This work was supported by the Molecular Pathology Shared Resource of the University of Colorado (National Cancer Institute Cancer Center Support Grant No. P30-CA046934) and by the Colorado Center for Personalized Medicine.

<ol>
	<li>Mitelman, F., Johansson, B., Mertens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+translocations+and+gene+fusions+on+cancer+causation.">The impact of translocations and gene fusions on cancer causation.</a> Nature Reviews Cancer. 7 (4), 233-245 (2007).</li><li>Daley, G. Q., Van Etten, R. A., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+chronic+myelogenous+leukemia+in+mice+by+the+P210bcr/abl+gene+of+the+Philadelphia+chromosome.">Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome.</a> Science. 247 (4944), 824-830 (1990).</li><li>Druker, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+a+specific+inhibitor+of+the+BCR-ABL+tyrosine+kinase+in+the+blast+crisis+of+chronic+myeloid+leukemia+and+acute+lymphoblastic+leukemia+with+the+Philadelphia+chromosome.">Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.</a> New England Journal of Medicine. 344 (14), 1038-1042 (2001).</li><li>Solomon, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-line+crizotinib+versus+chemotherapy+in+ALK-positive+lung+cancer.">First-line crizotinib versus chemotherapy in ALK-positive lung cancer.</a> New England Journal of Medicine. 371 (23), 2167-2177 (2014).</li><li>Shaw, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crizotinib+in+ROS1-rearranged+non-small-cell+lung+cancer.">Crizotinib in ROS1-rearranged non-small-cell lung cancer.</a> New England Journal of Medicine. 371 (21), 1963-1971 (2014).</li><li>Drilon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+Larotrectinib+in+TRK+Fusion-Positive+Cancers+in+Adults+and+Children.">Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children.</a> New England Journal of Medicine. 378 (8), 731-739 (2018).</li><li>Kawai, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SYT-SSX+gene+fusion+as+a+determinant+of+morphology+and+prognosis+in+synovial+sarcoma.">SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma.</a> New England Journal of Medicine. 338 (3), 153-160 (1998).</li><li>de Alava, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EWS-FLI1+fusion+transcript+structure+is+an+independent+determinant+of+prognosis+in+Ewing's+sarcoma.">EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing's sarcoma.</a> Journal of Clinical Oncology. 16 (4), 1248-1255 (1998).</li><li>Pierron, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+subtype+of+bone+sarcoma+defined+by+BCOR-CCNB3+gene+fusion.">A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion.</a> Nature Genetics. 44 (4), 461-466 (2012).</li><li>Papaemmanuil, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Classification+and+Prognosis+in+Acute+Myeloid+Leukemia.">Genomic Classification and Prognosis in Acute Myeloid Leukemia.</a> New England Journal of Medicine. 374 (23), 2209-2221 (2016).</li><li>Arber, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2016+revision+to+the+World+Health+Organization+classification+of+myeloid+neoplasms+and+acute.">The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute.</a> Blood. 127 (20), 2391-2405 (2016).</li><li>Sanders, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon+scanning+by+reverse+transcriptase-polymerase+chain+reaction+for+detection+of+known+and+novel+EML4-ALK+fusion+variants+in+non-small+cell+lung+cancer.">Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer.</a> Cancer Genetics. 204 (1), 45-52 (2011).</li><li>Camidge, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+the+Detection+of+Lung+Cancer+Patients+Harboring+Anaplastic+Lymphoma+Kinase+(ALK)+Gene+Rearrangements+Potentially+Suitable+for+ALK+Inhibitor+Treatment.">Optimizing the Detection of Lung Cancer Patients Harboring Anaplastic Lymphoma Kinase (ALK) Gene Rearrangements Potentially Suitable for ALK Inhibitor Treatment.</a> Clinical Cancer Research. 16 (22), 5581-5590 (2010).</li><li>Mino-Kenudson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+highly+sensitive+antibody+allows+for+the+routine+detection+of+ALK-rearranged+lung+adenocarcinomas+by+standard+immunohistochemistry.">A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.</a> Clinical Cancer Research. 16 (5), 1561-1571 (2010).</li><li>Suehara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+KIF5B-RET+and+GOPC-ROS1+fusions+in+lung+adenocarcinomas+through+a+comprehensive+mRNA-based+screen+for+tyrosine+kinase+fusions.">Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions.</a> Clinical Cancer Research. 18 (24), 6599-6608 (2012).</li><li>Davies, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Molecular+Testing+Modalities+for+Detection+of+ROS1+Rearrangements+in+a+Cohort+of+Positive+Patient+Samples.">Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples.</a> Journal of Thoracic Oncology. 13 (10), 1474-1482 (2018).</li><li>Reeser, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+Targeted+RNA+Sequencing+Assay+for+Kinase+Fusion+Detection+in+Solid+Tumors.">Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors.</a> Journal of Molecular Diagnostics. 19 (5), 682-696 (2017).</li><li>Beadling, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multiplexed+Amplicon+Approach+for+Detecting+Gene+Fusions+by+Next-Generation+Sequencing.">A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.</a> Journal of Molecular Diagnostics. 18 (2), 165-175 (2016).</li><li>Mamanova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-enrichment+strategies+for+next-generation+sequencing.">Target-enrichment strategies for next-generation sequencing.</a> Nature Methods. 7 (2), 111-118 (2010).</li><li>Zheng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anchored+multiplex+PCR+for+targeted+next-generation+sequencing.">Anchored multiplex PCR for targeted next-generation sequencing.</a> Nature Medicine. 20 (12), 1479-1484 (2014).</li><li>Davies, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+Response+to+Crizotinib+in+a+Patient+with+Lung+Cancer+Positive+for+an+HLA-DRB1-MET+Gene+Fusion.">Dramatic Response to Crizotinib in a Patient with Lung Cancer Positive for an HLA-DRB1-MET Gene Fusion.</a> JCO Precision Oncology. 2017 (1), (2017).</li><li>Vendrell, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+known+and+novel+ALK+fusion+transcripts+in+lung+cancer+patients+using+next-generation+sequencing+approaches.">Detection of known and novel ALK fusion transcripts in lung cancer patients using next-generation sequencing approaches.</a> Scientific Reports. 7 (1), 12510 (2017).</li><li>Agaram, N. P., Zhang, L., Cotzia, P., Antonescu, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+Spectrum+of+Genetic+Alterations+in+Pseudomyogenic+Hemangioendothelioma+With+Recurrent+Novel+ACTB-FOSB+Gene+Fusions.">Expanding the Spectrum of Genetic Alterations in Pseudomyogenic Hemangioendothelioma With Recurrent Novel ACTB-FOSB Gene Fusions.</a> American Journal of Surgical Pathology. 42 (12), 1653-1661 (2018).</li><li>Skalova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Profiling+of+Mammary+Analog+Secretory+Carcinoma+Revealed+a+Subset+of+Tumors+Harboring+a+Novel+ETV6-RET+Translocation:+Report+of+10+Cases.">Molecular Profiling of Mammary Analog Secretory Carcinoma Revealed a Subset of Tumors Harboring a Novel ETV6-RET Translocation: Report of 10 Cases.</a> American Journal of Surgical Pathology. 42 (2), 234-246 (2018).</li><li>Guseva, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anchored+multiplex+PCR+for+targeted+next-generation+sequencing+reveals+recurrent+and+novel+USP6+fusions+and+upregulation+of+USP6+expression+in+aneurysmal+bone+cyst.">Anchored multiplex PCR for targeted next-generation sequencing reveals recurrent and novel USP6 fusions and upregulation of USP6 expression in aneurysmal bone cyst.</a> Genes Chromosomes and Cancer. 56 (4), 266-277 (2017).</li></ol>

D.L.A. has received consulting fees from Bayer Oncology, Genentech, AbbVie, and Bristol Myers Squibb. K.D.D has received sponsored travel from ArcherDx.

Anchored multiplex PCR-based target enrichment and library preparation followed by next-generation sequencing is well suited for multiplexed gene fusion assessment in clinical tumor samples. By focusing on RNA input rather than genomic DNA, the need to sequence large and repetitive introns is avoided. Additionally, since this approach amplifies gene fusions regardless of the identity of the fusion partner, novel fusions are detected. This is a critical advantage in the clinical realm, and there have been many examples of...

The fusion of two or more genes into a single transcriptional entity can occur as the result of large scale chromosomal variations including deletions, duplications, insertions, inversions, and translocations. Through altered transcriptional control and/or altered functional properties of the expressed gene product, these fusion genes can confer oncogenic properties to cancer cells1. In many cases, fusion genes are known to act as primary oncogenic drivers by directly activating cellular proliferation and survival pathways.
The clinical relevance of gene fusions for cancer patients first became apparent with the discovery of the Philadelphia chromosome and the corresponding BCR-ABL1 fusion gene in chronic myelogenous leukemia (CML)2. The small molecule inhibitor imatinib mesylate was developed to specifically target this fusion gene and demonstrated remarkable efficacy in BCR-ABL1-positive CML patients3. Therapeutic targeting of oncogenic gene fusions has also been successful in solid tumors, with inhibition of ALK and ROS1 fusion genes in non-small cell lung cancer serving as primary examples4,5. Recently, the NTRK inhibitor larotrectinib was FDA approved for NTRK1/2/3 fusion-positive solid tumors, regardless of disease site6. Beyond therapy selection, gene fusion detection also has roles in disease diagnosis and prognosis. This is particularly prevalent in various sarcoma and hematologic malignancy subtypes that are diagnostically defined by the presence of specific fusions and/or for which presence of a fusion directly informs prognosis7,8,9,10,11. These are but a few of the examples of the clinical application of gene fusion detection for cancer patients.
Due to the critical role in clinical decision making, accurate gene fusion detection from clinical samples is of vital importance. Numerous techniques have been applied in clinical laboratories for fusion or chromosomal rearrangement analysis including: cytogenetic techniques, reverse transcription polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and 5&#8217;/3&#8217; expression imbalance analysis (among others)12,13,14,15. Presently, the rapidly expanding list of actionable gene fusions in cancer has resulted in the need to assess fusion status of multiple genes simultaneously. Consequently, some traditional techniques that can only query one or a few genes at a time are becoming inefficient approaches, especially when considering that clinical tumor samples are often very finite and not amenable to being divided among several assays. Next generation sequencing (NGS), however, is an analysis platform that is well suited for multi-gene testing, and NGS-based assays have become common in clinical molecular diagnostic laboratories.
Currently used NGS assays for fusion/rearrangement detection vary in regard to the input material used, the chemistries employed for library preparation and target enrichment, and the number of genes queried within an assay. NGS assays can be based on RNA or DNA (or both) extracted from the sample. Although RNA-based analysis is hampered by the tendency of clinical samples to contain highly degraded RNA, it circumvents the need to sequence large and often repetitive introns that are the targets of DNA-based fusion testing but have proven to be difficult for NGS data analysis16. Target enrichment strategies for RNA-based NGS assays can be largely divided into hybrid capture or amplicon-mediated approaches. While both strategies have been successfully utilized for fusion detection, each contains advantages over the other17,18. Hybrid capture assays generally result in more complex libraries and reduced allelic dropout, whereas amplicon-based assays generally require lower input and result in less off-target sequencing19. However, perhaps the primary limitation of traditional amplicon-based enrichment is the need for primers to all known fusion partners. This is problematic since many clinically important genes are known to fuse with dozens of different partners, and even if primer design allowed for detection of all known partners, novel fusion events would remain undetected. A recently described technique termed anchored multiplex PCR (or AMP for short) addresses this limitation20. In AMP, a &#8216;half-functional&#8217; NGS adapter is ligated to cDNA fragments that are derived from input RNA. Target enrichment is achieved by amplification between gene specific primers and a primer to the adapter. As a result, all fusions to genes of interest, even if a novel fusion partner is involved, should be detected (see Figure 1). This article describes the use of the ArcherDx FusionPlex Solid Tumor kit, an NGS-based assay that employs AMP for target enrichment and library preparation, for the detection of oncogenic gene fusions in solid tumor samples (see Supplementary Table 1 for complete gene list). The wet-bench protocol and data analysis steps have been rigorously validated in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory.

<table><tbody><tr><td>10 mM Tris HCl pH 8.0</td><td>IDT</td><td>11-05-01-13</td><td>Used for TNA dilution</td></tr><tr><td>1M Tris pH 7.0</td><td>Thermo Fisher</td><td>AM9850G</td><td>Used in library pooling</td></tr><tr><td>25 mL Reagent Reservoir with divider</td><td>USA Scientific</td><td>9173-2000</td><td>For use with multi-channel pipetters and large reagent volumes</td></tr><tr><td>96-well TemPlate Semi-Skirt 0.1mL PCR plate-natural</td><td>USA Scientific</td><td>1402-9700</td><td>Plate used for thermocycler steps</td></tr><tr><td>Agencourt AMPure XP Beads</td><td>Beckman Coulter</td><td>A63881</td><td>Used in purification after several assay steps</td></tr><tr><td>Agencourt Formapure Kit</td><td>Beckman Coulter</td><td>A33343</td><td>Used in TNA extraction</td></tr><tr><td>Archer FusionPlex Solid Tumor kit</td><td>ArcherDX</td><td>AB0005</td><td>This kit contains most of the reagents necessary to perform library preperation for Illumina sequencing (kits for Ion Torrent sequencing are also available)</td></tr><tr><td>Cold block, 96-well</td><td>Light Labs</td><td>A7079</td><td>Used for keeping samples chilled at various steps</td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>DSP-MD.43</td><td>Used for bead washes</td></tr><tr><td>Library Quantification for Illumina Internal Control Standard</td><td>Kapa Biosystems</td><td>KK4906</td><td>Used for library quantitation</td></tr><tr><td>Library Quantification Primers and ROX Low qPCR mix</td><td>Kapa Biosystems</td><td>KK4973</td><td>Used for library quantitation</td></tr><tr><td>Library Quantification Standards</td><td>Kapa Biosystems</td><td>KK4903</td><td>Used for library quantitation</td></tr><tr><td>Magnet Plate, 96-well (N38 grade)</td><td>Alpaqua</td><td>A32782</td><td>Used in bead purificiation steps</td></tr><tr><td>MBC Adapters Set B</td><td>ArcherDX</td><td>AK0016-48</td><td>Adapters that contain sample-specific indexes to enable multiplex sequencing</td></tr><tr><td>Micro Centrifuge</td><td>USA Scientific</td><td>2641-0016</td><td>Used for spinning down PCR tubes</td></tr><tr><td>MicroAmp EnduraPlate Optical 96 well Plate</td><td>Thermo-Fisher</td><td>4483485</td><td>Used for Pre-Seq QClibrary quantitation</td></tr><tr><td>Microamp Optical Film Compression Pad</td><td>Applied Biosystems</td><td>4312639</td><td>Used for library quantitation</td></tr><tr><td>Mini Plate Spinner</td><td>Labnet</td><td>MPS-1000</td><td>Used for collecing liquid at bottom of plate wells</td></tr><tr><td>MiSeq Reagent Kit v3 (600 cycle)</td><td>Illumina</td><td>MS-102-3003</td><td>Contains components necessary for a MiSeq sequencing run</td></tr><tr><td>MiSeqDx System</td><td>Illumina</td><td></td><td>NGS Sequencing Instrument</td></tr><tr><td>Model 9700 Thermocycler</td><td>Applied Biosystems</td><td></td><td>Used for several steps during assay</td></tr><tr><td>nuclease free water</td><td>Ambion</td><td>9938</td><td>Used as general diluent</td></tr><tr><td>Optical ABI 96-well PCR plate covers</td><td>Thermo-Fisher</td><td>4311971</td><td>Used for Pre-Seq QClibrary quantitation</td></tr><tr><td>PCR Workstation Model 600</td><td>Air Clean Systems</td><td>BZ10119636</td><td>Wet-bench assay steps performed in this &#039;dead air box&#039;</td></tr><tr><td>Proteinase K</td><td>Qiagen</td><td>1019499</td><td>Used in TNA extraction</td></tr><tr><td>QuantStudio 5</td><td>Applied Biosystems</td><td>LSA28139</td><td>qPCR instrument used for PreSeq and library quantitation</td></tr><tr><td>Qubit RNA HS Assay Kit</td><td>Life Technologies</td><td>Q32855</td><td>Use for determing RNA concentration in TNA samples</td></tr><tr><td>RNase Away</td><td>Fisher</td><td>12-402-178</td><td>Used for general RNase decontamination of work areas</td></tr><tr><td>Seraseq FFPE Tumor Fusion RNA Reference Material v2</td><td>SeraCare</td><td>0710-0129</td><td>Used as the assay positive control</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher</td><td>BP359-212</td><td>Used in clean-up steps and for sequencing setup</td></tr><tr><td>SYBR Green Supermix</td><td>Bio Rad</td><td>172-5120</td><td>Component of PreSeq QC Assay</td></tr><tr><td>TempAssure PCR 8-tube Strips</td><td>USA Scientific</td><td>1402-2700</td><td>Used for reagent and sample mixing etc.</td></tr><tr><td>Template RT PCR film</td><td>USA Scientific</td><td>2921-7800</td><td>Used for covering 96-well plates</td></tr><tr><td>U-Bottom 96-well Microplate</td><td>LSP</td><td>MP8117-R</td><td>Used during bead purification</td></tr></tbody></table>

oncogenic gene fusion detection using anchored multiplex polymerase chain reaction followed by next generation sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

Shown in Figure 3, Figure 4 and Figure 5 are screenshots from the analysis user interface demonstrating results from a lung adenocarcinoma sample. In Figure 3, the sample summary is shown (top) that lists the called strong evidence fusions, as well as the QC status (circled in red). The ADCK4-NUMBL fusion (of which 3 isoforms are listed) is immediately ignored because it is a persistent transcr...

Watch this Scientific Journal Video about Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing at JoVE.com

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

oncogenic-gene-fusion-detection-using-anchored-multiplex-polymerase

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9.4K Views.  University of Colorado - Anschutz Medical Campus. This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Video: Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

Behavior

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Medicine

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Biology

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Assessing Genomic Alteration in Non-Squamous Non-Small Cell Lung Cancer Patients

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly increased these last few years. The detection of molecular abnormalities is mandatory for the optimal care of advanced or metastatic NS-NSCLC patients, allowing targeted therapies to be administrated with an improvement in overall survival. Nevertheless, these tumors develop mechanisms of resistance that are potentially targetable using novel therapies. Some molecular alterations can also modulate the treatment response. The molecular characterization of NS-NSCLC has to be performed in a short turnaround time (TAT), in less than 10 working days, as recommended by the international guidelines. In addition, the origin of the tissue biopsies for genomic analysis is diverse, and their size is continuously decreasing with the development of less invasive methods and protocols. Consequently, pathologists are being challenged to perform effective molecular technics while maintaining an efficient and rapid diagnosis strategy. Here, we describe the ultra-fast amplicon-based next-generation sequencing (NGS) workflow used in daily routine practice at diagnosis for NS-NSCLC patients. We showed that this system is able to identify the current molecular targets used in precision medicine in thoracic oncology in an appropriate TAT.

Author Spotlight: Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer 

The increase of molecular biomarkers to be tested for non-squamous non-small cell lung cancer (NS-NSCLC) care management has prompted the development of fast and reliable molecular detection methods. We describe a workflow for genomic alteration assessment for NS-NSCLC patients using an ultra-fast-next generation sequencing (NGS) approach.

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly ...