Abstract
Background
The safety of using a cutting needle when performing a core-needle biopsy is of major concern, in particular for small lung tumors or tumors near the hilum.
Purpose
To investigate the usefulness of CT-guided fine needle aspiration biopsy (FNAB) of the lung in obtaining tumor tissue for epidermal growth factor receptor (EGFR) mutation analysis in advanced lung cancer patients.
Material and Methods
Forty-three patients with stage IIIB–IV lung cancer were enrolled. In all patients, CT-guided FNAB was performed using an 18-gauge or 20-gauge Chiba aspiration needle for histology diagnosis and EGFR mutation analysis. Complications associated with CT-guided FNAB were observed, and the specimen mutational assessments were recorded.
Results
The obtained tumor samples ranged from 0.5–1.5 cm in length and were adequate for histological and DNA analyses in all patients. No patient had a pneumothorax or hemoptysis. Minor needle tract bleeding appeared in eight patients. Mutation analysis was satisfactorily demonstrated in 23 mutations and 20 non-mutations. Ten and 13 mutations were identified by 18-gauge and 20-gauge needle biopsies, respectively. EFGR mutations, including 12 cases of EGFR exon 19 deletion and 11 cases of exon 21 point mutation, were present in 21 patients with adenocarcinomas, one with squamous cell carcinoma, and one with undifferentiated carcinoma.
Conclusion
CT-guided FNAB is a feasible and safe technique for obtaining lung tumor tissues for EGFR gene mutation analysis.
Lung cancer is the leading cause of cancer death in many countries of the world (1). Nearly 70% of non-small-cell lung cancer (NSCLC) patients present with advanced, unresectable disease at the time of presentation (2). The epidermal growth factor receptor (EGFR) is over-expressed in 40–80% of patients with NSCLC, with a much greater prevalence among East Asians than Caucasians (3). Targeted therapy using EGFR inhibitors has shown greater clinical response rates in the subgroup of patients with the following characteristics: women, non-smokers, adenocarcinoma histology, and East Asian origin (2). Thus, EGFR gene mutations may serve as biological markers for response to therapy (4), and the identification of active mutations of EGFR in tumor tissue is highly desirable for treatment stratification in this subpopulation of lung cancer patients (5, 6).
Determination of EGFR mutations has previously been preferred from surgically resected tumor specimens. However, surgical interventions to obtain specimens for DNA analysis are more invasive and more expensive, and they carry increased morbidity and mortality to the patients. Moreover, resected specimens may be unintentionally biased toward a minor group of patients eligible for resection, as close to 70% of NSCLC patients are at an advanced, unresectable stage at presentation (4). CT-guided coaxial core-needle biopsy of the lung has been shown to have great diagnostic accuracy, sensitivity, specificity, and negative predictive value (7) and has become the mainstay for non-surgical procurement of tissues from the lungs. Studies of surgical specimens and small tissue biopsies have shown up to 80% agreement in gene mutation analyses of lung cancer (8). Specimens obtained from CT-guided core needle biopsy were adequate for EGFR gene mutation analysis (9).
However, complications associated with CT-guided biopsy using a core needle, e.g. pneumothorax, hemoptysis or massive peritumoral hemorrhage, are of major concern (9). Significant and moderate degrees of pneumothorax, hemoptysis or peritumoral hemorrhage can be life-threatening and require urgent interventions. Specifically, the safety of performing core-needle biopsy when the lesion is close to the hilum of the lung is of considerable concern.
In this study, we prospectively investigated the feasibility and safety of CT-guided lung biopsy to obtain fresh lung cancer tissue for EGFR mutation detection in advanced lung cancer patients, using a fine aspiration needle, as opposed to the cutting needle used in core-needle biopsy.
Material and Methods
Patients
Forty-three consecutive patients with only advanced and unresectable stage IIIB or IV lung cancers were included between May 2008 and October 2010. Patients with operable lung cancer were excluded because EGFR mutation analysis in these patients would be performed on surgical specimens. There were 16 male and 27 female patients, aged from 38–80 years old with a mean of 62 years. No patients had received any chemotherapy before they underwent CT-guided fine needle aspiration biopsy (FNAB) to obtain tissue for EGFR demonstration. The risks and complications of the procedure were explained to the patients, and informed consent was obtained. The internal review board of our hospital approved this study.
CT-guided FNAB
All patients had received CT examinations prior to the CT-guided FNAB. All the lung tumors were located at the peripheral region of the lung (more than 1.0 cm away from the pulmonary hilum) on the CT images. The largest accessible lung mass was selected for biopsy, with additional consideration of the needle puncture pathway and the direction of the targeted mass. The transthoracic distance to the lung mass was chosen to be the shortest, without obvious emphysematous changes or bullae in the pathway of the needle entrance. Penetrating the pleura repetitively was avoided at the same site. Bleeding tendency was checked upon pre-biopsy CT examination.
CT-guided biopsies were performed with a dual-slice CT (Hispeed NX/i; GE Healthcare, Milwaukee, WI, USA) using a standardized protocol. The scan parameters were as follows: slice thickness 5 mm, 120 kV, 220 mA, 1.5 pitch, spiral acquisition, soft-tissue and lung kernel reconstructions. After disinfecting and sterile draping the patient's skin, local anesthesia was administered with 2% lidocaine solution. The standard percutaneous biopsy technique was performed by two experienced radiologists, both with a minimum of 10 years of experience, using a fine Chiba aspiration needle through a small skin incision. The 18-gauge and 20-gauge Chiba needles (Precisa; Latina, Italy) were used for the procedures, respectively, in 20 and 23 patients.
The solid parts of the tumors were assumed to be the targeted areas for biopsy. Limited scanning was performed to localize the lesion and to document the needle progression to the depth of the target. After the needle reached the targeted site, we separately obtained four tissue samples for each tumor by using four 50-mL syringes attached to the needle and tilting the needle tips in different directions. The tumor tissues were preserved in 10% formalin. After the diagnosis of primary lung cancer was confirmed by a conventional histological study, serial blank (unstained) tumor sections were sent for DNA analysis to detect EGFR mutations. Immediately after the biopsy, a CT scan was performed again to assess biopsy complications.
EGFR mutation analysis
The obtained tissues samples were sent to the laboratory center (TaKaRa Biotechnology Co. Ltd; Dalian, China) for EFGR mutation detection. EGFR mutational analysis of DNA was performed using polymerase chain reaction (PCR)–based direct sequencing of exons 18–21, as described previously (10). In brief, the EGFR reference sequence was obtained from the NCBI database. Genomic DNA was extracted from tumor samples with the TaKaRa DEXPATTM Kit (TaKaRa). PCR assays were then performed with obtained genomic DNA. Purified PCR products were sequenced in forward and reverse directions using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Version 3) and ABI PRISM 3730XL Genetic Analyzer (Applied Biosystems; Carlsbad, CA, USA). The chromatograms were analyzed by manual review.
Results
Patients and CT-guided FNAB
All patients were non-smokers (40 had never smoked, and three were ex-smokers) of East Asian descent. The clinical and tumor characteristics of the 43 patients are shown in Table 1. There were 14 patients with stage IIIB lung cancer and 29 patients with stage IV lung cancer. The obtained tumor samples ranged from 0.5–1.5 cm in length. Histological examinations of the needle biopsies diagnosed 32 adenocarcinomas, four squamous cell carcinomas, and seven undifferentiated carcinomas. The lesions were located in the left upper lobe in 12 patients and in the left lower lobe in six patients, in the right upper lobe in 16 patients, in the right middle lobe in four patients, and in the right lower lobe in six patients. The average longest cross-sectional diameter of the lesions was 4.0 cm (range 12–130 mm). Twenty-one patients had lesions smaller than 30 mm. The average depth of the lesion from the skin was 17 mm (range 0–50 mm).
Patient and tumor characteristics
All patients had successful CT-guided FNABs (Fig. 1). The tissue obtained from the biopsy was adequate for histological and DNA analyses in all patients. No patients had a pneumothorax, hemoptysis, or peritumoral hemorrhage. Eight patients showed needle tract bleeding, none of which progressed. All patients were placed under observation without any interventional management.
A 69-year-old woman with adenocarcinoma of the lung. A CT scan obtained during 20-gauge Chiba needle biopsy shows a 1.8-cm lung nodule. The patient is prone, and the targeted nodule is in the right upper lobe (a). A post-biopsy CT scan revealed minor needle tract bleeding (arrow) but without pneumothorax (b)
Mutational analysis
Mutation results were satisfactorily demonstrated in all patents, with 23 mutations found in 17 female patients and six male patients and 20 non-mutations. The observed mutations were EGFR exon 19 deletions in 12 patients and exon 21 L858R point mutations in 11 patients. Mutations appeared in 21 adenocarcinomas, one squamous cell carcinoma and one undifferentiated carcinoma and were identified by using 18-gauge and 20-gauge needle aspiration, respectively, in 10 and 13 patients.
Discussion
Chemotherapy is the mainstream therapy with the most benefits and best supportive care for advanced NSCLC patients (11). However, the therapeutic response of conventional chemotherapy is limited, as 17–22% of patients experience response rates with a median survival of 7–8 months achieved, even when treated with new chemotherapeutic regimens (12). It has been shown that EGFR mutations are commonly found in patients of East Asian origin (5, 6). Targeted therapeutic agents that inhibit the phosphorylation of the intracellular tyrosine kinase of EGFR have been shown to be beneficial for treating certain NSCLC mutations with EGFR over-expression (2). Mutational analysis is thus becoming an increasingly important component of clinical care. For patients with advanced lung cancer, molecular analysis using non-surgical biopsy samples is an important technique, which is helpful for predicting the outcome of the targeted treatment and is essential before starting the targeted treatment.
In this study, CT-guided FNAB was successfully applied to obtain tumor tissues for EGFR mutation analysis in all patients and achieved a comparable mutation rate to core needle biopsy (9). The overall EGFR mutation rate of non-small cell lung cancers in our series was 53% (23/43 cases). The reported mutation rate in Chinese patients has varied from 47% (13, 14) to 72.3% (9). Several demographic and pathological factors are related to EGFR mutation prevalence. A great number of EGFR mutations are observed, mainly among patients with adenocarcinoma, who have never smoked and who are of East Asian ethnicities. The overall incidence of EGFR mutations among Asian patients is approximately 30% overall: 47% among patients with adenocarcinoma and 56% among patients who have never smoked (15). The observed high prevalence of EGFR mutations in our study was likely related to the patients enrolled who were non-smokers. In addition, the mutation rate reported could be also have been affected by the biopsy procedures. In general, greater mutation results have been found with CT-guided core needle biopsy (9) than with blinded sampling methods.
Mutation status is routinely assessed in biopsies, but cytological specimens are frequently the only samples available. Boldrini et al. (16) found that cytological specimens of advanced/metastatic lung adenocarcinomas were perfectly adequate for the molecular analysis of EGFR and Kras mutations. Smouse et al. (17) further compared EGFR sequencing among 227 surgical and 12 cytological specimens and found that cytological specimens were suitable for EGFR sequencing and showed comparable sensitivity for mutations. Moreover, Lozano et al. (18) detected EGFR mutations simultaneously in biopsies and cytological samples from 20 patients and found that the mutation status was identical in patients who had both biopsies and cytological samples analyzed. Taken together, these observations suggest that the assessment of EGFR mutations in cytological samples should be also feasible (19).
CT-guided needle biopsy is simple and less invasive than surgical excision biopsy. Most patients can tolerate the procedure. However, the specimens taken by the needles are small. The procurement of such small biopsy samples ought to be sufficient to yield DNA for gene profiling. Contamination by non-cancer cells during the biopsy procedure should be avoided. Direct puncture using a biopsy needle of lung masses under image guidance could obtain specimens from the cancer center or from the solid parts. In our series, gene mutation demonstration was successfully achieved in all cases, whether using an 18-gauge or 20-gauge aspiration needle. This finding suggests that CT-guided FNAB could achieve the same success as when using small core needles, in terms of sufficient and reliable samples for mutational analysis of malignant lung tumors (20). However, FNAB could be favorable for lesions smaller than 2 cm, in particular those close to the hilum or major blood vessels. Almost half of the patients in our study had lesions smaller than 30 mm, and some patients had lesions close to the hilum of the lung.
The safety of CT-guided needle biopsy is another major concern in clinical settings. Common complications are pneumothorax and hemorrhage (hemoptysis or peritumor hemorrhagic parenchyma). It has been shown that the incidence of pneumothorax induced by 17-gauge coaxial needles was greater than by 19-gauge coaxial needles (21). However, Cheung et al. (9) observed a greater incidence of pneumothorax using 19-gauge coaxial needles (13.3%) than 17-gauge coaxial needles (12.5%). A multivariate analysis of 660 CT-guided coaxial cutting needle lung biopsies suggested that the greatest pneumothorax rates were related to lesion sizes ≤2 cm, lesion depths of 0.1–2 cm and procedures performed by less experienced radiologists (22). In contrast, we used fine aspiration needles for CT-guided lung biopsies. No patients had pneumothorax, whether using an 18-gauge or a 20-gauge fine needle.
Hemoptysis and massive peritumoral hemorrhage are two other important life-threatening complications. The greatest rate of hemoptysis was correlated with a lesion size ≤2 cm, a lesion depth ≥2.1 cm and the absence of pleural effusion (22). However, these predisposing risk factors are not absolute contraindications for CT-guided lung biopsy. In our series, all procedures followed the rules for choosing the shortest distances to the masses, as well as avoiding pleural penetration more than once, if possible. No patients had hemoptysis or focal hemorrhagic parenchyma. Only minor needle tract bleeding occurred in eight patients. Nevertheless, needle tract bleeding is unavoidable, no matter how minimally invasive the procedure is.
Previous studies using FNAB have reported a 52.7–62% incidence of pneumothorax in patients with small (≤1.0 cm in diameter) pulmonary lesions (23, 24). In a large series of 229 patients with lesions measuring <15 mm, the pneumothorax rate was 11.8% (25). However, Ohno et al. (26) documented a 28.4% pneumothorax incidence in 162 patients with pulmonary nodules measuring ≤20 mm, and they found that a percentage of predicted forced expiratory volume in 1 s greater than 70%, a single puncture, and a needle path length of 40 mm or less were associated with a low rate of pneumothorax. Oikonomou et al. (27) reported a 20.6% rate of pneumothorax using ultrathin needles (25 gauge) in 123 patients and found that pulmonary functional impairment was a risk factor for pneumothorax. These studies suggested that many factors might affect the incidence of pneumothorax, e.g. lesion size, lung functional impairment, and even the biopsy procedure. The absence of pneumothorax in this study might be assumed to be the result of the relatively larger pulmonary lesions (mean 40 mm) or the experience of the radiologists. It should be noted that the number of patients enrolled in the present study was relatively small. Further studies with greater numbers of patients or more patients with smaller pulmonary lesions are needed to observe the safety of CT-guided FNAB.
In conclusion, CT-guided lung FNAB was successfully used to obtain fresh cancer tissues for EGFR gene mutation analysis in lung cancer patients, with a relatively low incidence of complications. The obtained specimen quantity using this technique was adequate for gene demonstration. CT-guided FNAB is a feasible and safe technique that can be used to acquire lung cancer tissue for molecular analysis in lung cancer treatment, especially for patients who already have advanced lung cancer at the time of diagnosis.