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>
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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. 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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 actionable novel gene fusions identified through AMP reported in the literature21,22,23,24,25.
Since the assay is RNA based, it is critical to preserve RNA quality in samples during processing. It is also critical to determine which samples produced RNA sequencing that is too poor to trust negative fusion results. This is achieved by assessment of sequencing data from primers to four housekeeping genes. If sequencing of these genes is poor, then negative fusion results are deemed uninformative. In addition, given the complexity and multitude of wet-bench steps in the assay, it is important to include a fusion-positive control in every assay run. By doing so, compromised assay runs become apparent through analysis of expected fusion events in the control.
As with all amplicon-based approaches, AMP is highly reliant on individual primer performance. When assessing multiple exons of multiple genes, it is inevitable that some primers will not perform as well as others. Therefore, it is critical for users to know where the assay underperforms due to primer inefficiency. Additionally, NGS-based assays require complex bioinformatic data analysis. If the algorithms employed are not thoughtfully designed, false-negative and false-positive results are likely. It is very important that all gene fusions called by analysis algorithms be manually inspected by the user.
With an ever-growing list of actionable gene fusions that should be assessed in clinical tumor specimens, use of multiplexed assays like AMP will continue to increase in clinical laboratories. Future applications of the technique will likely focus on combining fusion and mutation assessment within a single assay. Regardless of the molecular assay approach, users must always be aware of assay limitations and should always establish quality control metrics to guide data interpretation.

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 &#39;dead air box&#39;</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 transcriptional readthrough event (noted by the broken circle icons next to the listings). The bottom of Figure 3 is a screenshot of the Read Statistics page. Particularly informative metrics are circled in green. The critical QC metric that determines the pass/fail nature of the sample is highlighted in red (this is the metric that dictates pass/fail status in the summary above). If this value is below 20, a negative fusion result is deemed &#8216;uninformative&#8217;. Figure 4 and Figure 5 are screenshots of the fusion schematics (top) and JBrowse views of fusion supporting reads (bottom) for the two fusions that warranted further investigation. The KIF5B-RET fusion (Figure 4) demonstrates a high number of supporting reads, a high % of reads from the primer supporting the fusion, and a high number of start sites. Additionally, several contiguous exons of the fusion partner (KIF5B) are included in the alignment, the fusion was verified to be in frame, and the reads supporting the fusion are generally free of mismatch. For these reasons, the fusion is deemed real and reportable. The LOC101927681-PDGFRB fusion (Figure 5) demonstrates lower supporting metrics. Furthermore, the portion of the reads mapping to the partner are relatively short and contain a high error rate, which strongly suggests misalignment and an artifactual fusion call. Finally, intronic sequence of PDGFRB is included, further suggesting an artifact (most, but not all, legitimate fusions are comprised solely of sequence that maps to coding regions of both genes). For these reasons, this fusion is deemed an artifact and not reportable.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59895/59895fig1.jpg" /> 
Figure 1: Schematic representation of AMP approach. Traditional amplicon-mediated approaches for target enrichment are limited by the fact that primers are needed for all fusion partners. Thus, novel fusion partners will not be detected. In AMP, the opposing primer is specific to the adapter, thus novel partners are detected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59895/59895fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59895/59895fig2.jpg" /> 
Figure 2: Correlation between PreSeq QC score and post-sequencing QC. The PreSeq Ct and average unique start sites per GSP2 Control values were correlated for a series of 100 samples. Generally, as expected, low PreSeq scores correlate with higher SS/GSP2 values (both are indicators of good quality RNA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59895/59895fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59895/59895fig3.jpg" /> 
Figure 3: Example sample summary and read statistic views. Top: view of a sample summary in the user interface with strong evidence fusions listed. Bottom: view of the read statistics page in the user interface. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59895/59895fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59895/59895fig4.jpg" /> 
Figure 4: Example of a legitimate fusion call. Top: view of the fusion schematic in the user interface. Bottom: JBrowse view of individual reads supporting the fusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59895/59895fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59895/59895fig5.jpg" /> 
Figure 5: Example of an artifactual fusion call. Top: view of the fusion schematic in the user interface. Bottom: JBrowse view of individual reads supporting the fusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59895/59895fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AKT3</td>
			<td>EWSR1</td>
			<td>NOTCH1</td>
			<td>PRKCA</td>
		</tr>
		<tr>
			<td>ALK</td>
			<td>FGFR1</td>
			<td>NOTCH2</td>
			<td>PRKCB</td>
		</tr>
		<tr>
			<td>ARHGAP26</td>
			<td>FGFR2</td>
			<td>NRG1</td>
			<td>RAF1</td>
		</tr>
		<tr>
			<td>AXL</td>
			<td>FGFR3</td>
			<td>NTRK1</td>
			<td>RELA</td>
		</tr>
		<tr>
			<td>BRAF</td>
			<td>FGR</td>
			<td>NTRK2</td>
			<td>RET</td>
		</tr>
		<tr>
			<td>BRD3</td>
			<td>INSR</td>
			<td>NTRK3</td>
			<td>ROS1</td>
		</tr>
		<tr>
			<td>BRD4</td>
			<td>MAML2</td>
			<td>NUMBL</td>
			<td>RSPO2</td>
		</tr>
		<tr>
			<td>EGFR</td>
			<td>MAST1</td>
			<td>NUTM1</td>
			<td>RSPO3</td>
		</tr>
		<tr>
			<td>ERG</td>
			<td>MAST2</td>
			<td>PDGFRA</td>
			<td>TERT</td>
		</tr>
		<tr>
			<td>ESR1</td>
			<td>MET</td>
			<td>PDGFRB</td>
			<td>TFE3</td>
		</tr>
		<tr>
			<td>ETV1</td>
			<td>MSMB</td>
			<td>PIK3CA</td>
			<td>TFEB</td>
		</tr>
		<tr>
			<td>ETV4</td>
			<td>MUSK</td>
			<td>PKN1</td>
			<td>THADA</td>
		</tr>
		<tr>
			<td>ETV5</td>
			<td>MYB</td>
			<td>PPARG</td>
			<td>TMPRSS2</td>
		</tr>
		<tr>
			<td>ETV6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">The assay includes gene-specific primers to various exons (and some introns) in the above 53 genes.</td>
		</tr>
	</tbody>
</table>
Supplementary Table 1: List of genes for which gene-specific primers are included in the assay (to various exons and introns).

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

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. ...

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.