FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2086939994> ?p ?o ?g. }

• W2086939994 endingPage "61" @default.
• W2086939994 startingPage "51" @default.
• W2086939994 abstract "Anaplastic lymphoma kinase gene (ALK) fusions have been identified in approximately 5% of non-small-cell lung carcinomas (NSCLCs) and define a distinct subpopulation of patients with lung cancer who are highly responsive to ALK kinase inhibitors, such as crizotinib. Because of this profound therapeutic implication, the latest National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology recommend upfront ALK screening for all patients with NSCLC. The Food and Drug Administration–approved companion diagnostic test (ie, fluorescence in situ hybridization) for identification of ALK-positive patients, however, is complex and has considerable limitations in terms of cost and throughput, making it difficult to screen many patients. To explore alternative screening modalities for detecting ALK fusions, we designed a combination of two transcript-based assays to detect for presence or absence of ALK fusions using NanoString’s nCounter technology. By using this combined gene expression and ALK fusion detection strategy, we developed a multiplexed assay with a quantitative scoring modality that is highly sensitive, reproducible, and capable of detecting low-abundant ALK fusion transcripts, even in samples with a low tumor cell content. In 66 archival NSCLC samples, our results were highly concordant to prior results obtained by fluorescence in situ hybridization and IHC. Our assay offers a cost-effective, easy-to-perform, high-throughput, and FFPE-compatible screening alternative for detection of ALK fusions. Anaplastic lymphoma kinase gene (ALK) fusions have been identified in approximately 5% of non-small-cell lung carcinomas (NSCLCs) and define a distinct subpopulation of patients with lung cancer who are highly responsive to ALK kinase inhibitors, such as crizotinib. Because of this profound therapeutic implication, the latest National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology recommend upfront ALK screening for all patients with NSCLC. The Food and Drug Administration–approved companion diagnostic test (ie, fluorescence in situ hybridization) for identification of ALK-positive patients, however, is complex and has considerable limitations in terms of cost and throughput, making it difficult to screen many patients. To explore alternative screening modalities for detecting ALK fusions, we designed a combination of two transcript-based assays to detect for presence or absence of ALK fusions using NanoString’s nCounter technology. By using this combined gene expression and ALK fusion detection strategy, we developed a multiplexed assay with a quantitative scoring modality that is highly sensitive, reproducible, and capable of detecting low-abundant ALK fusion transcripts, even in samples with a low tumor cell content. In 66 archival NSCLC samples, our results were highly concordant to prior results obtained by fluorescence in situ hybridization and IHC. Our assay offers a cost-effective, easy-to-perform, high-throughput, and FFPE-compatible screening alternative for detection of ALK fusions. Chromosomal aberrations targeting the anaplastic lymphoma kinase gene (ALK), which resides on the short arm of chromosome 2, at 2p23, have been identified in various cancer types, including anaplastic large-cell lymphoma,1Morris S.W. Kirstein M.N. Valentine M.B. Dittmer K.G. Shapiro D.N. Saltman D.L. Look A.T. Fusion of a kinase gene, ALK, to a nucleolar protein gene NPM, in non-Hodgkin’s lymphoma.Science. 1994; 263: 1281-1284Crossref PubMed Scopus (1964) Google Scholar non-small-cell lung carcinoma (NSCLC),2Soda M. Choi Y.L. Enomoto M. Takada S. Yamashita Y. Ishikawa S. Fujiwara S. Watanabe H. Kurashina K. Hatanaka H. Bando M. Ohno S. Ishikawa Y. Aburatani H. Niki T. Sohara Y. Sugiyama Y. Mano H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4322) Google Scholar and inflammatory myofibroblastic tumors.3Griffin C.A. Hawkins A.L. Dvorak C. Henkle C. Ellingham T. Perlman E.J. Recurrent involvement of 2p23 in inflammatory myofibroblastic tumors.Cancer Res. 1999; 59: 2776-2780PubMed Google Scholar In these neoplasms, chromosomal translocations (or inversions for that matter) result in the (generally up-regulated) expression of an oncogenic ALK fusion protein mediating aberrant signal transduction, leading to uncontrolled cell growth. ALK, a receptor tyrosine kinase belonging to the insulin receptor superfamily, is believed to play a normal physiological role in murine brain development; in adult humans, the endogenous wild-type ALK expression is low and generally confined to the central nervous system.4Mosse Y.P. Laudenslager M. Longo L. Cole K.A. Wood A. Attiyeh E.F. Laquaglia M.J. Sennett R. Lynch J.E. Perri P. Laureys G. Speleman F. Kim C. Hou C. Hakonarson H. Torkamani A. Schork N.J. Brodeur G.M. Tonini G.P. Rappaport E. Devoto M. Maris J.M. Identification of ALK as a major familial neuroblastoma predisposition gene.Nature. 2008; 455: 930-935Crossref PubMed Scopus (1024) Google Scholar As a result of the ALK-targeting tumorigenic chromosomal anomalies, a chimeric ALK protein containing the ALK tyrosine kinase domain fused to the N-terminal region of its fusion partner becomes expressed. Through ligand-independent activation, ALK fusion proteins constitutively transmit signals via phosphatidylinositol 3-kinase/Akt and RAS/RAF/extracellular signal–regulated kinase signaling pathways, leading to enhanced cell survival and proliferation.2Soda M. Choi Y.L. Enomoto M. Takada S. Yamashita Y. Ishikawa S. Fujiwara S. Watanabe H. Kurashina K. Hatanaka H. Bando M. Ohno S. Ishikawa Y. Aburatani H. Niki T. Sohara Y. Sugiyama Y. Mano H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4322) Google Scholar, 5Takezawa K. Okamoto I. Nishio K. Janne P.A. Nakagawa K. Role of ERK-BIM and STAT3-survivin signaling pathways in ALK inhibitor-induced apoptosis in EML4-ALK-positive lung cancer.Clin Cancer Res. 2011; 17: 2140-2148Crossref PubMed Scopus (118) Google Scholar These ALK-driven tumors rely specifically on the fusion oncoprotein for continued growth, and define a distinct patient subgroup that greatly benefits from targeted ALK inhibition. ALK fusions to echinoderm microtubule-like protein 4 (EML4) are found in approximately 2% to 5% of non-preselected NSCLC cases,6Perner S. Wagner P.L. Demichelis F. Mehra R. Lafargue C.J. Moss B.J. Arbogast S. Soltermann A. Weder W. Giordano T.J. Beer D.G. Rickman D.S. Chinnaiyan A.M. Moch H. Rubin M.A. EML4-ALK fusion lung cancer: a rare acquired event.Neoplasia. 2008; 10: 298-302Abstract Full Text PDF PubMed Google Scholar, 7Solomon B. Varella-Garcia M. Camidge D.R. ALK gene rearrangements: a new therapeutic target in a molecularly defined subset of non-small cell lung cancer.J Thorac Oncol. 2009; 4: 1450-1454Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar and were first identified in a lung adenocarcinoma from a Japanese patient harboring a paracentric chromosomal inversion of the short arm of chromosome 2.2Soda M. Choi Y.L. Enomoto M. Takada S. Yamashita Y. Ishikawa S. Fujiwara S. Watanabe H. Kurashina K. Hatanaka H. Bando M. Ohno S. Ishikawa Y. Aburatani H. Niki T. Sohara Y. Sugiyama Y. Mano H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4322) Google Scholar This inversion fused the 5′ end of EML4 (exons 1 to 13) to the 3′ end (beginning at exon 20) of ALK. The resulting fusion (designated variant 1) contained N-terminal portions of EML4 fused to the entire ALK cytoplasmic tyrosine kinase domain. Since then, several alternative oncogenic fusions have been identified, all containing variable truncations in EML4, invariably fused to ALK exon 20. In addition, ALK fusions involving KIF5B (residing at chromosome 10p11) and TFG (residing at chromosome 3q21) have also been reported in NSCLCs but are found at much lower frequencies.8Rikova K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li D. Zhou X. Gygi S.P. Gu T.L. Polakiewicz R.D. Rush J. Comb M.J. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.Cell. 2007; 131: 1190-1203Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar, 9Sasaki T. Rodig S.J. Chirieac L.R. Janne P.A. The biology and treatment of EML4-ALK non-small cell lung cancer.Eur J Cancer. 2010; 46: 1773-1780Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 10Takeuchi K. Choi Y.L. Togashi Y. Soda M. Hatano S. Inamura K. Takada S. Ueno T. Yamashita Y. Satoh Y. Okumura S. Nakagawa K. Ishikawa Y. Mano H. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer.Clin Cancer Res. 2009; 15: 3143-3149Crossref PubMed Scopus (630) Google Scholar Crizotinib (PF-02341066), a dual MET/ALK-specific kinase inhibitor, has previously shown its ability to induce apoptosis in ALK fusion-positive cancer cell line xenografts11Christensen J.G. Zou H.Y. Arango M.E. Li Q. Lee J.H. McDonnell S.R. Yamazaki S. Alton G.R. Mroczkowski B. Los G. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma.Mol Cancer Ther. 2007; 6: 3314-3322Crossref PubMed Scopus (573) Google Scholar and, after an impressive clinical efficacy in ALK-positive patients, has recently been approved by the Food and Drug Administration for the treatment of locally advanced or metastatic ALK-positive NSCLCs.12Kwak E.L. Bang Y.J. Camidge D.R. Shaw A.T. Solomon B. Maki R.G. Ou S.H. Dezube B.J. Janne P.A. Costa D.B. Varella-Garcia M. Kim W.H. Lynch T.J. Fidias P. Stubbs H. Engelman J.A. Sequist L.V. Tan W. Gandhi L. Mino-Kenudson M. Wei G.C. Shreeve S.M. Ratain M.J. Settleman J. Christensen J.G. Haber D.A. Wilner K. Salgia R. Shapiro G.I. Clark J.W. Iafrate A.J. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3831) Google Scholar Phase 3 clinical trials are under way in which clinical outcomes of crizotinib-treated patients are compared with those receiving standard first- and second-line therapies in advanced ALK-rearranged NSCLCs. Several clinically validated methods are available for the detection of ALK fusions, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and RT-PCR. Crizotinib-centered clinical trials use an FISH-based test that was recently approved by the Food and Drug Administration as the standard companion diagnostic test for crizotinib. This assay (the Vysis ALK Break Apart FISH Probe Kit; Abbott Molecular, Downers Grove, IL) uses neighboring, differentially labeled break-apart probes, which specifically detect the 5′ and 3′ ends of the ALK gene, respectively.13Shaw A.T. Yeap B.Y. Mino-Kenudson M. Digumarthy S.R. Costa D.B. Heist R.S. Solomon B. Stubbs H. Admane S. McDermott U. Settleman J. Kobayashi S. Mark E.J. Rodig S.J. Chirieac L.R. Kwak E.L. Lynch T.J. Iafrate A.J. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK.J Clin Oncol. 2009; 27: 4247-4253Crossref PubMed Scopus (1641) Google Scholar Normally, the corresponding red and green fluorescent signals are in close proximity, whereas any ALK rearrangement spatially separates the probes and, thereby, their signals, resulting in distinct and isolated red and green spots. At least 15% of all analyzed cells must be positive to score a break-apart signal.12Kwak E.L. Bang Y.J. Camidge D.R. Shaw A.T. Solomon B. Maki R.G. Ou S.H. Dezube B.J. Janne P.A. Costa D.B. Varella-Garcia M. Kim W.H. Lynch T.J. Fidias P. Stubbs H. Engelman J.A. Sequist L.V. Tan W. Gandhi L. Mino-Kenudson M. Wei G.C. Shreeve S.M. Ratain M.J. Settleman J. Christensen J.G. Haber D.A. Wilner K. Salgia R. Shapiro G.I. Clark J.W. Iafrate A.J. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3831) Google Scholar, 14Camidge D.R. Kono S.A. Flacco A. Tan A.C. Doebele R.C. Zhou Q. Crino L. Franklin W.A. Varella-Garcia M. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment.Clin Cancer Res. 2010; 16: 5581-5590Crossref PubMed Scopus (300) Google Scholar, 15Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar The FISH assay has undergone extensive validation in the clinical setting and is the gold standard for detection of ALK rearrangement. A disadvantage of this diagnostic assay lies in the fact that the signal can be subtle and, consequently, hard to interpret, requiring specialized technical expertise. It is also considerably more expensive compared with IHC and RT-PCR.16Atherly A. Camidge D. The cost-effectiveness of screening lung cancer patients for targeted drug sensitivity markers.Br J Cancer. 2012; 106: 1100-1106Crossref PubMed Scopus (83) Google Scholar IHC, on the other hand, detects expression of ALK protein. Because ALK expression is normally absent in the lung, the presence of ALK protein is indicative of a possible ALK rearrangement. Although IHC is relatively inexpensive, readily available in pathology laboratories, and suitable as a screening tool, it requires highly sensitive and specific ALK antibodies and the involvement of trained pathologists to interpret the staining results. ALK expression levels in NSCLCs are, for instance, much lower than in anaplastic large-cell lymphomas; consequently, antibodies used in the latter tumor type are not sensitive enough for routine use in NSCLCs.15Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar Methods are evolving to generate more sensitive and specific antibodies for IHC detection in NSCLCs.10Takeuchi K. Choi Y.L. Togashi Y. Soda M. Hatano S. Inamura K. Takada S. Ueno T. Yamashita Y. Satoh Y. Okumura S. Nakagawa K. Ishikawa Y. Mano H. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer.Clin Cancer Res. 2009; 15: 3143-3149Crossref PubMed Scopus (630) Google Scholar, 15Mino-Kenudson M. Chirieac L.R. Law K. Hornick J.L. Lindeman N. Mark E.J. Cohen D.W. Johnson B.E. Janne P.A. Iafrate A.J. Rodig S.J. A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.Clin Cancer Res. 2010; 16: 1561-1571Crossref PubMed Scopus (390) Google Scholar, 17Rodig S.J. Mino-Kenudson M. Dacic S. Yeap B.Y. Shaw A. Barletta J.A. Stubbs H. Law K. Lindeman N. Mark E. Janne P.A. Lynch T. Johnson B.E. Iafrate A.J. Chirieac L.R. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population.Clin Cancer Res. 2009; 15: 5216-5223Crossref PubMed Scopus (608) Google Scholar Both methods previously described indicate either the presence or absence of ALK fusion, regardless of the fusion partner. RT-PCR is a technique offering a unique advantage of variant detection with the possibility for precise EML4-ALK variant identification when combined with subsequent DNA sequencing. This approach relies on generating a PCR product using an array of primer pair combinations specifically designed to detect all known EML4-ALK variants.18Sanders H.R. Li H.R. Bruey J.M. Scheerle J.A. Meloni-Ehrig A.M. Kelly J.C. Novick C. Albitar M. Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer.Cancer Genet. 2011; 204: 45-52Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 19Takeuchi K. Choi Y.L. Soda M. Inamura K. Togashi Y. Hatano S. Enomoto M. Takada S. Yamashita Y. Satoh Y. Okumura S. Nakagawa K. Ishikawa Y. Mano H. Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts.Clin Cancer Res. 2008; 14: 6618-6624Crossref PubMed Scopus (434) Google Scholar Obviously, multiple primer sets and PCRs are necessary to reliably detect all possible fusion isoforms, and the availability of good-quality RNA is essential for optimal results. RNA from formalin-fixed, paraffin-embedded (FFPE) tissues poses additional technical challenges in some cases, precluding FFPE samples from analysis. The identification of patients with ALK fusion NSCLCs who are most likely to benefit from ALK inhibition is crucial to the clinical success of ALK inhibitors. In the early-phase trial of crizotinib, during which the drug achieved a 57% response rate, approximately 1500 patients were screened by FISH to identify 82 ALK-positive patients.12Kwak E.L. Bang Y.J. Camidge D.R. Shaw A.T. Solomon B. Maki R.G. Ou S.H. Dezube B.J. Janne P.A. Costa D.B. Varella-Garcia M. Kim W.H. Lynch T.J. Fidias P. Stubbs H. Engelman J.A. Sequist L.V. Tan W. Gandhi L. Mino-Kenudson M. Wei G.C. Shreeve S.M. Ratain M.J. Settleman J. Christensen J.G. Haber D.A. Wilner K. Salgia R. Shapiro G.I. Clark J.W. Iafrate A.J. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.N Engl J Med. 2010; 363: 1693-1703Crossref PubMed Scopus (3831) Google Scholar The many patients qualifying for screening underlie the need for a high-throughput and cost-effective screening modality. An optimal assay should be sensitive and specific but should also be economical, easy to perform, preferably automated, and readily adaptable to the workflow of clinical service laboratories. In this study, we explored a novel and alternative method for detecting ALK fusions by direct, multiplexed transcript profiling using NanoString’s gene expression platform. NSCLC samples (34 ALK-positive and 33 ALK-negative samples) were obtained from Seoul National University Hospital (SNUH) and Samsung Medical Center (SMC) (both in Seoul, South Korea) with prior full informed consent of the patients and with approval from the SNUH and SMC ethical committee/internal review board. Samples were selected based on ALK fusion status, as determined by FISH and/or IHC. Tumor cell content was assessed based on H&E-stained slides. Control NSCLC cell lines, NCI-H3122, NCI-H2228, and A549, were obtained from ATCC (Manassas, VA), xenografted, and preserved as FFPE tissue blocks. Sections (4 μm thick) were deparaffinized, dehydrated, immersed in 0.2 N HCl, and incubated in 1 mol/L NaSCN for 30 minutes at 80°C. Sections were then immersed in pepsin solution for 40 minutes. Dual-probe hybridization for ALK was performed according to the instructions of the supplier, using the LSI ALK break-apart probe set (Vysis, Downers Grove, IL). The probe mixture was applied to the slides, which were then incubated in a humidified atmosphere with Hybrite (Vysis) at 77°C for 5 minutes to simultaneously denature the probe and target DNA and subsequently at 37°C for 16 hours for hybridization. The slides were then immersed in 0.3% NP-40/0.4 times standard saline citrate for 5 minutes at room temperature, followed by 0.3% NP-40/0.4 times standard saline citrate for 5 minutes at 72°C. The nuclei were counterstained with DAPI. ALK FISH was considered positive when >15% of at least 50 tumor cells analyzed showed splitting apart of the fluorescent probes flanking the ALK locus. The FISH results were scored unbiased (ie, without prior knowledge of pre-existing IHC results). ALK IHC was performed using the Bond-max automated immunostainer (Leica Microsystems, Milton Keynes, UK). Paraffin sections (3 μm thick) were evaluated for IHC staining according to standard protocols. Each paraffin section was dewaxed, followed by heat-induced epitope retrieval: heating for 20 minutes at 100°C in Epitope Retrieval Solution pH 9.0 (Leica Microsystems). Subsequent antibody-specific steps were performed according to the manufacturer’s instructions. Slides were incubated with mouse monoclonal antibody for ALK (clone 5A4; Novocastra, Newcastle upon Tyne, UK) at 1:50 dilution. Antibody binding was detected by a standard detection kit (Bond-Polymer Refine Detection kit; Leica Microsystems). Mayer’s hematoxylin was used as the counterstain. Various normal and cancer TMA blocks were included as negative and positive controls. For ALK, IHC was interpreted as follows: negative, no staining; equivocal, faint cytoplasmic staining without any background staining; and positive, moderate to strong cytoplasmic staining in >10% of tumor cells. Total RNA was isolated from one to three FFPE tissue sections (10 μm thick) using Agencourt FormaPure-Nucleic Acid Extraction from FFPE Tissue kit (Beckman-Coulter, Indianapolis, IN). The manufacturer’s protocol for RNA extraction was followed with an additional DNase treatment step. RNA concentration was assessed using the Nanodrop 8000 (Thermo-Scientific, Wilmington, DE). nCounter assays were performed in duplicate, according to the manufacturer’s protocol (NanoString, Seattle, WA). Briefly, 500 ng of total RNA was hybridized to nCounter probe sets for 16 hours at 65°C. Samples were processed using an automated nCounter Sample Prep Station (NanoString Technologies, Inc., Seattle, WA). Cartridges containing immobilized and aligned reporter complex were subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies, Inc.) set at 1155 fields of view. Reporter counts were collected using NanoString’s nSolver analysis software version 1, normalized, and analyzed as described later. Data were normalized in two steps. First, six positive internal controls were used to remove potential systematic differences between individual hybridization experiments. Specifically, the sum of the intensity (Si) from the six positive control probes was calculated for sample (ie, replicate) i, individual probe intensity for sample i was then scaled by the normalization factor S/Si, where S = mean of Si. Second, the scaled intensity of sample i (obtained from step 1) was further normalized using housekeeping genes to remove any effect that might be attributed to, for instance, differences in the amount of input RNA. If Hi is the geometric mean of the intensity from the four housekeeping genes for sample i, the second step normalization factor was then defined as H/Hi. Background was determined from the eight excision repair cross-complementing–negative control probes. The mean and SD were calculated from the negative controls, and a threshold (B) was defined as the mean plus 2 SDs. A target with a normalized intensity value higher than this threshold was scored as present. The normalized intensity from sample replicates was averaged to obtain an averaged patient intensity for each probe and patient. To summarize ALK 3′ overexpression, we defined an ALK 3′ overexpression score (ie, ALK3′/5′ ratio) for each patient as follows: Alk3′/5′ = E3/max(A5, B), where E3 is the geometric mean of ALK 3′ probe expression, A5 is the average of the ALK 5′ probe expression, and B is the background threshold previously defined. ALK 3′ probes usually have a higher intensity and tend to follow a log normal distribution, whereas 5′ probes have a lower intensity and are more normally distributed. Thus, we used the geometric mean for ALK 3′ probes and the arithmetic mean for ALK 5′ probes. Using background threshold B to floor the denominator prevents an extremely small ALK 5′ expression value that could artificially inflate the score. For the fusion probe, we defined a fusion present call in a similar manner. The fusion probe for a tumor was called present if its normalized intensity was 2 SDs higher than the median, or B, whichever was larger. Herein, the median and SD were calculated from all fusion-negative samples. The SD was calculated from the median absolute deviation (MAD) from the median, which is a more robust measure of variability. Therefore, fusion present if intensity > max (B, median + 2*SMAD), where SMAD = 1.4826*MAD, is the standard deviation of normal distribution calculated using MAD. The percentage concordance was calculated between two platforms, and its CI was computed using Wilson’s score method. Cohen’s κ statistic was also calculated for concordance analysis. Data were analyzed using standard R software, version 2.13.1 (http://www.r-project.org, last accessed September 20, 2011). Concordance analysis was conducted in SAS 9.2 (SAS Institute, Cary, NC). The precise ALK fusion variant type from SNUH ALK-positive samples was determined by RT-PCR using an RNA UltraSense One-Step RT-PCR kit (Invitrogen, Carlsbad, CA), according to primers and conditions previously described by Sanders and coworkers.18Sanders H.R. Li H.R. Bruey J.M. Scheerle J.A. Meloni-Ehrig A.M. Kelly J.C. Novick C. Albitar M. Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer.Cancer Genet. 2011; 204: 45-52Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Wild-type ALK transcripts were detected by RT-PCR using ALK exon 18 forward primer (5′-TCGCTGATCCTCTCTGTGG-3′) and ALK exon 20 reverse primer (5′-CTTGCTCAGCTTGTACTCAGG-3′). The resulting PCR products were separated on a 2% size-select agarose E-gel (Invitrogen) and sequenced using a 3700 ABI Prism sequencer (Life Technologies, Foster City, CA). By using NanoString’s border probe approach,20Geiss G.K. Bumgarner R.E. Birditt B. Dahl T. Dowidar N. Dunaway D.L. Fell H.P. Ferree S. George R.D. Grogan T. James J.J. Maysuria M. Mitton J.D. Oliveri P. Osborn J.L. Peng T. Ratcliffe A.L. Webster P.J. Davidson E.H. Hood L. Dimitrov K. Direct multiplexed measurement of gene expression with color-coded probe pairs.Nature Biotechnol. 2008; 26: 317-325Crossref PubMed Scopus (1556) Google Scholar, 21Malkov V.A. Serikawa K.A. Balantac N. Watters J. Geiss G. Mashadi-Hossein A. Fare T. Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.BMC Res Notes. 2009; 2: 80Crossref PubMed Scopus (117) Google Scholar, 22Reis P.P. Waldron L. Goswami R.S. Xu W. Xuan Y. Perez-Ordonez B. Gullane P. Irish J. Jurisica I. Kamel-Reid S. mRNA transcript quantification in archival samples using multiplexed, color-coded probes.BMC Biotechnol. 2011; 11: 46Crossref PubMed Scopus (215) Google Scholar we designed two sequence-specific probe cocktails consisting of a mixture of 5′ capture and 3′ reporter probes, all containing sequences complementary to a contiguous target sequence (Figure 1A). Capture probes consisted of target-specific, approximately 50-mer oligonucleotides and were biotinylated to enable downstream capture of the mRNA-probe complex. Reporter probes also consisted of target-specific 50-mer oligonucleotides coupled to a unique, color-coded tag used for signal detection. The reporter tag consisted of four spectrally distinct fluorophores attached to seven segments along the reporter backbone. The order of the fluorescently labeled color tags dictated the formation of a unique molecular bar code for each reporter. Multiplex hybridization of probe sets to mRNA resulted in the formation of a tripartite complex of capture probe/RNA target/reporter probe. On removal of excess probes, the hybridization complexes were immobilized to a streptavidin-coated surface, where application of an electrical current aligned them in the same orientation. Reporter tags were digitally imaged and counted, where the number of specific reporter tags counted corresponded to the number of transcripts present. For our ALK fusion transcript assay, we designed a single-tube, multiplexed assay to simultaneously detect EML4-ALK fusion transcripts and measure specific ALK expression patterns for several ALK exons flanking the fusion break point. For fusion detection, EML4-specific 5′ capture probes and ALK-specific 3′ reporter probes were designed to hybridize to approximately 50 nucleotides of EML4 and ALK flanking the fusion junction, respectively (Table 1). EML4-ALK fusion isoforms were characterized by variable truncations in EML4, universally fused to the ALK kinase domain usually beginning at exon 20 (Table 1). Most EML4-ALK fusion variants shared the same downstream ALK exon 20 junction; thus, assignment of a unique reporter tag for each isoform was not possible because of use of the same molecular barcode to the downstream reporter probe. Thus, a common reporter probe designated as ALK exon 20, paired with the appropriate variant capture probe, would detect a preselected, expandable set of fusion transcripts containing ALK exon 20 sequences (Figure 1B). Capture probe sequences were designed to detect all major isoforms of EML4-ALK fusions (namely, variants 1, 2, and 3 with combined frequencies close to 70%).2Soda M. Choi Y.L. Enomoto M. Takada S. Yamashita Y. Ishikawa S. Fujiwara S. Watanabe H. Kurashina K. Hatanaka H. Bando M. Ohno S. Ishikawa Y. Aburatani H. Niki T. Sohara Y. Sugiyama Y. Mano H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature. 2007; 448: 561-566Crossref PubMed Scopus (4" @default.
• W2086939994 created "2016-06-24" @default.
• W2086939994 creator A5002187013 @default.
• W2086939994 creator A5008594172 @default.
• W2086939994 creator A5009075091 @default.
• W2086939994 creator A5016978421 @default.
• W2086939994 creator A5024070397 @default.
• W2086939994 creator A5028256943 @default.
• W2086939994 creator A5028509520 @default.
• W2086939994 creator A5034987867 @default.
• W2086939994 creator A5037066533 @default.
• W2086939994 creator A5052450087 @default.
• W2086939994 creator A5058521062 @default.
• W2086939994 creator A5066161983 @default.
• W2086939994 creator A5070181255 @default.
• W2086939994 creator A5075619842 @default.
• W2086939994 creator A5076271673 @default.
• W2086939994 creator A5076891866 @default.
• W2086939994 creator A5082025437 @default.
• W2086939994 creator A5087509589 @default.
• W2086939994 date "2013-01-01" @default.
• W2086939994 modified "2023-10-17" @default.
• W2086939994 title "Multiplexed Gene Expression and Fusion Transcript Analysis to Detect ALK Fusions in Lung Cancer" @default.
• W2086939994 cites W1517005017 @default.
• W2086939994 cites W1519176158 @default.
• W2086939994 cites W1995830102 @default.
• W2086939994 cites W2000397789 @default.
• W2086939994 cites W2009847758 @default.
• W2086939994 cites W2010978319 @default.
• W2086939994 cites W2031159738 @default.
• W2086939994 cites W2031494655 @default.
• W2086939994 cites W2038586807 @default.
• W2086939994 cites W2046911066 @default.
• W2086939994 cites W2050368996 @default.
• W2086939994 cites W2053541213 @default.
• W2086939994 cites W2055402151 @default.
• W2086939994 cites W2059336669 @default.
• W2086939994 cites W2066417528 @default.
• W2086939994 cites W2092036762 @default.
• W2086939994 cites W2094903176 @default.
• W2086939994 cites W2104830962 @default.
• W2086939994 cites W2121005606 @default.
• W2086939994 cites W2123727052 @default.
• W2086939994 cites W2135329354 @default.
• W2086939994 cites W2139110945 @default.
• W2086939994 cites W2149120912 @default.
• W2086939994 cites W2150424194 @default.
• W2086939994 cites W2153345506 @default.
• W2086939994 cites W2168691181 @default.
• W2086939994 doi "https://doi.org/10.1016/j.jmoldx.2012.08.006" @default.
• W2086939994 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23246132" @default.
• W2086939994 hasPublicationYear "2013" @default.
• W2086939994 type Work @default.
• W2086939994 sameAs 2086939994 @default.
• W2086939994 citedByCount "65" @default.
• W2086939994 countsByYear W20869399942013 @default.
• W2086939994 countsByYear W20869399942014 @default.
• W2086939994 countsByYear W20869399942015 @default.
• W2086939994 countsByYear W20869399942016 @default.
• W2086939994 countsByYear W20869399942017 @default.
• W2086939994 countsByYear W20869399942018 @default.
• W2086939994 countsByYear W20869399942019 @default.
• W2086939994 countsByYear W20869399942020 @default.
• W2086939994 countsByYear W20869399942021 @default.
• W2086939994 countsByYear W20869399942022 @default.
• W2086939994 countsByYear W20869399942023 @default.
• W2086939994 crossrefType "journal-article" @default.
• W2086939994 hasAuthorship W2086939994A5002187013 @default.
• W2086939994 hasAuthorship W2086939994A5008594172 @default.
• W2086939994 hasAuthorship W2086939994A5009075091 @default.
• W2086939994 hasAuthorship W2086939994A5016978421 @default.
• W2086939994 hasAuthorship W2086939994A5024070397 @default.
• W2086939994 hasAuthorship W2086939994A5028256943 @default.
• W2086939994 hasAuthorship W2086939994A5028509520 @default.
• W2086939994 hasAuthorship W2086939994A5034987867 @default.
• W2086939994 hasAuthorship W2086939994A5037066533 @default.
• W2086939994 hasAuthorship W2086939994A5052450087 @default.
• W2086939994 hasAuthorship W2086939994A5058521062 @default.
• W2086939994 hasAuthorship W2086939994A5066161983 @default.
• W2086939994 hasAuthorship W2086939994A5070181255 @default.
• W2086939994 hasAuthorship W2086939994A5075619842 @default.
• W2086939994 hasAuthorship W2086939994A5076271673 @default.
• W2086939994 hasAuthorship W2086939994A5076891866 @default.
• W2086939994 hasAuthorship W2086939994A5082025437 @default.
• W2086939994 hasAuthorship W2086939994A5087509589 @default.
• W2086939994 hasBestOaLocation W20869399941 @default.
• W2086939994 hasConcept C104317684 @default.
• W2086939994 hasConcept C111829193 @default.
• W2086939994 hasConcept C142724271 @default.
• W2086939994 hasConcept C150194340 @default.
• W2086939994 hasConcept C153911025 @default.
• W2086939994 hasConcept C2776256026 @default.
• W2086939994 hasConcept C2778274637 @default.
• W2086939994 hasConcept C502942594 @default.
• W2086939994 hasConcept C54355233 @default.
• W2086939994 hasConcept C70721500 @default.
• W2086939994 hasConcept C71924100 @default.
• W2086939994 hasConcept C86803240 @default.

• next