In the era of personalized cancer therapy, targeted therapy

Original Article Clinical and the Prognostic Characteristics of Lung Adenocarcinoma Patients with ROS1 Fusion in Comparison with Other Driver Mutations in East Asian Populations Yen-Fu Chen, MD,* Min-Shu Hsieh, MD, Shang-Gin Wu, MD,* Yih-Leong Chang, MD, Jin-Yuan Shih, MD, PhD, Yi-Nan Liu, PhD, Meng-Feng Tsai, PhD, Tzu-Hsiu Tsai, MD, Chong-Jen Yu, MD, PhD, James Chih-Hsin Yang, MD, PhD, and Pan-Chyr Yang, MD, PhD Introduction: The prevalence, demographic features, and clinical outcomes of lung adenocarcinoma patients with novel ROS1 oncogenic rearrangement in East Asian populations are not clear. This study aimed to investigate the clinical and prognostic characteristics of lung adenocarcinoma in patients with ROS1 fusion compared with other driver mutations. Methods: Multiplex reverse transcription-polymerase chain reaction was used to detect the ROS1 fusion gene in lung adenocarcinoma cases. Immunohistochemistry was used to confirm the expression of ROS1. The demographic data and clinical outcomes of patients with the ROS1 fusion gene were compared with those of patients without the ROS1 fusion gene, including those with the EGFR mutation, EML4-ALK fusion, KRAS mutation, and quadruple-negative patients. Results: Of 492 patients with lung adenocarcinoma, 12 (2.4%) had the ROS1 fusion gene. Their median age was 45.0 years, significantly younger than that of the ROS1 fusion-negative cohorts (p < 0.001). Acinar (including cribriform) and solid patterns were the two most common histologic subtypes in the ROS1 fusion tumors (7 of 12, 58.3%) and were predominantly seen in CD74-ROS1 fusion tumors (66.7%). There was no significant survival difference between the ROS1 fusion-positive and ROS1 fusion-negative cohorts in surgical group, but ROS1 fusion-positive patients might have worse outcomes than EGFR-mutant patients in the stage IV group. Conclusions: The ROS1 fusion gene can be successfully detected in East Asian patients with lung adenocarcinoma using multiplex *Department of Internal Medicine, National Taiwan University Hospital, Yun-Lin Branch, Yun-Lin; Department of Pathology; Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei; Department of Molecular Biotechnology, Da-Yeh University, Chang-Hua; Department of Oncology, National Taiwan University Hospital; and Graduate Institute of Oncology Cancer Research Centre, National Taiwan University, Taipei, Taiwan. *Hsieh MS and Wu SG contributed equally to this work. Disclosure: Dr. James Chih-Hsin Yang received honoraria from AstraZeneca and Boehringer Ingelheim for speeches and acts a consultant for Novartis, Pfizer, Clovis, Astrazeneca, Roche, Bayer, Merck, Optima and Innopharma. The other authors declare no conflict of interest. Address for correspondence: Jin-Yuan Shih, MD, PhD, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung- Shan South Road, Taipei 100, Taiwan. E-mail: jyshih@ntu.edu.tw Copyright 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/14/0908-1171 reverse transcription-polymerase chain reaction. These patients tend to be younger and have characteristic histologic subtypes. Due to the small number of ROS1 fusion patients, the prognostic value of ROS1 fusion need further studies to confirm. Key Words: EGFR mutation, EML4-ALK fusion, KRAS mutation, lung adenocarcinoma, ROS1 fusion, reverse transcription-polymerase chain reaction (J Thorac Oncol. 2014;9: 1171 1179) In the era of personalized cancer therapy, targeted therapy plays a critical role in non small-cell lung cancer (NSCLC) with identified oncogenic drivers. 1,2 Small molecular tyrosine kinase inhibitors (TKIs), such as epidermal growth factor receptor (EGFR) TKIs (gefitinib, erlotinib, and afatinib) and crizotinib, are known to be effective treatment and result in marked improvement of progression-free survival, response rate, and quality of life compared with chemotherapy in patients with lung adenocarcinoma harbouring the EGFR mutation 3 5 and the anaplastic lymphoma kinase (ALK) translocation patients, 6 respectively. Rapid and effective identification of driver genes in NSCLC is therefore an important issue in current practice. Among East Asians, NSCLC patients with EGFR mutation account for 30 50% of cases. They are usually individuals who never smoked, female, and have lung adenocarcinomas. 3,7 9 The targetable fusion oncokinase, the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene, is also found in NSCLC patients, with a 3 5% prevalence. It is most often detected in light or never smokers and in those with lung adenocarcinoma. 5,10 14 Thus, the clinical characteristics of lung cancer may provide more information that can effectively identify the most likely population who may benefit from specific targeted therapy. The novel ROS1 fusion gene has recently been described as a potential driver mutation in NSCLC 15 and crizotinib is active in ROS1 fusion-positive patients. 6,16,17 To date, a number of ROS1 fusion partners have been discovered, including CD74, SLC34A2, TPM3, SDC4, EZR, LRIG3, FIG, KDELR2, and CCDC6, with an overall prevalence of 0.7 2.6% in NSCLC 15,16,18 23 and 3.3% in lung adenocarcinoma. 21 Journal of Thoracic Oncology Volume 9, Number 8, August 2014 1171

Chen et al. Journal of Thoracic Oncology Volume 9, Number 8, August 2014 The clinical characteristics of patients with ROS1 fusion have been described in previous studies. They tend to be younger and never smokers, with various subtypes of histologic features of lung adenocarcinoma. 16,19 However, one Chinese study did not show specific clinicopathologic features of the ROS1 fusion patients. 22 The prognosis of ROS1 fusion-positive patients varied among reports. 16,19,22 These earlier case series only reported small numbers of patients, which may be due to the very low frequency of ROS1 fusions in NSCLC. As such, the definite clinical characteristics and prognosis of ROS1 fusion patients remain uncertain. Moreover, driver mutations may have different distributions and prevalences in different ethnicities. 16,24 27 To better understand this novel driver mutation that almost always occurs in lung adenocarcinoma, 16,23 the present study aimed to identify the ROS1 fusion gene in East Asian patients with lung adenocarcinoma. Tissue specimens of lung adenocarcinoma were collected for advanced molecular analysis, including the ROS1 fusion gene, EGFR mutation, EML4-ALK fusion gene, and KRAS mutation. The clinical characteristics were further investigated and the outcomes analyzed. METHODS Patient Selection and Tissue Procurement Consecutive patients receiving surgical resection for lung adenocarcinoma or pleurocentesis were identified for malignant pleural effusion (MPE) of lung adenocarcinoma at the National Taiwan University Hospital. One hundred and sixty surgical excision specimens of stage I IIIA primary lung adenocarcinoma and 332 MPE from stage IV lung adenocarcinoma were prospectively collected. Written informed consent was obtained from patients before specimen collection. Tissue sections were examined for adequacy by microscopy with hematoxylin and eosin staining. Adenocarcinoma histology was confirmed through pathology reports of the primary tumors or by cell blocks of MPEs with positive thyroid transcription factor-1 stains. 1,28 The clinical characteristics of the enrolled patients, including age, sex, smoking status, lung cancer histologic type, 1 cancer stage, 29 Eastern Cooperative Oncology Group performance status (ECOG), 30 initial metastatic sites, and systemic treatment were recorded for analysis. Patients who did not smoke or smoked less than 100 cigarettes in their lifetime were categorized as never smokers. 31 The rest were categorized as smokers. Overall survival (OS) was defined as the period from the date of first-line systemic treatment to the date of death. Relapse-free survival (RFS) was defined as the time from tumor resection to the earliest among the following outcomes: disease recurrence (local or metastatic), last follow-up without evidence of disease, or death without evidence of disease in surgical patients. The hospital s Institutional Review Board approved the study. Sequencing of EGFR Exons 18 21 The process of EGFR gene mutation analysis was as described previously. 32,33 Briefly, RNA was extracted from frozen tumor specimens or pleural effusion cell pellets using the RNeasy Mini Kit (QIAGEN). Exons 18 21 of EGFR were amplified with a forward (5 -GGATCGGCCTCTTCATGC3 ) and reverse primer (5 -TAAAATTGATTCCAATGCCATCC-3 ). Amplicons were purified and sequenced using the BigDye Terminator Sequencing Kit (Applied Biosystems, Foster City, CA). The sequencing products underwent electrophoresis on an automatic ABI PRISM 3700 genetic analyzer (Applied Biosystems). Both the forward and reverse sequences obtained were analyzed. Detection of EML4-ALK Fusion Gene For the screening of the EML4-ALK fusion gene, reverse transcription-polymerase chain reaction (RT-PCR) of the extracted RNA was performed for patients without identified EGFR mutations, using a Qiagen One-Step RT-PCR Kit (Qiagen). The primer set used for amplification was as the following: 5 -GTCAGCTCTTGAGTCACGAGTT-3 (forward primer, located on exon 2 of EML4), 5 -GTGCAGTGTTTAGCATTCTTGGGG-3 (forward primer, located on exon 13 of EML4), and 5 -TCTTGCCAGCAAAGCAGTAGTTGG-3 (reverse primer, located on exon 20 of ALK). 33 The PCR product was then electrophoresed to screen for the EML4-ALK fusion gene, with different variants subsequently confirmed by sequencing. Detection of KRAS Mutation Because the EGFR mutation, ALK alterations, and KRAS mutation are mutually exclusive in lung adenocarcinoma, 34 KRAS analysis was performed for patients without the EGFR mutation and EML4-ALK fusion gene. Briefly, exons 2 3 of the KRAS gene was amplified by RT-PCR using the following primers: forward, 5-GGCCTGCTGAAAATGACTGA-3 and 5-TCTTGCTAAGTCCTGAGCCTGTT-3. The KRAS reference sequence was based on NM_004985 from the National Centre for Biotechnology Information database. Identification of ROS1 Fusion Genes All tumor specimens without the EGFR mutation, EML4- ALK fusion, and KRAS mutation were investigated by multiplex RT-PCR for the known ROS1 fusion genes (CD74, SLC34A2, LRIG3, and SDC4). These four fusion genes could be detected in two separate multiplex RT-PCR. Total RNA was used as template for the RT-PCR amplification using a QIAGEN OneStep RT-PCR kit (Qiagen, Hilden, Germany). To detect the CD74- ROS1 and SLC34A2-ROS1 fusions, the forward primers were CD74 E5F (5 -CCTGAGACACCTTAAGAACACCA-3 ) and SLC34A2 E4F (5 -TCGGATTTCTCTACTTTTTCGTG-3 ). The reverse primer was ROS1 E34R (5 -TGAAACTTGTTTCTGGTATCCAA-3 ). To detect the SDC4-ROS1 and LRIG3-ROS1 fusions, the forward primers were SDC4-F (5 - CGAGTCGATCCGAGAGACTG-3 ) and LRIG3-F (5 -ACACAGATGAGACCAACTTGCC-3 ), whereas the reverse primer was ROS1-E36R (5 - AAGAGTATGTATTGCATAGCAGGCATTA-3 ). 1172 Copyright 2014 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 9, Number 8, August 2014 Lung Adenocarcinoma Patients with ROS1 Fusion Briefly, 50 100 ng total RNA was used as template. The following components were then added: 10 μl 5 reaction buffer, 2 μl dntp mix (10 mm each), 3 μl of 10 mm forward and reverse primer each, 2 μl QIAGEN OneStep RT-PCR enzyme mix, and RNase-free water to reach a total volume of 50 μl. The RT-PCR reaction was initiated at 50 C for 30 minutes, heated to 95 C for 15 minutes, and followed by 40 cycles of denaturation at 94 C for 50 seconds, annealing at 60 C for 50 seconds, extension at 72 C for 1 minute, and a final extension at 72 C for 10 minutes. The RT-PCR amplicons were purified and sequenced to confirm the translocation. Immunohistochemical Analysis for ROS1 Fusion Detection All tumor specimens without the EGFR mutation, EML4-ALK fusion, and KRAS mutation were also further analyzed by immunohistochemical (IHC) staining for ROS1 fusion. Formalin-fixed, paraffin-embedded tissue sections (5 μm) were dewaxed and rehydrated. After antigen retrieval (1.0 mm EDTA ph 9.0 at 121 C for 10 minutes) and blocking with 3% hydrogen peroxide, slides were allowed to react with a rabbit monoclonal antibody against ROS1 (clone D4D6, 1:250 dilution; Cell Signaling Technology, Beverly, MA) at room temperature for 1.5 hours. Slides were incubated with IHC stain detection kit (UltraVision Quanto Detection System, Thermo Scientific, Fremont, CA) and counterstained with hematoxylin. The background lung parenchyma was used as negative control. The staining scores were assessed as follows: 0, no staining; 1+, weak cytoplasmic staining; 2+, moderate cytoplasmic staining; and 3+, strong cytoplasmic staining. Statistical Analysis All categorical variables were analyzed with Pearson s χ 2 tests, except where a small size (<5) required the use of Fisher s exact test. One-way analysis of variance was used to analyze differences in the number of patient characteristics among the five different molecular cohorts. The OS and RFS were plotted using the Kaplan-Meier method and compared with log-rank test. Multivariate analysis for OS was performed using the Cox s proportional hazards model. Statistical significance was set at a two-sided p less than 0.05. All analyses were performed using the SPSS version 18.0 (SPSS Inc., Chicago, IL). RESULTS Clinical Characteristics A total of 492 lung adenocarcinoma patients were identified for analysis, including 160 patients in the surgical group (stage I IIIA) and 332 in the nonsurgical group (stage IV). Among them, 237 (48.2%) were male and approximately 30% were smokers. The median age of the patients was 65.3 years (range, 27 95 years). Based on the molecular analysis, the patients were further divided into five cohorts, which included the four ROS1 fusion-negative cohorts (EGFR mutation TABLE 1. Comparison of Clinical Characteristics of Lung Adenocarcinoma Patients with Driver Mutations Type of Mutation Total ROS1 Fusion EGFR Mutation EML4/ALK Fusion KRAS Mutation Quadruple Negative p Value Number 492 12 261 60 16 143 Age, median (range) 65.3 (27 95) 45.0 (32 72) 65.2 (29 95) 60.2 (27 85) 65.4 (50 74) 66.7 (27 91) 0.001 Age >65 years, n (%) 251 (51.0) 1 (8.3) 131 (50.2) 24 (40.0) 8 (50.0) 87 (60.8) 0.002 Gender Men, n (%) 237 (48.2) 6 (50.0) 102 (39.1) 30 (50.0) 8 (50.0) 91 (63.6) <0.001 Women, n (%) 255 (51.8) 6 (50.0) 159 (60.9) 30 (50.0) 8 (50.0) 52 (36.4) Smoking, n (%) 152 (30.9) 6 (50.0) 46 (17.6) 23 (38.3) 10 (62.5) 67 (46.9) <0.001 ECOG 0 1, n (%) 436 (86.6) 12 (100) 238 (91.2) 47 (78.3) 13 (81.3) 126 (88.1) 0.034 2 4, n (%) 56 (11.4) 0 (0) 23 (8.8) 13 (21.7) 3 (18.7) 17 (11.9) Clinical stage I IIIA, n (%) 160 (32.5) 5 (41.7) 100 (38.3) 11 (18.3) 8 (50.0) 36 (25.2) 0.003 IV, n (%) 332 (67.5) 7 (58.3) 161 (61.7) 49 (81.7) 8 (50.0) 107 (74.8) Stage IV group, n 332 7 161 49 8 107 0 1 128 (38.6) 5 (71.4) 65 (40.4) 18 (36.7) 0 (0) 40 (37.4) 0.073 2 204 (61.4) 2 (28.6) 96 (59.6) 31 (63.3) 8 (100) 67 (62.6) Pemetrexed containing therapy a, n (%) 164 (49.4) 6 (85.7) 75 (46.6) 28 (57.1) 1 (12.5) 54 (50.5) 0.044 Platinum-based doublet chemotherapy, n (%) 184 (55.4) 6 (85.7) 81 (50.3) 32 (65.3) 3 (37.5) 62 (57.9) 0.109 EGFR TKI use, n (%) 255 (76.8) 3 (42.9) 136 (84.5) 35 (71.4) 7 (87.5) 74 (69.2) 0.005 Crizotinib use, n (%) 10 (3.0) 0 (0) 0 (0) 10 (20.4) 0 (0) 0 (0) <0.001 a Single pemetrexed or platinum/pemetrexed doublet. EGFR TKI, epidermal growth factor receptor tyrosine kinase inhibitor; EML4-ALK, echinoderm microtubule-associated protein-like 4. Data are presented as n or n (%). Copyright 2014 by the International Association for the Study of Lung Cancer 1173

Chen et al. Journal of Thoracic Oncology Volume 9, Number 8, August 2014 TABLE 2. Clinical Characteristics of Patients with Lung Adenocarcinoma and the ROS1 Fusion (n = 12) No. Age Sex Ethnicity Smoking Distribution (Pack-Year) ROS1 Fusion a Stage Histologic Pattern b IHC Score c Pattern Surgical cohort 1 44 M Asian 0 CD74-ROS1 (6;34) II Acinar (with mucinous 3+ Diffuse cribriform), solid (signet-ring cell appearance) 2 45 F Asian 0 CD74-ROS1 (6;34) IIIA Acinar (with cribriform, 1+ Heterogeneous signet-ring cell appearance and eosinophilic secretion) 3 37 F Asian 0 CD74-ROS1 (6;34) II Solid (with signet-ring 3+ Diffuse cell appearance), acinar (with cribriform) 4 50 F Asian 0 SDC4-ROS1 (2; 34 + 35) II Acinar, lepidic, solid, 3+ Diffuse papillary 5 71 F Asian 0 SDC4-ROS1 (2;32) I Lepidic, papillary 1+ Heterogeneous Nonsurgical cohort 6 59 F Asian 0 CD74-ROS1 (6;34) IV Micropapillary, solid 3+ Diffuse 7 46 M Asian 10 CD74-ROS1 (6;34) IV Acinar (cribriform) 3+ Diffuse 8 36 M Asian 5 CD74-ROS1 (6;34) IV Solid (psammoma body) 3+ Diffuse 9 45 M Asian 17 CD74-ROS1 (6;34) IV Solid (with eosinophilic 3+ Diffuse secretion) 10 32 M Asian 15 CD74-ROS1 (6;34) IV Acinar, micropapillary 3+ Heterogeneous (with psammoma body) 11 59 M Asian 5 CD74-ROS1 (6;32 + 34) IV Acinar, solid (comedonecrosis), micropapillary 3+ Diffuse 12 44 F Asian 20 SLC34A2-ROS1 (4;32) IV Papillary Inadequate specimen a For ROS1 fusion, numbers represent fusion point (exon of partner; exon(s) of ROS1) b Arranged in order of predominance c Immunohistochemistry (IHC) score: 3+, strong cytoplasmic stain; 2+, moderate cytoplasmic stain; 1+, weak cytoplasmic stain; 0, negative. EGFR, epidermal growth factor receptor; EML4-ALK, echinoderm microtubule-associated protein-like 4; IHC, Immunohistochemistry; MPE, Malignant pleural effusion; NSCLC, non small-cell lung cancer; RT-PCR, reverse transcription-polymerase chain reaction. [n = 261, 53.0%], EML4-ALK fusion [n = 60, 12.2%], KRAS mutation [n = 16, 3.3%], and quadruple-negative [n = 143, 29.1%]); and the ROS1 fusion-positive cohort (n = 12, 2.4%; Table 1). ROS1 Fusion-Negative Cohorts In the ROS1 fusion-negative cohorts, patients with the EGFR mutation had significantly female predominance (60.9%) compared with the other molecular cohorts (p < 0.001). The EGFR mutation and EML4-ALK fusion patients tended to be never smokers (p < 0.001). There were no significant age differences among the ROS1 fusion-negative cohorts. Majority of patients had initial diagnosis at stage IV, which was most often seen in EML4-ALK fusion patients (81.7%; p = 0.003). Regarding systemic anticancer therapy in the stage IV group, a relatively lower proportion of ROS1 fusion-negative patients (49.4%, 164 of 332) received pemetrexed containing therapy (of those, 106 patients received single pemetrexed and 58 patients received platinum/pemetrexed doublet) and only half (55.4%, 184 of 332) received platinum-based doublet chemotherapy when compared with the ROS1 fusion-positive patients (87.5%, six of seven). Up to 84.5% (136 of 161) of EGFR mutation patients were exposed to EGFR TKI (p < 0.001), but only 20.4% (10 of 49) of the EML4-ALK fusion patients and none of the ROS1 fusion patients in the stage IV group received crizotinib (Table 1). ROS1 Fusions-Positive Cohort Among the 12 ROS1 fusion adenocarcinoma patients, the median age was 45.0 years (range, 32 71 years), which was significantly younger than those of the other molecular cohorts (p < 0.001). They also had relatively better performance status (ECOG 0 1) compared with the ROS1 fusionnegative cohorts (p = 0.034) at the time of lung cancer diagnosis. However, there was no sex predilection and up to 50% of patients were smokers (Table 1). Pathology and IHC of ROS1 Fusion Tumors The detailed ROS1 fusion patterns and their clinicopathologic features were summarized in Table 2. The CD74-ROS1 fusion was found in nine cases, SDC4-ROS1 fusion in two, and SLC34A2-ROS1 fusion in one case. Histologic analysis showed that ROS1 fusion tumors had heterogeneous patterns and up to 50% of ROS1 tumors were 1174 Copyright 2014 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 9, Number 8, August 2014 Lung Adenocarcinoma Patients with ROS1 Fusion FIGURE 1. Hematoxylin and eosin stain and Immunohistochemical staining (ROS1 D4D6) of ROS1 fusionpositive lung adenocarcinoma. (A, B) Case 3, surgical specimen with CD74-ROS1 fusion had diffuse and strongly (3+) cytoplasmic granular stain (100X). (C,D) Case 4, SDC4-ROS1 fusion tumors (100X) showed strong (3+), diffuse ROS1 expression in the cytoplasma. (E,F) Case 6, ROS1 protein was expressed diffusely in the cytoplasm of CD74-ROS1 fusion cell block specimen (400X). sub-classified as acinar-predominant morphology according to the International Association for the Study of Lung Cancer classification of lung adenocarcinomas.1 The acinar (including cribriform) pattern (detected in seven tumors, 58.3%) and solid pattern (detected in seven tumors, 58.3%) were the two most common histologic subtypes in ROS1 fusion-positive tumors. These two subtypes were both predominantly seen in the CD74-ROS1 fusion tumors (six of nine, 66.7%). Four cases exhibited signet-ring cell appearance and cribriform (including one mucinous cribriform) pattern, which were also characteristic features in ALK-rearranged lung cancers.35,36 Micropapillary, papillary, and lepidic patterns were detected in 25%, 25%, and 16.7%, respectively. Among the 12 RT-PCR-positive patients, immunohistochemical stain successfully detected ROS1 protein expression in 11 evaluable specimens (case 12 had no sufficient tissue for IHC analysis). Overexpression of the ROS1 protein was detected in cancerous part but not in normal lung parenchyma or lymphocytes. ROS1 protein expression could be identified in all fusion patterns, which included seven tumors with CD74-ROS1 fusion, one tumor with SDC4-ROS1 fusion had diffuse and strong cytoplasmic granular stain, one CD74-ROS1 fusion tumor with heterogeneous, strong cytoplasmic stain, one CD74-ROS1 fusion, and one SDC4-ROS1 fusion tumors showed weak ROS1 protein expression (Table 2 and Fig. 1). Copyright 2014 by the International Association for the Study of Lung Cancer 1175

Chen et al. Journal of Thoracic Oncology Volume 9, Number 8, August 2014 FIGURE 2. A, Kaplan-Meier survival curve of relapse-free survival for surgical patients with lung adenocarcinoma (n = 160) in different molecular cohorts. There was no significant survival difference among the ROS1 fusion-positive patients (dotted line), epidermal growth factor receptor (EGFR) mutation-positive patients (thick line), echinoderm microtubule-associated proteinlike 4 (EML4-ALK) fusion patients (thin dashed line), KRAS (thick dashed line) mutation, and quadruple-negative patients (thin line). (B) Kaplan-Meier survival curve of overall survival for surgical patients with lung adenocarcinoma (n = 160) with different molecular cohorts. There was no significant survival difference among ROS1 fusion-positive patients (dotted line), EGFR mutation-positive patients (thick line), EML4-ALK fusion patients (thin dashed line), KRAS (thick dashed line), and quadruple negative patients (thin line). MST, median survival time (months). The p value was calculated using the log-rank test. Among the 143 quadruple-negative patients, a total of 116 evaluable quadruple-negative samples were successfully analyzed by ROS1 antibody and 27 patients had insufficient materials for IHC analysis. Of them, 111 (95.7%) showed negative IHC stain. For clarifying the discrepancy between the ROS1 IHC stain and RT-PCR method, Fluorescence in situ hybridization (FISH) assay using ROS1 break apart FISH probe RUO kit (Abbott, IL) was performed according to the manufacturer s instruction, on the five positive IHC staining tumors. Of them, one tumor with strong positive (3+) stain had inadequate tissue for FISH study and three specimens with moderate (2+) ROS1 IHC stain showed negative result of FISH. Only one tumor with strong (3+) ROS1 IHC stain showed positive result of FISH study, which might contain other alternative ROS1 fusion partner not detected by RT-PCR. Clinical Outcomes In the surgical group, 116 (72.5%) deaths and 146 (91.2%) relapse events occurred during the follow-up period. The median duration of follow-up was 50.1 months. The median RFS for each molecular cohort was 24.6 months for the ROS1 fusion, 30.4 months for the EGFR mutation, 34.4 months for the EML4-ALK fusion, 8.1 months for the KRAS mutation, and 21.3 months for the quadruple negative (p = 0.771; Fig. 2A). There was no significant OS difference (p = 0.555) between the ROS1 fusion-positive and ROS1 fusion-negative cohorts in the surgical group (Fig. 2B). In the stage IV group, 271 (81.6 %) deaths occurred during the follow-up period. The median duration of follow-up was 14.1 months. The longest OS was patients with EGFR mutations (21.2 months), followed sequentially by those with EML4-ALK fusion gene (12.5 months), ROS1 fusion (11.9 months), quadruple-negative patients (11.1 months), and those with KRAS mutation (3.9 months; p < 0.001; Fig. 3). Moreover, after adjusting for clinical factors, multivariate analysis for OS showed that ECOG performance status 2 4 (Hazard ration [HR]: 2.41; p < 0.001) and patients with more than two metastasis sites on initial diagnosis (HR, 2.03; p < 0.001) were significantly associated with shorter OS. The presence of EGFR mutation (HR: 0.40; p < 0.001), use of EGFR TKI (HR: 0.46; p < 0.001), pemetrexed containing therapy (HR: 0.49; p < 0.001), crizotinib (HR: 0.20; p = 0.001), and platinum-based doublet chemotherapy (HR: 0.56; p < 0.001) were associated with longer OS (Table 3). DISCUSSION Twelve patients with the ROS1-fusion gene were identified from 492 patients with lung adenocarcinoma using multiplex RT-PCR methods. Eleven of twelve ROS1-fusion tumors were confirmed further by IHC. Patients with the ROS1-fusion genes tended to be younger and had no sex predilection. Acinar (including cribriform) and solid pattern were the two most common histologic subtypes in ROS1 fusion tumors and were predominant in the CD74-ROS1 fusion tumors. Compared with each ROS1 fusion-negative cohort, ROS1 fusion-positive patients had no significant survival difference in the surgical group, but might have worse outcome in stage IV groups compared with the EGFR mutation cohort. 1176 Copyright 2014 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 9, Number 8, August 2014 Lung Adenocarcinoma Patients with ROS1 Fusion FIGURE 3. Kaplan-Meier survival curve of overall survival (OS) for stage IV lung adenocarcinoma patients (n = 332) with different molecular cohorts. Epidermal growth factor receptor (EGFR) mutation patients (thick line) had longer OS than patients with the echinoderm microtubule-associated protein-like 4 (EML4-ALK) fusion (thin dashed line; p = 0.039), KRAS mutation (thick dashed line; p = 0.011), and the quadruple-negative patients (thin line; p < 0.001). The ROS1 fusion-positive patients (dotted line) had potentially worse outcome than EGFR mutation patients (green line; p = 0.139), but they did not have survival differences compared with the EML4-ALK fusion, KRAS mutation, and quadruple-negative cohorts (p = 0.656, 0.435, and 0.758, respectively). MST, median survival time (months). The p value was calculated using the log-rank test. In this study, the prevalence of ROS1 fusion is 2.4% in lung adenocarcinoma among East Asian patients. This is similar to previous reports. 16,18 22 Moreover, as a proposal of a diagnostic algorithm described by Go et al., 23 the data here shows that the prevalence of ROS1 fusion is 7.74% (12 of 155) in lung adenocarcinoma patients without EGFR/ALK/KRAS mutations. This can facilitate the detection of ROS1-fusion tumors. Unlike EGFR and KRAS with different frequencies between East Asians and Caucasians, 24 27 the prevalence of ROS1 fusion may be similar in different ethnic groups, with approximately 1 3% of NSCLC and mostly in lung adenocarcinoma. 16,18 22 Nevertheless, there are slight differences in different series, which may be due to different screening methods and strategies. FISH assay has been used for diagnosing cancers, 16,18,20 but it usually needs further confirmation by RT-PCR for definitive fusion partners. Furthermore, FISH assay requires expensive reagents, specialized equipment, larger tissue volume, longer processing time, and a high level of expertise compared with RT-PCR. The accuracy of RT-PCR results has been validated for ROS1 fusion gene detection in previous reports. 19,22 The present study demonstrates that the RT-PCR method can successfully identify the fusion partners of the ROS1 gene in both surgical and MPE specimens of lung adenocarcinoma without EGFR/ALK/KRAS mutation, and have a similar prevalence as in earlier reported series. 19,21 23 Consistent with previous reports, 16,19 patients with the ROS1 fusion gene are significantly younger (median age, 45.0 years) than those of other molecular cohorts, but without sex predilection. 22,37 However, up to 50% of ROS1 fusion patients are smokers and not predominant never smokers as previously reported. 16,19,22,23 Interestingly, in the surgical group here, the ROS1 fusion patients tend to be never smokers, with female predominance, as earlier study described. 19 The relationship among smoking status, sex, and ROS1 fusion gene remains unclear because of the very low percentage of such populations worldwide. Thus, further studies are needed. Nonetheless, knowledge of this clinical profile will help physicians select younger lung adenocarcinoma patients without EGFR/ALK/KRAS mutation to be most likely to harbor this genetic alteration. 5,16,23 The present study reveals that the acinar (including cribriform) structure and solid pattern are the common histologic subtypes of ROS1 fusion adenocarcinoma, which may correlate with the CD74-ROS1 fusion pattern. This has never been addressed before. Moreover, four studied cases also exhibit the signet-ring cell appearance and cribriform, which suggests that the ROS1 fusion tumors may share parts of similar characteristics of EML4-ALK lung cancer. 19,23,35,36 However, the patient number is too small for further inference. More studies are necessary to investigate the relationship between ROS1 fusion gene and specific histologic patterns. A novel ROS1 D4D6 rabbit monoclonal antibody IHC assay has been developed recently for detecting the ROS1 fusion gene. 19,21 In our study, all evaluable ROS1 fusionpositive tumors were also confirmed by this IHC assay successfully. Of them, most adenocarcinoma tumors expressed ROS1 protein diffusely in the cytoplasm, especially seen in CD74-ROS1 fusion pattern, which has the similar findings as Rimkunas et al. reported. 21 Moreover, by using the evaluable 116 quadruple-negative samples as negative controls, only five positive results were identified by ROS1 IHC stain with the specificity up to 95.7%, which was similar as Mescam- Mancini et al. reported. 38 Nevertheless, one RT-PCR negative tumor with strong positive (3+) of ROS1 IHC stain had positive result of FISH study, which might contain alternative ROS1 fusion partner. The correlation between ROS1 RT-PCR and IHC staining might need further studies to confirm. In present study, the surgical group has similar RFS and OS regardless of molecular status, which is similar to the report of Yoshida et al. 19 In contrast, Lee at al. 38 reported that ROS1 expression has worse prognosis for OS in stage I NSCLC. In addition, ROS1 fusion-positive patients in the nonsurgical group do not show significant survival difference compared with each ROS1 fusion-negative cohort (Fig. 3), which is consistent with a prior report. 16 However, longer survival in the ROS1-negative cohort has been reported by earlier studies using Chinese and Korea data. 22,37 This may result from a bias towards patients with EGFR mutation or EML4-ALK-positive receiving suitable target therapies with better prognosis. In the present study, multivariate analysis reveals that patients with the ROS1 fusion have worse outcome compared Copyright 2014 by the International Association for the Study of Lung Cancer 1177

Chen et al. Journal of Thoracic Oncology Volume 9, Number 8, August 2014 TABLE 3. Multivariate Analysis of Prognostic Factors for Overall Survival in Stage IV Cohort Clinical Factors No. of Patients Median OS (mo) Univariate Analysis Multivariate Analysis p Value HR (95% CI) p Value Age <65 157 18.6 0.002 0.937 65 175 12.8 0.99 (0.746 1.309) Sex Female 176 16.8 0.021 0.221 Male 156 13.1 1.21 (0.893 1.635) Smoking status Never smoker 234 17 <0.001 0.245 Smoker 92 11.8 1.23 (0.870 1.729) ECOG PS 0 1 276 17.9 <0.001 <0.001 2 4 56 3.9 2.41 (1.691 3.445) Metastasis sites 0 1 128 21.1 <0.001 <0.001 2 204 13.8 2.03 (1.517 2.722) Mutation status Quadruple negative 107 11.1 1 EGFR mutation 161 21.2 <0.001 0.40 (0.299 0.542) <0001 EML4-ALK fusion 49 12.5 0.051 0.98 (0.670 1.443) 0.931 KRAS mutation 8 3.9 0.709 0.66 (0.299 1.451) 0.3 ROS1 fusion 7 11.9 0.758 1.64 (0.729 3.677) 0.232 EGFR TKI use No 77 8.2 <0.001 <0001 Yes 255 17.3 0.46 (0.336 0.633) Platinum-based doublet chemotherapy use No 148 9.5 <0.001 <0.001 Yes 184 20 0.56 (0.410 0.768) Pemetrexed containing therapy a No 168 9.5 <0.001 <0.001 Yes 164 22.9 0.49 (0.365 0.658) Crizotinib use No 332 15.1 0.015 Yes 10 42.3 0.20 (0.076 0.514) 0.001 a Single pemetrexed or platinum/pemetrexed doublet. CI, confident interval; ECOG, Eastern Cooperative Oncology Group; EGFR, epidermal growth factor receptor; EML4-ALK, echinoderm microtubule-associated protein-like 4; HR, hazard ratio; mo, months; MPE, malignant pleural effusion; NSCLC, non small-cell lung cancer; PS, performance status; RT-PCR, reverse transcription-polymerase chain reaction. with the EGFR mutation cohort (Table 3). This is very likely that the most EGFR mutant patients received the treatment with EGFR TKIs, compared with the ROS1 fusion-positive patients without specific target therapy. However, the number of ROS1 fusion patients is still small in our cohort, further prospective clinical trials with large samples are necessary to clarify the impact of novel target therapy on ROS1 fusion patients and the role of ROS1 fusion gene on the prognosis of lung adenocarcinoma. The present study has several limitations. First, FISH assay was only performed in very limited samples. Nonetheless, the RT-PCR and IHC methods have good correlation with the FISH assay and can be used for definitive confirmation of ROS1 fusion partners. 19,21,22 Second, although crizotinib showed a high response rate and good safety profile among patients harboring the EML4-ALK 5 and ROS1 fusion, 16 only 20.4% of the EML4-ALK fusion patients and none of the ROS1 fusion patients received the crizotinib in this study. This may affect the survival of the EML4-ALK and ROS1 fusion cohorts. Third, this study screened samples by RT-PCR using only primers specific for CD74, SLC34A2, LRIG3, and SDC4. It is possible that additional patients in this cohort expressed the other known ROS1 fusions. Finally, we did not include the stage IV lung adenocarcinoma patients without MPE, which might have selection bias. In conclusion, approximately 2.4% of patients in the East Asian population with lung adenocarcinoma have the 1178 Copyright 2014 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 9, Number 8, August 2014 Lung Adenocarcinoma Patients with ROS1 Fusion ROS1 fusion. These patients tend to be younger, without sex predilection, and may effectively be detected by the RT-PCR and IHC methods. Although, stage IV ROS1 fusion-positive patients without specific targeted therapy might have worse outcome compared with the EGFR mutation cohort, who are mostly treated with EGFR TKIs, the prognostic value of ROS1 fusion still need further studies with large samples to confirm. ACKNOWLEDGMENTS The authors thank the Department of Medical Research at the National Taiwan University Hospital for providing the laboratory facility. This study was supported by grants from the National Science Council, Taiwan (NSC101-2314-B-002-169-MY3, NSC101-2314-B-002-170-MY3), Department of Health, Executive Yuan, Taiwan (DOH102-TD-PB-111-TM023), National Taiwan University (100C101-101, 10R71601-3), and National Taiwan University Hospital (102-S2158). 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