A total of 302 patients with suspicious NSCLC were enrolled from Department of Ultrasound, General Hospital of Daqing Oilfield between January 2014 and January 2015 in the present study. The age range of patients was between 43 and 72 years old with a median age of 52 years old and the male (148) to female (154) ratio was approximately equal. Patients suspected of having NSCLC were eligible for the present study. Inclusion criteria was as follows: Those confirmed with NSCLC, with stage IV disease at the time of study entry, those with measurable disease at the time of study entry, those with resolution to grade ≤1 adverse events, and those with adequate hematologic function, hepatic function, renal function and coagulation function. Exclusion criteria was as follows: Prior chemotherapy for relapsed and/or metastatic NSCLC, neo-adjuvant/adjuvant chemotherapy is permitted if at least 12 months has elapsed between the end of chemotherapy, prior treatment with Epidermal Growth Factor Receptor targeting small molecules or antibodies, radiotherapy or surgery (other than biopsy) within 4 weeks prior to enrollment, active brain metastases, any other current malignancy or malignancy diagnosed within the past 5 years, known pre-existing interstitial lung disease. The study protocol was approved by the Ethics Committee of General Hospital of Daqing Oilfield (QDGH: 2014061218; Daqing, China) and informed consent was obtained from all patients.

The ultrasound diagnosis system analyzed contrast-enhanced ultrasound clinical trials using a preprogrammed setting. The preprogrammed setting was optimized to reach the best image formation. The mechanical index was set between 0.2–0.4 to avoid destruction of the fragile microbubbles that contain nanoparticles. The details of principles and settings of the contrast-enhanced ultrasound were as previously described (22).

A novel ultrasound contrast agent was introduced with the aim of diagnosing NSCLC at an early stage. The multi-targeted tyrosine kinase inhibitor Lenvatinib (Selleck Chemicals LLC, Boston, MA, USA) was covalently bound with the nanoparticles of super paramagnetic iron oxide particles as previously described (23). The nanoparticle ultrasound contrast agent and Optison (GE Healthcare Life Sciences, Shanghai, China) entered the lung by breathing and an atomizer (GE Healthcare Life Sciences) was used to ensure the entire lung was covered. Following administration by respiration, the nanoparticle ultrasound contrast agent was distributed through the respiratory system. The microbubbles, within the nanoparticle, contained the multi-targeted tyrosine kinase inhibitor Lenvatinib and Lenvatinib was capable of transporting the target to tumor cells. Furthermore, the microbubbles targeted the lesion through the pulmonary circulation due to their small diameter. After 30 min, the nanoparticle ultrasound contrast agent could be visualized using an imaging system (General Electric GE Voluson S8; GE Healthcare Life Sciences). No side effects were observed in patients following its use.

Tumor cells were obtained from patients with NSCLC. The cells (1×10⁵) were cultured for 72 at 37°C in 6-well plates in Dulbecco's modified Eagle's minimal essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Biowhittaker; Lonza, Basel, Switzerland). The cell density was adjusted to 5×10⁴/ml. The cells were then incubated with primary anti-carcino embryonic antigen (catalogue no. ab4451; Abcam, Cambridge, MA, USA) or PBS (mock group) for 12 h at 4°C, then incubated with anti-rabbit immunoglobulin (Ig)G and anti-mouse IgG (catalogue nos. W4011 and W4021, respectively; Promega Corporation, Madison, WI, USA) for 2 h at 37°C at a dilution of 1:50 for squamous cell carcinoma antigen. Subsequently, cells were washed three times with PBS to remove the primary antibody at a dilution of 1:50 and the anti-goat IgG-horseradish peroxidase secondary antibody (catalogue no. A5420, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a dilution of 1:50 was added for 15 min at 4°C. The cells were then washed with PBS to completely remove the secondary antibody. Finally, cells were observed using a fluorescence microscope. The cells were fixed in neutral formalin for 15 min at 37°C and then embedded in paraffin and tissue sections were cut to 4 µm thick. Cells were then fixed with pre-warmed PBS + 4% paraformaldehyde for 30 min at 37°C and blocked with PBS + 0.3% bovine serum albumin (BSA, Sigma-Aldrich; Merck KGaA) for 30 min at 30°C. Conjugates [IgG-680-PTX (JZ200505), α-CEA-680 (JZ21834), and α-CEA-680-PTX (XT200514), Thermo Fisher Scientific, Inc.] were serially diluted 1:2 (60 µg/ml) in PBS + 0.3% BSA and 1.62 µM Hoechst 33342 dye (catalogue no. 62249; Thermo Fisher Scientific, Inc.) and incubated in 6-well plates for 1 h at room temperature. Immunofluorescence procedures were performed as previously reported (24). For histological staining, tumor sections were stained with hematoxylin and eosin and observed under light microscope, as previously reported (25).

A total of 302 patients with suspected NSCLC were enrolled in the test to confirm a final diagnosis of lung cancer. The mean age of the patients was 57.5±14.4 years old and there were fewer male patients than the female ones. The primary details of the characteristics of the patients are summarized (Table I). The tumor cells did not appear to show tumor metastasis in all patients, as observed by the modified contrast-enhanced ultrasound. Among these patients, 42 out of 302 (13.9%) suspicious NSCLC patients were diagnosed as tumor-free following analysis with contrast-enhanced ultrasound and subsequently confirmed by histological observation. Improved contrast-enhanced ultrasound demonstrated that 26.2% of patients (79/302) were diagnosed with squamous cell carcinoma, 28.8% of patients (87/302) were diagnosed with large cell carcinoma and 31.1% of patients (94/302) were diagnosed with adenocarcinoma according to the American Collegeof Chest Physicians evidence-based clinical practice guidelines (26). The diagnosis of NSCLC classification was summarized in Table II. These data indicated that the improved contrast-enhanced ultrasound of the present study was effective at diagnosing NSCLC.

Primary squamous cell carcinoma of the lung is a common type of lung cancer (26). Although ultrasound detection has many benefits in the diagnosis of cancer, the effectiveness of ultrasound in the diagnosis of cancer is limited, particularly for rough, irregular shaped tumors and heterogeneous tumor stroma. In the present study, a contrast-enhanced ultrasound was used to diagnose early stage squamous cell carcinoma. According to the imaging findings (Fig. 1A), 26.2% (79/302) of patients suspected of having NSCLC were diagnosed with squamous cell carcinoma at the primary sites of lung lesions (Table II). This diagnosis was confirmed by immunofluorescence and histological staining of squamous cell carcinoma (Fig. 1B). The observations of the present study exhibited high efficacy of the improved contrast-enhanced ultrasound combined with nanoparticle contrast agent in the diagnosis of squamous cell carcinoma, and all cases were consistent with the histological findings.

A previous study (8) has indicated that large cell carcinoma accounts for <10% in patients with lung cancer. Diagnosis of large cell carcinoma in the chest wall is challenging due to poor imaging quality of conventional ultrasound and the fact that features remain nonspecific in diagnostics. An accurate diagnosis in the clinic may provide an opportunity for tumor eradication and complete rehabilitation. In the present study, the effectiveness of an improved contrast-enhanced ultrasound combined with a nanoparticle contrast agent at detecting early-stage large cell carcinoma was investigated. The diagnostic results indicated that 28.8% (87/302) of patients suspected of having NSCLC were diagnosed with large cell carcinoma (Table II). In addition, the diagnosis was further confirmed by immunofluorescence and histological staining of the large cell carcinoma in all patients with large cell carcinoma (Fig. 2A and B). These observations indicate that a contrast-enhanced ultrasound may be an efficient and inexpensive way to detect early stage large cell carcinoma.

Primary adenocarcinoma of the lung tumors is one of the most common types of tumor, and this has been reported in previous study (4). Currently, contrast-enhanced ultrasonography is a potential and helpful medical protocol that may be used as a pain-free and non-invasive method of diagnosing adenocarcinoma. A previous study has documented the diagnosis of hepatocellular and cholangiocellular carcinomas or metastatic adenocarcinoma of the liver using contrast-enhanced ultrasonography and demonstrated contrast-enhanced ultrasonography was efficient at diagnosing tumors of the liver (27). However, to the best of our knowledge, there have been no studies analyzing the effectiveness of contrast-enhanced ultrasound used in the diagnosis of early-stage primary adenocarcinoma. The contrast-enhanced ultrasound data indicated that 31.1% (94/302) of patients with suspected NSCLC were confirmed to have early-stage adenocarcinoma by pathological analysis (Table II). The presence of adenocarcinomas in all patients was confirmed by immunofluorescence and histological staining (Fig. 3). Therefore, the results of contrast-enhanced ultrasound diagnosis of NSCLC patients were consistent with the immunofluorescence and histological staining results, indicating that contrast-enhanced ultrasound combined with nanoparticle contrast agent is a reliable protocol and effective at diagnosing adenocarcinomas.