Prospective multicenter validation of a next-generation sequencing panel using cytology specimens for lung cancer: cPANEL

Prospective multicenter validation of a next-generation sequencing panel using cytology specimens for lung cancer: cPANEL | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prospective multicenter validation of a next-generation sequencing panel using cytology specimens for lung cancer: cPANEL Kei Morikawa, Yuta Takashima, Masahide Oki, Akifumi Tsuzuku, Shuji Murakami, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6403904/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Oct, 2025 Read the published version in BMC Cancer → Version 1 posted 12 You are reading this latest preprint version Abstract Background There are no prospective studies to estimate whether cytology specimens can replace tissue samples using lung cancer gene panel analysis. We evaluated the success rate of gene panel testing and nucleic acid yield and quality when using cytology specimens for lung cancer over tissue specimens. Methods In this prospective study, clinical cytology specimens collected via transbronchial brushing, needle aspiration washing, and pleural effusion were stored in a nucleic acid stabilizer. The primary endpoint was the superior success rate of gene analysis using cytology specimens over the conventional success rate using tissue specimens. Results The full analysis set included 248 cases. The success rate for gene panel analysis using cytology specimens was 98.4% (95% confidence interval (CI), 95.9–99.6%) with a positive concordance rate of 97.4% (95% CI, 91.0–99.7%) by other companion diagnostic kits. The median value for nucleic acid yield and quality (DNA/RNA integrated number) of cytology specimens was 546.0/426.5 ng and 9.2/4.7 for DNA/RNA, respectively. The Pearson correlation coefficient of variant allele frequency between tissue formalin-fixed and paraffin-embedded (FFPE) sample and cytology specimens for mutant cases was 0.815. The ratio of double-stranded to total DNA showed that cytology specimens were of significantly higher quality than FFPE specimens. Conclusions The success rate of cytology specimensin gene analysis was significantly higher than conventional data. Because of the sufficient nucleic acid yield, high quality, and high correlation of mutant allele frequency compared to FFPE specimens, cytology specimens are suitable for panel testing as tissue substitutes. Clinical Trial Registration Trial Registration: UMIN Registry: UMIN000047215(cPANEL trial) https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000053766 Cytology specimen gene panel analysis next-generation sequencing non-small cell carcinoma variant allele frequency. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Personalized medicine, particularly molecularly targeted drugs, has considerably improved patient response rates and long-term prognosis. 1 – 2 This is particularly attributable to the expanded detectability of rare driver mutations at initial diagnosis beyond epidermal growth factor receptor ( EGFR ) and anaplastic lymphoma kinase ( ALK ). The Oncomine Dx Target Test Multi-CDx system (ODxTT, Thermo Fisher Scientific, San Jose, CA, USA), approved by the FDA in 2017, is a class-leading next-generation sequencing (NGS) panel for non-small cell lung cancer (NSCLC). 3 However, this system requires adequate amounts of malignant cells in tissue samples and meticulous sample handling, often leading to small sample sizes with compromised quality. 3 – 5 Previous global clinical trials and multicenter real-world data indicated that the panel had suboptimal success rates of 72.0–90.0%. 6–13 Moreover, the method requires increased time and effort for the collection of larger specimens and macro-dissection in specimen preparation. 7 – 8 Therefore, more efficient procedures with higher success rates and rapid analytical turnover are urgently required. Cytology specimens are commonly used in clinical practice since they require minimally invasive collection techniques and yield quick results. 14 – 16 However, their use in genetic panel testing is not widely endorsed in international guidelines, although occasionally used for a single-plex test as clinical practice. 17 – 20 Furthermore, cytology specimens are not applicable for comprehensive genome profile tests such as FoundationOne ®ฏ CDx (Foundation Medicine, Cambridge, MA, USA) and MSK-IMPACT ®ฏ (Memorial Sloan Kettering Cancer Center, New York, NY, USA). 21− 22 Although studies involving international collaboration have demonstrated high concordance in mutation detection and variant allele frequencies (VAF) across different NGS platforms using archival cytology samples, 23, 2 4 these studies typically utilized established cell lines rather than prospective clinical methods. 25 Therefore, this study primary aimed to confirm the success rate of panel tests using cytology samples, secondly aimed to compare the quantity and quality of nucleic acids yielded from cytology versus tissue samples to elucidate the value of cytology samples and their handling in gene panel tests. Both cytology and tissue samples should be triaged appropriately according to their intended use, but if panel testing cannot be performed due to insufficient tissue collection, patients' treatment options will be limited. Our results may aid in accelerating the application of gene panel tests for genetic mutation searches, thereby enhancing the development and application of personalized treatments. Methods Trial design The cPANEL prospective phase 3 multicenter trial evaluates the feasibility of performing gene panel tests using cytology specimens collected via brushing cytology, needle aspiration washing solution, and pleural effusion in clinical practice. The study was conducted in accordance with the Declaration of Helsinki and the study was approved by the Institutional Review Board of St. Marianna University School of Medicine, Kawasaki, Japan (approval number 5532). Written informed consent was obtained from all patients prior to study initialization. The study registered in the UMIN Registry (UMIN000047215). An independent data-monitoring committee reviewed the clinical data. Study Participants Eligible participants were adults aged ≥ 20 years who underwent cytopathological diagnosis for suspected primary lung cancer. Secondary registration excluded patients diagnosed with benign and metastatic lung cancer or whose paired cytology specimens showed no malignant cells. Diagnostic procedures Bronchoscopic evaluations were conducted using endobronchial ultrasonography (EU-ME2; Olympus, Tokyo, Japan), either with or without the assistance of a guide sheath kit (Olympus). EBUS-guided transbronchial needle aspiration (TBNA) or endoscopic ultrasound-guided fine-needle aspiration was performed via a flexible fiberscope, typically involving two to three passes with a 22-gauge needle. For CT or ultrasound (US)-guided core needle biopsies, a semi-automatic aspiration system (Temno Evolution, Care Fusion Japan, Tokyo, Japan) equipped with a 20-gauge needle (length 11 or 15 cm) was used in three attempts. When feasible, an adequate volume of tissue was obtained for further analysis. Cytology specimen collection Figure 1 outlines the process used for collecting cytology specimens. For transbronchial biopsies, lesion scraping was performed using a brush, with the collected material transferred onto a glass slide and agitated in 4 mL of normal saline two to three times. In the case of needle aspiration or biopsy, the core tissue was first harvested for histological analysis, followed by needle rinsing using approximately 1 mL of normal saline and air flushing (2–3 repetitions) to collect residual cells. The rinsing fluid was then equally split into two containers: one for routine sampling and the other for cytological examination (paired cytology samples). Patients with no malignant cells in the paired cytology samples were excluded from secondary registration. For pleural effusions, a minimum of 20 mL was collected, divided into two parts, centrifuged, and the cell pellets were preserved in containers for pathological assessment. Sample storage and transport conditions Cytology specimens for the NGS panel were collected in a sample container (GM tube, GeneMetrics, Osaka, Japan) containing 2 mL of a nucleic acid stabilizer to inhibit DNase/RNase activity. After storage in GM tube, no centrifugation or freezing was required. The sample containers were refrigerated and shipped to the inspection agency (DNA Chip Research, Tokyo, Japan). Sample analysis Sample purification Sample preparation and nucleic acid purification were conducted using commercially available kits following the manufacturer’s instructions, as previously described 25 . Specifically, cytology specimens were processed using the Maxwell® RSC Blood DNA and simplyRNA Cells Kits, while DNA and RNA from FFPE samples were extracted with the Maxwell® RSC DNA FFPE and RNA FFPE Kits (Promega, Madison, WI, USA). Nucleic acid quantification was performed using a Qubit™ fluorometer with dsDNA HS Assay Kits and NanoDrop® UV-spectrophotometry (Thermo Fisher Scientific, USA). DNA quality was evaluated using the Genomic DNA assay on a TapeStation system (Agilent) to determine the DNA Integrity Number (DIN). RNA quality was assessed using either the RNA HS assay on the TapeStation or the Bioanalyzer system (Agilent), providing RIN/eRIN values and DV200%. The ratio of double-stranded DNA to total DNA was also calculated to evaluate DNA integrity. Library preparation and NGS sequencing The Lung Cancer Compact panel™ (LCCP: DNA Chip Research) is an amplicon-based high-sensitivity NGS panel capable of measuring eight druggable genes in lung cancer, including EGFR , BRAF , KRAS , ERBB2 , ALK , ROS1 , MET , and RET . The LCCP was approved by the Ministry of Health, Labor, and Welfare as a multi-companion diagnostic kit (CDx) for lung cancer in Nobemver 2022 and is currently approved as a seven-gene CDx in Japan. The LCCP is characterized by highly sensitive mutation calls, with a limit of detection (LOD) of 0.14%, 0.20%, 0.48%, 0.24%, and 0.20% for driver mutations such as the EGFR exon-19 deletion, L858R, T790M, BRAF V600E, and KRAS G12C, respectively. Using purified nucleic acid, the LCCP assay along with library preparation, NGS sequencing (MiSeq; Illumina, San Diego, CA, USA), and data analysis were performed as previously described. 26 In brief, amplicon based multiplex PCR were performed to amplify targeting regions. As a design concept of the compact panel™, the sizes of target amplicon regions were optimized as narrow as possible to increase amplifiability of tumor-derived nucleic acids. Sequence libraries were constructed using the GenNext® NGS Library Prep Kit (Toyobo) from purified PCR products. Sequence data was obtained using MiSeq (Illumina, CA, USA) for the constructed sequence library (2 x 150 bp paired-end mode). Paired cytology specimen diagnosis Cytology specimen diagnosis was evaluated according to the World Health Organization’s (WHO) reporting system for lung cytopathology. 27 – 28 Among the five categories of lung cytopathological specimen types, atypical, suspicious for malignancy, and malignant diagnoses in paired specimens were considered for secondary registration, regardless of the tumor cell content. The cytological diagnoses and evaluations were confirmed by multiple pathologists and cytologists at each institution. Paired cytology samples from cases in which a genetic mutation was detected by the LCCP were sequentially collected for central evaluation. Pathological diagnosis and CDx Histopathological diagnosis was performed according to the 2015 WHO Classification of Tumors of the Lung. 29 A medical insurance-approved genetic test was performed as CDx. When sufficient samples could be collected, the samples were preferentially evaluated using the ODxTT or an Amoy 9-in-1 kit (Amoy Diagnostics, Xiamen, China). For single gene searches, a Cobas® EGFR mutation test was used to detect EGFR mutations; immunohistochemistry, Ventana OptiView ALK (D5F3; Roche Molecular Systems, Pleasanton, CA, USA) and Vysis® ALK Break Apart FISH probe kit (Abbott Japan, Tokyo, Japan) were used to detect ALK mutations; and Archer® MET (Invitae, San Francisco, CA, USA) was used to detect MET exon-14-skipping mutations. All other rare gene mutations were confirmed by ODxTT or Amoy detection. Concordance in variant allele frequencies (VAFs) between formalin-fixed paraffin-embedded (FFPE) tissue and cytology specimens The VAF concordance between the cytology panel and FFPE tissue panel assays was assessed in patients with genetic mutations to clarify the reliability of the gene mutation allele frequency in cytology samples. Liquid pleural effusion samples were used as cell blocks for tissue substitutes. Macrodissection of tissue samples was not specified in the protocol, it was performed at the discretion of each institution when the tumor content was low. Four 10-µm-thick FFPE slides, two for DNA extraction and two for RNA extraction, were prepared per case. The VAF of the primary oncogenic mutation was selected as the best indicator of the tumor cell content for each sample. Outcome Assessments The primary endpoint was to demonstrate the superiority of the LCCP when using cytology specimens, targeting a 90% success rate. This threshold was selected based on the upper bound of the 95% confidence interval (88.1%) observed in previous reports of multiple conventional panel tests using tissue samples (Supplementary eFigure 1). A successful result was defined by the extraction of at least 10 ng of both DNA and RNA, along with sufficient NGS sequencing depth: ≥ 5,000 reads for the DNA diagnostic module, ≥ 2,000 reads for the DNA research module, and ≥ 300 reads for the internal reference gene HPRT1 in the RNA module. Secondary endpoints included mutation detection in eight key lung cancer-related genes (EGFR, BRAF, ALK, ROS1, MET, RET, KRAS, and HER2), the concordance rate between LCCP results and established companion diagnostics, and a reduction in test result turnaround time when using cytology-based LCCP. Exploratory outcomes involved comparing nucleic acid yields, DNA Integrity Number (DIN), and RNA Integrity Number (RIN) across cytology collection methods. Additionally, for mutation-positive cases, variant allele frequency (VAF) concordance between cytology and FFPE tissue samples was evaluated. Sample Size We set the expected value of the success rate in this trial at 95%, the threshold success rate at 90%, the one-sided significance level at 2.5%, and power at 80%, and calculated the sample size using an exact test of binomial proportion (upper one-sided test). If the sample size was ≥ 243, the power of the test would always be ≥ 80%. Therefore, 243 patients were required for secondary registration (power: 0.839). Considering patient withdrawal of consent, we set the required number of secondary registrations to 248. Statistical analysis The Clopper and Pearson exact methods were used to calculate 95% CIs for binomial proportion testing. p < 0.05 was considered statistically significant. All analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA). Results In total, 320 patients were enrolled in the primary registration between March 23, 2022, and March 1, 2023. We excluded 66 patients from the secondary registration, and one patient was erroneously discarded by the physician. Therefore, 253 patients were enrolled in the secondary registration, of which five were excluded because more than 2 months had elapsed between specimen collection and submission. Finally, 248 patients were included in the full analysis set (FAS, Fig. 2 ). Baseline patient characteristics are summarized in Table 1 . The median age was 70 years (range: 31–90), with 158 patients (63.7%) being male. Clinical stages I, II, III, IV, and unknown were observed in 33 (13.3%), 25 (10.1%), 57 (23.0%), 132 (53.2%), and 1 (0.4%) cases, respectively. Histological classifications included adenocarcinoma in 153 patients (61.7%), squamous cell carcinoma in 42 (16.9%), small cell carcinoma in 28 (11.3%), and non-specified carcinoma in 25 (10.1%). Cytology specimens were collected using various procedures: 133 (53.6%) via transbronchial brushing (TBB), 56 (22.6%) via TBNA, 32 (12.9%) via ultrasound- or CT-guided puncture, 20 (8.1%) from pleural effusion, and 7 (2.8%) through other approaches (Table 1 ). Table 1 Patient characteristics and LCCP analysis. Patient characteristics n = 248, case (%) Pathological diagnosis n = 248, case (%) Sex Adenocarcinoma 153 (61.7) Male 158 (63.7) Squamous cell carcinoma 42 (16.9) Female 90 (36.3) Small cell carcinoma 28 (11.3) Median age , years (range) 70 (31–90) Not otherwise specified, other 25 (10.1) Clinical stage LCCP analysis success (rate) 244 (98.4) Ⅰ 33 (13.3) LCCP mutation detection case Ⅱ 25 (10.1) EGFR 59 Ⅲ 57 (23.0) KRAS G12X, G13X 24 Ⅳ 132 (53.2) KRAS G12C 15 Diagnostic procedure ALK 8 EBUS-TBB 133 (53.6) BRAF 4 EBUS-TBNA, EUS-FNA 56 (22.6) MET ex14 skip. 3 CT/US guided puncture 32 (12.9) RET 3 Pleural effusion 20 (8.1) HER2 2 Other 7 (2.8) ROS1 1 CT: computed tomography. EBUS-TBB: endobronchial ultrasonography-guided transbronchial brushing. EBUS-TBNA: endobronchial ultrasonography-guided transbronchial needle aspiration. EUS-FNA: endoscopic ultrasound-guided fine needle aspiration. LCCP: lung cancer compact panel. US: ultrasound. The success rate of LCCP genetic alteration testing, which was the primary endpoint, for all eight genes using cytology specimens was 98.4% (95% CI: 95.9–99.6%, Table 1 ), showing superiority over the 90% success rate of conventional NGS panel tests (p < 0.001). The positive concordance and predictive rates of the LCCP (secondary endpoint) were 97.4% (95% CI: 91.0–99.7%) and 93.8 (95% CI: 86.0–97.9%), respectively, for patients with genetic mutations using conventional CDx tests covered by public health insurance (Table S1 ). Among the 244 cases in the FAS in which genetic analysis was successful, driver gene mutations were detected in 103 (42.2%) using the LCCP (Table S2 ). In 150 cases of lung adenocarcinoma, the LCCP revealed driver gene mutations in 93 (62%) (Fig. 3 A) There was no difference in mutation detection rate between nucleic acid yields of 10–100 ng (60.7%) and those of more than 100 ng (62.3%). The proportions of mutation VAF% for <5%, 5-<10%, 10-<20%, 20-<30%, 30-<40%, 40-<50%, 50%≦, were 15.9%, 8.5%, 19.5%, 12.2%, 11.0%, 6.1%, 26.8%, respectively (Fig. 3 B). The median number of days from specimen submission to the reporting of analysis results (turnaround time) was 11.0 days. The LCCP success rate was analyzed for each specimen collection method and was 99.2% (95% CI: 95.9–100.0%) for TBB; 96.4% (95% CI: 87.7–99.6%) for TBNA; 100% (95% CI: 89.1–100.0%) for CT- or US-guided puncture; and 100% (95% CI: 83.2–100.0%) for pleural effusion. The median nucleic acid yield and DIN/RIN in the cytology specimens were 546.0/426.5 ng (Fig. 4 A) and 9.2/4.7 for DNA/RNA, respectively (Fig. 4 B, Table S3 ). Of 94 FFPE tissues, tumor content was recorded in 83 cases, with a median of 30% and a mean of 37% (range 2%-80%), all of which were successfully analyzed using LCCP. The Pearson correlation coefficient of VAFs between tissue FFPE samples and cytology specimens was 0.815 for the 85 cases where mutants were detected in cytology specimens (Fig. 5 A, Table S4 ). The ratio for double-stranded DNA: total DNA was 27.1/12.8% for cytology: FFPE specimens, indicating the higher quality of cytology specimens compared to the FFPE specimens (paired t -test, p < 0.001; Fig. 5 B, Table S5 ). Additionally, background noise during mutation analysis was significantly lower in cytology samples than in FFPE samples (Figure S2 , Table S6 ). Discussion This is the first multicenter study to prospectively evaluate the feasibility of an NGS lung cancer gene panel using cytology specimens. Our findings demonstrated a greater success rate for gene analysis using cytology specimens over conventional methods. Due to the adequate nucleic acid yield, high quality, and strong positive mutant allele frequency correlation with FFPE specimens, cytology specimens have the potential to replace tissue samples for detecting activating gene mutations. 30 The success rate of the LCCP using cytology specimens far surpassed that of conventional methods. Of 248 FAS cases, only four failed due to either defective HER2 gene amplification or insufficient DNA yield, whereas RNA analysis showed no failures, regardless of RNA being more degradation-prone. 31 – 33 In the 150 lung adenocarcinoma cases, mutations were detected in 62% cases, which is highly consistent with other CDx methods. 6 , 9 Sufficient nucleic acid yield and quality was confirmed regardless of the sample collection method. Moreover, the cytology specimens contained a significantly higher amount of double-stranded DNA compared to the FFPE samples collected during the same examination, suggesting higher-quality preservation. The cytology specimens are also less susceptible to external damage until nucleic acid extraction compared to FFPE specimens. 34 – 35 Notably, GM tube storage is highly versatile and useful for maintaining the quality of cytology specimens. Our results revealed a strong correlation between the VAFs detected in cytology and tissue specimens. In contrast, in approximately half of the cases, a higher allele ratio was detected in the cytology specimens. Furthermore, our findings are consistent with the fact that cytological diagnosis has the advantage of producing faster results than histological diagnosis, thereby shortening the time between specimen collection and sample submission. 36 Turnaround times have been shortened in actual clinical operations using the LCCP to a median of 8 days. We performed a few additional analyses to investigate the discordance in results between the LCCP and CDx tests (Table S7 ). In the two cases where there was no positive match with the LCCP, both harbored EGFR exon-21 L858R point mutations. In the first case, the LCCP detected a VAF of 0.1%, below the LOD of 0.14%. In the second case, a procedural error occurred, in which 2 mL of the pleural effusion was directly injected into the GM tube without centrifugation. Of the five cases with discordant positive predictive values, minor allelic frequency mutations were detected by the LCCP in two cases, while one case had a co-mutation of EGFR Ex19del and KRAS G12C. The other three cases, including two cases with an ALK fusion gene and one with a RET fusion gene, were associated with either false positives or sub-minor clones. In discordant cases, the signal remained around the LOD, and a large deviation between tumor content and the estimated VAF was estimated by the pathologist. This study had a few methodological limitations. First, this study did not perform a direct comparison but rather a verification of superiority based on a 90% success rate threshold calculated from pooled historical tissue panel data. 7 , 8 , 10 , 11 , 12 , 37 Some recent studies have reported high diagnostic rates using other NGS procedures, however, they were based on retrospective analyses using archived samples with known gene mutation information. 38 Second, the LCCP gene panel test is limited to eight druggable driver mutations but the amount of nucleic acid sample (both DNA and RNA) necessary for testing is equivalent to that used for ODxTT (10 ng). However, LCCP has a much larger variant search than other NGS or PCR panels (Figure S3 ). LCCP was designed to expand measurable driver mutations and variants in the near future. Third, this study involved prospective case enrollment in clinical practice, the low number of rare fusion gene mutations was a methodological limitation. Finally, the integration of an automated assay system can significantly reduce sample processing time and increase the efficiency of genetic analysis. This potential advance is expected to further shorten TAT. Research is currently underway to use less invasive specimen collection techniques and to utilize cytology specimens for other solid tumors. 39 – 40 In conclusion, our study demonstrated a high success rate for gene panel analysis using cytology specimens (98.4%). Based on the sufficient nucleic acid yield, high quality, and strong correlation in mutant allele frequency from FFPE specimens, we concluded that cytology specimens are suitable for gene panel testing as a substitute for tissue samples. Abbreviations FFPE formalin-fixed paraffin-embedded LCCP Lung Cancer Compact panel NGS next-generation sequencing NSCLC non-small cell lung cancer ODxTT,Oncomine Dx Target Test Multi-CDx system VAF variant allele frequencies Declarations Acknowledgments The authors thank Sae Yanagisawa from St. Marianna University School of Medicine, Center for Clinical and Translational Science, for data management; Jason Tonge from the St. Marianna University School of Medicine and Editage (www.editage.jp) for the language review of the manuscript; Miho Ishii, Mirai Yamago, Azusa Fujita, Mizuki Ono, Kentaro Ito, Hiroyuki Sato, and Motohiko Tanino of DNA Chip Research Inc. for their technical and experimental support; Prof. Kikuya Kato from the Nara Institute of Science and Technology for providing advice on this study; and Gene Metrics LCC and Yoko Takagi for providing GM tubes. Data Access, Responsibility, and Analysis: Kenichiro Tanabe (Pathology and Bioregulation, St. Marianna University Graduate School of Medicine, Japan) conducted and is responsible for the data analysis. Data are available upon reasonable request. Presentation of data: The phase 2 cPANEL study was presented at the IASLC 2022 World Conference on Lung Cancer (WCLC) press conference held in Vienna. The phase 3 cPANEL study was presented at the IASLC 2023 WCLC held in Singapore. Consent for Publication: All authors have contributed significantly, and that all authors are in agreement with the content of the manuscript. Disclosure Funding: The gene analyses were conducted and funded by DNA Chip Research, Inc., Tokyo, Japan. Conflicts of Interest: Kei Morikawa reports financial support was provided by DNA Chip Research Inc, and received lecture fees as honoraria from Eli-Lilly, Chugai Pharmaceutical, Takeda Pharmaceutical, AstraZeneca K.K., Daiichi Sankyo, MSD, Nippon Boehringer Ingelheim Co., Ltd., and Bristol-Myers Squibb Japan. Yoshiharu Sato, Seiji Nakamura, Yumi Ueda, Fumihiko Suzuki reports a relationship with DNA Chip Research Inc that includes: employment and equity or stocks. Ethics Statement ・The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of St. Marianna University School of Medicine (approval number 5532). ・Written informed consent was obtained from all participants prior to study initiation. ・The study registered in the UMIN Registry (UMIN000047215) on March 18, 2022. An independent data-monitoring committee reviewed the clinical data. ・Animal Studies: N/A. Author contributions: Conception and design: KM, MM, YS, and SN. 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J Int Med Res. 2021;49:300060520982687. Takeyasu Y, Yoshida T, Motoi N, Teishikata T, Tanaka M, Matsumoto Y, et al. Feasibility of next-generation sequencing (Oncomine™ DX Target Test) for the screening of oncogenic mutations in advanced non-small-cell lung cancer patients. Jpn J Clin Oncol. 2021;51:1114–122. Hofman V, Heeke S, Bontoux C, Chalabreysse L, Barritault M, Bringuier PP, et al. Ultrafast gene fusion assessment for nonsquamous NSCLC. JTO Clin Res Rep. 2022;4:100457. Morikawa K, Kinoshita K, Kida H, Inoue T, Mineshita M. Preliminary results of NGS gene panel test using NSCLC sputum cytology and therapeutic effect using corresponding molecular-targeted drugs. Genes. 2022;13:812. https:/ /center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000059594 Additional Declarations Competing interest reported. Kei Morikawa reports financial support was provided by DNA Chip Research Inc, and received lecture fees as honoraria from Eli-Lilly, Chugai Pharmaceutical, Takeda Pharmaceutical, AstraZeneca K.K., Daiichi Sankyo, MSD, Nippon Boehringer Ingelheim Co., Ltd., and Bristol-Myers Squibb Japan. Yoshiharu Sato, Seiji Nakamura, Yumi Ueda, Fumihiko Suzuki reports a relationship with DNA Chip Research Inc that includes: employment and equity or stocks. Supplementary Files TableS1.docx Tab. S1 Concordance of mutation call by CDx-tissue and LCCP-cytology. CDx: companion diagnostic test. LCCP: lung cancer compact panel. PPV: positive predictive value. NPV: negative predictive value. TableS2.xlsx Tab. S2 List of allele frequency LCCP CDx. CDx: companion diagnostic test. LCCP: lung cancer compact panel. TableS3.xlsx Tab. S3 Quality assessment result of purified nucleotide. TableS4.xlsx Tab. S4 List of allele frequency between cytology LCCP and tissue LCCP CDx. CDx: companion diagnostic test. LCCP: lung cancer compact panel. TableS5.xlsx Tab. S5 dsDNA/total DNA ratio for purified nucleotides. TableS6.xlsx Tab. S6 Comparison of background artifactural nucleotide substitution between FFPE and cytology (formaldehyde-free fixation). FFPE: formalin-fixed paraffin-embedded. TableS7.xlsx Tab. S7 List of discordant results between cytology LCCP and tissue CDx. CDx: companion diagnostic test. LCCP: lung cancer compact panel. Fig.S1.tif Fig. S1 Forest plot of success rates for historical data based on NGS lung cancer gene panels. NGS: next generation sequencing Fig.S2.tif Fig. S2 The background artifactural noise distribution analyzed for cytology and FFPE samples vizualized by box-violinplot. FFPE: formalin-fixed paraffin-embedded. VAF: variant allele frequency Fig.S3.tif Fig. S3 Venn diagram of variant detections of DNA (EGFR, HER2) by 3 gene panels. Amoy multi: Amoy Dx lung cancer multi-PCR gene panel. EGFR: epidermal growth factor receptor. HER2: human epidermal growth factor receptor 2. LCCP: Lung cancer compact panel. Oncomine: Oncomine Dx Target Test Multi-CDx system. Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2025 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 20 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviews received at journal 30 May, 2025 Reviewers agreed at journal 26 May, 2025 Reviews received at journal 23 May, 2025 Reviewers agreed at journal 22 May, 2025 Reviewers agreed at journal 20 May, 2025 Reviewers invited by journal 13 May, 2025 Editor assigned by journal 29 Apr, 2025 Editor invited by journal 11 Apr, 2025 Submission checks completed at journal 11 Apr, 2025 First submitted to journal 11 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6403904","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456103125,"identity":"f0d5bd1a-0b95-4b54-bfad-9fd574df8089","order_by":0,"name":"Kei 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LCCP: lung cancer compact panel. ROSE: rapid on-site cytologic evaluation. TBNA: transbronchial needle aspiration. US: ultrasound.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/a974ae10b8948a98167d11bd.png"},{"id":82798086,"identity":"c720d7a3-fa9b-4731-9da0-72db6478a0dd","added_by":"auto","created_at":"2025-05-15 10:48:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272048,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram of case registration\u003c/p\u003e\n\u003cp\u003eFAS: full analysis set. FFPE: formalin-fixed paraffin-embedded; LCCP: lung cancer compact panel.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/02e8e79fece119aa59ee539a.png"},{"id":82799757,"identity":"c0442113-0f04-49b9-8755-2cdbe81ed413","added_by":"auto","created_at":"2025-05-15 11:04:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":621296,"visible":true,"origin":"","legend":"\u003cp\u003ePie chart of mutation calls detected by LCCP assay for adenocarcinoma (A) and each mutation VAF% (B)\u003c/p\u003e\n\u003cp\u003eEBUS-TBB: endobronchial ultrasonography-guided transbronchial brushing. EBUS-TBNA: endobronchial ultrasonography-guided transbronchial needle aspiration. EUS-FNA: endoscopic ultrasound-guided fine needle aspiration. CT: computed tomography. US: ultrasound. VAF: variant allele frequency. FFPE: formalin-fixed paraffin-embedded.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/4b1f830e4a40cf2c5520b12f.png"},{"id":82798100,"identity":"2981b538-1cb5-471f-b762-c686039d8e65","added_by":"auto","created_at":"2025-05-15 10:48:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":746596,"visible":true,"origin":"","legend":"\u003cp\u003eNucleic acid yield (A) and quality (B) according to sample collection method\u003c/p\u003e\n\u003cp\u003eDIN: DNA Integrity Number. RIN: RNA Integrity Number.\u003c/p\u003e\n\u003cp\u003eEBUS-TBB: endobronchial ultrasonography-guided transbronchial brushing. EBUS-TBNA: endobronchial ultrasonography-guided transbronchial needle aspiration. EUS-FNA: endoscopic ultrasound-guided fine needle aspiration. CT: computed tomography. US: ultrasound.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/adf648b1a0df763eec6f0719.png"},{"id":82799301,"identity":"350411ef-cb0e-4265-a7e3-d2747a9bbdba","added_by":"auto","created_at":"2025-05-15 10:56:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":615167,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelations of VAF% (A) and purified DNA quality (B) between FFPE tissue and cytology samples\u003c/p\u003e\n\u003cp\u003eVAF: variant allele frequency. FFPE: formalin-fixed paraffin-embedded.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/e156c3f0d020db60c035467c.png"},{"id":93419713,"identity":"35ec3fcb-b4f8-435d-9dd0-dbe3a39182db","added_by":"auto","created_at":"2025-10-13 16:06:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4299025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/f5cc4712-dc86-4d32-b4ae-ca8021466db7.pdf"},{"id":82799297,"identity":"c7cfbc63-997f-4bb6-b3d9-c5d5c96b33d4","added_by":"auto","created_at":"2025-05-15 10:56:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":39608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTab. S1\u003c/strong\u003e Concordance of mutation call by CDx-tissue and LCCP-cytology.\u003c/p\u003e\n\u003cp\u003eCDx: companion diagnostic test. LCCP: lung cancer compact panel. PPV: positive predictive value. NPV: negative predictive value.\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/9fafca0cff75768c9cd14e1f.docx"},{"id":82799753,"identity":"71ae84a9-aee2-48f8-ab25-580d4c81f42b","added_by":"auto","created_at":"2025-05-15 11:04:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTab. S2\u003c/strong\u003e List of allele frequency LCCP CDx.\u003c/p\u003e\n\u003cp\u003eCDx: companion diagnostic test. LCCP: lung cancer compact panel.\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/8e93cb5c272678e204c52126.xlsx"},{"id":82798097,"identity":"49b2cb4a-4266-47ee-99d8-98d24959027e","added_by":"auto","created_at":"2025-05-15 10:48:02","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTab. 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LCCP: lung cancer compact panel.\u003c/p\u003e","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/24902455f9f75bf8f6996264.xlsx"},{"id":82800672,"identity":"e4163fef-eca5-43d7-aa08-84334c9c888e","added_by":"auto","created_at":"2025-05-15 11:12:02","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":17504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTab. 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S6 \u003c/strong\u003eComparison of background artifactural nucleotide substitution between FFPE and cytology (formaldehyde-free fixation).\u003c/p\u003e\n\u003cp\u003eFFPE: formalin-fixed paraffin-embedded.\u003c/p\u003e","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/d34faaa6ff472d34e96f65f9.xlsx"},{"id":82798101,"identity":"e337b6ba-fc5a-47ee-9302-e5a71f57cdf0","added_by":"auto","created_at":"2025-05-15 10:48:02","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":11831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTab. S7 \u003c/strong\u003eList of discordant results between cytology LCCP and tissue CDx.\u003c/p\u003e\n\u003cp\u003eCDx: companion diagnostic test. LCCP: lung cancer compact panel.\u003c/p\u003e","description":"","filename":"TableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/aeaf6d2cec25d55065274e35.xlsx"},{"id":82798113,"identity":"d3e4ca49-f527-4844-928e-1428bd4beb49","added_by":"auto","created_at":"2025-05-15 10:48:02","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":246128,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1\u003c/strong\u003e Forest plot of success rates for historical data based on NGS lung cancer gene panels.\u003c/p\u003e\n\u003cp\u003eNGS: next generation sequencing\u003c/p\u003e","description":"","filename":"Fig.S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/92ed7faeb7f0fd323d425094.tif"},{"id":82799309,"identity":"1b7a8873-2c35-48d2-a5df-a2b8f61e517e","added_by":"auto","created_at":"2025-05-15 10:56:02","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":106025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2\u003c/strong\u003e The background artifactural noise distribution analyzed for cytology and FFPE samples vizualized by box-violinplot.\u003c/p\u003e\n\u003cp\u003eFFPE: formalin-fixed paraffin-embedded. VAF: variant allele frequency\u003c/p\u003e","description":"","filename":"Fig.S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/d0a8934bb51c0fda26af8691.tif"},{"id":82799306,"identity":"883e65a9-631e-4b60-9970-8ea7c65fe053","added_by":"auto","created_at":"2025-05-15 10:56:02","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":33385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3\u003c/strong\u003e Venn diagram of variant detections of DNA (EGFR, HER2) by 3 gene panels.\u003c/p\u003e\n\u003cp\u003eAmoy multi: Amoy Dx lung cancer multi-PCR gene panel. EGFR: epidermal growth factor receptor. HER2: human epidermal growth factor receptor 2. LCCP: Lung cancer compact panel. Oncomine: Oncomine Dx Target Test Multi-CDx system.\u003c/p\u003e","description":"","filename":"Fig.S3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6403904/v1/b30716c5d64b179579ee8d20.tif"}],"financialInterests":"Competing interest reported. Kei Morikawa reports financial support was provided by DNA Chip Research Inc, and received lecture fees as honoraria from Eli-Lilly, Chugai Pharmaceutical, Takeda Pharmaceutical, AstraZeneca K.K., Daiichi Sankyo, MSD, Nippon Boehringer Ingelheim Co., Ltd., and Bristol-Myers Squibb Japan. Yoshiharu Sato, Seiji Nakamura, Yumi Ueda, Fumihiko Suzuki reports a relationship with DNA Chip Research Inc that includes: employment and equity or stocks.","formattedTitle":"Prospective multicenter validation of a next-generation sequencing panel using cytology specimens for lung cancer: cPANEL","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePersonalized medicine, particularly molecularly targeted drugs, has considerably improved patient response rates and long-term prognosis.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e This is particularly attributable to the expanded detectability of rare driver mutations at initial diagnosis beyond epidermal growth factor receptor (\u003cem\u003eEGFR\u003c/em\u003e) and anaplastic lymphoma kinase (\u003cem\u003eALK\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe Oncomine Dx Target Test Multi-CDx system (ODxTT, Thermo Fisher Scientific, San Jose, CA, USA), approved by the FDA in 2017, is a class-leading next-generation sequencing (NGS) panel for non-small cell lung cancer (NSCLC).\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e However, this system requires adequate amounts of malignant cells in tissue samples and meticulous sample handling, often leading to small sample sizes with compromised quality.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Previous global clinical trials and multicenter real-world data indicated that the panel had suboptimal success rates of 72.0\u0026ndash;90.0%.\u003csup\u003e6\u0026ndash;13\u003c/sup\u003e Moreover, the method requires increased time and effort for the collection of larger specimens and macro-dissection in specimen preparation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Therefore, more efficient procedures with higher success rates and rapid analytical turnover are urgently required.\u003c/p\u003e \u003cp\u003eCytology specimens are commonly used in clinical practice since they require minimally invasive collection techniques and yield quick results.\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e However, their use in genetic panel testing is not widely endorsed in international guidelines, although occasionally used for a single-plex test as clinical practice.\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Furthermore, cytology specimens are not applicable for comprehensive genome profile tests such as FoundationOne\u003csup\u003e\u0026reg;ฏ\u003c/sup\u003e CDx (Foundation Medicine, Cambridge, MA, USA) and MSK-IMPACT\u003csup\u003e\u0026reg;ฏ\u003c/sup\u003e (Memorial Sloan Kettering Cancer Center, New York, NY, USA).\u003csup\u003e21\u0026minus;\u0026thinsp;22\u003c/sup\u003e Although studies involving international collaboration have demonstrated high concordance in mutation detection and variant allele frequencies (VAF) across different NGS platforms using archival cytology samples,\u003csup\u003e23, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e4\u003c/sup\u003e these studies typically utilized established cell lines rather than prospective clinical methods.\u003csup\u003e25\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTherefore, this study primary aimed to confirm the success rate of panel tests using cytology samples, secondly aimed to compare the quantity and quality of nucleic acids yielded from cytology versus tissue samples to elucidate the value of cytology samples and their handling in gene panel tests. Both cytology and tissue samples should be triaged appropriately according to their intended use, but if panel testing cannot be performed due to insufficient tissue collection, patients' treatment options will be limited. Our results may aid in accelerating the application of gene panel tests for genetic mutation searches, thereby enhancing the development and application of personalized treatments.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTrial design\u003c/h2\u003e \u003cp\u003eThe cPANEL prospective phase 3 multicenter trial evaluates the feasibility of performing gene panel tests using cytology specimens collected via brushing cytology, needle aspiration washing solution, and pleural effusion in clinical practice. The study was conducted in accordance with the Declaration of Helsinki and the study was approved by the Institutional Review Board of St. Marianna University School of Medicine, Kawasaki, Japan (approval number 5532). Written informed consent was obtained from all patients prior to study initialization. The study registered in the UMIN Registry (UMIN000047215). An independent data-monitoring committee reviewed the clinical data.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Participants\u003c/h3\u003e\n\u003cp\u003eEligible participants were adults aged\u0026thinsp;\u0026ge;\u0026thinsp;20 years who underwent cytopathological diagnosis for suspected primary lung cancer. Secondary registration excluded patients diagnosed with benign and metastatic lung cancer or whose paired cytology specimens showed no malignant cells.\u003c/p\u003e\n\u003ch3\u003eDiagnostic procedures\u003c/h3\u003e\n\u003cp\u003eBronchoscopic evaluations were conducted using endobronchial ultrasonography (EU-ME2; Olympus, Tokyo, Japan), either with or without the assistance of a guide sheath kit (Olympus). EBUS-guided transbronchial needle aspiration (TBNA) or endoscopic ultrasound-guided fine-needle aspiration was performed via a flexible fiberscope, typically involving two to three passes with a 22-gauge needle. For CT or ultrasound (US)-guided core needle biopsies, a semi-automatic aspiration system (Temno Evolution, Care Fusion Japan, Tokyo, Japan) equipped with a 20-gauge needle (length 11 or 15 cm) was used in three attempts. When feasible, an adequate volume of tissue was obtained for further analysis.\u003c/p\u003e\n\u003ch3\u003eCytology specimen collection\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e outlines the process used for collecting cytology specimens. For transbronchial biopsies, lesion scraping was performed using a brush, with the collected material transferred onto a glass slide and agitated in 4 mL of normal saline two to three times. In the case of needle aspiration or biopsy, the core tissue was first harvested for histological analysis, followed by needle rinsing using approximately 1 mL of normal saline and air flushing (2\u0026ndash;3 repetitions) to collect residual cells. The rinsing fluid was then equally split into two containers: one for routine sampling and the other for cytological examination (paired cytology samples). Patients with no malignant cells in the paired cytology samples were excluded from secondary registration. For pleural effusions, a minimum of 20 mL was collected, divided into two parts, centrifuged, and the cell pellets were preserved in containers for pathological assessment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSample storage and transport conditions\u003c/h3\u003e\n\u003cp\u003eCytology specimens for the NGS panel were collected in a sample container (GM tube, GeneMetrics, Osaka, Japan) containing 2 mL of a nucleic acid stabilizer to inhibit DNase/RNase activity. After storage in GM tube, no centrifugation or freezing was required. The sample containers were refrigerated and shipped to the inspection agency (DNA Chip Research, Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample analysis\u003c/h2\u003e \u003cp\u003eSample purification\u003c/p\u003e \u003cp\u003eSample preparation and nucleic acid purification were conducted using commercially available kits following the manufacturer\u0026rsquo;s instructions, as previously described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Specifically, cytology specimens were processed using the Maxwell\u0026reg; RSC Blood DNA and simplyRNA Cells Kits, while DNA and RNA from FFPE samples were extracted with the Maxwell\u0026reg; RSC DNA FFPE and RNA FFPE Kits (Promega, Madison, WI, USA). Nucleic acid quantification was performed using a Qubit\u0026trade; fluorometer with dsDNA HS Assay Kits and NanoDrop\u0026reg; UV-spectrophotometry (Thermo Fisher Scientific, USA). DNA quality was evaluated using the Genomic DNA assay on a TapeStation system (Agilent) to determine the DNA Integrity Number (DIN). RNA quality was assessed using either the RNA HS assay on the TapeStation or the Bioanalyzer system (Agilent), providing RIN/eRIN values and DV200%. The ratio of double-stranded DNA to total DNA was also calculated to evaluate DNA integrity.\u003c/p\u003e \u003cp\u003eLibrary preparation and NGS sequencing\u003c/p\u003e \u003cp\u003eThe Lung Cancer Compact panel\u0026trade; (LCCP: DNA Chip Research) is an amplicon-based high-sensitivity NGS panel capable of measuring eight druggable genes in lung cancer, including \u003cem\u003eEGFR\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e, \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003eALK\u003c/em\u003e, \u003cem\u003eROS1\u003c/em\u003e, \u003cem\u003eMET\u003c/em\u003e, and \u003cem\u003eRET\u003c/em\u003e. The LCCP was approved by the Ministry of Health, Labor, and Welfare as a multi-companion diagnostic kit (CDx) for lung cancer in Nobemver 2022 and is currently approved as a seven-gene CDx in Japan. The LCCP is characterized by highly sensitive mutation calls, with a limit of detection (LOD) of 0.14%, 0.20%, 0.48%, 0.24%, and 0.20% for driver mutations such as the \u003cem\u003eEGFR\u003c/em\u003e exon-19 deletion, L858R, T790M, \u003cem\u003eBRAF\u003c/em\u003e V600E, and \u003cem\u003eKRAS\u003c/em\u003e G12C, respectively. Using purified nucleic acid, the LCCP assay along with library preparation, NGS sequencing (MiSeq; Illumina, San Diego, CA, USA), and data analysis were performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In brief, amplicon based multiplex PCR were performed to amplify targeting regions. As a design concept of the compact panel\u0026trade;, the sizes of target amplicon regions were optimized as narrow as possible to increase amplifiability of tumor-derived nucleic acids. Sequence libraries were constructed using the GenNext\u0026reg; NGS Library Prep Kit (Toyobo) from purified PCR products. Sequence data was obtained using MiSeq (Illumina, CA, USA) for the constructed sequence library (2 x 150 bp paired-end mode).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePaired cytology specimen diagnosis\u003c/h3\u003e\n\u003cp\u003eCytology specimen diagnosis was evaluated according to the World Health Organization\u0026rsquo;s (WHO) reporting system for lung cytopathology.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Among the five categories of lung cytopathological specimen types, atypical, suspicious for malignancy, and malignant diagnoses in paired specimens were considered for secondary registration, regardless of the tumor cell content. The cytological diagnoses and evaluations were confirmed by multiple pathologists and cytologists at each institution. Paired cytology samples from cases in which a genetic mutation was detected by the LCCP were sequentially collected for central evaluation.\u003c/p\u003e\n\u003ch3\u003ePathological diagnosis and CDx\u003c/h3\u003e\n\u003cp\u003eHistopathological diagnosis was performed according to the 2015 WHO Classification of Tumors of the Lung.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e A medical insurance-approved genetic test was performed as CDx. When sufficient samples could be collected, the samples were preferentially evaluated using the ODxTT or an Amoy 9-in-1 kit (Amoy Diagnostics, Xiamen, China). For single gene searches, a Cobas\u0026reg; EGFR mutation test was used to detect \u003cem\u003eEGFR\u003c/em\u003e mutations; immunohistochemistry, Ventana OptiView ALK (D5F3; Roche Molecular Systems, Pleasanton, CA, USA) and Vysis\u0026reg; ALK Break Apart FISH probe kit (Abbott Japan, Tokyo, Japan) were used to detect \u003cem\u003eALK\u003c/em\u003e mutations; and Archer\u0026reg; MET (Invitae, San Francisco, CA, USA) was used to detect \u003cem\u003eMET\u003c/em\u003e exon-14-skipping mutations. All other rare gene mutations were confirmed by ODxTT or Amoy detection.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eConcordance in variant allele frequencies (VAFs) between formalin-fixed paraffin-embedded (FFPE) tissue and cytology specimens\u003c/h2\u003e \u003cp\u003eThe VAF concordance between the cytology panel and FFPE tissue panel assays was assessed in patients with genetic mutations to clarify the reliability of the gene mutation allele frequency in cytology samples. Liquid pleural effusion samples were used as cell blocks for tissue substitutes. Macrodissection of tissue samples was not specified in the protocol, it was performed at the discretion of each institution when the tumor content was low. Four 10-\u0026micro;m-thick FFPE slides, two for DNA extraction and two for RNA extraction, were prepared per case. The VAF of the primary oncogenic mutation was selected as the best indicator of the tumor cell content for each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOutcome Assessments\u003c/h2\u003e \u003cp\u003eThe primary endpoint was to demonstrate the superiority of the LCCP when using cytology specimens, targeting a 90% success rate. This threshold was selected based on the upper bound of the 95% confidence interval (88.1%) observed in previous reports of multiple conventional panel tests using tissue samples (Supplementary eFigure 1).\u003c/p\u003e \u003cp\u003eA successful result was defined by the extraction of at least 10 ng of both DNA and RNA, along with sufficient NGS sequencing depth: \u0026ge; 5,000 reads for the DNA diagnostic module, \u0026ge; 2,000 reads for the DNA research module, and \u0026ge;\u0026thinsp;300 reads for the internal reference gene HPRT1 in the RNA module. Secondary endpoints included mutation detection in eight key lung cancer-related genes (EGFR, BRAF, ALK, ROS1, MET, RET, KRAS, and HER2), the concordance rate between LCCP results and established companion diagnostics, and a reduction in test result turnaround time when using cytology-based LCCP. Exploratory outcomes involved comparing nucleic acid yields, DNA Integrity Number (DIN), and RNA Integrity Number (RIN) across cytology collection methods. Additionally, for mutation-positive cases, variant allele frequency (VAF) concordance between cytology and FFPE tissue samples was evaluated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSample Size\u003c/h2\u003e \u003cp\u003eWe set the expected value of the success rate in this trial at 95%, the threshold success rate at 90%, the one-sided significance level at 2.5%, and power at 80%, and calculated the sample size using an exact test of binomial proportion (upper one-sided test). If the sample size was \u0026ge;\u0026thinsp;243, the power of the test would always be \u0026ge;\u0026thinsp;80%. Therefore, 243 patients were required for secondary registration (power: 0.839). Considering patient withdrawal of consent, we set the required number of secondary registrations to 248.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Clopper and Pearson exact methods were used to calculate 95% CIs for binomial proportion testing. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn total, 320 patients were enrolled in the primary registration between March 23, 2022, and March 1, 2023. We excluded 66 patients from the secondary registration, and one patient was erroneously discarded by the physician. Therefore, 253 patients were enrolled in the secondary registration, of which five were excluded because more than 2 months had elapsed between specimen collection and submission. Finally, 248 patients were included in the full analysis set (FAS, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBaseline patient characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age was 70 years (range: 31\u0026ndash;90), with 158 patients (63.7%) being male. Clinical stages I, II, III, IV, and unknown were observed in 33 (13.3%), 25 (10.1%), 57 (23.0%), 132 (53.2%), and 1 (0.4%) cases, respectively. Histological classifications included adenocarcinoma in 153 patients (61.7%), squamous cell carcinoma in 42 (16.9%), small cell carcinoma in 28 (11.3%), and non-specified carcinoma in 25 (10.1%). Cytology specimens were collected using various procedures: 133 (53.6%) via transbronchial brushing (TBB), 56 (22.6%) via TBNA, 32 (12.9%) via ultrasound- or CT-guided puncture, 20 (8.1%) from pleural effusion, and 7 (2.8%) through other approaches (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient characteristics and LCCP analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;248, case (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePathological diagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;248, case (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e153 (61.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e158 (63.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSquamous cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42 (16.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90 (36.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSmall cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28 (11.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMedian age\u003c/b\u003e, years (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70 (31\u0026ndash;90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNot otherwise specified, other\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25 (10.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClinical stage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eLCCP analysis success\u003c/b\u003e (rate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e244 (98.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅠ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33 (13.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eLCCP mutation detection\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅡ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 (10.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEGFR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅢ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57 (23.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eKRAS G12X, G13X\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅣ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e132 (53.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eKRAS G12C\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDiagnostic procedure\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eALK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEBUS-TBB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e133 (53.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBRAF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEBUS-TBNA, EUS-FNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56 (22.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMET ex14 skip.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCT/US guided puncture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32 (12.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRET\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePleural effusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (8.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHER2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (2.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eROS1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eCT: computed tomography. EBUS-TBB: endobronchial ultrasonography-guided transbronchial brushing. EBUS-TBNA: endobronchial ultrasonography-guided transbronchial needle aspiration. EUS-FNA: endoscopic ultrasound-guided fine needle aspiration. LCCP: lung cancer compact panel. US: ultrasound.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe success rate of LCCP genetic alteration testing, which was the primary endpoint, for all eight genes using cytology specimens was 98.4% (95% CI: 95.9\u0026ndash;99.6%, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), showing superiority over the 90% success rate of conventional NGS panel tests (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eThe positive concordance and predictive rates of the LCCP (secondary endpoint) were 97.4% (95% CI: 91.0\u0026ndash;99.7%) and 93.8 (95% CI: 86.0\u0026ndash;97.9%), respectively, for patients with genetic mutations using conventional CDx tests covered by public health insurance (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the 244 cases in the FAS in which genetic analysis was successful, driver gene mutations were detected in 103 (42.2%) using the LCCP (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In 150 cases of lung adenocarcinoma, the LCCP revealed driver gene mutations in 93 (62%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) There was no difference in mutation detection rate between nucleic acid yields of 10\u0026ndash;100 ng (60.7%) and those of more than 100 ng (62.3%). The proportions of mutation VAF% for \u0026lt;5%, 5-\u0026lt;10%, 10-\u0026lt;20%, 20-\u0026lt;30%, 30-\u0026lt;40%, 40-\u0026lt;50%, 50%≦, were 15.9%, 8.5%, 19.5%, 12.2%, 11.0%, 6.1%, 26.8%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The median number of days from specimen submission to the reporting of analysis results (turnaround time) was 11.0 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e The LCCP success rate was analyzed for each specimen collection method and was 99.2% (95% CI: 95.9\u0026ndash;100.0%) for TBB; 96.4% (95% CI: 87.7\u0026ndash;99.6%) for TBNA; 100% (95% CI: 89.1\u0026ndash;100.0%) for CT- or US-guided puncture; and 100% (95% CI: 83.2\u0026ndash;100.0%) for pleural effusion.\u003c/p\u003e \u003cp\u003eThe median nucleic acid yield and DIN/RIN in the cytology specimens were 546.0/426.5 ng (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and 9.2/4.7 for DNA/RNA, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Of 94 FFPE tissues, tumor content was recorded in 83 cases, with a median of 30% and a mean of 37% (range 2%-80%), all of which were successfully analyzed using LCCP. The Pearson correlation coefficient of VAFs between tissue FFPE samples and cytology specimens was 0.815 for the 85 cases where mutants were detected in cytology specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). The ratio for double-stranded DNA: total DNA was 27.1/12.8% for cytology: FFPE specimens, indicating the higher quality of cytology specimens compared to the FFPE specimens (paired \u003cem\u003et\u003c/em\u003e-test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Additionally, background noise during mutation analysis was significantly lower in cytology samples than in FFPE samples (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first multicenter study to prospectively evaluate the feasibility of an NGS lung cancer gene panel using cytology specimens. Our findings demonstrated a greater success rate for gene analysis using cytology specimens over conventional methods. Due to the adequate nucleic acid yield, high quality, and strong positive mutant allele frequency correlation with FFPE specimens, cytology specimens have the potential to replace tissue samples for detecting activating gene mutations.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe success rate of the LCCP using cytology specimens far surpassed that of conventional methods. Of 248 FAS cases, only four failed due to either defective \u003cem\u003eHER2\u003c/em\u003e gene amplification or insufficient DNA yield, whereas RNA analysis showed no failures, regardless of RNA being more degradation-prone.\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e In the 150 lung adenocarcinoma cases, mutations were detected in 62% cases, which is highly consistent with other CDx methods.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSufficient nucleic acid yield and quality was confirmed regardless of the sample collection method. Moreover, the cytology specimens contained a significantly higher amount of double-stranded DNA compared to the FFPE samples collected during the same examination, suggesting higher-quality preservation. The cytology specimens are also less susceptible to external damage until nucleic acid extraction compared to FFPE specimens.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Notably, GM tube storage is highly versatile and useful for maintaining the quality of cytology specimens.\u003c/p\u003e \u003cp\u003eOur results revealed a strong correlation between the VAFs detected in cytology and tissue specimens. In contrast, in approximately half of the cases, a higher allele ratio was detected in the cytology specimens. Furthermore, our findings are consistent with the fact that cytological diagnosis has the advantage of producing faster results than histological diagnosis, thereby shortening the time between specimen collection and sample submission.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Turnaround times have been shortened in actual clinical operations using the LCCP to a median of 8 days.\u003c/p\u003e \u003cp\u003eWe performed a few additional analyses to investigate the discordance in results between the LCCP and CDx tests (Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). In the two cases where there was no positive match with the LCCP, both harbored \u003cem\u003eEGFR\u003c/em\u003e exon-21 L858R point mutations. In the first case, the LCCP detected a VAF of 0.1%, below the LOD of 0.14%. In the second case, a procedural error occurred, in which 2 mL of the pleural effusion was directly injected into the GM tube without centrifugation. Of the five cases with discordant positive predictive values, minor allelic frequency mutations were detected by the LCCP in two cases, while one case had a co-mutation of \u003cem\u003eEGFR\u003c/em\u003e Ex19del and \u003cem\u003eKRAS\u003c/em\u003e G12C. The other three cases, including two cases with an \u003cem\u003eALK\u003c/em\u003e fusion gene and one with a \u003cem\u003eRET\u003c/em\u003e fusion gene, were associated with either false positives or sub-minor clones. In discordant cases, the signal remained around the LOD, and a large deviation between tumor content and the estimated VAF was estimated by the pathologist.\u003c/p\u003e \u003cp\u003eThis study had a few methodological limitations. First, this study did not perform a direct comparison but rather a verification of superiority based on a 90% success rate threshold calculated from pooled historical tissue panel data.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Some recent studies have reported high diagnostic rates using other NGS procedures, however, they were based on retrospective analyses using archived samples with known gene mutation information.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Second, the LCCP gene panel test is limited to eight druggable driver mutations but the amount of nucleic acid sample (both DNA and RNA) necessary for testing is equivalent to that used for ODxTT (10 ng). However, LCCP has a much larger variant search than other NGS or PCR panels (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). LCCP was designed to expand measurable driver mutations and variants in the near future. Third, this study involved prospective case enrollment in clinical practice, the low number of rare fusion gene mutations was a methodological limitation. Finally, the integration of an automated assay system can significantly reduce sample processing time and increase the efficiency of genetic analysis. This potential advance is expected to further shorten TAT. Research is currently underway to use less invasive specimen collection techniques and to utilize cytology specimens for other solid tumors.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrated a high success rate for gene panel analysis using cytology specimens (98.4%). Based on the sufficient nucleic acid yield, high quality, and strong correlation in mutant allele frequency from FFPE specimens, we concluded that cytology specimens are suitable for gene panel testing as a substitute for tissue samples.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFFPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eformalin-fixed paraffin-embedded\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCCP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLung Cancer Compact panel\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enext-generation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSCLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003enon-small cell lung cancer\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eODxTT,Oncomine Dx Target Test Multi-CDx system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVAF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evariant allele frequencies\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Sae Yanagisawa from St. Marianna University School of Medicine, Center for Clinical and Translational Science, for data management; Jason Tonge from the St. Marianna University School of Medicine and Editage (www.editage.jp) for the language review of the manuscript; Miho Ishii, Mirai Yamago, Azusa Fujita, Mizuki Ono, Kentaro Ito, Hiroyuki Sato, and Motohiko Tanino of DNA Chip Research Inc. for their technical and experimental support; Prof. Kikuya Kato from the Nara Institute of Science and Technology for providing advice on this study; and Gene Metrics LCC and Yoko Takagi for providing GM tubes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Access, Responsibility, and Analysis:\u0026nbsp;\u003c/strong\u003eKenichiro Tanabe (Pathology and Bioregulation, St. Marianna University Graduate School of Medicine, Japan) conducted and is responsible for the data analysis. Data are available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePresentation of data:\u0026nbsp;\u003c/strong\u003eThe phase 2 cPANEL study was presented at the IASLC 2022 World Conference on Lung Cancer (WCLC) press conference held in Vienna. The phase 3 cPANEL study was presented at the IASLC 2023 WCLC held in Singapore.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e All authors have contributed significantly, and that all authors are in agreement with the content of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe gene analyses were conducted and funded by DNA Chip Research, Inc., Tokyo, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e Kei Morikawa reports financial support was provided by DNA Chip Research Inc, and received lecture fees as honoraria from Eli-Lilly, Chugai Pharmaceutical, Takeda Pharmaceutical, AstraZeneca K.K., Daiichi Sankyo, MSD, Nippon Boehringer Ingelheim Co., Ltd., and Bristol-Myers Squibb Japan. Yoshiharu Sato, Seiji Nakamura, Yumi Ueda, Fumihiko Suzuki reports a relationship with DNA Chip Research Inc that includes: employment and equity or stocks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e・The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of St. Marianna University School of Medicine (approval number 5532).\u003c/p\u003e\n\u003cp\u003e・Written informed consent was obtained from all participants prior to study initiation.\u003c/p\u003e\n\u003cp\u003e・The study registered in the UMIN Registry (UMIN000047215) on March 18, 2022. An independent data-monitoring committee reviewed the clinical data.\u003c/p\u003e\n\u003cp\u003e・Animal Studies: N/A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Conception and design: KM, MM, YS, and SN. Sample collection: KM, YT, MO, AT, SM, DM, SF, NS, and FA. Pathological evaluation: TY. Experiment: YU; Sample analysis: KM, SN, YS, YU, and FS. Statistical analysis: KT, Interpretation: KM, MM. Drafting of the first version of the manuscript: KM, YS, and TY. Manuscript review and editing: KM and MM.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang M, Herbst RS, Boshoff C. Toward personalized treatment approaches for non-small-cell lung cancer. Nat Med. 2021;27(8):1345\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41591-021-01450-2\u003c/span\u003e\u003cspan address=\"10.1038/s41591-021-01450-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosele MF, Westphalen CB, Stenzinger A, Barlesi F, Bayle A, Bi\u0026egrave;che I, et al. 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Nonsmall cell lung carcinoma: diagnostic difficulties in small biopsies and cytological specimens: number 2 in the series Pathology for the clinician edited by Peter Dorfm\u0026uuml;ller and Alberto Cavazza. Eur Respir Rev. 2017;26:170007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHendry S, Mamotte L, Mesbah Ardakani N, Leslie C, Tesfai Y, Grieu-Iacopetta F, et al. Adequacy of cytology and small biopsy samples obtained with rapid onsite evaluation (ROSE) for predictive biomarker testing in non-small cell lung cancer. Pathology. 2023;55:917\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorii E, Hatanaka Y, Motoi N, Kawahara A, Hamakawa S, Kuwata T, et al. Guidelines for handling of cytological specimens in cancer genomic medicine. Pathobiology. 2023;90:289\u0026ndash;311.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilbury CA, Creeden J, Yip WK, Smith DL, Pattani V, Maxwell K, et al. 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J Int Med Res. 2021;49:300060520982687.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeyasu Y, Yoshida T, Motoi N, Teishikata T, Tanaka M, Matsumoto Y, et al. Feasibility of next-generation sequencing (Oncomine\u0026trade; DX Target Test) for the screening of oncogenic mutations in advanced non-small-cell lung cancer patients. Jpn J Clin Oncol. 2021;51:1114\u0026ndash;122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofman V, Heeke S, Bontoux C, Chalabreysse L, Barritault M, Bringuier PP, et al. Ultrafast gene fusion assessment for nonsquamous NSCLC. JTO Clin Res Rep. 2022;4:100457.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorikawa K, Kinoshita K, Kida H, Inoue T, Mineshita M. Preliminary results of NGS gene panel test using NSCLC sputum cytology and therapeutic effect using corresponding molecular-targeted drugs. Genes. 2022;13:812.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ehttps:/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000059594\u003c/span\u003e\u003cspan address=\"http:///center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000059594\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cytology specimen, gene panel analysis, next-generation sequencing, non-small cell carcinoma, variant allele frequency.","lastPublishedDoi":"10.21203/rs.3.rs-6403904/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6403904/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThere are no prospective studies to estimate whether cytology specimens can replace tissue samples using lung cancer gene panel analysis. We evaluated the success rate of gene panel testing and nucleic acid yield and quality when using cytology specimens for lung cancer over tissue specimens.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this prospective study, clinical cytology specimens collected via transbronchial brushing, needle aspiration washing, and pleural effusion were stored in a nucleic acid stabilizer. The primary endpoint was the superior success rate of gene analysis using cytology specimens over the conventional success rate using tissue specimens.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe full analysis set included 248 cases. The success rate for gene panel analysis using cytology specimens was 98.4% (95% confidence interval (CI), 95.9–99.6%) with a positive concordance rate of 97.4% (95% CI, 91.0–99.7%) by other companion diagnostic kits. The median value for nucleic acid yield and quality (DNA/RNA integrated number) of cytology specimens was 546.0/426.5 ng and 9.2/4.7 for DNA/RNA, respectively. The Pearson correlation coefficient of variant allele frequency between tissue formalin-fixed and paraffin-embedded (FFPE) sample and cytology specimens for mutant cases was 0.815. The ratio of double-stranded to total DNA showed that cytology specimens were of significantly higher quality than FFPE specimens.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe success rate of cytology specimensin gene analysis was significantly higher than conventional data. Because of the sufficient nucleic acid yield, high quality, and high correlation of mutant allele frequency compared to FFPE specimens, cytology specimens are suitable for panel testing as tissue substitutes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eClinical Trial Registration\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTrial Registration: UMIN Registry: UMIN000047215(cPANEL trial)\u003c/p\u003e\n\u003cp\u003ehttps://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000053766\u003c/p\u003e","manuscriptTitle":"Prospective multicenter validation of a next-generation sequencing panel using cytology specimens for lung cancer: cPANEL","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 10:47:57","doi":"10.21203/rs.3.rs-6403904/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-20T12:43:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T20:10:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-30T15:13:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246120359941771010322550957976285074093","date":"2025-05-26T13:07:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T10:45:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271663843187273154938824299849914291683","date":"2025-05-22T16:50:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"318287199110730904866710400574638089470","date":"2025-05-20T06:03:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T04:36:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-29T14:29:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-11T14:52:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-11T10:57:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-04-11T10:56:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3fefa6ab-2fe7-4d19-944b-f499c59f165d","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T16:01:14+00:00","versionOfRecord":{"articleIdentity":"rs-6403904","link":"https://doi.org/10.1186/s12885-025-14770-0","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2025-10-09 15:57:47","publishedOnDateReadable":"October 9th, 2025"},"versionCreatedAt":"2025-05-15 10:47:57","video":"","vorDoi":"10.1186/s12885-025-14770-0","vorDoiUrl":"https://doi.org/10.1186/s12885-025-14770-0","workflowStages":[]},"version":"v1","identity":"rs-6403904","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6403904","identity":"rs-6403904","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}