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Liquid Biopsy Analysis Unit, Translational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), Santiago de Compostela, SpainUniversidade de Santiago de Compostela (USC), Santiago de Compostela, SpainGalician Precision Oncology Research Group (ONCOGAL), Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), Santiago de Compostela, Spain
Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainGalician Precision Oncology Research Group (ONCOGAL), Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainEpigenomics Unit, Cancer Epigenomics, Translational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), Santiago de Compostela, SpainRoche-CHUS Joint Unit, Translational Medical Oncology Group (Oncomet), Health Research Institute of Santiago (IDIS), 15706 Santiago de Compostela, Spain
Galician Precision Oncology Research Group (ONCOGAL), Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainEpigenomics Unit, Cancer Epigenomics, Translational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), Santiago de Compostela, SpainCentro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Santiago de Compostela, Spain
Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Santiago de Compostela, SpainDepartment of Medical Oncology, Complexo Hospitalario Universitario de Santiago de Compostela (SERGAS), Santiago de Compostela, SpainTranslational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), Santiago de Compostela, Spain
Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainGalician Precision Oncology Research Group (ONCOGAL), Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainCentro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Santiago de Compostela, SpainDepartment of Medical Oncology, Complexo Hospitalario Universitario de Santiago de Compostela (SERGAS), Santiago de Compostela, SpainTranslational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), Santiago de Compostela, Spain
Galician Precision Oncology Research Group (ONCOGAL), Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainFundación Pública Galega de Medicina Xenómica, SERGAS; Grupo de Medicina Xenomica-USC, Health Research Institute of Santiago (IDIS), Santiago de Compostela, Spain
Liquid Biopsy Analysis Unit, Translational Medical Oncology (Oncomet), Health Research Institute of Santiago (IDIS), Santiago de Compostela, SpainGalician Precision Oncology Research Group (ONCOGAL), Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), Santiago de Compostela, SpainCentro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Santiago de Compostela, Spain
Target therapies, such as EGFR tyrosine kinase inhibitors (TKIs), are the first choice in NSCLC patients with specific EGFR mutations (presents in 17% of metastatic patients) leading to a longer survival.
Association of patient characteristics and tumor genomics with clinical outcomes among patients with non-small cell lung cancer using a clinicogenomic database.
Solid tumour samples are commonly analysed to determine the presence of EGFR mutations. However, tissue/cytological samples are not always available or evaluable. Circulating free DNA (cfDNA) analyses represent a good alternative strategy to assess the EGFR status using next generation sequencing (NGS) or digital polymerase chain reaction (PCR)-based technologies. However, these methods require technical expertise and long turnaround time. Idylla ctEGFR Mutation Assay (Biocartis, Belgium) is a fully integrated real-time PCR-based test with a one-day protocol that allows an easy implementation of liquid biopsy analyses into the clinical routine. Thus, in the current study we explored the accuracy and performance of the Idylla solution as a non-invasive test to detect EGFR mutations in plasma samples of advanced NSCLC patients.
With this purpose we evaluated the concordance between EGFR analyses on cfDNA using Idylla and tissue samples. In addition, the concordance among different liquid biopsy-based approaches, BEAMing (Sysmex Inostics, USA), AVENIO (Roche Diagnostics, Australia) and Idylla, was investigated to evaluate the performance of the Idylla solution versus the other approaches. Fifty-three blood samples from 40 advanced NSCLC patients treated between January 2018 and March 2022 at the Medical Oncology Service of Complexo Hospitalario Universitario de Santiago de Compostela were included in the study (Fig. 1, Fig. 2A ). The study was performed in accordance with the Declaration of Helsinki and all individuals signed informed consent forms approved by Santiago de Compostela and Lugo Ethics Committee (Ref: 2017/538) prior to enrolling in the study. Peripheral blood was obtained by direct venepuncture using CellSave (Menarini Silicon Biosystems, Italy), Streck Cell-Free DNA BCT (Streck, USA) or EDTA tubes.
Plasma and cellular components were separated by two sequential centrifugations and stored at –80°C until posterior analyses. cfDNA used for the analyses with BEAMing and AVENIO was isolated from 2 and 4 mL of plasma, respectively. For BEAMing technology, cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany) according to the manufacturer's instructions. In case of AVENIO analyses the cfDNA was isolated following the AVENIO ctDNA Expanded panel
(Roche, Switzerland) protocol. CfDNA levels were quantified by the fluorometric instrument Qubit 4 using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, USA).
Fig. 1Clinical and pathological characteristics of the patients included in the study. ADC, adenocarcinoma; F, female; M, male; NA, not applicable; NT, not tested; SCC, squamous cell carcinoma; WT, wild-type.
Fig. 2Feasibility study of Idylla ctEGFR Mutation Assay. (A) Study scheme. (B) Limit of detection (LOD) of seven EGFR alterations determined analysing different dilutions of control DNA (5, 1, 0.1%). (C) Proportion of patients with EGFR alterations or wild-type (WT) in total 53 plasma samples. (D) Concordance between Idylla EGFR Mutation Assay in cfDNA and Cobas test in tissue (n=40). Venn diagrams show the number of samples that present Del.ex19 or L858R mutation in tissue sample, blood sample or in both cases. (E) Concordance between BEAMing and Idylla assay in cfDNA samples (n=18). (F) Concordance between NGS and Idylla assay in cfDNA samples (n=15). (G) Variant allele frequencies (VAF) detected by BEAMing or NGS panel versus Idylla ΔCq. Red symbols represent discordant samples detected by BEAMing or NGS (n=3), green symbols represent concordant samples detected by both technologies.
The proposed technology, Idylla ctEGFR Mutation Assay, allows the qualitative detection of 49 EGFR mutations [four SNVs of exon 18 (G719A/C/S), two SNVs of exon 20 (T790M and S768I) and four SNVs of exon 21 (L858R and L861Q); 34 different exon 19 deletions, and five exon 20 insertions] in plasma samples. To explore in house the performance of the assay, accuracy and sensitivity were assessed. With this aim, we calculated the limit of detection (LOD) of the Idylla solution using commercial cfDNA standards that cover 10 EGFR variants with specific variant allele frequencies (VAF) of 5%, 1%, 0.1% and 0% (EGFR wild-type) (Cat no. HD825; Horizon Discovery, UK). Seven of the total 10 EGFR alterations present in the reference cfDNA are included in the panel analysed by the Idylla ctEGFR Mutation Assay [five SNVs including L861Q, L858R, S768I, T790M and G719S; one exon 20 insertion (V769-D770insASV) and one exon 19 deletion (del15)]. The LOD was defined for each of these seven alterations as the lowest mutant allele frequency yielding a positive result. Thus, LOD was determined as 5% VAF for L861Q and G719S; 1% for L858R, the insertion of exon 20 and T790M; and 0.1% VAF for Del15 of exon 19 and the point mutation S768I using a total of 160 ng of commercial cfDNA (Fig. 2B).
Once the experimental LOD was established, 53 samples from patients with NSCLC were analysed using the Idylla assay. Two mL of plasma together with 200 μL of proteinase K (20 mg/mL; Qiagen, Germany) were directly pipetted into the cartridge. The Idylla system allows cfDNA extraction, real time PCR amplification and detection of EGFR mutations in single samples. In parallel, the BEAMing method was performed with the OncoBEAM EGFR kit
Profiling of circulating tumor DNA in plasma of non-small cell lung cancer patients, monitoring of epidermal growth factor receptor p.T790M mutated allelic fraction using beads, emulsion, amplification, and magnetics companion assay and evaluation in fut.
(Roche, Switzerland), which covers alterations in 77 genes including all coding regions of EGFR, and sequenced on a NextSeq 500 (Illumina, USA). Moreover, tissue EGFR status was determined in the primary tumour obtained at diagnosis using Cobas EGFR Mutation Test v2 (Roche, Switzerland) in 39 patients (Fig. 2A). Statistical analyses were performed to evaluate the concordance between EGFR mutations based on tissue and the different plasma analysis technologies using R version 3.4.0 (The R Project, Austria) and GraphPad Prism 8 (GraphPad, USA).
Of the total samples analysed with the Idylla assay, 45.3% (24/53) presented at least one EGFR mutation. Among the 24 positive samples, two had more than one mutation, (harboured both L858R mutation and the T790M resistance mutation) and one presented two different exon 19 deletions, detecting 27 alterations in total. L858R mutation (48.1%) and Del.ex19 (33.3%) were the most frequent alterations detected (Fig. 2C). In addition, in 39 patients EGFR tissue status was determined using the Cobas EGFR Mutation Test v2. Overall concordance between the Cobas assay applied in tissue samples and the Idylla assay on plasma samples was 71.8% (28/39; Cohen's Kappa=0.472) reporting a moderate agreement (Fig. 2D). In six of 11 discordant patients the time between plasma sample collection and tissue biopsy was more than 3 months (Fig. 1). In addition, no clear association between discordant samples and clinical characteristics, such as metastatic localisations, was found. However, it is important to note that two discordant cases with a short time between liquid and solid biopsy (≤21 days) presented only brain metastasis. Of note, we observed a low concordance rate in cases with Del.ex19 (0.42), while a high concordance rate was found in the case of L858R mutation (0.8).
In addition, 17 paired plasma samples (before treatment or during therapy) were also analysed using OncoBEAM EGFR V2, reporting an overall concordance with the Idylla assay of 88.9% with a substantial agreement (16/18; Cohen's Kappa=0.78; Fig. 2E) in all samples. Nevertheless, three single determinations that showed VAFs equal or below 0.06% by OncoBEAM were undetectable on Idylla solution (Fig. 2G), in concordance with the results obtained with commercial cfDNA standards and the LOD observed. As well, 15 paired samples were analysed with both NGS and Idylla assay, reporting a concordance with the Idylla assay of 93.3% (14/15; Cohen's Kappa=0.865; Fig. 2F) in all samples. Similar to the above, results reported using BEAMing, determinations with low VAFs (below 0.13% in this case, n=2) and were undetectable on Idylla solution (Fig. 2G). Overall, taking into account both technologies (NGS and BEAMing), Idylla solution presents a high concordance rate (90.9% concordance, Cohen's Kappa= 0.820).
Finally, we also analysed the feasibility of using the Idylla ctEGFR Mutation Assay to monitor NSCLC patients during the treatment. Twenty-three samples from 10 NSCLC patients were analysed at different time points (Fig. 3A–D), and especially in one case (ID13), we observed that analysis using Idylla solution detects disease progression (Fig. 3B). A 62-year-old male was diagnosed in March of 2017 with NSCLC adenocarcinoma stage IIIA. Tissue sample analyses revealed the presence of the mutation L861Q in EGFR gene. In May, image analyses reported lesions into the central nervous system. The patient started chemotherapy and radiotherapy and showed a good response. In March 2018, the patient had a progression and he started gefitinib treatment (day 0). Before the treatment onset, a blood sample was collected and cfDNA analyses were performed by BEAMing technology and Idylla solution. Both reported a negative result for the mutation EGFR L861Q, probably due to location of the metastatic lesions and the consequent ctDNA low levels. Longitudinal analyses during gefitinib treatment (days 49, 103 and 201) in cfDNA reported the absence of L861Q mutation in plasma samples until day 355, when the patient presented progression of disease at different levels by image analyses. At progression, an increase in cfDNA levels and presence of the mutation L861Q using Idylla solution were determined, in accordance with the disease evolution. Other cases maintained mutation positivity at baseline and progression of disease and/or showed increased ctDNA levels as occurred in the patient ID19 (Fig. 3C).
Fig. 3Monitoring EGFR status in NSCLC patients using the Idylla EGFR Mutation Assay. (A) Swimmer plot on monitored patients (n=10). (B) Evolution of cfDNA levels and EGFR L861Q mutations in patient ID13 during gefitinib treatment. The L86Q1 mutation appeared at progression of disease (PD), which was confirmed by image analyses. (C,D) Evolution of cfDNA levels and EGFR L858R mutation in patient ID19 and ID11 during different TKI treatments. In both patients all determinations were positive for the mutation L858R. Patient ID19 progressed at 12 weeks and an increase in L858R mutation and cfDNA levels were found at this time, supporting the interest of using Idylla solution to detect progression of disease. Patient ID11 showed stable disease at 10 weeks by image analyses and a reduction of L858R mutation was found using Idylla solution despite an increase in cfDNA levels, suggesting an active response to gefitinib but probably a progression due to a different tumour clone. One month later, the patient showed clinical deterioration and died. In this case, imaging tests may be of limited value, while liquid biopsy could provide useful information on the disease evolution.
Regarding the interest in improving options to interrogate EGFR status in cfDNA, it is important to have in mind that tumour analyses have several limitations in NSCLC patients, such as access difficulty (mainly to biopsy metastasis), impossibility of characterising the disease in real time, and lack of representative tumour heterogeneity. To solve these limitations, in recent years liquid biopsy has emerged as a clear alternative and one of the main pillars for personalised oncology, reporting promising results mainly focused on the cfDNA analyses.
Rapid EGFR mutation detection using the Idylla platform: single-institution experience of 1200 cases analyzed by an in-house developed pipeline and comparison with concurrent next-generation sequencing results.
and its potential value to monitor the therapy response in NSCLC patients. Of note, a good concordance rate between analyses in tissue and plasma cfDNA with the Idylla BRAF Mutation Test has been previously described.
In our study, a moderate agreement between tissue and plasma results was found. This can be partially explained by the time between blood and tissue sample collection in the discordant patients, as longer time between collection of samples has a higher probability of molecular evolution of the tumour. On the other hand, although we reported a low concordance rate of exon 19 deletions in contrast with the high concordance of the point mutation L858R, the small size of our cohort could not allow us to reach solid conclusions regarding this. However, it's important to highlight that two of seven discordant samples presented a VAF lower than 0.13% by NGS or BEAMing assay and in five of seven discordant samples, the time between plasma sample collection and tissue biopsy was more than 5 months. Therefore, the lower ctDNA content and tumour evolution could explain this lower concordance.
Furthermore, we investigated the concordance rates among Idylla solution and other reference technologies, BEAMing and NGS, and found a good overall agreement determining EGFR status. In line with our study in lung cancer, the automated system demonstrated a good diagnostic performance for KRAS analyses in patients with colorectal cancer, reaching a high concordance with BEAMing.
Rapid EGFR mutation detection using the Idylla platform: single-institution experience of 1200 cases analyzed by an in-house developed pipeline and comparison with concurrent next-generation sequencing results.
Of note, we also reported good correlation with Idylla solution and AVENIO panel employing plasma samples but with a lower time-to-result with Idylla which can be crucial for metastatic patients.
Integration of rapid PCR testing as an adjunct to NGS in diagnostic pathology services within the UK: evidence from a case series of non-squamous, non-small cell lung cancer (NSCLC) patients with follow-up.
It is important to note that the Idylla solution allowed us to analyse the mutational profile of NSCLC patients in longitudinal plasma samples during treatment without the need to perform further tissue biopsies. This advantage is crucial in patients that showed treatment resistance. For example, the mutation T790M appears as a mechanism of resistance during TKI therapy. Its detection is crucial to select the patients that should be treated with a third generation of TKIs.
Here, although we only detected the T790M mutation in two plasma samples, our analysis with commercial cfDNA showed that the Idylla system allows us to detect the TKI resistance mutation with a LOD of 1%. Preliminary studies have reported a moderate T790M detection rate of 66.6–80.0% in plasma samples using Idylla solution,
according to our study. In addition, the paired plasma samples analysis showed that T790M mutations with a VAF lower than 0.06% using BEAMing/NGS cannot be detected by the Idylla system. However, analyses with a large cohort of patients harbouring the resistance mutation T790M are needed to validate the clinical value of the kit to specifically detect T790M mutations.
Overall, the results obtained in this pilot study with a real-world cohort of patients allowed us to conclude that Idylla ctEGFR mutation assay could be employed as a first screen to detect EGFR mutations fast and on demand. If no EGFR mutation is detected due to the low ctDNA content in plasma, another sensitive technique such as a ddPCR-based strategy should be employed to confirm the negative result. Some aspects should be taken into account for the selection of an optimal platform or analytic circuit to analyse EGFR alterations in ctDNA. For example, highly vascularised tumours with multiple metastatic locations will have higher levels of ctDNA; therefore, the genotyping of a clinically relevant mutation will be easier to detect.
Regarding its value as a monitoring tool, our preliminary results showed that changes in the mutational profile of EGFR samples using the Idylla solution could provide valuable information to monitor the response in patients receiving TKI therapies.
Acknowledgements
This project would not be possible without the kind collaboration of all patients.
Conflicts of interest and sources of funding
JG-C receives personal fees from AstraZeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, MSD, Novartis, Roche, Sanofi, Takeda, Pierre Fabre, Rovi, Lilly, MSD and Ippsen Pharma, outside the submitted work. LL-M. receives honoraria for lectures from Pfizer, Boehringer, Novartis, Astra Zeneca, Sanofi, Bristol, MSD, Takeda; for advisory boards from Sanofi, Lilly, Novartis, Boehringer, Amgen; and receives support for attending meetings from MSD and Astra Zeneca, outside the submitted work. The other authors declare no competing interests.
This study was financed by all the donors who participated in the Liquid Biopsy Crowdfunding campaign in 2017. LMR is supported by the Miguel Servet from ISCIII (CP20/00120) contract, funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union. RDP is supported by the Miguel Servet (CP21/00003) contract, funded by the ISCIII and co-funded by the European Union. AR-C is supported by the Roche-Chus Joint Unit (IN853B 2018/03) funded by GAIN, Axencia Galega de Innovación (GAIN), Vicepresidencia Segunda e Consellería de Economía, Empresa e Innovación”. ADL is funded by Consellería de Sanidade, Servizo Galego de Saúde (SERGAS).
Idylla reagents were provided free of charge by Biocartis. The funding source did not have any influence on the design, conduction, analysis and interpretation of data and report of the results for this study.
References
Sung H.
Ferlay J.
Siegel R.L.
et al.
Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
Association of patient characteristics and tumor genomics with clinical outcomes among patients with non-small cell lung cancer using a clinicogenomic database.
Profiling of circulating tumor DNA in plasma of non-small cell lung cancer patients, monitoring of epidermal growth factor receptor p.T790M mutated allelic fraction using beads, emulsion, amplification, and magnetics companion assay and evaluation in fut.
Rapid EGFR mutation detection using the Idylla platform: single-institution experience of 1200 cases analyzed by an in-house developed pipeline and comparison with concurrent next-generation sequencing results.
Integration of rapid PCR testing as an adjunct to NGS in diagnostic pathology services within the UK: evidence from a case series of non-squamous, non-small cell lung cancer (NSCLC) patients with follow-up.
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