AnalysisofReceptorTyrosineKinaseROS1-PositiveTumors in …clincancerres.aacrjournals.org/content/clincanres/18/16/4449.full.pdf · Predictive Biomarkers and Personalized Medicine

Predictive Biomarkers and Personalized MedicineSee commentary by Stumpfova and Jänne, p. 4222

Analysis of Receptor Tyrosine Kinase ROS1-Positive Tumorsin Non–Small Cell Lung Cancer: Identification of a FIG-ROS1Fusion

Victoria M. Rimkunas1, Katherine E. Crosby1, Daiqiang Li2, Yerong Hu3, Meghan E. Kelly1, Ting-Lei Gu1,Jennifer S. Mack1, Matthew R. Silver1, Xinmin Zhou3, and Herbert Haack1

AbstractPurpose: To deepen our understanding ofmutant ROS1 expression, localization, and frequency in non–

small cell lung cancer (NSCLC),we developed a highly specific and sensitive immunohistochemistry (IHC)-

based assay that is useful for the detection of wild-type and mutant ROS1.

Experimental Design:We analyzed 556 tumors with the ROS1 D4D6 rabbit monoclonal antibody IHC

assay to assess ROS1 expression levels and localization. A subset of tumors was analyzed by FISH to

determine the percentage of these tumors harboring ROS1 translocations. Using specific and sensitive IHC

assays, we analyzed the expression of anaplastic lymphoma kinase (ALK), EGFR L858R, and EGFR E746-

A750del mutations in a subset of lung tumors, including those expressing ROS1.

Results: InourNSCLCcohort ofChinese patients,we identified9 (1.6%) tumors expressingROS1and22

(4.0%) tumors expressing ALK. FISH identified tumors with ALK or ROS1 rearrangements, and IHC alone

was capable of detecting all cases with ALK and ROS1 rearrangements. ROS1 fusion partners were

determined by reverse transcriptase PCR identifying CD74-ROS1, SLC34A2-ROS1, and FIG-ROS1 fusions.

Some of the ALK and ROS1 rearranged tumors may also harbor coexisting EGFR mutations.

Conclusions:NSCLC tumors with ROS1 rearrangements are uncommon in the Chinese population and

represent a distinct entity of carcinomas. TheROS1 IHCassay describedhere is a valuable tool for identifying

patients expressing mutant ROS1 and could be routinely applied in clinical practice to detect lung cancers

that may be responsive to targeted therapies. Clin Cancer Res; 18(16); 4449–57. �2012 AACR.

IntroductionLung cancer is the leading causeof cancer relateddeaths in

the United States. Non–small cell lung cancer (NSCLC),which accounts for approximately 85% of all lung cancercases, is composed of adenocarcinoma, brochioloalveolar,squamous, and large cell carcinoma subtypes (1). Prognosisis poor for most patients with NSCLC even with the mostcurrent treatment regimens, which include surgery, chemo-therapy, and radiation. The EGF receptor (EGFR) ismutatedin10% to25%of thepatientswithNSCLC,most commonlyassociatedwith young, nonsmokingwomenofAsiandecent(2, 3). Survival time is significantly extended inpatientswith

EGFR mutations when treated with EGFR inhibitors gefiti-nib and erlotinib (2, 4–6).

Patients with EML4-ALK–positive tumors define a subsetof patients whomay respond to the dual anaplastic lympho-ma kinase (ALK) and MET inhibitor, Xalkori (crizotinib).Approximately 5% of patients with NSCLC contain theEML4-ALK fusion renderingALKconstitutively active, therebydriving tumor progression (7–11). Clinical trials with crizo-tinib report an overall response rate of 57% in patients withALK rearrangements (12). Patients with NSCLC lackingEGFR mutations or EML4-ALK are left with few options foreffectivemolecular targeted therapyandadismal 5-year survi-val rateof15%(13).For these reasons, thediscoveryandvalid-ation of new molecular targeted therapies is ever pressing.

ROS1 is a receptor tyrosine kinase (RTK) implicated intumor progression that has not been widely studied. Thefirst oncogenic fusion of ROS1 (FIG-ROS1) was discoveredin glioblastoma (14). An interstitial deletion of 240 kilo-bases on 6q21 results in expression of the FIG-ROS1 fusionprotein leading to constitutive tyrosine kinase activity. Inaddition to glioblastoma, short (S) and long (L) isoforms ofFIG-ROS1 have been identified as potential drivers in cho-langiocarcinoma (15). Expression of both isoforms of FIG-ROS1 induce tumorigenesis in xenograft models, and treat-ment with the small-molecule inhibitor TAE684 inhibits

Authors' Affiliation: 1Cell Signaling Technology, Danvers, Massachu-setts; 2Pathology and 3Cardiothoracic Surgery, Second Xiangya Hospital,Central South University, Changsha, Hunan, P.R. China

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Herbert Haack, Cell Signaling Technology, 3Trask Lane, Danvers, MA 01923. Phone: 978-867-2422; Fax: 978-867-2400; E-mail: [email protected] or Xinmin Zhou, Department ofCardiothoracic Surgery, Second Xiangya Hospital, Central South Univer-sity, Changsha 410011, P.R. China. Phone: 86-731-5295802; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-3351

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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on November 9, 2018. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 1, 2012; DOI: 10.1158/1078-0432.CCR-11-3351

http://clincancerres.aacrjournals.org/

cell growth of Baf3 cells overexpressing FIG-ROS1 (S) and(L)(15).

In addition to glioblastoma and cholangiocarcinoma,ROS1 fusions have also been identified in NSCLCs. Phos-phoproteomic studies surveying the tyrosine phosphoryla-tion space in 41NSCLC cell lines and 150 tumors identified1 cell line (HCC78) and 1 tumor with high ROS1 phos-phopeptides, implicating ROS1 as a possible oncogenicdriver. Two novel ROS1 fusion partners, CD74 and solutecarrier protein (SLC34A2), were identified from the tumorandHCC78 cell line, respectively. Unlike FIG-ROS1, CD74-ROS1 and SLC34A2-ROS1 result from translocation of theROS1 locus. Collectively, studies in glioblastoma, NSCLCs,and cholangiocarcinoma associate ROS1 gene rearrange-ments with the formation of solid tumors.

A recent report describes early evidence of clinicalresponse to crizotinib in ROS1-rearranged NSCLCs (16).With the current need to rapidly identify these patients forpotential therapies, we developed a highly sensitive andspecificROS1antibody to evaluateROS1protein expressioninNSCLCs. This study describes the validation of this ROS1antibody for immunohistochemistry (IHC) and our anal-ysis of ROS1 and ALK gene fusions in NSCLCs.

Materials and MethodsCell culture, antibodies, and Western blot

All cell culture reagents were purchased from Invitrogen.U-118 MG and HEK 293T cells were purchased from theAmerican Type Culture Collection. BaF3, Karpass-299, andHCC78 cells were purchased fromDSMZ (Deutsche Samm-lung vonMikroorganismenundZellkulturen). ALK (D5F3),XP (#3633; Cell Signaling Technology), and ROS1 (D4D6)rabbit monoclonal antibodies (mAb) were developed atCell Signaling Technology. ROS1 D4D6 recognizes aminoacids at the carboxy terminus of human ROS1.Western blotanalyses were conducted following Cell Signaling Technol-ogy protocols. All other antibodies and reagents forWesternblotting were from Cell Signaling Technology. Transfectionof myc/DDK-ROS1 (RC220652, Origene) was conducted

using FuGENE 6 (Roche) for more than 48 hours in HEK293T cells.

Human NSCLC tumor tissuesInstitutional Review Board approval was granted by the

Second Xiangya Hospital of Central South University(Chansha, Hunan, PR China). All NSCLC specimens werederived from tumor resections and subsequently embeddedin paraffin blocks provided by Second Xiangya hospital.Out of the 556NSCLC samples used in this study, 409 wereconstructed into tumor microarrays for IHC screening. Theremaining samples were screened as whole sections. AnyALK- or ROS-positive core from the tumor microarray wassubsequently confirmed using whole sections from theoriginal blocks. Two independent pathologists evaluatedall tumors as ALK and ROS1 IHC–positive tumors to con-firm diagnoses. When needed, IHC staining for p63, TTF-1,mucin, and CK 5/6 was conducted.

ImmunohistochemistryFour- to six-micrometer tissue sections were deparaffi-

nized, rehydrated, and then subjected to antigen retrieval inaDecloakingChamber (BiocareMedical) using 1.0mmol/LEDTA (pH 8.0). Slides were quenched in 3% H2O2 for 10minutes, washed in diH2O, and then blocked with TBS/0.1% Tween-20 (TBST)/5% goat serum. Slides were incu-bated overnight at 4�C with ROS1 (D4D6) rabbit mAb at0.19 mg/mL, or ALK (D5F3) XP rabbit mAb at 1.2 mg/mL,both diluted in SignalStain Antibody Diluent (#8112; CellSignaling Technology). Detection was conducted withEnVisionþ (Dako). Slides stained with EGFR (L858Rmutant–specific; 43B2) rabbit mAb (1.2 mg/mL); EGFR(E746-A750del–specific; 6B6) XP rabbit mAb (8.5 mg/mL);or EGFR (D38B1) XP rabbit mAb (0.28 mg/mL; #3197,#2085, and #4267, respectively; Cell Signaling Technolo-gy), all diluted in #8112, were incubated for 1 hour at roomtemperature, washed, and then incubated with SignalStainBoost IHC Detection Reagent [horseradish peroxidase(HRP), rabbit; Cell Signaling Technology, #8114] for 30minutes. All slides were exposed to NovaRED (Vector Lab-oratories)and coverslipsweremounted. Images (� 20)wereacquired using an Olympus CX41 microscope equippedwith anOlympusDP70 camera andDPController software.

FISHFISH was conducted on 4-mm thick formalin-fixed,

paraffin-embedded (FFPE) tissue sections. ROS1 break-apart probe was developed using bacterial artificial chro-mosomes: RP1-179P9, RP11-323017, RP11-213A17, andRP1-94G16 (Invitrogen). Bacteria artificial chromosomes(BAC) were labeled with spectrum orange and spectrumgreen dUTPS with the Nick Translation DNA Labeling Kit(Enzo Life Sciences). The LSI ALK Dual Color, ALK BreakApart Rearrangement Probe was purchased from AbbottMolecular. FISH-positive cases for both ALK and ROS1were defined as more than 15% split signals in tumorcells. At least 100 nuclei per sample were scored. TheNikon C1 confocal microscope, �60 objective, and trifilter

Translational RelevanceApproximately 2% of the non–small cell lung cancer

(NSCLC) tumors harbor ROS1 gene fusions, and emerg-ing preclinical and clinical data indicate that thesetumors are responsive to inhibitors that target anaplasticlymphoma kinase (ALK). However, the extent of ROS1protein expression in NSCLC and normal tissuesremains unknown. Using a novel ROS1 immunohisto-chemical assay, we determined that ROS1 proteinexpression is restricted to NSCLC tumors harboringROS1 fusions. This assay facilitates the routine identifi-cation of ROS1 rearranged NSCLCs in clinical practiceand detects lung cancers that may be responsive totargeted therapies.

Rimkunas et al.

Clin Cancer Res; 18(16) August 15, 2012 Clinical Cancer Research4450




[40, 6-diamidino-2-phenylindole (DAPI), tetramethyl rho-damine isothiocyanate (TRITC) and, fluorescein isothiocy-anate (FITC)] were used for scoring each case. For imageacquisition, the Olympus BX-51 widefield fluorescencemicroscope with �40 objective and Metamorph softwarewas used to generate multicolor images.

Reverse transcriptase PCR from FFPE tumor samplesRNA from three 10-mm sections was extracted following

standard protocols (RNeasy FFPE Kit, Qiagen). First strandcDNA was synthesized from 500 ng of total RNA with theuse of the SuperScript III First-Strand Synthesis System(Invitrogen) and gene specific primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ROS1 primerswere purchased from Qiagen. All primer sequences can befound in Supplementary Methods.

Transfection and cell proliferation assayTransfections were carried out using FuGENE 6 (Roche

Diagnostics), and retrovirus was harvested 48 hours aftertransfection. BaF3 cells were transduced with retroviralsupernatant containing either the MSCV-Neo/FIG-ROS(L)or MSCV-Neo/FIG-ROS1 (S) vector, and selected for G418(0.8 mg/ml). IL-3–independent growth was assessed by

plating transduced BaF3 cells in IL-3–free medium, afterthe cells were washed 3 times in PBS. For dose–responsecurves, cells were incubated for 72 hours in the presence ofcrizotinib (ChemieTek), and the number of viable cells wasdetermined with the CellTiter 96 AQueous One SolutionCell Proliferation Assay (Promega).

ResultsROS1 expression in cell lines, xenografts,and normal human tissues

To study ROS1 protein expression in NSCLCs, we devel-oped a ROS1 rabbit mAb clone D4D6. We first validatedROS1 D4D6 on cell lines and xenograft models and theninvestigated ROS1 expression in normal human tissues. Toour knowledge, the only cell lines that express ROS1 fusionproteins areHCC78 (SLC34A2-ROS1) andU-118MG(FIG-ROS1). ROS1 D4D6 recognizes ROS1 fusion proteins inboth cell lines by Western blot (Fig. 1A) and IHC (Fig. 1Band C). Specificity by Western blot was further confirmedusing antibodies recognizing ROS1 epitopes that are dis-tinct from ROS1 D4D6. In addition, Western blot signalsdetected in HCC78 and U-118 MG can be blocked whenROS1 D4D6 is incubated together with its immunogen(data not shown). Strong cytoplasmic andpunctate staining

HC

C78

U-1

18M

G

H31

22

ROS1 D4D6

kDa200140100

80605040

β-Actin

MK

N45

H44

1H

2347

H16

50

HC

C82

7

HC

C40

06

H35

8H

1975

H17

03H

3255

A B C

D E

F

H I J K L

M N O P Q

G

Figure 1. ROS1 expression in lung cancer cell lines, xenografts, and normal tissues. A, a panel of lung cell lines was analyzed by Western blot with the ROS1D4D6 antibody. ROS1 fusion proteins in HCC78 (SLC32A2-ROS1, 60–80 kDa) and U-118 MG (FIG-ROS1, 110 kDa) were detected. Lung cancer celllineswere screened to evaluate cross-reactivity with other RTKs. ROS1 or cross-reactive RTKswere not detected in these cell lines. All of these cell lineswerealso screened by IHC. ROS1 was detected in (B) HCC78, (C) U-118 MG, (D) HEK 293TþROS1-DDYK (FLAG) myc and, but absent, in (E) H3122.Xenografts were also examined by IHC to confirm cell pellet staining. F, strong cytoplasmic ROS1 staining was present in the HCC78 xenograft.G, as a negative control, a H3122 xenograft was stained with the ROS1 D4D6 antibody by IHC. Representative images of lung tissue at �20 and �40magnifications are shown in panels H and I, respectively. Other negative tissues include: heart (J), ovary (K), pancreas (L), testis (M), and stomach (N).Tissues staining with ROS1 rabbit mAb include: colon (O), kidney (P), and cerebellum (Q).

Analysis of RTK ROS1 and ALK in NSCLC Identifies FIG-ROS1 Fusion

www.aacrjournals.org Clin Cancer Res; 18(16) August 15, 2012 4451




was observed in the HCC78 cell line (Fig. 1B), whereascytoplasmic staining in U-118 MG cell line was relativelyweak (Fig. 1C), requiring the use of SignalStain Boost IHCDetection Reagent to enhance the signal.Wewere unable toidentify cell lines expressing full lengthROS1; however, full-length ROS1 is detectable by IHC when exogenouslyexpressed (Fig. 1D). To address RTK cross reactivity, wescreened a panel of lung cancer cell lines known to expressvarious RTKs including MET, Axl, EGFR, FGFR, PDGFR,ALK,DDR1, and IGF-1R.No cross-reactivitywas detected byWestern blot (Fig. 1A) or IHC (data not shown). We alsoexamined ROS1 expression by IHC in xenograft modelsincluding HCC78, H1299, H1975, HCC827, H1650,H2228, and H441. ROS1 staining was only observed inHCC827 andHCC78 xenografts. ROS1 protein in HCC827is undetectable by Western blotting (Fig. 1A), whereas theROS1 transcript is present (data not shown), suggesting thatlow levels of ROS1 protein can be detected using a sensitiveIHC assay. ROS1 is highly expressed in both the membraneand cytoplasmic compartments in HCC78 xenografts (Fig.1F). ROS1was not detected in theH3122 (EML4-ALKþ) cellpellet or xenograft (Fig. 1E and G).

Knowledge about wild-type ROS1 protein expressionand regulation in normal human tissues is limited. How-ever, expression of ROS1 transcripts in kidney, testis,lung, and intestines has been described (17–20). Toexamine the extent of wild-type ROS1 expression innormal tissues, we screened a normal human tissue arrayby IHC. ROS1 was not detected in normal lung (Fig. 1Hand I). ROS1 was absent in 22 tissues including heart,ovary, pancreas, and testis (Fig. 1J–M). ROS1 wasexpressed at low levels in parathyroid, eye, larynx, adrenalgland, and skeletal muscle tissues. Occasional strongstaining in cerebellum, peripheroneural tissue, stomach,

small intestine, and colon was observed (Fig. 1N, O, andQ). The highest levels of staining were observed in kidney(Fig. 1P).

ROS1 D4D6 rabbit mAb detects ROS1 fusion proteinsin formalin-fixed tumor samples

To address the frequency of ROS1 expression in NSCLCtumors and examine ROS1 cellular localization, wescreened 556 NSCLC tumors by IHC with ROS1 D4D6rabbit mAb. The NSCLC cohort is composed of 246 ade-nocarcinomas, 64 bronchioaveolar carcinomas, 226 squa-mous, and 20 large cell carcinomas. ROS1 was expressed in1.6% (9 of 556) of NSCLC tumors. Eighty-nine percent ofROS1-expressing tumors were adenocarinoma, similar towhat was observed in tumors expressing ALK and mutantEGFR proteins.One tumor expressing ROS1was of the largecell carcinoma subtype. Overall ROS1 was expressed in3.3% (8 of 246) of all adenocarcinoma tumors (Table 1).

To determine whether the ROS1 protein detected byROS1 D4D6 is due to a fusion or wild-type expression, weconducted FISH on all 9 ROS1-expressing tumors and asubset of negative tumors (n ¼ 138). The break-apart FISHassay previously described (21) detects all known ROS1fusions including FIG-ROS1, CD74-ROS1, and SLC34A2-ROS1. Negative tumor samples were completely devoid ofROS1protein expressionby IHC(Fig. 2AandB) andnormalby FISH. In 7 of 9 ROS1-expressing tumors, the FISH probeswere broken, indicative of translocation of the ROS1 locus(Fig. 2C). The probes 50 to the common breakpoint regionin ROS1 were not detected in one tumor, suggesting dele-tion of this chromosomal region equivalent to the FIG-ROS1 fusion pattern in U-118 MG (Supplementary Fig.S1F). We were unable to analyze one tumor due to highbackground and low signal intensity.

Table 1. Histopathology and genotypes of ROS1 IHC–positive samples

Tumor ID DiagnosisHistologicpattern (%) IHC Score ROS1 FISH ROS1 fusion

EGFR mutationstatus

147 Adenocarcinoma BAC (40), papillary(30), Acinar (20),Solid (10)

3þ cytoplasmic þ SLC34A2-ROS1 L858R

306 Adenocarcinoma Acinar (70), papillary(20), and solid (10)

3þ cytoplasmic þ CD74-ROS1 �

570 Adenocarcinoma Acinar (90), BAC (5),micropapillary (5)

3þ cytoplasmic,punctae

þ CD74-ROS1 �

760 Adenocarcinoma Signet cells 3þ cytoplasmic,membrane

þ Insufficientmaterial

Insufficientmaterial

400037 Adenocarcinoma Acinar 2þ cytoplasmic,punctae

þ CD74-ROS1 �

575 Large Cell 2þ cytoplasmic Not scoreable Unknown �668 Adenocarcinoma Solid (80), Acinar

(10), BAC (10)1þ cytoplasmic þ CD74-ROS1 �

702 Adenocarcinoma Papillary (40), Acinar(30), Solid (30)

1þ cytoplasmic þ SLC34A2-ROS1 E746-A750del

749 Adenocarcinoma Solid (80), Acinar (20) 1þ vesicular þ FIG-ROS1 (S) �

Rimkunas et al.





Although few differences were observed by FISH, a widevariety of ROS1 staining patterns were observed by IHC.ROS1 localized diffusely to the cytoplasm in 5 of 9 (55%)cases (Fig. 2D); however, strong perinuclear aggregates wereobserved in 2 of 9 tumors (Fig. 2E). In the large cellcarcinoma sample, ROS1 was localized to the cytoplasm(Fig. 2G and H). Both membrane (Fig. 2F) and vesicularlocalizationof ROS1 (Fig. 2J)were alsoobserved.Generally,normal adjacent tissue did not stain with ROS1 D4D6 (Fig.2K). In rare cases, nonneoplastic cells such as macrophagesand bronchial epithelial cells (Fig. 2L) stained with ROS1D4D6.

This cohort was also screened for ALK expression usingthe rabbit mAb ALK D5F3 IHC assay (11). In these tumors,ALK was expressed throughout the cytoplasmic compart-ment with varying intensities amongst tumors. Concurrentexpression of ALK and ROS1was never observed.We found22 of 556 (4.0%) tumors expressing ALK protein with 21confirming FISH positivity (Table 2). In all 21 cases, theFISH pattern was consistent with an inversion phenotype.Because of inadequate tissue availability, we were only ableto interrogate theALK fusionpartners for a small subset (n¼7) of samples. Themost common fusions inNSCLC, EML4-ALK variant 1 and variant 3 were assessed by FFPE reversetranscriptase PCR (RT-PCR). EML4-ALK variant 3was detec-ted in 5 of 7 samples, and neither EML4-ALK variant 1 nor 3were detected in the other 2 samples (data not shown).

Identification of ROS1 fusion partners by FFPERT-PCRBoth IHC and FISH are limited in their ability to distin-

guish one ROS1 fusion partner from another. To identifyROS1 fusion partners, we extracted total RNA from FFPEsections and conducted RT-PCR using primers specific toknown ROS1 fusions: CD74-ROS1, SCL34A2-ROS1, andFIG-ROS1. As a control, we amplified the 30 portionofROS1to detect any fusedorwild-typeROS1 transcript. All sampleswith ROS1 translocations were analyzed for SCL34A2-ROS1 (S) and (L) isoforms and CD74-ROS1. We identifiedfusion partners for 7 of 9 ROS1-expressing tumors (Table 1and Supplementary Table S1). CD74-ROS1 and SLC34A2-ROS1 fusions were detected with CD74-ROS1 being themost prevalent. All amplicons were sequence verified (datanot shown).Wewereunable to identify fusionpartners for 2tumors due to lack of material and low RNA quality.

One sample, #749, was tested for FIG-ROS1 fusionsbecause of the observed vesicular localization of ROS1(Fig. 2J) and deletion of green FISH probes (SupplementaryFig. S1F), which suggest the presence of a FIG-ROS1 fusion.RT-PCR followed by sequencing confirmed the presence ofFIG-ROS1 (S) (Fig. 3A). In addition, we devised an alter-native FISH assay to visualize the FIG-ROS1 fusion inwholesections from paraffin blocks (Supplementary Fig. S1). Toour knowledge, FIG-ROS1 fusions have not been previouslydescribed in NSCLCs.

Previously, FIG-ROS1 (S) was identified as a target of theALK inhibitor, TAE684 (15), suggesting that the recentlyFDA approved drug Xalkori (crizotinib) could also beeffective in inhibiting ROS1 kinase activity. To determine

A B

C D

E F

G H

I J

K L

Figure 2. Detection of ROS1 fusions in NSCLC tumors by IHC andFISH. ROS1-negative images of squamous cell carcinoma (A) andbronchioloavleolar carcinoma (B) tumors are seen. We used a break-apart FISH assay to detect ROS1 fusions. A representative FISH imagefrom one ROS1-positive adenocarcinoma is seen in C. Fused green andorange probe is indicative of the intactROS1 loci. The yellow arrowheadspoint to broken orange and green probes indicative of a ROS1translocation event. This broken orange and green phenotype waspresent in most ROS1-expressing tumors. ROS1 localized to differentcell compartments at varying intensities. In most adenocarcinomatumors, ROS1 was expressed diffusely in the cytoplasm. Examples ofROS1-positive samples: (D) tumor 306, 3þ; (E) tumor 570, 3þ; (F) tumor760, 3þ; (G) tumor 575, 2þ; (H) hematoxylin and eosin staining of largecell carcinoma sample 575; (I) tumor 668, 1þ; and (J) tumor 749, 1þ.K, lung 570 shows ROS1 positivity in tumor and no staining in adjacentnormal tissue. L, ROS1-negative tumor with staining in reactive bronchialepithelial cells.






whether the FIG-ROS1 (S) fusion could be a potentialtherapeutic target in NSCLC, we treated BaF3 cells over-expressing FIG-ROS1 (S) with crizotinib and evaluatedROS1 phosphorylation and cell growth. BaF3 cells expres-sing FIG-ROS1 (S) were treated with crizotinib ranging inconcentrations from 0 to 1 mmol/L. Cell growth was inhib-ited in Karpas-299 and Baf3-FIG-ROS1 upon crizotinibtreatment (Fig. 3B). ROS1 and ALK phosphorylation wasinhibited after 3 hours of crizotinib treatment at a concen-tration of 1 mmol/L for ROS1 and 0.3 mmol/L for ALK (Fig.3C). On the contrary, BaF3 cells expressing FLT3-ITD orempty vector (Neo-Myc) were not sensitive to crizotinib.The phosphorylation of critical signaling nodes p-STAT3and p-ERK in Karpas-299 and FIG-ROS1 (S)–expressingBaF3 cells were also inhibited by crizotinib (Fig. 3C). No

significant changes in the phosphorylation of FLT3, STAT3,or ERK were observed in the FLT3-Baf3 control (Fig. 3C).These data suggest that FIG-ROS1 (S) can be inhibited bycrizotinib.

Analysis of EGFR mutations in ROS1 and ALK-positiveNSCLC tumors

EGFR mutations and ALK fusions are thought to bemutually exclusive, although rare cases of coexpression inNSCLCs have been reported (22–24). Using IHC withmutation-specific EGFR antibodies (EGFR L858R and EGFRE746-A750del; ref. 25), we examined the mutational statusofROS1andALK-positive tumors inour cohortwhere tissuewas available (Tables 1 and 2). As expected, all ROS1- andALK-positive tumors expressed total EGFR. Unexpectedly,

Table 2. Histopathology and genotypes of ALK-positive cases

Tumor ID Diagnosis Histologic pattern (%) ALK FISHEGFR mutationstatus

187 Adenocarcinoma Solid focal signet cell ringfeatures

þ, split �

307 Adenocarcinoma BAC (30), Acinar (10),papillary (10), solid (50),clear cell and mucinousfeatures

þ, split �

587 Adenocarcinoma Acinar (85), solid (10),papillary (5)

Not scoreable L858R

618 Adenocarcinoma Solid þ, split Insufficient material645 Adenocarcinoma Solid (70), BAC (30) þ, split �652 Adenocarcinoma Papillary (60),

Micropapillary (40)þ, split �

663 Adenocarcinoma Papillary (50) BAC (50) þ, split �664 Adenocarcinoma Acinar þ, split L858R666 Adenocarcinoma Solid (90), Papillary (10) þ, split �670 Adenocarcinoma Solid (60), Papillary (40) þ, split �680 Adenocarcinoma Solid (70) and acinar (30)

with signet ring cellfeatures

þ, split �

759 Adenocarcinoma Solid with signet ring cells þ, split �580 Adenocarcinoma

(uncertain)þ, split �

70 Adenocarcinoma Solid þ, split Insufficient material383 Adenocarcinoma BAC (40), papillary (30),

Acinar (30)þ, split �

395 Adenocarcinoma Solid þ, split �278 Squamous; large

cell carcinoma(uncertain)

þ, split �

330 Large cellneuroendocrinecarcinoma

þ, split �

503 Squamous þ, split �615 Squamous þ, split Insufficient material644 Squamous þ, split �691 Squamous þ, split �

Rimkunas et al.





we identified 2 EGFR L858R/ALK–positive, 1 EGFR L858R/ROS1–positive, and 1 EGFR E746-A750del/ROS1–positivetumors (Fig. 4 and Supplementary Fig. S2). Immunohisto-chemical analysis of mutant EGFR and ROS1 on serial sec-tions revealed dual expression in identical areas of the tumor,suggesting coexpression in the same tumor cells (Fig. 4).The 2 ROS1-positive tumors express SLC34A2-ROS1

fusions, although their EGFR mutations are different.Sequencing confirmed the presence of EGFR mutations inthese 2 SLC34A2-ROS1–positive tumors (data not shown).We were unable to sequence the ALK-positive tumors forEGFR mutations.

DiscussionThe ability to administer molecular-targeted therapies to

the appropriate subset of patientswithNSCLC is essential tofulfill the promise of personalized cancer therapy. With anestimated 1.35 million newly diagnosed cases of lungcancer worldwide, the identification of novel oncogenicdrivers is imperative (1). ROS1 rearrangements are knownoncogenic drivers and define a unique clinically importantsubset of NSCLC (16, 21). To expand our understanding ofROS1 fusions in NSCLC, we developed ROS1 D4D6 rabbitmAb for IHC. With this antibody, we developed the ROS1D4D6 IHC assay and screened 556 primary NSCLC tumorsto evaluate the frequency of ROS1-expressing NSCLCtumors. We identified a small percentage of tumors expres-

sing ROS1 (1.6%); however, the presence of ROS1 maystratify patients with NSCLC into an important subset fortherapeutic response. The ROS1 IHC assay could possiblybe developed into a diagnostic tool to identify ROS1-positive patients who may respond to the kinase inhibitor,crizotinib (16).

Before our study, CD74-ROS1 and SLC34A2-ROS werethe only reported fusions of ROS1 known in primaryNSCLC tumors (16, 21, 26). In this study, we observedCD74-ROS1 as the most common ROS1 fusion, and for thefirst time identified FIG-ROS1 (S) in a primary NSCLCtumor. The FIG-ROS1 fusion previously described in glio-blastoma and cholangiocarcinoma had never beenobserved in NSCLC cell lines or tumors. All known ROS1fusions can be detected with the ROS1 D4D6 IHC assaymaking it a valuable tool for the identification of the lowfrequency of patients withNSCLC expressing ROS1 fusions.No correlation was observed between ROS1 localization orstaining intensity and the identification of ROS1 fusionpartner. The importance of ROS1 levels and localization aspredictive or prognostic biomarkers can only be evaluatedwith follow-up clinical studies in a larger set of ROS1patients.

Like other RTKs, oncogenic ROS1 is amenable to targetedtherapy. Selective and potent ROS1 kinase inhibitors haverecently been developed (27). These compounds have yet tobe evaluated in the ROS1 fusion–positive cell lines HCC78

A

B C

FIG-ROS (S)NSCLC

Cell growth inhibition by crizotinib140

120

100

80

60

40

20

00

μmol/L0.01

μmol/L0.03

μmol/L

BaF3-Neo-Myc

p-ROS

Crizotinib(μmol/L) 3 h

BaF

3-FI

G/R

OS

(S)

BaF

3-FL

T3/IT

D

Kar

pas-

299

0 0.1

0.3

1.0

0 0.1

0.3

1.0

0 0.1

0.3

1.0

ROS

p-STAT3

STAT3

p-ERK

p-ALK

ALK FLT3

p-FLT3

ERK

BaF3-FLT3-ITDBaF3-FIG_ROS(S)Karpas 299

0.1μmol/L

0.3μmol/L

1.0μmol/L

Crizotinib

Per

cen

tag

e o

f co

ntr

ol

Tumor ID 749GAPDH control

FIG ROS

T77

1T

749

NC

U-1

18M

G

Figure3. Cell proliferation inhibition andsignalingpropertiesof theFIG-ROS1 (S) fusion. A,RT-PCR resultswith primers specific to the (S) short isoformsofFIG-ROS1 for U-118 MG cell line (FIG-ROS1–positive), no template control (NC), tumor samples 749 (ROS1-positive), and 771 (ROS1-negative).GAPDH primerswereusedasa control. Sequencechromatograph confirms theFIG-ROS1 short breakpoint in tumor ID749.B, dose–response curveof crizotinib forBaF3cellsexpressing FIG-ROS1 (S). BaF3/FLT3-ITDandKarpas-299 (NPM-ALK) cells served asnegative andpositive controls, respectively. Results displayedare fromduplicate experiments. C, incubation of cells with different concentrations of crizotinib results in a decreased phosphorylation of the ROS1 kinase,accompanied by decreased phosphorylation of STAT3, ROS1, ALK, and ERK. Karpas-299 and BaF3-FLT3/ITD were used as positive and negative controls,respectively.






and U-118 MG. Our studies indicate that ROS1 expressionin NSCLC is mutually exclusive from ALK and represents asubset of patients for whom ROS1 may be the oncogenicdriver in tumorigenesis. Previous studies have identified thesmall-molecule ALK inhibitors, TAE684 and crizotinib, asROS1 kinase inhibitors where they suppressed proliferationof cell lines expressing ROS1 fusion protein (15, 16, 21). Inthis study, we have shown that the newly U.S. Food andDrug Administration (FDA)-approved drug crizotinib inhi-bits ROS1 kinase phosphorylation, which leads to an inhi-bition of cell proliferation in FIG-ROS1 (S)–expressingBaF3 cells. Crizotinib has also been shown to inhibit thecell line HCC78 expressing SLC34A2-ROS1 (16). Com-bined, these findings suggest that ROS1 is a promisingtarget for therapy in multiple cancer types includingNSCLC, cholangiocarcinoma, and glioblastoma whereROS1 fusion proteins have been identified.

We also identified 21 NSCLC cases expressing ALKfusions. The majority of cases were of the adenocarcinomasubtype, which is consistent with previous reports. ALK

expression in the large cell carcinoma and squamous cellcarcinoma subtypes has been less studied as most previousstudies are restricted to the adenocarcinoma subtype(8, 10, 28–30). We identified one large cell carcinoma and5 squamous cell carcinomas expressing ALK fusion protein.The significance of this finding is unclear and larger studiesare needed to confirm this finding. Like patients withadenocarcinoma, patients with large cell or squamous cellexpressing ALK fusions may benefit from ALK inhibitorssuch as crizotinib.

In our study, we identified a high percentage of ALK- orROS1-positive tumors also expressing mutant EGFR. Theethnicity of our cohort is exclusively Chinese, where EGFRmutations are more common than in non-Asian popula-tions. Mutations in EGFR were determined usingmutation-specific IHC antibodies, which together cover approximate-ly 80%of the EGFRmutations seen in patients withNSCLC.It is possible that there are additional cases of doublemutant NSCLC specimens, but they are likely to be few.Patients with both ALK and EGFR mutations may benefitfrom combination therapy targeting bothALK and EGFR. Ina recent report, Sasaki and colleagues identified 3 of 50treatment naive patients with NSCLC with ALK rearrange-ments and EGFR-activating mutations (22). Two of thesepatients underwent treatment with erlotinib and both haveachieved partial responses. Interestingly, these patientswerefound to expressmutant EGFR protein but not ALK protein.This observation is contrary to what we observed. In all 4cases, whereweobserved EGFRmutations andALKorROS1translocations, the proteins were expressed. The reason forthis discrepancy is unclear, but we suspect that the use ofdifferent ALK IHC assays on different NSCLC cohorts is aplausible explanation. Collectively, these data warrant fur-ther examination of the therapeutic options for ALK rear-ranged and EGFR mutant patients. We argue that ROS1fusion–positive patients expressing mutant EGFR shouldalso be evaluated for responsiveness to EGFR inhibitors.

In conclusion, we have identified ROS1-expressingtumors in 1.6% of NSCLCs by first screening with a ROS1IHC assay to capture any ROS1-expressing tumors. Subse-quent molecular analysis by FISH and RT-PCR identifiedROS1 rearrangements in these IHC-positive tumors. Theutility of a ROS1 IHC assay is underscored by the recentclinical response data of ROS1-rearranged patients to cri-zotinib (16). Patients withNSCLC expressing ROS1 fusionsare distinct from those expressing ALK fusions, furtherexpanding the clinical utility of crizotinib in NSCLC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank Cynthia Reeves for assistance with DNA sequencing.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 9, 2012; revised May 15, 2012; accepted May 21, 2012;published OnlineFirst June 1, 2012.

BA

DC

FE

HG

Figure 4. Mutant EGFR immunohistochemical staining of ROS1-expressing tumors. Images of ROS1/L858R–positive lungadenocarcinoma case 147 stainedwith (A) ROS1D4D6, (B) EGFRL858R,(C) EGFR A746-E750del, and (D) total EGFR antibodies. Images of ROS/EGFR A746-E750del–positive lung adenocarcinoma case 702 stainedwith (E) ROS1 D4D6, (F) EGFR A746-E750, (G) EGFR L858R, and (H) totalEGFR antibodies.

Rimkunas et al.





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2012;18:4449-4457. Published OnlineFirst June 1, 2012.Clin Cancer Res Victoria M. Rimkunas, Katherine E. Crosby, Daiqiang Li, et al.

Small Cell Lung Cancer: Identification of a FIG-ROS1 Fusion−Non Analysis of Receptor Tyrosine Kinase ROS1-Positive Tumors in

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