Detailed methods are provided in our previous studies. We created a fyve zinc finger domaintagged cd74 ros1 construct to relocalize the fusion protein to endosomes. Ampliseq for illumina rna fusion lung cancer research panel. The recent association of four oncogenic driver genes, alk, ros1, ret, and ntrk1, as lung tumor predictive biomarkers has increased the need for development of uptodate technologies for detection of these biomarkers in limited amounts of material. Among the 50 egfr wild type patients who detected fusion genes, 14 carried eml4alk fusion 28%, 2 had tpm3ros1 fusion 4%, and 3 harbored kif5bret fusion 6%. A fusion is when two different things are stuck together. In this case, part of the ros1 gene is fused to part of another gene. The transcriptional silencing in ros1 mutants can be released by the ddm1 mutation or the application of the dna. Multiplexed analysis of gene fusion events associated with leukemia and lung cancer. Fiftyfive patients harbored ros1 fusion gene that was detected by rtpcr, and 14 of them were treated with crizotinib. The gene fusion panels utilize ncounter elements reagents. Most laboratories currently rely on fluorescence in situ hybridisation fish assays using a. Ros1 fusions in chinese patients with nonsmallcell lung.
A ros1 fusion is a protein made from a gene fusion. A singletube multiplexed assay for detecting alk, ros1, and. Crizotinib in ros1rearranged nonsmallcell lung cancer nejm. Currently, the gold standard method for gene fusion detection is fluorescence in. Infection of 3t3 cells with the virus expressing ki f5balk, tpm3 ros1, sdc4 ros1, slc34a2 ros1, cd74 ros1, ezr ros1, lrig3 transcript varian t 2 ros1, or kif5bret both th e longer and shorter isoforms of ret led to the formation of multiple transformed. Ros1, a repressor of transcriptional gene silencing in. Ros1, a gene encoding for a receptor tyrosine kinase, is known to undergo rearrangement in many cancer types, including nsclc. Apr 05, 2018 t o verify detection of fusion variants, use commercially available celllines expressing the ros1 or ret fusion mrna of interest. Differential subcellular localization regulates oncogenic.
A fusion gene is a hybrid gene formed from two previously independent genes. An nsclc tissue microarray tma panel containing 447 samples was screened for ros1. Us9526722b1 us14744,042 us201514744042a us9526722b1 us 9526722 b1 us9526722 b1 us 9526722b1 us 201514744042 a us201514744042 a us 201514744042a us 9526722 b1 us9526722 b1 us 9526722b1 authority us united states prior art keywords ros1 compound formula cells present invention prior art date 20150619 legal status the legal status is an assumption and is not a legal. Unlikefigros1,cd74ros1 and slc34a2ros1 result from translocation of the ros1locus. Jul 25, 2017 although the failure rate for the ncounter rna gene fusion assay is not negligible 20%, 22 ncounter analysis is highly concordant with fish and ihc and thus is very specific. Ros1 rearrangement is a validated therapeutic driver gene in non. Aug 16, 2018 gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. Two novel ros1 fusion partners, cd74 and solute carrier protein slc34a2, were identi.
Analysisofreceptortyrosinekinaseros1positivetumors in. Detection of known and novel alk fusion transcripts in lung. Alk and ros1 testing on lung cancer cytologic samples. Aug 17, 2016 however, as the use of ros1 inhibitors becomes routine, accurate and timely detection of ros1 gene rearrangements will be critical for the optimal treatment of patients with nsclc. Identifying and targeting ros1 gene fusions in nonsmall cell. Archers alk ret ros1 kit offers a complete fusion detection solution, from library preparation through data analysis. Ros1 is a receptor tyrosine kinase encoded by the gene ros1 with structural similarity to the anaplastic lymphoma kinase alk protein. Clinicopathologic characteristics of patients with ros1. A bloodbased test for the detection of ros1 and ret. The protein encoded by this gene is a type i integral membrane protein with tyrosine kinase activity. Fusion genes have been found to be prevalent in all main types of human neoplasia. Ros1 genetic rearrangements, both inter and intrachromosomal, lead to constitutive activation of the ros1 kinase14. Sep 01, 2012 in order to confirm expression of a ros1 fusion gene and to identify the fusion partner in this patients tumor, we performed the same rtpcr assays as for the tma samples above. The former two appeared to occur in nsclc with higher frequency when compared with other fusion patterns.
Archers technology is uniquely suited to detecting these mutations because they involve many potential breakpoints and multiple possible fusion partners. All 18 studies contributed to the analysis of association between ros1 rearrangement and gender. In the ros1 mutants, the promoter of the silenced loci is hypermethylated, which may be triggered by small rnas produced from the transgene repeats. This was the first example of a genomic event that leads to the formation of a receptor tyrosine kinase rtk fusion protein in an astrocytoma. Japanese regulators approve amoydxs ros1 fusion cdx for lung. In the present cohort of patients, 109 15% were younger than 50 years of age. Nsclc patients, the identity of the oncogenic driver remains elusive. T o verify detection of fusion variants, use commercially available celllines expressing the ros1 or ret fusion mrna of interest. Importantly, recent studies have shown that rare fusion events such as those involving ntrk1, ntrk2, and ntrk3 can potently drive tumorigenesis.
Mutations in the arabidopsis ros1 locus cause transcriptional silencing of a transgene and a homologous endogenous gene. Approximately 2% of nsclc tumors harbor alterations in the ros1 gene. Now that we have introduced ros1, we will introduce some of its fusion partners. This page is a portal describing the biology of specific ros1 gene rearrangements ros1 fusions with other genes that result in the formation of cancer driving fusion proteins.
Identifying and targeting ros1 gene fusions in nonsmall. Ros1 fusions, which have been found to be potential driver mutations in a. Fusion positive droplets and gusb control gene droplets were observed for ros1 and ret in all 21 runs executed over the course of the study figure 3d,h. A fusion gene is a hybrid gene formed from two previously separate genes. The function of these house keeping proteins is difficult to understand for the non biologist. Collectively,studiesinglioblastoma,nsclcs, and cholangiocarcinoma associate ros1 gene rearrange. Oncogenic gene fusions involving the 3 region of ros1 kinase have. We created a fyve zinc finger domaintagged cd74ros1 construct to relocalize the fusion protein to endosomes.
It can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Entrez gene summary for ros1 gene this protooncogene, highlyexpressed in a variety of tumor cell lines, belongs to the sevenless subfamily of tyrosine kinase insulin receptor genes. Save time and sample material with multiplexed gene fusion detection in a single tube. Alk, ros1 and ret gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. A singletube multiplexed assay for detecting alk, ros1. The most common ros1 fusion partner that we identified was the gene encoding cd74, which was present in 11 of 25 samples 44%. C expression of the cd74 ros1 or sdc4 ros1 fusion gene products in tg mice. Junction sites are depicted as filled flags commonly occurring and unfilled flags less frequent. Comparison of in situ and extractionbased methods for the. Because little is known about these tumors, we examined the clinical characteristics and treatment outcomes of patients with nsclc with ros1 rearrangement. Fish reveals normal cells, which produce a yellow fusion signal, together with cancer cells, featuring green and red signals are separated by. Supplementary information ret, ros1, and alk fusions in lung.
Crizotinib, a highly effective inhibitor of ros1 kinase activity, is now fdaapproved for the treatment of patients with advanced ros1positive nsclc. Formaldehyde analysis gene fusion immunohistochemistry lung cancer, nonsmall cell messenger rna molecular diagnostic. Pdf a bloodbased test for the detection of ros1 and ret. Cd74 contains a 15 amino acid nterminal cytoplasmic extension, which anchors it into the er and which is included in the cd74 ros1 fusion gene used in our studies. Combined with direct digital counting on the ncounter analysis system and our patented probe design, the detection of multiple fusion genes is highly sensitive, quantitative and easy. The protein made by the fusion contains parts from each gene. The identification of these fusion genes play a prominent role in being a diagnostic and. These driver mutations can be categorized into mutations in or near the kinase domain, gene amplification or fusion. Part of the theme is that troubles ensues when a rarely expressed kinase gene gets fused to a house keeping gene. However, we found this patients sample to be negative for both the slc34a2ros1 and cd74ros1 fusions fig.
Breakpoint analysis of transcriptional and genomic. Approximately 7% of nonsmall cell lung carcinomas nsclcs harbor oncogenic fusions involving alk, ros1, and ret. Oncogenic gene fusions involving the 3 region of ros1 kinase have been identified in various human cancers. The recent association of four oncogenic driver genes, alk. The gene fusion is in frame, and preserves the tyrosine kinase domain of ros1, but removes its transmembrane and extracellular domains. Supplementary information ret, ros1, and alk fusions in lung cancer kengo takeuchi, 1,2 manabu soda,3 yuki togashi, 1,2 ritsuro suzuki,4 seiji sakata,1,2 satoko hatano,1,2 reimi asaka, 1,2 wakako hamanaka,2 hironori ninomiya,2 hirofumi uehara,5 young lim choi, 6 yukitoshi satoh,5 sakae okumura,5 ken nakagawa,5 hiroyuki mano,3,6 yuichi ishikawa. The fusion gene slc34a2ros1 solute carrier family 34 member 2 and. Ros1 gene rearrangements are associated with an elevated. Here we report the identification of alk, ros1, fgfr2 fusions in lung cancer pdx models and a nrg1 fusion in the ovarian cancer models by rna using sequencing data combined with the chimerascan and star fusion software for fusion calling the gene fusions, as validated by pcr and sanger sequencing, led to overexpression of the affected. We screened for known ros1 fusions, except figros1, kdelr2ros1 and ccdc6ros1, in nsclc using a multiplex rtpcr, and identified slc34a2ros1 fusion in four cases, cd74ros1 fusion in three cases and sdc4ros1 fusion in one case. Rearrangements of the ros1 gene occur in 12 % of nonsmall cell lung cancers nsclcs. The mrnas of ros1 and alk fusion were examined by using polymerase chain reaction technique and fusion gene detection kit. Formaldehyde analysis gene fusion immunohistochemistry lung cancer, nonsmall cell messenger rna molecular diagnostic techniques. Fusion proteins used to be called chimeras after the creature of greek mythology.
Purpose chromosomal rearrangements involving the ros1 receptor tyrosine kinase gene have recently been described in a subset of nonsmallcell lung cancers nsclcs. Full text high feasibility of cytological specimens for. In ros1 fusions, the kinase domain of ros1 is never silenced signal telling the cell to grow. Among the 50 egfr wild type patients who detected fusion genes, 14 carried eml4alk fusion 28%, 2 had tpm3 ros1 fusion 4%, and 3 harbored kif5bret fusion 6%. Amoydx ros1 fusion kit detects 14 ros1 gene fusions, which uses rtpcr technology to analyze tumor messenger rna from human tumor tissue or body fluids, and can provide results within 2. Association between ros1 fusion gene and gender in nsclc patients. Circular rna fcircsr derived from slc34a2ros1 fusion gene. Pcr helps find gene fusions in lung cancer xxpresspcr. Up to 36% of patients with ros1 fusion positive nsclc have brain metastases at the diagnosis of advanced disease. Schematic representation of alk a, ros1 c, and ret e mrnas and locations of gene expression probe sets relative to known fusion junction sites. Entrectinib in ros1 fusionpositive nonsmallcell lung. As ngs techniques are introduced into routine diagnostic practice, ros1 fusion gene testing will be provided as part of the initial testing package. Comparison of molecular testing modalities for detection. Alkretros1 gene fusion panel custom panels fusions of these genes are of high interest, as they represent an important class of genomic rearrangements in cancer.
Multiplexed transcriptome analysis to detect alk, ros1 and. In lung cancer, mutations in egfr 8, braf, kras, and pik3ca constitute important biomarkers to guide clinical care, and gene fusion events involving alk, ros1, and ret are also frequently identified. The identification of these fusion genes play a prominent role in being a diagnostic and prognostic marker. Ros1 gene rearrangements are associated with an elevated risk.
A bloodbased test for the detection of ros1 and ret fusion. Detection of fusion genes is critical for determining the best treatment. The ros1 gene is located on 6q22 and encodes a receptor tyrosine kinase of the insulin receptor family. Comparison of molecular testing modalities for detection of ros1. The panel is composed of 83 pairs of unique primers in a single pool that includes.
Although tumors harboring alk fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ros1 or ret fusions may also benefit from inhibitors targeting these kinases. Analysisofreceptortyrosinekinaseros1positivetumors in non. This research panel targets over 70 known lung cancerrelated fusion transcripts for the alk, ret, ros1, and ntrk genes community panel features. However, we found this patients sample to be negative for both the slc34a2 ros1 and cd74 ros1 fusions fig. Patients with nonsmall cell lung cancer harboring alk receptor tyrosine kinase alk, ros protooncogene 1 ros1, and ret protooncogene ret gene rearrangements can benefit from specific kinase inhibitors. Cd74 contains a 15 amino acid nterminal cytoplasmic extension, which anchors it into the er and which is included in the cd74ros1 fusion gene used in our studies. Detecting alk, ros1 and ret fusion genes in cell block samples. Archer sureshot alk ret ros1 controls are formalinfixed, paraffinembedded ffpe curls of homogeneously mixed, genetically defined alk, ret and ros1 fusionpositive cell lines. Ros1 rearrangements define a unique molecular class of.
Results females were more often inflicted by the egfr mutations, especially for the exon 19 deletion and l858r mutation. After identifying patients with the ros1 fusion gene, they then compared the demographic data and clinical outcomes of patients with patients without the gene, including those with the egfr mutation, eml4alk fusion, and kras mutation, and patients who were negative for all four genes. The transcriptional silencing in ros1 mutants can be released by the ddm1 mutation or the application of the dna methylation. Identification of alk, ros1, and ret fusions by a multiplexed mrnabased assay in formalinfixed, paraffinembedded samples from advanced nonsmallcell lung cancer patients. Rna was reversed to cdna, added to reaction buffer and then alk, ros1 and ret fusion genes were detected using eml4alk, ros1 and ret fusion gene detection kit amoydx, xiamen, china respectively by arms method as mentioned above. An nsclc tissue microarray tma panel containing 447 samples was screened for ros1 rearrangement by fish.
Molecular diagnostics and genetics, report by clinical chemistry. High feasibility of cytological specimens for detection of. Aug 08, 2018 ros1 positive cancers are caused by the gene ros1 fusing with one of a number of partner genes. All negative controls no rt and nrc yielded negative results across the entire 21 days data not shown. Ortenberg, germany was used to measure luminescence. Identification of alk, ros1, and ret fusions by a multiplexed. In smoking males, the chance to get an ros1 fusion is extremely rare 0 of 412 in our series. Simultaneous detection of gene fusions and base mutations. Combined with direct digital counting on the ncounter system and junction sequence probe design, the detection of fusion genes is highly sensitive, quantitative, and easy. Comparison of molecular testing modalities for detection of.
Entrectinib is a ros1 inhibitor that has been designed to effectively penetrate and remain in the cns. Breakpoint analysis of transcriptional and genomic profiles. Supplementary information ret, ros1, and alk fusions in. Crizotinib in ros1rearranged nonsmallcell lung cancer.
To drive cancer, ros1 fusion proteins must have the kinase domain of ros1 and a myriad of domains from their fusion part n ers. Gene fusions play an important role in the carcinogenesis of lung adenocarcinoma. Ros1positive cancers are caused by the gene ros1 fusing with one of a number of partner genes. The highest probability of detecting a fusion involving the ros1 gene was in nonsmoking females 17%, followed by smoking females 6. All the fusion positive samples were confirmed by dna sequencing. Patients with nonsmall cell lung cancer harboring alk receptor tyrosine kinase alk, ros protooncogene 1 ros1, and ret protooncogene ret gene rearrangements can benefit from speci. In this study, we sought to characterize ros1 fusion genes in nonsmall cell lung cancer nsclc and establish the fusion proteins as drug targets. A total of 5,409 male patients and 4,468 female patients were included, and ros1 rearrangement was detected in 85 males 1.
No prior knowledge of fusion partners and breakpoints required detection of known and novel gene fusions from as little as 20ng input nucleic acid. This cytologic smear was digitally scanned before fish analysis and reveals adenocarcinoma cells. Ros1 fusion proteins are constitutively active and drive downstream. Ros1 gene fusions are a wellcharacterized class of oncogenic driver found in approximately 1% to 2% of nsclc patients. Recurrent gene fusions, such as ros1 fusions, are oncogenic drivers of various cancers, including nonsmallcell lung cancer nsclc. The resulting ros1fig fusion protein is a constitutively activated tyrosine kinase.
Targeting ros1 fusion proteins with the small molecule inhibitor crizotinib is showing promise as an effective therapy in nsclc patients whose tumors are positive for these genetic abnormalities. Curls contain eml4alk, ccdc6ret, and slc34a2ros1 fusions. Up to 36% of patients with ros1 fusionpositive nsclc have brain metastases at the diagnosis of advanced disease. Ampliseq for illumina community panels are madetoorder. Mouse models for ros1fusionpositive lung cancers and their.
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