Inc. (South San Francisco, CA).
Financial support for third-party writing assistance for this article wasprovided by F. Hoffmann-La Roche Ltd. I. Wistuba was supported by theUT Lung Specialized Programs of Research Excellence grant (P50CA70907)and the MD Anderson Cancer Center Support grant CA016672.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received July 3, 2013; revised January 29, 2014; acceptedMarch 23, 2014;published OnlineFirst March 31, 2014.
Biomarkers for Onartuzumab þ Erlotinib Therapy in NSCLC
www.aacrjournals.org Clin Cancer Res; 20(17) September 1, 2014 4497
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2014;20:4488-4498. Published OnlineFirst March 31, 2014.Clin Cancer Res Hartmut Koeppen, Wei Yu, Jiping Zha, et al. BenefitLung Cancer: MET Expression Levels Are Predictive of Patient
Small Cell−Evaluating Erlotinib ± Onartuzumab in Advanced Non Biomarker Analyses from a Placebo-Controlled Phase II Study
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