The rise of molecular targeted chemotherapy has changed the fundamental concept of treatment in non–small-cell lung cancer (NSCLC). A relationship between epidermal growth factor receptor (EGFR) and EGFR–tyrosine kinase inhibitors was verified by large-scale clinical trials.1,2 Additional relationships between companion diagnoses are under investigation, and treatment with new agents are under development, including crizotinib for anaplastic lymphoma kinase (ALK), c-ros oncogene 1, or receptor tyrosine kinase (ROS1) fusion gene–positive lung cancer3,4; sunitinib for ret proto-oncogene (RET) fusion gene–positive lung cancer5,6; and vemurafenib for BRAF mutation (V600E)–positive lung cancer.7
Therefore, the existence of gene mutations or fusion genes becomes a key factor for treatment decisions in NSCLC. However, the methodology used to detect such gene mutations is still under development. Although operational specimens are suitable for genetic analysis with respect to sample assurance and containing massive tumor cells, genetic analysis before the operation is preferable for acquisition of genetic information for initial treatment planning, an issue in the application of companion biomarkers. Bronchoscopic samples are general samples that may provide definitive diagnoses and that can be obtained during preoperative stages of clinical evaluation. To overcome this issue, molecular analyses of microhistological samples (e.g., bronchoscopic histological samples) have been established, and we have reported the utility of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) sampling, which can be challenging, for molecular analysis.8,9 Thus, establishing a methodology for the molecular analysis of cytological specimens is the next step to developing personalized medicine by using genetic information and molecular targeted therapy.10
The aim of this study was to determine the utility of bronchoscopic ultra-microsamples (uMSs), which were obtained from rinsed fluid of the used needle as well as endoscopic histological samples (histological cores) for multiple molecular profiling analyses in NSCLC.
PATIENTS AND METHODS
The patient eligibility criteria were as follows: (1) patients who had undergone conventional bronchoscopy for the diagnosis of primary lesions or EBUS-TBNA for the diagnosis of metastatic lymph nodes; (2) patients from whom both histological and cytological specimens could be obtained; (3) patients with malignant cells proven by rapid onsite cytology in both cytological and histological specimens; and (4) patients providing informed consent for the study. A well-trained operator carried out all sampling procedures under local anesthesia and conscious sedation, without intubation. In general, all procedures were performed in the outpatient clinics of our hospitals.
Bronchoscopic Sampling from the Primary Tumor
For the diagnosis of primary lung lesions, including both peripheral and central lesions, transbronchial forceps biopsy (TBFB) was performed with a flexible bronchoscope (BFS-type260; Olympus, Tokyo, Japan). to obtain histological specimens after TBNA to obtain cytological specimens, and these procedure were repeated until malignant cells were confirmed by rapid onsite cytology with a touch smear (for TBFB) or smear (for TBNA) slide stained with Diff-Quik (Sysmex Corporation, Kobe, Japan). In eligible cases, a portion of the TBFB sample was preserved on ice immediately and stored at −80°C with 1 ml lysis buffer (MagNA Pure Compact RNA Isolation Kit; Roche Diagnostics, Mannheim, Germany); this sample was the control sample for molecular analysis (endoscopic histological core; core), whereas the remaining portion of the TBFB sample was used for pathological diagnosis. Used TBNA needles were rinsed in a 20-ml saline bottle and washed after every TBNA procedure. The remnant cells in the bottle were referred as considered the uMS in this study; these cells were well mixed and divided into three bottles: 5 ml for bacterial culture, 5 ml for the diagnosis, and 10 ml for molecular analysis. The uMS for molecular analysis was equally divided and immediately centrifuged for 2 minutes at 2000 rpm. The obtained cell pellet was stored at −80°C. This uMS preparation procedure was performed within 20 minutes of sample collection. Pathologists confirmed the final cytological diagnosis of the uMS, and the results, including those from molecular profiling, were compared with those of histological core samples as a control. Malignant diagnosis in cytological specimen was defined by findings of strongly suggestive of malignancy (class IV) or conclusive for malignancy (class V) according to Papanicolaou’s classification. This sampling sequence is described in the left (TBNA) and center (TBFB) columns of Figure 1.
EBUS-TBNA from Metastatic Nodes
For the diagnosis of metastatic lymph nodes, including both mediastinal and hilar nodes, a convex-probe EBUS (BF-UC260F-OL8, Olympus), dedicated ultrasound scanner (EU-C2000/EU-C60; Olympus), and dedicated 22-gauge needle equipped with an internal stylet (NA-201SX-4022; Olympus) were used to obtain histological and cytological specimens. Malignant cells in TBNA droplets from every puncture were confirmed by stained smears in onsite screening. Histological cores were preserved on ice immediately and stored at −80°C; these cores were used for control samples in molecular analyses. Used needles were rinsed in a 20-ml saline bottle for every puncture, and the uMSs were obtained and divided into three bottles for analysis using the same methods as those used for bronchoscopic samples. This sampling sequence is described in the right column of Figure 1.
DNA and RNA Extraction
The uMS pellet and tissue from the core were used to obtain DNA or RNA. These samples were homogenized with a MagNA Lyser for 60 seconds at 6500 rpm and placed on a cooling block for 1 minute. Supernatants (100 μl) were used for DNA extraction with a MagNA Pure Compact Nucleic Acid Isolation Kit. Supernatants (350 μl) from homogenates were centrifuged and RNA was extracted using a MagNA Pure Compact RNA Isolation Kit. cDNA cloning was performed with a First Strand cDNA Synthesis Kit (Roche). Extracted DNA and cDNA were stored at 4°C.
Detection and Sequencing of Mutations
For the screening of mutations or fusion genes, we applied high-resolution melting (HRM) analysis11 for both DNA and RNA analysis. We used a protocol and primer sequences that have been described previously to detect EGFR (4 primer sets),12 KRAS (2 primer sets),11,12 and BRAF (2 primer sets)13 mutations in DNA extracts in one run. For reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, optimized primer sets based on previously reported primer sets were used in two runs (ALK and ROS1, RET). For detection of the ALK fusion gene, a new forward primer (EML4-ALK v3b1; 5′-CAAGCATAAAGATGTCATCATCAAC-3′) was added to the eight previously reported primers,14 and multiplex RT-PCR was performed with these nine primer sets in one test. For ROS1 fusion gene detection, two optimized forward primers (TPM3-ROS1, 5′-GCTGAGTTTGCTGAGAGATCGGTAG-3′ and LRIG3-ROS1, 5′-CCAACACAGATGAGACCAACTTGC-3′) and four reported primers (SDC4-ROS1, SLC34A2-ROS1, CD74-ROS1, and EZR-ROS1)15 with an optimized reverse primer (5′-CGCAGCTCAGCCAACTCTTTGTC-3′) to avoid the amplification of nonspecific products. These six primer sets were used in multiplex RT-PCR in one test. The RET fusion gene was tested by multiplex RT-PCR with three reported primer sets in one test.15 After PCR amplification, purified products were then sequenced with a capillary sequencer (3130 Genetic Analyzer; Life Technologies, Carlsbad, CA), and the actual mutation sequence or fusion locus was defined. All mutation/fusion gene detections were repeated thrice to validate the results. In total, three runs, including 11 molecular tests, were needed for each sample.
Ethics Committee Approval
The bioethics committee of Chiba University Graduate School of Medicine approved this research (No. 275). Written consent was obtained from patients, and all samples were coded and managed independently.
Patients and Pathological Diagnosis
From November 2010 to October 2012, 146 patients (149 samples) were enrolled in this study; samples consisted of 52 primary tumors and 97 metastatic lymph nodes. By the revealed final pathological diagnosis, 15 samples were omitted and 134 samples were eligible for this study (107 from men and 27 from women). The reasons for the omitted cases included no malignant findings obtained in the histological core of the primary tumor (n = 4), metastasis of renal cell carcinoma (n = 1), gastric carcinoma (n = 1), small-cell lung cancer (n = 2), sarcoma (n = 1), malignant lymphoma (n = 1), and inflammation with severe atypia (n = 5). The latter 11 samples were diagnosed from core samples obtained by EBUS-TBNA. Final histological diagnoses in eligible samples of primary tumors and metastatic nodes consisted of adenocarcinomas in 30 and 50 (63% and 58%), squamous cell carcinoma in 16 and 27 (33% and 31%), and other cancers in 2 and 9 (4% and 10%) samples, respectively. Target lesion and size of primary tumor and metastatic node are listed in Supplementary Table 1 (Supplemental Digital Content 1, http://links.lww.com/JTO/A490). TBNA or EBUS-TBNA was attempted an average (±SD) of 2.37 ± 1.15 or 6.45 ± 3.26 times, and malignant cells were confirmed 1.71 times (81%) or 3.92 times (72%), respectively. No pathogens were cultured from uMSs in this study. Sample enrollment and pathological results are illustrated in Figure 2.
DNA and RNA Extraction
DNA was successfully extracted from both uMSs and histological cores for every sample. DNA was eluted in a final volume of 200 μl, and extracted DNA was obtained at concentrations of 9.3 ± 8.8 ng/μl from core samples and 3.7 ± 3.4 ng/μl from uMSs in bronchoscopic samples of primary tumors. In EBUS-TBNA samples of metastatic nodes, the extracted DNA concentrations were 24.8 ± 27.3 ng/μl from cores and 5.2 ± 4.1 ng/μl from uMSs. RNA was also successfully extracted from both uMSs and histological cores for every sample. RNA was eluted in a final volume of 50 μl. Extracted RNA was obtained at concentrations of 64.0 ± 45.0 ng/μl from cores and 12.8 ± 9.9 ng/μl from uMSs by bronchoscopy or 129.8 ± 140.3 ng/μl from cores and 18.1 ± 14.5 ng/μl from uMSs by EBUS-TBNA. These results are summarized in Supplementary Table 2 (Supplemental Digital Content 1, http://links.lww.com/JTO/A490).
Mutation/Fusion Gene Detection
We confirmed that our primer sets work properly by using following artificial sequences as the positive control; EGFR Exons 18–21 Genomic DNA Reference Standards, K-Ras Codons 12 & 13 Genomic DNA Reference Standards, B-Raf Codon 600 Genomic DNA Reference Standards (Horizon Diagnostics, Cambridge, United Kingdom), EML4-ALK (variant 1, 2, 3a), KIF5B-ALK, KIF5B-RET, CCDC6-RET, TPM3-ROS1, SDC4-ROS1, SLC34A2-ROS1, CD74-ROS1, EZR-ROS1, LRIG3-ROS1 (GenScript, Piscataway, NJ). HRM curve of these control sequences are illustrated in Supplementary Figure 1 (Supplemental Digital Content 3, http://links.lww.com/JTO/A492). In total, 73 genetic disorders were identified, including EGFR mutations (n = 21), KRAS mutations (n = 11), BRAF mutations (n = 1), ALK fusion genes (n = 5), ROS1 fusion genes (n = 1), RET fusion genes (n = 1), and silent mutations (n = 22). Double mutations (including silent mutation) were detected in 12 samples (Table 1), and HRM-PCR curves (melting peaks) of each mutation and fusion, along with the identified sequences, are shown in Figure 3.
Comparison of the Result from Histological Cores and uMSs
The results of comparisons between cores and uMSs are summarized in Table 2. Final cytological evaluations of uMSs revealed only 34% of malignant cells, despite that all eligible samples showed malignant findings in the final cytological and histological evaluation of cores. In uMSs from primary tumors, 25% (12 of 48) of malignant cells were confirmed, whereas 38% (33 of 86) of malignant cells were confirmed in uMSs from metastatic lymph nodes. In total, 1474 genetic tests were conducted, and complete concordance was confirmed in 1464 tests (99.3%). Major discrepancies occurred in six tests (0.4%); mutations were detected only in the histological core in four tests or only in uMSs in two tests. All major discrepancies occurred in samples from primary tumors. Minor discrepancies occurred in four tests (0.3%), and although these mutations were identified by HRM-PCR, their sequences were not identified by direct sequencing. This discrepancy occurred in uMSs, and the sequences were successfully identified in histological cores.
Through this study, we showed the utility of uMSs and bronchoscopic histological cores with respect to multiple molecular profiling analyses in NSCLC. Our data suggested that molecular analysis using uMSs provides accurate, easy-to-obtain data that can be used to replace conventional sampling methods in the development of molecular targeted therapies for patients with NSCLC.
The methodology was designed such that the uMS contained malignant cells, both through the sampling procedure itself and through onsite cytological evaluation. Generally, the primary objective of rinsing the biopsy needle is to clean the needle for the next biopsy procedure; usually this rinse liquid is considered waste. Our method allowed us to conduct molecular analysis using this waste without any additional biopsies or without repeated biopsies using other modalities. A comparison of the final pathological diagnosis and rapid onsite cytological evaluation revealed that only 34% (45 of 134) of samples were confirmed as malignant, an issue that has been previously reported;16 however, our study allowed us to perform molecular testing by a logically well-designed sampling procedure known as the “recycled method,” which enabled us to obtain malignant cells. uMS analysis may to increase the sensitivity to detect cancer through the molecular analysis in cases where we do not get enough tissue even for standard cytology.
In this study, 0.7% (10) of tests showed molecular profile discrepancies between histological cores and uMSs. Three minor discrepancies were caused by problems with sensitivity, which will be discussed later. We also observed about six major discrepancies between samples, and all these discrepancies occurred during sampling from primary lesions. For sampling from primary lesions, it consisted of two steps: TBFB and TBNA. In contrast, EBUS-TBNA was consisted of one step in which both histological and cytological specimens were obtained and no major discrepancies occurred in uMSs versus EBUS-TBNA samples. Thus, we assumed that the separate biopsy procedure was one cause of the discrepancies between bronchoscopic biopsy samples and uMSs. In addition, we retrospectively investigated the six major discrepancy cases; TBNA was attempted an average of 2 ± 1.09 times, and malignant cells were detected 1.33 times. These numbers are lower than those for EBUS-TBNA (6.45 ± 3.26 and 3.92 times, respectively). Multiple appropriate punctures may increase the total cell amount in uMS collected, and the collection of more cells may prevent the occurrence of major discrepancies. Notably, four of the major discrepancies were mutations detectable only in uMSs. Clinically, biopsy cores contain some normal lung tissue, and the rate of tumor occurrence is variable. Even when touch smear cytology shows malignant cells, whole core samples sometimes contain very small amounts of tumor cells, and this may be one cause for the faults observed in molecular analysis. In this regard, EBUS-TBNA is a preferable sampling procedure, avoiding quality discrepancies between core samples and uMSs.
The relationship between molecular biomarkers and molecular targeting agents has been well recognized, and the importance of molecular testing has increased. Therefore, a universal and accessible method through which to analyze samples is needed. Because of this, molecular testing costs have become an issue. The goal of molecular testing is to accomplish individualization and optimization of treatment using molecular targeting agents, which can be expensive. Obviously, less-expensive testing is favorable; in the United States, EGFR mutation gene tests cost approximately $700. Our PCR-based gene profiling method used a universal DNA/RNA extraction kit and conventional PCR methods and consisted of automated DNA/RNA extraction and three runs (1 for DNA and 2 for RNA) for all 11 molecular tests in each case. Thus, the HRM-PCR method can reduce the running cost because PCR primers are the only additional consumable required when a new target is discovered. The quality of the test result was assured by the combination of the biopsy procedure itself (containing malignant cells) and the high sensitivity of the method to detect mutations/fusion genes. Thus, the total cost for the present analyses was approximately U.S. $350 per patient in this study, suggesting that this method must be cost-effective. The actual molecular screening cost is listed and compared in Supplementary Table 3 (Supplemental Digital Content 1, http://links.lww.com/JTO/A490), and our institution absorbed all costs for molecular testing in this study because this survey was conducted for research purposes.
In the near future, molecular targeted therapy will become the mainstream treatment for anticancer chemotherapy, and new agents and corresponding new biomarkers will be found. Our screening method is able to accommodate newly discovered biomarkers with only the design of appropriate primer sets and may be easy to apply with high sensitivity for known mutations/fusion genes. In addition, using uMS screening methods allows for the preservation of small biopsy samples. If needed, preserved cores (frozen tissues) or paraffin-fixed cores may be used for secondary molecular marker investigations.
There were some limitations to this study. The first was the sensitivity of the PCR-based method. Our focus here was to show the quality of concordance between biopsy core samples and uMSs, and, as emphasized earlier in this article, these molecular tests need to be conducted conveniently and inexpensively. HRM analysis can achieve these goals by allowing us to perform multiple molecular analyses in one assay. For RNA testing, PCR-based methods have been reported to have sufficient sensitivity,17 whereas for DNA testing, previously reported methods (e.g., peptide nucleic acid–locked nucleic acid PCR-clamp methods,18 Scorpion amplification refractory mutation system methods,19 etc.) showed higher sensitivity, allowing detection of mutations with 1% of the tumor amount; HRM analysis requires greater than 5% of the tumor amount to achieve sufficient molecular analysis.20 For the verification of molecular analysis, we also surveyed the tumor ratio of core samples and uMSs in 20 randomly selected samples in our series, which could be evaluated both pathologically and cytologically. All these core samples and uMSs showed greater than 5% tumor content. Furthermore, we also analyzed EGFR mutations by the PCR-clamp method for verification in 15 cases, and the results were completely concordant with the results of HRM analysis. Therefore, we propose that this study limitation is acceptable, and we now need to explore the respective high-sensitivity methods for each genetic disorder to improve sensitivity. Further investigations are required to compare the sensitivity between mass samples and biopsy samples.
The second limitation of this study was the heterogeneity of the tumors. Bronchoscopic or EBUS-TBNA samples represented only partial sections and may not have been representative of the whole tumor in heterogeneous lung cancer. This issue is common in the analysis of microsamples and is difficult to overcome because this problem arises from the nature of the tumor itself. Metastatic lesions sometimes show different genetic profiles from the primary resected tumor,21 so this limitation may not even be overcome by using large operational specimens. Repeated biopsy of the same target lesion is one way to reduce this limitation, and from this point of view, analysis by uMS may lessen this limitation by representing accumulated malignant cells from multiple biopsy procedures.
The third limitation was that a single gene-analysis modality was applied for all uMSs. Especially for fusion gene detection, we have previously reported that multimodal analyses are desirable.9 This screening method using uMSs was specialized for molecular analysis, and, because only one third of uMSs showed malignant cells, pathological evaluation was limited by using uMS. For multimodal analysis, histological cores can be used for fluorescent in situ hybridization or immunohistochemistry if needed and may allow for improved sensitivity by additional molecular surveys.
In conclusion, appropriately prepared uMSs, in addition to histological core samples, are useful for multiple molecular profiling with respect to accuracy, cost, and convenience in NSCLC.
This study was supported by grants from the Chiba Foundation for Health Promotion and Disease Prevention (YS) and AstraZeneca Research Grant #201200639 (YS).
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Bronchoscopic ultrasound; Bronchoscope; Biomarker; Microsampling
Copyright © 2014 by the European Lung Cancer Conference and the International Association for the Study of Lung Cancer.