We thank Thermo Fisher Scientific for giving us the possibility to use their device and materials.

All procedures have been approved by the local ethics committee (Human Research Ethics Committee, Centre Hospitalier Universitaire de Nice, Tumoroth&#232;que BB-0033-00025). Informed consent was obtained from all patients to use samples and generated data. All samples were obtained from patients diagnosed with NS-NSCLC in LPCE (Nice, France) between 20 September and 31 January 2022 as part of the medical care.
1. Preparation of FFPE DNA and RNA samples using automated purification instrument ( API ) (Processing time: 5 h 15 min)
<ol>
	<li>Run planification on the instrument.
	<ol>
		<li>Power on the instrument and sign in with the user name and password (Table of Materials). Create a purification run plan by clicking Run, then Add Plan, and by giving a name to the run plan.</li>
		<li>Then, select the appropriate purification kit and protocol for sequentially purifying DNA and RNA from formalin-fixed paraffin-embedded (FFPE) samples. Enable quantitation after purification.</li>
		<li>Select the desired elution volume (50 &#181;L) from the dropdown list, then click Next. Select the number of samples that will be extracted.</li>
		<li>Then select each sample, click Edit, enter Sample ID, and then click Save.</li>
	</ol>
	</li>
	<li>Preparate FFPE DNA and RNA samples using GPI
	<ol>
		<li>Prepare FFPE tissue samples (4 cutting sections of 10 &#181;m with a microtome) or perform macrodissection directly on FFPE blocks. Place each FFPE tissue sample in separated and identified FFPE sample processing tubes (Table of Materials).</li>
		<li>Centrifuge at 2000 x g for 1 min to collect the tissue at the bottom of the tubes.</li>
		<li>Add to each FFPE sample processing tube a protease digest master mix prepared from a nucleic acid purification kit and incubate at 60 &#176;C for at least 60 min (Table of Materials). Incubate the samples at 90 &#176;C for 60 min.</li>
		<li>After incubation, let the samples cool down for 5 min at room temperature (RT).</li>
		<li>For each FFPE sample processing tube, lift the inner tube, lock it by turning left, and then centrifugate the samples at 2000 x g for 10 min.</li>
		<li>Unlock the inner tubes by turning right, separate them from the outer tubes, and discard them. Keep the samples in the outer tubes on ice until loading on the API.</li>
		<li>Prepare a DNase digestion master mix and load it in prefilled FFPE DNA and RNA purification plate 2. Then, load the prepared samples in FFPE DNA and RNA purification plate 1 (Table of Materials).</li>
	</ol>
	</li>
	<li>Start the purification run on API.
	<ol>
		<li>On the API screen, click Run, select the Run Plan, then click Next.</li>
		<li>Follow the on-screen indications to load the API with the required consumables and reagents needed for the purification run (Table of Materials).</li>
		<li>When all the reagents are loaded, click Next. Close the API door and click Start.</li>
		<li>At the end of the run, click &#171; unload &#187; on the touchscreen and immediately remove the 48-well nucleic acid archive plate containing the purified sample DNA and RNA.</li>
		<li>Export the quantitation results. Seal the plate and store purified samples at-80 &#176;C. Transfer the 96 well plate to the sequencer to be analyzed.</li>
	</ol>
	</li>
	<li>Create a sample and create a run.
	<ol>
		<li>Open and sign in to the software, then choose in the menu bar Samples and click Create Sample.</li>
		<li>Enter the sample&#39;s name, sex, and percentage of tumor cells, and complete the required fields and optional fields if needed.</li>
		<li>Save the details (the sex and the percentage of tumor cells are important for analyzing copy number variants [CNVs]).</li>
		<li>Choose Runs and Nucleic Acid to Result in the menu bar.</li>
		<li>Enter the run name in the Setup step. Click Next.</li>
		<li>Select assay in the Assays step. Click Next.</li>
		<li>Select the samples in the Samples step to run with the assay.</li>
		<li>Then, in the Selected Assays pane, click Assign. Click Next.</li>
		<li>Review the sample positions on the sample input plate. Click Next.</li>
		<li>Review the run plan summary, and then Save &#38; Print or Save.</li>
	</ol>
	</li>
</ol>
2. Automated NGS on the sequencer (Processing time: 30 min)
<ol>
	<li>Load the sample plate. 
	NOTE: The Optimal nucleic acid concentration is 0.5 ng/&#181;L. The validated nucleic acid concentration range is 0.33-1 ng/&#181;L. For DNA and RNA, 1 ng/&#181;L concentration was validated in the lab.
	<ol>
		<li>The sequencer requires 20 &#181;L total volume. Ensure there is sufficient volume for sample loading.</li>
		<li>Add positive controls (DNA and RNA) and NTC (DNA and RNA) to the 1st stage of the purification instrument without being extracted. Use DNA and RNA controls for each run to validate the run and validate the batches used. Add DNA and RNA controls to the plate at the end of the API run.</li>
	</ol>
	</li>
	<li>Load the sequencer and start a run.
	<ol>
		<li>Remove all the reagents from their boxes in the refrigerator or freezer and thaw at RT for a minimum period of 30 min (Table of Materials) and a maximum of 12 h.</li>
		<li>In the laboratory, keep the sequencer always turned ON following the advice given by the supplier during the on-site training. Sign in to the system.</li>
		<li>Load the plate from the purification instrument, which contains samples, after sealing the plate with a sheet of adhesive PCR plate foil (Table of Materials).</li>
		<li>Install all consumables in the deck. The sequencer provides step-by-step instructions to load each required consumable in a highlighted position (Table of Materials).</li>
		<li>Check that there are no precipitations on tube 3 of the Strip 2-HD. Flick the tube or gently vortex the strip to dissolve the precipitate if needed.</li>
		<li>Strip 1 and Strip 3 contain magnetic beads. This step is very critical: ensure that the beads are resuspended. There should be no beads left in the upper part of the tube.</li>
		<li>Turn the strip upside down and back 3-4 times to remove the beads. Hold the strip at one end with the strip seal oriented upward. Afterward, swiftly swing the strip downward using a quick, centrifugal arm movement, and finish by giving a sharp flick of the wrist.</li>
		<li>Centrifuge at 300 x g for 15 s. Repeat if needed. Centrifuge the strips with adapters and strip holders to minimize failures observed randomly in connection with residues of balls in the upper part of the strips.</li>
		<li>Close the system and click Next. Install the sequencing reagents bay doors (Table of Materials). Close the door. The sequencer automatically starts the run.</li>
	</ol>
	</li>
	<li>Cleaning
	<ol>
		<li>For cleaning after the run, when the run is complete, click Next.</li>
		<li>The door opens successively. Follow the on-screen instructions to empty the waste and remove all consumables. Click Next.</li>
		<li>Close the deck when finished, click Next. A 2 min UV cleaning starts. The sequencer is ready to start a new run.</li>
	</ol>
	</li>
</ol>
3. Analysis of the results using the integrated software (Analysis time by patient [samples ADN and ARN]: 15 min)
NOTE: The technique is accredited by the French Accreditation Committee (COFRAC) ISO 15189 (https://www.cofrac.fr/)
<ol>
	<li>Data and results
	<ol>
		<li>Select a run result or a sample result in the Results menu. In the Results/Run Results screen, all the runs are listed.</li>
		<li>Search the list of results by filtering in a column header.</li>
		<li>Click Results and click Sample Results. Then, click the name of the sample of interest in the sample name column to view sequencing results for a unique sample.</li>
		<li>Click Columns in the top left corner of the screen or deselect the columns from the table.</li>
	</ol>
	</li>
	<li>View the QC results and view the amplicon coverage.
	<ol>
		<li>Click the QC tab in the Results screen.</li>
		<li>For DNA, ensure that the median absolute pairwise differences (MAPDs) are between 0 and 0.5 to validate the copy number variants (CNV) analysis. The mapped reads must be greater than 1.5 million.</li>
		<li>For RNA, check if the mapped reads are between 150,000 and 400,000. Below this, there is a risk of false negatives, and beyond that, there is a risk of false positives.</li>
		<li>Click a sample name to open the Key Findings tab.</li>
		<li>Scroll to the amplicon Coverage Graphs.</li>
		<li>Review the coverage graphs. Define the minimal threshold for the amplicon coverage, as the supplier does not provide it. To define a range and a threshold, mix a control and a wild type (WT) sample. Then, observe the smallest value of the amplicon coverage for one or more selected mutations.</li>
	</ol>
	</li>
	<li>View variant results.
	<ol>
		<li>To export the data in tabular format, click Export in the upper right corner of the screen.</li>
		<li>To view the single nucleotid variant/insertion-deletion variant (SNV/INDEL) result, click the Variants tab, then click SNVs/Indels. Click Edit Filters and select No Filter. 
		NOTE: The sensitivity threshold has been defined at 5% according to the recommendations of the INCA (French National Cancer Institute) group (https://www.e-cancer.fr/Professionnels-de-sante/Les-therapies-ciblees/Les-plateformes-de-genetique-moleculaire-des-cancers/Le-programme-d-assurance-qualite-des-plateformes).</li>
		<li>View Fusion results and view RNA Exon Variants: Click the Variants tab, then click Fusions.</li>
		<li>In the top right, click Visualization and RNA Exon Variant, then review the RNA Exon Variants plot.</li>
		<li>With this kit, exon skipping of the EGFR, MET, and AR genes can be detected. Click Visualization; it is easier to analyze.</li>
		<li>View RNA exon tile fusion imbalance: Click the Variants tab, then click Fusions.</li>
		<li>In the top right, click Visualization and RNA Exon Tile Fusion Imbalance, then review the RNA Exon Tile Fusion Imbalance plots. An imbalance fusion is a fusion between a known partner and a partner that is not covered by the panel.</li>
		<li>View CNV results: Click CNVs in the Variants tab to display the data. 
		NOTE: It is necessary to be vigilant with the results of CNV by informing during the preparation of the samples of the gender and the percentage of tumor cells of the sample. The AR gene is only carried by the X chromosome. If the sex is not specified, the male sex could encounter losses, leading to false positive results in male patients.</li>
	</ol>
	</li>
	<li>Review plugin results.
	<ol>
		<li>Review Coverage Analysis plugin results: In the top right, click and select download files.</li>
		<li>The Coverage Analysis plugin generates a Coverage Analysis Report.</li>
		<li>Review molecular Coverage Analysis plugin results: In the top right, click and select download files. This analysis verifies if all the amplicons are covered and confirms the copy number variants (CNVs) results provided by the software.</li>
	</ol>
	</li>
	<li>View assay metrics and the run report
	<ol>
		<li>Click the Run Report tab in the Run Summary.</li>
		<li>To view the Run Report, select the Select Sample option in the dropdown menu.</li>
		<li>Assay metrics and the run report: Find the sequencing metrics at the top of the screen, followed by sample-specific metrics in the Run Samples table. 
		NOTE: Information in the customer support archive (CSA) file can help troubleshoot results, but they can&#39;t be directly analyzed. The CSA files summarize the data of the entire run data and highlight the causes of an issue in case of a failed run.</li>
		<li>Download a run report: Click the Reports tab.</li>
		<li>Click Download Report to download a run report summary in PDF format.</li>
	</ol>
	</li>
	<li>Generate variant report.
	<ol>
		<li>Enable Generate Report in the Setup step to automatically generate a variant report for each sample during data analysis of a run.</li>
		<li>After generating the variant report for a sample, the system makes the report available in two places: First, a link is made available in the Results/Sample Results screen. Click the link to download the PDF.</li>
		<li>Second, go to the Variant Report pane in the Reports tab and click on the Download Report button. Sign the variant reports electronically or manually. 
		NOTE: Electronically signed reports have (Sign off) after the sample name in the Sample Results screen.</li>
	</ol>
	</li>
</ol>

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

Christophe Bontoux participates in paid speaker activities and receives benefits from Thermo Fisher Scientific. Paul Hofman participates in paid speaker activities and receives benefits and funding from Thermo Fisher Scientific.

The development of an ultra-fast amplicon-based NGS approach as reflex testing for molecular alteration assessment at diagnosis of any stage NS-NSLC is an optimal option for the detection of all guideline-recommended and emerging biomarkers in NS-NSCLC5,22,23. While sequential methods (IHC, PCR, FISH) focus only on specific genes and can result in tissue material exhaustion, this NGS workflow allows a specific and sensitive evaluation of the status of 50 genes, including mutations, fusions, and amplifications, even with a low amount of nucleic acid (minimum 13 ng for each DNA and RNA sequencing according to this study).
In addition, this method provides quick and relevant results for the management of patients with NS-NSCLC. Compared to a bespoke approach, the absence of an initial selection of cases to be analyzed, depending on the tumor stage, and without waiting for the physician&#39;s request enables them to save precious time. In this line, it has been shown that NGS testing of advanced NS-NSCLC directly affects patient overall survival24,25. In addition, these molecular results are frequently obtained simultaneously from the IHC results, especially for the evaluation of the percentage of PD-L1 positive tumor cells26. The physician can get the &#34;molecular profile&#34; of the tumor with all the biomarkers required for the best therapeutic choice of first-line treatment27. In specific situations, the physician can enroll the patients into a clinical trial according to the genomic results28,29,30,31.
Both DNA and RNA analysis with the panel can be performed on 2-14 patients per run, excluding controls (1 positive and 1 negative control). It is possible to make three runs per week, which means that 42 patients can benefit from an ultra-fast NGS profiling of their tumor. The purificator system, which performs the fully automated extraction and quantification of DNA and RNA before library preparation, enables rapid evaluation of the feasibility of the sequencing analyses.
The ability to quantify the extracted DNA and RNA prior to library preparation, and the option to work with small amounts of nucleic acid (starting from a minimum of 10 ng of DNA or RNA, as per the specifications) allows a rapid assessment of sequencing analysis viability.
An NGS approach can help optimize the management of the sample workflow in thoracic oncology by being more comprehensive in the detection of actionable biomarkers. It has been shown that the PCR-based method has intrinsic limitations in detecting some rare mutations, especially EGFR exon 20 insertion mutations32. Thus, the NGS approach enables a higher proportion of patients to receive biomarker-focused therapies. In this context, it has been demonstrated that NGS reflex testing for all advanced NS-NSCLC is cost-effective33. Moreover, the fully automated nucleic acid extraction, library preparation, and sequencing integrated with this amplicon-based NGS System is a significant time saver for laboratory staff.
However, implementing ultra-fast next-generation sequencing (NGS) as a reflex testing for diagnosing NS-NSCLC could encounter certain constraints in daily clinical practice. The gene panel&#39;s scope (encompassing 50 genes) is fewer compared to the extensive panels that comprise several hundred genes34. The panel includes all the recommended genes that are associated with a targeted therapy in thoracic oncology. Furthermore, some genes of interest with a potential prognostic value are not included. For instance, KEAP1, STK11, SMARCA4, and RB1 are not present on the panel used in the present work. These genes can be important in the future for the selection of patients to benefit from the FDA-approved KRASG12C inhibitors sotorasib9. The use of ultra-fast NGS with amplicon sequencing technology may also introduce the potential for missing rare fusion gene partners35. In addition, the imbalance analyses revealed some false positive ALK rearrangement initially36. The threshold for detecting ALK imbalance fusions was increased in the new modified assay. However, it is recommended to validate an imbalance result with an orthogonal technique (IHC and/or FISH).
Above all, this workflow raises questions related to the economic sustainability and the reimbursement of the NGS test, which can vary greatly depending on the country&#39;s health system, especially in Europe37. For the cost-effectiveness approach, it is imperative to run enough samples at the same time to save on consumables, which is sometimes not possible, as it depends on the sample recruitment and the laboratory35. Moreover, the possibility of generating an unnecessary additional workload for the laboratory staff should be discussed and requires good communication between physicians, surgeons, pathologists, and technical staff.
The simultaneous use of both DNA and RNA NGS is ideal but remains debatable. Knowing that most of the genomic alterations for targeted therapy are mutually exclusive38, it could be possible to first perform DNA and then RNA sequencing analysis. However, this strategy would consequently lead to a longer TAT to generate a report.
The adequacy of both the quantity and quality of extracted nucleic acids could represent a limitation in ensuring the robustness of NGS analyses, especially in cases involving hybrid capture sequencing technology and extensive panels that require a higher amount of material with higher TAT6. In this regard, this amplicon-based NGS System offers the advantage of requiring only 13 ng of DNA and RNA for the panel and has a very low overall failure rate. This is highly valuable in thoracic oncology since the size of samples is getting smaller and smaller20,39,40.
The indications of molecular testing are continuously changing in thoracic oncology and, for some gene alterations, are now recommended in adjuvant settings15. In our opinion, it is highly probable that several additional targeted therapies will emerge for the treatment of early-stage NS-NSCLC in routine clinical practice, reinforcing the need to promptly search for biomarkers at diagnosis. Taken together, the current progress in precision medicine in lung cancer will ineluctably favor an ultra-fast NGS approach that allows a more comprehensive, rapid, and material-saving molecular profiling of tumors.

Over the last decade, the development of targeted and immuno-therapies has significantly increased the overall survival (OS) of non-squamous non-small cell lung cancer (NS-NSCLC)1,2. In this regard, the ,number of mandatory genes and molecular targets to analyze when treating NS-NSCLC has increased over the last few years3,4.
Current international guidelines recommend testing EGFR, ALK, ROS1, BRAF, NTRK, RET, and MET at diagnosis of advanced NS-NSCLC5. Moreover, as new drugs have recently given very promising results in clinical trials, additional genomic alterations will shortly be screened in a number of additional genes, notably KRAS and HER2, along with BRAC1/BRAC2, PI3KA, NRG1, and NUT6,7,8,9. In addition, the status of different associated genes, such as STK11, KEAP1, and TP53 may be of strong interest for a better prediction of the response or resistance to some targeted therapies and/or immune checkpoint inhibitors (ICIs)10,11,12.
Importantly, the molecular alterations must be reported without significant delay to ensure careful clinical decision-making. The absence of molecular characterization of a tumor may lead to the initiation of non-targeted therapies such as chemotherapy with/without immunotherapy, leading to a suboptimal treatment strategy, as chemotherapy response is limited in patients with actionable alterations, such as EGFR mutations or gene fusions13.
Moreover, the current development of targeted therapies/immunotherapies in neoadjuvant and/or adjuvant settings could lead to systematically looking for, at least, EGFR and ALK alterations in early-stage NS-NSCLC as ICIs should be administered only in tumors that are wild-type for EGFR and ALK14. It is now also mandatory to test for the presence of EGFR mutations in early-stage NS-NSCLC, since osimertinib (a third-generation EGFR tyrosine kinase inhibitor) can be used as adjuvant therapy in EGFR-mutant NS-NSCLC15.
The strategy for the assessment of the different biomarkers in predicting the response to different targeted therapies and/or immunotherapies in NS-NSCLC patients is moving fast, which makes the identification of these biomarkers sequentially difficult3,16. In this regard, Next-Generation Sequencing (NGS) is now the optimal approach for high throughput parallel assessment of gene alterations in NS-NSCLC5,17.
However, NGS workflow can be difficult to master and may conduct to longer TAT18,19. Thus, many centers still perform sequential approaches (immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and/or targeted sequencing). However, this strategy is limited in case of small sample size and, above all, because of the increased number of actionable mutations that are required to be tested in NS-NSCLC20. Thus, ultra-fast and straightforward testing methods allowing the rapid assessment of gene alterations have become increasingly important for optimal clinical decision-making. Moreover, approved and accreditated systems for molecular testing are becoming mandatory for the prescription of specific targeted therapies.
Here, we describe an ultra-fast and automated amplicon-based DNA/RNA NGS assay for molecular testing of NS-NSCLC that is used in the Laboratory of Clinical and Experimental Pathology Laboratory (LPCE), Nice University Hospital, France and is accredited according to the ISO 15189 norm by the French Accreditation Committee (COFRAC) (https://www.cofrac.fr/). The COFRAC certifies that the laboratory fulfills the requirements of the standard ISO 15189 and COFRAC rules of application for the activities of testing/calibration in molecular analysis in automated NGS on a sequencer with the panel performed by the laboratory. Accreditation per the recognized international standard ISO 15189 demonstrates the laboratory&#8217;s technical competence for a defined scope and the proper operation of an appropriate management system in this laboratory. The benefits and limitations of this workflow, starting from the preparation of tissue biopsy samples to obtaining the report, are discussed.

<table><tbody><tr><td>96 well hard shell plate clear</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>4483354</td><td></td></tr><tr><td>Adhesive PCR Plate Foil</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>AB0626</td><td></td></tr><tr><td>AutoLys M tube&amp;nbsp;</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A38738</td><td>FFPE sample processing tubes</td></tr><tr><td>Genexus Barcodes 1-32 HD</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A40261</td><td></td></tr><tr><td>Genexus GX5 Chip and Genexus Coupler</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A40269</td><td></td></tr><tr><td>Genexus Pipette Tips</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A40266</td><td></td></tr><tr><td>Genexus Purification Instrument</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A48148</td><td>Automated purification instrument (API)</td></tr><tr><td>Genexus Sequencing Kit</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A40271</td><td></td></tr><tr><td>Genexus Templating Strips 3-GX5 and 4</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A40263</td><td></td></tr><tr><td>Genexus Integrated Sequencer</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A45727</td><td></td></tr><tr><td>Ion Torrent&amp;nbsp; Genexus FFPE DNA/RNA Purification Combo Kit</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A45539</td><td></td></tr><tr><td>Oncomine&amp;nbsp; Precision Assay GX (OPA) Panel (included Strips 1 and 2-HD)</td><td>Thermo Fisher Scientific (Waltham, Massachusetts, USA)</td><td>A46291</td><td></td></tr></tbody></table>

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.

Using the procedure presented here, described in detail in our recent publications21, we developed an optimal workflow for the assessment of molecular alteration as a reflex testing in routinely performed clinical practice for diagnosis in patients with NS-NSCLC using an ultra-fast amplicon-based next-generation sequencing approach. The molecular workflow of the method is shown in Figure 1. The list of genes included in the panel is shown in Supplementary Figure 1.
Molecular analysis was performed in 259 patients with NS-NSCLC. It included 153 bronchial biopsies, 47 transthoracic biopsies, 39 surgical specimens, and 25 cellblocks from 13 pleural effusion and 7 EBUS (Table 1). The following driver mutant genes were observed in 242 valid or available NGS DNA/RNA analyses: KRAS (25.4%), EGFR (18.2%), BRAF (6.3%), ERBB2 (1.7%) and MET (1.7%). The following gene fusions were reported: ALK (4.3%), ROS1 (2.3%), RET (1.9%) and NTRK (0.8%). Following alterations were detected with an incidence below 1% (e.g., IDH1, CDKN2A, FGFR3, KIT, MTOR, FGFR4. NTRK3, NRAS, PIK3CA, IDH2, ERBB3, ERBB4, AR, CHECK2, SMO, PTEN). For DNA, 10 cases (4%) failed, whereas 5 cases (2%) failed for RNA. Failure was due to poor quality of DNA/RNA and/or low amount of DNA/RNA. Failed analysis occurred only with samples having less than 10% tumor cellularity. Thus, we defined the tumor cellularity cut-off at 10%. For DNA NGS results, 10 cases (4%) did not pass due to either the low quality of nucleic acids (6 cases) or insufficient nucleic acid amounts (4 cases with &#60;13 ng of DNA). Similarly, five cases (2%) failed for RNA sequencing results due to both inadequate RNA quality and a low quantity of RNA (&#60;13 ng of RNA). The average proportion of tumor cells in the unsuccessful cases was limited (15%, ranging from 10% to 20%), in contrast to the successful cases where tumor cell percentages ranged from 30% to 90%. Among the 15 failed samples, 11 originated from small tissue biopsies (with an average area of 2 mm2), while 4 were cytological specimens (such as endobronchial ultrasound [EBUS]).
The sensitivity of the method for the detection of SNVs and INDELs was 93.44%, for CNVs was 100%, and for fusions was 91.67% with a minimum tumor cell content of 10% (Table 1).
The mean TAT from endoscopy sampling from the molecular report was 72 h (range: 48-96 h), with a mean TAT from extraction to report of 24 h.
The analytical validation of the above-described NGS workflow was performed on 30 NS-NSCLC cases. The validation demonstrated 100% concordance with the gold standard methods previously used at our center and listed in detail in the original publication21: RT-PCR EGFR Mutation Test; RT-PCR KRAS Mutation Test; ALK IHC, ALK FISH; ROS1 IHC; ROS1 FISH; BRAFV600E; and DNA/RNA NGS methods.
The multi-step procedure from sampling to the molecular report of an EGFR/TP53-mutant lung adenocarcinoma is pictured in Figure 2. The representation of the mutation found in the biopsy sample using integrative genomic viewer (IGV) software is shown in Supplementary Figure 2. An example of a generated report from the reporting software is shown in Supplementary Figure 3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65190/65190fig01.jpg" /> 
Figure 1: Diagram of the panel workflow used in this study. This figure has been modified with permission from Ili&#233; et al.21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65190/65190fig01large.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/65190/65190fig02.jpg" /> 
Figure 2: Multiple steps from sampling to molecular biology report. (A) Three bronchial biopsy samples received in the laboratory from the endoscopy room. (B) Hematoxylin/Eosin/Safran-stained slide of the bronchial biopsy sample showing a lung adenocarcinoma with about 40%-50% of tumor cells (100x magnification). (C) Report from the reporting software showing an EGFR mutation associated with a TP53 mutation in the tumor sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65190/65190fig02large.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>Total number of patients</td>
			<td>259</td>
		</tr>
		<tr>
			<td>Bronchial biopsies</td>
			<td>153</td>
		</tr>
		<tr>
			<td>Transthoracic biopsies</td>
			<td>47</td>
		</tr>
		<tr>
			<td>Surgical specimens</td>
			<td>39</td>
		</tr>
		<tr>
			<td>Pleural effusion&#160;</td>
			<td>13</td>
		</tr>
		<tr>
			<td>EBUS</td>
			<td>7</td>
		</tr>
		<tr>
			<td>Driver mutant genes</td>
			<td></td>
		</tr>
		<tr>
			<td>KRAS&#160;</td>
			<td>25.40%</td>
		</tr>
		<tr>
			<td>EGFR&#160;</td>
			<td>18.20%</td>
		</tr>
		<tr>
			<td>BRAF</td>
			<td>6.30%</td>
		</tr>
		<tr>
			<td>ERBB2</td>
			<td>1.70%</td>
		</tr>
		<tr>
			<td>MET</td>
			<td>1.70%</td>
		</tr>
		<tr>
			<td>Gene fusions&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>ALK&#160;</td>
			<td>4.30%</td>
		</tr>
		<tr>
			<td>ROS1</td>
			<td>2.30%</td>
		</tr>
		<tr>
			<td>RET&#160;</td>
			<td>1.95%</td>
		</tr>
		<tr>
			<td>NTRK</td>
			<td>0.80%</td>
		</tr>
		<tr>
			<td>Failure - DNA cases</td>
			<td>10 cases (4%)</td>
		</tr>
		<tr>
			<td>Failure - RNA cases</td>
			<td>5 cases (2%)</td>
		</tr>
		<tr>
			<td>Tumor cellularity cut-off&#160;</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Percentage range of tumor cells in the failed cases&#160;</td>
			<td>10%&#8211;20%</td>
		</tr>
		<tr>
			<td>Percentage range of tumor cells in successful cases&#160;</td>
			<td>30%&#8211;90%</td>
		</tr>
		<tr>
			<td>Sensitivity of the method&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Detection of SNVs and INDELs&#160;</td>
			<td>93.44%</td>
		</tr>
		<tr>
			<td>Detection of CNVs&#160;</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>Detection of Fusions</td>
			<td>91.67%</td>
		</tr>
	</tbody>
</table>
Table 1: Outcomes of molecular analysis. Molecular analysis was performed in 259 patients with NS-NSCLC, including 153 bronchial biopsies, 47 transthoracic biopsies, 39 surgical specimens, and 25 cellblocks from 13 pleural effusion and 7 EBUS.
Supplementary Figure 1: The genes included in the NGS oncomine precision assay GX panel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65190/NGSOPAJove_Sup Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Representation of generated reads and mutations found in the biopsy sample using IGV software. (A) EGFR gene and (B) TP53 gene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65190/NGSOPAJove_Sup Figure 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Molecular report generated by the reporting software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65190/NGSOPAJove_Sup Figure 3.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer at JoVE.com

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.

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 ...

ultra-fast-amplicon-based-next-generation-sequencing-non-squamous-non

Nanyang Technological University

Research

JoVE Journal

Biology

1.0K Views.  Université Côte d’Azur, CHU de Nice. Tegenwoordig vereisen gerichte therapieën voor niet-kleincellige longkanker een snelle detectie van meerdere genomische veranderingen. Het is dus cruciaal voor een optimale zorg, aangezien thoracale biopsie vaak klein is en weinig tumorcellen bevat. Daarom presenteren we een ultrasnelle Next-Generation Sequencing-workflow die wordt geïmplementeerd voor routinematige diagnose van niet-kleincellige longkanker in het pathologielaboratorium.Momenteel gerichte NGS met behulp van verschill...

Video: Auteur in de schijnwerpers: vooruitgang in het testen van moleculaire biomarkers voor niet-plaveiselcel, niet-kleincellige longkanker 

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 ...

Cancer Research

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. ...

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

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

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.

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

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