Biomarker assessment is very important for the treatment of non-small cell lung cancer (NSCLC). For example, the importance of epidermal growth factor receptor (EGFR) gene mutation assessment before administration of EGFR tyrosine kinase inhibitors in patients with NSCLC is widely accepted. The European Commission approved gefitinib for first-line treatment of NSCLC in patients with EGFR mutations after a recent publication.1 The difference of mRNA expression level may also affect the treatment sensitivity or resistance. For example, excision repair cross-complementing 1 (ERCC1) and ribonuclease reductase subunit 1(RRM1) gene expressions may be correlated with shorter survival after cisplatin and gemcitabine treatment.2
A majority of the genetic alterations of NSCLC in surgically resected primary tumors have been previously analyzed; however, we often encounter difficulties in obtaining tissue samples, as many patients have advanced disease at the time of first presentation and are not eligible for surgery. Patients with lung cancer are often diagnosed by fine-needle aspiration (FNA). Samples obtained by FNA are usually adequate only for cytopathologic diagnosis. In fact, genetic diagnosis is currently not being performed as a routine clinical examination, except in a clinical research setting.3 However, the diagnostic approach using FNA-driven specimens have possibilities to add significant importance in patients with advanced NSCLC.
Molecular testing for gene mutation can be performed using DNA. DNA can be extracted from FNA cytologic samples.4–6 RNA can also be extracted from FNA samples7; however, the extraction of RNA is more difficult than DNA due to its instability. For mRNA expression analysis by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR, high-quality RNA is necessary. Tissue availability, however, is a major issue in lung cancer research. There is an urgent demand for a tumor banking system of samples obtained by FNA procedures. Optimizing the methodology for the processing and storing of FNA samples will open up possibilities of various molecular analyses. The diagnostic approach used for FNA samples can have a significant impact on patients with lung cancer.
MATERIALS AND METHODS
Surgically resected lobectomy samples were collected from 7 patients diagnosed with primary NSCLC. Each lobe had primary lung tumor of more than 3 cm in diameter. FNA using 21-gauge needle was performed by independent cytopathologists. A 10 mL syringe was used for applying negative pressure. The needle was moved back and forth 5 to 10 times within the tumor.
Sample Storage Protocol
The aspirates were stored according to the following protocol: in group 1, the aspirate was flushed out into a collection tube using air and then snap frozen with liquid nitrogen and in group 2, the aspirate was mixed with an RNA preservative solution (RNA later; Ambion, Austin, TX) and stored overnight at 4°C. After sample collection for both groups, these samples were stored at −80°C for 6 months.
Extraction of RNA
RNA was extracted from each sample and the quality and quantity were measured. Group 1 samples were kept at −80°C just before the RNA extraction. Group 2 samples were centrifuged at 3000 g for 10 minutes to make a pellet, and then the RNA preservative solution was removed completely using a pipette. The purification of total RNA for both groups was performed using an miRNeasy Mini Kit (QIAGEN Inc, Valencia, CA) following the manufacturer's instructions.
Measurement the Quantity and Quality of Extracted RNA
The amount of extracted RNA was measured using a spectrophotometer (NanoDrop: Thermo Scientific, Wilmington, DE). The ratio of absorptions at 260 nm versus 280 nm was measured simultaneously. Two microliters of RNA solution was applied to the NanoDrop. The quality of RNA was evaluated using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). An Agilent RNA 6000 Nano Kit (Agilent Technologies) was used, and the RNA Integrity Number (RIN) was measured. Bioanalyzer analysis was performed by The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada.
Reverse Transcription and Quantitative Real-time RT-PCR
As a reference standard, we used qPCR Human Reference Total RNA (Clontech, Mountain View, CA). A high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used for reverse transcription. The 500 ng sample of extracted RNA and reference standard RNA were used for reverse transcription. Quantitative real-time RT-PCR was performed in a LightCycler 480 (Roche Applied Science, Indianapolis, IN) using a QuantiFast SYBR Green PCR Kit (Qiagen) in a total volume of 20 mL. The samples were analyzed in duplicates. Human actin β (hACTB) was selected as a housekeeping gene, which was used for internal control of gene expression analysis. Keratin 19 (KRT19) was used as a target gene in this study, which is known as high expression of epithelial tumor cells. The sequences of primers used in this study are shown in Table 1. PCR amplification was carried out following the manufacturer's instructions. PCR products were subjected to electrophoresis on a 2% agarose gel.
The fluorescence intensity level was analyzed using LightCycler 480 Software (Roche). KRT19 mRNA expression was normalized to hACTB mRNA expression and presented as an expression ratio. Data were analyzed by Microsoft Excel and statistical analysis was performed using JMP 8.0 (SAS Institute, Cary, NC).
Quantity and Quality of Extracted RNA
RNA was extracted from all samples with a final elution volume of 50μl for both groups. The median amount of total RNA was 33.9 μg for group 1, and 35.8 μg for group 2. There was unevenness of the amount of extracted RNA because the amount of aspirates was not controlled. The mean ratio of absorptions at 260 nm versus 280 nm for the purity of RNA was 2.02 for group 1 and 1.97 for group 2. As for the quality of RNA, the mean RIN was 3.5 for group 1 and 6.3 for group 2 (Table 2). All RIN of each sample in group 2 was higher than that of group 1. The quality of RNA was significantly better in group 2 (P=0.0073, Mann-Whitney U test).
RT-PCR and Quantitative Real-time PCR
RT-PCR for hACTB and KRT19 was successfully performed in all samples. A clear single band could be observed at the appropriate area by electrophoresis for both genes (Fig. 1). The mRNA expression value of hACTB showed intrasample variation depending on the storage method. The lower RIN number samples tended to show lower mRNA expression of hACTB (Table 3). The relative mRNA expression ratio of KRT19 to hACTB also showed intrasample variation, except in 1 sample (sample No. 3) (Fig. 2). The mean relative expression value of KRT19 was 5.34 for group 1 and 3.92 for group 2. There were no significant difference for relative gene expression value of KRT19 between group 1 and group 2 (P=0.44, Mann-Whitney U test); however, the ratio of mRNA gene expression varied from 0.58 to 3.32 within the same sample.
We evaluated the different FNA sample storing methods for molecular testing using RNA. FNA samples could be stored by the following methods: (1) frozen with liquid nitrogen and (2) mixed with RNA preservative solution. Both of the samples could be stored at −80°C. RNA could be extracted from all samples stored in different conditions and extracted RNA can be used for RT-PCR. For molecular testing on FNA samples to become a routine clinical examination, the procedure must be simple, easy, and relatively inexpensive. In addition, the sample must be able to withstand long-term storage without degradation. However, Florell et al8 discovered that samples stored at −80°C for 6 months degraded the RNA within the tissue in both methods. As a pilot study, we extracted RNA from 2 FNA samples only 3 days after collection. We extracted RNA from the samples with high quality (RIN was 7.5 for both groups, data not shown). Hence, the RNA extraction protocol of this study was acceptable. The results demonstrate that the initial quality of the samples for RNA extraction is very good, and an optimal storage method is still needed.
RNA preservative solution has several advantages for clinical use. RNA later is a nontoxic aqueous sulfate salt solution and it can be kept at room temperature. The samples mixed with the RNA preservative solution can avoid RNA degradation and can undergo long-term storage.9 It is reported that high quality of RNA could be extracted from lung cancer biopsy samples that used RNA later for storage.10 In addition, RNA later does not affect the histologic structure and the results of immuhistochemistry; hence, it can be used for routine tissue banking protocol.11 FNA for thoracics targets many kinds of diseases such as lung cancer, metastatic diseases,12–13 lymphoma,14 and amyloidosis.15 Furthermore, multidirectional analysis will be required for specific molecular testing.16 From this point of view, the RNA preservative solution may have an advantage for FNA sample storage.
RNA integrity is very important to obtain reliable results. Highly degraded RNA will lead to a misconception of lower mRNA expression for specific genes.17 Purity of RNA can be assessed by using a spectrophotometer at wavelengths of 260/280 nm. High 260/280 nm absorption ratio means a high degree of purity; however, the degradation of RNA cannot be evaluated using a spectrophotometer. RNA integrity can be evaluated by visual inspection of the electropherogram, which shows 2 distinct peaks, 1 for 28 S and 1 for 18 S, and a flat baseline. It is known that the measurement of RIN using Bioanalyzer is superior to the manual of electropherogram ratio between the 28 S and 18 S peaks.17 In this study, all extracted RNA could be used for RT-PCR; however, the result of quantitative PCR for hACTB showed intrasample variation depending on the storage method. For obtaining reliable gene expression quantification data, high-quality RNA will be required.
In conclusion, RNA extracted from FNA samples can be used for molecular testing. The combination of FNA and novel approaches in molecular analysis and biomarker assessment holds promise for enhanced diagnosis and personalized management of lung cancer.
The authors thank Dr Terumoto Koike and Ms Guan Zehong for technical assistance with PCR procedures.
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