The past decade has witnessed significant success with personalized cancer therapy, and this paradigm shift has led to tremendous improvements in clinical outcomes in patients with molecularly defined subsets of tumors. Notably, the discovery of a variety of molecular and genetic alternations in non–small-cell lung cancer (NSCLC) has provided the opportunity for targeted therapy, such as epidermal growth factor receptortyrosine kinase inhibitors (gefitinib and erlotinib) for activating EGFR mutations in NSCLC, and anaplastic lymphoma kinase (ALK) inhibitor (crizotinib) for NSCLC harboring ALK gene fusion. The c-ros oncogene 1 (ROS1) receptor tyrosine kinase (RTK) has recently emerged as a potentially relevant therapeutic target in NSCLC. Here, our review focuses on the oncogenic role of aberrant ROS1 in NSCLC and targeting ROS1-rearranged NSCLC patients with ALK inhibitors.
ROS1 RECEPTOR STRUCTURE AND FUNCTION
v-ROS1 is an oncogene product of the avian sarcoma RNA tumor virus UR2.1 Wild-type ROS1 is located on chromosome 6 and encodes full-length ROS1, a 2,347 amino acid transmembrane tyrosine kinase (TK) receptor consisting of an extracellular ligand-binding domain composed of nine repeated fibronectin-like motifs, a short transmembrane domain, and an intracellular TK domain.2 Although very little is known of the extracellular domain function of this orphan receptor, the structural combination of ROS1 suggests its ability to directly couple extracellular adhesion-mediated events to tyrosine phosphorylation-based intracellular signaling. Using the Multiple Sequence Comparison by Log-Expression software, it has been found that there is a 49% amino acid homology between human ROS and ALK within the kinase domain3 and 77% identity at the adenosine triphosphate (ATP)-binding site.4
Dysregulated ROS1 may occur as a result of ROS1 gene fusion, overexpression, or mutations. Aberrant ROS1 kinase activity leads to activated downstream signaling of several oncogenic pathways including the phosphoinositide-3 kinase/v-akt murine thymoma viral oncogene homolog 1 (AKT)/mechanistic target of rapamycin (serine/threonine kinase) (mTOR), signal transducers and activators of transcription-3, RAS-MAPK/ERK, vav 3 guanine nucleotide exchange factor 1 (VAV3) and Src-homology 2 domain-containing phosphatase (SHP)-1 and -2 pathways (Fig. 1).5,6 With the activation of ALK fusion proteins, the downstream signaling pathways are similar to ROS1 RTK activation with the involvement of the RAS-MAPK/ERK, Janus kinase 3-signal transducers and activators of transcription-3, and phosphoinositide-3 kinase /Akt pathways, which control cell proliferation, survival, and cell cycling.7,8 However, unlike the echinoderm microtubule-associated protein-like 4 (EML4)-ALK gene fusion in which the coiled-coil domain within echinoderm microtubule-associated protein-like 4 mediates the constitutive dimerization resulting in aberrant constitutive activity,9 protein dimerization domains have not been described in ROS1 fusion receptor tyrosine kinases.5
ROS1 Fusion Genes
To date, seven distinct ROS1 gene fusions have been identified in solid tumors including fused in glioblastoma (FIG)-ROS1, CD74-ROS1, solute carrier family 34 (sodium phosphate), member 2 (SLC34A2)-ROS1, tropomyosin 3 (TPM3)-ROS1, syndecan 4 (SDC4)-ROS1, ezrin (EZR)-ROS1, leucine-rich repeats, and immunoglobulin-like domains 3 (LRIG3-ROS1) all of which encoded the same cytoplasmic portion of ROS1 TK domain. The break point of ROS1 with EZR is exon 34, TPM3, FIG, and LRIG3 is exon 35 whereas for CD74, SDC4, and SLC34A2 are exons 32 and 34. (Table1). All of the breakpoints in ROS1 allow the resulting fusion to retain the ROS1 kinase domain whereas with SDC4-ROS1, the ROS1 transmembrane domain is also retained.3 More recently, Govindan et al.9a have identified a novel ROS1 fusion, KDELR2 (endoplasmic reticulum protein retention receptor 2) in a never smoker patient with NSCLC.
FIG-ROS1 fusion was first described in glioblastoma multiforme6,10 and subsequently in 8.7% of cholangiocarcinoma tumors.11 The clinicopathologic characteristics of patients with glioblastoma multiforme and cholangiocarcinoma harboring FIG-ROS1 is unknown. In NSCLC, SLC34A2-ROS1 and CD74-ROS1 were initially discovered in the HCC78 cell line and in a patient tumor, respectively.12
Down-regulation of SLC34A2-ROS1 inhibited cellular proliferation in HCC78 cells12 whereas more recently, the transforming ability of FIG-ROS1 and TPM3-ROS1, SDC4-ROS1, SLC34A2-ROS1, CD74-ROS1, LRIG-ROS1, and EZR-ROS1 was demonstrated in vivo by Gu et al.11 and Takeuchi et al.13
In a screen using fluorescence in situ hybridization (FISH) for ROS1 rearrangements, 1.7% of NSCLC patients (18 of 1073) harbored CD74-ROS1 or SLC34A2-ROS1 fusions, and ROS1 fusions were more likely in younger patients, never smokers with adenocarcinoma subtype.14 Of the 18 cases reported in this study, no fusion partner was identified in eight samples. Takeuchi et al.13 have reported ROS1 fusion genes in 0.9% NSCLC (13 of 1476) and 1.2% of patients (13 of 1116) with adenocarcinomas. Four novel ROS1 gene fusions were reported: TPM3-ROS1, SDC4-ROS1, EZR-ROS1, and LRIG3-ROS1.13 In one case, no fusion partner was identified. In a study of East Asian never smokers with lung adenocarcinoma, 1% (2 of 202) harbored ROS1 fusion.15
Using immunohistochemistry, and confirming the findings by FISH and reverse transcriptase polymerase chain reaction (RT-PCR), Rimkunas et al.16 reported ROS1 was overexpressed in 1.6% of all lung NSCLC (9 of 556) and 3.3% of lung adenocarcinoma (8 of 246) from Chinese patients. The pattern of cellular localization of ROS1 protein was variable with diffuse cytoplasm, perinuclear aggregates, and membranous and vesicular localization reported. It is interesting that, this is the first study to report a case of FIG-ROS1 in NSCLC.
On the basis of the studies described above, it can be stated that ROS1 fusions represent a unique molecular subset of NSCLC with no overlap with other described oncogene drivers, and are rare with a frequency of 0.7% to 1.7% (Table 2) as compared with EGFR mutations (6%–33%)17 and ALK fusion (4%).18 There is overlap in the clinicopathologic characteristics between ROS1 and ALK rearrangement in NSCLC patients, namely in younger patients with a history of never smoking and adenocarcinoma subtype.
ROS1 Overexpressions and Mutations
Microarray analysis of NSCLC demonstrated significantly elevated ROS1 expressions in 20% to 30% of cases, and elevated ROS1 expression was found to be part of a molecular signature for lung adenocarcinoma subtype.19–21 ROS1 mutations have been reported in NSCLC22,23 and interestingly, they were in nonadenocarcinoma histologic subtypes. ROS1 somatic mutations were also reported in very low frequencies in cancers of the colon, ovary, breast, upper aerodigestive tract, and skin.22,24–26 The significance of these mutations is unknown. Overexpression of ROS1 has been reported in 33% to 56 % of glioblastoma and meningeal tumors.27–30
METHODS OF DETECTION FOR ALTERED ROS1
The first ROS1 translocations in human NSCLC (SLC34A2-ROS1 and CD74-ROS1) were discovered through a global survey of phosphotyrosine kinase signaling in 41 NSCLC cell lines and more than 150 NSCLC tumor samples using an immunoaffinity phosphoproteomic profiling strategy.12 More recently, a ROS1 break-apart FISH was used to screen for ROS1 rearrangements.14 As FISH provides no information about the translocation partner, rapid amplification of complementary DNA assays and fusion-specific RT-PCR combined with Sanger sequencing of the polymerase chain reaction products have been applied to identify ROS1 fusion variants in ROS1-positive screens. Validation of ROS1 fusions after their initial discovery was mostly performed using RT-PCR.10–12 Immunohistochemistry with the D4D6 rabbit monoclonal antibody (Cell Signaling, Danvers, MA) that recognizes amino acids at the carboxyl terminus of human ROS1 has recently been reported to detect ROS1 fusions identified by ROS1 FISH analysis with high sensitivity and no false-positive identification.16 ROS1 was not detectable using D4D6 in normal lung tissues, although in rare cases, non-neoplastic cells, such as macrophages and bronchial epithelial cells, displayed expression. This is in comparison to a previous report according to which high levels of ROS1 are expressed in lung normal tissue.5 As only 25% of cases (138 of 556) underwent ROS1 FISH analysis in this study, the false-negative rate of this assay remains to be clarified. Results from this study by Rimkunas et al.16 suggest ROS1 immunohistochemistry may be a useful method for screening for the low frequency of patients with NSCLC harboring ROS1 fusions. Advantages of an immunohistochemistry assay include a more rapid turnaround time and analysis can be performed on a small amount of tissue. In contrast, FISH requires more specialized equipment, is more costly, and signal fadeout occurs over time. In addition, next-generation sequencing31,32 and chromogenic in situ hybridization33,34 approaches may have further advantages in detecting ROS1 rearrangement, as shown in ALK-rearranged NSCLC.
Targeting ROS1-Rearranged NSCLC Patients with ALK Inhibitors
Given the high homology in the kinase domains of ROS1 and ALK, ALK inhibitors were tested efficacious against ROS1-positive cell lines and tumors (Table 3). In preclinical studies, TAE684, an ALK inhibitor, demonstrated in vitro activity against HCC78 cell lines,35 attenuated phosphorylation of downstream ROS1 signaling, and induced apoptosis in FIG-ROS–positive BaF cells.11 In silico modeling suggests the drug–amino acid interactions between TAE684 and ROS1 kinase domain is as strong as with the ALK kinase domain.3 A phase I trial (NCT00585195) of crizotinib was amended to include ROS1-positive advanced-stage NSCLC patients. Preliminary results in this subset of 14 evaluable patients reported a response rate of 57% with a disease control rate of 79% at 8 weeks.4 Other early-phase studies that include patients with tumors harboring ROS1 fusions of second-generation ALK inhibitors such as AP26113 (NCT01449461) and ASP3026 (NCT01284192) are ongoing (Table 4).
Perspective, Future Direction and Conclusion
Although rare, the evidence supports ROS1 fusions as a valid therapeutic target in a molecularly defined subset of NSCLC patients. Results from NCT00585195 are eagerly awaited. Future work will include testing the sensitivity of the ROS1 fusion variants to crizotinib. Further issues to be addressed include the optimal method to detect clinically relevant ROS1, exact definition of ROS1-positive tumors for treatment stratification and diagnosis, characterization of downstream signaling pathways in ROS1 fusion tumors, and understanding the resistance to ROS1 RTK inhibitors.
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Non–-small-cell lung cancer; Targeted therapy; ROS1; Anaplastic lymphoma kinase inhibitors; Crizotinib
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© 2012International Association for the Study of Lung Cancer