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 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.
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.
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)
2. Automated NGS on the sequencer (Processing time: 30 min)
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/)
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 <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 (<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.
Figure 1: Diagram of the panel workflow used in this study. This figure has been modified with permission from Ilié et al.21. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Total number of patients | 259 |
Bronchial biopsies | 153 |
Transthoracic biopsies | 47 |
Surgical specimens | 39 |
Pleural effusion | 13 |
EBUS | 7 |
Driver mutant genes | |
KRAS | 25.40% |
EGFR | 18.20% |
BRAF | 6.30% |
ERBB2 | 1.70% |
MET | 1.70% |
Gene fusions | |
ALK | 4.30% |
ROS1 | 2.30% |
RET | 1.95% |
NTRK | 0.80% |
Failure – DNA cases | 10 cases (4%) |
Failure – RNA cases | 5 cases (2%) |
Tumor cellularity cut-off | 10% |
Percentage range of tumor cells in the failed cases | 10%–20% |
Percentage range of tumor cells in successful cases | 30%–90% |
Sensitivity of the method | |
Detection of SNVs and INDELs | 93.44% |
Detection of CNVs | 100% |
Detection of Fusions | 91.67% |
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. Please click here to download this File.
Supplementary Figure 2: Representation of generated reads and mutations found in the biopsy sample using IGV software. (A) EGFR gene and (B) TP53 gene. Please click here to download this File.
Supplementary Figure 3: Molecular report generated by the reporting software. Please click here to download this File.
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'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 "molecular profile" 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'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'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.
The authors have nothing to disclose.
We thank Thermo Fisher Scientific for giving us the possibility to use their device and materials.
96 well hard shell plate clear | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | 4483354 | |
Adhesive PCR Plate Foil | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | AB0626 | |
AutoLys M tube | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A38738 | FFPE sample processing tubes |
Genexus Barcodes 1-32 HD | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40261 | |
Genexus GX5 Chip and Genexus Coupler | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40269 | |
Genexus Pipette Tips | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40266 | |
Genexus Purification Instrument | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A48148 | Automated purification instrument (API) |
Genexus Sequencing Kit | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40271 | |
Genexus Templating Strips 3-GX5 and 4 | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40263 | |
Genexus Integrated Sequencer | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A45727 | |
Ion Torrent Genexus FFPE DNA/RNA Purification Combo Kit | Thermo 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 |