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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160; 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&#160; 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.

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 ...