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 instru...

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

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...

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.

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ultra-fast amplicon-based next-generation sequencing in non-squamous non-small cell lung cancer

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

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

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

Nowadays, targeted therapies for non-small cell lung cancer require rapid detection of multiple genomic alterations. So it's crucial for an optimal care, as thoracic biopsy are often small and contain few tumor cells. So we present an ultra-fast Next-Generation Sequencing workflow that is implemented for routine non-small cell lung cancer diagnosis in the pathology laboratory.Currently targeted NGS using different panel sizes, one gene sequencing testing using RT-PCR, digital PCR, fluorescence in situ hybridization, and immunohistochemistry are the different methods used to detect molecular alterations. However, most of these methods can be time and tissue material consuming, leading to a delayed diagnosis, and a risk of tissue exertion. The challenge lies in efficiently detecting multiple molecular alterations while saving materials and answering a speeding diagnosis.The workflow provides thoracic oncologist with comprehensive tumor information, including histological diagnosis, PD-L1 status, and targetable genomic aberrations. It enables rapid treatment decision making for optimal therapy selection. Our protocol identify all key molecular targets in thoracic oncology based on ESMO, NCCN, and ASCO recommendations, delivers timely results even with small samples sizes and limited DNA/RNA.And so the automated system reduced technician workload with just three hours of wet lab manipulation. We aim to focus on the rapid and comprehensive genomic profiling characterizations of tumors by developing a large NGS panel for tissue and plasma samples in the future. However, increased strategies to detect numerous molecular alterations in small sample sizes and cytological samples using large panels are urgently needed.

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...

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

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

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

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Research

JoVE Journal

Biology

Preparation of FFPE DNA and RNA Samples for Sequencing Using an Automated Purification Instrument (API)

To begin, cut the formalin-fixed paraffin-embedded or FFPE DNA and RNA samples. Turn on the automated purification or API instrument and sign in with the username and password. Next, in the Integrated Genomic Viewer software, under samples, click Create Samples"enter the sample name and select the application category.After entering the sample details, click on Save"Then select Runs"click on Sample to Result"and enter the run name before clicking Next"Select the assay followed by a sample of interest and click Next"After selecting each sample, click Assign"then check the elution parameters and save the run. Place FFPE tissue samples and sample processing tubes. Centrifuge the tube at 2, 000 G for one minute.Add the Protease Digest master mix prepared from a nucleic acid purification kit into the tube. Incubate the sample at 60 degrees Celsius for one hour, followed by incubating it at 90 degrees Celsius for one hour. After cooling the samples, lift the inner tube of the sample processing tube and lock it by turning left.Centrifuge the inner tube at 2, 000 G for 10 minutes. Then unlock the tube by turning right and separating it from the other tube. Discard the inner tube and place the outer tube on ice until loading on the API.Prepare a DNA digestion master mix and load in prefilled FFPE DNA and RNA Purification plate two. Load the prepared samples in FFPE DNA and RNA purification plate one. On the API screen click Run"then select the run plan and click Next"Follow the on-screen instructions to load the required consumables and reagents for the purification run.Once all the reagents are loaded, click Next"close the API door and click Start"T the end of the run, click Unload"and immediately remove the 48-well nucleic acid archive plate containing the purified DNA and RNA. After the sample purification is over, seal the plate and store it at minus 80 degrees Celsius.

Amplicon-Based Next-Generation Sequencing of Non-Squamous Non-Small Cell Lung Cancer

To begin, thaw all the sequencing reagents at room temperature for 30 minutes, turn on the sequencer and sign into the system. Seal the 96 well plate containing purified FFPE DNA and RNA samples with a sheet of adhesive PCR plate foil and load it into the sequencer. According to the sequencer instructions, install all consumables in the respective deck.Inspect tube three of strip two HD for any precipitations. If the tube contains any precipitates, dislodge them by flicking the tube or gently vortexing the strip. Turn strips one and three upside down and back three to four times to remove beads, hold the strips at one end with the strip seal oriented upward.Then swing the strip downward using a quick centrifugal arm movement and finish by giving a sharp flick of the wrist centrifuge the strips at 300G for 15 seconds. After closing the system, install the sequencing reagents bay doors, and close the door.

Sequencing Analysis of Non-Squamous Non-Small Cell Lung Cancer Using Integrated Genomic Viewer Software 

To begin, open the integrated genomic viewer software. In the results menu, click Sample Results. Then click on the name of the sample of interest from the sample name column to view sequencing results.To review molecular coverage analysis plugin results, in the top right, click Download Files. Next in the run summary, click the Run Report Tab to view assay metrics and Run Report. To view the SNV and INDEL result, click the Variance tab, then click SNVs and INDELs.Click Edit Filters and select No Filter. Next, click the Variance tab, then Fusions to view fusion results in RNA exon variants. In the top right, click Visualization and RNA exon variant.Then review the RNA exon variance plot. Click the Variance tab, then select Fusions to view RNA exon tile fusion imbalance. In the top right, click Visualization, then RNA Exon Tile Fusion Imbalance and review the RNA Exon Tile Fusion Imbalance plots.In the Variance tab, click CNVs to display CNV results. Finally, generate a variant report by clicking the link to download the PDF. This study represents the molecular analysis performed in 259 patients with NS-NSCLC including various biopsy types and surgical specimens.The driver mutant genes and gene fusions were also observed in DNA-RNA analysis. The sensitivity of the method for detecting single nucleotide variance insertions and deletions, copy number variance and fusions was high ranging from 91.67%to 100%

712 Views.  Université Côte d’Azur, CHU de Nice.  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.

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

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