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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0000000000000118
Case Reports

Coexistence of Three Variants Involving Two Different Fusion Partners of ROS1 Including a Novel Variant of ROS1 Fusions in Lung Adenocarcinoma: A Case Report

Cai, Weijing MD, PhD*; Li, Wei MD, PhD*; Ren, Shengxiang MD, PhD*; Zheng, Limou PhD; Li, Xuefei PhD*; Zhou, Caicun MD, PhD*

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*Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Tongji University Medical School Cancer Institute, Shanghai, People’s Republic of China; and Translational Medical Center, Xiamen University, Xiamen, People’s Republic of China.

Disclosure: The authors declare no conflicts of interest.

W. Cai and W. Li contributed equally to this work.

Address for correspondence: Caicun Zhou, MD, PhD, Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Tongji University Medical School Cancer Institute, No. 507 Zheng Min Road, Shanghai 200433, People’s Republic of China. E-mail:

ROS1-rearranged non–small-cell lung cancer (NSCLC) is a unique molecular subtype of lung cancer. Similar to EGFR mutation and ALK rearrangement, ROS1 rearrangement is a key oncogenic driver in a subgroup of lung cancer.1 The determination of ROS1 rearrangement status is vitally important for clinical diagnosis and treatment options in lung cancer. A woman with lung adenocarcinoma harboring three variants of ROS1 fusions including a novel variant was reported here. This case report and relevant study were approved by the institutional review boards of the Shanghai Pulmonary Hospital.

A 61-year-old Chinese woman was admitted with a 2-month history of cough and dyspnea. A chest computed tomography revealed a 3-cm right middle lobe mass with multiple bilateral pulmonary nodules, multiple mediastinal lymph nodes, right pleural effusion, and a small amount of pericardial effusion (Figure 1A). Brain magnetic resonance imaging showed multiple brain metastases (Figure 1B). No metastasis was observed by abdominal ultrasound examination. The patient had no smoking history, but long-time exposure to fumes emitted from cooking oils. She had no family history of malignant tumor. Physical examination revealed left supraclavicular lymph nodes. A biopsy of left supraclavicular lymph nodes revealed metastatic lung adenocarcinoma. Meanwhile, adenocarcinoma cells were detected in the pleural fluids. The patient was clinically diagnosed with stage IV lung adenocarcinoma, T4N3M1b, with Eastern Cooperative Oncology Group performance status of 1.

Figure 1
Figure 1
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The biopsied specimen from this patient was tested for EGFR using amplification-refractory mutation system assay with AmoyDx EGFR 29 Mutations Detection Kit and was tested for ALK, ROS1, and RET status using reverse-transcriptase polymerase chain reaction (RT-PCR) assay with AmoyDx EML4-ALK Fusion Gene Detection Kit, AmoyDx ROS1 Fusion Gene Detection Kit, and AmoyDx KIF5B-RET Fusion Gene Detection Kit (Amoy Diagnostics, Xiamen, China). The ROS1 fusion variants screened by AmoyDx ROS1 Fusion Gene Detection Kit were seen in Table 1. The findings showed that the patient was negative for EGFR mutations, EML4-ALK fusions, and KIF5B-RET fusions and was positive for ROS1 fusions. Of interest, the results of RT-PCR for ROS1 fusion showed that both tube 1 and tube 2 were positive. Next, electrophoretic analyses of RT-PCR products in tube 1 and tube 2, respectively, on 1.5% agarose gel found two products (a long fragment and a short fragment) in tube 1 (Figure 1C) and only a product in tube 2. The long fragment product in tube 1 was validated as a novel variant of ROS1 fusions, CD74 exon 7 fused to ROS1 exon 32, and the short fragment product in tube 1 was SLC34A2 (exon 13 del);ROS1 (exon 32) by gel extraction and direct sequencing (Figure 1D, E). The product in tube 2 was CD74 (exon 6);ROS1 (exon 34) (Figure 1F).

Table 1
Table 1
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The patient was treated with pemetrexed plus cisplatin in the first-line treatment setting. After one cycle of this regimen, the patient discontinued chemotherapy for intolerable toxicity and then received best support care. In addition, the patient did not receive crizotinib due to economic reason.

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In NSCLC, some molecular aberrations, including EGFR mutation, and ALK, ROS1, and RET fusions, are shown to be associated with specific clinical characteristics and treatment benefits from corresponding targeted therapy. Of them, ROS1 fusions occur in about ~1% of NSCLC and more frequently in younger, nonsmoking patients with adenocarcinoma.1 A phase I trial has demonstrated that crizotinib has dramatic antitumor activity in patients with ROS1-rearranged NSCLC, with a high objective response rate of 56%.2 Therefore, the identification of ROS1 fusion variants is important for personalized therapy in lung cancer.

ROS1 rearrangement was first reported in lung adenocarcinoma by Rikova et al.3 in 2007. Three variants of ROS1 fusions involving two fusion patterns, SLC34A2 (exon 4);ROS1(exon 32), SLC34A2 (exon 4);ROS1(exon 34), and CD74 (exon 6);ROS1(exon 34), were identified in this study.3 Since then, the tumorigenicity of these two ROS1 fusions was validated by tumor formation of 3T3 cells transduced with a retrovirus containing SLC34A2-ROS1 or CD74-ROS1 in immunocompromised nude mice.4,5 Up to now, a total of nine fusion partners of ROS1 including SLC34A2, CD74, tropomyosin 3 (TPM3), syndecan (SDC4), ezrin (EZR), leucine-rich repeats and immunoglobulin-like domains (LRIG3), fused in glioblastoma (FIG), endoplasmic reticulum protein retention receptor 2 (KDELR2), and coiled-coil domain containing 6 (CCDC6) have been identified in NSCLC.1,6–11 Among them, CD74 and SLC34A2 are the most common fusion patterns of ROS1. All identified breakpoints of ROS1 are located in exon 32, 34, or 35, which ensure that the resulting fusion genes harbor the kinase domain of ROS1.6 The first report of all known variants of ROS1 fusions were seen in Table 2. CD74(exon 7);ROS1(exon 32) was first found as a ROS1 fusion variant in lung cancer in this case. Similar to CD74(exon 6);ROS1(exon 32), CD74 exon 7 fused to ROS1 exon 32 allows the fusion gene to contain promoter of CD74 and the kinase domain as well as the transmembrane domain of ROS1. Therefore, we consider that CD74-ROS1(C7;R32) may have potential oncogenic activity as a novel variant of ROS1 fusions. However, because of lack of DNA sequence of the fusion gene, we cannot determine whether C7;R32 is a resulting variant from different genomic rearrangement or just a splice variant in mRNA of CD74-ROS1.

Table 2
Table 2
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It was generally believed that oncogenic drivers were mutually exclusive in lung cancer.1,6,12 However, a concurrent occurrence of different oncogenic drivers has been described in rare cases.13–15 Rikova et al.3 found two variants of ROS1 fusions, SLC34A2-ROS1 (S4;R32) and SLC34A2-ROS1 (S4;R34), in a human NSCLC cell line (HCC78). We also identified that SLC34A2-ROS1 (S13del;R32) and SLC34A2-ROS1 (S13del;R34) coexisted in a female never-smoker with adenocarcinoma in our previous study.16 In addition, Rimkunas et al.9 described two cases with adenocarcinoma harboring coexisting EGFR mutation and SLC34A2-ROS1 fusion. In this case, three variants of ROS1 fusions were identified concurrently. The above-mentioned findings suggested the possibility of coexistence of different ROS1 fusion variants with or without other somatic genetic alteration. Furthermore, recent studies have found that clonal evolution plays an important role in tumor progression and development of metastasis.17,18 In this case, we first identified the coexistence of three variants involving two different fusion partners of ROS1 in a metastatic lesion and validated all identified ROS1 fusion variants by sequencing. Based on reliable results, this finding may be explained by high heterogeneity in tumor tissue.

In addition, break-apart fluorescence in situ hybridization (FISH) is the most commonly used diagnostic method for detecting ROS1 fusion genes to date. However, FISH cannot distinguish different variants of fusion genes using break-apart probe. For this case, RT-PCR may be a more efficient diagnosis method than FISH because FISH cannot identify the coexistence of different variants of ROS1 fusions. Although there is no clinical evidence on potential difference in sensitivity to ROS1 inhibitors among different variants with different fusion partners of ROS1 to date, a difference in sensitivity to crizotinib in vitro between Ba/F3 cells transduced with SDC4-ROS1 and HCC78 cell harboring SLC34A2-ROS1 was described by Davies et al.7 The inhibition of crizotinib in Ba/F3 cells transduced SDC4(exon 2);ROS1(exon 32) was more potent than in HCC78 cells that harbored SCL34A2-ROS1 (IC50 values, 31 nmol/L versus 775 nmol/L) in their study.7 Based on these preclinical data, we cannot exclude the possibility that patients with NSCLC harboring difference variants of ROS1 fusions may have different response to ROS1 inhibitors. Therefore, the diagnostic process for ROS1-rearranged lung cancer is worth further exploration.

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The case report first described a novel variant of ROS1 fusions, CD74 exon 7 fused to ROS1 exon 32, in a female never-smoker with lung adenocarcinoma. Meanwhile, this case harbored three variants involving two different fusion partners of ROS1, CD74, and SLC34A2. For this patient, crizotinib may be an effective treatment. However, the mechanism and frequency of this phenomenon remain unclear. The possible difference in response to ROS1 inhibitors among patients harboring different ROS1 fusion variants needs to be further investigated.

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1. Bergethon K, Shaw AT, Ou SH, et al. ROS1 rearrangements define a unique molecular class of lung cancers. J Clin Oncol. 2012;30:863–870

2. Ou SH, Bang YJ, Camidge DR, et al. Efficacy and safety of crizotinib in patients with advanced ROS1-rearranged non-small cell lung cancer (NSCLC). J Clin Oncol. 2013;31(suppl):abstr 8032

3. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 2007;131:1190–1203

4. Gu TL, Deng X, Huang F, et al. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma. PLoS One. 2011;6:e15640

5. Jun HJ, Johnson H, Bronson RT, de Feraudy S, White F, Charest A. The oncogenic lung cancer fusion kinase CD74-ROS activates a novel invasiveness pathway through E-Syt1 phosphorylation. Cancer Res. 2012;72:3764–3774

6. Takeuchi K, Soda M, Togashi Y, et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med. 2012;18:378–381

7. Davies KD, Le AT, Theodoro MF, et al. Identifying and targeting ROS1 gene fusions in non-small cell lung cancer. Clin Cancer Res. 2012;18:4570–4579

8. Rimkunas V, Crosby K, Kelly M, et al. Frequencies of ALK and ROS in NSCLC FFPE tumor samples utilizing a highly specific immunohistochemistry-based assay and FISH analysis. J Clin Oncol. 2010;28(15 suppl):abstr 10536

9. Rimkunas VM, Crosby KE, Li D, et al. Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion. Clin Cancer Res. 2012;18:4449–4457

10. Govindan R, Ding L, Griffith M, et al. Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell. 2012;150:1121–1134

11. Seo JS, Ju YS, Lee WC, et al. The transcriptional landscape and mutational profile of lung adenocarcinoma. Genome Res. 2012;22:2109–2119

12. Sun Y, Ren Y, Fang Z, et al. Lung adenocarcinoma from East Asian never-smokers is a disease largely defined by targetable oncogenic mutant kinases. J Clin Oncol. 2010;28:4616–4620

13. Boland JM, Jang JS, Li J, et al. MET and EGFR mutations identified in ALK-rearranged pulmonary adenocarcinoma: molecular analysis of 25 ALK-positive cases. J Thorac Oncol. 2013;8:574–581

14. Popat S, Vieira de Araújo A, Min T, et al. Lung adenocarcinoma with concurrent exon 19 EGFR mutation and ALK rearrangement responding to erlotinib. J Thorac Oncol. 2011;6:1962–1963

15. Miyanaga A, Shimizu K, Noro R, et al. Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4-ALK-rearranged non-small-cell lung cancer harbored coexisting EGFR mutation. BMC Cancer. 2013;13:262

16. Cai W, Li X, Su C, et al. ROS1 fusions in Chinese patients with non-small-cell lung cancer. Ann Oncol. 2013;24:1822–1827

17. Ding L, Ley TJ, Larson DE, et al. Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature. 2012;481:506–510

18. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883–892

Copyright © 2014 by the European Lung Cancer Conference and the International Association for the Study of Lung Cancer.


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