Method Article

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

DOI:

10.3791/65190

⸱

September 8th, 2023

 ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Laboratory of Clinical and Experimental Pathology, Centre Hospitalier Universitaire de Nice, Université Côte d’Azur, CHU de Nice, 2Hospital-Integrated Biobank (BB-0033-00025), Université Côte d'Azur, Hôpital Pasteur, CHU de Nice, 3Institut Hospitalo-Universitaire (IHU), RespirERA, Université Côte d'Azur, Hôpital Pasteur, CHU de Nice, 4FHU OncoAge, Université Côte d'Azur, 5Team 4, Institute of Research on Cancer and Aging (IRCAN), CNRS INSERM, Centre Antoine-Lacassagne, Université Côte d'Azur, 6Department of Pulmonary Medicine and Thoracic Oncology, Centre Hospitalier Universitaire de Nice, Université Côte d'Azur

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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’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.

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Protocol

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All procedures have been approved by the local ethics committee (Human Research Ethics Committee, Centre Hospitalier Universitaire de Nice, Tumorothèque 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)

  1. Run planification on the instrument.
    1. 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.
    2. 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.
    3. Select the desired elution volume (50 µL) from the dropdown list, then click Next. Select the number of samples that will be extracted.
    4. Then select each sample, click Edit, enter Sample ID, and then click Save.
  2. Preparate FFPE DNA and RNA samples using GPI
    1. Prepare FFPE tissue samples (4 cutting sections of 10 µm 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).
    2. Centrifuge at 2000 x g for 1 min to collect the tissue at the bottom of the tubes.
    3. Add to each FFPE sample processing tube a protease digest master mix prepared from a nucleic acid purification kit and incubate at 60 °C for at least 60 min (Table of Materials). Incubate the samples at 90 °C for 60 min.
    4. After incubation, let the samples cool down for 5 min at room temperature (RT).
    5. 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.
    6. 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.
    7. 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).
  3. Start the purification run on API.
    1. On the API screen, click Run, select the Run Plan, then click Next.
    2. Follow the on-screen indications to load the API with the required consumables and reagents needed for the purification run (Table of Materials).
    3. When all the reagents are loaded, click Next. Close the API door and click Start.
    4. At the end of the run, click « unload » on the touchscreen and immediately remove the 48-well nucleic acid archive plate containing the purified sample DNA and RNA.
    5. Export the quantitation results. Seal the plate and store purified samples at-80 °C. Transfer the 96 well plate to the sequencer to be analyzed.
  4. Create a sample and create a run.
    1. Open and sign in to the software, then choose in the menu bar Samples and click Create Sample.
    2. Enter the sample's name, sex, and percentage of tumor cells, and complete the required fields and optional fields if needed.
    3. Save the details (the sex and the percentage of tumor cells are important for analyzing copy number variants [CNVs]).
    4. Choose Runs and Nucleic Acid to Result in the menu bar.
    5. Enter the run name in the Setup step. Click Next.
    6. Select assay in the Assays step. Click Next.
    7. Select the samples in the Samples step to run with the assay.
    8. Then, in the Selected Assays pane, click Assign. Click Next.
    9. Review the sample positions on the sample input plate. Click Next.
    10. Review the run plan summary, and then Save & Print or Save.

2. Automated NGS on the sequencer (Processing time: 30 min)

  1. Load the sample plate.
    NOTE: The Optimal nucleic acid concentration is 0.5 ng/µL. The validated nucleic acid concentration range is 0.33-1 ng/µL. For DNA and RNA, 1 ng/µL concentration was validated in the lab.
    1. The sequencer requires 20 µL total volume. Ensure there is sufficient volume for sample loading.
    2. 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.
  2. Load the sequencer and start a run.
    1. 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.
    2. 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.
    3. 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).
    4. 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).
    5. 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.
    6. 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.
    7. 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.
    8. 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.
    9. Close the system and click Next. Install the sequencing reagents bay doors (Table of Materials). Close the door. The sequencer automatically starts the run.
  3. Cleaning
    1. For cleaning after the run, when the run is complete, click Next.
    2. The door opens successively. Follow the on-screen instructions to empty the waste and remove all consumables. Click Next.
    3. Close the deck when finished, click Next. A 2 min UV cleaning starts. The sequencer is ready to start a new run.

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/)

  1. Data and results
    1. Select a run result or a sample result in the Results menu. In the Results/Run Results screen, all the runs are listed.
    2. Search the list of results by filtering in a column header.
    3. 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.
    4. Click Columns in the top left corner of the screen or deselect the columns from the table.
  2. View the QC results and view the amplicon coverage.
    1. Click the QC tab in the Results screen.
    2. 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.
    3. 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.
    4. Click a sample name to open the Key Findings tab.
    5. Scroll to the amplicon Coverage Graphs.
    6. 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.
  3. View variant results.
    1. To export the data in tabular format, click Export in the upper right corner of the screen.
    2. 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).
    3. View Fusion results and view RNA Exon Variants: Click the Variants tab, then click Fusions.
    4. In the top right, click Visualization and RNA Exon Variant, then review the RNA Exon Variants plot.
    5. With this kit, exon skipping of the EGFR, MET, and AR genes can be detected. Click Visualization; it is easier to analyze.
    6. View RNA exon tile fusion imbalance: Click the Variants tab, then click Fusions.
    7. 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.
    8. 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.
  4. Review plugin results.
    1. Review Coverage Analysis plugin results: In the top right, click and select download files.
    2. The Coverage Analysis plugin generates a Coverage Analysis Report.
    3. 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.
  5. View assay metrics and the run report
    1. Click the Run Report tab in the Run Summary.
    2. To view the Run Report, select the Select Sample option in the dropdown menu.
    3. 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'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.
    4. Download a run report: Click the Reports tab.
    5. Click Download Report to download a run report summary in PDF format.
  6. Generate variant report.
    1. Enable Generate Report in the Setup step to automatically generate a variant report for each sample during data analysis of a run.
    2. 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.
    3. 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.

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Results

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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 Figu...

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Discussion

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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 eval...

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Disclosures

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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.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
96 well hard shell plate clearThermo Fisher Scientific (Waltham, Massachusetts, USA)4483354
Adhesive PCR Plate FoilThermo Fisher Scientific (Waltham, Massachusetts, USA)AB0626
AutoLys M tube Thermo Fisher Scientific (Waltham, Massachusetts, USA)A38738FFPE sample processing tubes
Genexus Barcodes 1-32 HDThermo Fisher Scientific (Waltham, Massachusetts, USA)A40261
Genexus GX5 Chip and Genexus CouplerThermo Fisher Scientific (Waltham, Massachusetts, USA)A40269
Genexus Pipette TipsThermo Fisher Scientific (Waltham, Massachusetts, USA)A40266
Genexus Purification InstrumentThermo Fisher Scientific (Waltham, Massachusetts, USA)A48148Automated purification instrument (API)
Genexus Sequencing KitThermo Fisher Scientific (Waltham, Massachusetts, USA)A40271
Genexus Templating Strips 3-GX5 and 4Thermo Fisher Scientific (Waltham, Massachusetts, USA)A40263
Genexus Integrated SequencerThermo Fisher Scientific (Waltham, Massachusetts, USA)A45727
Ion Torrent  Genexus FFPE DNA/RNA Purification Combo KitThermo Fisher Scientific (Waltham, Massachusetts, USA)A45539
Oncomine  Precision Assay GX (OPA) Panel (included Strips 1 and 2-HD)Thermo Fisher Scientific (Waltham, Massachusetts, USA)A46291

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Tags

Amplicon Based SequencingMolecular AlterationsGenomic ProfilingTargeted TherapyFFPE TissueDNA RNA PurificationVariant DetectionThoracic Oncology

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