Oncol Ther (2019) 7:131–139 https://doi.org/10.1007/s40487-019-00099-9 ORIGINAL RESEARCH Validation of Cell-Free DNA Collection Tubes for Determination of EGFR Mutation Status in Liquid Biopsy from NSCLC Patients Marta Sesé . Rosa Somoza . Inmaculada Maestu . Maria Martı́n Ureste . Alfredo Sanchez . Juan Felipe Cordoba . Irene Sansano . Griselda Venturas . Santiago Ramón y Cajal . Javier Hernández-Losa Received: May 31, 2019 / Published online: August 19, 2019 Ó The Author(s) 2019 ABSTRACT development of new techniques for studying tumor biomarkers in liquid biopsies, such as Introduction: Precision medicine has revolu- mutation analysis in cell-free DNA (cfDNA). tionized the understanding and treatment of cfDNA analysis can accurately determine tumor cancer by identifying subsets of patients who progression and prognosis and more effectively are amenable to specific treatments according identify appropriate targeted therapies. How- to their molecular characteristics, as exempli- ever, cfDNA is vulnerable, particularly during fied by epidermal growth factor receptor (EGFR) plasma sample shipping. mutations in non-small cell lung cancer Objective: We compared the cell- and DNA- (NSCLC). Although tissue biopsy is the gold stabilizing properties of cell-free DNA blood standard for determining molecular alterations collection tubes (BCTs) with those of the tradi- in tumors, its limitations have prompted the tional shipping method (frozen plasma) for EGFR mutation testing using the cobasÒ EGFR Mutation Test v2 in a prospective cohort of 49 patients from three different Spanish hospitals. Enhanced Digital Features To view enhanced digital features for this article, go to https://doi.org/10.6084/ Methods: In total, 98 NSCLC samples, two from m9.figshare.9213077. each patient, were studied; five of the 49 cases were considered invalid by cobasÒ with one of Electronic Supplementary Material The online the two shipping methods analyzed. After version of this article (https://doi.org/10.1007/s40487- 019-00099-9) contains supplementary material, which is excluding these samples, we analyzed 88 sam- available to authorized users. ples from 44 patients. Considering the current M. Sesé  R. Somoza  S. Ramón y Cajal  J. F. Cordoba J. Hernández-Losa (&) Department of Medical Oncology, Hospital Translational Molecular Pathology, Vall d’Hebron Universitari Arnau de Vilanova, Lleida, Spain Institute of Research (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain I. Sansano  G. Venturas  S. Ramón y Cajal  e-mail: jahernan@vhebron.net J. Hernández-Losa Department of Pathology, Hospital Universitari Vall I. Maestu  M. M. Ureste d’Hebron, Barcelona, Spain Department of Medical Oncology, Hospital Universitario Doctor Peset, Valencia, Spain M. Sesé  S. Ramón y Cajal  J. Hernández-Losa Spanish Biomedical Research Network Centre in A. Sanchez Oncology (CIBERONC), Barcelona, Spain Department of Medical Oncology, Consorci Hospitalari Provincial de Castelló, Castellón, Spain 132 Oncol Ther (2019) 7:131–139 methodology (frozen plasma) for sending sam- impact on molecular profiling of biomarker- ples as the gold standard, we evaluated the driven targeted therapies in NSCLC, as well as in sensitivity and specificity of cfDNA BCT many other tumors [8, 9]. shipment. Currently, tissue biopsy is the gold standard Results: The global agreement between the two for the determination of molecular alterations methods was 95.4%, with 100% sensitivity and in lung cancer tumors. However, it has limita- 94.6% specificity for the cfDNA BCTs. cfDNA tions. For example, most biopsies employ small- BCTs had a positive predictive value of 81.8% or fine-needle aspiration techniques that are and negative predictive value of 100%. often insufficient for genomic analysis after the Conclusion: cfDNA BCTs have the same sensi- initial histology diagnosis. The small size of the tivity for EGFR mutation analysis in liquid biopsy and cytology specimens also makes it biopsy as the current methodology and very impossible to determine the presence of residual high specificity. They also have some additional disease by classical hematoxylin and eosin advantages in terms of collection and further pathology and for accurate diagnosis at initial shipment. Therefore, cfDNA BCTs can be per- stages [10, 11]. In addition, both inter- and fectly incorporated into the routine practice for intratumoral heterogeneity may limit the EGFR mutation determination. genotyping tests results, which can have a sig- Funding: Roche Farma S.A., Spain. nificant impact on treatment, particularly when the mechanisms for resistance to EGFR inhibi- Keywords: Blood collection tubes; cfDNA; tors need to be evaluated [12]. In this regard, EGFR mutation analysis; NSCLC EGFR mutant-positive lung adenocarcinoma has been associated with moderate-to-high intratumoral heterogeneity in all NSCLC sub- INTRODUCTION types, which necessitates the analysis of EGFR mutations in circulating tumor DNA due to its Precision medicine has revolutionized the ability to overcome intratumoral heterogeneity understanding and treatment of cancer by [13–15]. Finally, it is worth mentioning the risks identifying subsets of patients who, according associated with biopsy in older lung cancer to their molecular characteristics, are amenable patients and patients with an inaccessible to specific treatments. Examples of predictive tumor location. biomarkers of treatment outcomes include Accordingly, new techniques have been mutations in RAS and BRAF in colorectal can- developed to study tumor biomarkers in liquid cer, BRAF in melanoma, c-KIT and PDGFRa in biopsies [16–18], such as mutation analysis in gastrointestinal stromal tumors, and mutations plasma-derived circulating cell-free DNA in epidermal growth factor receptor (EGFR) in (cfDNA), which is released by tumor cells in the non-small cell lung cancer (NSCLC). In the blood and which is considered the optimal particular case of lung cancer, patients are candidate for clinical application [19–22]. In screened for molecular alterations in EGFR, addition, cfDNA analysis can accurately deter- ALK, and ROS1 and for PD-L1 protein expres- mine tumor progression, diagnosis, and possi- sion to optimize treatment outcomes. The ble distant metastasis, it can predict prognosis identification of EGFR mutations as a key driver as well as more effectively individualize the event in lung adenocarcinoma patients led to targeted therapy, and it is less invasive for the the development of small molecule tyrosine patient. Moreover, liquid biopsy is the only kinase inhibitors (TKIs); including first-genera- sample source that has been officially approved tion (gefitinib [1, 2], erlotinib [3]), second-gen- for clinical use in lung cancer patients eration (afatinib [4]), and third-generation [16, 17, 23, 24]. (osimertinib [5, 6], rociletinib (CO1686) [7]) However, it is still difficult to preserve the inhibitors. Accordingly, the identification of integrity of the cfDNA population, particularly driver mutations in EGFR that in turn were during plasma sample shipping. Blood collec- actionable alterations has had a significant tion procedures have to be carefully considered Oncol Ther (2019) 7:131–139 133 to maintain cfDNA integrity until its analysis, sample to analyze EGFR mutational status for a especially temperature and preservative condi- first-line treatment or due to high heterogeneity tions [25]. Accordingly, the main objective of in NSCLC where tissue biopsy was negative, this work was to analyze the concordance in even though the clinical characteristics sug- cell- and DNA-stabilizing properties between gested, with high probability, the presence of cell-free DNA blood collection tubes (cfDNA EGFR mutant; and (b) patient treatment with BCTs) and the traditional shipping method, TKIs for monitoring and analysis of progression. comparing the performance of the two Duplicate blood samples were obtained and methodologies in the detection of the EGFR shipped to Vall d’Hebron Hospital for process- mutational state. EGFR testing was performed ing and storage using two distinct methods in using the cobasÒ EGFR Mutation Test v2 in all parallel: (1) via cell-free DNA blood collection liquid biopsy samples. This technique is a real- tubes (BCTs) (Roche), shipped at room temper- time polymerase chain reaction (RT-PCR)-based ature; and (2) via standard K2EDTA tubes con- test that can identify 42 different EGFR gene taining plasma (obtained via double mutations in exons 18, 19, 20, and 21 of the centrifugation of blood at 3000 rpm within gene; it has different detection limits for tissue- 20 min of blood extraction and frozen imme- derived DNA and plasma-derived cfDNA sam- diately at -80 °C), shipped frozen on dry ice ples [26]. Moreover, a secondary objective was (shipments were made weekly). Samples were to provide a proof of concept of an alternative collected within a 9-month period. shipment method for these specific blood sam- ples to an external laboratory as a real-life clin- Sample Processing and DNA Extraction ical practice experience. for Liquid Biopsy METHODS In total, 98 NSCLC liquid biopsy samples were included in the validation. BCTs were received Compliance with Ethics Guidelines at room temperature and were submitted to an initial centrifugation at 30009g for 10 min. Supernatants were collected and centrifuged at The Independent Ethics Committee of Arnau 16,0009g for 1 min to obtain the plasma; sev- Vilanova Hospital approved the study protocol, eral 1.5-mL aliquots were then made. Circulat- and all patients provided written informed ing cfDNA was then obtained via automated consent before their inclusion, in addition to DNA extraction from the 1.5-mL plasma sam- permission for the use of their blood samples ples using a QIAsymphony DSP Virus/Pathogen (EDTA tubes and BCTs) in EGFR mutation Kit (Qiagen) following the manufacturer’s pro- analysis. The study was carried out in accor- tocol on a QIAsymphony instrument. All sam- dance with the Declaration of Helsinki and its ples were eluted in 85 lL of elution buffer, 75 lL amendments and with all applicable regulatory of which was used in the mutation PCR requirements. protocol. Sample Collection EGFR Mutation Status Analysis in cfDNA A total of 49 patients diagnosed with advanced Mutation analysis was performed by qPCR using NSCLC from three Spanish centers (Hospital del a cobasÒ EGFR Mutation Test v2 kit (Roche). Doctor Peset, Hospital General de Castellon, Briefly, 75 lL of each DNA was used in each and Hospital Arnau de Vilanova) participated in assay on a cobasÒ Z480 analyzer. This kit detects this study. The diagnosis was consistent with 42 EGFR mutations (including G19A/S/C in the clinical practice of the centers, with oncol- exon 18, deletions and complex mutations in ogists including patients meeting either of the exon 19, S768I, T790 M, and exon 20 insertions, following criteria: (a) absence of a tissue biopsy and L858R in exon 21). A standard curve created 134 Oncol Ther (2019) 7:131–139 using the internal control Cp genomic DNA was the EGFR mutation status was analyzed by qPCR used to calculate the DNA concentration and using a cobasÒ EGFR Mutation Test v2 kit copy number, which is also represented as the (Fig. 1). Of the 49 cases, 5 were classified as semi-quantitative index of each EGFR mutant invalid by cobasÒ with one of the two shipping sample. For a sample to be classified as EGFR methodologies analyzed. The results of the mutation-positive in this analysis, it had to analysis, presented in Fig. 2a, revealed an 85.7% contain at least one activating mutation (i.e., concordance between the two techniques. The exon 19 deletion, L858R, G719x, or L861Q). rate improved to 95.4% after the exclusion of the five invalid samples (Fig. 2b). Concordance Statistical Analysis was defined as EGFR mutation-positive or EGFR mutation-negative, and the mutation type was Statistical analyses were performed with G-Stat the same in all EGFR-mutated samples, as version 2.0. The McNemar test was used to shown in Supplementary Table 1. analysis the EGFR mutation concordance Considering the current methodology (fro- between matched samples. zen plasma) for sending samples as the gold standard, we evaluated the sensitivity and specificity of the cfDNA BCT shipment. The RESULTS McNemar test showed that there was no sig- nificant difference between the two shipping Concordance of EGFR Mutation Detection methods (P = 0.157). in cfDNA Between cfDNA BCTs The global agreement between the two and the Routine Plasma Shipping shipping methods was 95.4%, with a sensitivity Procedure of 100% and specificity of 94.6% for the cfDNA BCTs. Further analysis of the cfDNA BCTs In total, 98 liquid biopsy samples were ana- determined a positive predictive value of 81.8% lyzed, corresponding to 49 patients from three and negative predictive value of 100% (Fig. 2b). different hospitals, with paired samples shipped Finally, we analyzed the discordant results. according to the two different shipping As mentioned above, five samples were consid- methodologies: (1) standard frozen plasma and ered invalid by cobasÒ in one of the two ship- (2) cfDNA BCTs. After the plasma was received ping procedures. The EGFR mutation statuses of in the laboratory and the DNA was extracted, the two discordant results in plasma were Fig. 1 Schematic representation of patients’ samples and the results of the EGFR mutation analysis Oncol Ther (2019) 7:131–139 135 Fig. 2 Comparison between cfDNA obtained from frozen analysis of the samples analyzed according to the two plasma and BCT samples for determining EGFR mutation different shipping methods status. a All samples included in this study. b Comparative compared with their corresponding results pre-analytical workflow for liquid biopsy from obtained via cobasÒ EGFR Mutation Test v1 kit blood collection and shipping to plasma analysis of formalin-fixed paraffin-embedded preparation, such as for EGFR mutation status (FFPE) biopsy samples; the findings confirmed testing, is simpler for cfDNA BCTs than for one of the results: an EGFR-del19 in the FFPE standard K2EDTA BCTs. For EGFR mutation results was found to be EGFR-del19 in the analysis, cfDNA BCTs in liquid biopsy have the cfDNA BCT results (Fig. 3). same sensitivity as the current methodology and very high specificity. Additionally, the semi-quantitative indices obtained by the two DISCUSSION different workflow approaches show high concordance. Plasma cfDNA is emerging as a clinically rele- The methods used for whole blood process- vant biomarker for the management and ing, preservation, and storage are important surveillance of cancer. Our results show that the considerations in circulating tumor DNA Fig. 3 Comparison of EGFR mutation status between cfDNA and FFPE samples 136 Oncol Ther (2019) 7:131–139 analysis [25, 27, 28]. Our results indicate that plasma processing and additional manipulation BCTs are preferable for the collection of circu- of samples by different individuals in the labo- lating DNA in a clinical setting due to their ratories, reducing the variability in the pre-an- favorable storage and shipping conditions. alytical steps and allowing standard room- Other studies indicated that BCTs are preferable temperature shipping to the testing laboratory; for the room-temperature preservation of (2) they provide a more standardized protocol cfDNA for up to 5 days and for preventing the that can be easily implemented in clinical lab- release of genomic DNA, both in oncology and oratory routine; and (3) the process is less prenatal diagnostics [29–32]. expensive. The price of EDTA tubes and dry ice Several pre-analytical steps are crucial to special courier services is about €78 per sample, obtain good concordant mutation results whereas BCTs and standard courier is about €11. between liquid biopsy and FFPE samples, Additionally, the dry ice service in this study beyond the natural behavior of the tumor, its has been established at once per week, which shedding capacity and clinical stage (III or IV), directly affects the EGFR test turnaround time. the inter- and intratumoral heterogeneity, and This study has some limitations, such as a tumor type. Some are related to the sample low number of samples included and a lack of handling, including plasma extraction proto- extra cfDNA to perform additional experiments cols, freeze/thaw cycles, sample shipment, and to elucidate the numbers of positive and nega- timing, which can affect the final mutation tives cases with both approaches. analysis of the EGFR gene in liquid biopsy. We analyzed all discordant cases in duplicate to rule out the presence of artifacts. However, we CONCLUSIONS did not include any additional methods to resolve discordant results because the protocol The use of cfDNA BCTs for the EGFR mutation used to analyze the EGFR mutations was analysis in liquid biopsy presents the same designed to consume all cfDNA obtained in each sensitivity as the current methodology, and extraction. We thus had no additional cfDNA to very high specificity. These cfDNA BCTs have perform further analysis with any high-sensi- additional advantages in the collection and tivity technique that might be recommended, further shipment processes. Therefore, due to such as droplet digital PCR or BEAMing. In line their high performance, these tubes can be with ASCO-CAP recommendations [18], we perfectly incorporated into the clinical routine compared the EGFR mutation results with those for this EGFR mutational determination. of some FFPE samples that had been previously analyzed in our laboratory with the cobasÒ EGFR Mutation Test v1, which is approved for muta- ACKNOWLEDGEMENTS tion analysis in FFPE samples [33]. In this com- parison, we only observed one EGFR mutant We thank the participants of the study. (Del19) in an FFPE sample, which was also detected by BCT analysis 2 months later. Funding. This study was sponsored by In addition, our results are consistent with Roche Farma S.A., Spain. The funder had no role others showing that cfDNA BCTs can replace in the study design, data collection and analy- the standard collection and transport method sis, decision to publish, preparation of the due to their strong ability to detect tumor- manuscript, or payment of the charges related specific mutations in key driver genes such as to article processing. All authors had full access BRAF, KRAS, and PIK3CA in various solid can- to all of the data in this study and take complete cers [34] and additional genes (EGFR and c-KIT) responsibility for the integrity of the data and tested by BEAMing or Safe-SeqS procedures [35]. accuracy of the data analysis. Roche Farma S.A., These cfDNA BCTs have some additional Spain also funded the journal’s Rapid Service advantages in the collection and shipment Fee. processes: (1) they eliminate the need for onsite Oncol Ther (2019) 7:131–139 137 Editorial Assistance. 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