Characterization of kinase fusions across cancer types
We analyzed 8,805 3’ kinase gene fusions curated from the COSMIC, focusing on the seven most common kinase fusions involving ALK, RET, ROS1, NTRK1, NTRK3, ABL1, and BRAF genes found in various types of cancers (Supplementary Table 1). In this study, we used 7,751 kinase fusions where the mRNA junction positions were validated (Supplementary Table 1).
We first analyzed the prevalence of the seven 3’ kinase fusions across 16 tissue types. Except for ABL1 fusions, all kinase fusions were identified in multiple tissue types at variable frequencies between the tissue types (Fig. 1a and Supplementary Table 2). We observed several common patterns of kinase fusions, in terms of partner genes and intron usage. ALK fusions were predominantly observed in lung cancer and lymphoma, while RET, ABL1, and BRAF fusions have been identified most frequently in thyroid cancer, leukemias, and pediatric low-grade gliomas, respectively. EML4 was the most frequent partner of ALK fusions in lung cancers, while NPM was the case in lymphomas (Fig. 1b and Supplementary Table 1). The other TK fusions, including RET, ROS1, BRAF, and ABL1 fusions, showed several frequent partner genes (Fig. 1b and Supplementary Table 3). Partner genes were largely specific to kinase genes, with several exceptions (Fig. 1c). For example, seven partner genes (9.6%), including KIF5B, TPM3, ERC1, and ETV6, were shared across the 73 kinase fusions (Fig. 1c, d and Supplementary Table 1). In contrast, most frequent partner genes, i.e., EML4, CCDC6, CD74, BCR and, KIAA1549 were exclusively associated with ALK, RET, ROS1, ABL1 and BRAF fusions, respectively (Fig. 1c, d and Supplementary Table 3). In summary, this analysis shows tissue type- and kinase gene-specific partnering in fusion oncogene formation.
Next, we analyzed the locations of fusion events between the kinase and partner genes. Notably, most fusion events in ALK, RET, and ABL1 occurred at the 5’ end of exon 20 (e20; 99.6%, 837/839), e12 (99.5%, 1,047/1,052), and e2 (99.1%, 5,040/5,087), respectively, regardless of their fusion partners. In contrast, ROS1 fusions occurred at variable locations, including e32 (20.5%), e34 (53.8%), e35 (14.1%), and e36 (11.5%) (Fig. 1e and Supplementary Table 3). A similar pattern was observed in the partner genes. Several partner genes showed exclusive preference in exon usage. For example, all NPM1-ALK and CD74-ROS1 fusions used a specific exon in the partner genes (e5 of NPM1 and e6 of CD74). In contrast, EML4 and KIF5B used several exons, including e6, e13, and e20 for EML4 and e15 and e16 for KIF5B (Fig. 1f and Supplementary Table 3). This indicates potential selective benefit of specific exon usage in creating kinase fusions.
Functionally Active Chromosomal Translocation Sequencing (FACTS) identifies oncogenic ALK fusions genome-wide
Genome-wide techniques identifying chromosomal translocations, such as HTGTS and TC-seq, have been widely used to study the mechanisms of translocation in normal and tumor cells by cloning chromosomal junctions generated within a few days of inducing a programmed DNA DSB at a specific site17–19. However, not all these translocations result in functional fusion genes, and their impact on oncogenesis cannot be determined by these technologies alone. To overcome this limitation, we developed FACTS to specifically map functional oncogenic fusions genome-widely in the setting of pharmaceutical selective pressure. Inspired by the recent reports indicating fusion oncogene formation as a mechanism of resistance to EGFR inhibitors, we applied FACTS into the in vitro model of EGFR inhibitor resistance using PC-9 cells, harboring EGFR-activating mutation (EGFR E746-A750del)20.
PC-9 cells have been extensively used to characterize various mechanisms of resistance to EGFR inhibitors. Under the EGFR inhibitor treatment, PC-9 cells typically undergo cell-cycle arrest, and a small number of cells undergo persistence. Emergence of fully resistant clones require additional genetic alterations, including secondary mutations in EGFR that prevent TKI binding21 or another oncogenic driver events that bypasses EGFR inhibition, such as MET amplifications or the acquisition of fusion oncogenes22. Therefore, we reasoned that PC-9 cells under selective pressure from a selective EGFR inhibitor osimertinib23 would be an ideal setting to test the functional outcome of various kinase fusions induced by genome-wide translocations.
ALK fusions are the most frequent TK fusions found in 3–7% of patients with NSCLC15 and drive resistance to targeted therapy in patients with EGFR-mutant or KRASG12C-mutant cancer7–9. Consistently, PC-9 cells expressing two sgRNAs targeting the relevant introns in EML4 (intron 6 or intron 13) and ALK (intron 19) to force the formation of typical EML4-ALK fusions generated resistant clones expressing the EML4-ALK fusions at a frequency of 1.16%-1.34% upon osimertinib selection (Extended Data Fig. 1a-d), which was consistent with the frequency of translocations induced by two DNA DSBs in previous studies24,25. Furthermore, EML4-ALK fusion expressed from the endogenous EML4 promoter rapidly induced osimertinib resistance in PC-9 cells by producing active and phosphorylated EML4-ALK fusion proteins (Extended Data Fig. 1e). Next, we applied FACTS to study ALK fusion formation from a single programmed DSB with any partners in the genome under the osimertinib treatment. We induced a programmed DSB in intron 19 of ALK, the hotspot of genomic rearrangements causing EML4-ALK fusions in NSCLC15 (Fig. 2a). Multiple osimertinib-resistant clones developed during selection, with an estimated frequency of ~ 1 clone/million cells (Fig. 2b). Analysis of single clones showed that each expressed ALK proteins with a wide range of sizes from approximately 60 kDa to 400 kDa, likely indicating formation of fusion oncoprotein in different sizes (Extended Data Fig. 1f). In contrast, when we introduced a DSB in intron 6 of EML4 gene, no ALK expression was observed in osimertinib-resistant clones (Extended Data Fig. 1g), indicating that the DSB at ALK is critical in fusion formation. The ALK fusions formed after the DSBs at ALK intron 19 showed a strong phosphorylation of the kinase domain, which resulted in sustained activation of the MAPK pathway despite the presence of osimertinib (Fig. 2c), explaining the mechanism of resistance. Both ALK and ERK1/2 phosphorylation were completely blocked by adding ALK-specific inhibitor lorlatinib (Fig. 2c). Consistently, the growth of osimertinib-resistant clones was inhibited by lorlatinib (Fig. 2d). These data showed that a DSB in ALK led to the formation of in-frame fusions, of which expression conferred resistance to osimertinib.
Next, we identified unknown 5’ partner genes of these ALK fusions by using a 3’ end-directed fusion assay26. Several in-frame ALK fusions were identified (Fig. 2e and Supplementary Table 4), and a subset of them was further validated in single clones using RT-PCR (Extended Data Fig. 1h). Three of these fusion partners, EML4, STRN, and ATIC, are on chromosome 2, where ALK is located, whereas others were spread in the genome (Fig. 2e and Extended Data Fig. 1i). Remarkably, several of these spontaneous ALK fusions were identical to those described in NSCLC27 or in other tumor types4 (Supplementary Table 4). For example, EML4-ALK fusions joined e2, e6, e13, or e18 of the EML4 gene to e20 of the ALK gene (Fig. 2e and Supplementary Table 4), exactly as seen in patients with NSCLC and other tumors28,29. In addition, some fusion partners showed exclusive exon usage, such as e3 in STRN and e31 in CLTC, while others showed variable usage (Fig. 2e and Supplementary Table 4). These exon usages were identical to the corresponding ALK fusions found in NSCLC and thyroid cancer (Supplementary Table 4). We also identified a list of new ALK fusions that have not been described in human tumors (Supplementary Table 4), which may indicate rare functional fusion events yet to be discovered. Several of them (e.g., QKI, TRAF2, TRAF3, and TP53BP1) were the genes previously reported in fusions with other kinases30–32 and contain dimerization or oligomerization domains, further supporting their functionality (Extended Data Fig. 1j and Supplementary Table 4). Taken together, FACTS demonstrated functional fusion oncogene formation through genome-wide translocations after a single DSB at ALK intron 19 and reproduced ALK fusion landscape in human cancers.
To test whether oncogenic ALK fusions can also be generated in non-cancerous cells, we applied FACTS to the bronchial epithelial BEAS-2B cells. These cells can grow in vivo once they are transformed by oncogenic drivers33. As a positive control, we injected mice with BEAS-2B cells where two DSBs were induced in EML4 intron 6 or 13 and ALK intron 19. As expected, all mice in this group developed tumors (Extended Data Fig. 1k-m). When we injected mice with BEAS-2B where a single DSB was introduced in ALK intron 19, we observed tumor formation at a lower rate and with a slower growth kinetics (Extended Data Fig. 1l, m). FACTS and RT-PCR validation revealed that tumors expressed various ALK fusions identical to those in PC-9 cells and patient samples (Extended Data Fig. 1n and Supplementary Table 4). Protein expression of these fusions was most likely determined by the fusion partner, with some fusions being expressed at higher levels than others (Extended Data Fig. 1o, p). Thus, by applying FACTS to immortalized normal-like bronchial epithelial cells, we demonstrated that a single DSB in ALK intron 19 produced functional ALK fusion oncogenes that led to a malignant transformation in vivo.
FACTS identifies oncogenic RET, ROS1, and NTRK1 fusions
We next applied FACTS to other kinase fusions. RET, ROS1, and NTRK family gene fusions are found in approximately 4% of patients with NSCLC34. We designed FACTS by introducing one DSB in their intron that is most frequently involved in chromosomal translocations, (i.e., intron 11 for RET, intron 33 for ROS1, and intron 11 for NTRK1; Extended Data Fig. 2a-d). Resistant clones developed after 4 weeks of osimertinib selection at a frequency comparable to what was observed from clones with ALK fusion (Fig. 2b and Extended Data Fig. 2e). FACTS identified several in-frame chimeric proteins with RET, ROS1, and NTRK1 and their joined partners across the genome (Extended Data Fig. 2f-n and Supplementary Table 4). We validated some of these acquired fusions by RT-PCR and Sanger sequencing and confirmed that they were identical to the RET fusions described in patients with NSCLC (Extended Data Fig. 2o, p). Other fusions were novel and not yet described in patients (Supplementary Table 4). We confirmed that acquired RET fusions conferred resistance to osimertinib, as demonstrated by the reversal of resistance phenotype by selpercatinib (Extended Data Fig. 2q). The fusion partner genes identified here also contained dimerization or oligomerization domains (Extended Data Fig. 2i-k and Supplementary Table 4). Intriguingly, FACTS identified exon fusion variants involving different ROS1 exons (e34, e35, and e36) or NTRK1 exons (e12 and e13) but only e12 of RET (Extended Data Fig. 2f-h and Supplementary Table 4), which is consistent with what was reported from patients27,35–37.
Gene transcription, rather than chromatin accessibility, dictates the selection of partner genes in TK fusions
Next, we investigated how ALK fusions are selected among many potential rearrangements, and which mechanistic factors dictate the choice of ALK fusion partners in the genome. Among all reported partner genes of ALK fusion from the patients with NSCLC, those identified by FACTS in our PC-9 model showed a significantly higher level of transcription (Extended Data Fig. 3a, b). In contrast, we found no difference in terms of chromatin accessibility measured by ATAC-seq or histone activation marks between the FACTS-identified and -unidentified genes (Extended Data Fig. 3c, d). Recurrent translocation partners consistently showed active chromatin marks (Extended Data Fig. 3e-j). These findings were consistent in partner genes of RET, ROS1, and NTRK1 fusions35–37 (Extended Data Fig. 3k-p). Taken together, the partner genes selected in FACTS were associated with higher level of transcription, compared to the other partner genes not identified by FACTS but reported in patients.
Next, we further explored whether gene transcription was sufficient to induce the formation of oncogenic fusions. PC-9 cells express very low to undetectable levels of HLA-DR molecules and the invariant chain CD74 that is essential for the assembly and subcellular trafficking of the MHC class II complex38 (Extended Data Fig. 4a-d). We hypothesized that this undetectable expression could explain why CD74 or HLA-DR fusions with kinases39 were not identified by FACTS in PC-9 cells. Because expression of both CD74 and HLA-DR can be induced by the Class II transactivator (CIITA)40 (Extended Data Fig. 4e), we asked whether induction of HLA-DR or CD74 expression by CIITA was sufficient to generate fusions of HLA-DR or CD74 with ROS1. FACTS was applied to PC-9 cells expressing CIITA that showed significantly increased HLA-DR and CD74 mRNA and protein levels (Extended Data Fig. 4f-m). By introducing DSBs in intron 33 of ROS1 (Extended Data Fig. 5a), we identified genome-wide oncogenic fusions including HLA-DRB1-ROS1 fusions in which the breakpoint in the HLA-DRB1 gene was identical to that observed in patients with HLA-DRB1-MET fusion39 (Extended Data Fig. 5b, c and Supplementary Table 5). We estimated the frequency of HLA-DRB1-ROS1 fusions at 6.7% using single clone analysis (Extended Data Fig. 5d). In contrast to HLA-DRB1-ROS1 fusions, CD74-ROS1 fusions were not detected, which suggests that induction of transcription for the CD74 gene was not sufficient to trigger CD74-ROS1 translocations. However, when we simultaneously introduced DSBs in both CD74 and ROS1 genes in either control PC-9 or CIITA-expressing PC-9 cells, resistant clones rapidly emerged only in CIITA-expressing PC-9 cells (Extended Data Fig. 5e, f). While DNA junctions were detected in both cells, CD74-ROS1 fusion transcripts were detected only in CIITA-expressing PC-9 cells (Extended Data Fig. 5g-i). These results suggest that increased gene expression of the partner gene is sufficient to induce the formation of TK fusions in loci such as HLA-DRB1, which is located on the same chromosome with ROS1, and point out that the detection of DNA junctions is insufficient to determine oncogenicity of resulting TK fusions without evidence of efficient transcription of the TK fusion.
Oncogenic TK fusions originate after selection of pools of rearrangements spontaneously occurring in fusion partner and TK genes
In some tumors such as lymphoma, recurrent translocations are the result of the activity of the activation-induced cytidine deaminase (AID) enzyme that targets specific regions of the genome41. Therefore, we asked whether the selection of specific partners or exons by TK fusions is mechanistically determined by the formation of DSBs in specific positions of genes or rather by the selection of random genomic DSBs. To this end, we generated libraries of DNA junctions by HTGTS which allows for an unbiased detection of genome-wide chromosomal rearrangements before selection17 (Extended Data Fig. 6a, b). HTGTS yielded 111,811 genomic translocation breakpoints before selection, which were distributed throughout the genome with enhanced clustering in the 2 Mbp regions surrounding the ALK DSB (Fig. 3a-c and Extended Data Fig. 6c). We identified 154 hotspots with significantly enriched breakpoint clustering (Fig. 3d and Supplementary Table 6). Only 2.6% (4/154; EML4, SQSTM1, TRAF2, and CLTC) of these hotspots occurred in genes that are known partners of ALK fusions (Fig. 3d and Supplementary Table 6). In sharp contrast, HTGTS performed with resistant clones after osimertinib selection yielded 5,005 DNA breakpoints with 81% hotspots (13/16) occurring in genes leading to the transcribed ALK fusions identified by FACTS (Fig. 2e and Fig. 3a and Supplementary Tables 4 and 6). Several strong genomic translocation hotspots observed before selections completely disappeared after selection (Fig. 3e-g and Extended Data Fig. 6d), most likely because the resulting rearrangements did not generate a functional ALK fusions. EML4 was the gene most frequently translocated with ALK after selection (Fig. 3h). Breakpoints before selection did not show a preferential strand bias, which is consistent with previous works of genome-wide cloning of unselected translocations17,42. In contrast, the breakpoints after selection showed a strong bias for an orientation of the gene leading to a functional fusion with ALK (Fig. 3i), with DNA breakpoints markedly enriched for junctions occurring in gene introns (Fig. 3j). Within individual partner genes, we observed a selective enrichment of breakpoints occurring in introns leading to in-frame functional fusions with ALK (Fig. 3k and Extended Data Fig. 6e-h). Overall, these data indicate that the formation of ALK fusion is the result of a functional selection of transcribed translocations based on the location and orientation, not just a reflection of DNA break frequency.
Next, we focused on TK genes and generated HTGTS libraries in BEAS-2B and PC-9 cells by inducing a DSB in EML4 as bait to capture breaks spontaneously occurring in TK genes (Extended Data Fig. 7a). We looked at the distribution of breakpoints in ALK, RET, ROS1, NTRK1, as well as other kinase genes known to generate oncogenic fusions in lung cancer, such as EGFR, ERBB4, MET, FGFR3, and EPHA243. Breakpoints identified in these kinases were evenly spread throughout the gene body including introns and exons without clear clusters (Extended Data Fig. 7b-j). In both BEAS-2B and PC-9 cells, more breakpoints were observed in ALK than in other kinases (Extended Data Fig. 7k, l and Supplementary Table 7), most likely because EML4 and ALK are proximally located on the same chromosome 244,45. More breakpoints in EGFR were detected in PC-9 cells than in BEAS-2B cells (Extended Data Fig. 7f, k, l), most likely due to the presence of > 4 copies of the EGFR gene in PC-922, and frequent breakpoints were observed also in EPHA2 gene, which is highly transcribed in these cells (Extended Data Fig. 7j, l). All combined, these data suggest that the preferential usage of specific partners or exons during oncogenic TK fusion formation is the result of a selection process among multiple combinations of junctions created by DSBs spontaneously generated in the genome, rather than due to the presence of pre-existing clusters of breakpoints like in the case of AID-initiated translocations.
Protein stability determines the selection of TK fusion partners
Next, we investigated the process of TK fusion selection. Consistent with the COSMIC analysis in patients, TK fusion partners obtained by FACTS were mutually exclusive in most cases30,40–42, with only a few partners shared by multiple TK fusions (Fig. 4a and Supplementary Table 4). Interestingly, some fusion partner genes, such as TPM3 and ETV6, used the same exons when they generated oncogenic fusions with different TKs (Fig. 4b). Thus, we explored functional basis of fusion-partner specificity to each kinase. We engineered all combinations of EML4 and CD74 fusions with ALK, RET, ROS1, and NTRK1 (Extended Data Fig. 8a, b). While all of the fusion junctions were detected equally at the genomic DNA levels (Extended Data Fig. 8c,d), some of the kinase fusion combinations did not yield resistant clones under osimertinib selection (Fig. 4c,d). While thousands of EML4-ALK, EML4-RET, or EML4-NTRK1 clones rapidly emerged, no clones with EML4-ROS1 fusions were observed (Fig. 4c). Likewise, while thousands of CD74-ROS1 clones emerged, no clones with CD74-ALK, CD74-RET, or CD74-NTRK1 fusions emerged in PC9 cells expressing CIITA (Fig. 4d). Next, we isolated single cell-derived clones harboring different fusions for further characterization (Extended Data Fig. 8e). Clones with CD74-ROS1 fusion displayed abundant protein that was phosphorylated as expected, but clones with EML4-ROS1 fusion showed very low abundance of the EML4-ROS1 protein that was also poorly phosphorylated (Fig. 4e). Treatment with proteasome inhibitor MG132 stabilized the EML4-ROS1 fusion protein and its phosphorylation substantially increased (Fig. 4e). Crizotinib, primarily a MET inhibitor with an activity on ROS1, inhibited the growth of clones harboring CD74-ROS1 fusions but not EML4-ROS1 fusions, suggesting that only stable and abundant kinase fusions could create oncogenic dependency (Fig. 4f).
Protein stability determines the specific exon usage of oncogenic TK fusions
An additional finding of COSMIC analysis was the preferential usage of specific exons in TK fusions (Fig. 1e, f). To better understand molecular basis of preferential exon usage in kinase fusions, we engineered EML4-ALK variants by CRISPR/Cas9 that fuse the same EML4 exon 6 to different ALK exons (e18, e19, or e20) (Extended Data Fig. 8f). All these fusions are predicted to be in frame, which could potentially lead to functional ALK fusions. However, these different fusion variants have been detected in different frequencies in patients, the EML4-ALK E6;A20 fusion being far more frequent than the E6;A18 or E6;A19 fusions (less than 1% among ALK fusions)46,47 (Fig. 1e), which was consistently observed in FACTS (Fig. 5a). To understand the cause of these differences, we generated clonal lines for each fusion variant. The mRNA transcription levels were comparable among the variants, likely due to their regulation by the same promoter (Fig. 5b and Extended Data Fig. 8g). However, the protein abundance and the level of phosphorylation were markedly different (Fig. 5c and Extended Data Fig. 8h). The E6;A20 fusion protein was highly expressed and phosphorylated, whereas the E6;A18 or E6;A19 fusions were much less abundant with barely detectable phosphorylation (Fig. 5c and Extended Data Fig. 8h). Consequently, the E6;A20 variant showed a greater potency in rescuing MAPK pathway activation compared to the E6;A18 or E6;A19 fusions in osimertinib-treated PC-9 cells (Fig. 5c). Treatment with MG132 stabilized the E6;A18 or E6;A19 fusions and led to their phosphorylation (Fig. 5d). Next, we investigated whether the different functional features of these EML4-ALK fusion variants were due to differences in subcellular localization, given recent evidence showing that the oncogenic activity of EML4-ALK is dependent on its subcellular localization and formation of protein granules in the cell cytoplasm48. The three EML4-ALK fusion variants showed comparable intracellular localization in confocal microscopy analysis (Extended Data Fig. 8i, j), with weaker signals with the E6:A18 or E6:A19 fusions, likely due to their low protein abundance. Functional assay showed that lorlatinib inhibited the growth of cells harboring E6;A20 fusions but not of the E6;A18 and E6;A19 fusions, suggesting that only E6;A20 fusions are stable enough to confer oncogenic dependence (Fig. 5e). These findings imply that the usage of specific exons in TK fusions is likely dictated by protein stability rather than transcription or subcellular localization of the resulting fusions, and that only an abundant expression of TK fusion proteins creates a dependency that might determine the efficacy of TKI treatment.
TKI therapy is less effective in patients with atypical ALK fusions
Since atypical ALK fusions showed reduced functionality and oncogenic signaling in PC-9 cell models (Fig. 5c, e), we investigated whether these findings were reflected in patients by studying clinical responses to ALK TKIs in patients carrying either typical or atypical ALK fusions. We analyzed 108 patients with metastatic NSCLC who tested positive for ALK fusions by next-generation sequencing (NGS) and received ALK TKI treatment and divided them into two groups based on the ALK gene fusion breakpoints: typical (breakpoints in ALK intron 19) and atypical (breakpoints in other ALK introns/exons or atypical fusion partner). There were 97 typical ALK fusions with ALK breakpoints in intron 19, and 11 atypical fusions cases with breakpoints in introns 16, 17, 18, and 20 or inside exon 20 (Extended Data Fig. 9a and Supplementary Table 8). Patients with atypical ALK fusions had clinical characteristics comparable to patients with typical ALK fusions in terms of age, gender, smoking history, ECOG performance status, and ALK inhibitor treatment (Extended Data Fig. 9b). The typical ALK fusion group had 88.7% (88/97) of EML4-ALK fusions or other known oncogenic ALK fusions, such as HIP1-ALK49, whereas the group of atypical ALK fusions was composed of 54.5% (6/11) of EML4-ALK fusions with non-intron 19 breakpoints (Extended Data Fig. 9c) or ALK fusions with atypical partners. Strikingly, the atypical group showed significantly lower objective response rate (ORR) to ALK TKI compared to the group of patients with typical ALK fusions (54.5% versus 88.7%, p = 0.01) (Extended Data Fig. 9d), resulting in a significantly shorter progression-free survival (PFS; 5 months versus 20.5 months, HR: 0.18 [95% CI: 0.08–0.38], p < 0.001) and overall survival (OS; 20.5 months versus 83.0 months, HR: 0.20 [95%CI: 0.09–0.45], p < 0.001) (Fig. 6a,b). We also confirmed that atypical ALK fusions retained a significant association with shorter PFS and OS after adjusting for potential confounders in multivariable Cox regression models (Extended Data Fig. 9e). We further examined co-occurring mutations in cases with typical or atypical ALK fusions. The most frequently mutated gene was TP53 in typical and atypical ALK fusions, with a significantly higher frequency in atypical fusions (62.5% versus 25.9%, p = 0.046), which may have also contributed to the worse outcomes to ALK TKIs observed in this subset of patients (Fig. 6c). In addition, atypical ALK fusions were associated with a higher rate of mutations in alternative oncogenic driver genes, including BLM, FLT4, RAF1, RB1, and TCF351, compared to typical ALK fusions (Fig. 6c and Extended Data Fig. 9f). Overall, these results demonstrate that atypical TK fusions are weaker oncogenic driver, are associated with increased co-mutation of other oncogenes, and respond poorly to ALK inhibition, providing a biomarker predictor for response to ALK TKI in patients.